Potent bradykinin B1 receptor antagonists: 4-Substituted phenyl cyclohexanes

Article abstract of DOI:10.1016/j.bmcl.2007.03.059

Selective bradykinin (BK) B₁ receptor antagonists have been shown to be antinociceptive in animal models and could be novel therapeutic agents for the treatment of pain and inflammation. Elucidation of the structure-activity relationships of the biphenyl moiety of the lead compound 1 provided a potent new structural class of BK B₁ receptor antagonists.

Full text of DOI:10.1016/j.bmcl.2007.03.059

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3006–3009
Potent bradykinin B1 receptor antagonists: 4-Substituted
phenyl cyclohexanes
Dai-Shi Su,a,* John L. Lim,a M. Kristine Markowitz,a Bang-Lin Wan,a Kathy L. Murphy,b
Duane R. Reiss,b C. Meacham Harrell,b Stacy S. O’Malley,b Rick W. Ransom,b
Raymond S. L. Chang,b Douglas J. Pettibone,b Cuyue Tang,c
Thomayant Prueksaritanont,c Roger M. Freidingera and Mark G. Bocka
aDepartment of Medicinal Chemistry, West Point, PA 19486, USA
bDepartment of Neuroscience Drug Discovery, West Point, PA 19486, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
Received 7 March 2007; revised 19 March 2007; accepted 19 March 2007
Available online 21 March 2007
Abstract—Selective bradykinin (BK) B1 receptor antagonists have been shown to be antinociceptive in animal models and could be
novel therapeutic agents for the treatment of pain and inflammation. Elucidation of the structure–activity relationships of the biphe-
nyl moiety of the lead compound 1 provided a potent new structural class of BK B1 receptor antagonists.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Bradykinin (BK) is an autacoid peptide produced by the
catalytic action of kallikrein enzymes on plasma and tis-
sue precursors termed kininogens. It plays an important
role in the pathophysiological processes accompanying
pain and inflammation. Its biological actions are medi-
ated by two known G-protein coupled receptors named
B1 and B2. The BK B2 receptor is constitutively
expressed in most cell types and evokes acute pain
responses following tissue injury, whereas the BK B1
receptor is induced during inflammatory insults or pain-
ful stimuli.1 In animal models, BK B1 receptor agonists,
such as des-Arg9-bradykinin (DABK) and des-Arg10-
kallidin (DAK), produce hyperalgesia, an effect that
can be blocked by peptide BK B1 receptor antagonists,
such as des-Arg9-Leu8-bradykinin (DALBK) and des-
Arg10-Leu9-kallidin (DALK).2 A study result from the
BK B1 receptor knockout mouse has implicated a role
for the BK B1 receptor in inflammation, algesia, and
neuropathic pain.3 In addition to the accepted periphe-
ral mode of action of the BK B1 receptor, the BK B1
receptor has also been accorded a central role on the ba-
sis of recent results which demonstrate that the BK B1
receptor is constitutively expressed in the central ner-
vous system (CNS) of mice and rats.4 Accordingly,
selective and effective BK B1 receptor antagonists hold
promise as novel therapeutic agents for the treatment
of pain and inflammation.5
The current study was initiated with the previously re-
ported biphenyl diaminopyridine lead compound 1
(Scheme 1).6 This paper reports the revelation and
SAR study results of a structural alternative, 4-substi-
tuted phenyl cyclohexane (2), that exhibits excellent
binding affinity and good receptor occupancy in an
ex vivo receptor occupancy assay which has been
designed to determine the extent of CNS penetration
of compound.
CF3
CF3
O
O
NH
NH
CO2Me
N
N
CO2Me
H
N
N
H
Keywords: Bradykinin; B1 receptor; B1 receptor antagonists; 4-Substi-
tuted phenyl cyclohexanes.
1
2
*
Corresponding author. Tel.: +1 215 652 6830; fax: +1 215 652
3971; e-mail: daishi_su@merck.com
Scheme 1. Lead compound and replacement of biaryl phenyl core.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.059

