Discovery and Structure-Activity Relationships of Novel Template, Truncated 1′-Homologated Adenosine Derivatives as Pure Dual PPARγ/δModulators

Article abstract of DOI:10.1021/acs.jmedchem.0c01874

Following our report that A3 adenosine receptor (AR) antagonist 1 exhibited a polypharmacological profile as a dual modulator of peroxisome proliferator-activated receptor (PPAR)γ/δ, we discovered a new template, 1′-homologated adenosine analogues 4a-4t, as dual PPARγ/δmodulators without AR binding. Removal of binding affinity to A3AR was achieved by 1′-homologation, and PPARγ/δdual modulation was derived from the structural similarity between the target nucleosides and PPAR modulator drug, rosiglitazone. All the final nucleosides were devoid of AR-binding affinity and exhibited high binding affinities to PPARγ/δbut lacked PPARα binding. 2-Cl derivatives exhibited dual receptor-binding affinity to PPARγ/δ, which was absent for the corresponding 2-H derivatives. 2-Propynyl substitution prevented PPARδ-binding affinity but preserved PPARγaffinity, indicating that the C2 position defines a pharmacophore for selective PPARγligand designs. PPARγ/δdual modulators functioning as both PPARγpartial agonists and PPARδantagonists promoted adiponectin production, suggesting their therapeutic potential against hypoadiponectinemia-associated cancer and metabolic diseases.

Full text of DOI:10.1021/acs.jmedchem.0c01874

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Article
Discovery and Structure−Activity Relationships of Novel Template,
Truncated 1′-Homologated Adenosine Derivatives as Pure Dual
PPARγ/δ Modulators
Seungchan An, Gyudong Kim, Hyun Jin Kim, Sungjin Ahn, Hyun Young Kim, Hyejin Ko,
Young Eum Hyun, Mai Nguyen, Juri Jeong, Zijing Liu, Jinhe Han, Hongseok Choi, Jinha Yu,
Ji Won Kim, Hyuk Woo Lee, Kenneth A. Jacobson, Won Jea Cho, Young-Mi Kim, Keon Wook Kang,
Minsoo Noh,* and Lak Shin Jeong*
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ABSTRACT: Following our report that A3 adenosine receptor (AR) antagonist 1
exhibited a polypharmacological profile as a dual modulator of peroxisome
proliferator-activated receptor (PPAR)γ/δ, we discovered a new template, 1′-
homologated adenosine analogues 4a−4t, as dual PPARγ/δ modulators without
AR binding. Removal of binding affinity to A3AR was achieved by 1′-
homologation, and PPARγ/δ dual modulation was derived from the structural
similarity between the target nucleosides and PPAR modulator drug, rosiglitazone.
All the final nucleosides were devoid of AR-binding affinity and exhibited high
binding affinities to PPARγ/δ but lacked PPARα binding. 2-Cl derivatives
exhibited dual receptor-binding affinity to PPARγ/δ, which was absent for the
corresponding 2-H derivatives. 2-Propynyl substitution prevented PPARδ-binding
affinity but preserved PPARγ affinity, indicating that the C2 position defines a
pharmacophore for selective PPARγ ligand designs. PPARγ/δ dual modulators
functioning as both PPARγ partial agonists and PPARδ antagonists promoted adiponectin production, suggesting their therapeutic
potential against hypoadiponectinemia-associated cancer and metabolic diseases.
been suggested with the expectation that the balanced PPAR
activation may reduce serious side effects.
INTRODUCTION
■
Metabolic diseases such as metabolic syndrome, diabetes, and
nonalcoholic steatohepatitis are associated with the disruption
of the cellular metabolic balance due to genetic or acquired
causes.1 Peroxisome proliferator-activated receptors (PPARs)
are nuclear transcriptional factors with a pivotal role in the
regulation of metabolic homeostasis. PPARs can sense the
cellular levels of fatty acids and activate the transcription of
various genes associated with the cellular response to changing
metabolic needs. Three PPAR isoforms, PPARα, PPARγ, and
PPARδ, have been identified in mammalian cells, and PPARs
show differentially expressed profiles in some tissues.2 PPARα is
highly expressed in liver, and its significant level is also detected
in all tissues. PPARγ is the major PPAR isoform in adipose
tissue, whereas PPARδ expression is mainly observed in skeletal
muscle. The heterodimeric form of PPARs with a retinoid X
receptor can bind to a peroxisome proliferator response element,
which is essential for the transcription of metabolic target genes.
PPARs regulate metabolic homeostasis, and their pharmaco-
logical ligands have been developed to treat human metabolic
diseases. However, as highly specific PPARγ or PPARδ agonists
have raised safety issues of hepatotoxicity and cardiovascular
adverse effects,3 PPAR dual- or pan-modulators have instead
Recently, we have reported that truncated 4′-thioadenosine
derivatives, acting as potent and selective A3 adenosine receptor
(AR) antagonists, also showed potent modulation of PPARs,
demonstrating their polypharmacological profiles.4 Among
these, compound 1 was discovered as a dual modulator acting
as PPARγ partial agonist and PPARδ antagonist, while
maintaining its binding affinity at the hA3AR (Figure 1).4 The
adiponectin secretion-promoting activity of 1 was found in the
phenotypic screening, exploiting the adipogenesis model of
human bone marrow mesenchymal stem cells (hBM-MSCs).
However, the effect of 1 and its analogues on adiponectin
production was not correlated to their A3AR-binding affinities.4
The target identification study revealed that 1 directly bound
both PPARγ and PPARδ. Interestingly, compound 1 signifi-
Received: October 29, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01874
© XXXX American Chemical Society
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Figure 1. Superimposed structures of rosiglitazone (magenta) and 4p
(green) inside binding pocket of PPARγ-LBD (PDB ID: 5YCP).
Amino acid residues close-contacting with 4p are presented, and
hydrogen-bonding residues are bold-highlighted.
cantly increased the nuclear receptor corepressor peptide
recruitment to the PPARδ ligand-binding domain (LBD),
whereas it did not affect the coactivator peptide recruitment, a
feature of PPARδ antagonists.4 Thus, if we can design the pure
PPAR modulators that lack binding affinity at ARs, it was
thought that the undesired effects arising from their AR activity
might be avoided, while retaining favorable effects from pure
PPAR modulation. To achieve this goal, we turned our attention
to the 1′-homologated 4′-thioadenosine analogues 2 because
they were totally devoid of binding affinity at any of the ARs.5
Furthermore, the homologated analogues showed a structural
similarity to a known thiazolidinedione PPARγ agonist,
rosiglitazone (3), which also contains one-carbon homologation
linker between the 1,3-thiazolidinedione ring and the aromatic
ring. Molecular modeling demonstrated that sugar ring and
purine ring of the target compound 2 mimic the binding mode of
the 1,3-thiazolidinedione ring and aromatic substituents of 3
against PPARγ, respectively (Figure 1).
Figure 2. Design of novel PPAR modulators lacking AR-binding affinity
by the homologation strategy.
such as troglitazone and rosiglitazone, while maintaining the
same high binding affinity as 1,3-thiazolidinediones.
As expected, our target compounds 4a−4t exhibited good
binding affinities to both PPARγ and PPARδ without binding
affinity to AR and a significant adiponectin secretion-promoting
activity during adipogenesis in hBM-MSCs through phenotypic
screening. Herein, we report the discovery and structure−
activity relationships (SARs) of novel template, truncated 1′-
homologated adenosine analogues 4a−4t as dual PPARγ/δ
modulators, and their potent adiponectin secretion-promoting
activity.
In this study, two hydroxyl groups from 4p were responsible
for generating several hydrogen bonds with Ser289, His323,
His449, and Tyr473 of PPARγ-LBD. These amino acid residues
were also involved in the binding interaction of rosiglitazone
against PPARγ-LBD. Unlike the phenyl moiety of rosiglitazone,
the nucleobase moiety can help increase the binding affinity by
forming the cation−π interaction and a noncovalent bond. A
docking score, calculated using AutoDock Vina software,
showed that 4p has a better binding affinity (−8.5 kcal/mol)
in comparison with that of rosiglitazone (−7.8 kcal/mol). The
detailed studies by using Flare are described in the Supporting
Information (Figures S28−S30).
Thus, to design the pure PPAR modulators devoid of the
binding affinity at the ARs, we synthesized the one-carbon
homologated 4′-oxo- and 4′-thioadenosine derivatives, 4a−4h
and 4m−4t with the same substituents with 1 and compared
their PPARs and AR binding affinities (Figure 2). As our
previous report4 that a hydrophobic group such as 3-halobenzyl
at the N6-position of adenine favored the adiponectin secretion-
promoting activity or PPAR-binding activity during adipo-
genesis in hBM-MSCs, we also synthesized 1-propynyl
derivatives 4i−4j to introduce a lipophilic substituent at the
C2 position of the purine ring to increase adiponectin secretion-
promoting activity during adipogenesis in hBM-MSCs. Another
reason to introduce a 1-propynyl group is to determine if steric
effects are important in binding to PPARγ and PPARδ. These
target compounds are expected to overcome the intrinsic
toxicity associated with 1,3-thiazolidinedione antidiabetic drugs
RESULTS AND DISCUSSION
■
Chemistry. The synthetic strategy for the synthesis of the
final nucleosides was first to synthesize the glycosyl donor and
then to condense it with the purine base. Truncated-
homologated 4′-thioadenosine derivatives, 4a−4h, were synthe-
sized according to our previously published procedures.5,6
As shown in Scheme 1, the glycosyl donor 105 was
synthesized, starting from D-mannose. D-Mannose was con-
verted to the diacetonide 5 by treating with 2,2-dimethox-
ypropane and acetone under acidic conditions. Reduction of 5
with sodium borohydride, followed by the treatment of the
resulting diol with mesyl chloride afforded the dimesylate 6.
Heating of 6 with sodium sulfide in dimethylformamide (DMF)
produced the 4-thiosugar 7. Regioselective hydrolysis of 5,6-
acetonide of 7 with 60% AcOH yielded 5,6-diol 8. Oxidative
cleavage of 8 with Pb(OAc)4 at 0 °C produced the aldehyde,
which without purification, was reduced with sodium borohy-
dride to give the primary alcohol 9. It is interesting to note that
oxidative cleavage using excess amounts of Pb(OAc)4 at room
temperature (rt) directly produced the 1-acetate, which was
formed via further oxidation of 8 to the acid followed by
successive oxidative decarboxylation. Treatment of 9 with
phosphorus oxychloride yielded the glycosyl donor 10.
The glycosyl donor 10 was condensed with 2,6-dichlor-
opurine and 6-chloropurine anion in DMF to give 11 and 12,
respectively, as shown in Scheme 2. Removals of the acetonide of
11 and 12 using 2 N HCl, followed by the treatment of the
B
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a
Scheme 1. Synthesis of Truncated-Homologated Glycosyl Donor 10
a
Reagents and conditions: (a) 2,2-dimethoxypropane, CSA, acetone, rt; (b) NaBH4, EtOH, rt; (c) MsCl, Et3N, DMAP, CH2Cl2, rt; (d) Na2S,
DMF, 80 °C; (e) 60% AcOH, rt; (f) Pb(OAc)4, EtOAc, 0 °C; (g) NaBH4, EtOH, 0 °C; and (h) POCl3, CH3CN, rt.
a
Scheme 2. Synthesis of Truncated 1′-Homologated 4′-Thioadenosine Derivatives, 4a−4h
a
Reagents and conditions: (a) NaH, 2,6-dichloropurine or 6-chloropurine, DMF, rt; (b) 2 N HCl, THF, rt; and (c) 3-halobenzylamines, Et3N,
EtOH, rt.
resulting diol with 3-halobenzylamines yielded the final
nucleosides, 4a−4d and 4e−4h, respectively.5
under acidic conditions to afford 14, which was subjected to
stannylation by treating with n-Bu3SnCl in the presence of
LiTMP to give the tin derivative 15. Treatment of 15 with iodine
at rt yielded 2-iodo derivative 16, which was treated with 10 mol
% CuCl2 to afford 17.
Next, we synthesized the truncated C2-propynyl-4′-thioade-
nosine derivatives 4i−4l to determine if bulky and lipophilic 1-
propynyl substituent might affect the adiponectin secretion-
promoting activity, PPAR-binding activity, or binding to PPARγ
and PPARδ. To functionalize the C2 position with the propynyl
group, using Sonogashira coupling, we synthesized the 6-chloro-
2-iodopurine 17 as the coupling substrate, as shown in Scheme
3.7 The N9 position of 6-chloropurine (13) was protected with
the tetrahydropyran group by treating with 2,3-dihydropyran
Condensation of 9 with 6-chloro-2-iodopurine (17) under
the standard Mitsunobu conditions yielded the protected
nucleoside 18 (Scheme 4). Sonogashira coupling of 18 with
propyne in the presence of CuI, Pd(PPh3)4, and Cs2CO3
produced C2-propynyl derivative 19 in 75% yield. Removal of
the acetonide of 19 with 2 N HCl, followed by treating the
resulting diol with 3-halobenzylamines yielded the truncated 1′-
homologated 2-propynyl-N6-(3-halobenzyl)adenosines 4i−4l.
Based on a bioisosteric rationale, we designed and synthesized
the corresponding 4′-oxo derivatives, 4m−4t from L-ribose, as
illustrated in Scheme 5. L-Ribose was converted to the 2,3-
acetonide 20 by treating with acetone under acidic conditions.
Protection of the primary hydroxyl group of 20 with trityl (Tr)
followed by reduction with NaBH4 afforded the diol 21.
Regioselective tosylation of the primary alcohol of 21 smoothly
converted the mono tosylate into 4-oxo sugar 22.8 Removal of
the Tr group of 22 was achieved by treating with Et2AlCl in
CH2Cl2 at −40 °C to produce the glycosyl donor 23. The
Mitsunobu condensation of 23 with 2,6-dichloropurine and 6-
chloropurine afforded the 2,6-dichloropurine nucleoside 24 and
the 6-chloropurine nucleoside 25, respectively. Treatment of 24
and 25 with 3-halobenzylamines followed by acetonide
a
Scheme 3. Synthesis of 6-Chloro-2-iodopurine 17
a
Reagents and conditions: (a) 2,3-dihydropyran, PTSA, THF, 80 °C;
(b) n-Bu3SnCl, LiTMP, THF/n-hexane (2:1), −78 °C; (c) I2, THF,
rt; and (d) 10 mol % CuCl2, EtOH/H2O (19:1), rt.
