Orally active adenosine A1 receptor agonists with antinociceptive effects in mice

Article abstract of DOI:10.1021/jm3004834

Adenosine A₁ receptor (A₁AR) agonists have antinociceptive effects in multiple preclinical models of acute and chronic pain. Although numerous A₁AR agonists have been developed, clinical applications of these agents have been hampered by their cardiovascular side effects. Herein we report a series of novel A₁AR agonists, some of which are structurally related to adenosine 5′-monophosphate (5′-AMP), a naturally occurring nucleotide that itself activates A ₁AR. These novel compounds potently activate A₁AR in several orthogonal in vitro assays and are subtype selective for A₁AR over A_2AAR, A_2BAR, and A₃AR. Among them, UNC32A (3a) is orally active and has dose-dependent antinociceptive effects in wild-type mice. The antinociceptive effects of 3a were completely abolished in A₁AR knockout mice, revealing a strict dependence on A₁AR for activity. The apparent lack of cardiovascular side effects when administered orally and high affinity (K_i of 36 nM for the human A₁AR) make this compound potentially suitable as a therapeutic.

Full text of DOI:10.1021/jm3004834

Article
pubs.acs.org/jmc
Orally Active Adenosine A1 Receptor Agonists with Antinociceptive
Effects in Mice
Ilia Korboukh,† Emily A. Hull-Ryde,† Joseph E. Rittiner,‡ Amarjit S. Randhawa,† Jennifer Coleman,‡
Brendan J. Fitzpatrick,‡ Vincent Setola,§ William P. Janzen,† Stephen V. Frye,† Mark J. Zylka,*,‡
and Jian Jin*,†
†Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman
School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
‡Department of Cell and Molecular Physiology, UNC Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599, United States
§Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
S
* Supporting Information
ABSTRACT: Adenosine A1 receptor (A1AR) agonists have antinociceptive
effects in multiple preclinical models of acute and chronic pain. Although
numerous A1AR agonists have been developed, clinical applications of these
agents have been hampered by their cardiovascular side effects. Herein we report
a series of novel A1AR agonists, some of which are structurally related to
adenosine 5′-monophosphate (5′-AMP), a naturally occurring nucleotide that
itself activates A1AR. These novel compounds potently activate A1AR in several
orthogonal in vitro assays and are subtype selective for A1AR over A2AAR,
A2BAR, and A3AR. Among them, UNC32A (3a) is orally active and has dose-
dependent antinociceptive effects in wild-type mice. The antinociceptive effects
of 3a were completely abolished in A1AR knockout mice, revealing a strict
dependence on A1AR for activity. The apparent lack of cardiovascular side effects when administered orally and high affinity (Ki
of 36 nM for the human A1AR) make this compound potentially suitable as a therapeutic.
dases that dephosphorylate adenosine 5′-monophosphate (5′-
AMP) to adenosine have potent, long-lasting, and entirely
A1AR-dependent thermal and mechanical antinociceptive
effects when administered intrathecally.20−22 Lastly, Goldman
and co-workers found that A1AR mediates the local
antinociceptive effects of acupuncture, suggesting that periph-
eral activation of A1ARs can contribute to analgesia.23
We recently found that 5′-AMP can directly activate A1AR
without being hydrolyzed to adenosine.24 In view of this
finding, we explored the structure−activity relationships (SAR)
of 5′-AMP analogues with respect to their A1AR agonist activity
in our studies below. Furthermore, clinical applications of the
available A1AR agonists have been hampered by their
cardiovascular side effects,15,25 and few orally bioavailable
compounds are known.11,12,26 Herein we report a novel series
of potent and selective A1AR agonists, which are orally active in
a mouse model of acute thermal nociception and lack apparent
cardiovascular side effects.
INTRODUCTION
■
The adenosine A1 receptor (A1AR) belongs to a family of four
G protein-coupled receptors that includes A1AR, A2AAR,
A2BAR, and A3AR.1 A1AR is expressed in the highest density
in the brain, adipose tissue, kidney, and heart atria and to a
lower extent in ventricles, lung, pancreas, liver, and GI tract.2−4
A1AR is also expressed in nociceptive dorsal root ganglia
neurons.5 A1AR and A3AR couple to inhibitory Gi/Go proteins,
which inhibit adenylate cyclase activity, thus reducing cellular
levels of cyclic adenosine monophosphate (cAMP). On the
other hand, A2AAR and A2BAR couple to stimulatory Gs
proteins and stimulate adenylate cyclase activity.6 Adenosine
receptors also modulate phospholipase C, influencing inositol
triphosphate and Ca2+ release from internal stores.7 In addition,
adenosine receptors regulate potassium and calcium channels.8
Many pathophysiological states are associated with changes in
adenosine levels, including asthma, neurodegenerative disor-
ders, psychosis, anxiety, and chronic inflammatory disease.9−12
A1AR has been linked to a variety of human conditions. For
example, adenosine is used clinically to treat supraventricular
tachycardia and targets A1AR in the atrioventicular node of the
heart. This clinical application prompted development of
selective A1AR agonists as antiarrythmic agents.13 Moreover,
adenosine can reduce allodynia and hyperalgesia associated
with chronic pain in rodents and humans.14−19 Ectonucleoti-
RESULTS AND DISCUSSION
■
Chemistry. Isopropylidene-protected 2-chlorocyclopentyl
adenosine 1a was synthesized according to the literature
Received: April 5, 2012
Published: June 27, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of Compounds 3a−f
a
Reagents and conditions: (i) R N,N-diisopropylphosphoramidite, tetrazole, CH2Cl2, 60 °C, 2 h; (ii) H2O2, 0 °C 1 h; (iii) p-TsOH, MeOH, 60 °C, 2
h; (iv) TFA, rt, 1 h; (v) NaOH, MeOH, rt, 30 min; (vi) TFA, CH2Cl2, rt, 1 h; (vii) oxalyl chloride, DMF, CH2Cl2, rt, 2 h; (viii) DIEA, glycine ethyl
ester HCl or phenethyl alcohol, rt, 4 h; (ix) p-TsOH, MeOH, 60 °C, 2 h.
a
Scheme 2. Synthesis of Compounds 5a−c
a
Reagents and conditions: (i) 10% Pd/C, H2, AcOH, MeOH, rt, 18 h; (ii) p-TsOH, MeOH, 60 °C, 2 h; (iii) R N,N-diisopropylphosphoramidite
tetrazole, CH2Cl2, 60 °C, 2 h; (iv) H2O2, 0 °C, 1 h; (v) p-TsOH, MeOH, 60 °C, 2 h; (vi) TFA, rt, 12 h; (vii) NaOH, MeOH, rt, 30 min.
procedure,27 reacted with methyl, ethyl, and tert-butyl N,N-
diisopropylphosphoramidite in the presence of tetrazole, and
subsequently treated with hydrogen peroxide to produce
phosphate diesters 2a−c (Scheme 1). Isopropylidene protect-
ing groups were then removed using p-TsOH in methanol to
give 3a, 3b, and 3c. Compound 3c was treated with TFA to
cleave the tert-butyl protecting groups, and the resulting free
phosphate was converted to its disodium salt 3d by NaOH
titration. Treatment of tert-butyl phosphate diester 2c with
TFA gave isopropylidene-protected phosphate 4. The
phosphate functionality in 4 was then converted to the
dichloride via oxalyl chloride treatment, and the product
reacted with glycine ethyl ester and phenethyl alcohol to
produce compounds 3e and 3f, respectively, following pTsOH-
assisted isopropylidene deprotection.
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a
Scheme 3. Synthesis of Compounds 12a−d
a
Reagents and conditions: (i) p-TsOH, mw; 10 min; (ii) 2-amino-3-phenylpropanol, EtOH, rt, 12 h; (iii) NH3/MeOH, rt, 12 h; (iv) R N,N-
diisopropylphosphoramidite, tetrazole, CH2Cl2, 60 °C, 2 h; (v) H2O2, 0 °C, 1 h; (vi) NH3/MeOH, rt, 12 h.
a
Pd/C and acetic acid-mediated dechlorination of 2a followed
by isopropylidene deprotection under standard conditions gave
5a (Scheme 2). Isopropylidene-protected N6-cyclopentyl
adenosine 6 was synthesized according to published proce-
dures28 and reacted with tert-butyl N,N-diisopropylphospor-
amidite to give intermediate 7, which was then taken through a
sequence similar to above to arrive at the disodium salt 5c.
