Synthesis and biological evaluation of 2-alkynyl-N6-methyl- 5′-N-methylcarboxamidoadenosine derivatives as potent and highly selective agonists for the human adenosine A3 receptor

Article abstract of DOI:10.1021/jm900754g

A new series of 2-aralkynyl-N⁶-methyl-MECAs 10-13 were synthesized and evaluated in radioligand binding studies and in a newEu-GTP functional assay that provides a powerful alternative to radioisotope use. The new compounds possess high affinit

Full text of DOI:10.1021/jm900754g

J. Med. Chem. 2009, 52, 7897–7900 7897
DOI: 10.1021/jm900754g
Synthesis and Biological Evaluation of 2-Alkynyl-N6-methyl-50-N-methylcarboxamidoadenosine
Derivatives as Potent and Highly Selective Agonists for the Human Adenosine A3 Receptor‡
Rosaria Volpini,† Michela Buccioni,† Diego Dal Ben,† Catia Lambertucci,† Carmen Lammi,† Gabriella Marucci,†
Anna T. Ramadori,† Karl-Norbert Klotz,§ and Gloria Cristalli*,†
†Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, I-62032 Camerino, Italy, and
§Institut fu€r Pharmakologie und Toxikologie, Universita€t Wu€rzburg, D-97078 Wu€rzburg, Germany
Received May 29, 2009
A new series of 2-aralkynyl-N6-methyl-MECAs 10-13 were synthesized and evaluated in radioligand
binding studies and in a new Eu-GTP functional assay that provides a powerful alternative toradioisotope
use. The new compounds possess high affinity and selectivity for the AA3R with N6-methyl-2-phenyl-
ethynylMECA (10) showing a subnanomolar affinity and about 100000-fold selectivity vs AA1R and
AA2AR. Furthermore, the new nucleosides showed to be full agonists, the N6-methyl-2-(2-pyridinyl)-
ethynylMECA (13) being the most potent in the series.
Introduction
Adenosine (Ado, Figure 1) is a naturally occurring nucleo-
side having a role in an ample variety of physiological and
pathophysiological processes through the activation of at
least four G-protein-coupled receptors (P1) cloned and classi-
fied as Ado A1, A2A, A2B, and A3 receptors (AA1R, AA2AR,
AA2BR, and AA3R, respectively).1,2 In particular, AA3R is
expressed in a broad range of tissues and couples to Gi/o
proteins leading to inhibition of adenylyl cyclase, increase of
intracellular Ca2þ concentration, and activation of phospho-
inositide 3-kinase.3 Since this subtype is involved in a variety
Figure 1. Structure of known potent and selective AA3R ligands.
ofkey physiological processes, experimental evidence suggests
that AA3R agonists could be employed as therapeutic agents
for the treatment of several pathologies.4 Cristalli and co-
workers have previously published the synthesis and biologi-
cal activity of 2-aralkynyl-N6-methoxyAdo compounds and
the corresponding 50-N-methyl and 50-N-ethylcarboxamido
derivatives (2-aralkynyl-N6-methoxyMECAs and NECAs,
respectively), which are endowed with good affinity and
different degrees of selectivity for the human AA3R.5-7
Among them, some MECA derivatives bearing an aromatic
ring directly linked to the 2-ethynyl group showed the best
profile for this receptor subtype (1-4, AA3R affinity in the
low nanomolar range and selectivity ranging from 1600- to
22000-fold; for details, see Table 1).8 Furthermore, in
a previous work by the same authors it was demonstrated
that the introduction of a methyl group in N6-position of
2-phenylethynylAdo (N6-methylPEAdo, Figure 1)9 favored
the interaction with the human AA3R, this compound having
comparable AA3R affinity and higher selectivity than the
corresponding N6-methoxy-2-phenylethynylAdo (N6-methoxy-
PEAdo, Figure 1) (N6-methylPEAdo Ki AA3R = 3.4 nM,
selectivity AA1R/AA3R=500 and AA2AR/AA3R = 2500 vs
N6-methoxyPEAdo, Ki AA3R = 3.8 nM, selectivity AA1R/
AA3R=300 and AA2AR/AA3R=1100).8
Starting from these observations and to find highly potent
and selective AA3R agonists, the synthesis of N6-methyl
analogues of 1-4 was undertaken. All of the new 2-aralkynyl-
N6-methyl-MECAs 10-13 were evaluated in radioligand
binding studies and in functional assays. In particular, a novel
time-resolved fluorometric (TRF) method, which exploits the
unique fluorescence properties of lanthanide chelates and
provides a powerful alternative to the assays that utilize
radioisotopes, was set up to evaluate the potency of the new
compounds.
