Acetonide protection of dopamine for the synthesis of highly pure N-docosahexaenoyldopamine

Article abstract of DOI:10.1016/j.tetlet.2010.02.089

Direct acetonide protection of the catechol of dopamine has proven to be problematic due to the formation of Pictet-Spengler isoquinolines. Here we report an efficient method for acetonide protection of dopamine, allowing the preparation of a dopamine prodrug without complications from the Pictet-Spengler reaction. Acetonide-protected dopamine was first synthesized by pre-protecting the amino group with phthaloyl followed by refluxing with 2,2-dimethoxypropane in the presence of TsOH. Further work demonstrated that Fmoc and trifluoroacetyl were also suitable N-protective groups, while Boc-protected dopamine gave an isoquinoline product. Acetonide-protected dopamine was coupled to DHA (all cis-4,7,10,13,16,19-docosahexaenoic acid) to produce the N-DHA-dopamine prodrug with high purity.

Full text of DOI:10.1016/j.tetlet.2010.02.089

Tetrahedron Letters 51 (2010) 2403–2405
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Acetonide protection of dopamine for the synthesis of highly pure
N-docosahexaenoyldopamine
*
Zhongqiang Liu, Bi-Huang Hu , Phillip B. Messersmith
Department of Biomedical Engineering, Northwestern University, Evanston, United States
Comprehensive Cancer Center of Northwestern University, IL, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Direct acetonide protection of the catechol of dopamine has proven to be problematic due to the forma-
tion of Pictet–Spengler isoquinolines. Here we report an efficient method for acetonide protection of
dopamine, allowing the preparation of a dopamine prodrug without complications from the Pictet–Spen-
gler reaction. Acetonide-protected dopamine was first synthesized by pre-protecting the amino group
with phthaloyl followed by refluxing with 2,2-dimethoxypropane in the presence of TsOH. Further work
demonstrated that Fmoc and trifluoroacetyl were also suitable N-protective groups, while Boc-protected
dopamine gave an isoquinoline product. Acetonide-protected dopamine was coupled to DHA (all
cis-4,7,10,13,16,19-docosahexaenoic acid) to produce the N-DHA-dopamine prodrug with high purity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 21 September 2009
Revised 15 February 2010
Accepted 16 February 2010
Available online 20 February 2010
In both vertebrate and invertebrate animals, 4-(2-amino-
ethyl)benzene-1,2-diol (dopamine) is the precursor of norepineph-
rine and epinephrine and also an important neurotransmitter
itself, essential to the normal functioning of the central nervous
system.1 Parkinson’s disease, affecting about 1% of the senior pop-
ulation, is characterized by a reduction of dopamine levels in the
striatum.2 At physiological pH dopamine is almost completely ion-
ized, resulting in low permeation across the blood brain barrier and
precluding it as a direct treatment for Parkinson’s disease.
halogenides or imidazolides gave less satisfactory results.5 The tar-
get products were obtained in lower yields and were contaminated
with the corresponding fatty acid imides.
One of the challenges in manipulating dopamine during the
reactions is that it is readily oxidized, especially under basic condi-
tions, to dopamine quinone, which undergoes self-polymerization
or nucleophilic addition reactions with amino or sulfhydryl
groups.6 Proper protection of the catechol group is often required
during the chemical modification of dopamine. Various catechol-
protecting groups have been reported, including methyl ether,7
cyclic ethyl orthoformate,8 t-butyldimethylsilyl,9 and acetonide.10
Easy protection/deprotection together with good stability to strong
bases and weak acids makes the acetonide protective group espe-
cially useful.11 Ideally, the only byproduct formed during deprotec-
tion is acetone which can be easily removed by evaporation. We
report herein a facile synthesis of N-DHA-dopamine prodrug with
high purity and good yield by coupling reaction between DHA
and acetonide-protected dopamine followed by removal of the
acetonide group.
