Synthesis and pharmacological evaluation of dual acting antioxidant a 2A adenosine receptor agonists

Article abstract of DOI:10.1021/jm300206u

A series of adenosine-5′-N-alkylcarboxamides and N ⁶-(2,2-diphenylethyl)adenosine-5′-N-alkylcarboxamides bearing antioxidant moieties in the 2-position were synthesized from the versatile intermediate, O⁶-(benzotriazol-1-yl)-2-fluoro

Full text of DOI:10.1021/jm300206u

Article
pubs.acs.org/jmc
Synthesis and Pharmacological Evaluation of Dual Acting
Antioxidant A2A Adenosine Receptor Agonists
Nicholas E. Hausler,† Shane M. Devine,† Fiona M. McRobb,† Lyndon Warfe,‡ Colin W. Pouton,‡
John M. Haynes,‡ Steven E. Bottle,§ Paul J. White,*,‡ and Peter J. Scammells*,†
†Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia
‡Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia
§Chemistry Discipline, Faculty of Science and Technology, Queensland University of Technology, QLD 4001, Australia
ABSTRACT: A series of adenosine-5′-N-alkylcarboxamides and N6-(2,2-
diphenylethyl)adenosine-5′-N-alkylcarboxamides bearing antioxidant
moieties in the 2-position were synthesized from the versatile
intermediate, O6-(benzotriazol-1-yl)-2-fluoro-2′,3′-O-isopropylideneino-
sine-5′-N-alkylcarboxamide (1). These compounds were evaluated as
A2A adenosine receptor (A2AR) agonists in a cAMP accumulation assay,
and a number of potent and selective agonists were identified. Three of
these compounds were evaluated further in an ischemic injury cell
survival assay and a reactive oxygen species (ROS) production assay whereby 15b and 15c were shown to reduce ROS activity
and cell death due to ischemia.
disease but unfortunately gave poor efficacy results in phase II
clinical trials.17 Apadenoson (ATL-146e) and Binodenoson
(MRE-0470), which are both highly selective A2AR agonists, are
being trialled for myocardial perfusion imaging, while
Sonedenoson (MRE-0094) has displayed promise for diabetic
foot ulcers and wound healing.18 The A2AR agonist
Regadenoson, licensed as Lexiscan, is a coronary vasodilator
which has found application diagnostically as a radionuclide
myocardial perfusion imaging agent.19 It is currently the only
selective agonist on the market.
Selective A2AR agonists have considerable promise in the
prevention and treatment of ischemia-reperfusion injury.20
After a myocardial infarction, the recruitment of immune cells
to the damaged myocardium and the death of cardiomyocytes
are two of the most important steps in the pathogenesis of the
disease. A2AR activation inhibits neutrophil accumulation and
reduces infarct size,21 and A2AR agonists are therefore of
interest as therapeutic agents post myocardial infarction.
Despite this promise, most inflammatory agents that have
been evaluated for the therapy of IR injury in clinical studies
have failed. Accordingly, it has been suggested that new
strategies to augment adenosine-mediated protective effects
may be more effective.20 Free radical production is well
established to increase massively during cardiac ischemia and
reperfusion, with a subsequent loss of cardiomyocytes. The use
of antioxidants as a strategy to reduce the loss of
cardiomyocytes has been of significant interest.22,23 The
combination of A2AR agonist and antioxidant functionalities is
conceptually attractive because it combines a mechanism to
inhibit both immune cell recruitment and direct loss of
INTRODUCTION
■
Adenosine is a ubiquitous endogenous nucleoside that mediates
a number of physiological processes.1,2 These effects are
mediated through binding interaction with adenosine receptors
(ARs) which are members of the G protein-coupled receptor
(GPCR) family. There are known to be four distinct AR
subtypes, which are termed A1, A2A, A2B, and A3. The A1 and
A3Rs couple to Gi inhibiting adenylate cyclase activity, thereby
prohibiting cyclic AMP production. Conversely, the A2A and
A2BRs couple to Gs and mediate the stimulation of adenylate
cyclase, promoting cyclic AMP production. Adenosine helps to
regulate a wide array of physiological processes and is involved
in cancer1 and cardioprotection.2 Specifically, a great deal of
interest has centered on the A2A receptor and its role in
inflammation.3−5
Recently, the emergence of GPCR crystal structure
elucidation and the discovery that the seven transmembrane
(7TM) topology is common between various family A GPCRs,
such as the CXCR4 chemokine receptor,6 D3 dopamine
receptor,7 and β1 and β2 adrenergic receptors,8,9 has led to a
paradigm shift in GPCR drug discovery. Specifically, the
illumination of the A2AR’s crystal structure with a bound
antagonist (ZM241385) has been reported.10 This initial report
led to increased efforts to crystallize the receptor with other
ligands, specifically A2AR selective agonists. Subsequently, the
A2AR selective agonist UK-432,097 has been used to probe the
internal structure of the active site.11 A number of
pharmacologically important agonists displaying A2AR selectiv-
ity have been synthesized and are shown in Figure 1. YT-146
and DPMA have shown cardioprotective effects,12,13 whereas
CGS-21680 has been used for studying neuronal trans-
mission.14−16 The drug candidate UK-432,097 was developed
by Pfizer for the treatment of chronic obstructive pulmonary
Received: February 15, 2012
Published: March 20, 2012
© 2012 American Chemical Society
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Figure 1. Pharmacologically important A2AR agonists.
a
Scheme 1
a
Reagents and conditions: (i) RNH2, DIPEA, MeCN, 25 °C; (ii) RNH2, DIPEA, EtOH, 70 °C; (iii) 1M HCl, MeCN, 50 °C.
cardiomyocytes. Accordingly, the central aim of this study is to
prepare dual acting agents which act as both A2AR selective
agonists and antioxidants.
The structure−activity relationships to date have suggested
three key points of the adenosine molecule that can be
modified to elicit A2AR selectivity, namely the 2-, 6-, and 5′-
positions. We have previously worked on the synthesis of
potent and selective A1R24 and A3R25 agonists and have now
expanded our approach to include A2AR agonists. As
demonstrated in Figure 1, A2AR selectivity can be enhanced
when bulky aromatic groups are incorporated in the 2-positon.
Less tolerance is exhibited at the N6-position apart from the
inclusion of a 2,2-diphenylethyl moiety as UK-432,097
demonstrates. The addition of a 5′-alkylcarboxamido group,
typically a methyl or an ethyl substituent, can also lead to
enhanced A2AR selectivity. Recently, we have reported
convergent syntheses of highly substituted 2-, 6-, and/or 5′-
substituted adenosines. With these modifications in mind, we
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a
Scheme 2
a
Reagents and conditions: (i) Boc2O, CH2Cl2, 0 °C; (ii) RCO2H, EDCI, HOBt, DIPEA, DMF, 25 °C; (iii) TFA, CH2Cl2, 25 °C; (iv) 2 or 3,
DIPEA, EtOH, 70 °C; (v) 1M HCl, MeCN, 50 °C.
a
Scheme 3
a
Reagents and conditions: (i) 6b or 7b, RCO2H, EDCI, HOBt, DIPEA, DMF, 25 °C; (ii) 1M HCl, MeCN, 50 °C.
have sought to incorporate this knowledge into a novel series of
adenosine-5′-N-alkylcarboxamides and N6-(2,2-diphenylethyl)-
adenosine-5′-N-alkylcarboxamides, which possess antioxidant
moieties in the 2-position. To this end, we have incorporated
an analogue of the recognized phenolic antioxidant, BHT,26
which is able to break radical chain reactions through atom
transfer as well as a lipoic acid analogue that can mediate in
glutathione redox couples.27,28 These antioxidant moieties are
likely to emulate many of the natural pathways which negate
free radicals as seen with vitamin E, resveratrol-type
compounds and endogenous lipoic acid.
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a
Scheme 4
a
Reagents and conditions: (i) 2,2-diphenylethylamine, DIPEA, MeCN, 25 °C; (ii) 12b, DIPEA, MeCN, 150 °C; (iii) 1M HCl, MeCN, 50 °C.
In addition to the 5′-N-ethylcarboxamido compounds, two
novel 5′-N-methylcarboxamides (18 and 20) containing the N6-
(2,2-diphenylethylamino) group were synthesized (Scheme 4)
and their properties studied. The influence of the 5′-N-
methylcarboxamide group was examined to determine any
impact on the selectivity arising from the incorporation of this
minor structural change. This synthetic sequence was achieved
in a similar fashion to that of the 5′-N-ethylcarboxamides
(Scheme 4).
cAMP Assays. Compound potency was assessed using a
cAMP accumulation assay in stably transfected CHO cell lines,
each overexpressing one of the human A1, A2A, A2B, and A3Rs.
The results of these assays are described in Table 1.
RESULTS AND DISCUSSION
Chemistry. The starting materials used in this study, O6-
■
(benzotriazol-1-yl)-2-fluoro-2′,3′-O-isopropylideneinosine-5′-N-
alkylcarboxamides (1a and 1b), were synthesized in six steps
from guanosine.29,30 The key features of these syntheses
include a BOP-mediated coupling and subsequent halogenation
via diazotization. The highly versatile intermediate 1a was first
subjected to N6-substitution with either ammonia or 2,2-
diphenylethylamine in the presence of DIPEA in MeCN to
afford 2 and 3, respectively (Scheme 1). The isopropylidene
groups on these molecules were deprotected with hydrochloric
acid at 50 °C to afford the 2-fluoro analogues 4 and 5.
We also envisaged utilizing a hydrophobic alkyl amine,
namely 4-(2-aminoethyl)aniline as a functionalized linker in the
2-position to incorporate known antioxidant groups. First, the
2-position of 2 and 3 were reacted with a small series of p-
substituted p-anilinophenethylamines and deprotected with
HCl to give the adenosine-5′-N-ethylcarboxamides 8a−c and
9a−c (Scheme 1).
Second, a series of phenethylamine linked amines containing
antioxidant moieties were synthesized and coupled to 2 or 3.
Reaction with Boc anhydride of the ethylamine portion of 4-(2-
aminoethyl)aniline in CH2Cl2 was carried out to give the Boc-
protected amine (11) (Scheme 2). This was then coupled with
the appropriate benzoic acid with EDCI, HOBt, and DIPEA in
DMF to give the corresponding amides, which were
subsequently treated with TFA to produce the amines 12a
and 12b. These primary amines included known antioxidant
groups utilized by our group in the past.29,30 Reaction of the
substituted amines 12a and 12b with 2-fluoroadenosine-5′-N-
ethyl carboxamides 2 and 3, followed by deprotection of the
isopropylidene group, afforded 15a, 15b, and 16b. Substitution
at the 2-position was achieved with a range of simple and
structurally diverse amines.
