Synthesis of photoreactive 2-phenethylamine derivatives-synthesis of adenosine derivatives enabling functional analysis of adenosine receptors by photoaffinity labeling

Article abstract of DOI:10.1002/ejoc.201201657

2-Phenylethylamine is well known as a substructure of many biologically active compounds, and the synthesis of its photoreactive derivatives to allow the analysis of biological functions is reported. This allowed us to synthesise ligands for adenosine rec

Full text of DOI:10.1002/ejoc.201201657

FULL PAPER
DOI: 10.1002/ejoc.201201657
Synthesis of Photoreactive 2-Phenethylamine Derivatives – Synthesis of
Adenosine Derivatives Enabling Functional Analysis of Adenosine Receptors by
Photoaffinity Labeling
Yuta Murai,[a] Katsuyoshi Masuda,[b] Yui Ogasawara,[a] Lei Wang,[a] Yasuyuki Hashidoko,[a]
Yasumaru Hatanaka,[c] So Iwata,[d] Takuya Kobayashi,[d] and Makoto Hashimoto*[a]
Keywords: Nucleosides / Photoaffinity labelling / Biological activity / Receptors / Adenosine
2-Phenylethylamine is well known as a substructure of many
biologically active compounds, and the synthesis of its photo-
reactive derivatives to allow the analysis of biological func-
tions is reported. This allowed us to synthesise ligands for
adenosine receptors, which have many functional roles in
biology and have been extensively studied for their many
roles in maintaining homeostasis. Adenosine is one of the
most common biochemical compounds, but photoaffinity la-
beling has not yet been used to study adenosine receptors.
Synthetic methods for producing photoreactive adenosine
derivatives that can be used with adenosine receptors were
established for the photophores phenyl azide and benzophe-
none. The effect of the introduction of the photoreactive com-
ponents was determined using an adenosine receptor assay.
2-PEA has also been used as a substructure of adeno-
sine-receptor ligands (Scheme 1). Adenosine receptors,
which have been cloned and categorized into four subtypes
(A1, A2A, A2B, and A3),[5] are G-protein-coupled receptors.
In the brain, A2A receptors are expressed at high densities
in the striatum. Descriptions of the crystal structures of
human A2A receptors in complexes with unselective
adenosine-receptor agonists (namely adenosine and N-eth-
Introduction
2-Phenylethylamine (2-PEA) skeletons are components
of many biologically active natural products, especially neu-
rotransmitters or neuromodulators in the central nervous
system.[1] Analysis of the biological functions of 2-PEAs
has been the subject of much research in the pharmaceuti-
cal field. Photoaffinity labeling is a method used to study
the interactions of low-molecular-weight biologically active
compounds with biomolecules.[2] It is suitable for the analy-
sis of biological interactions because it is based on the affin-
ity of the biologically active compound for the biomolecule.
But only a few photoreactive modifications of 2-PEA with
azide[3] or benzophenone[4] have been reported previously,
and the (trifluoromethyl)diazirinyl derivative of 2-PEA has
not yet been reported.
[a] Division of Applied Science, Graduate School of Agriculture,
Hokkaido University,
Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Japan
Fax: +81-117063849
E-mail: hasimoto@abs.agr.hokudai.ac.jp
Homepage: http://www.agr.hokudai.ac.jp/en/
[b] Suntory Institute for Bioorganic Research,
1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka
618-8503, Japan
[c] Graduate School of Medicine and Pharmaceutical Sciences,
University of Toyama,
2630 Sugitani, Toyama 930-0194, Japan
Scheme 1. Structures of adenosine-receptor ligands and designs of
photoreactive CGS-21680 derivatives. NECA (an unselective ago-
nist), ZM241385 (an A2A selective antagonist), and CGS-21680 (1,
an A2A-selective inverse agonist). The carboxyethyl group of CGS-
21680 was exchanged for photoreactive groups in this study.
[d] Department of Medical Chemistry and Cell Biology, Kyoto
University Faculty of Medicine,
Konoe-cho, Yoshdida, Sakyo-Ku, Kyoto 606-8501, Japan
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201657.
2428
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 2428–2433

Photoreactive 2-Phenethylamine Derivatives
yladenosine-5Ј-uronamide (NECA),[6]), and also with an
A
2A-selective antagonist (4-(2-[7-amino-2-(2-furyl)-[1,2,4]-
triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol
(ZM241385)[7]), have recently been published. However, the
crystal-structure studies with the agonists are not yet com-
plete.
3-{4-[2-({6-Amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-
3,4-dihydroxy-oxolan-2-yl]purin-2-yl}amino)ethyl]phenyl}-
propanoic acid (CGS-21680, 1) is a specific inverse agonist
for A2A receptors,[5] and has hypotensive activity in vivo.
Inverse agonists bind to the receptors and stabilize them,
and thus reduce their activity.[8] CGS-21680 and ZM241385
have 2-PEA substituents at the 2-position of adenine. Broad
acceptability has been observed for modifications at the p-
position of the 2-PEA moiety.[9] Modification at the p-posi-
tion of 2-PEA may be used for the introduction of photo-
phores into the ligand skeleton. For satisfactory results to
be obtained in photoaffinity labeling, the right choice of
photophore is critical, but there is no universal rule about
the best choice of photophore.[2e]
In this paper, the synthesis of photoreactive 2-PEA deriv-
atives with various photophores, phenyl azides, benzophe-
nones, and (trifluoromethyl)phenyldiazirines, and their in-
troduction into the NECA skeleton to give photoreactive
CGS-21680 derivatives (Scheme 1) are reported. We also re-
port the results of assays for determining the biological ac-
tivities of these compounds on the purified human adeno-
sine A2A receptor (A2AR), which had been expressed in
Pichia pastoris.[10]
Scheme 2. Synthesis of photoreactive phenylethylamine derivatives.
a) phenyl azide, b) (trifluoromethyl)phenyldiazirine, and c) benzo-
phenone.
