Synthesis and anti-renal fibrosis activity of conformationally locked truncated 2-hexynyl-N6-substituted-(N)-methanocarba-nucleosides as A3 adenosine receptor antagonists and partial agonists

Article abstract of DOI:10.1021/jm4015313

Truncated N⁶-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4′-thioadenosines. Hydrophobic N⁶ and/or C2 substituents were tolerated in A₃AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA _2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N ⁶-cycloalkyl group (4d) showed excellent binding affinity at the hA₃AR and was better than an unsubstituted free amino group (4a). A₃AR affinities of 3-halobenzylamine derivatives 4f-4i did not differ significantly, with K_i values of 7.8-16.0 nM. N⁶-Methyl derivative 4b (K_i = 4.9 nM) was a highly selective, low efficacy partial A₃AR agonist. All compounds were screened for renoprotective effects in human TGF-β1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-β1-induced collagen I upregulation, and their A₃AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC₅₀ = 0.83 μM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.

Full text of DOI:10.1021/jm4015313

Article
pubs.acs.org/jmc
Open Access on 01/24/2015
Synthesis and Anti-Renal Fibrosis Activity of Conformationally
Locked Truncated 2‑Hexynyl‑N6‑Substituted‑(N)‑Methanocarba-
nucleosides as A3 Adenosine Receptor Antagonists and Partial
Agonists
Akshata Nayak,†,‡ Girish Chandra,‡ Inah Hwang,‡ Kyunglim Kim,‡ Xiyan Hou,‡ Hea Ok Kim,‡
Pramod K. Sahu,‡ Kuldeep K. Roy,‡ Jakyung Yoo,‡ Yoonji Lee,‡ Minghua Cui,‡ Sun Choi,‡
Steven M. Moss,§ Khai Phan,§ Zhan-Guo Gao,§ Hunjoo Ha,‡ Kenneth A. Jacobson,§
and Lak Shin Jeong*,†,‡
†Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea
‡College of Pharmacy, Graduate School of Pharmaceutical Sciences and Global Top 5 Research Program, Ewha Womans University,
Seoul 120-750, Korea
§Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, and Digestive and Kidney
Disease, National Institutes of Health, Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: Truncated N6-substituted-(N)-methanocarba-adenosine deriva-
tives with 2-hexynyl substitution were synthesized to examine parallels with
corresponding 4′-thioadenosines. Hydrophobic N6 and/or C2 substituents were
tolerated in A3AR binding, but only an unsubstituted 6-amino group with a C2-
hexynyl group promoted high hA2AAR affinity. A small hydrophobic alkyl (4b and
4c) or N6-cycloalkyl group (4d) showed excellent binding affinity at the hA3AR
and was better than an unsubstituted free amino group (4a). A3AR affinities of 3-
halobenzylamine derivatives 4f−4i did not differ significantly, with Ki values of
7.8−16.0 nM. N6-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low
efficacy partial A3AR agonist. All compounds were screened for renoprotective effects in human TGF-β1-stimulated mProx
tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-β1-induced collagen I upregulation, and their
A3AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC50 = 0.83 μM), indicating its potential as
a good therapeutic candidate for treating renal fibrosis.
inhibitors7,8 is one of a few therapeutic options for the
INTRODUCTION
■
treatment of CKD. However, the efficacy of RAAS inhibitors is
Extracellular adenosine acts as a signaling molecule with a
limited;9 it is, therefore, highly desirable to develop new
generally cytoprotective function in the body. Adenosine
therapeutic agents to improve the prognosis of CKD patients.
mediates cell signaling through binding to four known subtypes
(A1, A2A, A2B, and A3) of adenosine receptors (ARs).1−4 A1, A2A,
Extracellular adenosine in the kidney dramatically increases in
response to renal hypoxia and ischemia, and increased
and A3ARs are activated by low levels of adenosine (EC50
0.01−1.0 μM) similar to physiological levels of adenosine,
whereas A2BAR is activated by high levels of adenosine (EC50
=
adenosine has been reported to be associated with CKD.10
ARs were upregulated in unilateral ureteral obstructed rat
kidneys, which is a well-characterized model of CKD,11 and
A3AR knockout mice were protected against ischemia- and
myoglobinuria-induced kidney failure.10 Therefore, A3AR
antagonists may become effective renoprotective agents for
the treatment of CKD.
=
24 μM).5 A1 and A3ARs are Gi-coupled G protein-coupled
receptors (GPCRs), and A2A and A2BARs are Gs-coupled
GPCRs. Binding of adenosine to the ARs modulates second
messengers such as adenosine 3′,5′-cyclic phosphate (cAMP),
inositol triphosphate (IP3), and diacylglycerol (DAG).1−5 For
example, the Gi-coupled A3AR inhibits adenylate cyclase (AC),
resulting in cAMP down-regulation, while it stimulates
phospholipase C (PLC), which increases the levels of IP3 and
DAG. Therefore, ARs have been attractive targets for the
development of new therapeutic agents related to cell signaling.
Chronic kidney disease (CKD) is characterized by kidney
fibrosis and is becoming a major health problem worldwide,6
and the use of renin−angiotensin−aldosterone system (RAAS)
Adenosine as a natural ligand has served as a good lead for
the development of new AR ligands.5 Extensive modifications
on the N6 and/or 4′-CH2OH of adenosine have been explored,
giving several potent and selective A3AR agonists12,13 such as
N6-(3-iodobenzyl)-5′-N-methylcarbamoyladenosine (IB-
Received: October 2, 2013
Published: January 24, 2014
© 2014 American Chemical Society
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MECA),14 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarbamoyl-
adenosine (Cl-IB-MECA),15 N6-(3-iodobenzyl)-5′-N-methyl-
carbamoyl-4′-thioadenosine (thio-IB-MECA),16 2-chloro-N6-
(3-iodobenzyl)-5′-N-methylcarbamoyl-4′-thioadenosine (thio-
Cl-IB-MECA),17 and 3′-amino-N6-{5-chloro-2-(3-methyl-
isoxazol-5-ylmethoxy)benzyl}-5′-N-methylcarbamoyladenosine
(CP-608039).18 These compounds contain the potency- and
efficacy-enhancing 5′-methyluronamide moiety and the N6-
hydrophobic moiety. Also, AR agonists that combined N6-alkyl
and 2-alkynyl substitutions proved useful in the identification of
A3 or A2B AR agonists with various selectivity profiles,
depending on the type of 2-alkynyl substitution.19 On the
other hand, the truncated nucleosides where the 5′-
methyluronamide of the A3AR agonists was deleted were
converted into potent and selective A3AR antagonists, because
there was no 5′-uronamide, which serves as the hydrogen
bonding donor required for receptor activation.20 Among these,
compound 1 showed potent antiglaucoma21 activity (Chart 1).
observed. For the synthesis of the target nucleoside 4, copper-
catalyzed23 and palladium-catalyzed24 cross-coupling reactions
were employed as key steps for functionalization of the C2-
position of 6-chloropurine nucleosides. Herein, we report the
synthesis of truncated C2-hexynyl-N6-substituted-(N)-metha-
nocarba-nucleosides 4 as potent and selective A3AR antagonists
and their renoprotective effects using TGF-β1-stimulated
mProx cells, a cell culture model for kidney fibrosis.25
RESULTS AND DISCUSSION
■
Chemistry. The desired C2-hexynyl-methanocarba-adeno-
sine derivatives 4a−4i were synthesized from our known
cyclopentenone intermediate 526 using a palladium-catalyzed
cross-coupling reaction as a key step (Scheme 1).
Scheme 1. Synthesis of Truncated (N)-Methanocarba-
a
Nucleosides
Chart 1. Design Strategy for Truncated (N)-Methanocarba-
a
Nucleosides in This Study
a
Ki values (nM) or % inhibition at 10 μM in binding to human A1,
A2A, and A3 adenosine receptors.
a
Reagents and conditions: (a) NaBH4, CeCl3−7H2O, methanol, 0 °C,
Introduction of the 2-hexynyl group on the C2-position of 1
but no substitution on the N6-position converted 1 into dually
acting A2AAR agonist and A3AR antagonist 2.22 Molecular
modeling and empirical structure activity studies in both the
ribose and the 4′-thioribose series indicated that the C2 binding
sites of A2AAR and A3AR were spacious enough to
accommodate the bulky substituent.