We also developed a protocol for solvent-free coupling of
2,6-dichloropurine (8) and β-D-ribofuranose 1,2,3,5-tetraacetate
(9) catalyzed by p-TsOH, carried out in a microwave reactor to
furnish 2,6-dichloropurineriboside triacetate (10) (Scheme 3).
Treatment of 10 with 2-amino-3-phenylpropanol in ethanol
overnight gave intermediate 11. Subsequently, acyl protecting
groups in 11 were removed by methanolic ammonia treatment
to give 12a as a mixture of two diastereoisomers inseparable by
column chromatography. Reaction of 11 with methyl and tert-
butyl N,N-diisopropylphosporamidite followed by addition of
hydrogen peroxide produced phosphate diesters, and the acyl
protecting groups were similarly removed via methanolic
ammonia treatment to furnish the desired products 12b and
12c. TFA treatment of 12b followed by titrating sodium
hydroxide into a methanolic solution of the resulting phosphate
yielded the desired disodium salt 12d as a mixture of two
diastereoisomers.
Reaction of 10 with (1R,2R)-2-aminocyclopentanol pro-
duced intermediate 13, which was phosphorylated using methyl
and benzyl phosphoramidite to furnish 14a and 14b,
respectively, after deprotection (Scheme 4). Benzyl protecting
groups in 14b were removed via Pd-mediated hydrogenation,
and the resulting phosphate was treated with NaOH to give the
desired phosphate disodium salt, compound 14c.
Biological Evaluation. The newly synthesized or commer-
cially available ligands were initially tested in a cAMP
accumulation assay, which measures inhibition of isoproterenol-
Scheme 4. Synthesis of Compounds 14a−c
a
Reagents and conditions: (i) (1R,2R)-2-aminocyclopentanol, EtOH,
rt, 12 h; (ii) R N,N-diisopropylphosphoramidite, tetrazole, CH2Cl2, 60
°C, 2 h; (iii) H2O2, 0 °C, 1 h; (iv) NH3/MeOH, rt, 12 h; (v) H2, Pd/
C, MeOH, rt, 12 h; (vi) NaOH, MeOH, rt, 30 min.
or forskolin-stimulated cAMP accumulation in human embry-
onic kidney 293T (HEK293T) cells transiently transfected with
human A1AR. Efficacies of the tested compounds were
compared to 2-chloro-N6-cyclopentyladenosine (3), a potent
full agonist of A1AR.29 All test compounds except 3b and 14a
were full agonists at the highest concentration tested in this
assay (Table 1). We previously reported that 5′-AMP is an
A1AR agonist,24 consistent with other studies30−32 showing that
A1AR can be activated by adenosine analogues with large and
negatively charged groups at the 5′-position.
Interestingly, we found that adenosine 2′-monophosphate
(2′-AMP) and adenosine 3′-monophosphate (3′-AMP) were
also full agonists of A1AR with similar potencies (EC50 values of
0.49 μM and 0.44 μM, respectively) (Table 1). This was not
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adenosine to 5′-AMP. Although the conversion of 5′-phosphate
(5c) to 5′-phosphate dimethyl ester (5a) reduced A1AR agonist
potency, this dimethyl ester modification could potentially
increase oral bioavailability of this series.
Table 1. cAMP Accumulation Assay Results
The 2-chloro-N6-cyclopentyl adenosine subseries was also
explored (Table 1). Compound 3 and its 5′-monophosphate
analogue 3d were equal to or slightly more potent than their
corresponding des-chloro analogues (5 and 5c). The 2-chloro-
N6-cyclopentyl adenosine 5′-phosphate dimethyl ester 3a,
which was designed to improve oral bioavailability, was a full
agonist in this assay with an EC50 value of 1.4 μM. The potency
rank for 5′-alcohol, phosphate, and phosphate dimethyl ester is
consistent for both the 2-chloro and des-chloro subseries.
These SAR results further support our recent finding that 5′-
AMP is a full agonist of A1AR.24 We next explored the SAR at
the 5′ position by examining diethyl and diphenethyl esters 3b
and 3f as well as phosphate diamide 3e. Interestingly,
phosphate diamide 3e was slightly more potent than dimethyl
ester 3a. On the other hand, diethyl ester 3b was significantly
less potent than 3a while diphenethyl ester 3f was equal to or
slightly less potent than 3a. These SAR findings suggest that
modifications to the ester or amide groups are tolerated in
general, resulting in full agonists of A1AR with moderate
potencies with the exception of compound 3b.
a
compd
R
EC50 (μM)
adenosine
2′-AMP
3′-AMP
5′-AMP
5
−
−
−
−
H
0.039
0.49
0.44
0.50
b
0.0063
5c
PO(OH)2
PO(OMe)2
H
0.10
4.4
5a
b
3
0.014/0.0046
0.072
3d
PO(OH)2
PO(OMe)2
PO(OEt)2
PO(OCH2CH2Ph)2
PO(NHCH2CO2Et)2
H
3a
1.4
3b
>10
3f
6.1
Two additional subseries which contain a phosphate or
phosphate dimethyl ester-modified hydroxyl moiety in the N6-
substituent of the purine (compounds 12b, 12d, 14a, and 14c)
were examined (Table 1). Consistent with the previously
published results,24,36−38 alcohols 12a and GR-79236 (14)
were potent A1AR agonists in the cAMP accumulation assay
(EC50 = 0.55 μM and 0.0059 μM). The corresponding
3e
0.53
0.55
1.5
12a
12d
12b
14
PO(OH)2
PO(OMe)2
H
5.4
b
0.0059
b
14c
14a
PO(OH)2
PO(OMe)2
0.12
b
>10
phosphates 12d and 14c were in general less potent (EC50
=
a
EC50 values are the average of at least two independent experiments
with SD values that are 3-fold less than the average. Forskolin
substituted for isoproterenol stimulation.
1.5 μM and 0.12 μM) than the alcohols 12a and 14, but still
showed significant agonist activity. The N6 position projects
toward the solvent-exposed extracellular face of A1AR, so it is
not surprising that a negatively charged phosphate group is
tolerated at this position. The phosphate diesters 12b and 14a
(EC50 = 5.4 μM and EC50 > 10 μM) were generally less potent
than the phosphates 12d and 14c, a SAR trend similar to that
observed in the two subseries described above.
We then selected compounds 3d (a representative
phosphate) and 3a (a representative phosphate ester) and
tested them in an orthogonal functional assay. This assay
utilizes chimeric G proteins to visualize human A1AR activation
in real-time and at a single cell resolution by measuring Ca2+
mobilization (Figure 1).24 The Emax of 3d and 3a were
b
due to hydrolysis to adenosine for the following reasons: (1)
Adenosine gives a characteristic “bimodal” response in the
HEK293T cAMP assay due to its activation of endogenous Gs-
coupled A2AAR in addition to the Gi-coupled ectopically
expressed A1AR.24 If AMP hydrolysis to adenosine were
occurring, we would expect to see a bimodal response for 2′-,
3′-, and 5′-AMP. However, we did not observe a bimodal
response for 2′-, 3′-, or 5′-AMP (data not shown). (2)
Inclusion of α,β-methylene adenosine 5′-diphosphate (αβ-met-
ADP), a potent inhibitor of ecto-5′-nucleotidase (NT5E/
CD73),33 did not significantly alter the potencies of 2′-AMP or
3′-AMP (Table S1, Supporting Information).
Recent X-ray crystallography studies using A2AAR have
identified positively charged histidine residues deep within the
agonist binding pocket, which are conserved in A1AR.34,35 One
of these residues, His278 in TM7, is proximal to the 2′ and 3′
hydroxyl groups in the ribose moiety of adenosine. Thus, a
phosphate group at the 2′ or 3′ positions could form a
stabilizing charge−charge interaction with His278, explaining
this novel activity of 2′- and 3′-AMP.
