Expeditious synthesis and biological evaluation of new C-6 1,2,3-triazole adenosine derivatives A1 receptor antagonists or agonists

Article abstract of DOI:10.1039/c0ob00017e

The synthesis of new C-6 1,2,3-triazole adenosine derivatives via microwave assisted 1,3-dipolar cycloaddition as key step is described. The binding on membranes of cells that over express A₁ adenosine receptors (A ₁AR) was also evaluated. Among them, four compounds increased cAMP production, in a dose-dependent manner acting as antagonists of the A ₁AR, while two compounds act as agonists.

Full text of DOI:10.1039/c0ob00017e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Expeditious synthesis and biological evaluation of new C-6 1,2,3-triazole
adenosine derivatives A1 receptor antagonists or agonists†
Smitha C. Mathew,a Youlet By,b Aure´lie Berthault,a Marie-Alice Virolleaud,a Louis Carrega,b Gae¨lle
Chouraqui,a Laurent Commeiras,a Jocelyne Condo,b Mireille Attolini,a Anouk Gaudel-Siri,a Jean Ruf,b Jean
Rodriguez,*a Jean-Luc Parrain*a and Re´gis Guieu*b,c
Received 19th April 2010, Accepted 11th June 2010
First published as an Advance Article on the web 9th July 2010
DOI: 10.1039/c0ob00017e
The synthesis of new C-6 1,2,3-triazole adenosine derivatives via microwave assisted 1,3-dipolar
cycloaddition as key step is described. The binding on membranes of cells that over express A1
adenosine receptors (A1AR) was also evaluated. Among them, four compounds increased cAMP
production, in a dose-dependent manner acting as antagonists of the A1AR, while two compounds act
as agonists.
In this context, we propose an expeditious approach based on
click-chemistry of specifically functionalized nucleoside analogues
Introduction
Adenosine, an ubiquitous nucleoside that comes from the de-
starting from commercially available inosine.
phosphorylation of ATP, acts on G protein-coupled adenosine
New methods for the synthesis of C6 purine derivatives
receptors (AR), named A1, A2A, A2B and A3, depending on
have been widely developed for two decades. Advances in this
the pharmacological properties of the receptor subtypes.1 Each
AR has a specific tissue distribution, ligand affinity, and signal
transduction mechanism. Because of their presence on nearly
every cell type, adenosine receptors have been obvious targets
for the development of new drugs for more than three decades.2
The A1 adenosine receptors (A1AR), highly expressed in heart,
brain, dorsal spinal cord, adipose tissue and kidney,3 particularly
raised our attention. According to the recent literature the best
approach for the discovery of new AR agonists consists of N6
modifications or introduction of selective functionalization at 2-
position of the adenine moiety and at the 3¢-, 4¢- or 5¢-position
of the ribose part. Among them N6-chlorocyclopentyladenosine
(CCPA) and N6-cyclopentyladenosine (CPA) have been reported
as being highly selective A1AR agonists.4 On the other hand,
reported AR antagonists are usually derivatives of xanthines such
as caffeine or theophylline.5 Thus, having drugs that modulate
A1AR at one’s disposal could be interesting in different therapeutic
areas, including heart failure, diuresis,6 heart arrhythmia,7 glucose
tolerance,8 cognitive diseases9 and contrast media-induced acute
renal failure.10
field employ nucleophilic displacement (SNAr),11 and the area
of organometallic cross-coupling12 such as Suzuki–Miyaura,13
Sonogashira,14 Stille,15 and Buchwald–Hartwig16 coupling reac-
tions. Alternatively, the discovery17 of the dramatic acceleration
of the copper(I) mediated Huisgen 1,3-dipolar cycloaddition of
alkyne and azides to yield 1,2,3-triazoles has generated the synthe-
sis of a plethora of new compounds and has accelerated advances
in areas ranging from drug discovery to material sciences.18
Although a number of 1,2,3-triazole nucleoside derivatives has
been described,19 most involve introduction of 1,2,3-triazole at
C2,20 and C821 or at the sugar moiety.22 To the best of our
knowledge, when we started the project, this “click reaction” was
unexploited for the synthesis of N6-aminopurine derivatives.23 As
part of our ongoing research towards the synthesis of biologically
active adenosine derivatives,24 we have developed this approach for
a rapid access to new N6 nucleoside derivatives. We describe herein
the synthesis, the A1 binding affinities and the biological evaluation
of a novel series of new C-6 1,2,3-triazole substituted purine
derivatives 1 via microwave assisted 1,3-dipolar cycloaddition.
Nevertheless, the modified adenosine approach is hampered
by the presence of adenosine deaminase (ADA) involved in
purines metabolism that irreversibly converts adenosine to the
less active inosine nucleoside by selective C-6 deamination. To
circumvent this drawback, the design of new analogues resistant
to ADA bearing a non metabolisable C-6 amino group is needed.
Results and discussion
As outlined retrosynthetically in Scheme 1, C-6 triazole substi-
tuted adenosines 1 could be readily obtained by “click chemistry”
between C-6 azido adenosine 2 and different substituted alkynes.
Our investigation started with the synthesis of 1,3-dipolar partner
2 (Scheme 2). As described in the literature,25,26 C-6 azido
aAix-Marseille Universite´, Institut des Sciences Mole´culaires de Marseille,
iSm2-UMR CNRS 6263, Centre Saint Je´roˆme, Service 532, 13397,
Marseille Cedex 20, France. E-mail: jl.parrain@univ-cezanne.fr; Fax: +33
4 91 28 91 87; Tel: +33 4 91 28 89 14
bUniversite´ de la Me´diterrane´e, UMR MD2 P2COE, Faculte´ de Me´decine,
Boulevard Jean Moulin, 13005, Marseille, France. E-mail: guieu.regis@
numericable.fr; Tel: +33 4 32 43 82
cAssistance Publique des Hoˆpitaux de Marseille, Boulevard Jean Moulin,
13005, Marseille, France
† Electronic supplementary information (ESI) available: HPLC chro-
matograms and NMR spectra. See DOI: 10.1039/c0ob00017e
Scheme 1 Retrosynthetic strategy for the synthesis of 1.
3874 | Org. Biomol. Chem., 2010, 8, 3874–3881
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Scheme 2 Preparation of C-6 azido adenosine 2. Reagents and conditions: (a) three steps, 90% overall yield; (b) LiN3, DMF, rt, 2 days (30%); (c)
phenylacetylene, conditions A: CuSO4·5H2O, sodium ascorbate, DMF, 80 ◦C, 12 h (45% of adenosine), conditions B: CuSO4·5H2O, sodium ascorbate,
DMF, MW, 100 ◦C, 30 min (80% of adenosine), conditions C: CuSO4·5H2O, sodium ascorbate, tBuOH : H2O, rt, 2 h (75% of adenosine) or conditions
D: Cu wire, CH3CN–H2O, 35 ◦C, 12 h (65% of adenosine).
adenosine 2 can be synthesized via nucleophilic displacement
reaction of the electrophilic nucleoside 3 by sodium azide. 6-
Chloropurine riboside (3) was prepared in three steps (90% overall
yield) according to a previously reported procedure from the
commercially available inosine.27
However, the SNAr reaction of 3 using the reported conditions
did not furnish the corresponding azide 2. Despite numerous
attempts to modify the reaction conditions (lower temperature,
Scheme 3 Synthesis of 4. Reagents and conditions: (a) PdCl2(PPh3)2, CuI,
NEt3, DMF, 60 ◦C, 3 h (69%); (b) TBAF, THF, 0 ◦C, 20 min (43%).
microwave irradiation or protection of the alcohol function on the
sugar part as an acetate or acetal), 2 was always detected only
with tetrabutylammonium fluoride31 to yield the terminal alkyne
4 in 43% yield.
in trace amount. Finally replacement of sodium azide by lithium
azide (DMF, rt, 2 days)28 led, in 30% yield, to the formation of 2
in equilibrium with its tetrazolo tautomeric form 2¢.25,28,29
The Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction was next
studied. In an initial experiment (Scheme 4), the alkynyl derivative
Even if 2 was obtained in low yield, the copper(I)-mediated Huis-
gen 1,3-dipolar cycloaddition was then investigated (Scheme 2).
