Structure-based design, synthesis, and in vivo antinociceptive effects of selective A1 adenosine receptor agonists

Article abstract of DOI:10.1021/acs.jmedchem.7b01399

Our previous work discovered that combining the appropriate 5'- and N⁶-substitution in adenosine derivatives leads to the highly selective human A₁ adenosine receptor (hA₁AR) agonists or highly potent dual hA₁AR agonists and hA₃AR antagonists. In order to explore novel dual adenosine receptor ligands, a series of N⁶-substituted-5'-pyrazolyl-adenosine and 2- chloro-adenosine derivatives were synthesized and assayed in vitro at all ARs. The N⁶-(±)-endo-norbornyl derivative 12 was the most potent and selective at A₁AR and effective as an analgesic in formalin test in mice, but none of the 5'-pyrazolyl series compounds showed a dual behavior at hA₁ and hA₃AR. Molecular modeling studies rationalized the structure-activity relationships and the selectivity profiles of the new series of A1AR agonists. Interestingly, an unexpected inverted binding mode of the N⁶-tetrahydrofuranyl derivative 14 was hypothesized to explain its low affinity at A₁AR.

Full text of DOI:10.1021/acs.jmedchem.7b01399

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Article
Structure-Based Design, Synthesis and in vivo Antinociceptive
Effects of Selective A1 Adenosine Receptor Agonists#
Riccardo Petrelli, Mirko Scortichini, Carmela Belardo, Serena Boccella, Livio
Luongo, Fabio Capone, Sonja Kachler, Patrizia Vita, Fabio Del Bello, Sabatino
Maione, Antonio Lavecchia, Karl-Norbert Klotz, and Loredana Cappellacci
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01399 • Publication Date (Web): 19 Dec 2017
Downloaded from http://pubs.acs.org on December 20, 2017
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Structure-Based Design, Synthesis and in vivo Antinociceptive Effects of Selective A
Adenosine Receptor Agonists
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#
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Riccardo Petrelli, Mirko Scortichini, Carmela Belardo, Serena Boccella, Livio Luongo, Fabio
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Capone, Sonja Kachler, Patrizia Vita, Fabio Del Bello, Sabatino Maione, Antonio Lavecchia,
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Karl-Norbert Klotz, and Loredana Cappellacci
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School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, 62032 Camerino, Italy
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Section of Pharmacology “L. Donatelli”, Department of Experimental Medicine, University of
Campania “L. Vanvitelli”, 80138 Naples, Italy
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Department of Pharmacy,“Drug Discovery” Laboratory, University of Naples Federico II,
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0131 Naples, Italy
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Institut für Pharmakologie and Toxikologie, Universität Würzburg, D-97078 Würzburg, Germany
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Keywords: A adenosine receptor agonists, antinociceptive activity, formalin test, N -substituted-
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-chloro-5-deoxy-adenosine derivatives, N -substituted-5-pyrazolyl-adenosine derivatives.
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ABSTRACT
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Our previous work discovered that combining the appropriate 5- and N -substitution in adenosine
derivatives leads to highly selective human A adenosine receptor (hA AR) agonists or highly
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potent dual hA AR agonists and hA AR antagonists. In order to explore novel dual adenosine
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receptor ligands, a series of N -substituted-5-pyrazolyl-adenosine and 2-chloro-adenosine
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derivatives were synthesized and assayed in vitro at all ARs. The N -(±)-endo-norbornyl
derivative 12 was the most potent and selective at A AR and effective as an analgesic in formalin
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test in mice, but none of the 5-pyrazolyl series compounds showed a dual behaviour at hA and
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hA AR. Molecular modeling studies rationalized the structure-activity relationships and the
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selectivity profiles of the new series of A AR agonists. Interestingly, an unexpected inverted
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binding mode of the N -tetrahydrofuranyl derivative 14 was hypothesized to explain its low
affinity at A AR.
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INTRODUCTION
Adenosine is an endogenous purine nucleoside that modulates a variety of physiological functions
as a result of its activation of specific G protein-coupled receptors defined as A , A , A , and A
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2A
2B
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adenosine receptors (ARs).
The A adenosine receptor (A AR) is the best characterized adenosine receptor subtype. Selective
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A AR agonists mediate neuro- and cardioprotective effects, reduce lipolysis in adipose tissue, and
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2
,3
intraocular pressure in glaucoma. The A AR is abundantly expressed in spinal cord and other
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neuronal tissue, and its activation produces pain-relieving effects in a number of preclinical animal
4-6
models.
Several selective A AR agonists have been developed as analgesics, e.g. N-cyclohexyl-2'-O-
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methyladenosine (SDZ WAG 994) , N-[(1S,2S)-2-hydroxycyclopentyl]adenosine (GR79236) ,
(2S,3S,4R,5R)-2-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(6-(4-chloro-2-fluoroanilino)purin-9-
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yl)tetrahydrofuran-3,4-diol (GW493838) . However, clinical trials of these nucleosides have been
discontinued, possibly due to their inability to penetrate CNS sufficiently to cause a substantial
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effect. Moreover, high doses may produce severe side effects, especially in the cardiovascular
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system (bradycardia and hypotension).
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N -substituted- and/or sugar-modified adenosine derivatives have been the subject of numerous
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publications on the structure activity relationship (SAR) at ARs.
The nature of the substituents
6
at the N - and 5-positions of adenosine derivatives has a major influence on AR affinity,
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selectivity and efficacy. Many N -substituted-5-modified adenosine derivatives turned out to be
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13,18
A AR agonists, others were found to be A AR partial agonists,
while others behaved as dual
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human (h) A AR agonists and A AR antagonists.
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Our recent works demonstrated that the substitution of OH at the 5-position of N -substituted
adenosine derivatives with a chlorine atom is not only well tolerated by the hA AR but even
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improves the A AR selectivity and affinity. 5-Chloro-5-deoxy-N -(±)-endo-norbornyl-adenosine
1
(
5Cl5d-(±)-ENBA, 3) turned out to be potent and the most selective human and mouse (m) A AR
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4,19
20
agonist vs A AR
with analgesic effects in a mouse model of neuropathic pain. Interestingly,
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at analgesic doses, 3 did not lower blood pressure or locomotor activity in mice.
Moreover, it
was found to reduce the dyskinesia caused by L-DOPA in a mouse model of Parkinson desease
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(PD).
A AR is the last of the adenosine receptor subtypes to be cloned. A AR agonists are in advanced
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3,24
clinical studies in the treatment of hepatocellular carcinoma, psoriasis and rheumatoid arthritis.
Recently, Salvemini and colleagues have reported that highly selective A AR agonists produce
3
antinociceptive effects at both central and peripheral levels without cardiovascular side effects
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,25,26
and, unlike opioids, without inherent reward.
Moreover, the authors proved that A AR
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agonists modified pathological but not normal protective nociception.
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Also, A AR antagonists are potentially useful in the treatment of various diseases such as
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glaucoma,
asthma, septic shock and other conditions.
Modification at the ribose moiety of adenosine derivatives was found to modulate both the affinity
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and efficacy at the A and A ARs. While 5-chloro-5-deoxy-N -substituted adenosine derivatives
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turned out to be potent hA AR agonists that were highly selective vs hA AR and mA AR, 5-
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C-ethyl-tetrazol-2-yl-N -substituted adenosine derivatives were found to be highly potent dual
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hA AR agonists and hA AR antagonists. Surprisingly, 5-C-ethyl-tetrazol-2-yl-N -substituted
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adenosine derivatives proved to be agonists at the rat (r) A AR that were endowed with strong
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analgesic activity in a formalin test in mice. Therefore, combining the appropriate 5-
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modification and N -substitution in adenosine derivatives leads to dual A AR and A AR ligands
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having different profiles of affinity and efficacy in human and other species. These dual acting
ligands might have the advantages of being a single molecule and associated pharmacokinetics,
but activating different signaling pathways, both leading to beneficial effects in the treatment of
various diseases, e.g. pain (dual A and A AR agonists), glaucoma and epilepsy (dual A agonist
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1
and A AR antagonist). For this reason, in order to explore novel combinations of 5-modification
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and N -substitution leading to dual A and A AR ligands, a series of 5-deoxy-5-pyrazolyl-
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adenosine and 2-chloroadenosine derivatives (compounds 9-16) was synthesized and evaluated for
affinity and selectivity at all cloned hAR subtypes. Some 2,5-bis-pyrazolyl-5-deoxy-adenosine
6
derivatives (compounds 17-20) and the intermediate 5-chloro-5-deoxy-N -substituted adenosine
derivatives, compounds 5-8, were also assayed at all AR subtypes. The most active compounds of
the series were tested for their in vivo analgesic activity in mice. A molecular modeling study
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rationalized the observed binding data of this series of 5,N -disubstituted adenosine derivatives.
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RESULTS AND DISCUSSION
Chemistry. The novel compounds 5-20 were synthesized according to the methods reported in
Schemes 1 and 2. The synthesis of compounds 5-8 is outlined in Scheme 1. Treatment of 6-chloro-
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or 2,6-dichloro-9H-(2,3,5-tri-O-acetyl--D-ribofuranosyl)-purine (21 and 22, respectively)
with 4-chloro-2-fluoroaniline in the presence of triethylamine in absolute ethanol, followed by
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sugar deblocking with methanolic ammonia, afforded compounds 25 and 26 in good yields. N -
[
(R)-3-Tetrahydrofuranyl]adenosine (Tecadenoson, 23), 2-chloro-tecadenoson (24) and the
corresponding 2′,3′-isopropylidene derivatives 27 and 28 respectively, were synthesized as
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previously reported.
Compounds 25 and 26 were then converted into their corresponding 2′,3′-isopropylidene
derivatives 29 and 30 using camphorsulfonic acid and 2,2-dimethoxypropane in acetone.
