Correlation study between A3 adenosine receptor binding affinity and anti-renal interstitial fibrosis activity of truncated adenosine derivatives

Article abstract of DOI:10.1007/s12272-018-1079-2

Truncated 4′-thionucleosides 1–4 and 4′-oxonucleosides 5–8 as potent and selective A₃AR antagonists were synthesized from d-mannose and d-erythronic acid γ-lactone, respectively. These nucleosides were evaluated for their anti-fibrotic renoprot

Full text of DOI:10.1007/s12272-018-1079-2

Arch. Pharm. Res.
Online ISSN 1976-3786
Print ISSN 0253-6269
https://doi.org/10.1007/s12272-018-1079-2
RESEARCH ARTICLE
Correlation study between A3 adenosine receptor binding affinity
and anti-renal interstitial fibrosis activity of truncated adenosine
derivatives
Jinha Yu1 Gyudong Kim1 Dnyandev B. Jarhad1 Hyuk Woo Lee2
•
•
•
•
Jiyoun Lee2 Chong Woo Park2 Hunjoo Ha3 Lak Shin Jeong1
•
•
•
Received: 8 June 2018 / Accepted: 7 September 2018
Ó The Pharmaceutical Society of Korea 2018
Abstract Truncated 40-thionucleosides 1–4 and 40-oxonu-
cleosides 5–8 as potent and selective A3AR antagonists
were synthesized from D-mannose and D-erythronic acid c-
lactone, respectively. These nucleosides were evaluated for
their anti-fibrotic renoprotective activity in TGF-b1-treated
murine proximal tubular (mProx) cells. Their antagonistic
activities for A3AR were proportional to their inhibitory
activities against TGF-b1-induced collagen I upregulation
in mProx cells. This result suggests that the binding affinity
of A3AR antagonists is closely correlated with their anti-
fibrotic activity. Thus, A3AR antagonists might be novel
therapeutic candidates for treating chronic kidney disease.
stimulate adenylate cyclase activity. For example, A2A and
2BARs are Gs-coupled receptors that can stimulate
A
adenylate cyclase while A1 and A3ARs are coupled to Gi
protein that can inhibit adenylate cyclase (Fredholm et al.
2001).
Extracellular adenosine concentrations can be rapidly
increased from 300 nM to 1200 nM in response to cellular
damage to protect tissue from damage due to hypoxia and
´
ischemia (Liang and Jacobson 1999; Hasko 2004). Extra-
cellular adenosine concentration is also dramatically
increased during renal hypoxia and ischemia compared to
that in normal kidneys (Rabadi and Lee 2015). It has been
recently reported that adenosine deaminase (ADA)
knockout mice show tubulointerstitial fibrosis, indicating
that adenosine might play an important role in controlling
chronic kidney disease (Dai et al. 2011). It has also been
reported that all four subtypes of ARs are up-regulated in
obstructed kidneys which is a well characterized model of
tubulointerstital fibrosis (Lee et al. 2012). We have repor-
ted the structure–activity relationship of truncated 40-oxo-
and 40-thionucleosides 1–8 as potent and selective A3AR
antagonists (Jeong et al. 2007, 2008; Pal et al. 2009). These
compounds have been found to be orally active and spe-
cies-independent A3AR antagonists suitable for efficacy
determination in animal model. Among these compounds,
truncated 2-chloro-N6-(3-iodobenzyl)-40-thioadenosine (1,
X = S) with Ki values 4.16 nm and 3.91 nM for human
(h) A3ARs and rat (r) A3ARs, respectively, exhibited
highly potent therapeutic effects on UUO-induced renal
fibrosis. Compound 1 inhibited UUO-induced renal fibrosis
and collagen I upregulation in a dose-dependent manner. It
also showed potent anti-tubulointerstitial fibrosis effect as
losartan at dose of 10 mg/kg, suggesting that compound 1
might be a good candidate for treating chronic kidney
disease (Lee et al. 2013).
Keywords A3 adenosine receptor Á Antagonist Á Binding
affinity Á Truncated adenosine Á Renal fibrosis
Introduction
Adenosine is an endogenous signaling molecule that
modulates many physiological processes through binding
to four subtypes of adenosine receptors (A1, A2A, A2B, and
A3ARs) (Ralevic and Burnstock 1998). These four sub-
types are classified based on their ability to either inhibit or
Jinha Yu and Gyudong Kim contributed equally to this work.