C
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PPARδ.4 Therefore, we investigated binding profiles of
compounds 4a−4t, which are structurally similar to 1, for
A3AR and PPARs (Table 1). First, their A3AR-binding affinity
was measured using the human A3AR expressed in the Chinese
hamster ovary cells. Although 1 showed high binding affinity (Ki
= 4.16 nM for A3AR), compounds 4a−4t did not significantly
bind A3AR up to 10 μM. Next, we determined whether
compounds 4a−4t directly bind PPARs by the time-resolved
fluorescence resonance energy transfer (TR-FRET)-based
nuclear receptor-binding assay (Table 1). In the TR-FRET-
based assays, compounds 4a−4t did not bind to PPARα as
compound 1 did, whereas several compounds showed high
receptor-binding activity for PPARγ and/or PPARδ. Com-
pounds 4a, 4c, 4d, and 4n−4p with 2-Cl exhibited a significant
dual receptor-binding activity against both PPARγ and PPARδ,
although their efficacy to PPARγ or PPARδ was not as potent as
positive control drugs, a PPARγ agonist pioglitazone (26), a
PPARδ agonist 2-[2-methyl-4-[[[4-methyl-2-[4-
(trifluoromethyl)phenyl]-5-thiazolyl]methyl]thio]phenoxy]-
acetic acid (27, GW501516), and a PPARδ antagonist 3-(((2-
methoxy-4-(phenylamino)phenyl)amino)sulfonyl)-2-thiophe-
necarboxylic acid methyl ester (28, GSK0660). Notably,
compounds 4i−4l with 1-propynyl substituent at the C2
position lost their binding activity to PPARδ, whereas they
retained their PPARγ-binding activity except 4j (Table 1),
indicating that the PPARδ ligand-binding pocket (LBP) was
unable to accommodate the relatively bulky substituent at the
C2 position. This result matches the previous report that the
LBP of PPARδ is smaller and narrower than that of PPARγ.9
Interestingly, there was no significant PPARγ- or PPARδ-
binding activity up to 30 μM for compounds 4e−4h and 4q−4t
with no substituent at the C2 position, indicating that proper
size of the lipophilic substituent is essential for binding activity
to PPARγ and PPARδ. In summary, 1′-homologation of 1
resulted in a dramatic loss of the A3AR-binding affinity, while
retaining the PPARγ/δ dual modulator potential (Table 1).
Effects of Compounds 4a−4t on Adiponectin Biosynthesis.
Next, the effect of compounds 4a−4t on adiponectin production
was evaluated with the adipogenesis model of hBM-MSCs
(Figure 3). hBM-MSCs have been used as a pharmacological
tool for screening adiponectin secretion-inducing compounds
by the phenotype.4,10 PPARs are known to contribute to insulin
sensitivity, in part through the regulation of synthesis and
secretion of adiponectin, an adipocyte-derived cytokine that
suppresses gluconeogenesis in the liver.11 Adiponectin has
received substantial attention because of its important role as an
integrative modulator for communication between adipose
tissue and other metabolism-related organs.11b,12 Adiponectin
has an antiatherogenic and anti-inflammatory activity as well as
an insulin-sensitizing activity.13 Thus, it has a potential in the
treatment of various diseases, including type 2 diabetes,
atherosclerosis, and cardiovascular disease.13 When 10 μM of
each compound was cotreated with the adipogenesis-inducing
medium consisting of insulin, dexamethasone, and 3-isobutyl-1-
methylxanthine (IDX medium) for 5 d, compounds 4a, 4c, 4d,
4i, 4k, 4l, 4n, 4o, and 4p significantly promoted adiponectin
production compared to the IDX vehicle control during
adipogenesis in hBM-MSCs, indicating the correlation with
PPAR-binding affinity (Figure 3A). As expected, compounds
4e−4h and 4q−4t with no substituent at the C2 position, which
are also inactive at PPARs, could not significantly promote
adiponectin production (Figure 3A). In phenotypic analysis
with potent adiponectin secretion-promoting PPARγ/δ dual
Scheme 4. Synthesis of Truncated 1′-Homologated 2-
a
Propynyl-4′-thioadenosine Derivatives, 4i−4l
a
Reagents and conditions: (a) PPh3, DIAD, THF, 0 °C, 1 h; 6-chloro-
2-iodopurine (17); (b) propyne, CuI, Pd(PPh3)4, Cs2CO3, DMF, rt;
(c) 2 N HCl, THF, rt; and (d) 3-halobenzylamines, Et3N, EtOH, rt.
Scheme 5. Synthesis of Truncated 1′-Homologated 4′-
a
Oxoadenosine Derivatives, 4m−4t
a
Reagents and conditions: (a) cH2SO4, acetone, rt, 3 h; (b) trityl
chloride, DMAP, pyridine, 80 °C, 3 h; (c) NaBH4, MeOH, rt, 1 h; (d)
TsCl, pyridine, rt, 12 h; (e) Et2AlCl, CH2Cl2, −40 °C, 3 h; (f) PPh3,
DIAD, THF, rt, 12 h; 2,6-dichloropurine or 6-chloropurine; (g) 3-
halobenzylamines, Et3N, THF, rt; and (h) 1 N HCl, MeOH, rt.
deprotection yielded the truncated 1′-homologated 4′-oxoade-
nosine derivatives, 4m−4t.
Biological Evaluation. PPAR-Binding Profiles of Com-
pounds 4a−4t. Compound 1 has a polypharmacological
receptor-binding profile recognizing A3AR, PPARγ, and
D
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Table 1. Receptor-Binding Profiles of Truncated 1′-Homologated Adenosine Derivatives 4a−4t
b
hA3AR Ki (nM) or %
PPARα % replacement at
30 μM (%)
PPARγ Ki (μM) or %
PPARδ Ki (μM) or %
a
b
b
cpd
X
Y
R
replacement
replacement
replacement
1
4.16
35.6
15.5
12.8
3.0
20.4
25.6
7.7
3.33 1.67
3.89 0.47
34.1% at 30 μM
4.07 0.86
1.23 0.25
24.4% at 30 μM
24.9% at 30 μM
33.5% at 30 μM
39.2% at 30 μM
7.66 0.65
41.7% at 30 μM
7.04 0.40
7.30 1.01
0.0106 0.015
1.91 0.39
7.8% at 30 μM
0.592 0.26
4a
4b
4c
4d
4e
4f
4g
4h
4i
S
S
S
S
S
S
S
S
S
S
S
S
O
O
O
O
O
O
O
O
Cl
Cl
Cl
Cl
H
H
H
H
F
17% at 10 μM
13% at 10 μM
21% at 10 μM
10% at 10 μM
9% at 10 μM
9% at 10 μM
6% at 10 μM
14% at 10 μM
2% at 10 μM
1% at 10 μM
12% at 10 μM
2% at 10 μM
2% at 10 μM
1% at 10 μM
14% at 10 μM
3% at 10 μM
1% at 10 μM
1% at 10 μM
14% at 10 μM
3% at 10 μM
ND
Cl
Br
I
0.106 0.031
0.8% at 30 μM
10.0% at 30 μM
9.9% at 30 μM
5.2% at 30 μM
13.8% at 30 μM
3.8% at 30 μM
15.3% at 30 μM
15.9% at 30 μM
7.3% at 30 μM
0.504 0.29
F
Cl
Br
I
5.4
9.4
−CCCH3
F
15.5
12.6
12.8
24.0
5.0
3.6
23.2
0.3
1.4
8.3
5.6
1.7
4j
4k
4l
−CCCH3
Cl
Br
I
−CCCH3
−CCCH3
4m
4n
4o
4p
4q
4r
4s
4t
Cl
Cl
Cl
Cl
H
F
9.0% at 30 μM
7.66 2.45
9.55 6.61
Cl
Br
I
1.43 0.67
3.56 0.75
0.374 0.036
14.6% at 30 μM
10.6% at 30 μM
16.5% at 30 μM
6.5% at 30 μM
14.7% at 30 μM
0.00608 0.0055
0.00465 0.0023
F
13.3% at 30 μM
0.8% at 30 μM
4.1% at 30 μM
16.0% at 30 μM
0.096 0.049
38.5% at 30 μM
59.9% at 30 μM
H
H
H
Cl
Br
I
26
27
28
3.5
44.9
12.4
ND
ND
a
b
Binding affinities of 4a−4h to human A3 AR were previously reported.6,23 Binding affinities to PPARs were determined based on TR-FRET-
based PPAR competitive binding assays. Ki, inhibition constant; ND, not determined.
modulators, compounds 4d and 4p significantly promoted lipid
droplet formation during adipogenesis in hBM-MSCs compared
to the IDX control (Figure 3B). In concentration-effect analysis
for the adiponectin production phenotypic assay, the half-
maximal effective concentration values (EC50) of compounds 4d
and 4p were 9.20 and 16.14 μM, respectively (Figure 3C).
Evaluation of a Pharmacological Profile of PPARγ/δ Dual
Modulators. One of the polypharmacological features of 1 is a
PPARγ partial agonist.4 To investigate how compounds 4a−4t
functionally affected PPARγ activation, a TR-FRET-based
PPARγ coactivator assay was performed for compounds 4a−
4d, 4i−4l, and 4m−4p, which showed a significant PPARγ-
binding activity (Figure 4). The level of interaction between the
PPARγ-LBD and a fluorescein-labeled coactivator peptide
thyroid hormone receptor-associated protein 220/vitamin D
receptor interacting protein (TRAP220/DRIP) derived from
MED1, a nuclear receptor coactivator encoded by MED1 gene,
was measured.14 MED1, a component of the mediator complex,
ligand dependently interacts with PPARγ and other nuclear
receptors and regulates transcription of RNA polymerase II-
dependent genes, by directly facilitating promoter recruitment
and function of RNA polymerase II.15 MED1 is also known to be
essential for PPARγ-mediated adipogenesis in mouse embryonic
fibroblasts.16 Compounds 4a, 4c, 4d, 4i−4l, 4n, 4o, and 4p
significantly increased the coactivator peptide recruitment.
However, the effects were less potent compared to those of
PPARγ agonist controls, N-(2-benzoylphenyl)-O-[2-(methyl-2-
pyridinylamino)ethyl]-L-tyrosine hydrochloride (29, GW1929)
and troglitazone (30) (Figure 4A). It is interesting to note that
the binding affinity of 4j was weaker than that of 4i, 4k, and 4l,
but 4j can still function as a PPARγ partial agonist. Because the
maximum binding efficacy of 4j was around 40% of GW1929
(PPARγ full agonist), this binding level was enough for 4j to
modulate the PPARγ function. It should be mentioned that
compounds showing weak activity tend to show more biological
variations, and 4j with weak activity had no statistical
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competitive antagonist against its full agonist.17 When
compound 4d was cotreated with a PPARγ full agonist 30 in
the TR-FRET-based PPAR coactivator assay, compound 4d
competitively inhibited PPARγ coactivation of 30 (Figure 4D).
Therefore, compound 4d and other PPARγ-binding compounds
function as a PPARγ partial agonist.
As the second polypharmacological feature of 1 is a PPARδ
antagonist,4 we next examined whether the PPARδ binding
compounds function as antagonists (Figure 5). To determine a
functional outcome for the PPARδ antagonism, a TR-FRET-
based PPARδ coactivator assay was performed. A fluorescein-
labeled peptide, the interaction domain 2 (ID2) of silencing
mediator of retinoid and thyroid hormone receptors (SMRT),
was used as a corepressor and peptide C33 was used as a
coactivator.18 SMRT, also known as nuclear receptor
corepressor 2, can control various adipogenic set points and
whole-body metabolic homeostasis by repressing nuclear
receptors.19 Compounds 4a, 4c, 4d, 4n, 4o, and 4p significantly
increased the interaction between PPARδ-LBD and the
corepressor peptide, SMRT-ID2 (Figure 5A), whereas these
compounds had no effect on the recruitment of coactivator
peptide (Figure 5B). Compounds 4d and 4p showed
concentration-dependent effects on the corepressor peptide
binding to PPARδ-LBD, and their EC50 values were 12.53 and
20.20 μM, respectively (Figure 5C). Importantly, the levels of
the corepressor peptide recruitment to PPARδ-LBD by
compounds 4a−4d and 4m−4p showed a significant correlation
to the adiponectin secretion-promoting activity (R2 = 0.80, p <
0.001) (Figure 5D).
Figure 3. Effects of truncated 1′-homologated adenosine derivatives
4a−4t on adiponectin biosynthesis. (A) hBM-MSCs were grown under
IDX conditions and cotreated with 26 or compounds 4a−4t. On the
5th day of adipogenesis induction, supernatants were harvested and
adiponectin ELISA was performed. (B) ORO staining was performed
to visualize lipid droplets after the 5-day adipogenesis induction. (C)
Concentration-effect analysis was performed for compounds 4d and 4p.
Results are the mean SD of three measurements using hBM-MSCs
from three independent cultures (n = 3, three independent experi-
ments); (*) p ≤ 0.05 and (**) p ≤ 0.01.
significance in coactivation analyses (Figure 4A). In the Pearson
correlation analysis, the levels of the coactivator peptide
recruitment on PPARγ-LBD by compounds 4a−4d, 4i−4l,
and 4m−4p were significantly correlated to their adiponectin
secretion-promoting activity (R2 = 0.71, p < 0.001) (Figure 4B).
When the maximal activity for the coactivator peptide
recruitment by a PPARγ full agonist 29 was taken as the 100%
effect, EC50 values of compounds 4d and 4p were calculated as
6.85 and 9.95 μM, respectively (Figure 4C). Next, we evaluated
whether compound 4d had characteristics of a PPARγ partial
agonist because 1 exhibited a PPARγ partial agonism among its
polypharmacological properties (Figure 4D). By pharmaco-
logical definition, a receptor partial agonist functions as a
Next, we investigated whether the PPARδ antagonism of 4d
was affected by a specific PPARδ agonist 27 (Figure 5E). The
recruitment of corepressor peptides by a PPARδ antagonist 28
was competitively inhibited by 27. The effect of 4d on the
PPARδ corepressor recruitment was also significantly inhibited
by 27 (Figure 5E). This result supported that 4d was a PPARδ
antagonist. In conclusion, compound 4d and other PPARγ/δ
compounds were dual modulators functioning as PPARγ partial
agonists and PPARδ antagonists.