Chlorination of 27-30 to the corresponding 5′-chloro derivatives 31-34 was performed by
treatment with a mixture of thionyl chloride, pyridine, and acetonitrile. Finally,
deisopropylidenation of 31-34 with 70% formic acid at 40 °C furnished the desired compounds 5-
6
8
. The reference compounds 5-chloro-5-deoxy-N -cyclopentyl-adenosine (5′-Cl-CPA, 1), 2-
6
chloro-5-chloro-5-deoxy-N -cyclopentyl-adenosine (5′-Cl-CCPA, 2), 3, and 2-chloro-5-chloro-
6
5
-deoxy-N -(±)-endo-norbornyl-adenosine (2-Cl-5′Cl5′d-(±)-ENBA, 4) were synthesized as
1
4
previously reported. As outlined in Scheme 2, direct substitution of 5′-chloro-5′-deoxy-
adenosine derivatives 1-8 with hydrazine monohydrate and subsequent condensation of the
resulting intermediates 35-46 with acetylacetone in ethanol afforded the target 5′-pyrazolyl and
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2
,5′-dipyrazolyl N -substituted adenosine derivatives 9-20.
Binding Affinity.
Compounds 9-20 were tested in radioligand binding assays for affinity at the human recombinant
ARs, stably transfected into Chinese hamster ovary (CHO) cells, utilizing radioligand binding
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assays (A , A , and A ) (Table 1, see also Supporting Information, Figure S1). Based on our
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previous results with 3,
the intermediate 5′-chloro-5′-deoxy-adenosine derivatives 5-8 were
also assayed at all AR subtypes.
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As shown in Table 1, 5′-deoxy-5′-(3,5-dimethyl)-pyrazolyl-N -substituted adenosine derivatives 9-
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6 showed hA AR affinity from the low nanomolar to half micromolar (4.35-491 nM), and some
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of them were highly selective vs A and A ARs. 5′-Deoxy-5′-pyrazolyl-N -(±)-endo-norborn-2-
2
A
3
yl-2-chloroadenosine (12) was the most potent and selective analogue (A AR K = 4.35 nM,
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selectivity A /A = 2168 fold and A /A = 333 fold, Table 2). As previously reported in the 5′-
2
A
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14
6
chloro-5′-deoxy-N -substituted adenosine derivatives series, in the 5′-pyrazolyl one, the N -(±)-
6
endo-norborn-2-yl substitution furnished the highest affinity at A AR. The N -tetrahydrofuranyl
1
substitution was not well tolerated, with compounds 13 and 14 (A AR K = 491 and 438 nM,
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i
respectively) being 100-fold less potent than compound 12. The 2-fluoro-4-chlorophenyl
substitution (compounds 15 and 16) was also less well tolerated (A AR K = 158 and 139 nM,
1
i
respectively) than the endo-norbornyl one (compounds 11 and 12, A AR K = 25.6 and 4.35 nM,
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i
respectively), though to a lesser extent. The substitution with a pyrazolyl group at the 2 position in
6
5
′-deoxy-5′-pyrazolyl-N -substituted adenosine derivatives (17-20) was well tolerated at A AR. K
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i
6
values of compounds 17-20 ranged from 8.02 to 91.4 nM at A AR with N -cyclopentyl-2,5′-bis-
1
pyrazolyl-5′-deoxy-adenosine (17) being the most potent and selective hA AR agonist among the
1
2
,6-bis-pyrazolyl- derivatives. However, the most potent compounds at hA AR turned out to be
1
the intermediate 5′-chloro-5′-deoxy-adenosine derivatives 5-8 (K values ranging from 1.87 to 11.2
i
nM). Compound 5 was found the most selective A vs A AR of the series (A AR/A AR = 635
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1
fold).
At hA AR, the 5′-deoxy-5′-pyrazolyl-, and the 5′-chloro-5′-deoxy-derivatives (9-20 and 5-8,
3
respectively) showed from moderate to very low affinity (K values ranging from 0.136 to 5.64
i

M). It is interesting to note that even though compound 16 displayed moderate affinity (K at
i
both A and A ARs = 139 nM), it turned out to be a dual hA and hA ligand that was highly
1
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selective vs A and A ARs (A /A = > 720, A /A = > 430).
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2B
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Adenylyl Cyclase Activity.
Due to the lack of a useful high-affinity radioligand for A AR, all novel compounds were tested
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in a functional A AR assay and the EC values are reported in Table 1 (see also Supporting
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Information, Figure S3). None of the compounds showed particularly high potency. The most
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potent compound 7 showed an EC comparable to NECA.
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Selected compounds were additionally tested for their functional effects at hA AR, and hA AR by
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measuring modulation of adenylyl cyclase activity. The ability of these compounds to inhibit
forskolin-stimulated cAMP production via the hA AR and the hA AR was studied in comparison
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37
with the full A agonist CCPA and the non-selective agonist NECA, respectively (Supporting
1
Information, Figures S2 and S4). Compounds were considered to be antagonists if they fully
reversed (>85%) the NECA-mediated inhibition of adenylyl cyclase activity.
As expected, all tested compounds were found to be agonists at hA ARs, whereas they were
1
antagonists at the hA AR subtype. EC values at A and/or IC values at A AR of selected
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compounds were also determined (Table 3). The most potent compounds at A AR were the 5′-
1
chloro-5′-deoxy-adenosine derivatives 6 and 7, showing EC50 values of 54.2 and 56 nM,
respectively (Table 3). Among the 5′-deoxy-5′-pyrazolyl-adenosine derivatives, compound 12
showed the best EC50 value at hA AR (EC =134 nM), while compound 16 showed the best IC
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value at hA AR (IC = 701 nM).
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Molecular Modeling.
The 5′-deoxy-5′-pyrazolyl-N -(±)-endo-norborn-2-yl adenosine compound 12 (A , K = 4.35 nM)
6
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i
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exhibited excellent binding affinity, and the N -(R)-3-tetrahydrofuranyl and N -2-fluoro-4-chloro-
phenyl analogues 14 (A , K = 438 nM) and 16 (A , K = 139 nM) showed, respectively, ≈100-fold
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and ≈32-fold less binding affinity at the hA AR. In addition, the presence of a chlorine atom in the
1

-D-ribofuranose ring in 6 led to a substantial increase in its binding affinity at the hA AR with a
1
K of 3.50 nM. In view of the observed variations in the hA AR binding affinities among these
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compounds, molecular docking experiments were carried out by the means of the GOLD 5.4.1
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program in combination with the ChemPLP scoring function (rescoring with ChemScore).
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Although two crystal structures are available now for the hA1AR,
both represent the inactive
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0
conformation of the receptor bound to covalent and non-covalent antagonists. Comparison of
agonist-bound and inactive conformations of the A AR crystal structures revealed several
2
A
conformational changes needed for receptor activation, especially within the ligand binding
region. These conformational changes include a tightening of hydrophilic residues in TM3, TM5
and TM7 around the ribose moiety, resulting in a significant contraction in the volume of the
4
4
binding site. Based on these observations, docking was performed considering the homology
model of the hA AR, built using the recently solved agonist-bound hA AR crystal structure
1
2A
4
5
(PDB ID: 3QAK) as a template.
The docking poses of 6 (CHEMscore fitness = 20.22 kJ/mol), 12 (CHEMscore fitness = 20.90
kJ/mol) and 16 (CHEMscore fitness = 20.11 kJ/mol) highlighted the crucial anchoring interactions
with the binding site of hA AR that are expected to be common among all the A AR agonists
1
1
6.55
(Figure 1). A strong H-bond interaction was observed between the carboxamide group of N254
4
6
6
7
(
using standard notation and the exocyclic N amino group and the N atom of the adenine ring
of three ligands. The C2-chlorine atom was within H-bonding distance from the NH group of
2
2.65
N70
side chain. The adenine core was anchored inside the binding site by a π−π stacking
6
.51
interaction with F171 (extracellular loop 2) and strong hydrophobic contacts with L250
and
7
.39
I274
.
7
.42
7.43
Moreover, the 3′- and 2′-OH groups of 6 were located in proximity to T277
and H278
,
respectively, and could form H-bonds with these residues. In contrast, the ribose ring of
compounds 12 and 16, as a result of the bulky and rigid 3,5-dimethyl-pyrazole substituent at the 5′
position, was subjected to a slight rotation about the N9-C1′ bond, thereby allowing only the 3′-
7.43
OH group to form a H-bond with H278 . In particular, while 6, which has a less bulky chlorine
atom in 5′, presented a glycosyl torsion angle  (defined by O-C1’-N9-C4 in Figure 2) clearly
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indicative of an anti-conformation ( = -152.4°), the same parameter for compounds 12 and 16
showed values still within the anti-conformation range but with a slight shift toward an anti/syn
intermediate state ( = 165.4,  = -164.3, respectively). Figure 2 illustrates the comparison of the
binding modes of compounds 6 and 12, highlighting the different orientation of the sugar moiety
in the adenosine derivatives.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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0
The 5′-(3,5-dimethyl)-pyrazole moiety of 12 (Figure 1B) and 16 (Figure 1C) was locked in the
3.36
cavity by a H-bond with T91 , with the threonine side chain acting as H-bond donator, while the
hydrophobic methyl groups attached at 2 and 5 positions of pyrazole were held by favorable vdW
3
.33
3.37
5.38
5.42
5.47
6.48
6.51
interactions with residues L88 , Q92 , M180 , N184 , V189 , W247 , L250 , and
6
.52
H251
.
The 5′ substituent of 6 consists of a chloromethyl group. A high flexibility of the Cl atom in the 5′
subpocket of A AR appeared in the docking results for chloromethyl agonist 6, with different
1
orientations of the Cl atom. Nevertheless, in 145 out of 200 cases GOLD found one recurring
6.52
solution in which the 5′-Cl atom of 6 was engaged in a H-bond interaction with H251 (Figure
1
A). Interestingly, the importance of this interaction in agonist recognition has been recently
4
7
demonstrated in the adenosine-bound A AR structure, where the interaction between the 5′-OH
2
A
6.52
5.42
group of the cocrystallized adenosine and the residues H250
and N181
is mediated by a
4
7
structured water molecule (wat2017 in the PDB entry 2YDO).