N6 substitution in the purine moiety has been shown to
improve binding affinities of ligands to A1AR.29 In our cAMP
accumulation assay, N6-cyclopentyl adenosine (5) was indeed
more potent than adenosine (EC50: 0.0063 μM versus 0.039
μM) (Table 1). The corresponding 5′-monophosphate
derivative 5c was less potent (EC50 = 0.10 μM) than compound
5. This result is consistent with the potency changes from
Figure 1. Compounds 3d and 3a are full agonists of A1AR in a Ca2+
mobilization assay. 3d: EC50 = 0.021 μM; and 3a: EC50 = 0.52 μM.
Adenosine (EC50 = 1.41 μM) was used as a positive control. 27−50
cells per condition. All data are presented as mean standard error.
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normalized to the Emax of adenosine in this assay. Both 3d and
3a were potent full agonists of A1AR with EC50 values of 0.021
μM and 0.52 μM, respectively. Therefore, using two distinct
and complementary assay platforms, we confirmed that
compounds 3d and 3a directly activated A1AR-mediated
signaling.
dose-dependently inhibited noxious thermal sensitivity (Figure
3A and 3B). Significant effects were observed even at a low
Compounds 3a and 3d were next evaluated in a radioligand
binding assay to determine their binding affinities to human
A1AR using tritiated compound 3 as the radioligand. Consistent
with their functional activities in the cAMP accumulation and
Ca2+ mobilization assays, compounds 3a and 3d had high
affinities for the human A1AR with Ki values of 36 nM and 25
nM, respectively (Figure 2).
Figure 3. Oral administration of compound 3a dose-dependently
decreases thermal sensitivity in wild-type mice. (A) Compound 3a and
(C) compound 5 were orally administrated at 50, 500, or 5000 nmol/
kg immediately after determination of the baseline time point. Paw
withdrawal latency was assessed at the indicated times, using
Hargreaves apparatus to deliver noxious thermal stimulus. Ten
C57BL/6 male mice per dose group. t tests were conducted relative
to the corresponding vehicle time point. (B, D) Area under curve
(AUC) analysis of data shown in (A, C). Five hour AUC
measurements were calculated relative to baseline paw withdrawal
latency for each mouse and averaged for each treatment condition. All
data are presented as mean standard error. * p < 0.05, ** p < 0.005,
*** p < 0.0005.
Figure 2. Compounds 3a and 3d have high binding affinities to human
A1AR. Concentration−response curves of 3a and 3d in the radioligand
binding assay using tritiated compound 3 as the radioligand, and 5′-N-
ethylcarboxamidoadenosine (NECA) as a positive control (data not
shown).
To evaluate subtype selectivity (selectivity for A1AR over
A2AAR, A2BAR, and A3AR) of compounds 3a and 3d, we tested
3a and 3d in human A2AAR, A2BAR, and A3AR radioligand
binding assays (Table 2). Both compounds had no binding
dose (50 nmol/kg). The effects of 3a were similar to 5 (Figure
3C and 3D), which was used as a positive control in this mouse
model. In addition, intrathecal (IT) or intravenous (IV)
injection of 3a produced marked and dose-dependent
inhibition of noxious thermal sensitivity (Figure S1, Supporting
Information), similar to oral administration. Importantly, the
thermal antinociceptive effect of 3a was completely abolished in
the A1AR knockout (A1AR−/−) mice even at a high dose (5
nmol, IT injection) (Figure 4), strongly demonstrating that the
biological effects of 3a are A1AR-mediated. Taken together,
these results reveal that compound 3a is an orally active
compound with potent A1AR-mediated antinociceptive activity
in mice. It is worth noting that carboxylic acid esters are
degraded rapidly in vivo by carboxylesterases; however, the
corresponding alkyl esters of phosphates are typically
metabolically stable.41 Therefore, it is unlikely that the observed
in vivo effects of the phosphate ester 3a are due to hydrolysis of
both alkyl esters to produce 3d or the dephosphorylated
product 3.
Clinical applications of the currently available A1AR agonists
are hampered by their cardiovascular side effects.32 To
determine if compound 3a affected cardiovascular function,
we monitored heart rate and body temperature in wild-type and
A1AR−/− mice following oral administration (5 was adminis-
tered as a positive control). At the highest dose tested (5000
nmol/kg, oral administration), compound 3a had negligible
effects on heart rate and body temperature in wild-type and
Table 2. Subtype Selectivity of Compounds 3a and 3d
Assessed Using Radioligand Binding Assays
binding affinity (Ki
binding
affinity ratio
(nM))
% inhibition/
[concentration] at A2B
A2A
/
A3/
A1
compound A1
A2A
A3
A1
3a
3d
36
25
4700
1600
450
7%/[50 μM]
0%/[10 μM]
130
640
12
52
1300
affinity to A2BAR (<10% inhibition at 50 μM and 10 μM,
respectively). In addition, compounds 3a and 3d were >100-
fold selective for A1AR over A2AAR and >10-fold selective for
A1AR over A3AR. Compounds 3a and 3d are “compound 3-
like” agonists, containing both 2-chloro and N6-cyclopentyl
groups, which convey strong subtype selectivity for A1AR.
Compound 3 itself displays >100-fold selectivity for A1AR over
A2AAR and A2BAR.29,39 As expected, compounds 3a and 3d
retained this A1AR selectivity after modification at the 5′
position. A3AR is known to be a low-affinity adenosine
receptor,40 so the modestly decreased binding affinity of 3a
and 3d at A3AR is not surprising.
Having established that compounds 3a and 3d are potent
and subtype selective A1AR agonists, we next evaluated the
antinociceptive effects of 3a in mice. We focused on 3a for in
vivo studies because of its enhanced potential to demonstrate
oral activity.41 Notably, we found that oral administration of 3a
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and uniquely lacks cardiovascular side-effects that are associated
with other A1AR agonists such as compound 5.
CONCLUSIONS
■
In summary, we designed and synthesized a novel series of
A1AR agonists which possess marked potency in several
orthogonal in vitro assays and are subtype selective for A1AR
over A2AAR, A2BAR, and A3AR. Surprisingly, our SAR studies
revealed that the addition of a phosphate or, in some cases a
phosphate ester group, to the 5′, 3′, 2′, or N6 moiety of
adenosine is tolerated. These findings reveal that A1AR can be
activated by diverse natural and non-natural nucleotide and
nucleoside analogues. Among our novel A1AR agonists,
compound 3a had potent, dose-dependent, and A1AR-depend-
ent antinociceptive activity in mice via oral administration. The
apparent lack of cardiovascular side effects in vivo and
nanomolar affinity at human A1AR makes this novel compound
potentially suitable for treating pain and other physiological
processes that are modulated by A1AR activation.
Figure 4. Effects of 3a on thermal sensitivity in wild-type mice are
completely abolished in A1AR−/− mice. Time course of effects of 3a on
on thermal paw withdrawal latency. 3a (5 nmol) was administered via
intrathecal injection immediately after determination of the baseline
time point. Thermal paw withdrawal latency in wild type and A1AR−/−
mice were monitored using Hargreaves apparatus. Ten C57BL/6 male
mice per group. All data are presented as mean standard error. t
tests were conducted relative to the baseline time point. ** p < 0.005.