In Vivo Study of 4p in Noninsulin-Dependent Type 2
Diabetes Mellitus. A leptin-deficient ob/ob mouse model is
commonly used for in vivo study of noninsulin-dependent type 2
Figure 4. Effects of truncated 1′-homologated adenosine derivatives on PPARγ coactivation. (A) TR-FRET-based PPARγ coactivation assay was
performed using fluorescein-TRAP220/DRIP peptide. PPARγ agonists 29 and 30 were used as positive controls. The degree of the recruiting
coactivator peptide to PPARγ-LBD was normalized to % coactivation. (B) Coefficient of determination (R2) between the PPARγ coactivation activity
and the level of relative adiponectin secretion-promoting activity was calculated using the Pearson correlation analysis. (C) Concentration-dependent
effects of 4d and 4p on the TRAP220/DRIP peptide-based PPARγ coactivation were investigated. (D) Evaluation of the PPARγ partial agonism of 4d.
Results are the mean SD of three measurements (n = 3); (*) p ≤ 0.05 and (**) p ≤ 0.01.
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Figure 5. Effects of truncated 1′-homologated adenosine derivatives on PPARδ coregulation. (A) TR-FRET-based PPARδ corepression assay was
performed using fluorescein-SMRT corepressor peptide. A PPARδ agonist 27 and a PPARδ antagonist 28 were used as positive or negative controls.
The degree of the recruiting corepressor to PPARγ-LBD was normalized to % corepression. (B) TR-FRET-based PPARδ coactivation assay was
performed using coactivator C33. (C) Concentration-dependent effects of 4d and 4p on the PPARδ corepression were investigated. (D) Pearson’s
correlation coefficient (R2) between the PPARδ corepression activity and the level of relative adiponectin secretion-promoting activity was calculated
using the Pearson correlation analysis. (E) Effects of PPARδ agonist 27 (1 μM) on PPARδ antagonism of 4d (10 μM) and 28 (1 μM). Results are the
mean SD of three measurements (n = 3); (*) p ≤ 0.05 and (**) p ≤ 0.01.
diabetes mellitus (T2D).20 We evaluated efficacy of 4p on
glucose tolerance and insulin sensitivity of obese−diabetic ob/ob
mice. 7 week old ob/ob mice were administered 30 mg/kg of 4p
six times per week. Mice orally administered with pioglitazone
were used as positive control. Glucose response curves during an
oral glucose tolerance test (OGTT) were plotted after 3 and 6
weeks. The area under the curve of OGTT showed no difference
between ob/ob and control mice, and ob/ob mice received 4p.
Unfortunately, 4p administration for 6 weeks did not result in
improving fasting glucose levels of ob/ob mice (see the
Supporting Information; Figure S1). To examine the effects of
4p on T2D in more detail, the serum insulin level was measured
through enzyme-linked immunosorbent assay (ELISA).
Although the blood glucose levels were not significantly
different, serum insulin levels of fed conditioned ob/ob mice
were decreased after administration of 4p (Figure 6A). In
addition, no weight gain was observed in the 4p administered
group (Figure 6B). Although 4p administration could not
normalize blood glucose levels of ob/ob mice, decreased serum
insulin levels demonstrate potential use of 4p as a molecular
entity for treatment of T2D.
Docking Analysis on the SAR of PPARγ/δ Dual Modulators.
The 1′-homologation of 1 resulted in the loss of the A3AR-
binding affinity. To investigate the structural basis of A3AR
activity of the target compounds, a homology model of the A3AR
was constructed based on the human A2AAR structure in an
active conformation (PDB ID: 5G53), and the optimum binding
mode of 1 was determined by protein−ligand docking analysis
(Figure 7B).21 A2AAR and A3AR showed 43.45% of sequence
identity, indicating that the structure of A3AR can be predicted
from the known A2AAR structure with an accuracy equivalent to
a low-resolution X-ray structure.22 In the optimum binding
mode, the adenine ring of 1 formed the π−π stacking interaction
with Phe168 of extracellular loop 2 and interacted with amino
acid residues Thr94, Ser271, and His272 through 4′-thiosugar
(Figure 7B,C). Such interaction patterns of ligands in the
binding pocket were observed in many cases including A2AAR-
ligand co-crystallized structures and conserved in other AR
subtypes.23,24 Notably, it was reported that the site-directed
mutagenesis on Thr94 or His272 residue can significantly
decrease the binding affinity of A3AR ligands regardless of their
agonistic or antagonistic features.24 In contrast to the case of 1, it
was difficult to determine the optimum docking model of
compound 4d against the homology-modeled A3AR because of
the lack of the A3AR-binding affinity. Among the various
docking modes of 4d to A3AR, we selected the docking model in
which the adenine ring of compound 4d formed a π−π stacking
with Phe168 for comparing with the docking model of 1 (Figure
7A,B). The binding stabilization of 1 and 4d with the A3AR,
calculated using AutoDock Vina, was −9.0 kcal/mol and −8.0
kcal/mol, respectively. The introduction of the methylene group
in 1 made it harder to achieve a close contact with Thr94 and
His272 (Figure 7C,D). In addition, the distance between the 3′-
OH of 4d and the donor hydrogen of His272 was 6.5 Å, too far
to form a hydrogen bonding interaction (Figure 7D), whereas
the distance in the model of 1 was 2.9 Å (Figure 7C). Therefore,
the lack of interaction with residues important for activity was
Figure 6. Effects of 4p on insulin sensitivity in ob/ob mice. (A) Fed
serum insulin levels were analyzed through the mouse insulin ELISA kit.
(B) Weight gain was calculated using body weights of mice prior to the
experiment and after 6 weeks of administration. **p < 0.01 compared to
lean mice; #p < 0.05 compared to vehicle-administered ob/ob mice.
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Figure 7. Molecular docking modes of compound 4d against A3AR, PPARγ, and PPARδ. (A) Optimum binding mode of compound 4d was generated
in the A3AR homology model constructed based on the A2AAR structure (PDB ID: 5G53). TM, transmembrane. (B) Selected docking mode of
compound 4d (green) for the comparison with A3AR antagonist 1 (cyan), forming π−π stacking with Phe168 with the adenine ring. Major amino acid
residues interacting with compound 4d in the optimum docking mode against the A3AR homology model were labeled. The amino acid residues
closely contacting with ligand (<van der Waals radii) or forming hydrogen bond with ligand are labeled. TM2 is shown transparently for clarity. The
residue numbers were assigned following Ballesteros−Weinstein notation.27 (C,D) Comparison of binding models of 1 (cyan, C) and 4d (green, D) in
the homology-modeled A3AR. Representative residues important for A3AR binding are shown in sticks. Distances between the hydroxyl group of
thiosugar of ligands and hydrogen of His272 were measured, given in Ångstrom. (E) Putative binding modes suggested by docking simulation of 3
(magenta) and 4d against PPARγ-LBD (PDB ID: 5YCP). Binding models of 4d and 3 were overlaid. (F−I) Putative binding modes suggested by
docking simulation of 4d (F), 4l (G), 4h (H) against PPARδ-LBD (PDB ID: 5U46). (I) Comparison of binding models of 27 (orange) and 4d in
PPARδ-LBP. H, helix. Possible hydrogen bonds are shown in yellow dashes. Binding free energies calculated by AutoDock Vina are presented in kcal/
mol. All molecular graphics works were conducted using PyMOL software.
responsible for the decrease of A3AR-binding affinity of 4d. One-
carbon homologation exhibited significant outcomes to induce
the loss of the A3AR-binding activity.
or maintain such binding models, resulting in weaker binding
affinities compared to 4d (Figures 7G,H). Therefore, the
docking simulation study further supported that compound 4d
and its congeners were PPARγ/δ dual modulators, agonists for
PPARγ and antagonists against PPARδ.
Next, to understand the molecular basis for the SAR of
PPARγ/δ dual modulators, the binding modes for compounds
4d, 4h, and 4l against PPAR-LBDs were compared (Figure 7).
The docking simulations of these compounds were performed
with receptor 3-dimensional coordinates prepared from the
PPARγ-LBD/3 complex structure (PDB ID: 5YCP).25 Notably,
one-carbon homologation of the truncated adenosine deriva-
tives was designed to improve PPARγ activity by structurally
mimicking PPARγ full agonists like pioglitazone and rosiglita-
zone. In the energy-minimized docking model for the PPARγ-
LBP, compound 3 or 4d interacted with amino acid residues in
both the hydrophilic and hydrophobic pockets interacting with
Tyr473 of helix (H) 12 (Figures 1 and 7E).
Similarly, PPARδ agonist 27-bound PPARδ-LBD was
processed for docking analysis (PDB ID: 5U46). In the docking
models against PPARδ-LBD, the 3-iodobenzyl group of 4d
interacted hydrophobically with Val312, the essential amino
acid residue significantly affecting the PPARδ ligand binding
(Figure 7F).26 In addition, when compared with those of well-
known PPARδ agonist 27, compound 4d in the optimum
binding mode for the PPARδ binding was preferentially located
between H3 and β-sheets, lacking an interaction with Tyr437,
the amino acid residue important for the potency of PPARδ
agonists through stabilizing H12 (Figure 7I).26 Meanwhile, 2-Cl
of 4d was used as an anchor, forming a halogen bond with
backbone nitrogen atoms of Lys229 and Gln230 on H2′−H3
loop of PPARδ-LBD. Compound 4d and its derivatives may
have acted as antagonists by tightly occupying the space between
H3 and the β-sheet, preventing PPARδ ligands from interacting
with Val312 and Tyr437. On the contrary, compound 4l with
bulky 2-propyne and compound 4h without 2-Cl could not form
Therapeutic Potential of Adiponectin Secretion-Promot-
ing PPARγ/δ Dual Modulators. Compound 4d and its related
compounds exhibited the unique PPAR dual modulator profile,
which functioned as both PPARγ partial agonists and PPARδ
antagonists. Most of PPAR dual- or pan-modulators have been
developed as co-agonists with the expectation that the balanced
PPAR activation may reduce serious side effects caused by
highly specific PPARγ or PPARδ agonists. In fact, many selective
PPARγ agonists and even PPARα/γ dual agonists were
discontinued from their therapeutic use or clinical development
because of their cardiovascular risks or other safety issues.3,28 In
addition, PPARδ agonists have raised the controversial issues
regarding to their increase in cancer risk.29 Compound 4d and
its related compounds with the dual pharmacological profile of a
partial agonist for PPARγ and an antagonist against PPARδ may
have therapeutic benefits which are different from those of other
dual- or pan-PPAR modulators.
Adiponectin secretion-promoting compound 4d may have
the potential to treat various diseases associated with
hypoadiponectinemia such as metabolic syndrome and cancer.
It has been reported that the exogenous treatment of
adiponectin improved the pathogenic phenotypes in animal
models for atherosclerosis and nonalcoholic fatty liver
diseases.30 Thus, the upregulation of adiponectin levels by
adiponectin secretion-promoting PPARγ/δ dual modulators
may provide therapeutic benefits to these metabolic conditions.
Interestingly, a PPARγ partial agonist and a PPARδ antagonist
may synergistically contribute to treat cancer. It has been
reported that the activation of PPARγ induced apoptosis of
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cancer cells.31 PPARδ antagonists have been suggested as new
drugs for antiproliferative therapeutics32 because a specific
PPARδ agonist GW501516 significantly induced tumors in
animal models.2a,29a Currently, the therapeutic potential of
PPARδ antagonists has not been yet fully investigated compared
with those of other PPAR modulators. Recently, it was reported
that PPARδ antagonists were effective to attenuate the psoriasis-
like pathogenic outcomes in a transgenic animal model.33
Psoriasis is a hyperproliferative skin disease, so the antipsoriatic
activity of PPARδ antagonists may be reasonably expected. In
this regard, the anticancer potential of a PPARγ/δ dual
modulator 4d should be further investigated.
(1.50 g, 5.76 mmol) in EtOH (25 mL) was added, cautiously in several
portions, sodium borohydride (440 mg, 11.53 mmol) at 0 °C under a
N2 atmosphere, and the mixture was stirred at rt for 2 h. The mixture
was neutralized with acetic acid and evaporated to remove solvent. The
mixture was partitioned between EtOAc and water, and the organic
layer was dried with anhydrous MgSO4, filtered, and evaporated. The
residue was purified by flash silica gel column chromatography (n-
hexanes/EtOAc = 1:1) to give the diol 11 (1.38 g, 92%) as a syrup: 1H
NMR (CDCl3, 400 MHz): δ 4.33 (dd, J = 1.6, 7.2 Hz, 1H), 4.24−4.28
(m, 1H), 4.06−4.13 (m, 2H), 3.92−3.97 (m, 1H), 3.76−3.85 (m, 2H),
3.59−3.61 (m, 1H), 1.48 (s, 3H), 1.38 (s, 3H), 1.36 (s, 3H), 1.33 (s,
3H); 13C NMR (CDCl3, 100 MHz): δ 110.5, 109.5, 79.2, 77.6, 77.5,
71.4, 68.4, 62.0, 27.1, 25.7, 25.4; [α]2D5 −3.88 (c 0.44, CH2Cl2); FAB-
MS m/z: 263 (M + H+); Anal. Calcd for C12H22O6: C, 54.95; H, 8.45.
Found: C, 54.90; H, 8.35.