In general, the series of 2-substituted adenosine-5′-N-
ethylcarboxamides (compounds 4, 8a−c, and 15a−c) proved
to be more potent agonists at the A2AR than the corresponding
N6-(2,2-diphenylethyl)adenosine-5′-N-ethylcarboxamide series
(5, 9a−c, and 16a−c). All members of the adenosine-5′-N-
ethylcarboxamide series had EC50 values less than 50 nM, and
five of the seven compounds in this series had EC50 values less
than 15 nM (Table 1, entries 1−7). More specifically, 2-
fluoroadenosine-5′-N-ethylcarboxamide (4) showed good
potency with an EC50 of 5.6 nM at the A2AR but relatively
poor selectivity versus the other receptor subtypes. Substitution
of the 2-fluoro with a 2-phenylethylamino (compound 8a) or p-
aminophenylethylamino moiety (8b) reduced the potency at
the A2AR but significantly improved receptor subtype
selectivity. Significantly, 8b retained reasonable potency (14
nM) and selectivity (∼10-fold vs A3R and ∼24-fold vs A1R)
selectivity. The amide derivatives of 8b, compounds 15a−c,
also proved to be potent and selective A2AR agonists. Notably,
the compounds bearing antioxidant functionality in the 2-
position (compounds 15b and 15c) demonstrated very good
potency for the A2AR, with EC50 values of 14 and 6.6 nM,
respectively. These compounds also exhibited high selectivity
for A2AR relative to the A1R (284- and 324-fold, respectively)
and reasonable selectivity relative to the A3R (∼18-fold in both
cases).
This sequence was unsuccessful for the preparation of 16a,
and an alternative route was sought. The inclusion of the lipoic
acid moiety into the analogous amide was also problematic,
therefore the more convergent approach from the 2-(2-(4-
aminophenyl))ethyl substituted adenosine-5′-N-ethylcarboxa-
mides 6b and 7b was employed (Scheme 3).
As noted above, the N6-(2,2-diphenylethyl)adenosine-5′-N-
ethylcarboxamide series (5, 9a−c, and 16a−c) were slightly less
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Table 1. Evaluation of the Adenosine Derivatives in cAMP Accumulation Assays at all Receptor Subtypes
potent at the A2AR (Table 1, entries 8−15). The p-
aminophenethylamino compound 9b showed very good
potency at the A2AR (EC50 = 6.3 nM), however the
phenethylamino (9a) and p-bromophenethylamino (9c)
analogues were less potent, with half-maximal inhibitory
concentrations of 66 and 31 nM, respectively. The selectivity
of 9b was markedly better as well with over 188-fold selectivity
(A2A/A1) but no significant increase over the A3R (A2A/A3 =
5.2). The compound 16a and the antioxidant containing
molecules 16b and 16c had relatively modest potency and
selectivity for the A2AR. The compound incorporating the 5′-N-
methylcarboxamide moiety (18) produced a highly potent
compound, exhibiting a 2.02 nM EC50 at the A2AR. This
compound showed some selectivity versus the A1R (60-fold)
but very modest selectivity relative versus the A3R (3.5-fold).
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In summary, a number of 2-substituted adenosine 5′-N-
alkylcarboxamides (8a−c and 15a−c) and N6-(2,2-dipheny-
lethyl), 2-substituted adenosine 5′-N-alkylcarboxamides (9a−c
and 16a−c) were synthesized and their pharmacology explored.
The N6-amino series (8a−c and 15a−c) showed overall greater
selectivity and potency when a functionalized linker joined to
an antioxidant group was attached. The N6-2,2-diphenylethy-
lamino series (9a−c and 16a−c) pharmacology was less
straightforward, however 9b was the most active compound at
the A2AR, demonstrating an EC50 of 6.3 nM. The retention of
the 2-fluoro groups (4, 5, 18) demonstrated highly potent
compounds, in particular 5 had an EC50 of 0.85 nM at the
A2AR.
two recent crystal structures (PDB ID: 2YDO and 2YDV),31
further supporting this binding mode.
Because of the open nature of the solvent exposed area of the
binding site, the 2-substituents of 8a, 8b, and 15b can dock in a
number of diverse orientations. These binding modes are also
influenced by the presence of water molecules. Interactions of
the 2-substituents with residues in extracellular loop 3 (ECL3)
and the extracellular side of transmembrane helix 7 (TM7,
Figure 2B) may explain selectivity observed between the
different subtypes. ECL3 is adjacent to the binding site and
shows a large degree of variability between adenosine receptor
subtypes, demonstrated by multiple sequence alignment (data
not shown). Analysis of the A2AR crystal structure31 in
conjunction with recent biophysical mapping of the A2AR
binding site32 has demonstrated that the 2-substituents may
interact with residues on the extracellular side of TM6 and
TM7, such as Tyr 7.36. Additionally, due to the variable nature
of residues in ECL3, interactions with this loop may also confer
subtype selectivity.
Molecular Modeling. The most potent A2AR ligands (8a,
8b, and 15b) identified in this study were docked into the
crystal structure of the A2AR (PDB ID: 3QAK, Figure 2A)11
On the basis of our docking studies, the 2-substituent of 15b
displays a range of conformations (Figure 2E), either
interacting with Thr 6.58, His 264, Leu 7.32, Met 7.35, Tyr
7.36, and Ile 7.39 (TM6, TM7 and ECL3, ligands shown in
yellow and orange) or with Ser 1.32, Ile 2.64, or Ser 2.65 (TM1
and TM2, ligands shown in green and blue), or protruding into
the bulk solvent with only limited interactions with the receptor
(ligand shown in magenta). The para-amino substitution in 8b
had increased selectivity over 8a, which is likely to be a result of
the ability to form additional hydrogen bonding interactions
with residues on the extracellular side of TM7 (Figure 2C,D).
Simulated Ischemia Assays. Compounds 15b and 15c
were selected for further characterization in a cell culture
hypoxia model developed in house and reported previously
using the rat atrial cardiomyoblast cell line H9c2.24,33−36 These
compounds showed good A2AR potency (with EC50 values of
14 and 6.6 nM), good selectivity for the A2AR over other
receptor subtypes, and both contained antioxidant functionality.
Compound 15a was also included in this study as a close
analogue of 15b, which lacks the antioxidant functionality. In
this assay, cells were exposed to media mimicking hypoxic
interstitial fluid, containing lactic acid to decrease the pH and
deoxy-glucose, and were then placed in an oxygen-free
environment (100% N2) for 12 h. After this time, around
40% of cells were dead, as evidenced by DAPI exclusion. This
level of cell death was normalized to 100%, and this provided
the baseline for the assay. Superoxide production was measured
using dihydroethidium staining as previously reported, at 20
μM for 45 min (see Figure 3). Compounds 15b and 15c were
shown to be protective in this model at submicromolar
concentrations (see Figure 3). In this assay, 15a was largely
inactive, demonstrating that the protective effects seen in this
cardiomyoblast model were due to the antioxidant hindered
phenol group. This observation was consistent with our
previous study, which found that antioxidants alone exhibit
protective effects in this simulated ischemia assay.35
Figure 2. Crystal structure of the adenosine A2A receptor in complex
with agonist UK-432,097 (A) and showing ECL3 residues highlighted
in green (B). Docked poses for compounds 8a (C) and 8b (D) and
15b (E), displaying range of poses observed in docking.
using the GOLD modeling software. In all of the top scoring
docked poses, the adenosine-5′-N-ethylcarboxamide (NECA)
component of each ligand docked in a similar way to that of
UK-432,097, with rms deviations of less than 1 Å to the
corresponding segment of UK-432,097. The NECA portion of
the docked ligands makes hydrogen bonding interactions to
residues Thr 3.36, His 6.52, Asn 6.55, Ser 7.42, and His 7.43,
which is consistent with the binding mode of UK-432,097 in
the A2AR crystal structure.11 The binding mode of adenosine
and the agonist NECA have subsequently been determined in
CONCLUSIONS
■
This study has identified a number of highly potent and
selective A2AR agonists, some of which possess antioxidant
functionality. Pleasingly, the incorporation of this antioxidant
functionality maintained high A2AR potency and promoted
improved selectivity for the A2AR over all other receptor
subtypes. A molecular modeling study, which utilized the
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Figure 3. Compounds 15b and 15c reduce ROS activity and cell death due to simulated ischemia. Top panel: confocal microscopy images showing
dihydroethidium staining for superoxide (red) and DAPI (blue) staining for dead cells. Bottom panel: quantitation of cell death and DHE
fluorescence intensity per cell after treatment. Cells were exposed to 95% N2 5% CO2 in HEPES buffer with lactic acid for 12 h followed by 1 h
incubation in HEPES glucose buffer. Both 15b and 15c were protective and reduced ROS production at 0.1 and 1 μM, while 15a had no significant
effect. *indicates p < 0.05; ANOVA n = 3 experiments conducted in quadruplicate.
recently reported X-ray crystal structure, was conducted to
rationalize the binding mode of these agonists. The study
identified key ligand−receptor interactions that may account
for the high level of A2AR selectivity observed for these
compounds. Interestingly, this modeling also suggests that the
appended antioxidant group projects beyond the receptor,
thereby placing it in a favorable position to interact with
reactive oxygen species present in the cytosol. The
cardioprotective effects of two of the most potent and selective
A2AR agonists (compounds 15b and 15c) were further
investigated in an ischemia model using rat atrial cardiomyo-
cytes. Both of these compounds showed strong protective
effects in this assay relative to the control. Compound 15a, a
closely related analogue of 15b lacking antioxidant function-
ality, was also evaluated in this assay and showed no significant
protective effects. This suggests that the antioxidant group is
the mediator of the protective effects observed for 15b. Two of
the best established mechanisms of cardioprotection are: (i)
A2AR activation and (ii) inactivation of damaging free radicals in
the heart using small molecule antioxidants. We believe that the
combination of these two modes of action within a single
molecule will provide additive or synergistic benefit. A2AR
activation is well-known to reduce the inflammatory drive to
remodel the heart, and we have shown that the antioxidant
functionality of our lead compounds prevents cell death in a
cardiac cell line after simulated ischemia. Compounds 15b and
15c are therefore promising candidates for development as
cardioprotective therapeutic agents. These two compounds will
now be evaluated in an in vivo ischemia-reperfusion model in
comparison to an A2AR agonist or antioxidant given alone.
EXPERIMENTAL SECTION
■
General Synthetic Procedures. Melting points were determined
on an Electrothermal melting point apparatus and are uncorrected. All
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microwave reactions took place in a Biotage Initiator microwave
synthesizer. All NMR spectra were recorded on a Bruker Avance DPX
300 MHz or Bruker Avance III 400 MHz Ultrashield Plus
2-Fluoro-N6-(2,2-diphenylethyl)adenosine-5′-N-ethylcarbox-
amide (5). General Procedure for Isopropylidene Deprotection.
