Structure-activity relationships of truncated C2- or C8-substituted adenosine derivatives as dual acting A2A and A3 adenosine receptor ligands

Article abstract of DOI:10.1021/jm201229j

Truncated N⁶-substituted-4′-oxo- and 4′- thioadenosine derivatives with C2 or C8 substitution were studied as dual acting A_2A and A₃ adenosine receptor (AR) ligands. The lithiation-mediated stannyl transfer and palladium-c

Full text of DOI:10.1021/jm201229j

Article
pubs.acs.org/jmc
Structure−Activity Relationships of Truncated C2- or C8-Substituted
Adenosine Derivatives as Dual Acting A2A and A3 Adenosine
Receptor Ligands
Xiyan Hou,† Mahesh S. Majik,† Kyunglim Kim,† Yuna Pyee,‡ Yoonji Lee,† Varughese Alexander,†
Hwa-Jin Chung,‡ Hyuk Woo Lee,† Girish Chandra,† Jin Hee Lee,† Seul-gi Park,† Won Jun Choi,†,§
Hea Ok Kim,† Khai Phan,∥ Zhan-Guo Gao,∥ Kenneth A. Jacobson,∥ Sun Choi,† Sang Kook Lee,‡
and Lak Shin Jeong*,†
†Laboratory of Medicinal Chemistry, College of Pharmacy, and Department of Bioinspired Science, Ewha Womans University, Seoul
120-750, Korea
‡College of Pharmacy, Seoul National University, Seoul 151-742, Korea
§College of Pharmacy, Dongguk University, Kyungki-do 410-774, Korea
∥Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: Truncated N6-substituted-4′-oxo- and 4′-thioadenosine derivatives
with C2 or C8 substitution were studied as dual acting A2A and A3 adenosine
receptor (AR) ligands. The lithiation-mediated stannyl transfer and palladium-
catalyzed cross-coupling reactions were utilized for functionalization of the C2
position of 6-chloropurine nucleosides. An unsubstituted 6-amino group and a
hydrophobic C2 substituent were required for high affinity at the hA2AAR, but
hydrophobic C8 substitution abolished binding at the hA2AAR. However, most of
synthesized compounds displayed medium to high binding affinity at the hA3AR,
regardless of C2 or C8 substitution, and low efficacy in a functional cAMP assay. Several compounds tended to be full hA2AAR
agonists. C2 substitution probed geometrically through hA2AAR docking was important for binding in order of hexynyl > hexenyl >
hexanyl. Compound 4g was the most potent ligand acting dually as hA2AAR agonist and hA3AR antagonist, which might be useful
for treatment of asthma or other inflammatory diseases.
of 1, to bind potently but with altered ability to induce the
conformational change essential for receptor activation. As
expected, members of the series of compound 2 proved to be
potent and selective A3AR antagonists.9 These A3AR antagonists 2
also showed species-independent binding affinity, making them
suitable for efficacy evaluation for drug development in small
animal models.9 Compound 2 (X = S, R = 3-iodobenzyl)
exhibited a potent antiglaucoma effect in vivo.10
Recently, truncated C2- or C8-substituted 4′-thioadenosine
derivatives 3 containing an appended hydrophobic hexenyl or
hexynyl chain at the C2 or C8 position of 2 were designed and
synthesized.11 Among these, a C2-hexynyl derivative 3 (R =
hexynyl) was discovered as a new template (Ki = 7.19 0.6 nM)
for the development of potent A2AAR agonists12 while maintaining
antagonism at the A3AR (Ki = 11.8 1.3 nM).11 However, C8-
substituted derivatives displayed greatly reduced binding affinity at
the A2AAR while maintaining high affinity at the A3AR, indicating
that the binding pocket for C8 substituents in the A2AAR was
relatively small when compared with that for C2 substituents,
INTRODUCTION
■
The structure of endogenous adenosine, which binds to four
subtypes (A1, A2A, A2B, and A3) of adenosine receptors (ARs)1,2
to induce physiological effects, has been extensively modified,
particularly on N6 and/or 4′-hydroxymethyl positions for the
development of potent and selective AR ligands.3,4 Among
these, 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarbamoylade-
nosine (Cl-IB-MECA, 1a, X = O, R = 3-iodobenzyl; Ki = 1.0
nM for hA3AR) is a versatile A3AR agonist, which is in clinical
trial as an anticancer agent (Figure 1).5 2-Chloro-N6-(3-
iodobenzyl)-5′-N-methylcarbamoyl-4′-thioadenosine (thio-Cl-
IB-MECA, 1b, X = S, R = 3-iodobenzyl; Ki = 0.38 nM for
hA3AR), which has a bioisosteric relationship to 1a, was also
found to be a potent and selective A3AR agonist.6 Compounds
1a and 1b showed antiproliferative effects in various cancer cell
lines, resulting from down-regulation of the Wnt signaling
pathway and other mechanisms.7
A molecular modeling study8 indicated that the NH of the 5′-
uronamide of 1 serves as a hydrogen bonding donor in the
A3AR binding site and is associated with the induced fit for the
receptor activation. On this basis, we designed and synthesized
truncated adenosine derivatives 2,9 removing the 5′-uronamide
Received: September 15, 2011
Published: December 5, 2011
© 2011 American Chemical Society
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Figure 1. Design of the target nucleosides acting dually at the A2A and A3ARs.
which could accommodate steric bulk in both A2A and A3ARs.11
This dual activity at both of these subtypes might be beneficial for
developing therapeutic drugs against diseases such as asthma and
inflammation.
Thus, it was of great interest to extend the structure−activity
relationship study of the truncated 4′-thio series 3 to the 4′-oxo
series 4 with various substituents at C2 or C8 positions. Truncated
4′-oxoadenosine derivatives containing C2-H or C2-Cl were
previously found to interact potently and selectively with the
A3AR.9c,d It was also interesting to determine the effects of N6
substituents on binding affinity to the A2AAR because 3-halobenzyl
substituents at the N6 position generally increased the binding
affinity and selectivity at the A3AR.3,4 Herein, we report a full
account of truncated C2-, C8-, or N6-modified adenosine
derivatives acting dually at the A2A and A3ARs.
n-tributyltin chloride produced C2-stannyl derivative 7 as a sole
regioisomer, which resulted from an anionic transfer of the
stannyl group from C8 to the C2 position (see Scheme 1S in
the Supporting Information for a reaction mechanism).13a
Treatment of 7 with iodine gave 2-iodo-6-chloropurine
derivative 8.13 Sonogashira14 coupling of 8 with 1-hexyne in
the presence of tetrakis(triphenylphosphine)palladium and
cesium carbonate yielded C2-hexynyl derivative, which was
hydrolyzed with 1 N HCl to give the 2-hexynyl-6-chloro
derivative 9. Compound 9 was converted into 2-hexynylade-
nosine derivative 4a and 2-hexynyl-N6-substituted-adenosine
derivatives 4b−e by treatment with ammonia and 3-halobenzyl
amines, respectively. 2-Hexynyladenosine derivative 4a was
subjected to hydrogenation with 10% Pd on carbon to give
saturated analogue 4f.
The corresponding 4′-thioadenosine derivatives 4g−k were
synthesized from glycosyl donor 10, which was easily
synthesized from D-mannose according to our previously
published procedure,9 as illustrated in Scheme 2.
RESULTS AND DISCUSSION
■
Chemistry. First, 2-hexynyl-N6-substituted-adenosine de-
rivatives 4a−f were synthesized from glycosyl donor 5,9
which was easily synthesized from commercially available 2,3-
O-isopropylidene-D-erythronic γ-lactone in two steps, as shown
in Scheme 1.
The lithiation-mediated stannyl transfer13 of 6-chloropurine
nucleosides used in Scheme 1 was first tried for the synthesis of
4′-thioadenosine derivatives 4g−k but resulted in poor
formation of 2-iodo-6-chloropurine derivative 11 (5%), due
to the presence of acidic 1′ and 4′ protons (conventional
nucleoside numbering) in the thiophene ring. Thus, alternative
method of directly condensing glycosyl donor 10 with freshly
prepared 2-iodo-6-chloropurine13b was attempted, as shown in
Scheme 2. Condensation of glycosyl donor 10 with silylated 2-
iodo-6-chloropurine in the presence of TMSOTf afforded
the β-anomer 11 (60%) along with a trace amount of its
α-anomer. The anomeric configuration was also confirmed
Condensation of glycosyl donor 5 with silylated 6-
chloropurine in the presence of trimethylsilyl trifluorometha-
nesulfonate (TMSOTf) as a Lewis acid yielded the β-anomer
69c (71%) as a single diastereomer. The anomeric configuration
1
in 6 was easily confirmed by a H NMR nuclear Overhauser
effect (NOE) experiment between H-8 and 3′-H. For the
functionalization at the C2 position of 6-chloropurine in 6,
lithiation-mediated stannyl transfer of 6-chloropurine nucleo-
sides reported by Tanaka and co-workers13a was utilized.
Treatment of 6 with a freshly prepared lithium 2,2,6,6-
tetramethylpiperidide (LiTMP) initially formed the C8-
lithiated species because the C8-hydrogen is more acidic than
the C2-hydrogen. Reaction of the C8-lithiated species with
1
by a H NMR NOE experiment between H-8 and 3′-H.
Sonogashira14 coupling reaction of 11 with 1-hexyne in the
presence of tetrakis(triphenylphosphine)palladium {(Ph3P)4Pd}
and cesium carbonate yielded the C2-hexynyl derivative 12.
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a
Treatment of 12 with 1 N HCl yielded the 2-hexynyl-6-chloro
derivative 13, which was treated with ammonia and 3-halobenzyl
amines to afford 2-hexynyl-4′-thioadenosine derivative 4g11 and
2-hexynyl-N6-substituted-4′-thioadenosine derivatives 4h−k,
respectively.
Scheme 1
After the successful introduction of the hexynyl group at the
C2 position, we decided to extend the synthesis to other
hydrophobic C2-hexenyl and C2-hexanyl derivatives. Synthesis
of 2-hexenyl derivatives was accomplished using a Suzuki15
coupling reaction as a key step (Scheme 3). Treatment of
2-iodo-6-chloro derivatives 8 and 11 with methanolic ammonia
gave 2-iodo-6-amino derivatives 14 and 15, respectively. Suzuki
coupling of C2-iodo derivatives 14 and 15 with (E)-1-
catecholboranylhexene,16 prepared by treating with 1-hexyne
and catecholborane in the presence of (Ph3P)4Pd, produced 2-
hexenyl derivatives 16 and 17, respectively. Treatment of 16
and 17 with 1 N HCl gave 2-hexenyl-adenosine derivative 4l
and 2-hexenyl-4′-thioadenosine derivative 4m,11 respectively.
By use of a synthetic strategy similar to Scheme 1, C8-
substituted adenosine derivatives 4n−p were synthesized from
glycosyl donors 5 and 10 (Scheme 4). Condensation of 59 with
silylated 6-chloropurine under Lewis acid conditions followed
by treatment with methanolic ammonia afforded adenine
derivative 18. Treatment of 18 with bromine and sodium
acetate in methanol yielded 8-bromo derivative 19. However, in
the case of 4′-thioadenosine derivatives, the same method was
tried but resulted in the extensive decomposition of starting
material. Thus, direct condensation of 10 with 8-bromoade-
nine17 under Lewis acid conditions provided 8-bromo
derivative 20 but only in 20% yield. Sonogashira coupling of
19 and 20 with 1-hexyne using bis(triphenylphosphine)
palladium dichloride {(Ph3P)4PdCl2} gave C8-hexynyl deriva-
tives 21 and 22, respectively. Treatment of 21 and 22 with 1 N
HCl yielded the final 8-hexynyl derivatives 4n and 4o,11
a
Reagents and conditions: (a) silylated 6-chloropurine, TMSOTf,
DCE, room temp to 80 °C, 5 h; (b) LiTMP, Bu3SnCl, HF, hexane,
−78 °C, 1 h; (c) I2, THF, room temp, 24 h; (d) 1-hexyne, Cs2CO3,
(Ph3P)4Pd, CuI, DMF, room temp, 3 h; (e) 1 N HCl, THF, room
temp, 15 h; (f) NH3/t-BuOH, 100 °C, 8 h or R1NH2, Et3N, EtOH,
room temp, 24−48 h; (g) Pd/C, H2, MeOH.
a
Scheme 2
a
Reagents and conditions: (a) 2-iodo-6-chloropurine, BSA, TMSOTf, CH3CN, room temp to 80 °C, 3 h; (b) 1-hexyne, Cs2CO3,
(Ph3P)4Pd, CuI, DMF, room temp, 3 h; (c) 1 N HCl, THF, room temp, 20 h; (d) NH3 in t-BuOH, 100 °C, 8 h or R1NH2, Et3N, EtOH,
room temp, 48 h.
