Carbon-14 labeling of Saxagliptin (BMS-477118)

Article abstract of DOI:10.1002/jlcr.1450

An efficient synthesis of carbon-14-labeled Saxagliptin (BMS-477118) is described. Initial synthesis of the key radiolabeled intermediate (S)-N-Boc-2-(3′-hydroxyadamantyl)glycine 2a utilized adamantanemethanol in a 10-step sequence. To shorten the sequenc

Full text of DOI:10.1002/jlcr.1450

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 1224–1229.
Published online 1 October 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1450
JLCR
Research Article
Carbon-14 labeling of Saxagliptin (BMS-477118)
KAI CAO1,*, SAMUEL J. BONACORSI JR1, BALU BALASUBRAMANIAN1, RONALD L. HANSON2, PERCY MANCHAND2,
JOLLIE D. GODFREY JR1, RITA FOX1, LISA J. CHRISTOPHER1, HONG SU1 and RAMASWAMY IYER1
1 Bristol-Myers Squibb Research & Development, Route 206 and Province Line Road, Princeton, NJ 08540, USA
2 Bristol-Myers Squibb Research & Development, 1 Squibb Drive, New Brunswick, NJ 08901, USA
Received 30 May 2007; Revised 17 July 2007; Accepted 25 July 2007
Abstract: An efficient synthesis of carbon-14-labeled Saxagliptin (BMS-477118) is described. Initial synthesis of the
key radiolabeled intermediate (S)-N-Boc-2-(30-hydroxyadamantyl)glycine 2a utilized adamantanemethanol in a 10-
step sequence. To shorten the sequence, 1-adamantylzinc bromide was reacted with ethyl [1, 2-14C]oxalyl chloride
catalyzed by [1,10-bis(diphenylphosphino)ferrocene]dichloropalladium (II). In five steps, 2b was synthesized in an
overall yield of 20% based on ethyl [1, 2-14C]oxalyl chloride. Compound 2b was subsequently converted to [14C]
BMS-477118 in a short sequence. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: Saxagliptin; DPP-IV inhibitor; carbon-14 synthesis
Introduction
HO
HO
DPP-IV (dipeptidyl-peptidase IV) inhibition has been
studied extensively as a treatment for Type 2 diabetes.1
Inhibition of DPP-IV results in preserved levels of
endogenous insulinotropic hormone, glucagon-like
peptide 1 (GLP-1), which in turn leads to enhanced
insulin efficiency.2,3
14C
1b
1a
N
N
14C
O
14C
O
H2N
H2N
N
N
Saxaglipin (BMS-477118) 1, a highly efficacious, stable
and long-acting DPP-IV inhibitor possessing a hydro-
xyadamantyl group was discovered by Bristol-Myers
Squibb scientists.4 The compound is currently under-
going clinical evaluation for the treatment of Type 2
diabetes. To support ongoing preclinical and clinical
studies, carbon-14-labeled BMS-477118 was needed for
metabolic and pharmacological studies of the molecule.
HO
HO
2a
2b
Boc
Boc
14C
14
OH
OH
14C
N
H
N
H
C
O
O
10-bis (diphenylphosphino)ferrocene]dichloropalladium
(II) (dppfPdCl2). This report describes both synthetic
approaches, with particular emphasis on the efficient
synthesis of [14C] BMS-477118 (1b).
Two different radiolabeled syntheses of
[
14C]
BMS-477118 are described in this paper. The first
synthesis of [14C] BMS-477118 (1a) required 13 steps
and utilized K14CN as the starting material. To shorten
the sequence, other labeling options were explored. A
five-step route was devised to prepare the key inter-
mediate 2b from the reaction of adamantylzinc bromide
with ethyl [1, 2-14C]oxalyl chloride in the presence of [1,
Results and discussion
Synthesis of (S)-N-Boc-[1-14C]-2-(30-Hydroxyadaman-
tyl)glycine (2a)
*Correspondence to: Kai Cao, Department of Chemical Synthesis,
As illustrated in Scheme 1, 1-adamantanemethanol 3
was tosylated, followed by SN2 reaction with K14CN to
introduce carbon-14 label. Hydrolysis of the resulting
Bristol-Myers Squibb Research
& Development, Route 206 and
Province Line Road, Mail-Stop: H14-02, Princeton, NJ 08543, USA.
E-mail: kai.cao@bms.com
Copyright # 2007 John Wiley & Sons, Ltd.

