Structure-activity relationships of adenine and deazaadenine derivatives as ligands for adenine receptors, a new purinergic receptor family

Article abstract of DOI:10.1021/jm9006356

Adenine derivatives bearing substituents in the 2-, N⁶-, 7-, 8-, and/or 9-position and a series of deazapurines were synthesized and investigated in [³H]adenine binding studies at the adenine receptor in rat brain cortical membrane preparations (rAde1R). Steep structure-activity relationships were observed. Substitution in the 8-position (amino, dimethylamino, piperidinyl, piperazinyl) or in the 9-position (2-morpholinoethyl) with basic residues or introduction of polar substituents at the 6-amino function (hydroxy, amino, acetyl) represented the best modifications. Functional evaluation of selected adenine derivatives in adenylate cyclase assays at 1321N1 astrocytoma cells stably expressing the rAde1R showed that all compounds investigated were agonists or partial agonists. A subset of compounds was additionally investigated in binding studies at human embryonic kidney (HEK293) cells, which also express a high-affinity adenine binding site. Structure-affinity relationships at the human cell line were similar to those at the rAde1R, but not identical. In particular, N ⁶-acetyladenine (25, K_i rat: 2.85 μM; K_i human: 0.515 μM) and 8-aminoadenine (33, K_i rat: 6.51 μM; K_i human: 0.0341 μM) were much more potent at the human as compared to the rat binding site. The new AdeR ligands may serve as lead structures and contribute to the elucidation of the functions of the adenine receptor family. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9006356

5974 J. Med. Chem. 2009, 52, 5974–5989
DOI: 10.1021/jm9006356
Structure-Activity Relationships of Adenine and Deazaadenine Derivatives as Ligands for Adenine
Receptors, a New Purinergic Receptor Family
Thomas Borrmann,† Aliaa Abdelrahman,† Rosaria Volpini,‡ Catia Lambertucci,‡ Edgars Alksnis,§ Simone Gorzalka,†
†
Melanie Knospe, Anke C. Schiedel, Gloria Cristalli, and Christa E. Muller*
†
‡
,†
€
†PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany,
‡Dipartimento di Scienze Chimiche, University of Camerino, Via S. Agostino 1, 62032 Camerino, Italy, and §Latvian Institute of Organic
Synthesis, Aizkraukles iela 21, Riga LV-1006, Latvia
Received May 14, 2009
Adenine derivatives bearing substituents in the 2-, N6-, 7-, 8-, and/or 9-position and a series of
deazapurines were synthesized and investigated in [3H]adenine binding studies at the adenine receptor
in rat brain cortical membrane preparations (rAde1R). Steep structure-activity relationships were
observed. Substitution in the 8-position (amino, dimethylamino, piperidinyl, piperazinyl) or in the
9-position (2-morpholinoethyl) with basic residues or introduction of polar substituents at the 6-amino
function (hydroxy, amino, acetyl) represented the best modifications. Functional evaluation of selected
adenine derivatives in adenylate cyclase assays at 1321N1 astrocytoma cells stably expressing the
rAde1R showed that all compounds investigated were agonists or partial agonists. A subset of
compounds was additionally investigated in binding studies at human embryonic kidney (HEK293)
cells, which also express a high-affinity adenine binding site. Structure-affinity relationships at the
human cell line were similar to those at the rAde1R, but not identical. In particular, N6-acetyladenine
(25, Ki rat: 2.85 μM; Ki human: 0.515 μM) and 8-aminoadenine (33, Ki rat: 6.51 μM; Ki human:
0.0341 μM) were much more potent at the human as compared to the rat binding site. The new AdeR
ligands may serve as lead structures and contribute to the elucidation of the functions of the adenine
receptor family.
Introduction
of the dorsal root ganglia, suggesting a role in nociception.7 In
vivo experiments showed that spinally administered adenine
facilitated electrically evoked neuronal responses in a rat
model of inflammation, indicating a pronociceptive role of
adenine in nociceptive sensory transmission.8 Rat AdeR
mRNA was also detected at lower expression levels in brain
cortex, hypothalamus, lung, peripheral blood leukocytes, and
ovaries,7 and binding sites for [3H]adenine were detected in
brain cortical and striatal membrane preparations9 and in
synaptosome-rich fractions.10 In studies at cultured rat cere-
bellar Purkinje cells, adenine showed concentration-dependent
neurotrophic effects; it was the most potent compound among
a series of physiological nucleobases, nucleosides, and nucleo-
tides.11,12 An involvement of adenine in renal function was
described by Wengert et al. who showed that adenine exhibited
a Gi protein-mediated and A1 adenosine receptor-independent
inhibitory effect on Naþ-ATPase activity in the basolateral
membranes of the proximal tubule isolated from adult pig
kidneys.13 Increased plasma adenine concentrations have been
observed to correlate with the progress of chronic renal failure
in humans,14 confirming the significance of adenine in renal
function.13
Purinergic receptors are membrane receptors subdivided
into the P1 (adenosine receptor) and the P2 (nucleotide
receptor) families.1-4 P1 receptors are G protein-coupled
receptors activated by the nucleoside adenosine, while P2
receptors, which are activated by adenine and/or uracil nucleo-
tides, are further subdivided into P2Y (G protein-coupled) and
P2X (ligand-gated ion channels) subtypes.2,3,5,6 Recently, a
new family of purinergic G protein-coupled receptors was
discovered that is activated by the nucleobase adenine.5
Because of the structural relationships between the physiolo-
gical agonists for the three families, adenine representing a
partial structure of the P1 agonist adenosine, which itself is
part of the P2 receptor agonist structures ATP and ADP, the
designation “P0 receptors” was suggested for the adenine
receptor (AdeRa) family.5 The first AdeR was discovered by
a reverse pharmacology approach: Bender and colleagues
identified the nucleobase adenine as the endogenous ligand
of an orphan rat Gi protein-coupled receptor.7 mRNA locali-
zation studies revealed highest expression in the small neurons
*To whom correspondence should be addressed. Phone: þ49-228-73-
2301. Fax: þ49-228-73-2567. E-mail: christa.mueller@uni-bonn.de.
a Abbreviations: AdeR, adenine receptor(s); AdoR, adenosine
receptor(s); DME, 1,2-dimethoxyethane; DMEM, Dulbecco’s modified
Eagle medium; FCS, fetal calf serum; HBSS, Hank’s balanced salt
solution; HEK, human embryonic kidney; HMDS, hexamethyldisila-
zane; mAde2R, mouse adenine 2 receptor; m/rAde1R, mouse/rat ade-
nine 1 receptor; MsCl, mesyl chloride; NBS, N-bromosuccinimide;
PTSA, p-toluenesulfonic acid; SAR, structure-activity relationships.
Very recently, a novel mouse AdeR subtype was cloned
from mouse brain tissue and the mouse neuroblastoma x rat
glioma hybrid cell line NG108-15, adding a second distinct
receptor subtype (mAde2R) besides the rat adenine receptor
(rAde1R).15 The obtained sequence was different from the
sequence of the putative mouse orthologue MrgA10 (putative
mAde1R) of the rat AdeR found by a bioinformatic search.7
r
pubs.acs.org/jmc
Published on Web 09/04/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5975
Chart 1. Structure and Numbering of Adenine
Expression analysis of the mRNA of the mAde2R revealed
highest expression in the brain and a lower expression level in
the spleen.15 Functional experiments showed that both the
rAde1R expressed in Chinese hamster ovary (CHO) cells7 and
the mAde2R expressed in human astrocytoma 1321N1 cells15
were coupled to adenylate cyclase via Gi protein. Further-
more, there is evidence for the existence of a human AdeR
because human cell and tissue membrane preparations have
shown high affinity binding sites for [3H]adenine.9 Because no
direct human orthologues of the rat and mouse adenine
receptors Ade1R and Ade2R appear to exist, it is likely, that
a distinct subtype or distinct subtypes are expressed in
humans. Nevertheless, the binding sites appear to be con-
servedasshownforthe already wellcharacterizedorthologues
rAde1R and mAde2R.15 In humans, extracellular adenine
levels can reach similar values as those for the well-known,
structurally related neuromodulator adenosine.16 Active and
passive transport systems for nucleobases have been described
to exist in various species including mammals.17,18 A saturable
transport system for adenine across basolateral membranes of
the sheep choroid plexus has been characterized.19 Recently,
Nagai et al. showed that [3H]adenine is taken up into primary
cultured rat cortical neurons via a saturable Naþ-independent
mechanism, indicating that efficient mechanisms for the
removal of adenine from the extracellular fluid exist. Such
systems are required for limiting the duration of receptor
activation.20
Syntheses. 1-Deazaadenine (3), 3-deazaadenine (5), 7-dea-
zaadenine (7), and 9-deazaadenine (10) were prepared ac-
cording to previously described procedures (see also
Supporting Information).36-40 20-Deoxyadenosine (99),
2-chloroadenosine (100), and 2-chloro-20-deoxyadenosine
(101) were commercially available. The 2-substituted ade-
nine derivatives 16 and 17 were synthesized in a first attempt
by reacting 12, prepared from 2,6-dichloropurine (11),41 with
2-phenylethanol, and 2-cyclohexylethanol, respectively
(Scheme 1). Because the reaction yield of 16 was below
10%, a new reaction pathway was developed for the synth-
esis of this compound. The reaction of 2,6-dichloropurine
(11) with 3,4-dihydropyran under acidic conditions led to the
protected derivative 13 in good yield. Compound 13 was
treated with ammonia to give 14, which was subsequently
reacted with 2-phenylethanol under basic conditions, yield-
ing 15. After deprotection of 15 with trifluoroacetic acid,
16 was obtained in about 60% yield.
The emerging evidence that adenine can act as a signaling
molecule activating G protein-coupled membrane receptors
now necessitates the development of pharmacological tools,
agonists and antagonists, to further elucidate the (patho)-
physiological functions of the receptors and to elicit their
potential as novel drug targets.
N6-Alkylated adenine derivatives 19-24 were obtained by
refluxing 6-chloropurine (18) with the appropriate amines
(Scheme 2).42-44 N6-Aminoalkyladenines 21a and 22a were
prepared by deprotection of the precursors 21 and 22 with
hydrogen chloride (4.0 M in dioxane). Reaction of adenine
(1) with vinyl acetate in DMSO at 60 °C yielded N6-acet-
yladenine (25).45
The only lead structure for the development of AdeR
ligands available so far is adenine itself. Non-nucleosidic
adenine derivatives have previously been found to bind to a
number of different targets depending on their substitution
pattern, including adenosine receptors,21-25 phosphodies-
terases,26 kinases,27-29 and heat shock protein (HSP) 90.30
Certainadeninederivatives havealso been foundtoinduce the
production of interferon31 and to promote the dedifferentia-
tion of committed cells into multipotent progenitor-type
cells.32 Deazaadenine derivatives have been developed as
adenosine receptor antagonists.33-35 However, structure-
activity relationships (SAR) for adenine derivatives appear
to be very different for the AdeR as compared to other targets:
the limited data available so far7,9,15 indicate very steep SAR
with only few and minor modifications tolerated by the AdeR.
