Discovery and optimization of 2-aryl oxazolo-pyrimidines as adenosine kinase inhibitors using liquid phase parallel synthesis

Article abstract of DOI:10.1016/j.bmcl.2004.01.082

Adenosine kinase inhibition is an attractive therapeutic approach for several conditions for example, neurodegeneration, seizures, ischemia, inflammation and pain. Several nucleosidic and non-nucleosidic inhibitors are available. Using a virtual screening approach, we have discovered that 2-aryl oxazolo-pyrimidines are adenosine kinase inhibitors. Subsequent high throughput derivatization enabled the optimization of this new inhibitor chemotype resulting in highly potent derivatives. A variety of analogues were produced by applying liquid phase parallel synthesis to vary the 7-amino residues as well as the 2-aryl moiety.

Full text of DOI:10.1016/j.bmcl.2004.01.082

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1997–2000
Discovery and optimization of 2-aryl oxazolo-pyrimidines as
adenosine kinase inhibitors using liquid phase parallel synthesis
M. Bauser,a,* G. Delapierre,a,y M. Hauswald,a T. Flessner,a D. D’Urso,b A. Hermann,b
B. Beyreuther,b J. De Vry,b P. Spreyer,b E. Reissmullerb and H. Meiera
aBAYER AG, Bayer Health Care, Division Pharma, Medicinal Chemistry, Germany
bBAYER AG, Bayer Health Care, Division Pharma, CNS Research, Germany
Received 29 September 2003; revised 4 December 2003; accepted 12 January 2004
Abstract—Adenosine kinase inhibition is an attractive therapeutic approach for several conditions for example, neurodegeneration,
seizures, ischemia, inflammation and pain. Several nucleosidic and non-nucleosidic inhibitors are available. Using a virtual screen-
ing approach, we have discovered that 2-aryl oxazolo-pyrimidines are adenosine kinase inhibitors. Subsequent high throughput
derivatization enabled the optimization of this new inhibitor chemotype resulting in highly potent derivatives. A variety of ana-
logues were produced by applying liquid phase parallel synthesis to vary the 7-amino residues as well as the 2-aryl moiety.
# 2004 Elsevier Ltd. All rights reserved.
Adenosine acts as an extracellular signaling molecule,
modulating cellular activity and local nutrition supply.
It binds to specific purinergic receptors,1 several sub-
types of which are described in the literature (A1, A2a,
A2b and A3).2 Adenosine levels are increased under
adverse conditions due to the intra- and extracellular
production of adenosine from adenosine phosphates.
Strong evidence suggests that this protective pathway is
involved in pathological processes including neurode-
generation, seizures, ischemia, inflammation and pain.3
Adenosine produces analgesic effects if applied intra-
thecally4 and mice lacking the A1 receptor exhibit
hyperalgesia.5 However, the short plasma half-life of
adenosine and possible side effects of systemic treatment
with A1 agonists limits their therapeutic use. Alter-
natively, analgesic effects can be achieved by increasing
local extracellular adenosine levels by inhibiting the
intracellular metabolizing enzyme adenosine kinase,
which is responsible for phosphorylating adenosine to
AMP, inside the cell. Since adenosine uptake is driven
by its concentration gradient, intracellular inhibition of
adenosine kinase decreases cellular re-uptake of adeno-
sine, leading to increased extracellular adenosine levels.
This approach, which acts selectively in tissues with
increased adenosine concentrations, offers some benefits
over the use of purinergic receptor agonists, because
purinergic receptors are widespread and undesirable
side effects could result from applying systemic treat-
ments. Consequently, adenosine kinase and its inhibi-
tors continue to be studied quite intensively.
A high-resolution crystal structure of the human
enzyme6 has been published and several inhibitors,
nucleosidic7 and non-nucleosidic,8 are known (Scheme
1). However, none of the currently available inhibitors
have proceeded to an advanced development stage so
far.
Therefore we were interested in finding new non-
nucleosidic chemotypes of adenosine kinase inhibitors.
Based on the available inhibitors (Scheme 1), the
* Corresponding author. Tel.: +49-(0)202-364981; e-mail: marcus.
bauser.mb@bayer-ag.de
CEA/LETI, Biology and Health Care Systems Department, Grenoble,
France.
Scheme 1. Structures of known nucleosidic and non-nucleosidic
adenosine kinase inhibitors and the proposed general structure of the
oxazolopyrimidine structure.
y
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.082