CARBON-14 LABELING OF SAXAGLIPTIN (BMS-477118) 1225
Scheme 1
cyano intermediate 5 yielded carboxylic acid 6. Methyl
ester 7 was prepared in 96% yield by treatment with
methanol and acetyl chloride, and a-hydroxylation with
trimethyl phosphite/oxygen gave 8 in 68% yield. Swern
oxidation gave the a-keto-ester 9. Hydroxylation of the
adamantyl ring was accomplished with 50% HNO3/
98% H2SO4 giving 10. Hydrolysis with 1 N NaOH gave
11.5 The yield for the combined hydroxylation/hydro-
lysis steps was 80%. Intermediate 11 was converted to
12 by reductive amination, using a liquid enzyme
concentrate from E. coli JM110 containing phenylala-
nine dehydrogenase (PDH) and formate dehydrogenase
(FDH).6 The bio-conversion mixture was treated with
(Boc)2O and 2a was isolated in 60% yield.
formation at the adamantyl bridgehead. There is
limited literature precedence describing synthetic ap-
plications of adamantyl bridgehead anions.7 Low yields
and poor reproducibility are common problems. The
most efficient way to introduce the a-keto-ester two-
carbon unit was through the use of an oxalic ester
derivative. Reagents and conditions have been reported
for the reaction of oxalic esters using organolithium
and Grignard reagents.8–10 Initial attempts using
adamantyl lithium and Grignard reagents with di-
methyl oxalate failed. Very low yields of the desired
product were observed for these reactions, due to the
poor reactivity and competing acyloin ester condensa-
tion. More reactive methyl oxalyl chloride has been
reported to react with organozinc reagents catalyzed
by Pd in moderate to excellent yields.11 Radiolabeled
ethyl oxalyl chloride was reacted with adamantylzinc
bromide prepared from Rieke1 Metals to obtain the
desired product 14. The use of adamantylzinc bromide
(Aldrich product) derived from the oxidative addition of
Rieke zinc to adamantyl bromide was critical to the
successful preparation of 2b.
The above sequence, although starting from cheap and
readily available K14CN, was lengthy and time consum-
ing. In addition, intermediate 5 was volatile and required
special precautions during hydrolysis to avoid losses.
Labeled [14C] BMS-477118 (1a) was prepared through
this route to support some preclinical studies, but the
liabilities of early label introduction and the length of
the synthesis were significant obstacles for a repeat
synthesis. A decision was made to pursue other labeling
approaches for the preparation of clinical supplies.
Methyl oxalyl chloride was used in unlabeled practice
acylation reactions with yields close to 80%. Unfortu-
nately, methyl[1, 2-14C]oxalyl chloride was not com-
mercially available as a labeled precursor. Ethyl[1,
2-14C]oxalyl chloride was substituted and gave some-
what lower yields. The synthesis of 2b began
by coupling 1-adamantylzinc bromide with ethyl
[1, 2-14C]oxalyl chloride in the presence of [1, 10-bis
Synthesis of (S)-N-Boc-[1, 2-14C]-2-(30-Hydroxyada-
mantyl)glycine (2b)
Introduction of a labeled glycine fragment was an
obvious option, but this approach required C–C bond
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1224–1229
DOI: 10.1002.jlcr

1226 K. CAO ET AL.
Scheme 2
Scheme 3
(diphenylphosphino)ferrocene]dichloropalladium
(II)
The target compound, 1b, a TFA salt was obtained
in 88% yield by deprotection using 2 N aqueous
HCl in isopropyl alcohol followed by preparative
high performance liquid chromatography (HPLC)
purification.
(Scheme 2). The a-keto-ester 14 was isolated in 42%
yield after purification. Hydroxylation of 14 in 50%
HNO3 and 98% H2SO4 followed by hydrolysis in 1 N
NaOH gave hydroxyadamantyl keto-acid 16, the sub-
strate for bioconversion. PDH/FDH-catalyzed reductive
amination, followed by Boc protection provided 2b in
87% yield.
