Regio- and stereoselective synthesis of truncated 3′- aminocarbanucleosides and their binding affinity at the A3 adenosine receptor

Article abstract of DOI:10.1039/c1ob05853c

The stereoselective synthesis of truncated 3′-aminocarbanucleosides 4a-dvia a stereo- and regioselective conversion of a diol 9 to bromoacetate 11a and their binding affinity towards the human A₃ adenosine receptor are described.

Full text of DOI:10.1039/c1ob05853c

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PAPER
Regio- and stereoselective synthesis of truncated 3¢-aminocarbanucleosides
and their binding affinity at the A3 adenosine receptor
Mun Ju Choi,a Girish Chandra,a Hyuk Woo Lee,a Xiyan Hou,a Won Jun Choi,a,b Khai Phan,c
Kenneth A. Jacobsonc and Lak Shin Jeong*a
Received 30th May 2011, Accepted 30th June 2011
DOI: 10.1039/c1ob05853c
The stereoselective synthesis of truncated 3¢-aminocarbanucleosides 4a–d via a stereo- and
regioselective conversion of a diol 9 to bromoacetate 11a and their binding affinity towards the human
A3 adenosine receptor are described.
Introduction
Adenosine binds to four subtypes (A1, A2A, A2B, and A3) of
adenosine receptors (ARs) located on cell membranes and regu-
lates many physiological functions by modulating cell signalling.1
Second messengers, such as inositol trisphosphate (IP3), diacyl-
glycerol (DAG), and cyclic AMP, are essential for cell signalling
and are controlled by ARs. Thus, ARs have been promising
targets for the treatment of several diseases associated with
these signalling pathways.2 For example, A3 AR agonists have
therapeutic potential against cancer,3 cerebral ischemia,4 and
myocardial ischemia,5 while A3 AR antagonists display anti-
inflammatory,6 anti-asthma,7 and anti-glaucoma activities.8
The locked methanocarba nucleosides 1,9 with fixed Northern
conformations were reported to be potent and selective A3 AR
agonists. However, their truncated analogues 210 were transformed
to potent and species-independent A3 AR antagonists due to the
absence of 5¢-uronamide, whose NH serves as a key hydrogen-
bonding donor essential for receptor activation. On the other
hand, 3¢-deoxy-3¢-aminoadenosine derivatives 311 have been dis-
covered to be potent and selective A3 AR agonists. The 3¢-
amino group of 3 improved its selectivity for other subtypes
and its aqueous solubility. Based on these findings, we designed
compounds 4 (Fig. 1), which combine the properties of truncated
nucleosides and 3¢-amino nucleosides to develop potent and
selective A3 AR antagonists. The 3¢-amino group was introduced
by the regioselective opening of the acylinium ion with bromide,
followed by treating the bromide with sodium azide. Herein,
we report the stereo- and regioselective synthesis of truncated
3¢-aminonucleosides 4 starting from D-ribose and their binding
affinity towards the human A3 AR.
Fig. 1 Rationale for the design of the target nucleosides.
Results and discussion
Synthesis of the desired nucleosides was achieved by first syn-
thesising the glycosyl donor and then condensing it with purine
bases.
Scheme 1 illustrates the synthesis of the key precursor 9 for the
introduction of a 3¢-amino group. D-Ribose was easily converted
to cyclopentenone 5 according to our previously published
procedure.12 Treatment of cyclopentenone 5 with NaBH4 in the
presence of CeCl3·7H2O gave the a-allylic alcohol 612 as a single
diastereomer, resulting from the attack of the hydride to the
less hindered convex face of the ketone.13 The stereoselective
cyclopropanation of the a-allylic alcohol 6 with diethyl zinc
and methylene iodide afforded the cyclopropyl-fused alcohol 7
as a single diastereomer due to the directing effect of the allylic
aLaboratory of Medicinal Chemistry, College of Pharmacy and Department
of Bioinspired Science, Ewha Womans University, Seoul, 120–750, Korea.
E-mail: lakjeong@ewha.ac.kr; Fax: +82-2-3277-2851; Tel: +82-2-3277-
3466
bCollege of Pharmacy, Dongguk University, Kyungki-do, 410-774, Korea
cMolecular Recognition Section, Laboratory of Bioorganic Chemistry,
National Institute of Diabetes, and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, Maryland, 20892, USA
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the pseudoequatorial direction (route b), yielding the desired
regioisomer 11a exclusively. Two regioisomers, 11a and 11b, were
unambiguously assigned by the diagnostic coupling constants typ-
ical of the boat conformation of the bicyclo[3.1.0]hexane system,
which has been extensively confirmed by X-ray crystallography
and NMR analysis.16 In the case of compound 11a, the coupling
constant of the JH2
,
was greater than zero because of the cis
H3
relationship, while, in the case of 11b, the corresponding coupling
constant was zero because a H2–C–C–H3 dihedral angle with a
trans relationships is close to 90◦.16 Thus, H-2 in 11a should be
split into a pseudo triplet or doublet of doublets, but H-2 in 11b
should appear as a doublet. Indeed, 1H NMR spectroscopy of 11a
indicated that H-2 showed a pseudo triplet at 5.95 ppm, confirming
the structure of 11a. Furthermore, irradiation of the H-2 pseudo
triplet at d 5.95 caused the H-3 doublet at d 5.60 to coalesce into
a singlet. This situation could not be the case for the alternative
regioisomer 11b. It should be noted that this type of assignment
works only in this system and could not be applied to the flexible,
carbocyclic counterparts.
