New 9-methyl-8-(4-hydroxyphenyl)adenine derivatives as A1 adenosine receptor antagonists

Article abstract of DOI:10.1135/cccc2011091

A new series of 9-methyladenines, bearing different bulky groups at the 8-position, were prepared and their affinity for the four human adenosine receptor subtypes were evaluated. All the synthesized compounds showed affinities at the A₁, A

Full text of DOI:10.1135/cccc2011091

A1 Adenosine Receptor Antagonists
1379
NEW 9-METHYL-8-(4-HYDROXYPHENYL)ADENINE DERIVATIVES AS
A1 ADENOSINE RECEPTOR ANTAGONISTS
b
Catia LAMBERTUCCIa1, Michela BUCCIONIa2, Barbara CACCIARI ,
c1
d
Diego DAL BENa3, Stephanie FEDERICO , Karl-Norbert KLOTZ ,
Gabriella MARUCCIa4, Rosaria VOLPINIa5, Giampiero SPALLUTO and
c2
a6,
Gloria CRISTALLI
*
a
School of Pharmacy, Medicinal Chemistry Unit, University of Camerino,
Via S. Agostino 1, 62032 Camerino (MC), Italy; e-mail: catia.lambertucci@unicam.it,
michela.buccioni@unicam.it, diego.dalben@unicam.it, gabriella.marucci@unicam.it,
rosaria.volpini@unicam.it, gloria.cristalli@unicam.it
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Ferrara,
via Fossato di Mortara 17–19, 44100 Ferrara, Italy; e-mail: barbara.cacciari@unife.it
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste,
Piazzale Europa 1, 34127 Trieste, Italy; stephanie.federico@phd.units.it, spalluto@units.it
Institut für Pharmakologie und Toxikologie, Universität Würzburg,
1
2
5
3
4
6
b
c
1
2
d
97078 Würzburg, Germany; e-mail: klotz@toxi.uni-wuerzburg.de
Received April 28, 2011
Accepted June 10, 2011
Published online November 20, 2011
Dedicated to Professor Antonín Holý on the occasion of his 75th birthday.
A new series of 9-methyladenines, bearing different bulky groups at the 8-position, were pre-
pared and their affinity for the four human adenosine receptor subtypes were evaluated. All
the synthesized compounds showed affinities at the A1, A2A, and A3AR subtypes ranging
from nanomolar to micromolar levels with different degrees of A1 selectivity, while they re-
sulted nearly inactive at A2BAR. In particular, 9-methyl-8-[4-(4-methylbenzyloxy)phenyl]-
adenine showed A1AR affinity in the nanomolar range and good levels of selectivity versus
the other receptor subtypes. Furthermore, a functional assay at mouse ileum allowed to as-
sess the potency of selected compounds at A1AR subtype. Results showed that all the tested
derivatives are neutral antagonists and their Kb values are in good agreement with the Ki
values from radioligand binding assay at human A1AR, confirming that the effect is due to
inhibition of this subtype.
Keywords: Adenosine receptors; Adenosine receptor antagonists; Adenine derivatives; A1AR
functional studies; Receptors; Nucleobases; Heterocycles; Ligand design; Ligand effects.
Adenosine is a naturally occurring nucleoside that mediates numerous
physiological and physio-pathological processes1 through the activation of
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1379–1393
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011091

1380
Lambertucci et al.:
four G protein-coupled receptors, which have been cloned2 and classified as
A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes3 on the bases of cou-
pling to second messengers and pharmacological profiles for agonists and
antagonists. Clarifying the physiological functions of the different subtypes
has been useful to detect possible targets of therapeutic intervention. At the
same time, lack of potent and selective ligands for a specific subtype could
be a drawback to the attribution of specific activity.
In the case of A2BAR, this lack precluded for many years a deep explora-
tion of its therapeutic prospective but the discovery of antagonists with
xanthine structure such as MRS 1754 (1) and CVT-6883 (2), and with non
xanthine structure such as OSIP 339391 (3) led to preclinical studies mainly
on inflammatory and angiogenic diseases (Fig. 1)4. Moreover, it is known
that in general adenosine antagonists could be promising therapeutic tar-
gets in a wide range of conditions, including cerebral and cardiac ischaemic
diseases, sleep disorders, immune and inflammatory disorders and cancer5.