Modified coupling of 2 with 20
EDC/HOBt-mediated coupling of 2b with 17 following
dehydration with TFAA in pyridine generated a fairly
complicated reaction mixture. Preparative HPLC se-
paration was required to purify the product. To improve
the yield and simplify isolation, a HATU-catalyzed
coupling of carboxylic acid 2b with nitrile 20 was
utilized (Scheme 4). The desired product 19 was
obtained in 90% yield, and was then converted to 1b
Completion of [14C] BMS-477118 synthesis
Carboxylic acid 2b was coupled with l-cis-4,
5-methanoprolinamide 17 using EDC/HOBt (Scheme
3). The product formed, 18, was treated with TFAA
in pyridine at ꢀ108C to dehydrate the amide. The
Boc-protected nitrile 19 was obtained in 53% yield.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1224–1229
DOI: 10.1002.jlcr

CARBON-14 LABELING OF SAXAGLIPTIN (BMS-477118) 1227
Scheme 4
by deprotection. This modification significantly im-
proved the overall radiochemical yield of 1b from 10%
for the EDC/HOBt coupling to 17% using this modified
procedure.
0.05% TFA; flow rate: 4 mL/min; gradient program:
100% (A) gradient change over 15 min to 100% (B), then
hold for 10 min at a 100% (B). TLC was carried out on
silica gel 60 F254 plates (Merck). Ethyl [1, 2–14C]oxalyl
chloride (97 mCi/mmol) was obtained from GE
Health Sciences. 1-Adamantylzinc bromide was
obtained from Rieke Metals (via Aldrich). All other
reagents were obtained from the Aldrich Chemical
Company of Milwaukee, WI, and were either ACS grade
or the highest quality material commercially available.
Identities of labeled compounds were established
by co-analysis with the fully characterized unlabeled
compounds using HPLC, NMR and mass spectrometry
when possible.
In summary, an efficient synthetic route for the
synthesis of [14C] BMS-477118 (1b) was developed.
Compared with the earlier synthesis of 1a, this improved
sequence was shorter and scalable to the long term
needs of the program. Carbon-14 labels were conveni-
ently introduced into the glycine moiety via a palladium-
mediated reaction of 1-admantylzinc bromide with ethyl
[1, 2-14C]oxalyl chloride. The success of this route
expands the known examples of adamantyl bridgehead
anion chemistry. An improved set of coupling conditions
were demonstrated for the reaction of Boc-protected
hydroxyladamantyl glycine 2b with 20 using HATU.
Multiple batches of [14C] BMS-477114 were prepared to
support preclinical and clinical studies.
Experimental
[1, 2-14C]-Ethyl 2-oxo-2-adamantylacetate (14)
Materials and methods
Ethyl [1, 2-14C]oxalyl chloride (200mCi, 97mCi/mmol)
was vacuum transferred to a 25mL flask. Anhydrous
THF (2mL) was added to the same flask under
Ar atmosphere. [1,10-Bis(diphenylphosphino)ferrocene]-
dichloropalladium (II) (8mg) was charged to second
flask under Ar, cooled to ꢀ478C, followed by addition of
1-adamantylzinc bromide in THF (13, 0.5 M, 7.4 mL).
The contents of the first flask were transferred to the
second flask through a cannula in 5 min. The reaction
mixture was stirred at ꢀ478C for 15min, warmed,
and held at ambient temperature for 3 h. Hydrochloric
acid (1N, 10mL) was added to the stirred mixture,
which was stirred for a total of 10min. The mixture was
extracted with t-butyl methyl ether (3ꢁ 10mL), and the
combined organic layers were washed with water
(10 mL ꢁ 2). After concentration under a steady stream
of nitrogen gas, the crude product was purified by flash
chromatography (hexanes/ethyl acetate 8/1 v/v).
Pooled fractions were evaporated to yield 190 mg 14
1H NMR and decoupled 13C NMR were recorded on
a
Bruker DRX-400 MHz or a Bruker Avance II
300 MHz spectrometer. Mass spectra were recorded
on either a Thermo LCQ or a LXQ spectrometer.
Radioactivity determinations were carried out with a
Wallac liquid scintillation counter, using standard
methods and Ecolite as the scintillation fluid. HPLC
purification and analysis were performed on
a
Rainin Dynamax HPLC system consisting of two SD-
200 pumps, a Rainin UV-1 detector, a ProStar PDA
detector and an IN/US b-RAM radioactive flowthrough
detector. The HPLC method for in-process analytical
HPLC analysis consisted of mobile phases A: 0.05%
TFA in water, and B: 0.05% TFA in acetonitrile; column:
YMC hydrosphere C18 (3 mm, 4.6 mm ꢁ 250 mm);
flow rate: 1 mL/min; gradient program: 10% (B) hold
for 5 min then ramp to 90% (B) over 10 min and hold for
5 min, then ramp back to 10% (B); detection: UV
(220 nm) or radiometric. Preparative HPLC was
conducted using a YMC AQ (10 mm ꢁ 250 mm); mobile
phase A: 10% MeOH in water containing 0.05%
TFA, mobile phase B: 90% MeOH in water containing
(83 mCi, yield 42%). 1H-NMR (300MHz, CDCl3)
d
(ppm): 1.36 (t, J¼ 7:2 Hz, 3H), 1.70–1.95 (m, 12H),
2.07 (br s, 3H), 4.31 (dd, J¼ 7:2 Hz, 2H); 13C-NMR
(75.48MHz, CDCl3) d (ppm): 201.7, 164.1, 61.5, 44.9,
37.2, 36.3, 27.6, 14.1.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1224–1229
DOI: 10.1002.jlcr

