Synthesis of [1,2-3H]ethylamine hydrochloride and [ 3H]-labeled apadenoson for a human ADME study

Article abstract of DOI:10.1002/jlcr.1495

Tritium-labeled [1,2-³H]ethylamine hydrochloride was prepared through a multiple-step sequence in high radioactive specificity. The labeled amine was isolated in high purity following cartridge filtration and used subsequently in the synthesis

Full text of DOI:10.1002/jlcr.1495

Research Article
Received 23 July 2007,
Revised 17 September 2007,
Accepted 30 November 2007
Published online 30 January 2008 in Wiley Interscience
(
www.interscience.wiley.com) DOI: 10.1002/jlcr.1495
3
Synthesis of [1,2- H]ethylamine hydrochloride
3
and [ H]-labeled apadenoson for a human
ADME study
a
Yang Hong, Samuel J. Bonacorsi Jr, Yuan Tian, Sharon Gong, Donglu
Ã
a
a
a
a
a
Zhang, W. Griffith Humphreys, Balu Balasubramanian, Edward H.
a
b
Cheesman, Zhiqin Zhang, James F. Castner, and Paul D. Crane
b
b
b
3
Tritium-labeled [1,2- H]ethylamine hydrochloride was prepared through a multiple-step sequence in high radioactive
specificity. The labeled amine was isolated in high purity following cartridge filtration and used subsequently in the
synthesis of [N-ethyl-1,2- H]apadenoson, an adenosine A2a receptor agonist. The overall yield for this transformation was
3
5
6% and the radiochemical purity of the final product was greater than 99%.
3
Keywords: apadenoson; adenosine A2a receptor agonist; tritium-NMR (T-NMR); [ H]ethylamine hydrochloride
Introduction
by hydrolysis. The product was conveniently isolated by rotary
evaporation and solid-phase extraction. The synthesis of tritium-
Apadenoson (Figure 1) is a C2 and 5’-substituted adenosine labeled apadenoson was designed around the use of this
3
analog with selective agonist activity for the adenosine A2a precursor, [1,2- H]ethylamine hydrochloride, which was incor-
1
,2
receptor.
This compound induces coronary vasodilatation porated into apadenoson. This route proved to be a concise and
during myocardial perfusion imaging. It is currently in clinical efficient method for the preparation of tritiated apadenoson.
development and undergoing testing as a pharmacological
stress agent. Owing to its high potency and associated low dose, Results and discussion
a tritium-labeled analog of apadenoson with high specific
3
The synthesis of [1,2- H]ethylamine hydrochloride began with
radioactivity was required for determination of the absorption,
distribution, metabolism, and excretion (ADME) properties of
the molecule. The primary goal of the ADME study was to obtain
a pharmacokinetic profile of the compound relative to the
profile of total drug-related materials and to determine major
clearance routes for the compound. The compound for human
clinical analysis was labeled in a metabolically stable pharma-
cophore of the molecule. The suitability of the ethylamine
moiety was established through a separate BDC rat study using
the same tritium-labeled substrate planned for the human
tritium reduction of commercially available N-vinylphthalimide
4
followed by the classical Gabriel synthesis (Scheme 1).
N-Vinylphthalimide was reduced in ethanol using carrier-free
tritium gas in the presence of a Pd/C catalyst. Pure [N-ethyl-
3
,2- H]phthalimide was obtained after catalyst removal by
1
filtration. The [N-ethyl-1,2- H]phthalimide was then refluxed
3
3
overnight in 6 N hydrochloric acid. The resulting [1,2- H]ethyl-
amine hydrochloride was isolated after solvent removal and
solid-phase extraction through C-18 SepPak cartridges. The
product, in ethanol, was determined to be 95% radiochemically
pure by HPLC, and stored as a solution until utilized in the
synthesis. The overall yield for these two steps was 82%, based
on the N-vinylphthalimide precursor utilized in the tritium
reduction.