Results and Discussion
Our synthetic methodology was based on the initial in-
stallation of the photophores into 2-PEA derivatives. 2-(4-
Bromophenyl)ethylamine (2) was selected as the starting method.[13] Deprotection with trifluoroacetic acid (TFA)
material because its Boc-protected derivative (i.e., 3; Boc = gave the desired product (i.e., 9) in moderate yield
tert-butoxycarbonyl) could be a common precursor for (Scheme 2, b).
both the phenyl azide and the (trifluoromethyl)diazirine.
The benzophenone derivative was synthesized from the
Compound 3 was subjected to substitution of bromide by corresponding N-acetylphenylethylamine derivative (i.e.,
azide (to give 4) in a CuI-catalyzed reaction. The yields were 10).[4] Friedel–Crafts benzoylation with aluminum chloride
influenced by the choice of ligands. A proline / NaOH sys- at 90 °C for 7 h gave 11, and this was followed by deprotec-
tem[11] resulted in the incomplete consumption of the tion of the acetyl moiety under acidic conditions to produce
haloarene. On the other hand, a N,NЈ-dimethylethylenedi- benzophenone derivative 12 in low yield (less than 30%
amine/sodium ascorbate system[12] gave the desired product over two steps). N-Trifluoroacetyl phenylalanine (13)[14] was
(i.e., 4) effectively. Acidic treatment to remove the Boc treated with benzoic anhydride in trifluoromethanesulfonic
group produced phenyl azide derivative 5 in moderate yield acid (TfOH) at room temperature. The Friedel–Crafts
(Scheme 2, a). The overall yield for the preparation of 5 was benzoylation was improved by using TfOH as catalyst and
identical to that from a sole previous report, in which the solvent.[15] Subsequent alkaline deprotection of the tri-
synthesis started from 2-(4-aminophenyl)ethylamine and fluoroacetyl group gave 12 in good yield (up to 82% for
used diazotization–azidation.[5]
two steps) (Scheme 2, c).
For the (trifluoromethyl)phenyldiazirine photophore,
The synthesis of the photoreactive CGS-21680 skeleton
compound 3 was subjected to trifluoroacetylation with was designed such that condensation reactions between 2-
CF3COOEt in the presence of tBuLi and KH to produce 6. chloro-N-ethyladenosine-5Ј-uronamide derivatives and
The trifluoroacetyl moiety was converted into a (trifluoro- photoreactive phenylethylamines would be used to con-
methyl)diazirinyl moiety (in 8) following
a
general struct adenosine derivatives modified at the 2-position.[9a,16]
Eur. J. Org. Chem. 2013, 2428–2433
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2429

M. Hashimoto et al.
FULL PAPER
The photoreactive 2-PEA derivatives were subjected to
condensation with 2-chloroadenosine derivative 15. The
original method, in which the reaction mixtures were heated
to 130 °C in ethanol, was used for the condensations.[9a] De-
tailed studies revealed that (trifluoromethyl)phenyl diaziri-
nyl derivative 9 did not tolerate the high-temperature condi-
tions. The ethyl moiety was always observed in the 1H
NMR spectra of the products with identical integrations.
In the 19F NMR spectra, the trifluoromethyl group in the
diazirine was observed at –66 ppm, and the peak was
shifted to –80 ppm after the condensations. These results
show that the (trifluoromethyl)diazirine moiety was broken
down during the reaction. Precursors of the diazirine pre-
cursor (i.e., Boc-deprotected 6 and 7) were also subjected
to the condensation conditions, but the desired reactions
were not observed.
On the other hand we can establish the condensation
conditions for other photophores (i.e., phenylazides 5 and
benzophenone 12) without decomposition. The reactions
were very slow (3 d), and care was taken regarding evapora-
tion of the solvent during the course of the reaction. De-
tailed studies revealed that 2 equiv. of the phenylethylamine
derivatives and a large excess (20 equiv.) of diisopropylethyl-
amine were required to maintain the nucleophilicity of the
phenylethylamines. A temperature of 110 °C was enough to
promote the reaction of compounds 5 and 15. It was ob-
served that compound 5 decomposed during the reaction at
130 °C, which was reported in the original paper. However,
no difference was observed between 110 and 130 °C for the
reaction of 12 and 15. The reaction required several days
to go to completion. The starting materials (i.e., 15 and 12)
were not consumed completely, but no significant further
reaction was observed after three days. After work-up, the
remaining starting materials 12 and 15 could be re-sub-
jected to the reaction conditions to give the products in the
Scheme 3. Synthesis of photoreactive CGS-21680 derivatives. DI-
PEA = diisopropylethylamine.
same yield. No improvements were observed when the Figure 1. Competitive inhibition assay for photoreactive CGS-
21680 derivatives 19 and 20 against [3H]-NECA (closed circle for
equivalent tertiary amine was used.
19 and closed square for 20) or [3H]-ZM24138 (open circle for 19
Azide derivative 16 was prepared in another way: p-
bromophenylethylamine (2) was condensed with adenosine
derivative 15 to give 18, and this was followed by azidation
and open square for 20).
Conclusions
with sodium azide in the presence of catalytic amounts of
CuI, N,NЈ-dimethylethylenediamine, and sodium ascorbate
We have developed a comprehensive synthesis of 2-PEA
derivatives containing three photophores for photoaffinity
at 100 °C for 6 h.[12] The azidation reactions went smoothly
and effectively. Compounds 16 and 17 were subjected to
ketal hydrolysis under acidic conditions at 30 °C to give
photoreactive adenosine derivatives 19 and 20 (Scheme 3).