1228 K. CAO ET AL.
[1, 2-14C]-Ethyl 2-oxo-2-(30-hydroxyadamantyl)
acetate (15)
hydroxide (5 N). The reaction flask was placed in a
temperature-controlled incubator with orbital oscillation
at 220 rpm while maintaining a constant temperature of
408C. The reaction was monitored by HPLC and the pH
was maintained at 8.0 with periodic additions of sodium
hydroxide (5 N) or sulfuric acid (5 N) until the reaction
was judged to be complete by HPLC (90% conversion at
100h). The reaction mixture was cooled to room
temperature, and the pH was adjusted to 9.5 with
sodium hydroxide (5N). Cellular debris was removed by
centrifugation (3 times at 2500rpm for 10min). The
resulting supernatant (ꢂ20 mL) was treated with NaOH
(10N) to adjust the pH to 13, then treated with (Boc)2O
(2.55g, 11.7 mmol) in THF (25 mL). After stirring at room
temperature for 3 h, THF was removed under a steady
stream of nitrogen gas. Additional protein precipitate
was removed by centrifugation. The basic solution (pH
11) was washed with isopropyl acetate twice (20 and
10 mL), then adjusted to pH 2.5–3 with hydrochloric acid
(1 N). The resulting aqueous solution was extracted with
isopropyl acetate (20mLꢁ 3), dried over Na2SO4, filtered
and evaporated to give 1.65 g of 2b (40 mCi, 24.2 mCi/mg,
yield 87%). 1H-NMR (300 MHz, DMSO-d6) d (ppm): 1.37
(s, 9H), 1.30–1.60 (m, 12H), 2.07 (br s, 2H), 3.67 and
6.79 (2 br d, 1 H, rotamers). 13C NMR (100.62MHz,
DMSO-d6) d (ppm): 28.2, 29.7, 29.8, 35.08, 37.1, 38.1,
40.4, 46.6, 62.5, 66.7, 78.0, 155.7, 172.3.
Sulfuric acid (1.9 mL) followed by nitric acid (50%,
175 mL) were added to a flask cooled to 08C. A separate
flask containing the a-keto-ester (14, 190 mg,
0.85 mmol, 83 mCi) was cooled to 08C, and the pre-
mixed sulfuric/nitric acid mixture was added all at
once. The reaction mixture was then stirred at 08C for
90 min. Ice–water (25 mL) was added to the mixture,
and it was then stirred for 30 min. The aqueous mixture
was extracted with ethyl acetate (10 mL ꢁ 3), and the
pooled ethyl acetate layers were evaporated. Flash
chromatography (hexanes/ethyl acetate 2/3 v/v) gave
140 mg 15 (54 mCi, 385.7 mCi/mg, yield 65%). 1H-NMR
(CDCl3) d (ppm): 1.34 (t, J¼ 7:1 Hz, 3H), 1.50–1.80 (m,
12H), 2.32 (m, 3H), 4.30 (dd, J¼ 7:2 Hz, 2H); 13C-NMR
(75.48 MHz, CDCl3) d (ppm): 14.1, 29.9, 34.8, 36.1,
44.1, 44.5, 48.2, 61.8, 68.1, 163.6, 199.9.