The most potent compounds described so far are 2-fluoro-
adenine (Ki = 0.62 μM), 8-thioadenine (Ki = 2.77 μM), and
N6-methyladenine (Ki = 3.64 μM), all of which were >20-
fold weaker than adenine itself (Ki = 0.0292 μM) in
[3H]adenine binding studies performed at rat brain cortical
membranes.9 At the mouse AdeR similar SAR were ob-
served.15 We have now synthesized a large series of deaza-
adenine and adenine derivatives with substituents in various
positions and studied their SAR and functional properties.
8-Substituted adenine derivatives were mostly prepared
according to published procedures with minor modifications
(Scheme 3). 8-Bromoadenine46 (26) was synthesized from
adenine (1) as a key precursor. The 8-oxo derivative 27 was
obtained by acetylation and subsequent hydrolysis of 26.46
Refluxing of 26 with thiourea in butanol led to the 8-thio
analogue 28.47 S-Alkylation of 8-thioadenine (28) was
achieved with alkyl iodides in 1.5 M potassium hydroxide
solution, affording compounds 29-31.47 The phenylsulfanyl
moiety was introduced via direct nucleophilic substitution of
8-bromoadenine (26) with thiophenol in the presence of
potassium carbonate, yielding 8-phenylsulfanyladenine
(32).30 8-Aminoadenine (33) was prepared by heating 26
with sodium azide in DMF at 100 °C, followed by hydro-
genation of the 8-azido function in the presence of 10%
palladium on charcoal.48 Treatment of 26 with benzylamine,
piperidine, pyrrolidine, or piperazine at 150 °C provided the
respective 8-aminoadenine derivatives 34-37.49 For cou-
pling of phenyl, styryl, and phenylethynyl substituents to
position 8 of adenine, Suzuki-Miyaura and Sonogashira
cross-coupling procedures, respectively, were applied. A
mixture of 26, potassium carbonate, tetrakis(triphenyl-
phosphine)palladium(0), and the appropriate boronic acid
in a mixture of water and 1,2-dimethoxyethane was subjected
to microwave irradiation yielding products 38-40.50 Sono-
Results and Discussion
The physiological receptor agonist adenine served as a lead
structure (Chart 1). A variety of modifications was introduced
at positions 2, N6, 7, 8, and/or 9. In addition deazapurine
derivatives were prepared and investigated.
gashira cross-coupling was performed according to the
51
method of Sagi et al., furnishing 8-phenylethynyladenine
ꢀ
(41). 8-Dimethylaminoadenine (42) was obtained as a side
product during the preparation of 8-hydroxyaminoadenine

5976 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Borrmann et al.
Scheme 1. Synthesis of 2-Substituted Adenine Derivatives 16 and 17a
a Reagents and conditions: (a) NH3/MeOH, 100 °C, 16 h; (b) (1) HMDS, (NH4)2SO4, reflux, 2 h; (2) R-OH, NaOH, 140 °C, 3.5 h; (c) 3,4-
dihydropyran, PTSA, acetonitrile, 60 °C, 1 h; (d) NH3, rt, 3 h; (e) phenethyl alcohol, NaOH, rt, 3 h; (f) trifluoroacetic acid, acetonitrile/H2O (1:1), rt, 2 h.
Scheme 2. Synthesis of N6-Substituted Adenine Derivativesa
a Reagents and conditions: (a) R1-NH2, EtOH, reflux, 20 h; (b) HCl (4.0 M in dioxane), rt, 15 min; (c) NH2OH, EtOH, reflux, 6 h; (d) NH2NH2,
30 min; (e) vinyl acetate, DMSO, 60 °C, 24 h.
Scheme 3. Synthesis of 8-Substituted Adenine Derivativesa
a Reagents and conditions: (a) Br2, rt, 5 h. (b) AcOH, NaOAc, reflux, 8 h; NaOCH3, H2O, 50 °C, 2 h; (c) thiourea, n-butanol, reflux, 28 h. (d) 1.5 M
KOH, alkyl iodide, rt, 1; (e) (1) K2CO3, thiophenol, DMF, rt, 1 h; (2) addition of 26 in DMF, 120 °C, 14 h; (f) (1) NaN3, DMF, 100 °C, 7 h; (2) 10%
Pd-C, H2 (4 bar), rt, overnight; (g) amine, 150 °C; (h) K2CO3, boronic acid, Pd(PPh3)4, water/DME, microwave, 30 W, 85 °C, 1 h; (i) Et3N,
Pd(PPh3)2Cl2, CuI, phenyl acetylene, DMF, argon, 50 °C, 2 d.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5977
by reaction of 26 with N,N-dimethylamine, probably formed
by degradation of DMF under the applied reaction condi-
tions (for structure of 42, see Table 1).48
Synthesis of 9-substituted adenine derivatives 60 and 62
was carried out as previously reported by reacting adenine
with the suitable alkyl halide in the presence of potassium
carbonate.57 Under these conditions, together with N-9
isomer 60, the N-7 isomer 63 was obtained as a side-product
(Scheme 6). Compound 61 was synthesized by treating
adenine with sodium hydride followed by reaction with
3-bromoheptane. 8-Bromo derivatives of 9-alkyladenines
(64 and 65) were obtained from 60 and 62 by reaction with
N-bromosuccinimide (NBS).57
9-Ethyladenine derivatives bearing an additional alkynyl
chain in position 2 or 8 (66, 68-70) were prepared as
previously described (see also Supporting Information).58,59
Compounds bearing an alkoxy or amino group in position 2
(74-76) were obtained by reacting 2-chloro-9-ethyladenine
(73)59,60 with the suitable alcohol under basic conditions, or
with phenethylamine, respectively, at high temperature
(Scheme 7). Reaction of 9-ethyl-2-phenethylaminoadenine
(76) with NBS yielded the 8-bromo derivative 77 as previously
described.61 Compound 72 was obtained by reaction of
2,6-dichloro-9-ethyl-9H-purine (71)60 with cyclopentylamine.
Ethoxy derivative 79 was prepared by reaction of 6-chloro-9-
9-Substituted adenine derivatives 46, 49-51, and 55 were
prepared by alkylation of adenine with alkyl bromides or
alkyl mesylates. The mesylates were synthesized from com-
mercially available hydroxyethylpyridines 43, 47, and 48 by
treatment with mesyl chloride and triethylamine in dichloro-
methane. Monitoring by thin-layer chromatography indi-
cated a nearly quantitative reaction, but extraction of the
mesylate 44 with dichloromethane from a saturated solution
of sodium carbonate provided a mixture of the desired
product 44 and the corresponding vinylpyridine (45)
(Scheme 4).
To circumvent such an elimination reaction, a mixture of
adenine, potassium carbonate, and crown ether (18-crown-
6) in dimethyl formamide was directly added to the mesylates
without prior workup. The temperature was raised to 100 °C,
dichloromethane was allowed to evaporate under the hood,
and the resulting solution was stirred for 4 h, affording the
9-substituted adenine derivatives 49-51 (Scheme 5). 9-
Phenethyladenine (46) was prepared by alkylation of 1 with
phenethyl bromide.52 For the preparation of the piperazine
analogue 55, protection of the secondary amino function in
hydroxyethylpiperazine (52) was required in order to selectively
mesylate the alcohol function.53 After mesylation and coupling
to adenine as described above, the protecting group was cleaved
off with hydrogen chloride (4.0 M in dioxane). 9-Substituted
adenine derivatives 56-59 were prepared according to pub-
lished procedures (for structures, see Table 2).54-56
Scheme 6. Synthesis of 7- and 9-Alkyladenines and Their 8-
Bromo Derivativesa
Scheme 4. Synthesis of the Mesylate 44 and Elimination of
Methylsulfonic Acida
a Reagents and conditions: (a) R-Br, K2CO3, DMF, rt, 16 h; (b) (1)
sodium hydride, DMF, 35 °C, 35 min; (2) 3-bromoheptane, rt, 21 h;
(c) NBS, DMF, rt, 12 h.
a Reagents and conditions: (a) Et3N, MsCl, CH2Cl2, 4 °C, 2 h;
(b) Na2CO3.
Scheme 5. Synthesis of 9-Substituted Adenine Derivativesa
a Reagents and conditions: (a) Phenethyl bromide, K2CO3, dimethyl acetamide, 110 °C 1 h; (b) (1) Et3N, MsCl, CH2Cl2, 4 °C, 2 h; (2) adenine, K2CO3,
18-crown-6, DMF, 100 °C, 4 h; (c) (1) Boc2O, CH2Cl2, 4 °C, 2 h; (2) rt, overnight; (d) HCl (4.0 M in dioxane), rt, 15 min.

5978 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Borrmann et al.
Scheme 7. Synthesis of 9-Ethyladenine Derivatives 72-77 and 9-Cyclohexyladenine Derivatives 79-80a
a Reagents and conditions: (a) cyclopentylamine, acetonitrile, rt, 15 h; (b) NH3, rt, 16 h; (c) R-OH, NaOH, 95 °C, 1-6 h; or R-NH2, 130 °C, 24 h;
(d) NBS, DMF, rt, 35 min; (e) EtOH, Et3N, 120 °C, 40 h; (f) NH3, rt, 24 h.
Scheme 8. Synthesis of 9-Ethyladenine derivatives 88 and 89a
Table 1. Affinities of Synthesized Deazapurines at Adenine Receptors
of Rat Brain Cortical Membrane Preparations
a Reagents and conditions: (a) ethylamine, 100 °C, 20 h; or cyclohex-
ylamine, 140 °C, 48 h.
Scheme 9. Synthesis of the 8-Phenyl-Substituted Adenine
Derivative 92a
a Reagents and conditions: (a) triethylorthobenzoate, methanesulfo-
nic acid, CH2Cl2, rt, 16 h; (b) butyl amine, rt, 3 h.
cyclohexyl-9H-purine (78)57 with ethanol under basic conditions.
