2-Phenylimidazo[2,1-i]purin-5-ones: Structure-activity relationships and characterization of potent and selective inverse agonists at human A3 adenosine receptors

Article abstract of DOI:10.1016/S0968-0896(02)00456-X

Structure-activity relationships of 2-phenyl-imidazo[2,1-i]purin-5-ones as ligands for human A₃ adenosine receptors (ARs) were investigated. An ethyl group in the 8-position of the imidazoline ring of 4-methyl-2-phenyl-imidazopurinone leading to chiral compounds was found to increase affinity for human A₃ ARs by several thousand-fold. Propyl substitution instead of methyl at N4 decreased A₃ affinity but increased A₁ affinity leading to potent A₁-selective AR antagonists. The most potent A₁ antagonist of the present series was (S)-8-ethyl-2-phenyl-4-propyl-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (S-3) exhibiting a K_i value of 7.4 nM at rat A₁ ARs and greater than 100-fold selectivity versus rat A_2A and human A₃ ARs. At human A₁ ARs 2-phenylimidazo[2,1-i]purin-5-ones were generally less potent and therefore less A₁-selective (S-3: K_i=98 nM). 2-, 3-, or 4-Mono-chlorination of the 2-phenyl ring reduced A₃ affinity but led to an increase in affinity for A₁ ARs, whereas di- (3,4-dichloro) or polychlorination (2,3,5-trichloro) increased A₃ affinity. The most potent and selective A₃ antagonist of the present series was the trichlorophenyl derivative (R)-8-ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-4,5,7,8-tetrahydro-1H- imidazo[2,1-i]purin-5-one (R-8) exhibiting a subnanomolar K_i value at human A₃ ARs and greater than 800-fold selectivity versus the other AR subtypes. Methylation of 4-alkyl-2-phenyl-substituted imidazo[2,1-i]purin-5-ones led exclusively to the N9-methyl derivatives, which exhibited largely reduced AR affinities as compared to the unmethylated compounds. [³⁵S]GTPγS binding studies of the most potent 2-phenyl-imidazo[2,1-i]purin-5-ones at membranes of Chinese hamster ovary cells expressing the human A₃ AR revealed that the compounds were inverse agonists at A₃ receptors under standard test conditions. Due to their high A₃ affinity, selectivity, and relatively high water-solubility, 2-phenyl-imidazo[2,1-i]purin-5-ones may become useful research tools.

Full text of DOI:10.1016/S0968-0896(02)00456-X

Bioorganic & Medicinal Chemistry 11 (2003) 347–356
2-Phenylimidazo[2,1-i]purin-5-ones:
Structure–Activity Relationships and Characterization of Potent
and Selective Inverse Agonists at Human A3 Adenosine Receptors
Vita Ozola,a Mark Thorand,a Martina Diekmann,a Ramatullah Qurishi,a
Britta Schumacher,a Kenneth A. Jacobsonb and Christa E. Mullera,*
aUniversity of Bonn, Pharmaceutical Institute Poppelsdorf, Kreuzbergweg 26, D-53115 Bonn, Germany
bLaboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland, USA
Received 3 July 2002; accepted 19 September 2002
Abstract—Structure–activity relationships of 2-phenyl-imidazo[2,1-i]purin-5-ones as ligands for human A3 adenosine receptors
(ARs) were investigated. An ethyl group in the 8-position of the imidazoline ring of 4-methyl-2-phenyl-imidazopurinone leading to
chiral compounds was found to increase affinity for human A3 ARs by several thousand-fold. Propyl substitution instead of methyl
at N4 decreased A3 affinity but increased A1 affinity leading to potent A1-selective AR antagonists. The most potent A1 antagonist
of the present series was (S)-8-ethyl-2-phenyl-4-propyl-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (S-3) exhibiting a Ki value
of 7.4 nM at rat A1 ARs and greater than 100-fold selectivity versus rat A2A and human A3 ARs. At human A1 ARs 2-phenyl-
imidazo[2,1-i]purin-5-ones were generally less potent and therefore less A1-selective (S-3: Ki=98 nM). 2-, 3-, or 4-Mono-chlorination
of the 2-phenyl ring reduced A3 affinity but led to an increase in affinity for A1 ARs, whereas di- (3,4-dichloro) or polychlorination
(2,3,5-trichloro) increased A3 affinity. The most potent and selective A3 antagonist of the present series was the trichlorophenyl
derivative (R)-8-ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (R-8) exhibiting a sub-
nanomolar Ki value at human A3 ARs and greater than 800-fold selectivity versus the other AR subtypes. Methylation of 4-alkyl-2-
phenyl-substituted imidazo[2,1-i]purin-5-ones led exclusively to the N9-methyl derivatives, which exhibited largely reduced AR
affinities as compared to the unmethylated compounds. [35S]GTPgS binding studies of the most potent 2-phenyl-imidazo[2,1-i]
purin-5-ones at membranes of Chinese hamster ovary cells expressing the human A3 AR revealed that the compounds were inverse
agonists at A3 receptors under standard test conditions. Due to their high A3 affinity, selectivity, and relatively high water-solubility,
2-phenyl-imidazo[2,1-i]purin-5-ones may become useful research tools.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
very low degree of water-solubility. More water-soluble
A3 antagonists are required as pharmacological tools
for in vitro and in vivo studies. A3 antagonists have
potential as novel drugs; postulated therapeutic appli-
cations include inflammatory diseases, glaucoma, and
stroke.2
A3 adenosine receptors (A3 ARs) are the youngest
member of the AR family of G-protein-coupled recep-
tors, which consists of four different subtypes, A1, A2A
,
A2B and A3.1 A number of selective antagonists for
human A3 ARs have been developed during the past
years.2 However, all of the potent and selective A3
antagonists developed so far, including triazoloquin-
azolines (e.g., MRS-1177, MRS-1220),3,4 pyrazolotri-
azolopyrimidines (e.g., MRE-3005F20, MRE-3008F20),5,6
isoquinolines (e.g., VUF-8504),7ꢀ9 and dihydropyridines
(e.g., MRS-1334),10,11 are highly lipophilic and display a
Imidazo[2,1-i]purinones and related tricyclic purine
derivatives derived from xanthines have been developed
as water-soluble AR antagonists with selectivity for A1 or
A2A ARs, respectively, depending on their substitution
pattern.12ꢀ14 Recently, we discovered that a 2-phenyl-
substituted imidazopurinone, namely (R)-8-ethyl-4-
methyl-2-phenyl-imidazo[2,1-i]purin-5-one (R-1, PSB-11,
Table 3) possesses high affinity for human A3 ARs
(Ki=2.3 nM) and is highly selective versus all other AR
subtypes.13 The present study was aimed at investigating
*Corresponding author. Tel.: +49-228-73-2301; fax: +49-228-73-
2567; e-mail: christa.mueller@uni-bonn.de
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00456-X

348
V. Ozola et al. / Bioorg. Med. Chem. 11 (2003) 347–356
the properties and the structure–activity relationships of
2-phenyl-imidazopurinones as adenosine receptor
ligands, particularly with regard to substituents at the
2-phenyl group, and at the nitrogen atoms N4 and N9.