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a
a
Scheme 3
Scheme 5
a
Reagents and conditions: (a) (E)-1-catecholboranylhexene,
(Ph3P)4Pd, Na2CO3, DMF, H2O, 90 °C, 15 h; (b) 1 N HCl, THF,
room temp, 15 h.
afforded the 8-hexenyladenosine derivative 4q and the 8-
hexenyl-4′-thioadenosine derivative 4r.11
Biological Evaluation. Binding assays were carried out
using standard radioligands and membrane preparations from
Chinese hamster ovary (CHO) cells stably expressing the
human (h) A1 or A3AR or human embryonic kidney (HEK)-
293 cells expressing the hA2AAR.18 Nonspecific binding was
defined using 10 μM of 5′-N-ethylcarboxamidoadenosine (25,
NECA).
a
Reagents and conditions: (a) NH3/MeOH, 80 °C, 2 h; (b) (E)-1-
catecholboranylhexene, (Ph3P)4Pd, Na2CO3, DMF, H2O, 90 °C, 15 h;
(c) 1 N HCl, THF, room temp, 15 h.
As shown in Table 1, 4′-thio derivatives generally exhibited
higher binding affinities to hA2A and hA3ARs than the
corresponding 4′-oxo derivatives. In case of N6-amino
derivatives, most of the C2-substituted analogues (R2 = hexynyl
and hexenyl) showed moderate to high binding affinities
at the hA2A and hA3ARs, while most of C8-substitutions
(R3 = hexynyl, hexenyl, and hexanyl) reduced binding affinity
at the hA2AAR but maintained high affinity at the hA3AR.
respectively. Compound 4n was converted to n-hexanyl
derivative 4p using catalytic hydrogenation.
For the synthesis of 8-hexenyl derivatives 4q and 4r, 8-bromo
derivatives 19 and 20 were coupled with (E)-1-catecholbor-
anylhexene16 under Suzuki conditions15 to give C2-hexenyl
derivatives 23 and 24, respectively (Scheme 5). Removal of the
isopropylidene group in 23 and 24 under acidic conditions
a
Scheme 4
a
Reagents and conditions: (a) silylated 6-chloropurine, TMSOTf, DCE, room temp to 80 °C, 5 h; (b) NH3, MeOH, 80 °C, 2 h; (c) Br2, 1 N
NaOAc, MeOH, room temp, 40 min; (d) silylated 8-bromoadenine, TMSOTf, DCE, room temp to 90 °C, 2 h; (e) 1-hexyne, CuI, TEA, DMF,
(PPh3)2PdCl2, room temp, 3 h; (f) 1 N HCl, THF, room temp, 15 h; (g) H2, Pd/C, MeOH, 15 h.
345
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Table 1. Binding Affinities of Known A3AR Antagonist 2 and Truncated C2- and C8-Substituted Derivatives 4a−r at Three
Subtypes of hARs and A3AR-Mediated Inhibition of cAMP Production
a
affinity (Ki SEM (nM) or % inhibition)
c
relative efficacy (% inhibition SEM of cAMP)
compd (R1, R2, R3)
hA1
hA2A
hA3
hA3
b
2 (X = S, R1 = 3-I-Bn, R2 = Cl, R3 = H)
2490 940
740 430
341 75
4.16 0.50
138 44
570 130
150 140
67 18
4.2 2.3
4a (X = O, R1 = H, R2 = 2-hexynyl, R3 = H)
4b (X = O, R1 = 3-F-Bn, R2 = 2-hexynyl, R3 = H)
4c (X = O, R1 = 3-Cl-Bn, R2 = 2-hexynyl, R3 = H)
4d (X = O, R1 = 3-Br-Bn, R2 = 2-hexynyl, R3 = H)
4e (X = O, R1 = 3-I-Bn, R2 = 2-hexynyl, R3 = H)
4f (X = O, R1 = H, R2 = 2-hexanyl, R3 = H)
63.2 15
25 3%
17.4 2.4
29.5 5.5
39.7 3.8
37.2 2.9
31.5 2.8
22.3 4.5
2.8 1.6
9
9
1%
5%
42 13%
27 4%
12 4%
18 2%
32 9%
220 50
39 5%
10 5%
2160 270
7.19 0.6
46 5%
d
4g (X = S, R1 = H, R2 = 2-hexynyl, R3 = H)
39 10%
24 5%
11.8 1.3
150 80
24.0 6.0
24.0 5.0
4h (X = S, R1 = 3-F-Bn, R2 = 2-hexynyl, R3 = H)
4i (X = S, R1 = 3-Cl-Bn, R2 = 2-hexynyl, R3 = H)
4j (X = S, R1 = 3-Br-Bn, R2 = 2-hexynyl, R3 = H)
4k (X = S, R1 = 3-I-Bn, R2 = 2-hexynyl, R3 = H)
4l (X = O, R1 = H, R2 = 2-hexenyl, R3 = H)
47.6 4.9
47.2 3.4
25.2 3.0
40.0 5.6
42.6 3.1
10.7 4.1
24.5 2.9
1.7 3.9
25 2%
3730 200
3910 970
4890 840
178 26
12 1%
15 3%
39
5
31.9 1.2%
16.2 8.4%
290 70
49.3 4.9%
18.1 5.0%
500 140
3.7 2.9%
218 79
d
4m (X = S, R1 = H, R2 = 2-hexenyl, R3 = H)
72.0 19.1
27.2 2.9%
46.5 4.3%
5.8 4.6%
27.3 6.3%
22.8 6.4%
13.2 0.8
31.7 7.4
20.0 4.0
24.7 2.1%
94.2 30.0
259 10
4n (X = O, R1 = H, R2 = H, R3 = 2-hexynyl)
d
4o (X = S, R1 = H, R2 = H, R3 = 2-hexynyl)
4p (X = O, R1 = H, R2 = H, R3 = 2-hexanyl)
4q (X = O, R1 = H, R2 = H, R3 = 2-hexenyl)
12.9 2.2
19.5 2.4
6.1 1.7
d
4r (X = S, R1 = H, R2 = H, R3 = 2-hexenyl)
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-(−)-N6-2-phenylisopropyladenosine (R-PIA), [3H]2-
[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (26, CGS21680, 10 nM), or 0.5 nM [125I]N6-(3-iodo-4-aminobenzyl)-5′-N-
methylcarboxamidoadenosine (27, 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 nonspecific binder 25 (10 μM). Values expressed as a percentage in italics refer to percent inhibition
b
c
of specific radioligand binding at 10 μM, with nonspecific binding defined using 10 μM 25. Reference 9. Maximal efficacy (at 10 μM) in an A3AR
functional assay, determined by inhibition of foskolin-stimulated cAMP production in AR-transfected CHO cells, expressed as percent inhibition
d
(mean standard error, n = 3−5) in comparison to effect (100%) of full agonist 1a at 10 μM. Binding data from ref 11.
These findings indicate that bulky hydrophobic pockets exist in
the binding sites of A2AAR and A3AR, allowing the C2-
substituent to form favorable hydrophobic interactions, but a
bulky hydrophobic group at C8 position could be tolerated
only at the binding site of the hA3AR but not hA2AAR. The
ability to enhance affinity at the A2AAR in the truncated series
by extending an unsaturated carbon chain at the C2 position
implies a mode of receptor binding in common with the
riboside series.19 However, introduction of a hydrophobic 3-
halobenzyl substituent on the N6-amino group of 4a and 4g,
resulting in 4b−e and 4h−k, respectively dramatically
decreased the binding affinity at the hA2AAR but maintained
the binding affinity at the hA3AR. These results indicate that an
unsubstituted primary amino group at the N6 position is
conducive to high binding affinity at the hA2AAR while a bulky
hydrophobic substituent at the N6 position is essential for high
affinity at the hA3AR. By correlation of pharmacological
behavior with the nucleoside substitution pattern, a hexynyl
substituent generally produced higher binding affinity than the
corresponding hexenyl substituent, and two hexanyl derivatives
(4f and 4p) were greatly reduced in binding affinity at the three
subtypes of hARs, indicating that the geometry of the
substituent is important for the binding affinity. All compounds
showed weak binding affinity at the hA1AR, although 4a, 4n,
and 4q displayed measurable Ki values less than 1 μM at this
subtype. Thus, in some cases hydrophobic substitution at the
C8 position permitted moderate binding affinity (Ki < 500 nM)
at the hA1AR. 4′-Oxo derivative 4d and 4′-thio derivatives 4i, 4j,
4k, and 4o displayed high selectivity for the hA3AR in
comparison to hA1AR and hA2AAR, with Ki values in the range
of 20−67 nM. From the SAR study, compound 4g was
discovered to be the most potent dually acting ligand, binding
with high affinity at both the hA2AAR (Ki = 7.19 0.6 nM) and
hA3AR (Ki = 11.8 1.3 nM) to the exclusion of the hA1AR.11
The functional screening protocol used at the hA3AR
consisted of examining the effects of a single concentration of
each compound (10 μM), which in each case except for 4f and
4p far exceeded the Ki values in binding and therefore was
interpreted as a roughly maximal effect under ligand saturating
conditions. As shown in Table 1, most of the derivatives are low
efficacy partial agonists at the hA3AR, with relative percent
inhibition of cyclic adenosine 5′-monophosphate (cAMP)
formation not exceeding 50%. The 4′-thio C2-substituted
derivatives 4i, 4j, and 4k appear to be hA3AR-selective partial
agonists. Some of the C2-substituted derivatives, 4c, 4h, 4i, 4k,
and 4l, displayed between 40% and 50% maximal efficacy in
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comparison to 1a. This was in contrast to another C2 derivative
4g, which did not have significant residual efficacy at the
hA3AR. The C8-substituted derivatives 4n−r did not exceed
25% of the maximal efficacy to 1a. A C8-hexynyl derivative 4o
in the 4′-thio series, which was highly potent in hA3AR binding
(Ki = 20 nM) and selective in comparison to A1AR and A2AAR
did not have significant residual efficacy at the hA3AR.
In a set of full concentration−response experiments,
compound 4g induced parallel right shifts of the concen-
tration−response curve of a full agonist 1a in the inhibition of
cAMP production at the hA3AR expressed in CHO cells,
indicating that it is as a full, competitive antagonist (KB = 1.69 nM)
at the hA3AR,11 as our previously reported truncated N6-substituted
4′-thioadenosine derivatives.9 Similarly, by the same criteria
compound 4m was a competitive antagonist (KB = 23.9 nM) at
the hA3AR (Figure 2).
Figure 3. Effects of compounds 4a (A) and 4m (B) in stimulation of
cAMP production at the hA2AAR expressed in CHO cells, compared to
NECA as reference full agonist (=100%).
adenosine derivative, which was also a hA2AAR agonist and
hA3AR antagonist.20
We then examined the effects of several compounds in
stimulation of cAMP production at the hA2BAR expressed in
CHO cells, compared to NECA as reference full agonist
(=100%). The most potent compound 4g was a weak partial
agonist in cyclic AMP accumulation (EC50 ≈ 10 μM).11 As
indicated by the percent stimulation at 10 μM, none of other
potent truncated derivatives (4′-oxo 4a 1.9 4.0%, 4l 1.8
4.9%; 4′-thio 4m 3.6
2.9%, 4o 7.0
4.4%) displayed
significant activity at this AR subtype.
Before examination of the pharmacological effect of the most
potent compound 4g, the partition coefficient (log P) was first
calculated using Tripos SYBYL X-1.2 for the druggability of 4g.
The log P of 4g was 0.51, which is sufficient to penetrate cell
membranes, suggesting the possibility of bioavailability by oral
as well as other administration routes.
Figure 2. Effects of compound 4m on inhibtion of cAMP production
induced by full agonist 1a at the hA3AR expressed in CHO cells (A).
The data were transformed to a linear Schild plot (B) indicating
competitive antagonism with a KB of 23.9 nM.