Phenylacetylene was chosen as a benchmark substrate and
the reaction was initially performed with sodium ascorbate,
CuSO4·5H2O in DMF for 12 h at 80 ◦C (conditions A). Un-
◦
4 was treated with benzyl azide 7a in dry DMF at 90 C in the
presence of copper(II) and sodium ascorbate under microwave con-
ditions for 30 min. We were pleased to find that these conditions
afforded the triazole derivative 8a in 58% yield thus validating
this new approach (Scheme 4). In order to examine the scope of
this approach, the [3+2]-cycloaddition was then performed with a
range of different azides 7b–j (Scheme 4). The latter were prepared
according to a safe literature procedure developed by Varma et al.32
based on a nucleophilic substitution reaction of alkyl halides by
sodium azide in water using microwave-assisted heating. The 1,3-
dipolar cycloaddition has then allowed the formation of several
new C-6 1,2,3-triazole adenosine derivatives 8b–j in good yields
(up to 82%) and with a total regioselectivity.
Interestingly, when the reaction was performed with 1,3-
diazidopropane 7k, a separable mixture of bis-C6-1,2,3-triazole
bis-adenosine 8k and mono cycloadduct derivative 8l were ob-
tained in moderate yield (respectively 24 and 16%, Scheme 5).
At last, deprotection of the acetate functions of the sugar part
under basic condition (NH3, MeOH) led to the formation of the
corresponding compounds 9a–k in excellent yields (up to 99%,
Scheme 4 and 5).
=
fortunately, the desired 1,4-disubstituted 1,2,3-triazole 1 (R Ph)
was never observed. Adenosine, resulting from the reduction
of the azido function of 2 into the corresponding amine, was
isolated in 45% yield as the main reaction product. This kind
of reduction was already described by Frieden et al. and can
be explain by a thermal decomposition of the azido function
to the corresponding nitrene, which evolved into the amine
formation.25 Whatever the conditions used to perform the 1,3-
dipolar cycloaddition (Scheme 2, conditions B: CuSO4·5H2O,
sodium ascorbate, DMF, MW, 100 ◦C, 30 min, conditions
C: CuSO4·5H2O, sodium ascorbate, tBuOH : H2O, rt, 2 h or
conditions D: Cu wire, CH3CN–H2O, 35 ◦C, 12 h), adenosine
was always the sole product obtained (65–80%). The presence of
the C-6 tretrazolyl tautomer could explain the lack of reactivity
in the [3+2]-cycloaddition process. This observation drove us to
envision a new strategy. Instead of installing the azide function on
the C-6 position of the adenosine moiety, it was decided to prepare
the corresponding C-6 acetylene 4. The readily available protected
chloroinosine 5 (Scheme 3) underwent a smooth Sonogashira
coupling with TMS-acetylene. The installation of the triple bond
at the C-6 position of the adenosine derivative was accomplished
in 69% yield.30 Deprotection of the TMS group was performed
Biological evaluation
These new C-6 1,2,3-triazole adenosine derivatives 9a–k were
then submitted to biological evaluation. The ability of these
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Scheme 4 Preparation of C-6 1,2,3-triazole substituted adenosines 9. Reagents and conditions: (a) CuSO4·5H2O, sodium ascorbate, DMF, mW, 90 ◦C,
30 min; (b) NH3, MeOH, rt, 12 h.
Scheme 5 Reactivity of 4 with 1,3-diazidopropane. (a) CuSO4·5H2O, sodium ascorbate, DMF, mW, 90 ◦C, 30 min, 1,3-diazodopropane 7k; (b) NH3,
MeOH, rt, 12 h.
unprecedented molecules to modulate cAMP production in
mammalian Chem-3 cells that over expressed A1ARs was first
In summary we have reported the synthesis of new C-6 1,2,3-
tested. Indeed, agonists of A1AR are believed to decrease the
cAMP production whereas antagonists display the opposite
effect.
As a matter of fact 9e, 9f, 9h and 9i increased cAMP production
in cell culture, in a dose-dependent manner acting as antagonists
Conclusions
triazole adenosine derivatives via microwave assisted 1,3-dipolar
cycloaddition as key step. Among them, four compounds ex-
pressed antagonist properties, while two expressed agonist prop-
erties, basing on cAMP production on A1AR. Their biological
effects were obtained at reasonable concentrations.
of the A1AR (Table 1), while 9j and 9k decreased cAMP
production acting as agonists (Table 1). The IC50 of compounds
was also evaluated, using binding assay on cell membranes. IC50
of antagonist compounds range between 0.29 mM and 1.5 mM and
Experimental
between 11 mM and 42 mM for agonist compounds (Table 1). Even
if these molecules have weaker affinity when compared to the well
known A1AR antagonist DPCPX, their biological effects occur at
reasonable concentrations.
Compounds 9a, 9b, 9c, 9d and 9g had an IC50 for A1AR
binding >100 mM and all compounds tested had an IC50 for A2A
adenosine receptor binding >50 mM (data not shown). While
nitroaromatic compounds have been reported to have A1AR
antagonist properties,33 interestingly, 9j exhibits agonist properties.
It is also interesting to note that nitroaromatic compounds were
known to act via the inhibition of NO synthase and cGMP
modulation.34
All reactions sensitive to oxygen and moisture were carried out
in oven-dried glassware under a slight positive pressure of argon
unless otherwise noted. Reagents and solvents were commercial
grades and were used as supplied. N,N-dimethylformamide was
distilled from calcium hydride and stored over calcium hydride.
THF was obtained from a Solvent Purification System. Triethy-
lamine was distilled from KOH prior to use. Analytical thin
layer chromatography (TLC) was performed on Merck precoated
analytical plates, 0.25 mm thick, silica gel 60 F254. Flash column
chromatography was performed on Merck Kieselgel 40–63 mm.
1H NMR and 13C NMR spectra were recorded on an AC300
using the deuterated solvent as internal deuterium lock. Chemical
3876 | Org. Biomol. Chem., 2010, 8, 3874–3881
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Table 1 Effects of compounds on cAMP production and on [3H] DPCPX displacement or [3H] CPA displacement on Chem 3 cells that overexpress the
A1 AR. IC50 for cAMP inhibition was defined as the concentration of compound that induces half of the maximal cAMP inhibition. EC50 for cAMP
production was defined as the concentration of compound that induces half of the maximal cAMP production. IC50 for DPCPX or CPA displacement
was defined as the concentration of competing ligand which displaces 50% of [3H] DPCPX or [3H] CPA used as specific radioligand
cAMP production
EC50/M
[3H]DPCPX displacement
IC50/M
[3H]CPA displacement
IC50/M
Compound
IC50/M
Properties
9a
>10-5
—
—
—
—
9b
>10-5
9c
>10-5
9d
>10-5
9e
5.5 0.9 10-7
7
2.9 0.8 10-7
15 4 10-7
>10-5
Antagonist
Antagonist
—
Antagonist
Antagonist
Agonist
Agonist
Antagonist
9f
2 10-7
9g
9h
6
7
2 10-6
2 10-7
6
4
1.8 10-7
2.9 10-7
9i
9j
1
0.6 10-6
42 11 10-6
11 4 10-6
9k
DPCPX
0.5 0.3 10-6
3.8 0.5 10-9
4
0.8 10-9
shift data are given in units d relative to residual protic solvent
where d(chloroform) = 7.26 ppm, d(DMSO) = 2.50 ppm and
d(methanol) = 3.31 ppm The multiplicity of a signal is indicated
as: br – broad, s – singlet, d – doublet, t – triplet, q – quartet,
m – multiplet, dd – doublet of doublets, etc. Coupling constants
(J) are quoted in Hz and recorded to the nearest 0.1 Hz. 13C
NMR spectra were recorded on an AC300 spectrometer using
the deuterated solvent as internal deuterium lock. Chemical
shift data are given in units d relative to residual protic solvent
where d(chloroform) = 77.16 ppm, d(DMSO) = 39.52 ppm and
d(methanol) = 49.00 ppm. High resolution mass spectra (HRMS)
have been performed using a mass spectrometer equipped with
a pneumatically assisted atmospheric pressure ionization. The
sample was ionized in positive mode electrospray.