& Lak Shin Jeong
lakjeong@snu.ac.kr
1
Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 151-742, Korea
2
Future Medicine Co., Ltd., Seoul 06665, Korea
3
College of Pharmacy, Ewha Womans University,
Seoul 03761, Korea
123

J. Yu et al.
Based on these findings, we wanted to establish the
relationship between binding affinity for A3AR and anti-
renal fibrosis activity of truncated 40-thio- and 40-oxoade-
nosine derivatives acting as A3AR antagonists (Fig. 1).
Herein, we report therapeutic potentials of A3AR antago-
nists on renal tubulointerstital fibrosis.
equiv) in 1,2-dichloroethane (10 mL) was added dropwise
to this mixture. Trimethylsilyl trifluoromethanesulfonate (2
equiv) was added dropwise to the mixture. The mixture
was stirred at 0 °C for 30 min, warmed to room tempera-
ture with stirring for 1 h, and then heated with stirring at
80 °C for 2 h. The mixture was cooled, diluted with
CH2Cl2, and washed with saturated NaHCO3 solution. The
organic layer was dried with anhydrous MgSO4 and
evaporated. The residue was subjected to a flash silica gel
column chromatography (CH2Cl2:MeOH = 50:1) to give
compound 17 or 18.
Materials and methods
1H NMR Spectra (CDCl3, CD3OD or DMSO-d6) were
recorded with Varian Unity Invoa 400 MHz Spectrometer.
1H NMR data were 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 were reported in Hertz. 13C
NMR spectra (CDCl3, CD3OD or DMSO-d6) were recor-
ded with Varian Unity Inova 100 MHz Spectrometer.
Chemical shifts were reported as parts per million (d) rel-
ative to solvent peak. Optical rotations were determined
with Jasco III in appropriate solvent. UV spectra were
recorded on U-3000 (Hitachi) in methanol or CH2Cl2.
Flash column chromatography was performed using silica
gel 60 (230–400 mesh, Merck). All anhydrous solvents
were distilled over CaH2, P2O5, or sodium/benzophenone
prior to reaction.
2,6-Dichloro-9-((3aR,4R,6aS)-2,2-dimethyltetrahydrothieno
[3,4-d][1,3]dioxol-4-yl)-9H-purine (17) Yield: 79%; UV
1
(CH2Cl2): kmax = 275.0 nm; H NMR (CDCl3): d 8.17 (s,
1H), 5.87 (s, 1H), 5.32 (pseudo t, 1H, J = 4.8 Hz), 5.21 (d,
1H, J = 5.6 Hz), 3.79 (dd, 1H, J = 4.4, 12.8 Hz), 3.26 (d,
1H, J = 13.2 Hz), 1.59 (s, 3H), 1.36 (s, 3H); 13C NMR
(CDCl3): d 153.3, 152.5, 152.4, 145.0, 131.8, 112.3, 89.8,
84.6, 70.6, 41.2, 26.6, 24.8; [a]2D5 - 42.04 (c 0.16, CH2-
Cl2); FAB-MS: m/z 347 [M ? H]?; Anal. Calcd for C12-
H12Cl2N4O2S: C, 41.51; H, 3.48; N, 16.14; S, 9.24. Found:
C, 41.84; H, 3.78; N, 15.99; S, 8.98.
2,6-Dichloro-9-((3aR,4R,6aS)-2,2-dimethyltetrahydrofuro
[3,4-d][1,3]dioxol-4-yl)-9H-purine (18) Yield: 69%;
1
UV (MeOH): kmax = 276.5 nm; H NMR (CDCl3): d 8.15
Synthesis
(s, 1H), 6.07 (s, 1H), 5.41 (d, 1H, J = 6.0 Hz), 5.29–5.26
(m, 1H), 4.31–4.25 (m, 2H), 1.57 (s, 3H), 1.41 (s, 3H);
[a]2D5 - 21.00 (c 0.10, DMSO); FAB-MS: m/z 331
[M ? H]?; Anal. Calcd for C12H12Cl2N4O3: C, 43.52; H,
3.65; N, 16.92. Found: C, 43.08; H, 3.61; N, 16.70.
Designed compounds 1–8 were synthesized as depicted in
Schemes 1, 2 and 3 following published procedures (Jeong
et al. 2007, 2008; Pal et al. 2009).
