N6-substituted C5′-modified adenosines as A1 adenosine receptor agonists

Article abstract of DOI:10.1016/j.bmc.2007.11.010

Adenosines bearing 5′-modification in conjunction with an N⁶-substituent have previously been shown to act as partial agonists at the A₁ adenosine receptor. Our current work investigates the effect of modifying the 5′-position in conjunction with efficacious bicyclic and tricyclic N⁶-substituents. Several highly potent agonists for the A₁ adenosine receptor were identified; however, all of these compounds behaved as full agonists. In keeping with previous reports, 5′-halogen and 5′-sulfide derivatives of N⁶-(endo-norborn-2-yl)adenosine were, in general, low nanomolar agonists of the A₁ adenosine receptor. The known partial agonist, N⁶-cyclopentyl-5′-deoxy-5′-ethylthioadenosine (2), also behaved as a full agonist in our assay.

Full text of DOI:10.1016/j.bmc.2007.11.010

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1861–1873
N6-substituted C50-modified adenosines as A1 adenosine
receptor agonists
T. D. Ashton,a Stephen P. Baker,b Sally A. Hutchinsonc and Peter J. Scammellsa,*
aDepartment of Medicinal Chemistry, Victorian College of Pharmacy, Monash University,
381 Royal Parade, Parkville, Vic. 3052, Australia
bDepartment of Pharmacology & Therapeutics, University of Florida College of Medicine, Box 100267, Gainesville, FL 32610, USA
cCSIRO Textile and Fibre Technology, Henry street, Belmont, Vic. 3216, Australia
Received 19 August 2007; revised 23 October 2007; accepted 1 November 2007
Available online 6 November 2007
Abstract—Adenosines bearing 50-modification in conjunction with an N6-substituent have previously been shown to act as partial
agonists at the A1 adenosine receptor. Our current work investigates the effect of modifying the 50-position in conjunction with
efficacious bicyclic and tricyclic N6-substituents. Several highly potent agonists for the A1 adenosine receptor were identified;
however, all of these compounds behaved as full agonists. In keeping with previous reports, 50-halogen and 50-sulfide derivatives
of N6-(endo-norborn-2-yl)adenosine were, in general, low nanomolar agonists of the A1 adenosine receptor. The known partial
agonist, N6-cyclopentyl-50-deoxy-50-ethylthioadenosine (2), also behaved as a full agonist in our assay.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
rate in cases of atrial flutter or fibrillation.2 These issues
have stimulated interest in the development of agents
which are both receptor subtype selective and have
improved metabolic stability. Achieving selectivity for
the A1AR is well documented; in general, a cycloalkyl
or heterocyclic substituent on the exo-cyclic nitrogen
of adenosine will confer a degree of subtype selectivity.
Certain 50-modifications and/or the incorporation of a
halogen in the 2-position are also known to improve
A1AR affinity and selectivity.3
Adenosine is an endogenous hormone that mediates a
variety of physiological and pathophysiological pro-
cesses. The functions of adenosine are mediated through
binding interactions with adenosine receptors (AR),
which are part of the super-family of G-protein coupled
receptors. Adenosine receptors consist of four known
subtypes; A1 and A3, which inhibit adenylate cyclase
activity, as well as A2A and A2B, which stimulate aden-
ylate cyclase leading to an increase in intracellular
cAMP. Adenosine receptors are widely distributed
throughout the body including the heart, central ner-
vous system, lymphocytes and kidneys.1 Adenosine,
marketed as Adenocardꢁ, is currently used in the treat-
ment of paroxysmal supraventricular tachycardia. How-
ever, adenosine also produces side effects as a result of
the widespread distribution of adenosine receptors and
its lack of receptor subtype selectivity. These side effects
are limited by adenosine’s short half-life which results
from its rapid breakdown in plasma by adenosine deam-
inase.2 However, the short half-life also limits adeno-
sine’s suitability for long-term control of ventricular
There has also been interest in the development of
partial agonists for the A1AR. By definition, a partial
agonist generates a sub-maximal response when there
is full receptor occupancy. This sub-maximal response
may prevent unwanted adverse events from occurring.
For example, a partial A1AR agonist may be able to
slow conduction in the atrio-ventricular (A-V) node
and provide ventricular rate control without producing
high degrees of A-V block that may occur with a full
agonist.4 Diminished receptor desensitisation has also
been observed for partial agonists.3
In the development of partial agonists, a series of
N6-substituted adenosines bearing carbamates and
thiocarbamates (e.g., CVT-2759, 1) (Fig. 1) in the
50-position showed partial agonist profiles.5,6 Further-
more, adenosine analogues possessing a 50-sulfide and
Keywords: Adenosine; A1AR agonist.
*
Corresponding author. Tel.: +61 3 9903 9542; fax: +61 3 9903
9582; e-mail: peter.scammells@vcp.monash.edu.au
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.010

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T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
ylthio (6), methylseleno (7), amino (8) and propylamino
(9) moieties in the 50-position. Continuation of this work
has seen further elaboration of this series.
O
HN
N
HN
N
N
N
N
N
N
N
20,30-Bis-O-(tert-butyldimethylsilyl)-50-chloro-50-deoxy-
N6-(endo-norborn-2-yl)adenosine (10a) was the starting
point for our N6-norbornyl series and was synthesised
as previously reported.15 When 10a was treated with so-
dium ethanethiolate (10.0 equiv) we observed good con-
version of the primary chloride; however, crude 1H
NMR and mass spectra indicated the formation of 11b
as well as concurrent deprotection (Scheme 1).15 With
this in mind we synthesised 50-deoxy-N6-(endo-nor-
born-2-yl)-50-ethylthioadenosine (12b) by the treatment
of 10a with sodium ethanethiolate in DMF at ambient
temperature. The TBS protecting groups were subse-
quently cleaved using ammonium fluoride in MeOH at
elevated temperature to give 12b in 50% yield over two
steps. We found the conversion to the propylsulfide
to also be problematic as full conversion was unable
to be achieved, and the starting material and product
were inseparable by chromatography (on silica). Conse-
quently, we adopted a complimentary approach which
involved the introduction and alkylation of a 50-thiol.
Thioacetates have been reported as a suitable precursor
for the inclusion of 50-sulfides.16 Substitution of the pri-
mary chloride, 10a, with potassium thioacetate in DMF
at ambient temperatures gives the masked thiol in very
good yield (74%). It is of note that performing this reac-
tion at elevated temperatures led to the formation of a
coloured impurity which was inseparable via column
chromatography. Treatment of the thioester 11d
with n-bromopropane in MeOH with the addition of
sodium deprotected the acetate in situ and subsequently
alkylated the thiol that was formed to give the desired
propylsulfide, 11e.
MeHN
O
S
O
O
O
HO OH
HO OH
1 CVT-2759
2
HO
HN
HN
N
N
N
N
F
RHN
S
HO
N
O
N
N
N
O
4a Me
4b Et
4c c-Pent
HO OH
HO OH
3 CVT-3619
Figure 1. Partial agonists at the A1 adenosine receptor.
N6-substituent (e.g., 2 and 3) have been identified as
A1AR selective partial agonists.7 In particular, CVT-
3619 (3), a modified adenosine bearing a 50-arylsulfide,
has been shown to reduce lipolysis in epididymal and
inguinal adipocytes whilst not effecting atrial rate.8
Compounds possessing an N6-cycloalkyl substituent
and 8-alkylamino motif (4a–c) were also shown to have
partial agonist activity at the A1AR in rat brain slices or
membranes.9 The synthesis of N6-norbornyl adenosine
derivatives has been reported previously and several
were shown to be potent, selective and full agonists for
the A1AR.10–12 In an attempt to take advantage of the
potency and selectivity conferred by an N6-norbornyl
group13 in the development of new partial A1AR
agonists, a series of 50-derivatives of N6-(endo-nor-
born-2-yl)adenosine and N6-(2S-exo-5,6-epithio-endo-
norborn-2-yl)adenosine were prepared and examined
for affinity, potency and intrinisic activity (IA, maximal
response) at the A1AR in DDT1 MF-2 cells.
Nitrogen analogues were also obtained from both nucle-
ophilic and electrophilic processes. The 50-methylamino
analogue 11a was synthesised in 30% yield by heating
10a in a sealed in tube in 40% methylamine in MeOH
solution. The bulk of the remaining material recovered
was unreacted starting material (63%). The 50-amino-
N6-substituted adenosine 13 was synthesised as previ-
ously reported and was used as the starting point for
the preparation of other nitrogen analogues.15 Reduc-
tive amination of 13, using an excess of formaldehyde
and sodium cyanoborohydride in MeCN at pH 7, gave
the 50-dimethyl analogue 14a in a 36% yield. The sec-
ondary amide 14b was established through treatment
of the primary amine with acetic anhydride in pyridine
at room temperature in excellent yield (93%).
2. Results and discussion
In previous work, we have described the synthesis of 50-
deoxy-N6-(endo-norborn-2-yl)-50-fluoroadenosine (5)14
(Fig. 2) and have also reported a convenient method
of generating 50-modified N6-substituted adenosines
from
20,30,50-tris-O-(tert-butyldimethylsilyl)inosine.15
This approach was applied to the synthesis of N6-
(endo-norbornyl)adenosine analogues containing meth-
Removal of the TBS-ethers was achieved by warming
with NH4F in MeOH to give the target compounds
12a, 12c–12e, 15a and 15b in moderate to very good
yields (49–82%). Reaction of 11d with NH4F led to the
removal of the acetate group and extended reaction
times allowed the 50-thiol 12d to be isolated as the sole
product.
HN
N
N
5 R = F
R
6 R = SMe
7 R = SeMe
8 R = NH2
9 R = NHPr
N
N
O
HO OH
Examples of 50-carbamates and thiocarbamates were
also prepared based on reports of this functionality lead-
Figure 2. 50-Modified N6-nornorn-2-yladenosines.

T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
1863
HN
N
HN
HN
N
R = TBS
(i)
N
N
N
N
N
N
N
N
N
(iii)
Cl
R
R
N
O
O
O
RO OR
R = TBS
TB SO OTBS
HO OH
10a
11a R = NHMe
11b R = SEt
11c R = S(2-F-Ph) 54%
30%
12a R = NHMe
12b R = SEt
12c R = S(2-F-Ph) 49%
58%
50%
(iii) 80%
10b R = H
11d R = SAc
74%
12d R = SH
12e R = SPr
82%
69%
(ii) 22%
11e R = SPr
R = TBS
(iv)
HN
N
HN
HN
N
N
N
N
N
N
N
N
(v) or (vi)
(iii)
H2N
R
R
N
N
N
O
O
O
TBSO OTBS
TB SO OTBS
HO OH
13
14a R = NMe2 36%
14b R = NHAc 93%
15a R = NMe2 78% H2
15b R = NHAc 51%
Scheme 1. Reagents and conditions: (i) for 11a, MeNH2, MeOH, 60 ꢂC; for 11b, NaSEt, DMF, rt; for 11c, 2-fluorothiophenol, NaH, THF then 10a,
0 ꢂC—rt; for 11d, KSAc, DMF, rt; (ii) n-PrBr, Na, MeOH, ꢀ20 ꢂC—rt; (iii) NH4F, MeOH, 50 ꢂC; (iv) Ref. 15; (v) H2CO, H2O, NaBH3CN, pH 7,
MeCN, rt; (vi) Ac2O, pyridine, 0 ꢂC—rt.
ing to attenuated intrinsic activities.5 Isopropylidenated
N6-(endo-norborn-2-yl)adenosine (18) was prepared in
two steps from 6-chloropurine riboside (16) (Scheme
2). Firstly, the N6-substituted adenosine 17 was pre-
pared in excellent yield (94%) from 16 using ( )-endo-
norborn-2-yl amine.HCl in the presence of Hunigs base
¨
[N(i-Pr)2Et] in refluxing t-BuOH. Protection of 17 as the
acetonide 18 was achieved using para-toluenesulphonic
acid and 2,2-dimethoxypropane in acetone at room tem-
perature in 92% yield. The carbamate derivative 19a was
then prepared via a sodium hydride generated alkoxide
which was treated with carbonyl-1,10-diimidazole
(CDI), followed by a MeNH2/MeOH solution to give
the methylcarbamate in very good yield (90%). Follow-
ing the method of Barma et al., the thiocarbamate deriv-
ative 19b was formed in 60% yield by treating 18 with
Cl
HN
HN
N
N
N
N
N
N
N
N
N
(i)
(ii)
HO
HO
HO
N
N
N
O
O
O
94%
92%
HO OH
HO OH
17
O
O
16
18
(iii)
HN
N
HN
N
N
N
N
N
N
N
(iv)
RO
RO
O
O
O
O
HO OH
20a R = C(O)NHMe 76%
20b R = C(S)NMe2 79%
19a R = C(O)NHMe
90%
60%
R = C(S)NMe2
19b
Scheme 2. Reagents and conditions: (i) ( )-endo-norborn-2-yl amineÆHCl, N(i-Pr)2Et, t-BuOH, 83 ꢂC; (ii) p-TsOH, 2,2-dimethoxypropane, acetone,
rt; (iii) for 19a, NaH, THF, CDI, MeNH2, MeOH, rt; for 19b, NaH, NaI (cat.), DMTC-Cl, solvent, 0 ꢂC—rt; (iv) 80% AcOH, 70–80 ꢂC.