Based on docking results, differences can be observed in the interactions formed by the ribose
moiety of compounds 6, 12 and 16; in fact, compounds 12 and 16 formed only two of the three H-
bonds predicted for binding of the full agonist 6 (EC = 54.2 nM). In particular, compounds 12
5
0
7
.43
3.36
7.42
and 16 (Figure 1B,C) established H-bonds with H278 , and T91
and not with T277
.
However, their ability to bridge between TM3 and TM7 likely correlates with the capacity to
induce the conformational changes required for receptor activation, such as an inward movement
of TM7.
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In contrast with 6, 12 and 16, derivative 14 (CHEMscore fitness = 15.35 kJ/mol) appeared to have
an inverted binding mode and did not maintain some key conserved interactions for binding
6
(
Figure 1D). The less bulky N -tetrahydrofuranyl substituent, mimicking the ribose moiety of the
0
1
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natural agonist adenosine, occupied a region close to ribose binding pocket but did not mimic the
7.42
7.43
H-bonding interactions of the 3′- and 2′-OH groups with T277 and H278 , respectively. This
6
result explains why appending a tetrahydrofuranyl group at the N position dramatically decreases
the A AR binding affinity.
1
6
Furthermore, the different nature of the substituents at the N position of the adenine ring of 6, 12
and 16 could influence the selectivity and the efficacy of the agonists at three hAR subtypes. The
6
N groups in the docking poses of the studied agonists accommodated in a pocket, which was
6
.54
6.58
7.35
5.38
located between TM6 and TM7 and delimited by residues L253 , T257 , T270 , M177
,
6.51
and at the bottom by L250 . Also note of the hA AR subtype accommodates more dramatic
1
differences compared to both hA AR and hA AR among residues controlling the upper region of
2A
3
6
the binding site, and this could affect the orientation and the interactions of the N -substituents for
42
each receptor subtype. For example, position 6.54 in A AR consists of a nonconserved bulky
1
and hydrophobic leucine residue, while a smaller isoleucine is present in the other AR subtypes.
7
.35
7.35
7.35
The corresponding residues of T270 in A AR are the bulky Met270 in A AR and L264
1
2A
6
6
in A AR. Another residue located in the N subpocket and involved in the anchoring of the N
3
substituent of the A AR agonists, but nonconserved among the adenosine receptors, is the
1
6
.58
T257 , which becomes a bulky leucine in hA AR.
3
The variation of the affinity and potency of this series of 5′-pyrazolyl adenosine analogues at the
6
A AR indicates that the N substituent could greatly affect these factors, in some cases
1
counterbalancing for the lack of H-bonding interactions in the ribose region. In particular, the
pocket at the upper part of the hA AR was found to have a shape more suitable to accommodate a
1
6
6
N -norbornyl (compound 12) or N -tetrahydrofuranyl (compound 6) substituent. On the other
6
hand, compound 16, which has a more flexible and extended 2-fluoro-4-chloro-phenyl group at N
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position, exhibits poor steric complementarity to the pocket as compared to the norbornyl or
4
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6
tetrahydrofuranyl groups and consequently has lower affinity at the A AR.
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Antinociceptive Effect.
Selected compounds endowed with high hA AR affinity and selectivity (5, 6 and 12) were
1
evaluated in vivo in mice by performing the formalin test.
4
8
Formalin injection induces a biphasic stereotypical nocifensive behavior. Nociceptive responses
are divided into an early, short lasting first phase (0-7 min) caused by a primary afferent discharge
produced by the stimulus, followed by a quiescent period and then a second, prolonged phase (15-
60 min) of tonic pain. Systemic administration of 5 (2 mg/kg, i.p.), 10 min before formalin,
slightly reduced only the early phase of the formalin test, while the highest dose of 5 used (4
mg/kg) reduced both the early and the late phases of the formalin test. This effect was prevented
4
9
by 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX, 50)
(1 mg/kg, i.p.), a selective A AR
1
antagonist (Figure 3).
Systemic administration of 6 (0.25, 0.5 and 2 mg/kg, i.p.), 10 min before formalin, reduced the
late nociceptive behavior induced by formalin in a dose-dependent manner (P < 0.005). The
highest dose of 6 used (2 mg/kg) significantly reduced only the late phase of the formalin test, and
this effect was prevented by 50 (1 mg/kg, i.p.) (Figure 4).
Systemic administration of 12 (1, 2 and 4 mg/kg, i.p.), 10 min before formalin, reduced the late
nociceptive behavior induced by formalin in a dose-dependent manner (P < 0.05) (Figure 5).
CONCLUSION
6
In conclusion, this study confirmed that N -substituted 5′-chloro-5′-deoxy-adenosine and 2-chloro-
adenosine derivatives are potent and selective A AR agonists potentially useful in the treatment of
1
pain. The substitution of a 5′-chlorine atom with a (3,5-dimethyl)-pyrazolyl moiety reduces both
the affinity and the selectivity at A AR with respect to the corresponding 5′-chloro-5′-deoxy-
1
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derivatives. As previously reported for 3, the N -(±)-endo-norbornyl substituent allowed to obtain
compound 12, the most potent and selective A AR agonist in the series of 5′-deoxy-5′-pyrazolyl
1
derivatives. Moreover, a molecular modeling study rationalized the unexpected low affinity at
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A AR of compounds 13 and 14.
1
While a 5′-C-tetrazolyl- moiety in adenosine derivatives was highly tolerated at both hA and
1
hA AR subtypes leading to potent dual acting A and A AR ligands, the 5′-(3,5-dimethyl)-
3
1
3
pyrazolyl moiety was tolerated at A AR but not at A AR.
1
3
6
In conclusion, in this work we discovered a new series of N ,5′-disubstituted-adenosine and 2-
chloro-adenosine derivatives as potent hA AR agonists useful in the treatment of pain.
1
EXPERIMENTAL SECTION
Chemical Synthesis. Materials and Instrumentation. All reagents and solvents were purchased
from Sigma-Aldrich Chemical Co, were analytical grade and were used as received. Thin layer
chromatography (TLC) was run on silica gel 60 F254 plates; column chromatography was run on
silica gel 60 (70−230 mesh, Merck and 200−400 mesh, Merck). Preparative thin layer
chromatography was run on silica gel GF (20 cm × 20 cm, 1000 μm, Analtech). The final
1
13
1
compounds were characterized by H NMR, C NMR, MS, and elemental analyses. H NMR and
1
3
C NMR spectra were recorded on 400 MHz NMR spectrometer (Varian Mercury AS400
instrument). The chemical shift values are expressed in δ values (ppm), and coupling constants (J)
are in hertz; tetramethylsilane (TMS) was used as an internal standard. Proton chemical data are
reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, dd = doublet of doublets,
pd = pseudo doublet, t = triplet, dt = doublet of triplets, q = quartet, dq = doublet of quartets, m =
multiplet, brs = broad singlet) coupling constant (s), integration. The presence of all exchangeable
protons was confirmed by addition of D O. The purity of final compounds was checked using an
2
Agilent 1100 series HPLC equipped with Gemini-NX 5µm C-18 100Å 250 x 4.6 mm column.
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Mobile phase consisted of a mixture of water/methanol (95:5) at a flow rate of 1 mL/min. Peaks
were detected by UV adsorption with a diode array detector (DAD) at 230, 254, and 280 nm. All
derivatives tested for biological activity showed ≥96% purity by HPLC analysis (Area % purity
was detected at 210 nm or 254 nm). Mass spectra were recorded on an HP 1100 series instrument.
All measurements were performed in the positive ion mode using atmospheric pressure
electrospray ionization (API-ESI). Elemental analyses (C, H, and N) were determined on
ThermoFisher Scientific FLASH 2000 CHNS analyzer and are within 0.4% of theoretical values.
4
5
6
7
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5
5
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5
5
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6
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0
3
Biological assays: [ H]CCPA was purchased from Amersham/GE Healthcare (58 Ci/mmol – 2.15
3
TBq/mmol; Purity 98.4%); [ H]NECA was purchased from American Radiolabeled Chemicals
3
Inc. (25 Ci/mmol – 0.93 TBq/mmol, Purity 99%); [ H]HEMADO was purchased from Tocris (24
Ci/mmol – 0.89 TBq/mmol, Purity > 97%). DPCPX (50) was purchased from Tocris (Bristol,
UK).
General Procedure for the Synthesis of Compounds 5-8.
Compounds 31-34 (1 equiv ) were treated with HCOOH 70% in water (10 mL), and the mixture
was stirred at 40 °C for the time reported below. The solvent was evaporated to dryness, and the
residues were coevaporated several times with CH OH and then purified by column
3
chromatography.
6
N -(R)-3-Tetrahydrofuranyl-9H-(5-chloro-5-deoxy--D-ribofuranosyl)adenine (5).
The title compound was synthesized from 31 (150 mg, 0.380 mmol). Chromatography on a silica
1
gel column (CHCl -MeOH, 97:3) gave 5 as a white foam (68 mg, 50% yield). HNMR (DMSO-
3
d6): δ 1.95-2.23 (2m, 2H, tetrahydrofuranyl), 3.60 (dd, J = 4.51, 8.80 Hz, 1H, tetrahydrofuranyl),
3.72 (q, J = 7.7 Hz, 1H, tetrahydrofuranyl), 3.80-3.96 (m, 4H, tetrahydrofuranyl, H-5'), 4.08 (q, J
=
5.32 Hz, 1H, H-4'), 4.20 (q, J = 4.71 Hz, 1H, H-3'), 4.58-4.71 (m, 1H, CHNH), 4.74 (q, J = 5.31
Hz, 1H, H-2'), 5.46 (d, J = 5.10 Hz, 1H, OH), 5.60 (d, J = 6.0 Hz, 1H, OH), 5.92 (d, J = 5.5 Hz,
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3
1
4
1
1
H, H-1'), 7.98 (brs, 1H, NH), 8.22 (brs, 1H, H-2), 8.38 (s, 1H, H-8). C NMR (DMSO-d ):
6
3.88, 52.35, 53.95, 67.55 (2C), 71.82, 72.89, 86.07, 96.21, 119.32, 140.21, 148.88, 152.24,
+
54.56. MS (API-ESI): m/z 356.8 [M+H] . Anal. calcd. For (C H ClN O ) C, 47.26; H, 5.10; N,
14 18
5
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9.68; Found: C, 47.24; H, 5.12; N, 19.67.
