NO-independent stimulators of soluble guanylate cyclase

Article abstract of DOI:10.1016/S0960-894X(01)00073-7

SARs around a novel type of guanylate cyclase stimulator which act by a mechanism different from classical NO-donors are described. Several pyrazolopyridinylpyrimidines are shown to relax aortic rings and revealed a long-lasting blood pressure lowering effect in rats after oral application.

Full text of DOI:10.1016/S0960-894X(01)00073-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 781–784
NO-Independent Stimulators of Soluble Guanylate Cyclase
a,
b
a
Alexander Straub, * Johannes-Peter Stasch, Cristina Alonso-Alija,
Jordi Benet-Buchholz, Bernhard Ducke, Achim Feurer and Chantal Fu
¨
rstnera
c
b
a
aInstitute of Medicinal Chemistry, Pharma Research Centre, Bayer AG, D-42096Wuppertal, FRG
bInstitute of Cardiovascular and Arteriosclerosis Research, Pharma Research Centre, Bayer AG, D-42096Wuppertal, FRG
cCentral Research, X-ray Laboratory, Division of Structural Research, Bayer AG, D-51368 Leverkusen, FRG
Received 11 December 2000; accepted 19 January 2001
Abstract—SARs around a novel type of guanylate cyclase stimulator which act by a mechanism different from classical NO-donors
are described. Several pyrazolopyridinylpyrimidines are shown to relax aortic rings and revealed a long-lasting blood pressure
lowering effect in rats after oral application. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
alternative sequence, for which a representative example
is shown in Scheme 1.
1
Soluble guanylate cyclase (sGC) is a heterodimeric (a/
b) heme-protein that converts GTP to cGMP, which is
an important messenger in signal transduction. Its nat-
ural stimulator is nitric oxide, which stimulates sGC via
the formation of a nitrosyl-heme complex. Organic
nitrates like glycerol trinitrate or isosorbide dinitrate
have been used for decades as a treatment for angina
pectoris. In vivo they are converted to NO which relaxes
vascular smooth muscles. The major drawback of this
therapy is the development of tolerance after repeated
applications. Recently an indazole derivative, YC-1, has
been described, which stimulates sGC directly via a dis-
tinct mechanism and sensitizes the enzyme towards its
Pharmacology4
For biological characterization we measured IC s for
5
the ability of the compounds to inhibit maximum con-
striction of phenylephrine-treated preconstricted
(3ꢀ10 g/mL) rabbit aortic rings. In order to prove the
mechanism of relaxation, the stimulatory effect on iso-
lated recombinant sGC alone and in the presence of NO
was measured. The in vivo efficacy was shown in
0
ꢁ8
4
anaesthetized rats after oral administration.
2
native activator NO. We used YC-1 as the lead structure
and selected the pyrazolopyridine Bay 41-2272 (40) as
the most promising one out of a series of thousands of
newly synthesized derivatives.
Results and Discussion
We started the optimization process with the variation
of the benzylic moiety of YC-1. At the methylene bridge
no substitution like a carbonyl-oxygen or alkyl groups is
tolerated. Omiting the methylene group with directly
linked aromatics leads to inactive compounds. Table 1
reflects the influence of substituents at the aromatic part
on the relaxation of preconstricted aortic rings. Fluorine
proved to be preferable leading to compounds which
show an increase in activity when changing the position
of the substituent from 4 (3)!3 (2)!2 (1). Other halo-
gens were less beneficial. The best activity was found
with a 5-pyrimidinylmethyl residue (10); however
because of its difficult synthetic accessibility and poor
solubility we focused on the 2-fluorophenyl moiety for
further optimization work.
Chemistry3
Pd-catalyzed biaryl-coupling is the method of choice for
the preparation of indazole-derived compounds. How-
ever this route gives low yields when applied to com-
pounds possessing more N-heteroatoms. Therefore, in
the case of pyrazolopyridinyl-pyrimidines we applied an
*
Corresponding author at current address. Bayer AG, Agricultural
Centre, Bldg. 6550, D-40789 Monheim. Tel.: +49-2173-384972; fax:
49-2173-384880; e-mail: alexander.straub.as@bayer-ag.de
+
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00073-7

