Development of an efficient and selective radioligand for bradykinin B 1 receptor occupancy studies

Article abstract of DOI:10.1016/j.bmcl.2004.09.074

We have developed an efficient and selective radioligand, the [ ³⁵S]-radiolabeled dihydroquinoxalinone derivative, 4, for an ex vivo receptor occupancy assay in transgenic rats over-expressing the human bradykinin B₁ receptor.

Full text of DOI:10.1016/j.bmcl.2004.09.074

Bioorganic& Medicinal Chemistry Letters 14 (2004) 6045–6048
Development of an efficient and selective radioligand
for bradykinin B1 receptor occupancy studies
Dai-Shi Su,a,* M. Kristine Markowitz,a Kathy L. Murphy,b Bang-Lin Wan,a
Matthew M. Zrada,a C. Meacham Harrell,b Stacy S. OÕMalley,b J. Fred Hess,b
Rick W. Ransom,b Ray S. Chang,b Michael A. Wallace,c Conrad E. Raab,c
Dennis C. Dean,c Douglas J. Pettibone,b Roger M. Freidingera and Mark G. Bocka
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Molecular Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA
cDrug Metabolism Label Compound Syntheses, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 30 June 2004; revised 23 September 2004; accepted 24 September 2004
Available online 12 October 2004
Abstract—We have developed an efficient and selective radioligand, the [35S]-radiolabeled dihydroquinoxalinone derivative, 4, for
an ex vivo receptor occupancy assay in transgenic rats over-expressing the human bradykinin B1 receptor.
Ó 2004 Elsevier Ltd. All rights reserved.
Bradykinin (BK) B1 and B2 receptors are G-protein cou-
pled receptors that mediate the action of kinin peptides
which are produced by the catalytic action of kallikrein
enzymes on plasma and tissue precursors termed kinin-
ogens.1 BK plays an important role in the pathophysio-
logical processes accompanying acute pain, chronic
pain, and inflammation. The BK B2 receptor is constitu-
tively expressed in most cell types, whereas the BK B1
receptor is induced during inflammatory insults or pain-
ful stimuli. In animal models, late phase hyperalgesia
can be attenuated by peptide BK B1 receptor antago-
nists, such as des-Arg9-Leu8-BK (DALBK) and des-
Arg10-Leu9-Kallidin (DALK).2 Recent data shows that
transgenicBK B 1 receptor knockout mice are hypoalge-
sic in chronic and acute pain models, implicating a role
for the BK B1 receptor in inflammation and algesia.3 In
addition to the accepted peripheral mode of action of
the BK B1 receptor, the BK B1 receptor has also been
accorded a central role on the basis of recent results
which demonstrate that the BK B1 receptor is constitu-
tively expressed in the central nervous system (CNS) of
mice and rats.4 To support our efforts toward the devel-
opment of human BK B1 receptor antagonists as novel
therapeuticagents for the treatment of pain and inflam-
mation,5 an efficient and selective radioligand was
needed for animal model studies. The commerc-
ially available peptide radioligands, [3H]DAK
(des-Arg10-Kallidin) and [3H]DALK, provided unsatis-
factory results in our occupancy studies (vide infra).
Herein we report the development of a novel [35S]-radi-
oligand for the study of human BK B1 receptor occu-
pancy in an ex vivo transgenic rat model.
In order to achieve good selectivity and an acceptable
signal to noise ratio in the animal model, a radioligand
candidate must have excellent binding affinity coupled
with well-balanced physicochemical properties (e.g.,
logP and protein binding). The radiolabeled compound
should also be readily accessible, preferably with intro-
duction of the radiolabel late in the synthesis.
The current study was initiated with the previously
reported, potent and selective BK B1 receptor antago-
nist, compound 1 (Table 1).5a Although compound 1
possesses excellent binding potency and offers the possi-
bility to introduce [35S] as a radiolabel atom, we judged
it to be unsuitable as a radioligand because of its chem-
ical intractability6 and also, in part, because of its
physiochemical properties. Therefore, subsequent work
focused on modifying the sulfonamide moiety and prop-
erties of compound 1 while maintaining potent binding
affinity. The protein binding and logP properties of 1
Keywords: Bradykinin B1 receptor; Antagonist; Radioligand; Ex vivo.
*
Corresponding author. Tel.: +1 215 652 6830; fax: +1 215 652
3971; e-mail: daishi_su@merck.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.074