Mesylation. To a stirred solution of diol X (38.52 g, 146.85 mmol)
and 4-DMAP (5.38 mg, 44.06 mmol) in a mixture of CH2Cl2 (300 mL)
and Et3N (163.75 mL, 1.17 mol) was added cautiously dropwise
methanesulfonyl chloride (47.59 mL, 587.42 mmol) at 0 °C under a N2
atmosphere. After being stirred for 1 h, at rt, the mixture was diluted
with CH2Cl2 and washed with saturated NaHCO3 solution. The
organic layer was dried with anhydrous MgSO4, filtered, and evaporated
to give the dimesylate compounds as brownish syrup. It was purified by
flash silica gel column chromatography (n-hexanes/EtOAc = 5:1) to
give a dimesylate 6 (57.83 g, 94%) as a syrup; 1H NMR (CDCl3, 400
MHz): δ 4.75 (pseudo t, J = 7.4 Hz, 1H), 4.33−4.45 (m, 4 H), 4.06−
4.20 (m, 3H), 3.12 (s, 3H), 3.07 (s, 3H), 1.51 (s, 3H), 1.43 (s, 3H), 1.37
(s, 3H), 1.33 (s, 3H); [α]D25 +38.32 (c 0.29, CH2Cl2); FAB-MS m/z:
419 (M + H+); Anal. Calcd for C14H26O10S2: C, 40.18; H, 6.26; S,
15.32. Found: C, 39.99; H, 6.65.
CONCLUSIONS
■
In this study, novel PPARγ/δ dual modulators functioning as
both PPARγ partial agonists and PPARδ antagonists were
designed and synthesized. These pure PPARγ/δ dual modu-
lators were discovered from the polypharmacology of 1 that can
simultaneously affect A3AR, PPARγ, and PPARδ, as well as the
structural similarity between the target compounds 4a−4t and
1,3-thiazolidinedione antidiabetic drugs. Compounds 4d and 4p
were discovered as the most promising compounds in 4′-thio-
and 4′-oxonucleosides, respectively, whose adiponectin secre-
tion-promoting PPARγ/δ dual modulators may have therapeu-
tic potential against cancer and metabolic diseases, and
pharmacological studies to demonstrate their therapeutic
benefits may be required in the near future.
(3aR,4S,6aS)-4-((R)-2,2-Dimethyl-1,3-dioxolan-4-yl)-2,2-
dimethyltetrahydrothieno[3,4-d][1,3]dioxole (7). To a stirred sol-
ution of dimesylate (933.80 mg, 2.23 mmol) in DMF (50 mL) was
added sodium sulfide (348.30 g, 4.46 mmol), and the mixture was
stirred with heating at 80 °C for overnight under a N2 atmosphere. After
the solvent was removed under reduced pressure, the residue was
partitioned between EtOAc and water. The organic layer was dried with
anhydrous MgSO4, filtered, and evaporated. The residue was purified
by flash silica gel column chromatography (n-hexanes/EtOAc = 8:1) to
give 7 (453.0 mg, 78%) as a syrup: 1H NMR (CDCl3, 400 MHz): δ 4.92
(dt, J = 1.8, 5.6 Hz, 1H), 4.72 (dd, J = 2.0, 6.0 Hz, 1H), 4.26−4.30 (m,
1H), 4.04 (s, 1H), 3.79 (t, J = 3.8 Hz, 1H), 3.31−3.32 (m, 1H), 3.19
(dd, J = 5.4, 12.0 Hz, 1H), 2.84 (dd, J = 1.6, 12.0 Hz, 1H), 1.51 (s, 3H),
1.43 (s, 3H), 1.32 (dd, J = 8.4 Hz, 6 H); 13C NMR (CDCl3): δ 111.5,
109.8, 87.3, 84.0, 78.0, 68.3, 56.3, 56.2, 38.8, 26.8, 26.3, 25.8, 24.9; [α]D25
−96.04 (c 0.20, CH2Cl2); FAB-MS m/z: 261 (M + H+); Anal. Calcd for
C12H20O4S: C, 55.36; H, 7.74; S, 12.32. Found: C, 55.29; H, 7.85; S,
12.20.
(R)-1-((3aR,4S,6aS)-2,2-Dimethyltetrahydrothieno[3,4-d][1,3]-
dioxol-4-yl)ethane-1,2-diol (8). A solution of 7 (21.78 g, 83.66 mmol)
in 60% aqueous AcOH (250 mL) was stirred at rt for 2 h. The reaction
mixture was evaporated under reduced pressure, and the resulting
residue was purified by flash silica gel column chromatography (n-
hexanes/EtOAc = 1:2) to give the diol 8 (14.85 g, 81%) as a white solid,
and 6.15 g of the starting material 13 was recovered after being repeated
for three times: mp 100.7−101.8 °C; 1H NMR (CDCl3, 400 MHz): δ
4.93 (dt, J = 1.8, 5.6 Hz, 1H), 4.76 (dd, J = 2.0, 5.6 Hz, 1H), 3.76−3.80
(m, 1H), 3.70−3.74 (m, 1H), 3.64−3.68 (m, 1H), 3.40 (dd, J = 1.6, 5.2
Hz, 1H), 3.16 (dd, J = 5.0, 12.8 Hz, 1H), 2.90 (dd, J = 1.6, 12.8 Hz, 1H),
1.52 (s, 3H), 1.33 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 111.6, 87.3,
84.0, 72.3, 65.3, 57.7, 32.0, 26.9, 24.9; [α]2D5 −52.38 (c 0.13, CH2Cl2);
FAB-MS m/z: 221 (M + H+); Anal. Calcd for C9H16O4S: C, 49.07; H,
7.32; S, 14.56. Found: C, 49.47; H, 7.72; S, 14.15.
EXPERIMENTAL SECTION
■
Chemical Synthesis. General Methods. Proton (1H) and carbon
(13C) NMR spectra were obtained on a Bruker AV 400 (400/100
MHz), Bruker AMX 500 (500/125 MHz), Jeol JNM-ECA600 (600/
150 MHz), or Bruker AVANCE III 800 (800/200 MHz) spectrometer.
Chemical shifts are reported as parts per million (δ) relative to the
solvent peak. Coupling constants (J) are reported in hertz (Hz). Mass
spectra were recorded on a Thermo LCQ XP instrument. 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 Fourier transform infrared
(FTS-135) made by Bio-Rad. Melting points were determined on a
Buchan B-540 instrument and are uncorrected. The crude compounds
were purified by column chromatography on a silica gel (Kieselgel 60,
70−230 mesh, Merck). 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. High-
performance liquid chromatography analysis was also performed to
determine the purity of the most promising compounds 4d and 4p,
confirming ≥95% purity. All animal experiments were performed in
compliance with by Institutional Animal Care and Use Committee
(IACUC, SNU-200605-5-1) in the Seoul National University.
(3aS,6R,6aS)-6-((R)-2,2-Dimethyl-1,3-dioxolan-4-yl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-ol (5). To a stirred
suspension of D-mannose (1.74 g, 6.52 mmol) and 2,2-dimethox-
ypropane (2.45 mL, 19.55 mmol) in acetone (50 mL) was added
portionwise camphosulfonic acid (0.45 g, 1.96 mmol) at 0 °C under a
N2 atmosphere. After being stirred for 24 h at rt, the mixture was
neutralized with Et3N and evaporated under reduced pressure. The
resulting residue was purified by flash silica gel column chromatography
(n-hexanes/EtOAc = 1:1) to give 5 (1.61 g, 95%) as a white solid: mp
120.3−120.5 °C; 1H NMR (CDCl3, 400 MHz): δ 5.34 (s, 1H), 4.76−
4.79 (m, 1H), 4.58 (d, 1H, J = 6.0 Hz), 4.34−4.39 (m, 1H), 4.15 (dd,
1H, J = 3.6, 7.2 Hz), 4.00−4.08 (m, 2H); [α]2D5 +11.71 (c 0.11,
CH2Cl2); FAB-MS m/z: 261 (M + H+); Anal. Calcd for C12H20O6: C,
55.37; H, 7.74. Found: C, 55.30; H, 7.75.
((3aR,4S,6aS)-2,2-Dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-
yl)methanol (9). To a stirred solution of 8 (20 g, 90.8 mmol) in ethyl
acetate (500 mL) was added Pb(OAc)4 (48.3 g, 109 mmol) at 0 °C
under a N2 atmosphere, and the reaction mixture was stirred for 10 min
at which time thin-layer chromatography (TLC) indicated the absence
of the starting material. The reaction mixture was filtered, and the
filtrate was diluted with EtOAc. The organic layer was washed with
((R)-2,2-Dimethyl-1,3-dioxolan-4-yl)((4R,5R)-2,2-dimethyl-5-
(((methylsulfonyl)oxy)methyl)-1,3-dioxolan-4-yl)methyl Methane-
sulfonate (6). Reduction. To a stirred solution of diacetonide 5
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saturated aqueous NaHCO3 solution, dried over anhydrous MgSO4,
and evaporated to give the aldehyde intermediate, which was used for
the next step without further purification. To a stirred solution of
aldehyde intermediate (5.6 g, 30.0 mmol) in EtOH (70 mL) was
carefully added sodium borohydride (1.3 g, 33.6 mmol) in several
portions at 0 °C, and the reaction mixture was stirred for 30 min at the
same temperature and neutralized with glacial AcOH. After the removal
of the solvent, the mixture was partitioned between EtOAc (150 mL)
and brine (100 mL). The organic layer was dried over anhydrous
MgSO4, filtered, and evaporated. The resulting residue was purified by
silica gel column chromatography (n-hexanes/EtOAc = 2:1) to give 9
(5.2 g, 91%) as a colorless syrup: 1H NMR (CDCl3, 400 MHz): δ 1.27
(s, 3H), 1.47 (s, 3H), 2.68 (br s, 1H), 2.84 (dd, J = 2.0, 12.6 Hz, 1H),
3.06 (dd, J = 5.2, 13.0 Hz, 1H), 3.38 (m, 1H), 3.56 (dd, J = 2.8, 8.8 Hz,
2H), 4.66 (dd, J = 1.6, 5.6 Hz, 1H), 4.86 (td, J = 1.6, 4.8 Hz, 1H); 13C
NMR (CDCl3): δ 111.2, 85.9, 83.7, 63.3, 56.6, 26.6, 24.7; FAB-MS m/
z: 191 (M + H+).
chromatography (CH2Cl2/EtOAc/MeOH = 10:10:1) to give the N6-
substituted amine derivatives 4a−h.
(2S,3R,4S)-2-((2-Chloro-6-((3-fluorobenzyl)amino)-9H-purin-9-
yl)methyl)tetrahydrothiophene-3,4-diol (4a). Yield = 82%; white
solid; mp 164−167 °C; UV (MeOH) λmax: 275 nm (pH 7); [α]2D5 +48.0
(c 0.50, MeOH); 1H NMR (DMSO-d6, 400 MHz): δ 8.83 (br s, 1H),
8.17 (s, 1H), 3.65 (m, 1H), 7.74 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.36
(d, J = 7.6 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 5.09 (d, J = 6.0 Hz, 1H),
5.03 (d, J = 4.8 Hz, 1H), 4.60 (br d, J = 5.2 Hz, 2H), 4.43 (dd, J = 6.4,
14.0 Hz, 1H), 4.16 (m, 2H), 3.78 (m, 1H), 2.89 (dd, J = 4.8, 10.4 Hz,
1H), 2.61 (dd, J = 4.8, 10.8 Hz, 1H); 13C NMR (DMSO-d6, 100 MHz):
δ 154.7, 152.9, 149.9, 141.9, 141.6, 136.0, 135.5, 130.5, 126.8, 118.0,
94.7, 77.1, 73.5, 48.3, 47.0, 42.5, 32.9; FAB-MS m/z: 410 (M + H+);
Anal. Calcd for C17H17ClFN5O2S: C, 49.82; H, 4.18; N, 17.09; S, 7.82.
Found: C, 49.94; H, 4.25; N, 17.12; S, 7.85.
(2S,3R,4S)-2-((2-Chloro-6-((3-chlorobenzyl)amino)-9H-purin-9-
yl)methyl)tetrahydrothiophene-3,4-diol (4b). Yield = 77%; white
solid; mp 154−158 °C; UV (MeOH) λmax: 275 nm (pH 7); [α]2D5 +77.5
(c 0.40, MeOH); 1H NMR (DMSO-d6, 400 MHz): δ 8.84 (br s, 1H),
8.17 (s, 1H), 7.55 (s, 1H), 7.42 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 7.6 Hz,
1H), 7.27 (t, J = 7.6 Hz, 1H), 5.09 (d, J = 5.6 Hz, 1H), 5.04 (d, J = 4.8
Hz, 1H), 4.63 (br d, J = 6.5 Hz, 2H), 4.44 (dd, J = 6.0, 14.0 Hz, 1H),
4.11−4.20 (m, 2H), 3.78 (m, 1H), 3.65 (m, 1H), 2.90 (dd, J = 4.8, 10.8
Hz, 1H), 2.62 (dd, J = 4.8, 10.6 Hz, 1H); 13C NMR (DMSO-d6, 100
MHz): δ 154.7, 152.9, 149.9, 142.0, 141.6, 130.5, 130.1, 129.7, 126.4,
121.5, 118.0, 77.1, 73.5, 48.3, 47.0, 42.6, 32.9; FAB-MS m/z: 426 (M+);
Anal. Calcd for C17H17Cl2N5O2S: C, 47.89; H, 4.02; N, 16.43; S, 7.52.
Found: C, 47.92; H, 4.05; N, 16.50; S, 7.55.
(3aR,4R,6aS)-4-(Chloromethyl)-2,2-dimethyltetrahydrothieno-
[3,4-d][1,3]dioxole (10). To a stirred solution of 8 (8.5 g, 40.7 mmol) in
anhydrous acetonitrile (100 mL) was added POCl3 (4.47 mL, 48.9
mmol) at 0 °C under a N2 atmosphere, and the reaction mixture was
stirred for 30 min at rt. The reaction mixture was dried in vacuo and
coevaporated with toluene twice to give 10, which was directly used for
1
the next step without further purification: H NMR (CDCl3, 400
MHz): δ 4.89 (m, 1H), 4.81 (dd, J = 0.8, 5.6 Hz, 1H), 2.88 (m, 2H),
3.65 (dd, J = 4.0, 8.2 Hz, 1H), 3.44 (m, 1H), 3.08 (dd, J = 4.8, 13.2 Hz,
1H), 1.46 (s, 3H), 1.27 (s, 3H); 13C NMR (CDCl3): δ 111.3, 86.1, 83.5,
55.3, 45.7, 37.8, 26.5, 24.7.