Direct protection of a catecholamine by acetonide is not easily
accomplished by refluxing with acetone or its ketal 2,2-dimethoxy-
propane (DMP) in the presence of a catalyst, for example, p-tolu-
enesulfonic acid (TsOH). Like other beta phenylamines, dopamine
readily undergoes Pictet–Spengler condensations with aldehydes
and ketones to produce tetrahydroisoquinolines. Acetonide
compounds such as 1-(2,2-dimethylbenzo[1,3]dioxol-5-yl)pro-
pan-2-amine are usually obtained by a complicated method
involving construction of the target molecule from an acetonide-
protected catechol subunit, introduction of a nitro group by a
nitration reagent such as nitroethane, and reduction of the –NO2
to –NH2 group with lithium aluminum hydride.12 Christian’s work
Prodrugs formed through conjugation of lipophilic molecules to
dopamine display enhanced uptake into the brain. DHA (all cis-
4,7,10,13,16,19-docosahexaenoic acid) is a major component of
polyunsaturated fatty acids in retinal and neuronal membranes.
Prodrug N-DHA-dopamine, a potentially useful appetite suppres-
sant, was found to have a Blood–Brain Barrier Index of 30%, compa-
rable with that of D
-glucose (33%).3,4 Unlike amphetamine-based
suppressants, which usually have cardiovascular and neuropsychi-
atric side effects and induce tolerance, N-DHA-dopamine was inac-
tive until it hydrolyzes to release dopamine in the brain and thus
produced no harmful side effects.4 Its synthesis was previously re-
ported by using symmetrical DHA anhydride and unprotected
dopamine. However, carboxylic anhydride is highly reactive to-
ward both the amino group and the free hydroxyl groups of dopa-
mine to give
a mixture. After time-consuming purifications
through mixed-bed ion exchange resins, a correct mass of N-
DHA-dopamine remained elusive.4 Other methods using fatty acyl
* Corresponding author. Tel.: +1 847 491 4928; fax: +1 847 467 5273.
E-mail address: philm@northwestern.edu (P.B. Messersmith).
Present address: College of Oceanography, Hainan University, 58 Renmin Avenue,
Haikou, Hainan Province 570228, China.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.02.089

2404
Z. Liu et al. / Tetrahedron Letters 51 (2010) 2403–2405
demonstrated that when dopamine was blocked as a glucoamide,
direct acetonide cyclization was readily accomplished by refluxing
in acetone.13,14 Considering that the cleavage of a glucoamide bond
to free up the dopamine may require harsh conditions, for exam-
ple, heating in strongly basic solution,15 we decided to explore a
strategy using an easily introducible and removable N-protecting
group to temporarily mask the amino group of dopamine followed
by refluxing with DMP in the presence of TsOH. A systematic study
was also carried out to screen the amino-protecting groups, includ-
ing cyclic imide, carbamate, and amide.
One of the simplest methods to differentiate the reactivity of
the amino and the catechol group is to form an ammonium salt
by protonation with strong acids. For example, selective acetyla-
tion of the catechol of 3,4-dihydroxyphenylalanine (DOPA) was
achieved by protonation of the amino group with hydrogen chlo-
ride or bromide.16,17 Unfortunately, refluxing dopamine hydrochlo-
ride with acetone gave a Pictet–Spengler isoquinoline instead of an
acetonide product (Scheme 1).18
Complete removal of the hydrogen atoms of the amino group
may provide sufficient protection for acetonide cyclization of a cat-
echolamine. For this purpose, the N-phthaloyl (Phth) protective
group was introduced to dopamine by N-carbethoxyphthalimide
in methanol. The resulting Phth-dopamine (1) was then refluxed
with DMP and a catalytic amount of TsOH in benzene. To shift
the reaction equilibrium, volatile byproducts were removed from
the reaction system by distillation. The condensed liquid was recy-
cled using a Soxhlet extractor, the thimble of which was filled with
anhydrous CaCl2 to absorb water and methanol. In addition, anhy-
drous solvent and argon protection were used to prevent the intro-
duction of water from external sources, shortening the reaction
time from 24 h to ca 1–2 h sometimes required for traditional
methods.21 The raw product was subjected to GC–MS analysis,
which gave a major peak in the chromatogram and a molecular
ion at 323 (calcd 323.12) in the mass spectrum, indicating that full
protection of the amino group results in acetonide cyclization
rather than isoquinoline formation. The identification of Phth-
dopamine(acetonide) (2) was further confirmed by high resolution
mass spectrometry (HRMS) and nuclear magnetic resonance
(NMR) spectroscopy, with the latter showing a peak at d ppm
117.8 for the quaternary carbon of the acetonide group. Aceto-
nide-protected dopamine (3)22 was obtained by deprotection of
the phthaloyl group of 2 with hydrazine in DCM. In a previous
application of this strategy, Phth-protected DOPA methyl ester
was converted to Phth-DOPA(acetonide)-OMe leading to the syn-
thesis of Fmoc-DOPA(acetonide)-OH, a useful intermediate to
incorporate DOPA into synthetic peptides.10
The use of hazardous hydrazine23 and the difficulty to remove
hydrazide byproduct24,25 during dephthalylation of Phth-
DOPA(acetonide)-OMe prompted us to examine other N-protecting
groups, such as the carbamates Boc and Fmoc and the amide triflu-
oroacetyl (Tfa), which remove only one of the two hydrogen atoms
of the amino group. The introduction of a carbonyl group adjacent
to the nitrogen atom followed by protonation of the amide nitro-
gen may form an N-acyliminium ion, which probably facilitates
Pictet–Spengler-type reactions due to the increased electrophilic
reactivity.26 However, acetonide cyclization may still be favored
if the reaction is carried out in an aprotic solvent with only cata-
lytic amount of acids.