2 h; (b) Et2Zn, CH2I2, CH2Cl2, rt, 5 h; (c) 2-iodo-6-chloropurine,
Ph3P, DIAD, THF, rt, 18 h; (d) 1-hexyne, (Ph3P)4Pd, Cs2CO3, CuI,
DMF, 50 °C, 6 h; (e) 2 N HCl/THF (1/1), 40 °C, 18 h; (f) R−NH2,
Et3N, ethanol, 90 °C, 18 h.
The cyclopentenone derivative 5 was converted to the
glycosyl donor 7 according to the reported procedure27
developed by our laboratory. Direct condensation of 7 with
6-chloro-2-iodopurine28 under the standard Mitsunobu con-
ditions in THF afforded the β-anomer 8 in 67% yield, similar to
a literature report.29 The anomeric β-configuration of 8 was
readily assigned by the diagnostic coupling constants typical of
the boat conformation of the bicyclo[3.1.0]hexane system,
which has been extensively confirmed by X-ray crystallography
Truncated (N)-methanocarba-nucleosides 320a were also
reported to be selective and potent A3AR antagonists,
indicating that compound 3 can also serve as a good template
for the development of A3AR ligands. Thus, we designed and
synthesized the truncated C2-hexynyl-(N)-methanocarba-nu-
cleosides 4, which hybridize the structure of C2-hexynyl
derivative 2 with that of (N)-methanocarba-nucleoside 3 to
determine if similar biological trends between 2 and 4 were
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and NMR analysis.30 The coupling constants of the JH1′,H2′ and
JH1′,H5′ should be zero, because both H1−C−C−H2 and H1−
C−C−H5 dihedral angles with trans relationships are close to
90°,30 indicating that 1′-H of 8 should appear as a singlet.
Indeed, 1H NMR of 8 showed that 1′-H appeared as a singlet at
5.03 ppm, confirming the structure of 8. Sonogashira31
coupling of 8 with 1-hexyne in the presence of palladium
catalyst yielded the C2-hexynyl derivative 9 (56%). Treatment
of 9 with 2 N HCl gave the 6-chloro derivative 10. Substitution
of the 6-position of 10 with ammonia and various primary
alkyl-, cycloalkyl-, and arylalkyl-amines afforded the final
nucleosides 4a−4i.
radioligand binding to the hA1AR and hA2AAR was determined
at 10 μM. Nonspecific binding was defined using 10 μM of 5′-
N-ethylcarboxamidoadenosine (NECA, 16).
Because binding affinity of similar (N)-methanocarba
compounds was reported to be very weak or absent at the
hA2BAR subtype,33 we did not include this receptor in the
radioligand binding assays. To confirm that activity of the
present chemical series is weak at the A2BAR, we performed a
functional assay in CHO cells expressing the hA2BAR.
Compound 4b at 10 μM produced only 15.7 12.6% of the
activation of cAMP production seen with full agonist 16.
As shown in Table 1, a variety of N6-alkyl, cycloalkyl, and
arylalkyl substituents in truncated (N)-methanocarba-nucleo-
side derivatives have produced nanomolar binding affinity at
the hA3AR subtype, indicating that bulky C2 and N6
substituents could be tolerable in the binding site of A3AR.
However, a hydrophobic substituent at the N6-position reduced
the binding affinity greatly at the hA2A AR subtype in the
presence of a hydrophobic C2-hexynyl group, and only an
unsubstituted 6-amino group showed good binding affinity (Ki
= 100 nM) at the hA2AAR, indicating that the N6 binding site of
hA2AAR is small. This trend is similar to that of truncated 2-
hexynyl-4′-thioadenosine (2),22 but truncated carbanucleoside
derivative 4a was 14-fold less potent than truncated 4′-
thioadenosine derivative 2. This result may be due to the fixed
conformation of (N)-methanocarba-nucleosides unlike the
flexible conformation of 4′-thioadenosine derivatives, hindering
them from forming a favorable hydrophobic interaction in the
binding site of A2AAR. However, all compounds showed very
weak binding affinity at the hA1AR, suggesting that the binding
sites may not be large enough to accommodate the bulky C2
and/or N6 substituent. Among compounds tested, 4b (R =
CH3) exhibited the highest binding affinity (Ki = 4.9 nM) at the
hA3AR subtype with high selectivity for the hA1 and hA2AARs.
The primary amine-substituted N6-alkyl- and N6-cycloalkyl-
derivatives (4b−4e) generally exhibited better binding affinity
at the hA3AR than the free amino derivative 4a, except
cyclopentyl derivative 4e. The order of compounds showing
high binding affinity at the hA3AR is as follows: 4b (R = CH3,
Ki = 4.6 nM) > 4c (R = ethyl, Ki = 6.7 nM) > 4d (R =
cyclopropyl, Ki = 9.2 nM) > 4a (R = H, Ki = 16.2 nM). The
binding affinities of 3-halobenzylamine derivatives 4f−4i at the
hA3AR did not differ significantly, with Ki values of 7.8−16.0
nM. The binding affinity at the hA3AR in this series decreased
in the following order: 3-Cl derivative 4h, 3-Br derivative 4g >
3-I derivative 4f > 3-F derivative 4i. All synthesized compounds
4a−4i have also produced nanomolar binding affinity at the
rA3AR, but they showed weaker binding affinity than that at the
hA3AR. The N6-alkyl derivatives 4b and 4c exhibited lower
binding affinity at the rA3AR than the free amino derivative 4a,
the N6-cycloalkyl derivatives 4d and 4e, and the 3-halobenzyl-
amine derivatives 4f−4i, which showed similar binding affinities
at the rA3AR, with Ki values in the range of 10.7−65 nM. The
3-chlorobenzyl derivative 4h exhibited the highest binding
affinity (Ki = 10.7 nM) at the rA3AR, unlike the N6-methyl
derivative 4b showing the highest affinity (Ki = 4.9 nM) at the
hA3AR.
The target nucleosides were also synthesized using a
lithiation-mediated stannyl transfer reaction28a and a copper-
catalyzed cross-coupling reaction23 as key steps for function-
alization of the C2-position (Scheme 2). The glycosyl donor 7
Scheme 2. Alternative Synthesis of Truncated (N)-
a
Methanocarba-Nucleosides
a
Reagents and conditions: (a) 6-chloropurine, Ph3P, DIAD, THF, rt,
18 h; (b) LiTMP, Bu3SnCl, THF, −78 °C, 5 h; (c) 1-iodohexyne, CuI,
DMF, 50 °C, 16 h.
was condensed with 6-chloropurine under the same conditions
used in Scheme 1 to give 6-chloropurine derivative 11.
Treatment of 11 with LiTMP followed by reacting the resulting
anion with tri-n-butyltin chloride afforded the C2-stannyl
derivative 12 exclusively.28a Copper-catalyzed coupling23 of 12
with 1-iodohexyne yielded the 2-hexynyl derivative 9, which
was converted to the same final nucleosides 4a−4i according
the same procedure used in Scheme 1.
Binding Affinity. 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, RBL-2H3 basophilic leukemia cells
expressing rat (r) A3AR, or human embryonic kidney (HEK)-
293 cells expressing the hA2AAR.32 Binding at the hA3AR or
rA3AR in this study was carried out using [125I]N6-(3-iodo-4-
aminobenzyl)-5′-N-methylcarboxamidoadenosine (I-AB-
MECA, 13) as a radioligand. Binding at the hA1AR using
[3H] (-)-N6-2-phenylisopropyl adenosine (R-PIA, 14) or
hA2AAR using [3H]CGS21680 (2-[p-(2-carboxyethyl)phenyl-
ethylamino]-5′-N-ethylcarboxamidoadenosine, 15) was carried
out. In cases of weak binding, the percent inhibition of
In a cAMP functional assay34 at the hA3AR expressed in
CHO cells, the most potent compound 4b behaved as a partial
agonist, in contrast to full antagonists 2 and 3 (Figure 1).