O
N
O
N
CF3
N
N
N
N
N
N
O
H
N
H
N
H
N
O
O
O
CN
CVT-6883 (2)
hA1 = 990 nM
hA2A = 690 nM
hA2B = 1.0 nM
hA3 = 1,000 nM
MRS 1754 (1)
hA1 = 403 nM
hA2A = 503 nM
hA2B = 2.0 nM
hA3 = 570 nM
H
H3C
N
NH2
N
NH
N
O
N
N
N
N
N
N
N
O
H
4
OSIP 339391 (3)
hA2B KD = 20 nM
selectivities > 70
hA1 = 27 nM
hA2A = 360 nM
hA3 = > 3,300 nM
FIG. 1
Structures and binding profile of some AR antagonists (Ki, nM)
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A1 Adenosine Receptor Antagonists
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A structural analysis of the xanthine derivatives 1 and 2 clearly points
out the importance for A2B affinity of bulky substituents containing phenyl
and heteroaromatic groups at the 8-position. On the other hand, we re-
ported that the introduction of different substituents at the 2-, 8-, and
9-position of the adenine moiety resulted in high-affinity antagonists pre-
senting distinct receptor selectivity profile6–10, with the derivative 9-ethyl-
8-phenyladenine (4) endowed with good affinity and selectivity for the
A1AR subtype11. Furthermore, Harada et al.12 some years ago found that
2-alkynyl-9-methyladenines bearing a substituted phenyl group in 8-position
inhibited agonist-induced glucose production in rat hepatocytes and showed
hypoglycemic activity in an animal model of noninsulin-dependent diabe-
tes mellitus, effects that are considered to be mediated by the interaction
with the A2BAR subtype.
Taking into account all the above reported observations and to check the
effect of introducing bulky substituents in 8-position of the purine core, we
designed and synthesized new 9-methyladenine derivatives 5–17 bearing in
8-position a para phenoxy moiety with an attached benzyl (Scheme 1) or
N-phenylacetamido group (Scheme 2, Fig. 2). The synthesized compounds
were tested at human AR subtypes expressed in Chinese hamster ovary
O
NH2
N
N
O
N
N
N
N
NH
O
N
H
O
N
CH3
R
R
MRS like molecules
Harada Compounds
NH2
N
N
N
N
O
R
CH3
general structure of designed molecules
FIG. 2
Rational design of synthesized compounds
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Lambertucci et al.:
(CHO) cells in radioligand binding assay with the aim at evaluating the ef-
fect of the introduced substituents on A1 and A2BARs binding affinity.
Introduction of alkynyl groups at the 2-position, suggested by Harada,
was avoided for two reasons. First of all, adenine derivatives with big
lipophilic substituents in 2- and 8-positions would be rather insoluble and,
secondly, we already proved that the introduction of an alkynyl group in
2-position of adenine brought about an improvement of affinity at all the
other receptor subtypes higher than that at the A2B subtype7,10
.
RESULTS AND DISCUSSION
Chemistry
The designed compounds 5–17 were synthesized as summarized in Schemes
1 and 2. Reaction of commercially available 5-amino-4,6-dichloro-
pyrimidine (18) with methylamine at 55 °C afforded derivative 19 as major
product13, which after a two-step coupling with 4-hydroxybenzadehyde
gave the purine derivative 20 in a good yield. The latter was then reacted
with liquid ammonia in a sealed tube at 70 °C to afford the desired
8-substituted adenine 5 (Scheme 1). Reaction of compound 5 with the ap-
Cl
Cl
Cl
N
N
NH2
NH
NH2
Cl
(i)
(ii)
N
N
N
OH
N
N
N
CH3
CH3
20
18
19
(iii)
NH2
N
NH2
N
N
N
N
(iv)
N
N
OH
O
N
CH3
CH3
R
6: R = H
5
7: R = 4-F
8: R = 4-CH3
9: R = 4-C(CH3)3
SCHEME 1
(i) CH3NH2, Et3N, 55 °C; (ii) 4-hydroxybenzaldehyde, CH3CO2H, r.t. then EtOH, FeCl3, 85 °C;
(iii) NH3, 70 °C, 24 h; (iv) substituted benzyl halide, K2CO3, dry DMF, r.t., 4 h
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1379–1393

A1 Adenosine Receptor Antagonists
1383
propriate substituted benzyl halides in dry dimethylformamide (DMF) in
the presence of potassium carbonate gave the corresponding O-benzyl de-
rivatives 6–9 (Scheme 1).
On the other hand, by reaction of 5 with α-bromo ethyl acetate under
the same conditions, the corresponding phenoxyacetate ethyl ester 10 was
obtained, which was converted into the corresponding acid derivative 11
by saponification with 5% NaOH. The final amido compounds 12–17 were
obtained by reaction of 11 with the suitable aniline derivatives in dry DMF
in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI;
Scheme 2).
The newly synthesized compounds 5–17 were tested at the human A1,
A
2A, and A3AR subtypes expressed in CHO cells in radioligand binding
using 2-chloro-N6-[3H]cyclopentyladenosine ([3H]CCPA) for A1AR, [3H]5′-
N-ethylcarboxamidoadenosine ([3H]NECA) for A2A, and [3H]2-hexyn-2-yl-
N6-methyladenosine ([3H]HEMADO) for A3ARs 14. In the case of A2BAR, Ki
values were calculated from IC50 values determined by inhibition of NECA-
stimulated adenylyl cyclase activity15. Ki values are expressed in nM, with
95% confidence intervals in parentheses. The results of binding and cyclase
activity studies are reported in Table I.