Compound 3 (175 mg, 0.32 mmol), MeCN (3 mL), 1 M HCl
(12 mL), 50 °C, 2 h, white solid (5) (45 mg, 28%), Rf = 0.19
1
spectrometer, and H and 13C NMR spectra were recorded at either
1
(1:5 petroleum spirits/EtOAc), mp 131−134 °C. H NMR
300.13 or 400.13 MHz and 75.4 or 100.62 MHz, respectively. Thin
layer chromatography was conducted on 0.2 mm plates using Merck
silica gel 60 F254. Column chromatography was achieved using Merck
silica gel 60 (article size 0.063−0.200 μm, 70−230 mesh). High
resolution mass spectra were obtained on a Waters LCT Premier XE
(TOF) mass spectrometer fitted with an ESI ion source, coupled to a
2795 Alliance separations module. LCMS were run to verify reaction
outcome and purity using an Agilent 6100 Series Single Quad coupled
to an Agilent 1200 Series HPLC. All compounds were of >95% purity.
The following buffers were used: buffer A 99.9% H2O, 0.1% formic
acid and buffer B 99.9% CH3CN, 0.1% formic acid. The following
gradient was used with a flow rate of 0.5 mL/min and total run time of
12 min: 0−4 min 95% buffer A and 5% buffer B, 4−7 min 0% buffer A
and 100% buffer B, 7−12 min 95% buffer A and 5% buffer B. Mass
spectra were acquired in positive and negative ion mode with a scan
range of 0−1000 m/z at 5 V. 2-Fluoro-O6-(benzotriazol-1-yl)-2′,3′-O-
isopropylideneinosine-5′-N-methylcarboxamide (1b),30 2-fluoro-O6-
(benzotriazol-1-yl)-2′,3′-O-isopropylideneinosine-5′-N-ethylcarboxa-
mide (1a),29 and 2-fluoro-2′,3′-O-isopropylideneadenosine-5′-N-ethyl-
carboxamide (2)29 were synthesized as previously published.
(400 MHz, CD3OD) δ: 1.20 (t, J = 7.2 Hz, 3H), 3.33−3.45 (m,
J = 7.2 Hz, 2H), 4.22 (dd, J = 8.2, 1.0 Hz, 2H), 4.32 (dd, J =
4.8, 1.0 Hz, 1H), 4.46 (d, J = 4.8 Hz, 1H), 4.51 (t, J = 7.8 Hz,
1H), 4.66 (dd, J = 7.8, 4.8 Hz, 1H), 5.93 (d, J = 7.8 Hz, 1H),
7.17−7.23 (m, 2H), 7.26−7.38 (m, 10H), 8.12 (s, 1H). 13C
NMR (75 MHz, CD3OD) δ: 13.7, 33.5, 44.8, 50.3, 71.9, 73.5,
85.0, 89.1, 118.4, 126.3, 127.9, 128.2, 140.9, 142.2, 149.1 (d, J =
19.2 Hz), 156.3 (d, J = 20.3 Hz), 159.0 (d, J = 207.2 Hz), 170.5.
HRMS calcd for C26H27N6O4F+ [M + H], 507.2151; found,
507.2161.
2-(2-Phenyl)ethylamino-2′,3′-O-isopropylideneadenosine-
5′-N-ethylcarboxamide14 (6a). General Procedure for 2-
Substitution. Compound 2 (120 mg, 0.33 mmol), 2-phenyl-
ethylamine (83 mg, 0.69 mmol), and DIPEA (250 μL) in
EtOH (2.5 mL) was stirred in a sealed reaction vessel at 70 °C.
After 7 days, the crude mixture was evaporated onto silica gel
and purified by column chromatography using EtOAc as the
eluent to give an off-white glassy solid (6a) (121 mg, 79%), Rf
1
= 0.40 (9:1 EtOAc/MeOH), mp 185−187 °C. H NMR (300
MHz, CD3OD) δ: 0.61 (t, J = 7.2 Hz, 3H), 1.38 (s, 3H), 1.59
(s, 3H), 2.71−2.83 (m, 1H), 2.84−2.98 (m, 3H), 3.43−3.56
(m, 1H), 3.67−3.79 (m, 1H), 4.61 (s, 1H), 5.58 (d, J = 6.0 Hz,
1H), 5.63 (d, J = 6.0 Hz, 1H), 6.22 (s, 1H), 7.13−7.20 (m,
1H), 7.21−7.32 (m, 4H), 7.88 (s, 1H). 13C NMR (75 MHz,
CD3OD) δ: 12.3, 23.8, 25.2, 33.1, 35.1, 42.3, 83.6, 84.1, 87.9,
91.1, 112.8, 113.0, 125.7, 128.0, 128.5, 138.1, 139.8, 151.0,
156.1, 159.3, 170.1. ESMS calcd for C23H30N7O4+ [M + H],
468.5; found, 468.4.
2-Fluoro-N6-(2,2-diphenylethyl)-2′,3′-O-isopropylideneade-
nosine-5′-N-ethylcarboxamide (3). General Procedure for N6-
Substitution. To a stirred solution of 1a (1.0 g, 2.07 mmol) and
DIPEA (1 mL) in MeCN (15 mL) at 0 °C was added 2,2-
diphenylethylamine (65 mg, 3.31 mmol). The solution was
allowed to rise to room temperature, and stirring was continued
for 16 h. The solvent was removed in vacuo, and the residue
partitioned between H2O and CH2Cl2. The aqueous phase was
extracted ×3 with CH2Cl2, the organic phase washed with H2O
and brine, and dried over MgSO4. The solution was filtered, the
solvent removed in vacuo, and the crude mixture loaded onto
silica gel and purified by column chromatography using a
gradient of 1:1 petroleum spirits/EtOAc to EtOAc as the eluent
to yield a pale-yellow solid 3 (825 mg, 73%), Rf = 0.41 (1:5
petroleum spirits/EtOAc), mp 85−91 °C. 1H NMR (300 MHz,
CD3OD) δ: 0.69 (t, J = 7.2 Hz, 3H), 1.42 (s, 3H), 1.59 (s, 3H),
2.84−2.95 (m, 2H), 4.09−4.28 (m, 2H), 4.50 (t, J = 8.1 Hz,
1H), 4.62 (s, 1H), 5.42 (d, J = 6.3 Hz, 1H), 5.56 (d, J = 6.3 Hz,
1H), 6.27 (s, 1H), 7.20−7.24 (m, 2H), 7.28−7.36 (m, 8H),
2-(2-(4-Aminophenyl))ethylamino-2′,3′-O-isopropylidenea-
denosine-5′-N-ethylcarboxamide (6b). General Procedure for
2-Substitution. Compound 2 (180 mg, 0.49 mmol), 4-amino-2-
phenylethylamine (147 mg, 1.08 mmol), DIPEA (300 μL), and
EtOH (5 mL), 70 °C, 3 days, off-white solid (6b) (128 mg,
54%), Rf = 0.23 (9:1 EtOAc/MeOH), mp 90−95 °C. 1H NMR
(300 MHz, CDCl3) δ: 0.74 (t, J = 7.2 Hz, 3H), 1.43 (s, 3H),
1.65 (s, 3H), 2.85 (t, J = 6.6 Hz, 2H), 2.89−3.09 (m, 2H),
3.48−3.61 (m, 1H), 3.62−3.73 (m, 1H), 4.71 (s, 1H), 4.99−
5.05 (m, 1H), 5.49 (br s, 2H), 5.57 (d, J = 6.0 Hz, 1H), 5.66 (d,
J = 6.0 Hz, 1H), 6.07 (br s, 2H), 6.69 (d, J = 7.2 Hz, 2H), 7.08
(d, J = 7.2 Hz, 2H), 7.55 (s, 1H). 13C NMR (75 MHz, CDCl3)
δ: 13.9, 25.0, 26.7, 33.8, 34.7, 43.0, 83.3, 84.0, 87.7, 91.3, 113.6,
114.1, 115.1, 129.3, 129.6, 137.0, 144.9, 151.3, 156.0, 159.5,
168.9. ESMS calcd for C23H31N8O4+ [M + H], 483.5; found,
483.3.
13
8.07 (s, 1H). C NMR (75 MHz, CD3OD) δ: 13.0, 24.1, 25.8
33.4, 44.8, 50.3, 83.4, 83.7, 87.0, 90.9, 113.6, 117.5, 126.3,
127.9, 128.3, 140.6, 142.2, 149.34 (d, J = 19.7 Hz), 156.10 (d, J
= 19.7 Hz), 159.10 (d, J = 208.0 Hz). ESMS calcd for
C29H32N6O4F+ [M + H], 547.6; found, 548.1.
2-(2-(4-Bromophenyl))ethylamino-2′,3′-O-isopropylidenea-
denosine-5′-N-ethylcarboxamide (6c). General Procedure for
2-Substitution. Compound 2 (140 mg, 0.38 mmol), 4-bromo-2-
phenylethylamine (169 mg, 0.84 mmol), DIPEA (300 μL), and
EtOH (3 mL), 70 °C, 6 days, off-white solid (6c) (199 mg,
2-Fluoroadenosine-5′-N-ethylcarboxamide (4). General
Procedure for Isopropylidene Deprotection. To a stirred solution
of 2 (250 mg, 0.68 mmol) in MeCN (4 mL) was added 1 M
HCl (20 mL). The flask was fitted with a reflux condenser, and
the solution was heated to 50 °C for 6 h. The solution was
allowed to cool and the pH adjusted to 8 with satd NaHCO3
solution, which produced a white precipitate. The solution was
extracted with EtOAc (×3), the organic phase washed with
H2O and brine, dried over MgSO4, filtered, and the crude
material dried onto SiO2 and purified via column chromatog-
raphy using a gradient of EtOAc to 9:1 EtOAc/MeOH to yield
a white solid (4) (96 mg, 43%), Rf = 0.26 (9:1 EtOAc/MeOH),
1
95%), Rf = 0.43 (9:1 EtOAc/MeOH), mp 113−118 °C. H
NMR (300 MHz, CDCl3) δ: 0.69 (t, J = 7.5 Hz, 3H), 1.40 (s,
3H), 1.63 (s, 3H), 2.80−3.02 (m, 4H), 3.48−3.61 (m, 1H),
3.63−3.77 (m, 1H), 4.71 (s, 1H), 5.21−5.25 (m, 1H), 5.55 (d, J
= 4.8 Hz, 1H), 5.68 (s, 1H), 5.76 (br s, 2H), 6.04 (br s, 1H),
6.08 (s, 1H), 7.16 (d, J = 7.5 Hz, 2H), 7.43 (d, J = 7.5 Hz, 2H),
7.54 (s, 1H). 13C NMR (100 MHz, CD3OD) δ: 14.0, 25.4, 27.0,
34.8, 36.2, 43.6, 79.5, 85.0, 85.5, 89.3, 92.5, 114.4, 114.5, 120.8,
131.9, 132.4, 139.6, 140.5, 152.4, 157.4, 160.8, 171.6. ESMS
calcd for C23H29N7O4Br+ [M + H], 546.5; found, 546.2.