Compound 4b at 10 μM displayed an EC50 of 45.8 nM and a
maximal stimulation of cAMP formation of 29.1 5.0% relative
to the full agonist 16 (= 100%). Similarly, other compounds
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Table 1. Binding Affinities and Anti-Renal Fibrosis Activity of Truncated 2-Hexynyl-N6-Substituted Derivatives 4a−4i and
Reference Nucleosides 2 and 3 at hARs and rA3AR
a
Ki (nM) or % inhibition at 10 μM
d
compd no.
R
hA1AR
hA2AAR
hA3AR
rA3AR
IC50 (μM)
b
e
e
2
39 10%
3040 610
29% 6%
14% 4%
31% 7%
2170 510
1580 240
48% 5%
38% 6%
19% 8%
21% 4%
7.19 0.6
1080 310
100 10
11.8 1.3
1.44 0.6
16.2 6.7
4.90 1.30
6.70 1.80
9.20 0.40
160 50
ND
ND
c
e
3
ND
18.6
6.12
0.83
0.84
11.8
>50
7.88
10.4
2.87
3.17
4a
4b
4c
4d
4e
4f
H
65 18
231 81
176 47
39 19
58 39
26 22
59 37
10.7 1.6
43 30
methyl
7490 590
2860 1060
2200 660
1760 410
2530 170
3150 170
3310 1220
27% 5%
ethyl
cyclopropyl
cyclopentyl
3-iodobenzyl
3-bromobenzyl
3-chlorobenzyl
3-fluorobenzyl
12.0 6.0
8.60 4.80
7.80 1.70
16.0 10.0
4g
4h
4i
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) or rA3AR expressed endogenously in RBL-2H3 cells. Binding was carried out using 1 nM [3H]14,
10 nM [3H]15, or 0.5 nM [125I]13 as radioligands for A1, A2A, and A3ARs, respectively. Values expressed as a percentage in italics refer to percent
inhibition of specific radioligand binding at 10 μM for 3 − 5 duplicate determinations, with nonspecific binding defined using 10 μM 16. ref 22. ref
20a. Concentration to inhibit the TGF-β1-induced collagen I mRNA expression by 50%. Not determined.
b
c
d
e
Molecular Docking Study. The truncated C2-substituted
thio-ribose compound 2 (A2A Ki = 7.19 nM) exhibited excellent
binding affinity, and the methanocarba analogue 4a (A2A Ki =
100 nM) showed ≈14-fold less binding affinity at the hA2A AR.
In addition, the presence of the 3-iodobenzyl group at the N6-
position in 4f led to a substantial decrease in its binding affinity
at the hA2AAR with a Ki of 2530 nM. In view of the observed
variations in the hA2AAR binding affinities among these
compounds, molecular docking and binding free energy
calculations were carried out considering the X-ray structure
of the hA2AAR complexed with an agonist, 16 (PDB code
2YDV35). The common interactions among N6-unsubstituted
compounds 2 and 4a at the hA2AAR includes: (i) the adenine
ring stabilized through π−π stacking interaction with Phe168
(extracellular loop 2) and a H-bonding interaction with
Asn2536.55, (ii) the exocyclic 6-amino group H-bonded with
Asn2536.55 and Glu169, and (iii) the projection of C2-hexynyl
group toward the extracellular side exhibiting hydrophobic
Figure 1. Effect of compound 4b on forskolin-induced stimulation of
cAMP production at the hA3AR expressed in CHO cells, compared to
16 as reference full agonist (= 100%). A representative curve from
three determinations is shown.
proved to be partial agonists of the hA3AR (% activation
relative to 16, triplicate determination): 4c, 15.5 6.7; 4d, 19.8
4.6; 4e, 27.1 14.6; 4i, 18.9 7.5. Compounds 4f, 4g, and
4h induced <5% of the activation seen with 16 and were
therefore antagonists.
Renoprotective Effects. All synthesized compounds were
tested for an antifibrotic effect in murine proximal (mProx)
cells, a cell line of mouse proximal tubular epithelial cells.25 As
shown in Table 1, most of the tested compounds strongly
inhibited transforming growth factor (TGF)-β1-induced
collagen I upregulation. Compound 4b showed the most
potent inhibitory activity (IC50 = 0.83 μM) against TGF-β1-
induced collagen I mRNA expression (Figure 2). The binding
affinity at the A3AR was almost proportional to the antifibrotic
activity, which indicates that the small N6-hydrophobic
substituent is also favored for renoprotective effects.
interaction with Phe168, Ile662.55, Leu2677.32, Met2707.35
,
Ile2747.39, and Tyr2717.36 residues (Figure 3).
In contrast, they exhibited different binding modes at the
ribose binding site formed by Val843.32, Leu853.33, Trp2466.48
,
Leu2496.51 and Ile2747.39, Ser2777.42, and His2787.43. The 2′-
and 3′-hydroxyl groups of 2 formed H-bonds with the two key
residues His2787.43 and Ser2777.42, respectively (Figure 3A),
whereas 4a lost one of the key H-bond interactions with
Ser2777.42 (Figure 3B). This residue Ser277 is a key residue
reported to be important for hA2AAR agonistic activity and
potency using site-directed mutagenesis.36−38 It appears that a
decrease in the binding affinity of 4a at the hA2AAR could be
due to the loss of H-bonding with Ser2777.42 at the ribose
binding site. The loss of H-bonding with Ser2777.42 may
particularly be attributed to the methanocarba ring (4a), being
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Figure 2. Inhibition of TGF-β1-induced COL1A1 gene expression in mProx24 cells, a cell line of mouse proximal tubular epithelial cells, by 4b. Data
are mean SE of three experiments. *p < 0.05 vs TGF-β1-stimulated mProx24 cells: arelative increase in COL1A1 gene expression (1.0 is the effect
b
of 5 ng/mL TGF-β1), at the concentration of 4b in μM indicated in column 1.
Figure 3. Predicted binding modes of N6-unsubstituted nucleosides 2 (A) and 4a (B) in the hA2AAR agonist-bound crystal structure. Compounds 2
and 4a are depicted in ball-and-stick, with carbon atoms in magenta and purple, respectively. The key amino acid residues are shown as capped-stick,
with carbon atoms in white. The Connolly surface of the receptor was generated by MOLCAD with green color and z-clipped for visual convenience.
The hydrogen bonds are shown as black dashed lines, and the nonpolar hydrogen atoms are not displayed for clarity.
less flexible than the thio-ribose ring (2). Furthermore, the
calculated prime MM-GBSA binding free energies (ΔGbind) for
2 and 4a were −104.16 and −90.37 kcal/mol, respectively,
which are in good agreement with their observed binding
affinities at the hA2AAR. However, 4f with a bulky group at the
N6-position did not fit well at the binding site of the hA2A AR.
These results show that the H-bond interactions with both
Ser2777.42 and His2787.43 at the ribose binding site are
important for high affinity and potency, and the bulky group
at the N6-position is unfavorable toward high binding affinity at
the hA2AAR.
In addition, we also performed the molecular docking studies
of the analogues 2 and 4a to hA3AR (see Figure 1S in
Supporting Information). Because the X-ray crystal structure of
hA3AR is not available yet, the homology model available in the
Protein Data Bank (PDB code 1OEA) was used. The docking
results showed that the binding modes of the analogues in
hA3AR are flipped compared to those in hA2AAR. In hA3AR,
the bulky C2-hexynyl group positions toward the middle of the
trans-membrane region exhibited hydrophobic interactions.
