Development of orally bioavailable and CNS penetrant biphenylaminocyclopropane carboxamide bradykinin B1 receptor antagonists

Article abstract of DOI:10.1021/jm061094b

A series of biphenylaminocyclopropane carboxamide based bradykinin B ₁ receptor antagonists has been developed that possesses good pharmacokinetic properties and is CNS penetrant. Discovery that the replacement of the trifluoropropionamide in t

Full text of DOI:10.1021/jm061094b

272
J. Med. Chem. 2007, 50, 272-282
Development of Orally Bioavailable and CNS Penetrant Biphenylaminocyclopropane
Carboxamide Bradykinin B1 Receptor Antagonists
Scott D. Kuduk,*,† Christina N. Di Marco,† Ronald K. Chang,† Michael R. Wood,† Kathy M. Schirripa,† June J. Kim,†
Jenny M. C. Wai,† Robert M. DiPardo,† Kathy L. Murphy,‡ Richard W. Ransom,‡ C. Meacham Harrell,‡ Duane R. Reiss,‡
Marie A. Holahan,‡ Jacquelynn Cook,‡ J. Fred Hess,‡ Nova Sain,§ Mark O. Urban,§ Cuyue Tang,| Thomayant Prueksaritanont,|
Douglas J. Pettibone,‡ and Mark G. Bock†
Departments of Medicinal Chemistry, Neuroscience Drug DiscoVery, Pain Research, and Drug Metabolism, Merck Research Laboratories,
Sumneytown Pike, P.O. Box 4, West Point, PennsylVania 19486
ReceiVed September 19, 2006
A series of biphenylaminocyclopropane carboxamide based bradykinin B1 receptor antagonists has been
developed that possesses good pharmacokinetic properties and is CNS penetrant. Discovery that the
replacement of the trifluoropropionamide in the lead structure with polyhaloacetamides, particularly a
trifluoroacetamide, significantly reduced P-glycoprotein mediated efflux for the series proved essential. One
of these novel bradykinin B1 antagonists (13b) also exhibited suitable pharmacokinetic properties and efficient
ex vivo receptor occupancy for further development as a novel approach for the treatment of pain and
inflammation.
Introduction
penetrant bradykinin B1 receptor antagonists are of considerable
interest as they may have superior efficacy relative to peripheral
B1 antagonists and might also find additional application in the
treatment of neuropathic pain.
It has been estimated that roughly 40 million Americans suffer
from chronic pain as a result of ailments ranging from
osteoarthritis and diabetic neuropathy to cancer pain. The
societal cost as a result is significant with depression, anxiety,
and insomnia as comorbidities. Accordingly, new and improved
treatments for pain represent a prominent area of unmet medical
needs.
In the past several years, non-peptide, small molecule
antagonists of the B1 receptor have been reported.14 For example,
we have recently shown that 2,3-diaminopyridine bradykinin
B1 antagonists exhibit a high affinity for the human B1 receptor,
good pharmacokinetic properties, and in vivo efficacy in rabbit
models of hyperalgesia and inflammation.15,16 However, the 2,3-
diaminopyridine core has proven to lead to high levels of
bioactivation in vitro and in vivo, prohibiting further develop-
ment of these compounds as clinical candidates.17
Bradykinin peptides (kinins) are rapidly produced from high
molecular weight kininogen precursor proteins following the
activation of kallikrein enzymes after tissue injury.1 Kinins
produce a variety of physiological effects, most notably, pain
and inflammation.2 These effects are transduced by two distinct
G-protein coupled receptors designated as B1 and B2.3 The B2
receptor is largely constitutively expressed in a number of tissues
and evokes the acute pain response following activation by the
kinin peptides bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-
Arg) and kallidin (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg).
Their corresponding metabolites, [des-Arg9]BK (DABK) and
[des-Arg10]kallidin, serve as agonists for the B1 receptor, which
is induced following pro-inflammatory and painful stimuli and
otherwise expressed at very low levels in healthy tissue.4 These
peptide agonists have been shown to produce hyperalgesia in
animal models that can be successfully blocked by peptide
derived B1 antagonists.5,6 In addition, B1 receptor knockout mice
have shown muted responses to thermal, chemical, and me-
chanical nociceptive stimuli, while appearing normal in all other
respects.7,8
It was recently disclosed that R-amino cyclopropylamides can
serve as effective surrogates for the 2,3-diaminopyridine
nucleus.18 Merging this new cyclopropylamide scaffold with the
critical components of diaminopyridine 1 (the trifluoropropi-
onamide and the B/C ring biphenyl) successfully led to
biphenylaminocyclopropane carboxamides such as 2a (Figure
1). While 2a showed excellent human B1 receptor binding
affinity and promising pharmacokinetic properties, it also proved
to be a significant substrate for human P-glycoprotein (P-gp)
mediated efflux,19 indicating that low levels of CNS penetration
in man would be expected. In this paper, we report our efforts
to decrease the P-gp mediated efflux of this cyclopropane
carboxamide series with the goal of discovering a suitably CNS
penetrant preclinical candidate for further development.
Many B1 receptor mediated effects, including pain, involve
peripheral mechanisms. The B1 receptor is also constitutively
expressed in the central nervous system (CNS) of rats and mice,
suggesting a central component in pain perception.9-11 This has
been supported by the demonstration of a hypoalgesic response
when B1 antagonists were administered centrally.12,13 Thus, CNS
* To whom correspondence should be addressed. Tel.: (215) 652-5147;
fax: (215) 652-3971; email: scott_d_kuduk@merck.com.
† Department of Medicinal Chemistry.
‡ Department of Neuroscience Drug Discovery.
§ Department of Pain Research.
| Department of Drug Metabolism.
Figure 1. Genesis of lead structure 2.
10.1021/jm061094b CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/23/2006

Biphenylaminocyclopropane B1 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 273
Scheme 1a
a(a) Rieke Zn, THF, 60°C. (b) Pd(Ph3P)4, 60°C, THF. (c) RaNi, H2, NH3-MeOH. (d) 1-(N-t-Boc-amino)-cyclopropane carboxylic acid, EDC, TEA,
HOBt, DMF. (e) HCl, EtOAc. (f) EDC, TEA, RCO2H, HOBt, CH2Cl2. (g) (S)-t-butyl sulfinamide, CH2Cl2, MgSO4. (h) MeMgBr, CH2Cl2, -48 °C. (i) HCl,
dioxane. (j) Boc2O, CH2Cl2. (k) pinacoldiboron ester, KOAc, DMSO, Pd(dppf)Cl2. (l) Pd(OAc)2, (tol)3P, K2CO3, THF-H2O, 80 °C.
Scheme 2a
Table 1. Bradykinin B1 Receptor Binding Affinities and P-gp
Susceptibility
P-gpb
Pappc
a
compound
R
hBK1
2a
7a
7b
7c
7d
7e
7f
CH2CF3
CF2CF3
CF3
CHF2
CH3
CHCl2
CCH3Cl2
CClF2
CHClF
0.81
2.95
1.47
13.5
11.6
0.54
119
2.5
18.4
2.2
4.1
3.4
6.3
2.8
nd
23
31
23
33
24
21
nd
29
nd
a(a) K2CO3, DMF, Mel or Etl (b) i. oxalyl chloride, CH2Cl2, ii. NH3,
CH2Cl2, iii. dimethylacetamide dimethylacetal, 120 °C, iv. NH2OH, AcOH.
(c) i. NH2OH, EtOH, 80 °C, ii. dimethylacetamide dimethylacetal, 120 °C.
(d) i. Me3SnN3, toluene, 120 °C, ii. Mel, K2CO3, DMF.
7g
7h
2.8
nd
2.8
a Values represent the numerical average of at least two experiments.
Interassay variability was (25% (Ki, nM). b MDR1 directional transport
ratio (B/A)/(A/B). Values represent the average of three experiments, and
interassay variability was (20%. c Passive permeability (10-6 cm/s).
Chemistry
Scheme 1 details the routes employed to access the biphe-
nylaminocyclopropane carboxamides used in this study. Negishi
cross coupling between bromide 3 and zinc reagent 4b followed
by nitrile reduction produced biphenylamine 5. Coupling 5 with
the Boc protected aminocyclopropane carboxylic acid and
consequent deprotection afforded amine 6. Acylation of amine
6 via EDC mediated coupling with the appropriate acid afforded
compounds 7a-h.
For compounds bearing a chiral center via the addition of a
methyl group at the benzylic position, requisite sulfinamide 9
could be prepared from aldehyde 8 using Ellman’s t-butyl-
sulfinimine methodology.20 Protecting group manipulation was
followed by conversion of bromide 9 to pinacolboronate 10.
Subsequent Suzuki coupling with the appropriate halide 14 was
followed by deprotection to produce chiral biphenylamines
11a-g. The biphenylamines could be processed as previously
described for 7a-h to afford final biphenylaminocyclopropane
carboxamides 12a-g and 13a-j. The preparation of requisite
halides 14a-e is illustrated in Scheme 2.
Chinese hamster ovary (CHO) cells stably expressing the hB1
or hB2 receptor. In vitro functional activity was assessed in
standard FLIPR experiments (IC50, nM) employing functionally
active hB1 receptors. Studies of P-gp mediated directional
transport were performed in LLC-PK1 cells expressing genes
for human P-gp (MDR1), and the ratio of transport from
basalateral to apical (B to A) direction to the ratio of transport
from apical to basalateral (A to B) direction was measured. Full
details for the previous experiments are described in the
Experimental Procedures.