1
mp 233−240 °C (dec). H NMR (400 MHz, DMSO-d6) δ:
1.03 (t, J = 4.8 Hz, 3H), 3.12−3.20 (m, 2H), 4.14 (br s, 1H),
4.29 (s, 1H), 4.53 (dd, J = 2.8, 4.8 Hz, 1H), 5.59 (d, J = 4.0 Hz,
1H), 5.72 (d, J = 2.8 Hz, 1H), 5.87 (d, J = 4.8 Hz, 1H), 7.81−
8.04 (m, 2H), 8.39 (s, 1H), 8.45 (m, 1H). 13C NMR(100 MHz,
DMSOd6) δ: 14.6, 33.2, 72.2, 73.0, 84.4, 87.6, 117.9, 150.3 (d, J
= 19.8 Hz), 157.7 (d, J = 20.9 Hz), 158.4 (d, J = 203.3 Hz),
170.0. HRMS calcd for C12H16N6O4F+ [M + H], 327.1212;
found, 327.1203.
2-(2-Phenyl)ethylamino-N6-(2,2-diphenylethyl)-2′,3′-isopro-
pylideneadenosine-5′-N-ethyl carboxamide (7a). General
Procedure for 2-Substitution. Compound 3 (200 mg, 0.37
mmol), 2-phenylethylamine (98 mg, 0.81 mmol), DIPEA (300
μL), and EtOH (3 mL), 70 °C, 4 days, white solid foam (7a)
(147 mg, 63%), Rf = 0.47 (1:5 petroleum spirits/MeOH), mp
58−64 °C. 1H NMR (400 MHz, CDCl3) δ: 0.68 (t, J = 7.2 Hz,
3528
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Journal of Medicinal Chemistry
Article
3H), 1.37 (s, 3H), 1.58 (s, 3H), 2.82−2.90 (m, 1H), 2.94−3.03
(m, 3H), 3.56−3.65 (br m, 1H), 3.68−3.76 (br m, 1H), 4.14−
4.28 (br m, 2H), 4.32−4.39 (br m, 1H), 4.64 (s, 1H), 4.93 (br
s, 1H), 5.46 (br s, 1H), 5.60−5.64 (m, 1H), 5.97 (s, 1H), 6.04
(br s, 1H), 7.18−7.34 (m, 16H), 7.39 (s, 1H). 13C NMR (75
MHz, CDCl3) δ: 14.3, 25.1, 26.8, 33.8, 36.1, 43.1, 45.0, 50.7,
83.4, 84.1, 87.7, 91.3, 113.6, 114.4, 126.3, 126.8, 128.2, 128.6,
128.8, 128.8, 128.9, 136.3, 139.6, 142.2, 154.9, 159.5, 169.1.
ESMS calcd for C37H42N7O4+ [M + H], 648.8; found, 648.4.
2-(2-(4-Aminophenyl))ethylamino-N6-(2,2-diphenylethyl)-
adenosine-2′,3′-isopropylidene-5′-N-ethylcarboxamide
(7b). General Procedure for 2-Substitution. Compound 3 (200
mg, 0.37 mmol), 2-(4-aminophenyl)ethylamine (110 mg, 0.81
mmol), DIPEA (300 μL), and EtOH (3 mL), 70 °C, pale-
yellow solid (7b) (195 mg, 80%), Rf = 0.35 (1:5 petroleum
spirits/EtOAc), mp 54−61 °C. 1H NMR (300 MHz, CDCl3) δ:
0.75 (t, J = 7.2 Hz, 3H), 1.44 (s, 3H), 1.65 (s, 3H), 2.88−3.10
(m, 4H), 3.53−3.63 (m, 1H), 3.65−3.76 (m, 1H), 4.26 (br s,
2H), 4.42 (t, J = 7.2 Hz, 1H), 4.70 (s, 1H), 5.01 (br s, 1H), 5.54
(br s, 1H), 5.65 (d, J = 5.7 Hz, 1H), 5.74 (br s, 1H), 6.02 (s,
1H), 6.16 (br s, 1H), 6.67 (d, J = 7.5 Hz, 2H), 7.07 (d, J = 7.5
Hz, 2H), 7.22−7.39 (m, 10H), 7.42 (s, 1H). 13C NMR (75
MHz, CDCl3) δ: 14.0, 25.1, 26.8, 33.8, 35.1, 43.4, 44.9, 50.7,
83.3, 84.0, 87.6, 91.4, 113.7, 114.4, 115.4, 126.8, 128.2, 128.7,
129.5, 129.7, 136.2, 142.2, 144.7, 150.5, 154.9, 159.6, 169.0.
2-(2-(4-Bromophenyl))ethylamino-N6-(2,2-diphenylethyl)-
2′,3′-isopropylideneadenosine-5′-N-ethylcarboxamide (7c).
General Procedure for 2-Substitution. Compound 3 (181 mg,
0.33 mmol), 2-(4-bromophenyl)ethylamine (140 mg, 0.70
mmol), DIPEA (300 μL), and EtOH (3 mL), 70 °C, 7 days,
white solid (7c) (142 mg, 59%), Rf = 0.55 (1:5 petroleum
spirits/EtOAc), mp 62−65 °C. 1H NMR (300 MHz, CDCl3) δ:
0.73 (t, J = 7.2 Hz, 3H), 1.43 (s, 3H), 1.65 (s, 3H), 2.95 (t, J =
6.6 Hz, 2H), 2.87−3.08 (m, 2H), 3.59−3.78 (m, 2H), 4.26 (br
s, 2H), 4.41 (t, J = 7.2 Hz, 1H), 4.71 (s, 1H), 5.00 (br s, 1H),
5.52 (br s, 1H), 5.68 (d, 1H), 6.04 (s, 1H), 7.15 (d, J = 7.5 Hz,
2H), 7.25−7.40 (m, 10H), 7.44 (d, J = 7.5 Hz, 2H), 7.45 (s,
1H). 13C NMR (75 MHz, CD3OD) δ: 14.0, 25.1, 26.8, 33.8,
35.5, 42.9, 44.9, 50.7, 83.5, 84.0, 87.8, 91.4, 113.6, 114.5, 120.1,
126.9, 128.1, 128.8, 130.6, 131.6, 136.3, 138.6, 142.1, 154.9,
159.4, 169.1. ESMS calcd for C37H41N7O4Br+ [M + H], 726.7;
found, 726.3.
2-(2-(4-Bromophenyl))ethylaminoadenosine-5′-N-ethylcar-
boxamide (8c). General Procedure for Isopropylidene Depro-
tection. Compound 6c (180 mg, 0.33 mmol), MeCN (2 mL),
and 1 M HCl (10 mL), 50 °C, 6 h, white solid (8c) (70 mg,
42%), Rf = 0.21 (9:1 EtOAc/MeOH), mp 80−85 °C. 1H NMR
(400 MHz, CD3OD) δ: 1.04 (t, J = 7.2 Hz, 3H), 2.88 (t, J = 7.2
Hz, 2H), 3.09−3.19 (m, 1H), 3.23−3.33 (m, 1H), 3.47−3.56
(m, 1H), 3.59−3.68 (m, 1H), 4.42 (d, J = 2.9 Hz, 1H), 4.48−
4.53 (m, 1H), 5.01 (dd, J = 5.2, 6.6 Hz, 1H), 5.96 (d, J = 6.6
Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H),
8.01 (s, 1H). 13C NMR (75 MHz, CD3OD) δ: 14.7, 35.2, 36.3,
44.0, 73.4, 74.7, 85.5, 90.0, 114.8, 120.8, 132.0, 132.5, 139.1,
140.5, 153.1, 157.5, 161.1, 172.0. HRMS calcd for
C20H25N7O4Br+ [M + H], 506.1146; found, 506.1165.
2-(2-Phenyl)ethylamino-N6-(2,2-diphenylethyl)adenosine-
5′-N-ethylcarboxamide (9a). General Procedure for Isopropyli-
dene Deprotection. Compound 7a (113 mg, 0.18 mmol),
MeCN (5 mL), and 1 M HCl (12 mL), 50 °C, 4 h, white solid
(9a) (23 mg, 21%), Rf = 0.07 (1:5 petroleum spirits/EtOAc),
1
mp 111−115 °C. H NMR (400 MHz, CDCl3) δ: 0.91 (t, J =
7.2 Hz, 3H), 2.79 (t, J = 7.2 Hz, 2H), 2.89−3.02 (m, 1H),
3.14−3.26 (m, 1H), 3.43−3.64 (m, 2H), 4.02−4.23 (m, 2H),
4.30 (t, J = 7.8 Hz, 1H), 4.39−4.43 (m, 1H), 4.54 (br s, 1H),
4.67−4.72 (m, 1H), 4.87 (br s, 1H), 5.67 (d, J = 6.0 Hz, 1H),
5.78 (br s, 1H), 7.05−7.24 (m, 16H), 7.35 (br s, 2H). 13C
NMR (100 MHz, CDCl3) δ: 14.6, 34.1, 36.0, 43.4, 44.9, 50.7,
72.3, 73.3, 84.2, 89.6, 114.7, 126.4, 126.8, 128.1, 128.6, 128.7,
136.3, 139.3, 142.1, 154.9, 159.3, 170.0. HRMS calcd for
C34H38N7O4+ [M + H], 608.2980; found, 608.3002.
2-(2-(4-Aminophenyl))ethylamino-N6-(2,2-diphenylethyl)-
adenosine-5′-N-ethylcarboxamide (9b). General Procedure for
Isopropylidene Deprotection. Compound 7b (190 mg, 0.29
mmol), MeCN (3 mL), and 1 M HCl (15 mL), 50 °C, 4 h,
light-tan foam (9b) (164 mg, 92%), Rf = 0.05 (1:5 petroleum
spirits/EtOAc), m: 90−96 °C. 1H NMR (300 MHz, CDCl3) δ:
1.05 (t, J = 7.2 Hz, 3H), 2.80 (br t, J = 6.9 Hz, 2H), 3.00−3.12
(m, 1H), 3.28−3.42 (m, 1H), 3.50−3.66 (br m, 2H), 4.11−4.36
(br m, 2H), 4.44 (t, J = 7.5 Hz, 1H), 4.54 (s, 1H), 4.62 (br s,
1H), 4.83−4.86 (m, 1H), 4.96−5.00 (m, 1H), 5.75 (d, J = 6.3
Hz, 1H), 5.99 (br s, 1H), 6.61 (d, J = 8.4 Hz, 2H), 6.98 (d, J =
8.4 Hz, 2H), 7.20−7.38 (m, 10H, Ar-H), 7.41 (s, 1H, NCHN).
13C NMR (75 MHz, CDCl3) δ: 14.7, 34.2, 35.1, 43.6, 45.0, 50.7,
71.8, 73.2, 84.3, 89.4, 114.6, 115.6, 126.8, 128.2, 128.7, 129.2,
129.6, 136.7, 142.2, 144.7, 150.5, 154.9, 159.5, 170.1. HRMS
calcd for C34H39N8O4+ [M + H], 623.3089; found, 623.3105.