afford 6 (800 mg, 69%). dH(300 MHz, CDCl3) 8.85 (s, 1H), 8.24 (s,
1H), 6.17 (d, J = 5.3 Hz, 1H), 5.88 (appt, J = 5.5 and 5.3 Hz, 1H),
5.58 (br appt, J = 4.9 and 5.1 Hz, 1H), 4.41–4.26 (m, 3H), 2.07 (s,
3H), 2.02 (s, 3H), 1.98 (s, 3H), 0.25 (s, 9H); dC(75 MHz, CDCl3)
170.2 (C), 169.5 (C), 169.3 (C), 153.7 (CH), 151.3 (C), 143.9 (CH),
141.7 (C), 134.9 (C), 106.0 (C), 98.2 (C), 86.5 (CH), 80.4 (CH), 73.0
(CH), 70.6 (CH), 62.9 (CH2), 20.7 (CH3), 20.5 (CH3), 20.3 (CH3),
-0.5 (3 ¥ CH3); HRMS (ESI) calcd for C21H27N4O7Si [M+H]+:
475.1644 found 475.1642
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-ethynyl-9H-purin-9-
yl)tetrahydrofuran-3,4-diyl diacetate 4
To a stirred solution of 6 (1.5 g, 3.1 mmol) in THF (20 mL) at
0 ◦C was added dropwise TBAF (3.4 mL, 3.4 mmol, 1M in THF)
and stirred for 20 min. The reaction mixture was quenched with
saturated aqueous ammonium chloride solution and the solvent
was removed under vacuum. The residue was extracted with
dichloromethane, dried over sodium sulfate then concentrated.
The resulting mixture was purified by column chromatography
on silica gel (94/6 dichloromethane–methanol) to give 4 (550 mg,
43%). dH(300 MHz, CDCl3) 8.78 (s, 1H), 8.25 (s, 1H), 6.12 (d, J =
5.1 Hz, 1H), 5.82 (br appt, J = 5.5 and 5.3 Hz, 1H), 5.51 (br appt,
J = 5.1 and 5.3 Hz, 1H), 4.33–4.17 (m, 3H), 3.73 (s, 1H), 1.97
(s, 3H), 1.92 (s, 3H), 1.90 (s, 3H); dC(75 MHz, CDCl3) 170.0 (C),
169.3 (C), 169.1 (C), 152.4 (CH), 151.0 (C), 144.2 (CH), 140.7 (C),
135.2 (C), 86.6 (C), 86.3 (CH), 80.1 (CH), 77.6 (CH), 72.8 (CH),
70.2 (CH), 62.7 (CH2), 20.4 (CH3), 20.2 (CH3), 20.1 (CH3); HRMS
(ESI) calcd for C18H19N4O7 [M+H]+: 403.1248 found 403.1241
(2R,3R,4S,5R)-2-(6-azido-9H-purin-9-yl)-5-(hydroxymethyl)-
tetrahydrofuran-3,4-diol 2
To a stirred solution of chloroinosine 3 (400 mg, 1.38 mmol) in
dry DMF was added lithium azide (750 mg, 15.2 mmol). The
reaction was stirred at room temperature for 60 h then the solvent
was removed under vacuum. The crude product was recrystallized
from methanol to give 2 (120 mg, 30%). dH(300 MHz, DMSO)
10.14 (s, 1H), 8.93 (s, 1H), 6.15 (d, J = 5.1 Hz, 1H), 5.66 (d, J
= 5.9 Hz, 1H), 5.33 (d, J = 5.3 Hz, 1H), 5.14 (appt, J = 5.3
and 5.5 Hz, 1H), 4.57 (q, J = 5.3 Hz, 1H), 4.21 (q, J = 4.7 Hz,
1H), 4.02 (q, J = 4.0 Hz, 1H), 3.76–3.69 (m, 1H), 3.65–3.57 (m,
1H); dC(75 MHz, DMSO) 145.4 (C), 142.6 (CH), 142.0 (C), 136.1
(CH), 120.3 (C), 88.4 (CH), 85.8 (CH), 74.6 (CH), 70.1 (CH),
61 (CH2).
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-((trimethylsilyl)ethynyl)-
9H-purin-9-yl)tetrahydrofuran-3,4-diyl diacetate 6
General procedure for the synthesis of compounds 8a–l.
A mixture of acetylene 4 (70 mg, 0.17 mmol), benzyl azide
(0.24 mmol), sodium ascorbate (10 mg, 0.051 mmol) and copper
sulfate pentahydrate (2 mg) in dry DMF (2 mL) was sub-
jected to microwave irradiation for 30 min at a temperature of
90 ◦C. The reaction mixture was then concentrated, extracted
with dichloromethane and the residue was purified by column
chromatography using dichloromethane–methanol as eluent to
afford the triazole derivatives 8a–l.
To a purged argon solution of chloroinosine 3 (1.1 g, 2.6 mmol),
trimethylsilyl acetylene (0.6 mL, 3.1 mmol), PdCl2(PPh3)2 (54 mg,
0.078 mmol) and copper iodide (30 mg, 0.156 mmol) in DMF
was added triethylamine (1 mL, 8.4 mmol). The mixture was
then heated at 60 ◦C for 3 h and the solvents were evaporated
under reduced pressure. The residue was purified by column
chromatography on silica gel (1/1 pet. ether–ethyl acetate) to
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(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-benzyl-1H -1,2,3-
triazol-4-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diyl diacetate 8a.
dH(300 MHz, CDCl3) 9.00 (s, 1H), 8.64 (s, 1H), 8.24 (s, 1H), 7.40–
7.31 (m, 5H), 6.25 (d, J = 5.3 Hz, 1H), 5.95 (t, J = 5.5 Hz, 1H),
5.68–5.65 (m, 3H), 4.47–4.32 (m, 3H), 2.13 (s, 3H), 2.09 (s, 3H),
2.04 (s, 3H); dC(75 MHz, CDCl3) 170.4 (C), 169.7 (C), 169.4 (C),
153.2 (CH), 151.8 (C), 148.0 (C), 143.9 (C), 143.2 (CH), 134.4
(C), 130.3 (C), 129.3 (2 ¥ CH), 129.0 (CH), 128.2 (2 ¥ CH), 126.9
(CH), 86.5 (CH), 80.5 (CH), 73.2 (CH), 70.7 (CH), 63.1 (CH2),
54.5 (CH2), 20.8 (CH3), 20.6 (CH3), 20.4 (CH3); HRMS (ESI)
calcd for C25H26N7O7 [M+H]+: 536.1888 found 536.1878.
(C), 143.1 (CH), 139.0 (C), 131.4 (C), 130.4 (C), 130.0 (2 ¥ CH),
128.3 (2 ¥ CH), 126.8 (CH), 86.5 (CH), 80.6 (CH), 73.3 (CH), 70.7
(CH), 63.1 (CH2), 53.4 (CH2), 21.3 (CH3), 20.9 (CH3), 20.6 (CH3),
20.5 (CH3); HRMS (ESI) calcd for C26H28N7O7 [M+H]+: 550.2045
found 550.2044.
