Novel small molecule bradykinin B1 receptor antagonists. Part 3: Hydroxyurea derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.11.121

Hydroxy urea moieties are introduced as a new class of bradykinin B₁ receptor antagonists. First, the SAR of the lead compound was systematically explored. Subsequent optimization resulted in the identification of several biaryl-based hydroxyurea bradykinin B₁ receptor antagonists with low-nanomolar activity and very high oral bioavailability in the rat.

Full text of DOI:10.1016/j.bmcl.2009.11.121

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1233–1236
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel small molecule bradykinin B1 receptor antagonists. Part 3: Hydroxyurea
derivatives
*
Karsten Schnatbaum , Marco Schaudt, Roland Stragies, Jochen R. Pfeifer, Christoph Gibson, Elsa Locardi,
Dirk Scharn, Uwe Richter, Holger Kalkhof, Klaus Dinkel, Gunther Zischinsky
Jerini AG, Invalidenstraße 130, 10115 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydroxy urea moieties are introduced as a new class of bradykinin B1 receptor antagonists. First, the SAR
of the lead compound was systematically explored. Subsequent optimization resulted in the identifica-
tion of several biaryl-based hydroxyurea bradykinin B1 receptor antagonists with low-nanomolar activity
and very high oral bioavailability in the rat.
Received 22 October 2009
Revised 23 November 2009
Accepted 24 November 2009
Available online 2 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Bradykinin B1
Antagonist
Oral bioavailability
Pharmacokinetic profile
The kinins bradykinin (BK) and kallidin (KD), along with their
active metabolites des-Arg-bradykinin (DABK) and des-Arg-kalli-
din (DAKD), are endogenous peptides whose actions are mediated
by the two G-protein coupled B1 and B2 receptors.1,2 While the B2
receptor is constitutively expressed under physiological conditions
and rapidly desensitized after stimulation, the B1 receptor is upreg-
ulated following tissue injury or inflammation and is not subject to
desensitization after repeated agonist stimulation.3 It is well estab-
lished that kinins contribute to the pathophysiological processes
accompanying inflammation and pain. While the B2 receptor has
been proposed to play a major role in acute disease states, the B1
receptor is the primary kinin receptor mediating chronic inflam-
mation and/or persistent pain.4,5
It has been shown that B1 receptor agonists produce hyperalge-
sia in animals under inflammatory conditions and this effect could
be blocked by B1 receptor antagonists.6 B1 receptor deficient mice
display a normal phenotype but show a reduced sensitivity to
chemical and thermal nociception.7 Furthermore, B1 receptor
antagonists effectively ameliorated inflammatory and neuropathic
pain in animal models.8,9 Animal studies suggesting a central role
for the B1 receptor, especially in neuropathic pain, have led to
the development of several B1 receptor antagonists with high
CNS penetration.10
sulfones,13 benzimidazoles14 and bromopyrazoles.15 The biaryl
derivative 1 (Fig. 1) which has been disclosed by Merck as one of
their clinical backup compounds is among the most advanced B1
receptor antagonists.11,16
The a-hydroxyacetamide moiety of 1 features the arrangement
of one hydrogen bond accepting and two hydrogen bond donating
functional groups which is the shared motif of all published biaryl-
based B1 receptor antagonists.17,18 We hypothesized that
a
hydroxyurea unit can effectively replace the required hydrogen
bond-forming groups to provide highly active B1 receptor antago-
nists featuring a reduced number of stereocenters (Fig. 1). In this
study, we disclose that hydroxyureas do indeed represent a novel
class of B1 receptor antagonists with high activity and favorable
pharmacokinetic properties.
The general method used for the preparation of the compounds
described in this study is outlined in Scheme 1. Benzyl amines 3a–
R6
N
CF3
H
N
H
N
R1
OH
OH
F
O
N
R4
O
N
X
O
R5
R3
Recently, a number of bradykinin B1 receptor antagonists have
N
been described. Examples include biaryls,11 sulfonamides,12 aryl
Cl
F
R2
* Corresponding author. Correspondence should be addressed to: Pericles Calias,
Shire HGT, 125 Spring Street, Lexington, MA 02421, USA. Tel.: +1 781 482 0701; fax:
+1 781 482 2961. E-mail: pcalias@shire.com.
1
2a-q
Figure 1. Design of new biaryl bradykinin B1 receptor antagonists.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.121