2-(2-(4-Bromophenyl))ethylamino-N6-(2,2-diphenylethyl)-
adenosine-5′-N-ethylcarboxamide (9c). General Procedure for
Isopropylidene Deprotection. Compound 7c (142 mg, 0.20
mmol), MeCN (3 mL), and 1 M HCl (10 mL), 50 °C, 2.5 h,
light-tan glassy solid (9c) (43 mg, 32%), Rf = 0.16 (1:5
2-(2-Phenyl)ethylaminoadenosine-5′-N-ethylcarboxamide14
(8a). General Procedure for Isopropylidene Deprotection.
Compound 6a (120 mg, 0.26 mmol), MeCN (2 mL), and 1
M HCl (20 mL), 50 °C, 12 h, pale-yellow solid (8a) (88 mg,
82%), Rf = 0.28 (9:1 EtOAc/MeOH), mp 110−114 °C (lit.
115−118 °C dec14). 1H NMR (300 MHz, CD3OD) δ: 1.06 (t, J
= 7.2 Hz, 3H), 2.90 (t, J = 7.2 Hz, 2H), 3.07−3.18 (m, 1H),
3.20−3.34 (m, 1H), 3.46−3.68 (m, 2H), 4.40 (d, J = 2.7 Hz,
1H), 4.48−4.52 (m, 1H), 5.00−5.05 (m, 1H), 5.93 (d, J = 6.3
Hz, 1H), 7.12−7.19 (m, 1H), 7.20−7.30 (m, 5H), 7.99 (s, 1H).
13C NMR (75 MHz, CD3OD) δ: 13.4, 33.8, 35.5, 42.9, 72.0,
1
petroleum spirits/EtOAc), mp 97−101 °C. H NMR (400
MHz, CD3OD) δ: 1.06 (t, J = 7.2 Hz, 3H), 2.88 (t, J = 7.3 Hz,
2H), 3.10−3.20 (m, 1H), 3.27−3.37 (m, 1H), 3.51−3.70 (m,
2H), 4.21 (br d, J = 6.9 Hz, 2H), 4.42 (d, J = 2.6 Hz, 1H),
4.44−4.52 (m, 2H), 4.95 (dd, J = 5.2, 6.3 Hz, 1H), 5.90 (d, J =
6.6 Hz, 1H), 7.06 (d, J = 8.3 Hz, 2H), 7.17−7.22 (m, 2H),
7.24−7.35 (m, 10H), 7.83 (s, 1H). 13C NMR (75 MHz,
CD3OD) δ: 13.5, 33.8, 73.4, 78.1, 84.2, 88.5, 113.6, 119.4,
126.2, 127.9, 128.3, 130.4, 131.1, 136.9, 139.1, 142.7, 150.9,
154.6, 159.7, 170.7. HRMS calcd for C34H37N7O4Br+ [M + H],
686.2085; found, 686.2090.
73.3, 84.0, 88.6, 113.4, 125.8, 128.1, 128.5, 137.7, 139.7, 151.7,
156.1, 159.7, 170.6. HRMS calcd for C20H26N7O4+ [M + H],
428.2041; found, 428.2058.
2-(2-(4-Aminophenyl))ethylaminoadenosine-5′-N-ethylcar-
boxamide (8b). General Procedure for Isopropylidene Depro-
tection. Compound 6b (128 mg, 0.27 mmol) and 1 M HCl (15
mL), 50 °C, 4 h, tan solid (8b) (46 mg, 39%), Rf = 0.1 (9:1
EtOAc/MeOH), mp 95−104 °C. 1H NMR (400 MHz,
CD3OD) δ: 1.05 (t, J = 7.2 Hz, 3H), 2.77 (t, J = 7.2 Hz,
2H), 3.08−3.18 (m, 1H), 3.24−3.33 (m, 1H), 3.42−3.51 (m,
1H), 3.55−3.64 (m, 1H), 4.42 (d, J = 2.8 Hz, 1H), 4.48−4.53
(m, 1H), 5.00−5.05 (m, 1H), 5.94 (d, J = 6.3 Hz, 1H), 6.70 (d,
J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 7.98 (s, 1H). 13C
NMR (400 MHz, CD3OD) δ: 14.7, 35.2, 36.1, 44.6, 73.2, 74.7,
85.5, 90.1, 114.7, 117.1, 130.5, 130.8, 139.2, 146.6, 153.1, 157.5,
N-tert-Butoxycarbonyl-2-(4-aminophenyl)ethylamine (11).
To a stirred solution of 2-(4-aminophenyl)ethylamine (1.20 g, 8.82
mmol) in CH2Cl2 (120 mL) cooled to −5 °C was added tert-
butoxycarbonyl anhydride (1.73 g, 8.09 mmol). The solution was
stirred for 45 min at 0 °C and then poured quickly into a 500 mL
separatory funnel charged with 80 mL ice/water. The organic phase
was washed ×3 with ice/water, twice with satd NaCl solution, dried
over MgSO4, filtered, and solvent removed in vacuo. The product was
purified via flash column chromatography (5:1 petroleum spirits/
EtOAc) to give an orange crystalline solid (11) (1.51 g, 72%), Rf =
+
161.2, 172.0. HRMS calcd for C20H27N8O4 [M + H],
443.2150; found, 443.2162.
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1
0.46 (1:1 petroleum spirits/EtOAc), mp 55−60 °C. H NMR (400
MHz, CDCl3) δ: 1.35 (s, 9H), 2.58 (t, J = 6.9 Hz, 2H), 3.21−3.25 (m,
2H), 3.53 (br s, 2H), 4.58 (br s, 1H), 6.53 (d, J = 8.2 Hz, 2H), 6.88 (d,
J = 8.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ: 28.4, 35.2, 42.0,
6.25 (s, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H),
7.69 (s, 1H), 7.81 (s, 2H), 7.87 (s, 1H).
2-(2-(4-N-(3,5-di-tert-Butyl-4-hydroxyphenylcarboxy)-
aminophenyl))ethylamino-2′,3′-isopropylideneadenosine-5′-
N-ethylcarboxamide (13b). General Procedure for 2-Substitu-
tion. Compound 2 (120 mg, 0.33 mmol), 12b (254 mg, 0.69
mmol), DIPEA (200 μL), and EtOH (2 mL), 3 days, 60 °C,
pale yellow−orange solid (13b) (151 mg, 64%), Rf = 0.27 (9:1
EtOAc/MeOH), mp 163−167 °C. 1H NMR (300 MHz,
CDCl3) δ: 0.73 (t, J = 7.2 Hz, 3H), 1.43 (s, 3H), 1.52 (s, 18H),
1.64 (s, 3H), 2.87−3.08 (m, 4H), 3.50−3.64 (m, 1H), 3.66−
3.78 (m, 1H), 4.71 (s, 1H), 5.09−5.15 (m, 1H), 5.52−5.73 (m,
4H), 6.08 (br s, 2H), 7.27 (d, J = 7.8 Hz, 2H), 7.53 (s, 1H),
+
79.0, 115.3, 128.8, 129.6, 144.9, 156.0. ESMS calcd for C13H21N2O2
[M + H], 237.3; found, 237.4
2-(4-N-(3,5-di-tert-Butylphenylcarboxy)aminophenyl)-
ethylamine (12a). General Procedure for Amide Coupling.
Compound 11 (250 mg, 1.06 mmol), 3,5-di-tert-butylbenzoic
acid (274 mg, 1.17 mmol), EDCI (230 mg, 1.20 mmol), HOBt
(160 mg, 1.20 mmol), and DIPEA (250 μL) were combined in
DMF (2.5 mL) and stirred at room temperature for 16 h. The
solution was poured into 50 mL of H2O and the resulting
precipitate filtered, rinsed with H2O, and dried in vacuo to give
N-tert-butoxycarbonyl-2-(4-N-(3,5-di-tert-butylphenylcarboxy)-
aminophenyl)ethylamine as an off-white solid (424 mg, 88%),
13
7.61 (d, J = 7.8 Hz, 2H), 7.75 (s, 2H), 8.03 (s, 1H). C NMR
(75 MHz, CDCl3) δ: 14.0, 25.1, 26.8, 30.2, 33.8, 34.5, 35.1,
42.8, 83.4, 84.0, 87.8, 91.4, 113.7, 114.2, 120.7, 124.4, 126.2,
129.3, 135.4, 136.2, 136.7, 137.1, 151.3, 155.8, 157.1, 159.4,
166.6, 168.9. ESMS calcd for C38H51N8O6+ [M + H], 715.9;
found, 715.3.
1
Rf = 0.79 (1:1 petroleum spirits/EtOAc), mp 177−185 °C. H
NMR (300 MHz, DMSO-d6) δ: 1.34 (s, 18H), 1.38 (s, 9H),
2.67 (t, J = 7.5 Hz, 2H), 3.04−3.16 (m, 2H), 6.89−6.95 (m,
1H), 7.17 (d, J = 8.1 Hz, 2H), 7.60 (s, 1H), 7.65 (d, J = 8.1 Hz,
2H), 7.73 (s, 2H), 10.10 (s, 1H). 13C NMR (75 MHz, CDCl3)
δ: 28.4, 28.5, 31.3, 31.4, 35.1, 77.3, 120.6, 121.2, 126.1, 129.4,
134.8, 135.2, 136.5, 151.6, 155.9, 166.9. ESMS calcd for
C28H41N2O3+ [M + H], 453.6; found, 453.6.