Acute inflammation is a short-term process that is
characterized by the typical signs of inflammation, such as
swelling, pain, and loss of function due to the infiltration of the
tissues by plasma and leukocytes. Among them, edema is one of
the fundamental actions of acute inflammation and it is an
essential parameter to be considered when evaluating com-
pounds with a potential anti-inflammatory activity.21 Therefore,
we tested the anti-inflammatory activity of 4g on carrageenan-
induced paw edema model in rats.22 Paw edema was induced
by the injection of carrageenan (0.1 mL in 1% solution), and
the volume of paw edema was monitored for 24 h. The paw
edema was increased and reached its maximum at 4 h after
treatment of carrageenan.
As shown in Figure 4, the treatment of 4g significantly (P < 0.01)
reduced the paw edema formation. The inhibition rate at 4 h was
51.1% with the treatment of 4g (20 mg/kg). In the same experi-
mental condition, indomethacin (20 mg/kg) was shown as 49.7%
inhibition at 4 h.
The potency of the anti-inflammatory effect of 4g was similar
to that of indomethacin. In this study, the formation of edema
reached a maximum at 4 h after carrageenan injection and the
However, in a cAMP functional assay at the hA2AAR
expressed in CHO cells, several of these nucleoside agonists
demonstrated a higher degree of relative efficacy than at the
hA3AR. For example, the most potent compound 4g behaved as
a full agonist compared to the standard CGS21680 and
displayed an EC50 of 12 nM,11 and compound 4a displayed a
maximal stimulation of cAMP formation of 89.9
3.8%,
relative to the full agonist NECA (=100%), with an EC50 of
80.3 nM (Figure 3A).
Compounds 4l and 4m were also agonists at the hA2AAR,
with maximal percent activity at 10 μM relative to NECA of
83.0
5.2% and 108.8
5.1%, respectively. In a full
concentration−response curve (Figure 3B), compound 4m
displayed an EC50 value of 20.6 1.58 nM in stimulation of
cAMP formation via the hA2AAR. As reported by us previously,
compound 4g was a potent full agonist in stimulation of cAMP
formation via the hA2AAR with an EC50 of 12 nM.11 The
findings that truncated nucleosides 4g and 4m serve as dual
ligands acting at the hA2A and hA3ARs are similar to the
pharmacological profile of a more heavily 2,5′-substituted
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Figure 4. Inhibitory effect of 4g on the carrageenan-induced paw edema. Paw edema was induced as described in the Experimental Section. The paw
volume was measured before (0 h) and at intervals of 0.5, 1, 2, 4, 6, and 24 h after carrageenan injection using a plethysmometer. Data represent the
mean SD (n = 6). ∗ indicates statistically significant differences from the control group (P < 0.01).
administration of 4g inhibited the paw edema induced by
carrageenan (Figure 4). These findings demonstrated that 4g
has a potent in vivo anti-inflammatory activity in an acute
inflammation model system.
structure−activity relationships of this class of nucleosides as
dual acting A2A and A3AR ligands. The corresponding 4′-thio-
and 4′-oxonucleosides were prepared starting from D-mannose
and D-erythronic γ-lactone, respectively. The highlight of our
synthetic endeavor is the functionalization at the C2 position of
6-chloropurine nucleosides using lithiation-mediated stannyl
transfer and palladium-catalyzed cross-coupling reactions. From
the study, it was revealed that an unsubstituted amino group at
the N6 position and a hydrophobic substituent at the C2
position were required for high binding affinity at the hA2AAR,
but hydrophobic substitution at the C8 position abolished
binding affinity at the hA2AAR. However, most of the
synthesized compounds displayed medium to high binding
affinity at the hA3AR, regardless of the C2- or C8-substitution.
Additionally, it was also found that the geometry of the C2-
substituent is crucial for the binding affinity in order of hexynyl >
hexenyl > hexanyl. From this study, compounds 4g and 4m
were discovered as a preferred ligand to act dually at the
hA2AAR and hA3AR with high affinity. This mixed activity as
A2AAR agonist/A3AR antagonist might be advantageous for
antiasthmatic activity. Moreover, these findings may facilitate
the discrimination of the modes of binding of nucleoside
derivatives at all subtypes of ARs.
Molecular Docking Study. The truncated C2-substituted
derivative 4g showed excellent binding affinity at the hA2AAR
with a Ki of 7.19 nM. Substituting the N6 position with a
chlorobenzyl group, however, dramatically decreased the
hA2AAR binding affinity of 4i by 520-fold in comparison to
4g. Our flexible docking study showed that 4g binds readily
into the antagonist-bound X-ray crystal structure of human A2A
AR (PDB code 3EML)23 (Figure 5A).11 Its adenine and sugar
moieties make tight interactions with the binding site residues
via the π−π stacking with Phe168 and H-bonding with Glu169,
Asn253, and Ser277. The C2-hexynyl group also contributes to
the ligand binding through favorable hydrophobic interactions.
In contrast, the N6-chlorobenzyl derivative 4i appeared to have
various binding modes and did not maintain some key
interactions for binding (Figure 5B). It seems that 4i would
lose important interactions at the binding site in order to
accommodate both the bulky N6-chlorobenzyl and C2-hexynyl
groups. This result could explain why appending a halobenzyl
group at the N6 position decreased the A2AAR binding affinity.
The binding modes of a series of the C2-substituted and N6-
unsubstitued derivatives (i.e., 4a, 4f, 4g, 4l, and 4m) were also
compared. As shown in Figure 6A, the adenine and sugar
moieties in this series of truncated nucleosides maintained
almost exactly the same binding interactions. This docking
study demonstrated that the different binding modes of the
C2-substituents directly influenced their activities and a
good correlation (r2 = 0.86) was shown between the binding
affinities and docking scores (Figure 6B). The activity of the
compounds decreased as the flexibility of their C2-
substituents increased. Containing the most flexible hexanyl
group at the C2 position, 4f showed a relatively low binding
affinity and docking score. Taken altogether, the appropriate
size of the functional group at the N6 position and rigidity
of the C2-substitutents appear to contribute to the ligand
binding.
EXPERIMENTAL SECTION
■
General Methods. 1H NMR spectra (CDCl3, CD3OD or DMSO-
d6) were recorded on Varian Unity Invoa 400 MHz instrument. The
1H NMR data are reported as peak multiplicities: s for singlet, d for
doublet, dd for doublet of doublets, t for triplet, q for quartet, br s for
broad singlet, and m for multiplet. Coupling constants are reported in
hertz. 13C NMR spectra (CDCl3, CD3OD, or DMSO-d6) were
recorded on Varian Unity Inova 100 MHz instrument. 19F NMR
spectra (CDCl3, CD3OD) were recorded on Varian Unity Inova 376
MHz instrument. The chemical shifts were reported as parts per
million (δ) relative to the solvent peak. Optical rotations were
determined on Jasco III in appropriate solvent. UV spectra were
recorded on U-3000 made by Hitachi in methanol or water. Infrared
spectra were recorded on FT-IR (FTS-135) made by Bio-Rad. Melt-
ing points were measured on B-540 made by Buchi. Elemental
analyses (C, H, and N) were used to determine the purity of all
synthesized compounds, and the results were within 0.4% of
the calculated values, confirming ≥95% purity. Reactions were
checked with TLC (Merck precoated 60F254 plates). Spots were
detected by viewing under a UV light, colorizing with charring after
dipping in anisaldehyde solution with acetic acid, sulfuric acid, and
methanol. Column chromatography was performed on silica gel 60
CONCLUSIONS
■
In summary, the series of truncated N6-substituted 4′-oxo- and
4′-thioadenosine derivatives, 4a−r with substitution at C2 or
C8 position, were synthesized in order to examine the
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Figure 5. Predicted binding modes of the C2-substituted derivative 4g and its N6-chlorobenzyl derivative 4i in an A2AAR crystal structure.23
(A) Compound 4g binds very well to the receptor, interacting with important residues at the binding site. (B) Compound 4i shows various binding
modes and does not maintain the key interactions. Compounds 4g and 4i are depicted in ball-and-stick with carbon atoms in magenta and gray,
respectively. The key residues are displayed as capped-stick with carbon atoms in white, and the hydrogen bonds are marked in black dashed lines.
The van der Waals surfaces of the ligands are colored by hydrogen bonding capability (red, H-bond donating region; blue, H-bond accepting
region). The Fast Connolly surface of the protein is Z-clipped, and the nonpolar hydrogens are undisplayed for clarity.
Figure 6. Predicted binding modes of the five C2-substituted and N6-unsubstituted derivatives in an hA2AAR crystal structure23 and the correlation
between their experimental binding affinities and docking scores. (A) The adenine and sugar moieties of the molecules showed almost exactly the
same binding modes maintaining the key interactions. Compounds 4a, 4f, 4g, 4l, and 4m are depicted as ball-and-stick with carbon atoms in purple,
light-blue, magenta, skyblue, and orange, respectively. (B) The scatter plot of the pKi values and GOLD fitness scores for the five compounds
showed a good correlation with r2 of 0.86.
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(230−400 mesh, Merck). Reagents were purchased from Aldrich
Chemical Co. Solvents were obtained from local suppliers. All the
anhydrous solvents were distilled over CaH2, P2O5, or sodium/
benzophenone prior to the reaction.
422.9715. Anal. Calcd for C12H12ClIN4O3: C, 34.10; H, 2.86; N, 13.26.
Found: C, 33.98; H, 2.77; N, 13.00.
(−)-(2R,3S,4S)-2-(6-Chloro-2-(hex-1-ynyl)-9H-purin-9-yl)-
tetrahydrofuran-3,4-diol (9). To a solution of 8 (1.00 g, 2.37
mmol) in anhydrous DMF (20 mL) were added tetrakis-
(triphenylphosphine)palladium (0.68 g, 0.59 mmol), copper iodide
(0.054 g, 0.28 mmol), cesium carbonate (0.77 g, 2.37 mmol), and 1-
hexyne (0.3 mL, 2.61 mmol) at room temperature, and the reaction
mixture was stirred at room temperature for 3 h. The reaction mixture
was evaporated, and the crude product was used in the next step
without further purification. To a stirred ice-cooled solution of above
crude product in THF (5 mL) was added 1 N HCl (5 mL), and the
mixture was stirred at room temperature for 15 h, neutralized with 1 N
NaOH solution, and then evaporated under reduced pressure. The
mixture was subjected to flash silica gel column chromatography
(CH2Cl2/MeOH = 30:1) to give 9 (0.42 g, 52%) as a white solid: mp
Synthesis. (−)-6-Chloro-9-((3aS,6R,6aS)-tetrahydro-2,2-
dimethylfuro[3,4-d][1,3]dioxol-6-yl)-9H-purine (6). 6-Chloro-
purine (0.618 g, 4.00 mmol), ammonium sulfate (0.079 g, 0.60 mmol),
and hexamethyldisilazane (HMDS) (15 mL) were refluxed for 15 h
under dry and inert conditions. The volatile was evaporated under
high vacuum, and the resulting solid was dissolved in 1,2-dichloro-
ethane (10 mL) and cooled at 0 °C. To this solution, a solution of 59
(0.404 g, 2.00 mmol) in 1,2-dichloroethane (10 mL) and TMSOTf
(0.72 mL, 4.00 mmol) were added dropwise, and the reaction mix-
ture was stirred at 0 °C for 30 min at room temperature for 1 h and
finally heated at 80 °C for 5 h. The mixture was cooled and diluted
with CH2Cl2. The organic layer was washed with saturated NaHCO3
solution, dried over anhydrous MgSO4, and filtered. The solvent
was evaporated under reduced pressure. The crude syrup was sub-
jected to flash silica gel column chromatography (hexane/EtOAc =
2:1) to give 6 (0.420 g, 71%) as a white solid: mp 118−119 °C;
UV (MeOH) λmax = 264 nm; 1H NMR (CDCl3) δ 8.73 (s, 1 H), 8.18
(s, 1 H), 6.10 (s, 1 H), 5.49 (d, 1 H, J = 6.0 Hz), 5.27−5.29 (m, 1 H),
4.25−4.31 (m, 2 H), 1.58 (s, 3 H), 1.41 (s, 3 H); 13C NMR (CDCl3) δ
152.3, 151.8, 151.4, 145.1, 132.5, 113.7, 92.2, 84.7, 81.5, 76.3, 26.6,
1
135−137 °C; UV (MeOH) λmax = 281.0 nm; H NMR (CD3OD) δ
8.70 (s, 1 H), 6.05 (d, 1 H, J = 6.4 Hz), 4.90−4.92 (m, 1 H), 4.55 (dd,
1 H, J = 4.0, 9.6 Hz), 4.43−4.45 (m, 1 H), 4.02 (dd, 1 H, J = 1.6, 9.6
Hz), 2.50 (t, 2 H, J = 7.2 Hz), 1.61−1.69 (m, 2 H), 1.50−1.58 (m, 2
H), 0.98 (t, 3 H, J = 7.2 Hz); 13C NMR (CD3OD) δ 153.1, 151.1,
147.9, 147.2, 132.1, 91.5, 91.1, 80.5, 76.6, 75.7, 72.3, 31.4, 23.1, 19.5,
13.9; [α]25 −65.83 (c 0.12, MeOH); (ESI+) (M + H+) m/z
D
337.1062. Anal. Calcd for C15H17ClN4O3: C, 53.50; H, 5.09; N, 16.64.
Found: C, 53.56; H, 5.01; N, 16.34.
