Novel Irreversible Agonists Acting at the A1 Adenosine Receptor

Article abstract of DOI:10.1021/acs.jmedchem.6b01561

The A₁ adenosine receptor (A₁AR) is an important G protein-coupled receptor that regulates a range of physiological functions. Herein we report the discovery of novel irreversible agonists acting at the A₁AR, which have the potential to serve as useful research tools for studying receptor structure and function. A series of novel adenosine derivatives bearing electrophilic substituents was synthesized, and four compounds, 8b, 15a, 15b, and 15d, were shown to possess similar potency and efficacy to the reference high efficacy agonist, NECA, in an assay of ERK1/2 phosphorylation assay. Insensitivity to antagonist addition in a real-time, label-free, xCELLigence assay was subsequently used to identify compounds that likely mediated their agonism through an irreversible interaction with the A₁AR. Of these compounds, 15b and 15d were more directly validated as irreversible agonists of the A₁AR using membrane-based [³H]DPCPX and [³⁵S]GTPγS binding experiments.

Full text of DOI:10.1021/acs.jmedchem.6b01561

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Article
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Novel Irreversible Agonists Acting at the A Adenosine Receptor
Manuela Jörg, Alisa Glukhova, Alaa Abdul-Ridha, Elizabeth A. Vecchio, Anh T.N. Nguyen,
Patrick M. Sexton, Paul J. White, Lauren T. May, Arthur Christopoulos, and Peter J. Scammells
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01561 • Publication Date (Web): 01 Dec 2016
Downloaded from http://pubs.acs.org on December 6, 2016
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Novel Irreversible Agonists Acting at the A Adenosine Receptor
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†
,§
‡,§
‡
‡
Manuela Jörg , Alisa Glukhova, , Alaa AbdulꢀRidha, Elizabeth A. Vecchio, Anh
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‡
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T.N. Nguyen, Patrick M. Sexton, Paul J. White, Lauren T. May, Arthur
‡,*
†,*
Christopoulos, Peter J. Scammells
†
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences; Monash University, Parkville,
‡
VIC 3052, Australia; Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences
Biology & Department of Pharmacology, Monash University, Parkville, VIC 3052, Australia
§
These authors contributed equally to this manuscript
ABSTRACT: The A
regulates a range of physiological functions. Herein we report the discovery of novel irreversible
agonists acting at the A AR, which have the potential to serve as useful research tools for studying
1 1
adenosine receptor (A AR) is an important G proteinꢀcoupled receptor that
1
receptor structure and function. A series of novel adenosine derivatives bearing electrophilic
substituents was synthesised and four compounds, 8b, 15a, 15b and 15d, were shown to posses
similar potency and efficacy to the reference high efficacy agonist, NECA, in an assay of ERK1/2
phosphorylation assay. Insensitivity to antagonist addition in a realꢀtime, labelꢀfree, xCELLigence
assay was subsequently used to identify compounds that likely mediated their agonism through an
irreversible interaction with the A AR. Of these compounds, 15b and 15d were more directly
1
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validated as irreversible agonists of the A
binding experiments.
1
AR using membraneꢀbased [ H]DPCPX and [ S]GTPγS
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ꢀ INTRODUCTION
Adenosine receptors (ARs) are a family of G proteinꢀcoupled receptors (GPCRs) that are distributed
1, 2
broadly throughout the body and are linked to a number of physiological functions.
The
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initiation of these functions occurs upon the activation of four different AR subtypes; namely the
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, 4
A
1
, A2A, A2B and A
3
ARs.
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The A AR is found in spinal cord, central nervous system, heart, and
adipose tissue, and is associated with a wide range of effects including the ability to slow down
1
, 5ꢀ8
heart rate, inhibit neurotransmitter release and inhibit lipolysis.
The A AR has been recognized
1
9, 10
as a promising drug target for a variety of disorders, such as neuropathic pain
and cardiac
1
1, 12
13ꢀ17
ischemiaꢀreperfusion injury.
Despite recent breakthroughs in GPCR structural biology,
and
numerous crystal structures solved for the A2AAR, there is surprisingly limited information
available about the structural and functional features of the A AR.
1
18ꢀ22
Irreversible probes/ligands are valuable tools for studying GPCRs both in vitro and in vivo.
Of
note, recent work has demonstrated advantages that covalent ligands could hold for GPCR
structural biology studies, which is based on the irreversible probe’s ability to form a stable and
2
3ꢀ27
long living ligandꢀreceptor complex with the targeted GPCR.
Irreversible agonists and
antagonists targeting the A AR subtype have been reported in the past, and the chemical structures
1
of the bestꢀstudied ligands are illustrated in Figure 1. The xanthineꢀbased irreversible antagonists
1,3ꢀdipropylꢀ8ꢀisothiocyanatophenyl(aminothiocarbonyl(2ꢀaminoethylaminocarbonyl(4ꢀmethyloxy
(phenyl)))xanthine (DITCꢀXAC) and 8ꢀcyclopentylꢀ3ꢀ(3ꢀ((4ꢀfluorosulfonylbenzoyl)oxy)propyl)ꢀ1ꢀ
28ꢀ30
propylxanthine (FSCPX), as well as variants thereof, have been part of extensive investigations.
Interrogation of linker attachment positions, irreversible groups, as well as a range of linker lengths
and types, suggested that the 3ꢀ and 8ꢀpostions of the xanthine scaffold are suitable for the
1
attachment of the irreversible group to maintain high affinity and selectivity for the A AR subtype.
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selective A AR irreversible antagonists
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O
N
H
N
O
N
O
NH
O
N
HN
S
NH
O
O
H
N
N
O
O
NCS
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O
N
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FO S
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m- or p-DITC-XAC
FSCPX
selective A AR irreversible agonists
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N
OH
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HN
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H
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NCS
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N
N3
HO
HO
O
O
OH OH
OH OH
R-AHPIA
m- or p-DITC-ADAC
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Figure 1. Chemical structures of irreversible xanthineꢀbased A AR antagonists DITCꢀXAC and
FSCPX as well as the adenosineꢀbased agonists DITCꢀADAC and RꢀAHPIA that haven been
previously described in the literature (blue: pharmacophore; red: reactive group; orange: linker).
1
Although not as wellꢀstudied, some irreversible probes capable of activating the A AR have also
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been developed; namely, N [4ꢀ[[[4ꢀ[[[[2ꢀ[[[(pꢀ(m)ꢀisothiocyanatophenyl)amino]thiocarbonyl]
amino]ethyl]amino]carbonyl]methyl]aniline]carbonyl]methyl]phenyl]adenosine (pꢀ and mꢀDITCꢀ
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1
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ADAC) and Rꢀ2ꢀazidoꢀN ꢀpꢀhydroxyphenylisopropyladenoisine (RꢀAHPIA).
Both probes are
based on an adenosine scaffold; however, they are structurally and functionally different from each
other. RꢀAHPIA forms its irreversible bond with the receptor upon photoactivation by UV light,
due to the incorporation of a photoreactive azide group in the 2ꢀpostion of the adenosine scaffold.
In contrast, pꢀ and mꢀDITCꢀADAC form a direct covalent bond with the receptor via a
chemoreactive isothiocyanate group attached to a lengthy 2ꢀ(4ꢀaminophenyl)acetic acidꢀbased
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linker that is incorporated into the N ꢀposition of the adenosine scaffold. Both pꢀ and mꢀDITCꢀ
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ADAC exhibited high (subꢀnanomolar) affinity at the A
1
AR, as well as, 200ꢀ to 600ꢀfold selectivity
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1
over the A ARs. In addition, both ligands were capable of activating the A AR to yield persistent
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cAMP responses in functional assays, but limited progress has been made since these early
studies, and it remains unknown as to whether such probes can also be used as useful tools to study
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AR structural biology.
RꢀAHPIA possesses an aryl azide reactive group, which needs to be activated by photolysis to
generate a covalent bond with the receptor. Even though pꢀ and mꢀDITCꢀADAC are known
irreversible probes based on a chemoreactive isothiocyanate group, these probes have a relatively
long and flexible linker. In view of the current interest in development of probes to facilitate GPCR
structural biology studies, we have chosen to investigate the effects of different linkers in length
and flexibility on the pharmacology of novel A
1
AR irreversible agonists, as well as their effects on
receptor thermostability. Specifically, we present the synthesis, characterization and
pharmacological evaluation of 15 novel adenosine analogs containing a reactive functional group
capable of forming a covalent bond with the receptor. A range of chemoreactive groups with
different types of action were incorporated; namely bicyclo[2.2.2]octaꢀ2,5ꢀdienes (Michael
acceptors), isothiocyanates and fluorosulfonylphenyl groups. The reactive groups were introduced
6
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via the N ꢀposition of the adenosine scaffold, since structureꢀactivity relationship data
as well as
the irreversible ligands p/mꢀDITCꢀADAC have shown that changes to this position are well
tolerated and have no detrimental effect on the affinity and selectivity at the A AR. Since the
3
1
1
formation of a covalent bond with chemoreactive irreversible probe is only possible by positioning
the chemoreactive functionality in close proximity to a nucleophilic amino acid residue, three series
of irreversible probes were synthesised incorporating linear linkers with 2ꢀ, 6ꢀ and 10ꢀcarbon in
length. In addition, given that many high affinity A
1
AR ligands have either an aromatic or another
6
35,
cyclic moiety at the N ꢀposition of the adenosine scaffold, an aromatic linker was also examined.