NH2
NH2
(i)
N
N
N
N
OEt
N
N
OH
O
N
N
O
CH3
CH3
5
10
(ii)
NH2
N
NH2
N
N
N
H
(iii)
OH
N
N
N
N
N
O
O
O
O
CH3
CH3
R
11
12: R = H
13: R = 4-I
14: R = 4-F
15: R = 4-CH3
16: R = 2,6-(CH3)2
17: R = 4-NO2
SCHEME 2
(i) α-bromoethyl acetate, K2CO3, dry DMF, r.t., 4 h; (ii) 5% NaOH, dioxane, r.t., 3 h; (iii) sub-
stituted aniline, EDCI, dry DMF, r.t., 24 h
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Lambertucci et al.:
TABLE I
Biological profile of synthesized compounds 5–17
NH2
N
N
OR
N
N
CH3
5-17
Ki, nM
Cpd
R
hA1ARa
hA2AARb
hA2BARc
>30000
hA3ARd
5
H
164
772
13600
(136–198)
(652-914)
(10300–18000)
831
(727–950)
24500
(23200–25800)
CH2Ph
6
7
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>30000
>40000
>40000
>40000
>40000
215
(183–252)
36100
(25800–50600)
CH2Ph-4-F
89
(62–127)
16800
(13400–21130)
CH2Ph-4-CH3
CH2Ph-4-C(CH3)3
CH2CO2Et
8
176
(104–300)
9
>100000
2990
(2150–4170)
4190
(3380–5200)
6630
(5590–7340)
10
11
12
13
14
15
26700
(24000–34300)
CH2CO2H
>100000
>40000
>40000
812
(777–849)
3380
(2420–4730)
CH2CONHPh
CH2CONHPh-4-I
CH2CONHPh-4-F
CH2CONHPh-4-CH3
145
(101–208)
1120
(911–1390)
2180
(1750–2720)
462
(294–727)
2600
(2270–2970)
>40000
>40000
>40000
222
(184–268)
1250
(1020–1520)
4070
(2380–7030)
12000
(9260–15500)
CH2CONHPh-2,6-(CH3)2
CH2CONHPh-4-NO2
16
17
>30000
>30000
1150
9550
12600
(1090–1200)
(7950–11500)
(10100–15900)
a
Displacement of specific [3H]CCPA binding at human A1AR expressed in CHO cells, (n =
3–6). Displacement of specific [3H]NECA binding at human A2AAR expressed in CHO cells.
b
c
Ki values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells ex-
pressing human A2BAR. Displacement of specific [3H]NECA binding at human A3AR ex-
d
pressed in CHO cells. Data are expressed as geometric means with 95% confidence limits in
parentheses.
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A1 Adenosine Receptor Antagonists
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All the synthesized compounds 5–17 showed affinities at the A1, A2A, and
A3AR subtypes ranging from nanomolar to micromolar levels with different
degrees of A1 selectivity, while they resulted nearly inactive at A2BAR. In
particular, the 8-(4-hydroxy)phenyl derivative 5 presented an A1AR affinity
of 164 nM with good selectivity vs A2B and A3ARs, while resulted to be
poorly selective vs the A2AAR (A2A/A1 = 4.7). Alkylation of the phenolic
function with benzyl groups (compounds 6–9) did not modify significantly
the potency at the A1AR but an increased selectivity could be observed,
since the interaction of these compounds with the other receptor subtypes
seems to be less favourable. More in detail, the unsubstituted benzyl deriva-
tive 6 resulted to be five-fold less potent at the A1AR but the affinity versus
the other receptor subtypes resulted dramatically reduced if compared with
the parent compound 5, with a significant increase of selectivity as a conse-
quence.
A general increase in affinity could be detected when a substitution at the
para-position of the phenyl ring was introduced (in 7–9). In particular, the
presence of a methyl group afforded the best compound of the series (deriv-
ative 8) with an A1AR affinity of 89 nM and good levels of selectivity versus
the other receptor subtypes (A2A/A1 = 191, A3/A1 > 454). Other substituents
at the para-position, such as fluorine (in 7) or tert-butyl (in 9) permitted re-
tention of affinity at the A1AR, comparable to that of the non alkylated de-
rivative 5, but the levels of selectivity versus the other receptors resulted to
increase (e.g. 7 A2A/A1 = 168, A3/A1 > 186 and 5 A2A/A1 = 4.7, A3/A1 > 83).
Alkylation of the phenolic function of compound 5 with an ethyl acetate
10 significantly reduced the affinity at the A1 and A2AARs, while a slight im-
provement was detected at the A3AR if compared with derivative 5. In con-
trast, the corresponding acid derivative 11 showed a complete loss of
affinity at the four human AR subtypes.
