8-Substituted 2-alkynyl-N9-propargyladenines as A2A adenosine receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2014.04.041

Structure-activity relationships of 2-alkynyladenine derivatives were explored by varying substituents at the 9-, 8- and 2-positions of the purine moiety in order to optimize A_2A adenosine receptor antagonist activity in vitro. A propargyl grou

Full text of DOI:10.1016/j.bmc.2014.04.041

Bioorganic & Medicinal Chemistry 22 (2014) 3072–3082
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
8-Substituted 2-alkynyl-N9-propargyladenines as A2A adenosine
receptor antagonists
⇑
Kazuki Endo, Kazuki Deguchi, Hirokazu Matsunaga, Kota Tomaya, Kohei Yamada
Biochemicals Division, Yamasa Corporation, 2-10-1 Araoicho, Choshi, Chiba 288-0056, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationships of 2-alkynyladenine derivatives were explored by varying substituents at
the 9-, 8- and 2-positions of the purine moiety in order to optimize A2A adenosine receptor antagonist
activity in vitro. A propargyl group at the 9-position was found to be important for A2A antagonist activ-
ity, and the introduction of a halogen, aryl, or heteroaryl at the 8-position further enhanced activity. A
series of 8-substituted 2-alkynyl-N9-propargyladenine derivatives exhibited potent antagonist activity,
with IC50 values in the low nM range. Compound 4a from this series was found to be orally active at a
dose of 3 mg/kg in a mouse catalepsy model and a 6-hydroxydopamine-lesioned rat model of Parkinson’s
disease.
Received 5 March 2014
Revised 15 April 2014
Accepted 18 April 2014
Available online 28 April 2014
Keywords:
Adenosine receptor
Agonist
Ó 2014 Elsevier Ltd. All rights reserved.
Antagonist
Parkinson’s disease
Structure–activity relationship
1. Introduction
The first relatively selective A2A AR antagonists reported were
8-styrylxanthines such as KW6002 (istradefylline, Fig. 1), which
Adenosine modulates a wide range of physiological functions by
interacting with specific cell surface G-protein-coupled receptors,
specifically A1, A2A, A2B, and A3 adenosine receptor (AR) sub-
types.1,2 All adenosine receptors are associated with the cAMP sec-
ond messenger system. A1 and A3 ARs are linked to inhibition of
adenylate cyclase, while A2A and A2B subtypes are linked to stimu-
lation of this enzyme.3 The A2A subtype is abundantly expressed in
the striatum, whereas A1 is widely distributed throughout the
brain.4 In recent years, A2A AR antagonists have emerged as an
attractive target for Parkinson’s disease therapy, primarily due to
localized expression in the striatum and the motor enhancement
function of this receptor.5
has a high affinity for the A2A AR and 50–100-fold selectivity for
A2A over the A1 subtype.8 More recently, istradefylline has been
approved for the treatment of PD in Japan. The second class of
antagonists characterized were the pyrazolo[4,3-e]-1,2,4-triazol-
o[1,5-c]pyrimidine derivatives, and SCH5826 is the prototype for
the tricyclic derivatives, possessing a low nM affinity for A2A AR
in vitro.9 However, low water solubility and consequent poor bio-
availability limited the pharmacological use of these compounds.
Neustadt et al. recently reported a series of arylpiperazine deriva-
tives of pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidines with
antagonist activity for the A2A.
10 Among these, SCH420814 (prelad-
enant) was demonstrated to be a potent, selective, and orally active
A2A AR antagonist. The third class of antagonists to appear, the
1,2,4-triazolo[4,5-e]-1,3,5-triazines, as typified by ZM241385, inhi-
bit A2A with sub nM affinity, but unfortunately showed some cross-
reactivity.11 Recently, a large series of derivatives bearing various
substituents at the 5-position on the triazolo-triazine nucleus,
and as well as the deaza analogues (triazoro-pyrimidines), were
synthesized.7 Several purine derivatives recently reported by
several groups appear to be very promising, such as the 2-amino-
6-(2-furanyl)purine derivatives and ST1535, which also exhibit
activity in the low nM range.12 The discovery and development
of potent and selective A2A AR antagonists as potential treatment
for PD and other neurodegenerative disorders has therefore been
an attractive field of research in the last decade.13
Parkinson’s disease (PD) is a hugely debilitating neurological
disorder, and current treatments fail to achieve long-term control.
Chronic over use of
such as wearing-off and dyskinesia, and new options to replace
or reduce -DOPA are much needed. A2A AR antagonists can restore
L-DOPA as a treatment causes complications
L
the deficiencies caused by degeneration of the striatonigral dopa-
mine system that result from loss of striatal neurons in PD, and
represent a potential novel therapeutic approach.6 A number of
potent and subtype-selective A2A antagonists, based on a variety
of structures have been reported, including xanthine and
nonxanthine compounds.7
⇑
Corresponding author. Tel.: +81 479 22 0095; fax: +81 479 22 9821.
E-mail address: k_yamada@yamasa.com (K. Yamada).
http://dx.doi.org/10.1016/j.bmc.2014.04.041
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

K. Endo et al. / Bioorg. Med. Chem. 22 (2014) 3072–3082
3073
NH2
O
N
Me
N
N
N
O
MeO
MeO
N
N
OMe
N
N
N
O
N
N
N
O
KW6002 (Istradefylline)
SCH420814 (Preladenant)
NH2
N
NH2
OH
N
N
N
O
N
N
N
N
N
N
Me
N
N
H
N
ST1535
ZM241385
Figure 1. Representative members of selective A2A AR antagonist classes.
In 1993, Barrett et al. reported that N6-cyclopentyl-N9-methyl-
adenine (N-0840) was a selective A1 antagonist, in contrast to
N6-cyclopentyladenosine, which was a known A1 agonist at the
time.14 This suggested that de-ribosylation and alkylation of the
N9-position could convert adenosine-based AR agonists into antag-
onists. These results, and the fact that 2-alkynyl adenosine deriva-
tives had been found to be potent A2A agonists (Scheme 1),15 led us
to design and synthesize the 2-alkynyladenine derivatives as
potential A2A AR antagonists. Cristalli et al. showed that 2- and
the pharmacophore included H-bonds at the 15-, 17-, and 25-posi-
tions, as well as hydrophobic interactions with residues at posi-
tions 11 and 20 (Fig. 2). These key pharmacophore interactions
were also present in another study.12c We noted that 2-alkynylad-
enine derivatives would also possess the appropriate functional
groups (N at the 7-position, amino group at the 6-position, and
hydrophobic residue at the 2-position) for potential antagonist
activity (Fig. 2). We hypothesized that introduction of an aryl or
heteroaryl group at the 8-position of the purine base may be
potent for both affinity and selectivity of the A2A AR subtype.
Furthermore, we speculated that the substituents on the N at the
9-position would be involved in switching the compounds from
agonists to antagonists, as mentioned above. Therefore, we first
tested the effects of N9-substituents on the 2-alkynyladenine
derivatives.
8-substituted N9-alkyladenine derivatives possessed good affinity
16
and selectivity for the A2A
.
On the other hand, Harada et al.
reported that 2-alkynyl-8-aryl-N9-methyladenine derivatives are
candidates for antidiabetic agents through their antagonistic
effects on the A2B AR.17 A three carbon linker between the adenine
core and the amide moiety of the alkyl residue at the 9-position
was important for A2B antagonistic activity, as shown by struc-
ture–activity relationship (SAR) studies on 9-alkyl-8-(3-fluoro-
We first synthesized some N9-alkylated 2-octyn-1-yladenine
derivatives, because the octynyl group at the 2-position was
expected to show a high affinity for the A2 subtype, based on 2-
octyn-1-yladenosine.15 It was reported that de-ribosylated 2-
octyn-1-yladenine (2) had no affinity for the A1 and A2 subtypes.15b
In contrast, introduction of a methyl group on the N at the 9-posi-
tion of 2-alkynyladenine derivatives was reported to increase the
affinity for these receptors.17 Therefore, the N9-substituent seemed
a logical place at which to manipulate A2A agonist/antagonist
activity.
phenyl)-2-(hydroxycyclohexyl-1-ethynyl)adenine
derivatives.18
Thus, 2-alkynyl-N9-alkyladenine derivatives have already been
evaluated as A2B antagonists, but not as A2A antagonists.
In this study, we describe the synthesis and SAR of novel 2-alk-
ynyladenine derivatives, which were evaluated for A2A antagonist
activity by investigating their inhibitory effects on 2-octyn-1-ylad-
enosine-induced vasodilation. We also report the discovery and
characterization of potent A2A antagonists. The efficacy of oral
administration was tested on PD models in mice (catalepsy) and
rats (turning behavior in unilateral 6-hydoxydopamine-lesioned
animals).