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ꢀ RESULTS AND DISCUSSION
Chemistry. The synthesis of the putative irreversible A AR agonists was based on a twoꢀstep
1
procedure; the attachment of a linker unit to the adenosine scaffold, followed by the introduction of
a chemoreactive group to the linker unit (Scheme 1). The linear linkers were introduced via a
substitution reaction of the respective diaminoalkane 2ꢀ4 with 6ꢀchloropurine riboside (1) in ethanol
and triethylamine. The resulting intermediates 5ꢀ7 were obtained in good yields (68ꢀ90%) after 3
hours at reflux. In the case of the 4ꢀaminobenzylamine linker (11), the more nucleophilic aliphatic
amino group was first protected with a Boc group under standard conditions. Next, the Bocꢀ
protected linker 12 was attached to the adenosine scaffold in 88% yield using the previously
described conditions for the substitution reaction with 6ꢀchloropurine riboside (13). The lower
nucleophilicity of this aryl amine contributed to the increased reaction time of 26 h compared to 3 h
for the alkyl linkers. The Boc protecting group was removed with trifluoroacetic acid in
dichloromethane to furnish 14, noting that it was important to avoid hydrochloric acid since it
initiated cleavage of the sugar moiety from the purine unit.
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The chemoreactive bicyclo[2.2.2]octaꢀ2,5ꢀdiene and 4ꢀfluorosulfonylphenyl moieties were
introduced using standard peptide bond coupling conditions (BOP and triethylamine). The
bicyclo[2.2.2]octaꢀ2,5ꢀdiene (8a-10a, 15a) analogs were obtained in reasonable yields ranging
between 11ꢀ42%, whereas the reactions with 4ꢀ(fluorosulfonyl)benzoic acid resulted in the
formation of several byꢀproducts which made the purification extremely challenging. Accordingly,
compounds 9d-10d and 15d were only isolated in very low yield (1ꢀ10%).
fluorosulfonylphenyl product with the diaminoethyl linker could not be isolated with high purity
>95%) due to interference from other side products. It is worth mentioning that the introduction of
The 4ꢀ
(
chemoreactive groups with acid halides instead of carboxylic acids failed due to side reactions on
the adenosine scaffold. Nevertheless, by simply changing the base from triethylamine to 2,6ꢀ
lutidine we were able significantly improve the yield of 15d from 1% to 68%, as no side products
were formed under this conditions and purification by column chromatography was not necessary.
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To afford the 1ꢀ(4ꢀisothiocyanatophenyl)thiourea (8b-10b, 15b) and isothiocyanate (8c-10c, 15c)
analogs, either 1,4ꢀphenylene diisothiocyanate or N,N'ꢀthiocarbonyldiimidazole was reacted with
the amine linker of intermediates 5-7 and 14 in dichloromethane at room temperature. In case of
the isothiocyanate analogs 8c-10c and 15c it was important to use only one equivalent of the
reagent to avoid the formation of further side products, while for the 1ꢀ(4ꢀ
isothiocyanatophenyl)thiourea analogs (8b-10b, 15b), an excess of reagent (3 equivalents) was used
without the earlier described implications. The yields of the synthesized isothiocyanates were
generally very modest, especially the two analogs with the 4ꢀaminobenzylamine linker (15b and
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5c) which were obtained in extremely poor yields, possibly due to the low solubility of
intermediate 14 in dichloromethane. The synthesis of 15b was optimized by using DMF as the
solvent to improve the conversion of the starting material to product as well as 1.1 equivalent of
reagent and a reaction concentration of 10 mg of starting material/mL of solvent to minimize the
formation of side products, including the dimer product. Preparative RPꢀHPLC was used for
purification avoiding silica gel column chromatography (normal phase), which contributed to the
degradation of 15b. The optimized protocol allowed the isolation of 15b in 17% yield (previously
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a
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Scheme 1. Synthesis of the adenosine A receptor irreversible agonists
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a
a
Reagents and conditions: (a) respective diaminoalkane, Et
3
N, EtOH, reflux, 3 h, 68ꢀ90%; (b )
b
bicyclo[2.2.2]octaꢀ2,5ꢀdieneꢀ2ꢀcarboxylic acid, BOP reagent, Et
3
N, DMF, rt, 20 h, 11ꢀ42%; (b )
c
1
,4ꢀphenylene diisothiocyanate, CH
Cl
2 2
, rt, 5ꢀ22 h, 18ꢀ35%; (b ) N,N'ꢀthiocarbonyldiimidazole,
d
CH
2
Cl
2
, rt, 43 h, 3ꢀ35%; (b ) 4ꢀ(fluorosulfonyl)benzoic acid, BOP reagent, Et
3
N, DMF, rt, 20 h, 8ꢀ
10%; (c) Boc anhydride, THF, rt, 5 h, 94%; (d) 12, Et
3
N, EtOH, reflux, 26 h, 88%; (e) TFA,
b
CH
2
Cl
2
, rt, 2 h, followed by 1 M NaOH, 70%; (f ) 1,4ꢀphenylene diisothiocyanate, DMF, rt, 2 h,
d
1
7%; (f ) 4ꢀ(fluorosulfonyl)benzoic acid, BOP reagent, 2,6ꢀlutidine, DMF, rt, 1 h, 68% .
Pharmacology. To assess the biological activity of the newly synthesised compounds, assays of
ERK1/2 phosphorylation (pERK1/2), a downstream signalling pathway linked to receptor
39
activation, were performed in FlpInCHO cells stably expressing the human A AR. No effects on
1
pERK1/2 were noted for any of the compounds in parental FlpInCHO cells not transfected with the
1
A AR (data not shown). Concentrationꢀresponse curves were constructed at the peakꢀresponse
time, identified from timeꢀcourse experiments as 5 min of agonist exposure. Analysis of
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concentrationꢀresponse curves allowed for the estimation of agonist potency and maximal pERK1/2
stimulated in the presence of each agonist (Table 1 and Figure 2). The maximal agonist response
was not significantly different from that of NECA for any of the compounds tested (p>0.05, oneꢀ
way ANOVA, Dunnett’s post hoc test), indicating that these ligands acted as full agonists of the
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A AR in the pERK1/2 assay. Despite most compounds being essentially equally effective, greater
variations were noted in their potencies. Only four compounds, 8b, 15a, 15b and 15d, exhibited
lowꢀnanomolar potencies that were not significantly different to NECA (Table 1 and Figure 2),
while the potencies of all other compounds were significantly lower (p<0.05, oneꢀway ANOVA,
Dunnett’s post hoc test). Interestingly, 8b, 15a, 15b and 15d all possess a rigid linker composed of
either an aromatic group (15a, 15b and 15d) or a short carbon chain (8b) between the adenosine
moiety and the irreversible reactive group. As such, results from the current series of compounds
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suggest compact chemical structures are beneficial for generating high potency A AR agonists.
Figure 2. Functional pERK1/2 concentrationꢀresponse curves to test compounds 15a, 15c, 15d and
NECA in A AR expressing FlpInCHO cells. Data points represent the mean and standard error of
three independent experiments performed in duplicate.
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Table 1: Effect of test compounds on ERK1/2 phosphorylation in human
A AR-expressing FlpIn CHO cells
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Maximal response
(% 1 M NECA)
Compound
pEC50
ꢀ
NECA
9.3 ± 0.4
NA
103 ± 2
NA
8
a
10
11
12
13
14
15
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17
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8b
8.5 ± 0.1
6.8 ± 0.1
7.8 ± 0.1
7.7 ± 0.1
8.2 ± 0.1
6.7 ± 0.2
7.6 ± 0.4
7.6 ± 0.1
7.1 ± 0.2
7.1 ± 0.2
8.8 ± 0.1
8.8 ± 0.1
7.7 ± 0.3
9.0 ± 0.1
82 ± 18
82 ± 9
93 ± 17
95 ± 5
*
***
8
c
****
9
a
*
*
*
***
9
b
*
9c
82± 13
75 ± 12
49 ± 23
87 ± 16
84 ± 21
119 ± 13
96 ± 14
113 ± 3
101 ± 18
119 ± 12
***
9
d
****
1
1
0a
0b
*
***
***
*
1
0c
****
1
1
0d
5a
15b
5c
5d
****
1
1
NA ꢀ not applicable; values represent the mean ± SEM from three independent
experiments performed in duplicate.
*
*
Significantly different (p≤0.001) compared to pEC50 or maximum response of
NECA (Oneꢀway ANOVA, Dunnett’s postꢀhoc test)
*
***
Significantly different (p≤0.0001) compared to pEC50 or maximum response of
NECA (Oneꢀway ANOVA, Dunnett’s postꢀhoc test)
1
The pERK1/2 assay identified all novel compounds to behave as agonists at the A AR, however
these experiments could not differentiate between reversible or irreversible receptor activators. To
address this, we next used a labelꢀfree xCELLigence assay capable of realꢀtime monitoring of
40
A
1
ARꢀmediated changes in cellular impedance over prolonged periods of time, including the
ability to determine whether activation of the receptor can be reversed by subsequent addition of an
TM
1 1
A AR antagonist. A AR expressing FlpInCHO cells were plated on an Eꢀplate one day before
the experiment and the change in impedance assessed for 1.5 h upon exposure to 1 ꢁM compound
or buffer as a control). The change in impedance in the presence of 1 ꢁM agonist or vehicle was
(
normalised within each experiment to the initial peak NECA response (5 min). The overall profile
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of the compound response over time was compared to that of the reference agonist, NECA (Figure
3
A). Similarly to NECA, each test compound stimulated a positive change in impedance relative to
the buffer control, further supporting the suggestion that these compounds promote A AR
1
activation. Of note, only 10c and 15b–15d promoted a maximal change in cellular impedance that
was not significantly different from that of NECA (oneꢀsample tꢀtest; Figure 3B). These results
suggest that within this assay, the remaining compounds were lower potency and/or partial agonists
relative to NECA.