1998
M. Bauser et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1997–2000
natural substrate, and the enzyme X-ray structure, a
virtual screening approach led to the proposal of 2-aryl
7-amino-oxazolo[5,4-d]pyrimidines as possible frame-
work for new lead structures.
Table 1. AKI inhibition by derivatives of 3
The target oxazolo-pyrimidines can be synthesized via
two major routes, either starting from 5-amino 4-cyano
oxazoles,9 or in three steps from commercially available
5-amino-4,6-dihydroxy-pyrimidine10 (Scheme 2). We
found the second route to be well suited for solution
phase parallel synthesis. 5-Amino-4,6-dihydroxy-pyr-
imidine was condensed with acid anhydrides, for example,
benzoic anhydride, at elevated temperature to form 2-
phenyloxazolo[5,4-d]pyrimidin-7-ones 2 in high yield
and purity. These intermediates were then transformed
quantitatively into the corresponding chloro derivatives
3 on treatment with phosphorous oxychloride at reflux
temperature. Displacement of the chloro substituent
was easily accomplished by heating an amine and 3 in
the presence of a base in DMF. The carboxylic acids
and amines required to probe the effect of the sub-
stituents at the 2 and 7 position of the oxazolo-pyr-
imidine scaffold are readily available in large numbers
and high diversity. The chloro derivatives of type 3 were
synthesized on a multi-gram scale. Derivatives 4
were produced in a library format by exchanging the
chlorine substituent with amines. We were able to
introduce primary and secondary amines including
sterically hindered amines, for example, tert-butyl
amine, and also anilines in yields varying from 20 to
90%. Library synthesis was performed in special glass
micro titer plates, enabling efficient mixing and heating.
Workup was performed by parallel filtration followed
by preparative HPLC-MS.11 In this manner, more than
800 compounds were made by varying the amine in
position 7 while keeping position 2 constant as phenyl.
These compounds were tested for adenosine kinase
inhibition in an assay similar to previously described
methods.12 The activity found for some derivatives
(Table 1) provided support for our pharmacophore
model. Specifically, the screening results indicate that an
additional aryl moiety in the amine residue at position 7
is necessary; simple alkyl derivatives 4a,b were inactive.
Moreover, in active compounds the aryl moiety must be
connected to the scaffold by a spacer with a well-defined
length and geometry.
Amine
AKI IC50 [nM]
Amine
AKI IC50 [nM]
1
ABT-702
10ꢁ3
4a
4c
>10.000#
4b
>10.000#
>10.000#
1.000#
>10.000#
>1.000#
270ꢁ80
>10.000#
4d
4f
4e
4g
4i
4h
230ꢁ60
4j
4l
>10.000#
4k
200ꢁ18
120ꢁ6
1.500ꢁ120
4m
most potent compounds in this series were the simple
open chain derivatives 4g and h and the racemic benzyl
bis-pyrrolidine 4k.13 Preparation of the enantiomeres of
4k, using both enantiomeres14 of the benzyl bis-pyrroli-
dine resulted in derivatives 4l and m, which exhibited a
difference in activity of one order of magnitude,
emphasizing the importance of the proper geometry for
activity. Subsequently, variation of the spacer length of
the more simple open chain derivatives, showed that
activity was lost with shorter chains between scaffold
and aryl moiety 4i,j. These results provide an illustra-
tion of the principle that synthesis of larger compound
libraries can be useful for generating lead structures in
early project stages using diverse starting materials. To
further optimize this compound class, the racemic bis-
pyrrolidine residue was kept constant as shown in 4k
and the phenyl moiety in position 2 was varied. We
added amino and bromo functionalities at the aryl resi-
due, thereby enabling further parallel derivatization
using amide bond forming and Suzuki coupling reac-
tions (Scheme 3).
The benzyl piperazine 4c, the bis-pyrrolidine 4d were as
well as 4-fluoro benzyl homopiperazine 4e were inactive.
Benzyl pyrrolo-piperidine 4f was slightly active. The
The derivatives of type 5 were prepared by using the
corresponding benzoic acid halides. The first step, acyl-
ation of the amino group in 5-amino 4,6-dihydroxy
pyrimidine, is followed by cyclization in polyphosphoric
acid at elevated temperature. The resulting 7-hydroxy
oxazolo-pyrimidines are transformed via phosphorous
oxychloride treatment to the corresponding chloro
derivatives 6. The benzyl bis-pyrrolidine residue is
introduced upon treating the chloro derivatives 6 with
the corresponding amine in DMF in the presence of
base. The nitro derivatives of type 7 are reduced using
catalytic hydrogenation. Parallel substitution of bro-
Scheme 2. Reagents and conditions: (a) neat, 140 ꢀC, 5 h; (b) POCl3,
reflux, 6 h; (c) (d) amine, DMF, Et3N, 110 ꢀC, 16 h.