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D.-S. Su et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6045–6048
Table 1. Receptor binding affinity and physicochemical properties of dihydroquinoxalinone analogs
H
O
R2
N
O
6
N
S
N
H
X
O
O
R1
Compound
X
H
R1
R2
hB1 Ki (nm)a,b
LogPc
Protein bindingd
36.0
N
1
3,4-Dichlorophenyl
0.034
0.48
N
H
2
3
H
H
3,4-Dichlorophenyl
3,4-Dichlorophenyl
1.09
3.73
3.35
2.35
NDe
82.5
N
N
N
N
N
N
4
5
H
H
2-Naphthyl
2-Naphthyl
0.02
0.07
0.25
3.32
79.4
98.5
N
H
N
N
H
N
6
7
8
Cl
Cl
Cl
2-Naphthyl
2-Naphthyl
2-Naphthyl
0.01
0.02
0.07
1.00
0.53
3.41
93.6
91.7
NDe
N
H
N
N
Me
N
N
H
a All Ki values are calculated for competition binding assays utilizing cloned human B1 and B2 receptors and are the average of at least two
experiments.
b The hB2 Ki values for all compounds are greater than 10,000nM.
c LogP values were determined by HPLC analysis in octanol/water.
d Human plasma protein.
e ND = not done: compound is insoluble in test medium.
were altered with the conversion of the imidazoline to an
imidazole (2), and triazole (3). However, the binding
affinities of these analogs were decreased 32- and 110-
fold, respectively.7 Replacement of the 3,4-dichlorophen-
ylsulfonamide moiety in 1 with the more readily acces-
sible [35S]-2-naphthylsulfonamide yielded compound 4,
which had 2-fold increased potency and increased pro-
tein binding. However, the logP value for compound 4
is not substantially different from compound 1. Oxida-
tion of the imidazoline in 4 to imidazole gave 5, which
is highly lipophilicand avidly protein bound. In a re-
lated pharmacokinetics study of an analog structurally
similar to 4, we discovered that the 6-position of the
dihydroquinoxalinone ring is subject to in vitro oxida-
tion (rat, dog, etc., liver microsomes) to form a phenol
derivative. This metabolicpathway acn be attenuated
with the introduction of a halogen atom at the corre-
sponding metabolicsite (data not shown). We speuc-
lated that the addition of halogen might also affect the
physicochemical properties of analogs. Therefore, a
chlorine atom was introduced in 4 to give the equipotent
derivative 6. Although compound 6 exhibits an accepta-
ble logP value, it is highly protein bound. Further mod-
ification of the imidazoline ring in 6 (e.g., methylation to
give 7 or oxidation to yield 8) did not result in significant
improvement in the properties. Taken together, these
data led us to re-assess compound 4. Based on its overall
profile (human B1 receptor binding potency, chemical
accessibility, as well as physicochemical properties) com-
pound 4 was evaluated as a potential radioligand.
Compound 4 was prepared according to the route de-
picted in Scheme 1. The preparative route is an adapta-
tion of our original synthesis to accommodate the
preparation of [35S]-4.5a Condensation of 2-fluoro nitro-
benzene (I) with commercially available methyl aspar-
tate ester (II) afforded the nitrobenzene adduct
(Scheme 1). Reduction of the nitro group resulted in
intramolecular cyclization to afford the key dihydroqui-
noxalinone methyl ester III. Saponification of ester III
followed by coupling of the resulting acid with 4-(2-
H
b
N
O
a
c
NO2
F
MeO
H2N
O
O
O
+
N
H
O
O
III
I
II
N
H
N
O
N
O
+
H2
H
Cl-
CN
N
N
N
O
O
H
O S O
N
H
N
H
4
IV
Scheme 1. Reagents and conditions: (a) 1. NaHCO3, MeOH, Reflux.
2. HCl, MeOH, 81% for two steps. 3. H2, Pd/C, EtOH, 98%. (b) 1. 1N
NaOH, MeOH; 2. 4-(2-aminoethyl)benzonitrile, EDCI, HOAt, Et3N,
CH2Cl2, 68% for two steps. (c) 1. 2-naphthyl-SO2Cl, pyridine, 96%; 2.
HCl, EtOH; 3. ethylene diamine, EtOH, 4. HCl, EtOAc, 0°C, 77% for
three steps.

