7-Deazaadenines bearing polar substituents: Structure - Activity relationships of new A1 and A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm000967d

A series of 28 new pyrrolo[2,3-d]pyrimidine-4-amines, pyrimido[4,5-b]indole-4-amines, and tetrahydropyrimido [4,5-b] indole-4-amines was synthesized and their adenosine receptor affinity determined in radioligand binding assays at rat A₁ and A(

Full text of DOI:10.1021/jm000967d

4636
J . Med. Chem. 2000, 43, 4636-4646
7-Dea za a d en in es Bea r in g P ola r Su bstitu en ts: Str u ctu r e-Activity Rela tion sh ip s
of New A1 a n d A3 Ad en osin e Recep tor An ta gon ists†
Sonja Hess,*,§ Christa E. Mu¨ller,‡,| Wolfram Frobenius,‡ Ulrike Reith,‡,| Karl-Norbert Klotz, and Kurt Eger§
Pharmaceutical Chemistry, Institute of Pharmacy, University of Leipzig, Bru¨derstrasse 34, D-04103 Leipzig, Germany,
Institute of Pharmacy and Food Chemistry, J ulius-Maximilians-Universita¨t Wu¨rzburg, Am Hubland,
D-97074 Wu¨rzburg, Germany, Institute of Pharmacology and Toxicology, J ulius-Maximilians-Universita¨t Wu¨rzburg,
Versbacher Strasse 9, D-97078 Wu¨rzburg, Germany, and Pharmaceutical Institute, University of Bonn, Kreuzbergweg 26,
D-53115 Bonn, Germany
Received J uly 17, 2000
A series of 28 new pyrrolo[2,3-d]pyrimidine-4-amines, pyrimido[4,5-b]indole-4-amines, and
tetrahydropyrimido[4,5-b]indole-4-amines was synthesized and their adenosine receptor affinity
determined in radioligand binding assays at rat A1 and A2A adenosine receptors (ARs). Selected
compounds were additionally investigated in binding assays at recombinant A3 ARs. The
2-phenyl residue in (R)-7-(1-methylbenzyl)-2-phenylpyrrolo[2,3-d]pyrimidine-4-amine (ADPEP,
1) and in the corresponding pyrimido[4,5-b]indole (APEPI, 3) could be bioisosterically replaced
by heterocyclic rings, such as 2-thienyl and 4-pyridyl. The resulting compounds retained high
affinity and selectivity for A1 ARs. J udging from the investigation of selected compounds, it
appears that they are also potent at human A1 ARs and selective not only versus A2A ARs but
also highly selective versus A2B and A3 ARs. The p-pyridyl-substituted derivatives 11 and 27
(APPPI) may be interesting pharmacological tools due to their fluorescent properties. Pyrrolo-
[2,3-d]pyrimidine-4-amine derivatives which were simultaneously substituted at N7 and N4,
combining the substitution pattern of ADPEP (1) and DPEAP (2), showed very low affinity for
A1 ARs. This finding supports our previously published hypothesis of different binding modes
for pyrrolopyrimidines, such as ADPEP (1) and DPEAP (2). DPEAP (2), a pyrrolo[2,3-d]-
pyrimidine-4-amine substituted at the amino group (N4), was found to exhibit high affinity for
human A3 ARs (Ki ) 28 nM), whereas N4-unsubstituted analogues were inactive. DPEAP (2)
and related compounds provide new leads for the development of antagonists for the human
A3 AR.
In tr od u ction
[1,5-a]pyridines.5-11 For the well-known A1 and A2A ARs
many agonists and antagonists have been developed
during the past decades.5-11
Adenosine receptors (ARs) belong to the superfamily
of G-protein-coupled receptors and are currently sub-
divided into the following subtypes: A1, A2A, A2B, and
A3.1,2 A1 and A3 AR activation can lead to an inhibition
of adenylate cyclase activity, while A2A and A2B AR
activation causes a stimulation of adenylate cyclase. In
addition, coupling to other second-messenger systems
has been described, including calcium or potassium ion
channels (A1) or phospholipase C (A1, A2B, A3).3
Agonists for ARs are all derived from the physiological
agonist adenosine. The ribose moiety appears to be
essential for agonistic activity.4 Adenine derivatives and
analogues lacking the ribose moiety have been shown
to act as antagonists at ARs.2,4 A variety of different
classes of heterocyclic compounds has been described
to possess antagonistic activity at ARs, including xan-
thines, adenines, 7-deazaadenines, 7-deaza-8-aza-
purines, triazolo[1,5-a]quinoxalines, and pyrazolo-
However, compounds which are believed to be highly
selective for A1 and A2A ARs, respectively, have to be
reevaluated, since (i) they may not be selective versus
A2B and/or A3 ARs and (ii) they may only be selective
in some species, such as rat but not humans. In fact,
several generally used “selective” ligands have recently
been found to be not very selective when tested in the
now available cell culture systems expressing the hu-
man AR subtypes, e.g., the A2A agonist CGS21680 is
only 2-fold selective for human A2A versus human A3
ARs.12-14 The A3 AR is the latest member of the AR
family. Recently, A3 AR agonists (adenosine derivatives)
and antagonists (including flavonoids, dihydropyridines,
triazolonaphthyridine, isoquinoline, quinazoline deriva-
tives) have been developed.13-19
Truly selective AR antagonists for the various sub-
types are needed as pharmacological tools and are also
of considerable interest as potential drugs. Possible
therapeutic applications for A1 antagonists include
cognitive deficits, renal failure, and cardiac arrhyth-
mias,5 while A2A antagonists may be beneficial for
patients suffering from Morbus Parkinson.20 A3 Antago-
nists are associated with cerebroprotective proper-
ties.14,21
† Preliminary results were presented in Ferrara at the 6th Inter-
national Symposium on Adenosine and Adenine Nucleotides, 1998;
abstract published in Drug Dev. Res. 1998, 43, 34.
* To whom correspondence should be addressed. Phone: +49-(0)-
341-97-36884. Fax: +49-(0)341-97-36709. E-mail: hess@rz.uni-leipzig.de.
‡ Institute of Pharmacy and Food Chemistry, J ulius-Maximilians-
Universita¨t Wu¨rzburg.
| University of Bonn.
Institute of Pharmacology and Toxicology, J ulius-Maximilians-
Universita¨t Wu¨rzburg.
During our search for novel AR antagonists, it was
found that 7-deazaadenines with the pyrrolo[2,3-d]-
§ University of Leipzig.
10.1021/jm000967d CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/11/2000

7-Deazaadenines Bearing Polar Substituents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4637
Ch a r t 1. Potent A1 AR Antagonists with
7-Deazaadenine Structure: Pyrrolo[2,3-d]pyrimidines 1
and 2 and Pyrimido[4,5-b]indole 3
Sch em e 1. Syntheses of Pyrrolo[2,3-d]pyrimidines,
Tetrahydropyrimido[4,5-b]indoles, and
Pyrimido[4,5-b]indoles (Method A)a
a
For R1-R4, see Chart 2 and Table 1. Reaction conditions: (a)
appropriate carbonitrile, sodium methylate, 2-propanol.
hydrogen bond donor (the exocyclic amino group in 1
and the pyrrole N7-H in 3) and a nearby hydrogen bond
acceptor (N3 in 1 and N1 in 3) are superimposed.11
Recently, Campbell et al. synthesized a series of
analogues of DPEAP (2), in which the substituent at
the exocyclic amino group was replaced by hydrophilic
residues.24 The new derivatives were investigated at
recombinant human A1 and A2A ARs. Some of them,
including the N-(trans-4-hydroxycyclohexyl) derivative
proved to be potent and selective antagonists at human
A1 ARs.24 We have now prepared further derivatives
substituted at the exocyclic amino group (analogues of
DPEAP, 2), including derivatives which are disubsti-
tuted at N4 and N7. These compounds were synthesized
and investigated at ARs in order to test our previously
designed pharmacophore model of different binding
modes.
pyrimidine and pyrimido[4,5-b]indole structure (see
Chart 1) were potent and selective AR antagonists.11,22,23
Pyrrolo[2,3-d]pyrimidine-4-amines bearing a 2-phenyl
substituent turned out to be particularly active at A1
ARs and highly selective for that receptor subtype.11,23
Thus, ADPEP (1) exhibited a Ki value at A1 ARs of rat
brain of 4.7 nM and ca. 800-fold selectivity as compared
to the high-affinity A2A ARs of rat striatum. APEPI (3)
showed a Ki value at A1 ARs of rat brain of 2.6 nM and
greater than 2000-fold selectivity toward A2A ARs.11,23
The compounds exhibited a high degree of stereoselec-
tivity, the (R)-configurated stereoisomers being 20-100-
fold more potent than the (S)-enantiomers.
In the present study we have prepared and investi-
gated a series of new 7-deazaadenine derivatives,
mainly with variation of the substituent in the 2-posi-
tion. Thus, pyrrolo[2,3-d]pyrimidines, tetrahydropy-
rimido[4,5-b]indoles, and pyrimido[4,5-b]indoles were
prepared bearing a substituted 2-phenyl group, or in
which the 2-phenyl substituent was bioisosterically
replaced by heteroaromatic rings, or a 1-methylbenzyl
residue. Our goal was to investigate the electronic and
steric requirements of the ARs with respect to the
2-substituent in these classes of compounds and to gain
further insight into their structure-activity relation-
ships (SARs). Furthermore, we tried to increase the
water solubility of the highly lipophilic parent com-
pounds by the introduction of basic heterocycles (e.g.,
pyridyl residues) which would allow for the formation
of more polar hydrochlorides.
Ch em istr y
Most of the products prepared were chiral, and in
most cases only the (R)-enantiomers, which had previ-
ously been shown to be the eutomers, were synthesized.
Ring-closure reactions (educts 4-8, see Chart 1) to
pyrrolo[2,3-d]pyrimidines and pyrimido[4,5-b]indoles
were performed either in a one-pot reaction (method A,
Scheme 1) or in two steps via acylamido derivatives and
subsequent ring closure (method B, Scheme 3).
Meth od A: On e-P ot Syn th esis. 5,6-Dimethylpyr-
rolo[2,3-d]pyrimidine-4-amines 9-17 were prepared
starting from the pyrrole derivative 4 (Chart 2 and
Scheme 1). Compound 4 was obtained in a one-pot
reaction developed by Eger et al. condensing 3-hydroxy-
2-butanone and (R)-1-methylbenzylamine in the pres-
ence of p-toluenesulfonic acid, followed by base-cata-
lyzed cyclization with malonodinitrile as described.25
Tetrahydroindoles 5 and 6 (Chart 2) were prepared
analogously.25e Ring closure was carried out using the
appropriate carbonitriles in the presence of sodium
methylate in 2-propanol (Scheme 1).
Reaction of pyrrolo[2,3-d]pyrimidine 11 with 2-pro-
piolic acid methyl ester at room temperature yielded 29.