General Procedure for the Condensation. To a solution of 6-
chloropurine (15.18 mmol) and 2,6-dichloropurine (15.18 mmol) in a
solution of anhydrous DMF (50 mL) was added NaH (15.18 mmol),
and the mixture was stirred at rt until the solution became clear. A
solution of 10 (12.64 mmol) in anhydrous DMF (7 mL) was added to
the resulting solution at rt and stirred overnight at rt. The reaction
mixture was diluted with EtOAc (70 mL) and washed with water several
times, dried with anhydrous MgSO4, and evaporated. The residue was
purified by flash silica gel column chromatography (n-hexanes/EtOAc
= 2:1) to give the condensed products 11 and 12, respectively.
2,6-Dichloro-9-(((3aR,4S,6aS)-2,2-dimethyltetrahydrothieno[3,4-
d][1,3]dioxol-4-yl)methyl)-9H-purine (11). Yield = 72%; white foam;
UV (MeOH) λmax: 275 nm (pH 7); [α]2D5 +173.3 (c 0.30, MeOH);
FAB-MS m/z: 361 (M + H+); 1H NMR (CDCl3, 400 MHz): δ 8.23 (s,
1H), 4.89 (m, 1H), 4.69 (dd, J = 2.0, 5.6 Hz, 1H), 4.47 (dd, J = 6.8, 14.2
Hz, 1H), 4.21 (dd, J = 8.8, 14.2 Hz, 1H), 3.74 (td, J = 2.0, 6.2 Hz, 1H),
2.98 (m, 2H), 1.47 (s, 3H), 1.27 (s, 3H); 13C NMR (CDCl3, 100
MHz): δ 153.37, 153.32, 152.1, 146.1, 145.5, 112.2, 86.2, 83.3, 54.1,
46.1, 37.0, 26.6, 24.8; Anal. Calcd for C13H14Cl2N4O2S: C, 43.22; H,
3.91; N, 15.51; S, 8.88. Found: C, 43.29; H, 3.96; N, 15.63; S, 8.82.
6-Chloro-9-(((3aR,4S,6aS)-2,2-dimethyltetrahydrothieno[3,4-d]-
[1,3]dioxol-4-yl)methyl)-9H-purine (12). Yield = 67%; white foam; UV
(MeOH) λmax: 265 nm (pH 7); [α]2D5 +128.0 (c 0.25, MeOH); FAB-MS
m/z: 327 (M + H+); 1H NMR (CDCl3, 400 MHz): δ 8.67 (s, 1H), 8.28
(s, 1H), 4.79 (m, 1H), 4.66 (dd, J = 2.0, 5.0 Hz, 1H), 4.59 (dd, J = 6.8,
14.0 Hz, 1H), 4.26 (dd, J = 8.0, 16.0 Hz, 1H), 3.72 (td, J = 1.2, 2.4 Hz,
1H), 2.88 (m, 2H), 1.38 (s, 3H), 1.19 (s, 3H); 13C NMR (CDCl3, 100
MHz): δ 152.1, 151.8, 151.0, 145.5, 131.1, 111.8, 86.0, 83.2, 54.1, 46.0,
36.9, 26.4, 24.7; Anal. Calcd for C13H15ClN4O2S: C, 47.78; H, 4.63; N,
17.14; S, 9.81. Found: C, 47.82; H, 4.65; N, 17.20; S, 9.85.
(2S,3R,4S)-2-((6-((3-Bromobenzyl)amino)-2-chloro-9H-purin-9-
yl)methyl)tetrahydrothiophene-3,4-diol (4c). Yield = 73%; white
solid; mp 165−169 °C; UV (MeOH) λmax: 274 nm (pH 7); [α]2D5 +70.0
(c 0.50, MeOH); 1H NMR (DMSO-d6, 400 MHz): δ 8.84 (br s, 1H),
8.17 (s, 1H), 7.02−7.30 (m, 4 H), 5.10 (d, J = 5.6 Hz, 1H), 5.04 (d, J =
4.8 Hz, 1H), 4.65 (br d, J = 5.2 Hz, 2H), 4.44 (dd, J = 6.4, 13.8 Hz, 1H),
4.13−4.21 (m, 2H), 3.79 (m, 1H), 3.66 (m, 1H), 2.89 (dd, J = 5.2, 10.6
Hz, 1H), 2.62 (dd, J = 4.4, 10.6 Hz, 1H); 13C NMR (DMSO-d6, 100
MHz): δ 163.3, 160.9, 154.8, 152.9, 149.9, 142.3, 141.6, 130.2, 130.2,
123.3, 118.0, 77.1, 73.6, 48.3, 47.0, 42.7, 32.9; FAB-MS m/z: 472 (M +
H+); Anal. Calcd for C17H17BrClN5O2S: C, 43.37; H, 3.64; N, 14.88; S,
6.81. Found: C, 43.40; H, 3.70; N, 14.90; S, 6.85.
(2S,3R,4S)-2-((2-Chloro-6-((3-iodobenzyl)amino)-9H-purin-9-yl)-
methyl)tetrahydrothiophene-3,4-diol (4d). Yield = 79%; white solid;
mp 174−180 °C; UV (MeOH) λmax: 274 nm (pH 7); [α]2D5 +136.4 (c
0.33, MeOH); 1H NMR (DMSO-d6, 400 MHz): δ 8.84 (br s, 1H), 8.17
(s, 1H), 7.27−7.40 (m, 4 H), 5.09 (d, J = 5.6 Hz, 1H), 5.04 (d, J = 4.8
Hz, 1H), 4.64 (br d, J = 4.8 Hz, 2H), 4.44 (dd, J = 6.0, 14.0 Hz, 1H),
4.13−4.20 (m, 2H), 3.78 (m, 1H), 3.65 (m, 1H), 2.89 (dd, J = 4.8, 10.4
Hz, 1H), 2.62 (dd, J = 4.8, 10.6 Hz, 1H); 13C NMR (DMSO-d6, 100
MHz): δ 154.7, 152.9, 149.9, 141.8, 141.6, 132.9, 130.1, 127.2, 126.8,
126.0, 118.0, 77.1, 73.6, 48.3, 47.0, 42.7, 32.9; FAB-MS m/z: 518 (M +
H+); Anal. Calcd for C17H17ClIN5O2S: C, 39.43; H, 3.31; N, 13.53; S,
6.19. Found: C, 39.52; H, 3.35; N, 13.58; S, 6.22.
(2S,3R,4S)-2-((6-((3-Fluorobenzyl)amino)-9H-purin-9-yl)methyl)-
tetrahydrothiophene-3,4-diol (4e). Yield = 75%; white solid; mp
117−120 °C; UV (MeOH) λmax: 274 nm (pH 7); [α]2D5 +53.3 (c 0.30,
MeOH); 1H NMR (DMSO-d6, 400 MHz): δ 8.36 (br s, 1H), 8.21 (s,
1H), 8.16 (s, 1H), 7.73 (s, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 7.6
Hz, 1H), 7.10 (t, J = 8.0 Hz, 1H), 5.09 (d, J = 5.6 Hz, 1H), 5.02 (d, J =
4.8 Hz, 1H), 4.66 (br s, 2H), 4.47 (dd, J = 6.0, 14.2 Hz, 1H), 4.19 (dd, J
= 7.6, 14.0 Hz, 1H), 4.14 (m, 1H), 3.78 (m, 1H), 3.68 (m, 1H), 2.88
(dd, J = 5.2, 10.4 Hz, 1H), 2.61 (dd, J = 5.2, 10.8 Hz, 1H); 13C NMR
(DMSO-d6, 100 MHz): δ 154.1, 152.3, 148.9, 142.9, 141.0, 135.7,
135.3, 130.4, 126.6, 118.9, 94.7, 76.8, 73.5, 48.7, 46.8, 42.2, 32.8; FAB-
MS m/z: 376 (M + H+); Anal. Calcd for C17H18FN5O2S: C, 54.39; H,
4.83; N, 18.65; S, 8.54. Found: C, 54.32; H, 4.96; N, 18.77; S, 8.62.
(2S,3R,4S)-2-((6-((3-Chlorobenzyl)amino)-9H-purin-9-yl)methyl)-
tetrahydrothiophene-3,4-diol (4f). Yield = 83%; white solid; mp 143−
147 °C; UV (MeOH) λmax: 275 nm (pH 7); [α]2D5 +71.8 (c 0.32,
MeOH); 1H NMR (DMSO-d6, 400 MHz): δ 8.38 (br s, 1H), 8.22 (s,
1H), 8.16 (s, 1H), 7.54 (s, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 7.6
General Procedure for the N6-Substitution Reaction. To a
stirred solution of 11 or 12 in THF was added dropwise 2 N HCl. The
acetonide group was deprotected by using HCl under a N2 atmosphere.
After being stirred at rt for 5 h, the reaction mixture was evaporated
under reduced pressure. The residue was purified by flash column
chromatography (reverse phase silica gel, H2O/MeOH, 10/1). To a
stirred solution of acetonide deprotected 6-chloropurine derivatives
(0.45 mmol) or 2,6-dichloropurine derivatives (0.45 mmol) and an
appropriate amine hydrochloride salts or free amines (0.90 mmol) in
EtOH (10 mL) was added Et3N (1.35 mmol), and the solution was
stirred overnight at rt. After removing the solvent under reduced
pressure, the residue was purified by flash silica gel column
J
https://dx.doi.org/10.1021/acs.jmedchem.0c01874
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 5.08 (d, J = 5.6 Hz, 1H), 5.01 (d, J =
4.8 Hz, 1H), 4.69 (br s, 2H), 4.46 (dd, J = 8.0, 14.0 Hz, 1H), 4.19 (dd, J
= 7.6, 14.0 Hz, 1H), 4.16 (m, 1H), 3.78 (m, 1H), 3.68 (m, 1H), 2.87
(dd, J = 5.2, 10.4 Hz, 1H), 2.61 (dd, J = 5.2, 10.4 Hz, 1H); 13C NMR
(DMSO-d6, 100 MHz): δ 154.1, 152.3, 148.9, 143.0, 141.0, 130.4,
129.8, 129.4, 126.2, 121.5, 118.9, 76.8, 73.5, 48.7, 46.8, 42.4, 32.8; FAB-
MS m/z: 392 (M + H+); Anal. Calcd for C17H18ClN5O2S: C, 52.10; H,
4.63; N, 17.87; S, 8.18. Found: C, 52.33; H, 4.64; N, 17.90; S, 8.16.
(2S,3R,4S)-2-((6-((3-Bromobenzyl)amino)-9H-purin-9-yl)methyl)-
tetrahydrothiophene-3,4-diol (4g). Yield = 78%; white solid; mp
134−139 °C; UV (MeOH) λmax: 270 nm (pH 7); [α]2D5 +72.0 (c 0.25,
MeOH); 1H NMR (DMSO-d6, 400 MHz): δ 8.37 (br s, 1H), 8.21 (s,
1H), 8.16 (s, 1H), 7.39 (s, 1H), 7.31 (m, 3H), 5.08 (d, J = 5.6 Hz, 1H),
5.02 (d, J = 5.2 Hz, 1H), 4.70 (br s, 2H), 4.47 (dd, J = 6.4, 14.0 Hz, 1H),
4.19 (dd, J = 7.6, 14.2 Hz, 1H), 4.14 (m, 1H), 3.78 (m, 1H), 3.67 (m,
1H), 2.87 (dd, J = 4.8, 10.2 Hz, 1H), 2.62 (dd, J = 5.2, 10.6 Hz, 1H); 13C
NMR (DMSO-d6, 100 MHz): δ 154.2, 152.3, 148.9, 142.8, 141.0,
132.8, 130.1, 126.9, 126.5, 125.8, 118.9, 76.8, 73.5, 48.7, 46.8, 42.4,
32.1; FAB-MS m/z: 436 (M + H+); Anal. Calcd for C17H18BrN5O2S: C,
46.80; H, 4.16; N, 16.05; S, 7.35. Found: C, 46.89; H, 4.21; N, 16.07; S,
7.41.
(2S,3R,4S)-2-((6-((3-Iodobenzyl)amino)-9H-purin-9-yl)methyl)-
tetrahydrothiophene-3,4-diol (4h). Yield = 75%; white solid; mp
172−175 °C; UV (MeOH) λmax: 272 nm (pH 7); [α]2D5 +68.5 (c 0.35,
MeOH); 1H NMR (DMSO-d6, 400 MHz): δ 8.36 (br s, 1H), 8.21 (s,
1H), 8.16 (s, 1H), 7.32 (m, 1H), 7.00−7.19 (m, 3H), 5.08 (d, J = 5.6
Hz, 1H), 5.02 (d, J = 4.8 Hz, 1H), 4.71 (br s, 2H), 4.46 (dd, J = 6.4, 14.0
Hz, 1H), 4.19 (dd, J = 7.6, 18.0 Hz, 1H), 4.14 (m, 1H), 3.78 (m, 1H),
3.67 (m, 1H), 2.87 (dd, J = 5.2, 10.6 Hz, 1H), 2.61 (dd, J = 5.2, 10.4 Hz,
1H); 13C NMR (DMSO-d6, 100 MHz): δ 163.3, 160.9, 154.2, 152.3,
148.9, 143.2, 141.0, 130.1, 130.0, 123.1, 118.9, 76.8, 73.5, 48.7, 46.8,
42.5, 32.8; FAB-MS m/z: 484 (M + H+); Anal. Calcd for
C17H18IN5O2S: C, 42.25; H, 3.75; N, 14.49; S, 6.63. Found: C,
42.34; H, 3.78; N, 14.51; S, 6.65.
6-Chloro-9-(((3aR,4S,6aS)-2,2-Dimethyltetrahydrothieno[3,4-d]-
[1,3]dioxol-4-yl)methyl)-2-iodo-9H-purine (18). 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) at 0 °C under a N2 atmosphere, and the mixture was stirred at
the same temperature for 15 min. To this solution was added a solution
of compound 9 (0.5 g, 2.62 mmol) in THF (10 mL) at 0 °C, and the
reaction mixture was stirred at rt for 1 h. The reaction mixture was
concentrated under reduced pressure, and the crude residue was
purified by flash silica gel column chromatography (n-hexanes/EtOAc
= 5:1) to give 18 (0.79 g, 67%) as a white solid: 1H NMR (CDCl3, 300
MHz): δ 8.10 (s, 1H), 4.86 (m, 1H), 4.71 (dd, J = 1.6, 5.6 Hz, 1H), 4.44
(dd, J = 6.6, 14.4 Hz, 1H), 4.22 (dd, J = 8.2, 14.0 Hz, 1H), 3.72 (t, J = 6.6
Hz, 1H), 2.94 (s, 1H), 2.94 (s, 1H), 1.47 (s, 3H), 1.26 (s, 3H); Anal.