The desired product 11a was treated with NaN3 in DMF at
100 ◦C to give the azido derivative 12a (44%), but its stereoisomer
12b (11%) was also obtained as a minor isomer (Scheme 3). As
illustrated in Scheme 3, the SN2 reaction of the bromide 11a with
sodium azide yielded the desired azido compound 12a as a major
isomer, but 11a also underwent an intramolecular SN2 reaction
with heating, forming the intermediate 10, which was attacked by
sodium azide at the C4 position (Scheme 2) to give 12b as a minor
isomer. The structures of 12a and 12b were also confirmed by the
diagnostic coupling constants typical of the boat conformation
of the bicyclo[3.1.0]hexane system, as shown in Scheme 3.16 In
the case of 12a, H-4 was split as a pseudo triplet at 4.12 ppm
because of two dihedral angles (H3–C–C–H4 and H4–C–C–H5)
with cis relationships, while, in the case of 12b, H-4 was split as a
singlet at 3.93 ppm because of two dihedral angles (H3–C–C–H4
and H4–C–C–H5) with trans relationships.16
Scheme 1
hydroxyl group.13,14 Benzoylation of 7, followed by hydrolysis of
the isopropylidene group in benzoate 8 with 50% trifluoroacetic
acid (TFA) yielded the diol 9, which is a key precursor to
azidation.
Treatment of the diol 9 with 2-acetoxyisobutyryl bromide15
gave bromoacetate 11a as a single regioisomer (75%) without the
formation of its regioisomer 11b (Scheme 2). In this reaction, after
the diol 9 was first converted to an acylinium ion 10, the bromide
attacked the C4 position of 10 in the pseudoaxial direction (route
a), which was more highly favoured than the same reaction in
Scheme 2
Scheme 3
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Table 1 Binding affinities of the known A3AR antagonist 2 and 3¢-amino derivatives 4a–d at three subtypes of hARs
Affinity (Ki, nM SEM, or % inhibition)a
Compound (X, R1, R2)
hA1
hA2A
hA3
2 (X = OH, R1 = Cl, R2 = 3-iodobenzyl)b
4a (X = NH2, R1 = Cl, R2 = 3-iodobenzyl)
4b (X = NH2, R1 = Cl, R2 = methyl)
4c (X = NH2, R1 = H, R2 = 3-iodobenzyl)
4d (X = NH2, R1 = H, R2 = methyl)
3040 610
15 8%
1080 310
12% 5%
5% 2%
24 3%
1.44 0.60
24.7% 0.6%
42.3% 7.4%
5460 1280
27.2% 5.3%
8
6%
10%
2%
3
5
2%
a All binding experiments were performed using adherent mammalian cells stably transfected with cDNA encoding the appropriate hAR (A1AR and
A3AR in CHO cells and A2AAR in HEK-293 cells). Binding was carried out using 1 nM [3H]R-PIA, 10 nM [3H]CGS21680, or 0.5 nM [125I]I-AB-MECA as
radioligands for A1, A2A, and A3ARs, respectively. Values are expressed as mean sem, n = 3-4 (outliers eliminated) and normalized against a non-specific
binder, 5¢-N-ethylcarboxamidoadenosine (NECA, 10 mM). Values expressed as a percentage refer to percent inhibition of specific radioligand binding at
10 mM, with nonspecific binding defined using 10 mM NECA. b Data from ref. 10.
Removal of the protecting groups of 12a with NaOMe afforded
the diol 13 (Scheme 4). Treatment of the diol 13 with thionyl
chloride followed by oxidation of the resulting cyclic sulfiphite 14
with RuCl3 and NaIO4 gave the cyclic sulphate 15 as an epoxide
surrogate.17
with methylamine produced 18b and 18d, respectively. Conversion
of the 3¢-azido group to the 3¢-amino group was achieved by
treating 18a–d with Ph3P and NH4OH in THF-H2O to give 4a–d,
respectively.11
Binding assays were performed using standard radioligands
and membrane preparations from Chinese hamster ovary (CHO)
cells stably expressing the human A1 or A3AR or from human
embryonic kidney cells (HEK-293) expressing human A2AAR.10
As shown in Table 1, all synthesised 3¢-amino derivatives dis-
played much lower binding affinities towards the human A3
AR than the reference A3 AR antagonist 2. This result was
unexpected because the 3¢-amino group can serve as the same
hydrogen bonding donor as the 3¢-hydroxyl group, and some 3¢-
amino nucleosides are reported to be potent A3AR agonists.11
Only compound 4c showed moderate binding affinity for the
human A3 AR.
Conclusions
In summary, we have achieved stereoselective synthesis of trun-
cated 3¢-aminocarbanucleosides, which are fixed in an (N) con-
formation. Their binding affinities towards human ARs were
also determined. The key step for the introduction of the 3¢-
amino group was to stereo- and regioselectively convert the diol
9 to the bromoacetate 11a via an acylinium ion 10. Finally, we
observed that azidation of the bromoacetate 11a with sodium
azide proceeded via an acylinium ion 10, in addition to direct a
SN2 reaction.
Scheme 4
The glycosyl donor 15 was transformed to the 3¢-deoxy-3¢-
aminoadenosine derivatives 4a–d as illustrated in Scheme 5. Con-
densation of 15 with 2,6-dichlorpurine and 6-chloropurine gave
the azido derivatives 16 and 17, respectively, after acid-catalysed
hydrolysis of the sulphates.18 The ring-opening reaction proceeded
with complete regioselectivity. Proton NMR spectroscopy of 16
confirmed the structure unambiguously by virtue of the diagnostic
coupling constants typical of the boat conformation of the
bicyclo[3.1.0]hexane system (Scheme 3). Indeed, irradiation of the
H-2¢ triplet at d 4.68 caused the H-3¢ doublet at d 4.20 to coalesce
into a singlet. Treatment of 16 and 17 with 3-iodobenzylamine
produced 18a and 18c, respectively. Treatment of 16 and 17
Experimental section
General methods
1H and 13C NMR Spectra (CDCl3 or CD3OD) were recorded on
1
400 and 100 MHz NMR, respectively. The H NMR data are
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Scheme 5
reported as peak multiplicities: s for singlet, d for doublet, dd for (+)-(1R,2S,3S,4R,5S)-Benzoic acid 3,4-O-Isopropylidene
doublet of doublets, t for triplet, q for quartet, br s for broad singlet
and m for multiplet. Coupling constants are reported in hertz.