2-Octyn-1-yladenosine (1), readily prepared from guanosine,15c
was hydrolyzed with 0.6 M HCl–1,4-dioxane at 100 °C for 6 h, to
afford 2-octyn-1-yladenine (2) in 92% yield.15b As shown in
Scheme 2 and Table 1, the adenine derivative (2) was alkylated
at the N9-position with 2 equiv of various alkylhalides in the
presence of K2CO3 in DMF, giving yields of 35–89%. All of these
alkylated 2-octyn-1-yladenines (3a–i) were obtained as a single
N9-isomer,20 as determined by 1H, 13C NMR and HMBC experi-
ments on the propargyl derivative 3e (Fig. 3). Cross-peaks from
H-10 to C-4 and H-N6 to C-5, were observed in the HMBC spectrum
of 3e.
2. Results and discussion
2.1. Synthesis and biological activities of 9-substituted 2-octyn-
1-yladenines
Recently, Jaakola et al. reported the crystal structure of a human
A2A AR bound to an antagonist, ZM241385.19 The key elements of
The A2A AR antagonist activities of N9-alkyl-2-octyn-1-ylade-
nine derivatives (3a–i) were evaluated in vitro by measuring inhi-
bition of 2-octyn-1-yladenosine-induced vasodilation, with
comparison against istradefylline as previously reported.21 Methyl
and ethyl derivatives (3a, 3b) showed moderate antagonist activity
(Table 1), albeit 30-fold weaker than istradefylline. The bulkiness
of the N9-alkyl groups (n-propyl, allyl, c-pentyl and benzyl)
appeared to reduce the activity. In contrast to the n-propyl
derivative 3c, propargyl derivative 3e showed comparable activity
to istradefylline. Interestingly, this activity disappeared upon sub-
stitution with a cyanomethyl group, which has a comparable steric
bulk to the propargyl group. These results indicated that the appro-
priate bulkiness at the N9-position was essential for A2A AR
NH2
N
N
NH2
N
N
N
N
O
R
N
R'
N
HO
OH
R
OH
2A Adenosine receptor agonist
(2-Alkynyladenosines)
A2A Adenosine receptorant agonist
(2-Alkynyladenines)
A
Scheme 1. Design of A2A AR antagonists based on A2A AR agonists.

3074
K. Endo et al. / Bioorg. Med. Chem. 22 (2014) 3072–3082
H-bond acceptor
H-bond donor
15NH2
NH2
17
N
6 N
25 O
OH
N7
N
N
R8
8
20
N
N
11
N
2
N
N
H
R9
R2
Hydrophobic interaction
2-Alkynyladenine derivatives
ZM241385
Figure 2. ZM241385 pharmacophore interactions and 2-alkynyladenine derivatives.
NH2
NH2
N
NH2
N
N
N
N
N
N
a)
b)
N
N
H
N
O
N
N
C6H13
R9
HO
C6H13
C6H13
OH
OH
2-Octyn-1-yladenosine (1)
2
3a-i
Scheme 2. Reagents and conditions: (a) 0.6 M HCl, 1,4-dioxane, 100 °C, (b) R–X (X = Br, I), K2CO3, DMF.
Table 1
Yields of N9-alkyl-2-octynyladenine derivatives and their inhibitory effects (IC50
values) on 2-octyn-1-yladenosine-induced vasodilation
and resulted in compounds with nM Ki.16b To explore this further,
we introduced halogen, aryl, and heteroaryl substituents at the
8-position.
a
Compounds
R
Yield (%)
IC50
(
lM)
N9-Propargyladenine derivative 3e was brominated with 1
equivalent of N-bromosuccinimide (NBS) in DMF to afford the
8-bromoadenine derivative 4a in 57% yield (Scheme 3). A large
excess of NBS increased the amount of 8- and 30-dibromo deriva-
tive 4b, which could not be separated. For example, the reaction
with 4 equiv of NBS gave a 1.4:1 mixture of 4a and 4b. We also
examined the chlorination and iodination at the 8-position using
N-chlorosuccinimide (NCS) or N-iodosuccinimide (NIS). Although
the reaction with NCS was slower than with NBS, 8-chloro deriva-
tive 4c was afforded in 55% yield without generation of the
dichloro byproduct. In contrast, iodination of 3e with NIS was very
rapid, but gave the undesired 30-iodo derivative 4e in 71% yield as a
sole product. Interestingly, the diiodo derivative was not produced
at all in this reaction. Halogen exchange of 4a with iodide, and
3a
3b
3c
3d
3e
3f
3g
3h
Me
Et
n-Pr
Allyl
Propargyl
CH2CN
CH2CH2OH
c-Pentyl
Bn
35
79
46
44
77
89
43
15
46
—
9.0 0.16
6.7 2.03
>10
>10
0.2 0.01
>10
>10
>10
>10
0.2 0.02
3i
Istradefylline
—
a
IC50: compound concentration that inhibits 2-octyn-1-yladenosine-induced
vasodilation by 50%.
H
5
NH
N
N
iodination of adenine derivative
success.
2 were attempted without
N
4
2
N
8-Aryl and 8-heteroaryl derivatives were prepared next
(Scheme 3). Phenyl, 3-fluorophenyl, 2-furyl and 1,2,3-triazol-2-yl
functional groups at the 8-position were tested as these were
reported to enhance the affinity for the A2A AR.12,17,19 8-Bromoad-
enine derivative 4a was reacted with phenylboronic acid in the
presence of Pd(PPh3)4 to afford 8-phenyl derivative 5a in 36% yield.
Under the same conditions, 8-(3-fluoro)phenyl and 2-furyl deriva-
tives (5b and 5c) were obtained in 30% and 53% yield, respectively.
The triazole derivative 5d was prepared by a substitution reaction
of the 8-bromo derivative 4a with 1,2,3-triazole in DMF according
to the literature.12 Unexpectedly, the reaction gave a complex mix-
ture that included the N1- and N2-triazolyl derivatives, with regio-
isomer yields of approximately were 23% (N1) and 17% (N2) as
estimated from HPLC analysis. Following complicated purification
procedures including ODS column chromatography, the desired
N2-triazolyl derivative 5d was obtained in 10% yield.
The effects of different substituents at the 8-position of
2-octyn-1-yl-N9-propargyladenine derivatives were evaluated
in vitro (Table 2). In all cases, A2A AR antagonist activity was
dramatically increased compared with the lead compound 3e.
The 8-bromo, 8-chloro, 8-(2-furyl) and 8-(1,2,3-triazol-2-yl)
derivatives (4a, 4c, 5c and 5d) were particularly effective, and were
50-fold more active than 3e.
C6H13
1'H
Figure 3. Assignment of the N7-, N9-isomer of 3e by HMBC experiments.
antagonist activity, and polar substituents are not compatible.
Based on these results, we decided to retain the propargyl substi-
tuent at the N9-position, and explored SAR at the 8- position.
2.2. Synthesis and biological activity of 8-substituted 2-octyn-1-
yl-N9-propargyladenines
According to molecular modeling studies on 5-amino-9-chloro-
2-(2-furyl)-1,2,4-triazolo[1,5-c]quinazoline (CGS15943), the pres-
ence of the furan ring at the 2-position of CGS15943, which is
equivalent to the 8-position of adenine, was apparently essential
for both affinity and selectivity towards the A2A subtype.22 In
contrast, 2-furyl and 3-fluorophenyl groups were the preferred
substituents at the 8-position of adenine for A2B antagonists.17
Recently, Lambertucci et al. introduced a bromine at the 8-position
of N9-ethyladenine derivatives, which increased the affinity for A2A

K. Endo et al. / Bioorg. Med. Chem. 22 (2014) 3072–3082
3075
NH2
N
NH2
N
NH2
N
N
N
N
N
a) or b)
c) or d)
N
X
R8
N
N
N
N
C6H13
C6H13
C6H13
Y
5a (R8=Ph)
3e
4a (X=Br, Y=H)
4b (X=Br, Y=Br)
4c (X=Cl, Y=H)
4d (X=I, Y=H)
4e (X=H, Y=I)
5b (R8=3-F-Ph)
5c (R8=2-furyl)
5d (R8=1,2,3-triazol-2-yl)
Scheme 3. Reagents and conditions: (a) NBS, AcOK, DMF, 57% for 4a, (b) NCS, AcOK, DMF, 55% for 4c, (c) R8-B(OH)2, Pd(PPh3)4 (10–40 mol %), K2CO3, dioxane–H2O, 80 °C, (5a:
36%, 5b: 30%, 5c: 53%), (d) 1,2,3-triazole, K2CO3, DMF, 100 °C, (5d: 10%).