Fmoc- and Boc-protected dopamine behaved quite differently
when refluxed with DMP and TsOH. Fmoc-dopamine, synthesized
by reacting dopamine with fluorenylmethyloxycarbonyl chloride,19
readily underwent acetonide cyclization to form Fmoc-dopa-
mine(acetonide)(4). The course of the reaction could be conveniently
monitored by the FeCl3 test on a TLC plate, which produced a black
spot for free catechols at room temperature. After acetonide protec-
tion, the spot was indistinguishable from yellow background, but
turned black after heating to 105 °C. In the case of Boc-dopamine20
,
even after a 4-h reflux with 5.5% TsOH and DMP, ferric chloride test
still produced a dark black dot on the TLC plate, indicating the pres-
ence of a significant amount of unprotected catechol. A second por-
tion of TsOH (1 equiv) was added and the reaction mixture was
stirred for another 1 h to give a precipitate, which was collected, puri-
fied, and subjected to intensive characterization. Positive mode LC–
MS data revealed a monoisotopic molecular ion of m/z 194.10, corre-
sponding to C11H16NO2 (MH+), suggesting the addition of a C2H6C
group to dopamine. Negative mode LC–MS data showed a peak at
171.00, indicating the presence of p-toluenesulfonate anions
((MÀH)À, calcd 171.01). Combining the data provided by HRMS and
NMR spectra, the product was identified as a sulfonic saltof a tetrahy-
droisoquinoline (5). The result is not entirely unexpected because the
acid-labile Boc protective group is readily removed when heated in
the presence of TsOH, releasing dopamine that subsequently under-
goes a Pictet–Spengler condensation.27
In contrast to the Boc protective group, Tfa is stable to acids, and
it is smaller in size than the Fmoc group. Although Tfa-dopamine
(6) could be isolated in low yield (19%) from the Bischler–Napier-
alski reaction of Tfa and methyl ether-protected dopamine,28 an
improved procedure was developed by treating dopamine hydro-
Scheme 1. Reagents and conditions: (a) Ref. 18; (b) N-carbethoxyphthalimide, Et3N, 74%; (c) Ref. 19; (d) Ref. 20; (e) CF3COOMe, Et3N in MeOH, 98%; (f) DMP, TsOH (4.5%),
benzene, reflux, yield, 95% (2), 90% (4), 89% (7); (g) DMP, TsOH (1.05 equiv), benzene, reflux, >10%; (h) H2NNH2, DCM, 97%; (i) LiOH, THF/H2O, 87%.

Z. Liu et al. / Tetrahedron Letters 51 (2010) 2403–2405
2405
completed at IMSERC, Northwestern University. The authors
thank Dominic Fullenkamp for his comments on the Letter.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.02.089.
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chloride with methyl trifluoroacetate in methanol in the presence
of triethylamine (Et3N), giving 6 in nearly quantitative yield. The
acetonide protection of compound 6 ran smoothly and was com-
pleted in 1.5 h with a yield of ca 89%. The advantage of using Tfa
protective group is that its deprotection does not require hazard-
ous hydrazine. Compound 3 was readily obtained by hydrolysis
of Tfa-dopamine(acetonide) (7) in lithium hydroxide solution fol-
lowed by simple extraction.