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(4-methoxybenzyl)-
1H -1,2,3-triazol-4-yl)-9H -purin-9-yl)tetrahydrofuran-3,4-diyl
diacetate 8f. dH(300 MHz, CDCl3) 9.00 (s, 1H), 8.60 (s, 1H), 8.24
(s, 1H), 7.28 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 6.26 (d,
J = 5.3 Hz, 2H), 5.95 (appt, J = 5.5 and 5.3 Hz, 1H), 5.67 (br dd,
J = 5.5 and 4.5 Hz, 1H), 5.59 (s, 2H), 4.48–4.34 (m, 3H), 3.78 (s,
3H), 2.14 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H); dC(75 MHz, CDCl3)
170.4 (C), 169.7 (C), 169.5 (C), 160.1 (C), 155.6 (C), 153.24 (CH),
153.19 (C), 151.8 (C), 143.2 (CH), 129.8 (2 ¥ CH), 126.6 (CH),
126.4 (2 ¥ C), 114.7 (2 ¥ CH), 86.5 (CH), 80.5 (CH), 73.2 (CH),
70.7 (CH), 63.1 (CH2), 55.4 (CH3), 54.1 (CH2), 20.9 (CH3), 20.6
(CH3), 20.5 (CH3); HRMS (ESI) calcd for C26H28N7O8 [M+H]+:
566.1994 found 566.1993.
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(4-bromobenzyl)-
1H-1,2,3-triazol-4-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diyl dia-
cetate 8b. dH(300 MHz, CDCl3) 8.99 (s, 1H), 8.65 (s, 1H), 8.24
(s, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.5 Hz, 1H), 6.24
(d, J = 5.1 Hz, 1H), 5.95 (appt, J = 5.5 and 5.3 Hz, 1H), 5.66
(br dd, J = 5.3 and 4.5 Hz, 1H), 5.60 (s, 2H), 4.47–4.28 (m, 3H),
2.12 (s, 3H), 2.08 (s, 3 H), 2.04 (s, 3 H); dC(75 MHz, CDCl3) 170.3
(C), 169.6 (C), 169.4 (C), 153.2 (CH), 151.8 (C), 147.8 (C), 144.0
(C), 143.3 (CH), 133.4 (C), 132.4 (2 ¥ CH), 130.3 (C), 129.8 (2 ¥
CH), 126.9 (CH), 123.1 (C), 86.5 (CH), 80.5 (CH), 73.2 (CH), 70.7
(CH), 63.1 (CH2), 53.7 (CH2), 20.8 (CH3), 20.6 (CH3), 20.4 (CH3);
HRMS (ESI) calcd for C25H25BrN7O7 [M+H]+: 614.0993 found
614.0994.
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(2-bromobenzyl)-
1H-1,2,3-triazol-4-yl)-9H -purin-9-yl)tetrahydrofuran-3,4-diyl
diacetate 8g. dH(300 MHz, CDCl3) d 9.02 (s, 1H), 8.74 (s, 1H),
8.26 (s, 1H), 7.62 (br d, J = 7.9 Hz, 1H), 7.33–7.19 (m, 3H), 6.27
(d, J = 5.5 Hz, 1H), 5.96 (t, J = 5.5 Hz, 1H), 5.80 (s, 2H), 5.68
(br dd, J = 5.5 and 4.4 Hz, 1H), 4.49–4.35 (m, 3H), 2.15 (s, 3H),
2.11 (s, 3 H), 2.06 (s, 3 H); dC(75 MHz, CDCl3) 170.4 (C), 169.7
(C), 169.5 (C), 153.3 (CH), 151.9 (C), 148.0 (C), 144.0 (C), 143.3
(CH), 134.0 (C), 133.4 (CH), 130.6 (CH), 130.4 (C and CH), 128.4
(CH), 127.2 (CH), 123.6 (C), 86.5 (CH), 80.6 (CH), 73.2 (CH),
70.7 (CH), 63.1 (CH2), 54.2 (CH2), 20.9 (CH3), 20.6 (CH3), 20.5
(CH3); HRMS (ESI) calcd for C25H25BrN7O7 [M+H]+: 614.0993
found 614.0992.
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(4-chlorobenzyl)-
1H-1,2,3-triazol-4-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diyl dia-
cetate 8c. dH(300 MHz, CDCl3) 9.02 (s, 1H), 8.65 (s, 1H), 8.25
(s, 1H), 7.35 (d, J = 8.7 Hz, 2H), 7.26 (d, J = 8.7 Hz, 1H), 6.26
(d, J = 5.3 Hz, 2H), 5.96 (t, J = 5.5 Hz, 1H), 5.68 (br dd, J = 5.5
and 4.5 Hz, 1H), 5.64 (s, 2H), 4.49–4.34 (m, 3H), 2.14 (s, 3H), 2.10
(s, 3 H), 2.06 (s, 3 H); dC(75 MHz, CDCl3) 170.4 (C), 170.0 (C),
169.5 (C), 153.3 (CH), 151.8 (C), 147.9 (C), 144.1 (C), 143.3 (CH),
135.1 (C), 132.9 (C), 130.4 (C), 129.6 (2 ¥ CH), 129.5 (2 ¥ CH),
126.8 (CH), 86.6 (CH), 80.6 (CH), 73.3 (CH), 70.7 (CH), 63.1
(CH2), 53.8 (CH2), 20.9 (CH3), 20.6 (CH3), 20.5 (CH3); HRMS
(ESI) calcd for C25H25ClN7O7 [M+H]+: 570.1499 found 570.1499.
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(3-chlorobenzyl)-
1H -1,2,3-triazol-4-yl)-9H -purin-9-yl)tetrahydrofuran-3,4-diyl
diacetate 8h. dH(300 MHz, CDCl3) 9.00 (s, 1H), 8.68 (s, 1H), 8.25
(s, 1H), 7.30–7.28 (m, 3H), 7.19–7.16 (m, 1H), 6.25 (d, J = 5.3 Hz,
2H), 5.95 (appt, J = 5.5 and 5.3 Hz, 1H), 5.67 (br dd, J = 5.5 and
4.5 Hz, 1H), 5.63 (s, 2H), 4.47–4.33 (m, 3H), 2.13 (s, 3H), 2.08 (s,
3H), 2.04 (s, 3H); dC(75 MHz, CDCl3) 170.4 (C), 169.7 (C), 169.5
(C), 153.2 (CH), 151.8 (C), 147.8 (C), 144.1 (C), 143.3 (CH), 136.4
(C), 135.1 (C), 130.6 (CH), 130.4 (C), 129.1 (CH), 128.2 (CH),
127.0 (CH), 126.2 (CH), 86.5 (CH), 80.5 (CH), 73.2 (CH), 70.7
(CH), 63.1 (CH2), 53.7 (CH2), 20.8 (CH3), 20.6 (CH3), 20.4 (CH3);
HRMS (ESI) calcd for C25H25ClN7O7 [M+H]+: 570.1499 found
570.1499.
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(4-fluorobenzyl)-
1H-1,2,3-triazol-4-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diyl dia-
cetate 8d. dH(300 MHz, CDCl3) 9.00 (s, 1H), 8.64 (s, 1H), 8.24
(s, 1H), 7.33–7.29 (m, 2H), 7.07–7.01 (m, 2H), 6.26 (d, J = 5.3 Hz,
1H), 5.95 (appt, J = 5.5 and 5.3 Hz, 1H), 5.67 (br dd, J = 5.5 and
4.5 Hz, 1H), 5.62 (s, 2H), 4.48–4.34 (m, 3H), 2.13 (s, 3H), 2.08 (s,
3H), 2.04 (s, 3H); dC(75 MHz, CDCl3) 170.4 (C), 169.7 (C), 169.4
(C), 163.0 (d, J = 248.7 Hz, C), 153.2 (CH), 151.8 (C), 147.9 (C),
144.0 (C), 143.3 (CH), 130.34 (C), 130.3 (d, J = 3.3 Hz, C), 130.1
(d, J = 8.2 Hz, 2 ¥ CH), 126.8 (CH), 116.3 (d, J = 22.0 Hz, 2 ¥
CH), 86.5 (CH), 80.5 (CH), 73.2 (CH), 70.7 (CH), 63.1 (CH2), 53.7
(CH2), 20.8 (CH3), 20.6 (CH3), 20.4 (CH3); HRMS (ESI) calcd for
C25H25FN7O7 [M+H]+: 554.1794 found 554.1793.