Results and Discussion
Chemistry. The 2-alkynyl-N6-methylMECA derivatives
10-13 were synthesized starting from 2-iodo-N6-methylAdo
(5), which was obtained from commercially available gua-
nosine in four steps.10 Protection of 20- and 30-hydroxyl
groups of 5, using acetone and p-toluenesulfonic acid at
room temperature for 16 h, gave 6, which was reacted with
KMnO4 in basic conditions to obtain the acid derivative 7.
Treatment of 7 with thionyl chloride in the presence of dry
DMF and subsequent reaction with methylamine at 0 °C for
1 h furnished the protected 2-iodo-N6-methylMECA 8.
‡ Contribution to celebrate the 100th anniversary of the Division of
Medicinal Chemistry of the American Chemical Society.
*To whom correspondence should be addressed. Phone: þ39-0737-
402252. Fax: þ39-0737-402295. E-mail: gloria.cristalli@unicam.it.
r
2009 American Chemical Society
Published on Web 10/19/2009
pubs.acs.org/jmc

7898 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Volpini et al.
Reaction of 8 with formic acid at 50 °C for 2 h gave the
deprotected 2-iodo-N6-methylMECA (9) that was in turn
treated with the suitable terminal alkynes, using a modifica-
tion of the classical palladium-catalyzed cross-coupling re-
action10,11 to give the desired 2-alkynyl-N6-methylMECA
derivatives 10-13 (Scheme 1).
Biological Data. All of the new compounds were evaluated
at the human recombinant adenosine receptors (ARs), stably
transfected into CHO cells, utilizing radioligand binding
studies (AA1R, AA2AR, AA3R) or adenylyl cyclase activity
assay (AA2BR) (Table 1). Receptor binding affinity was
determined using [3H]CCPA (2-chloro-N6-cyclopentyl-
adenosine) as radioligand for AA1R, whereas [3H]NECA
was used for AA2AR and AA3R subtypes.12 Most of the
tested compounds were inactive at AA2BR (Ki > 30 μM);
therefore, the AA2BR data are not shown in Table 1. Affinity
and selectivity data of 2-aralkynyl-N6-methoxyMECA deri-
vatives 1-4 are reported in Table 1 for comparison with the
corresponding new nucleosides 10-13.
The results of binding experiments showed that replace-
ment of the N6-methoxy group in 1-4 by a methyl induced
an increase of AA3R binding affinity ranging from 3-fold
(compare 13 to 4) to 8-fold (compare 11 to 2); in fact 10-13
are endowed with Ki in the subnanomolar range (0.44, 0.33,
0.43, and 0.40 nM, respectively).
Table 1. Affinity Data of New Derivatives in Radioligand Binding
Assays at Human AA1R, AA2AR, and AA3R
Furthermore, the same substitution led to a general,
although modest, decrease of affinity at the human AA1R
and AA2AR subtypes, resulting in a significant increase
of AA3R selectivity for the new compounds. In particular,
N6-methyl-2-phenylethynylMECA (N6-methylPEMECA,
10), showing Ki AA3R = 0.44 nM and a selectivity vs
AA1R and AA2AR of 77 000 and 97 000, respectively, is
one of the most potent and selective ligand of the human
AA3R with nucleoside structure reported so far.