2-(2-(4-N-(4-(1,2-di-Thiolane)pentanecarboxy)-
aminophenyl))ethylamino-2′,3′-isopropylideneadenosine-5′-
N-ethylcarboxamide (13c). General Procedure for Amide
Coupling. Compound 6b (59 mg, 0.12 mmol), EDCI (28 mg,
0.19 mmol), HOBt (19.8 mg, 0.15 mmol), 4-(1,2-dithiolane)-
pentanoic acid (30 mg, 0.15 mmol), DIPEA (350 μL), and
DMF (3.5 mL). Crude product evaporated onto SiO2, the
solvent removed and the product purified via column
chromatography using 85:15 EtOAc/MeOH as eluent to afford
a pale-yellow solid foam (13c) (50 mg, 61%), Rf = 0.41 (9:1
EtOAc/MeOH), mp 100−105 °C. 1H NMR (300 MHz,
CD3OD): 0.61 (t, J = 7.2 Hz, 3H), 1.38 (s, 3H), 1.43−1.60 (m,
2H), 1.57 (s, 3H), 1.62−1.80 (m, 4H), 1.82−1.96 (m, 1H),
2.39 (t, J = 6.6 Hz, 2H), 2.38−2.51 (m, 1H), 2.70−2.85 (m,
1H), 2.86−2.96 (m, 3H), 3.01−3.22 (m, 2H), 3.40−3.52 (m,
1H), 3.53−3.63 (m, 1H), 3.64−3.78 (m, 1H), 4.63 (s, 1H),
5.59 (d, J = 5.2 Hz, 1H), 5.75 (d, J = 5.2 Hz, 1H), 6.24 (s, 1H),
7.25 (d, J = 7.5 Hz, 2H), 7.50 (d, J = 7.5 Hz, 2H), 7.86 (s, 1H).
13C NMR (75 MHz, CD3OD): 12.6, 24.0, 25.3, 25.6, 28.6, 33.4,
N-tert-Butoxycarbonyl-2-(4-N-(3,5-di-tert-butylphenylcarboxy)-
aminophenyl)ethylamine (424 mg, 0.94 mmol) was dissolved in
CH2Cl2 (8.0 mL), and TFA (3.3 mL) was added slowly. The solution
was stirred for 3 h and followed by TLC. Upon completion, the pH of
the solution was adjusted to 8 via the dropwise addition of satd
NaHCO3 solution, then to pH = 10 with 4 M NaOH solution. The
resulting precipitate was removed via filtration, and the filtrate
extracted ×3 with EtOAc. The precipitate was added to the organic
phase, washed with water and satd NaCl, dried over MgSO4, filtered,
and the solvent removed to give a tan solid (12a) (386 mg, 98%), Rf =
0.00 (EtOAc), mp 175−180 °C (decomp). 1H NMR (300 MHz,
CD3OD) δ: 1.41 (s, 18H), 2.81 (t, J = 7.2 Hz, 2H), 2.91 (t, J = 7.2 Hz,
2H), 7.26 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 7.69 (s, 1H),
7.80 (s, 2H), 10.09 (s, 1H). 13C NMR (DMSO-d6, 75 MHz) δ: 31.6,
31.7, 33.5, 35.2, 121.4, 122.1, 123.2, 125.8, 129.3, 133.1, 135.0, 138.3,
151.0. ESMS calcd for C23H33N2O+ [M + H], 353.5; found, 353.3.
2-(4-N-(3,5-di-tert-Butyl-4-hydroxyphenylcarboxy)-
aminophenyl)ethylamine (12b). General Procedure for Amide
Coupling. Compound 11 (300 mg, 1.27 mmol), 3,5-di-tert-
butyl-4-hydroxybenzoic acid (362 mg, 1.45 mmol), EDCI (270
mg, 1.41 mmol), HOBt (1.41 mmol), DIPEA (300 μL), and
34.4, 34.9, 36.4, 38.0, 39.9, 42.5, 56.2, 83.6, 84.1, 87.9, 91.1,
113.1, 120.0, 128.8, 135.6, 136.6, 138.1, 151.0, 156.0, 159.4,
170.2, 172.8. ESMS calcd for C31H43N8O5S2+ [M + H], 671.9;
found, 671.3.
2-(2-(4-N-(3,5-di-tert-Butyl-4-hydroxyphenylcarboxy)-
aminophenyl))ethylamino-N6-(2,2-diphenylethyl)-2′,3′-isopro-
pylideneadenosine-5′-N-ethylcarboxamide (14b). General
Procedure for 2-Substitution. Compound 3 (250 mg, 0.46
mmol), 12b (370 mg, 1.01 mmol), DIPEA (350 μL), and
EtOH (3 mL), 70 °C, tan solid (14b) (186 mg, 45%), Rf = 0.61
1
DMF (3 mL), yellow−orange solid (475 mg, 80%). H NMR
(300 MHz, CDCl3) δ: 1.28 (s, 18H), 1.36 (s, 9H), 2.70 (t, J =
6.8 Hz, 2H), 3.22−3.34 (m, 2H), 4.51 (br s, 1H), 7.10 (d, J =
8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.53 (t, J = 1.8 Hz, 1H),
7.60 (s, 2H), 7.85 (s, 1H).
1
(1:5 petroleum spirits/EtOAc). H NMR (400 MHz, CDCl3)
δ: 0.62 (t, J = 7.2 Hz, 3H), 1.31 (s, 3H), 1.42 (s, 18H), 1.51 (s,
3H), 2.74−2.98 (m, 3H), 3.09−3.23 (m, 1H,), 3.46−3.58 (m,
1H), 3.58−3.70 (m, 1H), 4.08−4.21 (br m, 2H), 4.24−4.32 (m,
1H), 4.57 (d, J = 1.8 Hz, 1H), 4.85 (br s, 1H), 5.42 (br s, 1H),
5.53 (d, J = 1.4 Hz, 1H), 5.54 (s, 1H) 5.91 (s, 1H), 5.97 (br s,
1H), 7.10−7.29 (m, 12H), 7.33 (s, 1H), 7.47 (d, J = 8.2 Hz,
2H), 7.60 (s, 1H), 7.61 (s, 2H). ESMS calcd for C52H63N8O6+
[M + H], 896.1; found, 895.9.
2-(2-(4-N-(3,5-di-tert-Butylphenylcarboxy)aminophenyl))-
ethylaminoadenosine-5′-N-ethylcarboxamide (15a). General
Procedure for Isopropylidene Deprotection. Compound 13a (108
mg, 0.15 mmol) and 1 M HCl (12 mL), 60 °C, 8 h, off-white
solid foam (15a) (74 mg, 75%), Rf = 0.36 (9:1 EtOAc/MeOH),
mp 142−148 °C. 1H NMR (300 MHz, CD3OD) δ: 1.07 (t, J =
7.2 Hz, 3H), 1.41 (s, 18H), 2.93 (t, J = 7.2 Hz, 2H), 3.08−3.20
(m, 1H), 3.22−3.34 (m, 1H), 3.50−3.61 (m, 1H), 3.62−3.73
(m, 1H), 4.41 (d, J = 3.3 Hz, 1H), 4.49−4.56 (m, 1H), 5.07−
5.12 (m, 1H), 5.97 (d, J = 6.3 Hz, 1H), 7.29 (d, J = 8.4 Hz,
2H), 7.63 (d, J = 8.4 Hz, 2H), 7.70 (t, J = 1.5 Hz, 1H), 7.80 (d,
J = 1.5 Hz, 2H), 8.00 (s, 1H). 13C NMR (75 MHz, CD3OD) δ:
13.3, 30.4 (6C), 33.8, 34.6, 35.0, 42.9, 72.0, 73.3, 84.0, 88.7,
113.4, 121.4, 121.6, 125.6, 128.8, 134.4, 136.1, 136.6, 137.7,
N-tert-Butoxycarbonyl-2-(4-N-(3, 5-di-tert-butyl-4-
hydroxyphenylcarboxy)aminophenyl) ethylamine (475 mg, 1.02
mmol), CH2Cl2 (9.0 mL), and TFA (1.6 mL), red−orange glassy
solid (12b) (389 mg, 81%). 1H NMR (100 MHz, CD3OD) δ: 1.38 (s,
18H), 2.86 (t, J = 7.2 Hz, 2H), 3.09 (t, J = 7.2 Hz, 2H), 7.17 (d, J = 8.4
Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.68 (s, 2H), 7.81 (s, 1H). 13C
NMR (100 MHz, CD3OD) δ: 27.7, 30.6, 34.1, 35.7, 42.0, 123.2, 126.0,
+
126.8, 130.1, 133.9, 138.0, 138.7, 139.3. ESMS calcd for C23H33N2O2
[M + H], 369.5; found, 369.3.
2-(2-(4-N-(3,5-di-tert-Butylphenylcarboxy)aminophenyl))-
ethylamino-2′,3′-isopropylidene adenosine-5′-N-ethylcarbox-
amide (13a). General Procedure for 2-Substitution. Compound
2 (120 mg, 0.33 mmol), 12a (242 mg, 0.69 mmol), DIPEA
(250 μL), and EtOH (2.5 mL), 70 °C, 5 days, light-yellow solid
foam (13a) (108 mg, 47%), Rf = 0.48 (9:1 EtOAc/MeOH), mp
102−108 °C. 1H NMR (300 MHz, CD3OD) δ: 0.63 (t, J = 7.2
Hz, 3H), 1.41(s, 18H), 1.41 (s, 3H), 1.58 (s, 3H), 2.74−2.85
(m, 1H), 2.87−3.00 (m, 3H), 3.47−3.59 (m, 1H), 3.70−3.81
(m, 1H), 4.63 (s, 1H), 5.55−5.61 (m, 1H), 5.73−5.79 (m, 1H),
3530
dx.doi.org/10.1021/jm300206u | J. Med. Chem. 2012, 55, 3521−3534

Journal of Medicinal Chemistry
Article
151.0, 151.7, 156.1, 159.7, 168.4, 170.6. HRMS calcd for
C35H47N8O5+ [M + H], 659.3664; found, 659.3676.
128.4, 129.7, 130.6, 132.0, 132.6, 133.1, 139.3, 140.1, 140.5,
140.6, 146.1, 154.3, 158.7, 161.0, 163.4, 171.4, 174.2. HRMS
calcd for C49H59N8O6+ [M + H], 855.4552; found, 855.4538.
2-(2-(4-N-(4-(1, 2-Dithiolane)pentanecarboxy)-
aminophenyl))ethylamino-N6-(2,2-diphenylethyl)adenosine-
5′-N-ethylcarboxamide (16c). General Procedure for Amide
Coupling. Compound 7b (160 mg, 0.26 mmol), 4-(1,2-
dithiolane)pentanoic acid (79 mg, 0.38 mmol), EDCI (73
mg, 0.38 mmol), HOBt (52 mg, 0.38 mmol), DIPEA (250 μL),
and DMF (2.5 mL), white solid (16c) (31 mg, 15%), Rf = 0.24
2-(2-(4-N-(3,5-di-tert-Butyl-4-hydroxyphenylcarboxy)-
aminophenyl))ethylaminoadenosine-5′-N-ethylcarboxamide
(15b). General Procedure for Isopropylidene Deprotection.
