Identification and optimization of substituted 5-aminopyrazoles as potent and selective adenosine A1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.07.095

Potent and selective adenosine A₁ receptor antagonists were disclosed. SAR and pharmacological profile of selected compounds were discussed.

Full text of DOI:10.1016/j.bmcl.2010.07.095

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5891–5894
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and optimization of substituted 5-aminopyrazoles as potent
and selective adenosine A1 receptor antagonists
a
a
a
b
c
Nils Griebenow a, , Lars Bärfacker , Heinrich Meier , Dirk Schneider , Nicole Teusch , Klemens Lustig ,
*
Raimund Kast d, Peter Kolkhof d
a Bayer Schering Pharma AG, Global Drug Discovery, Medicinal Chemistry Wuppertal, Building 460, D-42096 Wuppertal, Germany
b Bayer Schering Pharma AG, Global Drug Discovery, Cell Biology, Building 456, D-42096 Wuppertal, Germany
c Bayer Schering Pharma AG, Global Drug Discovery, Research Pharmacokinetics, Building 468, D-42096 Wuppertal, Germany
d Bayer Schering Pharma AG, Global Drug Discovery, Cardiology Research, Building 500, D-42096 Wuppertal, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Potent and selective adenosine A1 receptor antagonists were disclosed. SAR and pharmacological profile
of selected compounds were discussed.
Received 10 July 2010
Revised 22 July 2010
Accepted 23 July 2010
Available online 1 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
5-Aminopyrazole
Adenosin A1 receptor antagonist
Heart failure
Diuresis
Natriuresis
Acute heart failure is one of the most common diseases in emer-
gency medicine and associated with a poor prognosis. The acute
decompensated heart failure registry (ADHERE) of 100,000 pa-
tients admitted with acute decompensated heart failure (ADHF) re-
veals that moderate and severe renal insufficiency, and even renal
failure, are common in this population, and that normal renal func-
tion is rare.1
Diuretics are the mainstay of treatment for HF associated with
volume overload. Diuretic therapy with loop diuretics can worsen
renal function (e.g. reduction of glomerular filtration rate) in ADHF
patients, and worsening renal function is associated with poorer
outcomes.2 Diuretic resistance in ADHF patients can also be con-
sidered as another indicator of poor prognosis in patients with
chronic heart failure. New diuretic drugs which increase diuresis
without worsening of renal function and, which overcome diuretic
resistance in ADHF patients are urgently needed for patients to im-
prove outcome and reduce re-hospitalization.
expressed in afferent arterioles, the inner medullar collecting
duct, the outer medullar collecting duct, the connecting tubule
and cortical collecting duct, and Henle’s loop (thin limb). It has
been found that tissue distributions and signaling transduction
pathways of the receptors show some species differences, but
the main effects of kidney A1 receptor activation are vasoconstric-
tion of arterioles, inhibition of diuresis and inhibition of sodium
transport. Inhibition of A1 receptor activity by A1 antagonists
results in inhibition of vasoconstriction of renal arterioles by
adenosine, increase of diuresis and natriuresis without worsening
of glomerular filtration rate (GFR) and diuretic activity in Furose-
mide resistance. This has been proven in animal experiments and
clinical studies with ADHF patients.4 A1 antagonists are, therefore,
a promising drug class for the improvement of renal function in
ADHF patients.
Our interest began with the identification of 1 as a moderate-
potent and selective adenosine A1 antagonist (Fig. 1). Amino-pyra-
zole 1 was discovered during the profiling and reevaluation of
compounds from an internal diabetes project.5 The development
of adenosine A1 receptor antagonists is an increasingly competitive
field6 and we were encouraged to find that 5-aminopyrazoles rep-
resented a novel chemotype.
Adenosine, a purine nucleoside, is present in all cells and re-
leased under a variety of different physiologic and pathophysio-
logic circumstances to affect various physiological actions. The
effects of adenosine are mediated by the stimulation of four
distinct receptors A1, A2a, A2b, and A3.3 Kidney A1 receptors are
However, inferior PK properties (see Table 3) and moderate
potency prevented 1 from showing consistent in vivo activity in
various diuresis models in rats.
* Corresponding author. Tel.: +49 202 36 5866; fax: +49 202 36 8149.
E-mail address: nils.griebenow@bayerhealthcare.com (N. Griebenow).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.095

5892
N. Griebenow et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5891–5894
O
congeners compared to mono substituted phenyl rings (comparing
8 to 7, 10 to 9, and 12 to 11).
This is particularly true for alkoxy substituents (see compound
12). Best potency resulted when the 3,4-substitution pattern is dis-
played by bicyclic ring systems, such as congeners 13 and 14.