Reaction of 78 with ammonia furnished the amino deriva-
tive 80.57
Compounds 82-84 and 86 were prepared from the
corresponding 6-iodo-9-ethyladenine derivatives according
to procedures described in literature (see also Supporting
Information).59,62 Reaction of 8-bromo-9-ethyladenine
(87)59 with the suitable amines at high temperature furni-
shed the 8-amino-9-ethyladenine derivatives 88 and 89
(Scheme 8).
Compound 92 bearing a phenyl group in the 8-position was
synthesized starting from 5-amino-4,6-dichloropyrimidine.
Reaction of 9057 with triethylorthobenzoate under acidic
conditions yielded 6-chloro-9-ethyl-8-phenyl-purine (91).63
Reaction of 91 with butylamine yielded the desired product
92 (Scheme 9). Differently substituted nucleosides (94, 95, 97,
and 98) were obtained according to published procedures (see
also Supporting Information).64-68
a Percent inhibition ( SEM of radioligand binding at 100 μM.
The structures of all final products were confirmed by 1H
NMR spectra and in some cases additionally by 13C NMR
and mass spectra (electrospray ionization). Purity was deter-
mined by elemental analysis and by RP-HPLC coupled to UV
detection at 254 nm.
Biological Evaluation. The affinities of the synthesized
compounds for the rat Ade1R were determined in radi-
oligand binding studies at rat brain cortical membrane

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5979
Table 2. Affinities of Synthesized Monosubstituted Adenine Derivatives at Adenine Receptors of Rat Brain Cortical Membrane Preparations
a Percent inhibition ( SEM of radioligand binding at 100 μM. b Percent inhibition ( SEM of radioligand binding at 30 μM. c Percent inhibition (
SEM of radioligand binding at 250 μM.
preparations using [3H]adenine (10 nM) as a radioligand.7,9,69 It
has clearly been shown that [3H]adenine is a suitable radioligand
for the labeling of AdeR if bacterial contaminations (which can
also bind [3H]adenine) in the applied buffer solutions are
avoided.69 Selected compounds were further investigated in
radioligand binding studies at membrane preparations of human
embryonic kidney (HEK) 293 cells in order to assess their affinity
for human adenine binding sites. Functional studies were per-
formed for selected compounds at 1321N1 astrocytoma cells
stably transfected with the rat Ade1R, which is coupled to
inhibition of adenylate cyclase,7,9 by investigating their potential
to inhibit isoproterenol-induced cAMP accumulation.15
binding studies at rat cortical membranes are collected in
Table 1 (deazapurines), Table 2 (monosubstituted adenine
derivatives), and Table 3 (di-, tri-, and tetra-substituted purine
derivatives).
All tested deazapurines were much less active at the
AdeR than the physiological ligand adenine (Ki value of
0.0292 μM). The 7- and 9-deazapurines 7, 8, and 10 showed
no significant affinity for the AdeR at concentrations up to
100 μM, whereas the 1- and 3-deazapurines 3 and 5 exhibited
AdeR affinities in the low micromolar range (>200-fold
decrease in affinity). This result indicated that the presence of
a hydrogen bond acceptor at position 7 is essential for
AdeR affinity. Interestingly, a similar effect was previously
observed for 7-deazaadenosine at adenosine receptors
Structure-Activity Relationships at Rat Adenine Receptors.
The Ki values of all compounds determined in radioligand

5980 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Borrmann et al.
Table 3. Affinities of Synthesized Di-, Tri-, and Tetrasubstituted Purine Derivatives at AdeRs of Rat Brain Cortical Membrane Preparations
a Percent inhibition ( SEM of radioligand binding at 100 μM.
(AdoRs), the 7-deazaadenosine being only weakly active or
inactive at A1, A2A, and A3 AdoR. 3-Deazaadenosine
had exhibited somewhat higher affinity for AdoR in the
micromolar range.70 The best tolerated deazaadenosine
derivatives in these previous studies had been the 1-deaza
compounds, which were similarly potent as the corresponding
adenosine derivatives.70 In contrast, the N1, as well as the N3,
are quite important for high AdeR affinity and appear to
strongly contribute to receptor binding probably as hydrogen
bond acceptors.
Binding studies of monosubstituted adenine derivatives
showed that modifications at the positions N6, 8, and 9 were

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5981
Figure 1. (A) Nonspecific and specific binding of 10 nM [3H]adenine to HEK293 cell membrane preparations (100 μg protein, n = 3; error bars
represent SEM values). (B) Homologous competition experiments with adenine vs [3H]adenine (10 nM) at membrane preparations of rat brain
cortical membrane and HEK293 cell membrane preparations, respectively. Data points are means ( SEM of three experiments performed in
triplicates.
best tolerated by the rat AdeR. The 2-substituted derivatives
16 and 17 as well as 7-benzyladenine (63) showed no measur-
able affinity for the AdeR, whereas several N6-, 8-, and
9-substituted adenine derivatives exhibited Ki values in the
low micromolar or even nanomolar range. At position N6
small, polar substituents as in compounds 23-25 (Ki values
of 0.500, 1.38, and 2.85 μM, respectively) led to higher AdeR
affinities than alkyl residues as in compounds 19-22a.
Adenine derivatives bearing aminoalkyl and N-t-boc-ami-
noalkyl substituents at position N6 (21, 21a, 22, and 22a)
exhibited no measurable AdeR affinity, whereas ethyl and
hydroxyethyl substitution at that position (19 and 20) was
still somewhat tolerated (Ki values of 16.4 and 23.9 μM,
respectively). Interestingly, the corresponding N6-acetyl-
substituted adenine 25 showed a higher affinity (Ki =
2.85 μM) than the corresponding alkyl derivative, consistent
with previous observations, that benzoyl substitution at
position N6 was better tolerated than benzyl substitution.9
These results indicated that N6-acylation is more favorable in
terms of increased AdeR affinity than N6-alkylation.
At position 8, particularly basic substituents as in 8-ami-
noadenine (33), 8-benzylaminoadenine (34), 8-(1-piperidinyl)-
adenine (35), 8-(1-piperazinyl)adenine (37), and 8-dimethyl-
aminoadenine (42) led to higher AdeR affinities than bulky,
lipophilic residues. Phenyl, 3-trifluoromethoxyphenyl, and
phenylethynyl substitution (38, 39, 41) resulted in a total loss
of affinity, whereas the more flexible 8-benzylaminoadenine
(34) exhibited a higher affinity in the low micromolar range
(Ki = 6.67 μM). The introduction of 8-alkyl- and 8-arylsulfa-
nyl residues (29-32) diminished the AdeR affinity when
compared to 8-thioadenine (Ki = 2.77 μM).9
9-Phenethyladenine (46) exhibited low affinity (Ki =
49.1 μM), whereas the introduction of one or two basic
nitrogen atoms to the 9-substituent could result in positive
or negative effects on the affinity, depending on the position
of the basic function. Both the piperazine derivative 55 and
the 4-pyridyl derivative 51 showed no measurable AdeR
affinity. 3-Pyridylethyl-substituted adenine 50 showed a
slight increase in affinity, while compound 49, featuring a
nitrogen atom in the ortho-position, had a more than 10-fold
higher affinity than the corresponding pyridyl derivatives
50 and 51 (Ki = 4.51 μM vs Ki values of 75.1 μM (50)
and >250 μM (51)). The morpholinyl derivative 58 also
showed good AdeR affinity in the low micromolar range.
The Ki values of 56, 57, 59, and 99 indicated that the
introduction of polar functions at position 9 positively
affected the AdeR affinity.
However, in general, the steep structure-activity relation-
ships that have been previously reported for the AdeR9
applied also for most of the monosubstituted adenine deri-
vatives described in the present study.
Of all tested di-, tri-, and tetra-substituted purine deriva-
tives, only compound 65 exhibited measurable AdeR affi-
nity. The introduction of a polar substituent at position 9 in
compound 65 was slightly more favorable when compared to
the benzyl and ethyl analogues 64 and 87, a finding that is
consistent with the observed affinities for 56, 57, 59, and 99,
which also bear polar substituents at position 9. The adenine
nucleosides and nucleoside analogues 94, 95, 97, 98, 100, and
101 showed no measurable AdeR affinity, which was in
agreement with results previously obtained for adenine
nucleosides by Gorzalka et al.9 All 9-ethyl-substituted ade-
nine derivatives showed no measurable AdeR affinity. The
fact that no additional substituent at positions 2, N6, or 8
could overcome the negative effect of the 9-ethyl substituent
on AdeR affinity demonstrated that the observed steep
structure-activity relationships applied particulary to posi-
tion 9.
Structure-Activity Relationships in Binding Studies at
Human Embryonic Kidney Cell Membranes. As already
pointed out, there is evidence showing that adenine may
play a role in human and pig renal function.13,14 Consistent
with these observations, we detected a specific binding site
for [3H]adenine in HEK293 cell membrane preparations
(Figure 1A). Homologous competition experiments with
adenine versus [3H]adenine (10 nM) at HEK293 cell mem-
branes yielded a Ki value of 47.1 nM, which is very similar to
the Ki value of 29.2 nM obtained for adenine at rat brain
cortical membrane preparations (Figure 1B).
We subsequently performed competition experiments to
investigate the structure-activity relationships of selected
adenine derivatives at the high-affinity adenine binding site
in HEK293 cell membranes. Experimental conditions for the
HEK membranes were the same as for the rat receptor
preparations. The determined Ki values are collected in
Table 4 and affinities are compared with those determined
for the rAde1R.
Like adenine, the deazaadenines 3 and 5 showed roughly
the same Ki values in both species (3: 7.89 μM (human),
10.6 μM (rat); 5: 6.68 μM (human), 6.81 μM (rat)). Most of
the investigated N6-substituted adenine derivatives (19, 20,
23, 24) exhibited higher affinity (4-19-fold) for the rat AdeR
than for the human adenine binding site. An exception was
N6-acetyladenine (25), which was 5.5-fold more potent

5982 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Borrmann et al.