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To probe for potential irreversibility of the agonistic effect of the test compounds, the sensitivity
of the cellular impedance response to the subsequent addition of a high concentration of A
1
AR
selective antagonist was assessed. After the cells were incubated with the compounds for 1.5 h, the
4
1
1
potent and selective A AR antagonist, SLV320 (16) was added at 1 ꢁM. Competition of 16 with
reversible ligands would be expected to result in a change (reduction) in impedance, whereas cells
incubated with irreversible compounds would not be expected to show a significant change in
impedance upon antagonist exposure. As expected, a significant decrease in cellular impedance
was observed upon antagonist addition to cells preꢀincubated with NECA (Figure 3C). Because the
maximal drop in NECA impedance values was observed 16 min after adding 16, all cellular
responses were normalised to those of NECA observed 16 min after antagonist addition. As shown
in Figure 3D, cells that had been exposed to 8b or 15b–15d were significantly resistant to reversal
of their effect on cellular impedance upon addition of 16 (p<0.05, oneꢀsample tꢀtest), consistent
with an irreversible mode of receptor activation.
Compounds containing
a
4ꢀ
fluorosulphonylbenzoyl moiety or an aryl isothocyanate (similar to that of DITCꢀADAC) appeared
to be the most successful in showing resistance to reversal by 16. Collectively, 15b and 15d
emerged as our most prominent compounds, because they were most potent and efficacious in the
pERK1/2 assay and could not be inhibited by 16 in the real time xCELLigence assay, suggesting
1
irreversible binding to the A AR.
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E
HO
NH
N
N
N
H
SLV320 (16)
Figure 3. Test for irreversible binding using a realꢀtime xCELLigence assay. (A) Changes in
cellular impedance over time caused by 1 ꢀM of 15a, 15d or NECA. Data points were normalised
to the NECA response observed at 5 min. (B) Normalised cell responses 5 min after ligand
addition. (C) Changes in impedance over time caused by the addition of 16 (1 ꢀM) to the cells preꢀ
treated with indicated compounds for 100 min. Data points were normalised to NECA response 16
min after 16 addition (indicated by the dashed line). (D) Normalised cell responses 16 min after 16
addition following preꢀtreatment with ligands from (B). Values represent the mean ± SEM from
*
**
***
****
three independent experiments performed in duplicate. p≤0.05,
p≤0.0001 (oneꢀsample tꢀtest, comparing means to 100%). (E) Chemical structure of 16.
p≤0.01,
p≤0.001,
To more directly validate the likelihood of an irreversible mode of binding to, and activation of,
the A AR by 15b and 15d, we performed additional biochemical assays. First, these compounds
1
were tested for their ability to covalently bind A ARs using equilibrium radioligand binding assays.
1
A
1
ARꢀFlpInCHO cell membranes were incubated with the compounds of interest for 1 h at 37°C,
3
and, after extensive washing, the specific binding of the orthosteric antagonist, [ H]DPCPX, was
determined (Figure 4A). As expected, following our washing protocol, membranes preꢀtreated with
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NECA bound [ H]DPCPX with a similar Bmax as membranes preꢀincubated with buffer (108 ± 11%
and 93 ± 10%, respectively; Figure 4A). In contrast, preꢀtreatment of membranes, followed by
42
extensive washout, with the irreversible A AR antagonist, FSCPX, prevented subsequent
1
3
[
H]DPCPX binding (2 ± 5% of the Bmax for membranes alone). Importantly, both 15b and 15d
0
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behaved similarly to FSCPX in preventing [ H]DPCPX binding after the same preꢀtreatment and
washout protocol (to 8 ± 4% and 16 ± 5%, respectively), indicating that these compounds remained
1
associated with the A AR after the extensive membrane washing procedure.
3
5
Figure 4. Radioligand (A) and [ S]GTPγS binding assays (B) on extensively washed membranes
following preꢀincubation with 10 ꢀM test compounds. In (B) membranes were stimulated with
buffer or 10 ꢀM NECA or 16 following preꢀincubation and washing procedure. Data points were
normalised to [ H]DPCPX Bmax (A) or 10 ꢀM NECAꢀinduced [ S]GTPγS binding (B) for
membranes preꢀincubated with buffer alone, then washed. Values represent the mean ± SEM from
three independent experiments performed in duplicates.
ANOVA, Dunnett’s post hoc, compare to [ S]GTPγS binding of membranes treated with NECA
following NECA preꢀincubation).
3
35
**
****
p≤0.001,
p≤0.0001 (Oneꢀway
35
Second, because formation of an irreversible bond could sometimes convert a compound
exhibiting agonist properties into an antagonist, e.g., as previously reported for acetylcholine
4
3
mustard at muscarinic acetylcholine receptors, we examined if 15b and 15d remained agonists
after the formation of a covalent bond, and, as such, promoted persistent A ARꢀmediated guanine
1
3
5
nucleotide exchange activity. To test this, we monitored the binding of [ S]GTPγS to activated G
3
proteins using the same A ARꢀFlpInCHO cell membranes as used in the [ H]DPCPX binding assay.
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After membranes were preꢀtreated with test compounds and washed extensively, they were
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stimulated with reaction buffer, NECA, or 16 (Figure 4B) and [ S]GTPγS incorporation was
35
measured. The amount of incorporated [ S]GTPγS was normalised to the response of control
membranes preꢀincubated with vehicle, subjected to the same washing protocol and then stimulated
with NECA (defined as 100%). As expected, membranes preꢀincubated with NECA followed by
washout responded to stimulation in the same way as vehicleꢀtreated membranes. The basal
response, measured in the absence of drug (41 ± 3% and 57 ± 9%, for the control and NECA preꢀ
treatments, respectively), could still be further increased by subsequent addition of 10 ꢁM NECA,
but not 10 ꢁM compound 16 (99 ± 7% and 37 ± 5%, respectively). Membranes preꢀtreated with
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FSCPX showed similar basal levels of [ S]GTPγS binding compared to vehicle controls (37 ± 6%),
but were completely unresponsive to NECA stimulation (32 ± 4%), as expected for an irreversible
35
antagonist occupying the A AR orthosteric site. In contrast, the basal level of [ S]GTPγS binding
1
in membranes preꢀtreated with 15b or 15d, followed by washout, was similar to that of control
samples exposed to 10 ꢁM NECA (124 ± 7 and 134 ± 13 %, respectively), indicating persistent and
maximal activation. Moreover, neither addition of NECA or 16 had any effect on this persistent
activity in 15b or 15d preꢀtreated membranes, indicating that the persistent effect is likely due to
irreversible binding.
Interestingly, evidence gathered from multiple GPCR crystallography studies over recent years
suggests a strong correlation between the ability of a ligand to stabilize a receptor and the ability to
44
form well diffracting crystals. In addition, receptor stabilisation by different classes of ligand
23, 45
could be very beneficial in assisting the discovery of conformationꢀspecific antibodies.
Thus,
to determine if our best compounds, 15b and 15d, enhanced A AR thermoꢀstability, we utilized a
1
4
6, 47
ThermoFluor assay
using purified A
1
ARs; as shown in the Supporting Information, the purified
AR was
receptor retained its native pharmacological properties. In the absence of drugs, the A
1
extremely unstable, as evidenced by high fluorescence at the start of the melting curve and a
shallow slope of the transition (Figure 5A). All tested compounds stabilised the A AR to some
1
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1 m
degree (Figure 5B). For instance, NECA modestly increased apoꢀA AR melting temperature (T )
by 4° (35.2 ± 1.6 and 38.7 ± 0.4°C, respectively), as did 8b, 9b, 9d, 10c and 15a. However, only
three compounds, 15b–15d significantly increased the receptor T substantially above the effect of
m
NECA (p<0.05, oneꢀway ANOVA, Dunnett’s post hoc test), to 46.6 ± 1.2, 42.7 ± 0.3 and 46.7 ±
0
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.7°C, respectively, highlighting the importance of an aromatic linker between the adenosine
moiety and an irreversible group ꢀ not only for achieving high agonist potency, but also for receptor
stabilisation; only FSCPX yielded a greater degree of thermoꢀstabilization. While only the binding
of G proteins or G proteinꢀmimicking nanobodies can achieve close to full activation of the
2
receptor, recent studies of the β adrenergic receptor activation using molecular dynamics
19
simulations, Fꢀfluorine NMR and double electronꢀelectron resonance spectroscopy have revealed
that there are multiple intermediate steps, and that these can indeed be stabilized by pseudoꢀ
48, 49
irreversible ligands.
2
Thus, it is reasonable to suggest that, similar to the β adrenergic receptor,
binding of our irreversible agonists, 15b or 15d, to the A AR increases the population of activeꢀlike
1
intermediate states, which explains the increase in observed thermostability and identifies these
compounds as useful tools for future structural biology studies of this important GPCR.
Figure 5. Test compounds induce A
curves were monitored by the increase in CPM dye fluorescence due to receptor unfolding. 10 ꢀM
of NECA and test compounds increased receptor melting temperature (T ), producing a rightꢀshift
1 1
AR stabilisation in a ThermoFluor assay. (A) A AR melting
m
m 1
of the melting curves. (B) T values of A AR in the presence of 10 ꢀM of test compounds. Data
points represent the mean ± SEM from three independent experiments performed in duplicates.