The synthesized photoreactive CGS 21680 derivatives
were subjected to competitive binding assays[17] with puri-
fied A2AR with an agonist or antagonist.[10] Competitive
inhibition assays with agonist ([3H]-NECA) revealed that
the affinities of both of the synthetic compounds (i.e., 19
and 20) were of the order of Ͻ1 μm, which is sufficient to
elucidate the biological function of the A2AR. Inhibition
assays with an A2AR-specific antagonist ([3H]-ZM24138)
suggested that the synthetic photoreactive compounds had
sufficient activity for functional analysis of A2ARs. The
modifications did not cause a significant decrease in the
affinity of the derivatives (Figure 1).
labeling. These derivatives were coupled with 2-Cl adenos-
ine derivatives to elucidate functional analysis of adenosine
receptors. Preliminary binding assays for the photoreactive
ligands indicated that they have enough activity to warrant
further analysis by photoaffinity labeling of the A2AR. Fur-
ther functional analysis of A2AR with these synthetic pho-
toreactive reagents is underway.
Experimental Section
General Remarks: NMR spectra were measured with JEOL EX-
280 or Bruker AMX500 spectrometers. All solvents were of reagent
grade and were distilled using the appropriate methods. ESI-TOF-
MS data were obtained with a Waters UPLC ESI-TOF mass spec-
trometer.
2430
www.eurjoc.org
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 2428–2433

Photoreactive 2-Phenethylamine Derivatives
tert-Butyl 4-Bromophenethylcarbamate (3): 2-(4-Bromophenyl)- 531 mg, 1.67 mmol) and hydroxylamine hydrochloride (346 mg,
ethylamine (1.41 g, 7.02 mmol) and NaOH (418 mg, 10.5 mmol)
were dissolved in dioxane (25 mL) and H2O (25 mL), and the mix-
ture was cooled to 0 °C. Di-tert-butyl dicarbonate (2.28 g,
10.53 mmol) in dioxane (12 mL) was added dropwise to the reac-
tion. The reaction was stirred at room temperature for 6 h, and
then the solvents were evaporated. The crude compound was puri-
fied by column chromatography (CH2Cl2/hexane, 1:4 to CH2Cl2)
to give 3 (2.05 g, 97%) as a colorless amorphous solid. Analytical
data were identical to those reported in the literature.[18]
5.02 mmol) in pyridine were stirred at 80 °C for 3 h. The pyridine
was removed on a rotary evaporator, and then the residue was dis-
solved in diethyl ether. This solution was washed with HCl (1 m)
and brine, dried with MgSO4, and the solvents were evaporated.
The crude product was purified by column chromatography
(CH2Cl2 to EtOAc/hexane, 1:2) to give tert-butyl 4-[2,2,2-trifluoro-
1-(hydroxyimino)ethyl]phenethylcarbamate (506 mg, 90%) as a col-
orless amorphous solid. 1H NMR (270 MHz, CDCl3): δ = 10.55
(s, 0.4 H), 10.23 (s, 0.6 H), 7.46 (d, J = 8.2 Hz, 0.8 H, Ar-H), 7.35
(d, J = 8.2 Hz, 1.2 H, Ar-H), 7.28 (d, J = 8.2 Hz, 0.8 H, Ar-H),
7.19 (d, J = 8.2 Hz, 1.2 H, Ar-H), 4.71 (br. s, 1 H, NH), 3.40 (br.
s, 2 H, CH2N), 2.82 (br. s, 2 H, PhCH2), 1.44 (s, 9 H, tBu) ppm.
tert-Butyl 4-Azidophenethylcarbamate (4): tert-Butyl 4-bromo-
phenethylcarbamate (3, 405 mg, 1.35 mmol), NaN3 (180 mg,
2.70 mmol), sodium ascorbate (13.2 mg, 0.067 mmol), CuI (26 mg,
13C NMR (68 MHz, CDCl3): δ = 156.3, 146.6 (q, 2JC,F = 32.2 Hz),
0.135 mmol),
and
N,NЈ-diethylethylenediamine
(22 μL,
1
141.4, 141.0, 128.9 (2 C), 128.8, 128.5, 124.6, 120.9 (q, JC,F
=
0.202 mmol) in EtOH (1.4 mL) and H2O (0.6 mL) were stirred for
4 h at 100 °C. The reaction mixture was poured into ice/water, and
the organic compound was extracted with EtOAc. The organic ex-
tract was washed with brine and dried with MgSO4, and the sol-
vents were evaporated. The crude product was purified by column
chromatography (CH2Cl2/hexane, 1:3 to CH2Cl2) to give 4 (291 mg,
82%) as a colorless amorphous mass. 1H NMR (270 MHz,
CDCl3): δ = 7.17 (d, J = 8.2 Hz, 2 H, Ar-H), 6.95 (d, J = 8.2 Hz,
2 H, Ar-H), 4.70 (br. s, 1 H, NH), 3.34 (q, J = 6.9 Hz, 2 H, CH2N),
2.76 (t, J = 6.9 Hz, 2 H, PhCH2), 1.43 (s, 9 H, tBu) ppm. 13C NMR
(68 MHz, CDCl3): δ = 155.7, 138.1, 135.7, 130.0, 119.0, 79.1, 41.7,
274.7 Hz), 80.0, 41.5, 36.1, 28.3 ppm.
tert-Butyl 4-[2,2,2-trifluoro-1-(hydroxyimino)ethyl]phenethylcarb-
amate (178 mg, 0.538 mmol) was dissolved in acetone (4 mL) and
cooled to 0 °C. Triethylamine (220 μL) and p-toluenesulfonyl chlor-
ide (205 mg, 1.07 mmol) were added, and the mixture was stirred
at the same temperature for 1 h. The solvents were evaporated, and
the crude residue was purified by column chromatography (CH2Cl2
to EtOAc/Hex, 1:3) to give tert-butyl 4-[2,2,2-trifluoro-1-(tosyloxyi-
mino)ethyl]phenethylcarbamate (220 mg, 84%) as
a colorless
amorphous solid. 1H NMR (270 MHz, CDCl3): δ = 7.90 (d, J =
35.5, 28.3 ppm. HRMS (ESI): calcd. for C13H18N4O2Na 285.1327; 8.2 Hz, 2 H, Ar-H), 7.43–7.22 (m, 6 H, Ar-H), 4.62 (br. s, 1 H),
found 285.1350.
