Synthesis of [2H4]oxymetazoline and [ 14C]oxymetazoline

Article abstract of DOI:10.1002/jlcr.1805

An efficient synthesis of [²H₄] and [ ¹⁴C]oxymetazoline has been developed. Both compounds follow the same synthetic route with the introduction of the label occurring at different synthetic steps. The synthesis of [²H₄]oxymetazoline from [²H₄]ethylene diamine was achieved in one step with a 40% yield. The synthesis of [¹⁴C]oxymetazoline from potassium [ ¹⁴C]cyanide was achieved in two steps with an overall radiochemical yield of 67%. Copyright

Full text of DOI:10.1002/jlcr.1805

Research Article
Received 25 May 2010,
Revised 2 June 2010,
Accepted 3 June 2010
Published online 16 September 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1805
Synthesis of [2H4]oxymetazoline
and [14C]oxymetazoline
Ã
Michelle L. Brugger, Scott Borges, Sumei Ren, and Paul McNamara
An efficient synthesis of [2H4] and [14C]oxymetazoline has been developed. Both compounds follow the same synthetic
route with the introduction of the label occurring at different synthetic steps. The synthesis of [2H4]oxymetazoline from
[2H4]ethylene diamine was achieved in one step with a 40% yield. The synthesis of [14C]oxymetazoline from potassium
[
14C]cyanide was achieved in two steps with an overall radiochemical yield of 67%.
Keywords: [2H4]oxymetazoline; [14C]oxymetazoline; Afrin; SCH 9384; carbon-14; synthesis
System 2
Introduction
Phenomenex Luna CN, 4.6 Â 100 mm, 3 mm, 280 nm; 20 mM
ammonium acetate (pH 5.0):MeCN (50:50) for 10 min followed
by a step gradient to MeCN, 1.0 ml/min.
Oxymetazoline is an over-the-counter nasal spray used to ease
nasal congestion caused by rhinitis, hay fever, allergies, colds, or
other sinus problems. It provides symptomatic relief of nasal
congestion by stimulating the a-adrenergic receptors in the
arterioles of the nasal mucosa and producing vasoconstriction.
The generic name is oxymetazoline hydrochloride. It is commonly
known as Afrins and marketed by Merck in the United States.
Specific activity of [14C]oxymetazoline was determined by LC/
MS on a Waters 2695 XE Separations module multisolvent
delivery system with Waters PDA 996 absorbance detector. Mass
spectra were acquired on the ZQ 2000 mass spectrometer
operating in electrospray positive mode (ES1). The following
system was used: Phenomenex NX C18, 4.6 Â 50 mm, 3 mm,
280 nm; 10 mM ammonium bicarbonate (pH 9.7):MeCN (55:45)
for 4 min, 1.0 ml/min.
Experimental
General
All reactions were carried out under an atmosphere of nitrogen
unless otherwise stated. Purifications were carried out by using
flash column chromatography on a Teledyne Isco CombiFlash Rf.
Synthesis of 3-(bromomethyl)-6-tert-butyl-2,4-dimethyl-
phenol (2)
2
[2H4]Ethylene diamine (98 atom% H) was purchased from MSD
To a round bottom flask containing 2,4-dimethyl-6-tert-butyl-
phenol 1 (4.56 g, 25.6 mmol) was added paraformaldehyde
(768 mg, 25.6 mmol) followed by the addition of AcOH (10 ml,
200 mmol). To this suspension was added 33% wt. HBr solution
in AcOH (4.63 ml, 25.6 mmol). This was stirred at 451C until no
starting material remained by TLC (10% ether in hexanes). The
reaction was cooled to room temperature and was poured into
H2O (30 ml) with vigorous stirring. The aqueous layer was
extracted with CH2Cl2 (2 Â 30 ml). The organics were pooled,
dried over Na2SO4, filtered, and concentrated under reduced
pressure to give an orange oil. This was purified by flash column
chromatography (silica gel, gradient from 100% hexanes to
100% ether) to give 2.86 g (41%) of compound 2 as a yellow oil.
1H-NMR (400 MHz, CDCl3):d6.97 (s, 1H), 4.74 (s, 1H), 4.56 (s, 2H),
2.36 (s, 3H), 2.29 (s, 3H), 1.42 (s, 9H).