*
****
p≤0.05,
m
p≤0.0001 (Oneꢀway ANOVA, Dunnett’s post hoc test, compared to T in the presence
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of NECA).
Finally, albeit not the major focus of the current study, we selected the three key irreversible
agonists that improved the thermostability of the A AR and assessed their activity at the other AR
subtypes (A2A, A2B and A ). For this purpose, we utilized a common downstream pathway linked to
1
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3
1
activation of all AR subtypes, namely cAMP accumulation, which is inhibited by the A and
3
A
3
ARs, but stimulated by the A2A and A2B ARs . As shown in Figure 6, all three compounds were
full agonists in this assay when compared to the reference agonist, NECA; no responses to any
compound were noted in parental FlpInCHO cells that did not express any AR subtype (not shown).
Despite displaying similar degrees of maximal agonist effect, differences in potencies were noted
between the compounds. In particular, 15d showed a significantly greater selectivity for the A
than any of the other subtypes, whereas 15b and 15c showed similar potencies across all subtypes
Table 2). Although beyond the scope of the current study, this finding suggests that these
1
AR
(
compounds can also be used for structural or chemical biology studies at other AR subtypes if an
irreversible mode of interaction can be validated at those subtypes. In contrast, the high selectivity
of 15d for the A
targeting the A
mixture of ARs.
1
AR identifies this compound as a novel tool that can be used specifically for
1
AR over other subtypes, perhaps even in native tissues or cells that express a
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Figure 6. Adenosine receptor subtype selectivity of selected agonists. Functional cAMP
concentrationꢀresponse curves to NECA or test compounds 15b, 15c or 15d at the indicated AR
subtype stably expressed in FlpInCHO cells. Data points represent the mean ± SEM of three to four
independent experiments performed in duplicate.
Table 2: AR receptor subtype potency (pEC50) values of selected test compounds on
cAMP accumulation or inhibition in human AR-expressing FlpIn CHO cells
Compound
AR Subtype
A
1
A
2A
A
2B
A
3
NECA
8.79 ± 0.09
7.26 ± 0.09
6.57 ± 0.11
9.04 ± 0.12**
8.60 ± 0.09
7.17 ± 0.11
6.19 ± 0.13
7.17 ± 0.09
7.37 ± 0.32*
6.65 ± 0.32
6.15 ± 0.32
6.93 ± 0.24
7.80 ± 0.15
7.35 ± 0.15
6.46 ± 0.17
6.87 ± 0.14
1
5b
5c
5d
1
1
Values represent the mean ± SEM from three to four independent experiments performed in
duplicates.
*
1
Significantly different (p < 0.05) from pEC50 value at the A and A2A ARs.
**Significantly different (p < 0.01) from pEC50 value at A2A, A2B and A
3
ARs. (Oneꢀway
ANOVA, Tukey’s postꢀhoc test)
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ꢀ
CONCLUSIONS
We synthesized and evaluated a series of novel adenosine analogs bearing electrophilic substituents,
aiming to identify novel agonists capable of irreversible binding and persistent activation the A AR.
Four compounds, 8b, 15a–15b and 15d, showed similar potency and efficacy to our reference
reversible) agonist, NECA, in a pERK1/2 assay. Sensitivity to antagonist addition in a realꢀtime,
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(
labelꢀfree xCELLigence assay was subsequently used to identify compounds that likely mediated
their agonism through an irreversible interaction with the A AR. Compounds 8b and 15b–15d
1
were insensitive to the addition of a high concentration of antagonist, compound 16, indicating
pseudoꢀ or complete irreversibility. Two compounds, 15b and 15d, which emerged as most
promising from both the pERK1/2 and xCELLigence assays, were more directly validated as
3
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1
irreversible agonists of the A AR using membraneꢀbased [ H]DPCPX and [ S]GTPγS binding
experiments. Both ligands bound tightly to A ARs in FlpInCHO cell membranes and could not be
1
3
displaced even after extensive washing, as evidenced by the inability of [ H]DPCPX to bind to
3
5
these membranes, and the persistent maximal binding of [ S]GTPγS to activated G proteins in a
manner resistant to both agonist or antagonist addition. To our knowledge, no A AR agonists have
1
been examined for the ability to promote receptor thermostability to date, although such a property
could prove extremely beneficial for structural biology studies. Thus, we also found that both 15b
and 15d stabilised purified, detergentꢀsolubilised A
1
ARs in a ThermoFluor assay to a significantly
AR
higher degree than NECA. Compound 15d also shows a high degree of selectivity for the A
1
subtype relative to other ARs, and thus may be prove useful for additional chemical biology
applications in native tissues or cells that express mixtures of AR subtypes.
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ꢀ
EXPERIMENTAL SECTION
Chemistry. Chemicals and solvents were purchased from standard suppliers and used without
®
further purification. Davisil silica gel (40ꢀ63 µm) for flash column chromatography was supplied
0
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by Grace Davison Discovery Sciences (Victoria, Australia) and deuterated solvents were purchased
from Cambridge Isotope Laboratories, Inc. (USA, distributed by Novachem PTY. Ltd, Victoria,
Australia).
Unless otherwise stated, reactions were carried out at ambient temperature. Reactions were
monitored by thin layer chromatography on commercially available preꢀcoated aluminiumꢀbacked
plates (Merck Kieselgel 60 F254). Visualisation was by examination under UV light (254 and 366
nm). All organic extracts collected after aqueous workꢀup procedures were dried over anhydrous
4 2 4
MgSO or Na SO before gravity filtering and evaporation to dryness. Organic solvents were
evaporated in vacuo at ≤ 40°C (water bath temperature). Purification using preparative layer
chromatography (PLC) was carried out on Analtech preparative TLC plates (200 mm x 200 mm x 2
mm).
1
13
H NMR and C NMR spectra were recorded on a Bruker Avance Nanobay III 400MHz
Ultrashield Plus spectrometer at 400.13 MHz and 100.62 MHz, respectively. Chemical shifts (δ)
are recorded in parts per million (ppm) with reference to the chemical shift of the deuterated
solvent. Coupling constants (J) and carbonꢀfluorine coupling constants (JCF) are recorded in Hz
and the significant multiplicities described by singlet (s), doublet (d), triplet (t), quadruplet (q),
broad (br), multiplet (m), doublet of doublets (dd), doublet of triplets (dt).
Preparative HPLC was performed using an Agilent 1260 infinity coupled with a binary
preparative pump and Agilent 1260 FCꢀPS fraction collector, using Agilent OpenLAB CDS
software (Rev C.01.04), and an Altima 5µM C8 22 x 250 mm column. The following buffers were
used; buffer A: H
2
O; buffer B: MeCN, with sample being run at a gradient of 30% buffer B to
1
00% buffer B over 10 min, at a flow rate of 20 mL/min.
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LCMS were run to verify reaction outcome and purity using an Agilent 6120 Series Single Quad
coupled to an Agilent 1260 Series HPLC. Unless stated otherwise, the compounds which
underwent pharmacological evaluation were >95% pure. The following buffers were used; buffer
10
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2
A: 0.1% formic acid in H O; buffer B: 0.1% formic acid in MeCN. The following gradient was
used with a Poroshell 120 ECꢀC18 50 x 3.0 mm 2.7 micron column, and a flow rate of 0.5 mL/min
and total run time of 5 min; 0–1 min 95% buffer A and 5% buffer B, from 1ꢀ2.5 min up to 0%
buffer A and 100% buffer B, held at this composition until 3.8 min, 3.8–4 min 95% buffer A and
5
% buffer B, held until 5 min at this composition. Mass spectra were acquired in positive and
negative ion mode with a scan range of 100–1000 m/z. UV detection was carried out at 214 and
2
54 nm. All retention times (
R
t ) are quoted in min. HRMS analyses were carried out on an Agilent
6
224 TOF LC/MS Mass Spectrometer coupled to an Agilent 1290 Infinity (Agilent, Palo Alto, CA).
All data were acquired and reference mass corrected via a dualꢀspray electrospray ionisation (ESI)
source. Acquisition was performed using the Agilent Mass Hunter Data Acquisition software
version B.05.00 Build 5.0.5042.2 and analysis was performed using Mass Hunter Qualitative
Analysis version B.05.00 Build 5.0.519.13. Melting points were measured on a Gallenkamp
Melting Point Apparatus.
6
General procedure A: N -Substituted adenosines. 6ꢀChloropurine riboside (1) (1.0 eq),
diaminoalkane (5.0 eq) and triethylamine (1.0 eq) were refluxed in ethanol (100 mL/ 2.5 g of the 6ꢀ
2
chloropurine riboside) for 3 h under N . Upon cooling to rt, a white solid precipitated was formed.
The suspension was filtered, washed with cold ethanol and dried under high vacuum to afford the
title product as a white powder.
General procedure B: Bicyclo[2.2.2]octa-2,5-diene irreversible ligands. Bicyclo[2.2.2]octaꢀ
2,5ꢀdieneꢀ2ꢀcarboxylic acid (1.50 eq), the respective amine (1.00 eq), BOP reagent (2.00 eq) and
triethylamine (4.00 eq) were dissolved in DMF (3 mL/ 100 mg of the amine). The mixture was
stirred at rt for 24 h. The mixture was diluted with water (50 mL) and extracted with ethyl acetate
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(2 × 50 mL). The combined organic layers were washed with water (2 × 50 mL) and brine (50 mL),
dried with Na SO , filtered, and concentrated under reduced pressure. The crude material was
2
4
purified by column chromatography (CH
General procedure C: 1-(4-isothiocyanatophenyl)thiourea irreversible ligands. 1,4ꢀ
Phenylene diisothiocyanate (3.0 eq) dissolved in CH Cl (3 mL) was dropwise added to a mixture
2 2
Cl
: MeOH 100:0 → 90:10) to yield the desired product.