Table 4. Affinities of Selected Adenine Derivatives at HEK293 Cell Membrane Preparations and Comparison with the Data Obtained for the Rat AdeR
Ki ( SEM [μM]
at HEK293
membranes vs
[3H]adenine
(n = 3)
ratio
Ki (HEK293)/
Ki (rat cortex)
compd
adenine (1)
1-deazaadenine (3)
0.0471 ( 0.0037
7.89 ( 1.86
1.6
0.7
3-deazaadenine (5)
N6-ethyladenine (19)
6.68 ( 1.30
1.0
ca. 100 (59 ( 2)a
ca. 100 (58 ( 1)a
9.53 ( 1.79
ca. 6
ca. 4
19
N6-(2-hydroxy)ethyladenine (20)
N6-hydroxyadenine (23)
N6-aminoadenine (24)
17.5 ( 1.7
0.515 ( 0.066
110 ( 4
0.0341 ( 0.0088
32.9 ( 2.9
13
N6-acetyladenine (25)
8-hydroxyadenine (27)
8-aminoadenine (33)
0.18
4.1
0.005
4.9
8.1
8-benzylaminoadenine (34)
8-(1-piperidinyl)adenine (35)
8-(1-pyrrolidinyl)adenine (36)
8-(1-piperazinyl)adenine (37)
8-styryladenine (40)
31.7 ( 4.8
>100 (18 ( 3)a
27.4 ( 4.6
>2.4
8.4
12.2 ( 1.8
34.1 ( 3.2
0.67
14.8
ca. 2.0
>22
ca. 1.3
8-dimethylaminoadenine (42)
9-phenethyladenine (46)
ca. 100 (57 ( 2)a
ca. 100 (58 ( 3)a
>100 (41 ( 2)a
>100 (44 ( 4)a
>100 (0 ( 4)a
6.23 ( 1.81
9-(2-(2-pyridyl)ethyl)adenine (49)
9-(2-(3-pyridyl)ethyl)adenine (50)
9-(2-(4-pyridyl)ethyl)adenine (51)
9-(2-(1-piperazinyl)ethyl)adenine (55)
9-(2-amino-2-carboxyethyl)adenine (56)
9-(2-carboxyethyl)adenine (57)
9-(2-morpholinoethyl)adenine (58)
20-deoxyadenosine (99)
0.58
0.90
1.8
18.5 ( 5.6
5.49 ( 1.42
>100 (29 ( 4)a
>9
a Percent inhibition ( SEM of radioligand binding at 100 μM.
at HEK293 membranes (Ki = 0.515 μM) as compared to the
rat receptor (Ki = 2.85 μM). At position 8, most of the
introduced substituents were less tolerated by the human
than by the rat binding site, while 8-styryladenine (40)
showed nearly the same affinity in both species. Again, there
was one striking exception: 8-aminoadenine (33) exhibited a
Ki value of 0.0341 μM at the human binding site and was thus
even more potent than adenine itself (Ki = 0.0471 μM). It
was 190-fold more potent at the human as compared to the
rat binding site. The 9-substituted adenine derivatives bear-
ing polar substituents (56, 57, and 58) showed almost iden-
tical affinities for the human and the rat binding sites. All
other 9-substituted compounds were weaker at human bind-
ing sites than at the rat adenine receptor. These results show
that there are many similarities between the high-affinity
binding sites in rat brain and in human embryonic kidney
cells, but there are also significant differences as would be
expected for different receptor subtypes.
Functional Studies. To investigate whether the potent
rAde1R ligands identified in this study were agonists or
antagonists, we performed functional studies with selected
compounds. Thus, 1321N1 astrocytoma cells were stably
transfected with the sequence for the rAde1R using a retro-
viral expression system.72 This cell line has previously been
proven to be suitable for the functional testing of the mouse
AdeR subtype mAde2.15 Adenine at 1 μM concentration
inhibited isoproterenol-induced cAMP formation in a dose-
dependent manner with a maximal inhibition of about
35-40% and an IC50 value of 8.29 ( 2.69 nM (Figure 2).
Selected adenine derivatives were tested for their effects
to inhibit isoproterenol-induced cAMP accumulation. To
obtain a full AdeR-activating effect, high concentrations of
test compounds (ca. 100 ꢀ Ki value; 50, 250, or 500 μM) were
applied. Compound 35 (8-(1-piperidinyl)adenine) was tested
at a lower concentration (30 μM, ca. 8-fold Ki, see Figure 2A)
because at 250 μM concentration it had led to a complete
inhibition of cAMP production in a preliminary experiment
(not shown), likely due to an AdeR-independent effect.
All compounds investigated showed agonistic effects at
rAde1R expressed in astrocytoma cells as shown in
Figure 2A. 8-(1-Piperidinyl)adenine (35), 8-aminoadenine
(33), and 9-(2-morpholinoethyl)adenine (58) showed about
the same efficacy as adenine. 8-Dimethylaminoadenine (42)
was even more efficacious than the physiological agonist,
showing 137% of the maximal effect of adenine. In contrast,
N6-hydroxyadenine (23, 51% efficacy), N6-acetyladenine
(25, 77% efficacy), and 8-(1-piperazinyl)adenine (37, 52%
efficacy) were partial agonists in the applied test system.
Compounds 25 and 37 were additionally tested for antago-
nistic effects. Concentration-response curves for adenine
were obtained in the absence and in the presence of 10 μM
(ca. 3-fold Ki value) of 25, or 37, respectively. Curves for
adenine in the presence of 25 in comparison with adenine
alone are shown in Figure 2B. Both compounds, 25 and 37,
had no statistically significant effects on the IC50 value or the
efficacy of adenine, indicating that they did not behave as
antagonists in this test system. Taken together, the results
from functional studies showed that minor structural mod-
ifications can have a significant impact on functional proper-
ties of the adenine derivatives.
Adenine Transporters. Adenine, which may act as an
extracellular neuromodulator by activation of G protein-
coupled adenine receptors, can be removed from its site of
action by cellular uptake via adenine transporters.17-20 We
therefore investigated whether 1321N1 astrocytoma cells
have an active uptake mechanism for adenine. Furthermore,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5983
Figure 3. Uptake of [3H]adenine (30 nM) by 1321N1 astrocytoma
cells stably expressing the rAde1R. Cells were incubated in HEPES-
buffered Ringer solution in the absence or presence of adenine or an
adenine derivative for 1 min at 37 °C, in one case at 0 °C. Data
represent means of two independent experiments performed in
triplicate.
Figure 2. (A) Inhibition of isoproterenol-stimulated cAMP accu-
mulation by adenine and selected adenine derivatives in 1321N1
astrocytoma cells stably expressing the rAde1R. Cellular cAMP
production was increased by preincubation with 3 nM isoproterenol
for 10 min at 37 °C; data are given as percentage of the mean
increases in cellular cAMP levels in the presence of isoproterenol
alone (% of control, n = 3). Adenine and compound 35 were tested
at 1 and 30 μM, respectively. Compounds 25, 37, 42, and 58 were
tested at 250 μM (100-fold Ki value). Compounds 23 and 33 were
tested at 50 and 500 μM, respectively. *p = 0.0151, ** p < 0.01,
*** p < 0.0001 indicates statistically significant differences from
control (isoproterenol); # p < 0.01, ## p = 0.0032 indicates
statistically significant differences between maximal effect of
adenine and effect of test compound at high concentration.
(B) Concentration-dependent inhibition of isoproterenol-stimu-
lated intracellular cAMP accumulation by adenine in the absence
and in the presence of compound 25 (10 μM). An IC50 value of
8.29 ( 2.69 nM was determined for adenine (n = 5). The presence of
25 (10 μM, 3-fold Ki) did not show any significant effect on the IC50
value or the efficacy of adenine (n = 3).
Conclusions
In summary, a large series of 2-, N6-, 7-, 8-, and 9-sub-
stituted adenine derivatives as well as deazapurines was
synthesized and SAR were analyzed in binding studies at
AdeR, a new purinergic receptor family. Steep SAR were
generally observed. Small, basic, or polar substituents were
best tolerated by the receptor, whereas more bulky and
lipophilic substituents, as well as alkyl substituents in the
9-position, were not well tolerated. Basic amino substituents
at position 8, and acylation at position N6 appeared to
represent favorable modifications of the adenine scaffold to
obtain potent AdeR ligands. SAR at the human high affinity
adenine binding site in HEK293 cells were similar but not
identical; N6-acetyladenine and in particular 8-aminoadenine
were much more potent at the human as compared to the rat
binding site. Functional evaluation of selected adenine deri-
vatives in cAMP assays at 1321N1 astrocytoma cells stably
expressing the rAde1R revealed that all compounds investi-
gated were agonists, however differing in efficacy. The new
AdeR ligands may serve as new lead structures. Although
most of them exhibited lower affinity than the physiological
agonist adenine, they may show a longer in vivo half-life than
adenine as the endogenous neurotransmitters and modulators
are generally removed fast from their sites of action. We
discovered that astrocytoma 1321N1 cells exhibit active trans-
port of adenine, and adenine derivatives that were found
to activate adenine receptors can also interfere with the
adenine transporter; SAR, however, are different. Thus the
best compounds of the present series may be useful for
elucidatingthe roles and functionsofthe new adeninereceptor
family.
we were interested in studying whether selected adenine
receptor ligands interfere with adenine uptake. This infor-
mation would be important for ligands that are used as
pharmacological tools in living systems. Uptake studies
revealed that 1321N1 astrocytoma, null cells as well as cells
expressing the rat adenine receptor, exhibited saturable,
sodium-independent transport of [3H]adenine. Km values
of ca. 30-60 μM and Vmax values of ca. 600-700 fmol/mg
protein were determined in homologous competition studies,
both in transfected and nontransfected cells (data not
shown). Uptake of 30 nM [3H]adenine was completely
inhibited by 1 mM unlabeled adenine and nearly abolished
at low temperature (0 °C) (Figure 3).
Selected adenine derivatives that had also been tested in
cAMP assays were investigated at a high concentration of
100 μM for their potential to inhibit [3H]adenine uptake. All
adenine derivatives inhibited [3H]adenine uptake at least to a
certain degree. N6-Acetyladenine (25), N6-hydroxyadenine
(23), 8-aminoadenine (33), and 9-(2-morpholinoethyl)-
adenine (58) were most potent, while compounds with a
larger 8-substituent (35, 37, 42) showed lower inhibitory
potency (Figure 3). SAR were clearly different from those
at the rat adenine receptor.
Experimental Section
All reagents were obtained from various suppliers (Acros,
Aldrich, Fluka, Merck, Sigma, and Novabiochem) and used
without further purification. Solvents were used without
additional purification or drying unless otherwise noted.

5984 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Borrmann et al.
Reactions were monitored by thin-layer chromatography (TLC)
using aluminum sheets coated with silica gel 60 F254 (Merck).