Calcd for C13H14ClIN4O2S: C, 34.49; H, 3.12; N, 12.38. Found: C,
34.29; H, 3.22; N, 12.49.
solution was stirred overnight at rt under a N2 atmosphere. After
removing the solvent under reduced pressure, the residue was purified
by flash silica gel column chromatography (CH2Cl2/MeOH) to give
the N6-substituted amine derivatives 4i−4l.
(2S,3R,4S)-2-((6-((3-Fluorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-
purin-9-yl)methyl)tetrahydrothiophene-3,4-diol (4i). Yield = 47%;
white solid; mp 118−121 °C; 1H NMR (CD3OD, 500 MHz): δ 8.10 (s,
1H), 7.31 (dd, J = 7.6, 13.9 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.11 (d, J
= 9.9 Hz, 1H), 6.96 (ddd, J = 2.2, 8.5, 10.5 Hz, 1H), 4.80 (br s, 2H),
4.53 (dd, J = 5.6, 14.2 Hz, 1H), 4.32 (dd, J = 7.5, 14.2 Hz, 1H), 4.23 (dd,
J = 4.1, 7.9 Hz, 1H), 3.87 (dd, J = 3.4, 6.6 Hz, 1H), 3.75 (dd, J = 6.6, 12.9
Hz, 1H), 2.98 (dd, J = 4.8, 11.0 Hz, 1H), 2.73 (dd, J = 4.1, 11.0 Hz, 1H),
2.05 (s, 3H); 13C NMR (CD3OD, 125 MHz): δ 166.1, 164.2, 156.5,
148.7, 144.2, 143.9, 132.0 (d, J = 8.0 Hz), 125.1, 120.3, 116.0 (d, J =
21.7 Hz), 115.6 (d, J = 21.3 Hz), 84.6, 81.3, 79.9, 76.3, 50.7, 48.8, 45.4,
34.7, 4.4; [α]2D0 −61.7 (c 0.08, MeOH); ESI-MS m/z: 414.1382 (M +
H+); Anal. Calcd for C20H20FN5O2S: C, 58.10; H, 4.88; N, 16.94.
Found: C, 58.40; H, 4.58; N, 16.64.
(2S,3R,4S)-2-((6-((3-Chlorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-
purin-9-yl)methyl)tetrahydrothiophene-3,4-diol (4j). Yield = 47%;
white solid; mp 125−128 °C; 1H NMR (CD3OD, 500 MHz): δ 8.10 (s,
1H), 7.39 (s, 1H), 7.27−7.34 (m, 2H), 7.21−7.26 (m, 1H), 4.78 (br s,
2H), 4.53 (dd, J = 5.7, 14.2 Hz, 1H), 4.32 (dd, J = 7.5, 14.2 Hz, 1H),
4.23 (dd, J = 4.1, 8.0 Hz, 1H), 3.87 (dd, J = 3.4, 6.6 Hz, 1H), 3.75 (dd, J
= 6.7, 13.0 Hz, 1H), 2.98 (dd, J = 4.8, 11.1 Hz, 1H), 2.73 (dd, J = 4.1,
11.0 Hz, 1H), 2.05 (s, 3H); 13C NMR (CD3OD, 125 MHz): δ 156.5,
148.7, 143.9, 143.7, 136.1, 131.8, 129.8, 129.4, 129.0, 127.8, 120.3, 84.6,
81.3, 79.9, 76.3, 50.4, 48.8, 45.3, 34.7, 4.3; [α]2D0 −125.0 (c 0.05,
MeOH); ESI-MS m/z: 430.1087 (M + H+); Anal. Calcd for
C20H20ClN5O2S: C, 55.87; H, 4.69; N, 16.29. Found: C, 55.57; H,
4.89; N, 16.59.
(2S,3R,4S)-2-((6-((3-Bromobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-
purin-9-yl)methyl)tetrahydrothiophene-3,4-diol (4k). Yield = 41%;
white solid; mp 124−126 °C; 1H NMR (CD3OD, 500 MHz): δ 8.10 (s,
1H), 7.56 (s, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.7 Hz, 1H),
7.23 (t, J = 7.8 Hz, 1H), 4.77 (br s, 2H), 4.53 (dd, J = 5.6, 14.2 Hz, 1H),
4.32 (dd, J = 7.5, 14.2 Hz, 1H), 4.23 (dd, J = 4.1, 8.0 Hz, 1H), 3.87 (dd, J
= 3.4, 6.6 Hz, 1H), 3.75 (dd, J = 6.6, 13.0 Hz, 1H), 2.98 (dd, J = 4.8, 11.0
Hz, 1H), 2.73 (dd, J = 4.1, 11.1 Hz, 1H), 2.06 (s, 3H); 13C NMR
(CD3OD, 125 MHz): δ 156.4, 151.0, 148.7, 143.9, 142.4, 132.4, 132.1,
132.0, 128.2, 124.2, 120.3, 84.6, 81.3, 79.9, 76.3, 50.4, 48.7, 45.1, 34.7,
4.4; [α]2D0 −75.4 (c 0.05, MeOH); ESI-MS m/z: 476.0559 (M + H+);
Anal. Calcd for C20H20BrN5O2S: C, 50.64; H, 4.25; N, 14.76. Found: C,
50.84; H, 4.45; N, 14.96.
(2S,3R,4S)-2-((6-((3-Iodobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-
purin-9-yl)methyl)tetrahydrothiophene-3,4-diol (4l). Yield = 51%;
white solid; mp 116−118 °C; 1H NMR (CD3OD, 500 MHz): δ 8.10 (s,
1H), 7.77 (s, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H),
7.08 (t, J = 7.8 Hz, 1H), 4.74 (br s, 2H), 4.53 (dd, J = 5.7, 14.2 Hz, 1H),
4.32 (dd, J = 7.5, 14.2 Hz, 1H), 3.87 (dd, J = 3.4, 6.6 Hz, 1H), 3.75 (dd, J
= 6.7, 13.0 Hz, 1H), 2.98 (dd, J = 4.8, 11.0 Hz, 1H), 2.73 (dd, J = 4.1,
11.0 Hz, 1H), 2.06 (s, 3H); 13C NMR (CD3OD, 125 MHz): δ 156.4,
151.0, 148.7, 143.9, 143.8, 138.6, 138.1, 132.1, 128.8, 127.1, 120.3, 95.7,
84.6, 81.3, 79.9, 76.3, 50.4, 48.8, 34.7, 4.4; [α]2D0 −99.6 (c 0.05, MeOH);
ESI-MS m/z: 522.0441 (M + H+); Anal. Calcd for C20H20IN5O2S: C,
46.07; H, 3.87; N, 13.43. Found: C, 46.27; H, 3.97; N, 13.63.
(3aS,6S,6aS)-6-(Hydroxymethyl)-2,2-dimethyltetrahydrofuro-
[3,4-d][1,3]dioxol-4-ol (20). To a stirred suspension of D-ribose (4 g,
26.64 mmol) in acetone (50 mL) was added dropwise conc. H2SO4
(0.12 mL) at rt, and the reaction mixture was stirred at rt for 3 h. The
mixture was neutralized with solid NaHCO3, filtered, and evaporated
under reduced pressure to give a colorless syrup. The residue was
purified by silica gel column chromatography using hexane and ethyl
acetate (1:2) as the eluent to afford 20 as a colorless syrup (4.3 g, 85%):
6-Chloro-9-(((3aR,4S,6aS)-2,2-dimethyltetrahydrothieno[3,4-d]-
[1,3]dioxol-4-yl)methyl)-2-(prop-1-yn-1-yl)-9H-purine (19). To a
stirred solution of 18 (315 mg, 0.70 mmol) in DMF (5 mL) were
added cesium carbonate (274 mg, 0.84 mmol, 1.2 equiv), tetrakis-
(triphenylphosphine)palladium(0) (81 mg, 0.07 mmol, 0.1 equiv), and
copper(I) iodide (16 mg, 0.084 mmol, 0.12 equiv) at rt under a N2
atmosphere. After being stirred at the same temperature for 3 h, the
reaction mixture was evaporated under reduced pressure. The residue
was purified by silica gel column chromatography (n-hexanes/EtOAc =
1
1:1) to give 19 as colorless oil (165 mg, 0.45 mmol, 65%): H NMR
(CDCl3, 500 MHz): δ 8.20 (s, 1H), 4.85 (quin, J = 3.2 Hz, 1H), 4.67
(dd, J = 1.6, 5.6 Hz, 1H), 4.45 (dd, J = 6.8, 14.4 Hz, 1H), 4.22 (dd, J =
8.2, 14.4 Hz, 1H), 3.74 (t, J = 8.2 Hz, 1H), 2.95 (s, 1H), 2.95 (s, 1H),
2.13 (s, 3H), 1.46 (s, 3H), 1.26 (s, 3H); Anal. Calcd for
C16H17ClN4O2S: C, 52.67; H, 4.70; N, 15.36. Found: C, 52.69; H,
4.77; N, 15.16.
[α]D24 +25.3 (c 1.05, CHCl3), lit.34 [α]D24 +21.3 (c 1.02, CHCl3); H
1
NMR (CD3OD, 400 MHz), δ 5.26 (s, 1H), 4.77 (d, J = 6.0 Hz, 1H),
4.52 (d, J = 6.0 Hz, 1H), 4.19 (irregular t, J = 4.4, 5.2 Hz, 1H), 3.63 (dd,
J = 4.8, 12.0 Hz, 1H), 3.59 (dd, J = 5.6, 12.0, 1H), 1.44 (s, 3H), 1.31 (s,
3H); Anal. Calcd for C8H14O5: C, 50.52; H, 7.42. Found: C, 50.48; H,
7.36.
General Procedure for the N6-Substitution Reaction. To a
stirred solution of 6-chloropurine derivative and an appropriate free
amines (3 equiv) in EtOH (0.2 M) was added Et3N (3 equiv), and the
K
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(S)-1-((4S,5R)-5-(Hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-
yl)-2-(trityloxy)ethanol (21). To a stirred solution of 20 (2.01 g, 10.56
mmol) in pyridine (36 mL, 0.3 M) were added trityl chloride (3.24 g,
11.62 mmol) and 4-dimethylaminopyridine (258 mg, 2.11 mmol) at 0
°C, and the mixture was stirred with heating at 80 °C under a N2
atmosphere. After being stirred for 3 h, the mixture was cooled, diluted
with EtOAc, and washed with saturated NH4Cl solution and brine and
evaporated to give the hemiacetal intermediate, which was used for the
next step without further purification. To a stirred solution of the
intermediate in methanol was added portionwise sodium borohydride
(1.19 g, 31.68 mmol) at 0 °C under a N2 atmosphere. After being stirred
for 1 h, the mixture was evaporated and the resulting white suspension
was diluted with EtOAc and washed with saturated NH4Cl solution and
brine. The separated organic layer was evaporated, and the residue was
purified by silica gel column chromatography (n-hexanes/EtOAc = 3:1)
to give diol 21 (2.93 g, 64% for 2 step) as a syrup: 1H NMR (CDCl3,
400 MHz): δ 7.40−7.46 (m, 6 H), 7.28−7.34 (m, 6 H), 7.22−7.28 (m,
3H), 4.29−4.36 (m, 1H), 4.10 (dd, J = 5.8, 9.6 Hz, 1H), 3.70−3.90 (m,
3H), 3.47 (dd, J = 3.0, 9.8 Hz, 1H), 3.31 (dd, J = 6.8, 9.8 Hz, 1H), 3.01
(dd, J = 5.0, 8.7 Hz, 1H), 2.89 (d, J = 3.9 Hz, 1H), 1.309 (s, 3H), 1.30 (s,
3H); 13C NMR (CDCl3, 100 MHz): δ 149.8, 147.6, 143.6, 135.9, 128.6,
127.9, 127.2, 123.7, 108.5, 87.1, 69.0, 65.0, 60.7, 27.8, 25.3; Anal. Calcd
for C27H30O5: C, 74.63; H, 6.96. Found: C, 74.53; H, 6.66.
(3aS,4S,6aR)-2,2-Dimethyl-4-((trityloxy)methyl)tetrahydrofuro-
[3,4-d][1,3]dioxole (22). To a cooled (0 °C) solution of 21 (14.18 g,
32.64 mmol) in anhydrous pyridine (110 mL, 0.3 M) were added 4-
toluenesulfonyl chloride (12.44 g, 65.28 mmol, 2 equiv) and 4-
dimethylaminopyridine (0.39 g, 3.26 mmol, 0.1 equiv) under a N2
atmosphere. After the reaction mixture was being stirred with heating
(80 °C) for 3 h, the reaction mixture was cooled and quenched with
saturated ammonium chloride solution. The organic layer was washed
with brine, dried with anhydrous MgSO4, and evaporated. The residue
was purified by silica gel column chromatography (n-hexanes/EtOAc =
30:1) to give 22 as a colorless syrup (11.41 g, 84%): 1H NMR (CDCl3,
500 MHz): δ 7.40 (d, J = 7.5 Hz, 6 H), 7.29 (dd, J = 7.3, 15.2 Hz, 6 H),
7.20−7.25 (m, 3H), 4.87 (t, J = 5.1 Hz, 1H), 4.64 (d, J = 6.1 Hz, 1H),
4.20 (dd, J = 3.8, 7.7 Hz, 1H), 4.15 (dd, J = 4.3, 10.2 Hz, 1H), 4.04 (d, J
= 10.2 Hz, 1H), 3.25 (dd, J = 4.0, 10.0 Hz, 1H), 3.09 (dd, J = 4.3, 10.0
Hz, 1H), 1.50 (s, 3H), 1.32 (s, 3H); 13C NMR (CDCl3, 125 MHz): δ
143.6, 128.6, 127.8, 127.0, 112.3, 87.0, 84.0, 83.0, 81.6, 74.3, 64.5, 26.6,
25.0; Anal. Calcd for C27H28O4: C, 77.86; H, 6.78. Found: C, 77.65; H,
6.59.