6
2-Chloro-N -(R)-3-tetrahydrofuranyl-9H-(5-chloro-5-deoxy--D-ribofuranosyl)adenine (6).
The title compound was synthesized from 32 (150 mg, 0.350 mmol). Chromatography on a silica
1
gel column (CHCl -MeOH, 98:3) gave 6 as a white foam (128 mg, 95% yield). HNMR (DMSO-
3
d6): δ 1.86-2.29 (m, 2H, tetrahydrofuranyl), 3.54-3.83 (m, 4H, tetrahydrofuranyl), 3.86-3.96 (m,
2
H, H-5′), 4.33-4.39 (m, 1H, H-4′), 4.62 (brs, 1H, CHNH), 5.11 (q, J = 3.2 Hz, 1H, H-3′), 5.41
13
(
dd, J = 2.99, 6.21 Hz, 1H, H-2′), 6.21 (s, 1H, H-1′), 8.38 (s, 1H, H-8), 8.62 (brs, 1H, NH). C
NMR (DMSO-d ): 43.91, 52.27, 53.85, 67.47 (2C), 71.73, 72.92, 86.12, 96.25, 119.15, 140.29,
6
+
1
48.93, 153.12, 154.71. MS (API-ESI): m/z 391.3 [M+H] . Anal. calcd. for(C H Cl N O ) C,
14 17
2
5
4
43.09; H, 4.39; N, 17.95; Found: C, 43.07; H, 4.38; N, 17.96.
6
N -(4-Chloro-2-fluorophenyl)amino-9H-(5-chloro-5-deoxy--D-ribofuranosyl)adenine (7).
The title compound was synthesized from 33 (130 mg, 0.286 mmol). Chromatography on a silica
1
gel column (CHCl -MeOH, 98:2) gave 7 as a white foam (81 mg, 68% yield). HNMR (DMSO-
3
d6): δ 3.85 (dd, J = 6.41, 11.54 Hz, 1H, H-5′), 3.95 (dd, J = 5.13, 11.54 Hz, 1H, H-5′), 4.11 (q, J =
5
.34 Hz, 1H, H-4′), 4.21 (q, J = 4.49 Hz, 1H, H-3′), 4.78 (q, J = 5.56 Hz, 1H, H-2′), 5.48 (d, J =
5.13 Hz, 1H, OH), 5.62 (d, J = 5.98 Hz, 1H, OH), 5.96 (d, J = 5.98 Hz, 1H, H-1′), 7.31 (dd, J =
.13, 8.98 Hz, 1H, arom.), 7.52 (dd, J = 2.35, 10.47 Hz, 1H, arom.), 7.62 (t, J = 8.55 Hz , 1H,
2
1
3
arom.), 8.31 (s, 1H, H-2), 8.52 (s, 1H, H-8), 9.68 (s, 1H, NH). C NMR (DMSO-d ): 45.22,
6
71.76, 73.41, 84.33, 88.03, 117.41, 119.45, 124.82, 129.65, 131.13, 142.13, 158.44, 153.11,
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53.95, 152.06, 157.97. MS (API-ESI): m/z 415.218. Anal. calcd. for (C H Cl FN O ) C, 46.39;
1
6
14
2
5
3
H, 3.41; N, 16.91; Found: C, 46.37; H, 3.43; N, 16.92.
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
0
1
2
3
4
5
6
7
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9
0
1
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0
2
-Chloro-N -(4-chloro-2-fluorophenyl)amino)-9H-(5-chloro-5-deoxy--D-ribofuranosyl)
adenine (8).
The title compound was synthesized from 34 (120 mg, 0.245 mmol). Chromatography on a silica
1
gel column (CHCl -MeOH, 98:2) gave 8 as a white foam (79 mg, 72% yield). HNMR (DMSO-
3
d6): δ 3.85 (dd, J = 6.41, 11.54 Hz, 1H, H-5′), 3.94 (dd, J = 5.13, 11.54 Hz, 1H, H-5′), 4.12 (q, J =
5
.55 Hz, 1H, H-4′), 4.19 (q, J = 4.91 Hz, 1H, H-3′), 4.67 (q, J = 5.55 Hz, 1H, H-2′), 5.51 (d, J =
5.13 Hz, 1H, OH), 5.65 (d, J = 5.98 Hz, 1H, OH), 5.89 (d, J = 5.55 Hz, 1H, H-1′), 7.32 (d, J =
1
3
8
.55 Hz,1H, arom.), 7.51-7.63 (m, 2H, arom.), 8.49 (s, 1H, H-8), 10.21 (s, 1H, NH). C NMR
(
DMSO-d ): 45.37, 71.88, 73.54, 84.65, 88.15, 117.44, 119.71, 125.42, 129.68, 131.37, 142.03,
6
+
1
48.78, 153.39, 154.05, 156.08, 158.57. MS (API-ESI): m/z 449.66 [M+H] . Anal. calcd. for
(C H Cl FN O ) C, 42.83; H, 2.92; N, 15.61; Found: C, 42.85; H, 2.91; N, 15.63.
1
6
13
3
5
3
General Procedure for the Synthesis of Compounds 9-20.
A solution of compounds 1-8 (1 equiv ) in EtOH/H O (1:1 v/v) and hydrazine monohydrate (10
2
equiv) was allowed to stir at room temperature for 24 h. The reaction mixture was then
concentrated in vacuo to yield crude compounds 35-46 which were used in the next step directly
without purification. To a suspension of 35-46 (1 equiv) in methanol (10 mL) containing 3 drops
of glacial acetic acid was added acetylacetone (2 equiv) and the mixture was heated at 80 °C for
the time reported below. After completion, the reaction mixture was evaporated to dryness and the
residue purified by column chromatography.
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N -Cyclopentyl-9H-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-5-deoxy--D-ribofuranosyl)adenine
(
9).
The title compound was synthesized from 1 (130 mg, 0.367 mmol, reaction time 5 h) following
0
1
2
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5
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8
9
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9
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the general procedure described above. Chromatography on silica gel column (CHCl –MeOH,
3
1
9
5:5) gave 9 as a white solid (53 mg, 35% yield). HNMR (DMSO-d ): δ 1.45-1.61 (m, 4H,
6
cyclopentyl), 1.64-1.77 (m, 2H, cyclopentyl), 1.82-1.95 (m, 2H, cyclopentyl), 2.03 (s, 3H, CH3),
.07 (s, 3H, CH ), 4.15-4.22 (m, 2H, H-5′), 4.27 (br s, 1H, H-4′), 4.48 (br s, 1H, CHNH), 4.52 (q,
2
3
J = 5.13 Hz, 1H, H-3′), 4.64 (s, 1H,H-2′), 5.33 (d, J = 5.13 Hz, 1H, OH), 5.51 (d, J = 5.99 Hz, 1H,
OH), 5.72 (s, 1H, pyrazoyl), 5.86 (d, J = 5.56 Hz, 1H, H-1′), 7.71 (br s, 1H, NH), 8.05 (s, 1H, H-
1
3
2), 8.2 (br s, 1H, H-8). C NMR (DMSO-d ): 11.23, 13.61, 23.86 (2C), 32.53 (2C), 52.83, 56.21,
6
7
2.09, 74.22, 84.27, 88.22, 104.91, 119.78, 140.38, 141.31, 147.93, 150.02, 152.48, 154.96 MS
+
(
API-ESI): m/z 414.48 [M+H] . Anal. calcd. for (C H N O ) C, 58.10; H, 6.58; N, 23.71; Found:
20
27
7
3
C, 58.11; H, 6.57; N, 23.72.
6
2
-Chloro-N -cyclopentyl-9H-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-5-deoxy--D-ribofuranosyl)
adenine (10).
The title compound was synthesized from 2 (140 mg, 0.360 mmol, reaction time 5 h) following
the general procedure described above. The residue was purified by chromatography on a silica
gel column with CHCl /MeOH (1-5%) as the eluent affording the fast migrating compound 10 as
3
1
a white solid (66 mg, 41% yield). H NMR (CDCl ) δ 1.58-1.71 (m, 4H, cyclopentyl), 1.73-1.82
3
(
m, 2H, cyclopentyl), 2.02-2.08 (m, 2H, cyclopentyl), 2.32 (s, 3H, CH ), 2.71 (s, 3H, CH ), 3.82
3 3
(
dq, J = 3.52, 11.96 Hz, 2H, H-5′), 4.41 (dd, J = 2.99, 5.56 Hz, 1H, H-4′), 4.46-4.54 (m, 2H, H-
′,CHNH), 4.57 (t, J = 5.77, 1H, H-2′),5.26 (d, J = 5.15 Hz, 1H, OH), 5.48 (d, J = 5.78 Hz, 1H,
OH), 5.96 (br s , 1H, NH), 6.02 (s, 1H, pyrazoyl), 6.38 (d, J = 5.13Hz, 1H, H-1′), 8.02 (s,1H. H-8).
3
1
3
C NMR (DMSO-d ): 11.21, 13.59, 23.81 (2C), 32.48 (2C), 52.79, 56.28, 72.11, 74.25, 84.32,
6
8
8.31, 105.02, 119.81, 140.41, 141.37, 147.98, 150.06, 152.43, 153.11 MS (API-ESI): m/z 448.92
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+
[
M+H] . Anal. calcd. for (C H ClN O ) C, 53.63; H, 5.55; N, 21.89; Found: C, 53.64; H, 5.86;
20 26 7 3
4
5
6
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8
9
1
1
1
1
1
1
1
1
1
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N, 21.88.