Introduction of an amido group between the methylene and the phenyl
ring did not modify substantially the A1 binding profile, derivatives 12–17
affinity being only slightly lower than that of the corresponding parent
compounds (compare 6, 7, and 8 with 12, 14, and 15, respectively). How-
ever, the amido derivatives presented a reduced selectivity, in particular vs
the A2AAR subtype since their A2A affinity was ten-fold higher than that of
the corresponding compounds 6–8. Interestingly, the presence of an iodine
at the 4-position of the phenyl ring (compound 13) induced affinity in the
µM range at the A3AR (Ki = 2180 nM) while all the other derivatives resulted
to be almost inactive at this subtype. The presence of a strong electron-
withdrawing group such as para-NO2 (in 17) or double substitution of the
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1379–1393

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Lambertucci et al.:
phenyl ring (in 16) led to a reduction of affinity at all the four human AR
subtypes.
Functional Studies
Since some adenine derivatives presented good affinity for the A1AR,
a functional assay at mouse ileum was performed to assess their potency at
this AR subtype. This assay was chosen since it is known that the inhibition
of rhythmic spontaneous contraction of ileum tissue is due to the activa-
tion of A1ARs 16
.
Concentration–response curves of NECA (Fig. 3) alone and in the pres-
ence of compounds 5, 8, 9, 13, and 15 at 10–6 M are reported in Fig. 4. In
the same figure, the NECA curve in the presence of the A1AR reference an-
NH2
N
N
O
N
N
O
N
N
H
N
N
O
N
O
H
HO
OH
NECA
DPCPX
FIG. 3
AR agonist (NECA) and antagonist (DPCPX) compounds used for biological evaluation analysis
FIG. 4
Concentration–response curves of NECA in the presence of compounds 5, 8, 9, 13, and 15 at
10–6 M at mouse ileum (A1AR) in comparison with DPCPX at the same concentration. Each
point represents the mean of four or five experiments with a maximum SEM lower than 10
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A1 Adenosine Receptor Antagonists
1387
tagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; Fig. 3), tested at the
same concentration, is shown. The obtained Kb values of the same com-
pounds with the addition of ligand 11 are reported in Table II.
TABLE II
Antagonist potency of selected compounds, expressed as Kb values, in the isolated mouse il-
eum (A1AR) in comparison with DPCPX (Ki values from binding experiments at A1AR are re-
ported for comparison)
Compoundsa
Kb, nM
Ki, nM (A1AR)
b
DPCPX
5
3.9
(4.9–5.1)
5
204
(181–227)
164
89
8
102
(90–114)
9
309
(274–344)
176
26700
11
22900c
(22600–23200)
13
15
371
(340–402)
145
222
562
(524–600)
a
b
Compounds (except 11) were tested at 10–6 M concentration. Kb values in functional tests
are geometric means of four or five experiments with 95% confidence limits in parentheses.
c
Values obtained at 10–4 M concentration.
FIG. 5
NECA effect on rhythmic spontaneous contractions of mouse ileum in the presence of an an-
tagonist
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Lambertucci et al.:
Results showed that all the selected compounds are neutral antagonists as
they were able to shift the NECA concentration–response curve (Fig. 5)
without affecting the rhythmic spontaneous contraction of ileum (data not
shown).
It is worthwhile to note that the Kb values obtained with this functional
assay at mouse ileum are in good agreement with the Ki values from
radioligand binding assay at human A1AR, confirming that the effect is due
to inhibition of the A1AR subtype (Table II).
CONCLUSIONS
In conclusion, the present study clearly demonstrated that introduction of
bulky substituents, similar to those present at the 8-position of A2B antago-
nists with xanthine structure, in the adenine nucleus does not induce any
A2BAR activity but good A1AR affinity and selectivity. Furthermore, a func-
tional study at mouse ileum showed that all the selected compounds are
neutral antagonists and their Kb values are in good agreement with the Ki
values from radioligand binding assay at human A1AR, confirming that the
effect is due to inhibition of this subtype. It is worthwhile to note that
A1AR antagonists may have therapeutic applications5 and some of them are
in clinical trials for the treatment of acute decompensated heart failure
(ADHF) with renal impairment4.
EXPERIMENTAL
Chemical Synthesis
Reactions were routinely monitored by thin-layer silica gel chromatography (TLC; precoated
F254 Merck plates) and products visualized by UV light and iodine or potassium permanga-
nate solution. IR spectra (ν, cm–1) were recorded on a Jasco FT-IR 200 spectrometer. 1H NMR
spectra were determined in DMSO-d6 solutions on a Bruker AC 200 spectrometer downfield
from tetramethylsilane as internal standard. Chemical shifts are given in ppm (δ-scale) and
coupling constants (J) in Hz. Light petroleum ether refers to the fractions boiling at 40–60
°C. Melting points were determined on a Buchi–Tottoli instrument and are uncorrected. Col-
umn chromatography was performed using Merck 60–200 mesh silica gel. All products re-
ported showed 1H NMR spectra in agreement with the assigned structures. Organic solutions
were dried over anhydrous magnesium sulfate.