General procedure for Vorbruggen condensation: synthesis
General procedure for hydrolysis: synthesis of compounds
of compounds 17 and 18
19 and 20
2,6-Dichloropurine (2 equiv), ammonium sulfate (0.3
equiv), and bis(trimethylsilyl)amine (20 mL) were refluxed
under inert and dry conditions until clear solution was
obtained. The solution was evaporated under high vacuum.
The resulting solid was dissolved in 1,2-dichloroethane
(10 mL) at 0 °C. The solution of compound 14 or 16 (1
A mixture of compound 17 or 18 (10 mmol) in THF
(20 mL) and 2 N HCl (10 mL) was stirred at room tem-
perature for 15 h. The mixture was neutralized with 1 N
NaOH solution. Volatiles were then carefully evaporated
under reduced pressure. The mixture was purified by flash
Fig. 1 A correlation study
between A3AR binding affinity
and anti-renal fibrosis activity
123

Correlation study between A3 adenosine receptor binding affinity…
Scheme 1 Synthesis of 40-thioglycosyl donor 14
Scheme 2 Synthesis of 40-
oxoglycosyl donor 16
(2R,3R,4S)-2-(2,6-Dichloro-9H-purin-9-yl)-tetrahydrofuran-
silica gel column chromatography (CH2Cl2:MeOH = 20:1)
3,4-diol (20) Yield: 57%; mp: 122.7–123.4 °C; UV
(MeOH): kmax = 276.5 nm; H NMR (DMSO-d6): d 8.98
to give compound 19 or 20.
1
(s, 1H), 5.96 (d, 1H, J = 6.4 Hz), 5.57 (d, 1H, J = 6.0 Hz),
5.32 (d, 1H, J = 4.0 Hz), 4.74–4.69 (m, 1H), 4.41 (dd, 1H,
J = 3.6, 9.2 Hz), 4.32–4.29 (m, 1H), 3.87 (dd, 1H, J = 2.0,
9.6 Hz); 13C NMR (DMSO-d6) d 153.2, 151.2, 150.0,
147.1, 131.2, 88.4, 74.8, 74.1, 70.1; [a]2D5 - 68.09 (c 0.14,
DMSO); FAB-MS: m/z 291 [M ? H]?; Anal. Calcd for
C9H8Cl2N4O3: C, 37.13; H, 2.77; N, 19.25. Found: C,
37.23; H, 3.11; N, 19.45.
(2R,3R,4S)-2-(2,6-Dichloro-9H-purin-9-yl)-tetrahydrothio-
phene-3,4-diol (19) Yield: 96%; mp: 198.3–200.3 °C;
UV (MeOH): kmax = 275.0 nm; 1H NMR (CD3OD) d 8.87
(s, 1H), 6.08 (d, 1H, J = 6.8 Hz), 4.69 (q, 1H, J = 3.2 Hz),
4.48 (q, 1H, J = 3.6 Hz), 3.56 (dd, 1H, J = 4.4, 11.2 Hz),
2.97 (dd, 1H, J = 3.4, 11.2 Hz); 13C NMR (DMSO-d6)
153.7, 151.1, 149.8, 147.5, 130.9, 78.8, 71.9, 62.4, 34.7;
[a]2D5 - 50.43 (c 0.12, DMSO); FAB-MS: m/z 307
[M ? H]?; Anal. Calcd for C9H8Cl2N4O2S: C, 35.19; H,
2.63; N, 18.24; S, 10.44. Found: C, 35.48; H, 2.40; N,
18.65; S, 10.84.
123

J. Yu et al.
Scheme 3 Synthesis of
truncated N6-(3-
halobenzyl)amino derivatives
1–8
General procedure for N6-amination: synthesis of 1–8
136.0, 135.6, 130.6, 126.8, 118.4, 94.7, 78.6, 72.1, 61.5,
42.5, 34.4; [a]2D5 - 78.91 (c 0.13, DMSO); FAB-MS: m/z
504 [M ? H]?; Anal. Calcd for C16H15ClIN5O2S: C,
38.15; H, 3.00; N, 13.90; S, 6.37 Found: C, 38.31; H, 2.96;
N, 13.98; S, 6.21.
Appropriate amine (1.5 equiv) was added to a solution of
compound 19 or 20 in EtOH (5 mL) at room temperature.
The mixture was stirred at room temperature for 2 h to 3 d
and evaporated. The residue was purified by a flash silica
gel column chromatography (CH2Cl2:MeOH = 20:1) to
give compounds 1–8.