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
2
2
the respective amine (1.0 eq) in CH
2
Cl
2
(5 mL/ 100 mg of the amine). The reaction mixture was
®
stirred at rt under nitrogen for 5ꢀ22 h. The reaction mixture was absorbed on Celite and purified
via column chromatography to obtain the title compound.
General procedure D: Isothiocyanate irreversible ligands.
N,N'ꢀThiocarbonyldiimidazole
(
TCDI) (1.00 eq) dissolved in CH Cl (2 mL) was dropwise added to a mixture of the respective
2
2
amine (1.00 eq) and CH
2
Cl
2
(5 mL/ per 100 mg of the amine). The reaction mixture was stirred for
®
4
2 h at rt under nitrogen. The reaction mixture was absorbed on Celite and purified via column
chromatography to obtain the title compound.
General procedure E: 4-Fluorosulfonylphenyl
irreversible
ligands.
4ꢀ
(Fluorosulfonyl)benzoic acid (1.50 eq), the respective amine (1.00 eq), BOP reagent (2.00 eq) and
triethylamine (4.00 eq) were dissolved in DMF (3 mL/ 100 mg of the amine). The mixture was
stirred at rt for 6 h. The mixture was diluted with water (50 mL) and extracted with ethyl acetate (2
×
50 mL). The combined organic layers were washed with water (2 × 50 mL) and brine (50 mL),
dried with Na SO , filtered, and concentrated under reduced pressure. The crude material was
purified by column chromatography to yield the title product.
2
4
(2R
,3
R
,4
S,5
R
)ꢀ2ꢀ(6ꢀ((2ꢀAminoethyl)amino)ꢀ9
H
ꢀpurinꢀ9ꢀyl)ꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ
1
3,4ꢀdiol (5). General procedure A. White powder (735 mg, 68%). H NMR (d6ꢀDMSO) δ 8.36 (d,
J
= 5.7 Hz, 1H), 8.21 (s, 1H), 7.82 (br s, 1H), 5.88 (d, J = 6.2 Hz, 1H), 5.70–5.00 (m, 2H), 4.69–
4
.56 (m, 1H), 4.15 (dd,
J
= 4.9, 3.0 Hz, 1H), 3.99–3.95 (m, 1H), 3.68 (dd, J = 12.1, 3.6 Hz, 1H),
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3.55 (dd,
J
= 12.1, 3.6 Hz, 1H), 3.50–3.43 (m, 2H), 3.32 (br s, 3H), 2.75 (t, J = 6.6 Hz, 2H); mp: 90ꢀ
+
+
9
2 °C; m/z MS (TOF ES ) 311.1 [M+H] ; LCꢀMS tR: 0.45.
(2
R
,3
R
,4
S,5
R
)ꢀ2ꢀ(6ꢀ((6ꢀAminohexyl)amino)ꢀ9
H
ꢀpurinꢀ9ꢀyl)ꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ
1
10
11
12
13
14
15
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17
18
19
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49
50
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53
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55
56
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59
60
3,4ꢀdiol (6). General procedure A. White powder (6.04 g, 90%). H NMR (
d
ꢀDMSO) δ 8.34 (s,
6
1
J
1
H), 8.21 (s, 1H), 7.90 (br s, 1H), 5.88 (d,
= 5.5, 5.5 Hz, 1H), 4.15 (dd, = 4.8, 3.0 Hz, 1H), 4.00–3.94 (m, 1H), 3.68 (dd,
H), 3.55 (dd, = 12.1, 3.5 Hz, 1H), 3.50 – 3.42 (m, 2H), 3.33 (br s, 3H), 2.50–2.46 (m, 2H), 1.65–
J
= 6.2 Hz, 1H), 5.46 (br s, 1H), 5.23 (br s, 1H), 4.62 (dd,
J
J
= 12.1, 3.6 Hz,
J
+
+
R
1.53 (m, 2H), 1.38–1.24 (m, 6H); mp: 163ꢀ166 °C; m/z MS (TOF ES ) 367.3 [M+H] ; LCꢀMS t :
2
.71.
(2
R
,3
R
,4
S,5
R
)ꢀ2ꢀ(6ꢀ((10ꢀAminodecyl)amino)ꢀ9
Hꢀpurinꢀ9ꢀyl)ꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ
1
3
,4ꢀdiol (7). General procedure A. White powder (1.26 g, 86%). H NMR (
d
6
ꢀDMSO) δ 8.34 (s,
= 5.5, 5.5
= 12.1, 3.5 Hz, 1H), 3.55
1
H), 8.20 (s, 1H), 7.89 (br s, 1H), 5.88 (d,
J
= 6.2 Hz, 1H), 5.70–4.80 (m, 2H), 4.61 (dd,
J
Hz, 1H), 4.15 (dd,
J
= 4.7, 3.1 Hz, 1H), 3.99–3.95 (m, 1H), 3.68 (dd,
J
(dd, J = 12.1, 3.5 Hz, 1H), 3.51–3.40 (m, 2H), 3.30 (br s, 3H), 2.50–2.45 (m, 2H), 1.66–1.50 (m,
+
+
2
H), 1.26 (d,
.75.
R
J = 22.1 Hz, 14H); mp: 153ꢀ156 °C; m/z MS (TOF ES ) 423.3 [M+H] ; LCꢀMS t :
2
Nꢀ(2ꢀ((9ꢀ((2R,3R,4S,5R)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
yl)amino)ethyl)bicyclo[2.2.2]octaꢀ2,5ꢀdieneꢀ2ꢀcarboxamide (8a). General procedure B. A second
silica column chromatography (EtOAc: MeOH 100:0 → 80:20) was performed to yield the desired
1
product as a white resin (15 mg, 11%). H NMR (
d
6
ꢀDMSO) δ 8.37 (s, 1H), 8.22 (s, 1H), 7.99–7.90
= 6.1 Hz, 1H), 5.45 (d, = 6.2 Hz, 1H),
= 4.7 Hz, 1H), 4.61 (dd, = 11.2, 6.1 Hz, 1H), 4.17–4.13
= 6.6, 3.4 Hz, 1H), 3.79–3.64 (m, 2H), 3.62–3.52 (m, 3H),
(
m, 2H), 6.96–6.92 (m, 1H), 6.37–6.25 (m, 2H), 5.89 (d,
5.41 (dd, = 7.2, 4.5 Hz, 1H), 5.20 (d,
m, 1H), 4.12–4.08 (m, 1H), 3.97 (dd,
J
J
J
J
J
(
J
+
+
3
ꢀ
R
.44–3.38 (m, 2H), 1.27–1.10 (m, 4H); m/z MS (TOF ES ) 443.1 [M+H] ; LCꢀMS t : 2.85; HRMS
+
C
21
H
27
6
N O
5
[M+H] calcd 443.2043; found 443.2046.
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1ꢀ(2ꢀ((9ꢀ((2
R
,3
R
,4
S
,5
R
)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
yl)amino)ethyl)ꢀ3ꢀ(4ꢀisothiocyanatophenyl)thiourea (8b). General procedure C. The purification
was performed via column chromatography (CH
2
Cl
2
: MeOH 100:0 → 80:20). The title compound
ꢀDMSO) δ 9.77 ( br s, 1H), 8.38 (s, 1H),
= 6.1 Hz,
= 4.7 Hz, 1H), 4.61 (dd,
J = 6.5, 3.3 Hz, 1H), 3.84–3.63 (m, 5H), 3.61–3.51
1
was obtained as a white solid (38 mg, 24%). H NMR (
d
6
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
8
.17 (s, 1H), 8.13 – 7.92 (m, 2H), 7.57 – 7.47 (m, 2H), 7.40 – 7.31 (m, 2H), 5.90 (d,
1H), 5.44 (d, = 6.3 Hz, 1H), 5.39 (dd, = 7.0, 4.6 Hz, 1H), 5.20 (d,
1.3, 6.0 Hz, 1H), 4.18–4.12 (m, 1H), 3.97 (dd,
J
J
J
J
J =
1
+
+
(
R
m, 1H); mp: 165ꢀ170 °C; m/z MS (TOF ES ) 503.1 [M+H] ; LCꢀMS t : 3.03; HRMS ꢀ
+
C
20
H
23
N
8
O
4
S
2
[M+H] calcd 503.1284; found 503.1287.
(2
R
,3
S,4
R,5
R
)ꢀ2ꢀ(Hydroxymethyl)ꢀ5ꢀ(6ꢀ((2ꢀisothiocyanatoethyl)amino)ꢀ9
H
ꢀpurinꢀ9ꢀ
yl)tetrahydrofuranꢀ3,4ꢀdiol (8c). General procedure D. The purification was performed twice via
column chromatography (CH Cl : MeOH 100:0 → 8:2). The title compound was obtained as a
2
2
1
white solid (17 mg, 15%). H NMR (CD
3
OD) δ 8.75 (s, 1H), 8.69 (s, 1H), 6.16 (d,
J = 5.6 Hz, 1H),
4
.77 (t,
J
= 5.3 Hz, 1H), 4.59–4.51 (m, 2H), 4.39 (dd, = 5.0, 3.7 Hz, 1H), 4.19 (m,
J
J
= 6.4, 3.1 Hz,
+
+
1
H), 3.92 (dd,
J
= 12.4, 2.9 Hz, 1H), 3.85–3.76 (m, 3H); m/z MS (TOF ES ) 353.1 [M+H] ; LCꢀMS
+
t
R
: 0.79; HRMS ꢀ C13
H
17
N
6
O
4
S [M+H] calcd 353.1032; found 353.1030.