3
study. The metabolic stability of [ H]apadenoson was examined
following administration of a single 3-mg/kg (250 mCi/kg)
intravenous dose of [ H]apadenoson. Radioactivity profiling of
3
plasma, urine, and fecal extract by high-performance liquid
chromatography (HPLC) separation, fraction collection, and
scintillation counting showed that no tritiated water was
formed. Total radioactivity was also measured in plasma and
urine samples before and after drying. The results of this study
a
Bristol-Myers Squibb Research and Development, Route 206 and Province Line
showed no loss of label due to either metabolism or chemical Road, Princeton, NJ 08540, USA
3
exchange.
b
Bristol-Myers Squibb Medical Imaging, 331 Treble Cove Road, Billerica, MA
3
The radiochemical synthesis of [ H]apadenoson required 01862, USA
3
efficient methodology to prepare [1,2- H]ethylamine hydro-
chloride. A product with high purity and specific radioactivity
was obtained in excellent yield by tritium reduction of a
*
Correspondence to: Yang Hong, Department of Chemical Synthesis, The Bristol-
Myers Squibb Research and Development, Route 206 and Province Line Road,
Princeton, NJ 08540, USA.
commercially available precursor, N-vinylphthalimide, followed E-mail: yang.hong@BMS.com
J. Label Compd. Radiopharm 2008, 51 113–117
Copyright r 2008 John Wiley & Sons, Ltd.

Y. Hong et al.
NH2
O
N
N
H
N
N
O
O
OMe
N
OH OH
1
Figure 1. Structure of apadenoson.
O
O
NH2 HCl
N
T2
N
HCl (6 N)
EtOH
T
Pd/C
T
T
O
O
T
2
3
4
Scheme 1
NH2
NH2
NH2
N
N
N
N
N
N
N
N
O
N
I
O
O
O
N
1. 9/1 TFA/H O
EEDQ
O
N
I
2
N
I
HO
O
N
N
H
2. MTBE
Ethylamine, 7.5 M
ethanolic solution
H
O
O
O
O
HO
OH
5
6
7
O
NH2
NH2
Pd(PPh ) Cl , CuI
O
N
H
3 2
2
N
N
OMe
N
H
N
Et N, DMF
N
3
N
O
N
O
O
O
+
OMe
N
I
N
OH OH
OH OH
8
9
1
Scheme 2
NH2
O
NH2
N
N
N
N
O
N
OMe
+
O
Pd(PPh ) Cl ,
3 2 2
HO
N
I
O
O
O
CuI, Et N, DMF
3
OMe
HO
N
N
HO OH
OH OH
10
8
5
NH2
NH2
O
O
N
N
N
O
N
N
HO
HN
O
OMe
3
.
OMe
[1,2- H]ethylamine HCl
PyBop, DIEA, DMF
N
N
T
N
O
O
T
OH OH
OH OH
1
0
1a
3
[
N-ethyl-1,2- H] Apadenoson
Scheme 3
3
The modified synthesis to produce [ H]apadenoson is
Scheme 2 shows the synthetic route for the preparation of
unlabeled apadenoson, in which the N-ethyl amide linkage is illustrated in Scheme 3. There were several advantages of this
formed prior to deprotection and Sonogashira coupling. In this route, including introduction of labeled ethylamine in the final
sequence an excess of ethylamine was used to drive the reaction synthetic step. In addition, complications of running a Sonoga-
to completion.
shira reaction on a micro-molar scale were avoided. A more
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 113–117

Y. Hong et al.
CPM
Table 2. Tritium NMR: multiple species of [N-ethyl-
1
3
,2- H]apadenoson
6
5
5
4
4
3
3
2
2
1
1
0000
5000
0000
5000
0000
5000
0000
5000
0000
5000
0000
Chemical shift d
Assignment
0.99, 1.01, 1.04, 1.07
3.20, 3.22, 3.24, 3.26, 3.28, 3.30
–CT , –CHT , –CH T
3
2
2
–CT
2
–, –CHT–
was critical for the success of the synthesis and avoided the use
of a volatile-free amine. The improved synthetic efficiency of
PyBop led to quantitative consumption of radiolabeled ethyla-
mine hydrochloride as the limiting reagent.