M. Bauser et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1997–2000
1999
mine using Suzuki coupling reactions was performed in
dioxane/water using Pd(PPh3)4 as the palladium source.
Amide bond formation was accomplished using car-
boxylic acids and standard coupling reagents or acid
halides in the presence of base. Over 100 amides and
more than 60 biaryl compounds were prepared in a
library and tested for inhibition of adenosine kinase
activity and results can be summarized as follows. Sub-
stituting the aryl ring in para position led to compounds
with slightly better activity than the corresponding meta
series. Substituting at the para or meta position with
amides gave compounds of higher potency than did
substitution at this position with aromatic rings. In the
series produced by adding aryl rings through the Suzuki
reaction, only heteroaryl substitution in the para posi-
tion, for example, 9a, resulted in derivatives with nota-
bly higher activity compared to the precursors 7a,b and
the unsubstituted phenyl derivative 4k (Table 2).
Those were prepared using the procedure depicted in
Scheme 3 using pyridyl-carboxylic chlorides. Addition-
ally, in this series we investigated amino-pyridines easily
prepared via the chloro intermediate 11 (Scheme 4).
While the 4-pyridyl derivative 11a, disappointingly
showed no activity, results from the 3-pyridyl deriva-
tives 11b/11c indicated room for optimization in this
sub-series. This was nicely confirmed by the N,N-dimethyl
derivative 12 leading to the first derivative in our pro-
gram with aꢂ10 nanomolar IC50 value for adenosine
Table 2. AK Inhibition of 2-aryl substituted 7-benzyl bispyrrolidino
oxazolo-pyrimidines
Substitution with a substituted phenyl led to inactive
compounds, for example, 9b. Adding benzoic acids on
the amine 8 resulted in poor activity, only smaller sub-
stituents like fluorine being tolerated on the aromatic
ring as in 10c,d. The benzamide itself was the most
potent example amoung the benzoyl series. However
phenylacetic acid derivatives are almost as potent as
phenyl derivatives 10e and f. Derivatives 10i–l were
twice as potent than the corresponding precursor 8 and
five times more potent than the unsubstituted 2-phenyl
oxazolo-pyrimidine 4k. Building on the activity of ani-
line 8a, the 5-aryl moiety was converted to pyridines
11a–c in order to present a more basic functionality.
Aryl residue AKI IC50 [nM]
Aryl residue AKI IC50 [nM]
1
ABT 702
10ꢁ3
7a
240/140
7b
260 / 60
9a
8a
90ꢁ40
50ꢁ6
9b
8b
>10.000#
n.d.
10a
>10.000# 10b
>10.000#
10c
10e
10g
10i
100/130
70/50
70/50
30/30
40/40
10d
10f
10h
10j
10l
190/200
150/210
50/90
60/50
10k
40/30
Scheme 3. Reagents and conditions: (a) Pyridine, 50 ꢀC, 16 h; (b)
PPA, 100 ꢀC, 6 h; (c) POCl3, reflux, 5 h; (d) DMF, Et3N, 110 ꢀC, 16 h;
(e) Pd/C H2, MeOH, 16 h; (f) boronic acid, Pd(PPh3)4, K2CO3, diox-
ane/water, 80 ꢀC, 16 h; (g) carboxylic acid, TBTU, DMF, Et3N, 16 h.
Scheme 4. Reagents and conditions: Excess dimethylamine, 2M in
THF, rt, 40 h.