Compounds were visualized under UV light (254 nm). Column
chromatography was carried out with silica gel 0.060-
0.200 mm (pore diameter ca. 6 nm). Preparative HPLC was
performed on a Knauer HPLC system using a Wellchrome
K-1800 pump, a WellChrome K-2600 spectrophotometer, and
a Eurospher 100 C18 column (250 mm ꢀ 20 mm, particle size
10 μm). A gradient of methanol in water was used as indicated
below with a flow rate of 20 mL/min. The purity of all tested
compounds was determined by elemental analysis and for some
compounds additionally by HPLC-UV obtained on an LC-MS
instrument (Applied Biosystems API 2000 LC-MS/MS, HPLC
Agilent 1100) using the following procedure: Compounds were
dissolved at a concentration of 0.5 mg/mL in methanol. Then,
10 μL of the sample were injected into a Phenomenex Luna C18
HPLC column (50 mm ꢀ 2.00 mm, particle size 3 μm) and were
chromatographed using a gradient of water/methanol (contain-
ing 2 mM ammonium acetate) from 90:10 to 0:100 for 30 min at a
flow rate of 250 μL/min, starting the gradient after 10 min. UV
absorption was detected from 200 to 950 nm using a diode array
detector. Purity of the compounds was determined at 254 nm and
proved to be g95%. Elemental microanalyses were performed
on a VarioEL apparatus, or a Fisons Instruments model EA 1108
CHNS-O model analyzer, respectively, and were generally
within (0.4% of the calculated values. Mass spectra were
recorded on an API 2000 mass spectrometer (electron spray ion
source, Applied Biosystems, Darmstadt, Germany) coupled with
an Agilent 1100 HPLC system. 1H- and 13C NMR spectra were
recorded on a Bruker Avance 500 MHz NMR spectrometer, or
on a Mercury 400 MHz spectrometer. CDCl3, DMSO-d6,
CD3OD, or D2O were used as solvents as indicated below.
NMR spectra were recorded at rt unless otherwise noted. Shifts
are given in parts per million (ppm) relative to the remaining
protons of the deuterated solvent used as internal standard.
Coupling constants J are given in hertz and spin multiplicities
are given as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br (broad). Elemental microanalyses were performed
on a VarioEL apparatus. Melting points were determined on a
General Procedure for the Synthesis of Compounds 35, 36, and
37. The appropriate amine (3 mL or 10 equiv) was added to 8-
bromoadenine (200 mg; 0.93 mmol) in a sealed vial and the
reaction mixture was heated at 150 °C. Volatiles were removed
under vacuum, the residue was coevaporated several times with
EtOH. Reaction times, purification methods, and yields are
given with the experimental data of the products.
General Suzuki-Miyaura Cross-Coupling Procedure to
Synthesize Compounds 38-40. The appropriate boronic acid
derivative (1.5 mmol), dissolved in 4 mL of DME, and Pd-
(PPh3)4 (0.1 mmol) were added to a solution of 8-bromoadenine
(26, 1.0 mmol) and K2CO3 (2.2 mmol) in 2 mL of water. The
mixture was stirred under microwave irradiation (30 W) at 85 °C
for 1 h. After filtration and washing with hot methanol, the
filtrate was evaporated to dryness and the residue was purified
by column chromatography eluting with 5% methanol in
dichloromethane.
General Procedure for the Synthesis of 9-Alkylated Adenine
Derivatives 49-51 and 55. A solution of the appropriate alcohol
(3.2 mmol) and triethylamine (5.0 mmol) in 10 mL of dry
dichloromethane was cooled to 4 °C. A solution of mesyl
chloride (3.8 mmol) in 5 mL of dry dichloromethane was added
dropwise and the mixture was stirred at 4 °C for 2 h. Adenine
(3.7 mmol), K2CO3 (4.8 mmol), and a catalytic amount of
18-crown-6, dissolved in 30 mL of dry DMF, were added, and
the mixture was stirred at 100 °C for 4 h while dichloromethane
was allowed to evaporate under the hood. DMF was evaporated
under reduced pressure, and the residue was purified by column
chromatography eluting with dichloromethane/methanol/
NH4OH (10:1:0.1).
2,6-Dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (13).
PTSA (2 mg) was added to 2,6-dichloro-9H-purine (100 mg;
0.53 mmol) in dry acetonitrile (2.0 mL). 3,4-Dihydropyran
(70 μL; 0.77 mmol) was added to the reaction mixture under
anhydrous conditions at 60 °C. After 1 h of stirring, aqueous
ammonia was added until a basic pH was reached and the
product was extracted from water with EtOAc. The organic
layers were collected and dried over Na2SO4, the solvent
was evaporated, and the residue was purified by column chro-
matography eluting with chloroform/cyclohexane/methanol
(60:38:2). Compound 13 was obtained as a pure product after
crystallization from methanol/Et2O in 92% yield; mp 113-
115 °C. 1H NMR (400 MHz, DMSO-d6) δ 1.61 (m, 2H,
H-tetrahydropyranyl), 1.75 (m, 1H, H-tetrahydropyranyl),
1.99 (m, 2H, H-tetrahydropyranyl), 2.17 (m, 1H, H-tetra-
hydropyranyl), 3.77 (m, 1H, H-CHO), 4.02 (m, 1H, H-CHO),
5.76 (dd, 1H, J = 2.24 and 10.66 Hz, CH-N), 8.98 (s, 1H, H-8).
2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (14).
Compound 13 (700 mg; 2.73 mmol) was added to 30 mL of liquid
ammonia in a steel vessel. The reaction was kept at rt for 3 h.
Volatiles were removed under vacuum, and compound 14 was
obtained by crystallization from methanol in 93% yield; mp
€
Buchi B-545 melting point apparatus and were uncorrected.
Lyophilization was performed with a Christ Alpha 1-4 LSC
freeze-dryer. Hydration was performed with a Hogen GC hydro-
gen generator from Proton Energy Systems. For microwave
reactions, a CEM Focused Microwave Synthesis type Discover
apparatus was used.
General Procedure for the Synthesis of N6-Alkylated Adenines
19-22. A mixture of 6-chloropurine (18) (for 19 and 20:
10 mmol; for 21 and 22: 2.6 mmol) and the appropriate amine
(for 19: 10 mL of ethylamine (70% in water); for 20: 10 mL of
ethanolamine; for 21 and 22: 6.0 mmol N-t-boc protected amine)
in 20 mL of ethanol was refluxed for 6 h. The solvent was
evaporated under reduced pressure, and the product was pur-
ified by column chromatography on silica gel using a gradient
from 10% methanol in dichloromethane to 20% methanol in
dichloromethane. A sample of each product was additionally
purified by RP-HPLC using a gradient from 10% methanol in
water to 100% methanol.
1
189-191 °C. H NMR (400 MHz, DMSO-d6) δ 1.58 (m, 2H,
H-tetrahydropyranyl), 1.71 (m, 1H, H-tetrahydropyranyl), 1.95
(m, 2H, H-tetrahydropyranyl), 2.21 (m, 1H, H-tetrahydropyranyl),
3.70 (m, 1H, H-CHO), 4.01 (m, 1H, H-CHO), 5.56 (dd, 1H, J =
2.16 and 10.86 Hz, CH-N), 7.84 (s (br), 2H, NH2), 8.38 (s, 1H, H-8).
2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (15).
Phenethyl alcohol (4.0 mL) and NaOH (400 mg) were added to
14 (385 mg; 1.62 mmol), and the reaction mixture was stirred
under anhydrous conditions for 3 h. The crude product was
purified by column chromatography using a flash silica gel
column and eluted with chloroform followed by chloroform/
cyclohexane/methanol (90:9.5:0.5). Compound 15 was obtained
as pure product after crystallization from methanol-Et2O in
77% yield; mp 133-135 °C. 1H NMR (400 MHz, DMSO-d6) δ
1.62 (m, 3H, H-tetrahydropyranyl), 1.94 (m, 2H, H-tetra-
hydropyranyl), 2.20 (m, 1H, H-tetrahydropyranyl), 3.02 (t,
3H, J = 7.0 Hz, CH2-Ph), 3.65 (m, 1H, H-CHO), 4.01 (m,
1H, H-CHO), 4.43 (t, 3H, J = 7.0 Hz, CH2-O), 5.50 (dd, 1H,
General Procedure for the Cleavage of the N-t-Boc Protection
Group. N-t-Boc-protected amine (0.10 mmol) was suspended in
2 mL of HCl solution (4.0 M in dioxane), and the mixture was
stirred at rt for 15 min. Diethyl ether (10 mL) was added, and the
resulting crystals were filtered off and washed with diethyl ether.
General Procedure for the Synthesis of 8-Alkylsulfanylade-
nines 29-31. A mixture of 6-amino-7,9-dihydropurin-8-thione
(28) (0.60 mmol), 1.5 M aq KOH solution (5 mL), and alkyl
iodide (0.70 mmol) was stirred at rt for 1 h in a closed flask. The
mixture was neutralized with acetic acid. The precipitated solid
was filtered off, washed with water, and purified by column
chromatography eluting with 10% methanol in dichloro-
methane.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5985
J = 2.1 and 10.9 Hz, CH-N), 7.33 (m, 7H, NH2 and H-Ph), 8.13
(s, 1H, H-8).
132.17 (C(phenyl)), 143.60 (C-8), 152.69 (C-2), 154.74 (C-6),
1 signal (C-4) not detectable. LC/ESI-MS: negative mode m/z
242 ([M - H]-), positive mode m/z 244 ([M þ H]þ). Purity
2-Phenethoxy-9H-purin-6-amine (16). Trifluoroacetic acid
(0.30 mL) was added to 15 (150 mg; 0.44 mmol) in a solution
of acetonitrile/H2O 1:1 (10 mL). The mixture was stirred at rt for
2 h. NaHCO3 was added to the reaction mixture until a neutral
pH was reached. The solvents were removed under reduced
pressure and were coevaporated three times with EtOH. The
residue was purified by column chromatography eluting with
chloroform/acetonitrile/methanol (86:7:7). Compound 16 was
(HPLC-UV 254 nm) 98.0%. Anal. (C11H9N5S H2O) C, H, N.
3
8-(Piperidin-1-yl)-9H-purin-6-amine (35). Compound 35 was
prepared from 26 according to the general procedure. Reaction
time: 16 h. 35 was obtained after crystallization from EtOH in
65% yield; mp > 250 °C. 1H NMR (400 MHz, DMSO-d6) δ 1.57
(s, 6H, H-piperidinyl), 3.45 (s, 4H, H-piperidinyl), 6.22 (s (br), 2H,
NH2), 7.90 (s, 1H, H-2), 10.75/11.77 (s, 1H, N9-H). LC/ESI-MS:
positive mode m/z 219 [M þ H]þ, 459 [2M þ Na]þ, negative mode
m/z 217 [M - H]-, 253 [M þ Cl]-. Anal. (C10H14N6) C, H, N.