Moreover, it was found that the 6–6 ring combination is more po-
tent compared to the 6–5 ring system (14 vs 13). The quinoxaline
15 exhibited greatly improved A1 potency. The downside of this
type of analog is the moderate to high clearance and low bioavail-
ability (see Table 3). It was found that 15 is rapidly metabolized at
the para-methoxy group of the aminobenzoic acid (O-demethyla-
tion) but also at the quinoxaline (hydroxylation) and the carboxylic
acid (glucuronidation). We had some concerns about the methoxy
substituent, because quinone imine could be formed after demeth-
ylation and oxidation of the hydroxyaniline, which could cause ad-
verse effects upon prolonged use.8 As a result of these concerns we
decided to proactively explore the feasibility to replace this substi-
tuent. Thus, replacement with chlorine, methyl, and hydrogen was
investigated resulting in a slight loss of potency (see Table 2: 16,
17, and 18).
O
IC50 A1
IC50 A2a
IC50 A2b >50 µM
40 nM
46 µM
N
N
N
H
O
OH
1
Figure 1. Initial lead structure from the reinvestigation of internal project
compounds.
Hence, an exploratory program was initiated to rapidly investi-
gate SAR around this novel chemotype as well as to identify an
analogue with a PK profile suitable for preliminary validation stud-
ies in in vivo preclinical models. A number of analogs were synthe-
sized according to the details in Scheme 1.
Compounds were build up in five linear steps. Initially, hydra-
zines 2 were condensed with 3-aminocrotononitrile to provide
the 5-aminopyrazoles 3. Subsequent Buchwald–Hartwig type
cross-coupling of the 5-aminopyrazoles with methyl bromobenzo-
ates yielded the N-aryl amino pyrazoles 4. Bromination of 4 fol-
lowed by Suzuki arylation with aryl boronic acids and
consecutively saponification of the methyl esters provided the tar-
get compounds 6.7
Early SAR exploration focused on aromatic variations at the C-4
of the pyrazole 5 which could be performed in a parallel library
synthesis format utilizing Suzuki cross-coupling methodology. A
list of selected compounds is shown in Table 1.
The SAR trends in Table 1 revealed that the combination of
meta- and para-substitution on the phenyl resulted in more potent
The SAR at the N-1 position of the pyrazole revealed that aro-
matic substituents, in particular ortho-substituted phenyl rings,
are clearly favored over alkyl groups, including cyclohexyl. The ser-
Table 1
SAR at the C-4 of the pyrazoles
Ar
O
4
N
N
N
H
O
OH
a
Compound
Ar
A1 IC50 [nM]
O
R2
1
40
Br
*
*
*
H2N
NH
O
O
N
a
7
8
3.160
568
NH2
N
R1
b
3
R1
2
F
F
9
10
11
12
13
14
15
1.390
294
306
233
231
27
Br
*
*
*
R2
R2
N
N
c
N
H
N
H
N
N
F
O
O
O
O
O
R1
R1
4
5
O
O
R3
*
*
R2
O
R3 B(OH)2
d, e
N
N
N
O
H
O
O
OH
R1
6
O
N
*
Scheme 1. (a) 1 N HCl (aq), 3-aminocrotononitrile, reflux, 16 h; (b) BINAP, Cs2CO3,
Pd2(dba)3, toluene, reflux, 16 h; (c) 1,3-dibromo-5,5-dimethylhydantoin, DCM, 0 °C
then rt, 2 h; (d) PdCl2(dppf), toluene, dioxane, 2 N Na2CO3 (aq), 75 °C, 16 h; (e)
NaOH, THF/MeOH/H2O (v/v = 3:1:1), rt, 16 h (96%). R-groups are defined according
to the examples in Table 1 and Table 2.
3.2
N
*
a
IC50 data were determined in a cAMP functional assay, see Ref. 1; values are
means of three experiments.

N. Griebenow et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5891–5894
5893
Table 2
Extended SAR of the pyrazoles
R2
3
R3
4
R4
N
N
1 N
R1
H
OH
O
a
a
a
a
Compd
R1
R2
R3
R4
A1 IC50 [nM]
3.2
A2a IC50
>50
[lM]
A2b IC50
>50
[l
M]
A3 IC50
13
[lM]
*
*
*
*
N
15
Me
OMe
Cl
N
N
*
*
*
*
*
*
*
*
*
*
*
16
17
18
19
20
21
22
23
24
25
Me
Me
Me
Me
Me
Me
Me
CF3
Et
16
>50
>50
>50
5.5
>50
>50
>50
1.1
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
N
N
Me
H
5.1
8.0
2.8
435
7.3
6.4
9.0
13
N
N
N
N
*
*
OMe
OMe
OMe
OMe
H
N
N
>50
>50
>50
>50
>50
>50
>50
>50
>50
19
N
N
*
Cl
N
N
*
N
N
*
*
*
*
N
N
H
>50
>50
N
N
H
H
277
N
F
N
N
26
Me
H
0.6
>50
>50
>10
*
nd = not determined.
a
IC50 data were determined in a cAMP functional assay, see Ref. 1; values are means of three experiments.
over the A2a and A2b receptors is also mediated by the methyl sub-
stituent (compare 15 and 19). Subsequently, SAR studies focused
on variations at the C-3 position. Replacement of the methyl group
by trifluoromethyl or ethyl was tolerated, however, introduction of
a hydrogen at this position resulted in a significant loss in potency
(see 25). Finally, SAR investigations were carried out at the quinox-
aline core. Fluorine-substitution at the C-5 position of the quinox-
aline gave highly potent compounds (see 26), compared with other
substitution patterns (data not shown).