The 7-unsubstituted pyrrolo[2,3-d]pyrimidine 30 was
prepared from racemic compound 17. Dealkylation in
the 7-position by heating with polyphosphoric acid
yielded racemic 30.25d Due to its low affinity at ARs,
pure enantiomers of 30 were not prepared. The hydroxy-
phenyl-substituted pyrrolo[2,3-d]pyrimidine 31 was pre-
pared from methoxyphenyl derivative 16 by cleavage
of the methyl ether using the Lewis acid boron tribro-
mide (Scheme 2).
An isomer of pyrrolo[2,3-d]pyrimidine 1 (ADPEP), in
which the (R)-1-methylbenzyl substituent was moved
from the pyrrole nitrogen atom to the exocyclic amino
group (DPEAP, 2), had proven to be a similarly potent
A1 AR antagonist as compared to ADPEP (1). On the
basis of these results, we had postulated different
binding modes for the two classes of compounds: 7-sub-
stituted pyrrolopyrimidines, such as ADPEP (1), on the
one hand and N4-substituted derivatives, such as DPEAP
(2), on the other hand.11 In our computer model, the two
compounds have been aligned in such a way that the
(R)-1-methylbenzyl substituents are overlapping and a
Indole 7 was prepared from tetrahydroindole 6 by
oxidation using dichlorodicyanoquinone (DDQ) in tet-

4638 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24
Ch a r t 2. Educts Synthesized
Hess et al.
Sch em e 2. Syntheses of Amino-Substituted
Pyrrolo[2,3-d]pyrimidines 29-31a
rahydrofuran (Chart 2). Reaction of tetrahydroindole 5
with the appropriate pyridinecarbonitriles in the pres-
ence of sodium methylate in 2-propanol yielded the
2-phenyltetrahydropyrimido[4,5-b]indoleamine deriva-
tives 18-20 following described procedures (Scheme
1).11,25 Analogously, compounds 21-24 were prepared
using the tetrahydroindole 6, and 25-28 were obtained
starting from indole 7 (Scheme 1).
Meth od B: Tw o-Step Syn th esis. Pyrroles 4 and 8
(Chart 2) were used as starting material to prepare N4-
a
Reaction conditions: (a) propiolic acid methylate, glacial acetic
acid; (b) phosphorus pentoxide; (c) boron tribromide, CH2Cl2.
substituted
5,6-dimethylpyrrolo[2,3-d]pyrimidines
(Scheme 3). Thus, the pyrroles were reacted with the
appropriate benzoyl chloride derivative to yield the
acylated pyrroles 32- 34, respectively. Pyrrolo[2,3-d]-
pyrimidine derivatives 35-37 were prepared by cycliza-
tion of 32-34 using phosphorus pentoxide in a mixture
of dimethylcyclohexylamine and water at 190 °C (Scheme
3). Reaction of 35-37 with phosphorus oxychloride
yielded chloro-substituted 5,6-dimethylpyrrolo[2,3-d]-
pyrimidines 38-40. The desired N4-substituted 5,6-
dimethylpyrrolo[2,3-d]pyrimidines 41-45 were pre-
pared from 38-40 by reaction with the appropriate
amines (Scheme 3).
Reagents, yields, and analytical data are given in
Table 1. 1H and 13C NMR spectral data were in ac-
cordance with the proposed structures. Selected NMR
data are given in the Experimental Section. Further
NMR data are available as Supporting Information.
Biologica l Eva lu a tion
The compounds were tested in radioligand binding
assays for affinity at A1 and A2A ARs in rat brain cortical
Sch em e 3. Syntheses of N4-Substituted Pyrrolo[2,3-d]pyrimidine Derivatives (Method B)a
a
Reaction conditions: (a) pyridine, CH2Cl2, 0 °C; (b) phosphorus pentoxide, N,N-dimethylcyclohexylamine, H2O, 190 °C; (c) POCl3; (d)
appropriate amine derivative.

7-Deazaadenines Bearing Polar Substituents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4639
Ta ble 1. Reagents, Yields, and Analytical Data of the New Compounds Synthesized
compd
reagent, g (mmol)
yield [g (%)]
formula
anal.a
TS-MS or EI-MS (70 eV) m/z mp (°C)
Type I: Pyrrolo[2,3-d]pyrimidine-4-amine Derivatives
9
10
11
12
13
14
15
16
17
29
30
31
34
37
39
40
41
42
43
44
45
2-pyridinecarbonitrile, 1.9 (18)
3-pyridinecarbonitrile, 1.9 (18)
4-pyridinecarbonitrile, 1.9 (18)
4-pyridinecarbonitrile N-oxide, 2.2 (18)
2-pyrazinecarbonitrile, 1.9 (18)
2-thiophenecarbonitrile, 2.0 (18)
2-furonitrile, 1.7 (18)
1.3 (21%)
2.8 (45%)
3.4 (55%)
4.1 (64%)
1.2 (20%)
3.6 (58%)
2.5 (42%)
1.8 (27%)
C
C
C
C
21H21N5
21H21N5
21H21N5
21H21N5O
C, H, N
C, H, N
C, H, N
C, H, N
C, H, Nb
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
TS-POS: 344 [M + H+]
TS-POS: 344 [M + H+]
TS-POS: 344 [M + H+]
EI-MS: 359 [M+]
233
249
171
264
210
168
193
125
144
175
165
154
180
266
133
158
145
176
122
232
70
C20H20N6
TS-POS: 345 [M + H+]
TS-NEG: 347 [M - H+]
TS-POS: 333 [M + H+]
EI-MS: 372 [M+]
C
C
C
C
C
C
C
20H20N4S
20H20N4O
23H24N4O
24H26N4
25H25N5O2
16H18N4
22H22N4O
2-methoxybenzonitrile, 2.4 (18)
(R,S)-1-methylphenylacetonitrile, 2.4 (18) 1.4 (38%)
EI-MS: 370 [M+]
TS-POS: 428
11, 0.70 (2)
17, 2.6 (7)
0.60 (70%)
1.4 (76%)
0.08 (21%)
4.4 (35%)
0.847 (69%)
1.6 (52%)
0.352 (54%)
1.003 (77%)
0.511 (83%)
0.704 (91%)
NCI: 265 [M - H+]
TS-POS: 359 [M + H+]
TS-POS: 308 [M + H+]
TS-POS: 308 [M + H+]
TS-POS: 397 [M++ 2]
TS-POS: 326 [M+]
16, 0.37 (1)
8, 2.5 (12.5)
34, 2.5 (8)
C19H21N3O
C
C
C
C
C
C
19H21N3O
22H19N3Cl2
19H20N3Cl
30H30N4
27H30N4
24H28N5
36, 3.0 (8)
37, 0.615 (2)
38, 1.085 (3)
38, 0.543 (1.5)
38, 0.732 (1.5)
39, 1.189 (3.0)
40, 0.326 (1.0)
TS-POS: 447 [M + H+]
TS-POS: 411 [M + H+]
TS-POS: 387 [M + H+]
TS-POS: 448 [M+]
0.687 (51%) C26H29N4OCl C, H, N
0.351 (93%)
C
23H30N4O
C, H, N
TS-POS: 379 [M+]
Type II: Tetrahydropyrimido[4,5-b]indole-4-amine Derivatives
18
19
20
21
22
23
24
2-pyridinecarbonitrile, 1.0 (10)
3-pyridinecarbonitrile, 1.0 (10)
4-pyridinecarbonitrile, 1.0 (10)
2-pyridinecarbonitrile, 2.6 (25)
3-pyridinecarbonitrile, 2.6 (25)
4-pyridinecarbonitrile, 2.6 (25)
4-pyridinecarbonitrile N-oxide, 3.0 (25)
2.1 (62%)
1.2 (35%)
0.7 (21%)
1.2 (13%)
1.0 (11%)
2.0 (22%)
6.0 (62%)
C
C
C
C
C
C
21H19N5
21H19N5
21H19N5
23H23N5
23H23N5
23H23N5
C, H, N
C, H, N
C, H, Nc
C, H, N
C, H, N
C, H, N
C, H, N
EI-MS: 341 [M+]
TS-POS: 342 [M + H+]
EI-MS: 342 [M + H+]
EI-MS: 369 [M+]
EI-MS: 369 [M+]
EI-MS: 369 [M+]
EI-MS: 385 [M+]
266
252
233
249
199
156
242
C23H23N5O
Type III: (R)-(1-Methylbenzyl)pyrimido[4,5-b]indole-4-amine Derivatives
25
26
27
28
2-pyridinecarbonitrile, 1.0 (10)
3-pyridinecarbonitrile, 1.0 (10)
4-pyridinecarbonitrile, 1.0 (10)
2-thiophenecarbonitrile, 1.1 (10)
1.2 (13%)
1.4 (38%)
1.2 (33%)
2.7 (73%)
C
C
C
C
23H19N5
23H19N5
23H19N5
22H18N4S
C, H, N
C, H, N
C, H, N
C, H, N
TS-NEG: 365 [M+]
EI-MS: 364 [M+]
EI-MS: 365 [M+]
EI-MS: 370 [M+]
111
197
170
110
a
b
Elemental analyses were within 0.4% of calculated values, unless otherwise noted. Calcd: 69.75; 5.85; 24.40. Found: 69.22; 5.67;
24.99. c Calcd: 73.88; 5.61; 20.51. Found: 73.77; 6.03; 19.93.
membrane and rat striatal membrane preparations,
respectively. [3H]N6-Cyclohexyladenosine ([3H]CHA, 1
nM) or [3H]-2-chloro-N6-cyclopentyladenosine ([3H]C-
CPA, 0.5 nM), respectively, was used as the A1 ligand
and CGS21680 ([3H]-2-[[[[4-(carboxyethyl)phenyl]ethyl]-
amino]-5′-N-(ethylcarbonyl)amino]adenosine, 5 nM) as
the A2A ligand. All compounds were dissolved in DMSO
and diluted into aqueous buffer solution. Compound 11
was additionally tested at recombinant human A1 ARs
expressed in Chinese hamster ovary (CHO) cells using
[3H]CCPA (0.5 nM). The A3 AR affinity was determined
at human recombinant A3 ARs expressed in CHO cells
using [3H]-5′-[(N-(ethylcarbonyl)amino]adenosine (NECA,
10 nM) as radioligand. The inhibition of NECA-
stimulated adenylate cyclase by test compounds was
measured using human recombinant A2B ARs expressed
in CHO cells.12
Th e 2-Su bstitu en t. 2-Substituted pyrrolo[2,3-d]py-
rimidine-4-amines (I), tetrahydropyrimido[4,5-b]indole-
4-amines (II), and pyrimido[4,5-b]indole-4-amines (III),
in which the 2-phenyl group was bioisosterically re-
placed by heterocyclic rings, were investigated (Table
2). Most of these compounds showed only low or
negligible affinity for the A2A ARs and were A1-selective.
However, these modifications led to a decrease in A1 AR
affinity compared to the parent, 2-phenyl-substituted
compounds ADPEP (1) and APEPI (3). While 2-furyl
(15), 2-thienyl (14), and 4-pyridyl (11) rings were well-
tolerated at the 2-position of pyrrolo[2,3-d]pyrimidine-
4-amines, 2- or 3-pyridyl (compounds 9 and 10), 4-pyridyl-
N-oxide (12), or 2-pyrazinyl (13) substituents in the
same position led to a large reduction in A1 AR affinity.