The trifluoropropionamide of lead structure 2 was replaced
with various amides to identify compounds that would not be
subject to human P-gp mediated efflux. After extensive screen-
ing, a series of polyhaloacetamides was found to be a promising
lead. Data for key compounds 7a-h are shown in Table 1. All
compounds were B1 selective (IC50 > 10 µM vs the B2 receptor).
While replacement of the 3,3,3-trifluoroproprionamide in lead
2 with a pentafluoropropionamide led to about a 3-fold loss in
Biological Results and Discussion. Ki values (nM) were
determined radiometrically using the appropriate radioligand and

274 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Kuduk et al.
Table 2. Bradykinin B1 Receptor Binding Affinities and Pgp Properties
Addition of a methyl group proved to have favorable effects
on both hB1 receptor affinity and P-gp susceptibility. For
example, the R-enantiomer 2b was about 5-fold more potent
(hKi ) 0.13 nM) than the des-methyl comparator 2a, while S-2c
gave a hKi of 22 nM. Furthermore, the P-gp susceptibility was
reduced 2-fold, establishing that the addition of a methyl group
with the R-configuration not only increased potency but reduced
the susceptibility to human P-gp mediated efflux. This proved
general for polyhaloacetamides 12a-e in Table 2. Among them,
trifluoroacetamide 12b represented a promising selection for
further evaluation. Although 12b was not significantly more
potent than haloacetamides 12c-e, nor did it possess the lowest
P-gp transport ratio as compared to 12a, it did embody the best
combination of desirable properties required in a single com-
pound.
a
compound
R
hBK1
P-gpb
Pappc
2b-(R)
2c-(S)
12a-(R)
12b-(R)
12c-(R)
12d-(R)
12e-(R)
CH2CF3
CH2CF3
CF2CF3
CF3
CHF2
CH3
0.13
22
1.6
0.57
0.4
0.93
0.81
8.6
nd
1.4
2.3
3.2
8.6
2.5
25
nd
31
28
31
21
34
CClF2
Having focused in on trifluoroacetamide as the key discovery
toward reducing the potential for P-gp mediated efflux, fine-
tuning the pharmacokinetic (PK) properties became paramount.
Drug metabolism studies have indicated that methyl ester
hydrolysis was a major metabolic clearance pathway for
compounds such as 12b. A number of alterations to the C-ring
ester and halogen substituents was made to scope their effects
with respect to binding affinity, human P-gp efflux, and
pharmacokinetic properties in the rat. Results are detailed in
Table 3.
a Values represent the numerical average of at least two experiments.
Interassay variability was (25% (Ki, nM). b MDR1 directional transport
ratio (B/A)/(A/B). Values represent the average of three experiments, and
interassay variability was (20%. c Passive permeability (10-6 cm/s).
hB1 receptor binding affinity, it provided a major advance in
that 7a showed significantly reduced susceptibility to human
P-gp medicated efflux (directional transport ratio of 2.2).
Removal of the difluoromethylene from the amide in 2 provided
trifluoroacetamide 7b, which improved the binding potency but
led to a slight increase in the P-gp susceptibility. Deletion of
one or all of the fluorines (7c-d) gave a 10-fold loss in hB1
receptor affinity without improvement in P-gp susceptibility.
Dichloroacetamide 7e provided the first subnanomolar com-
pound (hKi ) 0.54 nM) with a promising P-gp directional
transport ratio of 2.8. However, 7e proved to be quite unstable
and was eliminated from further consideration. Addition of a
methyl group provided 2,2-dichloropropionamide 7f, which had
a deleterious effect on hB1 binding. Mixed haloacetamides such
as 7g-h were similarly potent to 7a with reasonable P-gp
profiles.
Removal of the fluorine atom ortho to the ester (13a) on the
phenyl ring led to a to 20-fold loss in binding relative to 12b,
while having little impact on the P-gp profile. Replacement with
the larger chlorine (13b), however, afforded an improvement
in both P-gp efflux and Papp, while maintaining a very high
B1 receptor affinity with a hKi of 0.44 nM. The size of the ester
group is of consequence as the related ethyl ester 13c was about
3-fold less potent than the methyl ester 13b. Ester isosteres such
as oxadiazoles 13d-e maintained good receptor affinity but saw
moderate increases in P-gp efflux. A similar observation was
made with tetrazoles 13f-h, particularly for the weakly active
N-1 isomer 13h. It is likely that the increases in P-gp efflux are
a consequence of the enhanced hydrogen bond acceptor ability
resulting from the additional nitrogen atoms in those heterocyclic
rings. Other ester surrogates such as trifluoromethyl (13i) or an
ethyl ketone (13j) conserved good P-gp and permeability profiles
but are 2-3 times less active with respect to B1 receptor binding
affinity.
An additional factor that warrants attention with relation to
P-gp efflux is the passive permeability of a given compound,
which can also influence CNS levels.21 Compounds with low
permeability tend to diminish the reliability that a P-gp transport
assay can be used to predict CNS distribution. A passive
permeability value of 15 × 10-6 cm/s or greater is typical for
compounds that exhibit good levels of CNS penetration, and
haloacetamides 7a-h all met or exceeded this criterion.
The effects the polyhaloacetamides exert on P-gp transport
are dramatic, but the underlying reason is less evident. One
thought is that the increased lipophilicity due to the halogens
reduces P-gp mediated transport. However, P-gp substrate 2a
is equal to or more lipophilic (log P 2.9) than several low
transport compounds such as 7b (log P 2.7), 7c (log P 2.4),
and 7e (log P 2.8). In addition, the fewer hydrogen bond
acceptors a compound possesses, the less likely it is to be a
substrate for P-gp. Having the halogens alpha to the amide group
is vital, indicating that a strong electronic withdrawing effect
is active. We propose that the electron deficient amide is a
poorer hydrogen bond acceptor and is therefore less prone to
recognition by P-gp.22
In terms of the pharmacokinetic profiles, going from trifluo-
ropropionamide 2b to trifluoroacetamide 12b led to an increased
clearance with a shorter half-life in the rat. Theaddition of the
larger chlorine atom (13b) did increase bioavailability relative
to 12b, but no benefit in clearance or half-life was observed.
As expected, the larger ethyl ester 13c, which was assumed to
be less prone to hydrolysis, had an improved rat profile as
compared to the methyl ester. Surprisingly, the heterocyclic
isosteres 13d-g provided little benefit with regard to half-life
as compared to their ester counterparts. Trifluoromethyl com-
pound 13i had a good rat PK with the best half-life in the series
of 2.6 h. Taken as a whole, although a number of methyl ester
replacements proved to sustain good B1 receptor affinity, they
offered no significant advantage in terms of PK and were more
susceptible to P-gp mediated efflux. Consequently, the primary
focus remained on the esters 12b and 13b.
The addition of a methyl group at the benzylic methylene
has been shown to increase binding affinity in the aforesaid
2,3-diaminopyridine series.16 Accordingly, compounds bearing
this methyl group with select polyhaloacetamides from Table
1 were evaluated for their effects on hB1 binding and on human
P-gp transport (Table 2).
The pharmacokinetic behavior of 12b and 13b was evaluated
in the dog and monkey to further assess their potential for human
PK projection. Although the oxadiazoles did not offer an
advantage over the esters in terms of rat PK, oxadiazole 13d

Biphenylaminocyclopropane B1 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 275
Table 3. Bradykinin B1 Receptor Binding Affinities, P-gp Transport Properties, and Rat Pharmacokinetics
a Values represent the numerical average of at least two experiments. Interassay variability was (25% (Ki, nM) and (25% for the FLIPR experiments
(IC50, nM). b MDR1 directional transport ratio (B/A)/(A/B). Values represent the average of three experiments and interassay variability was (20%. c Passive
permeability (10-6 cm/s). d F% oral bioavailability, half-life is represented in hours, Cl in mL/min/kg. Sprague-Dawley rats (n ) 3). Oral dose ) 10 mg/kg,
IV dose ) 2 mg/kg. Interanimal variability was less than 20%.
Table 4. Rat and Dog Pharmacokinetics for Select Compounds
esters. However, since 13d was subject to human P-gp mediated
dog PKa
Rhesus PKb
efflux (P-gp ) 5.6), it was eliminated from further consideration.
Although compounds 12b and 13b exhibited only fair pharma-
cokinetic properties, they demonstrated superior stability in
human microsomes and hepatocytes (see Supporting Informa-
tion) relative to the three species, indicating that they were likely
to have adequate bioavailability in humans.
Cl
Cl
compound F (%) t1/2 (h) mL/min/kg F (%) t1/2 (h) mL/min/kg
12b
13b
13d
5
33
35
1.7
1.8
3.9
21
9
6
2
31
22
2.2
1.7
2.9
28
13
15
a Mongrel dogs (n ) 2). Oral dose 3 mg/kg and IV dose ) 1 mg/kg.
Interanimal variability was less than 20% for all values. b Rhesus monkies
(n ) 2). Oral dose 3 mg/kg and IV dose ) 1 mg/kg. Interanimal variability
was less than 20% for all values.
Pharmacodynamic Models. Previously, the 2,3-diaminopy-
ridine series of bradykinin B1 antagonists proved to be selective
for the human and rabbit over the rat receptor, thwarting efforts
to characterize lead compounds in classic rodent models of pain
and inflammation.15 Similarly, the biphenylaminocyclopropane
carboxamide series described here has shown a similar selectiv-
ity but also exhibits a high affinity for the rhesus bradykinin
B1 receptor as exemplified by 13b in Table 5.
was included to see if this ester surrogate offered improvement
in these species. Results are shown in Table 4.