Because of its favorable PK profile (Table 3: low clearance, high
bioavailability) compound 26 was progressed further to pharmaco-
logical in vivo investigations in rats. The diuretic and natriuretic
activity in vivo of the compound was investigated after oral as well
as intravenous application in two independent experiments each.9
Data from the first experiment after iv application of 0.3, 1.0, and
3.0 mg/kg of 26 revealed significant diuretic action at 0.3 mg/kg
Table 3
Pharmacokinetic results for compounds 1, 15 and 26 in Wistar ratsa
b
Compound
CLblood [LhÀ1 kgÀ1
]
T1/2 [h]
Fc [%]
1
15
26
4.00
2.62
0.57
1.5
0.4
1.8
20
20
97
a
Intravenous administration of 1 mg kgÀ1 in 1% DMSO and 99% plasma; oral
administration of 3 mg kgÀ1 in EtOH/PEG400/H2O (1:4:5).
b
Blood clearance.
Oral bioavailability.
c
ies of methyl derivatives shows that mono-ortho-substitution is
favored over disubstitution or the meta- and para-isomers (data
not shown), but methyl can be replaced with almost no loss in
potency by ethyl or chlorine (22 and 21). Additionally, selectivity

5894
N. Griebenow et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5891–5894
models after iv application up to 30 mg/kg.14 On the other hand,
***
12.5
10.0
7.5
compound 26, exhibited a significant higher selectivity versus
the A2b receptor compared with tonapophylline (A2b/A1 IC50 ratio:
26 >83.000, BG-9928 = 80, in-house data). One question in A2b
receptor antagonism is whether there are house-keeping pathways
driven by A2b receptor agonism whose disruption may lead to
unexpected toxicities.15
***
*
Beyond acute heart failure, other potential targets of adenosine
A1 blockade include drug-induced renal injury (e.g., due to antibi-
otics [gentamicin], immunosuppressive agents [cyclosporine], or
chemotherapy [cisplatin])4 and acute kidney injury after cardio-
pulmonary bypass. Furthermore A1 receptor antagonists may be
useful in minimizing or preventing endotoxin-induced organ
(e.g., lung) damage associated with sepsis as well as in the treat-
ment of osteoporosis, prosthetic joint loosening.16
5.0
2.5
0.0
V
0.1
0.3
1.0
3.0
compound 26 [mg/kg]
References and notes
Figure 2. Diuretic activity of 26 in Wistar rats.
1. Francis, G. C. Clin. J. Med. 2006, 73, S8.
2. Forman, D. E.; Butler, J.; Wang, Y.; Abraham, W. T.; O’Connor, C. M.; Gottlieb, S.
S.; Loh, E.; Massie, B. M.; Rich, M. W.; Stevenson, L. W.; Young, J. B.; Krumholz,
H. M. J. Am. Coll. Cardiol. 2004, 43, 61.
3. Fredholm, B. B.; Ijzerman, A. P.; Jacobson, K. A.; Klotz, K.-N.; Linden, J.
Pharmacol. Rev. 2001, 53, 527.
4. Dohadwala, M. M.; Givertz, M. M. Cardiovasc. Ther. 2008, 26, 276.
5. Rudolph, J.; Cantin, L.-D.; Magnuson, S.; Bullock, W.; Bullion, A.-M.; Chen, L.;
Chuang, C.-Y.; Liang, S.; Majumdar, D.; Ogutu, H.; Olague, A.; Qi, N.; Wickens, P.
L. PCT Int. Appl., WO 2004050650, 2004.
6. (a) Hobbs, R. E. Current Advances in Heart Disease, Proceedings of the World
Congress on Heart Disease, 14th International Academy of Cardiology Annual
Scientific Sessions, Toronto, ON, Canada, July 26–29, 2008, 273–276.; (b)
Doggrell, S. A. Curr. Opin. Invest. Drugs 2005, 6, 962.