Ki (nM)a
selectivity
AA1R/ AA2AR/
AA1Rb AA2ARc AA3Rd AA3R AA3R
compd
Ar
Moreover, the ability of the new compounds to activate
the human AA3R, stably transfected into CHO cells, was
evaluated in comparison with that elicited by the full agonist
Cl-IBMECA. This measurement was carried out by utilizing
the nonradioactive DELFIA Eu-GTP binding assay. The
new functional method is based on fluorescence emission
determination of the chelate Eu-GTP bound to AA3R, after
GDP/Eu-GTP exchange consequent to the receptor activa-
tion. Since it is the first report in which Eu-GTP functional
assay is used to evaluate the AR activation, some known
compounds such as the antagonist N6-methoxy-2-(2-pyridi-
nyl)ethynylAdo, the partial agonists N6-methoxyPEAdo
and N6-methylPEAdo, and the full agonists 1-4, whose
functional activity at AA3R has been already studied in
1
Ph 9140
p-CH3CO-Ph 53800
16300
10400
18700
18000
41700
7030
1.9
2.5
4800
21500
1600
8600
4200
2
3
p-F-Ph
2-Py
Ph
3000
3990
1.9
1.1
9800
4
3600
16400
97000
21500
29500
61000
10
11
12
13
32800
0.44
0.33
0.43
0.40
76500
31000
12500
22000
p-CH3CO-Ph 10200
p-F-Ph
2-Py
5310
8740
12700
24300
a 95% confidence intervals are reported in Table 2 of the Supporting
Information. b Displacement of specific [3H]CCPA binding in CHO
cells, stably transfected with human recombinant AA1R, expressed as
Ki (nM). c Displacement of specific [3H]NECA binding in CHO cells,
stably transfected with human recombinant AA2AR, expressed as Ki
(nM). d Displacement of specific [3H]NECA binding in CHO cells, stably
transfected with human recombinant AA3R, expressed as Ki (nM).
Scheme 1. Synthesis of Designed AA3R Ligandsa
a Conditions: (a) (CH3)2CO, PTSA; (b) KMnO4, KOH, H2O; (c) SOCl2, dry DMF, 50 °C; (d) CH3NH2, CH2Cl2, from -20 to 0 °C; (e) HCOOH 50%,
50 °C; (f) Ar-CtCH, CuI, (Ph3P)2PdCl2, Et3N, dry DMF, room temp.

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7899
Figure 2. Concentration-response curves in Eu-GTP functional assays by using CHO cells expressing the human AA3R. Each point
represents the mean of four to five experiments performed in triplicate with a maximum SEM lower than (10.
adenylyl cyclase experiments, were tested as reference com-
pounds. The results obtained with the Eu-GTP assay con-
firmed that the three Ado derivatives are antagonists
(N6-methoxy-2-(2-pyridinyl)ethynylAdo) or partial agonists
(N6-methoxyPEAdo and N6-methylPEAdo) showing lower
ability to activate the human AA3R than Cl-IBMECA
(Figure 2). In contrast, 1-4 were able to activate the receptor
with a relative fluorescence value comparable to that of Cl-
IBMECA, taken as 100% of response, thus behaving as full
agonists of the human AA3R (Figure 2). These results are in
close agreement with those obtained for the same com-
pounds in previously reported adenylyl cyclase experiments;
hence, they prove the high reliability of this nonradioactive
assay. Eu-GTP methods were finally used to evaluate the
new N6-methyl derivatives 10-13 that proved to evoke a
100% response in Eu-GTP functional assay, confirming that
50-N-alkylcarboxamidoAdo derivatives behave as full ago-
nists of the human AA3R.
selective AA3R ligands reported so far. In particular,
N6-methyl-2-phenylethynylMECA (10) showed a Ki AA3R
of 0.44 nM and a remarkable selectivity vs AA1R and AA2A
R
of 77000 and 97 000, respectively. Furthermore, these mole-
cules were found to behave as full agonists in a Eu-GTP
functional assay, which was set up to evaluate the activation
of ARs by their ligands. It is worthwhile to note that this
new functional assay avoids the use of any radiolabeled
compound.