((3aS,4S,6aR)-2,2-Dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-
yl)methanol (23). To a cooled (−40 °C) solution of 22 (1.04 g, 2.50
mmol) in anhydrous CH2Cl2 (12.5 mL, 0.2 M) was added dropwise
diethylaluminium chloride 1.0 M solution in hexanes (7.50 mL, 7.50
mmol, 3 equiv) under a N2 atmosphere. After the reaction mixture was
stirred at the same temperature for 3 h, the mixture was quenched with
saturated potassium sodium tartrate solution and stirred for additional
1 h. The organic layer was washed with brine, dried with anhydrous
MgSO4, and evaporated at 5 °C. The residue was purified by silica gel
column chromatography (n-hexanes/EtOAc = 4:1) to give 23 as a
colorless syrup (0.256 g, 59%): 1H NMR (CDCl3, 500 MHz): δ 4.79
(m, 1H), 4.58 (dd, J = 1.9, 6.3 Hz, 1H), 4.11 (ddd, J = 2.1, 3.9, 6.2 Hz,
1H), 3.95 (m, 2H), 3.65 (dd, J = 3.7, 11.6 Hz, 1H), 3.58 (dd, J = 6.7,
11.5 Hz, 1H), 1.50 (s, 3H), 1.32 (s, 3H); 13C NMR (125 MHz,
CDCl3): δ 112.9, 84.8, 81.8, 81.0, 72.8, 61.7, 26.6, 24.9; Anal. Calcd for
C8H14O4: C, 55.16; H, 8.10. Found: C, 55.36; H, 8.30.
General Procedure for the Synthesis of 4m−4t. To a stirred
solution of 6-chloropurine or 2,6-dichloropurine (1.7 equiv) and
triphenylphosphine (Ph3P) (1.7 equiv) in anhydrous THF (0.15 M)
was added DIAD (3.5 equiv) in anhydrous THF (0.3 M) at 0 °C under
a N2 atmosphere, and the mixture was stirred at the same temperature
for 15 min. To this solution was added a solution of compound 23 (1
equiv) in anhydrous THF (0.3 M) at 0 °C, and the reaction mixture was
stirred at rt for 1 h. The reaction mixture was concentrated under
reduced pressure, and the crude residue was purified by flash silica gel
column chromatography to give 24 and 25 contaminated with
triphenylphosphine oxide, respectively. To a stirred solution of 24 or
25 and an appropriate amine hydrochloride salts or free amines (3
equiv) in anhydrous THF (0.3 M) was added Et3N (3 equiv), and the
solution was stirred overnight at rt. After removing the solvent under
reduced pressure, the residue was purified by flash silica gel column
chromatography to give the triphenylphosphine oxide contaminated
with the N6-substituted amine intermediates. To a stirred solution of
N6-substituted amine intermediates in methanol (0.1 M) was added
dropwise 1 N HCl (0.1 equiv) at rt. After being stirred at rt at which
time TLC indicated the absence of the starting material, the reaction
mixture was evaporated under reduced pressure. The residue was
purified by flash column chromatography (reverse phase silica gel,
H2O/MeOH, 10/1) to give 4m−4t.
2,6-Dichloro-9-(((3aS,4S,6aR)-2,2-dimethyltetrahydrofuro[3,4-
1
d][1,3]dioxol-4-yl)methyl)-9H-purine (24). H NMR (CD3OD, 400
MHz): δ 8.51 (s, 1H), 4.83−4.90 (m, 1H), 4.31−4.39 (m, 3H), 4.06
(dd, J = 3.6, 10.8 Hz, 1H), 3.90 (d, J = 10.8 Hz, 1H), 1.41 (s, 3H), 1.30
(s, 3H).
6-Chloro-9-(((3aS,4S,6aR)-2,2-dimethyltetrahydrofuro[3,4-d]-
[1,3]dioxol-4-yl)methyl)-9H-purine (25). 1H NMR (CDCl3, 400
MHz): δ 8.77 (s, 1H), 8.22 (s, 1H), 4.78−4.81 (m, 1H), 4.55 (dd, J
= 2.5, 6.2 Hz, 1H), 4.47 (dd, J = 2.4, 12.8 Hz, 1H), 4.28−4.37 (m, 2H),
3.97−4.05 (m, 2H), 1.50 (s, 3H), 1.33 (s, 3H).
(2S,3R,4R)-2-((2-Chloro-6-((3-fluorobenzyl)amino)-9H-purin-9-
yl)methyl)tetrahydrofuran-3,4-diol (4m). Yield = 48%; white solid;
mp 185−186 °C; 1H NMR (CD3OD, 400 MHz): δ 8.00 (s, 1H), 7.30
(ddd, J = 6.0, 7.6, 13.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.10 (d, J =
10.0 Hz, 1H), 6.95 (ddd, J = 2.4, 8.4, 11.2 Hz, 1H), 4.41 (dd, J = 3.6,
14.8 Hz, 1H), 4.27 (dd, J = 6.8, 14.4 Hz, 1H), 3.94−4.04 (m, 3H), 3.78
(dd, J = 4.4, 7.2 Hz, 1H), 3.68 (dd, J = 2.0, 9.2 Hz, 1H); 13C NMR
(CD3OD, 100 MHz): δ 165.7, 164.5, 157.1, 156.3, 152.4, 144.1, 143.8,
132.0 (d, J = 8.1 Hz), 125.3, 119.7, 116.2 (d, J = 21.7 Hz), 115.7 (d, J =
21.2 Hz), 81.8, 75.6, 74.8, 73.1, 47.3; [α]2D0 −239.4 (c 0.05, MeOH);
ESI-MS m/z: 394.1065 (M + H+); Anal. Calcd for C17H17ClFN5O3: C,
51.85; H, 4.35; N, 17.78. Found: C, 51.75; H, 4.75; N, 17.68.
(2S,3R,4R)-2-((2-Chloro-6-((3-chlorobenzyl)amino)-9H-purin-9-
yl)methyl)tetrahydrofuran-3,4-diol (4n). Yield = 51%; white solid; mp
139−144 °C; 1H NMR (CD3OD, 400 MHz): δ 7.99 (s, 1H), 7.75 (s,
1H), 7.57 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 7.06 (t, J = 7.6
Hz, 1H), 4.68 (br s, 2H), 4.41 (dd, J = 3.6, 14.4 Hz, 1H), 4.26 (dd, J =
6.4, 14.4 Hz, 1H), 4.02 (dd, J = 3.6, 7.6 Hz, 1H), 3.93−4.00 (m, 2H),
3.78 (dd, J = 5.2, 7.6 Hz, 1H), 3.67 (dd, J = 2.0, 8.8 Hz, 1H); 13C NMR
(CD3OD, 100 MHz): δ 157.1, 156.3, 152.4, 144.1, 143.5, 138.7, 138.2,
132.1, 129.0, 119.7, 95.7, 81.7, 75.6, 74.8, 73.1, 47.3, 45.2; [α]2D0 −203.8
(c 0.05, MeOH); FAB-MS m/z: 410.0729 (M + H+); Anal. Calcd for
C17H17Cl2N5O3: C, 49.77; H, 4.18; N, 17.07. Found: C, 49.97; H, 4.38;
N, 17.37.
(2S,3R,4R)-2-((6-((3-Bromobenzyl)amino)-2-chloro-9H-purin-9-
yl)methyl)tetrahydrofuran-3,4-diol (4o). Yield = 53%; white solid; mp
130−132 °C; 1H NMR (CD3OD, 800 MHz): δ 8.01 (s, 1H), 7.55 (s,
1H), 7.36 (dd, J = 7.9, 25.9 Hz, 2H), 7.21 (t, J = 7.8 Hz, 1H), 4.71 (br s,
2H), 4.42 (dd, J = 3.5, 14.4 Hz, 1H), 4.27 (dd, J = 6.8, 14.4 Hz, 1H),
4.03 (ddd, J = 3.4, 6.9, 10.4 Hz, 1H), 3.97 (dd, J = 4.5, 9.6 Hz, 1H), 3.80
(dd, J = 5.0, 7.2 Hz, 1H), 3.69 (dd, J = 2.8, 9.6 Hz, 1H); 13C NMR
(CD3OD, 200 MHz): δ 157.0, 156.3, 152.4, 144.1, 143.5, 142.7, 132.5,
132.1, 128.4, 124.1, 119.6, 81.7, 75.6, 74.8, 73.1, 47.3, 45.2; [α]D20
−150.7 (c 0.1, MeOH); FAB-MS m/z: 454.0287 (M + H+); Anal. Calcd
for C17H17BrClN5O3: C, 44.90; H, 3.77; N, 15.40. Found: C, 44.90; H,
3.87; N, 15.60.
(2S,3R,4R)-2-((2-Chloro-6-((3-iodobenzyl)amino)-9H-purin-9-yl)-
methyl)tetrahydrofuran-3,4-diol (4p). Yield = 45%; white solid; mp
146−148 °C; 1H NMR (CD3OD, 400 MHz): δ 8.00 (s, 1H), 7.76 (s,
1H), 7.58 (d, J = 7.2 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.07 (t, J = 8.4
Hz, 1H), 4.68 (br s, 2H), 4.41 (dd, J = 3.6, 14.4 Hz, 1H), 4.27 (dd, J =
7.2, 14.8 Hz, 1H), 3.94−4.03 (m, 3H), 3.78 (dd, J = 5.2, 7.2 Hz, 1H),
3.68 (dd, J = 2.4, 9.2 Hz, 1H); 13C NMR (CD3OD, 100 MHz): δ 163.8,
157.1, 144.2, 143.5, 138.7, 138.2, 132.1, 130.3, 129.1, 129.0, 119.7,
108.8, 81.8, 75.6, 74.8, 73.1, 47.3; [α]2D0 −131.4 (c 0.1, MeOH); FAB-
MS m/z: 502.0151 (M + H+); Anal. Calcd for C17H17ClIN5O3: C,
40.70; H, 3.42; N, 13.96. Found: C, 40.90; H, 3.62; N, 13.96.
(2S,3R,4R)-2-((6-((3-Fluorobenzyl)amino)-9H-purin-9-yl)methyl)-
tetrahydrofuran-3,4-diol (4q). Yield = 52%; white solid; mp 140−141
L
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°C; 1H NMR (CD3OD, 400 MHz): δ 8.24 (s, 1H), 8.05 (s, 1H), 7.29
(ddd, J = 6.0, 8.0, 13.6 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.07−7.12 (m,
1H), 6.94 (ddd, J = 2.8, 8.8, 11.6 Hz, 1H), 4.54 (br s, 2H), 4.46 (dd, J =
3.2, 14.4 Hz, 1H), 4.33 (dd, J = 6.4, 14.4 Hz, 1H), 4.04 (ddd, J = 3.6, 6.8,
10.4 Hz, 1H), 3.93−4.01 (m, 2H), 3.78 (dd, J = 4.4, 7.2 Hz, 1H), 3.68
(dd, J = 2.0, 8.8 Hz, 1H); 13C NMR (CD3OD, 100 MHz): δ 156.2,
153.9, 143.6, 143.3, 135.2, 131.4, 131.3, 124.6, 115.3, 115.1, 115.0,
114.8, 81.2, 74.9, 74.2, 72.4, 46.5; [α]2D0 −391.6 (c 0.05, MeOH); ESI-
MS m/z: 360.1461 (M + H+); Anal. Calcd for C17H18FN5O3: C, 56.82;
H, 5.05; N, 19.49. Found: C, 56.92; H, 5.15; N, 19.59.
(2S,3R,4R)-2-((6-((3-Chlorobenzyl)amino)-9H-purin-9-yl)methyl)-
tetrahydrofuran-3,4-diol (4r). Yield = 56%; white solid; mp 150−153
°C; 1H NMR (CD3OD, 400 MHz): δ 8.24 (s, 1H), 8.06 (s, 1H), 7.37−
7.41 (m, 2H), 7.19−7.25 (m, 2H), 4.88 (br s, 2H), 4.47 (dd, J = 3.2,
14.0 Hz, 1H), 4.33 (dd, J = 6.4, 14.4 Hz, 1H), 4.04 (ddd, J = 3.6, 6.8,
10.4 Hz, 1H), 3.93−4.00 (m, 2H), 3.78 (dd, J = 4.8, 7.2 Hz, 1H), 3.68
(dd, J = 2.4, 9.2 Hz, 1H); 13C NMR (CD3OD, 100 MHz): δ 156.7,
154.6, 151.1, 143.9, 138.2, 135.2, 131.3, 130.9, 130.5, 128.9, 120.8,
108.8, 81.8, 75.6, 74.8, 73.1, 47.2; [α]2D0 +193.5 (c 0.04, MeOH); ESI-
MS m/z: 376.1182 (M + H+); Anal. Calcd for C17H18ClN5O3: C, 54.33;
H, 4.83; N, 18.64. Found: C, 54.53; H, 4.93; N, 18.84.
Enzyme-Linked Immunosorbent Assay. ELISA was conducted
to quantitatively evaluate the effects of the compounds on adiponectin
secretion. The Quantikine immunoassay kit (R&D Systems, Minne-
apolis, MN, USA) was used to measure adiponectin amount in cell
culture supernatants, according to the manufacturer instruction.
TR-FRET Assays. The TR-FRET-based receptor-binding assay was
performed using LanthaScreen competitive binding assay kits
(Invitrogen). LanthaScreen coactivator assay kits (Invitrogen) were
used to determine the receptor activation of PPARγ and PPARδ. The
PPARγ coactivator assay was performed using fluorescein-TRAP220/
DRIP coactivator peptide (sequence KVSQNPILTSLLQITGNGG).35
PPARδ coactivator and corepressor assays were performed using
fluorescein-C33 coactivator peptide (sequence HVEMHPLLMGLL-
MESQWGA) and SMRT-ID2 peptide (sequence HASTNM-
GLEAHRKALMGKYDQW), respectively.36 Assay plates were read
at wavelengths of 520 and 495 nm after 1 to 2 h of incubation, using a
CLARIOstar plate reader (BMG LABTECH, Ortenberg, Germany).
All procedures and instrument settings were in agreement with the
manufacturer instruction. PPAR ligands, 27, 28, and 29 were purchased
from Tocris Bioscience (Bristol, U.K.), and 30 was purchased from
Sigma-Aldrich.
