Synthesis of deuterium-labeled 3-O-methyldopa and 4-O-methyldopa

Article abstract of DOI:10.1002/jlcr.677

Concise methods for the synthesis of 4-hydroxy-3-[²H₃]-methoxyphenylalanine (3-O-[²H₃]-methydopa) and 3-hydroxy-4-[²H₃]-methoxyphenylalanine (4-O-[²H₃]-methydopa) are

Full text of DOI:10.1002/jlcr.677

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 389–394.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.677
Research Article
Synthesis of deuterium-labeled
3-O-methyldopa and 4-O-methyldopa
Aruna Perera1, Keith Hyland2, Hoa K. Nguyen1 and Robert R. Kane1,*
1 Department of Chemistry & Biochemistry and Center for Drug Discovery,
Baylor University, Waco, TX 76798-7348 USA
2 Institute of Metabolic Disease, Baylor University Medical Center , Dallas, TX,
75226, USA
Summary
Concise methods for the synthesis of 4-hydroxy-3-[2H3]-methoxyphenylalanine
(3-O-[2H3]-methydopa) and 3-hydroxy-4-[2H3]-methoxyphenylalanine (4-O-
[2H3]-methydopa) are described. The 3-O-[2H3]-methydopa is a valuable
internal standard for the tandem MS quantification of 3-O-methyldopa, a
metabolite of value in the diagnosis of aromatic l-amino acid decarboxylase
(AADC) deficiency. Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: 3-O-methyldopa; aromatic l-amino acid decarboxylase; tyrosine
Introduction
Aromatic l-amino acid decarboxylase (AADC) is the enzyme that
converts 3,4-dihydroxyphenylalanine (l-dopa) to dopamine and
5-hydroxytryptophan to serotonin. Inherited deficiency of this enzyme
leads to decreased levels of these two important neurotransmitters
resulting in a severe early onset neurological disorder.1 In the absence of
AADC activity, l-dopa is methylated to 4-hydroxy-3-methoxyphenyl-
alanine (3-O-methyldopa), which then accumulates in blood, urine and
*Correspondence to: Robert R. Kane, Department of Chemistry & Biochemistry and Center for
Drug Discovery, Baylor University, Waco, TX 76798-7348, USA. E-mail: bob_kane@baylor.edu
Copyright # 2003 John Wiley & Sons, Ltd.
Received 13 August 2002
Revised 27 September 2002
Accepted 17 October 2002