Isotopes. Potassium [14C]cyanide was purchased from GE Health-
care. 2,4-Dimethyl-6-tert-butyl phenol was purchased from May-
bridge and was purified before use. All other commercially
available reagents and solvents were purchased from Aldrich and
were used without further purification. Radioactivity measure-
ments were performed on a Packard 2200CA liquid scintillation
analyzer using Scintiverse BD as liquid scintillation cocktail. TLC
˚
was performed with Whatman LK6DF (silica gel 60 A) 5 Â 20 cm,
0.25-mm plates. The plates were scanned on a Bioscan 1000 linear
analyzer. Mass spectra were acquired on the JEOL MStation
magnetic sector mass spectrometer operating in the fast atom
bombardment (FAB) ionization mode.
[2H4]Oxymetazoline and [14C]oxymetazoline were analyzed by
HPLC conducted on a Waters 600 multisolvent delivery system
with Waters 2487 dual channel absorbance detector. Radio-
chemical purity of [14C]oxymetazoline was determined by using
a Radiomatic 525TR radioflow detector with Flo-Scint III liquid
scintillation cocktail (3:1). The following systems were used:
Merck Research Laboratories, Merck & Co., 2015 Galloping Hill Road, K-15-4545,
Kenilworth, NJ 07033, USA
System 1
Phenomenex Gemini C18, 4.6 Â 100 mm, 3 mm, 280 nm; MeOH:
MeCN:H2O:diethylamine (45:20:35:0.3) for 10 min followed by a
step gradient to MeCN, 1.0 ml/min.
*Correspondence to: Michelle L. Brugger, Merck Research Laboratories, Merck &
Co., 2015 Galloping Hill Road, K-15-4545, Kenilworth, NJ 07033, USA.
E-mail: michelle.brugger@merck.com
J. Label Compd. Radiopharm 2010, 53 793–795
Copyright r 2010 John Wiley & Sons, Ltd.

M. L. Brugger et al.
added thioacetamide (5.41 mg, 0.072 mmol) and ethylene
diamine 7 (483ml, 7.21 mmol). The tube was sealed and heated
at 1181C until the reaction was complete by TLC (10% 7 N NH3 in
MeOH in methylene chloride). The reaction was checked by TLC
every 2–3h. Additional thioacetamide (5.41 mg, 0.072 mmol) was
added until the reaction was complete. The total time for
completion was 16 h and a total of 37.9 mg of thioacetamide was
added. The reaction was poured into ice and extracted with
CH2Cl2 (3 Â 5 ml). The organics were pooled, washed with H2O
(25 ml), dried over Na2SO4, filtered, and concentrated under
reduced pressure to give 38.4 mCi (93.4%) of compound 8 as a
yellow solid with 97.8% RCP by TLC and 92.4% RCP by HPLC
system 1. Radio-TLC: Rf (7 N NH3 in MeOH: CH2Cl2 = 10:90) 0.203.
The crude was purified by flash column chromatography (silica
gel, gradient from 100% CH2Cl2 to 70% 7 N NH3 in MeOH in
CH2Cl2). For our purposes, the most pure fractions containing
product were combined and concentrated down under reduced
pressure to give 3.6mCi (8.8%) of compound 8 as a white solid
with RCP of 98.5 and 99.0% by HPLC system 1 and system 2,
respectively. The specific activity of [14C]oxymetazoline is
60.4 mCi/mmol as determined by LC/MS system. The less pure
fractions gave 31.2 mCi (76%) of compound 8 as a white solid
with 89.9% RCP by HPLC system 1. The total RCY for this step was
85%. The overall RCY of compound 8 over two steps was 67%.
2-(4-tert-butyl-3-hydroxy-2,6-dimethylphenyl)acetonitrile (3)
To a round bottom flask containing 3-(bromomethyl)-6-tert-butyl-
2,4-dimethylphenol 2 (2.86 g, 10.5 mmol) was added DMSO
(19.4ml). To this solution was added KCN (682 mg, 10.5mmol)
dissolved in H2O (9.7ml). The solution was stirred at 501C for 6 h
or until no starting material remained by TLC (10% ether in
hexanes). The reaction was cooled to room temperature and
diluted with H2O. The aqueous phase was extracted with ether
(3 Â 30ml). The organics were pooled, dried over Na2SO4, filtered,
and concentrated under reduced pressure to give an oily yellow
solid. Crystallization from hexanes gave 1.48g (65%) of compound
3 as an off-white powder. 1H-NMR (400 MHz, CDCl3):d6.99 (s, 1H),
4.78 (s, 1H), 3.63 (s, 2H), 2.33 (s, 3H), 2.28 (s, 3H), 1.41 (s, 9H).