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N -(±)-endo-Norbornyl-9H-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-5-deoxy--D-ribofuranosyl)
adenine (11).
The title compound was synthesized from 3 (135 mg, 0.355 mmol, reaction time 6 h) following
the general procedure described above. Chromatography on silica gel column (CHCl –MeOH,
3
1
9
5:5) gave 11 as a white solid (97 mg, 62 % yield). H NMR (DMSO-d ): δ 1.21-1.32 (m, 3H,
6
norbornyl), 1.42-1.53 (m, 3H, norbornyl), 1.52-1.65 (m, 1H, norbornyl), 1.83-1.96 (m, 1H,
norbornyl), 2.02 (s, 3H, CH ), 2.09 (s, 3H, CH ), 2.17 (brs, 1H, norbornyl), 2.33 (brs, 1H,
3
3
norbornyl), 4.11-4.39 (m, 5H, H-3′, H-4′, H-5′, CHNH), 4.55 (brs, 1H, H-2′), 5.35 (d, J = 5.13Hz,
1
1
1
1
H, OH), 5.54 (dd, J = 2.99, 5.76 Hz, 1H, OH), 5.72 (s, 1H, pyrazolyl), 5.86 (d, J = 5.56, 1H, H-
1
3
′), 7.79 (br s, 1H, CHNH), 8.08 (s, 1H, H-2), 8.21 (s, 1H, H-8). C NMR (DMSO-d ): 11.15,
6
3.57, 21.94, 29.64, 34.61, 35.77, 36.98, 38.42, 45.32, 52.96, 71.73, 73.46, 84.64, 87.92, 105.29,
+
19.43, 140.51, 141.75, 148.36, 150.22, 152.43, 155.81 MS (API-ESI): m/z 440.52 [M+H] . Anal.
calcd. for (C H N O ) C, 60.12; H, 6.65; N, 22.31; Found: C, 60.13; H, 6.66; N, 22.32.
2
2
29
7
3
6
2
-Chloro-N -(±)-endo-norbornyl-9H-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-5-deoxy--D-
ribofuranosyl)adenine (12).
The title compound was synthesized from 4 (140 mg, 0.338 mmol, reaction time 9 h) following
the general procedure described above. The residue was purified by chromatography on a silica
gel column with CHCl -MeOH (1-5%) as the eluent affording the fast migrating compound 12 as
3
1
a white solid (72 mg, 45% yield). H NMR (DMSO-d ): δ1.20-1.37 (m, 3H, norbornyl), 1.39-1.51
6
(m, 3H, norbornyl), 1.57-1.71 (m, 1H, norbornyl), 1.81-1.94 (m, 1H, norbornyl), 2.11 (brs, 1H,
norbornyl), 2.18 (s, 3H, CH ), 2.45 (brs, 1H, norbornyl), 2.49 (s, 3H, CH ), 3.81-4.24 (m, 4H, H-
3
3
3
′, H-4′, H-5′), 4.31 (brs, 1H, CHNH), 4.82 (brs, 1H, H-2′), 5.43 (d, J = 4.28 Hz, 1H, OH), 5.58 (s,
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H, pyrazolyl), 5.91 (d, J = 5.56 Hz, 1H, OH), ), 6.05 (d, J = 5.42, 1H, H-1′), 8.17 (br s, 1H,
13
CHNH), 8.32 (s, 1H, H-8). C NMR (DMSO-d ): 11.12, 13.53, 21.97, 29.62, 34.63, 35.74, 36.95,
6
3
1
5
8.46, 45.39, 52.91, 71.89, 73.42, 84.59, 87.95, 105.33, 119.47, 140.47, 141.72, 148.36, 150.25,
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53.95, 155.91 MS (API-ESI): m/z 474.96 [M+H] . Anal. calcd. for (C H ClN O ) C, 55.75; H,
2
2
28
7
3
.95; N, 20.69; Found: C, 55.76; H, 5.96; N, 20.68.
6
N -(R)-3-Tetrahydrofuranyl-9H-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-5-deoxy--D-
ribofuranosyl)adenine (13).
The title compound was synthesized from 5 (125 mg, 0.351 mmol, reaction time 5 h) following
the general procedure described above. Chromatography on silica gel column (CHCl -MeOH,
3
1
9
5:5) gave 13 as a white solid (85 mg, 58 % yield). H NMR (DMSO-d ): δ1.21-1.82 (2m, 2H,
6
tetrahydrofuranyl), 2.16 (s, 3H, CH ), 2.44 (s, 3H, CH ), 2.92 (dd, J = 4.22, 8.70 Hz, 1H,
3
3
tetrahydrofuranyl), 3.18 (m, 1H, tetrahydrofuranyl), 3.41-3.52 (m, 4H, tetrahydrofuranyl, H-5′),
.05 (t, J = 6.2Hz, 1H, H-4′), 4.07-4.11 (m, 1H, NHCH), 4.26 (t, J = 5.34 Hz, 1H, H-3′), 4.42 (s,
1H, H-2′), 5.22 (brs, 1H, OH), 5.38 (d, J = 5.98Hz, 1H, OH), 5.82 (s, 1H, pyrazolyl), 6.05 (d, J =
4
1
3
4
.71 Hz, 1H, H-1′), 7.61 (s, 1H, H-2), 7.83 (brs, 1H, H-8), 8.91 (brs, 1H, NH). C NMR (DMSO-
d6): 11.18, 13.49, 52.15, 52.92, 53.27, 67.53 (2C), 71.27, 73.69, 84.38, 87.98, 105.39, 119.72,
+
140.39, 141.89, 147.72, 149.82, 152.43, 155.81. MS (API-ESI): m/z 416.45 [M+H] . Anal. calcd.
for (C H N O ) C, 54.93; H, 6.07; N, 23.60; Found: C, 54.94; H, 6.06; N, 23.61.
1
9
25
7
4
6
2
-Chloro-N -(R)-3-tetrahydrofuranyl-9H-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-5-deoxy--D-
ribofuranosyl)adenine (14).
The title compound was synthesized from 6 (110 mg, 0.281 mmol, reaction time 9 h) following
the general procedure described above. The residue was purified by chromatography on a silica
gel column with CHCl -MeOH (1-3%) as the eluent affording the fast migrating compound 14 as
3
1
a white solid (65 mg, 51% yield). H NMR (DMSO-d ): δ 2.05-2.12 (m, 2H, tetrahydrofuranyl),
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.18 (s, 3H, CH ), 2.42 (s, 3H, CH ), 3.59-3.81 (2m, 4H, tetrahydrofuranyl, H-5′), 4.06 (m, 1H, H-
3
3
5
4
6
′), 4.11-4.22 (m, 3H, tetrahydrofuranyl, H-3′), 4.61-4.68 (m, 1H, NHCH), 4.81 (s, 1H, H-2′), 5.48
7
(d, J = 4.21 Hz, 1H, OH), 5.58 (d, J = 5.55 Hz, 1H, OH), 5.93 (d, J = 5.98 Hz, 1H, H-1′), 6.06 (s,
8
9
1
1
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8
9
0
1
5
1
H, pyrazolyl), 8.31 (brs, 1H, NH), 8.37 (brs, 1H, H-8). C NMR (DMSO-d ): 11.21, 13.53,
6
2.21, 52.94, 53.23, 67.59 (2C), 71.31, 73.72, 84.41, 88.01, 105.03, 119.68, 140.31, 141.73,
+
47.68, 149.91, 153.11, 154.83 MS (API-ESI): m/z 450.89 [M+H] . Anal. calcd. for
(C H ClN O ) C, 50.72; H, 5.38; N, 21.79; Found: C, 50.73; H, 5.37; N, 21.78.
1
9
24
7
4
6
N -(4-Chloro-2-fluorophenyl)amino)-9H-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-5-deoxy--D-
ribofuranosyl)adenine (15).
The title compound was synthesized from 7 (110 mg, 0.265 mmol, reaction time 7 h) following
the general procedure described above. Chromatography on silica gel column (CHCl -MeOH,
3
1
9
2
6:4) gave 15 as a white solid (52 mg, 41 % yield). H NMR (DMSO-d ): δ 2.03 (s, 3H, CH ),
6
3
.11 (s, 3H, CH ), 4.19-4.33 (m, 4H, H-3′, H-4′, H-5′), 4.61 (q, J = 5.34 Hz, 1H, H-2′), 5.36 (d, J =
3
5.12 Hz, 1H,OH), 5.55 (d, J = 5.98 Hz, 1H, OH), 5.72 (s, 1H, pyrazolyl), 5.93 (d, J = 5.56 Hz, 1H,
H-1′), 7.31 (dd, J = 1.5, 8.33 Hz, 1H, arom.), 7.49 (dd, J = 2.56, 10.26 Hz, 1H, arom.), 7.63 (t, J =
1
3
8
.55 Hz, 1H, arom.), 8.26 (s, 1H, H-2), 8.31 (s, 1H, H-8), 9.68 (s, 1H, NH). C NMR (DMSO-d ):
6
11.28, 13.78, 52.46, 72.09, 73.58, 85.22, 89.22, 105.38, 115.22, 118.37, 124.62, 126.23, 128.79,
1
30.09, 139.06, 140.64,144.67, 147.84, 148.95, 152.63, 156.64. MS (API-ESI): m/z 474.89
+
[
M+H] . Anal. calcd. for (C H ClFN O ) C, 53.23; H, 4.47; N, 20.69; Found: C, 53.24; H, 4.46;
21
21
7
3
N, 20.70.
6
2
-Chloro-N -(4-chloro-2-fluorophenyl)amino)-9H-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-5-
deoxy--D-ribofuranosyl) adenine (16).