Animal Study. Animals. 6 week old male C57BL/6J ob/ob mice
were obtained from SLC Inc. (Kotoh-cho, Japan). Mice were
acclimatized for 1 week prior to use and were housed in a 12 h light/
dark cycle at a temperature of 21 2 °C and 50 5% relative humidity.
Mice had free access to food except the time when they were fasted
before the measurement of blood glucose. The experimental
procedures were approved by IACUC (SNU-200605-5-1) in the
Seoul National University.
Drug Preparation and Administration. Pioglitazone and the test
drug (4p) were dissolved in 60% polyethylene glycol (PEG) containing
5% DMSO for an in vivo experiment. Pioglitazone (20 mg/kg) or 4p at a
dose of 30 mg/kg was orally administered 6 times per week for 3 weeks
followed by intraperitoneal administration for another 3 weeks. Lean
mice group and ob/ob mice control group were administered with an
equivalent volume of 60% PEG containing 5% DMSO. No adverse
effects were observed.
OGTT and Measurement of Fasting Blood Glucose Level. OGTT
and fasting blood glucose levels were measured after mice were fasted
for 10 h (starting from 11:00 pm, day before the experiment). Blood
glucose levels were determined in blood samples from the tail vein using
a G Care glucose analyzer (Green Cross, Inc., Seoul, South Korea). For
OGTT, the mice were administered with glucose (0.2 g/kg) and blood
samples were collected at t = 0, 15, 30, 60, 90, and 120 min.
Measurement of Serum Insulin Level. Fed serum insulin levels were
measured in blood samples obtained from cardiac puncture at final
sacrifice. Blood samples were centrifuged, and the serum part was used
to measure insulin levels through a LBIS mouse insulin ELISA kit
(Shibayagi, Gunma, Japan).
Statistical Analysis. The data are shown as the mean standard
deviation (SD). Comparisons of multiple groups were assessed by one-
way analysis of variance (ANOVA), followed by Tukey’s test. p value <
0.05 was considered to be statistically significant. GraphPad PRISM
software version 7.0 (GraphPad Software, San Diego, CA, USA) was
used for statistical analysis.
Molecular Modeling Study. Homology Modeling. An A3AR
homology model for molecular docking was built on the SWISS-
MODEL server.37 The crystal structure of the adenosine A2A receptor
bound to an engineered G protein in complex with AR agonist NECA
was downloaded from RCSB PDB (PDB ID: 5G53) and used as the
template for homology modeling.21,38 Human adenosine A3 receptor
target sequence for homology modeling was fetched from UniProtKB
(entry: P0DMS8).39
Computational Docking Simulation. Ligand-binding site for
docking was defined as a 30 Å3 size grid box centered on the centroid
of co-crystallized native ligands. The crystal structures of PPARγ and
PPARδ LBD were also downloaded from RCSB PDB (PDB ID: 5YCP,
5U46),25,26 and computational docking was performed using
AutoDock Vina version 1.1.2 (The Scripps Research Institute, La
Jolla, CA, USA).40 For each macromolecule−ligand pair, binding
(2S,3R,4R)-2-((6-((3-Bromobenzyl)amino)-9H-purin-9-yl)methyl)-
tetrahydrofuran-3,4-diol (4s). Yield = 47%; white solid; mp 148−151
°C; 1H NMR (CD3OD, 500 MHz): δ 8.25 (s, 1H), 8.07 (s, 1H), 7.53
(s, 1H), 7.35 (dd, J = 7.8, 16.8 Hz, 2H), 7.20 (t, J = 7.8 Hz, 1H), 4.78 (br
s, 2H), 4.47 (dd, J = 3.4, 14.4 Hz, 1H), 4.33 (dd, J = 6.6, 14.4 Hz, 1H),
4.05 (ddd, J = 3.4, 6.9, 10.4 Hz, 1H), 3.98−4.02 (m, 1H), 3.94−3.99
(m, 1H), 3.79 (dd, J = 5.0, 7.2 Hz, 1H), 3.69 (dd, J = 2.5, 9.6 Hz, 1H),
3.29−3.30 (m, 1H); 13C NMR (CD3OD, 125 MHz): δ 156.1, 153.9,
143.39, 143.33, 131.5 (d, J = 11.4 Hz), 131.3, 127.4, 123.5, 120.1, 108.5,
81.2, 74.9, 74.2, 72.4, 46.5; [α]2D0 +56.5 (c 0.04, MeOH); ESI-MS m/z:
422.0642 (M + H+); Anal. Calcd for C17H18BrN5O3: C, 48.58; H, 4.32;
N, 16.66. Found: C, 48.78; H, 4.52; N, 16.86.
(2S,3R,4R)-2-((6-((3-Iodobenzyl)amino)-9H-purin-9-yl)methyl)-
tetrahydrofuran-3,4-diol (4t). Yield = 38%; white solid; mp 177−179
°C; 1H NMR (DMSO-d6, 800 MHz): δ 8.35 (br s, 1H), 8.20 (br s, 1H),
8.07 (s, 1H), 7.72 (s, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.6 Hz,
1H), 7.10 (t, J = 7.7 Hz, 1H), 4.96 (d, J = 6.2 Hz, 1H), 4.83 (d, J = 4.6
Hz, 1H), 4.64 (br s, 2H), 4.34 (dd, J = 3.8, 14.2 Hz, 1H), 4.20 (dd, J =
7.3, 14.2 Hz, 1H), 3.95 (ddd, J = 3.9, 6.9, 10.7 Hz, 1H), 3.88−3.92 (m,
1H), 3.86 (dd, J = 4.8, 9.2 Hz, 1H), 3.71 (dd, J = 6.4, 11.5 Hz, 1H), 3.50
(dd, J = 3.3, 9.2 Hz, 1H), 3.29 (br s, 1H); 13C NMR (DMSO-d6, 200
MHz): δ 154.1, 152.2, 149.0, 142.9, 141.3, 135.7, 135.2, 130.4, 126.6,
94.6, 79.6, 73.2, 72.4, 70.2, 64.8, 45.2; [α]2D0 +29.8 (c 0.1, MeOH); FAB-
MS m/z: 468.0529 (M + H+); Anal. Calcd for C17H18IN5O3: C, 43.70;
H, 3.88; N, 14.99. Found: C, 43.90; H, 3.98; 14.99.
Cell Culture and Differentiation. hBM-MSCs (Lonza, Walkers-
ville, MD, USA) were maintained in low glucose (1 g/L) Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine serum
and supplemented with penicillin−streptomycin and Glutamax
(Invitrogen, Carlsbad, CA, USA). Cell culture media were exchanged
every 3 d. hBM-MSC were differentiated for 7 d under IDX conditions
(10 μg/mL insulin, 1 μM dexamethasone, 0.5 mM IDX) with DMEM
containing a high concentration of glucose (4.5 g/L) for cell growth
medium. 1, truncated 1′-homologated adenosine analogues (4a−4t) or
26 were cotreated with IDX conditions, and cell culture supernatants
were harvested on the 7th day in culture and 24 h after medium
exchange. 26 was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Oil Red O and Hematoxylin Staining. Oil red O (ORO) staining
was performed to evaluate effects of compounds on lipid accumulation
during differentiation. After the differentiation of hBM-MSC into
adipocytes, cells were cleansed twice with phosphate-buffered saline
(PBS) and fixed with 10% formalin in PBS (pH 7.4) for 1 h. Then, cells
were washed with 60% isopropanol and then dried at 25 °C. hBM-
MSCs were stained with 0.2% ORO solution (Sigma-Aldrich) for 20
min at 25 °C and then washed with tap water three times. After staining
lipid droplets, cells were further incubated for 1 min in hematoxylin
solution to stain the nucleus. Stained cells were photographed using an
Eclipse TS100 inverted microscope (Nikon Co., Tokyo, Japan).
M
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Article
models of ligands with the lowest binding free energy (kcal/mol) were
used for further analysis. All figures to show the molecular modeling
Young Eum Hyun − Research Institute of Pharmaceutical
Sciences, College of Pharmacy, Seoul National University,
Seoul 08826, Korea
Mai Nguyen − Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 08826,
Korea; College of Pharmacy and Research Institute of Drug
Development, Chonnam National University, Gwangju
61186, Korea
Juri Jeong − Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 08826,
Korea; College of Pharmacy and Research Institute of Drug
Development, Chonnam National University, Gwangju
61186, Korea
Zijing Liu − Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 08826,
Korea; College of Pharmacy and Research Institute of Drug
Development, Chonnam National University, Gwangju
61186, Korea
Jinhe Han − College of Pharmacy and Research Institute of Drug
Development, Chonnam National University, Gwangju
61186, Korea
Hongseok Choi − Research Institute of Pharmaceutical
Sciences, College of Pharmacy, Seoul National University,
Seoul 08826, Korea
Jinha Yu − Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 08826,
Korea
Ji Won Kim − Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 08826,
Korea
̈
results were visualized using PyMOL (Schrodinger, LLC, New York,
NY, USA).41
Statistical Analysis. Experimental values were presented in the
mean SD from three independent experiments. Statistical analyses
were conducted using R (R Foundation for Statistical Computing,
Vienna, Austria).42 Comparisons were performed using one-way
ANOVA followed by the posthoc Welch two sample t-test. The
threshold of statistical significance was set at (*) p ≤ 0.05 and (**) p ≤
0.01.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01874.
■
sı
1
HPLC data of 4d and 4p; H and 13C NMR copies;
computational docking study; and animal study (PDF)
Molecular formula strings (CSV)
A3AR/4d (PDB); A3AR/1 (PDB); PPARγ-LBD/4d
(PDB); PPARδ-LBD/4d (PDB); PPARδ-LBD/4h
(PDB); PPARδ-LBD/4l (PDB) (ZIP)
Accession Codes
The crystal structures of A2AAR (PDB ID: 5G53), PPARγ-LBD
(PDB ID: 5YCP), and PPARδ-LBD (PDB ID: 5U46) were
obtained from the Protein Data Bank. Authors will release the
atomic coordinates and experimental data upon article
publication.
AUTHOR INFORMATION
Corresponding Authors
■
Hyuk Woo Lee − Future Medicine Company Ltd., Seongnam,
Gyeonggi-do 13449, Korea
Minsoo Noh − Research Institute of Pharmaceutical Sciences,
College of Pharmacy and Natural Products Research Institute,
Seoul National University, Seoul 08826, Korea; orcid.org/
0000-0002-4020-5372; Phone: 82-2-880-2481;
Email: minsoo@alum.mit.edu, minsoonoh@snu.ac.kr
Lak Shin Jeong − Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 08826,
Korea; orcid.org/0000-0002-3441-707X; Phone: 82-2-
880-7850; Email: lakjeong@snu.ac.kr
Kenneth A. Jacobson − 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; orcid.org/0000-0001-8104-1493
Won Jea Cho − College of Pharmacy and Research Institute of
Drug Development, Chonnam National University, Gwangju
61186, Korea
Young-Mi Kim − Department of Pharmacy, College of
Pharmacy and Institute of Pharmaceutical Science and
Technology, Hanyang University, Ansan, Gyeonggi-do 15588,
Korea
Authors
Seungchan An − Research Institute of Pharmaceutical Sciences,
College of Pharmacy and Natural Products Research Institute,
Seoul National University, Seoul 08826, Korea; orcid.org/
0000-0002-5840-8987
Gyudong Kim − Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 08826,
Korea; College of Pharmacy and Research Institute of Drug
Development, Chonnam National University, Gwangju
61186, Korea
Hyun Jin Kim − Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 08826,
Korea
Sungjin Ahn − Research Institute of Pharmaceutical Sciences,
College of Pharmacy and Natural Products Research Institute,
Seoul National University, Seoul 08826, Korea
Hyun Young Kim − Research Institute of Pharmaceutical
Sciences, College of Pharmacy, Seoul National University,
Seoul 08826, Korea
Keon Wook Kang − Research Institute of Pharmaceutical
Sciences, College of Pharmacy, Seoul National University,
Seoul 08826, Korea
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01874
Author Contributions
Seungchan An and G.K. contributed equally to this work.
Minsoo Noh and Lak Shin Jeong designed and wrote the
manuscript. The manuscript was written through contributions
of all authors. All authors have given approval to the final version
of the manuscript.
Hyejin Ko − Research Institute of Pharmaceutical Sciences,
College of Pharmacy and Natural Products Research Institute,
Seoul National University, Seoul 08826, Korea
Notes
The authors declare no competing financial interest.
N
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ACKNOWLEDGMENTS
■
This work was supported by the National Research Foundation
( N R F ) g r a n t s ( N R F - 2 0 1 6 R 1 A 2 B 3 0 1 0 1 6 4 ,
2018R1A5A2024425, and 2019R1F1A1063905) of Korea.
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L. B.; Collins, J. L.; Oplinger, J. A.; Kliewer, S. A.; Gampe, R. T., Jr.;
McKee, D. D.; Moore, J. T.; Willson, T. M. Structural determinants of
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ABBREVIATIONS
■
NASH, nonalcoholic steatohepatitis; PPAR, peroxisome pro-
liferator-activated receptor; RXR, retinoid X receptor; PPRE,
peroxisome proliferator response element; AR, adenosine
receptor; hBM-MSCs, human bone marrow mesenchymal
stem cells; LBD, ligand-binding domain; SAR, structure−
activity relationship; TR-FRET, time-resolved fluorescence
resonance energy transfer; LBP, ligand binding pocket; EC50,
half-maximal effective concentration values; TRAP220/DRIP-1,
thyroid hormone receptor-associated protein 220/vitamin D
receptor interacting protein-1; ELISA, enzyme-linked immuno-
sorbent assay; EL, extracellular loop; NCoR2, nuclear receptor
corepressor 2; SMRT, silencing mediator of retinoid and thyroid
hormone receptor; SMRT-ID2, SMRT-interaction domain 2;
TM, transmembrane; DMEM, Dulbecco modified Eagle’s
medium; FBS, fetal bovine serum; ORO, oil red O; PBS,
phosphate buffered saline; ANOVA, one-way analysis of
variance; T2D, type 2 diabetes
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Adiponectin and metabolic syndrome. Arterioscler., Thromb., Vasc. Biol.
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influences of adiponectin on insulin resistance, type 2 diabetes, and
cardiovascular disease. Diabetes 2007, 56, 1198−1209.
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