However, in hA2AAR, there is limited space at the bottom of the
pocket, making the bulky hexynyl group face toward the
extracellular side. The NH2 group at N6-position forms the
hydrogen bonding with Asn6.55 in both hA2AAR (Asn253) and
hA3AR (Asn250). Interestingly, there is a relatively bigger space
near this region in hA3AR, whereas the NH2 group binds tightly
in the pocket of hA2AAR. It appears according to this docking
mode that this is why the N6-substituted derivatives (4b−4i)
maintained their binding affinity at the hA3AR, but not at the
hA2AAR.
CONCLUSIONS
■
The series of truncated N6-substituted-(N)-methanocarba-
adenosine derivatives, 4a−4i with 2-hexynyl group were
synthesized in order to examine if this class of nucleosides
behaves as the corresponding 4′-thioadenosine derivatives. The
functionalization at the C2-position of 6-chloropurine deriva-
tives was achieved using lithiation-mediated stannyl transfer
and copper- or palladium-catalyzed cross-coupling reactions. It
was revealed that all synthesized nucleosides showed very high
binding affinity at the hA3AR as well as at the rA3AR, as in the
case of the corresponding 4′-thioadenosine derivatives,
indicating that the hydrophobic N6 and/or C2 substituent
could be tolerable in the binding site of the A3AR. However,
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the anhydrous solvents used were redistilled over CaH, P2O5 or
sodium/benzophenone prior to the reaction.
only an unsubstituted 6-amino group in the presence of a bulky
C2-hexynyl group was associated with high binding affinity at
the hA2AAR (compound 4a). This trend is similar to that of the
corresponding 4′-thioadenosine derivatives, serving as dual
acting A2A and A3AR ligands. However, the binding affinity at
the hA2AAR of the truncated (N)-methanocarba-nucleoside 4a
is 14-fold less potent than the truncated 4′-thioadenosine
derivative 2. It is attributed to the loss of key hydrogen bonding
due to the rigid structure, which was confirmed by a hA2AAR
molecular docking study.
The specific structure−activity relationship for this series of
conformationally constrained nucleosides might arise from the
molecule of lacking in the flexibility required for optimal
interaction in the binding site because of the rigidity of (N)-
methanocarba-nucleosides. From this study, N6-methyl deriv-
ative 4b was discovered as a preferred hA3AR ligand (low
efficacy partial agonist) with high selectivity, whereas 3-
chlorobenzyl derivative 4h was discovered as the most
potent/selective rA3AR ligand in this series.
6-Chloro-9-((3aR,3bR,4aS,5R,5aS)-hexahydro-2,2dimethyl-
bicyclo[3.1.0]hex-1(5)-eno[3,2-d] [1,3]dioxol-5-yl)-2-iodo-9H-purine
(8). To a stirred solution of 2-iodo-6-chloropurine (1.23 g, 4.4 mmol)
and triphenylphosphine (Ph3P) (1.90 g, 4.4 mmol) in anhydrous THF
(20 mL) was added diisopropyl azodicarboxylate (DIAD) (1.44 mL,
9.16 mmol) in THF (10 mL) under N2 at 0 °C, and the mixture was
stirred at the same temperature for 15 min. To this solution was added
a solution of compound 719 (0.5 g, 2.93 mmol) in THF (10 mL) at 0
°C, and the reaction mixture was stirred at room temperature for 16 h.
The reaction mixture was concentrated under reduced pressure, and
the crude residue was purified by flash silica gel column
chromatography (hexane: EtOAc = 3:1) to give 8 (0.85 g, 67%) as
a white solid: mp 94−96 °C; UV (MeOH) λmax 282 nm. MS (ESI):
[M + H]+ calcd for C14H15ClIN4O2, 432.9923; found, 432.9931;
[α]25D = −10.4 (c 0.2, MeOH); 1H NMR (CDCl3) δ 0.95−1.01 (m, 2
H), 1.26 (s, 3 H), 1.55 (s, 3 H), 1.63−1.68 (m, 1 H), 2.12−2.18 (m, 1
H), 4.65−4.68 (m, 1 H), 5.03 (s, 1 H), 5.35−5.38 (m, 1 H), 8.12 (s, 1
H); 13C NMR (CDCl3) δ 9.4, 24.4, 25.6, 26.1, 26.5, 61.5, 81.6, 89.1,
112.8, 116.9, 132.1, 143.9, 150.9, 152.1. Anal. (C14H14ClIN4O2) C, H,
N.
The nature of the N6 substituent in this chemical series
modulates the level of hA3AR agonist efficacy (ranging from
nearly 0% to 29% of full agonist). For these assays, we used a
CHO cell with a high level of stable expression of the hA3AR,
which would tend to amplify partial agonist action. Because
even these partial agonists have a relatively low efficacy, they
can be expected to behave similarly to full antagonists in some
pharmacological models, especially in cases of low receptor
expression.39
A3AR antagonist 1 was recently shown to inhibit unilateral
ureteral obstruction-induced renal fibrosis and collagen I
upregulation.40 This suggests that A3AR antagonists might be
useful therapeutically to block the development and attenuate
the progression of renal fibrosis. All of the compounds
synthesized here were screened for renoprotective activity.
Among compounds tested, 4b exhibited the most potent
inhibitory activity (IC50 = 0.83 μM) against TGF-β1-induced
collagen I upregulation. These findings indicate that this series
of truncated (N)-methanocarba-nucleoside derivatives acting as
partial agonists of low efficacy or as antagonists, which show
high binding affinity at the human A3AR, can serve as a good
lead for the development of antirenal fibrosis agents.
6-Chloro-2-(hex-1-ynyl)-9-((3aR,3bR,4aS,5R,5aS)-hexahydro-2,2-
dimethylbicyclo [3.1.0]hex-1(5)-eno[3,2-d][1,3]dioxol-5-yl)-9H-pu-
rine (9). To a stirred solution of 8 (0.30 g, 0.69 mmol) in anhydrous
DMF (10 mL) were added tetrakis(triphenylphosphine)palladium
((Ph3P)4Pd) (0.20 g, 0.17 mmol), copper iodide (0.016 g, 0.08
mmol), cesium carbonate (0.226 g, 0.69 mmol), and 1-hexyne (0.07
mL, 0.62 mmol) at room temperature and the reaction mixture was
stirred at 50 °C for 5 h. The reaction mixture was cooled to room
temperature, quenched with saturated NaHCO3 (5 mL) solution, and
diluted with EtOAc (10 mL). The organic layer was separated and
aqueous layer was further extracted with EtOAc (3 × 5 mL). The
combined organic layers were washed with brine (10 mL) and water
(10 mL), dried over anhydrous MgSO4, and filtered. The solvent was
evaporated under reduced pressure and the crude residue was purified
by flash silica gel column chromatography (hexane: EtOAc = 3:1) to
give 9 (0.15 g, 56%) as a white foam: mp 105−107 °C; UV (MeOH)
λmax 284 nm. MS (ESI+): [M + H]+ calcd for C20H24ClIN4O2,
1
387.1582; found, 387.1586; [α]25 = +2.5 (c 0.2, MeOH). H NMR
D
(CDCl3, 400 MHz) δ: 0.94−1.03 (m, 5 H), 1.25 (s, 3 H), 1.47−1.54
(m, 2 H), 1.55 (s, 3 H), 1.63−1.70 (m, 3 H), 2.12−2.17 (m, 1 H),
2.48−2.51 (t, 2 H, J = 7.2 Hz), 4.63−4.65 (d, 1 H, J = 5.1 Hz), 5.13 (s,
1 H), 5.35−5.38 (t, 1 H, J = 6.00 Hz), 8.13 (s, 1 H). 13C NMR
(CDCl3, 100 MHz) δ: 9.3, 13.7, 19.2, 22.3, 24.4, 25.3, 26.0, 26.5, 30.2,
60.7, 79.7, 81.4, 89.1, 90.9, 112.6, 130.8, 144.2, 146.3, 151.1, 151.3.
Anal. (C20H23ClIN4O2) C, H, N.