Similarly, in the pyrimidoindole series (III), a 2-thienyl
(28) or 4-pyridyl (27) substituent in the 2-position was
much better tolerated than 2- or 3-pyridyl residues (25,
26). In general, the pyrimidoindoles (III) were some-
what more potent as compared to the corresponding
pyrrolopyrimidines (I), and similar SARs were observed,
with one striking exception: The 2-(3-pyridyl) derivative
in the pyrimidoindole series (III) retained A1 affinity
(26, Ki ) 92 nM), while the analogous pyrrolopyrimidine
10 was inactive at high concentrations (Table 2). The
rank order of potency for bioisosteric analogues of the
pyrrolopyrimidine ADPEP (1) was 2-furyl (15) g 2-thie-
nyl (14) g 4-pyridyl (11) > 2-pyrazinyl (13) > 2-pyridyl
(9) > 3-pyridyl (10). The rank order of potency for
analogues of the pyrimidoindole APEPI (3) was 4-py-
ridyl (27) g 2-thienyl (28) > 3-pyridyl (26) > 2-pyridyl
(25). The different SAR for the 3-pyridyl-substituted
Resu lts a n d Discu ssion
7-Deazaadenines have been investigated as antago-
nists at A1 and A2A ARs. In earlier studies we showed
that 7-deazaadenines are antagonists at A1 and A2A ARs
in adenylate cyclase assays.11,22,23 In contrast to the
7-deazaadenine derivatives presented, all AR agonists
and partial agonists described so far contain a (substi-
tuted) ribose or an analogous structure.14 The most
potent compound of this series, 27 (APPPI), has ad-
ditionally been investigated in [35S]GTPγS binding
studies at A1 ARs and clearly shown to be an A1 AR
antagonist (data not shown). On the basis of these
results, it is assumed that all 7-deaazaadenines de-
scribed herein are antagonists at ARs.

4640 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24
Hess et al.
Ta ble 2. AR Affinities of Pyrrolo[2,3-d]pyrimidine-4-amines, Tetrahydropyrimido[4,5-b]indole-4-amines, and
Pyrimido[4,5-b]indole-4-amines
Ki ( SEM (µM)
A2A AR vs
A1 AR vs [3H]CHA
or [3H]CCPA rat
brain cortex
A3 AR vs
[3H]CGS21680 [3H]NECA human
compd
R1
R2
R3
rat striatum
recomb receptors
Pyrrolo[2,3-d]pyrimidine-4-amines (I)
1 (ADPEP)
(R)-1-methylbenzyl phenyl
H
0.004711
3.7111
2 (DPEAP)d
H
phenyl
(R)-1-methylbenzyl
0.006711
23% (30 µM)11
43% (30 µM)b
3% (30 µM)b
1.3 ( 0.1
0.028 (10.4-77.8)c
9
10
11
(R)-1-methylbenzyl 2-pyridyl
(R)-1-methylbenzyl 3-pyridyl
(R)-1-methylbenzyl 4-pyridyl
H
H
H
2.1 ( 0.3
35% (100 µM)
0.046 ( 0.017
(0.0088 ( 0.0013e)
0.49 ( 0.025a
0.56 ( 0.12
12
13
14
15
16
17
29
30
31
41
42
43
44
45
(R)-1-methylbenzyl 4-pyridyl N-oxide
(R)-1-methylbenzyl 2-pyrazinyl
(R)-1-methylbenzyl 2-thienyl
H
H
H
H
H
H
5.9 ( 2.7
6.6 ( 0.5
0.019 ( 0.002a
0.016 ( 0.005a
38% (30 µM)a,b
33% (30 µM)a,b
5.4 ( 0.75
(R)-1-methylbenzyl 2-furyl
0.49 ( 0.08
14% (30 µM)b
38% (30 µM)b
2.3 ( 0.19
(R)-1-methylbenzyl 2-methoxyphenyl
(R)-1-methylbenzyl (R,S)-1-methylbenzyl
(R)-1-methylbenzyl 4-pyridyl
(R,S)-1-methylbenzyl
(R)-1-methylbenzyl 2-hydroxyphenyl
(R)-1-methylbenzyl phenyl
acrylic acid methylate 3.0 ( 0.25
H
H
(R)-1-methylbenzyl
cyclopentyl
aminoethyl
(R,S)-2-butan-1-ol
(R,S)-2-butan-1-ol
2.64 ( 0.32
H
6.84 ( 0.001a
0.24 ( 0.04a
18.3 ( 1.0
10% (10 µM)b
46% (30 µM)b
26% (30 µM)b
19% (30 µM)b
2% (30 µM)b
36% (30 µM)b
22% (30 µM)b
11.4 ( 2.6
8.65 ( 2.97
5.58 ( 0.53
0.57 ( 0.26
1.87 ( 0.23
(R)-1-methylbenzyl phenyl
(R)-1-methylbenzyl phenyl
(R)-1-methylbenzyl 4-chlorphenyl
41% (30 µM)b
3.58 ( 1.64a
2.6 ( 0.75a
cyclopentyl
phenyl
1.24 ( 0.41a
Tetrahydropyrimido[4,5-b]indole-4-amines (II)
18
19
20
21
22
23
24
phenyl
phenyl
phenyl
2-pyridyl
3-pyridyl
4-pyridyl
8.9 ( 2.0
42% (30 µM)b
44% (30 µM)b
28% (10 µM)b
38% (30 µM)b
46% (30 µM)b
2% (30 µM)b
50% (30 µM)b
7.9 ( 2.3
0.6 ( 0.3
(R)-1-methylbenzyl 2-pyridyl
(R)-1-methylbenzyl 3-pyridyl
(R)-1-methylbenzyl 4-pyridyl
12.5 ( 1.8
1.3 ( 0.03
8% (100 µM)a
>10
(R)-1-methylbenzyl 4-pyridyl N-oxide
1.85 ( 0.29a
Pyrimido[4,5-b]indole-4-amines (III)
3 (APEPI)d
(R)-1-methylbenzyl phenyl
(R)-1-methylbenzyl 2-pyridyl
(R)-1-methylbenzyl 3-pyridyl
0.002611
6.211
>1
25
26
0.54 ( 0.15a
0.092 ( 0.017a
0.021 ( 0.002
0.036 ( 0.006a
6% (1 µM)b
47% (10 µM)b
1.85 ( 0.2
32% (3 µM)b
27 (APPPI)d (R)-1-methylbenzyl 4-pyridyl
>10
28
(R)-1-methylbenzyl 2-thienyl
a
b
d
[3H]CCPA was used as radioligand. Percent inhibition at the indicated concentration. c 95% confidence limits. IC50 at human A2B
ARs > 10 µM. e Ki value at human recombinant A1 ARs expressed in CHO cells, determined with [3H]CCPA as radioligand.
compounds 10 and 26 could indicate different binding
modes for some members of the two series.
in the A1 AR affinity.11 In the present study, it was
found that the same substituent in the ortho-position
led to an even more drastic reduction in A1 AR affinity.
Thus, compound 16 showed an IC50 value of greater
than 30 µM (Table 2). The smaller hydroxyl substituent
(compound 31) was better tolerated (Ki A1 ) 0.24 µM)
but still led to a 50-fold reduction as compared to the
unsubstituted parent compound 1. The receptor does not
appear to accept large substituents in the ortho-position
of the 2-phenyl group, as it does not tolerate nitrogen
atoms (2-pyridyl substitution) at the same position
(compounds 9 and 21). A chlorine atom in the ortho-
position, however, only led to a 2-fold decrease in A1 AR
affinity.11
Tetrahydropyrimido[4,5-b]indole derivatives (II) ex-
hibited only weak A1 AR affinity as compared to the
corresponding pyrrolopyrimidines (I) and pyrimidoin-
doles (III); Ki values at the A1 AR were in the micro-
molar concentration range (compounds 18-24, Table 2).
It appears that a flat aromatic ring system, as in the
pyrimidoindoles, is well-accommodated by the A1 AR.
The tetrahydroyprimidoindole ring system (II) is unfa-
vorable, probably due to steric interference with the
receptor protein. The less spacious dimethyl substitu-
ents in the pyrrolopyrimidines (I) are better tolerated
by the receptor than the additional saturated ring in
the compound series II.
One compound bearing a 1-methylbenzyl substituent
in the 2-position instead of phenyl but lacking the (R)-
1-methylbenzyl residue at the pyrrole nitrogen in the
pyrrolopyrimidine series (I) was prepared (compound
In a recent publication, it has been shown that
methoxy substitution in the meta- or para-position of
the phenyl ring in ADPEP (1) led to ca. 20-fold reduction

7-Deazaadenines Bearing Polar Substituents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4641
30). It exhibited moderate A1 AR affinity (Ki ) 6.8 µM)
and thus was ca. 5-fold weaker as compared to the
analogous 2-phenyl derivative.23 Since the chiral com-
pound 30 was not superior to the 2-phenyl derivative,
the single enantiomers were not prepared.
substituent at the pyrrole nitrogen N7, either (R)-1-
methylbenzyl (29, 41-44) or cyclopentyl (45). Substitu-
tion at N4 comprised lipophilic residues, including (R)-
1-methylbenzyl (41) and cyclopentyl (42), as well as
more polar, hydrophilic residues, such as amino- and
hydroxyalkyl (43-45) and acrylic acid methyl ester (29).
Substituents in the 2-positions were those known to be
well-tolerated by the ARs (phenyl, p-chlorophenyl, p-
pyridyl). All of the compounds exhibited affinity to A3
ARs in the micromolar concentration range. The ad-
ditional (R)-1-methylbenzyl substituent at the pyrrole
nitrogen in compound 41 as compared to 2 (DPEAP)
caused a 400-fold decrease in A3 affinity. Thus, bis-
substitution at N4 and N7 appears to be unfavorable
for A3 affinity as it is for A1 AR affinity (see above). A
variety of different - polar and nonpolar - N4-substit-
uents was tolerated by the A3 AR. The most potent
compound, besides DPEAP (2), was the hydroxybutyl-
substituted derivative 44 showing a Ki value of 570 nM
at human A3 ARs; it was about 5-fold selective for A3
versus A1 ARs. Interestingly, the introduction of a
p-chloro substituent in the 2-phenyl group led to ca.
3-fold increase in A3 affinity, while it caused ca. 2-fold
reduction of the A1 affinity (Table 2).
One of the more potent compounds, the p-pyridyl-
substituted pyrrolopyrimidine 11 was additionally in-
vestigated at human recombinant A1 ARs. It was found
that compound 11 showed high affinity for the human
A1 AR exhibiting a Ki value of 8.8 nM.