782
A. Straub et al. / Bioorg. Med. Chem. Lett. 11 (2001) 781–784
Scheme 1. Reagents and conditions: (a) NC-CH¼C(ONa)CO
0% for steps (a) and (b); (c) NH /MeOH, rt, 2 days, quant.; (d) TFAA/pyridine, pyridine, rt, 8 h, quant.; (e) MeONa/MeOH, rt, 2 h, quant.; (f) 1.
Cl/HAc, MeOH, reflux, 8 h; 2. Na CO /H O, rt, 76.4%; (g) 1. HCO N] CHOt-Bu, neat,
Et, t-BuOK, THF, rt, 3 h; 2. HCl!pH 4; (h) [Me
2
Et/TFA, dioxane, reflux, 8 h; (b) Me
2
NCH¼CH-CHO/TFA/dioxane, reflux, 3 days,
5
3
NH
4
ꢂ
2
3
2
2
2
2
ꢂ
100 C, 46 h, 83.5%; (i) 7, neat, 100 C, 12 h, 23.4%; or 6, toluene, reflux, 8 h, 31.8%.
Concerning the pyrazol-type central moiety (see Table 2),
among all the core heterocycles investigated the pyr-
azolo[3,4-b]pyridin ring system (12) seems to be the
optimum with respect to in vitro effects, whereas inda-
zoles usually are somewhat weaker.
We next investigated the SARfor various substituents
at the aminopyrimidine (Table 4). A wide diversity of
substituents is tolerated in the 5-pyrimidine position
without considerably affecting the activity. For alkyl
residues, there seems to be an optimum at 2–5 carbon
As shown in Table 3, several heterocycles can replace
the furyl moiety. Besides other five-membered ring sys-
tems like oxazoles (20–22), we synthesized a 2-pyridyl
derivative (25) which had moderate activity. However,
after replacing it with a 2-pyrimidinyl-residue (26),
which displays rotational symmetry with respect to the
heteroatoms, a marked increase in potency could be
observed. In contrast to the furyl system no further
increase in activity was achieved by introduction of a
hydroxymethyl group (27). However, a 4-amino group
significantly enhanced the activity (28).
Table 2. Variation of the pyrazole type core heterocycle
Compounds
Heterocycle A
Relaxation of
preconstricted aortic rings
a
IC50, mM
Table 1. Variation of the N-substituent
1
1
1
1
1
1
2
3
4
5
4.9
1.7
6.932
Compounds
elaxation oR f Rp reconstricted
9.8b
a
aortic rings IC50, mM
YC-1
Ph
10
4.9
10
19
15
8.7
26
7.2
16
9.6
3.2
20.4 (27%)c
9.4d
1
2
3
4
5
6
7
8
9
2-F-Ph
3-F-Ph
4-F-Ph
2,6-F -Ph
2-CN-Ph
3-CN-Ph
2
16
2-(5-Chlorothiophene)
2-Pyridyl
3-Pyridyl
a
Values are standardized to YC-1=10 mM.
In combination with 5-hydroxymethyl-4-methyl-1,3-oxazol-2-yl instead
b
10
5-Pyrimidinyl
of 5-hydroxymethyl-2-furyl (cf. compound 20 in Table 3).
c
Percent of inhibition.
In combination with benzyl instead of 2-fluorobenzyl.
a
d
Values are standardized to YC-1=10 mM.

A. Straub et al. / Bioorg. Med. Chem. Lett. 11 (2001) 781–784
783
chain length. With increasing steric demand and lipo-
philicity, the activity decreases; the introduction of polar
residues generally leads to more potent compounds as
compared to alkyl chains with similar steric demand.
Table 3. Variation of the attached heterocycle
We selected 40 for further investigations. The X-ray
crystallographic analysis of 40 revealed a coplanar
arrangement of the biaryl system (Scheme 1). Since this
is a prerequisite for ligand function, we reasoned that
these molecules might display metal-complexing prop-
erties. Indeed we were able to prepare a silver complex
by adding AgNO to 40 in acetonitrile. The X-ray crys-
3
Compounds
B
Relaxation of preconstricted
a
aortic rings IC50, mM
tal structure is shown in Fig. 1. The relevance of this
phenomenon for the biological mechanism is still
unclear. However, substituting the core heterocycle by
an indole which lacks the ligand nitrogen leads to inactive
compounds.
17
18
19
1.7
9.3
8.0
Compound 40 concentration-dependently stimulated
isolated sGC from 0.1 nM–100 mM (Fig. 2). When given
Table 4. Variation of the pyrimidine substituents
2
2
0
1
0.9
1.2
2
2
2
2
2
2
2
2
3
4
5
6
7
8
1.5
Compounds
R1
R2
Relaxation of
preconstricted aortic
rings IC50 (nM)
3.2
a
3
3
1
2
H
Me
Et
n-Propyl
i-Propyl
n-Butyl
t-Butyl
n-Pentyl
n-Hexyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
265
675
272
252
207
218
709
200
1041
304
633
904
836
561
391
412
588
1294
343
243
261
378
517
414
306
478
508
650
1400
536
233
0.6
33
34
35
36
37
38
39
40
41
42
43
44
45
4.8
Cyclopropyl
Cyclobutyl
Cyclopentyl
1-Cyclopentenyl
Cyclohexyl
CN
2-Cyanoethyl
5-Cyanopentyl
6-Cyanoheptyl
OMe
1.25
1.3
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
SMe
0.27
SO
SO propyl
SO i-propyl
PO(OEt)
2
Me
2
2
2
2
3
9
0
1.8
8.6
N-Morpholino
F
CH
N-Thiomorpholino
CH NH
2 3
CF
2
2
H
Cyclopropyl
NH
2
a
a
Values are standardized to YC-1=10 mM.
Values are standardized to YC-1=10 mM.