Table 2
Inhibitory effects of 8-substituted 2-octyn-1-yl-N9-propargyladenine derivatives on
the 2-position also markedly enhanced the affinity. The antagonist
activity of 3e increased 50–100-fold by the introduction of a
halogen or 2-furyl group at the 8-position. In addition to the effects
of the 8-substituents, activity was elevated 400-fold by introduc-
tion of a 2-(1-hydroxycyclohexyl) group on the 2-ethnyl residue.
This result is concordant with an earlier report of the effects of
2-alkynyl resides on agonist activity.15c Although the most potent
antagonists were 12a and 13, we focused on compound 4a for bio-
logical evaluation as this was more easily prepared.
2-octyn-1-yladenosine-induced vasodilation
a
Compounds
R8
IC50 (nM)
3e
4a
4c
5a
5b
5c
5d
H
Br
213.0 10.23
5.1 0.32
Cl
6.0 0.50
Ph
3-F-Ph
2-Furyl
75.5 16.95
22.8 3.01
3.6 0.65
1,2,3-Triazol-2-yl
—
1.9 0.58
228.0 16.26
Istradefylline
2.5. Binding affinity of compound 4a for A1, A2A and A2B AR
subtypes
a
IC50: compound concentration that inhibits 2-octyn-1-yladenosine-induced
vasodilation by 50%.
The binding affinities [Ki (nM)] of 4a for human A1, A2A and A2B
subtypes (h-A1, h-A2A, and h-A2B) expressed in CHO-K1 (A1) and
HEK-293 (A2A, A2B) cells were determined in order to investigate
selectivity. Compound 4a showed a higher affinity for h-A2A
(Ki = 0.56 nM) than reported for purine-type compounds.12,16
Selectivity for A2B was 1300-fold higher, while this compound
was only 10-fold more selective towards the A1 subtype, which
was comparable to ST1535.
2.3. Synthesis and biological activity of 8-substituted 2-(1-
hydroxycyclohexyl)ethyn-1-yl-N9-propargyladenines
Finally, we examined the effects of alkynyl side chains at the
2-position. The effects of substituents at the 2-position of 2-
alkynyladenosines has been investigated, and the 2-(1-hydrox-
ycyclohexyl)ethyn-1-yl derivative showed 10-fold higher affinity
towards the A2A subtype than the 2-octyn-1-yl derivative.15c
Therefore, we synthesized the 2-[2-(1-hydroxycyclohexyl)ethyn-
1-yl]adenine derivatives to test the effects on antagonist activity.
2-[2-(1-Hydroxycyclohexyl)ethyn-1-yl]adenosine (9) was synthe-
2.6. Activity as measured using the haloperidol-induced
catalepsy model
To evaluate the in vivo activity of 2-alkynyl-N9-propargylade-
nine derivative 4a, the mouse catalepsy model was used. This
widely used rodent model for PD involves inducing catalepsy
(i.e., immobility) by subcutaneous injection with haloperidol
(1 mg/kg).24 The ability of A2A AR antagonists to reduce the cata-
leptic responses (on a scale of 1–5) was tested. Oral administration
of 4a (and the istradefylline positive control) at a concentration of
3 mg/kg led to a significant reduction in catalepsy (Fig. 4A). Both 4a
and istradefylline achieved this in a dose-dependent manner, with
a minimum effective dose (MED) of 3 mg/kg 5 h after oral admin-
istration (Fig. 4B).
sized by
a Sonogashira coupling reaction of 6, followed by
amination at the 6-position (Scheme 4),15c using the 6-pyridinium
salt as previously described.23 6-Chloropurine derivative 7 was
treated with pyridine–H2O (1:1) at 50 °C for 4 h to afford 6-pyrid-
inium intermediate 8, which was confirmed by HPLC analysis. 8
was treated with NH4OH–1,4-dioxane (2:1) at room temperature
to obtain 2-alkynyladenosine 9 in 61% yield from 7. Furthermore,
adenosine derivative
ycyclohexyl)ethyn-1-yl]-N9-propargyl
(11–13) using the methods described above.
9
was converted into 2-[2-(1-hydrox-
adenine derivatives
As expected, the in vitro A2A AR antagonist activity of the 2-[2-
(1-hydroxycyclohexyl)ethyn-1-yl] series was more potent than the
2-octyn-1-yl series, with 8-(2-furyl)-2-[2-(1-hydroxycyclohexyl)et
hyn-1-yl]-N9-propargyladenine (13) being the most potent com-
pound. It is noteworthy that 13 showed 570-fold higher activity
than the istradefylline positive control (Table 3).
2.7. Turning behavior in 6-OHDA-lesioned rats
Contralateral turning behavior in rats subjected to unilateral
6-hydroxydopamine (6-OHDA) lesions of the dopaminergic nigro-
striatal pathway is another well established animal model of
PD.25 In this model, a selective A2A AR antagonist reportedly poten-
tiated the contralateral turning behavior induced by direct dopa-
mine agonist,26 implying that the antagonists might be beneficial
for the treatment of PD. Morelli et al. reported that a selective
2.4. Structure–activity relationships of 2-alkynyl-N9-
propargyladenines
SAR of 2-alkynyl-N9-propargyladenines are summarized in
Scheme 5. The lead compound 3e showed comparable activity to
istradefylline in the in vitro assay. A propargyl group at the 9-posi-
tion was important for A2A AR antagonist activity, as mentioned
above, and substituents at the 8-position and alkynyl resides at
A
2A AR antagonist markedly increased the number of contralateral
rotations induced by a threshold dose of -DOPA, whereas an A2A
L
AR antagonist did not induce any turning behavior when adminis-
tered alone.25 We evaluated the effects of 2-alkynyladenine deriv-
ative 4a using this model. 4a potentiated the turning behavior

3076
K. Endo et al. / Bioorg. Med. Chem. 22 (2014) 3072–3082
Cl
Cl
N
N
N
N
N
N
AcO
N
AcO
b)
N
OH
I
a)
O
O
OAc
OAc
AcO
AcO
7
6
NH2
N
N
Cl-
N
+
N
N
N
c)
N
HO
AcO
N
N
OH
O
OH
O
OH
HO
OAc
AcO
9
8
NH2
N
NH2
N
N
N
N
e)
f) or g)
d)
N
H
N
OH
N
OH
10
11
NH2
NH2
N
N
N
N
N
h)
N
X
R8
N
OH
N
OH
13 (R8=2-furyl)
12a (X=Br)
12b (X=Cl)
Scheme 4. Reagents and conditions: (a) 1-ethynyl-1-cyclohexanol, (PPh3)2PdCl2 (10 mol %), CuI, Et3N, 1,4-dioxane, 98%, (b) pyridine–H2O (1:1), 50 °C, (c) NH4OH–1,4-
dioxane (2:1), 61% from 7, (d) 1 M HCl aq, 100 °C, 90%, (e) propargylbromide, K2CO3, DMF, 84%, (f) NBS, AcOK, DMF, 52%, (g) NCS, AcOK, DMF, 22%, (h) 2-furanboronic acid,
Pd(PPh3)4 (10 mol %), K2CO3, 1,4-dioxane–H2O, 100 °C, 59%.
Table 3
NH2
N
NH2
N
Inhibitory effects of 2-[2-(1-hydroxycyclohexyl)ethyn-1-yl]-N9-propargyladenine
derivatives on 2-octyn-1-yladenosine -induced vasodilation
N
N
N
N
50~100-fold
R8
Compounds
R8
IC50 (nM)
a
N
N
C6H13
C6H13
11
12a
12b
13
H
Br
97.3 33.24
0.5 0.02
3e (213.0 nM)
4a: R8=Br (5.1 nM)
Cl
3.0 1.67
4c: R8=Cl (6.0 nM)
2-Furyl
—
0.4 0.12
228.0 16.26
5c: R8=2-furyl (3.6 nM)
5d: R8=1,2,3-triazol-2-yl (1.9 nM)
2-fold
Istradefylline
a
IC50
: compound concentration that inhibits 2-octyn-1-yladenosine-induced
NH2
NH2
N
vasodilation by 50%.
N
N
N
~200-fold
N
R8
N
N
OH
N
OH
induced by L-DOPA without inducing stereotyped behavior when
administered on its own (Fig. 5). Although 4a alone had minimal
effect, turning behavior was induced significantly by administra-
tion of 100 mg/kg L-DOPA and 3 mg/kg 4a.