It was observed that the pyridyl-substituted pyrrol-
opyrimidines (9-11), tetrahydropyrimidoindoles (21-
23), and pyrimidoindoles (25-27) showed intensive
fluorescent properties. Acidification leading to a proto-
nation of the pyridyl nitrogen atom increased water
solubility of the compounds and also strongly enhanced
fluorescence of the compounds. A more detailed descrip-
tion of this effect is currently under investigation. The
p-pyridyl-substituted compounds 11 and 27, potent and
selective A1 AR ligands with fluorescent properties,
might be useful pharmacological tools.
Su bstitu tion of th e Exocyclic Am in o Gr ou p .
Since 2-phenylpyrrolopyrimidines bearing a 1-methyl-
benzyl substituent, either on the pyrrole nitrogen (N7,
e.g., 1, ADPEP) or on the exocyclic amino group (N4,
e.g., 2, DPEAP), were potent A1 AR antagonists, we
investigated whether a combination of N7- and N4-
substitution would be additive leading to an increase
in A1 AR affinity. However, the bis(1-methylbenzyl)-
substituted derivative 41 exhibited only low A1 AR
affinity (Ki ) 18.3 µM). All other bis-substituted com-
pounds (29, 41-45), including the N4-cyclopentyl de-
rivative 42, exhibited only low A1 AR affinity. These
results support our previously postulated pharmacoph-
ore model for the binding of AR antagonists.5,11 7-Sub-
stituted pyrrolopyrimidine derivatives and N4-substi-
tuted derivatives appear to exhibit different binding
modes at A1 ARs. It is very likely that the 7-substituent
in ADPEP (1) and the N4-substituent in DPEAP (2)
occupy the same receptor region.5,11
Wa ter Solu bility. Many high-affinity AR antagonists
exhibit low water solubility, which limits their useful-
ness as pharmacological tools.5,6d,26 Potent A1 AR an-
tagonists with the pyrrolo[2,3-d]pyrimidine or pyrimido-
[4,5-b]indole structure have been shown to be highly
lipophilic and soluble in water only in micromolar
concentrations.11 The present study was aimed at
improving the water solubility of the compounds by
introducing polar substituents in the 2-position (e.g.,
exchange of phenyl for pyridyl) and at the N4-amino
group (e.g., aminoethyl, hydroxybutyl substitution).
Water solubility of selected compounds was determined
by an HPLC/UV method (Table 3). Stock solutions of
the compounds were prepared in DMSO and diluted into
buffer to obtain a saturated solution containing 1%
DMSO. As expected, the replacement of the 2-phenyl
group in APEPI (3) by pyridyl residues resulted in a
ca. 6-10-fold increase in water solubility (compounds
25, 27, Table 3). Due to the lower affinities of compounds
25 and 27, their solubility-over-affinity ratio was not
improved as compared to APEPI (3). An even higher
increase in water solubility was achieved by the intro-
duction of polar substituents at the exocyclic amino
group (compounds 43-45). These compounds were 20-
50-fold better soluble than APEPI (3). The higher
solubility, however, was associated with lower AR
affinity. The best of the new compounds was the
p-pyridyl analogue of APEPI, compound 27, which
showed increased water solubility and a solubility-over-
affinity ratio of greater than 100. This has been postu-
lated as a prerequisite for compounds to be active in
vivo.26
A2B a n d A3 ARs. Initially, three representative
compounds, the N4-substituted pyrrolopyrimidine 2
(DPEAP), the 2-p-pyridyl-substituted pyrimidoindole 27
(APPPI), and the 2-phenyl-substituted pyrimidoindole
3 (APEPI) were investigated in radioligand binding
assays at human A3 ARs and in adenylate cyclase
assays at human A2B ARs. These experiments were
performed in order to assess A1 selectivity of the
compounds versus A2B and A3 ARs. All these compounds
did not show any antagonistic effects at A2B AR up to
concentrations of 10 µM. APPPI (27) and APEPI (3),
pyrimidoindole derivatives which are u n substituted at
the exocyclic amino group, did not show binding to A3
ARs at the investigated concentrations either (Table 2).
However, DPEAP (2), a pyrrolopyrimidine derivative
substituted at the amino group, exhibited surprisingly
high affinity for human A3 ARs (Ki ) 28 nM). Thus,
DPEAP (2) is only 4-fold selective for A1 versus A3 ARs.
Due to its high affinity for A3 ARs, DPEAP (2) has been
selected as a new lead compound for the development
of A3 AR antagonists. Further N4-substituted pyrrol-
opyrimidine derivatives (29, 41-45) of the present
series were investigated at A3 ARs. In contrast to
DPEAP (2), all these derivatives bore an additional
In conclusion, new potent, selective A1 AR antagonists
have been prepared, including 2-p-pyridyl-substituted
analogues of ADPEP (1) and APEPI (3), which may
prove to be useful pharmacological tools due to their
fluorescent properties. N4-Substituted pyrrolo[2,3-d]-
pyrimidine-4-amines provide new lead compounds for
the development of A3-selective AR antagonists.

4642 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24
Hess et al.
Ta ble 3. Solubility and Solubility-over-Affinity Ratios of Selected Pyrrolo[2,3-d]pyrimidines and Pyrimido[4,5-b]indoles
compd
R1
R2
phenyl
2-pyridyl
4-pyridyl
R3
Pyrimido[4,5-b]indoles (III)
solubilitya (µM)
A1 affinity
ratio solubility/A1 affinity
3 (APEPI)
25
27 (APPPI)
(R)-1-methylbenzyl
(R)-1-methylbenzyl
(R)-1-methylbenzyl
0.4811b
5.0
0.002611b
0.54
0.021
185
9
142
3.0b
Pyrrolo[2,3-d]pyrimidines (I)
43
44
45
(R)-1-methylbenzyl
(R)-1-methylbenzyl
cyclopentyl
phenyl
4-chlorphenyl
phenyl
aminoethyl
(R,S)-2-butan-1-ol
(R,S)-2-butan-1-ol
24.2
11.2
13.2
3.58
2.6
1.24
7
4
11
a
b
Determined in 50 mM TRIS-HCl buffer, pH 7.4, containing the indicated amount of DMSO. Estimated from the validation data.
The solubility is smaller than the limit of quantification (3.6 µM) but higher than the limit of detection (2.4 µM).
(DMSO-d6) δ 10.53 (CH3), 10.65 (CH3), 19.25 (CHCH3), 51.00
(CHCH3), 101.57 (C-4a), 105.71 (C-5), 122.69, 123.48 (C-3′ and
C-5′), 126.17, 126.89, 128.39 (aromatic CH), 129.79 (C-6),
136.44 (C-4′), 142.04 (aromatic ipso-C), 148.89 (C-6′), 150.83,
155.09, 156.32, 157.28 (C-2′, C-7a, C-4, C-2).
Exp er im en ta l Section
Ch em istr y. NMR spectra were measured on a Varian
Gemini 300 spectrometer (1H: 300 MHz; 13C: 75 MHz). The
chemical shifts of the remaining protons of the deuterated
solvents served as internal standard: δ (1H: DMSO-d6 ) 2.50,
CDCl3 ) 7.24, MeOD ) 3.35 and 4.78; 13C: DMSO-d6 ) 39.7,
CDCl3 ) 77.0, MeOD ) 49.3). All compounds were checked
for purity by TLC on 0.2-mm aluminum sheets with silica gel
60 F254 (Merck): hexane:ethyl acetate (2:1), toluene:acetone:
formic acid (60:39:1) or ethyl acetate, respectively, were used
as eluents.
Melting points were taken on a Bu¨chi 535 melting point
apparatus or a Gallenkamp variable heater and are uncor-
rected. Thermospray (negative and positive ionization) and
electron ionization mass spectra were recorded on an MS
Engine HP 5989A mass spectrometer (Hewlett-Packard).
Elemental analyses, NMR spectra, and mass spectra were
performed by the Institute of Organic Chemistry, University
of Leipzig. Additional NMR data are available as Supporting
Information.
(R)-5,6-Dim eth yl-7-(1-m eth ylben zyl)-2-(3′-p yr id yl)-7H-
p yr r olo[2,3-d ]p yr im id in e-4-a m in e (10): 1H NMR (CDCl3)
3
δ 2.03 (d, 3H, CHCH3, J ) 7.14 Hz), 2.09 (s, 3H, CH3), 2.36
(s, 3H, CH3), 5.16 (s, 2H, NH2), 6.34 (q, 1H, CHCH3, 3J ) 7.14
Hz), 7.22-7.34 (m, 6H, aromatic and pyridyl-H), 8.59 (dd, 1H,
pyridyl-H, J ) 1.6 and 3.3 Hz), 8.64 (dt, 1H, pyridyl-H, J )
8.0 Hz), 9.60 (d, 1H, pyridyl-H, J ) 1.4 Hz).
(R)-5,6-Dim eth yl-7-(1-m eth ylben zyl)-2-(4′-p yr id yl)-7H-
p yr r olo[2,3-d ]p yr im id in e-4-a m in e (11): 1H NMR (CDCl3)
3
δ 2.05 (d, 3H, CHCH3, J H,H ) 7.2 Hz), 2.06 (s, 3H, CH3), 2.35
3
(s, 3H, CH3), 5.22 (s, br, 2H, NH2), 6.37 (q, 1H, CHCH3, J )
7.2 Hz), 7.22-7.31 (m, 5H, phenyl), 8.27 (d, 2H, pyridyl, J )
6.0 Hz), 8.66 (d, 2H, pyridyl, J ) 5.8 Hz).
(R)-5,6-Dim eth yl-7-(1-m eth ylben zyl)-2-(4-p yr id yl-N-ox-
id e)-7H-p yr r olo[2,3-d ]p yr im id in e-4-a m in e (12): 1H NMR
(CDCl3) δ 2.00 (d, 3H, CHCH3, 3J ) 7.14 Hz), 2.09 (s, 3H, CH3),
2.36 (s, 3H, CH3), 5.13 (s, 2H, NH2), 6.33 (q, 1H, CHCH3, 3J )
7.14 Hz), 7.18-7.31 (m, 5H, aromatic), 8.21 (d, 2H, pyridyl-H,
J ) 7.2 Hz), 8.30 (d, 2H, pyridyl-H, J ) 7.2 Hz).
Compounds 4-6 and 8 were obtained as described.11,25
2-Am in o-1-(1-m eth ylben zyl)in d ole-3-ca r bon itr ile (7). A
solution of 6.6 g (25 mmol) of 6 and 11.4 g (50 mmol) of 2,3-
dichloro-5,6-dicyanoquinone (DDQ) in 50 mL of tetrahydrofu-
ran was refluxed for 3 h. After removing excess solvent in
vacuo the residue was dissolved in 1000 mL of ethyl acetate
and diethyl ether (1:1) and washed three times with 1000 mL
of 0.1 M aqueous NaOH solution and finally with water. The
organic phase was dried with anhydrous Na2SO4 and evapo-
rated in vacuo: 1H NMR (DMSO-d6) δ 1.84 (d, 3H, CHCH3, J
) 7 Hz), 5.88 (q, 1H, CHCH3, J ) 7 Hz), 6.68-6.76 (m, 2H,
H-7, H-6), 6.90-6.97 (m, 1H, H-5), 7.08 (s, 2H, NH2), 7.17 (d,
1H, H-4, J ) 7.7 Hz), 7.22-7.39 (m, 5H, aromatic).