1864
T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
S
NaH
followed
dimethylthiocarbamoyl
chloride
(DMTC-Cl) and catalytic sodium iodide.17 Deprotec-
tion was performed using 80% AcOH at 70–80 ꢂC to
give the target compounds, 20a and 20b, in good yield.
Cl
N
N
N
HN
N
N
N
N
EtHN
O
O
(i), (ii)
40%
N
Adenosine analogues with tricyclic N6-substituents
bearing an epithiirane in the 5,6-position of a norborn-
ane ring have proven to be highly potent A1AR ago-
nists.18 As a result we were interested in exploring the
effect of 50-modification on the activity of N6-(5,6-epith-
ionorborn-2-yl)adenosine. The key intermediate required
for this synthesis (compound 23a) was generated in 36%
yield using 9-(2,3-bis-O-(tert-butyldimethylsilyl)-5-chloro-
5-deoxy-b-D-ribofuranosyl)-6-chloropurine (21)15 and
2S-endo-amino-5,6-exo-epithiobicyclo[2.2.1]heptane (2S-
EtHN
O
O
O
O
HO OH
27
26
Scheme 4. Reagents and conditions: (i) Dowex-50W (H+), H2O, rt; (ii)
22, N(i-Pr)2Et, t-BuOH, 83 ꢂC.
the acetonide moiety was achieved using Dowex-50W
(H+) resin (Scheme 4). Substitution was performed on
the crude material with 2S-22 using the standard SNAr
conditions to give 27 in a 40% yield over two steps.
22)18 in the presence of Hunigs base [N(i-Pr) Et] in
¨
2
refluxing t-BuOH (Scheme 3). Similar to the N6-(endo-
norborn-2-yl)-series, the 50-chloro proved to be a suitable
precursor for nucleophilic displacement. Introduction of
the methylsulfide and concurrent TBS-deprotection was
achieved using sodium thiomethoxide in DMF at ambi-
ent temperatures to give 25a in a moderate yield of
47%. Potassium selenomethoxide was generated in situ
using potassium hydride and dimethyldiselenide in
DMF at 0 ꢂC,19 and subsequent addition of 23a led to
the formation of the desired dialkyl selenide 24b in low
yield (24%). Deprotection of 23a and 24 was achieved
using NH4F in MeOH to give the 50-chloride 23b and
50-methylselenide 25b in excellent yields (97% and
100%, respectively).
IJzerman and co-workers identified N6-cyclopentyl-50-
deoxy-50-ethylthioadenosine (2) as a high affinity partial
agonist at the A1AR.7 For use as a standard in our as-
say, we also required a sample of 2. 20,30-Bis-O-(tert-
butyldimethylsilyl)-50-chloro-50-deoxyinosine (28)15 was
converted to the corresponding 50-ethylthio analogue
29 using NaSEt in DMF in 44% yield (Scheme 5). We
were able to form the silylated adenosine analogue 30
in excellent yield (92%) using a procedure modified from
Wan et al.21 The silylated inosine 29 was heated at reflux
in the presence of cyclopentyl amine and bromo-tri-(1-
pyrrolidinyl)phosphonium hexafluorophosphate (PyB-
roP) and N(i-Pr)2Et in dichloroethane. Removal of the
protecting groups using NH4F in MeOH afforded 2 in
37% yield.
The N-ethyl-50-uronamide 26 was prepared as per the
method of Olsson and co-workers.20 Deprotection of
The 50-modified derivatives of N6-(endo-norborn-
2-yl)adenosines were tested for A1AR affinity by
displacement of specific [3H]CPX binding in DDT cell
membranes. These assays were performed in the
presence of 10 lM 50-guanylyl-imidodiphosphate to
maintain the receptors in the agonist low affinity state.
The potency and intrinsic activity of the compounds
were determined by their ability to inhibit (ꢀ)-isoprote-
renol-stimulated cAMP accumulation in DDT cells
(Table 1).
S
S
NH2
2S-22
Cl
N
HN
N
N
N
N
N
N
N
(i)
Cl
Cl
O
O
36%
TBSO OTBS
RO OR
21
23a R = TBS
23b R = H
(iv) 97%
As shown in Table 1, the 50-fluoro and chloro deriva-
tives (5 and 10b, respectively) have affinities and
potencies similar to the classical A1AR agonist, CPA.
R = TBS
(iii)
R = TBS
(ii) 23%
S
S
HN
N
HN
N
O
N
HN
N
N
N
N
N
N
N
N
NH
N
(iv)
(ii)
X
MeSe
O
X
EtS
N
N
N
O
O
O
92%
HO OH
TB SO OTBS
TB SO OTBS
RO OR
25a X = SMe
25b X = SeMe 100%
47%
24
28 X = Cl
30 R = TBS
2 R = H
(i) 44%
(iii) 37%
29 X = SEt
Scheme 3. Reagents and conditions: (i) 2S-22, N(i-Pr)2Et, t-BuOH,
83 ꢂC; (ii) Me2Se2, KH, DMF, then 23a, 0 ꢂC—rt; (iii) NaSMe, DMF,
rt; (iv) NH4F, MeOH, 50–60 ꢂC.
Scheme 5. Reagents and conditions: (i) NaSEt, DMF, rt; (ii) PyBroP,
c-PentNH2, N(i-Pr)2Et, DCE, 86 ꢂC; (iii) NH4F, MeOH, 50–60 ꢂC.

T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
1865
Table 1. Ki, IC50 and intrinsic activity (IA) of adenosine derivatives at the A1AR in DDT1 MF-2 cells
S
*
*
*
HN
N
HN
N
HN
N
N
N
N
N
N
N
N
N
N
R
R
R
O
O
O
HO OH
Entries 1-2
HO OH
Entries 3-17
HO OH
Entries 18-21
Entry
Compound
R
Ki (nM)
IC50 (nM)
IA
*
1
2
CPA
2
—
—
OH
SEt
F
21.5 5.7 (4)
173 35 (5)
17 6 (3)
2.7 0.6 (4)
21 8 (4)
1.00
1.02 0.02 (4)
0.97 0.04 (3)
1.03 0.03 (3)
1.00 0.01 (4)
0.95 0.01 (3)
1.04 0.03 (3)
0.92 0.03 (5)
0.90 0.01 (4)
0.89 0.02 (5)
0.92 0.06 (3)
0.83 0.07 (3)
0.91 0.12 (6)
0.97 0.04 (5)
0.97 0.04 (5)
1.03 0.02 (4)
0.96 0.02 (4)
0.98 0.02 (3)
1.05 0.03 (6)
0.93 0.04 (5)
0.96 0.03 (4)
3
5
endo
7
3 (3)
4
10b
12d
6
endo
endo
Cl
9.8 3.4 (3)
283 19 (4)
120 37 (5)
122 42 (5)
383 21 (3)
431 15 (3)
285 68 (3)
3933 1008 (3)
>10,000 (4)
2.6 1.6 (5)
224 69 (4)
68 13 (3)
5
SH
SMe
SEt
SPr
6
endo
7
12b
12e
12c
7
endo
28 15 (3)
15 4 (5)
8
endo
endo
9
S(2-F-Ph)
SeMe
NH2
21 13 (4)
48 13 (5)
10
11
12
13
14
15
16
17
18
19
20
21
endo
8
endo
351 135 (3)
867 135 (3)
4950 1800 (6)
605 209 (5)
4279 1196 (5)
410 50 (4)
710 200 (4)
0.12 0.03 (3)
0.3 0.1(6)
0.9 0.3 (5)
12 3 (4)
12a
9
endo
endo
NHMe
NHPr
7200 1700 (4)
5865 1309 (3)
11716 1563 (3)
590 110 (4)
2200 500 (4)
0.9 0.2 (3)
2.8 0.8 (3)
5.0 0.2 (3)
30 3 (3)
15b
15a
20a
20b
27
endo
NHAc
NMe2
endo
endo
OC(O)NHMe
OC(S)NMe2
C(O)NHEt
CH2Cl
endo
S-endo
S-endo
S-endo
S-endo
23b
25a
25b
CH2SMe
CH2SeMe
The Ki values were calculated from the concentration of the compounds that initiated [3H]CPX binding by 50%. The IC50 values are the concen-
tration of compounds that inhibited (ꢀ)-isoproterenol (1 lM) stimulated cAMP accumulation by 50% in DDT cells. The IA is the maximal inhibition
of (ꢀ)-isoproterenol-stimulated cAMP accumulation as compared to the maximum inhibition by CPA which was set at 1.00. Numbers in parentheses
are the n.
Furthermore, these two derivatives are full agonists as
they produced the same maximal response (inhibition
of cAMP formation) as CPA. The 50-thio derivatives
including the 50-thio (12d), methylthio (6), ethylthio
(12b), propylthio (12e) and (2-fluorophenyl)thio (12c)
compounds have affinities that ranged from 5.6- to 20-
fold lower and functional potencies that are 5.5- to 83-
fold lower than CPA. All of these derivatives retained
the same intrinsic activity as CPA. The 50-amino (8),
methylamino (12a), propylamino (9), acetamido (15b)
and dimethylamino (15a) analogues all have affinities
for the A1AR in the low to mid micromolar range and
potencies in the mid nanomolar to low micromolar
range. However, the intrinsic activities for the inhibition
of cAMP formation for all of these compounds did not
differ from CPA. The affinity of the 50-methylcarbamate
derivative 20a for the A1AR is 27-fold lower than CPA,
whereas the affinity for 50-dimethylthiocarbamate 20b is
102-fold lower. With respect to cAMP accumulation,
both of these derivatives had the same intrinsic activity
as CPA but with less potency. The 50-methylseleno
derivative 7 has an affinity and IC50 that are 13- and
18-fold lower than CPA, respectively, but with the same
intrinsic activity. Finally, the 50-modified N6-(2S-exo-
5,6-epithio-endo-norborn-2-yl)adenosine derivatives have
high affinity and potency for the A1AR. The N-ethyl-50-
uronamide 27 (Ki = 0.9 nM, IC50 = 0.12 nM), 50-chloro
23 (Ki = 2.8 nM, IC50 = 0.3 nM) and 50-methylthio 25a
(Ki = 5.0 nM, IC50 = 0.9 nM) derivatives have affinities
and potencies greater than that of CPA, whereas the
50-methylseleno derivative 25b has an affinity (30 nM)
and potency (12 nM) similar to that for CPA. All of
the N6-(2S-exo-5,6-epithio-endo-norborn-2-yl)adenosine
50-derivatives have the same intrinsic activity as CPA
indicating that they are full A1 agonists.
In general, the data from the present study show an
affinity (Ki) and potency (IC50) series for 50-modified
N6-(endo-norborn-2-yl)adenosines where halogens >
alkylthio > carbamate > alkylamino. With the exception
of the carbamates, which were not reported, this series is
similar to the corresponding 50-modified N6-cyclopentyl-
adenosines. However, the 50-substituted N6-cyclopentyl-
adenosines were largely partial agonists,7 whereas the
50-substituted N6-(endo-norborn-2-yl)adenosines are all
full agonists as compared to CPA. Furthermore,