5000
0
In the final step of the synthesis, ethanol was removed from
3
the solution of [1,2- H]ethylamine hydrochloride by rotary
0
:00
5:00
10:00
15:00mm:ss
3
Figure 2. Radio-HPLC analysis of the amidation reaction [N-ethyl-1,2- H]apadeno-
son (9.1 min component).
evaporation to dryness and the labeled ethylamine hydrochlor-
ide was dissolved in dry dimethyl formamide (DMF). An excess
of the intermediate acid 10 was added to the solution
along with PyBop and N,N-diisopropylethylamine (DIEA). Radio-
metric HPLC analysis showed concomitant consumption of
[1,2- H]ethylamine hydrochloride and the formation of
3
[N-ethyl-1,2- H]apadenoson. The reaction was complete in less
than 30 min as indicated by HPLC (Figure 2). The crude product
was purified by preparative reverse-phase HPLC giving 170 mCi
3
(56%) of pure [N-ethyl-1,2- H] apadenoson with high specific
Table 1. Tritium mass distribution and average specific
3
radioactivity of [N-ethyl-1,2- H]apadenoson
Unlabeled
Mono-tritiated analog
Di-tritiated
Tri-tritiated
3
17%
23%
26%
24%
Tetra-tritiated
Average specific radioactivity
9%
48 Ci/mmol
radioactivity.
The LC-MS analysis of the tritium-labeled product (Table 1
and Figure 3) shows a mixture of isotopic isomers containing
0
of this mixture was 48 Ci/mmol. Tritium NMR analysis of the
–4 tritons in each molecule. The average specific activity
3
N-ethyl-1,2- H] apadenoson (Figure 4 and Table 2) showed
[
multiple peaks for the isotopomers with a 5.6:1 ratio of methyl/
methylene tritium labeling. The observed distribution of tritium
was likely the result of T/H exchange that occurred during
heterogeneous catalytic reduction, which involves a compli-
5
cated mechanism. This ratio was consistent throughout the
multi-step reactions.
3
The synthesis of clinical supplies of [N-ethyl-1,2- H]apadeno-
son was carried under GLP conditions and was fully character-
ized for use in human clinical studies. Long-term stability and
formulation suitability studies were carried out to determine the
stability of the molecule. Overall, the compound was found to
be stable for several months if stored as a dilute solution in
absolute ethanol at low temperature.
Experimental
Materials: Compounds 6 and 7 were synthesized at Bris-
tol–Myers Squibb, Medical Imaging, Discovery Chemistry De-
partment. Compound 5 was prepared by a modification of the
6
procedure reported by Cristalli et al. Compound 8 was
prepared by a modification of the procedure reported by Rieger
7
et al. N-Vinylphthalimide and Pd/C, 10% were purchased from
Sigma-Aldrich. Carrier-free tritium gas was purchased from
American Radiolabeled Chemicals Inc. All other reagents were
obtained from commercial sources as ACS reagent grade or
3
Figure 3. Mass analysis of [N-ethyl-1,2- H]apadenoson.
s
higher. C18 Sep-Pak Cartridges were purchased from Waters
Corporation. HPLC was performed using an Agilent Chem-
Station HPLC system (Agilent Technologies) equipped with a IN/
US b-RAM detector (IN/US Systems). Tritium NMR spectra
efficient coupling reagent benzotriazol-1-yl-oxytripyrrolidino- were recorded on a Bruker DMX-300MHz spectrometer at
phosphonium hexafluorophosphate (PyBop) was also adopted 320 MHz. LC-MS was determined on an LXQ Mass Spectrometer
to improve the reactivity to form the amide. This modification (Thermo Electron Corp.). The tritiation manifold used in all
J. Label Compd. Radiopharm 2008, 51 113–117
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

Y. Hong et al.
NH2
O
N
N
N
O
OMe
Tn
N
O
Tm
NH
OH OH
Tm
1
0
0
0
0
0
0
0
0
0
.0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
1
0
0
0
.00
.75
.50
.25
0
0
.10
0.05
0
3.30
3.25
3.20
1.10
1.05
1.00
Chemical Shift (ppm)
Chemical Shift (ppm)
Tn
1.00
5.58
1.0
3
.0
2.5
2.0
Chemical Shift (ppm)
1.5
3
Figure 4. Tritium NMR (T–H decoupled) of [N-ethyl-1,2- H]apadenoson.
syntheses was purchased from IN/US Systems, Inc. (model: TRI- solutions were evaporated to dryness and dissolved in ethanol
SORBER ).