Compound 3 provides a convenient way to synthesize dopamine-
containing molecules including highly pure N-DHA-dopamine pro-
drug (Scheme 2). Commercially available DHA was activated with
dicyclohexylcarbodiimide (DCC) and converted to an N-hydroxy-
succinimide (NHS) ester, which was then stirred with dopamine(ace-
tonide) in toluene and chloroform. The resulting N-DHA-
dopamine(acetonide) (8) was quite stable to routine work-up and
was readily purified by flash chromatography (hexane/EtOAc) to give
a light yellowoil, yield 68% (90% pureby RP-HPLC). Themolecular for-
mulaofthis intermediatewasestablishedbyHRMS:C33H45NO3, MH+,
calcd 504.34722, found 504.34608. Further purification by semi-pre-
parativeRP-HPLC provides>98%purecompound 8, whichwas hydro-
lyzed in 30% trifluoroacetic acid (TFA) chloroform solution to
quantitativelyproduceDHA-dopamine(9),29 abrownoilresiduewith
a correct mass (HRMS): MH+, calcd 464.31592, found 464.31560.
Judging from analytical RP-HPLC chromatogram, the resultant com-
pound 9 was more than 97% pure without requiring any chromato-
graphic purification (see Supplementary data).
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22. 1H NMR (500 MHz, CDCl3): d ppm 6.66–6.59 (m, 3H), 2.92 (t, 2H), 2.66 (t, 2H),
1.91 (br, 2H), 1.67 (s, 6H). 13C NMR (125 MHz, CDCl3): 147.6, 145.9, 132.1,
121.1, 117.6, 108.9, 108.0, 42.8, 38.0, 25.8 (2C). DEPT: CH3, 25.8; CH2, 42.8,
38.0; CH, 121.1, 108.9, 108.0. GC–MS: m/z 193 (17%), 164 (80.6%), 163 (32.4%),
149 (100%), 124 (18.1%), 123 (75%), 121 (18.1%), 106 (23.6%). HRMS (ESI):
C11H15NO2, MH+, calcd 194.11756, found 194.11757.
23. Black, T. D.; Briggs, B. S.; Evans, R.; Muth, W. L.; Vangala, S.; Zmijewski, M. J.
Biotechnol. Lett. 1996, 18, 875–880.
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29. 1H NMR (500 MHz, CDCl3): d ppm 6.79–6.52 (m, 3H), 5.99 (s, catecholic
proton), 5.40–5.28 (m, 12H), 3.42 (m, 2H), 2.95–2.78 (m, 10H), 2.64 (t, 2H,
J = 6.8 Hz), 2.36 (q, 2H, J = 6.8 Hz), 2.21 (t, 2H, J = 6.8 Hz). 2.06 (m, 2H,
J = 7.3 Hz). 0.96 (t, 3H, J = 7.3 Hz). 13C NMR (125 MHz, CDCl3): d 174.2, 144.1,
142.8, 131.8, 130.1, 129.6, 128.3, 128.18, 128.10 (2C), 127.77 (2C),
127.59, 127.54, 127.17, 126.7, 120.3, 115.3, 115.2, 41.2, 36.3, 34.7, 25.6 (4C),
25.5, 23.4, 20.6, 14.3. LC–MS: MH+, calcd 464.31, found 464.30; (2M+H)+, calcd
927.63, found 927.60. HRMS: C30H41NO3, MH+, calcd 464.31592, found
464.31560.
In conclusion, by masking the amino group with a proper N-pro-
tecting group, acetonide-protected dopamine was first synthesized
in the presence of TsOH in anhydrous benzene. The resulting aceto-
nide derivative should facilitate the preparation of highly pure cate-
chol-containing compounds as demonstrated by the synthesis of a
lipophilic prodrug N-DHA-dopamine. This novel strategy is easily
generalized to acetonide protection of other catecholamines.
Acknowledgments
This research was supported by NIH Grants R37 DE 014193,
UL1 RR025741, and U54 CA119341. NMR and Mass Spectra were