3.43–3.33 (m, 2 H, CH2N), 2.88–2.80 (m, 2 H, PhCH2), 2.48–2.46
(m, 3 H, PhCH3), 1.43–1.42 (m, 9 H, tBu) ppm.
2-(4-Azidophenyl)ethanamine (5): TFA (200 μL) was added to tert-
butyl 4-azidophenethylcarbamate (4, 113 mg, 0.43 mmol) at 0 °C,
and the reaction mixture was stirred for 3 h at the same tempera-
ture. The mixture was basified with NaOH (1 m) and stirred for
10 min, then it was extracted with EtOAc. The organic phase was
washed with brine, dried with MgSO4, and evaporated to give 5
tert-Butyl 4-[2,2,2-trifluoro-1-(tosyloxyimino)ethyl]phenethylcarb-
amate (181 mg, 0.372 mmol) was dissolved in diethyl ether (5 mL).
The ether solution was added to excess liquid ammonia at –78 °C
in a pressure tube. The reaction mixture was warmed to room temp.
and then it was stirred for 6 h at that temperature. The excess am-
monia gas was removed in a draft chamber, and the residual solu-
1
(62.0 mg, 89%) as a colorless oil. H NMR (270 MHz, CDCl3): δ
= 7.19 (d, J = 8.2 Hz, 2 H, Ar-H), 6.97 (d, J = 8.2 Hz, 2 H, Ar- tion was concentrated. The crude residue was purified by column
H), 2.95 (t, J = 6.8 Hz, 2 H, CH2N), 2.73 (t, J = 6.8 Hz, 2 H, chromatography (EtOAc/hexane, 1:2) to give 7 (108 mg, 88%) as a
PhCH2), 1.39 (br. s, 2 H, NH2) ppm. 13C NMR (68 MHz, CDCl3): colorless amorphous solid. H NMR (270 MHz, CDCl3): δ = 7.55
1
δ = 137.9, 136.6, 130.1, 119.0, 43.4, 39.3 ppm. HRMS (ESI): calcd.
for C8H11N4 163.0984; found 163.0988.
(d, J = 8.2 Hz, 2 H, Ar-H), 7.25 (d, J = 8.2 Hz, 2 H, Ar-H), 4.61
(br. s, 1 H, CH2NH), 3.37 (q, J = 6.5 Hz, 2 H, CH2N), 2.81 (m, 3
H, PhCH2, CF3CNH), 2.23 (d, J = 9.6 Hz, 1 H, CF3CNH), 1.43
(s, 9 H, tBu) ppm. 13C NMR (68 MHz, CDCl3): δ = 155.8, 141.4,
tert-Butyl 4-(2,2,2-Trifluoroacetyl)phenethylcarbamate (6): tert-
Butyl 4-bromophenethylcarbamate (3, 612 mg, 2.04 mmol) in THF
(5 mL) was added dropwise to a suspension of potassium hydride
(30% suspension in mineral oil; 276 mg, 2.04 mmol) in THF
(10 mL) at 0 °C under N2. The reaction was cooled to –78 °C, and
tBuLi (1.7 m in pentane; 2.6 mL 416 mmol) was added dropwise
over a period of 10 min. Ethyl trifluoroacetate (1.2 mL, 10.3 mmol)
was added, and the mixture was stirred for 6 h at the same tempera-
ture, then ammonium chloride (satd. aq.; 5 mL) was added to the
reaction mixture. The mixture was extracted with diethyl ether. The
organic phase was washed with brine and dried with MgSO4, and
the solvents were evaporated. The crude product was purified by
column chromatography (EtOAc/hexane, 1:3) to give 6 (531 mg,
82%) as a colorless amorphous solid. 1H NMR (270 MHz,
CDCl3): δ = 8.01 (d, J = 8.2 Hz, 2 H, Ar-H), 7.39 (d, J = 8.2 Hz,
2
129.8, 129.2, 128.3, 123.5 (q, 1JC,F = 278.2 Hz), 79.4, 57.8 (q, JC,F
= 35.8 Hz), 41.5, 36.0, 28.3 ppm. 19F NMR (470 MHz, CDCl3): δ
= –75.54 ppm. HRMS (ESI): calcd. for C15H21F3N3O2 332.1586;
found 332.1615.
tert-Butyl
4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]phenethylcarb-
amate (8): Compound 7 (213 mg, 0.645 mmol) and activated MnO2
(500 mg) were suspended in diethyl ether (15 mL). The reaction
mixture was stirred at room temperature for 1 h, then the insoluble
material was removed by filtration. The filtrate was concentrated,
and the residue was purified by column chromatography (CH2Cl2)
1
to give 8 (197 mg, 93%) as a colorless amorphous solid. H NMR
(270 MHz, CDCl3): δ = 7.23 (d, J = 8.2 Hz, 2 H, Ar-H), 7.13 (d,
J = 8.2 Hz, 2 H, Ar-H), 4.52 (br. s, 1 H, CH2NH), 3.36 (q, J =
2 H, Ar-H), 4.84 (br. s, 1 H, NH), 3.40 (t, J = 6.8 Hz, 2 H, CH2N), 6.7 Hz, 2 H, CH2N), 2.80 (t, J = 6.7 Hz, 2 H, PhCH2), 1.42 (s, 9
2.91 (t, J = 6.8 Hz, 2 H, PhCH2), 1.42 (s, 9 H, tBu) ppm. 13C NMR
H, tBu) ppm. 13C NMR (68 MHz, CDCl3): δ = 155.8, 141.0, 129.2,
2
1
(68 MHz, CDCl3): δ = 179.9 (q, JC,F = 34.6 Hz), 155.8, 147.9,
127.1, 126.6, 122.1 (q, JC,F = 273.8 Hz), 79.2, 41.5, 35.9, 28.2 (q,
1
130.3, 129.5, 128.1, 116.6 (q, JC,F = 291.1 Hz), 79.3, 41.1, 36.4,
2JC,F = 40.2 Hz), 28.2 ppm. 19F NMR (470 MHz, CDCl3): δ =
–65.31 ppm. HRMS (ESI): calcd. for C15H18F3N3O2Na 352.1249;
found 352.1254.