Methyl ester 12b had extremely poor bioavailability (<5%)
and rapid clearance in both dog and rhesus. Replacement of
the fluorine with a chlorine ortho to the methyl ester (13b)
significantly improved the oral bioavailability and reduced the
clearance to an acceptable level in both species. Incorporation
of the oxadiazole (13d) showed good bioavailability and
improved the half-lives in dog and rhesus relative to the methyl
Accordingly, as one pharmacodynamic model, we induced
functional receptors in the rhesus vasculature by iv administra-
tion of lipopolysaccharide (LPS), an effect that is manifest by
a depressor blood pressure response to the B1 agonist DAK (1

276 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Kuduk et al.
Table 5. Species Differences and In Vivo Experiments for Compound
13b
human Ki (nM)a
0.4
1646
7.3
2.0
47 ( 10
520
rat Ki (nM)a
rabbit Ki (nM)a
Rhesus Ki (nM)a
Rhesus LPS AD90 (µg/Kg)b
transgenic rat Occ90 (nM)b
a Values represent the numerical average of at least two experiments.
Interassay variability was (25% for the binding assays (Ki, nM). b IV
dosing, see Experimental Procedures for full assay details.
Figure 3. Dose-response curve of 13b (p.o.) in hB1 knock-in mice.
efficacy produced by the maximum dose of 60 mg/kg (100%)
was similar to that observed with the NSAID naproxen in this
model (50%, data not shown). It should be noted that CFA
injection produced equivalent hyperalgesia in the wild-type mice,
which was unaffected by oral administration of 13b (Supporting
Information).
While 13b was not subject to human P-gp efflux (transport
ratio of 1.9), it was a substrate for mouse P-gp efflux in vitro
in LLC-PK1 cells (transport ratio of 9.3). To correlate how the
observed human P-gp transport ratios may affect CNS levels,
we investigated in vivo studies in P-gp deficient and competent
mice. P-gp had little impact on the plasma clearance of 13b
indicated by the AUCs [p(-)/p(++) ) 1.2], but a 9-fold
reduction in brain exposure was observed for P-gp competent
mice [AUCs b(-)/b(++) ) 9.1]. The ratio of brain to plasma
[b(-)/p(-)]for 13b in P-gp deficient mice is 1.05, demonstrating
excellent brain penetration in the absence of P-gp. We believe
that this will be similar to the case in man since 13b is not a
human P-gp substrate and should exhibit good brain penetration.
To validate this result with a primate model, CNS levels in a
Rhesus monkey were taken (2 mpk iv, 30 min) and found to
give an adequate brain/plasma ratio of 0.4.
Figure 2. Reversal of CFA-induced hypersensitivity by 13b in hB1
knock-in mice.
µg kg iv).23 Generation of antagonist dose-responses to the
depressor effects were created by pretreatment with rising doses
of 13b prior to treatment with DAK. In this model, 13b proved
highly potent to block the depressor effect with an AD90 of 47
µg/kg.
To measure the facility with which 13b binds to the human
B1 receptor expressed in the CNS, we employed the previously
described transgenic rat model.24 While the human B1 receptors
expressed in this particular transgenic rat model do not appear
to be functional, this model does serve as a useful tool to
demonstrate CNS receptor occupancy using an ex vivo binding
assay. Since there is currently no animal pain model with which
one can correlate CNS occupancy with efficacy, our goal was
to achieve high (i.e., 90%) brain receptor occupancy (Occ90) in
the transgenic rat. Compound 13b exhibited an Occ90 of 520
nM in the rat CNS, a reasonable brain level that should be
attainable in humans based on its low substrate activity at P-gp.
Recently, a human selective B1 antagonist has been shown
to be efficacious in reducing complete Freund’s adjuvant (CFA)-
induced mechanical hyperalgesia in humanized but not wild-
type mice.25 Accordingly, we decided to evaluate antagonist 13b
in a CFA-induced hyperalgesia model employing a similar
human B1 knock-in mouse. This particular mouse model utilized
gene targeting by homologous recombination to replace the
genomic sequence for the endogenous mouse B1 receptor with
that of the human B1 receptor.26 This B1 knock-in mouse has
been shown to possess the pharmacological characteristics
consistent with the human B1 receptor being expressed and
regulated physiologically.
As expected, injection of CFA into the left hind paw of the
human B1 knock-in mice produced inflammation and a decrease
in tactile withdrawal threshold (hyperalgesia) in the affected
hind paw when measured 24 h following CFA injection (Figure
2). The paw withdrawal thresholds of CFA injected mice were
unaffected by vehicle treatment. Compound 13b was dosed
orally at 6, 20, and 60 mg/kg, which resulted in plasma
concentrations of 1.0, 3.5, and 5.0 µM, respectively, at 1 h.
Oral administration of compound 13b dose dependently reversed
the hyperalgesia in hB1 knock-in mice with an ED50 (95% CL)
) 9.76 (6.0-15.8) mg/kg (Figures 2 and 3). Moreover, the
Conclusion
We have identified trifluoromethyl carboxamide 13b as a
potent antagonist for the human bradykinin B1 receptor that
demonstrated significantly reduced susceptibility to human P-gp
mediated efflux. The compound shows good potential for human
CNS penetration based on brain levels in CF-1 mice and
monkeys. Additionally, 13b also exhibited good CNS receptor
occupancy in the transgenic rat expressing the human B1
receptor and showed oral efficacy in reducing CFA-induced
hyperalgesia in a humanized mouse. On the basis of these
properties and the potential for good human pharmacokinetics,
compound 13b was selected as a development candidate for
the treatment of pain and inflammation.
Experimental Procedures
General. All commercially available chemicals and solvents were
used without further purification. 1H (400 MHz) NMR spectra were
recorded on a Varian VXR 400 spectrometer unless otherwise noted.
The chemical shifts are reported in δ (ppm) using the δ 0.00 signal
of Me4Si as an internal standard. High resolution MS data were
obtained on a Bruker Daltonics FTICR/MS. High resolution mass
spectral analysis was performed on a Bruker-daltonics BioApex
3T mass spectrometer. All animal studies described herein were
approved by the Merck Research Laboratories Institutional Animal
Care and Use Committee. HPLC spectra were recorded on a
Hewlett-Packard 1100 with a CombiScreen Pro C18 column. The
purity of compounds was assessed to be >95% by analytical
HPLC: (i) system 1: linear gradient over 10 min of CH3CN/0.1%
TFA and H2O/0.1% TFA 10:90 to 95:5 and 2 min at 95:5; flow

Biphenylaminocyclopropane B1 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 277
rate 1.0 mL/min; detection at 215 and 254 nm (YMC-Pack Pro
C18, 50 mm × 4.6 mm column). (ii) Linear gradient over 3.5 min
of CH3CN/0.1% TFA and H2O/0.1% TFA 5:95 to 95:5; flow rate
1.5 mL/min; detection at 215 nm (YMC-Pack Pro C18, 50 mm ×
4.6 mm column).
7.38 (t, J ) 8.0 Hz, 1H), 7.55 (q, J ) 8.0 Hz, 1H), 8.53 (s, 1H),
9.95 (s, 1H). MS m/z ) 507.25 (MH+). Anal. (C22H17F7N2O4•H2O)
C, H, N.
Methyl 3,3′-Difluoro-4′-{[({1-[(trifluoroacetyl)amino]cyclo-
propyl}carbonyl)amino]methyl}-1,1′-biphenyl-2-carboxylate (7b).
1
White solid: yield 66%. H NMR (400 MHz, CD3OD) δ 1.12 (q,
Methyl 4′-(Aminomethyl)-3,3′-difluoro-1,1′-biphenyl-2-car-
boxylate (5). To a heat-dried flask under N2 was added methyl
2-fluoro-6-iodobenzoate (5.00 g, 17.9 mmol) and Rieke Zn (Aldrich,
5 g in 100 mL of THF, 0.765 M THF, 24.5 mL, 18.8 mmol). The
reaction was heated to 90 °C for 4.5 h. To the crude zinc iodide
was added a solution of 4-bromo-2-fluorobenzonitrile (3.57 g, 17.9
mmol) in THF (5 mL) and Pd(PPh3)4 (0.100 g, 0.086 mmol). The
mixture was heated to reflux for 1 h, cooled to room temperature,
and concentrated in vacuo. The resultant residue was dissolved in
EtOAc and filtered through celite. The filtrate was washed with
H2O and brine, dried over Na2SO4, filtered, and concentrated. The
residue was subjected to silica gel chromatography (0-10% EtOAc
in hexanes) to provide methyl 4′-cyano-3,3′-difluoro-1,1′-biphenyl-
2-carboxylate as an orange solid.
J ) 4.2 Hz, 2H), 1.55 (q, J ) 4.2 Hz, 2H), 2.68 (s, 3H), 4.49 (s,
2H), 7.05-7.14 (m, 2H), 7.22-7.28 (m, 2H), 7.40 (t, J ) 7.8 Hz,
1H), 7.54-7.61 (m, 1H). HRMS Calcd for C21H18F5N2O4 (M+1):
457.1186. Found: 457.1182. Anal. (C21H17F5N2O4) C, H, N.
Methyl 4′-{[({1-[(Difluoroacetyl)amino]cyclopropyl}carbonyl)-
amino]methyl}-3,3′-difluoro-1,1′-biphenyl-2-carboxylate (7c).