1866
T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
compound 2 acted as a full A1AR agonist, whereas it
has previously been reported to be a partial A1 agonist
using different functional assays.7 The 50-substituted
carbamate and 50-substituted thiocarbamate which
showed reduced affinities and potencies at the A1AR
compared to CPA also acted as full agonists in the
cAMP inhibition assay. This contrasts to the reported
partial A1AR activity of CVT-2759 (1) which contains
a 50-substituted carbamate on a N6-substituted adeno-
sine.22 The difference between full and partial agonist
activity of the 50-substituted compounds may have sev-
eral explanations including differences in biological
preparations where receptor densities and/or coupling
efficiencies may be different and in the functional assays
used to determine the intrinsic activity. In the previous
reports,7,22 stimulation of [35S]GTPcS binding in rat
brain or DDT cell membranes was used as a functional
assay, whereas in the present study the inhibition of
cAMP accumulation in DDT cells was employed. The
50-(2-fluorophenyl)thio analogue of N6-(endo-norborn-
2-yl)adenosine (12c) is a full agonist for cAMP inhibi-
tion as compared to CPA. CVT-3619, which also
contains a 50-(2-fluorophenyl)thio substitutent, was also
reported to act as a full agonist for the inhibition of
cAMP accumulation in rat epididymal and inguinal adi-
pocytes, but as a partial agonist for the stimulation of
non-esterified fatty acid release from the same cells.6
Thus full or partial agonism may partly depend upon
the cellular response measured which suggests the possi-
bility that some of the 50-substituted N6-norbornylade-
nosines may have partial agonist activity at the A1AR
for responses other than cAMP accumulation. Finally,
N6-substituted adenosines have been shown to mainly
affect affinity and selectivity towards the A1AR,4,23
although it is possible that these substitutions in
conjunction with 50-alterations could also modify the
intrinsic activity of the molecule at the A1AR. Further
studies will be needed to specifically address the validity
of this issue.
tor reserve for the A1AR. Of particular interest are sev-
eral of the 50-modified N6-(2S-exo-epithio-endo-
norborn-2-yl)adenosine derivatives that have high affin-
ity and subnanomolar potency. All of the 50-derivatives
tested in the present study were full agonists for cAMP
inhibition as compared to CPA.
3. Experimental
3.1. General experimental
All 1H and 13C NMR spectra were recorded on a Bruker
300 WB spectrometer with Avance console. Unless
otherwise stated, spectra were acquired in CDCl3.
Chemical shifts are recorded in parts per million
1
(ppm). For H NMR spectra the residual solvent peak
was used as an internal reference (CHCl3 7.26). For
13C NMR spectra the central peak of the CDCl3 triplet
was used as the internal reference (77.0). Signal multi-
plicities are abbreviated as follows; s singlet, d doublet,
t triplet, q quartet, m multiplet, br broad, app. apparent.
Signal multiplicities and connectivities were assigned
using attached proton test (APT), homonuclear correla-
tion spectroscopy (COSY) and heteronuclear single-
quantum correlation (HSQC) experiments. 1H NMR
data were reported as follows: chemical shift (signal
multiplicity, coupling constant, integration, assign-
ment). 13C NMR data were reported as follows; chemi-
cal shift (C@O, C, CH, CH2, CH3). For instances when
fluorine is attached to carbon, chemical shift is followed
by multiplicity, and coupling constant instances where 2
resonances were observed for a particular carbon due to
diastereotopicity (1:1 mixtures), they are both reported
with a single characterisation for both. High resolution
electrospray mass spectra (HRMS) utilised electrospray
ionisation (ESI) and were obtained on a Bruker Bio-
Apex II FTMS. Low resolution electrospray mass
spectra (LRMS) using electrospray ionisation (ESI)
were obtained on a Micromass Platform II. Unless
otherwise stated, cone voltage was 20 V. MS data are
listed as mass-to-charge (m/z) with assignment (where
appropriate), isotopes and relative intensity in parenthe-
ses. Reactions were monitored by TLC on pre-coated
silica gel 60 F254 aluminium plates (Merck). Visualisa-
tions were achieved using UV light at 254 nm and
phosphomolbdic acid staining (4.8 g in 100 mL of
EtOH) followed by heating. Column chromatography
was carried out on silica gel 60 (0.063–0.200 mm;
Merck). Dry THF was distilled from a blue sodium
benzophenone ketyl solution under N2. Dry MeOH
was refluxed and distilled from Mg and I2 and stored
under N2. Dry acetone was refluxed over and distilled
from Drierite. Anhydrous DMF and DCE were pur-
chased from Sigma–Aldrich Chemical Co. Ltd. NaH
(60% dispersion in mineral oil) was used as supplied.
Unless otherwise stated, reagents were purchased and
used without further purification from Sigma–Aldrich
Chemical Co. Ltd.
The Ki/IC50 ratio can be used to indicate the presence of
a receptor reserve or as a relative indicator of the signal-
ling amplification between the receptor and the response
induced by the ligand. Thus a ratio of 1 would indicate
that the concentration of agonist that occupies half the
receptors produces the half-maximal response. Ratios
greater than one indicate that the half-maximal response
occurs with less than half-maximal receptor occupancy.
The latter case would indicate the presence of a receptor
reserve or a relatively higher signalling amplification.
For the 50-substituted N6-norbornyladenosines, the
Ki/IC50 ratios ranged from 1.3 to 25.5 with the 50-meth-
ylthio and 50-(2-fluorophenyl)thio analogues having the
largest ratio. Although there appears to be no obvious
structural pattern, the data suggest that substitutions
in the 50-position may contribute to a ligand’s receptor
reserve or signal amplification capability.
In summary, a range of 50-modified N6-(endo-norborn-
2-yl)adenosine derivatives were synthesised and
evaluated as A1AR agonists. The pharmacological data
indicate that modification of the 50-position can have
relatively large effects on the affinity, potency and recep-
3.1.1. 20,30-Bis-O-(tert-butyldimethylsilyl)-50-deoxy-N6-
(endo-norborn-2-yl)-50-methylaminoadenosine (11a).
sealed tube containing 10a (214.5 mg, 0.353 mmol) in
A

T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
1867
40% MeNH2 in MeOH (1 mL) was heated at 60 ꢂC for 8
days. The crude reaction mixture was concentrated un-
der reduced pressure and purified via column chroma-
tography (CHCl3 to CHCl3/MeOH; 95:5) to give 11a
(63 mg, 29.6%) and unreacted 10a (135 mg, 63.4%), both
as clear oils. 1H NMR d 8.30 (s, 1H, H-2/8), 7.80 (s, 1H,
H-8/2), 5.86, 5.84 (br s, 1H, N6H), 5.79, 5.78 (d,
J = 6.6 Hz, 1H, H-10), 4.96–4.94 (m, 1H, H-20), 4.50
(br s, 1H, H-200), 4.43–4.41 (m, 1H, H-30), 4.25–4.21
(m, 1H, H-40), 3.04 (m, 1H, H-5a0/b0), 2.91–2.90 (m,
1H, H-5b0/a0), 2.62 (br s, 1H, H-100), 2.54 (s, 3H,
NHCH3), 2.27 (br s, 1H, H-400), 2.23–2.22 (m, 1H, H-
3x00), 1.65–1.29 (m, 6H, H-500, H-600, H-700), 0.92 (s,
10H, H-3n00, t-Bu), 0.76 (s, 9H, t-Bu), 0.11 (s, 6H,
2 · CH3), ꢀ0.05 (s, 3H, CH3), ꢀ0.43, ꢀ0.44 (s, 3H,
CH3). 13C NMR d 155.2 (C), 152.8 (CH), 148.1 (C),
139.9, 139.8 (CH), 121.0 (C), 90.0 (CH), 84.9, 84.8
(CH), 73.7 (2 · CH), 53.0 (CH2), 52.2 (CH), 40.5
(CH), 38.3 (CH2), 38.1 (CH2), 36.8 (CH), 36.0 (CH3),
29.9 (CH2), 25.8 (CH3), 25.7 (CH3), 21.4 (CH2), 18.0
(C), 17.8 (C), ꢀ4.5 (CH3), ꢀ4.6 (CH3), ꢀ4.7 (CH3),
ꢀ5.6 (CH3). LRMS m/z (%): 603.5 (M+H+, 100),
604.5 (48), 605.5 (18). HRMS: C30H54N6O3 Si2 requires
[M+H]+ 603.3869. Found 603.3850.
3.1.3. 50-Acetylmercapto-20,30-bis-O-(tert-butyldimethyl-
silyl)-50-deoxy-N6-(endo-norborn-2-yl)adenosine (11d).
To a stirred solution of 10a (601.4 mg, 0.989 mmol) in
anhydrous DMF (6 mL) under a N2 atmosphere was
added KSAc (1.122 g, 9.820 mmol, 9.9 equiv) and the
resultant solution was stirred for 50 h. The crude reac-
tion mixture was taken up in EtOAc (100 mL), washed
with H2O (100 mL) which was then extracted with
EtOAc (100 mL), the combined organic phase was
washed using brine (2 · 100 mL), dried (MgSO4), fil-
tered and concentrated under reduced pressure. Subse-
quent column chromatography (EtOAc/hexane; 1:4)
1
gave 11d (476 mg, 74.3%) as a light tan oil. H NMR
d 8.25 (s, 1H, H-2/8), 7.77 (s, 1H, H-8/2), 5.97 (d,
J = 6.9 Hz,1H, NH), 5.76–5.74 (m, 1H, H-10), 5.20–
5.11 (m, 1H, H-20), 4.44 (br s, 1H, H-200),0 4.12–4.04
(m, 2H, H-30, H-40), 3.49–3.42 (m, 1H, H-5a /b0), 3.23–
3.18 (m, 1H, H-5b0/a0), 2.55 (br s, 1H, H-100), 2.24 (s,
3H, C(O)CH3), 2.16–2.11 (m, 1H, H-3x00, H-400), 1.60–
1.22 (m, 6H, H-500, H-600, H-700), 0.86 (s, 10H, t-Bu, H-
3n00), 0.70 (s, 9H, t-Bu), 0.05 (s, 3H, CH3), 0.04 (s, 3H,
CH3), ꢀ0.14, ꢀ0.14 (s, 3H, CH3), ꢀ0.37, ꢀ0.38 (s, 3H,
CH3). 13C NMR d 194.3 (C@O), 154.8 (C), 152.7
(CH), 148.4 (C), 139.6, 139.4 (CH), 120.6 (C), 89.5,
89.4 (CH), 84.1, 84.0 (CH), 74.6, 74.5 (CH), 73.4, 73.1
(CH), 51.9 (CH), 40.3 (CH), 38.0 (CH2), 37.9 (CH2),
36.6 (CH), 31.1 (CH2), 30.1 (CH3), 29.7 (CH2), 25.6
(CH3), 25.4 (CH3), 21.2 (CH2), 17.8 (C), 17.6 (C),
ꢀ4.7 (CH3), ꢀ4.9 (CH3), ꢀ5.0 (CH3), ꢀ5.5 (CH3).
LRMS m/z (%): 615.4 (20), 648.4 (M+H+, 100), 649.4
(62), 650.4 (27).
3.1.2. 20,30-Bis-O-(tert-butyldimethylsilyl)-50-deoxy-N6-
(endo-norborn-2-yl)-50-(2-fluorophenylthio)adenosine (11c).
To a solution containing NaH (133 mg, 3.325 mmol,
11.0 equiv) in dry THF (4 mL) held at 0 ꢂC under an
atmosphere of Ar was added 2-fluorothiphenol
(0.351 mL, 421 mg, 10.9 equiv) in a dropwise fashion over
10 min, then allowed to warm to room temperature over
1 h. The reaction mixture was then cooled to 0 ꢂC and 10a
(183.7 mg, 0.302 mmol) in THF (2 mL) was added in a
dropwise manner over 5 min. The resultant mixture was
stirred at rt for 52 h. before heating at reflux for a further
17 h. The resultant solution was taken up in sat. NH4Cl
(50 mL) and washed using EtOAc (2 · 50 mL). The com-
bined organic phase was then washed using H2O
(100 mL) and brine (100 mL), dried (MgSO4), filtered
and reduced in vacuo. Purification via column chroma-
tography using petroleum spirits/EtOAc (85:15) afforded
3.1.4. 20,30-Bis-O-(tert-butyldimethylsilyl)-50-deoxy-N6-
(endo-norborn-2-yl)-50-propylthioadenosine (11e). To a
stirred, degassed solution containing 11d (153.4 mg,
0.237 mmol) in anhydrous MeOH (5 mL) was added
n-PrBr (31.2 lL, 42.2 mg, 1.5 equiv) and the solution
was cooled to ꢀ20 ꢂC. Clean Na metal (14.5 mg,
0.631 mmol, 2.7 equiv) was added in one portion and
the reaction mixture was allowed to slowly come up to
rt and stirred overnight. The reaction mixture was taken
up in H2O (30 mL) and extracted using CHCl3
(3 · 30 mL), the combined organic phase was dried
(MgSO4), filtered and reduced in vacuo. Purification
via column chromatography (gradient elution; petro-
leum spirits/EtOAc (85:15) to EtOAc) gave 11e
1
11c (114 mg, 53.9%) as a clear oil. H NMR d 8.34 (s,
1H, H-2/8), 7.80, 7.79 (s, 1H, H-8/2), 7.44–7.40 (m, 1H,
ArH), 7.21–7.20 (m, 1H, ArH), 7.08–7.02 (m, 2H, ArH),
5.83–5.80 (m, 2H, H-10, NH), 5.31–5.21 (m, 1H, H-20),
4.54 (br s, 1H, H-200), 4.31–4.29 (m, 1H, H-30), 4.21 (br
s, 1H, H-40), 3.62–3.54 (m, 1H, H-5a0/b0), 3.40–3.31 (m,
1H, H-5b0/a0), 2.64 (br s, 1H, H-100), 2.29–2.21 (m, 2H,
H-3x00, H-400), 1.69–1.15 (m, 6H, H-500, H-600, H-700), 0.91
(s, 10H, t-Bu, H-3n00), 0.76 (s, 9H, t-Bu), 0.10 (s, 3H,
CH3), 0.07 (s, 3H, CH3), ꢀ0.11, ꢀ0.12 (s, 3H, CH3),
ꢀ0.33, ꢀ0.34 (s, 3H, CH3). 13C NMR d 161.2 (d,
1JCF = 244.0 Hz, C), 154.9 (C), 152.9 (CH), 148.5 (C),
1
(33.9 mg, 22.2%) as a clear oil. H NMR d 8.32 (s, 1H,
H-2/8), 7.94 (s, 1H, H-8/2), 6.21 (br s, 1H, NH), 5.88–
5.86 (m, 1H, H-10), 5.09–5.02 (m, 1H, H-20), 4.53 (br
s, 1H, H-200), 4.31 (app. t, Japp = 3.9 Hz, 1H, H-30),
4.24–4.19 (m, 1H, H-40), 3.10–3.02 (m, 1H, H-5a0/b0),
2.90–2.83 (m, 1H, H-5b0/a0), 2.63 (br s, 1H, H-100), 2.54
(t, J = 7.5 Hz, 2H, SCH2CH2), 2.27 (s, 1H, H-400),
2.21–2.17 (m, 1H, H-3x00), 1.75–1.22 (m, 8H, H-500, H-
600, H-700, SCH2CH2), 0.96–0.94 (m, 12H, H-3n00,
CH2CH3, t-Bu), 0.80 (s, 9H, t-Bu), 0.15 (s, 3H, CH3),
0.12 (s, 3H, CH3), ꢀ0.056, ꢀ0.060 (s, 3H, CH3),
ꢀ0.24, ꢀ0.25 (s, 3H, CH3). 13C NMR d 154.8 (C),
153.0 (CH), 148.7 (C), 139.6 (CH), 120.4 (C), 89.7,
89.6 (CH), 84.7, 84.6 (CH), 74.4 (CH), 74.0, 73.7
(CH), 52.2 (CH), 40.5 (CH), 38.3 (CH2), 37.9 (CH2),
36.8 (CH), 35.2 (CH2), 34.3 (CH2), 29.9 (CH2), 25.8
(CH3), 25.7 (CH3), 22.9 (CH2), 21.5 (CH2), 18.0 (C),
3
139.9, 139.7 (CH), 131.7 (CH), 128.5 (d, JCF = 7.7 Hz,
CH), 124.4 (CH), 122.6 (d, JCF = 17.2 Hz, C), 120.7
2
2
(C), 115.7 (d, JCF = 22.3 Hz, CH), 89.8, 89.7 (CH),
84.6, 84.4 (CH), 74.5, 74.5 (CH), 73.3, 73.1 (CH), 52.2
(CH2), 40.5 (CH), 38.2 (CH2), 38.0 (CH2), 36.7 (CH),
35.3 (CH2), 29.9 (CH2), 25.7 (CH3), 25.6 (CH3), 21.4
(CH2), 17.9 (C), 17.8 (C), ꢀ4.6 (CH3), ꢀ4.8 (2 · CH3),
ꢀ5.2 (CH3). LRMS m/z (%): 700.6 (M+H+, 100), 701.6
(55), 702.7 (25).