11 (97.3 nM)
12a: R8=Br (0.5 nM)
12b: R8=Cl (3.0 nM)
13: R8=2-furyl (0.4 nM)
3. Conclusion
Scheme 5. Structure–activity relationships for 2-alkynyl-N9-propargyladenines.
In summary, we synthesized various 2-alkynyl-N9-alkyladenine
derivatives and investigated their antagonist activity against the
A2A AR. 8-Substituted 2-alkynyl-N9-propargyladenine derivatives
were found to antagonize the receptor with IC50 values in the
low nM range. Furthermore, a selected member of this series (4a)
was shown to be an orally active A2A AR antagonist in the haloper-
idol-induced catalepsy PD model in mouse and the unilateral 6-
OHDA-lesioned PD model in rat. These results suggest that novel
2-alkynyl-N9-propargyladenine derivatives such as 4a may be
effective in the treatment of PD.
4. Experimental section
4.1. General
Physical data were measured as follows: Melting points were
determined on a Yanagimoto MP-500D micromelting point appa-
ratus, and were uncorrected. NMR spectra were recorded at

K. Endo et al. / Bioorg. Med. Chem. 22 (2014) 3072–3082
3077
4a
A
B
Istradefylline
* * *
* * *
Veh
5
4
5
4
3
3
2
1
0
2
1
0
Veh
0.3
1
3
10
0.3
1
3
10
1h
3h
5h
7h
Time after 4a (3mg/kg, p.o.) or Istradefylline (3mg/kg, p.o.)
4a (mg/kg, p.o.)
Istradefylline (mg/kg, p.o.)
Figure 4. Anti-cataleptogenic activity of 2-alkynyl-N9-propargyladenine derivative 4a. (A) Effects of 4a (3 mg/kg) and istradefylline (3 mg/kg) on catalepsy induced by
haloperidol in mice. Data show the mean catalepsy score ( SEM) 1, 3, 5, and 7 h after treatment (n = 7 per group). ⁄P < 0.001 versus vehicle. Statistical significance was
determined with the Repeated Two Way ANOVA test followed by the Tukey test. (B) Dose-dependence of the anti-cataleptogenic activity of 4a (0.3–10 mg/kg) and
istradefylline (0.3–10 mg/kg). Each column represents the mean catalepsy score ( SEM) 5 h after treatment (n = 7 per group). ⁄P < 0.05 versus vehicle. Statistical significance
was determined with the Kruskal–Wallis test followed by the Tukey test. The cataleptic responses were scored as follows: score 0, the cataleptic posture lasted for less than
5 s for both forelimbs and hind limbs; score 1, the cataleptic posture of forelimbs lasted for 5–10 s and that of hind limbs lasted less than 5 s; score 2, the cataleptic posture of
forelimbs lasted for more than 10 s and that of hind limbs lasted for less than 5 s; score 3, the cataleptic posture of forelimbs and hind limbs lasted for 5–10 s, or the cataleptic
posture of forelimbs lasted for less than 5 s but that of hind limbs lasted for more than 5 s; score 4, the cataleptic posture of forelimbs lasted for more than 10 s and that of
hind limbs lasted for 5–10 s, or the cataleptic posture of forelimbs lasted for 5–10 s and that of hind limbs lasted for more than 10 s; score 5, the cataleptic posture of both
forelimbs and hind limbs lasted for more than 10 s.
160
L-DOPA (100mg/kg, p.o.)
*
140
120
100
80
4a (3mg/kg, p.o.)
4a (3mg/kg, p.o.) + L-DOPA (100mg/kg, p.o.)
**
**
60
40
20
0
15
45
75
105
135
165
195
225
255
285
Time (min)
Figure 5. The effect of 4a (3 mg/kg) on
L
-DOPA-induced turning behavior in 6-OHDA-lesioned, hemiparkinsonian rats. Data show mean rotation counts for a 15 min duration
-DOPA (100 mg/kg) alone. Statistical significance was determined with the Repeated Two Way ANOVA test followed by the Tukey
( SEM). ⁄P < 0.05, ⁄⁄P < 0.01 compared with
L
test. Five animals were used in each group.
500 MHz (1H) and at 125 MHz
(
13C) on
a
Bruker AV-500
4.2. Representative syntheses
instrument in CDCl3 or DMSO-d6 as the solvent, with tetramethyl-
silane as the internal standards. UV spectra were recorded with a
Shimadzu UV-1800 spectrophotometer. ESI mass spectra were
recorded on a JEOL JMS-T100LP instrument. TLC was carried out
on Merck precoated plates (Kieselgel 60F254).
4.2.1. N9-Methyl-2-(1-octyn-1-yl)adenine (3a)
A mixture of 2 (200 mg, 0.82 mmol) and K2CO3 (180 mg,
1.3 mmol) in DMF (10 mL) was treated with CH3I (100
lL,
1.6 mmol), and the mixture was stirred at room temperature for

3078
K. Endo et al. / Bioorg. Med. Chem. 22 (2014) 3072–3082
23 h. The reaction was quenched with water, and the mixture was
extracted with AcOEt. The organic layers were dried over MgSO4,
filtered and concentrated. The residue was purified by column
chromatography (MeOH–CHCl3 = 1:16) to give 3a (73 mg, 35%).
1H NMR (CDCl3) d 7.77 (1H, s), 5.79 (2H, br s), 3.83 (3H, s), 2.45
(2H, t, J = 7.3 Hz), 1.69–1.63 (2H, m), 1.48–1.42 (2H, m), 1.35–
1.27 (4H, m), 0.89 (3H, t, J = 7.0 Hz); 13C NMR (CDCl3) d 155.1,
150.7, 147.0, 141.6, 118.9, 87.8, 80.3, 31.4, 30.0, 28.8, 28.3, 22.5,
and the mixture was stirred at room temperature for 6.5 h. The reaction
was quenched with water, and the mixture was extracted with AcOEt.
The organic layers were dried over MgSO4, filtered and concentrated.
The residue was purified by column chromatography (hexane/
AcOEt = 1:7) to give 3e (177 mg, 77%). 1H NMR (DMSO-d6) d 8.23
(1H, s), 7.39 (2H, br s), 5.02 (2H, d, J = 2.5 Hz), 3.48 (1H, t, J = 2.5 Hz),
2.41 (2H, t, J = 7.1 Hz), 1.57–1.51 (2H, m), 1.43–1.37 (2H, m), 1.32–
1.26 (4H, m), 0.88 (3H, t, J = 7.0 Hz); 13C NMR (DMSO-d6) d 155.7,
149.1, 145.9, 140.7, 117.8, 85.4, 81.1, 78.2, 75.9, 32.2, 30.7, 28.0, 27.8,
21.9, 18.1, 13.8; ESIMS-HR calcd for C16H19N5 282.1719, found
282.1733 (MH+); Anal. calcd for C16H19N5: C, 68.30; H, 6.81; N, 24.89.
found: C, 68.26; H, 6.90; N, 24.89.
19.4, 14.0; ESIMS-HR calcd for
C14H20N5 258.1719, found
258.1728 (MH+); Anal. calcd for C14H19N5: C, 65.34; H, 7.44; N,
27.22. found: C, 64.96; H, 7.46; N, 27.05.
4.2.2. N9-Ethyl-2-(1-octyn-1-yl)adenine (3b)
A
mixture of
2
(300 mg, 1.2 mmol) and K2CO3 (340 mg,
L, 2.5 mmol),
4.2.6. N9-Cyanomethyl-2-(1-octyn-1-yl)adenine (3f)
A mixture of 2 (200 mg, 0.82 mmol) and K2CO3 (230 mg,
1.6 mmol) in DMF (5 mL) was treated with bromoacetonitrile
2.5 mmol) in DMF (4 mL) was treated with EtBr (180
l
and the mixture was stirred at room temperature for 16 h. The reac-
tion was quenched with water, and the mixture was extracted with
AcOEt. The organic layers were dried over MgSO4, filtered and con-
centrated. The residue was purified by column chromatography
(EtOAc/MeOH = 15:1) to give 3b (263 mg, 79%). 1H NMR (CDCl3) d
7.82 (1H, s), 6.00 (2H, br s), 4.26 (2H, q, J = 7.3 Hz), 2.45 (2H, t,
J = 7.4 Hz), 1.69–1.63 (2H, m), 1.52 (3H, t, J = 7.3 Hz), 1.48–1.42
(2H, m), 1.34–1.28 (4H, m), 0.89 (3H, t, J = 6.9 Hz); 13C NMR (CDCl3)
d 155.2, 150.2, 146.8, 140.4, 119.0, 87.6, 80.4, 38.8, 31.4, 28.9, 28.3,
22.5, 19.4, 15.6, 14.0; ESIMS-HR calcd for C15H22N5 272.1875, found
272.1895 (MH+); Anal. calcd for C15H21N5ꢀH2O: C, 62.26; H, 8.01; N,
24.20. found: C, 62.15; H, 7.95; N, 24.23.