D.-S. Su et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6045–6048
6047
3500
3000
2500
2000
1500
1000
500
Total Binding
Nonspecific Binding (1uM DAK)
Specific Binding
2
1
KD=15pM
0
B
max=480fmol/mg protein
0
100
200
300
400
500
Bound (fmol/mg protein)
0
0
25
50
35S]-4 (pM)
75
100
[
Figure 1. [35S]-4 Saturation binding to human BK B1 receptor CHO membranes.
aminoethyl)benzonitrile delivered the desired amide IV.
Sulfonylation with 2-naphthylsulfonyl chloride was
followed by a three-step sequence which converted the
nitrile to the imidazoline ring leading to compound 4
as its HCl salt. The radiolabeled [35S]-4 was obtained
by this route using [35S]-2-naphthylsulfonyl chloride
on a 350mCi scale. The [35S]-2-naphthylsulfonyl chlo-
ride was obtained from commercially available [35S]-2-
naphthylsulfonic acid by the action of NaHCO3 and
oxalyl chloride.8
tors in the CNS (brain and spinal cord tissue), presum-
ably due to its inability to effectively cross the blood
brain barrier from the circulation or to diffuse through
the brain tissue from the lateral ventricles. Therefore,
this compound was not suitable for the development
of an in vivo receptor occupancy assay. Nonetheless,
the excellent in vitro binding properties of [35S]-4 led
us to develop an ex vivo method for the determination
of human B1 central receptor occupancy of BK B1
antagonists.13 The ex vivo assay involves analysis of
the rate of [35S]-4 association in membranes prepared
from tissues harvested from vehicle and BK B1 antago-
nist treated animals. In the assay, the association of
[35S]-4 is linear over 10min and receptor occupancy is
directly proportional to the decrease in the slope of
the association line compared to the control. The assay
works largely because of the high specific activity and
low nonspecific binding of [35S]-4 which allows the assay
to be run with minimal tissue over a short time frame.
Thus, the association and dissociation are kept to a min-
imum.14 This assay provides a rapid and reliable method
for determining the extent of a systemically-dosed test
compound to occupy the human BK B1 receptor over-
expressed in the CNS of transgenicrats.
In a panel of assays9 representing 170 enzymes, recep-
tors, and transporters, compound 4 exhibited over
2700-fold selectivity for the human BK B1 receptor.
Analog 4 demonstrated excellent functional antagonist
activity in a fluorescence imaging plate reader (FLIPR)
assay (human BK B1, IC50 = 0.04nM) that is in concert
with the receptor binding affinity. The binding of [35S]-4
to the human BK B1 receptor expressed in CHO mem-
branes was characterized.10 At 22°C, [35S]-4 reaches
equilibrium within 2h and dissociates slowly with a t1/2
value of ꢀ 200min. In saturation binding studies,
[35S]-4 labels a single site with a Kd value of 15pM
and Bmax value of 480fmol/mg protein, a density
equivalent to that labeled by [3H]DALK (Fig. 1). There
is no specific binding of [35S]-4 to CHO cell membranes
expressing the human B2 receptor. Data from competi-
tion binding studies indicates that the pharmacologies
of [35S]-4 and [3H]DALK binding to the human BK B1
receptor are similar (data not shown).
In summary, we have developed an efficient and selective
radioligand, [35S]-4, for the ex vivo human BK B1 recep-
tor occupancy study in transgenic rats. This assay pro-
vides important information in the selection of
nonpeptide BK B1 receptor antagonists for further
development.
Due to significant species differences observed for the
binding of BK B1 peptide and nonpeptide antagonists
to BK B1 receptors,5a,11,12 we generated and character-
ized transgenicrats in which the human BK B 1 receptor
is constitutively over-expressed.13 Administration of
[35S]-4 by either intravenous or intra-cerebroventricular
injection did not result in the labeling of BK B1 recep-
Acknowledgments
We are pleased to acknowledge the efforts of Drs. Z.
Zheng, P. Kunapuli, Mr. R. M. DiPardo, Mr. C. F.
Homnick, and Mrs. J. S. Murphy.