The chemical shifts are reported as parts per million (d) relative
to the solvent peak. Column chromatography was performed on
silica gel 60 (230–400 mesh). All the anhydrous solvents were
distilled over CaH2, P2O5 or sodium/benzophenone prior to the
reaction.
bicycle[3.1.0]hexa-2-yl-ester (8)
Benzoyl chloride (6.94 mL, 59.83 mmol) was added to a solution
of 7 (3.16 g, 19.94 mmol) in pyridine (20 mL), and the reaction
mixture was stirred at room temperature for 3.5 h. The mixture was
quenched with water and extracted with ethyl acetate. The organic
layer was dried (MgSO4), filtered, and evaporated. The residue
was purified by silica gel column chromatography (hexane : ethyl
acetate, 3 : 1) to give 8 (5.51 g, 99%) as a colourless syrup: MS
(ESI+) Found: 297.1090 [M+Na]+ for C16H18O4Na, calculated:
297.1098, [a]2D6.8 43.80 (c 4.37, CHCl3); 1H NMR (CDCl3) d 0.71–
0.77 (m, 1 H), 1.24 (s, 3 H), 1.31 (dd, J = 4.0, 8.0 Hz, 1 H), 1.47 (s,
3 H), 1.66–1.72 (m, 1 H), 1.89–1.95 (m, 1 H), 4.57 (pseudo t, J =
8.0 Hz, 1 H), 4.95 (pseudo t, J = 8.0 Hz, 1 H), 5.46 (pseudo t, J =
8.0 Hz, 1 H), 7.45–7.40 (m, 2 H), 7.55–7.51 (m, 1 H), 8.07–8.10 (m,
2 H); 13C NMR (CDCl3) d 7.5, 21.2, 24.7, 26.3, 74.3, 78.5, 80.9,
113.2, 128.4, 129.9, 130.4, 133.0, 166.5; Anal. calcd for C16H18O4:
C, 70.06; H, 6.61. Found: C, 70.08; H, 6.21.
(+)-(1R,2S,3S,4R,5S)-3,4-O-Isopropylidene
bicycle[3.1.0]hexa-2-ol (7)
Diethylzinc (64 mL, 64.07 mmol, 1.0 M solution in hexane)
and diiodomethane (10.34 mL, 128.14 mmol) were added to a
solution of 6 (5.00 g, 32.03 mmol) in methylene chloride (68 mL),
cooled in an ice bath, and the reaction mixture was stirred at
room temperature for 3 h. The mixture was quenched with a
cold, aqueous ammonium chloride solution and extracted with
methylene chloride. The organic layer was dried (MgSO4), filtered,
and evaporated. The residue was purified by silica gel column
chromatography (hexane : ethyl acetate, 4 : 1) to give 7 (3.20 g,
63%) as a colourless syrup: MS (ESI+) Found: 193.0836 [M+Na]+
for C9H14O3Na, calculated: 193.0844, [a]2D7.1 42.7 (c 3.72, CHCl3);
1H NMR (CDCl3) d 0.56–0.62 (m, 1 H), 0.94 (dd, J = 4.0, 8.0 Hz,
1 H), 1.25 (s, 3 H), 1.50 (s, 3 H), 1.57–1.64 (m, 1 H), 1.78–1.84 (m,
1 H), 2.43 (d, J = 4.0 Hz, 1 H, D2O exchangeable), 4.43 (pseudo t,
J = 8.0 Hz, 1 H), 4.46–4.52 (m, 1 H), 4.84 (pseudo t, J = 4.0 Hz,
1 H); 13C NMR (CDCl3) d 6.7, 21.4, 24.7, 26.2, 28.3, 71.5, 79.2,
81.1, 113.0; Anal. calcd for C9H14O3: C, 63.51; H, 8.29. Found: C,
63.53; H, 8.21.
(-)-(1R,2S,3S,4R,5S)-Benzoic acid
3,4-dihydroxy-bicyclo[3.1.0]hex-2-yl ester (9)
A solution of 8 (384.2 mg, 1.46 mmol) and 50% TFA (8 mL)
was stirred at room temperature overnight. The solvent was
evaporated and coevaporated with toluene to give a colourless
syrup. The syrup was purified by silica gel column chromatography
(methylene chloride : methanol, 19 : 1) to give 9 (311.2 mg, 91%)
as a white solid: mp 149.7–256.8 ◦C; MS (ESI+) Found: 491.1653
[2 M+Na]+ for C13H14O4Na, calculated: 491.1638, [a]2D7.1 -0.68
1
(c 2.79, CHCl3); H NMR (CDCl3) d 0.52–0.58 (m, 1 H), 1.29
(dd, J = 4.0, 8.0 Hz, 1 H), 1.71–1.83 (m, 2H), 2.73 (s, 1 H, D2O
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exchangeable), 2.75 (d, J = 3.2 Hz, 1 H, D2O exchangeable), 4.20
(dd, J = 8.0, 12.0 Hz, 1 H), 4.46–4.51 (m, 1 H), 5.38 (pseudo t, J =
4.0 Hz, 1 H), 7.41–7.45 (m, 2 H), 7.58–7.53 (m, 1 H), 8.05–8.08 (m,
2 H); 13C NMR (CDCl3) d 5.6, 19.9, 22.6, 68.7, 71.9, 76.1, 128.6,
129.9, 130.2, 133.4, 166.7; Anal. calcd for C13H14O4: C, 66.66; H,
6.02. Found: C, 66.56; H, 6.24.
Anal. calcd for C15H15N3O4: C, 59.79; H, 5.02; N, 13.95. Found:
C, 59.92; H, 5.20; N, 13.55.