(110 lL, 1.6 mmol), and the mixture was stirred at room tempera-
ture for 6.5 h. The reaction was quenched with water, and the mix-
ture was extracted with AcOEt. The organic layers were dried over
MgSO4, filtered and concentrated. The residue was purified by col-
umn chromatography (MeOH–CHCl3 = 1:19) to give 3f (206 mg,
89%). 1H NMR (CDCl3) d 7.76 (1H, s), 5.94 (2H, br s), 5.14 (2H, s),
2.46 (2H, t, J = 7.3 Hz), 1.70–1.64 (2H, m), 1.49–1.43 (2H, m),
1.34–1.29 (4H, m), 0.89 (3H, t, J = 7.1 Hz); 13C NMR (CDCl3) d
155.4, 149.8, 147.7, 139.2, 118.5, 113.2, 88.9, 79.9, 31.4, 30.9,
28.8, 28.2, 22.5, 19.4, 14.0; ESIMS-HR calcd for
C
15H19N6
283.1671, found 283.1686 (MH+); Anal. calcd for C15H18N6ꢀ0.1H2O:
C, 63.40; H, 6.46; N, 29.58. found: C, 63.25; H, 6.33; N, 29.46.
4.2.3. 2-(1-Octyn-1-yl)-N9-n-propyladenine (3c)
A mixture of 2 (200 mg, 0.82 mmol) and K2CO3 (180 mg,
4.2.7. N9-Hydroxyethyl-2-(1-octyn-1-yl)adenine (3g)
1.3 mmol) in DMF (10 mL) was treated with n-PrBr (150
l
L,
A mixture of 2 (200 mg, 0.82 mmol) and K2CO3 (230 mg,
1.6 mmol) in DMF (5 mL) was treated with 2-bromoethanol
(113 lL, 1.6 mmol), and the mixture was stirred at room tempera-
1.6 mmol), and the mixture was stirred at 50 °C for 7 h. The reac-
tion was quenched with water, and the mixture was extracted with
AcOEt. The organic layers were dried over MgSO4, filtered and con-
centrated. The residue was purified by column chromatography
(hexane/AcOEt = 1:7) to give 3c (107 mg, 46%). 1H NMR (CDCl3) d
7.79 (1H, s), 5.75 (2H, br s), 4.16 (2H, q, J = 7.3 Hz), 2.45 (2H, t,
J = 7.4 Hz), 1.92 (2H, tq, J = 7.3, 7.4 Hz), 1.69–1.63 (2H, m),1.48–
1.42 (2H, m), 1.35–1.28 (4H, m), 0.96 (3H, t, J = 7.4 Hz), 0.89 (3H,
t, J = 7.0 Hz); 13C NMR (CDCl3) d 155.1, 150.4, 146.8, 141.0, 119.0,
87.6, 80.4, 45.5, 31.4, 28.9, 28.3, 23.4, 22.5, 19.4, 14.0, 11.1;
ESIMS-HR calcd for C16H24N5 286.2032, found 286.2040 (MH+);
Anal. calcd for C16H23N5ꢀH2O: C, 63.34; H, 8.31; N, 23.08. found:
C, 63.14; H, 8.49; N, 23.01.
ture for 24 h. The reaction was quenched with water, and the mix-
ture was extracted with AcOEt. The organic layers were dried over
MgSO4, filtered and concentrated. The residue was purified by col-
umn chromatography (MeOH–CHCl3 = 1:13) to give 3g (102 mg,
43%). 1H NMR (CDCl3) d 7.80 (1H, s), 5.92 (2H, br s), 4.34 (2H, t,
J = 4.7 Hz), 4.19 (1H, br s), 4.03 (2H, t, J = 4.7 Hz), 2.43 (2H, t,
J = 7.3 Hz), 1.68–1.61 (2H, m), 1.45–1.42 (2H, m), 1.32–1.28 (4H,
m), 0.89 (3H, t, J = 6.9 Hz); 13C NMR (CDCl3) d 155.2, 150.1, 146.6,
141.8, 118.9, 88.1, 80.1, 61.2, 47.3, 31.4, 28.8, 28.3, 22.5, 19.4,
14.0; ESIMS-HR calcd for C15H22N5O 288.1746, found 288.1839
(MH+); Anal. calcd for C15H21N5ꢀ0.1H2O: C, 62.30; H, 7.39; N,
24.22. found: C, 62.19; H, 7.33; N, 24.34.
4.2.4. N9-Allyl-2-(1-octyn-1-yl)adenine (3d)
A mixture of 2 (200 mg, 0.82 mmol) and K2CO3 (230 mg,
4.2.8. N9-Cyclopentyl-2-(1-octyn-1-yl)adenine (3h)
1.6 mmol) in DMF (5 mL) was treated with allylbromide (138
l
L,
A mixture of 2 (200 mg, 0.82 mmol) and K2CO3 (230 mg,
1.6 mmol) in DMF (5 mL) was treated with cyclopentylbromide
(180 lL, 1.6 mmol), and the mixture was stirred at room tempera-
1.6 mmol), and the mixture was stirred at room temperature for
24 h. The reaction was quenched with water, and the mixture
was extracted with AcOEt. The organic layers were dried over
MgSO4, filtered and concentrated. The residue was purified by col-
umn chromatography (hexane/AcOEt = 1:7) to give 3d (103 mg,
44%). 1H NMR (CDCl3) d 7.80 (1H, s), 6.07–5.99 (1H, m), 5.85 (2H,
br s), 5.32 (1H, dd, J = 0.8, 10.2 Hz), 5.23 (1H, dd, J = 0.8, 17.1 Hz),
4.82 (2H, ddd, J = 1.3, 1.4, 5.8 Hz), 2.45 (2H, t, J = 7.4 Hz), 1.69–
1.63 (2H, m),1.48–1.42 (2H, m), 1.34–1.27 (4H, m), 0.89 (3H, t,
J = 7.0 Hz); 13C NMR (CDCl3) d 155.2, 150.2, 147.0, 140.9, 131.9,
119.2, 118.9, 87.8, 80.3, 45.7, 31.4, 28.9, 28.3, 22.5, 19.4, 14.0;
ESIMS-HR calcd for C16H22N5 284.1875, found 284.1881 (MH+);
Anal. calcd for C16H21N5: C, 67.82; H, 7.47; N, 24.71. found: C,
67.64; H, 7.60; N, 24.53.
ture for 20 h. The reaction was quenched with water, and the mix-
ture was extracted with AcOEt. The organic layers were dried over
MgSO4, filtered and concentrated. The residue was purified by col-
umn chromatography (AcOEt) to give 3h (203 mg, 79%). 1H NMR
(CDCl3) d 7.86 (1H, s), 5.73 (2H, br s), 5.02–5.00 (1H, m), 2.45
(2H, t, J = 7.3 Hz), 2.32–2.27 (2H, m), 2.05–1.89 (4H, m), 1.82–
1.78 (2H, m), 1.69–1.63 (2H, m), 1.48–1.42 (2H, m), 1.34–1.26
(4H, m), 0.89 (3H, t, J = 7.0 Hz); 13C NMR (CDCl3) d 155.1, 150.4,
146.6, 139.0, 119.1, 87.5, 80.5, 55.5, 33.2, 31.4, 28.9, 28.3, 23.9,
22.5, 19.4, 14.0; ESIMS-HR calcd for C18H26N5 312.2188, found
312.2190 (MH+); Anal. calcd for C18H25N5: C, 69.42; H, 8.09; N,
22.49. found: C, 69.07; H, 8.29; N, 22.28.