(1R,2R,3S,4R,5S)-4-(6-Chloro-2-(hex-1-ynyl)-9H-purin-9yl)bicyclo-
[3.1.0]hexane-2,3-diol (10). To a stirred ice-cooled solution of 9 (0.30
g, 0.77 mmol) in THF (3 mL) was added 2 N HCl (3 mL), and the
mixture was stirred at 40 °C for 16 h. The reaction mixture was
neutralized with saturated NaHCO3 (2 mL) solution and then diluted
with EtOAc (10 mL). The organic layer was separated, and the
aqueous layer was further extracted with EtOAc (2 × 5 mL). The
combined organic layers were washed with brine (10 mL), dried over
anhydrous MgSO4, filtered, and evaporated under reduced pressure.
The crude residue was purified by flash silica gel column
chromatography (hexane: EtOAc = 2:1) to give 10 (0.22 g, 73%) as
a white solid: mp 120−122 °C; UV (MeOH) λmax 285 nm. MS (ESI+):
EXPERIMENTAL SECTION
■
Chemical Synthesis. General Methods. 1H NMR spectra
(CDCl3, CD3OD, or DMSO-d6) were recorded on a Varian Unity
1
Invoa 400 MHz instrument. The H NMR data are reported as peak
multiplicities: s for singlet, d for doublet, dd for doublet of doublets, t
for triplet, q for quartet, brs for broad singlet, and m for multiplet.
Coupling constants are reported in hertz. 13C NMR spectra (CDCl3,
CD3OD, or DMSO-d6) were recorded on a Varian Unity Inova 100
MHz instrument. 19F NMR spectra (CDCl3, CD3OD) were recorded
on a 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. Melting points were determined on a Buchan B-540
instrument and are uncorrected. 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). Flash column chromatography was performed on
silica gel 60 (230−400 mesh, Merck). Reagents were purchased from
Aldrich Chemical Co. Solvents were obtained from local suppliers. All
[M + H]+ calcd for C17H20ClN4O2, 347.1269; found, 347.1274; [α]25
D
1
= +27.0 (c 0.2, MeOH). H NMR (CDCl3, 400 MHz) δ: 0.84−0.87
(m, 1 H), 0.94−0.97 (t, 3 H, J = 7.2 Hz), 1.29−1.32 (m, 1 H), 1.47−
1.53 (m, 2 H), 1.62−1.70 (m, 3 H), 2.10−2.14 (m, 1 H), 2.47−2.51 (t,
2 H, J = 7.2 Hz), 4.05−4.06 (d, 1 H, J = 6.0 Hz), 4.86−4.89 (t, 1 H, J =
6.0 Hz), 5.04 (s, 1 H), 8.20 (s, 1 H). 13C NMR (CDCl3, 100 MHz) δ:
7.8, 14.1, 19.6, 19.9, 22.7, 24.6, 30.6, 63.4, 72.3, 77.1, 79.9, 91.8, 131.3,
144.4, 146.5, 151.6, 151.7. Anal. (C17H19ClN4O2) C, H, N.
(1R,2R,3S,4R,5S)-4-(6-Amino-2-(hex-1-ynyl)-9H-purin-9-yl)-
bicyclo[3.1.0]hexane-2, 3-diol (4a). A solution of 10 (0.05 g, 0.42
mmol) in saturated NH3 in t-BuOH (5 mL) was stirred at 110 °C for
1349
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Journal of Medicinal Chemistry
Article
16 h. The reaction mixture was evaporated, and the residue was
purified by flash silica gel column chromatography (CH2Cl2: MeOH =
9: 1) to give 4a (0.103 g, 87%) as a white solid: mp 122−124 °C; UV
(MeOH) λmax 271 nm. MS (ESI+): [M + H]+ calcd for C17H22N5O2,
329.1796; found, 329.1797; [α]25D = +14.7 (c 1.75, MeOH). 1H NMR
(CD3OD, 400 MHz) δ: 0.76−0.78 (m, 1 H), 0.96−1.00 (t, 3 H, J =
7.2 Hz), 1.34−1.37 (m, 1 H), 1.50−1.70 (m, 5 H), 1.98−2.01 (m, 1
H), 2.45−2.48 (t, 2 H, J = 7.2 Hz), 3.86−3.88 (d, 1 H, J = 6.8 Hz),
4.66−4.69 (t, 1 H, J = 5.6 Hz), 4.83 (s, 1 H), 8.24 (s, 1 H). 13C NMR
(CD3OD) δ: 8.2, 14.1, 19.6, 19.7, 23.2, 24.7, 31.6, 64.0, 73.0, 77.4,
81.3, 88.6, 120.3, 141.4, 147.9, 157.2, 167.2. Anal. (C17H21N5O2) C, H,
N.
0.96−1.00 (t, 3 H, J = 7.2 Hz), 1.34−1.37 (m, 1 H), 1.50−1.58 (m, 2
H), 1.60−1.72 (m, 3 H), 1.96−2.01 (m, 1 H), 2.45−2.48 (t, 2 H, J =
7.2 Hz), 3.85−3.87 (d, 1 H, J = 6.4 Hz), 4.65−4.67 (t, 1 H, J = 5.6
Hz), 4.76 (brs, 2 H), 4.83 (s, 1 H), 7.07−7.11 (t, 1 H, J = 7.6 Hz),
7.39−7.41 (m, 1 H), 7.59−7.61 (m, 1 H), 7.80 (s, 1 H), 8.19 (s, 1 H).
13C NMR (CD3OD, 100 MHz) δ: 7.9, 14.0, 19.5, 19.6, 23.2, 24.6, 31.6,
44.4, 63.8, 73.1, 77.1, 81.7, 88.1, 94.9, 120.1, 128.2, 131.4, 137.4, 137.9,
140.7, 143.1, 147.9, 149.9, 155.7. Anal. (C24H26IN5O2) C, H, N.
(1R,2R,3S,4R,5S)-4-(6-(3-Bromobenzylamino)-2-(hex-1-ynyl)-9H-
purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (4g). Yield: 90%; white
solid; mp 124−126 °C; UV (MeOH) λmax 275 nm. MS (ESI+): [M
+ H]+ calcd for C24H27BrN5O2, 496.1343; found, 496.1350; [α]25
=
D
1
General Procedure for the Synthesis of 4b−4i. To a solution of 10
(1 equiv) in EtOH (10 mL) were added Et3N (3 equiv) and the
appropriate amine (1.5 equiv) at room temperature, and the mixture
was stirred at 90 °C for 18 h in a steel bomb. The reaction mixture was
evaporated and the residue was purified by flash silica gel column
chromatography (CH2Cl2/MeOH = 12:1) to give 4b−4i.
+21.2 (c 0.2, MeOH). H NMR (CD3OD, 400 MHz) δ: 0.75−0.77
(m, 1 H), 0.96−1.00 (t, 3 H, J = 7.2 Hz), 1.34−1.37 (m, 1 H), 1.50−
1.56 (m, 2 H), 1.60−1.71 (m, 3 H), 1.97−1.99 (m, 1 H), 2.45−2.48 (t,
2 H, J = 7.2 Hz), 3.85−3.87 (d, 1 H, J = 6.8 Hz), 4.65−4.68 (t, 1 H, J =
5.6 Hz), 4.80 (brs, 2 H), 4.83 (s, 1 H), 7.22−7.26 (m, 1 H), 7.36−7.41
(m, 2 H), 7.59 (s, 1 H), 8.19 (s, 1 H). 13C NMR (CD3OD, 100 MHz)
δ: 7.9, 14.0, 19.2, 19.6, 23.2, 24.6, 31.6, 44.5, 63.9, 73.1, 77.2, 81.6,
88.0, 120.1, 123.5, 127.6, 131.3, 131.4, 131.8, 140.7, 143.2, 148.0,
150.0, 155.8. Anal. (C24H26BrN5O2) C, H, N.