[2H4]Oxymetazoline (5)
To a pressure tube containing 2-(4-tert-butyl-3-hydroxy-2,6-dimethyl-
phenyl)acetonitrile 3 (350 mg, 1.60 mmol) was added thioacetamide
(12 mg, 0.16 mmol) and [2H4]ethylene diamine 4 (500 ml, 8.0 mmol).
The tube was sealed and heated at 1181C for 3.5 h or until no
starting material remained by TLC (10% 7 N NH3 in MeOH in CH2Cl2).
The reaction was poured into ice and was extracted with CH2Cl2
(3 Â 5 ml). The organics were pooled, dried over Na2SO4, filtered,
and concentrated under reduced pressure to give a dark yellow oil.
This was purified by flash column chromatography (silica gel,
gradient from 100% CH2Cl2 to 70% 7 N NH3 in MeOH in CH2Cl2) to
give 170.5 mg (40%) of compound 5 as a light yellow solid.
Results and discussion
[2H4]Oxymetazoline was prepared for use as an internal standard
in a bioanalytical liquid chromatography/tandem mass spectro-
metry method. [14C]Oxymetazoline was synthesized to support
drug disposition studies. [2H4] and [14C]oxymetazoline were
synthesized, using a modified literature procedure,1 in 40 and
67% yield, respectively.
The synthesis of the deuterium-labeled oxymetazoline is
shown in Scheme 1. 2,4-Dimethyl-6-tert-butylphenol was
bromomethylated by heating with paraformaldehyde and HBr
in acetic acid in 41% yield using modified literature proce-
dures.2,3 Cyanide was introduced via nucleophilic displacement
of bromine in 65% yield after crystallization from hexanes.4–6
Deuterium was incorporated by the cyclocondensation of
2-(4-tert-butyl-3-hydroxy-2,6-dimethylphenyl)acetonitrile with
[2H4]ethylene diamine in the presence of a catalytic amount of
thioacetamide in a pressure tube.7
Crystallization of [2H4]Oxymetazoline (5)
Several batches of yellow solid [2H4]Oxymetazoline 5 (329.4 mg)
were combined and suspended in acetone. Hexane was added
to further crystallize the compound. The suspension was placed
at À201C for 1 h. The white solid collected was dried to constant
weight to give 240 mg (73%) of 99.4% pure compound 5 as
determined by HPLC system 1. FAB-MS: m/z 265 (M1H)1. 1H-
NMR (600 MHz, CDCl3):d6.96 (s, 1H), 3.66 (s, 2H), 2.27 (s, 3H), 2.21
(s, 3H), and 1.40 (s, 9H).
2-(4-tert-butyl-3-hydroxy-2,6-dimethylphenyl)aceto[14C]ni-
trile (6)
To a round bottom flask containing 3-(bromomethyl)-6-tert-
butyl-2,4-dimethylphenol 2 (248 mg, 0.915 mmol) was added
DMSO (1.6 ml). To this solution was added potassium [14C]cya-
nide (51.8 mCi, 0.909 mmol) dissolved in H2O (200 ml). The vial
that contained the potassium [14C]cyanide was rinsed with H2O
(2 Â 205 ml) and the rinses were added to the reaction. The
yellow solution was stirred at 501C for 5.5 h or until no starting
material remained by TLC (10% ether in hexanes). The reaction
was cooled to room temperature and H2O was added. The
aqueous phase was extracted with ether (3 Â 5 ml). The organics
were pooled, dried over Na2SO4, filtered, and concentrated
under reduced pressure to give 41.1 mCi (79%) of compound 6
as a yellow oil with 98.1% RCP. Radio-TLC: Rf (ether:hex-
anes = 20:80) 0.146. The crude product was used directly in the
next step without further purification.