4.2.5. 2-(1-Octyn-1-yl)-N9-propargyladenine (3e)
4.2.9. N9-Benzyl-2-(1-octyn-1-yl)adenine (3i)
A mixture of 2 (200 mg, 0.82 mmol) and K2CO3 (230 mg, 1.6 mmol)
A mixture of 2 (200 mg, 0.82 mmol) and K2CO3 (180 mg,
in DMF (5 mL) was treated with propargylbromide (138
lL, 1.6 mmol),
1.3 mmol) in DMF (10 mL) was treated with BnBr (200 lL,

K. Endo et al. / Bioorg. Med. Chem. 22 (2014) 3072–3082
3079
1.6 mmol), and the mixture was stirred at 50 °C for 7 h. The reac-
4.2.13. 8-(3-Fluorophenyl)-2-(1-octyn-1-yl)-N9-
propargyladenine (5b)
tion was quenched with water, and the mixture was extracted with
AcOEt. The organic layers were dried over MgSO4, filtered and con-
centrated. The residue was purified by column chromatography
(hexane/AcOEt = 1:10) to give 3i (127 mg, 46%). 1H NMR (CDCl3)
d 7.71 (1H, s), 7.37–7.28 (5H, m), 5.79, (2H, br s), 5.37 (2H, s),
2.46 (2H, t, J = 7.3 Hz), 1.69–1.64 (2H, m), 1.47–1.44 (2H, m),
1.33–1.29 (4H, m), 0.89 (3H, t, J = 6.9 Hz); 13C NMR (CDCl3) d
155.2, 150.5, 147.1, 141.0, 135.5, 129.1, 128.5, 128.0, 87.8, 80.4,
47.2, 31.4, 28.9, 28.3, 22.5, 19.4, 14.0; ESIMS-HR calcd for
A mixture of 4a (100 mg, 0.28 mmol), 3-fluorophenylboronic
acid (78 mg, 0.56 mmol), Pd(PPh3)4 (64 mg, 0.055 mmol) and
K2CO3 (76 mg, 0.55 mmol) in 1,4-dioxane (3 mL) and H2O (2 mL)
was stirred at 80 °C for 30 min. Additional Pd(PPh3)4 was added
in twice, 0.13, 0.05 equiv, respectively and the reaction mixture
was stirred for further 1 h. Water was added, and the mixture
was extracted with AcOEt. The organic layers were dried over
MgSO4, filtered and concentrated. The residue was purified by col-
umn chromatography (hexane/AcOEt = 2:1) to give 5b (31 mg,
30%). 1H NMR (CDCl3) d 7.73–7.66 (2H, m), 7.55–7.54 (1H, m),
7.26–7.26 (1H, m), 5.70 (2H, br s), 5.00 (2H, d, J = 2.5 Hz), 2.47
(2H, t, J = 7.4 Hz), 2.42 (1H, t, J = 2.5 Hz), 1.70–1.65 (2H, m), 1.48–
1.45 (2H, m), 1.33–1.30 (4H, m), 0.90 (3H, t, J = 6.9 Hz); 13C NMR
(CDCl3) 163.9, 161.9, 154.9, 151.5, 150.0, 147.1, 131.2, 130.8,
130.8, 124.7, 124.7, 118.6, 117.7, 117.5, 116.3, 116.1, 88.2, 80.4,
74.2, 33.4, 31.4, 28.9, 28.3, 22.5, 19.4, 14.0; ESIMS-HR calcd for
C
C
20H24N5 334.2032, found 334.2026 (MH+); Anal. calcd for
20H25N5: C, 72.04; H, 6.95; N, 21.00. found: C, 71.99; H, 6.97; N,
21.01.
4.2.10. 8-Bromo-2-(1-octyn-1-yl)-N9-propargyladenine (4a)
A
mixture of 3e (281 mg, 1.0 mmol) and AcOK (29 mg,
0.30 mmol) in DMF (10 mL) was treated with NBS (178 mg,
1.0 mmol), and the mixture was stirred at room temperature for
2.5 h. The reaction was quenched with saturated Na2S2O3 solution,
and the mixture was extracted with AcOEt. The organic layers were
dried over MgSO4, filtered and concentrated. The residue was puri-
fied by column chromatography (hexane/AcOEt = 3:1) to give 4a
(204 mg, 57%). Analytical samples were crystallized from AcOEt/
hexane. Mp 146–147 °C; 1H NMR (CDCl3) d 5.64 (2H, s), 4.98 (2H,
d, J = 2.5 Hz), 2.45 (2H, t, J = 7.2 Hz), 2.36, (1H, t, J = 2.5 Hz), 1.69–
1.62 (2H, m), 1.47–1.42 (2H, m), 1.35–1.25 (4H, m), 0.89 (3H, t,
J = 7.2 Hz); 13C NMR (CDCl3) d 153.8, 151.0, 147.2, 127.4, 119.1,
88.6, 80.0, 73.8, 33.5, 31.4, 28.8, 28.2, 22.5, 19.4, 14.0; ESIMS-HR
calcd for C16H19BrN5 360.0824, found 360.0812 (MH+); Anal. calcd
for C16H18BrN5: C, 53.34; H, 5.04; N, 19.44. Found: C, 53.22; H,
4.94; N, 19.40.
C
22H23FN5 376.1937, found 376.1935 (MH+).
4.2.14. 8-(2-Furyl)-2-(1-octyn-1-yl)-N9-propargyladenine (5c)
A mixture of 4a (100 mg, 0.28 mmol), 2-furanboronic acid
(62 mg, 0.56 mmol), Pd(PPh3)4 (32 mg, 0.028 mmol) and K2CO3
(77 mg, 0.56 mmol) in 1,4-dioxane (3 mL) and H2O (2 mL) was stir-
red at 90 °C for 50 min. Water was added, and the mixture was
extracted with CHCl3. The organic layers were dried over MgSO4,
filtered and concentrated. The residue was purified by column
chromatography (hexane/AcOEt = 3/2 to 1:1) to give 5c (51 mg,
53%). 1H NMR (CDCl3) d 7.69 (1H, d, J = 1.5 Hz), 7.25 (1H, d,
J = 3.6 Hz), 6.64 (1H, dd, J = 1.5, 3.6 Hz), 5.94 (2H, br s), 5.24 (2H,
d, J = 2.4 Hz), 2.47 (2H, t, J = 6.7 Hz), 2.32 (1H, t, J = 2.5 Hz), 1.71–
1.65 (2H, m), 1.47–1.44 (2H, m), 1.33–1.29 (4H, m), 0.90 (3H, t,
J = 7.0 Hz); 13C NMR (CDCl3) d 154.7, 150.9, 146.9, 144.8, 144.1,
142.2, 118.6, 113.1, 112.2, 88.2, 80.3, 73.5, 33.2, 31.4, 28.9, 28.3,
22.5, 19.5, 14.0; ESIMS-HR calcd for C20H22N5O 348.1824, found
348.1823 (MH+); Anal. calcd for C20H21N5Oꢀ1.1H2O: C, 65.41; H,
6.37; N, 19.07. Found: C, 65.31; H, 6.07; N, 19.04.
4.2.11. 8-Chloro-2-(1-octyn-1-yl)-N9-propargyladenine (4c)
A
mixture of 3e (281 mg, 1.0 mmol) and AcOK (29 mg,
0.30 mmol) in DMF (10 mL) was treated with NCS (270 mg,
2.0 mmol), and the mixture was stirred at room temperature over-
night. The reaction was quenched with saturated Na2S2O3 solution,
and the mixture was extracted with CHCl3. The organic layers were
dried over MgSO4, filtered and concentrated. The residue was puri-
fied by column chromatography (hexane/AcOEt = 3:1) to give 4c
(173 mg, 55%). 1H NMR (CDCl3) d 6.00 (2H, s), 4.98 (2H, d,
J = 2.5 Hz), 2.45 (2H, t, J = 7.2 Hz), 2.37, (1H, t, J = 2.5 Hz), 1.69–
1.65 (2H, m), 1.48–1.42 (2H, m), 1.34–1.25 (4H, m), 0.89 (3H, t,
J = 7.2 Hz); 13C NMR (CDCl3) d 154.0, 150.5, 147.1, 138.5, 117.6,
88.6, 80.0, 73.8, 32.5, 31.4, 28.8, 28.2, 22.5, 19.4, 14.0; ESIMS-HR
calcd for C16H19ClN5 316.1329, found 316.1324 (MH+); Anal. calcd
for C16H18ClN5ꢀ0.3H2O: C, 59.83; H, 5.84; N, 21.80. Found: C, 60.09;
H, 5.73; N, 21.69.