1868
T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
17.9 (C), 13.4 (CH3), ꢀ4.4 (CH3), ꢀ4.6 (CH3), ꢀ4.7
(CH3), ꢀ5.1 (CH3). LRMS m/z (%): 648.4 (100), 649.4
(50), 650.3 (25).
ꢀ4.8 (CH3), ꢀ4.9 (CH3), ꢀ5.76, ꢀ5.83 (CH3). LRMS
m/z (%): 631.5 (M+H+, 100), 632.5 (40), 633.5 (20),
654.5 (15, M+Na+).
3.1.5. 20,30-Bis-O-(tert-butyldimethylsilyl)-50-deoxy-50-
dimethylamino-N6-(endo-norborn-2-yl)adenosine (14a).
To a solution containing 13 (72.7 mg, 0.123 mmol) in
MeCN (2 mL), stirred at ambient temperature, was added
formalin (35% CH2O(aq), 0.310 mL, 115 mg, 3.816 mmol,
30.9 equiv) in a single portion to give a cloudy solution.
After 5 min. the solution had cleared and NaBH3CN
(31.8 mg, 0.506 mmol, 4.1 equiv) was added to again give
a cloudy solution which clears with stirring. After 15 min.
AcOH (5 drops) was added to adjust pH to 7 and stirred
for 24 h. The reaction mixture was reduced in vacuo, ta-
ken up in CHCl3 (50 mL) and washed using 2.5 M NaOH
(50 mL), dried (MgSO4), filtered and concentrated under
reduced pressure. Subsequent column chromatography
gave the title compound (27.4 mg, 36.0%) as a clear oil.
1H NMR d 8.33 (s, 1H, H-2/8), 7.84 (s, 1H, H-8/2), 5.83,
5.83 (d, J = 4.8 Hz, 1H, H-10), 5.74, 5.71 (br s, 1H, NH),
5.02–4.96 (m, 1H, 0H-20), 4.52 (br s, 1H, H-200), 4.23–4.19
(m, 2H, H-30, H-4 ), 2.88 (br s, 1H, H-5a0/b0), 2.63 (br s,
2H, H-5b0/a0, H-100), 2.34 (s, 6H, N(CH3)2), 2.28–2.18
(m, 2H, H-400, H-3x00), 1.71–1.27 (m, 6H, H-500, H-600, H-
700), 0.94 (s, 10H, H-3n00, t-Bu), 0.82 (s, 9H, t-Bu), 0.13
(s, 3H, CH3), 0.11 (s, 3H, CH3), ꢀ0.03, ꢀ0.04 (s, 3H,
CH3), ꢀ0.20, ꢀ0.22 (s, 3H, CH3). 13C NMR d 155.0 (C),
152.9 (CH), 148.5 (C), 139.6, 139.5 (CH), 120.8 (C), 90.0
(CH), 82.5, 82.4 (CH), 74.3 (CH), 74.0, 73.8 (CH), 61.4
(CH2), 52.1 (CH), 45.9 (CH3), 40.5 (CH), 38.2 (CH2,
CH2), 36.8 (CH), 29.9 (CH2), 25.8 (CH3), 25.7 (CH3),
21.4 (CH2), 18.0 (C), 17.8 (C), ꢀ4.4 (CH3), ꢀ4.7
(2 · CH3), ꢀ5.0 (CH3). LRMS m/z (%): 617.7 (M+H+,
100), 618.6 (45), 619.6 (19).
3.1.7. N6-(endo-Norborn-2-yl)adenosine (17). A solution
containing 16 (1.005 g, 3.504 mmol), N(i-Pr)2Et
(1.50 mL, 1.113 g, 8.611 mmol, 2.5 equiv), and ( )-
endo-norborn-2-yl amine.HCl (0.618 g, 4.186 mmol,
1.2 equiv) in t-BuOH was heated at reflux for 14 h.
The reaction mixture was reduced in vacuo and purified
via column chromatography (CHCl3/MeOH, 9:1) to
give 17 (1.192 g, 94.1%) as a tan foam. 1H NMR d
8.17 (s, 1H, H-2/8), 7.88 (s, 1H, H-8/2), 6.26 (br s, 1H,
NH), 5.83 (s, 1H, H-10), 4.93 (s, 1H, H-20), 4.45 (br s,
2H, H-200, H-30), 4.27 (s, 1H, H-40), 3.89 (d,
J = 11.9 Hz, 1H, H-5a0/b0), 3.71 (d, J = 11.9 Hz, 1H,
H-5b0/a0), 2.55 (s, 1H, H-100), 2.25–2.17 (m, 2H, H-3x00,
H-400), 1.57–1.21 (m, 6H, H-500, H-600, H-700), 0.88 (br s,
1H, H-3n00). 13C NMR d 154.7 (C), 153.2 (CH), 146.9
(C), 139.8 (CH), 120.3 (C), 90.6 (CH), 86.4 (CH), 73.9
(CH), 72.0 (CH), 62.8 (CH2), 52.2 (CH), 40.3 (CH),
38.1 (CH2), 37.6 (CH2), 36.7 (CH), 29.8 (CH2), 21.4
(CH2). LRMS m/z (%): 362.2 (M+H+, 100).
3.1.8. 20,30-O-Isopropylidene-N6-(endo-norborn-2-yl)aden-
osine (18). To a magnetically stirred solution of 17
(1.125 g, 3.113 mmol) in dry acetone (40 mL) was added
2,2-dimethoxypropane (6.5 mL, 5.506 g, 52.861 mmol,
17.0 equiv) and p-TsOH (0.645 g, 3.745 mmol, 1.2 equiv).
The resultant mixture was stirred at ambient temperature
for 1.5 h. before being concentrated under reduced pres-
sure, taken up in H2O (30 mL) and extracted using CHCl3
(2 · 50 mL). The combined organic phase was washed
using sat. NaHCO3 (30 mL), dried (MgSO4), filtered and
reduced in vacuo to give 18 (1.150 g, 92.0%) as a tan
foam. 1H NMR d 8.28 (s, 1H, H-2/8), 7.76 (s, 1H, H-8/2),
6.68 (br s, 1H, OH), 6.01 (br s, 1H, NH), 5.82 (d,
J = 4.5 Hz, 1H, H-10), 5.20–5.07 (m, 1H, H-20), 5.08 (d,
J = 6.0 Hz, 1 H, H-30), 4.49 (s, 2 H, H-40, H-200), 3.93 (d,
J = 12.6 Hz, 1 H, H-5a0/b0), 3.75 (d, J = 12.6 Hz, 1H, H-
5b0/a0), 2.59 (br s, 1H, H-100), 2.24 (br s, 1H, H-400), 2.18–
2.14 (m, 1H, H-3x00), 1.60–1.23 (m, 6H, H-500, H-600, H-
700), 1.60 (s, 3H, CH3), 1.34 (s, 3H, CH3), 0.91–0.87 (m,
1H, H-3n00). 13C NMR d 155.0 (C), 152.5 (CH), 146.9
(C), 139.1 (CH), 121.0 (C), 113.6 (C), 93.9 (CH), 85.9
(CH), 82.8 (CH), 81.4 (CH), 63.1 (CH2), 51.9 (CH), 40.2
(CH), 38.0 (CH2), 37.7 (CH2), 36.5 (CH), 29.7 (CH2),
27.4 (CH3), 25.0 (CH3), 21.3 (CH2). LRMS m/z (%):
402.4 (M+H+, 100), 403.3 (20).
3.1.6. 50-Acetamido-20,30-bis-O-(tert-butyldimethylsilyl)-
50-deoxy-N6-(endo-norborn-2-yl)adenosine (14b). To a
stirred solution containing 13 (118 mg, 0.201 mmol) in
anhydrous pyridine (1 mL) held at 0 ꢂC, was added
Ac2O (19 lL, 21 mg, 0.201 mmol, 1 equiv). The reaction
mixture was then stirred at rt for 1.5 h. after which time
it was poured into EtOAc (20 mL) and washed with
0.5 M HCl (2 · 20 mL). The organic layer was then
dried (MgSO4), filtered and reduced in vacuo. Column
chromatography was performed using CHCl3/MeOH
(95:5) as the eluent. Concentration of the appropriate
fractions gave 14b (118 mg, 93.3%) as an orange oil.
1H NMR d 9.06 (br s, 1H, NHAc), 8.33 (s, 1H, H-2/
8), 7.77 (s, 1H, H-8/2), 6.00 (br s, 1H, N6H), 5.71 (d,
J = 7.8 Hz, 1H, H-10), 4.91ꢀ4.84 (m, 1H, H-20), 4.47
(br s, 1H, H-200), 4.23ꢀ4.15 (m, 2H, H-5a0, H-5b0),
4.08 (d, J = 3.6 Hz, 1H, H-30), 3.10ꢀ3.06 (m, 1H, H-
40), 2.62 (br s, 1H, H-100), 2.27ꢀ2.17 (m, 2H, H-400, H-
3x00), 2.12 (s, 3H, CH3), 1.68ꢀ1.25 (m, 6H, H-500, H-600,
H-700), 0.92 (s, 10H, t-Bu, H-3n00), 0.69 (s, 9H, t-Bu),
0.11 (s, 3H, CH3), 0.09 (s, 3H, CH3), ꢀ0.20 (s, 3H,
CH3), ꢀ0.53 (s, 3H, CH3). 13C NMR d 170.3 (C@O),
155.2 (C), 152.4 (CH), 147.6 (C), 140.5 (CH), 121.4
(C), 90.1 (CH), 87.0 (CH), 73.4, 73.3 (CH), 52.1 (CH),
40.6 (CH2), 40.4 (CH), 38.2 (CH2), 37.8 (CH2), 36.7
(CH), 29.8, 29.8 (CH2), 25.7 (CH3), 25.5 (CH3), 24.2
(CH3), 22.6 (CH2), 17.9 (C), 17.7 (C), ꢀ4.6 (CH3),
3.1.9.
20,30-O-Isopropylidene-50-O-methylaminocarba-
moyl-N6-(endo-norborn-2-yl)adenosine (19a). To a solu-
tion containing18 (15.2 mg, 0.038 mmol) in dry THF
(1 mL) under N2 was added CDI (22.3 mg, 0.138 mmol,
3.6 equiv). The reaction mixture was stirred for 1.5 h.
after which time 2 drops of H2O was added followed
by 40% MeNH2 in MeOH (0.5 mL), then the reaction
vessel was sealed and left to stir overnight. The crude
reaction mixture was reduced in vacuo and purified via
column chromatography (CHCl3/MeOH; 95:5) to give
1
19a (15.7 mg, 90.4%) as a clear oil. H NMR d 8.34 (s,
1H, H-2/8), 7.82 (s, 1H, H-8/2), 6.07 (d, J = 1.8 Hz,
1H, H-10), 5.87 (br s, 1H, N6H), 5.49–5.47 (m, 1H, H-