D.-S. Su et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3006–3009
3007
Table 1. Binding affinities of select BK B1 antagonists
substituents led to the trifluoromethyl compound 16
with subnanomolar binding affinity. Compound 16
shows good functional antagonist potency in a Fluores-
cence Imaging Plate Reader assay (FLIPR) with an IC50
of 1.15 nM. Compound 16 was examined in a rat phar-
macokinetic experiment.9 However, it exhibits high plas-
ma clearance (37.7 mL/min/kg), a short half-life (0.8 h),
and poor bioavailability.
CF3
O
R1
R2
NH
R3
R4
N
N
H
Compound
R1
R2
R3
R4
Kia (nM)
Incorporation of alternatives to the trifluoroethyl amide
side chains from the SAR study of the original series
into compound 16 led to a potent isoxazole analogue,
17, with a Ki of 0.6 nM (Scheme 2). Compound 17 dem-
onstrates excellent functional antagonist potency in a
FLIPR assay with an IC50 of 0.45 nM which is in accord
with the receptor binding affinity. Although compound
17 shows improvement versus 16 in terms of pharmaco-
kinetic parameters, it exhibits modest clearance (16.1
and 21.3 mL/min/kg) and short half-life (1 and 1.3 h)
in rat and dog, respectively.10
2
3
CO2Me
OAc
OH
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
H
H
H
H
H
H
H
H
F
1645
1451
478
14
H
H
4
H
H
5
H
H
6
F
H
18
7
H
F
3.7
60
8
H
H
CF3
9
H
H
H
H
Cl
H
H
H
H
24.5
49
10
11
12
13
14
15
16
H
CO2Me
F
F
2
30
H
Cl
Cl
H
H
H
Cl
Cl
OCF3
CF3
4.9
3.7
3.6
0.4
To determine the extent to which the compounds in this
series occupy the human BK B1 receptor, 17 was exam-
ined in an ex vivo receptor occupancy study in trans-
genic rats in which the human BK B1 receptor is
constitutively over-expressed.11 In this study, 17 showed
dose-dependent occupancy following iv infusion over
30 min (Table 2). At a dose of 6 mg/kg, compound 17
shows 77% and 84% of receptor occupancy in both brain
and spinal cord with concentrations of 666 and 468 nM,
respectively. These data suggest that this compound
penetrates the blood–brain barrier and occupies the
receptor in the CNS which is in accordance with the
finding that compound 17 is not a substrate for P-glyco-
a Values represent the numerical average of at least two experiments.
Interassay variability was 25%.
The central ring of the lead compound 1 can be replaced
with a number of alternative heterocycles and cycloalk-
yls.7 We found that the bis-substitution at the 4-position
of cyclohexane ring is well tolerated (analogue 2, Table
1).8 The methyl ester of compound 2 was replaced with
several other functional groups as shown in Table 1.
Replacement of the methyl ester with an amide,
hydroxymethyl, or 3-methyl-1,2,4-oxidiazole proved to
be ineffective (data not shown). However, replacement
of the ester with acetoxy yielded an equipotent com-
pound (3). Hydrolysis of the acetate derivative gave
the alcohol, 4, with a threefold binding affinity enhance-
ment. Replacement of hydroxyl to a cyano group im-
proved the binding affinity about 30-fold (compound
5). The importance of stereochemistry of the 4-substi-
tuted phenyl cyclohexane was also investigated with
the cis isomer (5) imparting greater potency (about
100-fold) than the trans isomer (data not shown).
CF3
N
O
O
O
CF3
CF3
NH
NH
CN
CN
N
N
N
N
H
H
17
16
Scheme 2.
With the potency enhancing cis cyano group at 4-posi-
tion, we then examined substitution on the phenyl ring.
The fluorine atom was used as a probe. Among the three
positions on the substituted phenyl ring, meta-fluoro
compound 7 showed fourfold improvements over the
lead compound 5. Other substituents were introduced
at the meta position, but with significant loss of potency
(compounds 9 and 10). Difluoro analogue 11 was
slightly more potent, but multiple chlorine substitution
on the phenyl ring did not increase the binding affinity
(compounds 12 and 13). Although the ortho-fluoro ana-
logue (6) was not as potent as meta-fluoro compound
(7), the ortho chlorine analogue, 14, was about fourfold
more potent than parent analogue 5 and equally potent
as compound 7. Further modification of the ortho
Table 2. Ex vivo receptor occupancy study results of compound 17
Occupancy (%)
Concn (nM)
0.06 mpk
Plasma
Brain
—
19
3
24
<26
<26
Spinal cord
0.6 mpk
Plasma
Brain
—
58
50
274
62
Spinal cord
35
6 mpk
Plasma
Brain
Spinal cord
—
77
84
3216
666
468