8-(Pyrrolidin-1-yl)-9H-purin-6-amine (36). Compound 36 was
prepared from 26 according to the general procedure. Reaction
time: 16 h. After evaporation of volatiles, the residue was
purified by column chromatography eluting with chloroform/
methanol (98:2 to 96:4). 36 was obtained in 71% yield after
crystallization from acetonitrile; mp 228-232 °C (dec.). 1H
NMR (400 MHz, DMSO-d6) δ 1.96 (m, 4H, H-pyrrolidinyl),
3.45 (m, 4H, H-pyrrolidinyl), 6.73 (s (br), 2H, NH2), 8.06 (s, 1H,
H-2), 11.62 (s (br), 1H, N9-H). LC/ESI-MS: positive mode m/z
205 [M þ H]þ, negative mode m/z 203 [M - H]-, 283 [M þ Br]-.
Anal. (C9H12N6) C, H, N.
1
obtained as a pure product in 92% yield; mp > 250 °C. H
NMR (400 MHz, DMSO-d6) δ 3.00 (t, 3H, J = 6.6 Hz, CH2-
Ph), 4.38 (t, 3H, J = 6.6 Hz, CH2-O), 7.13 (s (br), 2H, NH2), 7.31
(m, 5H, H-Ph), 7.90 (s, 1H, H-8), 12.56 (s (br), 1H, NH). Anal.
(C13H13N5O) C, H, N.
2-(2-Cyclohexylethoxy)-9H-purin-6-amine (17). (NH4)2SO4
(10 mg) was added to 12 (0.515 mg; 3.04 mmol) in HMDS
(22 mL). The mixture was heated for 2 h under reflux, and
HMDS was removed by coevaporation with toluene. 2-Cyclo-
hexylethanol (5.0 mL) and NaOH (250 mg) were added to the
residue, and the reaction mixture was heated at 140 °C for 3.5 h.
Volatiles were removed under vacuum, and the residue was
purified by column chromatography eluting with chloroform/
methanol (90:10). Compound 17 was obtained in 42% yield; mp
> 250 °C. 1H NMR (400 MHz, DMSO-d6) δ 0.91 (m, 2H,
H-cyclohexyl), 1.10 (m, 3H, H-cyclohexyl), 1.40 (m, 1H,
H-cyclohexyl), 1.54 (m, 2H, H-cyclohexyl), 1.67 (m, 5H,
H-cyclohexyl), 4.18 (t, 2H, J = 6.6 Hz, CH2-O), 7.05 (s (br),
2H, NH2), 7.84 (s, 1H, H-8), 12.55 (s (br), 1H, NH). Anal.
(C13H19N5O) C, H, N.
8-(Piperazin-1-yl)-9H-purin-6-amine (37). Compound 37 was
prepared from 26 according to the general procedure. Reaction
time: 4 h. The residue obtained after evaporation of volatiles was
chromatographed over a reversed phase silica gel column eluting
with 2-propanol/NH4OH/H2O (85:10:5). 37 was obtained in 63%
1
yield; mp > 250 °C. H NMR (400 MHz, DMSO-d6) δ 3.21 (s
(br), 4H, H-piperazinyl), 3.70 (s (br), 4H, H-piperazinyl), 6.55 (s
(br), 2H, NH2), 7.96 (s, 1H, H-2), 8.99 (s (br), 1H, NH-
piperazinyl), 12.20 (s (br), 1H, N9-H). LC/ESI-MS: positive
mode m/z 220 [M þ H]þ, negative mode m/z 218 [M - H]-, 254
[M þ Cl]-, 300 [M þ Br]-. Anal. (C9H13N7) C, H, N.
tert-Butyl 3-(9H-purin-6-ylamino)propylcarbamate (22).
Compound 22 was prepared from 18 according to the general
procedure in 93% yield; mp 201 °C. 1H NMR (500 MHz,
CD3OD) δ 1.45 (s, 9H, CH3), 1.86 (m, 2H, CH2-CH2-CH2),
3.19 (m, 2H, CH2-NHCO), 3.66 (s (br), 2H, CH2-N6-H), 8.08 (s,
1H, 8-H), 8.24 (s, 1H, 2-H). 13C NMR (125 MHz, CD3OD) δ
29.06 (CH3), 31.18 (CH2-CH2-CH2), 39.07, 39.37 (CH2-N), 80.23
(C(CH3)3), 119.55 (C-5), 140.81 (C-8), 151.26 (C-4), 154.04 (C-2),
156.13 (C-6), 158.87 (CdO). LC/ESI-MS: negative mode m/z 291
([M - H]-), positive mode m/z 293 ([M þ H]þ). Purity (HPLC-
8-(3-(Trifluoromethoxy)phenyl)-9H-purin-6-amine (39). Com-
pound 39 was prepared from 26 according to the general
1
procedure in 68% yield; mp > 300 °C (dec). H NMR (500
MHz, DMSO-d6) δ 7.20 (s (br), 2H, NH2), 7.47 (m, 1H, CH-
(phenyl)), 7.68 (m, 1H, CH(phenyl)), 8.10 (s, 1H, 2-H),
8.14-8.16 (m, 2H, CH(phenyl)), 13.47 (s (br), 1H, NH). 13C
NMR (125 MHz, DMSO-d6) δ 118.48 (CH(phenyl)), 119.49 (C-
5), 120.24 (q, J = 255.26 Hz, CF3), 122.31, 125.22, 131.33
(CH(phenyl)), 132.29 (C(phenyl)), 146.98, 149.00 (C-8, O-C-
(phenyl)), 152.41 (C-4), 153.02 (C-2), 155.61 (C-6). LC/ESI-MS:
negative mode m/z 294 ([M - H]-), positive mode m/z 296 ([M þ
UV 254 nm) 100.0%. Anal. (C13H20N6O2 0.25H2O) C, H, N.
3
8-(Ethylthio)-9H-purin-6-amine (30). Compound 30 was pre-
pared from 28 according to the general procedure in 56% yield;
mp 261 °C. 1H NMR (500 MHz, DMSO-d6) δ 1.32 (t, 3H, J =
7.25 Hz, CH3), 3.23 (q, 2H, J = 7.25 Hz, CH2), 6.90 (s (br), 2H,
NH2), 8.03 (s, 1H, 2-H), 12.92 (s (br), 1H, NH). 13C NMR
(125 MHz, DMSO-d6) δ 15.23 (CH3), 25.80 (CH2), 119.47 (C-5),
146.95 (C-8), 151.75 (C-2), 152.36 (C-4), 153.91 (C-6). LC/ESI-
MS: negative mode m/z 194 ([M - H]-), positive mode m/z 196
([M þ H]þ). Purity (HPLC-UV 254 nm) 98.5%. Anal. (C7H9-
H]þ). Purity (HPLC-UV 254 nm) 99.8%. Anal. (C12H8F3N5O
0.35H2O) C, H, N.
3
8-(Phenylethynyl)-9H-purin-6-amine (41). Compound 41 was
prepared from 26 according to a procedure published for
analogues in 35% yield.51 The product was purified by column
chromatography eluting with 5% methanol in dichloro-
N5S 1.25H2O) C, H, N.
3
1
8-(Propylthio)-9H-purin-6-amine (31). Compound 31 was
prepared from 28 according to the general procedure in 39%
yield; mp 249 °C. 1H NMR (500 MHz, DMSO-d6) δ 0.97 (t, 3H,
J = 7.25 Hz, CH3), 1.68 (m, 2H, CH2-CH3), 3.21 (t, 2H, J =
7.10 Hz, S-CH2), 6.89 (s (br), 2H, NH2), 8.02 (s, 1H, 2-H), 12.87
(s (br), 1H, NH). 13C NMR (125 MHz, DMSO-d6) δ 13.13
(CH3), 22.75 (CH2-CH3), 33.30 (S-CH2), 119.18 (C-5), 147.26
(C-8), 151.72 (C-2), 152.94 (C-4), 153.60 (C-6). LC/ESI-MS:
negative mode m/z 208 ([M - H]-), positive mode m/z 210
([M þ H]þ). Purity (HPLC-UV 254 nm) 98.1%. Anal. (C8H11-
methane; mp > 235 °C (dec). H NMR (500 MHz, DMSO-
d6) δ 7.31 (s (br), 2H, NH2), 7.49 (m, 3H, CH(phenyl)), 7.62 (m,
2H, CH(phenyl)), 8.14 (s, 1H, 2-H), 13.47 (s (br), 1H, NH). 13C
NMR (125 MHz, DMSO-d6) δ 80.75 (CꢁC-C(phenyl)), 90.67
(CꢁC-C(phenyl)), 119.41 (C-5), 120.57 (C(phenyl)), 129.17,
130.14, 131.84 (CH(phenyl)), 132.00 (C-8), 150.45 (C-4), 153.64
(C-2), 155.65 (C-6). LC/ESI-MS: negative mode m/z 234 ([M -
H]-), positive mode m/z 236 ([M þ H]þ). Purity (HPLC-UV
254 nm) 95.4%. Anal. (C13H9N5 H2O) C, H, N.
3
9-(2-(Pyridin-3-yl)ethyl)-9H-purin-6-amine (50). Compound
50 was prepared from 47 according to the general procedure in
46% yield; mp 215 °C. 1H NMR (500 MHz, DMSO-d6) δ 3.17 (t,
2H, J =7.09 Hz, CH2-C(pyridyl)), 4.41 (t, 2H, J =7.09Hz, CH2-
N), 7.12 (s (br), 2H, NH2), 7.25 (m, 1H, CH(pyridyl)), 7.53 (m,
1H, CH(pyridyl)), 7.95 (s, 1H, 8-H), 8.12 (s, 1H, 2-H), 8.30 (m,
1H, CH(pyridyl)), 8.37 (m, 1H, CH(pyridyl)). 13C NMR (125
MHz, DMSO-d6) δ 32.34 (CH2-C (pyridyl)), 43.84 (N-CH2),
118.86 (C-5), 123.54 (CH(pyridyl)), 133.66 (C(pyridyl)), 136.31
(CH(pyridyl)), 140.77 (C-8), 147.92 (CH-N(pyridyl)), 149.63
N5S 1.4H2O) C, H, N.
3
8-(Phenylthio)-9H-purin-6-amine (32). Compound 32 was
prepared from 26 according to a procedure published for
analogues in 37% yield.30 The product was purified by column
chromatography eluting with 10% methanol in dichloro-
1
methane; mp > 260 °C (dec). H NMR (500 MHz, DMSO-
d6) δ 7.17 (s (br), 2H, NH2), 7.31-7.42 (m, 5H, CH(phenyl)),
8.09 (s, 1H, 2-H), 13.24 (s (br), 1H, NH). 13C NMR (125 MHz,
DMSO-d6) δ 119.67 (C-5), 128.01, 129.73, 130.47 (CH(phenyl)),

5986 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Borrmann et al.