[1, 2-14C] 2-Oxo-2-(30-hydroxyadamantyl)acetic
acid (16)
a-Keto-ester (15, 140mg, 54mCi, 0.63mmol) and un-
labeled 15 (223 mg, 1.44 mmol) were dissolved in THF
(2 mL), cooled to 08C, and sodium hydroxide (1 N, ꢂ2mL)
was added to give a final solution pH of 14. After stirring
at 08C for 60 min, water (20 mL) was added, followed by
extraction with ethyl acetate (20 mL). The aqueous phase
was acidified to pH 3 with 1 N hydrochloric acid, and then
extracted with ethyl acetate (15 mL ꢁ 3). Combined ethyl
acetate layers were dried over sodium sulfate, filtered and
evaporated. The resulting product 16 (274 mg, 46 mCi)
was diluted with unlabeled 16 (1.03 g), giving 1.31 g of 16
(46 mCi, 35.1 mCi/mg, yield 85%). 1H-NMR (300 MHz,
DMSO-d6) d (ppm): 1.45–1.70 (m, 12H), 2.17 (br s, 3H);
13C-NMR (75.48 MHz, DMSO-d6) d (ppm): 29.4, 34.6,
36.0, 44.0, 44.7, 47.0, 66.0, 166.1, 202.5.
(S)-N-Boc-[1,2-14C]-2-(30-Hydroxyadamantyl)glycine-
l-cis-4,5-methanoprolinenitrile (19)
Procedure A. Coupling reaction between N-Boc-hydro-
xyadamantyl glycine (2b, 1.65g, 40mCi, 5.1 mmol) and
l-cis-4,5-methanoprolinamide 17 (1.23 g, 5.8 mmol) in
MeCN (6mL) was carried out using general peptide
coupling conditions: HOBt (784mg, 5.8 mmol), EDC
(1.55 g, 8.1 mmol) and DIPEA (1.88g, 14.6mmol) at
room temperature overnight. One normality hydrochlo-
ric acid (6mL) and brine (6mL) were added to the
reaction mixture. The reaction mixture was extracted
with EtOAc (20 mL). The EtOAc layer was washed with
20% KHCO3. After evaporation, the crude amide was
redissolved in EtOAc (3.2 mL), cooled to ꢀ58C, and
treated with pyridine (1.7mL). Trifluoroacetic anhydride
(1.8mL) was added over 10min, then the mixture was
warmed to room temperature over 40min. The solvent
was removed under reduced pressure and the inter-
mediate trifluoroacetate nitrile was hydrolyzed with 25%
K2CO3 (9.6mL) in methanol (6mL) at 35–408C for 1 h.
Methanol was removed and the aqueous layer was
extracted with EtOAc (20 mLꢁ 2). The extracts were
washed with hydrochloric acid (1N) and brine, dried
over Na2SO4, filtered and concentrated. The crude
product was recrystallized from isopropanol/water,
(S)-N-Boc-[1, 2-14C]-2-(30-Hydroxyadamantyl)
glycine (2b)
[1, 2-14C] 2-Oxo-2-(30-hydroxyadamantyl)acetic acid
16 (1.31 g, 46 mCi, 5.8 mmol) and ammonium formate
(740 mg, 11.7 mmol) were dissolved in water (10.5 mL).
Sodium hydroxide (10 N) was added to adjust the pH to
8.0, then NADþ (3.5 mg) and dithiothreitol (DTT,
2.0 mg) were added and the total volume was brought
to 12 mL with water. The reaction mixture was warmed
to 408C, and the pH was readjusted to 8.0 with sodium
hydroxide (5 N). PDH/FDH enzymes from E. coli extract
(1300 units, 2.6 g of the liquid enzyme extract) were
added and the pH was again adjusted to 8.0 with sodium
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1224–1229
DOI: 10.1002.jlcr

CARBON-14 LABELING OF SAXAGLIPTIN (BMS-477118) 1229
and then further purified by preparative HPLC. 19
(21 mCi, 19.1 mCi/mg, 1.1 g) was obtained in 53% yield.
1H-NMR (400MHz, CDCl3) d (ppm): 1.04–1.07 (m, 2H),
1.42 (s, 9H), 1.35–1.91 (m, 13H), 2.24 (m, 2H), 2.36 (dd,
J ¼ 13:2, 2.3 Hz, 1H), 2.56 (m, 1H), 3.82 (dd, J ¼ 11,
4.4 Hz), 4.45 (d, J¼ 9:6 Hz, 1H), 5.03 (dd, J ¼ 10:4,
2.3 Hz), 5.29 (br dd, J¼ 9:7 Hz, 1H). 13C-NMR
(100.62 MHz, CDCl3) d (ppm): 13.5, 17.8, 28.4, 30.2,
30.4, 35.2, 37.0, 37.5, 38.0, 41.2, 44.3, 44.4, 45.1,
46.3, 58.6, 68.6, 80.0, 119.3, 155.8, 169.9. MS (ESþ)
4135–4141; (c) Reimer MK, Holst JJ, Ahre´n B.