(R)-5,6-Dim et h yl-7-(1-m et h ylb en zyl)-2-(2′-p yr a zin yl)-
7H-pyr r olo[2,3-d]pyr im idin e-4-am in e (13): 1H NMR (CDCl3)
3
δ 2.03 (d, 3H, CHCH3, J ) 7.41 Hz), 2.10 (s, 3H, CH3), 2.36
(s, 3H, CH3), 5.38 (s, 2H, NH2), 6.34 (q, 1H, CHCH3, 3J ) 7.41
Hz), 7.19-7.30 (m, 5H, aromatic), 8.52-8.70 (m, 2H, pyrazinyl-
H), 9.66-9.68 (q, 1H, pyrazinyl-H).
(R)-5,6-Dim eth yl-7-(1-m eth ylben zyl)-2-(2′-th ien yl)-7H-
p yr r olo[2,3-d ]p yr im id in e-4-a m in e (14): 1H NMR (DMSO-
d6) δ 1.99 (d, 3H, CHCH3, 3J ) 7.35 Hz), 2.09 (s, 3H, CH3),
3
2.28 (s, 3H, CH3), 6.04 (q, 1H, CHCH3, J ) 7.35 Hz), 6.47 (s,
2-Su bstitu ted Der iva tives of 5,6-Dim eth yl-7H-7-(1-m e-
th ylben zyl)pyr r olo[2,3-d]pyr im idin es 9-17. Gen er al P r o-
ced u r e: A suspension of 4.3 g (18 mmol) of 4, 1.9 g (36 mmol)
of sodium methylate, and 18 mmol of the appropriate carbo-
nitrile derivative (see Table 1) in 50 mL of 2-propanol was
refluxed for 8 h. After cooling to about 30 °C, the mixture was
diluted with 20 mL of EtOH to keep sodium methylate in
solution. After cooling, the precipitated crystals were filtered
off and recrystallized from 50 mL of EtOH:H2O (1:1). Reagents,
yields and selected analytical data are given in Table 1.
(R)-5,6-Dim eth yl-7-(1-m eth ylben zyl)-2-(2′-p yr id yl)-7H-
p yr r olo[2,3-d ]p yr im id in e-4-a m in e (9): 1H NMR (DMSO-
d6) δ 1.99 (d, 3H, CHCH3, 3J ) 7.14 Hz), 2.08 (s, 3H, CH3),
2H, NH2), 7.08 (dd, 1H, 4′-thienyl, 3J ) 4.95 and 3.51 Hz),
7.24-7.29 (m, 5H, phenyl), 7.49 (d, 1H, thienyl, 3J ) 4.95 Hz),
3
7.73 (d, 1H, thienyl, J ) 3.51 Hz).
(R)-5,6-Dim eth yl-2-(2′-fu r yl)-7-(1-m eth ylben zyl)-7H-pyr -
r olo[2,3-d ]p yr im id in e-4-a m in e (15): 1H NMR (DMSO-d6)
δ 1.95 (d, 3H, CHCH3, 3J ) 7.1 Hz), 2.03 (s, 3H, CH3), 2.28 (s,
3H, CH3), 6.16 (q, 1H, CHCH3), 6.51 (s, br, NH2), 6.57 (s, 1H,
3
3
furyl, J ) 7.1 Hz), 7.00 (d, 1H, furyl, J ) 2.9 Hz), 7.16-7.32
(m, 5H, phenyl), 7.72 (s, 1H, furyl).
(R)-5,6-Dim eth yl-2-(2-m eth oxyp h en yl)-7-(1-m eth ylben -
zyl)-7H-p yr r olo[2,3-d ]p yr im id in e-4-a m in e (16): 1H NMR
3
(DMSO-d6) δ 1.94 (d, 3H, CHCH3, J ) 7.14 Hz), 2.08 (s, 3H,
3
2.32 (s, 3H, CH3), 6.24 (q, 1H, CHCH3, J ) 7.14 Hz), 6.66 (s,
CH3), 2.29 (s, 3H, CH3), 3.71 (s, 3H, OCH3), 6.07 (q, 1H,
3
br, NH2), 7.17-7.39 (m, 6H, phenyl, 5′-pyridyl), 7.85 (t, 1H,
4′-pyridyl, J ) 7.6 Hz), 8.31-8.34 (d, 1H, 3′-pyridyl, J ) 7.7
Hz), 8.62-8.63 (d, 1H, 6′-pyridyl, J ) 4.0 Hz); 13C NMR
CHCH3, J ) 7.14 Hz), 6.39 (s, 2H, NH2), 6.96 (t, 1H, J ) 7.4
Hz), 7.04 (d, 1H, J ) 8.3 Hz), 7.21-7.30 (m, 5H, aromatic),
7.40 (dd, 2H, aromatic, J ) 1.6 and 5.9 Hz).

7-Deazaadenines Bearing Polar Substituents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4643
5,6-Dim eth yl-2(R,S)-(1-m eth ylben zyl)-7(R)-(1-m eth yl-
ben zyl)-7H-p yr r olo[2,3-d ]p yr im id in e-4-a m in e (17): 1H
NMR (CDCl3) δ 1.70 (d, 2 × 3H, CHCH3, J ) 7.14 Hz), 2.04
(d, 3H, CHCH3, J ) 7.14 Hz), 2.07 (d, 3H, CHCH3, J ) 7.14
Hz), 2.10 (s, 3H, CH3), 2.14 (s, 3H, CH3), 2.26 (s, 2 × 3H, CH3),
4.2 (m, 2 × 1H, CHCH3, J ) 7.14 Hz), 5.32 (s, 2 × 2H, NH2),
6.11 (q, 1H, CHCH3, J ) 7.14 Hz), 6.20 (q, 1H, CHCH3, J )
7.14 Hz), 7.24-7.30 (m, 20 H, aromatic).
2-Su b st it u t ed Der iva t ives of 5,6,7,8-Tet r a h yd r o-9-
p h en yl-9H-p yr im id o[4,5-b]in d oles 18-20. Gen er a l P r o-
ced u r e: A suspension of 2.4 g (10 mmol) of 5, 1.0 g (20 mmol)
of sodium methylate, and 10 mmol of the appropriate carbo-
nitrile derivative (see Table 1) in 20 mL of 2-propanol was
refluxed for 6 h. After cooling to about 30 °C, the mixture was
diluted with 10 mL of EtOH to keep sodium methylate in
solution. After cooling, the precipitated crystals were filtered
off and recrystallized from 100 mL of EtOH. Reagents, yields
and selected analytical data are given in Table 1.
2.81 (m, 2H, CH2), 6.06 (q, 1H, CHCH3, 3J ) 7.14 Hz), 6.52 (s,
2H, NH2), 7.24-7.33 (m, 5H, aromatic), 8.19-8.30 (m, 4H,
pyridyl-H).
2-Su bstitu ted Der iva tives of (R)-9-(1-Meth ylben zyl)-
9H-p yr im id o[4,5-b]in d ole-4-a m in es 25-28. Gen er a l P r o-
ced u r e: A suspension of 2.4 g (10 mmol) of 7, 1.1 g (20 mmol)
of sodium methylate, and 10 mmol of the appropriate carbo-
nitrile derivative (see Table 1) in 20 mL of 2-propanol was
refluxed for 8 h. After cooling to about 30 °C, the mixture was
diluted with 10 mL of EtOH to keep sodium methylate in
solution. After cooling, the precipitated crystals were filtered
off and recrystallized from 50 mL of EtOH:H2O (1:1). Reagents,
yields and selected analytical data are given in Table 1.
(R)-9-(1-Meth ylben zyl)-2-(2′-p yr id yl)-9H-p yr im id o[4,5-
b]in d ole-4-a m in e (25): 1H NMR (DMSO-d6) δ 2.09 (d, 3H,
CHCH3, 3J ) 7.14 Hz), 6.74 (q, 1H, CHCH3, 3J ) 7.14 Hz),
7.26-7.37 (m, 9H, aromatic), 7.81-7.84 (m, 2H, pyridyl-H),
8.63 (d, 1H, pyridyl-H, J ) 7.95 Hz), 8.83 (d, 1H, pyridyl-H, J
) 3.84 Hz).
(R)-5,6,7,8-Tet r a h yd r o-9-p h en yl-2-(2′-p yr id yl)-9H -p y-
r im id o[4,5-b]in d ole-4-a m in e (18): 1H NMR (MeOD) δ 1.81-
1.91 (m, 4H, CH2CH2), 2.49-2.50 (d, 2H, CH2, J ) 5.2 Hz),
2.87-2.89 (d, 2H, CH2, J ) 5.6 Hz), 7.40-7.42 (m, 1H,
5′-pyridyl), 7.50-7.57 (m, 5H, phenyl), 7.87 (dt, 1H, 4′-pyridyl,
J ) 1.2 and 7.1 Hz), 8.05 (d, 1H, 3′-pyridyl, J ) 7.95 Hz), 8.66
(d, 1H, 6′-pyridyl, J ) 4.32 Hz).
(R)-9-(1-Meth ylben zyl)-2-(3′-p yr id yl)-9H-p yr im id o[4,5-
b]in d ole-4-a m in e (26): 1H NMR (DMSO-d6) δ 2.11 (d, 3H,
CHCH3, J ) 7.2 Hz), 6.68 (q, 1H, CHCH3, J ) 7.14 Hz), 7.24-
7.40 (m, 9H, aromatic CH), 7.79-7.82 (m, 1H, pyridyl-H), 8.68
(br, s, 1H, pyridyl-H), 8.79 (d, 1H, pyridyl-H, J ) 7.98 Hz),
9.75 (br, s, 1H, pyridyl-H).
(R)-9-(1-Meth ylben zyl)-2-(4′-p yr id yl)-9H-p yr im id o[4,5-
b]in d ole-4-a m in e (27): 1H NMR (MeOD) δ 2.10 (d, 3H,
CHCH3, 3J ) 7.14 Hz), 6.57 (q, 1H, CHCH3, 3J ) 7.14 Hz),
7.22-7.44 (m, 9H, aromatic, indole-H), 8.32 (d, 2H, pyridyl-
H, 3J ) 6.0 Hz), 8.40 (d, 1H, NH, J ) 7.4 Hz), 8.72 (d, 2H,
(R)-5,6,7,8-Tet r a h yd r o-9-p h en yl-2-(3′-p yr id yl)-9H -p y-
r im id o[4,5-b]in d ole-4-a m in e (19): 1H NMR (MeOD) δ 0.50
(m, 4H, CH2-CH2), 1.16 (t, 2H, CH2, 3J ) 5.3 Hz), 1.52 (t, 2H,
3
CH2, J ) 5.6 Hz), 6.01-6.16 (m, 5H, aromatic), 6.27 (m, 1H,
5′-pyridyl), 7.22 (d, 1H, 6′-pyridyl, 3J 5′,6′ ) 4.2 Hz), 7.39 (d, 1H,
3
3
pyridyl-H, J ) 6.0 Hz).