28.2 ppm. 19F NMR (470 MHz, CDCl3): δ = –71.34 ppm. HRMS
(ESI): calcd. for C15H19F3NO3 318.1317; found 318.1326.
tert-Butyl 4-[3-(Trifluoromethyl)diaziridin-3-yl]phenethylcarbamate
(7): tert-Butyl 4-(2,2,2-trifluoroacetyl)phenethylcarbamate (6,
2-{4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]phenyl}ethanamine (9):
TFA (250 μL) was added to compound 8 (159 mg, 0.482 mmol) at
Eur. J. Org. Chem. 2013, 2428–2433
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2431

M. Hashimoto et al.
FULL PAPER
0 °C. The reaction mixture was stirred for 2 h at the same tempera-
ture, and then the TFA was removed on a rotary evaporator. The
residue was dissolved in MeOH (1 mL) and NaOH (1 m; 1 mL),
and the mixture was stirred for 1 h at room temperature. The reac-
tion mixture was extracted with EtOAc (30 mL), and the extract
was washed with brine, dried with MgSO4, and evaporated to give
9 (105 mg, 95%) as a yellow oil. 1H NMR (270 MHz, CDCl3): δ =
7.22 (d, J = 8.6 Hz, 2 H, Ar-H), 7.12 (d, J = 8.6 Hz, 2 H, Ar-H),
2.94 (t, J = 6.8 Hz, 2 H, CH2N), 2.74 (t, J = 6.8 Hz, 2 H, PhCH2),
1.23 (br. s, 2 H, NH2) ppm. 13C NMR (68 MHz, CDCl3): δ = 141.7,
129.2, 126.9, 126.5, 122.1 (q, 1JC,F = 273.2 Hz), 43.0, 39.4, 28.2 (q,
39.8 ppm. HRMS (ESI): calcd. for C15H16NO 226.1232; found
226.1215.
(3aR,4S,6R,6aS)-6-[6-Amino-2-(4-azidophenethylamino)-9H-purin-
9-yl]-N-ethyl-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carb-
oxamide (16)
Method 1, from 13: A mixture of 5 (98.1 mg, 0.604 mmol), 15
(45.9 mg, 0.119 mmol), and DIPEA (166 μL, 0.956 mmol) in EtOH
(2 mL) was stirred at 110 °C for 3 d. The solvent was removed, and
the crude product was purified by column chromatography (EtOAc
to EtOAc/MeOH, 10:1) to give 16 (9.9 mg, 16 %) as a white
amorphous solid.
2JC,F
= δ =
40.4 Hz) ppm. 19F NMR (470 MHz, CDCl3):
–65.32 ppm. HRMS (ESI): calcd. for C10H11F3N3 230.0905; found
230.0889.
Method 2, from 18: Compound 18 (75.4 mg, 0.138 mmol), NaN3
(22.4 mg, 0.345 mmol), sodium ascorbate (3.5 mg, 0.017 mmol),
CuI (6.7 mg, 0.035 mmol), and N,NЈ-dimethylethylenediamine
(5.5 μL, 0.051 mmol) in EtOH (1.4 mL) and H2O (0.6 mL) were
stirred at 100 °C for 6 h. The reaction mixture was poured into ice/
water (15 mL) and extracted with EtOAc (30 mL). The organic
phase was washed with brine and dried with MgSO4, and the sol-
vents were evaporated. The crude product was purified by column
chromatography (EtOAc to EtOAc/MeOH, 10:1) to give 16
(34.4 mg, 49 %) as a colorless amorphous solid. 1H NMR
(270 MHz, CD3OD): δ = 7.88 (s, 1 H, 8-H), 7.34 (d, J = 8.2 Hz, 2
H, Ar-H), 6.99 (d, J = 8.2 Hz, 2 H, Ar-H), 6.24 (s, 1 H, 1Ј-H), 5.74
(d, J = 6.3 Hz, 1 H, 2Ј-H), 5.58 (d, J = 6.3 Hz, 1 H, 3Ј-H), 4.63 (s,
1 H, 4Ј-H), 3.78–3.62 (m, 1 H, NCH2CH2Ph), 3.57–3.42 (m, 1 H,
NCH2CH2Ph), 3.00–2.70 (m, 4 H, CH2Ph, CH2CH3), 1.58 (s, 3 H,
CH3), 1.38 (s, 3 H, CH3), 0.61 (t, J = 7.4 Hz, 3 H, CH2CH3) ppm.
13C NMR (68 MHz, CD3OD): δ = 171.6, 160.8, 157.4, 152.4, 139.5,
139.2, 138.3, 131.4, 119.9, 114.5, 114.3, 92.5, 89.3, 85.5, 85.0, 43.8,
36.1, 34.8, 27.0, 25.4, 14.0 ppm. HRMS (ESI): calcd. for
C23H29N10O4 509.2373; found 509.2357.
N-(4-Benzoylphenethyl)acetamide (11): Benzoyl chloride (0.862 g,
7.11 mmol) and N-acetyl-β-phenethylamine (10, 1.0 g, 6.13 mmol)
were dissolved in dry nitrobenzene (3 mL), and AlCl3 (1.63 g,
12.2 mmol) was added in small portions to the solution at 0 °C.