T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
1869
20), 5.02 (dd, J = 5.7 and 3.0 Hz, 1H, H-30), 4.72 (br s,
1H, NHCH3), 4.51–4.43 (m, 2H, H-40, H-200), 4.32 (dd,
J = 11.7 and 3.9 Hz, 1H, H-5a0/b0), 4.20 (dd, J = 11.7
and 5.7 Hz, 1H, H-5b0/a0), 2.72 (d, J = 4.5 Hz, 3H,
NHCH3), 2.60 (br s, 1H, H-100), 2.26–2.15 (m, 2H, H-
3x00, H-400), 1.68–1.24 (m, 12H, H-500, H-600, H-700,
C(CH3)2), 0.93–0.87 (m, 1H, H-3n00). 13C NMR d
156.3 (C@O), 155.0 (C), 153.3 (CH), 148.3 (C), 138.8
(CH), 120.4 (C), 114.4 (C), 91.1 (CH), 85.3 (CH), 84.0
(CH), 81.7 (CH), 64.4 (CH2), 52.1 (CH), 42.4 (CH),
38.2 (CH2), 38.1 (CH2), 36.8 (CH), 29.9 (CH2), 27.5
(CH), 27.1 (CH3), 25.3 (CH3), 21.4 (CH2). LRMS m/z
(%): 459.4 (M+H+, 100), 460.5 (25).
(CH). LRMS m/z (%): 209.1 (55), 279.2 (100), 391.5
(50), 419.4 (M+H+, 70). HRMS: C19H26N6O5 requires
[M+H]+ 419.2037. Found 419.2024.
3.1.12. 50-O-Dimethylaminothiocarbamoyl-N6-(endo-nor-
born-2-yl)adenosine (20b). A solution of 19b (113.9 mg,
0.233 mmol) in 80% AcOH (4 mL) was heated at 80 ꢂC
for 17 h. The reaction mixture was reduced in vacuo
and purified via column chromatography eluting with
CHCl3/MeOH (95:5) to give 20b (83 mg, 79.0%) as a
1
clear oil. H NMR d 8.24 (s, 1H, H-2/8), 7.92 (s, 1H,
H-8/2), 6.19 (br s, 1H, NH), 5.96 (d, J = 4.5 Hz, 1H,
H-10), 4.76–4.69 (m, 2H, H-5a0, H-5b0), 4.62–4.58 (m,
1H, H-20), 4.51–4.45 (m, 3H, H-200, H-30, H-40), 3.31
(s, 3H, CH3), 2.99 (d, J = 2.7 Hz, 3H, CH3), 2.58 (br s,
1H, H-100), 2.26–2.15 (m, 2H, H-3x00, H-400), 1.67–1.24
(m, 6H, H-500, H-600, H-700), 0.92 (m, 1H, H-3n00). 13C
NMR d 187.7 (C@S), 154.7 (C), 152.8 (C), 147.6 (C),
137.9 (CH), 119.9 (C), 90.2 (CH), 83.4 (CH), 75.3
(CH), 71.1 (CH), 70.1 (CH2), 52.2 (CH), 43.0 (CH3),
40.4 (CH), 38.2 (CH2), 38.0 (CH2), 37.9 (CH3), 36.8
(CH), 29.9 (CH2), 21.5 (CH2). LRMS m/z (%): 449.2
(M+H+, 100). HRMS: C20H28N6O4S requires [M+H]+
449.1966. Found 449.1968.
3.1.10. 50-O-Dimethylaminothiocarbamoyl-20,30-O-iso-
propylidene-N6-(endo-norborn-2-yl)adenosine (19b). To a
magnetically stirred solution of NaH (21.1 mg,
0.528 mmol, 1.1 equiv) in anhydrous THF (4 mL) held
at 0 ꢂC under an Ar atmosphere was added a solution
of 18 (192.6 mg, 0.480 mmol) in dry THF (2 mL). After
30 min. NaI (4 mg, 0.027 mmol, 0.06 equiv) and
DMTC-Cl (73.1 mg, 0.591 mmol, 1.2 equiv) were added
successively and the resultant solution was allowed to
warm to rt, then stirred overnight. The reaction mixture
was quenched using sat. NH4Cl (5 mL) and then ex-
tracted using Et2O (3 · 10 mL). The combined organic
phase was washed using H2O (30 mL) and brine
(30 mL), dried (Na2SO4), filtered and reduced in vacuo.
Subsequent column chromatography gave the title com-
pound (140 mg, 59.8%) as a white foam. 1H NMR d 8.32
(s, 1H, H-2/8), 7.80 (s, 1H, H-8/2), 6.04 (d, J = 1.8 Hz,
1H, H-10), 5.88 (br s, 1H, NH), 5.48 (d, J = 6.3 Hz,
1H, H-20), 5.08 (dd, J = 6.3 and 3.3 Hz, 1H, H-30),
4.74 (dd, J = 11.3 and 3.9 Hz, 1H, H-5a0/b0), 4.60–4.44
(m, 3H, H-40, H-5b0/a0, H-200), 3.28 (s, 3H, NCH3),
3.00 (d, J = 1.2 Hz, 3H, NCH3), 2.59 (br s, 1H, H-100),
2.24–2.15 (m, 2H, H-3x00, H-400), 1.63–1.22 (m, 6H, H-
500, H-600, H-700), 1.58 (s, 3H, CCH3), 1.36 (s, 3H,
CCH3), 0.92–0.85 (m, 1H, H-3n00). 13C NMR d 187.4
(C@S), 154.8 (C), 153.2 (CH), 148.1 (C), 138.7 (CH),
120.4 (C), 114.5 (C), 90.9 (CH), 84.9 (CH), 84.0 (CH),
81.5 (CH), 70.2 (CH2), 52.0 (CH), 42.8 (CH3), 40.4
(CH), 38.1 (CH2), 38.0 (CH2), 37.7 (CH3), 36.7 (CH),
29.8 (CH2), 27.0 (CH3), 25.3 (CH3), 21.4 (CH2). LRMS
m/z (%): 489.5 (M+H+, 100), 490.4 (30).
3.1.13. 20,30-Bis-O-(tert-butyldimethylsilyl)-50-chloro-50-
deoxy-N6-(2S-exo-5,6-epithio-endo-norborn-2-yl)adeno-
sine (23a). A mixture of 21 (255 mg, 0.48 mmol), 22
(132 mg, ꢁ0.46 mmol) and N(i-Pr)2Et (0.3 mL,
1.7 mmol) in t-BuOH (4 mL) was heated to reflux for
20 h. The reaction mixture was reduced in vacuo on
SiO2 and purification by column chromatography
(petroleum ether/EtOAc, 10:1) afforded title compound
23a (109 mg, 36%) as a transparent solid (mp 82–84 ꢂC).
1H NMR d 8.35 (br s, 1H, H-2/8), 7.94 (br s, 1H, H-8/2),
6.07 (br s, 1H, NH), 5.86 (d, 1H, H-10), 5.09 (t, 1H, H-20),
4.72 (br s, 1H, H-200), 4.37 (t, 1H, H-30), 4.26–4.30 (m,
1H, H-40), 4.08 (dd, 1H, H-5a0/H-5b0), 3.72 (dd, 1H,
H-5a0/H-5b0), 3.03 (d, 1H, H-500/H-600), 2.96 (d, 1H,
H-100/H-400), 2.93 (d, 1H, H-500/H-600), 2.51 (d, 1H,
H-100/H-400), 2.41–2.32 (m, 1H, H-3x00), 1.66–1.63 (m,
1H, H-7s00), 1.20–1.10 (m, 1H, H-3n00), 0.98–0.92 (m,
1H, H-7a00), 0.92 (s, 9H, t-Bu), 0.79, (s, 9H, t-Bu), 0.13
(s, 3H, CH3), 0.11 (s, 3H, CH3), ꢀ0.05 (s, 3H, CH3),
ꢀ0.24 (s, 3H, CH3). 13C NMR d 154.6, 154.6, 152.9,
139.6, 120.6, 89.8, 84.0, 73.5, 72.6, 53.0, 43.5, 41.2,
38.1, 37.3, 36.2, 33.2, 27.5, 25.7, 25.6, 17.9, 17.8, ꢀ4.5,
ꢀ4.7, ꢀ4.8, ꢀ5.1.
3.1.11. 50-O-Methylaminocarbamoyl-N6-(endo-norborn-
2-yl)adenosine (20a). A solution of 19a (49.9 mg,
0.109 mmol) in 80% AcOH (2 mL) was heated at 70–
80 ꢂC for 43 h. The reaction mixture was reduced in va-
cuo, co-evaporated using H2O to give 20a (35 mg,
3.1.14. 20,30-Bis-O-(tert-butyldimethylsilyl)-50-deoxy-N6-
(2S-exo-5,6-epithio-endo-norborn-2-yl)-50-methylselenoad-
enosine (24). To a suspension of KH (20 mg, in oil) in
DMF (0.5 mL) was added a solution of Me2Se2
(15 mg, 0.11 mmol) in DMF (1 mL). The reaction was
stirred at rt for 2 h, then a solution of 23a (35 mg,
0.055 mmol) in DMF (1 mL) was added. After 4 h. the
reaction was diluted with water (10 mL) and extracted
with EtOAc (3 · 10 mL). The organic phase was reduced
in vacuo and purification by column chromatography
(CHCl3/MeOH; 98:2) afforded the title compound 24
1
76.1%) as a clear oil. H NMR d 8.27 (s, 1H, H-2/8),
7.95 (s, 1H, H-8/2), 6.05 (br s, 1H, N6H), 5.96 (d,
J = 5.1 Hz, 1H, H-10), 4.93 (br s, 1H, NHCH3), 4.51
(app. d, Japp = 4.2 Hz, 1H, H-20), 4.41–4.30 (m, 4H,
H-20, H-30, H-40, H-5a0, H-5b0), 4.08 (br s, 1H, H-200),
2.76, 2.74 (s, 3H, NHCH3), 2.61 (br s, 1H, H-100),
2.28–2.18 (m, 2H, H-3x00, H-400), 1.65–1.21 (m, 6H, H-
500, H-600, H-700), 0.95–0.90 (m, 1H, H-3n00). 13C NMR
d 156.7 (C@O), 154.7 (C), 152.6 (CH), 147.7 (C), 138.0
(CH), 119.8 (C), 89.9 (CH), 83.9 (CH), 75.6 (CH), 71.4
(CH), 64.0 (CH2), 52.3 (CH), 40.6 (CH), 38.3 (CH2),
37.9 (CH2), 36.8 (CH), 29.9 (CH2), 27.6 (CH), 21.5
1
(6 mg, 23%) as a transparent solid (mp 47–49 ꢂC). H
NMR d 8.39 (s, 1 H, H-2/8), 7.91 (s, 1H, H-8/2), 5.89
(d, 1H, H-10), 5.77 (br s, 1H, NH), 5.13 (t, 1H, H-20),

