Bradykinin B1 receptor antagonists: An α-hydroxy amide with an improved metabolism profile

Article abstract of DOI:10.1016/j.bmcl.2008.07.126

A series of carbo- and heterocyclic α-hydroxy amide-derived bradykinin B₁ antagonists was prepared and evaluated. A 4,4-difluorocyclohexyl α-hydroxy amide was incorporated along with a 2-methyl tetrazole in lieu of an oxadiazole to afford a suitable compound with good pharmacokinetic properties, CNS penetration, and clearance by multiple metabolic pathways.

Full text of DOI:10.1016/j.bmcl.2008.07.126

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5107–5110
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Bradykinin B1 receptor antagonists: An
with an improved metabolism profile
a
-hydroxy amide
a
a
a
Scott D. Kuduk a, , Ronald K. Chang , Robert M. DiPardo , Christina N. Di Marco ,
Kathy L. Murphy b, Richard W. Ransom b, Duane R. Reiss b, Cuyue Tang c,
Thomayant Prueksaritanont c, Douglas J. Pettibone b, Mark G. Bock a
*
a Department of Medicinal Chemistry, Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
b Department of Neuroscience Drug Discovery, Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
c Drug Metabolism Merck Research Laboratories, Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of carbo- and heterocyclic
and evaluated. A 4,4-difluorocyclohexyl
zole in lieu of an oxadiazole to afford a suitable compound with good pharmacokinetic properties, CNS
penetration, and clearance by multiple metabolic pathways.
a
-hydroxy amide-derived bradykinin B1 antagonists were prepared
-hydroxy amide was incorporated along with a 2-methyl tetra-
Received 9 July 2008
Revised 29 July 2008
Accepted 29 July 2008
Available online 5 August 2008
a
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Bradykinin
Pain
GPCR
Tetrazole
One component of the response of humans and animals to tis-
sue injury is mediated by bradykinin (BK) peptides which are pro-
duced in plasma soon after injury occurs. The physiological effects
of these peptides, including pain and inflammation,1,2 are regu-
lated by the G-protein-coupled receptors, B1 and B2.3–5 The B2
receptor is constitutively expressed, and the current thinking is
that it is responsible for the incipient acute pain response follow-
ing injury. The native ligands for the B2 receptor are the peptides
bradykinin (BK = Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) and kalli-
din (Lys-BK). The metabolites of these peptides, [des-Arg9] brady-
kinin and [des-Arg10] kallidin, are agonists for the B1 receptor,
which is induced subsequent to the occurrence of the injury.
Animal models have demonstrated that bradykinin B1 receptor
antagonists attenuate the response to painful stimuli.6–9 It has
been further shown that transgenic B1 knockout mice exhibit re-
duced sensitivity to painful stimuli while appearing normal in all
other respects.10 In addition, experimental evidence has been gath-
ered that indicates that the B1 receptor is constitutively expressed
in the central nervous system of rats and mice.11 This would imply
a role for the B1 receptor not only in the periphery but also in the
CNS. Therefore, CNS penetrant bradykinin B1 receptor antagonists
are highly desirable because they may be much more efficacious
than peripheral B1 antagonists.12
Recently, we reported a series of a-hydroxy amide-based bra-
dykinin BK1 receptor antagonists.13 For example, compound 1
bearing an oxadiazole ester surrogate had sub-nanomolar affinity
for the B1 receptor, good pharmacokinetics across three species,
and exhibited efficient receptor occupancy in an ex vivo transgenic
mouse model. However, 1 was metabolized exclusively by a single
P450 in the form of CYP2B6 raising the possibility for drug–drug
interactions (Fig. 1). In addition, this oxidation by CYP2B6 led to
a major circulating metabolite in vivo (2), which also showed
excellent hBK1 binding affinity and good pharmacokinetic proper-
ties. Accordingly, we sought to prepare compounds that would be
oxidized by multiple CYP enzymes and/or have alternative clear-
F3C
O
F3C
O
OH
NH
OH
NH
HO
O
N
O
CYP2B6
N
N
N
N
N
F
F
F
F
Cl
Cl
1: hB1 Ki =0.5 nM
2: hB1 Ki =0.8 nM
* Corresponding author.
E-mail address: scott_d_kuduk@merck.com (S.D. Kuduk).
Figure 1. Metabolism of compound 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.126