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(3,4-dichlorobenzyl)-
1H -1,2,3-triazol-4-yl)-9H -purin-9-yl)tetrahydrofuran-3,4-diyl
diacetate 8i. dH(300 MHz, CDCl3) 9.00 (s, 1H), 8.69 (s, 1H),
8.25 (s, 1H), 7.43–7.40 (m, 2H), 7.14 (br dd, J = 8.3 and 2.3 Hz,
1H), 6.25 (d, J = 5.3 Hz, 1H), 5.95 (appt, J = 5.5 and 5.3 Hz, 1H),
5.67 (br dd, J = 5.5 and 4.7 Hz, 1H), 5.62 (s, 2H), 4.47–4.33 (m,
3H), 2.13 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H); dC(75 MHz, CDCl3)
170.4 (C), 169.7 (C), 169.5 (C), 153.2 (CH), 151.8 (C), 147.7 (C),
144.2 (C), 143.4 (CH), 134.6 (C), 133.4 (C), 133.2 (C), 131.3 (CH),
130.4 (C), 130.0 (CH), 127.3 (CH), 126.9 (CH), 86.6 (CH), 80.5
(CH), 73.2 (CH), 70.7 (CH), 63.1 (CH2), 53.1 (CH2), 20.8 (CH3),
20.6 (CH3), 20.4 (CH3); HRMS (ESI) calcd for C25H24N7O7Cl2
[M+H]+: 604.1109 found 604.1118.
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(4-methylbenzyl)-
1H-1,2,3-triazol-4-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diyl dia-
cetate 8e. dH(300 MHz, CDCl3) 9.01 (s, 1H), 8.62 (s, 1H), 8.24
(s, 1H), 7.23 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 8.3 Hz, 2H), 6.26
(d, J = 5.3 Hz, 1H), 5.95 (t, J = 5.5 Hz, 1H), 5.68 (br dd, J =
5.5 and 4.5 Hz, 1H), 5.62 (s, 2H), 4.48–4.34 (m, 3H), 2.33 (s, 3H),
2.15 (s, 3H), 2.10 (s, 3H), 2.06 (s, 3H); dC(75 MHz, CDCl3) 170.4
(C), 169.7 (C), 169.5 (C), 153.3 (CH), 151.8 (C), 148.1 (C), 144.0
3878 | Org. Biomol. Chem., 2010, 8, 3874–3881
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(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(4-nitrobenzyl)-1H-
1,2,3-triazol-4-yl)-9H -purin-9-yl)tetrahydrofuran-3,4-diyl dia-
cetate 8j. mp 142.5 ◦C; dH(300 MHz, CDCl3) 9.03 (s, 1H), 8.75
(s, 1H), 8.26 (s, 1H), 8.22 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.8 Hz,
2H), 6.26 (d, J = 5.1 Hz, 1H), 5.96 (appt, J = 5.5 and 5.3 Hz, 1H),
5.79 (s, 2H), 5.68 (br dd, J = 5.3 and 4.7 Hz, 1H), 4.49–4.34 (m,
3H), 2.15 (s, 3H), 2.10 (s, 3 H), 2.06 (s, 3 H); dC(75 MHz, CDCl3)
170.4 (C), 169.7 (C), 169.5 (C), 153.3 (CH), 151.8 (C), 148.3 (C),
147.6 (C), 144.4 (C), 143.4 (CH), 141.4 (CH), 130.5 (C), 128.8
(2 ¥ CH), 127.2 (CH), 124.5 (2 ¥ CH), 112.9 (C), 86.7 (CH), 80.5
(CH), 73.3 (CH), 70.7 (CH), 63.1 (CH2), 20.8 (CH3), 20.6 (CH3),
20.5 (CH3); HRMS (ESI) calcd for C25H25N8O9 [M+H]+: 581.1739
found 581.1743
(2R,3R,4S,5R)-2-(6-(1-(4-bromobenzyl)-1H -1,2,3-triazol-4-
yl)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 9b.
mp 126–127 ◦C; dH(300 MHz, DMSO) 9.24 (s, 1H), 8.98 (s, 1H),
8.92 (s, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H),
6.08 (d, J = 5.5 Hz, 1H), 5.79 (s, 2H), 5.60 (br s, 1H), 5.29 (br s,
1H), 5.15 (br s, 1H), 4.66 (appt, J = 5.3 and 5.1 Hz, 1H), 4.22 (br
t, J = 4.3 Hz, 1H), 4.00 (br q, J = 3.8 Hz, 1H), 3.73 (br dd, J
= 12.1 and 3.4 Hz, 1H), 3.60 (br dd, J = 12.1 and 3.6 Hz, 1H);
dC(75 MHz, DMSO) 152.2 (CH), 151.6 (C), 146.7 (C), 145.2 (CH),
142.4 (C), 135.3 (C), 131.8 (2 ¥ CH), 130.4 (2 ¥ CH), 129.7 (C),
128.4 (CH), 121.6 (C), 87.8 (CH), 85.7 (CH), 73.7 (CH), 70.2 (CH),
61.2 (CH2), 52.3 (CH2); HRMS (ESI) calcd for C19H19N7O4Br
[M+H]+ 488.0676 found 488.0678; HPLC purity grade. 254 nm:
92%; 300 nm: 99%
8k. dH(300 MHz, CDCl3) 9.02 (s, 2H), 8.86 (s, 2H), 8.27 (s,
2H), 6.28 (d, J = 5.3 Hz, 1H), 5.99 (appt, J = 5.5 and 5.3 Hz, 1H),
5.70 (br dd, J = 5.3 and 4.7 Hz, 1H), 4.62–4.58 (m, 4H), 4.50–4.36
(m, 8H), 2.15 (s, 3H), 2.12 (s, 3 H), 2.08 (s, 3 H); dC(75 MHz,
CDCl3) 170.5 (2 ¥ C), 169.7 (2 ¥ C), 169.5 (2 ¥ C), 153.3 (2 ¥ CH),
151.8 (2 ¥ C), 147.8 (2 ¥ C), 143.7 (2 ¥ C), 143.4 (2 ¥ CH), 130.5
(2 ¥ C), 127.8 (2 ¥ CH), 86.6 (2 ¥ CH), 80.6 (2 ¥ CH), 73.3 (2 ¥
CH), 70.7 (2 ¥ CH), 61.1 (2 ¥ CH2), 47.2 (2 ¥ CH2), 30.7 (CH2),
20.9 (2 ¥ CH3), 20.7 (2 ¥ CH3), 20.5 (2 ¥ CH3); HRMS (ESI) calcd
for C39H43N14O14 [M+H]+: 931.3078 found 931.3080
(2R,3R,4S,5R)-2-(6-(1-(4-chlorobenzyl)-1H -1,2,3-triazol-4-
yl)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 9c.