25.0; [α]25 −50.69 (c 0.80, MeOH); (ESI+) (M + H+) m/z
D
(−)-6-Chloro-9-((3aS,4R,6aR)-2,2-dimethyltetrahydrothieno-
[3,4-d][1,3]dioxol-4-yl-2-iodo-9H-purine (11). To a stirred sol-
ution of 2-iodo-6-chloropurine (1.90 g, 6.88 mmol) in dry CH3CN
(30 mL) was added N,O-bis(trimethylsilyl)acetamide (BSA, 2.24 mL,
9.16 mmol) under dry and inert conditions, and the reaction mixture
was stirred at 40 °C for 45 min to obtain a clear solution. To this
solution, a solution of 109 (1.00 g, 4.58 mmol) in dry CH3CN (5 mL)
was added dropwise, followed by the addition of TMSOTf (0.91 mL)
at room temperature, and the mixture was heated to 80 °C for
3 h. The mixture was cooled to room temperature, quenched with
saturated NaHCO3 solution (40 mL), and diluted with EtOAc
(50 mL), and then organic layers were separated. The aqueous layer
was again extracted with EtOAc (3 × 30 mL). The combined organic
layers were dried over anhydrous MgSO4 and filtered. The solvent was
evaporated under reduced pressure. The residue was subjected to flash
silica gel column chromatography (hexane/EtOAc = 3:1) to give
297.0752. Anal. Calcd for C12H13ClN4O3: C, 48.58; H, 4.42; N, 18.88.
Found: C, 48.59; H, 4.12; N, 18.08.
(−)-2-(Tributylstannyl)-6-chloro-9-((3aS,6R,6aS)-tetrahydro-
2,2-dimethylfuro[3,4 -d][1,3]dioxol-6-yl)-9H-purine (7). To
a stirred solution of 2,2,6,6-tetramethylpiperidine (TMP, 10.7 mL,
63.53 mmol) in dry hexane (15 mL) and dry THF (30 mL) was added
n-butyllithium (41.6 mL, 1.5 M solution in hexanes, 66.7 mmol)
dropwise at −78 °C over 30 min, and the mixture was stirred at the
same temperature for 1 h. To this mixture, a solution of 6 (3.77 g,
12.7 mmol) in dry THF (30 mL) was added dropwise, and the mixture
was stirred at −78 °C for 30 min. n-Tributyltin chloride (17.23 mL,
63.53 mmol) was added dropwise to the dark reaction mixture, and
the mixture was stirred at the same temperature for 1 h. The resulting
dark solution was quenched by dropwise addition of a saturated
aqueous NH4Cl solution (50 mL). After the mixture was stirred
at room temperature for 15 h, the mixture was diluted with CH2Cl2
(100 mL). The organic layer was washed with saturated NaHCO3
solution, dried over anhydrous MgSO4, and filtered. The solvent was
evaporated under reduced pressure. The crude syrup was subjected to
flash silica gel column chromatography (hexane/EtOAc = 5:1) to give
7 (7.10 g, 95%) as a colorless syrup: UV (MeOH) λmax = 269 nm;
1H NMR (CDCl3) δ 8.06 (s, 1 H), 6.06 (s, 1 H), 5.58 (d, 1 H, J =
6.0 Hz), 5.30−5.32 (m, 1 H), 4.24−4.30 (m, 2 H), 1.55−1.66 (m,
9 H), 1.41 (s, 3 H), 1.27−1.38 (m, 7 H), 1.19−1.23 (m, 5 H), 0.86−
0.92 (m, 9 H); 13C NMR (CDCl3) δ 182.4, 150.6, 150.1, 144.1, 131.1,
113.5, 91.9, 84.6, 81.9, 76.3, 29.3, 29.2, 29.1, 27.8, 27.5, 27.2, 26.6,
compound 11 as a white foam (1.20 g, 60%): UV (CH2Cl2) λmax
=
282.0 nm; 1H NMR (CDCl3) δ 8.06 (s, 1 H), 5.84 (s, 1 H), 5.33 (t, 1
H, J = 4.8 Hz), 5.21 (d, 1 H, J = 5.6 Hz), 3.80 (dd, 1 H, J = 4.4, 12.8
Hz), 3.25 (d, 1 H, J = 12.8 Hz), 1.59 (s, 3 H), 1.37 (s, 3 H); 13C NMR
(CDCl3) δ 151.9, 151.2, 144.4, 132.7, 116.7, 112.1, 90.0, 84.8, 70.7,
41.5, 26.6, 24.8; [α]25 −66.33 (c 0.10, CH2Cl2); (ESI+) (M + H+)
D
m/z 438.9486. Anal. Calcd for C12H12ClIN4O2S: C, 32.86; H, 2.76;
N, 12.77; S, 7.31. Found: C, 32.89; H, 2.89; N, 12.47; S, 7.01.
(−)-6-Chloro-9-((3aS,4R,6aR)-2,2-dimethyltetrahydrothieno-
[3,4-d][1,3]dioxol-4-yl-2-(hex-1-ynyl)-9H-purine (12). Com-
pound 11 (0.60 g, 1.37 mmol) was converted to 12 (0.46 g, 86%)
as a thick syrupy liquid, using the same Sonogashira conditions used in
the preparation of 9: UV (MeOH) λmax = 285.0 nm; 1H NMR
(CDCl3) δ 8.16 (s, 1 H), 5.90 (s, 1 H), 5.32 (t, 1 H, J = 4.8 Hz), 5.21
(d, 1 H, J = 5.2 Hz), 3.79 (dd, 1 H, J = 4.4, 12.8 Hz), 3.23 (d, 1 H, J =
12.8 Hz), 2.48 (t, 2 H, J = 7.2 Hz), 1.61−1.70 (m, 2 H), 1.58 (s, 3 H),
1.47−1.54 (m, 2 H), 1.35 (s, 3 H), 0.95 (t, 3 H, J = 7.2 Hz); 13C NMR
(CDCl3) δ 151.3, 151.2, 146.3, 144.7, 130.8, 112.1, 91.3, 89.9, 843.7,
24.9, 13.9, 12.7, 12.6, 10.9, 9.3, 9.2; [α]25 −8.18 (c 0.33, MeOH);
D
(ESI+) (M + H+) m/z 587.1804. Anal. Calcd for C24H39ClN4O3Sn: C,
49.21; H, 6.71; N, 9.56. Found: C, 49.03; H, 6.66; N, 9.16.
(−)-6-Chloro-9-((3aS,6R,6aS)-tetrahydro-2,2-dimethylfuro-
[3,4-d][1,3]dioxol-6-yl)-2-iodo-9H-purine (8). To a stirred sol-
ution of 7 (7.10 g, 12.1 mmol) in dry THF (100 mL) was added
iodine (4.60 g, 18.2 mmol), and the reaction mixture was stirred for
24 h under a nitrogen atmosphere. The mixture was diluted with
saturated sodium metabisulfite, stirred for 1 h, and then extracted with
CH2Cl2 (3 × 30 mL). The organic layer was dried over anhydrous
MgSO4 and filtered. The solvent was evaporated under reduced
pressure. The crude syrup was subjected to flash silica gel column
chromatography (hexane/EtOAc = 3:1) to give 8 (4.30 g, 84%) as a
white foam: UV (CH2Cl2) λmax = 282.5 nm; 1H NMR (CDCl3) δ 8.07
(s, 1 H), 6.05 (s, 1 H), 5.40 (d, 1 H, J = 6.0 Hz), 5.25−5.28 (m, 1 H),
4.27−4.28 (m, 2 H), 1.57 (s, 3 H), 1.42 (s, 3 H); 13C NMR (CDCl3) δ
152.1, 151.3, 145.1, 132.4, 116.8, 113.8, 92.1, 84.7, 81.5, 76.8, 26.6,
79.7, 70.2, 41.2, 30.3, 26.6, 24.8, 22.3, 19.3, 13.8; [α]25 −57.35 (c
D
0.469, MeOH); (ESI+) (M + H+) m/z 393.1145. Anal. Calcd for
C18H21ClN4O2S: C, 55.02; H, 5.39; N, 14.26; S, 8.16. Found: C, 55.41;
H, 5.21; N, 14.47; S, 8.11.
(−)-(2R,3S,4R)-2-(6-Chloro-2-(hex-1-ynyl)-9H-purin-9-yl)-
tetrahydrothiophene-3,4-diol (13). Compound 12 (0.92 g, 2.34
mmol) was converted to 13 (0.58 g, 70%) as a white solid, using the
same hydrolysis conditions used in the preparation of 9: mp 76−
78 °C; UV (MeOH) λmax = 284.5 nm; 1H NMR (CD3OD) δ 8.87 (s, 1
H), 6.11 (d, 1 H, J = 6.4 Hz), 4.69 (dd, 1 H, J = 3.2, 6.4 Hz), 4.48 (dd,
1 H, J = 3.6, 7.6 Hz), 3.56 (dd, 1 H, J = 4.4, 10.8 Hz), 2.97 (dd, 1 H,
25.1; [α]25 −21.88 (c 0.16, MeOH); (ESI+) (M + H+) m/z
D
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J = 3.6, 10.8 Hz), 2.51 (t, 2 H, J = 6.8 Hz), 1.61−1.67 (m, 2 H), 1.50−
1.56 (m, 2 H), 0.98 (t, 3 H, J = 7.2 Hz); 13 C NMR (CD3OD) δ 153.6,
151.0, 148.0, 147.1, 131.9, 91.5, 80.9, 80.5, 74.4, 64.6, 35.5, 31.4, 23.1,
3.87 (dd, 1 H, J = 4.0, 12.4 Hz), 3.14 (d, 1 H, J = 12.4 Hz), 1.60
(s, 3 H), 1.38 (s, 3 H); 13C NMR (CDCl3) δ 154.3, 153.0, 150.7,
127.8, 120.4, 111.4, 89.0, 86.0, 71.9, 41.4, 26.6, 24.7; [α]25 −77.07
D
19.5, 14.0; [α]25 −32.49 (c 0.634, MeOH); (ESI+) (M + H+) m/z
(c 0.16, CH2Cl2); (ESI+) (M + H+) m/z 373.0151. Anal. Calcd for
C12H14BrN5O2S: C, 38.72; H, 3.79; N, 18.81; S, 8.61. Found: C, 38.88;
H, 3.88; N, 18.48; S, 8.41.
D
353.0835. Anal. Calcd for C15H17ClN4O2S: C, 51.06; H, 4.86; N,
15.88; S, 9.09. Found: C, 51.09; H, 4.89; N, 15.47; S, 9.21.
