Efficient and facile synthesis of novel stable monodeuterium labeled ractopamine

Article abstract of DOI:10.1002/jlcr.3355

A novel synthetic route to stable deuterium labeled ractopamine was disclosed with 6.49% total yield and 97.7% isotopic abundance. Its structure and the isotope-abundance were confirmed according to ¹H-NMR and high-resolution mass spectrometry. A novel synthetic route to stable deuterium labeled ractopamine was disclosed with 9 steps, 6.49% total yield and 97.7% isotopic abundance. Its structure and the isotope-abundance were confirmed according to ¹H-NMR and high-resolution mass spectrometry.

Full text of DOI:10.1002/jlcr.3355

Research article
Received 12 August 2015,
Revised 30 September 2015,
Accepted 5 October 2015
Published online 2 November 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3355
Efficient and facile synthesis of novel stable
monodeuterium labeled ractopamine
a
a
a
a,b
a
Feifei Su, Fulong Wu, He Tang, Zhonghua Wang, * and Fanhong Wu *
A novel synthetic route to stable deuterium labeled ractopamine was disclosed with 6.49% total yield and 97.7% isotopic
1
abundance. Its structure and the isotope-abundance were confirmed according to H-NMR and high-resolution mass
spectrometry.
Keywords: deuterium labeling; drug standards; ractopamine; β-adrenergic agonists
Introduction
double bond, corresponding saturated ketone 3 was obtained
with 61% yield.
Reduction of 3 using 99% sodium borodeuteride gave 4 in
Ractopamine, a β-adrenergic agonist, can improve the efficiency
of feed utilization and enhance the meat leanness in livestock.
1,2
93.1% yield. Efficient tosylation was achieved in the presence
However, because of the residue of the ractopamine, the meat
products obtained from treated animals may pose a potentia3l
risk for consumer health, such as asthma or angiocardiopathy.
Thus, it had been prohibited as a growth promoter of farm
animals in several countries, such as the European Union, Japan,
of 4-dimethylaminopyridine and triethylamine in 30 min at room
temperature. Substitution of the tosyloxy group of 5 with
sodium azide furnished azide 6, which was subsequently
reduced to the corresponding amine 7 under reduction
conditions with 95% yield over two steps.
4
,5
and China.
Compound 7 coupled with 2-bromo-4′-methoxyacetophenone
in the presence of N, N-diisopropylethylamine in tetrahydrofuran
to furnish compound 8, which existed in its enol form as
Recently, many methods had been developed for the analysis
of ractopamine, including high-performance liquid chromatography,
gas chromatography-mass spectrometry, and liquid chromatography-
mass spectrometry. But these methods had some defects for
determining the residue of ractopamine in animal tissues
because of interference from other impurities and losses in
the sample purification process. More prominent and reliable
1
confirmed by H-NMR of non-deuterium labeled compound 8′.
In Figure 1, the chemical shift of H , attached to the enol, was
A
found at 8.02 ppm as a single peak, while no characteristic signals
of its corresponding keto-tautomer (the CO-CH -NH signal might
2
occur at 3.00–4.00 ppm with two hydrogen atoms) was found in
this spectrum.
6
–8
analytical methods had aroused the researchers’ interest,
in
which employing isotopically labeled compounds as internal
standards could adjust for the losses in sample pretreatment
and purification, offset the error caused by ion suppression in
the mass spectrometric detection process, and make the results
closer to the true value efficiently.
Subsequently, quick reduction of crude 8 in the presence of
catalyst 10% Pd/C under an atmosphere of hydrogen gave the
desired product 9. After the demethylation of 9 by treatment
with borontribromide at À60 °C, the desired compound 10 was
obtained with 70% yield.
This deuterium labeled ractopamine could be used for the
preliminary and qualitative screening of veterinary drug residues
and metabolic mechanism study of ractopamine as its metabolic
1
From the H-NMR spectrum Figure 2, the H
B
of 10′, non-
deuterium labeled ractopamine, was observed at 3.27–3.30 ppm,
while no corresponding signal was observed in the spectrum
of 10. So the deuterium atom was successfully introduced to
the desired position, and the isotopic abundance was 97.7%
according to its high-resolution mass spectrometry (HRMS) of
Figure 3.
9
stability. To date, there were very few procedures described in
the literature for the preparation of deuterium labeled
ractopamine. Li’s group applied a patent for the preparation of
10
3 4
d - and d -ractopamine. Therefore, a promising and practical
route is reported in this article.