(C-4), 149.90 (CH-N(pyridyl)), 152.53 (C-2), 156.05 (C-6). LC/
ESI-MS: negative mode m/z 239 ([M - H]-), positive mode
m/z 241 ([M þ H]þ). Purity (HPLC-UV 254 nm) 100.0%. Anal.
2-Chloro-N-cyclopentyl-9-ethyl-9H-purin-6-amine (72). 2,6-
Dichloro-9-ethyl-9H-purine (71) (300 mg; 1.38 mmol) was dis-
solved in acetonitrile (10 mL), and cyclopentylamine (2 mL;
20.25 mmol) was added. The solution was stirred at rt for 15 h.
Volatiles were removed under vacuum, and the residue was
purified by column chromatography eluting with cyclohexane/
ethyl acetate (55:45). Compound 72 was obtained in 88% yield;
(C12H12N6 0.9H2O) C, H, N.
3
9-(2-(Pyridin-4-yl)ethyl)-9H-purin-6-amine (51). Compound
51 was prepared from 48 according to the general procedure
in 56% yield; mp 240 °C. 1H NMR (500 MHz, DMSO-d6) δ 3.18
(t, 2H, J = 7.09 Hz, CH2-C(pyridyl)), 4.43 (t, 2H, J = 7.09 Hz,
CH2-N), 7.14-7.16 (m, 4H, CH(pyridyl), NH2), 7.96 (s, 1H, 8-
H), 8.13 (s, 1H, 2-H), 8.40-8.41 (m, 2H, CH(pyridyl)). 13C
NMR (125 MHz, DMSO-d6) δ 34.37 (CH2-C(pyridyl)), 43.19
(N-CH2), 118.85 (C-5), 124.24 (CH(pyridyl)), 140.78 (C-8),
147.09 (C(pyridyl)), 149.63 (C-4), 149.69 (CH-N(pyridyl)),
152.56 (C-2), 156.07 (C-6). LC/ESI-MS: positive mode
m/z 241 ([M þ H]þ). Purity (HPLC-UV 254 nm) 100.0%. Anal.
1
mp 79-83 °C. H NMR (400 MHz, DMSO-d6) δ 1.38 (t, 3H,
J = 7.2 Hz, CH3), 1.55 (m, 4H, H-cyclopentyl), 1.72 (m, 2H,
H-cyclopentyl), 1.92 (m, 4H, H-cyclopentyl), 4.12 (q, 2H, J =
7.2 Hz, CH2-N), 4.42 (m, 1H, m, CH-N), 8.18 (m, 2H, NH and
H-8). Anal. (C12H16ClN5) C, H, N.
2-Butoxy-9-ethyl-9H-purin-6-amine (75). NaOH (300 mg)
was added to 2-chloro-9-ethyl-9H-adenine (300 mg; 1.52 mmol)
in 1-butanol (6 mL), and the reaction mixture was stirred at
90 °C for 1 h. The reaction mixture was neutralized with HCl,
and the product was extracted from H2O with chloroform. The
organic layers were collected, dried over Na2SO4, and concen-
trated under vacuum. The residue was purified by column
chromatography eluting with ethyl acetate/cyclohexane/methanol
(60:33:7) to obtain 75 in 75% yield; mp 113-115 °C. 1H NMR
(400 MHz, DMSO-d6) δ 0.91 (t, 3H, J = 7.4 Hz, (CH2)2CH3),
1.35 (t, 3H, J = 7.4 Hz, N-CH2CH3), 1.39 (m, 2H, CH2-
CH2CH3), 1.64 (m, 2H, CH2CH2-O), 4.04 (q, 2H, J = 7.2 Hz,
CH2-N), 4.18 (t, 2H, J = 6.6 Hz, CH2-O), 7.12 (s (br), 2H, NH2),
7.92 (s, 1H, H-8). Anal. (C11H17N5O) C, H, N.
9-Cyclohexyl-6-ethoxy-9H-purine (79). Et3N was added to a
solution of 7857 (100 mg; 0.42 mmol) in EtOH (5 mL), and the
mixture was stirred at 120 °C for 40 h. Volatiles were removed
under vacuum, and the residue was purified by column chro-
matography eluting with chloroform/methanol (99.5:0.5), pro-
viding 79 in 51% yield; mp 86-89 °C. 1H NMR (DMSO-d6) δ
1.27 (m, 2H, H-cyclohexyl), 1.41 (t, 3H, J = 7.1 Hz, CH3), 1.47
(m, 1H, H-cyclohexyl), 1.70 (m, 1H, H-cyclohexyl), 1.87 (m, 3H,
H-cyclohexyl), 2.00 (m, 3H, H-cyclohexyl), 4.45 (m, 1H, CH-N),
4.59 (q, 2H, J = 7.1 Hz, CH2-O), 8.48 (s, 1H, H-8), 8.50 (s, 1H,
H-2). Anal. (C13H18N4O) C, H, N.
(C12H12N6 0.5H2O) C, H, N.
3
tert-Butyl 4-(2-(6-Amino-9H-purin-9-yl)ethyl)piperazine-1-
carboxylate (54). Compound 54 was prepared from 53 accord-
ing to the general procedure in 38% yield; mp 207 °C. 1H NMR
(500 MHz, DMSO-d6) δ 1.37 (s, 9H, CH3), 2.38 (m (br), 4H,
CH2(piperazinyl)), 2.70 (t, 2H, J = 6.30 Hz, CH2-CH2-N-
(purinyl)), 3.23 (m (br), 4H, CH2(piperazinyl)), 4.24 (t, 2H,
J = 6.30 Hz, CH2-N(purinyl)), 7.11 (s (br), 2H, NH2), 8.10 (s,
1H, 8-H), 8.12 (s, 1H, 2-H). 13C NMR (125 MHz, DMSO-d6,
343 K) δ 28.19 (CH3), 40.11 (CH2-N(purinyl)), 43.44 (CH2-
(piperazinyl)), 52.35 (CH2-CH2-N(purinyl)), 56.76 (CH2(pip-
erazinyl)), 78.88 (C(CH3)3), 118.70 (C-5), 141.27 (C-8), 149.72
(C-4), 152.41 (C-2), 153.95 (CdO), 156.04 (C-6). LC/ESI-MS:
negative mode m/z 346 ([M - H]-), positive mode m/z 348 ([M þ
H]þ). Purity (HPLC-UV 254 nm) 100.0%. Anal. (C16H25N7O2
0.1H2O) C, H, N.
3
9-(2-(Piperazin-1-yl)ethyl)-9H-purin-6-amine Dihydrochlor-
ide (55). Compound 55 was prepared by deprotection of 54
according to the general procedure in 91% yield; mp > 255 °C
(dec). 1H NMR (500 MHz, D2O) δ 3.52 (s (br), 8H, CH2-
(piperazinyl)), 3.63 (t, 2H, J = 6.52 Hz, CH2-CH2-N(purinyl)),
4.76 (t, 2H, J = 6.62 Hz, CH2-N(purinyl)), 8.42 (s, 1H, CH-
(purinyl)), 8.48 (s, 1H, CH(purinyl)). 13C NMR (125 MHz,
D2O) δ 42.18 (CH2-CH2-N(purinyl)), 44.27 (CH2(piperazinyl)),
51.83 (CH2(piperazinyl)), 58.40 (CH2-N(purinyl)), 121.09 (C-5),
147.52, 147.58 (C-2, C-8), 151.86, 152.81 (C-4, C-6). LC/ESI-
MS: positive mode m/z 248 ([M þ H]þ). Purity (HPLC-UV
254 nm) 99.8%. Anal. (C11H17N7 2HCl H2O) C, H, N.
N8,9-Diethyl-9H-purine-6,8-diamine (88). Ethylamine (10 mL)
was added to 8-bromo-9-ethyl-9H-purin-6-amine (87) (200 mg,
0.83 mmol) in a sealed vial, and the mixture was heated at 100 °C
for 20 h. Volatiles were removed under vacuum, and the residue
was purified by column chromatography eluting with chloro-
form/methanol (95:5). Compound 88 was obtained in 85% yield;
mp 189-191 °C. 1H NMR (400 MHz, DMSO-d6) δ 1.22 (m, 6H,
2ꢀ CH3), 3.42 (q, 2H, J = 5.7 Hz, CH2-NH), 4.01 (q, 2H, J =
7.0 Hz, CH2-N), 6.37 (s (br), 2H, NH2), 6.70 (t, 1H, J = 5.7 Hz,
NH), 7.91 (s, 1H, H-2). Anal. (C9H14N6) C, H, N.
3
3
4-(2-(6-Amino-9H-purin-9-yl)ethylamino)butan-1-ol
(59).
Compound 59 was prepared according to a published proce-
dure describing the synthesis of analogues.56 The product was
crystallized from ethanol/acetonitrile in 24% yield; mp 133 °C.
1H NMR (500 MHz, DMSO-d6) δ 1.36-1.40 (m, 4H, CH2-
CH2-CH2-O), 2.49 (t, 2H, NH-CH2(butyl), overlapping with
solvent signal), 2.90 (t, 2H, J = 6.15 Hz, CH2-CH2-N-
(purinyl)), 3.34 (m, 2H, CH2-OH), 4.17 (t, 2H, J = 6.30 Hz,
CH2-CH2-N(purinyl)), 7.11 (s (br), 2H, NH2), 8.08 (s, 1H,
CH(purinyl)), 8.12 (s, 1H, CH(purinyl)). 13C NMR (125 MHz,
DMSO-d6) δ 26.17 (CH2-CH2-O), 30.40 (CH2-CH2-CH2-O),
42.99, 48.40, 48.73 (CH2-N), 60.73 (O-CH2), 118.72 (C-5),
141.20 (C-8), 149.62 (C-4), 152.26 (C-2), 155.93 (C-6).