*** p < 0.0005.
A1AR−/− mice (Figure 5A and 5C). On the other hand,
compound 5 elicited a statistically significant decrease in heart
EXPERIMENTAL SECTION
■
General Procedure for Chemical Synthesis. HPLC data of all
compounds were acquired using an Agilent 6110 Series system with
the UV detector set to 220 nm. Samples were injected (<10 μL) onto
an Agilent Eclipse Plus 4.6 × 50 mm, 1.8 μM, C18 column at room
temperature. Mobile phases consisting of H2O + 0.1% acetic acid (A)
and MeOH + 0.1% acetic acid (B) were used. A linear gradient from
10% to 100% B during 5.0 min was used followed by 100% B for
another 2 min with a flow rate of 1.0 mL/min. Mass spectra (MS) data
were acquired in positive ion mode using an Agilent 6110 single
quadrupole mass spectrometer with an electrospray ionization (ESI)
source. High-resolution mass spectra (HRMS) were acquired using a
Shimadzu LCMS-IT-TOF time-of-flight mass spectrometer. Nuclear
magnetic resonance (NMR) spectra were recorded on a Varian
Mercury spectrometer at 400 MHz for proton (1H NMR) and 100
MHz for carbon (13C NMR); chemical shifts are reported in ppm (δ)
relative to the solvent peaks.44 Preparative HPLC was performed using
an Agilent Prep 1200 series with the UV detector set to 220 nm.
Samples were injected onto a Phenomenex Luna 75 × 30 mm, 5 μM,
C-18 column at room temperature. Mobile phases consisting of H2O +
0.1% TFA (A) and MeOH (B) were used at a flow rate of 30 mL/min.
A linear gradient from 10% to 100% B in 17.0 min was followed by
100% B for another 3 min. Adenosine, 5′-AMP, 3′-AMP, 2′-AMP, 3, 5,
and 14 were purchased from Sigma-Aldrich. All synthesized
compounds have >95% purity by the above analytical HPLC method
unless specific purities are noted below.
Preparation of ((2R,3S,4R,5R)-5-(2-Chloro-6-(cyclopentylamino)-
9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl) Phosphates
(3a,b). To a solution of 2-chloro-N6-cyclopentyl-2′,3′-O-(1-methyl-
ethylidene)-(9-Cl)adenosine (1) (0.73 mmol) in CH2Cl2 (15 mL)
were added the appropriate phosphoramidite (1.5 mmol) and tetrazole
(2.4 mmol). The reaction mixture was heated at 60 °C for 2 h and
cooled to 0 °C, and hydrogen peroxide (0.3 mL, of 30% w/w solution)
was added dropwise. The reaction mixture was stirred for 1 h at rt,
diluted with CH2Cl2 (100 mL), and washed with 10% aqueous sodium
metabisulfite (25 mL × 2), saturated aqueous sodium bicarbonate (25
mL × 2), water (25 mL × 2), and brine (25 mL × 2). The solvent was
then removed under reduced pressure, and the residue was purified by
preparative HPLC to afford the TFA salts of 2a,b. To a solution of
phosphates 2a,b (0.32 mmol) in methanol (10 mL) was then added p-
toluenesulfonic acid (0.03 mmol), the mixture was heated at 60 °C for
2 h, and the solvent was removed under reduced pressure. The
residues were then purified by reverse phase preparative HPLC to
afford the TFA salts of the title compounds.
Figure 5. Compound 3a does not have long-lasting effects on heart
rate or body temperature while compound 5 causes a significant
decrease in heart rate and body temperature in wild-type mice. (A, C)
Effects of 3a on (A) heart rate and (C) body temperature in wild-type
(red) and A1AR−/− (black) mice. (B, D) Effects of 5 on (B) heart rate
and (D) body temperature in wild-type (red) and A1AR−/− (black)
mice. Compounds were orally administered at 5000 nmol/kg
immediately before telemetry recording began. Eight C57BL/6 male
mice per group for A1AR−/− body temperature measurements, and six
male mice per group for all other conditions. Red bar: p < 0.05.
rate and body temperature which lasted for 4 to 6 h in wild-type
mice (Figure 5B and 5D) but not in A1AR−/− mice. Compound
5 caused a modest increase in heart rate in A1AR−/− mice,
possibly reflecting known off-target activation of stimulatory
A2AR.42,43 Collectively, our results indicate that our novel A1AR
agonist 3a has potent antinociceptive effects but minimal to no
cardiovascular side effects when administered orally at a high
5000 nmol/kg dose. In contrast, the same high dose of 5 has
antinociceptive effects and significant cardiovascular side effects.
These data in turn suggest 3a has a large therapeutic window
((2R,3S,4R,5R)-5-(2-Chloro-6-(cyclopentylamino)-9H-purin-9-yl)-
3,4-dihydroxytetrahydrofuran-2-yl)methyl Dimethyl Phosphate
1
(3a). (22%) white solid. H NMR (300 MHz, DMSO-d6): δ 8.30 (s,
1H), 5.86 (d, J = 5.0 Hz, 1H), 5.75−5.50 (m, 1H), 4.58−4.48 (m,
6472
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1H), 4.50−4.35 (m, 1H), 4.30−4.40 (m, 4H), 3.72−3.53 (m, 6H),
2.11−1.89 (m, 2H), 1.89−1.51 (m, 6H); 13C NMR (101 MHz,
DMSO-d6) δ 155.12, 153.78, 149.95, 139.89, 118.88, 87.92, 83.01,
82.94, 73.54, 70.35, 67.33, 54.64, 54.58, 54.52, 52.14, 33.45, 32.27,
23.93; HRMS calcd for C17H25ClN5O7P + H: 478.1253; found:
478.1243 [M + H]+.
was then removed under reduced pressure, and the residue was
dissolved in DMF (10 mL) followed by addition of N,N-
diisopropylethylamine (121 mg, 0.92 mmol) and phenylethyl alcohol
(119 mg, 0.78 mmol). The reaction mixture was stirred at rt for 4 h,
and the solvent was removed under reduced pressure. The residue was
dissolved in water, extracted with ethyl acetate (3 × 50 mL), dried
over Na2SO4, filtered, and concentrated in vacuo. To this were added
methanol (10 mL) and p-toluenesulfonic acid (1 mg), and the mixture
was heated at 60 °C for 2 h. The solvents were removed under
reduced pressure, and the residue was purified by preparative HPLC to
provide the TFA salt of the title compound 3f (15 mg, 8%) as a white
((2R,3S,4R,5R)-5-(2-Chloro-6-(cyclopentylamino)-9H-purin-9-yl)-
3,4-dihydroxytetrahydrofuran-2-yl)methyl Diethyl Phosphate (3b).
1
(22%) white solid. H NMR (400 MHz, CD3OD): δ 8.21 (s, 1H),
5.97 (d, J = 4.3 Hz, 1H), 4.72−4.62 (m, 1H), 4.50 (brs, 1H), 4.44 (t, J
= 5.2 Hz, 1H), 4.38−4.33 (m, 1H), 4.32−4.26 (m, 1H), 4.26−4.20 (m,
1H), 4.15−4.02 (m, 4H), 2.18−2.01 (m, 2H), 1.92−1.74 (m, 2H),
1.73−1.52 (m, 4H), 1.37−1.24 (m, 6H); HRMS calcd for
C19H29ClN5O7P + H: 506.1566; found: 506.1551 [M + H]+.
1
solid. H NMR (400 MHz, CDCl3): δ 7.93 (s, 1H), 7.36−7.07 (m,
10H), 6.20 (brs, 1H), 5.89 (d, J = 4.5 Hz, 1H), 4.56 (brs, 1H), 4.49−
4.43 (m, 1H), 4.39−4.35 (m, 1H), 4.30−4.25 (m, 1H), 4.17−4.03 (m,
4H), 4.03−3.95 (m, 1H), 2.98−2.76 (m, 5H), 2.22−2.04 (m, 2H),
1.87−1.61 (m, 4H), 1.60−1.45 (m, 2H), 1.39−1.13 (m, 2H); HRMS
calcd for C31H37ClN5O7P + H: 658.2192; found: 658.2173 [M + H]+.
((3aR,4R,6R,6aR)-6-(2-Chloro-6-(cyclopentylamino)-9H-purin-9-
yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl Dihy-
drogen Phosphate (4). To a solution of phosphate 3c (150 mg,
0.25 mmol) in CH2Cl2 (10 mL) was added TFA (0.3 mL, 2.63 mmol)
in CH2Cl2 (10 mL), and the reaction mixture was stirred for 1 h at rt.
The solvent was removed under reduced pressure to afford the title
compound 4 (93 mg, 76% yield) as a white solid, carried on to the
next step without further purification.