1
White solid: yield 34%. H NMR (400 MHz, CD3OD) δ 1.10 (q,
J ) 4.5 Hz, 2H), 1.53 (q, J ) 4.5 Hz, 2H), 3.68 (s, 3H), 4.49 (s,
2H), 6.03 (t, J ) 54.0 Hz, 1H), 7.05-7.14 (m, 2H), 7.19-7.25
(m, 2H), 7.39 (t, J ) 7.8 Hz, 1H), 7.51-7.58 (m, 1H). HRMS
Calcd for C21H19F4N2O4 (M+1): 439.1281. Found: 439.1271.
Anal. (C21H18F4N2O4•0.85 CH2Cl2) C, H, N.
Methyl 4′-[({[1-(Acetylamino)cyclopropyl]carbonyl}amino)-
methyl]-3,3′-difluoro-1,1′-biphenyl-2-carboxylate (7d). White
solid: yield 54%. 1H NMR (400 MHz, CD3OD) δ 1.04 (q, J ) 4.5
Hz, 2H)c 4.49 (q, J ) 4.5 Hz, 2H), 3.70 (s, 3H), 4.51 (d, J ) 5.9
Hz, 2H), 7.06-7.15 (m, 2H), 7.21-7.27 (m, 2H), 7.42 (t, J ) 7.9
Hz, 1H), 7.52-7.60 (m, 1H), 8.52 (t, J ) 5.9 Hz, 1H). HRMS
Calcd for C21H21F2N2O4 (M + 1): 403.1469. Found: 403.1459.
Anal. (C21H20F2N2O4•0.35 CH2Cl2) C, H, N.
To a solution of the methyl 4′-cyano-3,3′-difluoro-1,1′-biphenyl-
2-carboxylate (1.30 g, 4.76 mmol) in NH3 in MeOH (2.0 M, 20
mL, 40 mmol) was added Raney Nickel (suspension in H2O, ∼0.25
g). The solution was placed under an atmosphere of H2 (balloon)
and stirred at room temperature overnight. The mixture was filtered
through glass filter paper and concentrated in vacuo. The residue
was azeotroped 3 times with toluene and partitioned between 1 N
HCl and EtOAc. The organic layer was washed 2 times with 1 N
HCl. Aqueous NaOH (1 M) was added to pH ∼10, and the product
was extracted 3 times with EtOAc. The combined organic fractions
were dried over Na2SO4, filtered, and concentrated to provide 5
Methyl 4′-{[({1-[(Dichloroacetyl)amino]cyclopropyl}carbonyl)-
amino]methyl}-3,3′-difluoro-1,1′-biphenyl-2-carboxylate (7e).
1
White solid: yield 71%. H NMR (400 MHz, CD3OD) δ 1.08 (q,
J ) 4.6 Hz, 2H), 1.54 (q, J ) 4.6 Hz, 2H), 3.68 (s, 3H), 4.50 (s,
2H), 6.24 (s, 1H), 7.05-7.14 (m, 2H), 7.19-7.25 (m, 2H), 7.39 (t,
J ) 7.8 Hz, 1H), 7.51-7.58 (m, 1H). HRMS Calcd for C21H19-
Cl2F2N2O4 (M + 1): 471.0690. Found: 471.0681. Anal. (C21H18-
Cl2F2N2O4•0.4 H2O) C, H, N.
1
(0.89 g, 68%). H NMR (400 MHz, CDCl3) δ 1.53 (s, 2H), 3.74
(s, 3H), 3.95 (s, 2H), 7.05-7.18 (m, 4H), 7.35-7.49 (m, 2H).
1-[({[3,3′-Difluoro-2′-(methoxycarbonyl)-1,1′-biphenyl-4-yl]-
methyl}amino)carbonyl]cyclopropanaminium Chloride (6). Com-
pound 5 (0.620 g, 2.24 mmol) was dissolved in EtOAc (10 mL),
cooled to 0 °C, and saturated with HCl (g). After 30 min at room
temperature, the mixture was concentrated in vacuo. A solution of
this crude salt, (∼0.70 g, 2.2 mmol), tert-butoxycarbonyl-1-
aminocyclopropane-1-carboxylic acid (0.494 g, 2.45 mmol), 1-ethyl-
(3-dimethylaminopropyl)carbodiimide hydrochloride (0.855 g, 4.46
mmol), 1-hydroxy-7-azabenzotriazole (0.010 g, 0.15 mmol), and
triethylamine (1.35 g, 13.4 mmol) in CH2Cl2 (15 mL) was stirred
at room temperature overnight. The solution was washed with
aqueous sodium bicarbonate and brine, dried over Na2SO4, filtered,
and concentrated. The residue was subjected to silica gel chroma-
tography (10-50% EtOAc in hexanes) to provide methyl 4′-{[({1-
[(tert-butoxycarbonyl)amino]cyclopropyl}carbonyl)amino]methyl}-
3,3′-difluoro-1,1′-biphenyl-2-carboxylate (1.04 g, 100%) as a white
solid. This Boc protected amine was dissolved in EtOAc (10 mL),
cooled to 0 °C, and saturated with HCl (g). After 30 min at room
temperature, the mixture was concentrated in vacuo. The residue
was azeotroped 3 times with toluene to provide the hydrochloride
Methyl 4′-{[({1-[(2,2-Dichloropropanoyl)amino]cyclopropyl}-
carbonyl)amino]methyl}-3,3′-difluoro-1,1′-biphenyl-2-carboxy-
late (7f). White solid: yield 23%. 1H NMR (400 MHz, CD3OD) δ
1.11 (q, J ) 4.8 Hz, 2H), 1.54 (q, J ) 4.8 Hz, 2H), 2.28 (s, 3H),
3.68 (s, 3H), 4.51 (s, 2H), 7.06-7.14 (m, 2H), 7.20-7.26 (m, 2H),
7.40 (t, J ) 7.8 Hz, 1H), 7.51-7.58 (m, 1H). HRMS Calcd for
C22H21Cl2F2N2O4 (M+1): 485.0846. Found: 485.0835.
Methyl 4′-({[(1-{[Chloro(difluoro)acetyl]amino}cyclopropyl)-
carbonyl]amino}methyl)-3,3′-difluoro-1,1′-biphenyl-2-carboxy-
1
late (7g). White solid: yield 34%. H NMR (400 MHz, CD3OD)
δ 1.11 (q, J ) 4.6 Hz, 2H), 1.55 (q, J ) 4.6 Hz, 2H), 3.68 (s, 3H),
4.49 (s, 2H), 7.05-7.14 (m, 2H), 7.19-7.26 (m, 2H), 7.38 (t, J )
7.8 Hz, 1H), 7.51-7.58 (m, 1H). HRMS Calcd for C21H18ClF4N2O4
(M + 1): 473.0891. Found: 473.0880. Anal. (C21H17ClF4N2-
O4•0.5H2O) C, H, N.
Methyl 4′-({[(1-{[Chloro(fluoro)acetyl]amino}cyclopropyl)-
carbonyl]amino}methyl)-3,3′-difluoro-1,1′-biphenyl-2-carboxy-
1
late (7h). White solid: yield 32%. H NMR (400 MHz, CD3OD)
δ 1.04-1.15 (m, 2H), 1.58-1.69 (m, 2H), 3.73 (s, 3H), 4.51 (d, J
) 6.1 Hz, 2H), 6.29 (d, J ) 50.5 Hz, 1H), 6.78 (t, J ) 6.0 Hz,
1H), 7.02-7.16 (m, 4H), 7.27-7.49 (m, 3H). MS m/z ) 455.11
(MH+). Anal. (C21H18ClF3N2O4) C, H, N.
1
salt of 6 as a white solid. H NMR (400 MHz, CD3OD) δ 1.37-
1.40 (m, 2H), 1.53-1.57 (m, 2H), 3.68 (s, 3H), 4.47 (s, 2H), 7.07-
7.14 (m, 2H), 7.21-7.25 (m, 2H), 7.37 (t, J ) 7.7 Hz, 1H), 7.52-
7.57 (m, 1H). MS m/z ) 361.22 (MH+).
N-[(1R)-1-(4-Bromo-2-fluorophenyl)ethyl]-2-methylpropane-
2-sulfinamide (9). To a solution of (S)-(-)-2-methyl-2-propane-
sulfinamide (20.20 g, 0.167 mol) in 350 mL of CH2Cl2 was added
4-bromo-2-fluorobenzaldehyde (35.53 g, 0.1750 mol), pyridinium
p-toluenesulfonate (2.09 g, 8.33 mmol), and magnesium sulfate
(200.6 g, 1.667 mol). The reaction was stirred at room temperature
for 48 h. More magnesium sulfate (100.3 g, 0.833 mol) was added,
and the reaction was stirred for an additional 24 h. The mixture
was filtered through celite, washed with CH2Cl2, and concentrated
in vacuo. The resulting residue was subjected to silica gel
chromatography (0-10% ethyl acetate in hexanes) to provide
N-[(1E)-(4-bromo-2-fluorophenyl)methylidene]-2-methylpropane-
2-sulfinamide (24.83 g, 48%) as a white solid: 1H NMR (400 MHz,
CDCl3) δ 1.27 (s, 9H), 7.35-7.41 (m, 2H), 7.87 (t, J ) 7.8 Hz,
1H), 8.83 (s, 1H). MS m/z 308 (MH+).