mp 118–119 ◦C; dH(300 MHz, DMSO) 9.24 (s, 1H), 8.98 (s, 1H),
8.91 (s, 1H), 7.53–7.39 (m, 4H), 6.08 (d, J = 5.5 Hz, 1H), 5.80 (s,
2H), 5.57 (br s, 1H), 5.25 (br s, 1H), 5.15–5.11 (br t, J = 5.5 Hz,
1H), 4.68–4.63 (m, 1H), 4.24–4.19 (m, 1H), 4.02–3.98 (q, J =
3.8 Hz, 1H), 3.77–3.68 (m, 1H), 3.63–3.56 (m, 1H); dC(75 MHz,
DMSO) 152.2 (CH), 151.6 (C), 146.7 (C), 145.1 (CH), 142.4 (C),
134.9 (C), 133.0 (C), 130.1 (2 ¥ CH), 129.7 (C), 128.9 (2 ¥ CH),
128.4 (CH), 87.8 (CH), 85.7 (CH), 73.7 (CH), 70.2 (CH), 61.2
(CH2), 52.2 (CH2); HRMS (ESI) calcd for C19H19N7O4Cl [M+H]+
444.1182 found 444.1181; HPLC purity grade. 254 nm: 96%; 300
nm: 99%
(2R,3S,4R,5R)-2-(acetoxymethyl)-5-(6-(1-(3-azidopropyl)-1H-
1,2,3-triazol-4-yl)-9H -purin-9-yl)tetrahydrofuran-3,4-diyl dia-
cetate 8l. dH(300 MHz, CDCl3) 9.04 (s, 1H), 8.77 (s, 1H), 8.28 (s,
1H), 6.28 (d, J = 5.3 Hz, 1H), 5.98 (appt, J = 5.5 and 5.3 Hz, 1H),
5.68 (br dd, J = 5.5 and 4.5 Hz, 1H), 4.60 (t, J = 6.8 Hz, 1H),
4.50–4.35 (m, 3H), 3.41 (t, J = 6.3 Hz, 2H), 2.31–2.22 (m, 2H),
2.15 (s, 3H), 2.12 (s, 3 H), 2.08 (s, 3 H); dC(75 MHz, CDCl3) 170.4
(C), 169.7 (C), 169.5 (C), 153.2 (CH), 151.8 (C), 147.8 (C), 143.5
(C), 143.3 (CH), 130.4 (C), 127.4 (CH), 86.6 (CH), 80.6 (CH),
73.3 (CH), 70.7 (CH), 63.1 (CH2), 48.1 (CH2), 47.7 (CH2), 29.6
(CH2), 20.9 (CH3), 20.7 (CH3), 20.5 (CH3); HRMS (ESI) calcd for
C21H25N10O7 [M+H]+: 529.1902 found 529.1907
(2R,3R,4S,5R)-2-(6-(1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)-
9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol
9d.
◦
mp 97–98 C; dH(300 MHz, CDCl3–MeOD = 1/1) 8.88 (s, 1H),
8.77 (s, 1H), 8.50 (s, 1H), 7.40–7.35 (m, 2H), 7.09–7.03 (m, 2H),
6.04 (d, J = 6.2 Hz, 1H), 5.65 (s, 2H), 4.80 (br dd, J = 5.9 and
5.5 Hz, 1H), 4.38 (dd, J = 5.1 and 2.7 Hz, 1H), 4.24–4.22 (m, 1H),
3.93 (dd, J = 12.7 and 2.3 Hz, 1H), 3.76 (dd, J = 12.7 and 2.3
Hz); dC(75 MHz, CDCl3–MeOD = 1/1) 163.4 (d, J = 248.2 Hz,
C), 152.3 (CH), 151.8 (C), 145.9 (CH), 143.7 (C), 130.8 (d, J =
3.3 Hz, C), 130.6 (d, J = 8.3 Hz, 2 ¥ CH), 127.6 (CH) 116.5 (d, J
= 22.1 Hz, 2 ¥ CH), 90.8 (CH), 87.3 (CH), 74.7 (CH), 71.7 (CH),
62.7 (CH2), 54.0 (CH2); HRMS (ESI) calcd for C19H19N7O4F
[M+H]+ 428.1477 found 428.1481; HPLC purity grade. 254 nm :
100%; 300 nm : 100%
General procedure for the synthesis of compounds 9a–9k
A solution of NH3/MeOH (0.7 mL) was added over triacetate 9
(70 mmol). The mixture was stirred overnight at room temperature
and followed by TLC. Once reaction was complete by TLC,
the mixture was evaporated under reduced pressure and purified
by column chromatography using dichloromethane–methanol
(85/15) as eluent to afford the triazole derivatives 9a–9k.
(2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-(1-(4-methylbenzyl)-
1H-1,2,3-triazol-4-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diol 9e.
mp 90–91 ◦C; dH(300 MHz, DMSO) d 9.17 (s, 1H), 8.97 (s, 1H),
8.90 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H),
6.07 (d, J = 5.5 Hz, 1H), 5.73 (s, 2H), 5.58 (br s, 1H), 5.27 (br s,
1H), 5.15–5.12 (m, 1H), 4.65 (br s, 1H), 4.21 (br s, 1H), 4.02–
3.98 (m, 1H), 3.75–3.69 (m, 1H), 3.63–3.56 (m, 1H), 2.28 (s, 3H);
dC(75 MHz, DMSO) 152.2 (CH), 151.6 (C), 146.8 (C), 145.1 (CH),
142.4 (C), 137.7 (C), 132.8 (C), 129.7 (C), 129.4 (2 ¥ CH), 128.2
(2 ¥ CH), 128.1 (CH), 87.8 (CH), 85.7 (CH), 73.7 (CH), 70.2
(CH), 61.2 (CH2), 52.8 (CH2), 20.7 (CH3); HRMS (ESI) calcd
for C20H22N7O4 [M+H]+ 424.1728 found 424.1726; HPLC purity
grade. 254 nm: 88%; 300 nm: 97%.
(2R,3R,4S,5R)-2-(6-(1-benzyl-1H-1,2,3-triazol-4-yl)-9H-purin-
9-yl)-5-(hydroxymethyl)-tetrahydrofuran-3,4-diol 9a. mp 76–
77 ◦C; dH(300 MHz, DMSO) 9.21 (s, 1H), 8.98 (s, 1H), 8.90 (s,
1H), 7.31–7.50 (m, 5H), 6.07 (d, J = 5.5 Hz, 1H), 5.79 (s, 2H),
5.61 (br s, 1H), 5.26 (br s, 1H), 5.15–5.12 (m, 1H), 4.64 (br s, 1H),
4.21 (br s, 1H), 4.99 (br s, 1H), 3.75–3.68 (m, 1H), 3.62–3.56 (m,
1H); dC(75 MHz, CDCl3) 151.9 (CH), 150.8 (C), 146.8 (C), 145.3
(CH), 142.8 (C), 134.3 (C), 130.6 (C), 129.3 (2 ¥ CH), 129.1 (CH),
128.2 (2 ¥ CH), 127.4 (CH), 91.5 (CH), 87.7 (CH), 74.0 (CH), 72.6
(CH), 63.2 (CH2), 54.6 (CH2); HRMS (ESI) calcd for C19H20N7O4
[M+H]+ 410.1571 found 410.1570; HPLC purity grade. 254 nm:
98.5%; 300 nm: 100%.
(2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-(1-(4-methoxybenzyl)-
1H-1,2,3-triazol-4-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diol 9f.
mp 167–168 ◦C; dH(300 MHz, DMSO) 9.15 (s, 1H), 8.97 (s, 1H),
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8.90 (s, 1H), 7.40 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H),
6.07 (d, J = 5.3 Hz, 1H), 5.70 (s, 2H), 5.57 (br s, 1H), 5.26 (br s,
1H), 5.14 (br s, 1H), 4.65 (br s, 1H), 4.21 (br s, 1H), 3.99 (br s, 1H),
3.74–3.70 (m, 4H), 3.61–3.57 (m, 1H); dC(75 MHz, DMSO) 159.3
(C), 152.2 (CH), 151.6 (C), 146.8 (C), 145.1 (CH), 142.3 (C), 129.9
(2 ¥ CH), 129.7 (C), 127.9 (CH), 127.7 (C), 114.2 (2 ¥ CH), 87.8
(CH), 85.7 (CH), 73.7 (CH), 70.2 (CH), 61.2 (CH2), 55.2 (CH3),
52.6 (CH2); HRMS (ESI) calcd for C20H22N7O5 [M+H]+ 440.1677
found 440.1677; HPLC purity grade. 254 nm: 95%; 300 nm: 100%.
(CH), 73.71 (CH), 70.2 (CH), 61.2 (CH2), 52.1 (CH2); HRMS
(ESI) calcd for C19H19N8O6 [M+H]+ 455.1422 found 455.1424;
HPLC purity grade: 254 nm: 98%; 300 nm: 100%
Drugs, reagents and cell culture medium
RPMI 1640, glucose, fetal calf serum (10%), geneticin (G418),
penicillin, streptomycin (100 IU mL-1), DMEM/Nut mix F12,
Glutamax were purchased from Invitrogen (Cergy Pontoise,
France). Isobutyl methyl xanthine (IBMX), 8 cyclopentyl-1,3-
dipropylxanthine (DPCPX), and methanol were from SIGMA
Aldrich (St Quentin Fallavier, France). Chem 3 growth supplement
was from Millipore (Billerica MA USA). [H3] DPCPX were from
Amersham (Orsay, France).