N-Ethyl-1,2- H]phthalimide (3): Compound
s
(5 mL) to give 322.5 mCi of 4 (95% radiochemically pure by
(3.4 mg, HPLC). Tritium distribution of the compound was determined by
3
[
2
3
2
0 mmol) was dissolved in EtOH (0.7 mL) and 4 mg of 10% Pd/
H NMR (320 MHz, d -methanol, T–H decoupled) d ppm
4
3
C added. The mixture was then reduced under an atmosphere of 1.17–1.25 (methyl, H), 2.93–3.01 (methylene, H), methyl/
3
3
2
000 mCi of carrier-free tritium gas using a commercial tritiation methylene H ratio, 5.5:1.
manifold at room temperature for 2 h. Labile tritium was Analytical HPLC conditions: Column: BDS Hypersil C18,
removed by distillation and rotary evaporation of the ethanol 250 Â 4.6 mm, 5 mm; solvent: A, water with 0.1% TFA; B,
3
R
solution. The [N-ethyl-1,2- H]phthalimide product was dissolved acetonitrile with 0.1% TFA; flow: 1 ml/min; T = 4 min; gradient
in EtOH (5 mL) and assayed by liquid scintillation counting to table: 0–2 min 0–5% B; 2–8 min 5% B; 8–12 min 5–100% B,
give 550 mCi of the tritium-labeled product (99% by analytical 15 min 100% B.
HPLC). Tritium distribution of the compound was determined by
3
(2S,3R,4S,5R)-5-(6-amino-2-(3-((1r,4r)-4-(methoxycarbonyl)cyclo-
H NMR (320 MHz, chloroform-d, T-H decoupled) d ppm hexyl)prop-1-ynyl)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
3
.21–1.29 (methyl, H), 3.69–3.75 (methylene, H), methyl/ carboxylic acid (10): Compounds 5 (710 mg, 1.75 mmol) and 8
3
1
methylene H ratio, 5:1.
3
(471 mg, 2.6 mmol) were dissolved in dry DMF (1 mL) and
Analytic HPLC method: Column: BDS Hypersil C18, triethylamine (2.5 mL) under nitrogen. The solution was vacuum
50 Â 4.6 mm, 5 mm; solvent: A, water with 0.1% TFA; B, purged twice with nitrogen flushing followed by the addition of
2
acetonitrile with 0.1% TFA; flow: 1 ml/min; wavelength 255 nm; Pd(Ph ) Cl (92.3 mg, 0.13 mmol) and CuI (50 mg, 0.262 mmol).
3
2
2
T
R
= 8.7 min. The column was eluted with 40% acetonitrile in The solution was stirred for 1.5 h until 5 was consumed. At this
water with 0.1% TFA for 10 min and eluted with acetonitrile with point, the solvents were removed under vacuum and the
.1% TFA afterwards. residual oil run through a short silica plug (HOAc/MeOH/THF
1,2- H]Ethylamine hydrochloride (4): Compound 3 (400 mCi 1:10:90) to afford 532 mg of crude amber solid after solvent
0
3
[
in 4 ml EtOH) was mixed with 4 ml HCl (6 N). The solution was removal. This was purified by preparative HPLC: Phenomenex
heated to 1051C and refluxed overnight to achieve complete Luna C-18 (10 m, 100 A, 41.2 Â 250 mm) A: 0.1% TFA; B: 90%
hydrolysis. The [1,2- H]ethylamine hydrochloride solution was ACN/0.1% TFA; flow rate = 20 mL/min.
3
concentrated by rotary evaporation to 2 mL by rotary evapora-
tion. The solution was then filtered successively through two
Waters C18 SepPak cartridges connected in series. The
cartridges were rinsed with water (2 Â 2 mL) to achieve
complete recovery of the radioactivity. The combined aqueous
Time (min)
0
4
5
20
50
%
B
15
15
30
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 113–117

Y. Hong et al.
The product-containing fractions were combined and lyophi-
Analytic HPLC condition: Column: BDS Hypersil C18,
lized to afford product 10 as a white powder (255 mg, 32%). 250 Â 4.6 mm, 5 mm; solvent: A, water with 0.1% TFA;
1
HRMS: calculated m/z = 482.16462 (M1Na) , observed B, acetonitrile with 0.1% TFA; flow: 1 mL/min; UV = 254 nm;
1
m/z = 482.16467 (M1Na) ; H NMR (DMSO-d
1
1
6
, 300.13 MHz):
.10 (dddd, 2H, J = 3.3, 12.8, 12.8, 12.8 Hz), 1.36 (dddd, 2H, J = 3.3,
T
R
= 8.7 min; 30% B isocratic.