General Procedure for the Preparation of Compounds 7a-
h. Methyl 3,3′-Difluoro-4′-{[({1-[(2,2,3,3,3-pentafluoropropanoyl)-
amino]cyclopropyl}carbonyl)amino]methyl}-1,1′-biphenyl-2-
carboxylate (7a). A solution of 6 (48.4 mg, 0.12 mmol), pentafluoro-
propionic acid (40.0 mg, 0.24 mmol), 1-ethyl-(3-dimethylamino-
propyl)carbodiimide hydrochloride (46.8 mg, 0.24 mmol), 1-hy-
droxy-7-azabenzotriazole (0.010 g, 0.15 mmol), and triethylamine
(74.1 mg, 0.73 mmol) in DMF (2 mL) was stirred at room
temperature overnight. The mixture was diluted with EtOAc,
washed with aqueous sodium bicarbonate and brine, dried over Na2-
SO4, filtered, and concentrated. The residue was purified via
reversed phase HPLC to provide 7a (10 mg, 16%) as a white solid.
1H NMR (400 MHz, CD3OD) δ 1.06-1.09 (m, 2H), 1.55-1.58
(m, 2H), 3.68 (s, 1H), 7.20-7.26 (m, 2H), 7.07-7.13 (m, 2H),

278 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Kuduk et al.
To a solution of the aforementioned imine (32.6 g, 0.106 mol)
in CH2Cl2 (550 mL) at -48 °C was added methylmagnesium
chloride (3.0 M solution in ether, 53.3 mL, 0.160 mol) dropwise
over 1 h. The reaction was quenched with aqueous ammonium
chloride, and the aqueous layer was extracted with CH2Cl2. The
combined organics were dried over Na2SO4, filtered, and concen-
trated in vacuo. The resulting residue was subjected to silica gel
chromatography (10-50% EtOAc in hexanes) to provide 9 (22.6
g, 65%) as a white solid: 1H NMR (400 MHz, CDCl3) δ 1.19 (s,
9H), 1.56 (d, J ) 6.8 Hz, 3H), 3.33 (d, J ) 4.2 Hz, 1H), 4.77-
4.85 (m, 1H), 7.21-7.29 (m, 3H). MS m/z 324.11 (MH+).
tert-Butyl (1R)-1-[2-Fluoro-4-(4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)phenyl]ethylcarbonate (10). To a solution of 9
(26.29 g, 81.58 mmol) in MeOH (40 mL) was added HCl in a
dioxane (4 M, 40.8 mL, 0.163 mol) solution. The reaction mixture
was concentrated in vacuo, and ether was added. The white solid
was collected, washed with cold ether, and dried in vacuo to provide
(1R)-1-(4-bromo-2-fluorophenyl)ethanaminium chloride (14.2 g,
98%) as a white solid: 1H NMR (400 MHz, CD3OD) δ 1.64 (d, J
) 6.8 Hz, 3H), 4.70 (q, J ) 6.8 Hz, 1H), 7.41-7.52 (m, 3H). MS
m/z 203.14 (MH+).
To a solution of (1R)-1-(4-bromo-2-fluorophenyl)ethanaminium
chloride (14.3 g, 55.9 mmol) in CH2Cl2 (300 mL) at 0 °C was
added di(tert-butyl) dicarbonate (17.98 g, 82.40 mmol) and tri-
ethylamine (8.25 g, 81.6 mmol). The solution was washed with
water and brine, dried over Na2SO4, filtered, and concentrated in
vacuo to provide crude tert-butyl (1R)-1-(4-bromo-2-fluorophenyl)-
ethylcarbamate (30.2 g, 100%) as a white solid: 1H NMR (400
MHz, CDCl3) δ 1.41 (br s, 9H), 1.53 (s, 3H), 4.91 (s, 1H), 7.13-
7.24 (m, 3H).
A mixture of tert-butyl (1R)-1-(4-bromo-2-fluorophenyl)ethyl-
carbamate (26.4 g, 83.1 mmol), bis(pinacolato)diboron (31.6 g,
0.125 mol), potassium acetate (24.5 g, 0.249 mol), and [1,1′-bis-
(diphenylphosphino)ferrocene]palladium(II) dichloride (0.26 g, 0.36
mmol) in DMSO (80 mL) was heated to 90 °C under N2 for 3 h.
The mixture was then cooled to room temperature and partitioned
between EtOAc and water. The organic extract was washed with
water and brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was subjected to silica gel chromatography (0-
10% EtOAc in hexanes) to provide 10 (32.0 g, 100%) as a beige
solid: 1H NMR (400 MHz, CDCl3) δ 1.26 (s, 12H), 1.33 (s, 9H),
1.52 (s, 3H), 4.97 (s, 1H), 7.27-7.29 (m, 1H), 7.44 (d, J ) 11.2
Hz, 1H), 7.53 (d, J ) 7.5 Hz, 1H).
General Procedure for the Preparation of Compounds 11a-
g. Methyl 4′-{(1R)-1-[(tert-Butoxycarbonyl)amino]ethyl}-3-
chloro-3′-fluoro-1,1′-biphenyl-2-carboxylate (11b). A mixture of
methyl 2-bromo-6-chlorobenzoate (2.25 g, 9.03 mmol), tert-butyl
(1R)-1-[2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phe-
nyl]ethylcarbonate 10 (3.00 g, 8.21 mmol), potassium carbonate
(2.84 g, 20.5 mmol), tri-o-tolylphosphine (0.10 g, 0.33 mmol), and
palladium acetate (0.018 g, 0.08 mmol) in THF (40 mL) and water
(4 mL) was heated in a sealed flask at 100 °C for 4 h. The mixture
was then cooled and concentrated in vacuo. The resulting residue
was dissolved in EtOAc, washed with water and brine, dried over
Na2SO4, filtered, and concentrated in vacuo to provide methyl 4′-
{(1R)-1-[(tert-butoxycarbonyl)amino]ethyl}-3-chloro-3′-fluoro-1,1′-
biphenyl-2-carboxylate as a white solid.
The Boc protected amine was dissolved in EtOAc (20 mL),
cooled to 0 °C, and saturated with HCl (g). After 30 min at room
temperature, the mixture was concentrated in vacuo. The residue
was azeotroped 3 times with toluene to provide (1R)-1-[3′-chloro-
3-fluoro-2′-(methoxycarbonyl)-1,1′-biphenyl-4-yl]ethanaminium chlo-
ride. The product was partitioned between EtOAc and aqueous
sodium bicarbonate. The organic layer was washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo to provide 11b
(1.79 g, 70%) as a yellow oil: 1H NMR (400 MHz, CDCl3) δ 1.44
(d, J ) 6.6 Hz, 3H), 1.58 (s, 2H), 3.74 (s, 3H), 4.41 (q, J ) 6.6
Hz, 1H), 7.04-7.16 (m, 2H), 7.27-7.30 (m, 1H), 7.37-7.47 (m,
3H). The crude compounds were then taken directly onto 12a-e
and 13a-j.
General Procedure for the Preparation of Compounds 12a-e
and 13a-j. Methyl 3-Chloro-3′-fluoro-4′-{(1R)-1-[({1-[(trifluo-
roac-etyl)amino]cyclopropyl}carbonyl)amino]ethyl}-1,1′-biphe-
nyl-2-carboxylate (13b). A solution of the HCl salt of 11b (1.00
g, 2.91 mmol), tert-butoxycarbonyl-1-aminocyclopropane-1-car-
boxylic acid (0.614 g, 3.05 mmol), 1-ethyl-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (1.11 g, 5.81 mmol), 1-hydroxy-
7-azabenzotriazole (0.010 g, 0.15 mmol), and triethylamine (1.76
g, 17.4 mmol) in CH2Cl2 (20 mL) was stirred at room temperatue
overnight. The solution was washed with aqueous sodium bicarbon-
ate and brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was subjected to silica gel chromatography (10-
40% EtOAc in hexanes) to provide methyl 4′-{(1R)-1-[({1-[tert-
butoxycarbonyl)amino]cyclopropyl}carbonyl)amino]ethyl}-3-chloro-
3′-fluoro-1,1′-biphenyl-2-carboxylate (1.38 g, 96%) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 0.98-1.06 (m, 2H), 1.46 (s,
9H), 1.51-1.61 (m, 5H), 3.73 (s, 3H), 5.07 (br s, 1H), 5.22-5.32
(m, 1H), 7.00 (br s, 1H), 7.05-7.13 (m, 2H), 7.23-7.26 (m, 1H),
7.29-7.37 (m, 1H), 7.40-7.45 (m, 2H). MS m/z 491.30 (MH+).
The product was dissolved in EtOAc (10 mL) and cooled to 0
°C, and the solution was saturated with HCl (g). After 30 min at
room temperature, the mixture was concentrated in vacuo. The
residue was azeotroped 3 times with toluene to provide 1-[({(1R)-
1-[3′-chloro-3-fluoro-2′-(methoxycarbonyl)-1,1′-biphenyl-4-yl]ethyl}-
amino)carbonyl]cyclopropanaminium chloride as a white solid: MS
m/z 391.21 (MH+).
To a solution of the previous product (13.60 g, 31.83 mmol) in
CH2Cl2 (150 mL) and triethylamine (6.44 g, 6.36 mmol) at 0 °C
was added trifluoroacetic anhydride (6.68 g, 3.18 mmol). The
solution was diluted with additional CH2Cl2 and washed with
aqueous sodium bicarbonate and brine, dried over Na2SO4, filtered,
and concentrated. The residue was subjected to silica gel chroma-
tography (10-40% EtOAc in hexanes) to provide 13b (13.5 g,
87.1%) as a white solid: 1H NMR (400 MHz, CD3OD) δ 1.03-
1.27 (m, 2H), 1.49-1.52 (m, 5H), 3.69 (s, 3H), 5.31 (q, J ) 7.1
Hz, 1H), 7.07-7.17 (m, 2H), 7.33-7.51 (m, 4H). MS m/z ) 487.22
(MH+). Anal. (C22H19ClF4N2O4) C, H, N.