Sodium ((2R,3S,4R,5R)-5-(2-Chloro-6-(cyclopentylamino)-9H-
purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl Phosphate
(3d). To a solution of 1 (300 mg, 0.73 mmol) in CH2Cl2 (15 mL)
were added tert-buytl N,N-diisopropylphosphoramidite (405 mg, 1.5
mmol) and tetrazole (5 mL of 0.45 M solution in ACN, 2.4 mmol),
and the reaction mixture was heated at 60 °C for 2 h. The temperature
was decreased to 0 °C, and hydrogen peroxide (0.3 mL, of 30% w/w
solution) was added. The reaction mixture was stirred for 1 h at rt,
CH2Cl2 (100 mL) was added, and the organic layer was washed with
10% sodium metabisulfite (25 mL × 2), saturated sodium bicarbonate
(25 mL × 2), water (25 mL × 2), and brine (25 mL × 2). The solvent
was removed under reduced pressure and purified by preparative
HPLC to afford the TFA salt of compound 2c (268 mg, 61%). To a
solution of phosphate 2c (44 mg, 0.09 mmol) in methanol (10 mL)
was added p-toluenesulfonic acid (5 mg, 0.03 mmol), and the mixture
was heated at 60 °C for 2 h. The solvent was removed under reduced
pressure, trifluoroacetic acid (0.3 mL, 2.63 mmol) was added in
CH2Cl2 (10 mL), and the reaction mixture was stirred for 1 h at rt.
The solvent was removed under reduced pressure, and the residue was
purified by preparative HPLC to afford the TFA salt of compound 3d.
The phosphate was then dissolved in methanol (5 mL), NaOH (0.90
mmol, 36 mg) in water (5 mL) was added, and the mixture was stirred
for 30 min at rt. The solvent was removed under reduced pressure to
((2R,3S,4R,5R)-5-(6-(Cyclopentylamino)-9H-purin-9-yl)-3,4-dihy-
droxytetrahydrofuran-2-yl)methyl Dimethyl Phosphate (5a). To a
solution of phosphate 2a (600 mg, 1.16 mmol) in methanol (50 mL)
were added 10% Pd/C (300 mg, 50% w/w) and acetic acid (50 mL),
and the reaction flask was fitted with a hydrogen balloon. The reaction
mixture was then stirred for 6 h at rt and filtered through Celite. The
solvent was removed under reduced pressure, the residue was
dissolved in methanol (10 mL), and p-toluenesulfonic acid (17 mg,
0.16 mmol) was added. The reaction mixture was heated at 60 °C for
2 h, the solvent was removed under reduced pressure, and the residue
was purified via preparative HPLC to afford the TFA salt of the title
1
compound 5a (200 mg, 38% yield) as a white solid. H NMR (300
1
MHz, DMSO-d6): δ 8.41 (brs, 1H), 8.30 (s, 1H), 5.86 (d, J = 5.2 Hz,
1H), 4.61 (t, J = 5.4 Hz, 1H), 4.35−4.05 (m, 2H), 3.70−3.62 (m, 6H),
2.05−1.91 (m, 2H), 1.81−1.55 (m, 7 H); HRMS calcd for
C17H26N5O7P + H: 444.1643; found: 444.1630 [M + H]+.
afford the title compound 3d (18 mg, 42% yield) as a white solid. H
NMR (300 MHz, CD3OD): δ 8.47 (s, 1H), 6.02 (d, J = 5.7 Hz, 1H),
4.65−4.55 (m, 1H), 4.55−4.45 (m, 1H), 4.41−4.35 (m, 1H), 4.25−
4.20 (m, 1H), 4.12−4.05(m, 3H), 2.23−2.07 (m, 2H), 1.86−1.69 (m,
7H); HRMS calcd for C15H21ClN5O7P + H: 450.0945; found:
450.0911 [M + H]+.
Sodium ((2R,3S,4R,5R)-5-(6-(Cyclopentylamino)-9H-purin-9-yl)-
3,4-dihydroxytetrahydrofuran-2-yl)methyl Phosphate (5c). To a
solution of 6 (281 mg 0.75 mmol) in CH2Cl2 (15 mL) were added
tert-buytl-N,N-diisopropylphosphoramidite (405 mg, 1.5 mmol) and
tetrazole (5 mL of 0.45 M solution in ACN, 2.4 mmol). The reaction
mixture was heated at 60 °C for 2 h and then cooled to 0 °C, and
hydrogen peroxide (0.3 mL, of 30% w/w solution) was added
dropwise. The reaction mixture was then stirred for an additional 1 h,
diluted with CH2Cl2 (100 mL), and washed with 10% sodium
metabisulfite (25 mL × 2), saturated sodium bicarbonate (25 mL × 2),
water (25 mL × 2), and brine (25 mL × 2). The solvent was removed
under reduced pressure to afford compound 7 (217 mg, 51% yield),
which was used in the following steps without further purificaiton. To
a solution of phosphate 7 (145 mg, 0.26 mmol) in methanol (10 mL)
was added p-toluenesulfonic acid (28 mg, 0.03 mmol), and the
reaction mixture was heated at 60 °C for 2 h. The solvent was removed
under reduced pressure, TFA (0.3 mL, 2.63 mmol) in CH2Cl2 (10
mL) was added, and the reaction mixture was stirred at rt for an
additional 1 h. The solvent was again removed under reduced pressure,
and the residue was purified by preparative HPLC to afford the TFA
salt of the title compound. To a solution of the TFA salt (50 mg, 0.12
mmol) in methanol (5 mL) was added NaOH (9.6 mg, 0.24 mmol) in
water (5 mL), and the reaction mixture was stirred for 30 min at rt.
The solvent was removed under reduced pressure to afford the title
Ethyl 2-(((((2R,3S,4R,5R)-5-(2-Chloro-6-(cyclopentylamino)-9H-
purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)((2-oxo-2-
ethyloxyethyl)amino)phosphoryl) amino)acetate (3e). To a solution
of phosphate 4 (150 mg, 310 mmol) in CH2Cl2 (5 mL) were added
oxalyl chloride (155 mg, 1.23 mmol) and DMF (1 drop), and the
mixture was stirred at rt for 2 h. The solvent was removed under
reduced pressure, and the residue was dissolved in DMF (10 mL)
followed by addition of N,N-diisopropylethylamine (121 mg, 0.92
mmol) and ethyl 2-aminoacetate (119 mg, 0.78 mmol). The reaction
mixture was then stirred for 4 h at rt, and the solvent was removed
under reduced pressure. The residue was dissolved in water, extracted
with ethyl acetate (3 × 50 mL), and dried over Na2SO4. The solvent
was removed under reduced pressure, the residue was dissolved in
methanol (10 mL), p-toluenesulfonic acid (1 mg, 0.009 mmol) was
added, and the mixture was heated at 60 °C for 2 h. The solvent was
removed under reduced pressure, and the residue was purified by
preparative HPLC to afford the TFA salt of the title compound 3e (15
1
mg, 7%) as a white solid. H NMR (400 MHz, CDCl3): δ 8.54 (s,
1H), 7.39−7.26 (m, 1H), 6.06 (s, 1H), 5.62−4.82 (m, 5H), 4.66−4.45
(m, 3H), 4.40−4.20 (m, 2H), 4.16−4.05 (m, 3H), 3.75−3.60 (m, 4H),
2.16−2.05 (m, 2H), 1.84−1.44 (m, 5H), 1.23 (m, 6H); HRMS calcd
for C23H35ClN7O9P + H: 620.1995; found: 620.2008 [M + H]+.