The title compound was synthesized from 8 (120 mg, 0.267 mmol, reaction time 5 h) following
the general procedure described above. The residue was purified by chromatography on a silica
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6
gel column with CHCl -MeOH (1-5%) as the eluent affording the fast migrating compound 16 as
3
1
a white solid (84 mg, 62% yield). H NMR (DMSO-d ): δ 2.12 (s, 3H, CH ), 2.19 (s, 3H, CH ),
6
3
3
3
.78-3.95 (m, 1H, H-5′), 4.06-4.18 (2m, 2H, H-4′, H-5′), 4.25 (q, J = 7.26 Hz, 1H, H-3′), 4.82 (q,
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J = 5.76 Hz, 1H, H-2′), 5.52 (d, J = 5.13 Hz, 1H, OH), 5.61 (d, J = 5.98 Hz, 1H, OH), 5.74 (s, 1H,
pyrazolyl), 5.96 (d, J = 6.26 Hz, 1H, H-1′), 7.32 (dd, J = 2.12, 9.83 Hz, 1H, arom.), 7.51-7.58 (m,
1
3
2
H, arom.), 8.48 (s, 1H, H-8), 10.05 (s, 1H, NH). C NMR (DMSO-d ): 11.31, 13.72, 52.51,
6
72.11, 73.53, 85.31, 89.41, 105.27, 115.43, 118.51, 124.47, 126.36, 128.85, 130.13, 139.13,
+
1
40.82, 144.73, 147.89, 149.02, 153.27, 156.91. MS (API-ESI): m/z 509.33 [M+H] . Anal. calcd.
for (C H Cl FN O ) C, 49.62; H, 3.97; N, 19.29; Found: C, 49.63; H, 3.96; N, 19.30.
2
1
20
2
7
4
6
2
-(3,5-Dimethyl-1H-pyrazol-1-yl)-N -cyclopentyl-9H-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-5-
deoxy--D-ribofuranosyl)adenine (17).
Compound 17 was obtained from the same reaction of compound 10 after chromatography on a
silica gel column with CHCl -MeOH (1-5%) as the slowest migrating compound (white solid, 22
3
1
mg, 12% yield). H NMR (CDCl ) δ 1.53-1.81 (m, 4H, cyclopentyl), 1.83-2.02 (m, 2H,
3
cyclopentyl), 2.04-2.12 (m, 2H, cyclopentyl), 2.13 (s, 3H, CH ), 2.24 (s, 3H, CH ), 2.32 (s, 3H,
3
3
CH ), 2.68 (s, 3H, CH ), 4.06 (brs, 1H, H-4′), 4.35 (brs, 2H, H-5′), 4.45 (q, J = 5.22 Hz, 1H, H-3′),
3
3
4
.48 (brs, 1H, CHNH), 4.64 (t, J = 5.56, 1H, H-2′), 5.33 (d, J = 5.11 Hz, 1H, OH), 5.51 (d, J =
.65 Hz, 1H, OH), 5.83 (brs, 2H, pyrazolyl, NHCH), 6.02 (s, 1H, pyrazolyl), 6.31 (brs, 1H, H-1′),
5
1
3
7.42 (s, 1H, H-8). C NMR (DMSO-d ): 11.21, 12.38, 13.55 (2C), 23.86 (2C), 32.51 (2C), 52.83,
6
5
6.31, 72.26, 74.32, 84.29, 88.36, 104.87, 105.25, 119.59, 140.71, 141.63 (2C), 148.12 (2C),
+
1
49.86, 152.22, 154.72. MS (API-ESI): m/z 508.59 [M+H] . Anal. calcd. for (C H N O ) C,
2
5
33
9
3
59.16; H, 6.55; N, 24.84; Found: C, 59.15; H, 6.56; N, 24.85.
6
2
-(3,5-Dimethyl-1H-pyrazol-1-yl)-N -(±)-endo-norbornyl-9H-(5-(3,5-dimethyl-1H-pyrazol-1-
yl)-5-deoxy--D-ribofuranosyl) adenine (18).
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Compound 18 was obtained from the same reaction of compound 12 after chromatography on a
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silica gel column with CHCl -MeOH (1-5%) as the slowest migrating compound (white solid, 39
3
1
mg, 22% yield). H NMR (DMSO-d ): δ1.19-1.31 (m, 3H, norbornyl), 1.39-1.44 (m, 3H,
6
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norbornyl), 1.52-1.67 (m, 1H, norbornyl), 1.81-1.94 (m, 1H, norbornyl), 2.02 (brs, 6H, CH ), 2.11
3
(
brs, 6H, CH ), 2.21 (brs, 1H, norbornyl), 2.49 (brs, 1H, norbornyl), 4.11-4.41 (m, 5H, H-3′, H-4′,
3
H-5′,CHNH) 4.57 (brs, 1H, H-2′), 5.37 (d, J = 5.25 Hz, 1H, OH), 5.52 (d, J = 5.45 Hz, 1H, OH),
5.72 (s, 1H, pyrazolyl), 5.88 (s, 1H, pyrazolyl), 6.03 (s, 1H, H-1′), 8.05 (brs, 1H, CHNH), 8.09 (s,
13
1
H, H-8). C NMR (DMSO-d ): 11.18, 12.33, 13.45 (2C), 22.08, 29.33, 34.52, 35.82, 36.34,
6
3
8.55, 45.22, 53.01, 71.77, 73.82, 84.67, 87.78, 104.96, 105.34, 119.61, 140.68, 141.58 (2C),
+
148.23 (2C), 149.91, 152.37, 154.77. MS (API-ESI): m/z 534.63 [M+H] . Anal. calcd. for
(
C H N O ) C, 60.77; H, 6.61; N, 23.62; Found: C, 60.76; H, 6.62; N, 23.61.
27 35 9 3
6
2
-(3,5-Dimethyl-1H-pyrazol-1-yl)-N -(R)-3-tetrahydrofuranyl-9H-(5-(3,5-dimethyl-1H-
pyrazol-1-yl)-5-deoxy--D-ribofuranosyl) adenine (19).
Compound 19 was obtained from the same reaction of compound 14 after chromatography on a
silica gel column with CHCl -MeOH (1-3%) as the slowest migrating compound (white solid, 29
3
1
mg, 20% yield). H NMR (CDCl ) δ 1.91-2.03 (m, 2H, tetrahydrofuranyl), 2.11 (s, 3H, CH ), 2.23
3
3
(
s, 3H, CH ), 2.29 (s, 3H, CH ), 2.51-2.63 (m, 1H, tetrahydrofuranyl), 2.67 (s, 3H, CH ), 3.81-
3
3
3
4
.03 (m, 4H, tetrahydrofuranyl, H-5′), 4.11 (m, 1H, tetrahydrofuranyl), 4.33 (s, 1H, H-4′), 4.45 (m,
1H, H-3′), 4.71 (t, J = 5.40 Hz, 1H, H-2′), 4.77-4.83 (m, 1H, NHCH), 5.85 (brs, 1H, OH), 6.02
(brs, 1H, OH), 6.19 (brs, 2H, pyrazolyl), 6.32 (s, 1H, H-1′), 7.55 (brs, 1H, CHNH), 8.03 (s, 1H, H-
1
3
8
). C NMR (DMSO-d ): 11.23, 12.34, 13.58 (2C), 52.26, 53.03, 53.28, 67.62 (2C), 71.31, 73.78,
6
84.48, 88.18, 104.92, 105.22, 119.73, 140.52, 141.77 (2C), 147.79 (2C), 149.96, 152.06, 154.89.
+
MS (API-ESI): m/z 510.57 [M+H] . Anal. calcd. for (C H N O ) C, 56.57; H, 6.13; N, 24.74;
2
4
31
9
4
Found: C, 56.56; H, 6.12; N, 24.75.
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-(3,5-Dimethyl-1H-pyrazol-1-yl)-N -(4-chloro-2-fluorophenyl)amino)-9H-(5-(3,5-dimethyl-
H-pyrazol-1-yl)-5-deoxy--D-ribofuranosyl) adenine (20).
Compound 20 was obtained from the same reaction of compound 16 after chromatography on a
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silica gel column with CHCl -MeOH (1-5%) as the slowest migrating compound (white solid, 27
3
1
mg, 18% yield). H NMR (DMSO-d ): δ 2.12 (s, 3H, CH ), 2.21 (s, 3H, CH ), 2.27 (s, 3H, CH ),
6
3
3
3
2.48 (s, 3H, CH ), 4.11-4.39 (m, 4H, H-3′, H-4′, H-5′), 4.58-4.71 (m, 1H, H-2′), 5.28 (d, J = 5.32
3
Hz, 1H, OH), 5.62 (d, J = 5.78 Hz, 1H, OH), 5.83 (s, 1H, pyrazolyl), 5.96 (s, 1H, pyrazolyl), 6.11
(
d, J = 5.45 Hz, 1H, H-1′), 7.09-7.16 (m, 2H, arom.), 7.61-7.72 (m, 1H, arom.), 8.27 (s, 1H, H-8),
1
3
9
.36 (s, 1H, NH). C NMR (DMSO-d ): 11.32, 12.39, 13.78 (2C), 52.57, 72.19, 73.58, 85.43,
6
89.52, 104.93, 105.32, 115.22, 119.02, 124.49, 126.42, 128.74, 130.13, 139.22, 140.68 (2C),
+
1
44.76, 147.78 (2C), 149.32, 152.93, 157.08. MS (API-ESI): m/z 569.02 [M+H] . Anal. calcd. for
(
C H ClFN O ) C, 54.98; H, 4.79; N, 22.19; Found: C, 54.99; H, 4.77; N, 22.18.
26 27 9 3
6
General procedure for the N -Amination of 21 and 22 into compounds 25 and 26.
3
1
To a stirred solution of 6-chloro-9H-(2,3,5-O-acetyl--D-ribofuranosyl)purine (21) or 2,6-
32
dichloro-9H-(2,3,5-O-acetyl-D-ribofuranosyl)purine (22) (1 equiv) in absolute ethanol (20 mL),
4
-chloro-2-fluoroaniline (1.6 equiv) was added. The reaction mixture was refluxed for the time
reported below and concentrated in vacuo. The residue was dissolved in methanolic ammonia (10
mL) and stirred at room temperature overnight. The solution was evaporated to dryness and the
residue was purified by chromatography on a silica gel column.