6-Chloro-9-methyl-8-(4-hydroxyphenyl)purine (20)
A methanolic (8 ml) solution of compound 19 (300 mg, 1.89 mmol), acetic acid (0.25 ml),
and 4-hydroxybenzaldehyde (231 mg, 1.89 mmol) was stirred at room temperature for 24 h.
Then the solvent was removed under reduced pressure and to the residue dissolved in abso-
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1379–1393

A1 Adenosine Receptor Antagonists
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lute EtOH (8 ml), FeCl3 (307 mg) was added. The resulting mixture was heated at 85 °C for
6 h. After cooling to room temperature, the pure product 20 was obtained after filtration as
a white solid (yield 62%); m.p. 206 °C. IR (KBr): 3450–3090, 1610, 1515, 1430. 1H NMR
(DMSO-d6): 3.92 (s, 3 H, CH3); 6.98 (d, 2 H, H-Ph, J = 8.4); 7.84 (d, 2 H, H-Ph, J = 8.4); 8.75
(s, 1 H, H-2); 10.23 (s, 1 H, OH). For C12H9ClN4O (260.68) calculated: 55.29% C, 3.48% H,
21.49% N; found: 55.41% C, 3.52% H, 21.34% N.
9-Methyl-8-(4-hydroxyphenyl)adenine (5)
In a steel vial cooled to –80 °C, compound 20 (1.55 g, 5.95 mmol) and an excess of liquid
ammonia were poured. The vial was sealed and heated at 70 °C for 24 h. Then the mixture
was cooled to room temperature and, after evaporation of ammonia, the crude product was
purified by flash chromatography (CHCl3–cyclohexane–MeOH 75:25:5) to give compound 5
as a white solid (yield 91%); m.p. > 250 °C. IR (KBr): 3430–3055, 1615, 1510, 1425. 1H NMR
(DMSO-d6): 3.75 (s, 3 H, CH3); 6.92 (d, 2 H, H-Ph, J = 8.0); 7.18 (s, 2 H, NH2); 7.68 (d, 2 H,
H-Ph, J = 8.4); 8.13 (s, 1 H, H-2); 9.97 (s, 1 H, OH). For C12H11N5O (241.25) calculated:
59.74% C, 4.60% H, 29.03% N; found: 59.82% C, 4.57% H, 29.07% N.
Synthesis of O-Alkylated Compounds 6–10. General Procedure
A mixture of the adenine derivative 5 (50 mg, 0.207 mmol), K2CO3 (31.5 mg, 0.228 mmol),
and the appropriate halide (1.2 eq) in dry DMF (4 ml) was stirred at room temperature for
6 h. The solvent was removed under reduced pressure and the residue was poured into water
(10 ml) and extracted with EtOAc (3 × 15 ml). The combined organic layers were dried and
concentrated under vacuum. The crude product was purified by flash chromatography
(EtOAc–light petroleum 1:1) to yield the final compounds 6–10 as solids.
9-Methyl-8-(4-benzyloxyphenyl)adenine (6). Yield 75%, white solid, m.p. 225 °C. IR (KBr):
3330–3120, 1615, 1510, 1425. 1H NMR: 4.05 (s, 3 H, CH3); 5.19 (s, 2 H, O-CH2); 7.18 (d,
2 H, H-Ph, J = 9.0); 7.22–7.26 (m, 2 H, H-Ph); 7.30 (bs, 2 H, NH2); 7.38–7.45 (m, 3 H, H-Ph);
7.83 (d, 2 H, H-Ph, J = 9.0); 8.37 (s, 1 H, H-2). For C19H17N5O (331.37) calculated: 68.87% C,
5.17% H, 21.13% N; found: 69.01% C, 5.21% H, 21.07% N.
9-Methyl-8-[4-(4-fluorobenzyloxy)phenyl]adenine (7). Yield 66%, white solid, m.p. 237 °C.
IR (KBr): 3335–3120, 1605, 1515, 1420. 1H NMR: 4.07 (s, 3 H, CH3); 5.16 (s, 2 H, O-CH2);
7.15 (d, 2 H, H-Ph, J = 9.0); 7.21 (d, 2 H, H-Ph, J = 9.0); 7.27 (bs, 2 H, NH2); 7.42 (d, 2 H,
H-Ph, J = 9.0); 7.89 (d, 2 H, H-Ph, J = 9.0); 8.40 (s, 1 H, H-2). For C19H16FN5O (349.36) calcu-
lated: 65.32% C, 4.62% H, 20.05% N; found: 65.21% C, 4.55% H, 20.07% N.
9-Methyl-8-[4-(4-methylbenzyloxy)phenyl]adenine (8). Yield 72%, white solid, m.p. 225 °C.