The cyclocondensation reaction with the deuterium label
proved to be the most challenging step. Literature conditions7
require a ten-fold excess of the ethylene diamine, as it also acts
as solvent in the reaction. In an effort to reduce cost and
conserve the labeled reagent, the [2H4]ethylene diamine was
dropped to a five-fold excess. However, under these conditions,
we were unable to drive the reaction to completion. At best,
there was a 20% yield of product and 57% recovery of unreacted
starting material. Dash et al. reported smooth transformations of
various nitriles to their corresponding 2-imidazolines via
thioacetamide catalysed cyclocondensations.7 Their proposed
mechanistic pathway involves the initial reaction of thioaceta-
mide with ethylene diamine producing 2-methyl-2-imidazoline
and H2S; with the H2S acting as the actual catalyst for the
cyclocondensation reaction. Based on this theory, we ran this
reaction in a pressure tube in order to contain the H2S and drive
the reaction to completion. As shown in Table 1, this change
doubled the yield of [2H4]oxymetazoline to 40% after column
purification, all starting material was consumed, and it allowed
[
14C]Oxymetazoline (8)
To a pressure tube containing 2-(4-tert-butyl-3-hydroxy-2,6-
dimethylphenyl)aceto[14C]nitrile 6 (41.1 mCi, 0.721 mmol) was
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M. L. Brugger et al.
(CH2O)n,
HBr in AcOH
Br
AcOH,
HO
HO
°
45 C
41%
2
1
2H
2H
NH2
2H
H2N
2H
S
2H
2H
2H
KCN
DMSO/H2O
NH2
5.0 equiv
2H
N
N
N
H
HO
°
118 C
HO
°
50 C
40%
65%
3
5
4
[2H4]Oxymetazoline
Scheme 1. Synthesis of [2H4]oxymetazoline.
Table 1. Optimization of reaction to produce [2H4]oxymetazoline
Reaction
1
Eq. of 4
5.0
Comments
Yield (%)a
20
Refluxed (RB flask, condensor), 1181C, 3 h. TLC analysis of reaction mixture
showed incomplete reaction and six major components
2
3
5.0
5.0
Refluxed (RB flask, condensor), 1181C, 16 h. TLC analysis of reaction mixture
showed incomplete reaction and six major components. Additional thioacetamide
did not change the reaction profile
Heated (Sealed tube), 1181C, 3.5 h. TLC analysis of the crude reaction showed
complete consumption of 3 and formation of product 5 and two additional components
18
40
aAfter silica gel chromatography.
The [14C] label was incorporated one step earlier, as in
Scheme 2, by nucleophilic displacement of bromine from
3-(bromomethyl)-6-tert-butyl-2,4-dimethylphenol with [14C]cya-
nide from potassium [14C]cyanide.4–6 The remaining steps were
identical to the SIL synthesis.
K14CN
DMSO/H2O
Br
HO
°
50 C
79%
2
Acknowledgements
S
The authors thank Dr Tze-Ming Chan and Dr Pradip Das from
Merck Research Laboratories for the NMR and MS analysis of the
final compounds as well as Dr David Hesk from Merck Research
Laboratories for helpful and insightful discussions.
H2N
NH2
NH2
N
14C
N
14C
HO
°
118 C
N
H
HO
85%
8
7
6
[
14C]Oxymetazoline
References
Scheme 2. Synthesis of [14C]oxymetazoline.
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[4] B. Raju, G. S. Krishna Rao, Indian J. Chem. B. 1987, 26, 469–470.
[5] D. L. Holmes, D. A. Lightner, Tetrahedron 1995, 51, 1607–1622.
[6] M. Gates, J. Org. Chem. 1982, 47, 578–582.
for the conservation of the label by using a five-fold rather than
a ten-fold excess. Additionally, TLC showed a much cleaner
reaction.
Further attempts to optimize the reaction were set aside due
to an accelerated delivery timeline requested by the customer
group for the deuterated standard. A 40% yield was acceptable
to deliver sufficient material.
[7] P. Dash, D. P. Kudav, J. A. Parihar, J. Chem. Res. 2004, 490–491.
J. Label Compd. Radiopharm 2010, 53 793–795
Copyright r 2010 John Wiley & Sons, Ltd.
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