784
A. Straub et al. / Bioorg. Med. Chem. Lett. 11 (2001) 781–784
Figure 1. Single crystal X-ray structure of the silver (Ag(I)) complex of
ꢁ
4
0 (NO
3
-counterion not shown).
Figure 3. Effect of 40 on blood pressure of anesthetized rats: -*-
Control (n=6); -!- 40: 0.3 mg/kg po (n=4); - - 40: 1.0 mg/kg po
(
n=5); - - 40: 3.0 mg/kg po (n=4).
References
. (a) For recent reviews see: Hobbs, A. J. Trends Pharmacol.
1
Sci. 1997, 18, 484. (b) Moncada, S.; Palmer, R. M.; Higgs,
E. A. Pharmacol. Rev. 1991, 43, 109. (c) Furchgott, R. F.
Angew. Chem., Int. Ed. Engl. 1999, 38, 1870. (d) Murad, F.
Angew. Chem., Int. Ed. Engl. 1999, 38, 1857. (e) Ignarro, L. J.
Angew. Chem., Int. Ed. Engl. 1999, 38, 1882.
2
. (a) Ko, F. N.; Wu, C.-C.; Kuo, S.-C.; Lee, F.-Y.; Teng, C.-
M. Blood 1994, 84, 4226. (b) Wu, C. C.; Ko, F. N.; Kuo, S. C.;
Lee, F. Y.; Teng, C. M. Br. J. Pharmacol. 1995, 116, 1973. (c)
Friebe, A.; Schultz, G.; Koesling, D. EMBO J. 1996, 15, 6863.
(
R.; Busse, R. Br. J. Pharmacol. 1997, 120, 681. (e) Becker,
E. M.; Wunder, F.; Kast, R.; Robyr, C.; Hoenicka, M.; Gerzer,
¨
¨
d) Mulsch, A.; Bauersachs, J.; Schafer, A.; Stasch, J. P.; Kast,
Figure 2. Stimulation of purified sGC by 40 (0.0001–100 mM) in the
absence (-*-) and presence of SIN-1 (-!- 0.01, -&- 0.1, -^- 1, and -
~- 10 mM) and ODQ (- - 10 mM). Stimulation of - - heme-free sGC.
R.; Schro
Hoenicka, M.; Becker, E. M.; Apeler, H.; Sirichoke, T.; Schro
der, H.; Gerzer, R.; Stasch, J. P. J. Mol. Med. 1999, 77, 14.
. (a) All final compounds were characterized by NMRand
¨
der, H.; Stasch, J. P. Nitric Oxide 1999, 3, 55. (f)
The specific activity of sGC is expressed as x-fold stimulation versus
basal activity (basal activity in the presence of Mg2+ 152 nmol/mg/
¨
-
min). The data presented represent meansꢃSEM, from four indepen-
3
dent experiments performed in duplicate.
mass spectrometry. Crystallographic data for 40 and the Ag
complex of 40 have been deposited at the Cambridge Crystal-
lographic Data Centre (40: CCDC 157014; 40-complex:
CCDC 157015). For experimental details see Straub, A.;
Feurer, A.; Alonso-Alija, C.; Stasch, J.-P.; Perzborn, E.; Hut-
¨
ter, J.; Dembowsky, K.; Stahl, E. WO 2,000,006,568, 2000.
Chem. Abstr. 2000, 132, 122629. (b) Gordon, D. W. Synlett
in combination, 40 and the NO donor SIN-1 revealed a
strong potentiating effect over a wide range of con-
centrations. As shown in Fig. 2, 40 does not activate the
heme-free enzyme. The sGC inhibitor ODQ completely
inhibited the effect of 40 at intact sGC. Investigations
concerning the molecular mechanism of action are pub-
lished elsewhere. After oral administration to anaes-
thetized rats it dose-dependently produced a long-lasting
decrease in blood pressure (Fig. 3).
1
998, 1065. (c) Collot, V.; Dallemagne, P.; Bovy, P. R.; Rault,
S. Tetrahedron 1999, 55, 6917.
. Becker, E. M.; Wunder, F.; Kast, R.; Robyr, C.; Hoenicka,
M.; Gerzer, R.; Schroder, H.; Stasch, J.-P. Nitric Oxide 1999,
4
5
¨
3
5
, 55.
. Stasch, J.-P.; Becker, E. M.; Alonso-Alija, C.; Apeler, H.;
Dembowsky, K.; Feurer, A.; Gerzer, R.; Minuth, T.; Perz-
born, E.; Pleiß, U.; Schroder, H.; Schroeder, W.; Stahl, E.;
Steinke, W.; Straub, A.; Schramm, M. Nature 2001, 409 (in
Our data demonstrate SARs of NO-independent sGC
stimulators which may offer a novel approach for
treatment of cardiovascular diseases.
¨
press).