Results and discussion
a
School of Chemical and Environmental Engineering, Shanghai Institute of
Technology, Shanghai, China
The nine-step synthesis of the labeled ractopamine 10 started with
commercially available p-methoxybenzaldehyde as depicted in
b
Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic
Scheme 1. First of all, in the presence of NaOH as the catalyst Chemistry, Chinese Academy of Sciences, Shanghai, China
and at 25 °C, p-methoxybenzaldehyde was reacted with the excess
*
Correspondence to: Zhonghua Wang and Fanhong Wu, School of
amount of acetone to furnish α, β-unsaturated ketone 2 with 67%
yield via aldol condensation and subsequent dehydration.
Chemical and Environmental Engineering, Shanghai Institute of Technology,
Shanghai, China.
Through the subsequent hydrogenation of the carbon-carbon E-mail: wzhsit@163.com; wfh@sit.edu.cn
J. Label Compd. Radiopharm 2015, 58 479–482
Copyright © 2015 John Wiley & Sons, Ltd.

F. Su et al.
Scheme 1. Synthesis of monodeuterium labeled ractopamine. Reagents and conditions: (i) 10% NaOH(aq), rt, 1.5 h, 67%; (ii) H2, Raney Ni, EA, rt, 4 h, 61%; (iii) NaBD4, THF,
rt, 5 h, 93%; (iv) TsCl, NEt3, DMAP, DCM, rt, 30 min, 64.0%; (v) NaN3, DMF, rt, 10 h, 96.8%; (vi) H2, 10% Pd/C, MeOH, rt, 10 h, 98%; (vii) 2-bromo-4’-methoxyacetophenone,
iPr2NEt, THF, rt, 3 h; (viii) H2, 10% Pd/C, MeOH, rt, 4 h; and (ix) BBr3, DCM, rt, 6 h, 70%.
1
Figure 1. H-NMR spectrum of 8′.
1
Figure 2. H-NMR spectrum of 10 and 10′.
Figure 3. HRMS spectrum of 10.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 479–482

F. Su et al.
(
2
500 MHz, CDCl
3
) δ ppm 1.28 (s, 3H), 1.73–1.93 (m, 2H), 2.43 (s, 3H),
Conclusion
.42–2.58 (m, 2H), 3.78 (s, 3H), 6.79 (d, J = 8.5, 2H), 6.98 (d, J = 8.5, 2H),
+
In summary, the stable isotope labeled ractopamine had been 7.33 (d, J = 8.1, 2H), 7.79 (d, J = 8.2, 2H). HRESIMS: [M + Na] m/z
prepared with 6.49% total yield and 97.7% isotopic abundance. 358.1190 (Calcd for [C18
+
H21DO
4
S+Na] 358.1199)
1
Its structure was confirmed by H-NMR and HRMS.
1
-(3-azidobutyl)-4-methoxybenzene (6)
Experimental
To a stirred solution of 5 (2.52 g, 7.51 mmol) in DMF (100 mL) under
nitrogen atmosphere was added sodium azide (1.52 g, 23.4 mmol). After
stirring for 10 h at room temperature, the reaction mixture was quenched
with water and extracted with ethyl acetate. The combined organic
extracts were dried over anhydrous Na SO and concentrated to give
2 4
the crude product, which was purified by flash column chromatograph
General
All reactions were carried out under an atmosphere of nitrogen. All
commercially available reagents and solvents were used without
purification. The stable labeled raw material, sodium borodeuteride,
was purchased from CIL. H-NMR spectra were recorded on Bruker
3 2 6
Avance III 500 MHz spectrometers using CDCl , D O, and DMSO-d unless
otherwise stated. HRMS data were obtained from Bruker solanX 70
equipped with Electrospray ionization source. Column chromatography
and preparative thin-layer chromatography were carried out by using
Merck silica gel 60 (230–400 mesh), respectively. Purity was determinated
by high-performance liquid chromatography (Essentia LC-15C) that used
1
on silica gel using petroleum ether/ethyl acetate (19:1) as eluent to yield
1
azide 6 (1.50 g, 96.8%). H NMR (500 MHz, CDCl
3
) δ ppm 1.28 (s, 3H),
1
7
.69–1.82 (m, 2H), 2.58–2.73 (m, 2H), 3.79 (s, 3H), 6.84 (d, J = 8.6 Hz, 2H),
+
.11 (d, 2H, J = 8.5 Hz). HRESIMS: [M + H] m/z 207.1288 (Calcd for
+
[
C
11
H14DN
3
O+H] 207.1356 )
4
-(4-methoxyphenyl)butan-2-amine (7)
WondaSil C18-WR 4.6 * 150 mm, 5 μm column, and the solvent which To a solution of 6 (1.50 g, 7.27 mmol) in methanol (50 mL) was added
contained water and acetonitrile.