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D.-S. Su et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6045–6048
R.; Kim, J. J.; Han, W.; Dorsey, B. D.; Homnick, C. F.;
Supplementary data
DiPardo, R. M.; Kuduk, S. D.; MacNeil, T.; Murphy, K.
L.; Lis, E. V.; Ransom, R. W.; Stump, G. L.; Lynch, J. J.;
OÕMalley, S. S.; Miller, P. J.; Chen, T.-B.; Harrell, C. M.;
Chang, R. S. L.; Punam, S.; Ellis, J. D.; Bondiskey, P. J.;
Pettibone, D. J.; Freidinger, R. M.; Bock, M. G. J. Med.
Chem. 2003, 46, 1803–1806.
Experimental details for [35S]-4 and characterization of 4
can be found, in the online version, at doi:10.1016/
j.bmcl.2004.09.074.
Reference and notes
6. During the time of this study, vialble methods to incor-
porate [35S] into the requisite starting material were
unavailable.
1. (a) Marceau, F.; Regoli, D. Nat. Rev. Drug Discov. 2004,
3(10), 845–852; (b) Bock, M. G.; Hess, J. F.; Pettibone, D.
J. In Annual Reports in Medicinal Chemistry; Doherty, A.
M., Ed.; Elsevier: USA, 2003; Vol. 38, pp 111–120; (c)
Couture, R.; Harrisson, M.; Vianna, R. M.; Cloutier, F.
Eur. J. Pharmacol. 2001, 429, 161–176; (d) Bock, M. G.;
Longmore, J. Bradykinin antagonists: new opportunities.
Curr. Opin. Chem. Biol. 2000, 4, 401–406; (e) Marceau, F.
Kinin Immunopharmacology 1995, 30, 1–26; (f) Regoli, D.;
Barabe, J. Pharmacol. Rev. 1980, 32, 1–46.
2. Rupniak, N. M. J.; Longmore, J.; Hill, R. G. In Molecular
Basis of Pain Induction; Wood, J., Ed.; John Wiley, 2000;
pp 149–174.
3. (a) Pesquero, J. B.; Araujo, R. C.; Heppenstall, P. A.;
Stucky, C. L.; Silva, J. A., Jr.; Walther, T.; Oliveira, S. M.;
Pesquero, J. L.; Paiva, A. C. M.; Calixto, J. B.; Lewin, G.
R.; Bader, M. Proc. Natl. Acad. Sci. U.S.A 2000, 97, 8140–
8145; (b) Ferreira, J.; Campos, M. M.; Araujo, R.; Bader,
M.; Pesquero, J. B.; Calixto, J. B. Neuropharmacology
2002, 43, 1188–1197.
4. (a) Seabrook, G. R.; Bowery, B. J.; Heavens, R.; Brown,
N.; Ford, H.; Sirinathsinghi, D. J. S.; Borkowski, J. A.;
Hess, J. F.; Strader, C. D.; Hill, R. G. Neuropharmacology
1997, 36, 1009–1017; (b) Ma, Q.-P.; Hill, R. G.; Sir-
inathsinghi, D. J. S. Neuroreport 2000, 11, 4003–4005; (c)
Wotherspoon, G.; Winter, J. Neurosci. Lett. 2000, 294,
175–178.
5. (a) Su, D.-S.; Markowitz, M. K.; DiPardo, R. M.;
Murphy, K. L.; Harrell, C. M.; OÕMalley, S. S.; Ransom,
R. W.; Chang, R. S. L.; Ha, S.; Hess, F. J.; Pettibone, D.
J.; Mason, G. S.; Boyce, S.; Freidinger, R. M.; Bock, M.
G. J. Am. Chem. Soc. 2003, 125, 7516–7517; (b) Wood, M.
7. For the assay protocol, see Ref. 5a.
8. See Supplementary data for the preparation of [35S]-4. The
shelf life of radioligand 4 is limited by the t1/2 of 35S
(ꢀ87 days).
9. Panlabs (MDS Pharma Services, Bothell, WA).
10. Ransom, R. W.; Harrell, C. M.; Reiss, D. R.; Murphy, K.
L.; Chang, R. S.-L.; Hess, J. F.; Miller, P. J.; OÕMalley, S.
S.; Hey, P. J.; Kunapuli, P.; Su, D.-S.; Markowitz, M. K.;
Wallace, M. A.; Jones, A. N.; Dean, D. C.; Pettibone, D.
J.; Freidinger, R. M.; Bock, M. G. Eur. J. Pharmacol.
2004, 499, 77–84.
11. MacNeil, T.; Feighner, S.; Hreniuk, D. L.; Hess, J. F.; Van
der Ploeg, L. H. Can. J. Physiol. Pharmacol. 1997, 75, 735–
740.
12. For compound 4, BK B1 Ki: 0.02nM (human), 0.05nM
(rabbit), 0.5nM (dog), 34.0nM (rat), and 0.02 (rhesus
monkey).
13. Hess, J. F.; Ransom, R. W.; Zeng, Z.; Chang, R. S. L.;
Hey, P. J.; Warren, L.; Harrell, C. M.; Murphy, K. L.;
Chen, T.-B.; Miller, P. J.; Lis, E.; Reiss, D.; Gibson, R. E.;
Markowitz, M. K.; Dipardo, R. M.; Su, D.-S.; Bock, M.
G.; Gould, R. J.; Pettibone, D. J. J. Pharmacol. Exp.
Therapeut. 2004, 310(2), 488–497.
14. The commercially available [3H]DAK and [3H]DALK,
two peptide radioligands with high affinity (0.1–0.2nM)
for the human BK B1 receptor but relatively low specific
activity (75Ci/lmol), were also tested in the same assay
format. Both required long incubation times (leading to
dissociation artifacts) and high tissue concentrations
(association artifacts) and could not be used for the
development of a reliable assay.