((2R,3S,4R,5R)-5-(2-Chloro-6-(cyclopentylamino)-9H-purin-9-yl)-
3,4-dihydroxytetrahydrofuran-2-yl)methyl Diphenethyl Phosphate
(3f). To a solution of phosphate 4 (150 mg, 310 mmol) in CH2Cl2 (5
mL) were added oxalyl chloride (155 mg, 1.23 mmol) and DMF (1
drop), and the reaction mixture was stirred for 2 h at rt. The solvent
1
compound 5c (55 mg, 99% yield) as a white solid. H NMR (300
MHz, DMSO-d6): δ 8.52 (s, 1H), 8.30 (s, 1H), 7.47 (d, J = 6.1, 1H),
7.17 (d, J = 6.0, 1H), 5.95 (d, J = 6.0 Hz, 1H), 5.48 (brs, 2H), 4.55 (t, J
= 6.0, 2.4 Hz, 1H), 4.40 (m, 1H), 4.29 (m, 1H), 2.19−1.92 (m, 2H),
1.80−1.54 (m, 7H); MS(ESI) m/z 460.4[M - H]−
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1
(2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(2,6-dichloro-9H-purin-9-yl)-
tetrahydrofuran-3,4-diyl Diacetate (10). 2,6-Dichloro-9H-purine
(2.38 g, 12.6 mmol), (2S,3R,4R,5R)-5-(acetoxymethyl)-
tetrahydrofuran-2,3,4-triyl triacetate (4.00 g, 12.6 mmol) and p-
toluenesulfonic acid monohydrate (10 mg, 0.06 mmol) were ground to
a smooth consistency using a mortar and pestle. The powder was
transferred to a microwave vial and heated in the microwave reactor
for 5 min at 100 °C at a 50 W power setting. The resulting brown oil
was dissolved in methanol (25 mL) and stirred for 2 h. The precipitate
was collected and washed with methanol to afford the title compound
10 (2.87 g, 51%) as a tan solid. 1H NMR (300 MHz, DMSO): 8.92 (s,
1 H), 6.32 (d, J = 5.0 Hz, 1 H), 5.90 (t, J = 5.4 Hz, 1H), 5.62 (t, J = 5.5
Hz, 1H), 4.46−4.25 (m, 4H), 2.11(s, 1H), 2.05(s, 1H), 2.02(s, 1H);
MS(ESI) m/z 447 [M + H]+.
methyl Phosphate (12b). (38%) (clear oil) H NMR (400 MHz,
CDCl3): δ 8.28 (d, J = 51.4 Hz, 1H), 7.98 (dd, J = 23.7, 7.3 Hz, 1H),
7.35−7.15 (m, 5H), 5.83 (m, 1H), 4.79 (d, J = 29.4 Hz, 2H), 4.40 (s,
1H), 4.33−4.20 (m, 2H), 4.18−4.05 (m, 1H), 4.01−3.88 (m, 1H),
3.83−3.64 (m, 6H), 3.14−2.94 (m, 3H); 13C NMR (126 MHz,
CDCl3) δ 160.9, 160.6, 155.7, 154.0, 148.5, 139.9, 139.6, 136.8, 129.4,
128.8, 127.1, 116.8, 116.5, 116.0, 113.9, 91.6, 87.9, 74.2, 72.1, 71.8,
67.9, 62.9, 62.6, 55.0, 52.5, 36.7; MS(ESI) m/z 544 [M + H]+.
Di-tert-butyl (2-((2-Chloro-9-(3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)-3-phenylpropyl) Phos-
1
phate (12c). (61%) (white solid). H NMR (400 MHz, CD3OD): δ
8.18 (s, 1H), 7.27−7.10 (m, 5H), 5.90 (m, 1H), 4.78 (br s, 1H), 4.64−
4.56 (m, 1H), 4.26−4.20 (m, 1H), 4.15−4.05 (m, 2H), 4.03−3.90 (m,
1H), 3.85−3.80 (m, 1H), 3.74−3.65 (m, 1H), 3.30−3.22 (m, 2H),
3.02−2.90 (m, 2H) 1.37 (s, 9H), 1.33 (s, 9H); 13C NMR (101 MHz,
CD3OD) δ 156.39, 155.20, 150.68, 141.78, 138.93, 130.40, 129.50,
129.39, 127.68, 127.51, 91.03, 90.97, 87.85, 84.64, 84.56, 75.55, 75.47,
72.35, 72.33, 68.23, 68.17, 63.31, 63.30, 53.35, 49.64, 49.43, 49.28,
49.21, 49.07, 49.00, 48.79, 48.57, 48.36, 37.93, 30.13, 30.09, 30.09,
30.05; HRMS calcd for C27H39ClN5O8P + H: 628.2298; found:
628.2289 [M + H]+.
Sodium 2-((2-Chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)-3-phe-
nylpropyl Phosphate (12d). To a solution of phosphate 12c (100 mg,
0.159 mmol) in CH2Cl2 (16 mL) was added trifluoroacetic acid (0.2
mL, 1.75 mmol), and the reaction mixture was stirred at rt for 1 h. The
solvent was then removed under reduced pressure, and the residue was
purified by preparative HPLC to afford the TFA salt of the title
compound. The phosphate salt was then dissolved in methanol (5
mL), NaOH (9.6 mg, 0.24 mmol) in water (5 mL) was added, and the
reaction mixture was stirred for 30 min at rt. The solvent was then
removed under reduced pressure to afford the title compound 12d (55
(2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(2-chloro-6-((1-hydroxy-3-
phenylpropan-2-yl)amino)-9H-purin-9-yl)tetrahydrofuran-3,4-diyl
Diacetate (11). To a solution of 2-amino-3-phenylpropanol (67.6 mg,
0.45 mmol) in ethanol (10 mL) were added 2,6-dichloropurine
riboside (10) (100 mg, 0.22 mmol) and triethylamine (25 mg, 0.25
mmol). The reaction mixture was stirred at 70 °C for 12 h and
partitioned between ethyl acetate (100 mL) and water (50 mL). The
aqueous layer was extracted with ethyl acetate (100 mL × 2). The
combined organics were washed with brine (10 mL) and dried over
Na2SO4. The solvent was removed under reduced pressure, and the
residue was purified by flash column chromatography on silica gel
(50% EtOAc/hexanes) to afford the title compound 11 (clear oil) as
1
an inseparable mixture of diastereomers (242 mg, 86%). H NMR
(400 MHz, CDCl3): δ 7.84−7.82 (m, 1H), 7.24−7.13 (m, 5H), 6.10−
6.07 (m, 1H), 5.73−5.70 (m, 1H), 5.56−5.52 (m, 1H), 4.70−4.49 (m,
2H), 4.34−4.28 (m, 3H), 3.90−3.60 (m, 2H), 3.03−2.90 (m, 2H),
2.12−1.96 (m, 9H); 13C NMR (126 MHz, CDCl3) δ 170.4, 169.7,
169.6, 169.5, 169.4, 154.8, 138.1, 137.9, 129.4, 128.5, 126.5, 85.6, 85.7,
70.6, 67.1, 63.1, 62.4, 53.8, 37.2, 37.1, 20.8, 20.7, 20.6, 20.5, 20.5, 20.4;
MS(ESI) m/z 562 [M + H]+.
1
mg, 99%) as a white solid. H NMR (400 MHz, CD3OD): δ 8.16 (s,
1H), 7.22−7.14 (m, 2H), 7.14−7.06 (m, 2H), 7.06−6.98 (m, 1H),
5.80−5.76 (m, 1H), 4.64 (br s, 1H), 4.52 (t, J = 5.5 Hz, 1H), 4.22−
4.16 (m, 1H), 4.08−3.91 (m, 3H), 3.80−3.72 (m, 1H), 3.64−3.60 (m,
1H), 3.22−3.15 (m, 2H), 3.00−2.82 (m, 2H); 13C NMR (126 MHz,
D2O) δ 155.7, 153.7, 148.6, 140.3, 138.4, 129.9, 128.1, 126.5, 118.8,
88.6, 85.9, 73.7, 70.7, 65.5, 61.7, 53.4, 37.6; HRMS calcd for
C19H23ClN5O8P + H: 516.1051; found: 516.1019 [M + H]+.