Compound 13b (150 mg, 0.21 mmol), MeCN (2 mL), and
1 M HCl (20 mL), 65 °C, 12 h, white solid (15b) (25 mg,
18%), Rf = 0.10 (9:1 EtOAc/MeOH), mp 230−235 °C (dec).
1H NMR (300 MHz, CD3OD) δ: 1.06 (t, J = 7.2 Hz, 3H), 2.90
(t, J = 7.2 Hz, 2H), 3.08−3.21 (m, 1H), 3.22−3.36 (m, 1H),
3.48−3.59 (m, 1H), 3.60−3.73 (m, 1H), 4.43 (s, 1H), 4.49−
4.54 (m, 1H), 5.02−5.06 (m, 1H), 5.96 (d, J = 6.3 Hz, 1H),
7.26 (d, J = 7.8 Hz, 2H), 7.59 (d, J = 7.8 Hz, 2H), 7.78 (s, 2H),
8.00 (s, 1H). 13C NMR (100 MHz, CD3OD) δ: 14.8, 30.6, 35.2,
35.7, 36.4, 44.3, 73.3, 74.7, 85.5, 90.1, 114.8, 122.8, 125.9, 12
7.0, 130.2, 137.3, 138.3, 138.7, 139.1, 153.1, 157.5, 158.9, 161.2,
1
(1:5 petroleum spirits/EtOAc), mp 107−112 °C. H NMR
(400 MHz, CD3OD) δ: 0.96 (t, J = 7.2 Hz, 3H), 1.37−1.52 (m,
2H), 1.55−1.72 (m, 4H), 1.75−1.86 (m, 1H), 2.28 (t, J = 7.4
Hz, 2H), 2.31−2.41 (m, 1H), 2.80 (t, J = 7.2 Hz, 2H), 2.93−
3.11 (m, 3H), 3.17−3.28 (m, 1H), 3.45−3.53 (m, 2H), 3.52−
3.64 (m, 1H), 4.12 (d, J = 7.1 Hz, 2H), 4.31 (d, J = 2.6 Hz,
1H), 4.32−4.36 (br m, 1H), 4.39 (t, J = 7.8 Hz, 1H), 4.82 (dd, J
= 5.2, 6.4 Hz, 1H), 5.76 (d, J = 6.6 Hz, 1H), 7.04 (d, J = 8.5 Hz,
2H), 7.07−7.13 (m, 2H), 7.15−7.24 (m, 8H), 7.35 (d, J = 8.5
Hz, 2H), 7.68 (s, 1H). 13C NMR (100 MHz, CD3OD) δ: 17.3,
29.2, 32.5, 37.7, 38.3, 38.9, 40.3, 41.9, 43.8, 46.8, 60.1, 75.8,
77.2, 88.0, 92.6, 117.3, 124.0, 132.8, 139.5, 140.5, 141.7, 155.6,
160.0, 163.6, 174.6, 176.9. HRMS calcd for C42H51N8O5S2+ [M
+ H], 811.3418; found, 811.3427.
+
170.1, 172.1. HRMS calcd for C35H47N8O6 [M + H],
675.3613; found, 675.3632.
2-(2-(4-N-(4-(1,2-Dithiolane)pentanecarboxy)-
aminophenyl))ethylaminoadenosine-5′-N-ethylcarboxamide
(15c). General Procedure for Isopropylidene Deprotection.
Compound 13c (50 mg, 0.08 mmol), MeCN (4 mL), and 1
M HCl (10 mL), 55 °C, 2 h, pale-yellow solid (15c) (13 mg,
28%), Rf = 0.10 (9:1 EtOAc/MeOH), mp 82−85 °C. 1H NMR
(400 MHz, CD3OD) δ: 1.05 (t, J = 7.2 Hz, 3H), 1.47−1.60 (m,
2H), 1.64−1.83 (m, 4H,), 1.86−1.96 (m, 1H), 2.39 (t, J = 7.2
Hz, 2H), 2.43−2.53 (m, 1H), 2.87 (t, J = 7.2 Hz, 2H), 3.07−
3.23 (m, 3H), 3.24−3.33 (m, 1H), 3.48−3.58 (m, 1H), 3.58−
3.69 (m, 2H), 4.42 (d, J = 2.8 Hz, 1H), 4.49−4.54 (m, 1H),
5.01 (t, J = 6.4 Hz, 1H), 5.95 (d, J = 6.4 Hz, 1H), 7.22 (d, J =
2-Fluoro-N6-(2,2-diphenylethyl)-2′,3′-O-isopropylideneade-
nosine-5′-N-methylcarboxamide (17). General Procedure for
N6-Substitution. Compound 1b (225 mg, 0.48 mmol), 2,2-
diphenylethylamine (142 mg, 0.72 mmol), DIPEA (250 μL),
and MeCN (15 mL), yellow solid (17) (203 mg, 80%), Rf =
1
0.38 (1:5 petroleum spirits/EtOAc); mp 92−95 °C. H NMR
13
(400 MHz, CD3OD) δ: 1.38 (s, 3H), 1.56 (s, 3H), 2.34 (s,
3H), 4.13−4.21 (m, 2H), 4.47 (t, 1H, J = 8.0 Hz), 4.60 (d, J =
1.2 Hz, 1H), 5.34 (dd, J = 6.1, 1.5 Hz, 1H), 5.46 (d, J = 1.7 Hz,
1H), 6.20 (d, J = 1.2 Hz, 1H), 7.14−7.19 (m, 2H), 7.22−7.33
(m, 8H), 8.00 (s, 1H). 13C NMR (101 MHz, CD3OD) δ: 25.3,
25.8, 27.1, 46.1, 51.7, 84.7, 85.0, 88.1, 92.4, 115.1, 118.9 (d, J =
4.0 Hz), 127.3 (×2), 129.3 (×4), 129.6 (×4), 142.0 (d, J = 3.0
Hz), 143.6 (d, J = 1.1 Hz), 149.8 (d, J = 20.5 Hz), 155.6 (d, J =
20.7 Hz), 160.5 (d, J = 210.0 Hz), 172.0. ESMS calcd for
C28H30N6O4F+ [M + H], 533.6; found, 533.8.
8.2 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 8.00 (s, 1H). C NMR
(100 MHz, CD3OD) δ: 17.3, 29.2, 32.5, 37.7, 38.3, 38.9, 40.3,
41.9, 43.8, 46.8, 60.1, 75.8, 77.2, 88.0, 92.6, 117.3, 124.0, 132.8,
134.9, 139.5, 140.5, 141.7, 155.6, 160.0, 163.6, 174.6, 176.9.
HRMS calcd for C28H39N8O5S2+ [M + H], 631.2479; found,
631.2475.
2-(2-(4-N-(3,5-di-tert-Butylphenylcarboxy)aminophenyl))-
ethylamino-N6-(2,2-diphenylethyl) adenosine-5′-N-ethylcar-
boxamide (16a). General Procedure for Amide Coupling.
Compound 7b (69 mg, 0.11 mmol), 3,5-di-tert-butylbenzoic
acid (29 mg, 0.13 mmol), EDCI (24 mg, 0.13 mmol), HOBt
(17 mg, 0.13 mmol), DIPEA (26 μL), and DMF (265 μL), tan
glassy solid (16a) (39 mg, 42%), Rf = 0.19 (1:5 petroleum
spirits/EtOAc), mp 124−128 °C. 1H NMR (300 MHz,
CD3OD) δ: 1.07 (t, J = 6.9 Hz, 3H), 1.40 (s, 18H), 2.92 (t,
J = 6.9 Hz, 2H), 3.07−3.39 (m, 2H), 3.52−3.68 (m, 2H), 4.22
(d, J = 6.6 Hz, 2H), 4.42 (s, 1H), 4.48 (s, 1H), 4.49−4.52 (m,
1H), 4.98−5.02 (m, 1H), 5.91 (s, J = 6.3 Hz, 1H), 7.19 (d, J =
7.5 Hz, 2H), 7.12−7.20 (m, 2H), 7.21−7.44 (m, 8H), 7.59 (d, J
= 7.5 Hz, 2H), 7.69 (s, 1H), 7.81 (s, 2H), 7.91 (s, 1H). 13C
NMR (100 MHz, CD3OD) δ: 14.8, 31.8, 35.2, 36.0, 36.8, 44.7,
46.0, 51.9, 73.1, 74.7, 85.6, 90.0, 114.9, 122.8, 122.9, 127.1,
127.7, 129.3, 129.7, 130.2, 135.8, 137.6, 138.0, 138.4, 144.1,
152.5, 156.1, 161.2, 170.0, 172.1. HRMS calcd for C49H59N8O5+
[M + H], 839.4603; found, 839.4642.
2-Fluoro-N6-(2,2-diphenylethyl)adenosine-5′-N-methylcar-
boxamide (18). General Procedure for Isopropylidene Depro-
tection. Compound 17 (60 mg, 0.11 mmol), MeCN (2 mL),
and 1 M HCl (10 mL), 50 °C, 2 h, white solid (18) (22 mg,
40%), Rf1= 0.29 (89:10:1 CH2Cl2/MeOH/NH4OH), mp 128−
131 °C. H NMR (400 MHz, CD3OD) δ: 2.87 (s, 3H), 4.15−
4.25 (m, 2H), 4.29 (dd, 1H), 4.46 (s, 1H), 4.49 (t, J = 8.1 Hz,
1H), 4.62 (dd, J = 7.7, 4.8 Hz, 1H), 5.91 (d, J = 7.8 Hz, 1H),
7.17−7.23 (m, 2H), 7.24−7.36 (m, 8H), 8.09 (s, 1H). 13C
NMR (101 MHz, CD3OD) δ: 25.8, 46.2, 51.7, 73.2, 78.9, 86.5,
90.6, 119.8 (d, J = 4.0 Hz), 127.2 (×2), 129.3 (×4), 129.6 (×4),
142.2 (d, J = 2.7 Hz), 143.6 (d, J = 1.1 Hz), 150.5 (d, J = 19.8
Hz), 157.8 (d, J = 20.3 Hz), 160.4 (d, J = 208.5 Hz), 172.6.
HRMS calcd for C25H26N6O4F+ [M + H], 493.1994; found,
493.2010.
2-(2-(4-N-(3,5-di-tert-Butyl-4-hydroxyphenylcarboxy)-
aminophenyl))ethylamino-N6-(2,2-diphenylethyl)adenosine-
5′-N-methylcarboxamide (20). General Procedure for 2-
Substitution. Compound 17 (188 mg, 0.35 mmol), 12b (169
mg, 0.46 mmol), DIPEA (250 μL), and MeCN, 150 °C (MW),
2 h, yellow solid (19) (229 mg, 74%).