4.2.15. 2-(1-Octyn-1-yl)-N9-propargyl-8-[2-(1,2,3-
triazolyl)]adenine (5d)
A mixture of 4a (100 mg, 0.28 mmol), 1,2,3-triazole (19 mg,
0.28 mmol) and K2CO3 (77 mg, 0.56 mmol) in DMF (1 mL) was stir-
red at room temperature overnight. The reaction was quenched
with water, and the mixture was extracted with AcOEt. The organic
layers were washed with brine, dried over MgSO4, filtered and con-
centrated. The residue was purified by column chromatography
(hexane/AcOEt = 4:1 to 1:1) to give isomer mixture. Then, the mix-
ture was purified by ODS column chromatography (MeCN/
H2O = 3:7 to 2:3) to give 5d (10 mg, 10%). 1H NMR (CDCl3) d 8.03
(2H, s), 6.11 (2H, br s), 5.47 (2H, d, J = 2.4 Hz), 2.48 (2H, t,
J = 7.3 Hz), 2.16 (1H, t, J = 2.4 Hz), 1.74–1.65 (2H, m), 1.50–1.44
(2H, m), 1.35–1.26 (4H, m), 0.90 (3H, t, J = 7.0 Hz); 13C NMR (CDCl3)
d 155.1, 150.4, 147.6, 141.9, 137.5, 116.3, 88.6, 80.2, 73.1, 34.2,
31.4, 29.7, 28.9, 28.3, 22.5, 19.4, 14.0; ESIMS-HR calcd for
4.2.12. 2-(1-Octyn-1-yl)-8-phenyl-N9-propargyladenine (5a)
A mixture of 4a (100 mg, 0.28 mmol), phenylboronic acid
(68 mg, 0.52 mmol), Pd(PPh3)4 (64 mg, 0.055 mmol) and K2CO3
(76 mg, 0.55 mmol) in 1,4-dioxane (3 mL) and H2O (2 mL) was stir-
red at 80 °C for 30 min. Additional Pd(PPh3)4 (32 mg, 0.028 mmol)
was added, and the reaction mixture was stirred for further 1 h.
Water was added, and the mixture was extracted with AcOEt.
The organic layers were dried over MgSO4, filtered and concen-
trated. The residue was purified by column chromatography (hex-
ane/AcOEt = 2:1) to give 5a (36 mg, 36%). 1H NMR (CDCl3) d 7.90
(2H, m), 7.57–7.55 (3H, m), 5.84 (2H, br s), 5.01 (2H, d,
J = 2.3 Hz), 2.47 (2H, t, J = 7.4 Hz), 2.40 (1H, t, J = 2.5 Hz), 1.71–
1.65 (2H, m), 1.48–1.45 (2H, m), 1.33–1.30 (4H, m), 0.90 (3H, t,
J = 6.9 Hz); 13C NMR (CDCl3) d 154.8, 151.5, 146.8, 130.5, 129.2,
129.1, 118.6, 88.0, 80.4, 73.9, 33.4, 31.4, 28.9, 28.3, 22.5, 19.5,
14.1; ESIMS-HR calcd for C22H24N5 358.2032, found 358.2025
(MH+).
C
18H21N8 349.1889, found 349.1895 (MH+).
4.2.16. 9-(2,3,5-Tri-O-acetyl-1-b-D-ribofuranosyl)-6-chloro-2-[2-
(1-hydroxycyclohexyl)ethyn-1-yl]purine (7)15c
A mixture of 6 (108 mg, 0.20 mmol),ethynyl-1-cyclohexanol
(30 mg, 0.24 mmol), (PPh3)2PdCl2 (3 mg, 0.0040 mmol) and CuI
(1.5 mg, 0.0080 mmol) in 1,4-dioxane (2 mL) was treated with
Et3N (56 lL, 0.40 mmol), and the mixture was stirred at room tem-
perature for 8.5 h. Additionalethynyl-1-cyclohexanol (30 mg,
0.24 mmol) was added, and the mixture was stirred for further
15 h. The reaction was quenched with aqueous EDTA, and the mix-
ture was extracted with CHCl3. The organic layers were washed

3080
K. Endo et al. / Bioorg. Med. Chem. 22 (2014) 3072–3082
with aqueous EDTA and brine, dried over Na2SO4, filtered and con-
centrated. The residue was purified by column chromatography
(CHCl3 to MeOH–CHCl3 = 1:49) to give 7 (105 mg, quant). 1H
NMR (CDCl3) d 8.28 (1H, s), 6.21 (1H, d, J = 5.4 Hz), 5.90 (1H, t,
J = 5.4 Hz), 5.74 (1H, dd, J = 4.1, 5.4 Hz), 4.51–4.43 (3H, m), 2.97
(1H, s), 2.18 (3H, s), 2.12 (3H, s), 2.10 (3H, s), 2.08–2.05 (2H, m),
1.78–1.56 (7H, m), 1.35–1.33 (1H, m); 13C NMR (CDCl3) d 170.5,
169.5, 169.3, 151.5, 151.2, 146.0, 144.2, 131.5, 93.0, 87.1, 82.3,
80.9, 73.3, 70.8, 68.6, 63.1, 39.4, 25.2, 23.0, 20.8, 20.5, 20.4;
mixture was extracted with CHCl3. The organic layers were dried
over Na2SO4, filtered and concentrated. The residue was purified
by column chromatography (AcOEt) to give 12a (405 mg, 52%).
Analytical samples were crystallized from 1,4-dioxane/hexane.
mp. 225–229 °C; 1H NMR (DMSO-d6) d 7.63 (2H, s), 5.59 (1H, s),
4.95 (2H, d, J = 2.3 Hz), 3.49 (1H, t, J = 2.3 Hz), 1.84–1.82 (2H, m),
1.65–1.47 (7H, m), 1.27–1.23 (1H, m); 13C NMR (DMSO-d6) d
154.6, 150.2, 146.0, 126.7, 118.2, 89.6, 82.7, 77.3, 75.7, 66.7, 66.3,
33.0, 24.8, 22.5; ESIMS-HR calcd for C16H17BrN5O 374.0616, found
374.0618 (MH+); Anal. calcd for C16H16BrN5Oꢀ0.3H2O: C, 50.62; H,
4.41; N, 18.45. Found: C, 50.54; H, 4.16; N, 18.35.
ESIMS-HR calcd for
C24H28ClN4O8 535.1596, found 535.1597
(MH+).
4.2.17. 2-[2-(1-Hydroxycyclohexyl)ethyn-1-yl]adenosine (9)15c
A solution of 7 (1.61 g, 3.0 mmol) in pyridine (6 mL) and H2O
(6 mL) was stirred at 50 °C for 4 h. The solvent was removed in
vacuo, and the a solution of the resulting residue in NH4OH
(6 mL) and 1,4-dioxane (3 mL) was stirred at for 4 h. The solvent
was removed in vacuo and the residue was purified by column
chromatography (MeOH–CHCl3 = 1:19 to 1:9) to give 9 (708 mg,
61%). 1H NMR (DMSO-d6) d 8.42 (1H, s), 7.47 (2H, br s), 5.88 (1H,
d, J = 6.1 Hz), 5.54 (1H, s), 5.47 (1H, t, J = 6.2 Hz), 5.20–5.17 (2H,
m), 4.51 (1H, dd, J = 6.1, 6.2 Hz), 4.14–4.10 (1H, m), 3.96–3.94
(1H, m), 3.68–3.65 (1H, m), 3.59–3.56 (1H, m), 1.86–1.83 (2H,
m), 1.65–1.47 (7H, m), 1.26–1.24 (1H, m); 13C NMR (DMSO-d6) d
155.8, 149.3, 145.5, 140.0, 118.5, 89.1, 87.1, 85.6, 82.8, 73.8, 70.4,
4.2.21. 8-Chloro-2-[2-(1-hydroxycyclohexyl)ethyn-1-yl]-N9-
propargyladenine (12b)
A
mixture of 11 (200 mg, 0.67 mmol) and AcOK (20 mg,
0.20 mmol) in DMF (7 mL) was treated with NCS (180 mg,
1.3 mmol), and the mixture was stirred at room temperature for
28.5 h. The reaction was quenched with saturated Na2S2O3 solu-
tion, and the mixture was extracted with CHCl3. The organic layers
were dried over Na2SO4, filtered and concentrated. The residue was
suspended in CHCl3, and resulting precipitate was crystallized from
1,4-dioxane/H2O to give 12b (49 mg, 22%). mp. 225–235 °C; 1H
NMR (DMSO-d6)
d 7.60 (2H, s), 5.55 (1H, s), 4.98 (2H, d,
J = 2.3 Hz), 3.48 (1H, t, J = 2.3 Hz), 1.85–1.82 (2H, m), 1.66–1.48
(7H, m), 1.28–1.25 (1H, m); 13C NMR (DMSO-d6) d 155.4, 150.5,
146.9, 137.6, 117.5, 90.4, 83.4, 77.8, 76.5, 67.4, 67.1, 32.9, 25.5,
23.2; ESIMS-HR calcd for C16H17ClN5O 330.1122, found 330.1134
(MH+); Anal. calcd for C16H16ClN5Oꢀ0.3H2O: C, 57.33; H, 4.99; N,
20.89. Found: C, 57.39; H, 4.87; N, 20.58.
66.6, 61.4, 24.8, 22.5, 22.3; ESIMS-HR calcd for
C18H24N5O5
390.1777, found 390.1766 (MH+); Anal. calcd for C18H23N5O5-
ꢀ1.6H2O: C, 51.69; H, 6.31; N, 16.75. Found: C, 51.94; H, 6.16; N,
16.35.