LC/ESI-MS: negative mode m/z 249 ([M - H]-). Purity
N8-Cyclohexyl-9-ethyl-9H-purine-6,8-diamine (89). Cyclo-
hexylamine (15 mL) was added to 8-bromo-9-ethyl-9H-purin-
6-amine (87) (200 mg, 0.83 mmol) in a sealed vial, and the
mixture was heated at 140 °C for 48 h. Volatiles were removed
under vacuum, and the residue was purified by column
chromatography eluting with chloroform/cyclohexane/
methanol (85:10:5). Compound 89 was obtained in 18% yield
1
(38 mg); mp 193 °C (dec). H NMR (400 MHz, DMSO-d6)
δ 1.18 (t, 3H, J = 7.1 Hz, CH3), 1.32 (m, 5H, H-cyclohexyl),
1.65 (m, 1H, H-cyclohexyl), 1.75 (m, 2H, H-cyclohexyl),
1.99 (m, 2H, H-cyclohexyl), 3.77 (m, 1H, CH-N), 4.00
(q, 2H, J = 7.1 Hz, CH2-N), 6.33 (s (br), 2H, NH2), 6.42
(d, 1H, J = 8.1 Hz, NH), 7.90 (s, 1H, H-2). Anal. (C13H20N6)
C, H, N.
(HPLC-UV 254 nm) 97.9%. Anal. (C11H18N6O 0.4H2O)
3
C, H, N.
9-(3-Heptyl)adenine (61). Adenine (200 mg, 1.48 mmol) was
dissolved in 30 mL of dry DMF, and after addition of 1.5 mmol
of NaH, the mixture was stirred at 35 °C for 30 min. 3-Bromo-
heptane (1.55 mmol) was added, and the mixture was stirred at 30
°C for 21 h. The solvent was evaporated, and the residue was
purified by column chromatography eluting with chloroform/
methanol (97:3) yielding the pure product in 71% yield; mp
N-Butyl-9-ethyl-8-phenyl-9H-purin-6-amine (92). Butylamine
(5 mL) was added to 9163 (40 mg; 0.16 mmol), and the reaction
mixture was stirred at rt for 3 h. Volatiles were removed under
vacuum, and the residue was purified on a silica gel preparative
TLC eluting with chloroform/methanol (98:2). 92 was obtained
in 85% yield; mp 93-95 °C. 1H NMR (400 MHz, DMSO-d6) δ
0.91 (t, 3H, J = 7.2 Hz, (CH2)3CH3), 1.30 (m, 5H, N-CH2CH3
and CH2CH2CH2CH3), 1.60 (m, 2H, CH2CH2CH2CH3), 3.51
(m, 2H, CH2-N6), 4.26 (q, 2H, J = 7.2 Hz, CH2-N), 7.58 (m, 3H,
1
151-153 °C. H NMR (CDCl3) δ 0.84 (m, 6H, CH-CH2-CH3,
CH2-CH2-CH3), 1.25 (m, 4H, CH2-CH2-CH3), 1.93 (m, 4H, CH2-
CH-CH2), 4.41 (m, 1H, CH2-CH-CH2), 5.66 (s (br), 2H, NH2),
7.78 (s, 1H, H-2), 8.36 (s, 1H, H-8). Anal. (C12H19N5) C, H, N.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5987
H-Ph), 7.79 (m, 3H, H-Ph and NH), 8.25 (s, 1H, H-2). Anal.
(C17H21N5) C, H, N.
were washed with Hank’s balanced salt solution (HBSS) con-
sisting of 20 mM Hepes, 130 mM NaCl, 5.5 mM glucose, 0.83
mM MgSO4, 0.5 mM MgCl2, 1.4 mM CaCl2 2H2O,
Radioligand Binding Assays. Competition assays with
[3H]adenine (27 Ci/mmol; GE Healthcare) at rat brain cortical
membranes9,71 were carried out as previously described.9,15,69 In
brief, a rat brain cortical membrane preparation (100 μg of
protein) was incubated with 10 nM [3H]adenine in 50 mM Tris-
HCl buffer pH 7.4 in a total volume of 200 μL. All aqueous
solutions were autoclaved or heated at >90 °C for at least
10 min prior to use in order to destroy potential bacterial
contaminations that may express binding sites for [3H]adenine.69
Compounds were initially tested at a high concentration of 30,
100, or 250 μM, respectively, depending on their solubility.
Concentration-inhibition curves were determined of those com-
pounds that showed >50% inhibition of radioligand binding at
100 μM using 6-9 different concentrations of the tested com-
pound spanning at least 3 orders of magnitude. Three separate
experiments were performed each in triplicate. Nonspecific bind-
ing was determined in the presence of 100 μM adenine. Incuba-
tions were carried out for 1 h at rt and were terminated by rapid
filtration through GF/B glass fiber filters (Whatman, Dassel,
Germany). Filters were washed three times each with 3 mL of
ice-cold 50 mM Tris-HCl buffer pH 7.4. Filter-bound radio-
activity was measured by liquid scintillation counting. Competi-
tion assays with [3H]adenine at HEK293 cell membranes69 were
carried out the same way.
Functional Assays. Retroviral Transfection of 1321N1 Astro-
cytoma Cells. The coding sequence for the rAde1R was obtained
from imaGenes GmbH, Berlin (Germany) and inserted into the
retroviral vector pLXSN using the restriction enzymes EcoRI
and XhoI. Escherichia coli cells were transformed with the
plasmid DNA, and DNA was isolated from individual clones.
Sequencing was performed by GATC Biotech (Konstanz,
Germany). 1321N1 astrocytoma cells were transfected using
a retroviral system essentially as described.72 In brief, GPþ
envAM12 packaging cells were plated at a density of 1.5 ꢀ
106 into 25 cm2 flasks using Dulbecco’s modified Eagle medium
(DMEM) containing 10% FCS, 100 U/mL penicillin G,
100 μg/mL streptomycin, and 1% ultraglutamine 24 h before
transfection. The cells were transfected using Lipofectamine
2000 (Invitrogen, Karlsruhe, Germany). To increase infection
efficiency, plasmids containing receptor DNA (63%) were
mixed with plasmids containing vesicular stomatitis virus G
protein (VSV-G) DNA (37%) prior to transfection. After 16 h,
the medium was replaced by 3 mL of medium containing 5 mM
sodium butyrate. The transfected GPþenvAM12 cells were
cultured at 32 °C, 5% CO2, and 96% air for 48 h. The super-
natant of the GPþenvAm12 cells containing the recombinant
viruses was harvested and filtered. The virus solution (2 mL) was
mixed with 4 μL of Polybrene (4 mg/mL) and added to 1321N1
astrocytoma cells, which were incubated for 2.5 h at 37 °C. After
48 h, these cells were selected for Geneticin resistance in the
presence of 800 μg/mL G418. The cells were grown adherently
and maintained DMEM containing 10% fetal calf serum, peni-
cillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (2 mM),
and Geneticin (G418, 800 μg/mL) at 37 °C in 5% CO2/95% air.
Cell Culture. 1321N1 astrocytoma cells were cultured at
37 °C, 96% humidity and 5% CO2 in DMEM supplemented
with 10% FCS, 100 U/mL penicillin G, 100 μg/mL streptomy-
cin, and L-glutamine (2 mM). After stable transfection, G418
(800 μg/mL) was added. GPþenvAM12 packaging cells were
maintained in HXM media containing DMEM supplemented
with 10% FCS, 100 U/mL penicillin G, 100 μg/mL streptomy-
cin, 1% ultraglutamine, 15 μg/mL hypoxanthine, 250 μg/mL
xanthine, 25 μg/mL mycophenolic acid, and 200 μg/mL hygro-
mycin B under the same conditions.
3
4.17 mM NaHCO3, 5.36 mM KCl, 0.4 mM KH2PO4, and 0.3
mM NaHPO4, pH 7.3 and then incubated with HBSS for 2 h at
37 °C. Cellular cAMP production was then stimulated by the
addition of 3 nM isoproterenol at 37 °C. Solvent, adenine, or the
adenine derivative, respectively, was added together with iso-
proterenol. In antagonist experiments, adenine in different
concentrations (0.1-1000 nM) and test compound (10 μM)
were added to the cells at the same time. The reaction was
stopped after 10 min by removal of the reaction buffer followed
by the addition of a hot lysis solution (500 μL; 90 °C; Na2EDTA
4 mM; Triton X-100 0.01%). cAMP levels in the supernatant
were then quantified by incubation of an aliquot with [3H]cAMP
and cAMP binding protein prepared from calf adrenal glands.73
The mixture was incubated at 4 °C for 60 min, then the samples
were harvested by filtration through GF/B filters using a
harvester (Brandel, Gaithersburg, MD). Each filter was rinsed
3 times each with 3 mL of 50 mM Tris-HCl, pH 7.4. The filters
were punched out into scintillation vials and counted in a liquid
scintillation counter after adding 2.5 mL of scintillation fluid
and waiting for at least 6 h. Amounts of cAMP per well were
calculated from a cAMP standard curve (0-40 pmol) deter-
mined for each experiment. The isoproterenol-induced cAMP
production in the presence of test compound was
expressed as percentage of the isoproterenol-induced cAMP
production in the absence of test compound (= 100%).
[3H]Adenine Uptake Assays. Uptake assays were performed
essentially as previously described.20,74 1321N1 astrocytoma
cells stably expressing the rAde1R, or null cells, respectively,
were cultured on 24-well plates (SARSTEDT AG & Co.,
€
Numbrecht, Germany). All uptake experiments were performed
in triplicates in a total volume of 250 μL. Stock solutions of
adenine derivatives were prepared in DMSO. After removal of
the culture medium, cells were washed with HEPES-buffered
Ringer solution (containing in mM 135 NaCl, 5 KCl, 3.33
NaH2PO4, 0.83 Na2HPO4, 1.0 CaCl2, 1.0 MgCl2, 10 glucose,
and 5 HEPES, pH 7.4). In some experiments, NaCl was replaced
by choline chloride. Test compounds and radiolabeled
[3H]adenine (30 nM) were added at the same time to start the
assay. After 1 min, the uptake was stopped by washing the cells
with ice-cold PBS containing 5 mM unlabeled adenine. Cells
were subsequently solubilized in 5% aq Triton X-100 solution.
After addition of 2.5 mL of scintillation fluid and waiting for at
least 6 h, radioactivity was measured in a liquid scintillation
counter.
Acknowledgment. T.B. was supported by a stipend pro-
€
€
vided by the Bischofliche Studienforderung Cusanuswerk. A.
A., C.E.M., M.K. and S.G. are grateful for support by the
Deutsche Forschungsgemeinschaft (DFG, GRK804), R.V.,
C.L., and G.C. were supported by the Fondo di Ricerca di
Ateneo (University of Camerino) and the Ministero della
Salute Progetto Ordinario (RF-CNM-2007-662855).
Supporting Information Available: Elemental analysis data of
all tested compounds; information and data on syntheses,
yields, melting points, NMR, and LC/ESI-MS spectra of the
tested compounds that have previously been described in the
literature. This material is available free of charge via the
Internet at http://pubs.acs.org.
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