(2R,3R,4S,5R)-2-(6-(1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl)-
9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol
9g.
mp 80–81 ◦C; dH(300 MHz, DMSO) d 9.17 (s, 1H), 8.99 (s, 1H),
8.90 (s, 1H), 7.72 (br d, J = 7.6 Hz, 1H), 7.47–7.42 (m, 1H),
7.37–7.29 (m, 1H), 6.07 (d, J = 5.5 Hz, 1H), 5.89 (s, 2H), 5.56
(br s, 1H), 5.25 (br s, 1H), 5.14–5.11 (m, 1H), 4.64 (br s, 1H), 4.22
(br s, 1H), 4.02–3.98 (m, 1H), 3.75–3.68 (m, 1H), 3.63–3.56 (m,
1H); dC(75 MHz, CDCl3) 151.9 (CH), 150.8 (C), 146.7 (C), 145.5
(CH), 142.8 (C), 133.7 (C), 133.4 (CH), 130.7 (CH), 130.6 (C),
130.4 (CH), 128.4 (CH), 127.8 (CH), 91.4 (CH), 87.7 (CH), 74.0
(CH), 72.5 (CH), 63.1 (CH2), 54.3 (CH2); HRMS (ESI) calcd for
C19H19N7O4Br [M+H]+ 488.0676 found 488.0676; HPLC purity
grade. 254 nm: 93%; 300 nm: 96%
Cell culture
Stable cell line human recombinant A1AR, was obtained from
Millipore (CHO, CHEM 3 cells; Millipore, Billerica MA, USA).
◦
Cells were cultured (5% CO2, 37 C) in RPMI 1640, containing
4.5 g L-1 of glucose, supplemented with 10% of fetal calf serum,
0.8 mg mL-1 of geneticin (G418), 100 IU mL-1 of penicillin
and streptomycin and diluted (1/100) with Chem 3 growth
supplement. Cells were cultured (5% CO2, 37 ◦C) in DMEM/Nut
mix F12 with fetal calf serum and glutamax, according to the
recommendations of the manufacturer.
(2R,3R,4S,5R)-2-(6-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)-
H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 9h. mp
67–68 ◦C; dH(300 MHz, DMSO) 9.28 (s, 1H), 8.98 (s, 1H), 8.92 (s,
1H), 7.52 (s, 1H), 7.45–7.56 (m, 3H), 6.08 (br d, J = 5.3 Hz, 1H),
5.82 (br s, 2H), 5.58 (br s, 1H), 5.26 (br s, 1H), 5.16–5.13 (m, 1H),
4.68–4.63 (m, 1H), 4.22 (br s, 1H), 4.00 (br s, 1H), 3.76–3.69 (m,
1H), 3.64–3.55 (m, 1H); dC(75 MHz, DMSO) 152.2 (CH), 151.6
(C), 146.7 (C), 145.2 (CH), 142.4 (C), 138.2 (C), 133.3 (C), 130.8
(CH), 129.7 (C), 128.5 (CH), 128.3 (CH), 128.1 (CH), 126.9 (CH),
87.8 (CH), 85.7 (CH), 73.7 (CH), 70.2 (CH), 61.2 (CH2), 52.2
(CH2); HRMS (ESI) calcd for C19H19N7O4Cl [M+H]+ 444.1182
found 444.1183; HPLC purity grade. 254 nm: 96%; 300 nm: 99%.
cAMP concentration determination
Cells were incubated (2 ¥ 105 per well) 30 min with 1 mM of IBMX,
and increasing concentration of tested drugs. Total amount of
cAMP was determined by immunoassay (cAMP Biotrak Enzyme
immunoasay, EIA system, Amersham Pharmacia Biotrak Orsay,
France). EC 50 is the concentration of drug causing half maximal
inhibition. Data obtained from the dose–response curved were
analyzed by non linear regression analysis (GraphPad Prism,
Software, San Diego USA).
(2R,3R,4S,5R)-2-(6-(1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-
yl)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 9i.
mp 117–118 ◦C; dH(300 MHz, DMSO) 9.29 (s, 1H), 8.98 (s, 1H),
8.92 (s, 1H), 7.75 (d, J = 2.1 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H),
7.40 (dd, J = 8.3 and 2.1 Hz, 1H), 6.08 (d, J = 5.5 Hz, 1H), 5.81
(s, 2H), 5.57 (br s, 1H), 5.26 (br s, 1H), 5.15–5.12 (m, 1H), 4.65
(br s, 1H), 4.21 (br s, 1H), 4.02–3.98 (m, 1H), 3.76–3.69 (m, 1H),
3.63–3.56 (m, 1H); dC(75 MHz, DMSO) 152.2 (CH), 151.6 (C),
146.7 (C), 145.2 (CH), 142 5 (C), 136.8 (C), 131.3 (C), 131.1 (CH),
131.09 (C), 130.3 (CH), 129.7 (C), 128.6 (CH), 128.5 (CH), 87.8
(CH), 85.7 (CH), 73.7 (CH), 70.2 (CH), 61.2 (CH2), 51.6 (CH2);
HRMS (ESI) calcd for C19H18N7O4Cl2 [M+H]+ 478.0792 found
478.0789; HPLC purity grade. 254 nm: 96%; 300 nm: 99%.
Binding assay
Frozen CHO◦cells were subjected to three freeze-thaw cycles
◦
◦
(-80 C, +20 C), centrifuged (4 C, 5000 g, 20 min), suspended
in 4.5 mL of binding buffer (50 mM Tris-HCl, 10 mM MgCl2,
pH 7.4), and homogenized just prior to the binding assay. We used
[3H] DPCPX selective A1 adenosine receptors (A1ARs) ligand.
Homogenates of CHO cell membranes (200 ml for a total volume
of 250 ml) were incubated (90 min at room temperature) with
[3H] DPCPX at the concentration of 5nM (10X KD) and in
the presence of increasing concentration of drugs tested (10-8 to
10-4 M), to obtained displacement curves. Binding experiments
were performed in triplicate. Bound and free radioactivities were
separated by vacuum-filtration of the sample through Whatman
(Breenford, UK) GF/C glass-fiber filters. Cold binding buffer
(1 mL) was added to the sample before filtering. The filter was
washed three times and bound radioactivity was determined with
a Beckman LS-1800 liquid scintillation spectrometer. A weighted
non-linear least-square curve fitting program, Graph Pad Prism
(Graph Pad Software Inc., San Diego, CA, USA) was used for the
computer analysis. Non-specific binding was defined as binding in
(2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-(1-(4-nitrobenzyl)-1H-
1,2,3-triazol-4-yl)-9H-purin-9-yl)tetrahydrofuran-3,4-diol
9j.
dH(300 MHz, DMSO) d 9.33 (s, 1H), 8.99 (s, 1H), 8.92 (s, 1H),
8.26 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H), 6.08 (d, J =
5.5 Hz, 1H), 5.99 (s, 2H), 5.57 (br d, J = 5.7 Hz, 1H), 5.25 (br
d, J = 4.7 Hz, 1H), 5.13 (br t, J = 5.4 Hz, 1H), 4.68–4.63 (m,
1H), 4.24–4.19 (m, 1H), 4.02–3.98 (m, 1H), 3.76–3.69 (m, 1H),
3.63–3.56 (m, 1H); dC(75 MHz, DMSO) d 152.2 (CH), 151.6 (C),
147.3 (C), 146.6 (C), 145.2 (CH), 143.3 (C), 142.5 (C), 129.7 (C),
129.2 (2 ¥ CH), 128.8 (CH), 124.0 (2 ¥ CH), 87.8 (CH), 85.7
3880 | Org. Biomol. Chem., 2010, 8, 3874–3881
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the presence of 10 mM of unlabeled ligand. EC 50 was defined as
the concentration of drug that induce the half maximal effects.