(2R,3R,4S)-2-(2-Chloro-6-(3-fluorobenzylamino)-9H-purin-9-yl)
tetrahydrothiophene-3,4-diol
(2) Yield:
80%;
mp:
1
183.2–183.5 °C; UV (MeOH): kmax = 275.0 nm; H NMR
(DMSO-d6): d 8.91 (t, 1H-NH, J = 5.8 Hz), 8.51 (s, 1H),
7.33–7.39 (m, 1H), 7.13–7.18 (m, 2 H), 7.06 (dt, 1H,
J = 2.8, 11.6 Hz), 5.82 (d, 1H, J = 7.2 Hz), 5.56 (d, 1H-
OH, J = 6.0 Hz), 5. 37 (d, 1H-OH, J = 4.4 Hz), 4.65 (d,
1H, J = 6.0 Hz), 4.60 (m, 1H), 4.33–4.35 (m, 1H), 3.41
(dd, 1H, J = 4.0, 10.8 Hz), 2.79 (dd, 1H, J = 2.8, 10.8 Hz);
13C NMR (DMSO-d6): d 182.8, 165.3, 162.9, 156.6, 155.3,
142.5, 132.4 (J = 8.0 Hz; CF), 125.1, 119.9, 115.9, 115.6,
80.5, 73.9, 63.4, 44.5, 36.1; [a]2D5 - 96.21 (c 0.12, DMSO);
FAB-MS: m/z 396 [M ? H]?; Anal. Calcd for
(2R,3R,4S)-2-(2-Chloro-6-(3-iodobenzylamino)-9H-purin-
9-yl)-tetrahydrothiophene-3,4-diol (1) Yield: 84%; mp:
1
198.7–199.9 °C; UV (MeOH): kmax = 274.0 nm; H NMR
(DMSO-d6): d 8.90 (t, 1H –NH, J = 6.4 Hz), 8.51 (s, 1H),
7.74 (s, 1H), 7.60 (d, 1H, J = 7.6 Hz), 7.35 (d, 1H,
J = 7.6 Hz), 7.13 (t, 1H, J = 8.0 Hz), 5.82 (d, 1H,
J = 7.6 Hz), 5.56 (d, 1H, J = 6.4 Hz), 5.37 (d, 1H,
J = 4.0 Hz), 4.60 (d, 3H, J = 4.4 Hz), 4.34 (brs, 1H), 3.38
(dd, 1H, J = 4.0, 10.8 Hz), 2.80 (dd, 1H, J = 4.0, 10.8 Hz);
13C NMR (DMSO-d6) d 154.8, 153.0, 150.3, 141.9, 140.6,
123

Correlation study between A3 adenosine receptor binding affinity…
C16H15ClFN5O2S: C, 48.55; H, 3.82; N, 17.69; S, 8.10.
Found: C, 48.47; H, 3.75; N, 17.57; S, 7.70.
J = 6.0 Hz), 5.22 (d, 1H, J = 4.0 Hz), 4.74–4.64 (m, 3 H),
4.41 (dd, 1H, J = 3.6, 9.2 Hz), 4.25 (br s, 1H), 3.80 (dd,
1H, J = 1.2, 9.6 Hz); 13C NMR (DMSO-d6): d 163.4,
163.9, 154.9, 153.1, 149.9, 142.2 (d, J = 26.8 Hz), 140.7,
130.3 (d, J = 34.8 Hz; CF), 123.2, 118.8, 113.8 (dd,
J = 82.4, 122.0 Hz; C-F), 87.5, 74.4, 73.7, 70.2, 42.7; [a]D25
- 73.40 (c 0.094, DMSO); FABMS: m/z 380 [M ? H]?;