4.2.18. 2-[2-(1-Hydroxycyclohexyl)ethyn-1-yl]adenine (10)
A solution of 9 (24.6 g, 63 mmol) in 1 M aqueous HCl (320 mL)
was stirred at 100 °C for 1 h. After cooling, 1 M aqueous NaOH was
added to the mixture to adjust the pH to 8.5, and the mixture was
stirred at room temperature for 1.5 h. Then, the resulting precipi-
tate was corrected to give 10 (14.6 g, 90%). 1H NMR (DMSO-d6) d
12.86 (1H, br s), 8.13 (1H, br s), 7.24 (2H, br s), 5.50 (1H, s),
1.85–1.82 (2H, m), 1.66–1.46 (7H, m), 1.28–1.22 (1H, m); 13C
NMR (DMSO-d6) d 155.5, 150.2, 145.4, 139.7, 117.9, 88.2, 83.3,
74.0, 66.6, 34.7, 24.8, 22.5, 21.3; ESIMS-HR calcd for C13H16N5O
258.1355, found 258.1374 (MH+).
4.2.22. 8-(2-Furyl)-2-[2-(1-hydroxycyclohexyl)ethyn-1-yl]-N9-
propargyladenine (13)
A
mixture of 12a (1.00 g, 2.7 mmol), 2-furanboronic acid
(598 mg, 5.3 mmol), Pd(PPh3)4 (309 mg, 0.27 mmol) and K2CO3
(738 mg, 5.3 mmol) in 1,4-dioxane (30 mL) and H2O (20 mL) was
stirred at 100 °C for 25 min. The reaction was quenched with
water, and the mixture was extracted with CHCl3. The organic lay-
ers were dried over Na2SO4, filtered and concentrated. The residue
was purified by column chromatography (CHCl3 to MeOH–
CHCl3 = 1:32) to give 13 (567 mg, 59%). Analytical samples were
crystallized from 1,4-dioxane. mp. 220–229 °C (dec.); 1H NMR
(DMSO-d6) d 8.03 (1H, d, J = 1.7 Hz), 7.60 (2H, br s), 7.27 (1H, d,
J = 3.4 Hz), 6.80 (1H, dd, J = 1.7 Hz, 3.4 Hz), 5.58 (1H, s), 5.21 (2H,
d, J = 2.4 Hz), 3.42 (1H, t, J = 2.4 Hz), 1.87–1.84 (2H, m), 1.67–1.47
(7H, m), 1.28–1.27 (1H, m); 13C NMR (DMSO-d6) d 155.4, 150.1,
145.8, 145.3, 143.7, 140.8, 117.8, 112.6, 112.3, 89.4, 82.9, 78.2,
75.5, 66.6, 66.3, 32.7, 24.8, 22.5; ESIMS-HR calcd for C20H20N5O2
362.1617, found 362.1631 (MH+); Anal. calcd for C20H19N5O2-
ꢀ0.2H2O: C, 65.81; H, 5.36; N, 19.19. Found: C, 65.76; H, 5.31; N,
19.02.
4.2.19. 2-[2-(1-Hydroxycyclohexyl)ethyn-1-yl]-N9-
propargyladenine (11)
A mixture of 10 (2.10 g, 8.2 mmol), K2CO3 (2.25 g, 16 mmol) in
DMF (16.5 mL) was treated with propargylbromide (1.23 mL,
16 mmol), and the mixture was stirred at room temperature for
3 h. The reaction mixture was poured into water (80 mL), and the
mixture was stirred at room temperature for 2 h. Then, the result-
ing precipitate was corrected to give 11 (2.04 g, 84%). 1H NMR
(DMSO-d6) d 8.24 (1H, s), 7.43 (2H, br s), 5.59 (1H, s), 5.03 (2H, d,
J = 2.3 Hz), 3.55–3.43 (1H, m), 1.84–1.83 (2H, m), 1.65–1.46 (7H,
m), 1.28–1.26 (1H, m); 13C NMR (DMSO-d6) d 155.7, 149.1, 145.7,
140.9, 117.9, 89.0, 82.9, 78.1, 75.9, 66.6, 32.3, 24.7, 22.4; ESIMS-
HR calcd for C16H18N5O 296.1511, found 296.1526 (MH+); Anal.
calcd for C16H17N5Oꢀ1.6H2O: C, 59.28; H, 6.28; N, 21.60. Found: C,
59.48; H, 5.95; N, 21.25.
4.3. Inhibitory effects of the 2-alkynyladenine derivatives on 2-
octyn-1-yladenosine-induced vasodilation
4.3.1. Vascular preparations
Male Wistar rats, 10 weeks old weighing 305–355 g, were pur-
chased from Clea Japan (Tokyo, Japan). The rats were killed, under
ether anesthesia, by bleeding from the abdominal aorta, and the
femoral vein were isolated from each rat. The vessels were placed
in physiological salt solution (PSS), composed of 118 mM NaCl,
4.7 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3,
1.2 mM KH2PO4, and 11 mM glucose, and continuously bubbled
with 95% O2–5% CO2. The vessels were cleaned of loosely adhering
connective tissue and cut into rings (5 mm in width) under a dis-
secting microscope. Special care was taken not to damage the
endothelium. Each ring was mounted between two stainless steel
4.2.20. 8-Bromo-2-[2-(1-hydroxycyclohexyl)ethyn-1-yl]-N9-
propargyladenine (12a)
A
mixture of 11 (620 mg, 2.1 mmol) and AcOK (62 mg,
0.63 mmol) in DMF (21 mL) was treated with NBS (374 mg,
2.1 mmol), and the mixture was stirred at room temperature for
2.5 h. Additional NBS was added in twice, 0.2, 0.1 equiv, respec-
tively and the reaction mixture was stirred for further 2.5 h. The
reaction was quenched with saturated Na2S2O3 solution, and the

K. Endo et al. / Bioorg. Med. Chem. 22 (2014) 3072–3082
3081
hooks and placed in 10-ml organ baths containing PSS at 37 °C. The
isometric tension of the rings was measured by a force–displace-
ment transducer (TB-611, Nihon-Kohden, Japan) and recorded on
a pen recorder (FBR-252A, TOA, Japan). The rings were equilibrated
for 60 min at a resting tension of 0.5 g for femoral vein ring, the
optimal tension for inducing maximal contraction. During the
equilibration time, the PSS in the bath was replaced every 20 min.
placed in the center of open chambers (50 cm long ꢂ 50 cm
wide ꢂ 30 cm high) located in a sound-attenuating behavioral test
room. Illumination of test room was the same as the rat colony
room. The animal’s behavior was observed for 300 min. After each
test, the field was cleaned with an water. The following behavioral
categories were examined: distance of path, time the animal spent
moving. Turning Behaviors were automatically analyzed with com-
puter-based video tracking system (Muromachi kikai Co., Ltd,
Japan).
4.3.2. Vasodilation inhibition study
We previously reported 5-hydroxytryptamine (10ꢁ7 to
3 ꢂ 10ꢁ5 M) induced concentration-dependent contraction in the
isolated rat femoral vein.27 The contractile response to 10ꢁ5 M 5-
hydroxytryptamine (5-HT) in the vein was stable during the time
period required to construct the 10ꢁ7 M 2-octyn-1-yladenosine
induced relaxing response. And the vein was completely dilated
at 2-octyn-1-yladenosine concentrations as low as 10ꢁ7 M. There-
fore following the 1 h equilibration period, the rings were precon-
Acknowledgments
We are grateful to Dr. T. Noguchi of Yamasa Corporation for
helpful discussions and for supporting this work, and to Dr. S.
Kohgo and Mr. A. Kawasaki (also of Yamasa Corporation) for help-
ful discussions.
tracted with 10ꢁ5 M 5-HT. After the contraction induced by 10ꢁ5
M
References and notes
5-HT had reached a plateau, 10ꢁ7 M 2-octyn-1-yladenosine were
added to the bath for the maximum vasodilation response.27 To
examine the inhibitory efficacy of 2-alkynyladenine derivatives
on 2-octyn-1-yladenosine induced vasodilation, 2-alkynyladenine
derivatives were incubated for 10 min prior to the addition of 5-
HT.
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