5108
S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5107–5110
oxidative de-methylation
leading to inactive NH
oxidative
N N
metabolism
NHBoc
N
F3C
N
N
OH
NH
NH2
OH
NH
a-c
( )n
O
Br
F
Br
F
+
O
O
N
O
F
B
N
N
N
N
O
N
N
N
Cl
Cl
F
F
F
F
7
8
9
d, e
Cl
X
Cl
OH
Compound 1
( )n
N N
NH2
N
Figure 2. Strategy to address metabolism.
N N
O
NH
F
f
N
N
N
N
N
ance pathways, while avoiding the production of a major circulat-
ing active metabolite.
F
F
F
The strategy to overcome these issues is shown in Fig. 2. Previ-
ous work on a series of related biaryl bradykinin B1 antagonists has
shown that an N-2-methyl tetrazole was an effective isostere for a
methyl ester.14 Oxidative dealkylation led to the acidic N–H-tetra-
zole which had no significant affinity for the B1 receptor. Therefore,
we wanted to incorporate a tetrazole in lieu of the oxadiazole to
address the circulating metabolite issue. To confer additional oxi-
dation potential to the molecule, hopefully by P450’s other than
CYP2B6, we planned to remove the metabolism resistant trifluoro-
methyl group and incorporate an aliphatic cyclopentane ring.
Cl
Cl
11a-i
10
Scheme 2. Reagents and conditions: (a) NOBF4, KCN. (b) TMSnN3, toluene, 120 °C.
(c) MeI, K2CO3, DMF. (d) PdCl2Ádppf, DMSO, K2CO3, H2O. (e) HCl, EtOAc. (f) 4a–g,
EDC, TEA, HOBt, DMF.
Compounds 11a–i were evaluated for human bradykinin B1
binding affinity and susceptibility to efflux by the transporter P-
glycoprotein (P-gp). In addition, pharmacokinetic properties in
dog were screened to further triage analogs for additional testing.17
Results are shown in Table 1.
The methods used to prepare the
a-hydroxy carboxylic acids
4a–g are shown in Scheme 1. When the ketone starting materials
were available (3a–d), the compounds were prepared via hydroly-
sis of the intermediate cyanohydrins. Alternatively, the compounds
could be prepared from the corresponding esters, via hydroxyl-
ation using the method described by Davis.15
The requisite tetrazole 8 was prepared in a three-step sequence
(diazotization–cyanation, tetrazole formation, and methylation)
from commercially available aniline 7 (Scheme 2). The N-2-methyl
tetrazole 8 was formed as a 1:1 mixture with the N-1 isomer (not
shown), but was readily separable. Suzuki coupling of 8 with bor-
onate 9,16 followed by removal of the Boc group with HCl, provided
amine 10. Final acylation with the appropriate acid 4a–g, afforded
target molecules 11a–i. Compound 11d was prepared via TPAP oxi-
dation of 11b.
Cyclopentane derivative 11a proved to have high affinity for the
B1 receptor, similar to that for 1, and was not a substrate for human
P-gp (BA/AB ratio = 2.1). This compound also exhibited low dog
clearance, but a shorter half-life relative to 1. Incorporation of a sul-
fide (11b) was similarly well tolerated with only a slight increase in
P-gp susceptibility, but sulfoxide and sulfone analogs 11c-d proved
to be impressive substrates for P-gp. This is likely due to incorpora-
tion of the additional heteroatom(s), as the related ketone 11e,
which despite having impressive binding and dog pharmacokinetic
properties, also proved to be subject to P-gp-mediated efflux.
In order to avoid the undesired effects on P-gp and to improve the
pharmacokinetic profile of 11a, a difluoromethylene unit was incor-
porated into the cyclopentane ring. This led to a loss of symmetry to
produce diastereomers 11f and 11g after chromatographic resolu-
tion. Both diastereomers maintained good B1 binding affinity, but
it was fortuitous that the modestly more potent 11g also exhibited
a markedly better half-life in the dog (t1/2 = 19 h) with a more pref-
erableP-gpprofile. Homologationtothesymmetrical six-membered
ring analogs was also investigated. Tetrahydropyran 11h exhibited
good affinity, but continued the trend of increasing P-gp efflux
(BA/AB = 8.2) with additional heteroatom incorporation. However,
difluorocyclohexane 11i exhibited excellent binding affinity
(hKi = 0.41 nM), P-gp transport, and dog pharmacokinetic profiles
(F = 91%) comparable to difluorocyclopentane 11g.
Analogs 11a, 11g, and 11i were selected for further triage via
determination of rat pharmacokinetics and brain penetration in
African green monkey. In addition, ex vivo receptor binding exper-
iments were investigated using a transgenic rat construct, which
expresses the hBK B1 receptor broadly throughout the CNS, includ-
ing the brain and spinal cord.18 The binding efficiency of a com-
pound to the hBK B1 receptor expressed in the CNS can therefore
be determined with this rat model. Results are shown in Table 2.
With respect to rat pharmacokinetics, difluoro derivatives 11g
and 11i exhibited better bioavailability (>60%) and lower clearance
relative to cyclopentane 11a (F = 26%, Cl = 10 mL/min/kg). The
pharmacokinetic advantage of the difluoro analogs is across spe-
a, b
OH
O
X
( )n
X
( )n
CO2H
3a; X = CH2, n = 1
3b; X = CH2, n = 2
3c; X = S, n = 2
3d; X = O, n = 2
4a; X = CH2, n = 1
4b; X = CH2, n = 2
4c; X = S, n = 2
4d; X = O, n = 2
F
O
HO
e-g
c,d
F
OH
( )n
CO2H
( )n
CO2Bn
CO2H
5a; n = 0
5b; n = 1
4e; n = 0
4f; n = 1
TBSO
HO
c, h, i
f, g, j
OH
CO2H
CO2Bn
CO2H
6
4g
Scheme 1. Reagents and conditions: (a) NaCN, H2O, Na2S2O3. (b) HCl, reflux. (c)
BnBr, K2CO3, DMF. (d) PCC, DCM. (e) Deoxo-fluor, CH2Cl2, rt. (f) KHMDS, THF,
À78 °C, Davis Oxaziridine. (g) Pd/C, H2, EtOAc. (h) i—BH3, THF. ii—NaBO3, H2O. (i)
TBSCl, imidazole, DMF. (j) 1 N HCl, THF.