1870
T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
4.75 (br s, 1H, H-200), 4.35–4.24 (m, 2H, H-30, H-40),
3.10–2.89 (m, 5H, H-100, H-500, H-600, H-5a0, H-5b0),
2.55 (s, 1H, H-400), 2.46–2.37 (m, 1H, H-3x00), 2.04 (s,
3H, SeCH3), 1.69 (m, 1H, H-7s00), 1.15–1.10 (m, 1H, H-
3n00), 0.91–0.87 (m, 1H, H-7a00), 0.80 (s, 9H, t-Bu), 0.95
(s, 9H, t-Bu), 0.16 (s, 3H, CH3), 0.13 (s, 3H, CH3),
ꢀ0.06 (s, 3H, CH3), ꢀ0.27 (s, 3H, CH3). 13C NMR d
154.7, 152.9, 140.0, 128.8, 120.8, 89.7, 85.0, 75.1, 73.7,
53.0, 41.2, 38.1, 37.3, 36.5, 33.1, 29.7, 27.6, 25.8, 25.7,
18.0, 17.9, 5.5, ꢀ4.4, ꢀ4.5, ꢀ4.7, ꢀ5.1.
sation using MeOH gave 29 (240 mg, 0.444 mmol,
44.4%) as clear prisms. H NMR d 8.57 (s, 1H, H-2/8),
1
8.27 (s, 1H, H-8/2), 5.97 (d, J = 4.8 Hz, 1H, H-10), 4.82
(br s, 1H, H-20), 4.26 (br s, 2H, H-30, H-40), 3.02 (dd,
J = 14.1 and 6.3 Hz, 1H, H-5a0/b0), 2.91 (dd, J = 14.1
and 3.9 Hz, 1H, H-5b0/a0), 2.61 (q, J = 7.4 Hz, 2H,
CH2), 1.27 (t, J = 7.4 Hz, 3H, CH3), 0.95 (s, 9H, t-Bu),
0.81 (s, 9H, t-Bu), 0.16 (s, 3H, CH3), 0.13 (s, 3H,
CH3), ꢀ0.02 (s, 3H, CH3), ꢀ0.19 (s, 3H, CH3). 13C
NMR d 157.7 (C), 148.4 (C), 146.0 (CH), 139.9 (CH),
123.5 (C), 89.7 (CH), 84.9 (CH), 75.0 (CH), 74.2 (CH),
34.0 (CH2), 27.1 (CH2), 25.8 (CH3), 25.7 (CH3), 18.0
(C), 17.9 (C), 14.7 (CH3), ꢀ4.4 (CH3), ꢀ4.5 (CH3),
ꢀ4.6 (CH3), ꢀ5.0 (CH3). LRMS m/z (%): 541.3
(M+H+, 100), 405.4 (40).
3.1.15. 50-Deoxy-N6-(2S-exo-5,6-epithio-endo-norborn-2-
yl)-50-methylthioadenosine (25a). The thiirane 23a
(32 mg, 0.05 mmol) and NaSCH3 (18 mg, 0.26 mmol)
were dissolved in DMF (1 mL) and stirred at rt for
41 h. The reaction mixture was packed on SiO2 and
purification by column chromatography (CHCl3 to
CHCl3/MeOH, 98:2) afforded the title compound 25a
3.1.18. N6-Cyclopentyl-20,30-bis-O-(tert-butyldimethylsi-
lyl)-50-deoxy-50-ethylthioadenosine (30). To a magneti-
cally stirred solution containing 29 (37.3 mg, 0.069
mmol) and PyBroP (66.3 mg, 0.142 mmol, 2.1 equiv) in
DCE (1 mL) was added N(i-Pr)2Et (48.6 lL, 36.1 mg,
0.279 mmol, 4.0 equiv). The reaction mixture was stirred
at ambient temperature for 10 min. after which time c-
PentNH2 (13.6 lL, 11.7 mg, 0.138 mmol, 2.0 equiv)
was added and the resultant solution was heated at re-
flux for 22 h. The reaction mixture was reduced in vacuo
then purified via column chromatography, eluting using
CHCl3/MeOH (98:2) to give the title compound
1
(10 mg, 47%) as a transparent solid (mp 69–71 ꢂC). H
NMR d 8.33 (s, 1H, H-2/8), 8.01 (s, 1H, H-8/2), 6.27
(br s, 1H, NH), 5.93 (d, 1H, H-10), 4.70 (br s, 1H, H-
200), 4.56 (t, 1H, H-20), 4.47–4.43 (m, 1H, H-400), 4.40–
4.38 (m, 1H, H-30), 3.05 (d, 1H, H-6n00), 2.98–2.97 (m,
2H, H-100, H-5n00), 2.83 (d, 2H, H-5a0/b0), 2.55 (s, 1H,
H-400), 2.39–2.34 (m, 1H, H-3x00), 2.18 (s, 3H, SCH3),
1.68 (d, 1H, H-7s00), 1.19–1.14 (m, 1H, H-3n00), 0.97 (d,
1H, H-7a00). 13C NMR d 154.7, 152.6, 147.8, 137.9,
119.9, 90.2, 84.9, 75.4, 73.4, 53.0, 41.2, 38.1, 37.3, 36.8,
36.1, 33.1, 27.5, 16.7. HRMS: C18H23N5O3S2 requires
[M+H]+ 422.1315. Found 422.1316.
1
(38.4 mg, 91.6%) as a clear oil. H NMR d 8.32 (s, 1H,
H-2/8), 7.91 (s, 1H, H-8/2), 5.86 (d, J = 5.4 Hz, 2H, H-
10, NH), 5.04 (br s, 1H, H-20), 4.61 (br s, 1H, H-100),
4.29 (br s, 1H, H-30), 4.21 (br s, 1H, H-40), 3.06 (dd,
J = 14.1 and 6.6 Hz, 1H, H-5a0/b0), 2.87 (dd, J = 14.1
and 5.7 Hz, 1H, H-5b0/a0), 2.58 (q, J = 7.2 Hz, 2H,
SCH2CH3), 2.14–2.10 (m, 2H, H-2a00, H-5a00), 1.77–1.57
(m, 6H, H-2b00, H-3a00, H-3b00, H-4a00, H-4b00, H-5b00),
1.24 (t, J = 7.2 Hz, 3H, SCH2CH3), 0.93 (s, 9H, t-Bu),
0.78 (s, 9H, t-Bu), 0.14 (s, 3H, CH3), 0.11 (s, 3H, CH3),
ꢀ0.07 (s, 3H, CH3), ꢀ0.28 (s, 3H, CH3). 13C NMR d
154.2 (C), 152.7 (CH), 148.7 (C), 139.8 (CH), 120.5 (C),
89.5 (CH), 84.6 (CH), 74.3 (CH), 73.7 (CH), 52.4 (CH),
33.8 (CH2), 33.4 (CH2), 26.9 (CH2), 25.8 (CH3), 25.6
(CH3), 23.7 (CH2), 18.0 (C), 17.8 (C), 14.6 (CH2), ꢀ4.4
(CH3), ꢀ4.6 (CH3), ꢀ4.7 (CH3), ꢀ5.1 (CH3). LRMS m/
z (%): 608.4 (M+H+, 100), 609.6 (45).
3.1.16. N6-(2S-exo-5,6-epithio-endo-norborn-2-yl)-N-eth-
yladenosine-50-uronamide (27). Compound 26 (74 mg,
0.200 mmol) was suspended in water (2 mL) with Dow-
ex-50W (H+) resin (148 mg) and stirred at rt for 45.5 h.
The reaction was then filtered and reduced in vacuo to
afford a viscous oil which was taken up in t-BuOH
(4 mL). To this mixture was added 21 (43 mg,
0.15 mmol) and N(i-Pr)2Et (0.1 mL, 0.600 mmol) and
was then heated at reflux for 15.5 h. The reaction mix-
ture was reduced in vacuo on SiO2 and purification by
column chromatography (CHCl3/MeOH; 95:5) afforded
1
27 (25 mg, 40%) as a white solid (mp 206–207 ꢂC). H
NMR d 8.33 (s, 1H, H-2/8), 8.28 (s, 1H, H-8/2), 6.02
(d, 1H, H-10), 4.76 (dd, 1H, H-20), 4.72–4.65 (m, 1H,
H-200), 4.47 (d, 1H, H-40), 4.32 (dd, 1H, H-30), 3.37 (q,
2H, CH2CH3), 3.08 (d, 1H, H-600), 3.04 (d, 1H, H-500),
2.89 (d, 1H, H-100), 2.51 (d, 1H, H-400), 2.40–2.31 (m,
1H, H-3x00), 1.68 (d, 1H, H-7s00), 1.31–1.24 (m, 1H, H-
3n00), 1.23 (t, 3H, CH2CH3), 0.97 (d, 1H, H-7a00). 13C
NMR d 172.0, 156.2, 153.9, 149.5, 142.2, 121.5, 90.5,
86.4, 75.0, 73.4, 54.4, 42.5, 39.4, 38.0, 36.3, 35.1, 33.8,
28.2, 15.1. HRMS: C19H24N6O4S requires [M+H]+
433.1653. Found 433.1649.
3.2. General procedure for TBS deprotection
50-Modified compounds and NH4Cl (P10 equiv) were
heated to ꢁ60 ꢂC in dry methanol (1–4 mL) for 24–
48 h. under a N2 atmosphere. The reaction mixture
was then packed onto SiO2 and purified by column
chromatography (CHCl3:MeOH).
3.2.1. N6-Cyclopentyl-50-deoxy-50-ethylthioadenosine (2).
Compound 30 (43 mg, 0.071 mmol) was reacted with
NH4F (25 mg, 0.683 mmol, 9.6 equiv) to afford 2 as a
clear oil (10 mg, 37%). CHCl3/MeOH (85:15). 1H
NMR d 8.28 (s, 1H, H-2/8), 8.06 (s, 1H, H-8/2), 5.97
(br s, 1H, H-10), 4.59 (br s, 2H, H-20, H-100), 4.40 (s,
2H, H-30, H-40), 2.87 (br s, 2H, H-5a0, H-5b0), 2.60 (q,
J = 7.2 Hz, 2H, SCH2CH3), 2.16–2.11 (m, 2H, H-200,
H-500), 1.79–1.58 (m, 6H, H-200, H-300, H-400, H-500), 1.25
3.1.17. 20,30-Bis-O-(tert-butyldimethylsilyl)-50-deoxy-50-
ethylthioinosine (29). To a solution of 28 (515 mg,
1.000 mmol) in anhydrous DMF (10 mL) under N2
was added NaSEt (480 mg, 5.706 mmol, 5.7 equiv) and
stirred vigorously at ambient temperature overnight.
The resultant solution was taken up in EtOAc (50 mL)
and washed using 4% NaHCO3 (50 mL) and H2O
(50 mL), dried (Na2SO4), filtered and reduced. Crystalli-