Fractions containing pure product were pooled and concen-
1
2.8, 12.8, 12.8 Hz), 1.52 (m, 1H), 1.91 (m, 4H), 2.25 (dddd, 1H, trated by rotary evaporation. The final product was reconsti-
J = 3.4, 3.4, 12.2, 12.2 Hz), 2.36 (d, 2H), 3.58 (s, 3H), 4.28 (dd, 1H, tuted in ethanol to afford 170 mCi (56% yield) of 1a with a
J = 2.5, 4.5 Hz ), 4.41 (d, 1H), 4.46 (dd, 1H, J = 4.5, 6.5 Hz), 6.02 radiochemical purity 499%. The specific radioactivity was
1
3
(
d, 1H), 5.5–6.7 (b, 3H) 7.67 (bs, 2H), 8.50 (s, 1H) C NMR: 25.54, measured to be 48.07 Ci/mmol by LC-MS and tritium distribution
3
2
8
8.22, 30.98, 35.77, 42.05, 51.24, 73.30, 74.06, 81.12, 82.62, 85.65, of the compound was determined by H NMR (320 MHz,
3
6.77, 118.07, 139.87, 144.74, 149.51, 155.10, 171.91, 175.35.
3
d6-DMSO, T–H decoupled) d ppm 0.99–1.07 (methyl, H),
3 3
[
N-Ethyl-1,2- H]apadenoson, [(1R,4R)-methyl 4-(3-(6-amino-9- 3.20–3.30 (methylene, H), methyl/methylene H ratio, 5.58:1.
3
(
(2R,3S,4R,5S)-5-(1,2- H ethylcarbamoyl)-3,4-dihydroxytetrahydro-
furan-2-yl)-9H-purin-2-yl)prop-2-ynyl)cyclohexanecarboxylate]
1a): Compound 4 (300 mCi, 6.25 mmol) was dried under vacuum
in a 50-mL round-bottomed flask. Compound 10 (6 mg, The authors wish to thank Mr Robert Espina for the assistance in
3 mmol), (benzotriazol-1-yloxy)tripyrrolidino phosphonium hex- tritium NMR analysis and Mr Michael Lago for the assistance in
Acknowledgement
(
1
afluorophosphate (10 mg, 19.2 mmol), and DMF (5 mL) were GLP documentation.
added to the reaction flask at room temperature giving a clear
solution. Excess DIEA (0.2 mL) was then added and the solution
was stirred at room temperature. After 1.5 h HPLC indicated
References
3
complete consumption of the [ H]ethylamine and formation of
compound 1a. At this stage, the reaction was terminated by
addition of water (20 mL). The resulting solution was passed
through four Sep-Pak C18 cartridges (1 mL size) connected in
series. The cartridges were washed with 10 mL of water first and
[
1] D. C. Cassada, C. G. Tribble, S. M. Long, V. E. Laubach, A. K. Kaza,
J. Linden, B. N. Nguyen, J. M. Rieger, S. M. Fiser, I. L. Kron, J. Kern,
J. Vasc. Surg. 2002, 35, 994–998.
[
2] D. K. Glover, L. M. Riou, M. Ruiz, G. W. Sullivan, J. Linden, J. M. Rieger,
T. L. Macdonald, D. D. Watson, G. A. Beller, Am. J. Physiol. Heart Circ.
Physiol. 2005, 288, H1851–H1858.
the compound was eluted afterward with ethanol (4 Â 2 mL). [3] D. Zhang, W.G. Humphreys, Unpublished results.
The ethanol fractions were combined and concentrated to a [4] S. Gabriel, Ber. 1887, 20, 2224.
[
[
5] I. Horiuti, M. Polanyi, Trans. Faraday Soc. 1934, 30, 1164–1172.
6] G. Cristalli, A. Eleuteri, S. Vittori, R. Volpini, M. J. Lohse, K. N. Klotz,
J. Med. Chem. 1992, 32, 2363–2368.
small volume for HPLC purification.
Preparative HPLC conditions: LUNA 5m C18(2) (10 Â 250);
solvent: A, water 1% TEAA; B, acetonitrile; flow: 4.7 mL/min;
UV = 254 nm; gradient: 0–35 min 25% B; 35–45 min 25–100% B.
[7] J. M. Rieger, M. L. Brown, G. W. Sullivan, J. Linder, R. L. Macdonald,
J. Med. Chem. 2001, 44, 531–539.
J. Label Compd. Radiopharm 2008, 51 113–117
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org