3008
D.-S. Su et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3006–3009
O
O
O
O
CO2Me
MeO
OMe
d
c
b
a
CN
CF3
CN
CF3
CN
CN
CF3
(I)
CF3
(II)
(III)
NO2
NH2
CN
N
O
CF3
NO2
CN
O
e
g
f
CF3
NH
CN
N
N
H
CN
N
N
CF3
(IV)
~ 6:1 cis to trans
CF3
H
(V)
(VI)
17
Scheme 3. Reagents and conditions: (a) methyl acrylate, Triton B, AcCN, reflux, 83%; (b) NaH, DME, reflux, 88%; (c) DMSO, NaCl, 150 ꢁC, 61%;
(d) CH3NO2, ethylene diamine (cat.), reflux, 86%; (e) Raney Ni, H2, MeOH; (f) 2-chloro-3-nitro pyridine, TEA, THF, silica gel chromatography,
50% for two steps; (g) Raney Ni, H2, MeOH, then isoxazole-5-carbonyl chloride, 50% for two steps.
protein (P-gp) mediated efflux (MDR1, (B/A)/(A/B): 2.5,
References and notes
Passive permeability (Papp): 36 · 10À6cm/s).12,13
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Compound 17 was prepared according to the route
depicted in Scheme 3. All other compounds were syn-
thesized in an analogous fashion. Double Michael
reactions of commercially available benzoacetonitrile
with methyl acrylate afforded adduct I. Dieckmann
cyclization followed by decarboxylation of II yielded
cyclohexanone, III. Henry reaction and reduction of
the resulting vinyl nitro intermediate IV provided
the amine derivative (V) as a mixture of cis and trans
isomers (ratio: 6:1). Nucleophilic aromatic substitution
of 2-chloro-3-nitro pyridine by the amine (V) delivered
the desired aminopyridine compound. At this stage
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separated by flash chromatography. Reduction of
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ole-5-carbonyl chloride provided the target compound
17.
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In summary, we have successfully identified alternative
isosteres for the biphenyl moiety of the lead compound
1. The compounds disclosed in this paper represent a
new structural class of BK B1 receptor antagonists.
Compound 17 is an optimal member of the series to
emerge from this study and exhibits promising
ex vivo receptor occupancy in transgenic rats. Further
evaluation and modification of these compounds to im-
prove their pharmacokinetic properties are in progress.
The results from these studies will be disclosed in due
course.
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Murphy, K. L.; Harrell, C. M.; O’Malley, S. S.; Ransom,
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Acknowledgments
We thank Dr. Neville J. Anthony for proofreading this
manuscript and Dr. Steve M. Pitzenberger for NMR
assistance. We are grateful to Drs. Bruce D. Dorsey,
Scott D. Kuduk, Michael R. Wood, and Dong-Mei
Feng for useful discussions.

D.-S. Su et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3006–3009
3009
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8. For the assay protocols, please see Refs. 5a–d.
9. iv, 2 mg/kg, vehicle: DMSO; po, 10 mg/kg, vehicle: 1%
methyl cellulose.
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13. P-gp mediated directional transport was performed in
LLC-PK1 cells expressing genes for human P-gp (MDR1),
and the ratio of transport from basolateral to apical (B to
A) direction to the ratio of transport from apical to
basolateral (A to B) direction was measured. For the assay
protocols, please see Ref. 5d.