Experimental Procedures
€
Chemistry. Melting points were determined with a Buchi
apparatus and are uncorrected. 1H NMR spectra were obtained
with a Varian Mercury 400 MHz spectrometer; δ is in ppm and J
in Hz. All exchangeable protons were confirmed by addition of
D2O. Mass spectra were recorded on an HP 1100-MSD series
instrument. All measurements were performed using electro-
spray ionization (ESI-MS) on a single quadrupole analyzer.
Thin layer chromatography (TLC) was carried out on precoated
TLC plates with silica gel 60 F-254 (Fluka). For column
chromatography, silica gel 60 (Merck) and silica RP-18
(Aldrich) were used. Elemental analyses were determined on
Fisons Instruments model EA 1108 CHNS-O model analyzer
and are within 0.4% of theoretical values. Purity of the com-
pounds was g95% according to elemental analysis data.
2-Iodo-20,30-O-isopropylidene-N6-methyladenosine (6). To 5
(5.21 g, 12.80 mmol) in dry acetone (280 mL) p-toluenesulfonic
acid was added (24.35 g; 141.57 mmol), and the mixture was left
for 16 h under stirring at room temperature. Saturated solution
of NaHCO3 was added to neutralize the pH, and the reaction
was left under stirring 1 h. Acetone was removed under vacuum
and the residue partitioned between CHCl3 and H2O. Organic
extracts were dried over Na2SO4, filtered, and concentrated to
dryness. Compound 6 was obtained, after crystallization from
CH3OH, as a white solid: 93% yield.
EC50 values elicited by 1-4 and 10-13 were in general
comparable to that of the reference compound Cl-IB-
MECA, with the exception of N6-methoxy-2-(2-pyridi-
nyl)ethynylMECA
nyl)ethynylMECA (13), which are 7- to 8-fold more potent
(4)
and
N6-methyl-2-(2-pyridi-
than Cl-IBMECA itself (EC50=5.2 ꢀ 10-8 and 4.6 ꢀ 10-8
M
versus EC50=3.6 ꢀ 10-7 M, respectively). All data concern-
ing percentage of response and EC50 of the tested ligands are
reported in Table 3 of Supporting Information. These results
emphasize that in the full agonist disubstituted MECA
derivatives, the nature of the substituent in 2-position plays
an important role in the activation of AA3R, the presence of
a 2-pyridinylethynyl group leading to an increase of potency.
Conclusion
2-Iodo-20,30-O-isopropylidene-N6-methyl-40-carboxylic Acid
Adenosine (7). To a suspension of 6 (4.0 g, 8.94 mmol) in 500
mL of H2O, KOH (1.45 g) and a solution of KMnO4 (4.09 g) in
120 mL of H2O were added dropwise under stirring. The mixture
was set aside in the dark at room temperature for 40 h. After
cooling at 5-10 °C, the mixture was decolorized by a 35% H2O2
solution (10.2 mL) in 49.2 mL of H2O, maintaining the tem-
perature under 10 °C in an ice-salt bath. The mixture was
filtered through Celite, the filtrate concentrated in vacuo to
In summary, new disubstituted MECA derivatives bearing
a methyl group in the N6-position and an aralkynyl substi-
tuent in the 2-position were designed and synthesized to find
potent and selective AA3R ligands. Binding experiments,
performed in CHO cells, stable transfected with human
ARs, demonstrated that the new compounds 10-13 possess
affinity in the subnanomolar range and a very high selectivity
for the target subtype, being among the most potent and

7900 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Volpini et al.
about 50 mL and then acidified to pH 4 with 2 N HCl. The
resulting precipitate was filtered off and washed with water to
give 7 as a white solid: 62% yield.
Functional Assay. Compounds in Table 1 (1-4 and 10-13)
were evaluated for receptor activity at human AA3R, stably
transfected into CHO cells, and CHO wild type utilizing DEL-
FIA Eu-GTP (DELFIA GTP-binding kit, Perkin-Elmer Life
Science) binding assay. The bound Eu-GTP was measured with
time-resolved fluorometer, GENIosPro TECAN.