10% palladium on carbon, and hydrogenated under hydrogen
atmosphere at room temperature for 10 h. The reaction mixture was
(
E)-4-(4-methoxyphenyl)but-3-en-2-one (2)
1
filtered, and the filtrate was concentrated to yield 7 (1.29 g, 98%).
NMR (500 MHz, CDCl
H), 3.78 (s, 3H), 6.82 (d, J = 8.5 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H). HRESIMS:
H
) δ ppm 1.09 (s, 3H), 1.61 (m, 2H), 2.57–2.62 (m,
A 5% aqueous sodium hydroxide (2 mL) was added gradually to a
solution of p-methoxybenzaldehyde (64.58 g, 0.47 mol) and acetone
3
2
+
+
[
M+H] m/z 181.1434 (Calcd for [C11H16DNO+H] 181.1451 )
(177 mL, 2.38 mol) in water at 0 °C. The reaction mixture was stirred at
room temperature for 1.5 h and then neutralized with 1 N hydrochloric
acid to form a yellow solid. The crude product was filtered, washed with (Z)-1-(4-methoxyphenyl)-2-((4-(4-methoxyphenyl)butan-2-yl)
cold water, and dried then purified by column chromatography on a amino)ethanol (8)
silica gel column using petroleum ether/ethyl acetate (7:1) as eluent to
1
N, N-diisopropylethylamine (1.70 mL, 9.73 mmol) was added dropwise to
a mixture of 2-bromo-4′-methoxyacetophenone (72.6 mg, 0.317 mmol),
powdered molecular sieves (4 Å, 0.5 g) and 7 (114 mg, 0.632 mmol) in
tetrahydrofuran (10 mL) and stirred for 3 h under nitrogen. The reaction
mixture was filtered, and the solvent removed under reduced pressure
to afford crude 8 which was used in the next step without further
purification.
yield 2 (55.79 g, 67%). H NMR (500 MHz, CDCl
2
3
3
) δ ppm 7.50 (d, J = 8.7 Hz,
H), 7.47 (d, J = 16.8 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 16.2 Hz, 1H),
.84 (s, 3H), and 2.36 (s, 3H).
4
-(4-methoxyphenyl)butan-2-one (3)
To a stirred solution of 2 (7.20 g, 0.04 mol) in ethyl acetate, (35 mL) was
added Raney nickel (0.8 mL), the solution was stirred for 4 h under
hydrogen atmosphere at room temperature. The reaction mixture was
filtered, washed with ethyl acetate, and the crude product obtained by
the evaporation of the solvent was chromatographed over a silica gel
1
-(4-methoxyphenyl)-2-((4-(4-methoxyphenyl)butan-2-yl)amino)
ethanol (9)
column using petroleum ether/ethyl acetate (15:2) as eluent to furnish
To a stirred solution of 8 in methanol (10 mL) was added palladium (10%
on carbon, 20 mg), the solution was stirred for 4 h under hydrogen
atmosphere at room temperature. The reaction mixture was filtered,
washed with methanol, and the crude product obtained by the
evaporation of the solvent was chromatographed over a silica gel
1
3
(4.47 g, 61%). H NMR (500 MHz, CDCl
3
) δ ppm 7.09 (d, J = 8.6 Hz, 2H),
6.82 (d, J = 8.6 Hz, 2H), 3.77 (s, 3H), 2.83 (t, J = 7.3 Hz, 2H), 2.71 (t, J = 7.2 Hz,
2H), and 2.12 (s, 3H).