IR (KBr): 3340–3110, 1610, 1515, 1410. 1H NMR: 2.40 (s, 3 H, Ph-CH3); 4.09 (s, 3 H,
N9-CH3); 5.16 (s, 2 H, O-CH2); 7.18 (d, 2 H, H-Ph, J = 9.0); 7.20 (d, 2 H, H-Ph, J = 9.0); 7.25
(bs, 2 H, NH2); 7.38 (d, 2 H, H-Ph, J = 9.0); 7.87 (d, 2 H, H-Ph, J = 9.0); 8.42 (s, 1 H, H-2).
For C20H19N5O (345.40) calculated: 69.55% C, 5.54% H, 20.28% N; found: 69.61% C,
5.51% H, 20.22% N.
9-Methyl-8-[4-(4-tert-butylbenzyloxy)phenyl]adenine (9). Yield 69%, white solid, m.p. 238 °C.
IR (KBr): 3335–3110, 1600, 1505, 1410. 1H NMR: 1.36 (s, 9 H, C(CH3)3); 4.01 (s, 3 H, CH3);
5.15 (s, 2 H, O-CH2); 7.20 (d, 2 H, H-Ph, J = 9.0); 7.24 (d, 2 H, H-Ph, J = 9.0); 7.33 (bs, 2 H,
NH2); 7.43 (d, 2 H, H-Ph, J = 9.0); 7.78 (d, 2 H, H-Ph, J = 9.0); 8.35 (s, 1 H, H-2). For
C
23H25N5O (387.48) calculated: 71.29% C, 6.50% H, 18.07% N; found: 71.23% C, 6.42% H,
18.02% N.
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Ethyl[4-(9-methyladenin-8-yl)phenoxy]acetate (10). Yield 83%, white solid, m.p. 155 °C.
IR (KBr): 3350–3120, 1725, 1605, 1515, 1420. 1H NMR: 1.2 (t, 3 H, CH2CH3, J = 7.0); 4.00 (s,
3 H, N9-CH3); 4.34 (q, 2 H, CH2CH3, J = 7.0); 4.75 (s, 2 H, O-CH2); 7.11 (d, 2 H, H-Ph, J =
9.0); 7.26 (bs, 2 H, NH2); 7.79 (d, 2 H, H-Ph, J = 9.0); 8.35 (s, 1 H, H-2). For C16H17N5O3
(327.34) calculated: 58.71% C, 5.23% H, 21.39% N; found: 58.83% C, 5.28% H, 21.33% N.
[4-(9-Methyladenin-8-yl)phenoxy]acetic Acid (11)
Ester 10 (500 mg, 1.53 mmol) was dissolved in dioxane (3 ml) and 5% aqueous NaOH (3 ml).
The resulting mixture was stirred at room temperature for 4 h. The solution was diluted
with water (5 ml) and acidified with 10% aqueous HCl solution to pH 2. The precipitated
acid 11 was collected by filtration. Yield 93%, grey solid, m.p. > 300 °C. IR (KBr):
3550–3020, 1715, 1610, 1505, 1410. 1H NMR: 3.78 (s, 3 H, CH3); 4.76 (s, 2 H, O-CH2); 7.09
(d, 2 H, H-Ph, J = 9.0); 7.25 (bs, 2 H, NH2); 7.81 (d, 2 H, H-Ph, J = 9.0); 8.16 (s, 1 H, H-2);
12.3 (bs, 1 H, COOH). For C14H13N5O3 (299.28) calculated: 56.18% C, 4.38% H, 23.40% N;
found: 56.26% C, 4.32% H, 23.47% N.
Preparation of Phenylacetamido Derivatives 12–17. General Procedure
A mixture of acid 11 (50 mg, 0.167 mmol), appropriate aniline (1.1 eq.), and EDCI (64 mg,
0.338 mmol) in dry DMF (4 ml) was stirred at room temperature for 24 h. Then the solvent
was removed under reduced pressure and the residue was suspended in water (5 ml) and ex-
tracted with EtOAc (3 × 5 ml). The combined organic layers were dried and concentrated at
reduced pressure. The crude product was then purified by flash chromatography (EtOAc–
light petroleum 7:3) to yield the desired final compounds 12–17 as solids.
2-[4-(9-Methyladenin-8-yl)phenoxy]-N-phenylacetamide (12). Yield 74%, pale yellow solid,
m.p. 195 °C. IR (KBr): 3330–3120, 1700, 1615, 1525, 1430. 1H NMR: 3.79 (s, 3 H, CH3); 4.82
(s, 2 H, O-CH2); 7.17–7.28 (m, 5 H, H-Ph); 7.32 (bs, 2 H, NH2); 7.65 (d, 2 H, H-Ph, J = 9.0);
7.85 (d, 2 H, H-Ph, J = 9.0); 8.17 (s, 1 H, H-2); 10.20 (bs, 1 H, CONH). For C20H18N6O2
(374.40) calculated: 64.16% C, 4.85% H, 22.45% N; found: 64.23% C, 4.82% H, 22.49% N.