Methyl 3,3′-Difluoro-4′-{(1R)-1-[({1-[(2,2,3,3-tetrafluoro-
propanoyl)amino]cyclopropyl}carbonyl)amino]ethyl}-1,1′-bi-
phenyl-2-carboxylate (12a). White solid: yield 7%. 1H NMR (400
MHz, CD3OD) δ 1.01-1.15 (m, 2H), 1.41-1.55 (m, 5H), 3.68 (s,
3H), 5.22-5.32 (m, 1H), 6.38 (tt, J ) 5.1, 52.5 Hz, 1H), 7.06-
7.14 (m, 2H), 7.20-7.26 (m, 2H), 7.39 (t, J ) 7.9 Hz, 1H), 7.51-
7.58 (m, 1H), 7.77(d, J ) 7.6 Hz, 1H), 9.73 (s, 1H). MS m/z )
503.26 (MH+). Anal. (C23H20F6N2O4•0.45 CH2Cl2) C, H, N.
Methyl 3,3′-Difluoro-4′-{(1R)-1-[({1-[(trifluoroacetyl)amino]-
cyclopropyl}carbonyl)amino]ethyl}-1,1′-biphenyl-2-carboxy-
1
late (12b). White solid: 95.1%. H NMR (400 MHz, CD3OD) δ
1.02-1.16 (m, 2H), 1.49-1.51 (m, 5H), 3.68 (s, 3H), 4.86 (s, 2H),
5.30 (q, J ) 7.1 Hz, 1H), 7.05-7.14 (m, 2H), 7.19-7.26 (m, 2H),
7.41 (t, J ) 7.9 Hz, 1H), 7.51-7.58 (m, 1H). MS m/z ) 471.23
(MH+). Anal. (C22H19F5N2O4•0.5 H2O) C, H, N.
Methyl 4′-{(1R)-1-[({1-[(Difluoroacetyl)amino]cyclopropyl}-
carbonyl)amino]ethyl}-3,3′-difluoro-1,1′-biphenyl-2-carboxy-
1
late (12c). White solid: 51%. H NMR (400 MHz, CD3OD) δ
1.01-1.15 (m, 2H), 1.45-1.52 (m, 5H), 3.69 (s, 3H), 5.30-5.34
(m, 1H), 6.08 (t, J ) 54.0 Hz, 1H), 7.06-7.15 (m, 2H), 7.20-
7.26 (m, 2H), 7.43 (t, J ) 7.9 Hz, 1H), 7.52-7.59 (m, 1H), 8.21
(d, J ) 7.6 Hz, 1H). MS m/z ) 453.19 (M + H+). Anal.
(C22H20F4N2O4•0.2 H2O) C, H, N.
Methyl 4′-[(1R)-1-({[1-(Acetylamino)cyclopropyl]carbonyl}-
amino)ethyl]-3,3′-difluoro-1,1′-biphenyl-2-carboxylate (12d). White
1
solid: 36%. H NMR (400 MHz, CD3OD) δ 0.98-1.06 (m, 2H),
1.39-1.49 (m, 2H), 1.50 (d, J ) 7.1 Hz, 3H), 2.02 (s, 3H), 3.69
(s, 3H), 5.27-5.35 (m, 1H), 7.05-7.15 (m, 2H), 7.20-7.26 (m,
2H), 7.45 (t, J ) 8.1 Hz, 1H), 7.52-7.59 (m, 1H), 8.10 (d, J ) 8.1
Hz, 1H). MS m/z ) 417.23 (MH+). Anal. (C22H22F2N2O4•0.7 H2O)
C, H, N.
Methyl 4′-((1R)-1-{[(1-{[Chloro(difluoro)acetyl]amino}cyclo-
propyl)carbonyl]amino}ethyl)-3,3′-difluoro-1,1′-biphenyl-2-car-
1
boxylate (12e). White solid: yield: 70%. H NMR (400 MHz,

Biphenylaminocyclopropane B1 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 279
CD3OD) δ 1.02-1.16 (m, 2H), 1.46-1.55 (m, 5H), 3.68 (s, 3H),
5.29 (q, J ) 7.1 Hz, 1H), 7.05-7.24 (m, 2H), 7.20-7.26 (m, 2H),
7.41 (t, J ) 7.9 Hz, 1H), 7.51-7.58 (m, 1H). MS m/z ) 487.14
(MH+). Anal. (C22H19ClF4N2O4•0.3 H2O) C, H, N.
Methyl 3′-Fluoro-4′-{(1R)-1-[({1-[(trifluoroacetyl)amino]-
cyclopropyl}carbonyl)amino]ethyl}-1,1′-biphenyl-2-carboxy-
late (13a). White solid: yield 22%. 1H NMR (400 MHz, CD3OD)
δ 1.02-1.16 (m, 2H), 1.47-1.56 (m, 5H), 3.64 (s, 3H), 5.32 (q, J
) 7.1 Hz, 1H), 6.97-7.06 (m, 2H), 7.35-7.40 (m, 2H), 7.46 (dot,
J ) 1.2, 7.6 Hz, 1H), 7.58 (dot, J ) 1.5, 7.6 Hz, 1H), 7.78 (dd, J
) 1.3, 7.6 Hz, 1H). HRMS Calcd for C22H24F4N3O4 (M+NH+):
470.1703. Found: 470.1694. Anal. (C22H20F4N2O4•0.5 H2O) C, H,
N.
Ethyl 3-Chloro-3′-fluoro-4′-{(1R)-1-[({1-[(trifluoroacetyl)-
amino]cyclopropyl}carbonyl)amino]ethyl}-1,1′-biphenyl-2-car-
boxylate (13c). White solid: yield 63%. 1H NMR (400 MHz,
CDCl3) δ 1.04-1.20 (m, 5H), 1.49 (J ) 7.1 Hz, 3H), 1.53-1.66
(m, 2H), 4.20 (q, J ) 7.1 Hz, 2H), 5.18-5.28 (m, 1H), 6.64 (d, J
) 8.3 Hz, 1H), 7.05-7.13 (m, 2H), 7.22-7.28 (m, 2H), 7.35-
7.45 (m, 3H). MS m/z ) 501.1 (MH+). Anal. (C23H21ClF4N2O4)
C, H, N.
White solid: yield 12%. 1H NMR (400 MHz, CDCl3) δ 0.93 (t, J
) 7.1 Hz, 3H), 1.07-1.20 (m, 2H), 1.49 (d, J ) 7.1 Hz, 3H), 1.56-
1.58 (m, 2H), 2.39 (q, J ) 7.1 Hz, 2H), 5.18-5.28 (m, 1H), 6.60
(d, J ) 8.1 Hz, 1H), 7.00-7.06 (m, 2H), 7.21-7.30 (m, 2H), 7.34-
7.44 (m, 2H). MS m/z ) 485.3 (MH+). HRMS Calcd for C23H21-
ClF4N2O3 (M + 1): 485.1242. Found: 485.1250.
Methyl 2-Fluoro-6-iodobenzoate (14a). To a solution of
2-fluoro-6-iodobenzoic acid (3.20 g, 12.0 mmol) in MeOH (25 mL)
was added (trimethylsilyl)diazomethane (2.0 M in hexanes, 9.02
mL, 18.0 mmol). The reaction was stirred at room temperature for
2 h and then concentrated in vacuo. The residue was partitioned
between CH2Cl2 and aqueous sodium bicarbonate. The organic layer
was washed with aqueous sodium bicarbonate and brine, dried over
Na2SO4, filtered, and concentrated. The residue was subjected to
silica gel chromatography (0-5% EtOAc in hexanes) to provide
1
14a (3.41 g, 100%) as a clear oil. H NMR (300 MHz, CDCl3) δ
3.98 (s, 3H), 7.10-7.15 (m, 2H), 7.65 (t, J ) 4.5 Hz, 1H). MS m/z
) 281.2 (MH+).
Ethyl 2-Fluoro-6-iodobenzoate (14b). A mixture 2-fluoro-6-
iodobenzoic acid (3.20 g, 12.0 mmol), potassium carbonate (1.14
g, 8.27 mmol), and iodoethane (1.76 g, 11.3 mmol) in DMF (5
mL) was stirred at room temperature for 3 h. The mixture was
partitioned between EtOAc and aqueous sodium bicarbonate, and
the organic extract was washed with water and brine, dried over
Na2SO4, filtered, and concentrated. The residue was subjected to
silica gel chromatography (0-5% EtOAc in hexanes) to provide
14b (2.11 g, 95.3%) as a yellow oil. 1H NMR (300 MHz, CDCl3)
δ 1.42 (t, J ) 7.1 Hz, 3H), 4.45 (q, J ) 7.1 Hz, 2H), 7.08-7.12
(m, 2H), 7.62-7.65 (m, 1H). MS m/z ) 295.0 (MH+).
N-{(1R)-1-[3,3′-Difluoro-2′-(3-methyl-1,2,4-oxadiazol-5-yl)-
1,1′-biphenyl-4-yl]ethyl}-1-[(trifluoroacetyl)amino]cyclopro-
1
panecarboxamide (13d). White solid: yield 78%. H NMR (400
MHz, CD3OD) δ 1.01-1.29 (m, 2H), 1.45-1.51 (m, 5H), 2.36 (s,
3H), 5.27 (q, J ) 7.1 Hz, 1H), 6.91-6.95 (m, 2H), 7.30-7.40 (m,
3H), 7.70-7.77 (m, 1H). HRMS Calcd for C23H20F5N4O3 (M +
1): 495.1455. Found: 495.1460. Anal. (C23H19F5N4O3•0.6CH2Cl2)
C, H, N.