2000
M. Bauser et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1997–2000
Table 3. AK Inhibition of 2-pyridyl substituted 7-benzyl bispyrroli-
dino oxazolo-pyrimidines
3. Kowaluk, E. A.; Bhagwat, S. S.; Jarvis, F. J. Current
Pharm. Design 1998, 4, 404.
4. Gomes, J. A.; Li, X.; Pan, H. L.; Eisenach, J. C. Anes-
thesiology 1999, 1072.
5. Johannson, B.; Halldner, L.; Dunwiddie, T. V.; Masino,
S. A.; Poelchen, W.; Gimenez-Llort, L.; Escorihuela,
R. M.; Fernandez-Teruel, A.; Wiesenfeld-Hallin, Z.;
Xu, X. J.; Hardemark, A.; Betsholz, C.; Herlenius, E.;
Fredholm, B. B. PNAS 2001, 98, 9407.
6. Mathews, I. I.; Erion, M. D.; Ealick, S. E. Biochemistry
1998, 37, 15607.
7. Zhu, C. Z.; Mikusa, J.; Chu, K. L.; Cowart, M.; Kowaluk,
E. A.; Jarvis, M. F.; McGaraughty, S. Brain Research
2001, 905, 104.
8. (a) Cowart, M.; Lee, C.-H.; Gfesser, G. A.; Bayburt,
E. K.; Bhagwat, S. S.; Stewart, A. O.; Yu, H.; Kohlhaas,
K. L.; McGaraughty, S.; Wismer, C. T.; Mikusa, J.; Zhu,
C.; Alexander, K. M.; Jarvis, M. F.; Kowaluk, E. A.
Bioorg. Med. Chem. Lett. 2001, 11, 84. (b) Gomtsyan, A.;
Didomenico, S.; Lee, C. H.; Matulenko, M. A.; Kim, K.;
Kowaluk, E. A.; Wismer, C. T.; Mikusa, J.; Yu, H.;
Kohlhaas, K.; Jarvis, M. F.; Bhagwat, S. S. J. Med.
Chem. 2002, 45, 3639.
Aryl residue
ABT 702
AKI
IC50 [nM]
Aryl residue
AKI
IC50 [nM]
1
10ꢁ3
>1.000#
11a
11b
12
90/130
10/6
11c
70#
kinase inhibiton, comparing favorably with the reference
ABT 702 1 in our assay (Table 3).
9. Tolman, R. L.; Robins, R. K.; Townsend, L. B. J. Am.
Chem. Soc. 1969, 91, 2102.
In summary, we have discovered that oxazolo pyr-
imidines are a novel class of structurally novel adeno-
sine kinase inhibitors. High throughput derivatization
of the oxazolo-pyrimidine scaffold was performed using
liquid phase parallel synthesis techniques. This led to
the discovery of 12, an adenosine kinase inhibitor with
aꢂ10 nanomolar IC50 value.
10. (a) Patil, V. D.; Towsend, L. B. J. Heterocyclic Chem.
1971, 503. (b) Bold, G.; Manley, P. W.; Martin-Kohler,
A.; Sequin, U. WO Patent WO 02/092604 2002.
11. (a) Zeng, L.; Burton, L.; Yung, K.; Sushan, B.; Kassel,
D. B. J. Chromatogr. A 1998, 794, 4. (b) Bauser, M. J.
Chrom. Sci. 2002, 40, 292.
12. Human Adenosine Kinase (AK) was cloned and expres-
sed in E. coli. Inhibition of AK was determined in an in-
vitro assay measuring the conversion of radioactive
[14C]Adenosine (c=4 mM 16.7 nCi) to its corresponding
monophosphate derivative in the presence of ATP
(c=410 mM), MgCl2 (c=833 mM) and appropriate con-
centrations of test compound. IC50 values are determined
from the read out of three titration’s showing a regular
curve. They are given with SEM if this was performed in
at least 3 individual experiments. If only two experiments
have been performed both IC50 values are given. If only
one experiment was performed the IC50 values are marked
with a # Yamada, Y.; Goto, H.; Ogasawara, N. Biochim.
Biophys. Acta 1980, 616, 199.
Acknowledgements
We thank Dr. R. Grosser for separating 4k into the
enantiomeres, A. Grawinger, L. Jansen, R. Muller, C.
Oschwald, T.-O. Repp, D. Schneider, C. Wilkop, D.
Zehner, I. Pluschkell, J. Micksch, D. Huttebrauker for
preparing and purifying the compound libraries and I.
Flocke for performing the AK assay.
References and notes
13. Schenke, T.; Petersen, U.; Eur. Pat. Appl. EP 393424A2
1990.
1. All compounds prepared were chromatographically pur-
ified and analyzed using either HPLC-MS or NMR
Jacobsen, M. A. Expert Opin. Ther. Patents 2002, 12, 489.
2. Sebastiao, A. M.; Ribeiro, J. A. TIPS 2000, 21, 341.
14. The racemic bis-pyrrolidine was separated into the enan-
tiomeres by chromatography, using Daicelphase Chiralcel
OD as stationary phase and iso-hexane/iso-propanole (80/
20 v/v and 0,05% Diethylamine) as mobil phase.