(1R,2R,3S,4R,5S)-4-(2-(Hex-1-ynyl)-6-(methylamino)-9H-purin-9-
bicyclo[3.1.0]hexane-2,3-diol (4b). Yield: 75%; white solid; mp 118−
120 °C; UV (MeOH) λmax 273 nm. MS (ESI+): [M + H]+ calcd for
C18H24N5O2, 342.1925; found, 342.1925; [α]25 = +35.5 (c 0.2,
(1R,2R,3S,4R,5S)-4-(6-(3-Chlorobenzylamino)-2-(hex-1-ynyl)-9H-
purin-9-yl)bicyclo[3.1.0] hexane-2,3-diol (4h). Yield: 90%; white
solid; mp 109−111 °C; UV (MeOH) λmax 274 nm. MS (ESI+): [M +
D
MeOH). 1H NMR (CD3OD, 400 MHz) δ: 0.73−0.79 (m, 1 H),
0.96−1.00 (t, 3 H, J = 7.2 Hz), 1.34−1.38 (m, 1 H), 1.49−1.71 (m, 5
H), 1.95−2.01 (m, 1 H), 2.45−2.49 (t, 2 H, J = 7.2 Hz), 3.11 (brs, 3
H), 3.84−3.86 (d, 1 H, J = 6.8 Hz), 4.64−4.67 (t, 1 H, J = 5.6 Hz),
4.82 (s, 1 H), 8.16 (s, 1 H). 13C NMR (CD3OD, 100 MHz) δ: 7.9,
13.9, 19.6, 19.6, 23.2, 24.6, 27.8, 31.6, 63.8, 73.0, 77.2, 81.6, 87.9,
120.2, 140.3, 148.1, 149.4, 156.6. Anal. (C18H23N5O2) C, H, N.
(1R,2R,3S,4R,5S)-4-(6-(ethylamino)-2-(hex-1-ynyl)-9H-purin-9-yl)-
bicyclo[3.1.0]hexane-2,3-diol (4c). Yield: 76%; white solid; mp 98−
100 °C; UV (MeOH) λmax 273 nm. MS (ESI+): [M + H]+ calcd for
H]+ calcd for C24H27ClN5O2, 452.1848; found, 452.1842; [α]25
=
D
1
+13.1 (c 0.2, MeOH). H NMR (CD3OD, 400 MHz) δ: 0.73−0.79
(m, 1 H), 0.90−1.00 (t, 3 H, J = 7.2 Hz), 1.34−1.37 (m, 1 H), 1.50−
1.72 (m, 5 H), 1.96−2.04 (m, 1 H), 2.44−2.48 (t, 2 H, J = 7.2 Hz),
3.85−3.87 (d, 1 H, J = 6.8 Hz), 4.65−4.68 (t, 1 H, J = 5.6 Hz), 4.80
(brs, 2 H), 4.83 (s, 1 H), 7.24−7.34 (m, 3 H), 7.42−7.43 (m, 1 H),
8.19 (s, 1 H). 13C NMR (CD3OD, 100 MHz) δ: 7.9, 14.0, 19.5, 19.6,
23.1, 24.6, 31.6, 44.6, 63.8, 73.0, 77.1, 81.7, 88.1, 120.5, 127.1, 128.3,
128.8, 131.1, 135.4, 140.7, 142.9, 147.9, 149.9, 155.8. Anal.
(C24H26ClN5O2) C, H, N.
C19H26N5O2, 356.2081; found, 356.2083; [α]25 = +8.5 (c 0.2,
D
MeOH); 1H NMR (CD3OD, 400 MHz) δ: 0.74−0.77 (m, 1 H),
0.96−1.00 (t, 3 H, J = 7.2 Hz), 1.27−1.31 (t, 3 H, J = 7.2 Hz), 1.34−
1.37 (m, 1 H), 1.50−1.70 (m, 5 H), 1.96−2.00 (m, 1 H), 2.45−2.50 (t,
2 H, J = 7.2 Hz), 3.62 (brs, 2 H), 3.84−3.85 (d, 1 H, J = 6.8 Hz),
4.64−4.67 (t, 1 H, J = 5.6 Hz), 4.81 (s, 1 H), 8.16 (s, 1 H). 13C NMR
(CD3OD, 100 MHz) δ: 7.9, 14.0, 15.1, 19.5, 19.2, 23.2, 24.6, 31.6,
36.6, 63.8, 73.1, 77.1, 81.6, 87.9, 119.9, 140.3, 148.1, 149.5, 155.9. Anal.
(C19H25N5O2) C, H, N.
(1R,2R,3S,4R,5S)-4-(6-(3-Fluorobenzylamino)-2-(hex-1-ynyl)-9H-
purin-9-yl)bicyclo[3.1.0] hexane-2,3-diol (4i). Yield: 70%; white solid;
mp 99−101 °C; UV (MeOH) λmax 273 nm. MS (ESI+): [M +
H]+ calcd for C24H27FN5O2, 436.2143; found, 436.2141; [α]25D = +2.5
1
(c 0.2, MeOH). H NMR (CD3OD, 400 MHz) δ: 0.73−0.79 (m, 1
H), 0.96−1.00 (t, 3 H, J = 7.2 Hz), 1.35−1.37 (m, 1 H), 1.49−1.71
(m, 5 H), 1.95−2.01 (m, 1 H), 2.44−2.47 (t, 2 H, J = 7.2 Hz), 3.85−
3.87 (d, 1 H, J = 6.4 Hz), 4.65−4.67 (t, 1 H, J = 5.6 Hz), 4.80 (brs, 2
H), 4.83 (s, 1 H), 6.94−6.99 (m, 1 H), 7.12−7.15 (m, 1 H), 7.20−7.22
(m, 1 H), 7.30−7.35 (m, 1 H), 8.18 (s, 1 H). 13C NMR (CD3OD, 100
MHz) δ: 7.9, 13.9, 19.5, 19.6, 23.1, 24.6, 31.6, 44.6, 63.8, 73.0, 77.1,
81.7, 88.0, 115.3, 115.5, 120.0, 124.5, 131.2, 131.3, 140.7, 143.5, 148.0,
150.0, 155.8. Anal. (C24H26FN5O2) C, H, N.