The reaction mixture was heated at 90 °C for 7 h, then it was
poured into HCl (conc.), and the mixture was concentrated. The
residue was extracted with diethyl ether. The organic phase was
washed with sodium hydroxide (10% aq.) and with water, dried
with sodium sulfate, filtered and concentrated. The residue was
subjected to silica gel chromatography (CHCl3/MeOH, 20:1) to
give 11 (1.08 g, 66%) as a pale yellow oil. 1H NMR (270 MHz,
CDCl3): δ = 7.83–7.28 (m, 9 H, Ar-H), 5.48 (br. s, 1 H, NH), 3.56
(q, J = 6.6 Hz, 2 H, CH2N), 2.92 (t, J = 6.6 Hz, 2 H, PhCH2), 1.96
(s, 3 H, COCH3) ppm. 13C NMR (68 MHz, CDCl3): δ = 196.3,
170.1, 143.9, 137.6, 136.0, 132.4, 130.5, 129.9, 128.7, 128.3, 40.4,
35.7, 23.3 ppm. HRMS (ESI): calcd. for C17H18NO2 268.1338;
found 268.1330.
N-(4-Benzoylphenethyl)-2,2,2-trifluoroacetamide (14): TfOH (1 mL)
was added to N-trifluoroacetyl-β-phenethylamine 13[13] (299 mg,
1.38 mmol) and benzoic anhydride (624 mg, 2.76 mmol) at 0 °C.
The reaction mixture was allowed to warm to room temperature
and stirred for 4 h. The reaction was quenched with ice/water/
EtOAc. The organic phase was washed brine, and the solvents were
evaporated. The crude product was purified by column chromatog-
raphy (EtOAc/hexane, 1:4) to give 14 (382 mg, 86%) as a colorless
oil. 1H NMR (270 MHz, CDCl3): δ = 7.75–7.17 (m, 10 H, Ar-H
and NH), 3.64 (q, J = 6.7 Hz, 2 H, CH2N), 2.98 (t, J = 7.3 Hz, 2
H, PhCH2) ppm. 13C NMR (68 MHz, CDCl3): δ = 196.56, 157.38
(q, 2JC,F = 37.1 Hz), 142.87, 137.34, 135.92, 132.45, 130.45, 129.79,
(3aR,4S,6R,6aS)-6-[6-Amino-2-(4-benzoylphenethylamino)-9H-
purin-9-yl]-N-ethyl-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-
4-carboxamide (17): A mixture of 12 (114 mg, 0.51 mmol), 15
(62.8 mg, 0.163 mmol), and DIPEA (284 μL, 1.63 mmol) in EtOH
(2 mL) was stirred at 120 °C for 3 d. The solvent was removed, and
the crude product was purified by column chromatography (EtOAc
to EtOAc/MeOH, 10:1) to give 15 (34.1 mg, 37%) as a colorless
1
amorphous solid. H NMR (270 MHz, CD3OD): δ = 7.84 (s, 1 H,
8-H), 7.76–7.40 (m, 9 H, Ar-H), 6.21 (s, 1 H, 1Ј-H), 5.72 (d, J =
5.6 Hz, 1 H, 2Ј-H), 5.53 (d, J = 5.6 Hz, 1 H, 3Ј-H), 4.60 (s, 1 H,
4Ј-H), 3.84–3.70 (m, 1 H, NCH2 CH2Ph), 3.62–3.48 (m, 1 H, NCH2
CH2Ph), 3.02 (t, J = 7.3 Hz, 2 H, CH2CH3), 2.94–2.68 (m, 2 H,
NCH2CH2Ph), 1.54 (s, 3 H, CH3), 1.34 (s, 3 H, CH3), 0.59 (t, J =
7.3 Hz, 3 H, CH2CH3) ppm. 13C NMR (68 MHz, CD3OD): δ =
198.4, 171.6, 160.8, 157.4, 152.4, 147.1, 139.6, 139.1, 136.6, 133.6,
131.3, 130.9, 130.1, 129.5, 114.5, 114.3, 92.5, 89.3, 85.5, 85.0, 43.4,
36.9, 34.78, 27.0, 25.4, 14.0 ppm. HRMS (ESI): calcd. for
C30H34N7O5 572.2621; found 572.2646.
1
128.56, 128.23, 115.73 (q, JC,F = 288.1 Hz), 40.81, 34.89 ppm.
HRMS (ESI): calcd. for C17H15F3NO2 322.1055; found 322.1068.
[4-(2-Aminoethyl)phenyl](phenyl)methanone (12)
Method 1, from 11: Compound 11 (0.524 g, 1.96 mmol) was dis-
solved in HCl (6 m; 3.2 mL) and the mixture was heated at 120 °C
for 7 h. The solution was extracted with EtOAc twice. The aqueous
phase was basified with NaOH and extracted with EtOAc three
times. The organic phase was concentrated to give 12 (0.194 mg,
44%) as a yellow oil.
(3aR,4S,6R,6aS)-6-[6-Amino-2-(4-bromophenethylamino)-9H-
purin-9-yl]-N-ethyl-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-
4-carboxamide (18): A mixture of 2 (141 μL, 0.993 mmol), 15
(70.7 mg, 0.184 mmol), and DIPEA (320 μL, 1.84 mmol) in EtOH
Method 2, from 14: NaOH (1 m aq.; 4 mL) was added to 14
(264 mg, 0.821 mmol) in methanol (1 mL) at 0 °C, and the reaction (2 mL) was stirred at 130 °C for 3 d. The solvent was removed, and
mixture was stirred for 2 h at the same temperature. The compound
was extracted with EtOAc, and washed with brine. The organic
phase was evaporated to give 12 (177 mg, 95%) as a colorless oil.