Nꢀ(6ꢀ((9ꢀ((2 ,3 ,4
R
R
S
,5
R
)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9
Hꢀpurinꢀ6ꢀ
yl)amino)hexyl)bicyclo[2.2.2]octaꢀ2,5ꢀdieneꢀ2ꢀcarboxamide (9a). General procedure B. White
1
solid (53 mg, 39%). H NMR (
d
6
ꢀDMSO) δ 8.34 (s, 1H), 8.20 (br s, 1H), 7.87 (br s, 1H), 7.75 (br t,
= 6.2 Hz, 1H), 5.50–5.40 (m,
= 11.2, 5.8 Hz, 1H), 4.18–4.13 (m, 1H), 4.12–4.08 (m,
J
2
1
2
= 5.5 Hz, 1H), 6.97–6.87 (m, 1H), 6.36–6.26 (m, 2H), 5.88 (d,
J
H), 5.18 (d, = 4.6 Hz, 1H), 4.62 (dd,
J
J
H), 4.00–3.95 (m, 1H), 3.73–3.64 (m, 2H), 3.60–3.53 (m, 1H), 3.51–3.39 (m, 2H), 3.15–3.02 (m,
H), 1.66–1.54 (m, 2H), 1.51–1.38 (m, 2H), 1.36–1.09 (m, 8H); mp: 108ꢀ111 °C; m/z MS (TOF
+
+
+
ES ) 499.3 [M+H] ; LCꢀMS
R 35 6 5
t : 2.98; HRMS ꢀ C25H N O [M+H] calcd 499.2669; found
4
99.2673.
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1ꢀ(6ꢀ((9ꢀ((2
R
,3
R
,4
S
,5
R
)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
yl)amino)hexyl)ꢀ3ꢀ(4ꢀisothiocyanatophenyl)thiourea (9b). General procedure C. The purification
was performed via column chromatography (CH
2
Cl
2
: MeOH 100:0 → 90:10). The title compound
ꢀDMSO) δ 9.62 (br s, 1H), 8.34 (s, 1H),
= 6.2 Hz, 1H),
= 11.3, 6.1 Hz, 1H), 4.18–4.11 (m, 1H),
= 6.5, 3.3 Hz, 1H), 3.74–3.65 (m, 1H), 3.63–3.53 (m, 1H), 3.53–3.38 (m, 4H), 1.69–1.46
1
was obtained as a white solid (80 mg, 35%). H NMR (
d
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
.21 (br s, 1H), 8.00–7.83 (m, 2H), 7.60–7.50 (m, 2H), 7.44–7.29 (m, 2H), 5.88 (d,
5.48–5.40 (m, 2H), 5.20 (d, = 4.6 Hz, 1H), 4.62 (dd,
.97 (dd,
J
J
J
3
J
+
+
(
R
m, 4H), 1.42–1.27 (m, 4H); mp: 131ꢀ135 °C; m/z MS (TOF ES ) 559.2 [M+H] ; LCꢀMS t : 3.14;
+
HRMS ꢀ C24
,3 ,4
yl)tetrahydrofuranꢀ3,4ꢀdiol (9c). General procedure D. The purification was performed via column
chromatography (CH Cl : MeOH 100:0 → 80:20). The title compound was obtained as a white
H
31
N
8
O
4
S
2
[M+H] calcd 559.1910; found 559.1913.
(2R S R,5R)ꢀ2ꢀ(Hydroxymethyl)ꢀ5ꢀ(6ꢀ((6ꢀisothiocyanatohexyl)amino)ꢀ9Hꢀpurinꢀ9ꢀ
2
2
1
solid (10 mg, 9%). H NMR (CD
dd, = 6.2, 5.3 Hz, 1H), 4.22 (dd,
Hz, 1H), 3.64 (dd, = 12.6, 2.5 Hz, 1H), 3.59–3.40 (m, 4H), 1.68–1.53 (m, 4H), 1.46–1.32 (m, 4H);
3
OD) δ 8.15 (s, 1H), 8.12 (br s, 1H), 5.85 (d,
J
= 6.5 Hz, 1H), 4.64
(
J
J
= 5.1, 2.4 Hz, 1H), 4.12–4.03 (m, 1H), 3.79 (dd,
J
= 12.6, 2.4
J
+
+
+
m/z MS (TOF ES ) 409.1 [M+H] ; LCꢀMS
R 25 6 4
t : 3.04; HRMS ꢀ C17H N O S [M+H] calcd
409.1658; found 409.1663.
4ꢀ((6ꢀ((9ꢀ((2
R,3R,4S,5R)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
yl)amino)hexyl)carbamoyl)benzenesulfonyl fluoride (9d). General procedure E. The purification
was performed via column chromatography (CH
2
Cl
2
: methanol 100:0 → 8:2) and (EtOAc: MeOH
1
1
00:0 → 80:20). The title compound was obtained as a white solid (50 mg, 10%). H NMR (d6
ꢀ
DMSO) δ 8.85 (br t,
J
= 5.5 Hz, 1H), 8.34 (s, 1H), 8.28–8.23 (m, 2H), 8.22–8.13 (m, 3H), 7.95–
= 6.2 Hz, 1H), 5.47–5.41 (m, 2H), 5.19 (d, = 4.6 Hz, 1H), 4.61 (dd,
11.3, 6.1 Hz, 1H), 4.17–4.12 (m, 1H), 3.97 (dd, = 6.5, 3.4 Hz, 1H), 3.71–3.63 (m, 1H), 3.59–3.51
m, 1H), 3.60–3.51 (m 2H), 3.28 (dd, = 12.9, 6.7 Hz, 2H), 1.67–1.48 (m, 4H), 1.42–1.30 (m, 4H);
7
.88 (m, 1H), 5.88 (d,
J
J
J =
J
(
J
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4
4
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5
5
5
5
5
5
5
5
5
5
6
+
+
+
m/z MS (TOF ES ) 553.2 [M+H] ; LCꢀMS
t
R
: 3.33; HRMS ꢀ C23
H
30FN
6
O
7
S [M+H] calcd
5
53.1881; found 553.1888.
ꢀ(10ꢀ((9ꢀ((2R,3R,4S,5R)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
N
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
yl)amino)decyl)bicyclo[2.2.2]octaꢀ2,5ꢀdieneꢀ2ꢀcarboxamide (10a). General procedure B. White
1
solid (53 mg, 34%). H NMR (
d
6
ꢀDMSO) δ 8.34 (s, 1H), 8.20 (br s, 1H), 7.94–7.84 (m, 1H), 7.75
= 6.3, 1.7 Hz, 1H), 6.38–6.25 (m, 2H), 5.88 (d, = 6.2 Hz, 1H),
= 4.6 Hz, 1H), 4.61 (dd, = 11.3, 6.0 Hz, 1H), 4.16–4.12 (m, 1H),
4.12–4.07 (m, 1H), 4.01–3.92 (m, 1H), 3.74–3.63 (m, 2H), 3.60–3.51 (m, 1H), 3.50–3.39 (m, 2H),
.14–2.99 (m, 2H), 1.64–1.52 (m, 2H), 1.46–1.36 (m, 2H), 1.35–1.10 (m, 16H); mp: 88ꢀ90 °C; m/z
(br t,
J
= 5.5 Hz, 1H), 6.91 (dd,
J
J
5
.49–5.39 (m, 2H), 5.19 (d,
J
J
3
+
+
+
MS (TOF ES ) 555.3 [M+H] ; LCꢀMS
R 43 6 5
t : 3.15; HRMS ꢀ C29H N O [M+H] calcd 555.3296;
found 555.3301.
1ꢀ(10ꢀ((9ꢀ((2
R,3R,4S,5R)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
yl)amino)decyl)ꢀ3ꢀ(4ꢀisothiocyanatophenyl)thiourea (10b). General procedure C. The purification
was performed twice via column chromatography (CH
2
Cl
: MeOH 100:0 → 80:20). The title
2
1
compound was obtained as a white solid (26 mg, 18%). H NMR (
d
6
ꢀDMSO) δ 9.64 (br s, 1H),
8
.34 (s, 1H), 8.20 (br s, 1H), 7.99–7.80 (m, 2H), 7.58–7.51 (m, 2H), 7.41–7.33 (m, 2H), 5.88 (d,
.2 Hz, 1H), 5.47–5.40 (m, 2H), 5.19 (d, = 4.6 Hz, 1H), 4.61 (dd, = 11.3, 6.0 Hz, 1H), 4.17–4.12
= 6.5, 3.4 Hz, 1H), 3.72–3.63 (m, 1H), 3.63–3.52 (m, 1H), 3.51–3.39 (m, 4H),
J =
6
J
J
(m, 1H), 3.97 (dd,
J
+
+
1.66–1.46 (m, 4H), 1.39–1.22 (m, 12H); mp: 88ꢀ93 °C; m/z MS (TOF ES ) 615.2 [M+H] ; LCꢀMS
+
t
R
: 3.33; HRMS ꢀ C28
,3 ,4 ,5
yl)tetrahydrofuranꢀ3,4ꢀdiol (10c). General procedure D. The purification was performed twice via
H
39
N
8
O
S
4 2
[M+H] calcd 615.2536; found 615.2566.