((2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(2-chloro-6-(((1R,2R)-2-
hydroxycyclopentyl)amino)-9H-purin-9-yl)tetrahydrofuran-3,4-diyl
Diacetate (13). To a solution of (1R,2R)-2-aminocyclopentanol (346
mg, 3.42 mmol) in ethanol (3 mL) were added 2,6-dichloropurine
riboside (10) (305 mg, 0.68 mmol) and triethylamine (686 mg, 6.80
mmol). The reaction mixture was heated at 70 °C for 12 h and
partitioned between ethyl acetate (100 mL) and water (50 mL). The
aqueous layer was extracted with ethyl acetate (100 mL × 2). The
combined organics were washed with brine (10 mL) and dried over
Na2SO4. The solvent was removed under reduced pressure, and the
residue was purified by flash column chromatography on silica gel
(50% EtOAc/hexanes) to afford the title compound 13 (clear oil)
(323 mg, 92%). 1H NMR (400 MHz, CDCl3): δ 8.30 (s, 1H), 6.90 (s,
1H), 5.80−5.77 (m, 1H), 5.57−5.54 (m, 1H), 4.48−4.46 (m, 1H),
4.44−4.38 (m, 2H), 4.15−4.01 (m, 1H), 3.47−3.40 (m, 2H), 3.32−
2.89 (m, 2H), 2.19 (s, 3H), 2.15−2.12 (m, 5H), 2.09−2.07 (m, 4H),
2.13−2.08 (m, 1H), 2.05−1.65 (m, 2H), MS(ESI) m/z 5.12 [M +
H]+.
(1R,2R)-2-((2-Chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)-
cyclopentyl Dimethyl Phosphate (14a). To a solution of acetate 13
(60 mg, 0.12 mmol) in CH2Cl2 (5 mL) were added dimethyl
diisopropylphosphoramidite (45 mg, 0.23 mmol) and tetrazole (0.45
M solution in THF, 0.83 mL, 0.37 mmol), and the mixture was heated
at 60 °C for 2 h. The reaction was then cooled to 0 °C, H2O2 (0.1 mL
of 30% solution, 0.12 mmol) was added, and the reaction mixture was
stirred for an additional 1 h. The reaction was then diluted with
CH2Cl2 (100 mL) and washed with 10% sodium metabisulfite,
saturated sodium bicarbonate, water, and brine. The solvent was
removed under reduced pressure, and the residue was purified by flash
column chromatography on silica gel (10% methanol/CH2Cl2) to
(2R,3R,4S,5R)-2-(2-Chloro-6-((1-hydroxy-3-phenylpropan-2-yl)-
amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol
(12a). To acetate 11 (65 mg, 0.116 mmol) was added methanolic
ammonia (30 mL) at 0 °C, the reaction vessel was sealed, and the
reaction mixture was stirred for 5 h at rt. The solvent was then
removed under reduced pressure, and the residue was purified by
preparative HPLC to afford the TFA salt of the title compound 12a
(30 mg, 60%) (white solid) as an inseparable mixture of diastereomers.
1H NMR (400 MHz, DMSO): δ 8.42−8.34 (m, 1H), 8.08 (d, J = 8.3
Hz, 1H), 7.35−7.20 (m, 4H), 7.17−7.07 (m, 1H), 5.80 (d, J = 5.8 Hz,
1H), 4.55−4.34 (m, 2H), 4.15−4.06 (m, 1H), 3.98−3.89 (m, 1H),
3.65 (dd, J = 12.0, 4.0 Hz, 1H), 3.60−3.42 (m, 3H), 3.04−2,71 (m,
2H); 13C NMR (126 MHz, DMSO) δ 158.91, 158.54, 155.91, 155.44,
153.49, 149.90, 140.22, 139.95, 139.65, 139.46, 129.49, 128.73, 128.53,
126.36, 118.89, 117.72, 87.86, 87.64, 86.16, 74.06, 70.81, 63.59, 62.82,
61.80, 56.27, 54.36, 40.58, 40.37, 40.16, 39.95, 39.74, 39.53, 39.32,
37.64, 36.71, 31.11.
Preparation of 2-((2-Chloro-9-(3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)-3-phenylpropyl Phos-
phates (12b,c). To a solution of 11 (0.35 mmol) in CH2Cl2 (5
mL) were added the appropriate phosphoramidite (0.70 mmol) and
tetrazole (0.45 M solution in THF, 2.22 mL, 1.02 mmol), and the
mixture was heated for 2 h at 60 °C. The reaction was then cooled to 0
°C, H2O2 (0.2 mL of 30% solution, 0.225 mmol) was added, and the
reaction mixture was stirred for an additional 1 h. The reaction mixture
was diluted with CH2Cl2 (100 mL) and washed with 10% aq sodium
metabisulfite (20 mL), sat. aq sodium bicarbonate (20 mL), water (20
mL), and brine (20 mL). The solvent was removed under reduced
pressure, and the residue was purified by flash column chromatography
on silica gel (10% methanol/CH2Cl2) to afford the acetate. To the
acetate (0.135 mmol) was added methanolic ammonia (20 mL) at 0
°C, the reaction vessel was sealed, and the reaction mixture was stirred
for 5 h at rt. The solvent was then removed under reduced pressure,
and the residue was purified by preparative HPLC to afford
compounds 12b,c.
2-((2-Chloro-9-(3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)-3-phenylpropyl Di-
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afford the phosphate as a clear oil. To the phosphate (60 mg, 0.097
mmol) was then added methanolic ammonia (30 mL) at 0 °C, the
reaction vessel was sealed, and the reaction mixture was stirred for 5 h
at rt. The solvent was removed under reduced pressure, and the
residue was purified by flash column chromatography (10% EtOAc/
hexanes) to afford the title compound 14a (white solid) (18 mg, 40%).
1H NMR (400 MHz, CD3OD): δ 8.30 (s, 1H), 5.92 (d, J = 6.0 Hz,
1H), 4.88−4.87 (m, 1H), 4.85−4.75 (m, 1H), 4.74−4.60 (m, 2H),
4.33−4.31 (m, 1H), 4.15 (q, J = 2.8 Hz, 1H), 3.89 (dd, J = 12.5, 2.7
Hz, 1H), 3.80−3.69 (m, 6H), 2.31−2.23 (m, 1H), 2.20−2.03 (m, 1H),
2.00−1.83 (m, 3H), 1.80−1.62 (m, 1H); HRMS calcd for
C17H25ClN5O8P + H: 494.1208; found: 494.1219 [M + H]+.
compounds in the presence or absence of 10 μM final αβ-met-ADP
(Sigma #M3763) were brought up to 8× final concentration in Hanks’
Balanced Salt Solution (Gibco #14175) supplemented with 2 mM
HEPES, pH 7.4, and immediately added to the cell plates with a
Multimek automated liquid handling device (Nanoscreen, Charleston,
SC). Following a 10 min incubation at room temperature, 100 μM
final 3-isobutyl-1-methylxanthine (Sigma) and 175 nM (−)-isoproter-
enol bitartrate (Sigma) or 1.5 μM forskolin (Tocris) were added by
Multimek. Seven minutes later, GloSensor cAMP reagent (Promega
#E1291) containing 0.1% final luciferin and supplemented with 0.3%
final NP40 (Tergitol, Sigma #NP40S) to permeabilize the cells was
added by Multimek along with a final 5 μL addition of 100% ethanol
(Decon Laboratories) to eliminate bubbles. Luminescence was read on
an Envision platereader (Perkin-Elmer) for 15 min. Data from
approximately 95% of the maximal response for isoproterenol or
forskolin (10−14 min post GloSensor reagent addition) were
normalized for scale to 100% response equivalent to the response of
1 μM 3 and 0% response equal to the response from isoproterenol or
forskolin alone. Normalized data were fit in GraphPad Prism with four
parameter curves for EC50 determinations. Because HEK293T cells
endogenously express A2AR,45,46 the cAMP response for adenosine
and 5, known agonists of both A1AR (Gi coupled) and A2AR (Gs
coupled), was bimodal.24 Concentrations below 1 μM were used to
calculate potencies for adenosine, 5, 14, and 14c, against the A1
receptor.