2-[4-(9-Methyladenin-8-yl)phenoxy]-N-(4-iodophenyl)acetamide (13). Yield 67%, pale yellow
solid, m.p. 192 °C. IR (KBr): 3350–3150, 1695, 1620, 1522, 1410. 1H NMR: 3.78 (s, 3 H,
CH3); 4.82 (s, 2 H, O-CH2); 7.18 (d, 2 H, H-Ph, J = 9.0); 7.23 (bs, 2 H, NH2); 7.51 (d, 2 H,
H-Ph, J = 9.0); 7.68 (d, 2 H, H-Ph, J = 9.0); 7.84 (d, 2 H, H-Ph, J = 9.0); 8.16 (s, 1 H, H-2);
10.29 (bs, 1 H, CONH). For C20H17IN6O2 (500.29) calculated: 48.01% C, 3.42% H, 16.80% N;
found: 47.88% C, 3.37% H, 16.75% N.
2-[4-(9-Methyladenin-8-yl)phenoxy]-N-(4-fluorophenyl)acetamide (14). Yield 70%, grey solid,
m.p. 260 °C. IR (KBr): 3340–3135, 1695, 1615, 1500, 1420. 1H NMR: 3.79 (s, 3 H, CH3); 4.81
(s, 2 H, O-CH2); 7.16 (d, 2 H, H-Ph, J = 9.0); 7.21 (d, 2 H, H-Ph, J = 9.0); 7.27 (bs, 2 H, NH2);
7.66 (d, 2 H, H-Ph, J = 9.0); 7.85 (d, 2 H, H-Ph, J = 9.0); 8.17 (s, 1 H, H-2); 10.25 (bs, 1 H,
CONH). For C20H17FN6O2 (392.39) calculated: 61.22% C, 4.37% H, 21.42% N; found:
61.33% C, 4.42% H, 21.37% N.
2-[4-(9-Methyladenin-8-yl)phenoxy]-N-(4-tolyl)acetamide (15). Yield 56%, pale yellow solid,
m.p. 218 °C. IR (KBr): 3340–3125, 1692, 1615, 1510, 1410. 1H NMR: 2.26 (s, 3 H, Ph-CH3);
3.79 (s, 3 H, N9-CH3); 4.80 (s, 2 H, O-CH2); 7.12 (d, 2 H, H-Ph, J = 9.0); 7.19 (d, 2 H, H-Ph,
J = 9.0); 7.25 (bs, 2 H, NH2); 7.54 (d, 2 H, H-Ph, J = 9.0); 7.84 (d, 2 H, H-Ph, J = 9.0); 8.16
(s, 1 H, H-2); 10.10 (bs, 1 H, CONH). For C21H20N6O2 (388.42) calculated: 64.94% C,
5.19% H, 21.64% N; found: 65.03% C, 5.22% H, 21.75% N.
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A1 Adenosine Receptor Antagonists
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2-[4-(9-Methyladenin-8-yl)phenoxy]-N-(2,6-dimethylphenyl)acetamide (16). Yield 74%, pale
yellow solid, m.p. 236 °C. IR (KBr): 3330–3100, 1694, 1620, 1500, 1430. 1H NMR: 2.13 (s,
6 H, PH-(CH3)2); 3.79 (s, 3 H, N9-CH3); 4.85 (s, 2 H, O-CH2); 7.07–7.09 (m, 2 H, H-Ph);
7.20–7.22 (m, 1 H, H-Ph); 7.25 (d, 2 H, H-Ph, J = 9.0); 7.28 (bs, 2 H, NH2); 7.65 (d, 2 H,
H-Ph, J = 9.0); 8.17 (s, 1 H, H-2); 9.59 (bs, 1 H, CONH). For C22H22N6O2 (402.45) calculated:
65.66% C, 5.51% H, 20.88% N; found: 65.59% C, 5.43% H, 21.00% N.
2-[4-(9-Methyladenin-8-yl)phenoxy]-N-(4-nitrophenyl)acetamide (17). Yield 73%, yellow solid,
m.p. 150 °C. IR (KBr): 3345–3120, 1697, 1620, 1550, 1510, 1430, 1360. 1H NMR: 3.81 (s,
3 H, CH3); 4.80 (s, 2 H, O-CH2); 7.18 (d, 2 H, H-Ph, J = 9.0); 7.25 (d, 2 H, H-Ph, J = 9.0);
7.33 (bs, 2 H, NH2); 7.71 (d, 2 H, H-Ph, J = 9.0); 7.88 (d, 2 H, H-Ph, J = 9.0); 8.19 (s, 1 H,
H-2); 10.02 (bs, 1 H, CONH). For C20H17N7O4 (419.39) calculated: 57.28% C, 4.09% H,
23.38% N; found: 57.19% C, 4.13% H, 23.29% N.