(-)-(1R,2S,3S,4R,5S)-4-Azido-bicyclo[3.1.0]hexane-2,3-diol (13)
Sodium methoxide (0.2 g, 3.78 mmol) was added to a solution of
12a (0.38 g, 1.26 mmol) in methanol (20 mL), and the reaction
mixture was stirred at room temperature for 2 h. The solvent
was evaporated and the residue was partitioned between ethyl
acetate and water. The organic layer was dried (MgSO4), filtered,
and evaporated. The residue was purified by silica gel column
chromatography (methylene chloride:methanol, 10 : 1) to give 13
(0.20 g, 99%) as a colourless syrup: [a]2D6.7 -0.15 (c 2.68, MeOH);
IR (KBr) 2101.2 cm-1; 1H NMR (DMSO-d6) d 0.37–0.42 (m, 1 H),
1.32 (dd, J = 4.0, 8.0 Hz, 1 H), 1.44–1.48 (m, 1 H), 1.55–1.59 (m, 1
H), 3.62 (pseudo t, J = 4.0 Hz, 1 H), 3.73–3.77 (m, 1 H), 4.14–4.19
(m, 1 H), 4.37 (d, J = 8.0 Hz 1 H, D2O exchangeable), 5.19 (d,
J = 4.0 Hz, 1 H, D2O exchangeable); 13C NMR (MeOH-d4) d 6.5,
19.7, 21.7, 63.5, 71.5, 73.4; Anal. calcd for C6H9N3O2: C, 46.45;
H, 5.85; N, 27.08. Found: C, 46.42; H, 5.88; N, 27.56.
(+)-(1R,2S,3S,4S,5S)-Benzoic acid
3-acetoxy-4-bromo-bicyclo[3.1.0]hex-2-yl ester (11a)
A solution of 2-acetoxyisobutyryl bromide (1.86 mL, 12.81 mmol)
in anhydrous acetonitrile (10 mL) was added dropwise to a
suspension of 9 (1.00 g, 4.27 mmol) in anhydrous acetonitrile
(43 mL) at 0 ◦C, and the reaction mixture was stirred at
0
◦C for 3.5 h. A saturated sodium bicarbonate solution was
added dropwise at 0 ◦C until the pH of the solution reached
8. The solution was then neutralised with acetic acid to pH 5.
The mixture was extracted with ethyl acetate and washed with
sodium bicarbonate (x2) and brine. The organic layer was dried
(MgSO4), filtered, and evaporated. After evaporation, the residue
was purified by silica gel column chromatography (hexane : ethyl
acetate, 10 : 1) to give 11a (1.08 g, 75%) as a colourless syrup: MS
(ESI+) Found: 361.0040 [M+Na]+ for C15H15BrO4Na, calculated:
(-)-(1R,2S,3S,4R,5S) 4-Azido-bicyclo[3.1.0]hexane-
2,3-O-sulphite (14)
1
361.0048, [a]2D7.1 0.18 (c 15.1, CHCl3); IR (KBr) 2101.9 cm-1; H
Triethylamine (0.18 mL, 0.54 mmol) and thionyl chloride
(0.04 mL, 0.54 mmol) were added to a solution of 13 (55.2 mg,
0.36 mmol) in methylene chloride (3 mL) at 0 ◦C, and the reaction
mixture was stirred at 0 ◦C for 10 min. The reaction mixture was
partitioned between methylene chloride and water. The organic
layer was dried (MgSO4), filtered, and evaporated. The residue
was purified by silica gel column chromatography (hexane : ethyl
acetate, 2 : 1) to give 14 (66.6 mg, 94%) as a colourless syrup: [a]D27.1
NMR (CDCl3) d 0.72–0.78 (m, 1 H), 1.21–1.25 (m, 1 H), 1.94–1.96
(m, 1 H), 2.0 (s, 3 H), 2.11–2.15 (m, 1 H), 4.28 (s, 1 H), 5.60 (d, J =
6.8 Hz, 1 H), 5.95 (pseudo t, J = 6.7 Hz, 1 H), 7.40–7.44 (m, 2 H),
7.53–7.57 (m, 1 H), 7.96–7.99 (m, 2 H); 13C NMR (CDCl3) d 10.6,
20.9, 25.1, 51.9, 73.7, 76.5, 128.5, 129.6, 133.2, 165.8, 168.8; Anal.
calcd for C15H15BrO4: C, 53.12; H, 4.46. Found: C, 53.52; H, 4.21.
(+)-(1R,2S,3S,4R,5S)-Benzoic acid 3-acetoxy-4-azido-bicyclo
[3.1.0]hex-2-yl ester (12a) and (+)-(1R,2S,3S,4S5S)-Benzoic acid
3-acetoxy-4-azido-bicyclo[3.1.0]hex-2-yl ester (12b)
1
-2.33 (c 3.31, CHCl3); IR (KBr) 2104.7 cm-1; H NMR (CDCl3)
d 0.87–0.93 (m, 1 H), 1.0–1.03 (m, 1 H), 1.83–1.89 (m, 1 H), 1.96–
2.03 (m, 1 H), 4.30 (pseudo t, J = 4.0 Hz, 1 H), 5.21–5.25 (m, 1
H), 5.65 (pseudo t, J = 8.0 Hz, 1 H); 13C NMR (CDCl3) d 8.3,
20.4, 25.3, 60.4, 82.2, 87.5; Anal. calcd for C6H7N3O3S: C, 35.82;
H, 3.51; N, 20.88. Found: C, 35.53; H, 3.22; N, 20.98.