N-{(1R)-1-[3,3′-Difluoro-2′-(5-methyl-1,2,4-oxadiazol-3-yl)-
1,1′-biphenyl-4-yl]ethyl}-1-[(trifluoroacetyl)amino]cyclopro-
5-(2-Fluoro-6-iodophenyl)-3-methyl-1,2,4-oxadiazole (14c). To
a solution of 2-fluoro-6-iodobenzoic acid (15.0 g, 56.4 mmol) in
CH2Cl2 (150 mL) containing DMF (0.1 mL) was added oxalyl
chloride (9.30 g, 73.3 mmol) dropwise. The solution was stirred at
room temperature for 75 min and then concentrated in vacuo. The
residue was redissolved in 150 mL of CH2Cl2, and the solution
was saturated 3 times with ammonia gas. The solution was
concentrated in vacuo and dried under vacuum overnight. The
residue was dissolved in N,N-dimethylacetamide dimethyl acetal
(24.7 mL, 0.169 mol) and heated to 120 °C for 5 h. Additional
N,N-dimethylacetamide dimethyl acetal (25 mL, 0.17 mol) was
added over the course of the reaction to drive it to completion.
The solution was cooled to room temperature, concentrated in
vacuo, and dried under vacuum overnight. To a solution of the crude
material in dioxane (57 mL) was added hydroxylamine hydrochlo-
ride (4.70 g, 67.7mmol), 5 N NaOH (13.5 mL, 67.7 mmol), and
70% acetic acid (57 mL). The mixture was stirred at 60 °C for 2
h and then at 90 °C for 3 h. The resulting solution was cooled to
room temperature, diluted with EtOAc, and neutralized with
aqueous sodium bicarbonate. The organic layer was washed with
aqueous sodium bicarbonate and brine, dried over Na2SO4, filtered,
and concentrated. The residue was filtered through silica gel (10%
EtOAc in hexanes) to provide 14g (8.1 g, 47%) as orange yellow
1
panecarboxamide (13e). Off-white solid: yield 45%. H NMR
(400 MHz, CD3OD) δ 1.07-1.18 (m, 2H), 1.17 (d, J ) 7.0 Hz,
3H), 1.60-1.67 (m, 2H), 2.59 (s, 3H), 5.16-5.21 (m, 1H), 6.56
(d, J ) 8.2 Hz, 1H), 6.94-6.99 (m, 3H), 7.15 (t, J ) 7.8 Hz, 1H),
7.20-7.24 (m, 2H), 7.51-7.56 (m, 1H). MS m/z ) 495.3 (MH+).
Anal. (C23H19F5N4O3•0.2CH2Cl2) C, H, N.
N-{(1R)-1-[3,3′-Difluoro-2′-(2-methyl-2H-tetraazol-5-yl)-1,1′-
biphenyl-4-yl]ethyl}-1-[(trifluoroacetyl)amino]cyclopropanecar-
1
boxamide (13f). White solid: yield 44%. H NMR (400 MHz,
CD3OD) δ 1.01-1.18 (m, 2H), 1.44 (d, J ) 7.1 Hz, 3H), 1.49 (d,
J ) 3.7 Hz, 2H), 4.86 (s, 3H), 5.23 (d, J ) 7.1 Hz, 1H), 6.83-
6.89 (m, 2H), 7.21-7.34 (m, 3H), 7.61-7.68 (m, 1H). MS m/z )
495.32 (MH+). Anal. (C22H19F5N6O2•0.2 CH3CO2C2H5) C, H, N.
N-{(1R)-1-[3′-Chloro-3-fluoro-2′-(2-methyl-2H-tetrazol-5-yl)-
biphenyl-4-yl]ethyl}-1-[(trifluoroacetyl)amino]cyclopropanecar-
1
boxamide (13g). White foam: yield 84%. H NMR (400 MHz,
CDCl3) δ 1.04-1.15 (m, 2H), 1.42 (d, J ) 7.0 Hz, 3H), 1.51-
1.62 (m, 2H), 4.33 (s, 3H), 5.14 (q, J ) 7.5 Hz, 1H), 6.59 (d, J )
8.2 Hz, 1H), 6.83 (m, 2H), 7.06 (t, J ) 7.8 Hz, 1H), 7.33 (d, J )
7.5 Hz, 1H), 7.48-7.56 (m, 3H). HRMS Calcd for C22H20ClF4N6O2
(M + 1): 511.1272. Found: 511.1276.
1
N-{(1R)-1-[3,3′-Difluoro-2′-(1-methyl-1H-tetraazol-5-yl)-1,1′-
biphenyl-4-yl]ethyl}-1-[(trifluoroacetyl)amino]cyclopropanecar-
crystals. H NMR (300 MHz, CDCl3) δ 2.55 (s, 3H), 7.19-7.30
(m, 2H), 7.79 (d, J ) 6.3 Hz, 1H). MS m/z ) 305.06 (MH+).
5-(2-Fluoro-6-iodophenyl)-2-methyl-2H-tetraazole (14d) and
5-(2-Fluoro-6-iodophenyl)-1-methyl-1H-tetraazole (14e). A solu-
tion of commercially available (Aldrich) 2-fluoro-6-iodobenzonitrile
(17.8 g, 72.2 mmol) and azidotrimethyltin (15.0 g, 72.9 mmol) in
toluene (150 mL) was heated to 125 °C for 72 h. The solution was
cooled to room temperature and partitioned between EtOAc and
0.5 N HCl. The organic layer was washed with water and brine,
dried over Na2SO4, filtered, and concentrated to provide 5-(2-fluoro-
6-iodophenyl)-1H-tetraazole as a white solid. A mixture of 5-(2-
fluoro-6-iodophenyl)-1H-tetraazole (20.0 g, 81.0 mmol), potassium
carbonate (16.1 g, 0.113 mol), and iodomethane (16.1 g, 0.113 mol)
in DMF (25 mL) was stirred at room temperature for 3 h. The
mixture was partitioned between EtOAc and water, and the organic
extract was washed with water and brine, dried over Na2SO4,
filtered, and concentrated. The residue was subjected to silica gel
chromatography (0-10% EtOAc in hexanes) to provide 14d (3.93
g, 26.2%) as a white solid and 14e (9.77 g, 39.7%) as a yellow
1
boxamide (13h). White solid: yield 22%. H NMR (400 MHz,
CD3OD) δ 1.04-1.19 (m, 2H), 1.43 (d, J ) 7.1 Hz, 3H), 1.53-
1.65 (m, 2H), 3.70 (s, 3H), 5.14-5.19 (m, 1H), 6.61 (d, J ) 8.0
Hz, 1H), 6.76-6.87 (m, 2H), 7.14 (t, J ) 7.8 Hz, 1H), 7.28-7.36
(m, 2H), 7.64-7.71 (m, 2H). MS m/z ) 495.21 (MH+). Anal.
(C22H19F5N6O2•0.1 CH2Cl2) C, H, N.
N-{(1R)-1-[3,3′-Difluoro-2′-(trifluoromethyl)-1,1′-biphenyl-4-
yl]ethyl}-1-[(trifluoroacetyl)amino]cyclopropanecarboxamide (13i).
White solid: yield 79%. 1H NMR (400 MHz, DMSO-d6) δ 0.942-
1.06 (m, 2H), 1.34-1.40 (m, 2H), 1.42 (d, J ) 7.1 Hz, 3H), 5.22-
5.29 (m, 1H), 7.13 (d, J ) 8.1 Hz, 1H), 7.17 (d, J ) 11.2 Hz, 1H),
7.25 (d, J ) 7.7 Hz, 1H), 7.44 (t, J ) 8.0 Hz, 1H), 7.52-7.57 (m,
1H), 7.73-7.79 (m, 1H), 8.29 (d, J ) 8.1 Hz, 1H), 9.80 (s, 1H).
HRMS Calcd for C21H16F8N2O2 (M + 1): 418.1157. Found:
481.1173.
N-[(1R)-1-(3′-Chloro-3-fluoro-2′-propionyl-1,1′-biphenyl-4-
yl)ethyl]-1-[(trifluoroacetyl)amino]cyclopropanecarboxamide (13j).

280 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Kuduk et al.
solid. 14d: 1H NMR (300 MHz, CDCl3) δ 4.48 (s, 3H), 7.18-
7.24 (m, 2H), 7.77-7.80 (m, 1H). MS m/z ) 305.05 (MH+). 14e:
1H NMR (300 MHz, CDCl3) δ 3.97 (s, 3H), 7.27-7.40 (m, 2H),
7.82-7.85 (m, 1H). MS m/z ) 305.05 (MH+).
were then washed 4 times with a dye-free salt solution containing
probenecid, and then 100 µL of salt solution with 1 mM probenecid
was added to each well. Des-arg10 kallidin-induced elevation of
cytosolic calcium was determined using a Fluorescence Imaging
Plate Reader (FLIPR, Molecular Devices Corp., Sunnyvale, CA).
All assays were conducted at 37 °C. Antagonist was added to the
appropriate wells in a volume of 50 µL of Hank’s solution 2 min
prior to the addition of 3 nM des-arg10 kallidin in a 50 µL volume.
Changes in cellular fluorescence due to increased cytosolic calcium
ion concentrations in response to agonist were determined using
an excitation wavelength of 488 nm and a 510-570 nm bandwidth
emission filter. Curve fitting and IC50 calculations were performed
using GraphPad Prism software. At least eight concentrations of
antagonist were used to generate each inhibition curve.