(2R S R R)ꢀ2ꢀ(Hydroxymethyl)ꢀ5ꢀ(6ꢀ((10ꢀisothiocyanatodecyl)amino)ꢀ9Hꢀpurinꢀ9ꢀ
column chromatography (CH
2
Cl
2
: MeOH 100:0 → 80:20). The title compound was obtained as a
OD) δ 8.27 (s, 1H), 8.23 (br s, 1H), 5.97 (d, = 6.5 Hz,
= 5.1, 2.4 Hz, 1H), 4.19 (dd, = 5.0,
1
white solid (53 mg, 35%). H NMR (CD
3
J
1
H), 4.76 (dd,
J
= 6.5, 5.1 Hz, 1H), 4.62 (br s, 1H), 4.34 (dd,
J
J
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2.5 Hz, 1H), 3.91 (dd,
J
= 12.6, 2.4 Hz, 1H), 3.76 (dd,
J = 12.6, 2.6 Hz, 1H), 3.64–3.57 (m, 1H),
3
.56 (t, J = 6.5 Hz, 2H), 1.77–1.64 (m, 4H), 1.51–1.30 (m, 12H); mp: 111ꢀ113 °C; m/z MS (TOF
+
+
+
ES ) 456.2 [M+H] ; LCꢀMS
t
R
: 3.29; HRMS ꢀ C21
H
33
N
6
O
4
S [M+H] calcd 456.2284; found
4
56.2287.
ꢀ((10ꢀ((9ꢀ((2
yl)amino)decyl)carbamoyl)benzenesulfonyl fluoride (10d). General procedure E. The purification
was performed via column chromatography (CH Cl : MeOH 100:0 → 80:20) and (EtOAc: MeOH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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46
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49
50
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54
55
56
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59
60
4
R,3R,4S,5R)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
2
2
1
100:0 → 80:20). The title compound was obtained as a white solid (11 mg, 8%). H NMR (
DMSO) δ 8.84 (br t, = 5.5 Hz, 1H), 8.34 (s, 1H), 8.28–8.23 (m, 2H), 8.23–8.13 (m, 3H), 7.93–
= 6.2 Hz, 1H), 5.49–5.39 (m, 2H), 5.19 (d, = 4.6 Hz, 1H), 4.61 (dd,
1.3, 6.1 Hz, 1H), 4.16–4.12 (m, 1H), 3.97 (dd, = 6.5, 3.4 Hz, 1H), 3.75–3.62 (m, 1H), 3.62–3.52
6
d ꢀ
J
7
.85 (m, 1H), 5.88 (d,
J
J
J =
1
J
(m, 1H), 3.50–3.40 (m, 2H), 3.31–3.22 (m, 2H), 1.67–1.49 (m, 4H), 1.34–1.22 (m, 12H); m/z MS
+
+
+
R 6 7
(TOF ES ) 609.2 [M+H] ; LCꢀMS t : 3.19; HRMS ꢀ C27H38FN O S [M+H] calcd 609.2507; found
6
09.2517.
tertꢀButyl (4ꢀaminobenzyl)carbamate (12). A solution of diꢀtertꢀbutyl dicarbonate (3.75 g,
7.2 mmol, 1.05 eq) in THF (30 mL) was added to a solution 4ꢀaminobenzylamine (11) (2.00 g,
6.4 mmol, 1.00 eq) in THF (50 mL). The reaction was stirred at rt for 5 h. Ethyl acetate (400 mL)
1
1
was added and the organic layer was washed with 1 M HCl (100 mL), 1 M NaOH (100 mL), sat.
NaHCO (100 mL) and brine (100 mL). The water layer was extracted with ethyl acetate (3 x 100
mL). The combined organic layers were dried with Na SO , filtered and the solvent was removed
3
2
4
under reduced pressure. The crude material was purified by column chromatography (PET → PET:
1
EtOAc 1:1) to give the desired product was a yellow solid (3.42 g, 94%). H NMR (CDCl ) δ 7.15–
3
7
.03 (m, 2H), 6.71–6.60 (m, 2H), 4.77 (br s, 1H), 4.20 (d,
J
= 5.4 Hz, 2H), 3.74 (br s, 2H), 1.48 (s,
+
+
9H); mp: 70ꢀ73 °C; m/z MS (TOF ES ) 223.1 [M+H] ; LCꢀMS : 2.73.
t
R
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2
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tertꢀButyl
(4ꢀ((9ꢀ((2R,3R,4S,5R)ꢀ3,4ꢀdihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀ
purinꢀ6ꢀyl)amino)benzyl)carbamate (13). Triethylamine (714 ꢁL, 5.23 mmol, 1.00 eq) was added
to a mixture of 6ꢀchloropurine riboside (1) (1.50 g, 5.23 mmol, 1.00 eq) and tertꢀbutyl (4ꢀ
aminobenzyl)carbamate (12) (3.49 g, 15.7 mmol, 3.00 eq) in ethanol (50 mL). The reaction mixture
was stirred at reflux for 26 h, before it was cooled to rt. Upon cooling, a white solid precipitated
was formed. The suspension was filtered, washed with cold ethanol and dried under high vacuum
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
1
to afford the title product as a white powder (2.17 g, 88%). H NMR (d6ꢀDMSO) δ 9.93 (s, 1H),
8
.54 (s, 1H), 8.39 (s, 1H), 7.90–7.77 (m, 2H), 7.36 (t,
6.0 Hz, 1H), 5.50 (d, = 6.2 Hz, 1H), 5.31 (dd,
4.64 (dd, = 11.2, 6.0 Hz, 1H), 4.18 (dd, = 8.1, 4.8 Hz, 1H), 4.09 (d,
J = 6.1 Hz, 1H), 7.27–7.13 (m, 2H), 5.96 (d, J
=
J
J
= 6.8, 4.8 Hz, 1H), 5.23 (d,
J
= 4.7 Hz, 1H),
J
J
J
= 6.1 Hz, 2H), 3.99 (dd, J
=
6.5, 3.6 Hz, 1H), 3.77–3.64 (m, 1H), 3.62–3.50 (m, 1H), 1.40 (s, 9H); mp: 178ꢀ181 °C; m/z MS
+
+
(
TOF ES ) 473.2 [M+H] ; LCꢀMS tR: 3.03.
(2 ,3 ,4 ,5 )ꢀ2ꢀ(6ꢀ((4ꢀ(Aminomethyl)phenyl)amino)ꢀ9
hydroxymethyl)tetrahydrofuranꢀ3,4ꢀdiol (14). tertꢀButyl (4ꢀ((9ꢀ((2
hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9 ꢀpurinꢀ6ꢀyl)amino)benzyl)carbamate (1.00 g, 2.12 mmol,
.00 eq) (13) was dissolved in CH Cl (20 mL)and trifluoroacetic acid (2 mL) was added and the
R
R
S
R
H
ꢀpurinꢀ9ꢀyl)ꢀ5ꢀ
R,3R,4S,5R)ꢀ3,4ꢀdihydroxyꢀ5ꢀ
(
(
H
1
2
2
reaction was stirred at rt. After 6 h DCM (50 mL) was added, followed by the addition of 1 M
NaOH until the reaction mixture basic (pH= 12). The layers were separated and the water layer
reduced under vacuo. The residue was purified via reversed phase chromatography (C18 column)
(H
2
O: MeOH 95:5 → 0:100) to obtain the titled compound as a white solid (450 mg,
1
70%). H NMR (d6ꢀDMSO) δ 9.88 (s, 1H), 8.53 (s, 1H), 8.38 (s, 1H), 7.87 – 7.81 (m, 2H), 7.31 –
7.25 (m, 2H), 5.96 (d, J = 6.0 Hz, 1H), 5.66 (br s, 1H), 5.40 (br s, 1H), 5.35 – 5.28 (m, 1H), 4.63 (t,
J
= 5.4 Hz, 1H), 4.24 – 4.14 (m, 1H), 3.99 (q,
J
= 3.6 Hz, 1H), 3.75 – 3.66 (m,
J
= 13.7 Hz, 3H),
R
: 1.38. Remark:
+
+
3
.62 – 3.53 (m, 1H), 1.72 (br s, 2H); m/z MS (TOF ES ) 373.0 [M+H] ; LCꢀMS
t
Amine 14 was successfully used as the TFA salt for further reactions, however, in case of the
synthesis of compounds 15b and 15d better results were obtained when using the free amine.
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9
N
ꢀ(4ꢀ((9ꢀ((2
R
,3
R
,4
S
,5
R
)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
yl)amino)benzyl)bicyclo[2.2.2]octaꢀ2,5ꢀdieneꢀ2ꢀcarboxamide (15a). General procedure B. White
1
solid (113 mg, 42%). H NMR (
d
6
ꢀDMSO) δ 9.93 (s, 1H), 8.54 (s, 1H), 8.38 (s, 1H), 8.35 (br t,
= 6.3, 1.9 Hz, 1H), 6.40–6.28 (m,
= 6.1 Hz, 1H), 5.30 (dd, = 6.7, 4.8 Hz, 1H), 5.23 (d,
= 5.9 Hz, 2H), 4.20–4.14 (m, 2H), 3.99 (dd,
J =
6
.1 Hz, 1H), 7.87–7.81 (m, 2H), 7.25–7.17 (m, 2H), 7.02 (dd, J
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
H), 5.96 (d,
J
= 6.0 Hz, 1H), 5.50 (d,
J
J
J =
4.7 Hz, 1H), 4.64 (dd,
J
= 11.1, 5.8 Hz, 1H), 4.28 (d, J
J
= 7.0, 3.6 Hz, 1H), 3.77–3.66 (m, 2H), 3.61–3.52 (m, 1H), 1.30–1.14 (m, 4H); mp: 195ꢀ200 °C;
+
+
+
R 29 6 5
m/z MS (TOF ES ) 505.2 [M+H] ; LCꢀMS t : 3.04; HRMS ꢀ C26H N O [M+H] calcd 505.2199;
found 505.2203.