T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
1871
(t, J = 7.2 Hz, 3H, CH3). Data obtained for 2 were in
accordance with the literature.7
3.2.5. 50-Deoxy-N6-(endo-norborn-2-yl)-50-(2-fluorophenyl-
thio)adenosine (12c). Compound 11c (118.7 mg,
0.170 mmol) was reacted with NH4F (64.9 mg,
1.752 mmol) to afford the title compound 12c (39.1 mg,
48.9%) as a clear oil. 1H NMR d 8.25 (s, 1H, H-2/8), 7.78
(s, 1H, H-8/2), 7.41 (app. t, Japp = 7.5 Hz, 1H, ArH),
7.23–7.18 (m, 1H, ArH), 7.06–7.00 (m, 2H, ArH), 6.52
(br s, 1H, OH), 6.05, 6.03 (br s, 1H, NH), 5.87 (d,
J = 5.7 Hz, 1H, H-10), 4.63 ꢀ4.58 (m, 1H, H-20), 4.42 (br
s, 3H, H-200, H-30, H-40), 3.96 (br s, 1H, OH), 3.29–3.17
(m, 2H, H-5a0, H-b0), 2.60 (br s, 1H, H-100), 2.27–2.17 (m,
2H, H-3x00, H-400), 1.65–1.16 (m, 6H, H-500, H-600, H-700),
0.94–0.85 (m, 1H, H-3n00). 13C NMR d 161.7 (d,
1JCF = 244.3 Hz, C), 154.9 (C), 152.7 (CH), 147.7 (C),
3.2.2. 50-Chloro-50-deoxy-N6-(endo-norborn-2-yl)adeno-
sine (10b). Compound 10a (37.9 mg, 0.062 mmol) was re-
acted with NH4F (24.0 mg, 0.648 mmol, 10.4 equiv) to
give 10 b (18.9 mg, 79.8%) as a clear oil. CHCl3/MeOH
1
(95:5). H NMR d 8.27 (s, 1H, H-2/8), 8.03 (s, 1H, H-8/
2), 6.13 (br s, 1H, NH), 5.99 (d, J = 4.8 Hz, 1H, H-10),
4.59 (app. t, Japp = 5.1 Hz, 1H, H-20), 4.53–4.43 (m, 1H,
H-30, H-40, H-200), 3.84 (dd, J = 12.0 and 4.5 Hz, 1H, H-
5a0/b0), 3.77 (dd, J = 12.0 and 3.9 Hz, 1H, H-b0/a0), 2.62
(br s, 1H, H-100), 2.29 (br s, 1H, H-400), 2.23–2.19 (m, 1H,
H-3x00), 1.67–1.22 (6H, H-500, H-600, H-700), 0.98–0.93 (m,
1H, H-3n00). 13C NMR d 154.6 (C), 152.7 (CH), 147.6
(C), 137.7 (CH), 119.6 (C), 89.6, 89.5 (CH), 84.2 (CH),
75.0, 74.9 (CH), 52.0 (CH), 44.1 (CH2), 40.3 (CH), 38.1
(CH2), 37.7 (CH2), 36.6 (CH), 29.7 (CH2), 21.3 (CH2).
LRMS m/z (%): 380.3 (M+H+, 35Cl, 100), 382.3 (37Cl,
25). HRMS: C17H22ClN5O3 requires [M+H]+ 380.1484.
Found 380.1485.
3
137.8 (CH), 133.1 (CH), 129.3 (d, JCF = 7.8 Hz, CH),
124.6 (d, JCF = 3.2 Hz, CH), 121.9 (d, JCF = 17.3 Hz,
4
2
2
C), 119.9 (C), 115.9 (d, JCF = 22.4 Hz, CH), 90.2, 90.1
(CH), 84.9 (CH), 75.3, 75.2 (CH), 73.3 (CH), 52.2 (CH2),
40.5 (CH), 38.2 (CH2), 37.9 (CH2), 36.8 (CH), 36.4
(CH2), 29.9 (CH2), 21.5 (CH2). LRMS m/z (%): 472.3
(M+H+, 100), 473.2 (30). HRMS: C19H28N6O3 requires
[M+H]+ 472.1813. Found 472.1821.
3.2.3. 50-Deoxy-N6-(endo-norborn-2-yl)-50-methylamino-
adenosine (12a). Compound 11a (42 mg, 0.070 mmol)
was reacted with NH4F (29 mg, 11.2 mmol) to afford
the title compound (15 mg, 57.7%) as a clear oil.
3.2.6. 50-Deoxy-N6-(endo-norborn-2-yl)-50-mercaptoade-
nosine (12d). Compound 11d (50.6 mg, 0.078 mmol) was
reacted with NH4F (33.3 mg, 0.899 mmol) to afford the ti-
tle compound 12d (24.1 mg, 81.8%) as a clear oil. CHCl3/
1
CHCl3/MeOH (8:2). H NMR d 8.24 (s, 1H, H-2/8),
1
8.22 (s, 1H, H-8/2), 5.96 (d, J = 5.4 Hz, 1H, H-10),
ꢁ4.8 (1H, H-20), 4.27–4.19 (m, 2H, H-30, H-40), 3.04
(dd, J = 12.8 and 7.5 Hz, 1H, H-5a0/b0), 2.95 (dd,
J = 12.8 and 3.6 Hz, 1H, H-5b0/a0), 2.59 (br s, 1H, H-
100), 2.46 (s, 3H, NHCH3), 2.28 (br s, 1H, H-400), 2.23–
2.13 (m, 1H, H-3x00), 1.67–1.33 (m, 6H, H-500, H-600, H-
700), 1.09–1.02 (m, 1H, H-3n00). 13C NMR d 156.3 (C),
154.1 (CH), 149.8 (C), 141.4 (CH), 121.2 (C), 91.1
(CH), 84.5 (CH), 74.9 (CH), 73.5 (CH), 54.5 (CH2),
53.7 (CH), 41.9 (CH), 39.3 (CH3), 38.3 (2 · CH2), 36.1
(CH), 30.9 (CH2), 22.5 (CH2). LRMS m/z (%): 375.3
(M+H+, 100), 376.3 (20). HRMS: C18H26N6O3 requires
[M+H]+ 375.2139. Found 375.2111.
MeOH (9:1). H NMR (CD3OD) d 8.24 (s, 1H, H-2/8),
8.22 (s, 1H, H-8/2), 6.00 (d, J = 5.4 Hz, 1H, H-10),
ꢁ4.85 (coincides with DOH, H-20) 4.43 (br s, 1H, H-200),
4.34–4.27 (m, 2H, H-30, H-40), 3.27–3.11 (m, 2H, H-5a0.
H-5b0), 2.61 (br s, 1H, H-10), 2.29 (br s, 1H, H-40), 2.24–
2.14 (m, 1H, H-3x00), 1.79–1.36 (m, 6H, H-500, H-600, H-
700), 1.09–1.02 (m, 1H, H-3n00). 13C NMR (CD3OD) d
156.7 (C), 154.6 (CH), 149.6 (C), 141.4 (CH), 121.4 (C),
90.8 (CH), 85.2 (CH), 75.4 (CH), 74.8 (CH), 54.1 (CH),
43.7 (CH2), 42.3 (CH), 39.7 (CH2), 38.8 (CH), 38.7
(CH2), 31.3 (CH2), 23.0 (CH2).
3.2.7. 50-Deoxy-N6-(endo-norborn-2-yl)-50-propylthioade-
nosine (12e). Compound 11e (34 mg, 0.052 mmol) was re-
acted with NH4F (21 mg, 0.562 mmol, 10.9 equiv) to
afford the title compound 12e (15 mg, 69.1%) as a clear
3.2.4. 50-Deoxy-N6-(endo-norborn-2-yl)-50-ethylthioade-
nosine (12b). A solution of 10a (108.6 mg, 0.178 mmol)
and NaSEt (150.4 mg, 1.788 mmol, 10.0 equiv) in anhy-
drous DMF (2 mL) under an N2 atmosphere was stirred
at ambient temperature for 44 h. The resultant crude
solution was taken up in sat. NH4Cl (90 mL) and ex-
tracted using EtOAc (2 · 60 mL), the combined organic
phase was washed using H2O (3 · 90 mL), dried
(Na2SO4), filtered and reduced in vacuo. The crude mate-
rial was reacted with NH4F (77.3 mg, 2.087 mmol,
ꢁ11.7 equiv) to afford 12b (36.0 mg, 49.9%) as a clear
1
oil. H NMR d 8.28 (s, 1H, H-2/8), 7.99 (s, 1H, H-8/2),
6.07 (br s, 1H, NH), 5.93 (d, J = 5.1 Hz, 1H, H-10), 4.57–
4.38 (m, 4H, H-20, H-200, H-30, H-40), 2.84 (app. d,
Japp = 5.7 Hz, 2H, H-5a0, H-b0), 2.62 (br s, 1H, H-100),
2.55 (t, J = 7.2 Hz, 2H, SCH2CH2), 2.28–2.19 (m, 2H, H-
3x00, H-400), 1.67–1.26 (m, 8H, H-500, H-600, H-700,
SCH2CH2), 0.97 (t, J = 7.2 Hz, 4H, H-3n00, CH2CH3).
13C NMR d 155.0 (C), 152.7 (CH), 147.8 (C), 137.8
(CH), 120.0 (C), 90.2 (CH), 85.2 (CH), 75.5 (CH), 73.4
(CH), 52.2 (CH), 40.5 (CH), 38.3 (CH2), 38.0 (CH2), 36.8
(CH), 35.2 (CH2), 34.7 (CH2), 29.9 (CH2), 22.9(CH2),
21.5 (CH2), 13.3 (CH3). HRMS: C20H29N5O3S requires
[M+H]+ 420.2064. Found 420.2074.
1
oil. CHCl3/MeOH (95:5). H NMR d 8.27 (s, 1H, H-2/
8), 7.99 (s, 1H, H-8/2), 6.09 (br s, 1H, NH), 5.94 (d,
J = 5.1 Hz, 1H, H-10), 4.59 (app. t, Japp = 3.6 Hz, 1H,
H-20), 4.44–4.37 (m, 3H, H-200, H-30, H-40), 2.87 (s, 1H,
H-5a0/b0), 2.85 (s, 1H, H-5b0/a0), 2.63–2.56 (m, 3H, H-
100, SCH2CH3), 2.28 (s, 1H, H-400), 2.22–2.18 (m, 1H, H-
3x00), 1.66–1.30 (m, 6H, H-500, H-600, H-700), 1.25 (t,
J = 7.5 Hz, 3H, SCH2CH3), 0.97–0.90 (m, 1H, H-3n00).
LRMS m/z (%): 406.4 (M+H+, 30). HRMS:
C19H27N5O3S requires [M+H]+ 406.1907. Found
406.1906.
3.2.8. 50-Deoxy-50-dimethylamino-N6-(endo-norborn-2-yl)-
adenosine (15a). Compound 14a (52.4 mg, 0.085 mmol)
was reacted with NH4F (37 mg, 1.004 mmol, 11.8 equiv)
1
to afford 15a as a clear oil (26 mg, 78.2%). H NMR d
8.30 (s, 1H, H-2/8), 7.95 (s, 1H, H-8/2), 6.06 (br s, 1H,
NH), 5.92 (d, J = 4.5 Hz, 1H, H-10), 4.53 (br s, 1H, H-20),