Sodium azide (225.6 mg, 3.47 mmol) was added to a suspension of
11a (235.4 mg, 0.69 mmol) in DMF (8 mL) at room temperature,
and the reaction mixture was stirred at 100 ◦C for 3 h. The solvent
was evaporated and the residue was quenched with water and
extracted with ether. The organic layer was dried (MgSO4), filtered,
and evaporated. After evaporation, the residue was purified by
silica gel column chromatography (hexane : ethyl acetate, 9 : 1) to
give 12a (92.7 mg, 44%) as a colourless syrup and 12b (23.2 mg,
11%) as a colourless syrup.
(-)-(1S,2R,3S,4R,5R)-2-Azido-4-(2,6-dichloro-purin-9-yl)-
bicyclo[3.1.0]hexan-3-ol (16)
Sodium metaperiodate (2.04 g, 6.5 mmol) and ruthenium chloride
trihydrate (71.9 mg) were added to a solution of 14 (0.68 g,
3.40 mmol) in carbon tetrachloride, acetonitrile, and water
(1 : 1 : 1.5, 17.5 mL), and the reaction mixture was stirred at room
temperature for 10 min. The reaction mixture was partitioned
between methylene chloride and water and the organic layer was
dried (MgSO4), filtered, and evaporated. The residue was purified
by silica gel column chromatography (hexane : ethyl acetate, 1 : 1)
to give 15 as a colourless syrup, which was immediately used for
the next reaction.
A suspension of 2,6-dichloropurine (0.96 g, 5.1 mmol), sodium
hydride (0.2 g, 5.1 mmol, 60% dispersion in mineral oil), and
18-crown-6 (1.35 g, 5.1 mmol) in tetrahydrofuran (10 mL) was
stirred at 80 ◦C for 3 h. To this solution, a solution of 15 in
tetrahydrofuran (10 mL) was added, and the mixture was stirred
at room temperature for 4 h. The mixture was cooled to 0 ◦C,
and 30% sulphuric acid was added carefully to lower the pH to 2.
The reaction mixture was stirred at room temperature overnight
Compound 12a. MS (ESI+) Found: 324.0949 [M+Na]+ for
C15H15N3O4Na, calculated: 324.0957, [a]2D6.5 0.33 (c 3.29, CHCl3);
1
IR (KBr) 2101.3 cm-1; H NMR (CDCl3) d 0.76–0.81 (m, 1 H),
1.49–1.53 (m, 1 H), 1.84–1.85 (m, 1 H), 1.89–1.94 (m, 1 H), 2.08
(s, 3 H), 4.12 (pseudo t, J = 4.0 Hz, 1 H), 5.47 (pseudo t, J =
8.0 Hz, 1 H), 5.63 (pseudo t, J = 4.0 Hz, 1 H), 7.41–7.46 (m, 2
H), 7.54–7.58 (m, 1 H), 7.98–8.01 (m, 2 H); 13C NMR (CDCl3) d
6.3, 18.7, 19.2, 20.7, 61.0, 69.4, 73.6, 128.6, 129.7, 133.36, 165.87,
169.41; Anal. calcd for C15H15N3O4: C, 59.79; H, 5.02; N, 13.95.
Found: C, 60.12; H, 5.21; N, 13.56.
Compound 12b. MS (ESI+) m/z 301.1057 [M]+; [a]2D6.5; 0.30◦ (c
1.1, CHCl3); IR (KBr) 2101.3 cm-1;1H NMR (CDCl3) d 0.77–0.83
(m, 1 H), 1.07–1.11 (m, 1 H), 1.60–1.65 (m, 1 H), 2.0 (s, 3H), 2.01–
2.06 (m, 1 H), 3.93 (s, 1 H), 5.26 (d, J = 8.0 Hz, 1 H), 5.71–5.74 (m,
1 H), 7.42–7.42 (m, 2 H), 7.55–7.59 (m, 1 H), 7.97– 8.01 (m, 2 H);
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and then neutralised with a sodium bicarbonate solution. The
reaction mixture was partitioned between ethyl acetate and water.
The organic layer was dried (MgSO4), filtered, and evaporated.
The residue was purified by silica gel column chromatography
(methylene chloride : methanol, 30 : 1) to give 16 (0.68 g, 61%)
as a colourless syrup: MS (ESI+) Found: 326.0320 [M+H]+ for
C11H10Cl2N7O, calculated: 326.0347, [a]2D6.2 -7.68 (c 1.15, CHCl3);
(-)-(1S,2R,3S,4R,5R)-2-Azido-4-(2-chloro-6-methylamino-purin-
9-yl)-bicyclo[3.1.0]hexan-3-ol (18b)
◦
Yield: 91%; white solid; mp 228.1–228.9 C; MS (ESI+) Found:
321.0974 [M+H]+ for C12H14ClN8O, calculated: 321.1001, [a]D27.2
-0.34 (c 5.8, MeOH); UV (MeOH) lmax 272.0 nm; IR (KBr)
1
2105.38 cm-1; H NMR (MeOH-d4) d 0.90–0.96 (m, 1 H), 1.54–
1.57 (m, 1 H), 1.72–1.77 (m, 1 H), 2.06–2.12 (m, 1 H), 3.09 (s, 3
H), 4.04 (d, J = 6.4 Hz, 1 H), 4.15 (pseudo t, J = 5.2 Hz, 1 H),
4.79 (s, 1 H), 7.98 (s, 1 H); 13C NMR (MeOH-d4) d 8.7, 18.6, 21.7,
27.8, 63.1, 63.8, 77.9, 119.5, 139.2, 149.8, 155.3, 156.8; Anal. calcd
for C12H13ClN8O: C, 44.94; H, 4.09; N, 34.94. Found: C, 44.79; H,
4.24; N, 35.10.