(1R,2R,3S,4R,5S)-4-(6-(Cyclopropylamino)-2-(hex-1-ynyl)-9H-
purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (4d). Yield: 68%; white
solid; mp 94−96 °C; UV (MeOH) λmax 275.0 nm. MS (ESI+): [M
+ H]+ calcd for C20H26N5O2, 356.2081; found, 368.2078; [α]25
=
D
1
+20.2 (c 0.2, MeOH). H NMR (CD3OD, 400 MHz) δ: 0.61−0.65
(m, 2 H), 0.73−0.79 (m, 1 H), 0.86−0.91 (m, 2 H), 0.96−1.00 (t, 3 H,
J = 7.2 Hz), 1.33−1.38 (m, 1 H), 1.48−1.71 (m, 5 H), 1.95−2.01 (m,
1 H), 2.46−2.50 (t, 2 H, J = 7.2 Hz), 3.05 (brs, 1 H), 3.84−3.86 (d, 1
H, J = 6.8 Hz), 4.65−4.67 (t, 1 H, J = 5.6 Hz), 4.83 (s, 1 H), 8.18 (s, 1
H). 13C NMR (CD3OD, 100 MHz) δ: 7.7, 7.9, 14.0, 19.6, 19.7, 23.2,
24.6, 24.8, 31.6, 63.8, 73.0, 77.2, 81.6, 88.2, 120.1, 140.7, 148.0, 149.8,
157.1. Anal. (C20H25N5O2) C, H, N.
6-Chloro-9-((3aR,3bR,4aS,5R,5aS)-Hexahydro-2,2-
dimethylbicyclo[3.1.0]hex-1(5)-eno [3,2-d][1,3] dioxol-5-yl)-9H-pu-
rine (11). Compound 6 (0.50 g, 2.93 mmol) was converted to 11
(0.63 g, 70%) as a white solid according to the same procedure used in
the preparation of 8: mp 84−86 °C; UV(CH2Cl2) λmax 265 nm. MS
(ESI+): [M + H]+ calcd for C14H16ClN4O2, 307.0956; found,
1
307.0951; [α]25 = −40.5 (c 0.2, MeOH). H NMR (CDCl3, 400
(1R,2R,3S,4R,5S)-4-(6-(Cyclopentylamino)-2-(hex-1-ynyl)-9H-
purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (4e). Yield: 65%; white solid;
mp 95−97 °C; UV (MeOH) λmax 275 nm. MS (ESI+): [M+H]+ calcd
D
MHz) δ: 0.96−1.04 (m, 2 H), 1.24 (s, 3 H), 1.56 (s, 3 H), 1.70−1.75
(m, 1 H), 2.13−2.19 (m, 1 H), 4.68−4.70 (m, 1 H), 5.08 (s, 1 H),
5.36−5.39 (t, 1 H, J = 6.8 Hz), 8.18 (s, 1 H), 8.78 (s, 1 H). 13C NMR
(CDCl3, 100 MHz) δ: 9.3, 24.3, 25.5, 26.0, 26.1, 61.4, 81.4, 89.1,
112.6, 132.1, 143.9, 151.3, 151.4, 152.3. Anal. (C14H15ClN4O2) C, H,
N.
for C22H30N5O2, 396.2394; found, 396.2400; [α]25 = +21.4 (c 0.2,
D
MeOH). 1H NMR (CD3OD, 400 MHz) δ: 0.74−0.78 (m, 1 H),
0.96−1.00 (t, 3 H, J = 7.2 Hz), 1.34−1.37 (m, 1 H), 1.50−1.83 (m, 10
H), 1.96−1.99 (m, 2 H), 2.06−2.12 (m, 2 H), 2.45−2.48 (t, 2 H, J =
7.2 Hz), 3.83−3.85 (d, 1 H, J = 6.4 Hz), 4.60 (brs, 1 H), 4.64−4.67 (t,
1 H, J = 5.6 Hz), 4.81(s, 1 H), 8.18 (s, 1 H). 13C NMR (CD3OD, 100
MHz) δ: 7.9, 13.9, 19.55, 19.60, 23.2, 24.6, 24.7, 31.6, 34.0, 53.5, 63.8,
73.0, 77.1, 81.7, 87.9, 119.8, 140.3, 148.1, 149.5, 155.5. Anal.
(C22H29N5O2) C, H, N.
2-(Tributylstannyl)-6-chloro-9-((3aR,3bR,4aS,5R,5aS)-hexahydro-
2,2-dimethylbicyclo[3.1.0] hex-1(5)-eno[3,2-d][1,3]dioxol-5-yl)-9H-
purine (12). To a stirred solution of 2,2,6,6-tetramethylpiperidine
(TMP, 1.36 mL, 8.00 mmol) in dry hexane (5 mL) and dry THF (10
mL) was added n-butyllithium (5.6 mL, 1.5 M solution in hexanes,
8.47 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
11 (0.50 g, 1.60 mmol) in dry THF (10 mL) was added dropwise, and
the mixture was stirred at −78 °C for 30 min. Tributyltin chloride
(1.74 mL, 8.0 mmol) was successively added dropwise to the dark
(1R,2R,3S,4R,5S)-4-(6-(3-Iodobenzylamino)-2-(hex-1-ynyl)-9H-
purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (4f). Yield: 88%; white solid;
mp 128−130 °C; UV (MeOH) λmax 274 nm. MS (ESI+): [M + H]+
calcd for C24H27IN5O2, 544.1204; found, 544.1212; [α]25 = +30.3 (c
D
1
0.2, MeOH). H NMR (CD3OD, 400 MHz) δ: 0.73−0.79 (m, 1 H),
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reaction mixture, and the mixture was stirred at the same temperature
for another 2 h. The resulting dark solution was quenched by dropwise
addition of a saturated aqueous NH4Cl solution (15 mL). After the
mixture was stirred at room temperature for 15 h, the mixture was
diluted with CH2Cl2 (15 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 purified by flash silica gel column chromatography
(hexane/EtOAc = 5:1) to give 12 (0.68 g, 70%) as a colorless syrup:
UV (MeOH) λmax 269 nm. MS (ESI+): [M + H]+ calcd for
Nonspecific binding was determined using 10 μM of 16 in the buffer.
The mixtures were incubated at 25 °C for 60 min. Binding reactions
were terminated by filtration through Whatman GF/B filters under
reduced pressure using a MT-24 cell harvester (Brandell, Gaithers-
burgh, MD, USA). Filters were washed three times with 9 mL ice-cold
buffer. Filters for A3AR binding were counted using a PerkinElmer
Cobra II γ-counter.
Cyclic AMP Accumulation Assay. Intracellular cAMP levels were
measured with a competitive protein binding method.34 CHO cells
that expressed the recombinant hA2BAR or hA3AR were harvested by
trypsinization. After centrifugation and resuspension in medium, cells
were plated in 24-well plates in 0.5 mL medium. After 24 h, the
medium was removed, and cells were washed three times with 1 mL
DMEM, containing 50 mM N-(2-hydroxyethyl)piperazine-N′-2-
ethanesulfonic acid (HEPES), pH 7.4. Cells were then treated with
agonists and/or test compounds in the presence of rolipram (10 μM)
and adenosine deaminase (3 units/mL). For assay of the hA3AR but
not the hA2BAR, forskolin (10 μM) was added to the medium after 45
min. After the addition of forskolin, the incubation was continued an
additional 15 min. The reaction was terminated upon removal of 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, protein kinase A (PKA) was
incubated with [3H]cAMP (2 nM) in K2HPO4/EDTA buffer
(K2HPO4, 150 mM; EDTA, 10 mM), 20 μL of the cell lysate, and
30 μL 0.1 M HCl or 50 μL of cAMP solution (0−16 pmol/200 μL for
standard curve). Bound radioactivity was separated by rapid filtration
through Whatman GF/C filters and washed once with cold buffer.
Bound radioactivity was measured by liquid scintillation spectrometry.
Statistical Analysis. Binding and functional parameters were
calculated using Prism 5.0 software (GraphPAD, San Diego, CA,
USA). IC50 values obtained from competition curves were converted
to Ki values using the Cheng−Prusoff equation.43 Data were expressed
as mean standard error of the mean.
C26H42ClN4O2Sn, 597.2011; found, 595.2020; [α]25 = −36.5 (c 0.2,
D
1
MeOH). H NMR (CDCl3) δ: 0.85−0.97 (m, 11 H), 1.13−1.38 (m,
15 H), 1.52−1.67 (m, 10 H), 2.03−2.13(m, 1 H), 4.79−4.81 (d, 1 H, J
= 7.2 Hz), 4.96 (s, 1 H), 5.43- 5.47 (m, 1 H), 8.01 (s, 1 H). 13C NMR
(CD3OD) δ: 9.4, 10.9, 24.3, 25.8, 26.1, 26.7, 27.4, 29.1, 62.1, 81.9,
89.1, 112.4, 131.0, 142.9, 149.8, 150.5, 182.1.
6-Chloro-2-(hex-1-ynyl)-9-((3aR,3bR,4aS,5R,5aS)-hexahydro-2,2-
dimethylbicyclo[3.1.0]hex-1(5) -eno[3,2-d][1,3]dioxol-5-yl)-9H-pu-
rine (9). To a stirred solution of 12 (0.40 g, 0.60 mmol) and copper
iodide (0.013 g, 0.06 mmol) in anhydrous DMF (5 mL) was added 1-
iodohexyne (0.124 mL, 0.6 mmol) in DMF (4 mL) dropwise via
syringe pump over a period of 1 h at room temperature, and the
reaction mixture was stirred at 50 °C for 5 h. The reaction mixture was
cooled to room temperature, quenched with saturated NaHCO3 (5
mL) solution, and diluted with EtOAc (10 mL). The organic layer was
separated, and the aqueous layer was further extracted with EtOAc (3
× 5 mL). The combined organic layers were washed with brine (5
mL) and water (5 mL), dried over anhydrous MgSO4, and filtered.