4′-pyridyl, J 4′,5′ ) 8.0 Hz), 7.91 (s, 1H, 2′-pyridyl).
(R)-9-(1-Meth ylben zyl)-2-(th ien yl)-9H-p yr im id o[4,5-b]-
in d ole-4-a m in e (28): 1H NMR (CDCl3) δ 2.10 (d, 3H, CHCH3,
3J ) 7.14 Hz), 5.43 (br, s, 2H, NH2), 6.62 (q, 1H, CHCH3, 3J )
7.14 Hz), 7.14 (dd, 1H, 4′-thienyl-H), 7.18-7.44 (m, 9H,
aromatic, indole-H), 7.74 (m, 1H, thienyl-H), 8.04 (dd, 1H,
thienyl-H).
(R)-5,6,7,8-Tet r a h yd r o-9-p h en yl-2-(4′-p yr id yl)-9H -p y-
r im id o[4,5-b]in d ole-4-a m in e (20): 1H NMR (CDCl3) δ 1.88-
3
1.95 (m, 4H, CH2), 2.63 (t, 2H, CH2, J ) 6.0 Hz), 2.95 (t, 2H,
CH2, 3J ) 6.0 Hz), 5.13 (s, 2H, NH2), 7.45-7.55 (m, 5H,
aromatic), 8.18 (dd, 2H, pyridyl-H, J ) 1.4 and 3.2 Hz), 8.62
(dd, 2H, pyridyl-H, J ) 1.1 and 4.7 Hz).
(R)-3-[5,6-Dim eth yl-7-(1-m eth ylben zyl)-2-p yr id in -4-yl-
7H-p yr r olo[2,3-d ]p yr im id in -4-yla m in o]a cr ylic Acid Me-
th yla te (29). A solution of 11 (700 mg, 2 mmol) and propiolic
acid methylate (600 mg, 7 mmol) in 5 mL of glacial acetic acid
was stirred at room temperature for 12 h. After evaporation
in vacuo the residual oil was treated with 5 mL of acetone to
yield crystalline 29: 1H NMR (CDCl3) δ 2.06 (d, 3H, CH-CH3,
3J ) 7.14 Hz), 2.20 (s, 3H, CH3), 2.44 (s, 3H, CH3), 3.70 (s, 3H,
OCH3), 5.19 (d, 1H, 3J ) 8.73 Hz), 6.18 (q, 1H, CH-CH3, 3J )
7.14 Hz), 7.25-7.32 (m, 5H, aromatic), 8.26 (d, 2H, pyridyl-H,
3J ) 5.5 Hz), 8.46 (dd, 1H, 3J ) 8.73 and 11.5 Hz), 8.67 (d,
2-Su bstitu ted Der iva tives of 5,6,7,8-Tetr a h yd r o-9-(1-
m eth ylben zyl)-9H-p yr im id o[4,5-b]in d oles 21-24. Gen -
er a l P r oced u r e: A suspension of 6.6 g (25 mmol) of 6, 2.7 g
(50 mmol) of sodium methylate, and 25 mmol of the appropri-
ate carbonitrile derivative (see Table 1) in 50 mL of 2-propanol
was refluxed for 6 h. After cooling to about 30 °C, the mixture
was diluted with 20 mL of EtOH to keep sodium methylate in
solution. After cooling, the precipitated crystals were filtered
off and recrystallized from 50 mL of EtOH. Reagents, yields
and selected analytical data are given in Table 1.
(R)-5,6,7,8-Tetr ah ydr o-9-(1-m eth ylben zyl)-2-(2′-pyr idyl)-
9H-p yr im id o[4,5-b]in d ole-4-a m in e (21): 1H NMR (MeOD)
δ 1.68-1.74 (m, 4H, CH2-CH2), 1.97 (d, 3H, CHCH3, 3J ) 7.14
Hz), 2.19-2.26 (m, 1H, CH2), 2.55-2.61 (m, 1H, CH2), 2.87
(br, s, 2H, CH2), 6.35 (q, 1H, CHCH3, 3J ) 7.14 Hz), 7.18-
7.28 (m, 5H, aromatic), 7.39-7.41 (m, 1H, 5′-pyridyl), 7.87 (m,
2H, pyridyl-H, 3J ) 4.7 Hz), 10.84 (d, 1H, NH, 3J
) 11.5
cis
Hz); 13C NMR (CDCl3) δ 10.14, 10.67 (2 x CH3), 19.21 (CHCH3),
51.00 (CHCH3), 52.20 (OCH3), 91.89 (CHCdO), 103.68 (C-4a),
104.72 (C-5), 121.27 (C-3′ and C-5′), 126.32, 127.15, 128.46
(aromatic CH), 133.96 (C-6), 140.19, 141.42 (C-4′, aromatic
ipso-C), 145.21 (CH-NH), 149.28, 151.66, 153.39 (C-4, C-7a,
C-2), 150.04 (C-2′ and C-6′), 169.66 (CdO).
3
1H, 4′-pyridyl), 8.40 (d, 1H, 3′-pyridyl, J ) 8.0 Hz), 8.63 (d,
3
1H, 6′-pyridyl, J ) 4.2 Hz).
(R,S)-5,6-Dim eth yl-2-(1-m eth ylben zyl)-7H-p yr r olo[2,3-
d ]p yr im id in e-4-a m in e (30). A mixture of 17 (2.6 g, 7 mmol)
in 35 g of polyphosphoric acid was stirred for 3 h at 70-80 °C.
The mixture was poured into ice-cold water and sufficient,
concentrated ammonia solution was subsequently added to
obtain a pH value of 10. The precipitated crystals were
collected by filtration, washed with water, and recrystallized
from ethanol: 1H NMR (DMSO-d6) δ 1.56 (d, 3H, CHCH3 3J
) 7.14 Hz), 2.16 (s. 3H, CH3), 2.21 (s, 3H, CH3), 4.20 (q, 1H,
(R)-5,6,7,8-Tetr ah ydr o-9-(1-m eth ylben zyl)-2-(3′-pyr idyl)-
9H-p yr im id o[4,5-b]in d ole-4-a m in e (22): 1H NMR (DMSO-
3
d6) δ 1.67-1.71 (m, 4H, CH2-CH2), 1.98 (d, 3H, CHCH3, J )
7.14 Hz), 2.49 (m, 2H, CH2), 2.82 (m, 2H, CH2), 6.05 (q, 1H,
3
CHCH3, J ) 7.14 Hz), 6.51 (s, 2H, NH2), 7.20-7.29 (m, 5H,
9-phenyl), 7.44 (m, 1H, 5′-pyridyl, 3J 5′,6′ ) 4.65 Hz), 8.55-8.60
3
(m, 2H, 4′-pyridyl, 6′-pyridyl, J 5′,6′ ) 4.65 Hz), 9.4 (s, 1H, 2′-
pyridyl).
3
(R)-5,6,7,8-Tetr ah ydr o-9-(1-m eth ylben zyl)-2-(4′-pyr idyl)-
9H-p yr im id o[4,5-b]in d ole-4-a m in e (23): 1H NMR (MeOD)
δ 1.66-1.71 (4H, CH2-CH2), 1.98 (d, 3J ) 7.08 Hz, 3H,
CHCH3), 2.49-2.51 (m, 2H, CH2), 2.83 (br, s, 2H, CH2), 6.07
(q, 1H, 3J ) 7.14 Hz, CHCH3), 6.56 (s, 2H, NH2), 7.20-7.29
(m, 5H, 9-phenyl), 8.22 (dd, 2H, pyridyl), 8.70 (t, 2H, pyridyl).
CHCH3, J ) 7.14 Hz), 6.95 (s, 2H, NH2), 7.11-7.35 (m, 5H,
aromatic), 11.44 (s, 1H, NH); 13C NMR (DMSO-d6) δ 10.12,
10.48 (2 × CH3), 20.38 (CHCH3), 45.93 (CHCH3), 100.39 (C-
4a), 104.61 (C-5), 125.97, 127.16, 127.22, 127.40, 128.04, 128.22
(aromatic C and C-6), 144.76 (aromatic ipso-C), 149.75, 155.10,
161.53 (C-2, C-4, C-7a).
(R)-5,6,7,8-Tetr ah ydr o-9-(1-m eth ylben zyl)-2-(4′-pyr idyl-
N-oxid e)-9H-p yr im id o[4,5-b]in d ole-4-a m in e (24): 1H NMR
(DMSO-d6) δ 1.63-1.72 (m, 4H, CH2-CH2), 1.97 (d, 3H,
(R)-5,6-Dim eth yl-2-(2-h yd r oxyp h en yl)-7-(1-m eth ylben -
zyl)-7H-p yr r olo[2,3-d ]p yr im id in e-4-a m in e (31). A solution
of 16 (373 mg, 1 mmol) in CH2Cl2 was cooled to 0 °C. Boron
tribromide (1.0 mL, 10 mmol) was added and the mixture was
3
CHCH3, J ) 7.14 Hz), 2.37 (m, 1H, CH2), 2.55 (m, 1H, CH2),

4644 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24
Hess et al.
stirred at 0 °C for 1 h and subsequently at room temperature
for 24 h. After the mixture had cooled to -10 °C, 10 mL of
water was slowly added. The mixture was then extracted three
times with 20 mL of CH2Cl2 each, and the organic phase was
dried over anhydrous Na2SO4 and evaporated in vacuo. The
precipitated crystals were recrystallized from 10 mL of etha-
nol: 1H NMR (CDCl3) δ 2.02 (d, 3H, CHCH3, 3J ) 7.3 Hz),
2.11 (s, 3H, CH3), 2.37 (s, 3H, CH3), 5.14 (s, 2H, NH2), 6.21 (q,
1H, CHCH3, 3J ) 7.3 Hz), 6.89-6.97 (m, 2H, aromatic), 7.22-
7.32 (m, 7H, aromatic), 8.44 (dd, 1H, aromatic, J ) 1.6 and
6.3 Hz), 13.81 (br, s, 1H, OH); 13C NMR (CDCl3) δ 10.98 (2 ×
CH3), 19.15 (CHCH3), 51.75 (CHCH3), 101.45 (C-4a), 105.87
(C-5), 117.34, 118.56 (aromatic CH), 120.34 (C-2′), 126.37,
127.24, 128.60, 128.76 (aromatic CH), 130.59 (C-6), 131.21
(aromatic CH), 141.38 (aromatic ipso-C), 155.02, 156.89, 159.75
(C-7a, C-4, C-2).
Der iva tives of (R)-2-Ben za m id o-4,5-d im eth yl-1-(1-m e-
th ylben zyl)-1H-p yr r ole-3-ca r bon itr ile (32 a n d 33). Gen -
er a l P r oced u r e: To an ice-cold solution of 30 mmol of 4 in 20
mL of CH2Cl2 was added 5 mL of pyridine followed by the
addition of 32 mmol of the appropriate benzoyl chloride
derivative. After 1 h of stirring the mixture in an ice bath, 10
mL of petroleum ether (bp 40-60 °C) was added to complete
the precipitation of the product. The precipitate was collected
by filtration and recrystallized from EtOH/H2O.