Yields, melting points and analytical data of the new
1
compounds are collected in Table 1. H NMR spectral
data were in accordance with the proposed structures
(see Experimental).
Analysis of enantiomeric purity
Results and Discussion
Chemistry
A recently developed capillary electrophoresis (CE)
method13 was used to determine the enantiomeric purity
of compounds 3–8 (see Table 2). As chiral dis-
criminators cyclodextrins were added to the inlet buffer
(b-cyclodextrin or 2-hydroxypropyl-b-cyclodextrin plus
sulfated b-cyclodextrin), and sulfated b-cyclodextrin
was added to the outlet buffer. The separations were
performed at a pH value of 4.5 (phosphate buffer), at
which the compounds were protonated. The compounds
were detected and identified by their UV spectra using a
diode array detector. Conditions were optimized for
each compound (Table 2). Typical separations are
shown in Figure 2 for the racemate RS-8 (A) and the
enantiomer S-3 (B). All compounds investigated exhib-
ited a high degree of enantiomeric purity; contamina-
tion by the other enantiomer was lower than 1.5% in all
cases.
2-Phenylimidazo[2,1-i]purin-5-one derivatives were syn-
thesized from the corresponding 3-methyl- or 3-propyl-
substituted 8-phenylxanthine derivatives, respectively, in
analogy to described procedures.13ꢀ16 Imidazopurinones
S-3 and R-8 were methylated using methyl iodide and
sodium hydride in dry dimethylformamide to yield
N9-methylated derivatives S-9 and R-10 (Fig. 1). Methyl-
ation of a 2-unsubstituted imidazo[2,1-i]purin-5-one had
been reported to yield the N1-methylated product.15
For the 2-phenyl-substituted imidazo[2,1-i]purinones,
we found methylation to occur predominantly or exclu-
sively at N9. If the methylation was performed in
dimethylformamide using methyl iodide in the presence
of potassium carbonate N9- and N1-methylated pro-
ducts were formed in 85 and 15% yield, respectively.14
The use of sodium hydride instead of potassium car-
bonate led to the exclusive formation of N9-methylated
products S-9 and R-10 (Fig. 1).
Biological activity
The new compounds were investigated in radioligand
binding assays at A1 ARs of rat brain cortical mem-
branes using the A1-selective radioligand [3H]2-chloro-
N6-cyclopentyladenosine ([3H]CCPA], and at A2A
The position of the methyl group could unambiguously
be assigned by NMR spectroscopy. While a methyl
group at N1 of 2-phenyl-substituted imidazo[2,1-i]pur-
inones was found at ca. 4.05 ppm (in DMSO-d6),13,14
the signal for a methyl group at N9 was at 3.54 (S-9), or
3.60 ppm (R-10), respectively. Hetero-multiple quantum
coherence (HMQC) experiments with compound R-10
showed a correlation between the methyl group and C8
(at 62.0 ppm). However, neither a correlation with C2
(at 160.6 ppm) nor with C9b (at 109.6 ppm), the C
atom neighboring N1, could be observed. Therefore,
the methyl group is clearly attached to N9 rather than
N1.
ARs of rat brain striatal membranes using the A2A
-
selective radiolig and [3H]3-(3-hydroxypropyl)-7-methyl-
8-(m-methoxystyryl)-1-propargylxanthine ([3H]MSX-2).
The compounds were additionally investigated at native
human A1 and A2A ARs using membrane preparations
of post-mortem human brain. [3H]CCPA was used as a
radioligand for the human A1 and [3H]-2-[[4-(carboxy-
ethyl)phenyl]ethyl]amino] - 50 - N - (ethylcarbonyl)amino]
adenosine ([3H]CGS21680) for the human A2A ARs. All
compounds were investigated at recombinant human A3
Figure 1. Tautomeric structures and alkylation of imidazo[2,1-i]purin-5-ones.

V. Ozola et al. / Bioorg. Med. Chem. 11 (2003) 347–356
349
Table 1. Yields, melting points and analytical data of synthesized 2-phenylimidazo[2,1-i]purin-5-one derivatives
20
D
a
Compd
R2
R4
R8
R9
Yield
(%)
mp
(ꢁC)
½ꢀŠ
Concentration
(mg/mL)
Formula
Analyses
R-1
2
Phenyl
Phenyl
Phenyl
Methyl Ethyl
Methyl
H
H
H
H
H
H
H
H
51
60
79
53
42
60
42
70
76
58
>250
303
4.5b
—
10.0
—
C16H17N5O
C14H13N5O*H2O
C18H21N5O*2H2O
C18H19N5OCl2
C16H16N5OCl*H2O
C16H16N5OCl
C16H16N5OCl*2H2O
C16H14N5O C 3l
C19H21N5OCl2
295.1423c
Cd, H, Nd
C
C , H, N
H
S-3
S-4
S-5
S-6
S-7
R-8
S-9
R-10
Propyl Ethyl
Propyl Ethyl
Methyl Ethyl
Methyl Ethyl
Methyl Ethyl
232 ꢀ27.3
283 ꢀ11.8
125 ꢀ15.3
283 ꢀ10.5
262 ꢀ19.5
5.1
5.1
7.7
4.4
5.7
3.7
5.7
3.6
e, N, H
3,4-Dichlorophenyl
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
2,3,5-Trichlorophenyl Methyl Ethyl
3,4-Dichlorophenyl
2,3,5-Trichlorophenyl Methyl Ethyl Methyl
C
C, H, N
, H, N
C
f, N, H
160
215
12.3
3.9
397.0259c
405.1123c
Propyl Ethyl Methyl
214 ꢀ10.7
C17H16N5OCl3*0.5H2O
C
, H, N
aDetermined in DMSO.
bDetermined for the hydrochloride in methanol.13
cDetermined by high resolution mass spectroscopy.
dC: calcd, 58.94; found, 58.48; N: calcd, 24.55; found, 23.86.
eH: calcd, 7.01; found, 6.40.
fH: calcd, 5.51; found, 4.94.