Anal. Calcd for C16H15ClFN5O3: C, 50.60; H, 3.98; N,
18.44. Found: C, 50.30; H, 4.01; N, 18.04.
(2R,3R,4S)-2-(2-Chloro-6-(3-chlorobenzylamino)-9H-purin-
9-yl)-tetrahydrothiophene-3,4-diol (3) Yield: 82%; mp:
1
163.3–165.3 °C; UV (MeOH): kmax = 274.5 nm; H NMR
(CD3OD): d 8.34 (s, 1H), 7.41 (s, 1H), 7.24–7.34 (m, 3 H),
5.94 (d, 1H, J = 6.4 Hz), 4.75 (brs, 2 H), 4.61 (q, 1H,
J = 3.2 Hz), 4.45 (q, 1H, J = 4.0 Hz), 3.51 (dd, 1H,
J = 4.8, 11.2 Hz), 2.95 (dd, 1H, J = 3.6, 10.8 Hz); 13C
NMR (CD3OD): d 141.8, 135.5, 131.2, 128.9, 128.5, 127.3,
80.9, 74.5, 64.2, 44.7, 35.3; [a]2D5 - 69.92 (c 0.13, DMSO);
FABMS: m/z 411 [M]?; Anal. Calcd for C16H15Cl2N5O2S:
C, 46.61; H, 3.67; N, 16.99; S, 7.78. Found: C, 46.65; H,
3.67; N, 16.74; S, 7.39.
(2R,3R,4R)-2-(2-Chloro-6-(3-chlorobenzylamino)-9H-purin-
9-yl)-tetrahydrofuran-3,4-diol (7) Yield: 76%; mp:
1
196.4–197.0 °C; UV (MeOH): kmax = 271.5 nm; H NMR
(DMSO-d6): d 8.92 (t, 1H, J = 6.0 Hz), 8.43 (s, 1H), 7.39
(s, 1H), 7.37–7.28 (m, 3H), 5.80 (d, 1 H, J = 6.8 Hz), 5.47
(d, 1H, J = 6.4 Hz), 5.22 (d, 1H, J = 4.0 Hz), 4.73–4.64
(m, 3H), 4.32 (dd, 1H, J = 3.6, 9.2 Hz), 4.24 (br s, 1H),
3.79 (dd, 1H, J = 1.6, 9.2 Hz); 13C NMR (DMSO-d6): d
154.8, 153.1, 149.9, 141.8, 140.7, 132.9, 130.2, 127.1,
126.8, 125.9, 118.7, 87.5, 74.4, 73.7, 70.2, 42.7; [a]D25
- 78.19 (c 0.133, DMSO); FAB-MS: m/z 396 [M ? H]?;
Anal. Calcd for C16H15Cl2N5O3: C, 48.50; H, 3.82; N,
17.68. Found: C, 48.56; H, 3.89; N, 17.12.
(2R,3R,4S)-2-(2-Chloro-6-(3-bromobenzylamino)-9H-purin-
9-yl)-tetrahydrothiophene-3,4-diol (4) Yield: 83%; mp:
1
184.0–185.0 °C; UV (MeOH): kmax = 274.0 nm; H NMR
(DMSO-d6): d 8.91 (brs, 1H –NH), 8.51 (s, 1H), 7.55 (s,
1H), 7.43 (d, 1H, J = 7.6 Hz), 7.33–7.35 (m, 1H),
7.26–7.30 (m, 1H), 5.82 (d, 1H, J = 7.2 Hz), 5.57 (d, 1H –
OH, J = 6.0 Hz), 5.38 (d, 1H –OH, J = 4.0 Hz), 4.60–4.63
(m, 3 H), 4.34 (s, 1H), 3.41 (dd, 1H, J = 4.4, 11.2 Hz), 2.80
(dd, 1H, J = 2.8, 10.8 Hz); 13C NMR (DMSO-d6): d 154.8,
153.0, 150.3, 142.1, 140.6, 130.6, 130.1, 129.8, 126.4,
121.6, 118.5, 78.6, 72.1, 61.5, 42.6, 34.5; [a]2D5 - 83.60
(c 0.13, DMSO); FAB-MS: m/z 456 [M ? H]?; Anal.
Calcd for C16H15BrClN5O2S: C, 42.07; H, 3.31; N, 15.33;
S, 7.02 Found: C, 42.23; H, 3.37; N, 15.19; S, 6.98.