1872
T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
4.46 (br s, 1H, H-200), 4.30 (br s, 2H, H-30, H-40), 2.63–2.61
(m, 3H, H-100, H-5a0, H-5b0), 2.32 (s, 6H, N(CH3)2), 2.28–
2.18 (m, 2H, H-3x00, H-400), 1.66–1.22 (m, 6H, H-500, H-600,
H-700), 0.95–0.91 (m, 1H, H-3n00). 13C NMR d 154.9 (C),
152.8 (CH), 147.7 (C), 138.0 (CH), 120.1 (C), 90.3 (CH),
83.0 (CH), 75.0 (CH), 73.1 (CH), 61.8 (CH2), 52.1 (CH),
46.2 (CH3), 40.4 (CH), 38.2 (CH2), 37.9 (CH2), 36.7
(CH), 29.9 (CH2), 21.5 (CH2). LRMS m/z (%): 389.3
(M+H+, 100), 390.3 (25). HRMS: C19H28N6O3 requires
[M+H]+ 389.2296. Found 389.2292.
1.69 (d, 1H, H-7s00), 1.27–1.24 (m, 1H, H-3n00), 0.98 (d,
1H, H-7a00). 13C NMR (400 MHz) d 154.9, 152.7, 148.2,
138.2, 120.2, 90.6, 85.9, 75.9, 74.2, 53.2, 41.4, 38.3, 37.4,
36.5, 33.1, 29.8, 27.7, 5.6. HRMS: C18H23N5O3SSe re-
quires [M+H]+ 470.0760. Found 470.0770.
3.3. Cell culture and cAMP assay
DDT1MF-2 cells were grown in 48-well culture plates
using Dulbecco’s modified Eagle’s medium containing
2.5 lg/mL amphotericin B, 100 U/ml penicillin G,
0.1 mg/mL streptomycin sulfate and 5% foetal bovine
serum in a humidified atmosphere of 95% air and 5%
CO2. Cells were routinely used at one day preconfluence.
To begin an experiment, the culture medium was
removed from each well, and warm Hank’s Balanced Salt
solution (HBSS) was added. This wash solution was
removed after 6 min at 37 ꢂC and replaced with HBSS
containing adenosine deaminase (0.5 U/mL), 20 lM
rolipram, with or without 1 lM (ꢀ)-isoproterenol and
varying concentrations of the test compounds in the pres-
ence of (ꢀ)-isoproterenol. After a 6-min incubation at
37 ꢂC, the incubation solution was aspirated, and HCl
(0.5 mL, 50 mM) was added to terminate drug action.
3.2.9. 50-Acetamido-50-deoxy-N6-(endo-norborn-2-yl)aden-
osine (15b). Compound 14b (118 mg, 0.187 mmol) was re-
acted with NH4F (70 mg, 1.895 mmol, 10.1 equiv) to
afford 15b (39 mg, 51.3%) as an opaque oil. CHCl3/MeOH
0
(97:3). 1H NMR d8.79(brs,1H, N5 H), 8.26(s, 1H, H-2/8),
7.85 (s, 1H, H-8/2), 6.19 (s, 1H, N6H), 5.78 (d, J = 6.6 Hz,
1H, H-10), 4.80–4.75 (m, 1H, H-20), 4.42 (br s, 1H, H-200),
4.32 (br s, 1H, H-5a0/b0), 4.22 (app. d, Japp = 6.0 Hz, 1H,
H-5a0/b0), 4.08–4.00 (m, 1H, H-30), 3.17 (app. d,
Japp = 13.5 Hz, 1H, H-40), 2.57 (br s, 1H, H-100), 2.25 (br
s, 1H, H-400), 2.19–2.16 (m, 1H, H-3x00), 2.03 (s, 3H,
CH3), 1.62–1.18 (m, 6H, H-500, H-600, H-700), 0.91–0.87 (m,
1H, H-3n00). 13C NMR d 171.6 (C@O), 155.1 (C), 152.5
(CH), 147.7 (C), 140.2 (CH), 121.0 (C), 90.9 (CH), 85.0
(CH), 73.1 (CH), 71.4 (CH), 52.2 (CH), 41.1 (CH2), 40.4
(CH), 38.2 (CH2), 37.9 (CH2), 36.8 (CH), 29.9 (CH2),
22.8 (CH3), 21.4 (CH2). LRMS m/z (%): 403.2 (100),
404.3 (25). HRMS: C19H26N6O4 requires [M+H]+
403.2088. Found 403.2102.
The cAMP content in each well was determined by
radioimmunoassay. Briefly, a standard was prepared in
duplicate with tubes containing 100 lL cAMP (0.001–
10 pmol) in 50 mM HCl. The well plates containing
the samples were gently agitated and a 5-lL aliquot
was transferred to tubes containing 100 lL of 50 mM
HCl. Each sample was then acetylated by the addition
of 4.5 lL of a 3.5:1 mixture of triethylamine and acetic
anhydride and immediately vortexed. A 10-lL aliquot
3.2.10. 50-Chloro-50-deoxy-N6-(2S-exo-5,6-epithio-endo-
norborn-2-yl)adenosine (23b). Compound 23a (34 mg,
0.053 mmol) and NH4F (53 mg, 1.431 mmol, 27.0 equiv)
were heated to ꢁ60 ꢂC in MeOH (4 mL) for 14 h. The
reaction mixture was then packed on SiO2 and purified
by column chromatography (CHCl3/MeOH; 98:2) to
yield 23b (21 mg, 97%) as a white solid (mp 122–124
of
[
125I]-ScAMP-TME containing 20,000 cpm was
added followed by 100 lL cAMP antibody in a solution
of 50 mM sodium–acetate buffer, pH 4.75, containing
0.125% BSA. After incubation at room temperature,
50 lL hydroxyapatite in a 1:1 suspension with water
was added and incubated for 10 min at room tempera-
ture. Using a Brandell cell harvester, the samples were
then aspirated through Whatman GF/B glass fibre filters
under reduced pressure, and the filters rinsed with an
additional 6 mL of ice-cold 10 mM Tris buffer at pH
7.0. The radioactivity retained by the filters was deter-
mined using a Beckman gamma counter.
1
ꢂC). H NMR (CD3OD) d 8.29 (s, 1H, H-2/8), 8.27 (s,
1H, H-2/8), 6.04 (d, 1H, H-10), 4.77 (t, 1H, H-20), 4.71
(br s, 1H, H-200), 4.39 (s, 1H, H-30), 4.28 (d, 1H, H-40),
3.95 (dd, 1H, H-5a0/b0), 3.85 (dd, 1H, H-5b0/a0), 3.07
(s, 1H, H-600), 3.04 (s, 1H, H-500), 2.87 (s, 1H, H-100),
2.50 (s, 1H, H-400), 2.41–2.32 (m, 1H, H-3x00), 1.66 (d,
1H, H-7s00), 1.34–1.24 (m, 1H, H-3n00), 0.96 (d, 1H, H-
7a00). 13C NMR (CD3OD) d 156.0, 154.0, 149.7, 140.6,
120.8, 89.9, 85.2, 75.0, 72.6, 54.3, 45.2, 42.4, 39.4, 38.0,
36.3, 33.8, 28.1. HRMS: C17H20ClN5O3S requires
[M+H]+ 410.1048. Found 410.1054.
Acknowledgements
3.2.11. 50-Deoxy-N6-(2S-exo-5,6-epithio-endo-norborn-2-
yl)-50-methylselenoadenosine (25b). Compound 24b
(7 mg, 0.010 mmol) and NH4F (81 mg, 2.187 mmol,
21.9 equiv) were heated to ꢁ60 ꢂC in MeOH (2 mL) for
40 h. The reaction mixture was then packed on SiO2 and
purified by column chromatography (CHCl3/MeOH;
98:2) to yield the title compound 25b (5 mg, 100%) as a
white solid (mp 82–84 ꢂC). 1H NMR d 8.36 (s, 1H, H-2/
8), 8.00 (s, 1H, H-8/2), 5.90 (d, 1H, H-10), 4.74 (br s, 1H,
H-200), 4.58 (t, 1H, H-20), 4.54–4.49 (m, 1H, H-40), 4.40–
4.38 (m, 1H, H-30), 3.05 (d, 1H, H-600), 2.98–2.97 (m,
2H, H-100, H-500), 2.83 (dd, 2H, H-5a0, H-5b0), 2.56 (s,
1H, H-400), 2.53–2.37 (m, 1H, H-3x00), 2.08 (s, 3H, CH3),
The authors thank Mr. Stuart Thompson, Dr. Simon
Egan and Dr. Martin Scanlon for assistance with the
acquisition of NMR and mass spectra. We also thank
the ARC Centre for Free Radical Chemistry and Bio-
technology for funding this research, in part.
References and notes
1. Yan, L.; Burbiel, J. C.; Maaß, A.; Muller, C. E. Expert
¨
Opin. Emerg. Drugs 2003, 8, 537–576.
2. Lerman, B. B.; Belardinelli, L. Circulation 1991, 83, 1499–
1509.

T. D. Ashton et al. / Bioorg. Med. Chem. 16 (2008) 1861–1873
1873
3. Hutchinson, S. A.; Scammells, P. J. Curr. Pharm. Design
2004, 10, 2021–2039.
13. Trivedi, B. K.; Bridges, A. J.; Patt, W. C.; Priebe, S. R.;
Bruns, R. F. J. Med. Chem. 1989, 32, 8–11.
14. Ashton, T. D.; Scammells, P. J. Bioorg. Med. Chem. Lett.
2005, 15, 3361–3363.
15. Ashton, T. D.; Scammells, P. J. Bioorg. Med. Chem. Lett.
2006, 16, 4564–4566.
4. Dhalla, A. K.; Shryock, J. C.; Shreeniwas, R.; Belardinelli,
L. Curr. Top. Med. Chem. 2003, 3, 369–385.
5. Palle, V. P.; Varkhedkar, V.; Ibrahim, P.; Ahmed, H.; Li,
Z.; Gao, Z.; Ozcek, M.; Wu, Y.; Zeng, D.; Wu, L.; Leung,
K.; Chu, N.; Zablocki, J. A. Bioorg. Med. Chem. Lett.
2004, 14, 535–539.
16. Pignot, M.; Pljevaljcic, G.; Weinhold, E. Eur. J. Org.
Chem. 2000, 3, 549–555.
6. Song, Y.; Wu, L.; Shryock, J. C.; Belardinelli, L. Circu-
lation 2002, 105, 118–123.
7. van der Wenden, E. M.; Carnielli, M.; Roelen, H. C. P. F.;
17. Barma, D. K.; Bandyopadhyay, A.; Capdevila, J. H.;
Falck, J. R. Org. Lett. 2003, 5, 4755–4757.
18. Hutchinson, S. A.; Baker, S. P.; Linden, J.; Scammells, P.
J. Bioorg. Med. Chem. 2004, 12, 4877–4884.
19. Krief, A.; Trabelsi, M.; Dumont, W. Synthesis 1992, 10,
933–935.
20. Olsson, R. A.; Kusachi, S.; Thompson, R. D.; Ukena, D.;
Padgett, W.; Daly, J. W. J. Med. Chem. 1986, 29, 1683–
1689.
Lorenzen, A.; von Frijtag Drabbe Kunzel, J. K.; IJzer-
¨
man, A. P. J. Med. Chem. 1998, 41, 102–108.
8. Fatholahi, M.; Xiang, Y.; Wu, Y.; Li, Y.; Wu, L.; Dhalla,
A. K.; Berlardinelli, L.; Shryock, J. C. J. Pharmacol. Exp.
Ther. 2006, 317, 676–684.
9. Roelen, H.; Veldman, N.; Spek, A. L.; von Frijtag Drabbe
ˆ
Kunzel, J.; Mathot, R. A. A.; IJzerman, A. P. J. Med.
¨
Chem. 1996, 39, 1463–1471.
21. Wan, Z.-K.; Binnun, E.; Wilson, D. P.; Lee, J. Org. Lett.
2005, 7, 5877–5880.
10. Elzein, E.; Kalla, R.; Li, X.; Perry, T.; Marquart, T.;
Micklatcher, M.; Li, Y.; Wu, Y.; Zeng, D.; Zablocki, J.
Bioorg. Med. Chem. Lett. 2007, 17, 161–166.
11. Hutchinson, S. A.; Baker, S. P.; Scammells, P. J. Bioorg.
Med. Chem. Lett. 1999, 9, 933–936.
22. Morrison, C. F.; Elzein, E.; Jiang, B.; Ibrahim, P. N.;
Marquart, T.; Palle, V.; Shenk, K. D.; Varkhedkar, V.;
Maa, T.; Wu, L.; Wu, Y.; Zeng, D.; Fong, I.; Lustig, D.;
Leung, K.; Zablocki, J. A. Bioorg. Med. Chem. Lett. 2004,
14, 3793–3797.
12. Hutchinson, S. A.; Baker, S. P.; Scammells, P. J. Bioorg.
Med. Chem. 2002, 10, 1115–1122.
23. de Ligt, R. A. F.; IJzerman, A. P. Curr. Pharm. Design
2002, 8, 2333–2344.