6
N -(4-Chloro-2-fluorophenyl)amino-9H-(β-D-ribofuranosyl)adenine (25).
Reaction of 21 (300 mg, 0.726 mmol) with 4-chloro-2-fluoroaniline (169.1 mg, 129 L, 1.16
mmol) for 6 h followed by deprotection gave 25, which was purified by chromatography on a
1
silica gel column (CHCl -MeOH, 99:1) as a white solid (201 mg, 70% yield). HNMR (DMSO-
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d6):  3.52-3.77 (m, 2H, H-5′), 3.95 (q, J = 3.71 Hz, 1H, H-4′), 4.15 (q, J = 4.7 Hz, 1H, H-3′), 4.6
q, J = 5.99 Hz, 1H, H-2′), 5.21 (d, J = 4.7 Hz, 1H, OH), 5.25 (q, J = 4.91 Hz, 1H, OH), 5.48 (d, J
= 6.42 Hz, 1H, OH), 5.94 (d, J = 5.98 Hz, 1H, H-1′), 7.31 (dd, J = 1.28, 8.55 Hz, 1H, arom.), 7.53
4
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9
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(
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(dd, J = 2.09, 10.26 Hz, 1H, arom.), 7.62 (t, J = 8.55 Hz, 1H, arom.), 8.32 (s, 1H, H-2), 8.51 (s,
1
3
1
H, H-8), 9.71 (br s, 1H, NH). C NMR (DMSO-d ): 61.77, 70.96, 74.33, 86.39, 88.22, 117.31,
6
119.63, 125.38, 125.62, 129.55, 131.29, 141.83, 151.69, 152.27, 154.12, 156.02, 158.52. MS
+
(
API-ESI): m/z 396.77 [M+H] . Anal. calcd. For (C H ClFN O ) C, 48.56; H, 3.82; N, 17.70;
1
6
15
5
4
Found: C, 48.55; H, 3.83; N, 17.71.
6
2-Chloro-N -(4-chloro-2-fluorophenyl)amino-9H-(β-D-ribofuranosyl)adenine (26).
Reaction of 22 (250 mg, 0.558 mmol) with 4-chloro-2-fluoroaniline (130 mg, 99.1 L, 0.892
mmol) for 10 h followed by deprotection gave 26, which was purified by chromatography on a
1
silica gel column (CHCl -MeOH, 99:1) as a white solid (156 mg, 65% yield). H NMR (DMSO-
3
d6): δ 3.51-3.72 (2m, 2H, H-5′), 3.94 (d, J = 3.63 Hz, 1H, H-4′), 4.05-4.15 (m, 1H, H-3′), 4.52 (q,
J = 5.77 Hz, 1H, H-2′), 5.31 (t, J = 5.55 Hz, 1H, OH), 5.21 (d, J = 5.13 Hz, 1H, OH), 5.51 (d, J =
5.98 Hz, 1H, OH), 5.86 (d, J = 5.99 Hz, 1H, H-1′), 7.32 (d, J = 8.55 Hz, 1H, arom.), 7.52 (t, J =
13
4
.28 Hz, 1H, arom.), 7.55 (d, J = 2.13 Hz, 1H, arom.), 8.53 (s, 1H, H-8), 10.21 (brs, 1H, NH). C
NMR (DMSO-d ):61.92, 70.98, 74.45, 86.41, 88.15, 117.43, 119.69, 125.41, 125.57, 129.68,
6
+
131.32, 141.91, 151.73, 153.27, 154.01, 156.08, 158.58. MS (API-ESI): m/z 431.25 [M+H] .
Anal. calcd. for (C H Cl FN O ) C, 44.67; H, 3.28; N, 16.28; Found: C, 44.68; H, 3.27; N,
1
6
14
2
5
4
1
6.29.
General procedure for the Synthesis of 2′,3′-O-Isopropylidene derivatives 29, and 30.
A mixture of 25 or 26 (1 equiv), 2,2-dimethoxypropane (18.1 equiv) and camphorsulfonic acid (1
equiv) in anhydrous acetone (10 mL) was stirred at 55 °C for the time reported below. The solvent
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was removed in vacuo, and the residue was purified by column chromatography to afford the
desired compounds.
6
N -(4-Chloro-2-fluorophenyl)amino-9H-(2,3-O-isopropylidene-β-D-ribofuranosyl)adenine
0
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(29).
The title compound was synthesized from 25 (200 mg, 0.505 mmol, reaction time 6 h).
Chromatography on a silica gel column (CHCl -MeOH, 98:2) gave 29 as a white foam (209 mg,
3
1
9
5% yield). HNMR (DMSO-d ): δ 1.36 (s, 3H, CH ), 1.52 (s, 3H, CH ), 3.41-3.62 (m, 2H, H-
6
3
3
5′), 4.24 (dt, J = 2.70, 5.10 Hz, 1H, H-4′), 4.95 (dd, J = 2.40 Hz, 1H, H-3′), 5.25 (t, J = 5.60 Hz,
1
9
H, OH), 5.36 (dd, J = 3.22, 6.30 Hz, 1H, H-2′), 6.16 (d, J = 2.4 Hz, 1H, H-1′), 7.3 (dd, J = 4.50,
.20 Hz, 1H, arom.), 7.5 (dd, J = 6.50, 10.40 Hz, 1H, arom.), 7.61 (t, J = 8.40 Hz, 1H, arom.), 8.29
+
(s, 1H, H-2), 8.52 (s, 1H, H-8), 9.71 (s, 1H, NH). MS (API-ESI): m/z 436.837 [M+H] . Anal.
calcd. for (C H ClFN O ) C, 52.36; H, 4.39; N, 16.07; Found: C, 52.37; H, 4.38; N, 16.06.
1
9
19
5
4
6
2-Chloro-N -(4-chloro-2-fluorophenyl)amino-9H-(2,3-O-isopropylidene--D-ribofuranosyl)-
adenine (30).
The title compound was obtained starting from 26 (150 mg, 0.348 mmol, reaction time 2 h).
Chromatography on a silica gel column (CHCl -MeOH, 97:3) gave 30 as a white foam (136 mg,
3
1
8
4
3% yield). H NMR (DMSO-d ): δ 1.32 (s, 3H, CH ), 1.51 (s, 3H, CH ), 3.51 (m, 2H, H-5′),
6
3
3
.22 (pd, J = 3.0 Hz, 1H, H-4′), 4.93 (dd, J =1.93, 6.20 Hz, 1H, H-3′), 5.06 (t, J = 5.13 Hz, 1H,
OH), 5.3 (dd, J = 2.56, 5.98 Hz, 1H, H-2′), 6.11 (d, J = 2.13 Hz, 1H, H-1′), 7.31 (dd, J = 1.71,
8
.12 Hz, 1H, arom.), 7.48-7.56 (m, 2H, arom.), 8.47 (s, 1H, H-8), 10.22 (s, 1H, NH). MS (API-
+
ESI): m/z 471.28 [M+H] . Anal. calcd. For (C H Cl FN O ) C, 48.53; H, 3.86; N, 14.89; Found:
1
9
18
2
5
3
C, 48.54; H, 3.87; N, 14.87.
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General procedure for the Synthesis of Compounds 31-34.
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Compounds 27-30 (1 equiv) in dry acetonitrile (10 mL) under nitrogen atmosphere were stirred
with cooling to -5 °C. SOCl (3 equiv) was added portionwise followed by dry pyridine (2 equiv)
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and allowed to react for 30 min at -5º C, then allowed to warm to room temperature and stirred for
6
h. The procedure was repeated after 6 h, and the mixture was stirred at room temperature
overnight. Water was added (5 mL), and the solution was neutralized with NaHCO (1 M) and
3
extracted with CH Cl (3 × 10 mL). The organic layer was dried (Na SO ), and the solvent was
2
2
2
4
evaporated to dryness. The residue was purified by column chromatography as reported below.
6
N -(R)-3-Tetrahydrofuranyl-9H-(2,3-O-isopropylidene-5-chloro-5-deoxy-β-D-ribofuranosyl)
adenine (31).
The title compound was synthesized from 27 (290 mg, 0.768 mmol). Chromatography on a silica
1
gel column (CHCl -MeOH, 99:1) gave 31 as a white foam (167 mg, 55% yield). H NMR
3
(DMSO-d ): δ 1.38 (s, 3H, CH ), 1.55 (s, 3H, CH ), 1.95-2.23 (2m, 2H, tetrahydrofuranyl), 3.60
6 3 3
(dd, J = 4.52, 8.80 Hz, 1H, tetrahydrofuranyl), 3.72 (q, J = 7.70 Hz, 1H, tetrahydrofuranyl), 3.80-
3
3
7
.96 (m, 4H, tetrahydrofuranyl, H-5′), 4.08 (q, J = 5.30 Hz, 1H, H-4′), 4.20 (q, J = 4.70 Hz, 1H, H-
′), 4.58-4.71 (m, 1H, NHCH), 4.74 (q, J = 5.30 Hz, 1H, H-2′), 5.92 (d, J = 5.50 Hz, 1H, H-1′),
+
.98 (brs, 1H, NH), 8.22 (brs, 1H, H-2), 8.38 (s, 1H, H-8). MS (API-ESI): m/z 396.8 [M+H] .
Anal. calcd. for (C H ClN O ) C, 51.58; H, 5.60; N, 17.69; Found: C, 51.57; H, 5.61; N, 17.68.
1
7
22
5
4
6
2-Chloro-N -(R)-3-tetrahydrofuranyl-9H-(2,3-O-isopropylidene-5-chloro-5-deoxy-β-D-
ribofuranosyl)adenine (32).