(2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(2-chloro-6-((2-((di-tert-
butoxyphosphoryl)oxy)cyclopentyl)amino)-9H-purin-9-yl)-
tetrahydrofuran-3,4-diyl Diacetate (14b). To a solution of 13 (429
mg, 0.838 mmol) in CH2Cl2 (5 mL) were added dibenzyl
diisopropylphosphoramidite (579 mg, 1.68 mmol) and tetrazole
(0.45 M solution in THF, 6.0 mL, 2.68 mmol) and the reaction
mixture was heated for 2 h at 60 °C. The temperature was lowered to
0 °C, H2O2 (0.5 mL of 30% solution, 0.19 mmol) was added, and the
reaction mixture was stirred for an additional 1 h. The reaction was
then diluted with CH2Cl2 (100 mL) and washed with 10% aq sodium
metabisulfite (30 mL), saturated aq sodium bicarbonate (30 mL),
water (30 mL), and brine (30 mL). The solvent was removed under
reduced pressure, and the residue was purified by flash column
chromatography on silica gel (10% methanol/CH2Cl2) to afford the
Ca+ Mobilization. Assays of Ca+ mobilization followed a published
1
title compound 14b (clear oil) (100 mg, 15%). H NMR (400 MHz,
procedure.24
CDCl3): δ 8.01 (s, 1H), 7.35−7.11 (m, 10H), 6.09 (d, J = 5.5 Hz, 1H),
5.65 (t, J = 5.5 Hz, 2H), 5.49−5.46 (m, 1H), 5.01−4.89 (m, 4H),
4.80−4.70 (m, 1H), 4.65−4.50 (m, 1H), 4.43−4.36 (m, 1H), 4.35−
4.36 (m, 2H), 2.25−2.15 (m, 1H), 2.13−2.04 (m, 6H), 2.01 (s, 3H),
1.99−1.90 (m, 1H), 1.86−1.67 (m, 3H), 1.63−1.44 (m, 1H);
MS(ESI) m/z 772 [M + H]+.
Radioligand Binding. Radioligand binding assays were performed
by Cerep France http://www.cerep.fr/
Behavior Assay. All procedures and behavioral experiments
involving vertebrate animals were approved by the Institutional
Animal Care and Use Committee at the University of North Carolina
Sodium (1R,2R)-2-((2-Chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)-
cyclopentyl Phosphate (14c). To a solution of phosphate 14b (32
mg, 0.038 mmol) in methanol (10 mL) was added Pd−C (10%, 30
mg), and the reaction mixture was stirred for 12 h at rt in a pressure
vessel under 60 psi of hydrogen gas. After 12 h, the mixture was
filtered through Celite to remove the catalyst. The solvent was
removed under reduced pressure, and 6 N methanolic ammonia (25
mL) was added at 0 °C. The reaction was again sealed in a pressure
vessel and stirred for 12 h at rt. The volatiles were removed under
reduced pressure, and the residue was purified via preparative HPLC
chromatography to give the TFA salt of the title compound. NaOH
(13.5 mg, 0.34 mmol) in water (5 mL) was added, and the reaction
mixture was stirred for 30 min at rt. The solvent was then removed
under reduced pressure to afford the disodium salt of the title
at Chapel Hill. C57BL/6 mice were purchased from Jackson
47,48
Laboratories. A1AR−/−
mice were backcrossed to C57BL/6 mice
(Jackson) for 12 generations. Male mice, 2−4 months old, were
acclimated to the testing room, equipment, and experimenter for 1−3
days before behavioral testing. Noxious thermal sensitivity was
measured by heating one hindpaw with a Plantar Test apparatus
(IITC) following the Hargreaves method.49 The radiant heat source
intensity was calibrated so that a paw-withdrawal reflex was evoked in
∼10 s, on average, in wild-type C57BL/6 mice. Cutoff time was 20 s.
One measurement was taken from each paw at the indicated time
points to determine paw withdrawal latency. Compounds (10 mM,
dissolved in DMSO) were diluted in 0.9% saline then administered to
unanesthetized mice via acute intrathecal injection (5 μL, direct
lumbar puncture method50), intravenous tail vein injection, or oral
gavage. The final DMSO concentration was 5% or less.
1
compound 14c (80 mg, 99%) as a white solid. H NMR (400 MHz,
Telemetry. Data Sciences International ETA-F20 transmitters were
implanted as follows: A 2 cm midline abdominal incision was made in
anesthetized mice. The transmitter was placed intraabdominally on top
of the intestines, parallel with the long axis of the body and the two
leads pointing caudally. A large (14 gauge) needle was used to pass
through the abdominal muscles on either side of the incision. The
leads were passed through the lumen of the needle, one on each side,
and the needle was withdrawn. The leads were placed (positive by the
xiphoid and negative on the right pectoral) and anchored in place. The
abdomen was closed with absorbable sutures and the skin with
nonabsorbable sutures.
D2O): δ 8.10 (s, 1H), 5.83 (d, J = 5.5 Hz, 1H), 4.72−4.69 (m, 1H),
4.46−4.30 (m, 1H), 4.30−4.24 (m, 1H), 4.24−4.06 (m, 2H), 3.82−
3.62 (m, 2H), 2.17−2.07 (m, 1H), 2.05−1.90 (m, 1H), 1.77−1.53 (m,
3H), 1.46−1.29 (m, 1H); HRMS calcd for C15H21ClN5O8P + H:
466.0895; found: 466.0862 [M + H]+.
cAMP Accumulation Assay. cAMP determinations were made
using a modified GloSensor Luciferase detection system (Promega).
Low passage, subconfluent HEK293T/17 cells (ATCC CRL-11268)
were grown in Dulbecco’s Modified Eagle’s Medium without phenol
red (Gibco #31053) and supplemented with 10% fetal bovine serum
(Hyclone ‘Characterized’ #SH30071.03). Cells were reverse trans-
fected by spotting a calcium phosphate DNA complex mixture
containing 12.5 ng each of GloSensor 22F plasmid (Promega #E2301)
and human adenosine A1 receptor plasmid (Adora1, GenBank
accession #AY136746, Missouri S&T Clone Collection (www.cdna.
org) in 25 mM HEPES at pH 7.1, 140 mM sodium chloride, 0.75 mM
disodium monophosphate, and 250 mM calcium chloride. Cells were
immediately added at a density of 20 000 cells per well using a
Multidrop 384 (Titertek) to 384-well white, clear-bottom tissue
culture plates (Corning #3707). Cell plates were incubated for 24 h at
37 °C and 5% CO2. Sixteen-point, 1:3 dilution curves of test
ASSOCIATED CONTENT
■
S
* Supporting Information
Cyclic AMP responses for 2′-, 3′-, and 5′-AMP in absence or
presence of αβ-met-ADP. Effects of compound 3a in the
antinociceptive mouse model via IT and IV administrations.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Journal of Medicinal Chemistry
Article
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 919-843-8459. Fax: 919-843-8465. E-mail: jianjin@
unc.edu (J.J.). Phone: 919-966-2540. E-mail: zylka@med.unc.
edu (M.J.Z.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Mauricio Rojas at the UNC Mouse
Cardiovascular Models Core for implanting telemetry devices,
Dr. Lindsey Ingerman James for critical reading the
experimental section and review of spectra, and Dr. Bryan
Roth and the National Institute of Mental Health, Psychoactive
Drug Screen Program, for assay support. We also acknowledge
the assistance of Adam Cheely for his contributions in the
development of the assay techniques. The research described
here was supported by a grant from NINDS (R01NS067688).
ABBREVIATIONS USED
■
A1AR, Adenosine A1 receptor; A2AAR, adenosine A2A receptor;
A2BAR, adenosine A2B receptor; A3AR, adenosine A3 receptor;
cAMP, cyclic adenosine monophosphate; 5′-AMP, adenosine
5′-monophosphate; SAR, structure−activity relationships; 2′-
AMP, adenosine 2′-monophosphate; 3′-AMP, adenosine 3′-
monophosphate; αβ-met-ADP, α,β-methylene adenosine 5′-
diphosphate; NT5E, ecto-5′-nucleotidase/CD73; IT, intra-
thecal; IV, intravenous
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