(−)-9-((3aS,6R,6aS)-Tetrahydro-2,2-dimethylfuro[3,4-d][1,3]-
dioxol-6-yl)-2-iodo-9H-purin-6-amine (14). A solution of 8
(0.303 g, 0.72 mmol) in methanolic ammonia (5 mL) was stirred
for 2 h at 80 °C. The reaction mixture was evaporated and the crude
residue was subjected to flash silica gel column chromatography
(hexane/EtOAc = 1:1) to give 15 (0.205 g, 71%) as a colorless syrup:
UV (CH2Cl2) λmax = 262.0 nm; 1H NMR (DMSO-d6) δ 8.16 (s, 1 H),
7.74 (br s, 2 H, D2O exchangeable), 6.11 (s, 1 H), 5.27 (d, 1 H, J = 5.6
Hz), 5.13−5.15 (m, 1 H), 4.06−4.12 (m, 2 H), 1.47 (s, 3 H), 1.33 (s,
3 H); 13C NMR (DMSO-d6) δ 155.9, 149.3, 139.8, 120.9, 118.9, 112.0,
(−)-(2R,3S,4S)-2-(6-Amino-2-(hex-1-ynyl)-9H-purine-9-yl)-
tetrahydrofuran-3,4-diol (4a). A solution of 9 (0.125 g, 0.42 mmol)
in NH3/tBuOH (5 mL) was stirred at 100 °C for 8 h. The reaction
mixture was evaporated and the residue was subjected to flash silica gel
column chromatography (CH2Cl2/MeOH = 20:1) to give 4a (0.103 g,
87%) as a white solid: mp 164−166 °C; UV (MeOH) λmax = 269.5
1
nm; H NMR (CD3OD) δ 8.26 (s, 1 H), 5.94 (d, 1 H, J = 6.4 Hz),
4.86−4.87 (m, 1H), 4.51 (dd, 1 H, J = 4.0, 9.6 Hz), 4.40−4.42
(m, 1 H), 3.98 (dd, 1 H, J = 2.0, 9.6 Hz), 2.45 (t, 2 H, J = 7.2 Hz),
1.61−1.65 (m, 2 H), 1.49−1.55 (m, 2 H), 0.98 (t, 3 H, J = 7.6 Hz);
13C NMR (CD3OD) δ 157.2, 151.1, 148.1, 142.3, 120.1, 99.4, 88.4,
89.2, 84.0, 80.7, 74.5, 26.2, 24.6; [α]25 −13.25 (c 0.08, CH2Cl2);
D
(ESI+) (M + H+) m/z 404.0225. Anal. Calcd for C12H14IN5O3: C,
35.75; H, 3.50; N, 17.37. Found: C, 35.89; H, 3.89; N, 17.47.
(−)-9-((3aS,4R,6aR)-Tetrahydro-2,2-dimethylthieno[3,4-d]-
[1,3]dioxol-4-yl)-2-iodo-9H-purin-6-amine (15). Compound 11
(0.535 g, 1.22 mmol) was converted to 15 (0.439 g, 85%) as a
colorless syrup, according to the same procedure used in the
preparation of 14: UV (CH2Cl2) λmax = 267.0 nm; 1H NMR
(CD3OD) δ 8.20 (s, 1 H), 5.97 (s, 1 H), 5.30 (pseudo t, 1 H, J = 5.2
Hz), 5.23 (d, 1 H, J = 5.6 Hz), 3.80 (dd, 1 H, J = 4.4, 12.8 Hz), 3.14
(d, 1 H, J = 12.8 Hz), 1.54 (s, 3 H), 1.35 (s, 3 H); 13C NMR
(CD3OD) δ 156.1, 150.4, 141.9, 120.0, 118.0, 112.7, 91.2, 86.4, 71.3,
41.6, 26.8, 24.9; [α]25D −61.90 (c 0.08, CH2Cl2); (ESI+) (M + H+) m/
z 419.998. Anal. Calcd for C12H14IN5O2S: C, 34.38; H, 3.37; N, 16.70;
O, 7.63; S, 7.65. Found: C, 34.11; H, 3.22; N, 16.40; S, 7.45.
81.4, 76.6, 75.4, 72.3, 31.7, 23.2, 19.6, 14.1; [α]25 −39.50 (c 0.16,
D
MeOH); (ESI+) (M + H+) m/z 318.1557. Anal. Calcd for
C15H19N5O3: C, 56.77; H,6.03; N, 22.07. Found: C, 56.98; H, 5.88;
N, 22.01.
General Procedure for the Synthesis of 4b−e. To a solution
of diol 9 in EtOH (5 mL) were added Et3N (3 equiv) and 3-
halobenzylamine (1.5 equiv) at room temperature, and the mixture
was stirred at room temperature for 24−48 h. The solvent was
evaporated and the residue was purified by a flash silica gel column
chromatography (CH2Cl2/MeOH = 20:1) to give 4b−e as white
solids.
(−)-(2R,3S,4S)-2-(6-(3-Fluorobenzylamino)-2-(hex-1-ynyl)-
9H-purin-9-yl)tetrahydrofuran-3,4-diol (4b). Yield, 78%; mp
1
206−207 °C; UV (MeOH) λmax = 272.0 nm; H NMR (DMSO-d6)
(−)-9-((3aS,6R,6aS)-Tetrahydro-2,2-dimethylfuro[3,4-d][1,3]-
dioxol-6-yl)-9H-purin-6-amine (18). A solution of 6 (0.37 g,
1.25 mmol) in methanolic ammonia (5 mL) was heated for 2 h at
80 °C. The reaction mixture was evaporated and the crude residue was
subjected to flash silica gel column chromatography (hexane/EtOAc =
δ 8.47 (brs, 1 H, D2O exchangeable), 8.44 (s, 1 H), 7.32−7.37 (m, 1 H),
7.12−7.17 (m, 2 H), 7.02−7.07 (m, 1 H), 5.85 (d, 1 H, J = 6.8 Hz),
5.45 (d, 1 H, J = 6.4 Hz, D2O exchangeable), 5.20 (d, 1 H, J = 4.0 Hz,
D2O exchangeable), 4.69−4.75 (m, 3 H), 4.33 (dd, 1 H, J = 3.6,
9.2 Hz), 4.24−4.25 (m, 1 H), 3.79 (dd, 1 H, J = 1.6, 9.2 Hz), 2.41 (t,
2 H, J = 7.2 Hz), 1.49−1.56 (m, 2 H), 1.38−1.47 (m, 2 H), 0.91 (t, 3 H,
J = 7.2 Hz); 13C NMR (DMSO-d6) δ 163.4, 161.0, 154.0, 149.1, 145.8,
142.9, 140.6, 130.2 (d), 123.1, 119.0, 113.6 (q), 87.1, 85.6, 81.6, 74.4,
1:1) to give 18 (0.27 g, 78%) as a white foam: UV (CH2Cl2) λmax
=
255 nm; 1H NMR (CDCl3) δ 8.32 (s, 1 H), 7.89 (s, 1 H), 6.19 (brs, 2 H),
6.03 (s, 1 H), 5.48 (d, 1 H, J = 6.0 Hz), 5.27 (dd, 1 H, J = 3.2, 5.6 Hz),
4.24−4.31 (m, 2 H), 1.58 (s, 3 H), 1.41 (s, 3 H); 13C NMR (CDCl3) δ
154.7, 151.3, 149.5, 141.1, 120.3, 113.5, 91.9, 84.8, 81.7, 76.1, 26.6,
73.5, 70.2, 42.4, 29.8, 21.5, 17.9, 13.4; [α]25 −58.87 (c 0.23, MeOH);
D
(ESI+) (M + H+) m/z 426.1948. Anal. Calcd for C22H24FN5O3: C, 62.11;
H, 5.69; N, 16.46. Found: C, 61.98; H, 5.89; N, 16.23.
25.1; [α]25 −35.48 (c 0.09, CH2Cl2); (ESI+) (M + H+) m/z
D
278.1257. Anal. Calcd for C12H15N5O3: C, 51.98; H, 5.45; N, 25.26.
Found: C, 51.98; H, 5.87; N, 25.47.
(−)-(2R,3S,4S)-2-(6-(3-Chlorobenzylamino)-2-(hex-1-ynyl)-
9H-purin-9-yl)tetrahydrofuran-3,4-diol (4c). Yield, 78%; mp
1
(−)-8-Bromo-9-((3aS,6R,6aS)-tetrahydro-2,2-dimethylfuro-
[3,4-d][1,3]dioxol-6-yl)-9H-purin-6-amine (19). To a solution of
18 (89 mg, 0.32 mmol) in MeOH (10 mL) and 1 N sodium acetate
(1.7 mL) was added bromine (0.033 mL, 0.64 mmol), and the mixture
was stirred at room temperature for 40 min. The mixture was diluted
with saturated sodium metabisulfite solution and stirred until the red
color disappeared. The volatile was evaporated, and the resulting
aqueous layer was extracted with EtOAc (3 × 30 mL). The combined
organic layers were washed with water, dried over anhydrous MgSO4,
and filtered. The solvent was evaporated to give a pale yellowish
residue. The residue was purified by flash silica gel column
chromatography (hexane/EtOAc = 2:1) to give 19 (63 mg, 55%) as
a white foam: UV (CH2Cl2) λmax = 262 nm; 1H NMR (CDCl3) δ 8.26
(s, 1 H), 6.20 (brs, 2 H), 6.16 (s, 1 H), 5.61 (d, 1 H, J = 5.6 Hz),
5.36−5.38 (m, 1 H), 4.20−4.21 (m, 2 H), 1.59 (s, 3 H), 1.42 (s, 3 H);
13C NMR (CDCl3) δ 153.8, 151.9, 150.9, 128.8, 120.0, 113.2, 92.4,
210−212 °C; UV (MeOH) λmax = 271.5 nm; H NMR (DMSO-d6)
δ 8.47 (brs, 1 H, D2O exchangeable), 8.44 (s, 1 H), 7.27−7.39 (m,
4 H), 5.84 (d, 1 H, J = 6.8 Hz), 5.45 (d, 1 H, J = 6.4 Hz, D2O
exchangeable), 5.20 (d, 1 H, J = 4.0 Hz, D2O exchangeable), 4.67−
4.75 (m, 3 H), 4.33 (dd, 1 H, J = 3.6, 9.2 Hz), 4.24−4.25 (m, 1 H),
3.79 (dd, 1 H, J = 1.6, 9.2 Hz), 2.41 (t, 2 H, J = 6.8 Hz), 1.49−1.57 (m,
2 H), 1.38−1.47 (m, 2 H), 0.91 (t, 3 H, J = 7.2 Hz); 13C NMR
(DMSO-d6) δ 154.0, 149.2, 145.8, 142.5, 140.6, 132.9, 130.1, 128.3,
127.0, 126.6, 125.8, 87.1, 85.7, 81.6, 74.4, 73.5, 70.2, 42.3, 29.8, 21.5,
17.9, 13.4; [α]25 −57.46 (c 0.13, MeOH); (ESI+) (M + H+) m/z
D
442.1647. Anal. Calcd for C22H24ClN5O3: C, 59.79; H, 5.47; N, 15.85.
Found: C, 59.99; H, 5.55; N, 15.90.
(−)-(2R,3S,4S)-2-(6-(3-Bromobenzylamino)-2-(hex-1-ynyl)-
9H-purin-9-yl)-tetrahydrofuran-3,4-diol (4d). Yield, 73%; mp
1
217−218 °C; UV (MeOH) λmax = 273.0 nm; H NMR (DMSO-d6)
δ 8.47 (brs, 1 H, D2O exchangeable), 8.44 (s, 1 H), 7.54 (s, 1 H), 7.42
(d, 1 H, J = 7.6 Hz), 7.25−7.34 (m, 2 H), 5.84 (d, 1 H, J = 6.4 Hz),
5.45 (d, 1 H, J = 6.4 Hz, D2O exchangeable), 5.20 (d, 1 H, J = 4.0 Hz,
D2O exchangeable), 4.67−4.74 (m, 3 H), 4.32 (dd, 1 H, J = 3.6,
9.2 Hz), 4.24−4.25 (m, 1 H), 3.79 (dd, 1 H, J = 1.6, 9.2 Hz), 2.41 (t,
2 H, J = 7.2 Hz), 1.49−1.56 (m, 2 H), 1.38−1.47 (m, 2 H), 0.91
(t, 3 H, J = 7.2 Hz); 13C NMR (DMSO-d6) δ 154.0, 145.8, 142.8,
140.6, 130.5, 130.0, 129.6, 126.3, 121.5, 87.1, 85.7, 81.6, 78.8; 74.4,
73.5, 70.2, 42.3, 29.9, 21.5, 17.9, 13.4; [α]25D −49.63 (c 0.14, MeOH);
(ESI+) (M + H+) m/z 486.1136. Anal. Calcd for C22H24BrN5O3: C,
54.33; H, 4.97; N, 14.40. Found: C, 54.23; H, 5.01; N, 14.21.