POCl3 was refluxed for 1 h. Excess reagent was removed in
vacuo. The residue was poured on water, collected by filtration,
washed until neutral reaction, and recrystallized from 500 mL
of acetone or 750 mL of ethanol, respectively.
(R)-4-Ch lor o-2-ph en yl-5,6-dim eth yl-7-(1-m eth ylben zyl)-
7H-p yr r olo[2,3-d ]p yr im id in e (38): IR (KBr) ν 3440, 2940,
2344, 1654, 1600, 1560, 1530, 1452, 1424, 1170, 1042; 1H NMR
3
(CDCl3) δ 2.08 (d, 3H, CHCH3, J H,H ) 7.2 Hz), 2.16 (s, 3H,
3
CH3), 2.42 (s, 3H, CH3), 6.40 (q, 1H, CHCH3, J H,H ) 7.2 Hz),
7.23-7.47 (m, 8H, phenyl-H), 8.48-8.50 (d, 2H, phenyl-H); 13
C
NMR (CDCl3) δ 10.14 (CH3), 11.44 (CH3), 19.29 (CHCH3), 52.14
(CHCH3), 107.19 (C-4a), 114.71 (C-5), 126.47, 127.42, 128.02,
128.38, 128.66 (aromatic CH), 129.77 (C-6), 135.40, 141.20
(aromatic ipso-C), 150.63, 152.60, 156.16 (C-7a, C-4, C-2).
(R)-4-Ch lor o-2-(4-ch lor op h en yl)-5,6-d im eth yl-7-(1-m e-
th ylben zyl)-7H-p yr r olo[2,3-d ]p yr im id in e (39): IR (KBr) ν
2924, 1664, 1526, 1452, 1428, 1392, 1382, 1090, 842, 786, 702;
1H NMR (CDCl3) δ 2.06 (d, 3H, CHCH3, 3J H,H ) 7.35 Hz), 2.16
3
(s, 3H, CH3), 2.41 (s, 3H, CH3), 6.37 (q, 1H, CHCH3, J H,H
)
7.35 Hz), 7.21-7.33 (m, 5H, phenyl-H), 7.40-7.46 (m, 2H,
phenyl-H), 8.42 (d, 2H,phenyl-H, J ) 8.7 Hz); 13C NMR (CDCl3)
δ 10.12 (CH3), 11.45 (CH3), 19.26 (CHCH3), 52.14 (CHCH3),
107.32 (C-4a), 114.83 (C-5), 126.43, 127.46, 128.57, 128.68,
129.31 (aromatic CH), 135.70, 135.81, 136.38 (C-6, ipso- und
para-C), 141.07 (aromatic ipso-C), 150.62, 152.44, 155.08 (C-
7a, C-4, C-2).
2-Ben za m id o-1-cyclop en t yl-4,5-d im et h yl-1H -p yr r ole-
3-ca r bon itr ile (34). To an ice-cold solution of 12.5 mmol of 8
in 10 mL of CH2Cl2 was added 2.5 mL of pyridine followed by
the addition of 1.6 mL (12.5 mmol) of benzoyl chloride. After
1 h of stirring the mixture in an ice bath, 5 mL of petroleum
ether (bp 40-60 °C) was added. The resulting hygroscopic
residue was purified by flash chromatography. The product
was eluted with petroleum ether:ethyl acetate (3:1). After
evaporation of the solvent, the precipitate was recrystallized
from ethyl acetate. Reagents, yields and some analytical data
are given in Table 1: IR (KBr) ν 3216, 2214, 1644, 1510, 1486,
1456, 1394, 1302, 1288, 1246; 1H NMR (DMSO-d6) δ 1.51-
1.55 (m, 2H, CH2), 1.68-1.70 (m, 2H, CH2), 1.91-2.00 (m, 4H,
CH2), 2.03 (s, 3H, CH3), 2.19 (s, 3H, CH3), 4.55 (quint, 1H, CH),
7.54-7.64 (m, 3H, phenyl-H), 8.00 (d, 2H, phenyl-H, J ) 7.0
Hz), 10.18 (s, 1H, NH); 13C NMR (DMSO-d6) δ 9.77, 10.53
(CH3), 24.90, 30.85 (CH2), 55.69 (CH), 90.41 (C-3), 113.24,
115.77 (C-4, CN), 124.26 (C-5), 127.72, 128.60, 132.26 (aro-
matic CH), 130.63 (C-2), 132.85 (aromatic ipso-C), 167.01 (Cd
O).
Der iva tives of 7H-P yr r olo[2,3-d ]p yr im d in -4(3H)-on e
(35-37). Gen er a l P r oced u r e: H2O (4.5 g, 250 mmol) was
added dropwise to a mixture of 22.7 g (160 mmol) of phospho-
rus pentoxide and 20.2 g (160 mmol) of N,N-dimethylcyclo-
hexylamine with stirring and ice cooling. The viscous mixture
was heated (to ca. 220 °C) until a homogeneous solution was
obtained. After cooling to 190 °C, 8 mmol of the appropriate
2-benzamido-4,5-dimethyl-1-1H-pyrrole-3-carbonitrile (32-34)
was added with stirring. The temperature was kept at 190-
200 °C for 4 h. After allowing to cool to 90 °C, a pH value of
12 was adjusted by the addition of 2 N NaOH (ca. 150 mL), to
obtain a separated amine phase. The precipitate was collected
by filtration, washed with H2O and subsequently with acetone,
and recrystallized from 800 mL of EtOH. Reagents, yields and
some analytical data are given in Table 1.
4-Ch lor o-7-cyclop en tyl-5,6-d im eth yl-2-p h en yl-7H-p yr -
r olo[2,3-d ]p yr im id in e (40). A mixture of 2 mmol (615 mg)
of 37 and 3 mL of POCl3 was refluxed for 1 h. Excess reagent
was removed in vacuo. The residue was poured on water,
collected by filtration, washed until neutral reaction, and
recrystallized from 500 mL of acetone: IR (KBr) ν 2956, 2870,
1602, 1530, 1472, 1452, 1440, 1424, 1400, 1198, 702; 1H NMR
(DMSO-d6) δ 1.74 (m, 2H, CH2), 2.10 (m, 4H, CH2-CH2), 2.38
(s, 3H, CH3), 2.42 (s + m, 3H, CH3, 2H, CH2), 4.93 (quint, 1H,
CH), 7.50 (m, 3H, phenyl-H), 8.34 (d, 2H, phenyl-H, J ) 6.57
Hz); 13C NMR (DMSO-d6) δ 9.91, 10.54 (CH3), 24.98, 30.56
(CH2), 55.74 (CH), 104.75 (C-4a), 114.70 (C-5), 127.28, 128.60,
129.92 (aromatic CH), 137.26, 137.31 (C-6, aromatic ipso-C),
149.36, 151.08, 154.29 (C-7a, C-4, C-2).
(R,R)-5,6-Dim et h yl-7-(1-m et h ylb en zyl)-2-p h en yl-7H -
p yr r olo[2,3-d ]p yr im id in e-4-(1-m eth ylben zyl)a m in e (41).
A solution of 1.1 g (3 mmol) of 38 and 3.6 g (30 mmol) of (R)-
1-methylbenzylamine in 20 mL of ethanol was refluxed for 70
h. After cooling, the precipitated crystals were collected by
filtration and recrystallized from ethanol: IR (KBr) ν 3448,
1
1592, 1572, 1458, 1444, 1426, 1408, 1388, 1374, 766, 700; H
NMR (DMSO-d6) δ 1.63 (d, 3H, NH-CH-CH3, 3J H,H ) 6.9 Hz),
3
2.00 (d, 3H, CHCH3, J H,H ) 7.1 Hz), 2.11 (s, 3H, CH3), 2.42
(s, 3H, CH3), 5.63 (m, 1H, NH-CH-CH3, J ) 6.9 and 7.4 Hz),
3
3
6.16 (q, 1H, CH-CH3, J H,H ) 7.1 Hz), 6.35 (d, 1H, NH, J H,H
) 7.4 Hz), 7.21-7.39 (m, 11H, phenyl-H), 7.55 (d, 2H, phenyl-
H, J ) 7.38 Hz), 8.25 (dd, 2H, phenyl-H, J ) 1.6 and 6.4 Hz);
13C NMR (DMSO-d6) δ 10.53 (CH3), 10.87 (CH3), 19.33
(CHCH3), 22.69 (CHCH3), 49.58 (CHCH3), 51.30 (CHCH3),
101.40 (C-4a), 105.14 (C-5), 126.12, 126.23, 126.31, 126.87,
127.16, 127.99, 128.11, 128.35, 128.84 (aromatic CH), 129.18
(C-6), 139.19, 142.17, 145.93 (aromatic ipso-C), 150.58, 154.64,
155.08 (C-7a, C-4, C-2).
(R)-5,6-Dim eth yl-7-(1-m eth ylben zyl)-2-p h en yl-7H-p yr -
r olo[2,3-d ]p yr im id in e-4-cyclop en tyla m in e (42). A solution
of 543 mg (1.5 mmol) of 38 in 1.48 mL (15 mmol) of cyclopen-
tylamine was refluxed for 5 h. After cooling, the precipitated
crystals were collected by filtration and recrystallized from
ethanol: IR (KBr) ν 3456, 2942, 1592, 1574, 1544, 1496, 1458,
1442, 1428, 774, 704; 1H NMR (DMSO-d6) δ 1.63-1.64 (m, 6H,
cyclopentyl-H), 2.00-2.03 (d, 3H, CHCH3, 3J H,H ) 7.2 Hz), 2.10
(s, 3H, CH3), 2.33 (s, 3H, CH3), 2.50 (m, 2H, CH2), 4.63 (q, 1H,
CH-cyclopentyl), 5.86 (d, 1H, NH, J ) 6.6 Hz), 6.17 (q, 1H,
CH-CH3, 3J H,H ) 7.2 Hz), 7.20-7.43 (m, 8H, phenyl-H), 8.35-
8.38 (d, 2H, phenyl-H); 13C NMR (DMSO-d6) δ 10.51 (CH3),
10.71 (CH3), 19.35 (CHCH3), 23.67 (CH2CH2), 32.44 (CH2-
CHCH2), 51.26 (CHCH3), 52.19 (CH2CHCH2), 101.46 (C-4a),
105.09 (C-5), 126.22, 126.84, 127.13, 128.05, 128.34, 128.83
7-Cyclop en tyl-5,6-d im eth yl-2-p h en yl-7H-p yr r olo[2,3-d ]-
p yr im id in -4(3H)-on e (37): IR (KBr) ν 3434, 3144, 3060, 2954,
2868, 1660, 1550, 1522, 1394, 692, 528; 1H NMR (DMSO-d6) δ
1.67 (m, 2H, CH2), 2.02 (m, 4H, CH2-CH2), 2.25 (s, br, 5H,
CH3 + CH2), 2.26 (s, 3H, CH3), 4.81 (quint, 1H, CH), 7.50 (m,
3H, phenyl-H), 8.10 (m, 2H, phenyl-H), 11.84 (s, br, 1H, NH);
13C NMR (DMSO-d6) δ 9.80, 10.00 (CH3), 24.94, 31.18 (CH2),
55.17 (CH), 105.61 (C-4a), 108.93 (C-5), 127.05, 130.38 (C-6 +
aromatic ipso-C), 128.49, 128.56 (aromatic CH), 133.18 (aro-
matic CH), 146.34, 148.40 (C-7a, C-2), 159.65 (CdO).