The solvent was evaporated under reduced pressure, and the residue
was purified by flash silica gel column chromatography (hexane/
EtOAc = 3: 1) to give 9 (0.155 g, 60%) as a white foam, whose
spectral data were identical to those of authentic sample.
Biological Assays. Cell Culture and Membrane Preparation.
CHO cells expressing the recombinant hA1 or A3R and HEK-293 cells
expressing the hA2AAR were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) and F12 (1:1) supplemented with 10% fetal bovine
serum, 100 U/ml penicillin, 100 μg/mL streptomycin, and 2 μmol/mL
glutamine. RBL-2H3 cells endogenously expressing rA3AR were
cultured as described.41 Cells were harvested by trypsinization. After
homogenization and suspension, cells were centrifuged at 500g for 10
min, and the pellet was resuspended in 50 mM Tris·HCl buffer (pH
7.4) containing 10 mM MgCl2. The suspension was homogenized with
an electric homogenizer for 10 s and was then recentrifuged at 20 000g
for 20 min at 4 °C. The resultant pellets were resuspended in buffer
containing 3 U/mL adenosine deaminase, and the suspension was
stored at −80 °C until the binding experiments. The protein
concentration was measured using the Bradford assay.42
Binding Assays at the hA1 and hA2AARs. For binding to the
hA1AR, 50 μL of increasing concentrations of a test ligand and 50 μL
of [3H]14 (2 nM, PerkinElmer, Boston, MA) were incubated with
membranes (40 μg/tube) from CHO cells stably expressing the hA1
AR 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.32 Nonspecific binding was
determined using 10 μM of N6-cyclopentyladenosine (CPA, 17). For
hA2AAR binding, membranes (20 μg/tube) from HEK-293 cells stably
expressing the hA2AAR were incubated at 25 °C for 60 min with a final
concentration of 15 nM [3H]15 (American Radiolabeled Chemicals,
Inc., St. Louis, MO) in a mixture containing 50 μL of increasing
concentrations of a test ligand and 200 μL of 50 mM Tris·HCl, pH
7.4, containing 10 mM MgCl2. Compound 16 (10 μM) was used to
define nonspecific binding. The reaction was terminated by filtration
with GF/B filters. Filters for A1 and A2AAR binding were placed in
scintillation vials containing 5 mL of Hydrofluor scintillation buffer
and counted using a PerkinElmer Tricarb 2810TR Liquid Scintillation
Analyzer.
Antifibrosis Assay. Immortalized murine proximal tubular cells
(mProx24) derived from microdissected proximal tubular segments of
C57BL6/J adult mouse kidneys were supplied from Dr. Sugaya at St.
Marianna University School of Medicine, Kanagawa, Japan. mProx24
were maintained in DMEM supplemented with 10% fetal calf serum
(FCS; Gibco), 100 U/ml penicillin, 100 μg/mL streptomycin, and 44
mM NaHCO3 under 5% CO2 environment at 37 °C. Cells were
cultured in 6-well plate for mRNA analysis. At next day after seeding
cell on 6-well plate, the cultured cells were growth-arrested with a
DMEM medium containing 0.15% FCS for 24h. Each synthesized
compound was dissolved in DMSO to 50 mM and it was diluted to 20
mM, 10 mM, and 1 mM. After cells were pretreated with the
synthesized compound dissolved in DMEM containing 0.15% FCS for
1 h, treated with recombinant human transforming growth factor-β1
(hTGF β1, R&D Systems) 5 ng/mL for 6 h. Total RNA was extracted
from mProx24 using Trizol (Invitrogen) according to the standard
protocol. mRNA expressions were measured by real-time PCR using
StepOnePlus (Applied Biosystems) with 20 μL reaction volume
consisting of cDNA transcripts, primer pairs, and SYBR Green PCR
Master Mix (Applied Biosystems). Quantifications were normalized to
18S. The sequences of mouse collagen Iα1 primer pairs are 5′-
GAACATCACCTACCA CTGCA-3′ and 5′-GTTGGGATGGAGG-
GAGTTTA-3′.
Molecular Modeling. The X-ray crystal structure of the human A2A
AR in complex with an agonist, 16 (PDB ID: 2YDV)34 was retrieved
from the protein data bank (PDB) and prepared using the Protein
Preparation Wizard in Maestro v9.2 (Schrodinger, LLC, NY, U.S.A.),
̈
where water and ions were removed, hydrogen atoms were added and
optimized, and then the protein was minimized using the Optimized
Potentials for Liquid Simulations-all atom (OPLS-AA) 2005 force
field. The structures of the molecules were sketched in the Maestro
Binding Assay at the hA3AR and rA3AR. Each tube in the
competitive binding assay contained 100 μL membrane suspension
(20 μg protein), 50 μL [125I]13 (1.0 nM, PerkinElmer, Boston, MA),
and 50 μL of increasing concentrations of the test ligands in Tris·HCl
buffer (50 mM, pH 8.0) containing 10 mM MgCl2, 1 mM EDTA.32
and energy minimized using Impact v5.7 (Schrodinger, LLC, NY,
̈
U.S.A.) considering conjugant gradient algorithm with the maximum
minimization cycles of 1000 and convergence gradient of 0.001 kJ/
mol-Å. The four docking programs Glide-SP (standard precision),
Glide-XP (extra precision), GOLD, and Surflex-dock showed
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Journal of Medicinal Chemistry
Article
consistent results, and the Glide-XP docking results are presented. The
receptor grid box with 10 Å around the centroid of the cocrystallized
NECA was generated. The best binding poses of 2, 4a, and 4f were
selected for the calculation of the receptor−ligand binding free energy
(ΔGbind) using Prime molecular mechanics-generalized Born surface
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ASSOCIATED CONTENT
■
S
* Supporting Information
Elemental analyses, molecular docking studies in the hA3AR
1
homology model, and H and 13C NMR copies of 8−12 and
4a−4i. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: lakjeong@snu.ac.kr. Fax: 82-2-888-9122. Tel.: 82-2-
880-7850.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This research was supported by grants from Midcareer
Research Program (2010-0026203 and 370C-20130120) and
the National Leading Research Lab (NLRL) Program (2011-
0028885) through the Ministry of Science, ICT & Future
Planning (MSIP) and the National Research Foundation,
Korea and in part by the Intramural Research Program of the
NIH, National Institute of Diabetes and Digestive and Kidney
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ABBREVIATIONS:
■
AR, adenosine receptor; TGF, transforming growth factor;
mProx, murine proximal; cAMP, cyclic adenosine-5′-mono-
phosphate; IP3, inositol triphosphate; DAG, diacylglycerol; AC,
adenylate cyclase; PLC, phospholipase C; CKD, renin−angio-
tensin−aldosterone system; RAAS, Chronic kidney disease; Cl-
IB-MECA, 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarbamoy-
ladenosine; thio-Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-5′-
N-methylcarbamoyl-4′-thioadenosine; LiTMP, lithium tetrame-
thylpiperidide; CHO, Chinese hamster ovary; HEK, human
embryonic kidney; I-AB-MECA, N6-(3-iodo-4-aminobenzyl)-
5′-N-methylcarboxamidoadenosine; R-PIA, (-)-N6-2-phenyliso-
propyl adenosine; CGS21680, 2-[p-(2-carboxyethyl)-
phenylethylamino]-5′-N-ethylcarboxamidoadenosine; NECA,
5′-N-ethylcarboxamidoadenosine; DMEM, Dulbecco′s modi-
fied Eagle’s medium; HEPES, N-(2-hydroxyethyl)piperazine-
N′-2-ethanesulfonic acid; OPLS-AA, optimized potentials for
liquid simulations-all atom; MM-GBSA, molecular mechanics-
generalized born surface area; TM, transmembrane
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