(2R,3R,4R)-2-(2-Chloro-6-(3-bromobenzylamino)-9H-purin-
9-yl)-tetrahydrofuran-3,4-diol (8) Yield: 81%; mp:
1
181.5–181.7 °C; UV (MeOH): kmax = 274.5 nm; H NMR
(DMSO-d6): d 8.92 (t, 1H, J = 6.0 Hz), 8.43 (s, 1H), 7.55
(s, 1H), 7.44 (d, 1H, J = 8.0 Hz), 7.35–7.33 (m, 1H), 7.30–
7.26 (m, 1H), 5.81 (d, 1H, J = 6.4 Hz), 5.47 (d, 1H,
J = 6.4 Hz), 5.22 (d, 1H, J = 4.0 Hz), 4.69–4.66 (m, 1H),
4.62 (s, 2H), 4.32 (dd, 1H, J = 3.6, 9.2 Hz), 4.25 (br s, 1H),
3.80 (dd, 1H, J = 1.6, 9.2 Hz); 13C NMR (DMSO-d6): d
154.9, 153.2, 149.9, 142.1, 140.7, 130.6, 130.1, 129.8,
126.4, 121.6, 118.8, 87.5, 74.4, 73.7, 70.2, 42.6; [a]D25
- 62.75 (c 0.10, DMSO); FAB-MS: m/z 440 [M ? H]?;
Anal. Calcd for C16H15ClBrN5O3: C, 43.61; H, 3.43; N,
15.89. Found: C, 43.92; H, 3.44; N, 16.05.
(2R,3R,4R)-2-(2-Chloro-6-(3-iodobenzylamino)-9H-purin-
9-yl)-tetrahydrofuran-3,4-diol (5) Yield: 78%; mp:
1
195.5–195.8 °C; UV (MeOH): kmax = 274.0 nm; H NMR
(DMSO-d6): d 8.91 (t, 1H, J = 6.4 Hz), 8.44 (s, 1H), 7.75
(s, 1H), 7.61 (d, 1H, J = 8.0 Hz), 7.36 (d, 1H, J = 7.6 Hz),
7.13 (t, 1H, J = 4.0 Hz), 5.81 (d, 1H, J = 6.8 Hz), 5.47 (d,
1H, J = 6.8 Hz), 5.23 (d, 1H, J = 4.0 Hz), 4.72 (dd, 1H,
J = 6.4, 10.8 Hz), 4.61 (d, 1H, J = 6.0 Hz), 4.34 (dd, 1H,
J = 3.6, 9.2 Hz), 3.81 (dd, 1H, J = 1.2, 9.2 Hz); 13C NMR
(DMSO-d6): d 154.8, 153.2, 149.9, 141.9, 140.7, 136.0,
135.6, 130.6, 126.8, 118.8, 94.8, 87.5, 74.4, 73.7, 70.2,
42.5; [a]2D5 - 68.07 (c 0.12, DMSO); FAB-MS: m/z 488
[M ? H]?; Anal. Calcd for C16H15ClIN5O3: C, 39.41; H,
3.10; N, 14.36. Found: C, 39.66; H, 3.08; N, 14.53.
Binding assay for hA3 and rA3 adenosine receptor
Binding affinities of final nucleosides 1–8 for human
(h) A3AR and rat (r) A3AR were measured using standard
radioligands and membrane preparations (Jeong et al.
2007, 2008). First, hA3AR or rA3AR was expressed in
Chinese hamster ovary (CHO) cells. [125I]N6-(4-amino-3-
(2R,3R,4R)-2-(2-Chloro-6-(3-fluorobenzylamino)-9H-purin-
iodobenzyl)-50-N-methylcarboxamidoadenosine
(I-AB-
9-yl)-tetrahydrofuran-3,4-diol (6) Yield: 83%; mp:
1
MECA) was used to measure the binding affinity. Values
are expressed as mean SEM (n = 3–4 after outliers were
eliminated). They were normalized against NECA.
187.0–187.9 °C; UV (MeOH): kmax = 271.0 nm; H NMR
(DMSO-d6): d 8.92 (t, 1H, J = 6.0 Hz), 8.43 (s, 1H),
7.39–7.33 (m, 1H), 7.18–7.13 (m, 2 H), 7.06 (dt, 1H,
J = 2.0, 8.4 Hz), 5.81 (d, 1H, J = 6.8 Hz), 5.47 (d, 1H,
123

J. Yu et al.
Table 1 Binding affinities and anti-renal fibrosis activity of A3AR antagonists 1–8
Compound
Affinity, Ki, nM SEM
IC50 (lM)a
rA3
hA3
1 (X = S, R = 3-iodobenzyl)
2 (X = S, R = 3-fluorobenzyl)
3 (X = S, R = 3-chlorobenzyl)
4 (X = S, R = 3-bromobenzyl)
5 (X = O, R = 3-iodobenzyl)
6 (X = O, R = 3-fluorobenzyl)
7 (X = O, R = 3-chlorobenzyl)
8 (X = O, R = 3-bromobenzyl)
3.9 1.1
36.2 10.7
6.2 1.8
6.1 1.8
ND
4.16 0.50
7.4 1.3
20.9
8.8
1.66 0.90
8.99 5.17
42.9 8.9
284 37
8.1
25.8
73.2
ND
[ 100
[ 100
[ 100
ND
75.0 11.7
13.0 6.9
ND
ND not determined
aConcentration to inhibit TGF-b1-induced collagen I mRNA expression by 50%
normalized to 18S. Mouse a-SMA primers were 50-
GTCCCAGACATCAGGGAGTAA-30 and 50-TCGGA-
TACTTCAGCGTCAGGA-30.