4
-(4-methoxyphenyl)butan-2-ol (4)
column using dichloromethane/methanol (7:3) as eluent to furnish 9
1
(
84 mg, 40.2%). H NMR (500 MHz, CDCl
3
,) δ ppm 1.25 (d, J = 6.7 Hz, 3H),
Reduction of 3 (2.25 g, 12.6 mmol) was carried out with sodium
borodeuteride (0.82 g, 19.6 mmol) in tetrahydrofuran (100 mL) for 5 h at
1
.73–1.99 (m, 2H), 2.55–2.69 (m, 2H), 2.72–3.05 (m, 2H), 3.77 (d,
J = 0.9 Hz, 3H), 3.79 (s, 3H), 4.90 (d, J = 7.8 Hz, 1H), 6.80 (d, J = 8.5 Hz, 2H),
2
5 °C. After completion of the reaction, the reaction mass was neutralized
with dilute HCl (5%) and extracted with dichloromethane. The organic
layer was dried over Na SO and evaporated under vacuum. Compound
was obtained as colorless oil in 93% yield. H NMR (500 MHz, CDCl
δ ppm 1.21 (s, 3H), 1.44 (s, 1H), 1.68–1.77 (m, 2H), 2.59–2.72 (m, 2H),
6
2
.85 (d, J = 8.2 Hz, 2H), 7.08 (dd, J = 8.5, 4.0 Hz, 2H), 7.28 (dd, J = 8.7,
+ +
3
.0 Hz, 2H). HRESIMS: [M+H] m/z 331.2122 (Calcd for [C20H26DNO +H]
2
4
1
331.2132)
4
3
)
4-(1-hydroxy-2-((4-(4-hydroxyphenyl) butan-2-yl) amino)ethyl)phenol
3.79 (s, 3H), 6.83 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H). HRESIMS: [M
+
+
(10)
+
2
Na] m/z 204.1111 (Calcd for [C11H15DO +Na] 204.1111)
To a solution of 9 (62 mg, 0.19 mmol) in dichloromethane (5 mL) cooled
at À60 °C under nitrogen was slowly added boron tribromide (0.5 mL,
4
-(4-methoxyphenyl)butan-2-yl 4-methylbenzenesulfonate (5)
To a stirred solution of 4 (2.13 g, 11.7 mmol) in dichloromethane (21 mL), 5.3 mmol). The reaction mixture was stirred for 1 h at À60 °C and then
containing a catalytic amount of 4-dimethylaminopyridine (3.12 g, allowed to warm to room temperature. After quenching with 10%
2
5.5 mmol) and triethylamine (21 mL, 151 mmol), was added dropwise NaHCO
3
at 0 °C, the mixture was extracted with ethyl acetate. The
and concentrated under reduced
30 mL) at 0 °C under nitrogen. The reaction was monitored by thin-layer pressure to give 10 (40 mg, 70%). H NMR (500 MHz, D O) δ ppm 1.32
the p-toluensulfonyl chloride (9.05 g, 47.47 mmol) in dichloromethane organic phases were dried over MgSO
(
4
1
2
chromatography. At the end of the reaction, the reaction mixture was
(d, J = 6.4 Hz, 3H), 1.77–2.03 (m, 2H), 2.50–2.74 (m, 2H), 3.17 (m, 2H),
concentrated to give the crude product, which was purified by flash
4.86 (m, 1H), 6.83 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.3 Hz, 2H), 7.14 (d,
+
column chromatograph on silica gel using petroleum ether/ethyl acetate J = 6.9 Hz, 2H), 7.24 (t, J = 8.2 Hz, 2H). HRESIMS: [M+H] m/z 303.1820
1
+
(
3
7:1) as eluent to yield 5 (2.53 g, 64.0%) as a pale yellow liquid. H NMR (Calcd for [C18H22DNO +H] 303.1819)
J. Label Compd. Radiopharm 2015, 58 479–482
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F. Su et al.
[
[
4] Commission of the European Communities. Council Directive
Conflict of interest
7
0/534/EEC, 1997.
5] Announcement Number 176. People’s Republic of China, Agriculture
Ministry.
The authors did not report any conflict of interest.
[6] D. C. Suo, G. L. Zhao, J. Chromatogr. Sci. 2013, 52, 1–5.
[7] C. Li, Y. L. Wu, J. Chromatogr. A. 2010, 1217, 7873–7877.
[8] V. V. De Almeida, V. S. Miyada, Braz. Arch. Biol. Technol. 2012, 55,
445–456.
References
[1] K. J. Mimbs, T. D. Pringle, M. J. Azain, S. A. Meers, T. A. Armstrong,
J. Anim. Sci. 2005, 83, 1361–1369.
2] D. J. Smith, J. Anim. Sci. 1998, 76, 173–176.
[9] A. E. Mutlib, Chem. Res. Toxicol. 2008, 21, 1672–1689.
[10] J. Li, Z. Xu, R. Yu, Y. Chen, H. Wang, CN 104311436 A, 2014.
[
[3] J. Blanca, P. Muñoz, M. Morgado, N. Méndez et al., Anal. Chim. Acta.
2
005, 529, 199–205.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 479–482