1
UV (CHCl3) lmax 274.5 nm; IR (KBr) 2104.4 cm-1; H NMR
(CDCl3) d 1.00–1.06 (m, 1 H), 1.24–1.28 (m, 1 H), 1.41–1.44 (m,
1 H), 1.73–1.78 (m, 1 H), 2.18–2.24 (m, 1 H), 2.63 (br s, 1 H, D2O
exchangeable), 4.20 (d, J = 6.4 Hz, 1 H), 4.68 (pseudo t, J = 5.6 Hz,
1 H), 4.97 (s, 1 H), 8.17 (s, 1 H); 13C NMR (MeOH-d4) d 9.1, 19.1,
22.3, 63.9, 65.4, 78.6, 132.2, 147.4, 152.1, 153.8, 155.3; Anal. calcd
for C11H9Cl2N7O: C, 40.51; H, 2.78; N, 30.06. Found: C, 40.63; H,
2.44; N, 30.16.
(-)-(1S,2R,3S,4R,5R)-2-Azido-4-[6-(3-iodo-benzylamino)-purin-
9-yl]-bicyclo[3.1.0]hexan-3-ol (18c)
Yield: 76%; white foam; MS (ESI+) Found: 489.0640 [M+H]+ for
(-)-(1S,2R,3S,4R,5R)-2-Azido-4-(6-chloro-purin-9-yl)-
bicyclo[3.1.0]hexan-3-ol (17)
C18H18IN8O, calculated: 489.0667; [a]2D7.0 -1.08 (c 1.98, CHCl3);
1
UV (CHCl3) lmax 267.5 nm; IR (KBr) 2104.3 cm-1; H NMR
Compound 15 (288.0 mg, 1.44 mmol) was condensed with 6-
chloropurine to give compound 17 (228.1 mg, 54%) as a foam
according to a procedure similar to that was used to prepare 16:
MS (ESI+) Found: 292.0706 [M+H]+ for C11H11ClN7O, calculated:
292.0734, [a]2D6.4 -1.65 (c 1.03, CHCl3); UV (MeOH) lmax 264.0 nm;
IR (KBr) 2110.76 cm-1; 1H NMR (CDCl3) d 1.00–1.06 (m, 1 H),
1.43–1.46 (m, 1 H), 1.77–1.81 (m, 1 H), 2.17–2.23 (m, 1 H), 2.99
(pseudo t, J = 2.8 Hz, 1 H, D2O exchangeable), 4.23 (d, J = 6.6 Hz,
1 H), 4.69 (pseudo t, J = 5.6 Hz, 1 H), 5.01 (s, 1 H), 8.20 (s, 1 H),
8.76 (s, 1 H); 13C NMR (CDCl3) d 8.9, 19.8, 21.9, 63.8, 64.1, 77.9,
132.3, 143.8, 151.4, 151.7, 152.3; Anal. calcd for C11H10ClN7O: C,
45.29; H, 3.46; N, 33.61. Found: C, 45.69; H, 3.24; N, 33.56.
(CDCl3) d 0.99 (dd, J = 7.6, 14.0 Hz, 1 H), 1.54–1.59 (m, 1 H),
1.72–1.76 (m, 1 H), 2.07–2.13 (m, 1 H), 2.30 (br s, 1 H, D2O
exchangeable), 4.16 (d, J = 5.6 Hz, 1 H), 4.32 (dd, J = 5.6, 11.2 Hz,
1 H), 4.83 (s, 2 H), 4.92 (s, 1 H), 6.65 (s, 1 H, D2O exchangeable),
7.04 (pseudo t, J = 8.0 Hz, 1 H), 7.32 (d, J = 7.6 Hz, 1 H), 7.59 (d,
J = 8.0 Hz, 1 H), 7.71 (s, 1 H), 7.78 (s, 1 H), 8.39 (s, 1 H); 13C NMR
(CDCl3) d 8.8, 18.9, 21.2, 24.1, 29.9, 43.8, 62.9, 63.2, 94.8, 120.3,
127.4, 130.6, 136.6, 136.7, 138.1, 141.2, 153.4, 154.9; Anal. calcd
for C18H17IN8O: C, 44.28; H, 3.51; N, 22.95. Found: C, 44.45; H,
3.29; N, 22.58.
(-)-(1S,2R,3S,4R,5R)-2-Azido-4-(6-methylamino-purin-9-yl)-
bicyclo[3.1.0]hexan-3-ol (18d)
General Procedure for the Synthesis of 18a–d
Yield: 88%; white foam; MS (ESI+) Found: 287.1365 [M+H]+ for
C12H15N8O, calculated: 287.1393, [a]2D7.1 -17.60 (c 2.21, MeOH);
An appropriate amount of amine (1.5 equiv.) was added to a
solution of 16 and 17 in EtOH (5 mL) at room temperature, and
the mixture was stirred at room temperature for a time period
ranging from 20 min to 1 d and then evaporated. The residue was
purified by flash silica gel column chromatography (hexane : ethyl
acetate, 1 : 1) to give 18a,18b,18c, and 18d, respectively.