7. (a) For experimental details and characterization of the compounds see: (a)
Baerfacker, L.; Kast, R.; Griebenow, N.; Meier, H.; Kolkhof, P.; Albrecht-Kuepper,
B.; Nitsche, A., Stasch, J.-P.; Schneider, D.; Teusch, N.; Rudolph, J.; Whelan, J.;
Bullock, W.; Pleasic-Williams, S. PCT Int. Appl., WO 2010020363, 2010.; (b)
Baerfacker, L.; Kast, R.; Griebenow, N.; Meier, H.; Kolkhof, P.; Albrecht-Kuepper,
B.; Nitsche, A.; Stasch, J.-P.; Schneider, D.; Teusch, N. PCT Int. Appl., WO
2010020366, 2010.; (c) Baerfacker, L.; Kast, R.; Griebenow, N.; Meier, H.;
Kolkhof, P.; Albrecht-Kuepper, B.; Nitsche, A.; Stasch, J.-P.; Schneider, D.;
Teusch, N. Ger. Offen. DE 102008039083, 2010.; (d) Baerfacker, L.; Kast, R.;
Griebenow, N.; Meier, H.; Kolkhof, P.; Albrecht-Kuepper, B.; Nitsche, A., Stasch,
J.-P.; Schneider, D.; Teusch, N. Ger. Offen. DE 102008039082, 2010.
8. Chen, W.; Koenigs, L. L.; Thompson, S. J.; Peter, R. M.; Rettie, A. E.; Trager, W. F.;
Nelson, S. D. Chem. Res. Toxicol. 1998, 11, 295.
9. After seven days of acclimatization, male Wistar rats (body mass 200–350 g,
Charles River Breeding or Harlan) were placed in metabolic cages (Tecniplast
Deutschland GmbH, Hohenpeißenberg, Germany) on water ad libitum. The test
compounds were administered either orally or intravenously in 1 ml/kg vehicle
solution (40% PEG400 and 60% Tris buffer) to 6–8 animals per dosage group.
The urine of the animals was collected at the bottom of the cages in special
collecting tubes. The usual duration of urine collection was 4 h. The urine
samples were analyzed for volume as well as sodium and potassium on a flame
photometer.
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
***
***
***
*
V
0.1
0.3
1.0
3.0
compound 26 [mg/kg]
Figure 3. Natriuretic activity of 26 in Wistar rats.
(Fig. 2) as well as a significant increase in natriuresis at 0.1 mg/kg
(Fig. 3).
In conclusion, we have identified a novel series of substituted 5-
aminopyrazoles as potent and selective adenosine A1 receptor
antagonists. Lead optimization resulted in the identification of 26
with favorable PK properties suitable for in vivo studies. Lead 26
showed the expected profile of an A1 receptor antagonist regarding
diuresis and natriuresis. However, the recent failure of rolofylline
(KW-3902) in the PROTECT trial10 and the adjustment of tonap-
ophylline (BG-9928) from phase III to phase II11 have raised some
concerns about the use of A1 antagonists for the treatment of acute
heart failure. Since these compounds are xanthine derivatives in
contrast to our discovered class of 5-aminopyrazoles, it will be
interesting to see whether non-xanthine-like A1 antagonists will
have a future in the treatment of acute heart failure.
10. (a) Weatherley, B. D.; Cotter, G.; Dittrich, H. C.; DeLucca, P.; Mansoor, G. A.;
Bloomfield, D. M.; Ponikowski, P.; O’Connor, C. M.; Metra, M.; Massie, B. M. J.
Card. Fail. 2010, 16, 25; (b) Details on the design of PROTECT are available at
http://www.clinicaltrials.gov, identifiers NCT00328692 and NCT00354458.
11. A global phase III trial began in August 2008; the trial was closed in December
2009. By March 2010, the drug was no longer listed on Biogen’s pipeline and
the program was presumed to have been discontinued.
12. Pronounced blood–brain-barrier penetration of KW-3902 has been found in rat
(in house data).
13. Observations from clinical trials suggest that KW-3902 might lower the seizure
threshold. See: (a) Givertz, M. M.; Massie, B. M.; Fields, T. K.; Pearson, L. L.;
Dittrich, H. C. J. Am. Coll. Cardiol. 2007, 50, 1551; (b) Givertz, M. M. Circ. Heart
Fail. 2009, 2, 519.
Furthermore compound 26 may have a potentially superior
safety profile compared to rolofylline and tonapophylline. On the
one hand, rolofylline is able to cross the blood–brain barrier,12
a
14. Inhouse data from MES and PTZ models. MES: maximal electroshock. PTZ:
pentylenetetrazol.
15. Zablocki, J.; Elzein, E.; Kalla, R. Expert Opin. Ther. Patents 2006, 16, 1347.
16. Giorgi, I.; Nieri, P. Expert Opin. Ther. Patents 2008, 18, 677.
fact that might be associated with pro-convulsive effects via inhi-
bition of A1 receptors in the central nervous system.13 Whereas,
compound 26 showed no pro-convulsive effects in relevant mouse