Table 2. Analysis of enantiomeric purity of chiral compounds by capillary electrophoresis
Enantiomer
Cyclodextrin(s) in inlet buffera
Cyclodextrin in
outlet buffera
Capillary
effective length
Determined purity (%) Determined impurityc (%)
ÆR.S.D.b (%)
ÆR.S.D.b (%)
S-3
S-4
S-5
S-6
S-7
R-8
HPCD (6 mM)+s-b-CD (1 mg/mL)
HPCD (6 mM)+s-b-CD (1 mg/mL)
HPCD (6 mM)+s-b-CD (1 mg/mL)
HPCD (6 mM)+s-b-CD (0.5 mg/mL) s-b-CD (0.5 mg/mL)
HPCD (6 mM)+s-b-CD (0.5 mg/mL) s-b-CD (0.5 mg/mL)
s-b-CD (1 mg/mL)
s-b-CD (0.5 mg/mL)
s-b-CD (1 mg/mL)
50 cm
50 cm
50 cm
50 cm
50 cm
40 cm
98.90Æ0.06 (n=4)
98.80Æ0.08 (n=3)
99.32Æ0.05 (n=4)
99.80Æ0.04 (n=3)
99.29Æ0.10 (n=4)
98.75Æ0.22 (n=8)
1.10Æ0.06
1.20Æ0.08
0.68Æ0.05
0.20Æ0.04
0.71Æ0.10
1.25Æ0.22
b-CD (6 mM)
s-b-CD (2.5 mg/mL)
aPhosphate buffer, 50 mM, pH 4.5; b-CD=b-cyclodextrin; HPCD=2-hydroxypropyl-b-cyclodextrin; s-b-CD=sulfated b-cyclodextrin.
bR.S.D.=relative standard deviation.
cContamination by the other enantiomer.
ARs expressed in CHO or HEK cells using [125I]AB-
enantioselectivity at ARs.13 For the analysis of the
structure–activity relationships of compounds 3–10 only
one single enantiomer was synthesized. The ethyl resi-
due in the 8-position at the imidazoline ring of
4-methyl-2-phenyl-imidazopurinone proved to be
essential for high affinity at A3 ARs. Imidazopurinone
2, lacking the ethyl group, was several thousand-fold
less potent at A3 ARs than the analogous ethyl deriva-
tives R-1 and S-1. At A1 and A2A ARs the decrease in
potency by removal of the ethyl group was much less
pronounced (only 4–19-fold). An isomer of 2, in which
the 4-methyl group was (formally) moved to the N1-
position (1-methyl-2-phenyl-imidazo[2.1-i]purin-5-one)
had previously been shown to exhibit high A3 affinity
(Ki=47 nM, 400-fold as potent as 2) but similar A1 and
A2A affinity as 2. The corresponding 1,4-dimethyl deri-
vative, however, had shown reduced A3 affinity.13 These
results indicate that imidazopurinones can exhibit dif-
ferent binding modes at A3 ARs depending on the sub-
stitution pattern. This conclusion is corroborated by
recently published data on another imidazopurinone
MECA as an A3 radioligand. In
a few cases,
[3H]NECA, or the novel radioligand [3H]PSB-11, was
used for A3 binding assays. Ki values determined with
different A3 radioligands were very similar (see Table 3,
compounds R-1 and R-8).
Structure–activity relationships
2-Phenylimidazopurinone R-1 (PSB-11) had been found
in a previous study to possess high affinity for human
A3 ARs (Ki=2.3 nM).13 The compound had been
shown to be 190-fold selective versus rat A1 ARs, and
exhibited even higher selectivity (>900-fold) versus rat
A2A and mouse A2B ARs. A similar Ki value at human
recombinant A3 ARs was found using a different radi-
oligand, [3H]NECA (Ki=3.5 nM, Table 3). R-1 has now
been shown to possess similarly high affinity versus the
human A1 and A2A receptor subtypes (Table 3). Chiral
2-phenyl-substituted imidazopurinones, such as com-
pounds R-1 and S-1 had exhibited only a low degree of

350
V. Ozola et al. / Bioorg. Med. Chem. 11 (2003) 347–356
Figure 2. Typical electropherograms of chiral separation of two chiral compounds. A. Racemate RS-8. Inlet buffer: phosphate 50 mM, b-cyclo-
dextrin 6 mM, pH 4.5. Outlet buffer: phosphate 50 mM, sulfated b-cyclodextrin 2.5 mg/mL, pH 4.5. Fused silica capillary, 40 cm effective lengthÂ75
mm I.D.Â375 O.D.; constant current: 90 mA; injection time: 5 s with 0.5 p.s.i.; B. Enantiomer S-3. Inlet buffer: phosphate 50 mM, hydroxypropyl-b-
cyclodextrin 6 mM, sulfated b-cyclodextrin 1 mg/mL, pH 4.5. Outlet buffer: phosphate 50 mM, sulfated b-cyclodextrin 1 mg/mL, pH 4.5. Fused
silica capillary, 50 cm effective lengthÂ75 mm I.D.Â375 O.D.; constant current: 90 mA; injection time: 20 s with 0.5 p.s.i.
derivative unsubstituted in the 8-position, KF26777 (2-
(p-bromophenyl)-4-propyl-substituted imidazo[2,1-i]
purin-5-one), which has been reported to be a potent
and selective antagonist at human A3 ARs.16,17
of fact, compound S-3 is a very potent and selective A1
AR antagonist with a Ki value of 7.4 nM at rat A1 ARs
and over 100-fold selectivity versus rat A2A ARs. The
compound is less potent at human A1 ARs (Ki=98 nM)
and less selective (36-fold versus human A2A, 9-fold
versus human A3 ARs).