UV (CHCl3) lmax 269.5 nm; IR (KBr) 2104.9 cm-1; H NMR
1
(MeOH-d4) d 0.90–0.96 (m, 1 H), 1.49–1.52 (m, 1 H), 1.74–1.79
(m, 1 H), 2.06–2.12 (m, 1 H), 3.11 (s, 3 H), 4.10 (dd, J = 0.8, 6.4 Hz,
1 H), 4.22 (pseudo t, J = 5.2 Hz, 1 H), 4.86 (s, 1 H), 8.14 (s, 1 H),
8.26 (s, 1 H); 13C NMR (MeOH-d4) d 8.9, 19.3, 21.9, 27.9, 63.8,
64.2, 78.8, 120.9, 139.9, 153.8, 156.8; Anal. calcd for C12H14N8O:
C, 50.34; H, 4.93; N, 39.14. Found: C, 50.67; H, 4.94; N, 38.99.
(-)-(1S,2R,3S,4R,5R)-2-Azido-4-[2-chloro-6-(3-iodo-
benzylamino)-purin-9-yl]-bicyclo[3.1.0] hexan-3-ol (18a)
General Procedure for the Synthesis of 4a–d
Yield: 89%; white foam; MS (ESI+) Found: 523.0238 [M+H]+ for
C18H17ClIN8O, calculated: 523.0266, [a]2D6.0 -7.21 (c 2.62, CHCl3);
Triphenylphosphine was added to a solution of 18a–d in THF
(1 mL), and the solution was cooled to 0 ◦C. After 30 min, an
ammonium hydroxide solution (0.1 mL) and water (0.02 mL) were
added, and the reaction mixture was stirred at room temperature
overnight. The reaction mixture was concentrated, and 1 N HCl
was added to adjust to pH 1. The resultant solution was then
partitioned between diethyl ether and water. The aqueous layer
was further neutralised with sodium bicarbonate. After complete
removal of water, the solid obtained was filtered and washed with
a methylene chloride : methanol (6 : 1) solution. The residue was
recrystallised using methylene chloride and methanol to give pure
4a–d as a white solid.
1
UV (MeOH) lmax 272.0 nm; IR (KBr) 2105.6 cm-1; H NMR
(CDCl3) d 0.93–0.98 (m, 1 H), 1.43–1.46 (m, 1 H), 1.66–1.70 (m,
1 H), 2.05–2.11 (m, 1 H), 4.18 (d, J = 6.8 Hz, 1 H), 4.36 (t, J =
6.0 Hz, 1 H), 4.68 (s, 1 H, D2O exchangeable), 4.77 (s, 2 H), 4.84
(s, 1 H), 7.0 (br s, 1 H, D2O exchangeable), 7.04 (pseudo t, J =
8.0 Hz, 1 H), 7.32 (d, J = 7.6 Hz, 1 H), 7.58 (dt, J = 1.2, 9.2 Hz,
1 H), 7.70 (pseudo t, J = 1.2 Hz, 1 H), 7.72 (s, 1 H); 13C NMR
(CDCl3) d 8.7, 14.4, 19.1, 21.4, 43.9, 60.6, 63.2, 77.5, 94.7, 118.9,
127.3, 130.6, 136.8, 138.5, 140.6, 149.9, 154.8, 155.2, 171.4; Anal.
calcd for C18H16ClIN8O: C, 41.36; H, 3.09; N, 21.44. Found: C,
41.76; H, 3.24; N, 21.57.
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(-)-(1S,2R,3S,4R,5R)-2-Amino-4-[2-chloro-6-(3-iodo-
benzylamino)-purin-9-yl]-bicyclo [3.1.0]hexan-3-ol (4a)
in DMEM and F12 (1 : 1) supplemented with 10% foetal bovine
serum, 100 units/mL penicillin, 100 mg/mL streptomycin, and
2 mmol mL-1 glutamine. In addition, 800 mg mL-1 Geneticin and
500 mg mL-1 hygromycin were added to the A2A media and the A1
and A3 media, respectively. After harvesting the cells, they were
homogenised for 10 s with an electric homogeniser, pipetted into
1-mL vials, and stored at -80 ◦C until the binding experiments were
conducted. The concentration of protein was determined using a
BCA Protein Assay Kit from Pierce Biotechnology (Rockford,
IL).19
◦
Yield: 86%; mp 255.8–262.2 C; MS (FAB) Found: 497.0354 for
C18H19ClIN6O, calculated: 497.7460, [a]2D6.9 -7.5 (c 0.32, AcOH);
UV (MeOH) lmax 272.0 nm; 1H NMR (AcOH-d4) d 0.87–0.94 (m,
1 H), 1.24–1.31 (m, 1 H), 1.84–1.89 (m, 1 H), 2.17–2.21 (m, 1 H),
4.40 (d, J = 6.4 Hz, 1 H), 4.60 (pseudo t, J = 5.2 Hz, 1 H), 4.75
(s, 2 H), 5.0 (s, 1 H), 7.07 (pseudo t, J = 7.6 Hz, 1 H), 7.40 (d, J =
7.6 Hz, 1 H), 7.60 (d, J = 8.0 Hz, 1 H), 7.81 (s, 1 H), 8.22 (s, 1 H);
13C NMR (AcOH-d4) d 8.0, 21.8, 21.9, 30.6, 44.2, 55.1, 64.8, 75.4,
94.8, 118.3, 128.2, 131.2, 137.9, 140.7, 142.3, 150.5, 155.8, 156.0;
Anal. calcd for C18H18ClIN6O: C, 43.52; H, 3.65; N, 16.92. Found:
C, 43.12; H, 3.88; N, 16.58.
Radioligand membrane binding studies
Radioligand binding assays with A1, A2A, and A3ARs were
performed according to the procedures described previously.20–22
Each tube contained 100 mL of membrane suspension (20 mg of
protein), 50 mL of a stock solution of agonist radioligand, and
50 mL of increasing concentrations of the test ligands in Tris-HCl
buffer (50 mM, pH 7.5) containing 10 mM MgCl2. Nonspecific
binding was determined using a final concentration of 10 mM
NECA, a non-specific agonist, diluted with the buffer.