1234
K. Schnatbaum et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1233–1236
icantly, whereas the chlorine atom in R2 position turned out to be
essential as shown by decreased potency of the chlorine-lacking
compound 2d.
R6
N
H
N
R1
NH2
X
R1
OH
R4
R4
O
R7
HN
Table 2 shows the SAR of B ring modifications of 2a. A fluorine
atom proved to be the preferred substituent in R4 position, as the
corresponding methyl derivative 2e (vs 2a) and unsubstituted 2g
(vs 2f) revealed lower activity. In addition, we found that the fluo-
ropyridine ring produces more active compounds as compared to
the fluorophenyl counterpart 2f (vs 2b).
a
X
+
OH
4a-j
R5
R5
R3
R2
R3
R2
Optimization of the urea substituent R6 is described in Table 3.
To increase the aqueous solubility, we replaced the methyl oxadi-
azole ring by methyl tetrazole.11,22
3a-l
2a-q
b
F
F
F
F
H2N
NH
OH
O
+
OH
Table 2
4a
5
Effect of B ring modifications on antagonistic activity of hydroxyurea 2a,b,e–g
H
F
F
F
F
R1
N
N
F
F
d
c
OH
H
+
R4
O
N
N
Boc
Boc
O
X
HN
N
Boc
OH
B
A
OH
Boc
O
O
4c
7
N
Scheme 1. Reagents and conditions: (a) N,N0-disuccinimidyl carbonate, DIPEA,
MeCN, 0 °C?rt; (b) NaBH3CN, MeOH/H2O; (c) PPh3, DEAD, THF, 0 °C; (d) HCl/MeOH.
Cl
F
R1
R4
X
CAM IC50 (nM)
a
Compound
l10,19,20 were activated with N,N0-disuccinimidyl carbonate (DSC)
and converted with the respective hydroxylamines, in a one pot
reaction, to the hydroxyurea 2a–q. All hydroxylamines were
commercially available with the exception of 4a, 4b and N-cyclope-
ntylhydroxylamine, which were prepared as previously described.21
Briefly, 4a was prepared by reductive amination of cyclohexanone 5
with hydroxylamine. A Mitsunobu reaction between 1,3-difluoro-
propan-2-ol and N,O-bis-Boc-hydroxylamine followed by Boc
deprotection furnished 4c.
2a
2b
2e
2f
CH3
H
CH3
H
F
F
CH3
F
H
N
N
N
C
1.2
17
3.6
32
2g
H
C
>100
a
Numerical average of at least two experiments.
Table 3
Initial evaluations were conducted with hydroxyurea 2a (Table
1). The in vitro functional activity at the B1 receptor was assessed
in a DAKD-mediated calcium mobilization (CAM) assay using hu-
man lung fibroblasts IMR-90 pretreated with IL-1 beta. Compound
2a (IC50 = 1.2 nM) proved to be as potent as the corresponding
hydroxyacetamide derivative 1 (IC50 = 1.1 nM) while containing
one less stereocenter and having a lower molecular mass. This
encouraging result prompted the exploration of the structure–
activity relationship (SAR) of 2a. As described previously,11 re-
moval of the benzylic methyl group (2b, IC50 = 17 nM) significantly
reduced the potency. Replacement of the fluorine atom with a
chlorine atom (2c, IC50 = 2.3 nM) did not affect the potency signif-
Antagonistic activity of hydroxyurea 2h–p
R6
N
H
N
OH
F
O
N
N
N
N
N
Cl
F
R6
CAM IC50 (nM)
a
Compound
2h
2i
2j
2k
2l
H
59
17
4.6
1.0
1.4
Me
Et
iPr
tBu
Table 1
Antagonistic activity of hydroxyurea 2a–d
H
R1
N
N
2m
2n
0.80
3.9
OH
F
O
N
N
B
A
O
N
F
F
F
R2
R3
2o
2p
0.19
0.50
a
Compound
R1
R2
R3
CAM IC50 (nM)
2a
2b
2c
2d
CH3
H
CH3
CH3
Cl
Cl
Cl
H
F
F
Cl
F
1.2
17
2.3
31
F
a
a
Numerical average of at least two experiments.
Numerical average of at least two experiments.