1H NMR (270 MHz, CDCl3): δ = 7.83–7.29 (m, 9 H, Ar-H), 3.04
(br. s, 2 H, NH2), 2.85 (t, J = 6.8 Hz, 2 H, CH2N), 1.25 (d, J =
the crude product was purified by column chromatography (EtOAc
to EtOAc/MeOH, 10:1) to give 18 (74.1 mg, 74%) as a colorless
1
amorphous solid. H NMR (270 MHz, CD3OD): δ = 7.90 (s, 1 H,
8-H), 7.45 (d, J = 8.6 Hz, 2 H, Ar-H), 7.26 (d, J = 8.6 Hz, 2 H,
Ar-H), 6.27 (s, 1 H, 1Ј-H), 5.76 (d, J = 6.3 Hz, 1 H, 2Ј-H), 5.61 (d,
6.8 Hz, 2 H, PhCH2) ppm. 13C NMR (68 MHz, CDCl3): δ = 196.4, J = 6.3 Hz, 1 H, 3Ј-H), 4.66 (s, 1 H, 4Ј-H), 3.80–3.70 (m, 1 H,
144.8, 137.7, 135.7, 132.3, 130.4, 129.9, 128.7, 128.2, 43.0,
NCH2 CH2Ph), 3.58–3.48 (m, 1 H, NCH2CH2Ph), 2.88 (m, 4 H,
2432
www.eurjoc.org
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 2428–2433

Photoreactive 2-Phenethylamine Derivatives
CH2Ph, CH2CH3), 1.61 (s, 3 H, CH3), 1.40 (s, 3 H, CH3), 0.64 (t,
J = 7.3 Hz, 3 H, CH2CH3) ppm. 13C NMR (68 MHz, CD3OD): δ
= 168.8, 159.3, 155.8, 151.1, 138.4, 137.0, 131.4, 130.5, 112.0, 114.1,
113.5, 91.2, 87.8, 83.8, 83.3, 42.4, 35.0, 33.7, 26.6, 24.8, 14.1 ppm.
HRMS (ESI): calcd. for C23H29BrN7O4 546.1464 and 548.1444;
found 546.1464 and 548.1456.
Acknowledgments
This research was partially supported by the Ministry of Educa-
tion, Science, Sports and Culture (Grant-in-Aid for Scientific Re-
search (C), grant numbers 19510210 and 21510219).
[1] P. R. Paetsch, A. J. Greenshaw, Neurochem. Res. 1993, 18,
1015–1022.
(2S,3R,4S,5R)-5-[6-Amino-2-(4-azidophenethylamino)-9H-purin-9-
yl]-N-ethyl-3,4-dihydroxytetrahydrofuran-2-carboxamide (19): A
solution of 16 (33.8 mg, 0.066 mmol) in HCl (1 m; 10 mL) and
MeCN (2 mL) was stirred for 3 h at 40 °C. The solution was cooled
to 0 °C, basified with NaHCO3 (satd. aq.), and extracted with
EtOAc. The organic phase was washed with brine, dried with
MgSO4, and evaporated to give 19 (26.5 mg, 86%) as a colorless
[2] a) J. Brunner, Annu. Rev. Biochem. 1993, 62, 483–514; b) Y.
Hatanaka, H. Nakayama, Y. Kanaoka, Rev. Heteroat. Chem.
1996, 14, 213–243; c) G. Dormán, G. D. Prestwich, Trends Bi-
otechnol. 2000, 18, 64–77; d) T. Tomohiro, M. Hashimoto, Y.
Hatanaka, Chem. Rec. 2005, 5, 385–395; e) M. Hashimoto, Y.
Hatanaka, Eur. J. Org. Chem. 2008, 2513–2523; f) L. Dubinsky,
B. P. Krom, M. M. Meijler, Bioorg. Med. Chem. 2012, 20, 554–
570.
1
amorphous solid. H NMR (270 MHz, CD3OD): δ = 8.00 (s, 1 H,
8-H), 7.25 (d, J = 8.2 Hz, 2 H, Ar-H), 6.96 (d, J = 8.2 Hz, 2 H,
Ar-H), 5.94 (d, J = 6.3 Hz, 1 H, 1Ј-H), 4.99 (t, J = 5.9 Hz, 1 H,
2Ј-H), 4.53–4.48 (m, 1 H, 3Ј-H), 4.41 (d, J = 2.6 Hz, 1 H, 4Ј-H),
3.67–3.39 (m, 2 H, NCH2 CH2Ph), 3.29–3.05 (m, 2 H, CH2Ph),
2.86 (t, J = 7.1 Hz, 2 H, CH2CH3), 1.03 (t, J = 7.1 Hz, 3 H,
CH2CH3) ppm. 13C NMR (68 MHz, CD3OD): δ = 172.0, 161.1,
157.5, 153.0, 139.2, 139.0, 138.2, 131.4, 120.0, 114.7, 90.0, 85.4,
74.7, 73.4, 44.2, 36.3, 35.1, 14.7 ppm. HRMS (ESI): calcd. for
C20H25N10O4 469.2060; found 469.2065.
[3] D. W. Romanini, M. B. Francis, Bioconjugate Chem. 2008, 19,
153–157.
[4] H. Rudolf, R. Manfred, S. Robert, G. Wolfgang, R. Eckhard,
DE Patent 1988, 3718638.
[5] B. B. Fredholm, A. P. IJzerman, K. A. Jacobson, J. Linden,
C. E. Müller, Pharmacol. Rev. 2011, 63, 1–34.
[6] a) F. Xu, H. Wu, V. Katritch, G. W. Han, K. A. Jacobson, Z. G.
Gao, V. Cherezov, R. C. Stevens, Science 2011, 332, 322–327;
b) G. Lebon, T. Warne, P. C. Edwards, K. Bennett, C. J. Lang-
mead, A. G. Leslie, C. G. Tate, Nature 2011, 474, 521–525.
[7] A. S. Doré, N. Robertson, J. C. Errey, I. Ng, K. Hollenstein,
B. Tehan, E. Hurrell, K. Bennett, M. Congreve, F. Magnani,
C. G. Tate, M. Weir, F. H. Marshall, Structure 2011, 19, 1283–
93.