General Procedure for Isopropylidene Deprotection. Compound
19 (200 mg, 0.23 mmol), MeCN (10 mL), and 1 M HCl (15 mL), 2 h,
white solid (20) (45 mg, 24%), Rf = 0.30 (89:10:1 CH2Cl2/MeOH/
NH4OH), mp 120−123 °C. 1H NMR (400 MHz, CD3OD) δ: 1.47 (s,
18H), 2.71 (s, 3H), 2.89 (t, J = 6.9 Hz, 2H), 3.53−3.60 (m, 1H),
3.65−3.72 (m, 1H), 4.20 (d, J = 6.4 Hz, 2H), 4.40 (s, 2H), 4.48 (t, J =
7.8 Hz, 1H), 4.97−4.99 (m, 1H), 5.85 (d, J = 7.0 Hz, 1H), 7.13−7.17
(m, 4H), 7.23−7.31 (m, 8H), 7.52 (d, J = 8.4 Hz, 2H), 7.76−7.78 (m,
3H). 13C NMR (101 MHz, CD3OD) δ: 26.3, 30.6, 35.6, 36.7, 44.6,
46.0, 51.9, 72.6, 74.7, 85.8, 90.0, 115.0, 122.8, 125.9, 127.0, 127.6,
2-(2-(4-N-(3,5-di-tert-Butyl-4-hydroxyphenylcarboxy)-
aminophenyl))ethylamino-N6-(2,2-diphenylethyl)adenosine-
5′-N-ethylcarboxamide (16b). General Procedure for Isopropy-
lidene Deprotection. Compound 14b (175 mg, 0.20 mmol),
MeCN (5 mL), and 1 M HCl (15 mL), 50 °C, 6 h, white solid
(16b) (20 mg, 12%), Rf = 0.23 (1:5 petroleum spirits/EtOAc),
1
mp 118−124 °C. H NMR (400 MHz, CDCl3) δ: 0.93 (t, J =
7.2 Hz, 3H), 1.37 (s, 18H), 2.75 (br t, J = 6.0 Hz, 2H), 2.91−
3.04 (m, 1H), 3.18−3.32 (m, 1H), 3.39−3.62 (br m, 2H), 4.13
(br s, 2H), 4.29 (t, J = 7.6 Hz, 1H), 4.39 (d, J = 2.4 Hz, 1H),
4.50 (br s, 1H), 4.69 (t, J = 5.5 Hz, 1H), 4.84−4.87 (m, 1H),
5.54 (br s, 1H), 5.63 (d, J = 6.0 Hz, 1H), 5.72 (br s, 1H), 7.03
(d, J = 8.2 Hz, 2H), 7.10−7.16 (m, 2H), 7.17−7.25 (m, 10H),
7.29 (br s, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.61 (s, 2H), 7.94 (s,
1H). 13C NMR (100 MHz, CDCl3/CD3OD) δ: 18.3, 33.9, 38.0,
38.3, 39.4, 47.1, 48.8, 54.5, 75.3, 76.8, 88.3, 93.4, 118.3, 124.9,
3531
dx.doi.org/10.1021/jm300206u | J. Med. Chem. 2012, 55, 3521−3534

Journal of Medicinal Chemistry
Article
129.2, 129.3, 129.5, 129.6, 130.1, 137.2, 138.2, 138.6, 138.7, 144.0,
152.2, 156.0, 158.9, 161.2, 170.0, 172.8. HRMS calcd for C48H57N8O6
[M + H], 841.4396; found, 841.4397.
excitation. The SI assay was repeated in at least three different
passages. PI-positive cells were quantified using ImageJ (NIH Image;
National Institutes of Health, USA). The normalized dead cell
percentage was calculated by dividing the number of DAPI-positive
cells per well by the average number of DAPI-positive cells in the SI
treated wells for that experiment. DHE staining was performed by
incubating the cells at 20 μM for 45 min, prior to imaging, to
determine superoxide levels within the H9c2 cells.
Statistical Analysis. The effects of compounds 15a, 15b, and 15c
on cardiomyoblast cell death during hypoxia were determined using a
two-way analysis of variance (ANOVA), with one factor being
antioxidant type and one factor being concentration. Identification of
individual group-to-group differences was performed using Bonferroni
posthoc analysis.
+
Cell Culture and cAMP Assay. Four stably transfected CHO cell
lines, each overexpressing one of the human A1, A2A, A2B, and A3Rs,
were used for cAMP functional assays. The reporter lines were a kind
gift from Prof. A. Christopoulos, Monash Institute of Pharmaceutical
Sciences. Cells were cultured in high glucose Dulbecco’s Modified
Eagle’s Medium plus GlutaMAX containing 1 mM sodium pyruvate,
15 mM HEPES, 1 mg/mL hygromycin B, and 5% fetal bovine serum
in a humidified atmosphere of 95% air and 5% CO2. All cell culture
reagents were purchased from Invitrogen, Australia. Cells were grown
to ∼80% confluence, split (0.01% trypsin), then seeded into 96-well
plates, at 20000 cells/well, and grown overnight. Cyclic AMP
functional assays were performed using a Perkin-Elmer Alphascreen
kit according to manufacturer’s instructions. Briefly, media was
removed from wells and replaced with a cAMP acceptor bead solution
consisting of phenol red free DMEM/F-12 media containing 15 mM
HEPES and L-glutamine, supplemented with 100 μM Rolipram
(Tocris Biosciences) and anti-cAMP acceptor beads at 2 units/well.
This was followed by the addition of test compounds. The acceptor
bead solution and compound mix were left to incubate for 30 min
prior to addition of the donor bead solution, containing 0.1% BSA,
0.3% final 10% Tween-20, 5 mM HEPES, 2 units/well of streptavidin
coated donor beads, and 2 units/well of biotinylated cAMP. After
addition of the donor bead solution, the cells were incubated for 2 h in
the dark at 37 °C and 5% CO2 prior to being read on a Perkin-Elmer
Wallac, 2101 multiplate reader. Vehicle controls consisted of 0.1%
DMSO and 0.9% ethanol, final concentration.
For the A2A and A2B assays, cells were incubated with compounds in
the range 0.1 nM through to 100 μM. The assay is essentially a
competitive binding, proximity assay with endogenous cAMP and
exogenously added biotinylated cAMP competing for binding to an
antibody conjugated to the acceptor beads. For the A1R and A3R
assays, cells were incubated with 10 μM forskolin (final concentration,
to activate adenylate cyclase) and compounds in the range 0.1 nM
through to 100 μM. Results were expressed either as an elevation of
cAMP (A2A and A2BR assays generating EC50 values) or an A1 or A3R
mediated inhibition of the forskolin-stimulated elevation of cAMP
(IC50 values).
Molecular Modeling. Ballesteros−Weinstein nomenclature37 was
used to identify GPCR residues, except in loop regions, where crystal
structure numbering was used. Ligands were prepared using LigPrep
2.438 at physiological pH. The crystal structure of the adenosine A2A
receptor (A2AR) extracted from its complex with the agonist UK-
432,097 (PDB: 3QAK) was used in docking studies. The T4-lysozyme
was deleted, and the protein was prepared in Maestro 9.139 with the
ProteinPrep wizard. Water molecules within 5 Å of the crystal
structure ligand were retained (residue numbers 1204 and 1209).
Ligands were docked into the crystal structure using GOLD.40,41
The wizard in the HERMES interface was used to generate the input
files for GOLD. The centroid of the binding site was defined by the
center of the ligand UK-432,097, including all atoms within 12 Å of
the centroid. The “goldscore_p450_csd” template was used with the
following modifications. Each ligand was subjected to 200 genetic
algorithm runs, generating 200 poses per ligand. The genetic algorithm
search options were set to “very flexible”. Early termination of docking
was allowed if the two top scoring poses were within 2.0 Å of each
other, and the option to generate diverse solutions was selected,
clustering to an rmsd of 2.0 Å.
The resulting docked poses were clustered in Maestro, based on
their similarity to the adenosine-5-N-ethylcarboxamide (NECA)
portion of UK-432,097 in the A2AR crystal structure.
AUTHOR INFORMATION
■
Corresponding Author
Cell Culture and Simulated Ischemia Assay. The H9c2(2−1)
embryonic rat atrial cell line (American Type Culture Collection,
ATCC, Manassas, VA, USA) was used for this study. The cell line was
grown in Dulbecco’s Modified Eagle’s Medium containing 4 mM L-
glutamate, 4.5 g/L glucose, 3.7 g/L sodium bicarbonate, 100 U/mL
penicillin, and 100 mg/mL streptomycin supplemented with 10% fetal
bovine serum (Invitrogen, Mount Waverley, VIC, Australia) in a 5%
CO2 incubator. Cells were used at 60−70% confluence and plated one
day prior to assay at 40000 cells per well of 96-well plate. Simulated
ischemia was induced using conditions developed in our lab and
described previously.24,33−36 In short, ischemia was achieved by
incubating the cells in hypoxic simulated ischemia (SI) medium at pH
6.4 containing (in mM): 137 NaCl, 3.5 KCl, 0.88 CaCl2·2H2O, 0.51
MgSO4·7H2O, 5.55 D-glucose, 4 HEPES, 10 2-deoxy-D-glucose, and 20
DL-lactic acid (Sigma, Castle Hill, NSW, Australia) plus 2% fetal bovine
serum. Cells were incubated under nitrogen (100% N2 gas
atmosphere) for 12 h at 37 °C. Fresh simulated ischemia medium
was prepared for each experiment and sterile filtered prior to
experimentation. Agonists were dissolved in SI buffer and added to
the wells at concentrations between 0.5 and 20 μM. Each treatment
was repeated in three independent assays performed in triplicate wells.
Cell Viability (DAPI) Assay and DHE Imaging of H9C2 (2−1)
Cells. Detection of nonviable cells resulted from ischemia was
achieved by 4′,6-diamidino-2-phenylindole (DAPI) exclusion assay.
This was performed as previously except that DAPI was used rather
than propidium iodide in order to allow coimaging with DHE. Twelve
hours post simulated ischemia, cells were first washed with PBS and
stained with 400 nM DAPI (Sigma) for 5 min, followed by PBS rinse
twice prior to imaging. Images were taken using a confocal microscope
(Nikon A1; Nikon Instruments, Tokyo, Japan) using 405 nm laser
*For P.J.S.: phone, +61 (0)3 9903 9542; E-mail, Peter.
Scammells@monash.edu. For P.J.W.: phone, +61 (0)3 9903
9074; E-mail, Paul.White@monash.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Elizabeth Yuriev and Dr. David Chalmers for
assistance with the molecular modelling studies as well as the
ARC Centre of Excellence for Free Radical Chemistry and
Biotechnology for financial support.
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