In xanthine derivatives the exchange of a 3-methyl
group (corresponding to the 4-position in imidazo[2,1-i]
purinones) by a propyl group generally results in a large
increase in affinity for human A3 ARs.2,18 However, the
same modification in imidazopurinones, such as the
4-propyl derivatives S-3 and S-4, appeared to decrease
A3 affinity to a large extent. The propyl derivative S-3,
for example, exhibited 88-fold lower affinity for A3 ARs
than its methyl homologue S-1. Conversely, A1 (16-fold,
rat; 3-fold, human receptors) and A2A affinity (4-fold,
rat; 8-fold, human receptor) were increased by the
exchange of methyl (S-1) for propyl (S-3). As a matter
Chloro-substitution of the 2-phenyl group was explored.
Chlorine atoms at different positions of the phenyl ring
did not have much influence on A2A affinity, which was
generally found to be rather low with all of the
2-phenylimidazopurinones, typically in the micromolar
concentration range. At A1 ARs, chlorination in the 2-,
3- or 4-position of the phenyl ring caused a moderate
(2–7-fold) increase in affinity, with the following rank
order of potency: 2-chlorophenyl (S-5)>3-chlorophenyl
(S-6)>4-chlorophenyl (S-7)>phenyl (S-1). However,

V. Ozola et al. / Bioorg. Med. Chem. 11 (2003) 347–356
351
Table 3. Adenosine receptor affinities and selectivities of 2-phenylimidazo[2,1-i]purin-5-one derivatives
Compd
R2
R4
R8
Ki (nM)ÆSEM or % displacement at concentration indicated (in brackets)
A1 Affinity rat
brain cortical
membranes
[3H]CCPA
A
1 Affinity human
brain cortical
membranes
[3H]CCPA
A
2A Affinity rat
brain striatal
membranes
[3H]MSX-2
A
2A Affinity human
A
3 Affinity human
recombinant
receptors
brain caudate-putamen
membranes
[3H]CGS-21680
[
125I]AB-MECA
N1/N9-unsubstituted imidazopurinones
R-1 (PSB-11)
Phenyl
Methyl (R)-Ethyl
44013a
1640Æ50
210013b
330013b
1280Æ430
2.313
3.5Æ0.39c
9.813
S-1
2
Phenyl
Phenyl
Phenyl
Methyl (S)-Ethyl
Methyl
11513a
2230Æ200
7.4Æ1.4
46Æ1
305Æ25
440Æ80
H
n.d.d
14,100Æ4,460
n.d.
3400Æ2400
n.d.
18,900Æ3200
860Æ640
116Æ17
S-3
S-4
S-5
S-6
S-7
Propyl (S)-Ethyl
Propyl (S)-Ethyl
Methyl (S)-Ethyl
Methyl (S)-Ethyl
Methyl (S)-Ethyl
98Æ12
n.d.
863Æ203
3,4-Dichlorophenyl
2-chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
26%e (2.5 mM)
4170Æ350
2530Æ240
2630Æ320
6040Æ260
17Æ1
90Æ11
2500Æ1200
420Æ340
184Æ129
12Æ1
30Æ2
n.d.
n.d.
n.d.
n.d.
63Æ20
R-8 (PSB-10) 2,3,5-Trichlorophenyl Methyl (R)-Ethyl 805Æ55
1700Æ200
2700Æ500
0.433Æ0.045c
0.997Æ0.311f
N9-methylated derivatives
S-9
R-10
3,4-Dichlorophenyl
2,3,5-Trichlorophenyl Methyl (R)-Ethyl 12%Æ1e (10 mM)
Propyl (S)-Ethyl 2800Æ1,100g
36,000Æ16,500g
35,500Æ18,500g
13,000Æ2,000g
6600Æ600g
2350Æ1150f,g
17%Æ1e (10 mM)
0%e (10 mM)
395Æ55f
aDetermined versus [3H]CHA.
bDetermined versus [3H]CGS21680.
cDetermined versus [3H]NECA.
dn.d.=not determined.
ePercentage inhibition at concentration indicated.
fDetermined versus [3H]PSB-11.
gEstimated value; full curve could not be determined due to limited water solubility.
the introduction of two or three chlorine atoms as in
compounds S-4 (3,4-dichlorophenyl) and R-8 (2,3,5-tri-
chlorophenyl) resulted in a moderate decrease or no
change in A1 affinity (compare S-4/S-3 and R-1/R-8). At
A3 ARs the effect of chlorination was different (opposite
to the effects at A1 ARs) and much more pronounced.
The affinity decreased within the following sequence:
phenyl (S-1)ꢂ4-chlorophenyl (S-7)>> 3-chlorophenyl
(S-6)>2-chlorophenyl (S-5). However, polychlorination
led to an increase in A3 affinity. Thus, the 3,4-dichloro-
phenyl derivative S-4 was 7-fold more potent than the
corresponding phenyl derivative S-3, and the 2,3,5-tri-
chlorophenyl derivative R-8 exhibited 8-fold higher A3
affinity as compared to the corresponding unsubstituted
phenyl derivative R-1. Meanwhile, the trichlorophenyl
derivative R-8 has successfully been also used as a pre-
cursor for the preparation of a novel A3 antagonist
radioligand ([3H]PSB-11), in which the chlorine atoms
were replaced by tritium.19 Compound R-8 is the most
potent A3 antagonist of the present series. It exhibits a
subnanomolar Ki value (Ki=0.433 nM versus
[3H]NECA) and therefore belongs to the most potent A3
antagonists described to date. In addition, R-8 is highly
selective (>800-fold) versus the other AR subtypes.
400-fold). This result confirms the important role of a
hydrogen bond donor/hydrogen bond acceptor motif
(N1-H/N9, or N9-H/N1, respectively, in imidazo[2,1-i]
purinones) for A1 and also for A3 receptor ligands.20 By
methylation this structure is destroyed.
8-Ethyl-2-phenylimidazopurinones
were
generally
weaker (2–13-fold) at human as compared to rat A1
ARs. In contrast, the compounds appeared to be some-
what more potent at human than at rat A2A receptors.