4-Ch lor o-Su bstitu ted 5,6-Dim eth yl-2-p h en yl-7-(1-m e-
th ylben zyl)-7H-pyr r olo[2,3-d]pyr im idin es 38 an d 39. Gen -
er a l P r oced u r e: A mixture of 8 mmol of the appropriate 7H-
pyrrolo[2,3-d]pyrimidin-4(3H)-one (35 or 36) and 20 mL of

7-Deazaadenines Bearing Polar Substituents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4645
(aromatic CH), 128.89 (C-6), 139.37, 142.24 (aromatic ipso-
C), 150.42, 154.81, 155.88 (C-7a, C-4, C-2).
parameters: selectivity, precision, accuracy, linearity, range,
limit of detection (LOD), and limit of quantification (LOQ).
Detailed data are available as Supporting Information.
Ra d ioliga n d Bin d in g Assa ys. The compounds were tested
in radioligand binding assays for affinity to A1 and A2A ARs
in rat cortical membrane and rat striatal membrane prepara-
tions, respectively. An A1-selective agonist radioligand, either
[3H]-N6-cyclohexyladenosine (CHA, 1 nM) or [3H]-2-chloro-N6-
cyclohexyladenosine (CCPA, 0.5 nM), respectively, was used
as the A1 ligand and the A2A-selective agonist [3H]-2-[[[[4-
(carboxyethyl)phenyl]ethyl]amino]-5′-N-(ethylcarbonyl)amino]-
adenosine (CGS21680, 5 nM) as the A2A ligand, as previously
described.9b,27,28 Inhibition of receptor-radioligand binding was
determined by a range of 5-7 concentrations of the compounds
in triplicate in at least three separate experiments.
(R)-N1-[5,6-Dim eth yl-7-(1-m eth ylben zyl)-2-p h en yl-7H-
p yr r olo[2,3-d ]p yr im id in -4-yl]eth a n ed ia m in e (43). A solu-
tion of 732 mg (1.5 mmol) of 38 in 5.0 mL of ethylenediamine
was refluxed for 6 h. After cooling, 20 mL of an ice-cold mixture
of water and ethanol was added. The precipitated crystals were
collected by filtration and recrystallized from 200 mL of
ethanol: IR ν 3396, 1592, 1576, 1542, 1496, 1444, 1426, 1382,
1324, 700; 1H NMR (DMSO-d6) δ 2.01 (d, 3H, CHCH3, 3J H,H
)
7.14 Hz), 2.09 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.89 (t, 2H, CH2,
J ) 5.7 Hz), 3.63 (q, 2H, CH2, J ) 5.7 Hz), 6.16 (q, 1H, CHCH3,
3J H,H ) 7.14 Hz), 6.49 (s, 1H, NH), 7.21-7.42 (m, 9H, Phenyl-
H), 8.37 (d, 2H, NH2, J ) 7.4 Hz); 13C NMR (DMSO-d6) δ 10.50
(CH3), 10.73 (CH3), 19.35 (CHCH3), 40.92 (CH2), 43.08 (CH2),
51.25 (CHCH3), 101.36 (C-4a), 105.11 (C-5), 126.22, 126.84,
127.18 (aromatic CH), 127.99 (C-6), 128.03, 128.34, 128.83
(aromatic CH), 139.33, 142.25 (aromatic ipso-C), 150.39,
154.84, 156.09 (C-7a, C-4, C-2).
A3 AR affinities to human recombinant receptors expressed
in CHO cell membranes were determined as described using
[3H]-5′-[(N-(ethylcarbonyl)amino]adenosine (NECA) as the A3
radioligand.12
The Cheng-Prusoff equation and KD values of 1 nM for [3H]-
CHA (A1), 0.2 nM for [3H]CCPA (A1), 14 nM for [3H]CGS21680
(A2A), and 6 nM for [3H]NECA (A3) were used to calculate the
Ki values from IC50 values, determined by the nonlinear curve-
fitting program Prism, version 2.0 (GraphPad, San Diego,
CA).29
(R,S)-2-[4-Ch lor op h en yl-5,6-d im et h yl-7(R)-(1-m et h yl-
ben zyl)-7H-p yr r olo[2,3-d ]p yr im id in -4-yla m in o]bu ta n -1-
ol (44). A solution of 1.2 g (3.0 mmol) of 39 in 10 mL of freshly
distilled 2-aminobutanol was refluxed for 7 h. The resulting
oil was mixed with silica gel and the mixture was put on a
silica gel column. After purification by column chromatography
with petroleum ether:ethyl acetate (2:1), the precipitate was
recrystallized from 200 mL of methanol: IR (KBr) ν 3428,
Ra d ioliga n d Bin d in g a t Hu m a n Recom bin a n t A1 ARs.
Mem br a n e p r ep a r a tion : After growing CHO cells express-
ing human A1 ARs, they were washed and frozen in the dishes
at -80 °C.12 For the preparation of membranes, the cells were
thawed followed by scraping them off the Petri dishes using
TRIS buffer (pH 7.4, 50 mM). The cell suspension was
homogenized on ice (polytron, 10 s with high speed) and then
centrifuged for 20 min at 4 °C and 40000g. After resuspending
the pellet in TRIS puffer, it was washed twice and stored in
aliquots at -80 °C with a protein concentration of 2.3 mg/mL.
Bin d in g a ssa y: Radioligand binding assays were per-
formed essentially as described.12,30 [3H]CCPA was used as the
A1 ligand in a concentration of 0.5 nM. The protein concentra-
tion in the assays was 70-100 µg/mL. A Kd value of 0.7 nM
was used to calculate Ki values.30 The nonspecific binding
amounted to 10% of the total binding.
1
2938, 1660, 1600, 1486, 1458, 1090, 1064, 728, 696, 540; H
3
NMR (CDCl3) δ 0.96 (t, 3H, CH2CH3, J H,H ) 7.4 Hz), 1.31 (m,
1H, CH2, J ) 6.9 Hz), 1.47 (m, 1H, CH2, J ) 7.0 Hz), 2.03 (d,
3H, CHCH3, J ) 7.14 Hz), 2.06 (s, 3H, CH3), 2.45 (s, 3H, CH3),
2.77 (m, br, 1H, CH, J ) 4.11 Hz), 3.30 (dq, 1H, CH2OH, J )
8.0 Hz), 3.61 (dd, 1H, CH2OH, J ) 3.7 + 6.8 Hz), 6.28 (q, 1H,
CHCH3, 7.14 Hz), 7.20-7.32 (m, 5H, phenyl-H), 7.44 (d, 2H,
pCl-phenyl-H, J ) 8.5 Hz), 8.31 (d, 2H, pCl-phenyl-H, J ) 8.5
Hz); 13C NMR (CDCl3) δ 9.86 (CH2CH3), 10.49 (CH3), 10.96
(CH3), 19.42 (CHCH3), 27.33 (CH2CH3), 51.83 (CHCH3), 54.53
(CH), 66.43 (CH2OH), 105.56 (C-4a), 111.60 (C-5), 126.41,
127.20, 128.55, 128.75, 128.81 (aromatic CH), 129.27, 131.79
(C-6, para-Cl-C), 136.65, 141.57 (aromatic ipso C), 148.07,
148.22, 161.74 (C-7a, C-4, C-2).
(R,S)-2-[7-Cyclop en tyl-5,6-d im eth yl-2-p h en yl-7H-p yr -
r olo[2,3-d ]p yr im id in -4-yla m in o]bu ta n -1-ol (45). A solution
of 326 mg (1.0 mmol) of 40 in ca. 5 mL of freshly distilled
2-aminobutanol was refluxed for 1 h. The resulting oil was
mixed with silica gel and the mixture was put on a silica gel
column. After purification by column chromatography with
petroleum ether:ethyl acetate (2:1), the resulting oil was
lyophilized: IR (KBr) ν 3452, 2960, 2866, 1594, 1574, 1542,
1460, 1446, 1428, 1412, 1378, 1324, 774, 702; 1H NMR (CDCl3)
δ 1.09 (t, 3H, CH3, J ) 7.5 Hz), 1.74 (m, 4H, CH2CH2), 2.02-
2.17 (m, 2H, CH2), 2.33 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.48-
2.52 (m, 2H, CH2), 3.72 (dd, 1H, CH2-O, J ) 2.5 + 8.0 Hz),
3.94 (dd, 1H, CH2-O, J ) 2.3und 8.2 Hz), 4.29 (m, 1H,
CH2CH*CH2), 4.83 (quint, 1H, CH, J ) 8.7 Hz), 5.16 (d, 1H,
NH, J ) 5.8 Hz), 7.38-7.48 (m, 3H, phenyl-H), 8.36 (d, 2H,
phenyl-H, J ) 7.14 Hz); 13C NMR (CDCl3) δ 10.56 (CH2CH3),
11.01 (CH3), 11.37 (CH3), 25.13 (CH2CH3), 25.49 (2 × CH2),
31.14 (CH2), 31.25 (CH2), 55.83 (CH), 56.10 (C*H), 68.68 (CH2-
OH), 102.19 (C-4a), 104.01 (C-5), 127.67, 128.28, 129.02
(aromatic CH), 130.01 (C-6), 139.44 (aromatic ipso-C), 150.47,
155.46, 157.13 (C-7a, C-4, C-2).
Solu bility Deter m in a tion . The solubility of selected com-
pounds was determined using HPLC/UV. A 10 mM solution
of the compounds in DMSO was prepared, diluted 1:100 in
TRIS-HCl buffer, 50 mM, pH 7.4, and allowed to reach
equilibrium by shaking overnight at room temperature. After
centrifugation (1000g), the supernatant was filtered through
cotton. Several dilutions of these saturated stock solutions
were made in the buffer, on the basis of the estimated
solubility. These dilutions were injected into the HPLC system
and analyzed by UV detection. The solubility of the compounds
was then calculated using calibration curves, previously car-
ried out. The method was validated according to the following
Ack n ow led gm en t. The authors thank Dr. J . Hipp
for performing a few of the binding assays. S. Hess and
K. Eger are grateful for support from the Deutsche
Forschungsgemeinschaft (DFG) within the Innovation-
skolleg Chemisches Signal und Biologische Antwort
(Chemical Signal and Biological Response) and from the
Fonds der Chemischen Industrie. C. E. Mu¨ller is grate-
ful for support from the Fonds der Chemischen Indus-
trie.
Su p p or tin g In for m a tion Ava ila ble: IR and 1H and 13C
NMR data of intermediate and final products synthesized;
validation data of solubility determination. This material is
available free of charge via the Internet at http://pubs.acs.org.
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