Anti-fibrosis assay
Anti-fibrosis assay for compound 1–8 was performed fol-
lowing published procedures (Lee et al. 2013). Briefly,
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 cells were maintained in
DMEM supplemented with 10% fetal calf serum (FCS;
Gibco), 100 U/ml penicillin, 100 lg/mL streptomycin, and
44 mM NaHCO3 under 5% CO2 environment at 37 °C.
Cells were cultured in 6-well plates for mRNA analysis. On
the next day after seeding cells onto 6-well plates, cultured
cells were growth-arrested with DMEM medium contain-
ing 0.15% FCS for 24 h. Each synthesized compound was
dissolved in DMSO to 50 mM and 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, they were treated with recombinant
human transforming growth factor-b1 (hTGF b1, R&D
Systems) at 10 ng/mL for 6 h. Total RNA was extracted
from mProx24 cells using Trizol (Invitrogen) according to
the standard protocol. mRNA expression level was mea-
sured by real-time PCR using StepOnePlus (Applied
Biosystems) with reaction volume of 20 lL consisting of
cDNA transcripts, primer pairs, and SYBR Green PCR
Master Mix (Applied Biosystems). Quantifications were
Results
All final compounds 1–8 showed high binding affinities for
hA3AR. They were synthesized as shown in Schemes 1, 2
and 3 according to our previously published procedures
(Jeong et al. 2007, 2008; Pal et al. 2009). First, 40-thio-
glycosyl donor 14 (Jeong et al. 2007) was synthesized
starting from D-mannose in six steps (Scheme 1). Briefly,
D-mannose was converted to diacetonide 9 under acidic
conditions. Reduction of diacetonide with sodium boro-
hydride gave diol 10 which was mesylated followed by
cyclization with anhydrous sodium sulfide to afford
4-thiosugar (12). Selective hydrolysis of 5,6-acetonide of
4-thiosugar with 60% aqueous acetic acid followed by
treatment of diol with Pb(OAc)4 afforded 40-thioglycosyl
donor 14. Then 40-oxoglycosyl donor 16 (Pal et al. 2009)
was synthesized, starting from commercially available 2,3-
O-isopropylidene-D-erythronolactone (Scheme 2). Lactone
was reduced to lactol (15) which was treated with acetic
anhydride to yield 40-oxoglycosyl donor 16.
Glycosyl donors 14 and 16 were condensed with sily-
lated 2,6-dichloropurine in the presence of TMSOTf to
give 2,6-dichloropurine derivatives 17 and 18 (Scheme 3).
123

Correlation study between A3 adenosine receptor binding affinity…
Science Research Program (2018R1D1A1B07049982) of the National
Research Foundation (NRF), Republic of Korea.
Compounds 17 and 18 were hydrolyzed with 1 N HCl to
give 19 and 20, respectively. Then 2,6-dichloropurine
derivatives 19 and 20 were treated with 3-halobenzylamine
in EtOH at room temperature to yield final nucleosides 1–4
and 5–8, respectively.
Compliance with ethical standards
Conflict of interest The authors declared that they have no conflict
of interest.
Discussion
References
All final compounds were potent and selective A3AR
antagonists. They were evaluated for anti-fibrotic reno-
protective activity in TGF-b1-treated murine proximal
tubular (mProx) cells (Lee et al. 2013). As potent A3AR
antagonists, 40-thio analogues 1–4 with Ki’s of
1.66–8.99 nM strongly inhibited TGF-b1-induced collagen
I upregulation in mProx cells, with IC50 values of
8.1–25.8 lM (Table 1). The anti-renal fibrosis activities of
40-thio analogues 1–4 decreased in the following order:
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Acknowledgements This research was supported by grants from
Mid-career Research Program (2016R1A2B3010164) and Basic
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