Compounds, such as R-1 and R-8, were highly A3-selec-
tive in a comparison at the human receptor subtypes.
pH-Dependence of AR binding
The pKa value of imidazo[2,1-i]purinones had been
determined to be around 7, close to physiological pH
value.13,14 At physiological pH the compounds are
expected to be partly protonated, which is believed to be
the reason for their improved water-solubility in com-
parison with xanthine derivatives. The cation is stabi-
lized by delocalization of the positive charge. In order to
investigate whether the neutral molecule or the cation
binds preferably to the receptors, and which species will
bind at pH 7.4 (the physiologic pH value used in all of
our experiments), we performed pH-dependent radi-
oligand binding experiments at A1 ARs using com-
pound S-3, a potent, selective A1 antagonist. The results
are shown in Figure 3. At an alkaline pH of 8.0, at
which the compound is unprotonated, the same low Ki
Methylation at N9 of 2-phenylimidazopurinones S-4
and R-8 led to a large decrease in affinity at A1 and A3
ARs (compare S-4/S-9 and R-8/R-10). The affinity
decrease was most pronounced in compound R-10 as
compared to R-8 with respect to the A3 AR (almost

352
V. Ozola et al. / Bioorg. Med. Chem. 11 (2003) 347–356
Figure 3. pH-Dependence of A1 adenosine receptor affinity of imidazopurinone derivative S-3; Ki values at pH 7.4 and 8.0 were not statistically
significantly different from each other; both Ki values were statistically significantly different from Ki value at pH 6.0 (unpaired t-test).
value is obtained as at physiologic pH of 7.4. If the pH
value is reduced to 6 in order to produce the protonated
compound, the Ki value is statistically significantly
reduced by about 2-fold. This may indicate that the
neutral molecule binds preferably to the receptor and
that it is probably the species binding to the receptors
under physiological conditions. An alternative explana-
tion would be that our results reflect different ionization
states of the receptor protein at pH 8.0 and 7.4 versus
pH 6.0.
reduction in [35S]GTPgS binding.24 While agonists are
believed to bind preferentially to the active receptor
conformation, neutral antagonists are thought to have
no preference for either conformation, and inverse ago-
nists to bind preferentially to an inactive conforma-
tion.25 In the present [35S]GTPgS binding study we have
found that the new A3-selective imidazopurinones R-1
and R-8 caused a concentration-dependent reduction in
[35S]GTPgS binding to 84 and 81%, respectively, of
control binding. The EC50 values of this effect corre-
sponded with the Ki values of the compounds at A3
ARs, compound R-8 being several-fold more potent
[35S]GTPꢀS binding studies
Selected imidazo[2,1-i]purin-5-one derivatives have ear-
lier been shown to act as antagonists at A1-, A2A-, and
A2B ARs in adenylate cyclase assays.13 We have now
investigated two potent A3-selective imidazopurinones,
R-1 and R-8, in [35S]GTPgS binding studies using
recombinant human A3 ARs expressed in Chinese
hamster ovary (CHO) cells. The results are shown in
Figure 4. The AR agonist NECA and the A3-selective
AR antagonist MRS-1523, a pyridine derivative (Ki
value at human A3 ARs=19 nM)21 were included as
standard compounds for comparison. The binding of
agonists to certain G-protein-coupled receptors (GPCR)
may be modulated by GTP, which binds to the G-protein.
Thus, the addition of GTP has been shown to cause a
rightward shift of the competition curve for agonists but
not for antagonists at A1 and A3 ARs, both of which
are Gi-coupled receptors.6,22 While GTP modulates
agonist binding, conversely, agonists can also modulate
GTP binding to the G-protein. Thus, agonists increase
the binding of [35S]GTPgS, a radiolabelled stable ana-
logue of GTP.23 This is shown in Figure 4 at the A3 AR
for the agonist NECA, which caused a concentration-
dependent increase in [35S]GTPgS binding with an EC50
value of 77 nM. This corresponds well with the Ki
value of NECA obtained in radioligand binding
experiments at human A3 ARs (Ki=35 nM).6 Neutral
antagonists would not have any influence on
[35S]GTPgS binding, while inverse agonists lead to a
Figure 4. Stimulation (by NECA) and inhibition (by 2-phenylimi-
dazo[2,1-i]purin-5-one derivatives R-1 and R-8) of [35S]GTPgS binding
to membranes prepared from Chinese hamster ovary cells expressing
the human A3 adenosine receptor; NECA (agonist): EC50=77Æ10
nM, max. stimulation (control=100%): 147%; compound R-1:
IC50=36Æ3 nM, max. inhibition: to 81%; compound R-8:
IC50=4.0Æ0.3 nM, max. inhibition: to 84% of control [35S]GTPgS
binding. MRS 1523 caused no significant inhibition of [35S]GTPgS
binding.

V. Ozola et al. / Bioorg. Med. Chem. 11 (2003) 347–356
353
than R-1 (see Fig. 4 and Table 3). Based on these
results, the new A3 antagonists could be characterized
as inverse agonists under the applied test conditions.
In contrast, the standard A3 antagonist MRS-152321
did not have any effect on [35S]GTPgS binding and
thus behaved as a neutral antagonist in this test sys-
tem. Also, the recently described imidazopurinone
derivative KF26777 (2-(p-bromophenyl)-7,8-dihydro-
Additional NMR data of the tricyclic purinone deriva-
tives are available as Supporting Information. Analysis
of enantiomeric purity was performed as described.13
(R)- and (S)-aminobutanol of high optical purity was
obtained from Merck; (R)-aminobutanol: [a]2D0= ꢀ9.5–
10.5, (S)-aminobutanol: [a]2D0=+9ꢀ+10.
4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one
dihydro-
As an example, the preparation of 8-ethyl-4-methyl-2-
(2,3,5-trichlorophenyl)-(8R)-4,5,7,8-tetrahydro-1H-imi-
dazo[2,1-i]purin-5-one (R-8) is described in detail. The
other compounds were synthesized analogously.
chloride), structurally related to R-1 (PSB-11) and R-8
(PSB-10), had no effect on [35S]GTPgS binding and
behaved as a neutral, competitive antagonist at human
A3 adenosine receptors expressed in human embryonic
kidney (HEK) cells.17
8-Ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-(8R)-4,5,7,8-
tetrahydro-1H-imidazo[2,1-i]purin-5-one (R-8). 6-Amino-
1-methyl-5-[(2,3,5-trichlorobenzylidene)amino]-1H-pyri-
midin-2,4-dione. 5,6-Diamino-1-methyluracil (1.88 g, 12
mmol) and 2,3,5-trichlorobenzaldehyde (2.72 g, 13
mmol) were suspended in 50 mL of ethanol and refluxed
for 3 h. The precipitate was collected by filtration,
washed with ethanol and dried in vacuo. Yield: 87%.