84.3, 82.2, 76.5, 26.6, 24.9; [α]25 −28.08 (c 0.26, CH2Cl2); (ESI+)
D
(M + H+) m/z 356.0365. Anal. Calcd for C12H14BrN5O3: C, 40.47; H,
3.96; N, 19.66. Found: C, 40.12; H, 3.88; N, 19.48.
( − ) - 8 - B r o m o - 9 - ( ( 3 a R , 6 R , 6 a S ) - t e t r a h y d r o - 2 , 2 -
dimethylthieno[3,4-d][1,3]dioxol-6-yl)-9H-purin-2-amine (20).
8-Bromoadenine (0.40 g, 1.84 mmol) and 10 (0.20 g, 0.92 mmol)
were condensed to give 20 (69 mg, 20%) as colorless syrup, according
to similar procedure used in the preparation of 6: UV (MeOH) λmax
=
263.5 nm; 1H NMR (CDCl3) δ 8.25 (s, 1 H), 5.91 (s, 1 H), 5.81 (brs,
2 H, NH2), 5.54 (d, 1 H, J = 5.2 Hz), 5.50 (pseudo t, 1 H, J = 5.2 Hz),
351
dx.doi.org/10.1021/jm201229j | J. Med. Chem. 2012, 55, 342−356

Journal of Medicinal Chemistry
Article
(−)-(2R,3S,4S)-2-(6-(3-Iodobenzylamino)-2-(hex-1-ynyl)-9H-
purin-9-yl)tetrahydofuran-3,4-diol (4e). Yield, 85%; mp 224−
199−201 °C; UV (MeOH) λmax = 275.0 nm; 1H NMR (DMSO-d6) δ
8.52 (s, 1 H), 8.46 (br s, 1 H, D2O exchangeable), 7.27−7.39 (m, 4 H),
5.87 (d, 1 H, J = 7.6 Hz), 5.54 (d, 1 H, J = 6.4 Hz, D2O exchangeable),
5.35 (d, 1 H, J = 4.4 Hz, D2O exchangeable), 4.58−4.67 (m, 3 H), 4.33−
4.35 (m, 1 H), 3.41 (dd, 1 H, J = 4.0, 10.8 Hz), 2.80 (dd, 1 H, J =
3.2, 10.8 Hz), 2.41 (t, 2 H, J = 7.2 Hz), 1.50−1.57 (m, 2 H), 1.38−1.47
1
226 °C; UV (MeOH) λmax = 273.0 nm; H NMR (DMSO-d6) δ 8.47
(brs, 1 H, D2O exchangeable), 8.43 (s, 1 H), 7.73 (pseudo t, 1 H, J =
1.6 Hz), 7.59 (d, 1 H, J = 7.6 Hz), 7.34 (d, 1 H, J = 8.0 Hz), 7.11
(pseudo t, 1 H, J = 8.0 Hz), 5.84 (d, 1 H, J = 6.4 Hz), 5.44 (brs, 1 H,
D2O exchangeable), 5.19 (d, 1 H, J = 2.8 Hz, D2O exchangeable),
4.63−4.72 (m, 3 H), 4.33 (dd, 1 H, J = 3.6, 9.2 Hz), 4.25 (brs, 1 H),
3.79 (dd, 1 H, J = 1.6, 9.2 Hz), 2.41 (t, 2 H, J = 7.2 Hz), 1.50−1.57
(m, 2 H), 1.38−1.47 (m, 2 H), 0.91 (t, 3 H, J = 7.2 Hz); 13C NMR
(DMSO-d6) δ 153.9, 149.1, 145.8, 142.6, 140.6, 135.9, 135.4, 130.5,
126.6, 118.9, 94.7, 87.1, 85.7, 81.6, 74.4, 73.5, 70.2, 42.2, 29.8, 21.5,
13
(m, 2 H), 0.91 (t, 3 H, J = 7.2 Hz); C NMR (DMSO-d6) δ 153.9,
149.4, 145.7, 142.5, 140.5, 132.9, 130.1, 127.0, 126.6, 125.8, 118.7, 85.7,
81.6, 78.6, 72.2, 61.1, 42.3, 34.4, 29.8, 21.5, 17.9, 13.4; [α]25 −46.67
D
(c 0.30, MeOH); (ESI+) (M + H+) m/z 458.1415. Anal. Calcd for
C22H24ClN5O2S: C, 57.70; H, 5.28; N, 15.29; S, 7.00. Found: C, 57.41;
H, 5.68; N, 15.47; S, 7.00.
17.9, 13.4; [α]25 −58.09 (c 0.14, MeOH); (ESI+) (M + H+) m/z
(−)-(2R,3S,4R)-2-(6-(3-Bromobenzylamino)-2-(hex-1-ynyl)-
9H-purin-9-yl)tetrahydrothiophene-3,4-diol (4j). Yield, 81%;
mp 206−208 °C; UV (MeOH) λmax = 275.5 nm; 1H NMR
(DMSO-d6) δ 8.52 (s, 1 H), 8.46 (br s, 1 H, D2O exchangeable),
7.54 (s, 1 H), 7.42 (d, 1 H, J = 8.0 Hz), 7.33 (d, 1 H, J = 7.6 Hz), 7.27
(t, 1 H, J = 7.6 Hz), 5.87 (d, 1 H, J = 7.2 Hz), 5.84 (d, 1 H, J = 6.4 Hz,
D2O exchangeable), 5.36 (d, 1 H, J = 4.0 Hz, D2O exchangeable),
4.60−4.66 (m, 3 H), 4.34−4.35 (m, 1 H), 3.40 (dd, 1 H, J = 4.0, 10.8
D
534.0998. Anal. Calcd for C22H24IN5O3: C, 49.54; H, 4.54; N, 13.13.
Found: C, 49.14; H, 4.13; N, 13.01.
(−)-(2R,3S,4S)-2-(6-Amino-2-hexy1-9H-purine-9-yl)-
tetrahydrofuran-3,4-diol (4f). A mixture of 4a (22 mg, 0.069 mmol),
absolute ethanol (5 mL), and 10% palladium on carbon (5 mg) was
hydrogenated on a Parr apparatus at 40 psi for 15 h. The mixture was
filtered and the solvent was evaporated to give a residue. The residue
was subjected to flash silica gel column chromatography (CH2Cl2/
MeOH = 20:1) to give 4f (15 mg, 68%) as a white solid: mp 105−
Hz), 2.80 (dd, 1 H, J = 3.2, 10.8 Hz), 2.41 (t, 2 H, J = 7.2 Hz), 1.51−
13
1.55 (m, 2 H), 1.39−1.45 (m, 2 H), 0.91 (t, 3 H, J = 7.2 Hz);
C
1
107 °C; UV (MeOH) λmax = 261.5 nm; H NMR (CD3OD) δ 8.19
NMR (DMSO-d6) δ 153.9, 149.4, 145.6, 142.7, 140.5, 130.4, 129.9,
(s, 1 H), 5.95 (d, 1 H, J = 6.4 Hz), 4.93−4.96 (m, 1 H), 4.52 (dd, 1 H,
J = 4.0, 9.6 Hz), 4.42−4.44 (m, 1 H), 3.99 (dd, 1 H, J = 1.6, 9.6 Hz),
2.74 (t, 2 H, J = 7.6 Hz), 1.74−1.80 (m, 2 H), 1.30−1.39 (m, 6 H),
0.88−0.92 (m, 3 H); 13C NMR (CD3OD) δ 167.2, 157.1, 151.8, 141.5,
129.5, 126.2, 121.5, 118.6, 85.7, 81.5, 78.6, 72.2, 61.1, 42.3, 34.4, 29.8,
21.5, 17.9, 13.4; [α]25 −52.17 (c 0.207, MeOH); (ESI+) (M + H+)
D
m/z 504.0890. Anal. Calcd for C22H24BrN5O2S: C, 52.59; H, 4.81; N,
13.94; S, 6.38. Found: C, 52.22; H, 4.89; N, 13.57; S, 5.99.
119.0, 90.5, 76.6, 75.5, 72.5, 40.0, 33.0, 30.3, 30.0, 23.8, 14.6; [α]25
(−)-(2R,3S,4R)-2-(6-(3-Iodobenzylamino)-2-(hex-1-ynyl)-9H-
purin-9-yl)tetrahydrothiophene-3,4-diol (4k). Yield, 77%; mp
225−227 °C; UV (MeOH) λmax = 274.5 nm; 1H NMR (DMSO-d6) δ
8.52 (s, 1 H), 8.45 (br s, 1 H, D2O exchangeable), 7.73 (s, 1 H), 7.58
(d, 1 H, J = 8.0 Hz), 7.34 (d, 1 H, J = 7.6 Hz), 7.11 (t, 1 H, J =
7.6 Hz), 5.87 (d, 1 H, J = 7.2 Hz), 5.54 (d, 1 H, J = 6.0 Hz, D2O
exchangeable), 5.36 (d, 1 H, J = 4.0 Hz, D2O exchangeable), 4.57−
4.62 (m, 3 H), 4.32−4.36 (m, 1 H), 3.40 (dd, 1 H, J = 4.0, 10.8 Hz),
2.80 (dd, 1 H, J = 3.2, 10.8 Hz), 2.42 (t, 2 H, J = 7.2 Hz), 1.50−1.57
D
−54.05 (c 0.11, MeOH); (ESI+) (M + H+) m/z 322.1883. Anal. Calcd
for C15H23N5O3: C, 56.06; H, 7.21; N, 21.79. Found: C, 56.44; H,
7.02; N, 21.49.
(−)-(2R,3S,4R)-2-(6-Amino-2-(hex-1-ynyl)-9H-purin-9-yl)-
tetrahydrothiophene-3,4-diol (4g). Compound 13 (0.065 g,
0.18 mmol) was converted to 4g (0.051 g, 83%) as a white solid,
using the same procedure in the preparation of 4a: mp 234−235; UV
(MeOH) λmax = 271.5 nm; 1H NMR (DMSO-d6) δ 8.47 (s, 1 H), 7.35
(br s, 2 H, D2O exchangeable), 5.85 (d, 1 H, J = 7.2 Hz), 5.53 (d, 1 H,
J = 6.4 Hz, D2O exchangeable), 5.34 (d, 1 H, J = 4.0 Hz, D2O
exchangeable), 4.57−4.61 (m, 1 H), 4.33−4.35 (m, 1 H), 3.40 (dd,
1 H, J = 4.4, 10.8 Hz), 2.80 (dd, 1 H, J = 2.8, 10.8 Hz), 2.41 (t, 2 H, J =
6.8 Hz), 1.49−1.55 (m, 2 H), 1.40−1.46 (m, 2 H), 0.91 (t, 3 H, J =
7.2 Hz); 13C NMR (DMSO-d6) δ 155.7, 149.9, 145.7, 140.4, 118.3,
13
(m, 2 H), 1.38−1.47 (m, 2 H), 0.91 (t, 3 H, J = 7.2 Hz); C NMR
(DMSO-d6) δ 153.9, 149.4, 145.6, 142.6, 140.5, 135.9, 135.4, 130.5,
126.6, 118.6, 94.7, 85.7, 81.5, 78.6, 72.1, 61.1, 42.2, 34.3, 29.8, 21.5,
17.9, 13.4; [α]25 −43.33 (c 0.18, MeOH); (ESI+) (M + H+) m/z
D
550.0764. Anal. Calcd for C22H24IN5O2S: C, 48.09; H, 4.40; N, 12.75;
S, 5.84. Found: C, 48.09; H, 4.21; N, 12.47; S, 6.01.