[8] G. Milligan, Mol. Pharmacol. 2003, 64, 1271–1276.
[9] a) A. J. Hutchison, M. Williams, R. deJesus, R. Yokoyama,
H. H. Oei, G. R. Ghai, R. L. Webb, H. C. Zoganas, G. A.
Stone, M. F. Jarvis, J. Med. Chem. 1990, 33, 1919–1924; b) J. E.
Francis, R. L. Webb, G. R. Ghai, A. J. Hutchison, M. A. Mos-
kal, R. deJesus, R. Yokoyama, S. L. Rovinski, N. Contardo, R.
Dotson, B. Barclay, G. A. Stone, M. F. Jarvis, J. Med. Chem.
1991, 34, 2570–2579; c) N. E. Hausler, S. M. Devine, F. M.
McRobb, L. Warfe, C. W. Pouton, J. M. Haynes, S. E. Bottle,
P. J. White, P. J. Scammells, J. Med. Chem. 2012, 55, 3521–3534.
[10] T. Yurugi-Kobayashi, H. Asada, M. Shiroishi, T. Shimamura,
S. Funamoto, N. Katsuta, K. Ito, T. Sugawara, N. Tokuda, H.
Tsujimoto, T. Murata, N. Nomura, K. Haga, T. Haga, S. Iwata,
T. Kobayashi, Biochem. Biophys. Res. Commun. 2009, 380, 271–
276.
[11] W. Zhu, D. Ma, Chem. Commun. 2004, 888–889.
[12] J. Andersen, U. Madsen, F. Björkling, X. Liang, Synlett 2005,
2209–2213.
[13] Y. Murai, K. Masuda, Y. Sakihama, Y. Hashidoko, Y. Hat-
anaka, M. Hashimoto, J. Org. Chem. 2012, 77, 1092–1098.
[14] S. G. Stewart, C. H. Heath, E. L. Ghisalberti, Eur. J. Org.
Chem. 2009, 1934–1943.
[15] R. Murashige, Y. Hayashi, S. Ohmori, A. Torii, Y. Aizu, Y.
Muto, Y. Murai, Y. Oda, M. Hashimoto, Tetrahedron 2011, 67,
641–649.
[16] a) R. C. Foitzik, S. M. Devine, N. E. Hausler, P. J. Scammells,
Tetrahedron 2009, 65, 8851–8857; b) S. M. Devine, P. J. Scam-
mells, Eur. J. Org. Chem. 2011, 1092–1098.
(2S,3R,4S,5R)-5-[6-Amino-2-(4-benzoylphenethylamino)-9H-purin-
9-yl]-N-ethyl-3,4-dihydroxytetrahydrofuran-2-carboxamide (20): A
solution of 17 (24.9 mg, 0.0435 mmol) in HCl (1 m; 10 mL) and
MeCN (3 mL) was stirred for 3 h at 40 °C. The solution was cooled
to 0 °C, basified with NaHCO3 (satd. aq.), and extracted with
EtOAc. The organic phase was washed with brine, dried with
MgSO4, and evaporated to give 20 (20.6 mg, 89 %) as a white
1
amorphous solid. H NMR (270 MHz, CD3OD): δ = 8.03 (s, 1 H,
8-H), 7.74–7.38 (m, 9 H, Ar-H), 5.95 (d, J = 6.6 Hz, 1 H, 1Ј-H),
4.88 (t, J = 5.9 Hz, 1 H, 2Ј-H), 4.51–4.45 (m, 1 H, 3Ј-H), 4.41 (d,
J = 3.0 Hz, 1 H, 4Ј-H), 3.79–3.53 (m, 2 H, NCH2 CH2Ph), 3.27–
3.10 (m, 2 H, CH2Ph), 3.00 (t, J = 7.6 Hz, 2 H, CH2CH3), 1.04 (t,
J = 7.6 Hz, 3 H, CH2CH3) ppm. 13C NMR (68 MHz, CD3OD): δ
= 198.5, 172.1, 146.8, 139.5, 139.0, 136.7, 133.7, 131.4, 131.0, 130.1,
129.5, 90.1, 85.4, 79.5, 74.7, 73.9, 43.8, 37.0, 35.3, 32.8, 23.7, 14.7,
14.4 ppm. HRMS (ESI): calcd. for C27H30N7O5 532.2308; found
532.2324.
Ligand Binding Assays for Adenosine A2A Receptor (A2AAR): Com-
pounds 17 and 18 were subjected to ligand binding assays with
purified human A2AAR, which had been expressed in Pichia pas-
toris,[7] using radioligands of the antagonist [3H]-ZM241385 and
the agonist [3H]-NECA as described previously.[10] Briefly, GF/F
glass filters were pre-soaked with polyethyleneamine (0.3%). The
binding experiments were carried out as single-point binding mea-
surements in duplicate using radioligand (20 nM) in Hepes (20 mm;
pH 7.0) containing NaCl (100 mm) at 25 °C for 30 min. The incu-
bation was terminated by the addition of Tris-HCl (pH 7.4; 20 mm;
2 mL), and the mixture was rapidly filtered through the GF/F fil-
ters. The filters were then washed three times with the above buffer
(5 mL).
[17] T. Hino, T. Arakawa, H. Iwanari, T. Yurugi-Kobayashi, C.
Ikeda-Suno, Y. Nakada-Nakura, O. Kusano-Arai, S. Weyand,
T. Shimamura, N. Nomura, A. D. Cameron, T. Kobayashi, T.
Hamakubo, S. Iwata, T. Murata, Nature 2012, 482, 237–240.
[18] K.-I. Tanaka, S. Yoshifuji, Y. Nitta, Chem. Pharm. Bull. 1988,
36, 3125–3129.
Supporting Information (see footnote on the first page of this arti-
1
cle): H and 13C NMR spectra.
Received: December 7, 2012
Published Online: March 5, 2013
Eur. J. Org. Chem. 2013, 2428–2433
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2433