The yields for analogous compounds ranged from 87–
Conclusion
In summary, structure–activity relationships of 2-phenyl-
imidazo[2,1-i]purinones as AR ligands have been explored
with respect to the 8-phenyl ring (chlorine substitution),
the N4-alkyl residue (methyl versus propyl), 8-ethyl sub-
stitution versus hydrogen, and N9-methylation. 8-Ethyl-
4-methyl-2-(2,3,5-trichlorophenyl)-(8R)-4,5,7,8-tetra-
hydro-1H-imidazo[2,1-i]purin-5-one (R-8) was developed
as a very potent and selective ligand for human A3 ARs.
The compounds R-1 and R-8 were shown to exhibit
inverse agonistic activity in [35S]GTPgS binding studies at
human recombinant A3 ARs expressed in Chinese ham-
ster ovary cells under standard conditions. Due to their
assumed relatively good water-solubility 2-phenylimi-
dazopurinones may become useful research tools for
studying A3 adenosine receptors and their functions.
1
89%. H NMR (DMSO-d6): d (ppm) 3.34 (s, 3H, CH3),
7.57 (s, 2H, NH2), 7.68 (d, JHH=2.5 Hz, ar), 8.46 (d,
4
4JHH=2.6 Hz, ar), 10.04 (s, 1H, CH), 10.74 (s, 1H,
NH).
3-Methyl-8-(2,3,5-trichlorophenyl)-3,7-dihydro-1H-purin-
2,6-dione. 6-Amino-1-methyl-5-[(2,3,5-trichlorobenzyl-
idene)amino]-1H-pyrimidin-2,4-dione (12.2 g, 35 mmol)
was dissolved in 200 mL of thionyl chloride. The solu-
tion was stirred for 8 h at rt. Excess SOCl2 was removed
by distillation. To the residue saturated aq NaHCO3
solution was added until a pH value of 7 was obtained.
The product was collected by filtration. Yield: 93%. The
yields for analogous compounds ranged from 91–93%.
1H NMR (DMSO-d6): d (ppm) 3.40 (s, 3H, CH3), 7.80
Supporting information available. Additional 1H and 13
NMR data of synthesized compounds.
C
4
4
(d, JHH=2.4 Hz, 1H, ar), 8.01 (d, JHH=2.5 Hz, 1H,
ar), 11.17 (s, 1H, NH).
Experimental
3-Methyl-6-thioxo-8-(2,3,5-trichlorophenyl)-2,3,6,7-tetra-
hydro-1H-purin-2-one. A mixture of 3-methyl-8-(2,3,5-
trichlorophenyl)-3,7-dihydro-1H-purin-2,6-dione (1.3 g,
3.9 mmol) and phosphorus pentasulfide (1.4 g, 6.3
mmol) in 200 mL of dry pyridine was refluxed for 8 h.
After cooling the mixture to rt, 400 mL of H2O was
added and the solution was left standing for 2 h. Then it
was reduced in vacuo to 1/3 of its volume. The formed
precipitate was collected by filtration and suspended in
100 mL of 2 N NaOH. The solution was filtered and the
product was precipitated by adding dilute HCl solution
to achieve a pH value of 4, and the precipitate was col-
lected by filtration. Yield: 86%. 1H NMR (DMSO-d6): d
(ppm) 3.42 (s, 3H, CH3), 7.83 (d, 4JHH=2.5 Hz, 1H, ar),
Synthetic procedures. NMR spectra were performed on a
Varian XL-300 (1H: 300 MHz, 13C : 75 MHz), a Bruker
AMX 500 (1H: 500 MHz, 13C: 125 MHz), and a Bruker
DRX 500 (1H: 500 MHz, 13C: 125 MHz). The chemical
shifts of the deuterated solvent served as internal standard:
d (ppm) DMSO: 1H: 2.50; 13C: 39.1; CHCl3: 1H: 7.24; 13C:
1
77.0; CH2Cl2: H: 5.32; 13C: 53.5. Coupling constants (J)
are given in Hertz (Hz). All compounds were checked for
purity by TLCusing aluminium sheets with s ilica gel 60
F254 (Merck). Preparative HPLCwas performed with a
Knauer HPLCpump 64 using a Eurospher column (type
100-C18: 7mm, 250*20 mm). The melting points were
determined by a Buchi 545 melting point apparatus and
are uncorrected. Optical rotation was determined on a
Perkin–Elmer 241 polarimeter using solutions in DMSO.
4
8.03 (d, JHH=2.4 Hz, 1H, ar), 12.36 (s, 1H, NH).
3-Methyl-6-methylthio-8-(2,3,5-trichlorophenyl)-3,7-di-
hydro-2H-purin-2-one.
3-Methyl-6-thioxo-8-(2,3,5-tri-
Elemental analyses were performed by the Institute of
Pharmaceutical Chemistry, University of Bonn. The
mass spectra were collected on an MS-50 A.E.I. mass
spectrometer. Only selected NMR data for one repre-
sentative member of each class of compounds is given.
chlorophenyl)-2,3,6,7-tetrahydro-1H-purin-2-one (1.0 g,
2.8 mmol) was suspended in 16 mL of 0.5 N NaOH.
EtOH (ca. 5 mL) was added until a clear solution was
obtained. At rt, 4.2 mmol (0.25 mL) of CH3I was added
slowly. After stirring for 1 h, the product was collected

354
V. Ozola et al. / Bioorg. Med. Chem. 11 (2003) 347–356
by filtration and washed with H2O (10 mL). Yield: 86%.
1H NMR (DMSO-d6): d (ppm) 2.65 (s, 3H, S-CH3), 3.50
(s, 3H, N-CH3), 7.88 (d, 4JHH=2.5 Hz, 1H, ar), 8.03 (d,
4JHH=2.4 Hz, 1H, ar).
3JHH=1.9 Hz, 1H, ar). 13CNMR (CDCl 3): d (ppm) 7.9
(CH3); 11.1 (CH3); 21.2 (CH2); 24.3 (CH2); 31.8
(N9CH3); 45.4 (N4CH2); 48.6 (C7); 62.0 (C8); 110.2
(C9b); 125.9 (Car); 128.2 (Car); 130.2 (Car); 131.3 (Car);
132.1 (Car); 135.8 (Car); 147.4 (C3a); 147.5 (C9a); 157.9
(C5); 160.9 (C2).