85.4, 81.3, 78.5, 72.2, 61.1, 34.3, 29.9, 21.5, 17.9, 13.4; [α]25 −28.36
(−)-(2R,3S,4S)-2-(6-Amino-2-((E)-hex-1-enyl)-9H-purin-9-yl)-
tetrahydrofuran-3,4-diol (4l). A mixture of 14 (0.096 g, 0.24 mmol),
tetrakis(triphenylphosphine)palladium (28 mg, 0.024 mmol), sodium
carbonate (76 mg, 0.72 mmol), and (E)-1-catecholboranylhexene
(67 mg, 0.33 mmol) in DMF and H2O (8:1, 5 mL) was stirred at
90 °C for 15 h. The reaction mixture was filtered over a Celite bed,
and the residue was evaporated to give the crude compound 16. To a
solution of 16 in THF (5 mL) was added 1 N HCl (5 mL), and the
mixture was stirred at room temperature for 15 h. The mixture was
neutralized with 1 N NaOH and then carefully evaporated under
reduced pressure. The crude residue was subjected to flash silica gel
column chromatography (CH2Cl2/MeOH = 20:1) to give 4l (48 mg,
63%) as a white solid: mp 165−167 °C; UV (MeOH) λmax = 293.0
nm; 1H NMR (CD3OD) δ 8.20 (s, 1 H), 6.99−7.06 (m, 1 H), 6.36 (tt,
1 H, J = 1.6, 15.6 Hz), 5.97 (d, 1 H, J = 6.4 Hz), 4.94 (dd, 1 H, J = 4.8,
6.0 Hz), 4.53 (dd, 1 H, J = 4.0, 9.6 Hz), 4.43−4.46 (m, 1 H), 3.99 (dd,
1 H, J = 2.0, 9.6 Hz), 2.25−2.31 (m, 2 H), 1.47−1.54 (m, 2 H), 1.36−
1.45 (m, 2 H), 0.95 (t, 3 H, J = 7.2 Hz); 13C NMR (CD3OD) δ 160.9,
156.9, 151.7, 141.6, 141.3, 130.7, 90.3, 76.4, 75.4, 72.4, 57.9, 33.3, 32.3,
23.4, 14.3; [α]25 −47.05 (c 0.12, MeOH); (ESI+) (M + H+) m/z
D
(c 0.20, MeOH); (ESI+) (M + H+) m/z 334.1336. Anal. Calcd for
C15H19N5O2S: C, 54.04; H, 5.74; N, 21.01; S, 9.62. Found: C, 54.44;
H, 5.89; N, 21.21; S, 9.41.
General Procedure for the Synthesis of 4h−k. To a solution
of diol 13 in EtOH (20 mL) were added Et3N (3 equiv) and 3-
halobenzylamine (1.5 equiv) at room temperature, and the mixture
was stirred at room temperature for 48 h. The solvent was evaporated
and the residue was purified by a flash silica gel column
chromatography (CH2Cl2/MeOH = 20:1) to give 4h−k as white
solids.
(−)-(2R,3S,4R)-2-(6-(3-Fluorobenzylamino)-2-(hex-1-ynyl)-
9H-purin-9-yl)tetrahydrothiophene-3,4-diol (4h). Yield, 79%;
mp 200−202 °C; UV (MeOH) λmax = 274.0 nm; 1H NMR
(DMSO-d6) δ 8.52 (s, 1 H), 8.45 (br s, 1 H, D2O exchangeable),
7.32−7.37 (m, 1 H), 7.12−7.17 (m, 2 H), 7.02−7.07 (m, 1 H), 5.87
(d, 1 H, J = 7.2 Hz), 5.54 (d, 1 H, J = 6.0 Hz, D2O exchangeable), 5.36
(d, 1 H, J = 4.4 Hz, D2O exchangeable), 4.68 (br s, 2 H), 4.58−4.62
(m, 1 H), 4.34−4.35 (m, 1 H), 3.41 (dd, 1 H, J = 4.4, 10.8 Hz), 2.80
(dd, 1 H, J = 2.8, 10.8 Hz), 2.40 (t, 2 H, J = 7.2 Hz), 1.49−1.55
(m, 2 H), 1.39−1.47 (m, 2 H), 0.91 (t, 3 H, J = 7.2 Hz); C NMR
320.1747; [α]25D −44.80 (c 0.12, MeOH). Anal. Calcd for
13
D
(DMSO-d6) δ 163.4, 160.9, 154.0, 149.4, 145.6, 143.0, 140.5, 130.2
C15H21N5O3: C, 56.41; H, 6.63; N, 21.90. Found: C, 56.78; H, 6.89;
N, 21.77.
(d), 123.1, 118.6, 113.6 (q), 85.7, 81.5, 78.6, 72.2, 61.1, 42.4, 34.4,
29.8, 21.5, 17.9, 13.4; [α]25 −48.91 (c 0.184, MeOH); (ESI+) (M +
(−)-(2R,3S,4R)-2-(6-Amino-2-(E)-hex-1-enyl)-9H-purine-9-yl)-
tetrahydrothiophene-3,4-diol (4m). Compound 15 (46 mg,
0.11 mmol) was converted to 4m (23 mg, 63%) as a white solid, accord-
ing to the same procedure used in the preparation of 4l: mp 209−210 °C;
UV (MeOH) λmax = 274.5 nm; 1H NMR (DMSO-d6) δ 8.37 (s, 1 H),
D
H+) m/z 442.1708. Anal. Calcd for C22H24FN5O2S: C, 59.85; H, 5.48;
N, 15.86; S, 7.26. Found: C, 59.84; H, 5.88; N, 15.47; S, 7.21.
(−)-(2R,3S,4R)-2-(6-(3-Chlorobenzylamino)-2-(hex-1-ynyl)-
9H-purin-9-yl)tetrahydrothiophene-3,4-diol (4i). Yield, 72%; mp
352
dx.doi.org/10.1021/jm201229j | J. Med. Chem. 2012, 55, 342−356

Journal of Medicinal Chemistry
Article
7.11 (brs, 2 H, NH2), 6.87−6.94 (m, 1 H), 6.29 (d, 1 H, J = 15.2 Hz),
5.89 (d, 1 H, J = 7.2 Hz), 5.52 (d, 1 H, OH, J = 6.0 Hz), 5.36 (d, 1 H,
OH, J = 4.0 Hz), 4.63−4.67 (m, 1 H), 4.37 (pseudo t, 1 H, J = 3.2
Hz), 3.42 (dd, 1 H, J = 4.4, 10.8 Hz), 2.80 (dd, 1 H, J = 2.8, 10.8 Hz),
2.20−2.26 (m, 2 H), 1.41−1.48 (m, 2 H), 1.29−1.38 (m, 2 H), 0.90 (t,
3 H, J = 7.2 Hz); 13C NMR (DMSO-d6) δ 158.2, 155.5, 150.6, 139.6,
Celite, and the volatiles were evaporated to give the crude com-
pound 23. To a solution of 23 in THF (3 mL) was added 1 N HCl
(3 mL), and the mixture was stirred at room temperature for 15 h. The
mixture was neutralized with 1 N NaOH solution and then carefully
evaporated under reduced pressure. The mixture was subjected
to flash silica gel column chromatography (CH2Cl2/MeOH = 20:1)
to give 4q (41 mg, 58%) as a white solid: mp 204−206 °C; UV
138.2, 130.4, 117.7, 78.5, 72.3, 61.0, 34.4, 31.4, 30.5, 21.7, 13.8; [α]25
D
1
−44.80 (c 0.12, MeOH); (ESI+) (M + H+) m/z 336.1499. Anal. Calcd
for C15H21N5O2S: C, 53.71; H, 6.31; N, 20.88; S, 9.56. Found: C,
53.89; H, 6.71; N, 20.49; S, 9.32.
(MeOH) λmax = 296.5 nm; H NMR (DMSO-d6) δ 8.08 (s, 1 H),
7.18 (brs, 2 H, D2O exchangeable), 6.84−6.92 (m, 1 H), 6.58 (tt, 1 H,
J = 1.6, 15.6 Hz), 5.91 (d, 1 H, J = 6.8 Hz), 5.36 (d, 1 H, J = 6.8 Hz,
D2O exchangeable), 5.21 (d, 1 H, J = 4.0 Hz, D2O exchangeable),
5.05−5.06 (m, 1 H), 4.37 (dd, 1 H, J = 3.6, 9.6 Hz), 4.28 (d, 1 H, J =
3.6 Hz), 3.82 (dd, 1 H, J = 1.2, 9.6 Hz); 2.28−2.34 (m, 2 H), 1.43−
1.50 (m, 2 H), 1.31−1.40 (m, 2 H), 0.91 (t, 3 H, J = 7.6 Hz); 13C
NMR (DMSO-d6) δ 155.4, 151.9, 150.2, 148.0, 140.8, 118.8, 116.6,
(−)-(2R,3S,4S)-2-(6-Amino-8-(hex-1-ynyl)-9H-purine-9-yl)-
tetrahydrofuran-3,4-diol (4n). Compound 19 (0.605 g, 0.19 mmol)
was dissolved in Et3N (10 mL) and DMF (10 mL). After the solu-
tion was purged with N2, (Ph3P)2PdCl2 (0.238 g, 0.34 mmol), CuI
(0.065 g, 0.34 mmol), and 1-hexyne (0.49 mL, 4.25 mmol) were
subsequently added dropwise. The mixture was stirred at room
temperature for 3 h. The volatiles were evaporated to give the crude
compound 21. To a solution of 21 in THF (10 mL) was added 1 N
HCl (10 mL), and the mixture was stirred at room temperature for
15 h. The mixture was neutralized with 1 N NaOH and then evapo-
rated under reduced pressure. The crude residue was subjected to flash
silica gel column chromatography (CH2Cl2/MeOH = 10:1) to give 4n
87.6, 74.1, 73.3, 70.5, 32.0, 30.3, 21.7, 13.7; [α]25 −24.76 (c 0.11,
D
MeOH); (ESI+) (M + H+) m/z 320.1717. Anal. Calcd for
C15H21N5O3: C, 56.41; H, 6.63; N, 21.93. Found: C, 56.32; H,
6.85; N, 21.89.
(−)-(2R,3S,4R)-2-(6-Amino-8-(hex-1-enyl)-9H-purine-9-yl)-
tetrahydrothiophene-3,4-diol (4r). Compound 20 (42 mg, 0.12 mmol)
was converted to 4r (25 mg, 65%) as a white solid, according to the
same procedure used in the preparation of 4q: mp 248−249 °C; UV
(MeOH) λmax = 297.5 nm; 1H NMR (CD3OD) δ 8.15 (s, 1 H), 6.94−
7.01 (m, 1 H), 6.60 (d, 1 H, J = 15.2 Hz), 6.15 (d, 1 H, J = 8.0 Hz),
5.27 (dd, 1 H, J = 3.6, 8.0 Hz), 4.48−4.50 (m, 1 H), 3.65 (dd, 1 H, J =
3.6, 11.6 Hz), 2.93 (dd, 1 H, J = 1.6, 11.6 Hz), 2.36−2.42 (m, 2 H),
1.53−1.58 (m, 2 H), 1.42−1.50 (m, 2 H), 0.98 (t, 3 H, J = 7.2 Hz);
13C NMR (CD3OD) δ 153.5, 152.9, 151.8, 151.2, 143.9, 120.1,
(0.334 g, 62%) as a white solid: mp 228−232 °C; UV (MeOH) λmax
=
1
291.5 nm; H NMR (CD3OD) δ 8.19 (s, 1 H), 6.14 (d, 1 H, J = 7.2
Hz), 5.32 (dd, 1 H, J = 4.8, 6.8 Hz), 4.57 (dd, 1 H, J = 3.2, 9.6 Hz),
4.43−4.45 (m, 1 H), 3.99 (dd, 1 H, J = 1.2, 9.6 Hz), 2.59 (t, 2 H, J =
6.8 Hz), 1.64−1.70 (m, 2 H), 1.52−1.58 (m, 2 H), 1.00 (t, 3 H, J =
7.2 Hz); 13C NMR (CD3OD) δ 156.9, 154.4, 150.6, 136.7, 120.3, 99.9,
91.1, 76.1, 75.1, 72.9, 70.8, 31.2, 23.1, 19.7, 13.9; [α]25 −42.75
D
117.4, 78.8, 74.4, 63.9, 36.3, 32.0, 32.1, 23.4, 14.3; [α]25 −71.56
(c 0.14, MeOH); (ESI+) (M + H+) m/z 318.1568. Anal. Calcd for
C15H19N5O3: C, 56.77; H, 6.03; N, 22.07. Found: C, 56.89; H, 6.23;
N, 22.47.
D
(c 0.10, MeOH); (ESI+) (M + H+) m/z 336.1494. Anal. Calcd for
C15H21N5O2S: C, 53.71; H, 6.31; N, 20.88; S, 9.56. Found: C, 53.56;
H, 6.33; N, 20.67; S, 9.45.