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5107–5110
5109
Table 1
Bradykinin B1 receptor binding affinities, P-gp transport properties, and dog pharmacokinetics
X
OH
( )n
N N
O
NH
F
N
N
N
F
Cl
P-gpb
Papp (10À6 cm/s)c
Dog F%
Dog t1/2
Dog CL
a
d
d
d
Compound
X
n
hBK1 (nM)
1
—
CH2
S
S(O)
S(O)
C@O
CF2
CF2
O
—
0
0
0
0
0
0
0
1
1
0.66
0.48
0.39
1.6
0.69
0.67
1.0
0.67
0.96
0.41
1.9
2.1
2.9
14.3
29
27
29
31
24
22
31
29
32
24
31
84
nd
nd
nd
nd
nd
nd
85
nd
91
11
0.5
0.6
3.2
2.3
0.5
0.3
0.3
0.2
nd
11a
11b
11c
11d
11e
11f
11g
11h
11i
4.7
5.4
3.1
9.9
9.8
3.0
19
2
16
2.6
1.9
8.2
2.0
nd
20.3
CF2
1.0
a
Values represent the numerical average of at least two experiments. Interassay variability was 25% (Ki, nM).
b
c
MDR1 directional transport ratio (B to A)/(A to B). Values represent the average of three experiments and interassay variability was 20%.
Passive permeability (10À6 cm/s).
d
F% oral bioavailability, half-life is represented in hours, CL in mL/min/kga Mongrel dogs (n = 2). Oral dose 3 mg/kg in methocel, IV dose = 1 mg/kg in DMSO. Interanimal
variability was less than 20% for all values.
(Fig. 3). N-Demethylation was mediated by CYP2B6 similar to the
hydroxylation of 1 into metabolite 2. However, unlike the case
for 2, the N-desmethyl metabolite had 30 fold weaker affinity
(hKi = 21 nM) for the hB1 receptor. In addition, CYP3A4 mediated
hydroxylation of the difluorocyclohexane ring was seen as a sec-
ond oxidative pathway. Direct conversion of the tertiary hydroxyl
of 11i into the acyl glucuronide was also observed, although this
pathway is much more significant in rat hepatocytes than other
species. Hydrolysis of the amide bond was also noted as a fourth
biotransformation pathway.
Table 2
Rat pharmacokinetics and transgenic rat receptor occupancy for select compounds
Rat F%a
Rat t1/2 (h)a
Rat CLa
Occ90
B/Pc
b
Compound
11a
11g
11i
26
96
62
5.5
4.1
5.6
10
5.2
2.2
214
120
140
nd
0.55
0.63
a
F% oral bioavailability, half-life is represented in hours, CL in mL/min/kg.
Sprague–Dawley rats (n = 3). Oral dose = 10 mg/kg in methocel, IV dose = 2 mg/kg
in DMSO. Interanimal variability was less than 20%.
b
Values represent the numerical average of at least two experiments. Interassay
variability was 25% (Ki, nM).
In conclusion, a series of 5- and 6-membered carbocyclic and
c
African Green Monkeys (n = 2). IV dose = 2 mg/kg. Interanimal variability was
heterocyclic
a-hydroxy amide-derived bradykinin B1 antagonists
less than 20% for all values.
bearing an N-2 methyl tetrazole as an oxadiazole replacement
was prepared and evaluated. A number of compounds with excel-
lent B1 binding affinity, good pharmacokinetic properties, and
desirable P-gp transport properties was elucidated. In particular,
4,4-difluorocyclohexane 11i possessed similar properties to oxadi-
azole 1, but was found to undergo biotransformation by multiple
pathways lowering the risk of any potential drug–drug interac-
tions. In addition, no major active circulating metabolites were ob-
served. Accordingly, 11i was selected for additional pre-clinical
development as a backup to compound 1.
cies and consistent with high microsomal and hepatocyte stability
for these three compounds (data not shown).
Both difluoro analogs proved to be very efficient in the ex vivo
receptor occupancy assay compared to the cyclopentane deriva-
tive. In addition, 11g and 11i were found to be brain penetrant in
African green monkey with brain to plasma ratios of 0.55 and
0.63, respectively. However, the symmetrical difluorocyclohexane
11i showed better dose proportionality in rat and dog, and was se-
lected for more in depth metabolic studies.
Metabolic profiling revealed that compound 11i was metabo-
lized by multiple pathways in human microsomes and hepatocytes
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N
O
-demethylation
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N
N
N
Hydroxylation
(CYP3A4)
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F
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Figure 3. Metabolic profile of 11i.

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