15 M. Hocek, M. Masoj´ıdkova´ and A. Holy, Tetrahedron, 1997, 53, 2291–
2302; G. Langli, L.-L. Gundersen and F. Rise, Tetrahedron, 1996, 52,
5625–5638.
16 M. K. Lakshman, Curr. Org. Synth., 2005, 2, 83–112.
17 C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 2002,
67, 3057–3064; V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B.
Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596–2599.
18 J. E. Moses and A. D. Moorhouse, Chem. Soc. Rev., 2007, 36, 1249–
1262.
Acknowledgements
The authors thank the Agence Nationale pour la Recherche
(ANR-05-BLAN-0175-01), the CNRS, the Universite´ Paul
Ce´zanne, the Universite´ de la Me´diterrane´e, the Faculte´
de Me´decine, the Assistance Publique des Hoˆpitaux de Marseille,
the Fondation Me´rieux and the Conseil Re´gional Provence Alpes
Coˆte d’Azur for fundings. The Conseil Ge´ne´ral des Bouches du
Rhoˆne is also gratefully acknowledged.
19 F. Amblar, J. H. Cho and R. F. Schinazi, Chem. Rev., 2009, 109, 4207–
4220.
20 C. Dyrager, K. Bo¨rjesson, P. Dine´r, A. Elf, B. Albinsson, L. M.
Wilhelmsson and M. GrØtli, Eur. J. Org. Chem., 2009, 1515–1521;
A. Gupte, H. I. Boshoff, D. J. Wilson, J. Neres, N. P. Labello, R. V.
Somu, C. Xing, C. E. Barry and C. C. Aldrich, J. Med. Chem., 2008,
51, 7495–7507; L. Cosyn, K. K. Palaniappan, S.-K. Kim, H. T. Duong,
Z.-G. Gao, K. A. Jacobson and S. V. Calenbergh, J. Med. Chem., 2006,
49, 7373–7383.
21 H. Gunji and A. Vasella, Helv. Chim. Acta, 2000, 83, 3229–3245; G.
O’Mahony, E. Ehrman and M. Grotli, Tetrahedron Lett., 2005, 46,
6745–6748.
22 A. M. Jawalekar, N. Meeuwenoord, J. G. O. Cremers, H. S. Overkleeft,
G. A. Van Der Marel, F. P. J. T. Rutjes and F. L. van Delft, J. Org.
Chem., 2008, 73, 287–290and references cited therein.
23 During the preparation of the manuscript, an efficient preparation of C-
6 azidopurine ribonucleoside and their ligation reactions with alkynes
was published by M. K. Lakshman, M. K. Lakshman, M. K. Singh,
D. Parrish, R. Balachandran and Bi. W. Day, J. Org. Chem., 2010, 75,
2461–2473.
Notes and references
1 V. Ralevic and G. Burnstock, Pharmacol Rev., 1998, 50, 413–492; J. C.
Shryock and L. Belardinelli, Am. J. Cardiol., 1997, 79, 2–10.
2 K. A. Jacobson and Z.-G. Gao, Nat. Rev. Drug Discovery, 2006, 5,
247–264.
3 S. M. Reppert, D. R. Weaver, J. H. Stehle and S. A. Rivkees, Mol.
Endocrinol., 1991, 5, 1037–1048; A. K. Dixon, A. K. Gubitz, D. J.
Sirinathsinghji, P. J. Richardson and T. C. Freeman, Br. J. Pharmacol.,
1996, 118, 1461–1468; R. Guieu, B. Dussol, G. Halimi, G. Bechi, F.
Sampieri, Y. Berland, J. Sampol, F. Couraud and H. Rochat, Gen.
Pharmacol., 1998, 31, 553–561.
24 S. C. Mathew, N. Ghosh, Y. By, A. Berthault, M.-A. Virolleaud, L.
Carrega, G. Chouraqui, L. Commeiras, J. Condo, M. Attolini, A.
Gaudel-Siri, J. Ruf, J.-L. Parrain, J. Rodriguez and R. Guieu, Bioorg.
Med. Chem. Lett., 2009, 19, 6736–6739.
25 M. Frieden, A. Avino and R. Eritja, Nucleosides, Nucleotides Nucleic
Acids, 2003, 22, 193–202.
26 C. Mathe´, T. Lioux and G. Gosselin, Nucleosides, Nucleotides Nucleic
Acids, 2003, 22, 605–609.
27 H. Zhao, A. R. Pagano, W. M. Wang, A. Shallop, B. L. Gaffney and
R. A. Jones, J. Org. Chem., 1997, 62, 7832–7835.
4 J. A. Angus, L. B. Cobbin, R. Einstein and H. M. Maguire, Br. J. Phar-
macol., 1971, 41, 592–599.
5 S. Moro, Z.-G. Gao, K. A. Jacobson and G. Spatdullo, Med. Res. Rev.,
2006, 26, 131–159.
6 M. M. Givertz, B. M. Massie, T. K. Fields, L. L. Pearson and H. C.
Dittrich, J. Am. Coll. Cardiol., 2007, 50, 1551–1560.
7 R. C. Wesley, D. Porzio and M. Sadeghi, Cardiovasc. Res., 1993, 27,
129–133.
8 B. Xu, D. A. Berkich, G. H. Crist and K. F. LaNoue, Am. J. Physiol.,
1998, 274, 271–279.
28 L. P. Kotra, K. K. Manouilov, E. Cretton-Scott, J.-P. Sommadossi,
F. D. Boudinot, R. F. Schinazi and C. K. Chu, J. Med. Chem., 1996,
39, 5202–5207.
29 J. A. Johnson, H. J. Thomas and H. J. Schaeffer, J. Am. Chem. Soc.,
1958, 80, 699–702.
30 P. Turek, P. Nova´k, R. Pohl, M. Hocek and M. Kotora, J. Org. Chem.,
2006, 71, 8978–8981.
31 S. Eppacher, P. K. Bhardwaj, B. Bernet, J. L. Bravo Gala, T. Kno¨pfel
and A. Vasella, Helv. Chim. Acta, 2004, 87, 2969–2986.
32 Y. Ju, D. Kumar and R.S. Varma, J. Org. Chem., 2006, 71,
6697–6700.
9 T. Maemoto, M. Tada, T. Mihara, N. Ueyama, H. Matsuoka, K.
Harada, T. Yamaji, K. Shirakawa, S. Kuroda, A. Akahane, A. Iwashita,
N. Matsuoka and S. Mutoh, J. Pharmacol. Sci., 2004, 96, 42–52.
10 A. Pflueger, T. S. Larson, K. A. Nath, B. F. King, J. M. Gross and F. G.
Knox, Mayo Clin. Proc., 2000, 75, 1275–1283.
11 S. Bae and M. Lakshman, J. Am. Chem. Soc., 2007, 129, 782–789;
G.-R. Qu, R. Xia, X.-N. Yang, J.-G. Li, D.-C. Wang and H. M. Guo,
J. Org. Chem., 2008, 73, 2416–2419; M. K. Lakshman and J. Franck,
Org. Biomol. Chem., 2009, 7, 2933–2940, and references cited therein.
12 M. Hocek, Eur. J. Org. Chem., 2003, 245–254.
13 M. K. Lakshman, J. H. Hilmer, J. Q. Martin, J. C. Keeler, Y. Q. V.
Dinh, F. N. Ngassa and L. M. Russon, J. Am. Chem. Soc., 2001, 123,
7779–7787.
33 O. Yuzenko and K Kiec-Kononowicz, Curr. Med. Chem., 2006, 13,
3609–3625.
34 A. M. Pelletier, S. Venkataramana, K. G Miller, B. M Bennet, D. G Nair,
S. R Louressen and M. G Blennerhassett, Am. J. Physiol. Gastrointest
Liver Physiol., 2010, DOI: 10.1152/ajpgi.00259.2009.
14 A. Matsuda, M. Shinozaki, T. Yamaguchi, H. Homma, R. Nomoto,
T. Miyasaka, Y. Watanabe and T. Abiru, J. Med. Chem., 1992, 35,
241–252.
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The Royal Society of Chemistry 2010
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