6-(1-Hydroxymethyl-(1R)-propylamino)-3-methyl-8-(2,3,5-
trichlorophenyl)-3,7-dihydro-2H-purin-2-one. A mixture
of 3-methyl-6-methylthio-8-(2,3,5-trichlorophenyl)-3,7-
dihydro-2H-purin-2-one (0.37 g, 1 mmol) and 0.44 g (5
mmol) of (R)-2-aminobutanol in 1 mL of DMSO was
heated for 1 h at 150 ꢁC. The solvent and the excess
amount of alcohol were removed in vacuo. The residue
was purified by silica gel column chromatography using
a linear gradient CH2Cl2!CH2Cl2/MeOH (93/7, v/v)
R-10: 1H NMR (CDCl3): d (ppm) 1.00 (t, 3JHH=7.5 Hz,
3H, CH3); 1.74 (ddq, JHH=14.8 Hz, JHH=7.5 Hz,
2
3
2
3
1H, CH2); 1.96 (ddq, JHH=14.8 Hz, JHH=7.5 Hz,
3JHH=3.2 Hz, 1H, CH2); 3.60 (s, 3H, CH3); 3.66 (s, 3H,
2
3
CH3); 3.94 (dd, JHH=11.4 Hz, JHH=7.2 Hz, 1H,
2
CH2); 4.07 (m, 1H, CH); 4.37 (dd, JHH=11.34 Hz,
4
3JHH=9.8 Hz, CH2); 7.43 (d, JHH=2.5 Hz, 1H, ar);
4
followed by recrystallization from
a
mixture of
7.85 (d, JHH=2.5 Hz, 1H, ar). 13C NMR (CDCl3): d
1
H2O:EtOH (20:80). Yield: 43%. H NMR (DMSO-d6):
d (ppm) 0.93 (m, 3H, CH3), 1.40–1.82 (m, 2H, CH2),
3.43 (s, 3H, CH3), 3.48–3.73 (m, 2H, CH2), 4.10 (br, 1H,
(ppm) 8.0 (CH3); 24.4 (CH2); 30.3 (N4CH3); 32.1
(N9CH3); 48.6 (C7); 62.0 (C8); 109.6 (C9b); 129.4 (Car);
129.9 (Car); 130.0 (Car); 132.2 (Car); 134.7 (Car); 137.1
(Car); 147.7 (C3a); 147.8 (C9a); 157.4 (C5); 160.4 (C2).
3
OH), 4.97 (m, 1H, CH), 7.51 (d, JHH=7.9 Hz, NH),
7.73, (s, 1H, ar), 7.99 (s, 1H, ar).
Receptor binding studies
8-Ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-(8R)-4,5,7,8-
tetrahydro-1H-imidazo[2,1-i]purin-5-one (R-8). To 30
mL of thionyl chloride (cooled to 0 ꢁC), 0.3 mmol of
6-(1-hydroxymethyl-1R-propylamino)-3-methyl-8-(2,3,5-
trichlorophenyl)-3,7-dihydro-2H-purin-2-one was added.
The mixture was refluxed for 60 min. Excess thionyl
chloride was removed by vacuum distillation. The residue
was purified by silica gel chromatography using a linear
gradient CH2Cl2!CH2Cl2/MeOH (93/7, v/v) followed
by recrystallization from a mixture of H2O:EtOH
(10:90). Yield: 43%. 1H NMR (DMSO-d6): d (ppm)
Radioligand competition experiments were performed
as previously described using rat brain cortical mem-
brane preparations as a source of A1 ARs, and rat brain
striatal membrane preparation for A2A AR
assays.13,26,27 Frozen rat brains (unstripped) were
obtained from Pel-Freez, Rogers, Arkansas, USA. For
assays at human A3 ARs, CHO or HEK cell membranes
recombinantly expressing the human A3 receptor sub-
type were used as described.13,28 Human post-mortem
brain membrane preparations were used for radioligand
binding assays at human A1 and A2A ARs; human
frontal cortex was used as A1 AR source, caudate-
putamen as A2A AR-containing tissue. The latter was
prepared as previously described.29 Human frontal cor-
tex membranes were prepared analogously. Frontal
cortex was pooled from three male subjects (36, 40 and
44 years old), caudate-putamen was from five subjects (1
female, 50 years, 4 male, 23, 35, 35, and 40 years old) with
no history of brain diseases (‘control brains’). The brains
were dissected no longer than 24–48 h post mortem.
3
0.93 (t, JHH=7.3 Hz, 3H, CH3), 1.67 (m, 2H, CH2),
2
3
3.46 (s, 3H, CH3), 3.78 (dd, JHH=9.7 Hz, JHH=5.4
Hz, 1H, CH2); 4.28–4.15 (m, 2H, CH, CH2); 7.77 (d,
4
4JHH=2.6H, 1H, ar); 7.94 (d, JHH=2.6 Hz, 1H, ar).
2-(3,4-Dichlorophenyl)-8-ethyl-9-methyl-4-propyl-(8S)-
4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (S-9),
4,9 - dimethyl - 8 - ethyl - 2 - (2,3,5 - trichlorophenyl) - (8R) -
4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (R-10).
To a solution of the imidazo[2,1-i]purin-5-one (0.2
mmol) S-4, or R-8, respectively, in 5 mL of DMF
sodium hydride (20–30 mg) was added portionwise.