Biology
All pharmacological methods followed the procedures as described earlier14. In brief, mem-
branes for radioligand binding were prepared from CHO cells stably transfected with human
adenosine receptor subtypes in a two-step procedure. In a first low-speed step (1000 × g) cell
fragments and nuclei were removed. The crude membrane fraction was sedimented from the
supernatant at 100,000 × g. The membrane pellet was resuspended in the buffer used for the
respective binding experiments, frozen in liquid nitrogen and stored at –80 °C. For the mea-
surement of adenylyl cyclase activity, only one high speed centrifugation of the homoge-
nate was used. The resulting crude membrane pellet was resuspended in 50 mM Tris-HCl,
pH 7.4 and immediately used for the cyclase assay.
For radioligand binding at A1AR 1 nM [3H]CCPA, at A2AAR 10 nM [3H]NECA and at A3AR
1 nM [3H]HEMADO were used, respectively. Non specific binding of [3H]CCPA was deter-
mined in the presence of 1 mM theophylline, in the case of [3H]NECA 100 pM N6-(R)-phenyl-
isopropyladenosine (R-PIA) was used. Ki values from competition experiments were
calculated with the program SCTFIT 17. Radioligand binding at A2BAR is problematic as no
high-affinity ligand is available for this subtype. Therefore, inhibition of NECA-stimulated
adenylyl cyclase activity was determined as a measurement of affinity of compounds. EC50
values from these experiments were converted to Ki values with the Cheng and Prusoff
equation15
.
Functional antagonism in mouse ileum. Animal testing was carried out according to the
European Communities Council Directive of 24 November 1986 (86/609/EEC). Experiments
were performed on male mice (BALB/C; 25.5 0.5 g body weight, 8 weeks old) reared at the
School of Pharmacy building B, University of Camerino (Camerino, Italy). They were housed
in a room on a reverse 12-h light/dark cycle, temperature of 20–22 °C, and humidity of
45–55% with free access to food and water. Animals were sacrificed by cervical dislocation,
ileum was isolated, freed from adhering connective tissue and placed in Krebs solution of
the following composition (in mM): NaCl 119, KCl 4.5, MgSO4 2.5, NaHCO3 25, KH2PO4 1.2,
CaCl2 2.5, glucose 11.1. Segments (20 mm in length) were suspended in organ baths con-
taining 10 ml of Krebs solution kept at 37 °C and aerated with 5% CO2:95% O2.
Recording of mechanical activity. The experiments were performed according to a reported
procedure16. Ileum segments were connected to isometric force transducers (Ugo Basile,
Biological Research Apparatus, VA, Italy) and mechanical activity was visualized, recorded
and analyzed on a personal computer using the PowerLab/400 system (Ugo Basile, Italy).
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Longitudinal preparations were subjected to an initial tension of 200 mg and were allowed
to equilibrate for at least 30 min. Rhythmic spontaneous contractions of varying amplitude
developed in all preparations. Concentration–response curve for NECA was constructed by
cumulative addition of the drug. All compounds were solubilized in DMSO to prepare 10–2
M
stock solutions, which were then diluted with the suitable buffer. The concentration of ago-
nist in the organ bath was increased approximately 3-fold at each step, with each addition
being made only after the response to the previous addition had attained a maximal level
and remained steady.
Antagonism study was carried out incubating tissues for 1 h with a dose of compound un-
der study before repeating the curve of NECA. Time control experiments showed that a sec-
ond curve to the agonist was reproducible. Parallel experiments in which tissues did not
receive any antagonist were run in order to check any variation in sensitivity.
Statistical analysis. Values of concentration–response curves are given as mean standard
error of four or five independent observations. Kb values are expressed as geometric means
with 95% confidence limits. Student’s t-test was used to assess the statistical significance of
the difference between two means. Statistical significance was set at p < 0.05.
Inhibitory effects induced by NECA were estimated as the decrease in the amplitude of
the spontaneous contraction where 100% corresponding to maximum amplitude. Curves
were fitted by a non-linear regression using a Prism 4.0 program (GraphPAD Software, San
Diego (CA), USA).
To quantify agonist (NECA) potency, EC50 values were calculated. EC50 is the concentra-
tion of agonist required to produce 50% of the maximum effect.
To quantify antagonist potency, Kb values were calculated from the equation Kb = (DR – 1) – [B],
where DR is the ratio of EC50 values of agonist after and before treatment with antagonist
concentration [B]18
.
This work was supported by Fondo di Ricerca di Ateneo (University of Camerino) and grants
from the Italian Ministry for Health (Progetto Ordinario Neurolesi, RF-CNM-2007-662855) and
Italian Ministry for University and Research (PRIN 2008). We thank S. Kachler for expert technical
assistance.
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