(-)-(1S,2R,3S,4R,5R)-2-Amino-4-(2-chloro-6-methylamino-purin-
9-yl)-bicyclo[3.1.0] hexan-3-ol (4b)
◦
Yield: 85%; mp 249.2–259.6 C; MS (ESI+) Found: 295.1069
[M+H]+ for C12H16ClN6O, calculated: 295.1096, [a]2D6.3 -2.0 (c 1.3,
AcOH); UV (MeOH) lmax 272.0 nm; 1H NMR (AcOH-d4) d 0.93–
0.88 (m, 1 H), 1.28–1.30 (m, 1 H), 1.84–1.88 (m, 1 H), 2.17–2.20
(m, 1 H), 2.54 (s, 3 H), 4.39 (d, J = 8 Hz, 1 H), 4.59 (pseudo t, J =
4 Hz, 1 H), 4.98 (s, 1 H), 8.19 (s, 1 H); 13C NMR (AcOH-d4) d 8.0,
21.9, 27.7, 55.0, 64.7, 75.4, 118.3, 139.9, 140.1, 149.8, 156.2, 156.5;
Anal. calcd for C12H15ClN6O: C, 48.90; H, 5.13; N, 28.51. Found:
C, 48.79; H, 5.24; N, 28.58.
The mixtures were incubated at 25 ◦C for 60 min. Binding
reactions were terminated by filtration through Whatman GF/B
filters under reduced pressure using a MT-24 cell harvester
(Brandell, Gaithersburg, MD). The filters were washed three times
with 5 mL of 50 mM ice-cold Tris-HCl buffer (pH 7.5). The
radioactive agonists [3H]R-PIA and [3H]CGS21680 were used for
the A1 and A2AAR assays, respectively, while [125I]AB-MECA was
used for the A3AR assay. All of the filters were washed three
times with Tris-HCl, pH 7.5. Filters for A1 and A2AAR binding
were placed in scintillation vials containing 5 mL of Hydrofluor
scintillation buffer and counted using a PerkinElmer Tricarb
2810TR Liquid Scintillation Analyzer. Filters for A3AR binding
were counted using a PerkinElmer Cobra II g-counter.
(-)-(1S,2R,3S,4R,5R)-2-Amino-4-[6-(3-iodo-benzylamino)-purin-
9-yl]-bicyclo[3.1.0]hexan-3-ol (4c)
◦
Yield: 84%; mp 239.9–244.7 C; MS (ESI+) Found: 463.0723
[M+H]+ for C18H20IN6O, calculated: 463.07651, [a]2D6.3 -1.90 (c
1.38, AcOH); UV (MeOH) lmax 268.0 nm; 1H NMR (AcOH-d4) d
1.0–0.90 (m, 1 H), 1.60–1.80 (m, 1 H), 1.98–2.0 (m, 1 H), 2.20–2.25
(m, 1 H), 4.36 (d, J = 8.0 Hz, 1 H), 4.51 (pseudo t, J = 8.0 Hz 1
H), 4.81 (s, 2 H), 5.08 (s, 1 H), 7.06 (pseudo t, J = 8.0 Hz, 1 H),
7.39 (d, J = 8.0 Hz, 1 H), 7.60 (d, J = 8.0 Hz, 1 H), 7.79 (s, 1 H),
8.34 (s, 1 H), 8.46 (s, 1 H); 13C NMR (AcOH-d4) d 8.1, 20.0, 21.7,
44.3, 54.8, 64.4, 75.3, 94.8, 119.4, 127.7, 127.8, 131.3, 137.5, 140.2,
142.1, 148.4, 154.0, 155.2; Anal. calcd for C18H19IN6O: C, 46.77;
H, 4.14; N, 18.18. Found: C, 46.77; H, 4.24; N, 18.11.
Data analysis
Binding and functional parameters were calculated using the
Prism 5.0 software (GraphPAD, San Diego, CA, USA). IC50 values
obtained from competition curves were converted to Ki values
using the Cheng-Prusoff equation.23 Data were expressed as mean
standard error.
(-)-(1S,2R,3S,4R,5R)-2-Amino-4-(6-methylamino-purin-9-yl)-
bicyclo[3.1.0]hexan-3-ol (4d)
◦
Yield: 86%; mp 238.7–242.4 C; MS (ESI+) Found: 261.1460
[M+H]+ for C12H17N6O, calculated: 261.1488, [a]2D6.1 -2.30 (c 1.43,
MeOH); UV (MeOH) lmax 267.0 nm; 1H NMR (AcOH-d4) d 0.66–
0.72 (m, 1 H), 1.23 (dd, J = 4.0, 8.0 Hz, 1 H), 1.66–1.71 (m, 1 H),
1.89–2.0 (m, 1 H), 2.87 (s, 3 H), 3.73–3.75 (m, 1 H), 3.78–3.80 (m,
1 H), 4.90 (s, 1 H), 8.17 (s, 1 H), 8.27 (s, 1 H); 13C NMR (AcOH-
d4) d 7.3, 19.9, 25.4, 55.0, 64.7, 77.1, 139.8, 153.8; Anal. calcd for
C12H16N6O: C, 55.37; H, 6.20; N, 32.29. Found: C, 55.38; H, 6.21;
N, 32.11.
Acknowledgements
This work was supported by the grant from the Korea Research
Foundation (NRF-2008-314-E00304, R31-2008-000-10010-0, and
R15-2006-020) and the NIDDK Intramural Research program.
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