After stirring for 15 min at 0 ꢁC , 93mL (1.5 mmol) of
methyl iodide was added, and the mixture was stirred
for 45 min at rt. The excess sodium hydride was filtered
off and the product was extracted with CHCl3, washed
with water and brine and subsequently concentrated by
rotary evaporation. The product was purified by silica
[3H]2-Chloro-N6-cyclopentyladenosine ([3H]CCPA) was
used as A1 radioligand, [3H]3-(3-hydroxypropyl)-7-
methyl - 8 - (m - methoxystyryl) - 1 - propargylxanthine
([3H]MSX-2) as A2A ligand at rat receptors, and [3H]-2-
[[4 - (carboxyethyl)phenyl]ethyl]amino] - 50 - N - (ethyl-
carbonyl)amino]adenosine ([3H]CGS21680) as A2A
radioligand at human A2A ARs.26ꢀ29
3,4-dihydroxy-5-[6-[3-iodobenzyl)amino]-9H-9-purinyl)-
3-tetrahydro-2-furancarboxamide
([125I]AB-MECA),
[3H]N2-ethyl-3,4-dihydroxy-5-[6-amino]-9H-9-purinyl)-3-
tetrahydro-2-furancarboxamide ([3H]NECA), or [3H]2-
phenyl-8-ethyl-4-methyl-(8R)-4,5,7,8-tetrahydro-1H-
imidazo[2,1-i]purin-5-one ([3H]PSB-11), respectively,
were employed as radioligands for the human recombi-
nant A3 ARs.13,18,19,28
[
125I]N2-methyl-
gel chromatography using
a
linear gradient:
CH2Cl2!CH2Cl2/MeOH (95:5, v/v). Further purifi-
cation of compound R-10 could be achieved by pre-
parative HPLCusing a linear gradient: MeOH/H 2O
(60:40)!MeOH/H2O (90:10) over 30 min.
1
S-9: H NMR (CDCl3): d (ppm) 0.90 (m, 6H, 2*CH3),
1.73 (m, 1H, CH2), 1.85 (m, 2H, CH2), 1.95 (m, 1H,
2
CH2);3.57 (s, 3H, NCH3), 3.87 (dd, JHH=10.5 Hz,
3JHH=7.25 Hz, 1H, CH2), 4.00 (m, 1H, CH); 4.12 (t,
Curves were determined using 6–7 different concen-
trations of test compounds spanning 3 orders of magni-
tude. At least two to three separate experiments were
performed each in duplicate or triplicate.
2
3JHH=7.6 Hz, 2H, CH2); 4.28 (dd, JHH=10.5 Hz, 1H,
3
3JHH=10.5 Hz, CH2); 7.45 (d, JHH=8.3 Hz, 1H, ar);
3
8.07 (dd, JHH=8.3 Hz, 3JHH=1.9 Hz, 1H, ar); 8.34 (d,

V. Ozola et al. / Bioorg. Med. Chem. 11 (2003) 347–356
355
Data were analyzed using Graph Pad PRISM version
3.0 (San Diego, CA, USA). For statistical comparison
between groups, data were subjected to analysis of var-
iance followed by unpaired t-test.
gemeinschaft within the Graduiertenkolleg GRK 677
(scholarship for B. Schumacher). C. E. Muller is
grateful for support by the Fonds der Chemischen
Industrie and the Bundesministerium fur Bildung und
Forschung (BMBF). The post mortem human brain
material was kindly provided by Professor Dr. P.
Falkai, University of Bonn, Clinic for Psychiatry and
Psychotherapy.
Radioligand binding assays at human post-mortem brain
cortical A1 adenosine receptors
The assays were performed essentially as the assays at
rat A1 ARs using 0.5 nM of [3H]CCPA (KD=3.8 nM),
2-chloroadenosine (400 mM) to define nonspecific bind-
ing in 50 mM Tris–HCl buffer (pH 7.4) in a final volume
of 1 mL. Membrane suspensions were washed with the
buffer before starting the assay by centrifugation at
35,000g for 20 min (4 ꢁC) and subsequently pre-
incubated with 0.12 I.U./mL of adenosine deaminase
for 30 min. Protein concentration in the assay was ca. 75
mg/mL. Incubation was at 23 ꢁCfor 90 min in a shaking
water-bath and was terminated by rapid filtration
through glass fiber GF/B filters.
References and Notes
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12. Suzuki, F.; Shimada, J.; Nonaka, H.; Ishii, A.; Shiozaki,
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14. Hipp, J. Dissertation, University of Wurzburg, 1997.
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16. Tsumuki, H.; Saki, M.; Nonaka, H.; Ichimura, M.; Shi-
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[35S]GTPꢀS-binding studies
The incubation mixture contained in a total volume of 200
mL, 50 mM Tris–HCl pH 7.4, 1 mM EDTA, 100 mM
NaCl, 0.2 I.U./ mL adenosine deaminase, 5 mM MgCl2, 1
mM DTT, 10 mM GDP, 0.5% (w/v) bovine serum albu-
min, 0.1–0.5 nM [35S]GTPgS (about 30,000 cpm) and test
compound diluted in DMSO. To start the incubation, 7.5
mg of membrane protein were added to the glass tubes and
kept for 45 min at 25ꢁCon a shaking water bath. Incu-
bation was terminated by adding 4 ꢁCcold rinse buffer (50
mM Tris–HCl pH 7.4, 5 mM MgCl2) and rapid filtration
through glass fiber filters (Whatman GF/B) using a Bran-
dell cell harvester. Nonspecific binding was determined in
the presence of 10 mM of unlabelled GTPgS and was less
than 0.5% of total [35S]GTPgS binding. Filters were pun-
ched out and transferred to minivials and 3 mL of Ultima
Gold scintillation cocktail (Canberra Packard) was
added. After 180 min of incubation, the samples were
counted in an LSCTricarb 2100TR (Canberra Packard).
EC50 and IC50 values were calculated from dose–
response curves using the program Graph Pad Prism
3.00, and the inbuilt equations for sigmoidal dose
response with variable slope. Experiments were car-
ried out in triplicates in at least two independent
experiments.
1H and 13CNMR spectral data of intermediate and final
products
18. Hayallah, A. M.; Sandoval-Ramirez, J.; Reith, U.; Scho-
bert, U.; Preiss, B.; Schumacher, B.; Daly, J. W.; Muller, C. E.
J. Med. Chem. 2002, 45, 1500.
Additional NMR spectral data are provided for most
intermediate and all final products.
19. Muller, C. E.; Diekmann, M.; Thorand, M.; Ozola, V.
Bioorg. Med. Chem. Lett. 2002, 12, 501.
20. Muller, C. E. Exp. Opin. Ther. Patents 1997, 7, 419.
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Acknowledgements
We thank Stefanie Weyler, Ulrike Reith and Sonja Hinz
for performing some of the radioligand binding assays.
V. Ozola was on leave from the Latvian Institute of
Organic Synthesis in Riga, Latvia and was supported by
a Humboldt fellowship (Roman Herzog program). This
work was supported by the Deutsche Forschungs-
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