K. Schnatbaum et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1233–1236
1235
Table 4
The comparison of the hydrogen substituted 2h (IC50 = 59 nM)
Pharmacokinetic parameters in rat for selected compounds
and the methylated analogue 2i (IC50 = 17 nM) shows that methyl
substitution at R6 is clearly preferred. This prompted the investiga-
tion of larger hydrophobic groups in this position. Extension from
an ethyl substituent (2j, IC50 = 4.6 nM) to an isopropyl group (2k,
IC50 = 1.0 nM) gradually increased the activity to the low-nanomo-
lar level. The bulky tert-butyl group (2l, IC50 = 1.4 nM), however,
did not result in higher activity, indicating that N-substitution with
a tertiary C-atom is optimal in terms of activity and molecular
weight. Based on this finding, the cyclopentyl analogue 2m
(IC50 = 0.80 nM) was synthesized and, as expected, had a moderate
increase on activity compared to the isopropyl compound. It was
rather surprising that the cyclohexyl analogue 2n (IC50 = 3.9 nM)
was significantly less active whereas the difluoro counterpart 2o
(IC50 = 0.19 nM) exhibited excellent potency. The reason for the in-
creased activity by the difluoro substitution is likely due to a spe-
cific interaction with the receptor, as exemplified by modelling
studies (see below). Finally, based on the beneficial effect of fluo-
rine substitution in this position, the 1,3-difluoro-2-propyl ana-
logue 2p (IC50 = 0.50 nM) was synthesized and shown to also
have high functional activity.
The reported affinity trends can be rationalized through model-
ling studies. Figure 2 depicts compound 2o docked into a homology
model of the B1 receptor. The receptor model was generated on the
basis of the X-ray structure of bovine rhodopsin and the complex
was relaxed by means of molecular dynamics simulations and
geometry optimization. The employed GPCR modelling and dock-
ing strategy followed closely the procedure described in the sup-
plementary material of Gibson et al.23 The formation of two
hydrogen bonds, one between the carboxyl group of the urea moi-
ety and the side chain of Asn114 and one between the hydroxyl
group and Asn298, can be considered the key interactions for this
series. Apparently, the difluorocyclohexyl substituent engages
most effectively in hydrophobic interactions with the B1 receptor,
namely with the side chains of Ile117 and Phe302.
a
Compound
t1/2 (min)
CLa (mL/min kg)
Fb (%)
2a
2o
2p
12
47
80
58
13
8
8
78
86
a
1 mg/kg iv.
1 mg mg/kg po.
b
2o and 2p. These compounds are characterized by considerable ter-
minalhalflives(t1/2 >45 min), lowclearance(CL<14 mL/min/kg)and
high oral bioavailability (F >75%). Compared to previously described
hydroxyacetamide derivatives18 they have a slightly higher clear-
ance, but also higher oral bioavailability in the rat.
Compound 2o was further characterized and shown to have
good solubility in aqueous PBS buffer (39 lM) as well as high
chemical stability (>99% remaining after incubation in 0.05 N HCl
or PBS buffer at 50 °C for 5 days). Metabolic stability of 2o is also
high after incubation with human and rat liver microsomes for
1 h (61% and 84%, respectively).25 The human plasma protein bind-
ing of 2o was 98.7%. We found no cytotoxicity in human hepato-
cytes up to a concentration of 100 l
M26 and no mutagenic
potential in an Ames test27 for this class of compounds. The efflux
ratio of 2o in a bidirectional permeability assay through Caco-2 cell
monolayers was found to be 1.8 (Papp, A–B = 24 ꢀ 10ꢁ6 cm/s; Papp, B–A
=
43 ꢀ 10ꢁ6 cm/s). Since an efflux ratio of <3.0 is not considered
significant, 2o is not a substrate for P-gp.28
In conclusion, novel bradykinin B1 receptor antagonists have
been developed based on a hydroxyurea scaffold. Several highly ac-
tive antagonists were identified by systematic SAR examination
and optimization that display high oral bioavailability in the rat.
In-depth characterization of one compound, 2o, revealed that it
has high stability towards various conditions and is not a substrate
for P-gp.
Pharmacokinetic data for selected compounds in male Wistar
rats are shown in Table 4.24 The lead compound 2a revealed a poor
PK profile, as it is rapidly cleared after iv administration
(CL = 58 mL/min kg) and has low oral bioavailability (F = 8 %). How-
ever, the microsomal stabilityof the B1 receptor antagonists could be
improved by replacing the oxadiazole with tetrazole and by increas-
ing the steric bulk of R6. These modifications led to substantially im-
proved pharmacokinetic properties as exemplified by compounds
Acknowledgements
We thank Pericles Calias and Brigitte Hoch for proofreading the
manuscript and for their consent to handle any future correspon-
dence. We also thank Jessica Bald, Anja Borrmann, Antje Burian,
Anett Hauser, Birgit Hollmann, Bianka Knopp, Stephanie Köpke,
Ricarda Lange, Nicole Liebelt, Corinna Mewes, Daniel Ohlendorf,
Katy Pesta, Frank Polster, Dagmar Riexinger, Beatrice Rosskopp,
Jana Rother, Edith Weigt, Rocco Weise, Daniela Wulf and Ariane
Zwintscher for their skillful technical assistance.
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