Eur J Endocrinol 2002; 146: 717–727; (d) Pospisilik
JA, Stafford SG, Demuth H-U, Brownsey R,
Parkhouse W, Finegood DT, McIntosh CHS,
Pederson RA. Diabetes 2002; 51: 943–950; (e)
Pospisilik JA, Martin J, Doty T, Ehses JA,
Pamir N, Lynn FC, Piteau S, Demuth H-U, McIn-
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741–750.
2. (a) Holst JJ. Diabetologia 2006; 49: 253–260; (b)
Brubaker PL, Drucker DJ. Endocrinology 2004;
145: 2653–2659; (c) Holst JJ. Horm Metab Res
2004; 36: 747–754.
14
m/z: 416, 420 (for C2) ½M þ Hꢃþ.
Procedure B. N-Boc-hydroxyadamantyl glycine (2b,
150 mg, 6.8 mCi, 45.3mCi/mg, 0.46mmol), l-cis-4, 5-
methanoprolinenitrile (20, 80mg, 0.55mmol) and HATU
(213mg, 0.56mmol) were dissolved in anhydrous DMF
(4.8 mL), placed under an Ar atmosphere and cooled to
08C. DIPEA (213mg, 1.65mmol) was added over 5 min,
and the mixture was stirred at 0–108C for 5 h. The crude
product was purified by preparative HPLC to give
171 mg 19 (6.1 mCi, 35.7 mCi/mg, yield 90%).
3. Kim YB, Kopcho LM, Kirby MS, Hamann LG,
Weigelt CA, Metzler WJ, Marcinkeviciene J. Arch
Biochem Biophys 2006; 445: 9–18.
4. Augeri DJ, Robl JA, Betebenner DA, Magnin DR,
Khanna A, Robertson JG, Wang A, Simpkins LM,
Taunk P, Huang Q, Han S-P, Abboa-Offei B, Cap M,
Xin L, Tao L, Tozzo E, Welzel GE, Egan DM,
Marcinkeviciene J, Chang SY, Biller SA, Kirby MS,
Parker RA, Hamann LG. J Med Chem 2005; 48:
5025–5037.
(S)-[1, 2-14C]-2-(30-Hydroxyadamantyl)glycine-l-cis-4,
5-methanoprolinenitrile TFA salt (1b)
5. Vu TC, Brzozowski DB, Fox R, Godfrey Jr JD,
Hanson RL, Kolotuchin SV, Mazzullo Jr JA,
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20040624 CAN 141:54618 AN 2004:515478
CAPLUS.
Nitrile 19 (6.8 mCi, 0.41 mmol, 35.7 mCi/mg, 190 mg) was
dissolved in isopropyl alcohol (3 mL) and heated to 708C
under N2. Hydrochloric acid (2 N, 2.8 mL) was added, and
the reaction mixture stirred for 90 min at 708C. The
mixture was cooled to room temperature, and the solvent
was evaporated. The crude was purified by preparative
HPLC to give desired product as a TFA salt, 6 mCi,
155 mg, yield 88%. Specific activity: 38.7 mCi/mg and
radiochemical purity: 99.8%. 1H NMR (400 MHz, CD3OD)
d (ppm): 0.97 (ddd, J ¼ 11:6, 7.2, 2.8 Hz, 1H), 1.10 and
1.13 (ABq, JAB ¼ 6:6 Hz, 1H), 1.55–1.85 (m, 12H), 2.01
(ddd, J ¼ 14:3, 11.6, 5.5 Hz, 1H), 2.28 (s, 2H), 2.35 (dd,
J ¼ 13:7, 2.2 Hz, 1H), 2.62 (ddd, J ¼ 13:7, 11.0, 5.5 Hz,
1H), 3.92 (ddd, J ¼ 8:8, 6.0, 2.8 Hz, 1H), 4.28 (s, 1H),
5.19 (dd, J ¼ 11:0, 2.2Hz, 1H); 13C NMR (100.62 MHz,
CD3OD) d (ppm): 14.3, 19.2, 31.4, 31.4, 31.5, 36.0, 38.2,
39.3, 40.9, 44.8, 44.9, 46.6, 47.0, 60.0, 68.6, 120.4,
167.4. MS (ESþ) m/z: 316, 320 (for 14C2) ½M þ Hꢃþ.
6. Hanson RL, Goldberg SL, Brzozowski DB, Tully TP,
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Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1224–1229
DOI: 10.1002.jlcr