(−)-(2R,3S,4R)-2-(6-Amino-8-(hex-1-ynyl)-9H-purine-9-yl)-
tetrahydrothiophene-3,4-diol (4o). Compound 20 (69 mg, 0.19 mmol)
was converted to 4o (36 mg, 58%) as a white solid, according to the same
procedure used in the preparation of 4n: mp 234−235 °C; UV (MeOH)
λmax = 294.0 nm; 1H NMR (DMSO-d6) δ 8.16 (s, 1 H), 7.40 (brs, 2 H,
NH2), 6.03 (d, 1 H, J = 7.6 Hz), 5.44 (d, 1 H, OH, J = 6.4 Hz), 5.33
(d, 1 H, OH, J = 4.0 Hz), 5.24−5.29 (m, 1 H), 4.40 (dd, 1 H, J = 1.6,
3.2 Hz), 3.42 (dd, 1 H, J = 3.6, 11.2 Hz), 2.80 (dd, 1 H, J = 2.0, 11.2 Hz),
2.59 (t, 2 H, J = 6.8 Hz), 1.55−1.62 (m, 2 H), 1.44−1.51 (m, 2 H), 0.93
(t, 3 H, J = 7.2 Hz); 13C NMR (DMSO-d6) δ 155.7, 153.1, 149.1, 133.7,
Pharmacology. In Vitro Assays. Cell Culture and Membrane
Preparation. CHO cells stably expressing either the recombinant hA1
or hA3AR and HEK-293 cells stably expressing the human A2AAR were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) and F12
(1:1) supplemented with 10% fetal bovine serum, 100 units/mL
penicillin, 100 μg/mL streptomycin, and 2 μmol/mL glutamine. In
addition, we added 800 μg/mL Geneticin to the hA2AAR medium and
500 μg/mL hygromycin B to the hA1AR, hA2BAR, and hA3AR media.
After harvesting the cells, we centrifuged them at 250g for 5 min at
4 °C. The pellet was resuspended in 50 mM Tris-HCl buffer (pH 7.5),
containing 10 mM MgCl2. The suspension was homogenized with an
electric homogenizer for 10 s and was then recentrifuged at 20000g
for 30 min at 4 °C. The resultant pellet was homogenized again,
resuspended in the buffer mentioned above in the presence of 3 U/mL
adenosine deaminase, finally 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).24
Radioligand Binding Assay. Radioligand binding assays with A1,
A2A, and A3ARs were performed according to the procedures described
previously.25−27 Briefly, for binding to human A1 receptors, [3H]PIA
(1 nM) was incubated with membranes (40 μg/tube) from CHO cells
stably expressing human A1 receptors at 25 °C for 60 min in 50 mM
Tris·HCl buffer (pH 7.4; MgCl2, 10 mM) in a total assay volume of
200 μL. Nonspecific binding was determined using 10 μM 25. For
human A2A receptor binding, membranes (20 μg/tube) from HEK-293
cells stably expressing human A2AARs were incubated with 15 nM
[3H]26 at 25 °C for 60 min in 200 μL of 50 mM Tris-HCl, pH 7.4,
containing 10 mM MgCl2. Reaction was terminated by filtration with
GF/B filters. For competitive binding assay to human A3ARs, each
tube contained 100 μL suspension of membranes (20 μg of protein)
from CHO cells stably expressing the human A3AR, 50 μL of [125I]27
(0.5 nM), and 50 μL of increasing concentrations of the nucleoside
derivative in Tris-HCl buffer (50 mM, pH 7.4) containing 10 mM
MgCl2 and 1 mM EDTA. Nonspecific binding was determined using
10 μM 25 in the buffer. The mixtures were incubated at 25 °C for 60 min.
Binding reactions were terminated by filtration through Whatman
118.9, 98.0, 76.5, 72.3, 70.7, 63.2, 35.5, 29.6, 21.4, 18.3, 13.4; [α]25
D
−112.2 (c 0.14, MeOH); (ESI+) (M + H+) m/z 334.1339. Anal. Calcd
for C15H19N5O2S: C, 54.04; H, 5.74; N, 21.01; S, 9.62. Found: C, 54.43;
H, 5.88; N, 19.94; S, 9.31.
(−)-(2R,3S,4S)-2-(6-Amino-8-hexyl-9H-purine-9-yl)-
tetrahydrofuran-3,4-diol (4p). A mixture of 4n (0.116 g, 0.36
mmol), absolute ethanol (20 mL), and 10% palladium on carbon (20
mg) was hydrogenated on a Parr apparatus at 40 psi for 15 h. The
mixture was filtered, and the solvent was evaporated. The mixture was
subjected to flash silica gel column chromatography (CH2Cl2/MeOH =
20:1) to give 4p (81 mg, 69%) as a white solid: mp 228−229 °C;
UV (MeOH) λmax = 259.5 nm; 1H NMR (DMSO-d6) δ 8.14 (s, 1 H),
7.41 (brs, 2 H, D2O exchangeable), 5.76 (d, 1 H, J = 6.8 Hz), 5.37
(brs, 1 H, D2O exchangeable), 5.25 (brs, 1 H, D2O exchangeable),
5.13−5.15 (m, 1 H), 4.40 (dd, 1 H, J = 3.2, 9.2 Hz), 4.28 (pseudo t,
1 H, J = 3.2 Hz), 3.83 (d, 1 H, J = 9.2 Hz), 2.82−2.87 (m, 2 H), 1.69−
1.77 (m, 2 H), 1.32−1.39 (m, 2 H), 1.26−1.31 (m, 4 H), 0.85−0.88
(m, 3 H); 13C NMR (DMSO-d6) δ 154.3, 152.9, 150.5, 150.1, 118.1,
88.3, 74.3, 73.2, 70.7, 30.9, 28.4, 27.6, 27.4, 22.0, 13.9; [α]25 −36.50
D
(c 0.13, MeOH); (ESI+) (M + H+) m/z 322.1883. Anal. Calcd for
C15H23N5O3: C, 56.06; H, 7.21; N, 21.79. Found: C, 56.45; H, 7.22;
N, 21.77.
(−)-(2R,3S,4S)-2-(6-Amino-8-(hex-1-enyl)-9H-purine-9-yl)-
tetrahydrofuran-3,4-diol (4q). A mixture of 19 (78.8 mg, 0.22 mmol),
tetrakis(triphenylphosphine)palladium(0) (26 mg, 0.022 mmol),
sodium carbonate (70 mg, 0.66 mmol), and (E)-1-catecholboranyl-
hexene (134 mg, 0.66 mmol) in DMF and H2O (8: 1, 5 mL) was
stirred for 15 h at 90 °C. The reaction mixture was filtered by a bed of
353
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Journal of Medicinal Chemistry
Article
GF/B filters under reduced pressure using a MT-24 cell harvester
(Brandell, Gaithersburg, MD, U.S.). Filters were washed three times
with 9 mL of ice-cold buffer. Radioactivity was determined in a
Beckman 5500B γ-counter.
For binding at all three subtypes, Ki values are expressed as the
mean SEM, n = 3−5 (outliers eliminated), and normalized against a
nonspecific binder, 10 μM 25. Alternatively, for weak binding a
percent inhibition of specific radioligand binding at 10 μM, relative to
inhibition by 10 μM 25 assigned as 100%, is given.
cAMP Accumulation Assay. Intracellular cAMP levels were
measured with a competitive protein binding method.28 CHO cells
that expressed the recombinant hA2AAR, hA2BAR, or hA3AR were
harvested by trypsinization. After centrifugation and resuspension in
medium, cells were planted in 24-well plates in 1.0 mL of medium.
After 24 h, the medium was removed and cells were washed three
times with 1 mL of DMEM containing 50 mM HEPES, pH 7.4. Cells
were then treated with the test agonist in the presence of rolipram
(10 μM) and adenosine deaminase (3 units/mL). For the hA2AAR and
the hA2BAR, incubation with agonist was performed for 30 min. For
the hA3AR, after 30 min forskolin (10 μM) was added to the medium,
and incubation was continued for an additional 15 min. The reaction
was terminated by removing the supernatant, and cells were lysed
upon the addition of 200 μL of 0.1 M ice-cold HCl. The cell lysate was
resuspended and stored at −20 °C. For determination of cAMP
production, 100 μL of the HCl solution was used in the Sigma Direct
cAMP enzyme immunoassay following the instructions provided with
the kit. The results were interpreted using a Bio-Tek Instruments
ELx808 Ultra microplate reader at 405 nm.
Data Analysis. Binding and functional parameters were calculated
using the Prism 5.0 software (GraphPAD, San Diego, CA, U.S.). IC50
values obtained from competition curves were converted to Ki values
using the Cheng−Prusoff equation.29 Data were expressed as the mean
standard error.
In Vivo Assay of Anti-Inflammatory Effect. Animals. Male
Sprague−Dawley (SD) rats (140−160 g, 5 weeks old) were purchased
from Central Laboratory Animal Inc. (Seoul, Korea). Animals were
housed under standard laboratory conditions with free access to food
and water. The temperature was thermostatically regulated to 22 2 °C,
and a 12 h light/dark schedule was maintained. Prior to their use, they
were allowed 1 week for acclimatization within the work area
environment. All animal experiments were carried out in accordance
with Institutional Animal Care and Use Committee Guidelines of
Seoul National University (SNU-201110-4).
Carrageenan-Induced Paw Edema. Carrageenan-induced hind
paw edema model in rats was used for the assessment of anti-
inflammatory activity.30 Test compounds 4g (20 mg/kg) and
indomethacin (20 mg/kg) were administered by an intraperitoneal
injection dissolved in 5% Cremophor and 5% ethanol in PBS, and
solvent alone was served as a vehicle control. Thirty minutes after the
administration of 4g, vehicle, or indomethacin, paw edema was
induced by subplantar injection of 0.1 mL of 1% freshly prepared
carrageenan suspension in normal saline into the right hind paw of
each rat. The left hind paw was injected with 0.1 mL of normal saline.
The paw volume was measured before (0 h) and at intervals of 0.5, 1,
2, 4, 6, and 24 h after carrageenan injection using a plethysmometer
(Ugo Basile, Comerio, Italy).
and partial protein flexibility. The region of 9 Å around the
cocrystallized ligand was defined as the binding site. The side chains
of the eight residues (i.e., Thr88, Phe168, Glu169, Trp246, Leu249,
Asn253, Ser277, and His278), which are important for ligand
binding, were set to be flexible with “crystal mode”. GoldScore
scoring function was used, and other parameters were set as default
except the number of GA runs, set as 30. The Fast Connolly surface
of the receptor and the van der Waals surface of each ligand were
generated by MOLCAD in SYBYL. All computational calculations
were undertaken on Intel Xeon Quad-core 2.5 GHz workstation with
Linux Cent OS, release 5.5.
ASSOCIATED CONTENT
* Supporting Information
■
S
Scheme 1S for the mechanism of lithiation mediated stannyl
transfer for the formation of 7. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 82-2-3277-3466. Fax: 82-2-3277-2851. E-mail:
lakjeong@ewha.ac.kr.
■
ACKNOWLEDGMENTS
■
This work was supported by the grants from Basic Science Research
(Grant 2008-314-E00304), the National Core Research Center
(Grant 2011-0006244), the World Class University (Grant R31-
2008-000-10010-0), National Leading Research Lab Program
(Grant 2011-0028885), and Brain Research Center of the 21st
Century Frontier Research (Grant 2011K000289) from National
Research Foundation (NRF), Korea, and the Intramural Research
Program of NIDDK, NIH, Bethesda, MD, U.S.
ABBREVIATIONS USED
■
AR, adenosine receptor; Cl-IB-MECA, 2-chloro-N6-(3-iodoben-
zyl)-5′-N-methylcarbamoyladenosine; thio-Cl-IB-MECA, 2-
chloro-N6-(3-iodobenzyl)-5′-N-methylcarbamoyl-4′-thioadeno-
sine; TMSOTf, trimethylsilyl trifluoromethanesulfonate; NOE,
nuclear Overhauser effect; R-PIA, (−)-N6-2-phenylisopropylade-
nosine; I-AB-MECA, N6-(3-iodo-4-aminobenzyl)-5′-N-methylcar-
boxamidoadenosine; NECA, 5′-N-ethylcarboxamidoadenosine;
HMDS, hexamethyldisilazane; BSA, N,O-bis(trimethyl-
silyl)acetamide; DMEM, Dulbecco’s modified Eagle’s medium;
CHO, Chinese hamster ovary; HEK, human embryonic kidney;
cAMP, cyclic adenosine 5′-monophosphate
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