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A. PERERA ET AL.
cerebrospinal fluid in infants and children with a deficiency of this
enzyme.2 3-O-methyldopa therefore provides a biochemical marker that
can be used to screen for this disease.
The analysis of multiple compounds eluted from dried blood spots
collected from neonates using tandem mass spectrometry (MS) can be
used to screen for over 30 inherited diseases.3 The tandem MS analysis
includes amino acids, and 3-O-methyldopa can be incorporated into the
screening process to help identify patients with AADC deficiency.
Accurate quantification of metabolites by tandem MS, however,
requires incorporation of stable isotope internal standards for each of
the compounds analyzed. We have therefore developed syntheses of
stably labeled 3-O-[2H3]-methyldopa (1) and its isomer 4-O-[2H3]-
methyldopa (2).
Initial attempts at the synthesis of these compounds involved various
methods for the direct methylation of 3,4-dihydroxyphenylalanine
(dopa). This ‘simple’ approach was problematic, and instead the target
compounds were synthesized starting with the selectively protected dopa
precursors 3 and 5. These compounds are readily available from
tyrosine by a sequence of reactions described by Jung and Lazarova
involving a Reimer–Tiemann formylation and a Dakin oxidation.4
Similar strategies for the synthesis of selectively protected dopa
derivatives had previously been described by the Boger group,5 and
more recent modifications of this general strategy have also appeared in
the literature.6 We now report that 3-O-[2H3]-methyldopa (1) and its
isomer 4-O-[2H3]-methyldopa (2) have been synthesized from tyrosine in
overall yields of 20% (6 steps) and 24% (5 steps) (Scheme 1).
Discussion
The straightforward synthesis of labeled 3-O-methyldopa 1 began with
the selectively protected dopa analogue 3 (Scheme 2), which is available
Scheme 1.
Copyright # 2003 John Wiley & Sons, Ltd.
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DEUTERIUM-LABELED 3-O-METHYLDOPA AND 4-O METHYLDOPA
391
Scheme 2.
in 4 steps from tyrosine in 33% yield.4 The deuterium label was
incorporated by alkylation of the monoprotected catechol 3 with excess
CD3I, which was followed by removal of both protecting groups (N-
tBOC and O-benzyl) by treatment with trifluoroacetic acid in CH2Cl2.
Crude 1 was purified by preparative reverse phase TLC (water/
methanol/sodium octylsulfonate 9:1:1 mM), and the ion-pairing reagent
was subsequently removed by filtration through H+-form Dowex-
50 Â 4 (NH4OH eluant).
Preparation of the labeled 4-O-methyldopa 2 began with hydroxy-
benzaldehyde 5, which can be synthesized in two steps from tyrosine
(59% overall).4 Introduction of the label was again accomplished by
alkylation with excess CD3I. Diphenyl diselenide-mediated oxidation of
the alkylated benzaldehyde 6, followed by aminolysis of the resulting
formate ester, afforded the protected dopa derivative 7, which was de-
protected and purified in analogy with the 3-O-methyldopa derivative.
Conclusion
We have found that selectively protected l-dopa derivatives 3 and 5,
(Scheme 3) which are reliably available in good yield, are useful
intermediates for the synthesis of deuterium-labeled methyldopa
Scheme 3.
Copyright # 2003 John Wiley & Sons, Ltd.
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derivatives. The deuterium label incorporation is accomplished by a
phenol alkylation, which is performed using a large excess of deuterated
methyl iodide in order to minimize reaction time and avoid racemization.
Experimental
N-[(1,1- Dimethylethoxy)carbonyl 4-benzyloxy-3-[2H3]-methoxyphenyl-
alanine (4)
A suspension of anhydrous potassium carbonate (0.5 g, 3.6 mmol, 7 eq)
in chloroform/methanol (2/1; 6 ml) was refluxed under N2 for 15 min
and then cooled. Compound 3 (0.20 g, 0.52 mmol, 1 eq) and CD3I (1 ml,
4.5 mmol, 8.7 eq) were added and the mixture was refluxed under N2 for
an additional 14 h. The reaction mixture was diluted with water and
ethyl acetate, and the aqueous layer was acidified to pH 1 with 1 N HCl,
the layers separated, and the aqueous layer back extracted with ethyl
acetate (3 Â 100 ml). The combined organic extracts were washed with
brine and water, dried over MgSO4, and concentrated under vacuum.
Flash column chromatography (75 g silica gel, 22:1 CH2Cl2/MeOH 1%
acetic acid) afforded the desired product (0.17 g, 0.42 mmol, 81%) as a
brown oil.
4-hydroxy-3-[2H3]-methoxyphenylalanine (1)
Compound 4 (0.2 g, 0.50 mmol, 1 eq) was dissolved in 2 ml of
dichloromethane and 2 ml of triflouroacetic acid was carefully added.
The reaction was stirred under N2 for 4 h at room temperature. The
reaction mixture was then concentrated under vacuum and the product
dissolved in 1 ml water. Preparative reverse phase TLC (1 mM
1-octanesulfonic acid [sodium salt] in water/methanol [9/1], pH 2)
afforded an oil that was dissolved in 10% acetic acid (3 ml), loaded on
an ion exchange column (Dowex 50 Â 4-400, acid form, 5 g) that was
then washed extensively with water. Elution of the resin with
concentrated ammonium hydroxide followed by concentration under
vacuum afforded the desired product (0.08 g, 0.37 mmol, 74%) as a light
brown power.
N-[(1,1-dimethylethoxy) carbonyl] 3-formyl-4-[2H3]-methoxyphenyl-
alanine (6)
A suspension of anhydrous potassium carbonate (0.5 g, 3.6 mmol,
4.4 eq) in chloroform/methanol (2/1; 6 ml) was refluxed under N2 for
Copyright # 2003 John Wiley & Sons, Ltd.
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DEUTERIUM-LABELED 3-O-METHYLDOPA AND 4-O METHYLDOPA
393
15 min and then cooled. Compound 5 (0.25 g, 0.81 mmol, 1 eq) and
CD3I (1 ml, 4.5 mmol, 5.5 eq) were added and the mixture was refluxed
under N2 for an additional 14 hrs. The reaction mixture was diluted with
water and ethyl acetate, and the aqueous layer was acidified to pH 1
with 1 N HCl, the layers separated, and the aqueous layer back
extracted with ethyl acetate (3 Â 50 ml). The combined organic extracts
were washed with brine and water, dried over MgSO4, and concentrated
under vacuum. Flash column chromatography (75 g silica gel, 1% acetic
acid in 22:1 CH2Cl2/MeOH) afforded the desired product (0.17 g,
0.45 mmol, 56%) as a brown oil.
N-[(1,1-dimethylethoxy)carbonyl] 3-hydroxy-4-[2H3]-methoxyphenyl-
alanine (7)
Diphenyl diselenide (0.003 g, 0.01 mmol, 0.03 eq) and aqueous hydrogen
peroxide (0.5 ml of a 35% aqueous solution, 4.6 mmol, ꢀ15 eq) was
added to a solution of compound 6 (0.1 g, 0.31 mmol, 1 eq) in
dichloromethane (3 ml). The reaction mixture was stirred under N2 at
room temperature for 24 h and then diluted with water (5 ml) and ethyl
acetate (20 ml). The layers were separated and the organic layer washed
with brine, dried over MgSO4, and concentrated under vacuum. The
crude residue was redissolved in 1 ml methanol, 3 ml of 2 M ammonia in
methanol was added, and the reaction stirred at room temperature for
2 h. The mixture was then concentrated and diluted with water (5 ml)
and ethyl acetate (10 ml). The layers were separated and then the
aqueous layer was acidified to pH 1 with 1 N HCl and back extracted
with ethyl acetate (3 Â 25 ml). The combined organic extracts were
washed with brine, dried over MgSO4, and concentrated to afford 0.08 g
(0.25 mmol, 81%) of reasonably pure 7.
3-hydroxy-4-[2H3]-methoxyphenylalanine (2)
Compound 7 (0.1 g, 0.32 mmol, 1 eq) was dissolved in 2 ml of
dichloromethane and then 2 ml of triflouroacetic acid was carefully
added. The reaction was stirred for 2 h at room temperature and then
concentrated under vacuum. Preparative reverse phase TLC (water/
methanol [9/1] with 1 mM 1-octanesulfonic acid sodium salt, pH 2)
afforded an oil that was dissolved in 10% acetic acid (3 ml), loaded on
an ion exchange column (Dowex 50 Â 4-400, acid form, 5 g) that was
then washed extensively with water. Elution of the resin with
concentrated ammonium hydroxide followed by concentration under
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vacuum afforded the desired product (0.06 g, 0.28 mmol, 88%) as a light
brown power.
Acknowledgements
This research was supported by a grant from the Robert A. Welch
Foundation (Grant #AA-1355).
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mental and characterization details (NMR spectra, mass spectra, and
HPLC traces) are available from the authors upon request.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 389–394