1ꢀ(4ꢀ((9ꢀ((2R,3R,4S,5R)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
yl)amino)benzyl)ꢀ3ꢀ(4ꢀisothiocyanatophenyl)thiourea (15b). General procedure C. 1,4ꢀPhenylene
diisothiocyanate (23 mg, 118 µmol, 1.10 eq) was added to a solution of (2R,3R,4S,5R)ꢀ2ꢀ(6ꢀ((4ꢀ
(aminomethyl)phenyl)amino)ꢀ9
Hꢀpurinꢀ9ꢀyl)ꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ3,4ꢀdiol
(14)
(
40 mg, 107 µmol, 1.00 eq) in DMF (4 mL). The reaction mixture was stirred at rt for 2 h, then
water was added until a white precipitate formed. The solid was allowed to settle to the ground and
the liquid phase was decanted. The residue was taken up in DMF (2 mL) and purified via
preparative RPꢀHPLC (30ꢀ100% ACN) to obtain the titled compound as a white solid (10 mg,
1
1
7%). H NMR (
d
6
ꢀDMSO) δ 10.02 (s, 1H), 9.83 (br s, 1H), 8.60 (s, 1H), 8.45 (s, 1H), 8.41–8.30
(
m, 1H), 7.99–7.92 (m, 2H), 7.68–7.61 (m, 2H), 7.49–7.43 (m, 2H), 7.41–7.34 (m, 2H), 6.02 (d,
.0 Hz, 1H), 5.54 (d, = 6.2 Hz, 1H), 5.35 (dd, = 6.7, 4.8 Hz, 1H), 5.28 (d, = 4.7 Hz, 1H), 4.75
(d, = 5.0 Hz, 2H), 4.72–4.67 (m, 1H), 4.27–4.21 (m, 1H), 4.04 (dd, = 3.6 Hz, 1H), 3.80–3.72 (m,
1H), 3.69–3.60 (m, 1H); H NMR (d6ꢀDMSO) δ 180.9, 152.6, 152.4, 149.8, 141.2, 139.7, 138.9,
J =
6
J
J
J
J
J
1
1
1
5
33.3, 133.1, 128.2, 126.7, 125.3, 123.9, 121.5, 120.8, 88.3, 86.3, 74.1, 71.0, 62.0, 47.4; mp: 172ꢀ
+
+
+
R 25 8 4 2
76 °C; m/z MS (TOF ES ) 565.1 [M+H] ; LCꢀMS t : 3.22; HRMS – C25H N O S [M+H] calcd
65.1440; found 565.1447.
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(2R,3S,4R,5R)ꢀ2ꢀ(Hydroxymethyl)ꢀ5ꢀ(6ꢀ((4ꢀ(isothiocyanatomethyl)phenyl)amino)ꢀ9Hꢀpurinꢀ9ꢀ
yl)tetrahydrofuranꢀ3,4ꢀdiol (15c). General procedure D. The purification was performed via
column chromatography (CH
2
Cl
: MeOH 100:0 → 8:2) and preparative TLC (CH
Cl : MeOH
2 2 2
1
9
5:5). The title compound was obtained as a white solid (5 mg, 3%). H NMR (
d
6
ꢀDMSO) δ 10.08
= 6.0 Hz, 1H),
= 4.8 Hz, 1H), 4.89 (s, 2H), 4.67–
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
(
s, 1H), 8.57 (s, 1H), 8.43 (s, 1H), 8.04–7.97 (m, 2H), 7.40–7.33 (m, 2H), 5.97 (d, J
5.51 (d,
J
= 6.2 Hz, 1H), 5.28 (dd,
J
= 6.6, 4.9 Hz, 1H), 5.23 (d,
J
4
.61 (m, 1H), 4.21–4.15 (m, 1H), 4.01–3.97 (m, 1H), 3.73–3.68 (m, 1H), 3.61–3.54 (m, 1H); m/z
+
+
+
MS (TOF ES ) 415.1 [M+H] ; LCꢀMS
t
R
: 3.15; HRMS – C18
H
19
N
6
O
4
S [M+H] calcd 415.1188;
found 415.1184.
4
ꢀ((4ꢀ((9ꢀ((2
yl)amino)benzyl)carbamoyl)benzenesulfonyl fluoride (15d). (Fluorosulfonyl)benzoic acid (24 mg,
18 µmol, 1.1 eq), (2R,3R,4S,5R)ꢀ2ꢀ(6ꢀ((4ꢀ(aminomethyl)phenyl)amino)ꢀ9 ꢀpurinꢀ9ꢀyl)ꢀ5ꢀ
(hydroxymethyl)tetrahydrofuranꢀ3,4ꢀdiol (14) (40 mg, 107 µmol, 1.00 eq), BOP reagent (95 mg,
24 µmol, 2.00 eq) and 2,6ꢀlutidine (62 µL, 537 µmol, 5.00 eq) were dissolved in DMF (8 mL).
R,3R,4S,5R)ꢀ3,4ꢀDihydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀ
1
H
2
The mixture was stirred at rt for 1 h. The mixture was diluted with water (50 mL) and extracted
with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water (2 × 50 mL)
and brine (50 mL), dried with Na SO , filtered, and concentrated under reduced pressure. The
2
4
residue was suspended in a mixture of MeOH and DCM (10 mL). The solid was allowed to settle
on the ground and the liquid layer was decanted. The solid was dried under a nitrogen flow to give
1
the titled compound as a white powder (41 mg, 68%). H NMR (
d
6
ꢀDMSO) δ 9.96 (s, 1H), 9.42 (br
t,
J
= 5.6 Hz, 1H), 8.54 (s, 1H), 8.38 (s, 1H), 8.30–8.20 (m, 4H), 7.91–7.85 (m, 2H), 7.36–7.27 (m,
= 6.0 Hz, 1H), 5.51–5.42 (m, 1H), 5.32–5.15 (m, 2H), 4.64 (t, = 5.6 Hz, 1H), 4.49
= 5.7 Hz, 2H), 4.20–4.14 (m, 1H), 4.00–3.96 (m, 1H), 3.70 (dd, = 11.8, 3.6 Hz, 1H), 3.58
= 12.5, 4.2 Hz, 1H), strong concentration dependent effects of nonexchangeable proton
2H), 5.96 (d,
J
J
(d,
J
J
(
dd,
J
50, 51
1
resonances
6
were observed for the multiplicity of the signal at 8.30–8.20 ppm.; H NMR (d ꢀ
DMSO) δ 164.8, 152.6, 152.4, 149.8, 141.9, 141.1, 138.8, 133.9, 133.8, 129.7, 129.1, 128.1, 121.5,
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+
120.7, 88.3, 86.3, 74.1, 71.0. 62.0, 43.1; mp: >350 °C (degraded/ turned black); m/z MS (TOF ES )
+
+
5
59.1 [M+H] ; LCꢀMS tR: 3.08; HRMS ꢀ C H FN O S [M+H] calcd 559.1411; found 559.1387.
24 24 6 7
TM
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Pharmacology. ERK 1/2 phosphorylation assays. FlpIn Chinese hamster ovary (FlpInCHO)
1 1
cells stably expressing the human A AR (A ARꢀFlpInCHO) cells were seeded into 96ꢀwell culture
4
plates at a density of 4 × 10 cells/well in DMEM containing 10% FBS and maintained at 37°C in a
humidified incubator containing 5% CO
2
. After 6 h, cells were washed with serum free DMEM
before assaying.
and maintained in serum free DMEM for approximately 18 h at 37°C in 5% CO
2
Agonist concentrationꢀresponse curves were generated at the peakꢀresponse time point (5 min
exposure). In all cases, stimulation with 10% FBS for 5 min was used as a positive control.
TM
Stimulation was terminated by the rapid removal of media and addition of 100 ꢀL SureFire lysis
buffer to each well. Plates were then agitated for 5 min and detection performed as previously
39
described.
Data were baselineꢀcorrected and then expressed as a percentage the pERK1/2
response in the presence of 1 ꢀM NECA (defined as 100%) and fitted to “log (agonist) vs.
response” equation in Prism 6.0 (GraphPad Software).
xCELLigence realꢀtime cell analysis assays. Cellular impedance was measured over time using the
labelꢀfree technology, xCELLigence RealꢀTime Cell Analyzer (RTCA) singleꢀplate (SP) instrument
(
Roche Diagnostics GmbH and ACEA Biosciences). A
1
ARꢀFlpInCHO cells were seeded into 96ꢀ
well Eꢀplates plates at a density of 4 × 10 cells/well in DMEM containing 10% FBS and
maintained at 37°C in a humidified incubator containing 5% CO . After 6 h, cells were washed
with serum free DMEM and maintained in serum free DMEM for approximately 18 h at 37°C in
5% CO before assaying. The Eꢀplate was then plated into the RTCA reader and allowed to
equilibrate for at least 1 h at 37°C in 5% CO , during which time the cellular impedance was
TM
4
2
2
2
measured at 10 min intervals. Following the equilibration period, cells were exposed to 1 ꢁM
agonist or vehicle and changes in cellular impedance measured at 15 s intervals. After 1.7 h, the
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