2-Iodo-20,30-O-isopropylidene-N6-methyl-50-N-methylcarbox-
amidoadenosine (8). To 7 (0.300 g, 0.65 mmol), SOCl2 (1 mL) and
DMF (24 μL, 0.31 mmol) were added at 0 °C in nitrogen
atmosphere. The mixture was stirred at 50 °C for 2 h. Solvent
was removed under vacuum and the residue coevaporated three
times with dry toluene. Then dry CH2Cl2 (2 mL) was added and
the mixture cooled at -20 °C. CH3NH2 (1 mL) was added, and
the mixture was left for 1 h at 0 °C. Volatiles were removed, and
the residue was partitioned between H2O and CH2Cl2. The
organic extracts were dried over Na2SO4, filtered, and concen-
trated to dryness. The residue was purified on a flash silica gel
column, eluting with cHex-CHCl3-CH3OH (70:29:1 to
70:20:10). After crystallization from CH3OH, 8 was obtained
as a white solid in 44% yield.
Acknowledgment. This work was supported by Fondo di
Ricerca di Ateneo (University of Camerino) and Ministero
della Salute Progetto Ordinario Neurolesi (Grant RF-CNM-
2007-662855).
Supporting Information Available: Chemical-physical data
of synthesized compounds 6-13; detailed biological protocols.
This material is available free of charge via the Internet at http://
pubs.acs.org.
2-Iodo-N6-methyl-50-N-methylcarboxamidoadenosine (9). To
8 (0.191 g, 0.40 mmol), a 50% solution of HCOOH (15 mL) was
added, and the mixture was heated at 50 °C for 2 h. The solvent
was removed under vacuum, and the residue was coevaporated
three times with H2O and then EtOH to give 9 as a white solid
(crystallization from MeOH): yield 85%.
References
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General Procedure for the Synthesis of 2-Alkynyl-N6-methyl-
50-N-methylcarboxamidoadenosine Derivatives 10-13. To a so-
lution of 9 (0.120 g, 0.28 mmol) in dry DMF (7.5 mL),
triethylamine (1.12 mL), bis(triphenylphosphine)palladium
dichloride (5.0 mg, 0.007 mmol), CuI (0.3 mg, 0.0015 mmol),
and the suitable terminal alkyne (1.68 mmol) were added. The
mixture was stirred under nitrogen atmosphere at room tem-
perature and for the time reported in the specific description.
After evaporation under vacuum and chromatography over
silica gel column eluting with the suitable mixture of solvents,
MECA derivatives 10-13 were obtained.
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N6-Methyl-2-phenylethynyl-50-N-methylcarboxamidoadeno-
sine (10). Reaction of 9 with phenylethyne for 40 min, followed
by flash chromatography eluting with CHCl3-CH3OH (95:5)
and further purification by RP-18 chromatography eluting with
CH3OH-H2O (50:50), gave 10 as a white solid (crystallization
from EtOH): 42% yield.
2-(4-Acetylphenyl)ethynyl-N6-methyl-50-N-methylcarboxami-
doadenosine (11). Reaction of9 with4-ethynylacetophenonefor 2
h followed by flash chromatography eluting with CHCl3-
cHex-CH3OH (80:15:5) gave 11 as a white solid (crystalli-
zation from EtOH), 93% yield.
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doadenosine (12). Reaction of 9 with 1-ethynyl-4-fluorobenzene
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from EtOH, as a white solid: 85% yield.
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adenosine (13). Reaction of 9 with 2-ethynylpyridine for 3 h,
chromatography eluting with CHCl3-CH3CN-CH3OH (80:
10:10), and crystallization from EtOH gave 13 as white solid,
64% yield.
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Biological Evaluation. Binding Studies. Dissociation con-
stants of unlabeled compounds (Ki) were determined in compe-
tition experiments in 96-well microplates as described recently.12