The title compound was synthesized from 28 (300 mg, 0.728 mmol). Chromatography on a silica
1
gel column (CHCl -MeOH, 99:1) gave 32 as a white foam (204 mg, 65% yield). H NMR
3
(
DMSO-d ): δ 1.35 (s, 3H, CH ), 1.52 (s, 3H, CH ), 1.85-2.28 (m, 2H, tetrahydrofuranyl), 3.55-
6 3 3
3
.81 (m, 4H, tetrahydrofuranyl), 3.81-3.92 (m, 2H, H-5′), 4.3-4.36 (m, 1H, H-4′), 4.62 (brs, 1H,
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NHCH), 5.1 (q, J = 3.02 Hz, 1H, H-3′), 5.37 (dd, J = 2.99, 6.20 Hz, 1H, H-2′), 6.19 (s, 1H, H-1′),
+
8
.38 (s, 1H, H-8), 8.62 (brs, 1H, NH). MS (API-ESI): m/z 431.28 [M+H] . Anal. calcd. for
(
C H N O C ) C, 47.45; H, 4.92; N, 16.28; Found: C, 47.46; H, 4.93; N, 16.27.
17 21 5 4 l2
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N -(4-Chloro-2-fluorophenyl)amino-9H-(2,3-O-isopropylidene-5-chloro-5-deoxy-β-D-
ribofuranosyl)adenine (33).
The title compound was synthesized from 29 (300 mg, 0.688 mmol). Chromatography on a silica
1
gel column (CHCl ) gave 33 as a white foam (134 mg, 43% yield). HNMR (DMSO-d ): δ 1.32 (s,
3
6
3H, CH ), 1.52 (s, 3H, CH ), 3.75 (dd, J = 6.42, 11.12 Hz, 1H, H-5′) 3.85 (dd, J = 6.84, 11.12 Hz,
3
3
1
H, H-5′), 4.36 (dt, J = 2.99, 6.41 Hz, 1H, H-4′), 5.08 (dd, J = 2.99, 6.41 Hz, 1H, H-3′), 5.51 (dd,
J= 2.35, 6.21 Hz, 1H, H-2′), 6.23 (d, J = 2.56 Hz, 1H, H-1′), 7.29 (dd, J = 1.28, 8.55 Hz, 1H,
arom.), 7.52 (dd, J = 2.14, 10.26 Hz, 1H, arom.), 7.61 (t, J = 8.55 Hz, 1H, arom.), 8.33 (s, 1H, H-
+
2
), 8.48 (s, 1H, H-8), 9.76 (s, 1H, NH). MS (API-ESI): m/z 455.28 [M+H] . Anal. calcd. for
(
C H Cl FN O ) C, 50.23; H, 3.99; N, 15.42; Found: C, 50.21; H, 3.97; N, 15.43.
19 18 2 5 3
6
2
-Chloro-N -(4-chloro-2-fluorophenyl)amino-9H-(2,3-O-isopropylidene-5-chloro-5-deoxy-β-
D-ribofuranosyl)adenine (34).
The title compound was synthesized from 30 (270 mg, 0.574 mmol). Chromatography on a silica
1
gel column (CHCl ) gave 34 as a white foam (216 mg, 77% yield). HNMR (DMSO-d ): δ 1.38 (s,
3
6
3
1
H, CH ), 1.58 (s, 3H, CH ), 3.77 (dd, J = 6.19, 11.32 Hz, 1H, H-5′), 3.86 (dd, J = 6.83, 11,12 Hz,
3 3
H, H-5′), 4.35 (dt, J = 3.02, 6.41 Hz, 1H, H-4′), 5.03 (dd, J = 2.99, 5.99 Hz, 1H, H-3′), 5.41 (dd, J
= 2.14, 5.98 Hz, 1H, H-2′), 6.21 (d, J = 1.71 Hz, 1H, H-1′), 7.31 (dd, J = 2.14, 8.55 Hz, 1H,
arom.), 7.53 (m, 2H, arom.), 8.48 (s, 1H, H-8), 10.22 (s, 1H, NH). MS (API-ESI): m/z 489.73
+
[
M+H] . Anal. calcd. for (C H Cl FN O ) C, 46.69; H, 3.51; N, 14.33; Found: C, 46.67; H, 3.53;
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17
3
5
3
N, 14.35.
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Journal of Medicinal Chemistry
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Membrane preparation. Membranes for radioligand binding were prepared as described
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previously. In brief, after homogenization of CHO cells stably transfected with the hAR subtype
membranes were prepared in a two-step procedure. A first low-speed centrifugation (1,000 x g)
was used to remove cell fragments and nuclei and was followed by a high-speed centrifugation
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(
100,000 x g) of the supernatant in order to sediment a crude membrane fraction. The resulting
membrane pellets were resuspended in the buffer used for the respective binding experiments,
frozen in liquid nitrogen and stored in aliquots at -80°C. Adenylyl cyclase activity was measured
in a membrane fraction obtained in a simplified procedure with only one high-speed centrifugation
of the homogenate. The resulting crude membrane pellet was resuspended in 50 mM Tris/HCl, pH
7.4 and used immediately for the cyclase assay.
Radioligand binding and Adenylyl Cyclase Assay. In competition experiments the following
3
3
radioligands were used: 1 nM [ H]CCPA for hA ARs, 10 nM [ H]NECA for hA ARs, 1 nM
1
2A
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35,38
3
[
H]HEMADO for hA ARs.
Nonspecific binding of [ H]CCPA was determined in the
3
3
3
presence of 1 mM theophylline, while nonspecific binding of [ H]NECA and [ H]HEMADO was
6
estimated in the presence of 100 µM (R)-N phenylisopropyladenosine (R-PIA). Dissociation
constants (K -values) were calculated from radioligand competition experiments utilizing the
i
program Prism (GraphPad, San Diego, CA, USA).
Due to the lack of a useful high-affinity radioligand for A2B adenosine receptors, stimulation of
3
5
adenylyl cyclase activity was measured to determine agonist potency (EC50 values). If only
partial agonistic activity was observed, efficacy was compared to 100 µM NECA as a full agonist.
All values are given as geometric means with 95% confidence intervals (n  3). The functional
activity at the hA , A , and A receptors was determined in adenylyl cyclase experiments. The
1
2A
3
inhibition of forskolin-stimulated adenylyl cyclase via hA and A receptors was measured as
1
3
4
9
described earlier. As reference agonists (efficacy = 100%), CCPA and NECA, respectively, were
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used. Compounds were considered to be A antagonists if they fully reversed (>85%) the NECA-
3
mediated inhibition of adenylyl cyclase activity (IC values in Table 3).
50
Computational Chemistry. Molecular modeling and graphics manipulations were performed
using MOE (Molecular Operating Environment, version 2013.08, Chemical Computing Group,
Toronto, Canada) and UCSF-CHIMERA 1.8.1 (http://www.cgl.ucsf.edu/chimera) software
packages, running on an E4 Computer Engineering E1080 workstation provided with an Intel
Xeon processor. GOLD Suite 5.4.1 docking package (CCDC Software Limited: Cambridge, U.K.,
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2008) was used for all docking calculations. Figures were generated using Pymol 1.8.2
(Schrödinger, LLC, New York, NY, 2016).
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Residue Indexing. The Ballesteros−Weinstein double-numbering system was used to describe
the transmembrane (TM) location of the amino acids. Along with numbering their positions in the
primary amino acid sequence, the residues have numbers in parentheses (X.YZ) that indicate their
position in each transmembrane (TM) helix (X), relative to a conserved reference residue in that
TM helix (YZ).
Three-Dimensional Structure of hA AR. In this study we used a previously published hA AR
1
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50
homology model, built by means of the homology modeling tools implemented in the MOE
suite. In particular, the crystal structure of the hA AR cocrystallized with the agonist UK-432097
2
A
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4
(PDB ID: 3QAK), was selected as a template for the entire hA AR structure. The hA AR
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1
sequence was retrieved from the publicly available sequence database www.uniprot.org and
aligned against the sequence of the respective A AR template, taking into account the highly
2
A
conserved residues in each TM domain and following the numbering scheme by Ballesteros and
46
Weinstein. Then, a homology model was built using the automated Homology Modeling
protocol implemented in the MOE suite.
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Docking Simulations of 5′-pyrazolyl adenosine derivatives at the hA AR Model. Structures of
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compounds 6, 12, 14, and 16 were built using the builder tool implemented in the MOE suite and
−
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subjected to a MMFF94x energy minimization until a rms gradient was <0.05 kcal mol Å .
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Molecular docking of the ligands at the hA AR model was performed by means of the GOLD
1
software, which uses a genetic algorithm and considers full ligand conformational flexibility and
partial protein flexibility, i.e. the flexibility of side chain residues only. The coordinates of four
6
.55
6.48
7.43
key residues in the binding pocket of hA AR model, that is F (EL2), N , W and H , were
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3.36
chosen as active-site origin. The active-site radius was set equal to 13 Å. The mobility of T
,
6.48
6.52
6.55
7.42
7.43
W
, H , N , T , and H side chains was set up using the flexible sidechains option in the
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1
GOLD front end, which incorporates the Lovell rotamer library. Each GA run used the default
parameters of 100 000 genetic operations on an initial population of 100 members divided into
five subpopulations, with weights for crossover, mutation, and migration being set to 95, 95, and
1
0, respectively. GOLD allows a user-definable number of GA runs per ligand, each of which
starts from a different orientation. For these experiments, the number of GA runs was set to 200
without the option of early termination, and scoring of the docked poses was performed with the
4
1
original ChemPLP scoring function followed by rescoring with ChemScore. The final receptor–
ligand complex for each ligand was chosen interactively by selecting the highest scoring pose that
was consistent with experimentally-derived information about the binding mode of the ligand.
Formalin Test. Adult male BLC57/6 mice (25-30 g) were housed three per cage under controlled
illumination (12 h light/12 h dark cycle; light on 06:00 h) and standard environmental conditions
(room temperature 20-22 °C, humidity 55-60%) for at least 1 week before the beginning of the
experiments. Chow and tap water were available ad libitum. All surgery and experimental
procedures were performed during the light cycle and were approved by the Animal Ethics
Committee of the University of Campania “L. Vanvitelli”. Animal care was in compliance with
European regulations on the protection of laboratory animals (O.J. of E.C. L358/1 18/12/86). All
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