Transepithelial Transport Assay of P-gp. A transepithelial
transport study was conducted as described27 with minor modifica-
tions. The cells were maintained in M199 media supplemented with
10% FBS. The cells at density of 5 × 104 cells/mL were plated
onto a 96-well filter with 150 µL/well (pore size 3.0 µm, 0.11 cm2
surface areas; Millipore Corp., Bedford, MA). Cells were supple-
mented with fresh media on the third day and used for the transport
studies on the fourth day after plating. The concentrations of all
test compounds were at 5 µM unless otherwise indicated. Verapamil
at a 1 µM concentration was used as a marker Pgp substrate for a
positive control (Sigma-Aldrich, St. Louis, MO). Hanks’ Balanced
Salt Solution (HBSS, Gibco-Invitrogen Corporation, Carlsbad, CA),
a serum/protein free medium, was used through the experiments
to eliminate any differences in protein binding among the com-
pounds tested. Before the addition of testing compounds, the media
were first replaced with HBSS media containing 1 mM HEPES to
equilibrate cells into experimental conditions for 30 min. The
transport experiment was then initiated by replacing the medium
in each compartment with 150 µl of fresh HBSS with or without
the test compound. After a 3- h incubation, 100 µL aliquots were
taken from the opposite compartment as receiver samples and from
the loading compartment as donor samples. The samples of the
radioactive compounds were placed in scintillation vials containing
5 mL of scintillation cocktail, and the total radioactivity was
measured by a liquid scintillation counter. The appearance of
radioactivity in the opposite compartment was presented as a
fraction of the total radioactivity added at the beginning of the
experiment. Directional transport was measured in triplicate and
presented as mean ( SD. The samples of unradiolabeled test
compounds were analyzed by LC/MS/MS.
African Green Monkey Brain/Plasma Experiments. The test
compound was prepared in 100% PEG 200 and administered iv as
a bolus injection at 2 mg/kg to conscious African green monkeys
(n ) 2). A pre-dose sample of blood was taken as well as samples
at 15 and 30 min post-dosing for determination of plasma drug
concentrations. At 30 min post-dose, the animal was euthanized
with pentobarbital, and the brain and spinal cord were removed.
Several brain and spinal cord regions were dissected, frozen in liquid
nitrogen, and stored at -70 °C until analysis by LC/MS/MS.
Rhesus LPS Model. Rhesus monkeys (male or female, 5.5-
10.3 kg) were fasted overnight, placed in primate restraint chairs,
and transported to the laboratory. Conscious monkeys were treated
with lipopolysaccharide (LPS, 20 µg/kg of iv bolus) and allowed
2 h for hemodynamic equilibration and B1 receptor expression. The
baseline depressor response to the B1 receptor agonist, des-Arg10-
kallidin (DAK, 0.3 µg/kg of iv bolus), was then measured twice
with a 20 min recovery period between measurements. Pilot studies
established that the depressor response to DAK remained consistent
(20-23 mmHg decrease in mean arterial pressure, MAP) in this
model when measured at 20 min intervals over 10 challenges. The
dose-response effects of the B1 receptor antagonist were evaluated
with increasing iv bolus doses (3, 10, 30, and 100 µg/kg) given 5
min prior to each subsequent DAK administration. Following the
final test of antagonist effects on DAK-induced MAP decrease,
DAK challenges were repeated 4 times at 20 min intervals to
investigate recovery to baseline DAK response. Blood samples were
taken at 2 min after each iv dose to determine plasma concentration
3-(2-Fluoro-6-iodophenyl)-5-methyl-1,2,4-oxadiazole (14f). A
mixture of 2-fluoro-6-iodobenzonitrile (12.4 g, 50.2 mmol), hy-
droxylamine hydrochloride (4.53 g, 65.2 mmol), and ethanol (50
mL) was stirred vigorously. To this reaction mixture was added
sodium tert-butoxide (7.23 g, 75.3 mmol). The reaction was refluxed
at 100 °C for 5 h. Additional hydroxylamine hydrochloride (4.53
g, 65.2 mmol) and sodium tert-butoxide (7.23 g, 75.3 mmol) were
added over the course of the reaction to drive it to completion.
The reaction was concentrated with ethanol under vacuum. The
crude residue was dissolved in CH2Cl2 and washed with 1 N HCl.
The aqueous layer was extracted with CH2Cl2 twice, basified with
ammonium hydroxide slowly (pH ∼9), and re-extracted with CH2-
Cl2 3 times. The organic layers were combined, dried over Na2-
SO4, filtered, and concentrated in vacuo. The residue was subjected
to silica gel chromatography (0-50% EtOAc in hexane) to afford
2-fluoro-N-hydroxy-6-iodobenzenecarboximidamide as a yellow oil.
1H NMR (400 MHz, CDCl3) δ 7.67 (t, J ) 4.5 Hz, 1H), 7.12 (m,
2H), 4.78 (bs, OH). MS m/z ) 281.4 (MH+). The 2-fluoro-N-
hydroxy-6-iodobenzenecarboximidamide (2.14 g, 7.64 mmol) was
dissolved in N,N-dimethylacetamide dimethyl acetal (1.12 mL, 7.64
mmol). After 1 h and 15 min of stirring at room temperature, the
reaction solution was concentrated in vacuo. The residue was
subjected to silica gel chromatography (0-15% EtOAc in hexane)
1
to provide 14f (1.36 g, 59%) as a clear oil. H NMR (300 MHz,
CDCl3) δ 7.73 (t, J ) 4.3 Hz, 1H), 7.16-7.20 (m, 2H), 2.70 (s,
3H). MS m/z ) 305.1 (MH+).
Biological Methods. Receptor Binding Assays. Radioligand
binding assays were performed using membranes from CHO cells
that stably express the human or rat bradykinin B1 receptors or
CHO cells that express the human bradykinin B2 receptor. For all
receptor types, cells were harvested from culture flasks in PBS/1
mM EDTA and centrifuged at 1000g for 10 min. The cell pellets
were homogenized with a polytron in ice-cold 20 mM HEPES, pH
7.4 and 1 mM EDTA (lysis buffer) and centrifuged at 50 000g for
20 min. The membrane pellets were rehomogenized in lysis buffer
and centrifuged again at 50 000g, and the final pellets were
resuspended at 5 mg of protein/mL in an assay buffer (20 mM
HEPES, pH 7.4, 120 mM NaCl, 5 mM KCl) supplemented with
1% BSA and frozen at -80 °C.
On the day of assay, membranes were centrifuged at 14 000g
for 5 min and resuspended to the desired protein concentration (0.2
mg/mL for typical membrane preparations) in assay buffer contain-
ing 100 nM enaliprilat, 140 µg/mL bacitracin, and 0.1% BSA. 3H-
des-arg10 leu9 kallidin (0.2 nM) was the radioligand used for the
3
human bradykinin B1 receptors; H-des-arg10 kallidin (1 nM) was
used for the rat bradykinin B1 receptors; and 3H-bradykinin (1 nM)
was used to label the human bradykinin B2 receptor.
For all assays, compounds were diluted from DMSO stock
solutions with 4 µL added to assay tubes for a final DMSO
concentration of 2%. This was followed by the addition of 100 µL
of the radioligand and 100 µL of the membrane suspension.
Nonspecific binding for the bradykinin B1 receptor binding assays
was determined using 1 µM des-arg10 kallidin, while nonspecific
binding for the bradykinin B2 receptor was determined with 1 µM
bradykinin. Tubes were incubated at room temperature (22 °C) for
60 min followed by filtration using a Tomtec 96-well harvesting
system. Radioactivity retained by the filter was counted using a
Wallac beta-plate scintillation counter.
CHO Human and Bradykinin B1 FLIPR Protocol. CHO cells
engineered to stably express the human or rat bradykinin B1 receptor
were seeded at a density of 25 000 cells per well in a 96-well plate
in 200 µL of cell culture media (Iscove’s modified DMEM
containing 1 mg/mL G418 and 10% heat inactivated fetal calf
serum). After overnight incubation at 37 °C, the cell plates were
washed twice with Hank’s buffered salt solution, and the cells were
incubated for 60 min at 37 °C with Hank’s solution containing 4
µM fluo-3 acetoxymethyl ester and 1 mM probenecid. The cells

Biphenylaminocyclopropane B1 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 281
of test compounds. Crossover studies were conducted with test
compound versus vehicle in the same monkeys (n ) 3), and a
minimum of 2 weeks was allowed between limbs of the study.
Supporting Information Available: Microsomal/hepatocyte
stability data, CF-1 mouse data, CFA wt mouse data, and PK
procedures. This material is available free of charge via the Internet
at http://pubs.acs.org.
Ex Vivo Occupancy Experiments. Transgenic hB1 rats were
dosed intravenously by infusion over a 30 min period at various
doses with test compound and sacrificed. In time course studies,
animals were also sacrificed at 150 and 600 min following the end
of infusion. The test compound was also dosed orally, and rats were
sacrificed from 0.5 to 6 h post-dose. Samples of brain and cord
were quickly removed and frozen for use in the ex vivo occupancy
assay, while a second set of tissue samples and a plasma samples
was frozen for LCMS determination of test compound levels. For
the ex vivo assay, approximately 35 mg of cord or brain was
homogenized in 2000 volumes of ice-cold assay buffer (20 mM
HEPES, 120 mM NaCl, 5 mM KCl, pH 7.4) and centrifuged at
75 000g for 10 min. The pellets were resuspended in ice-cold buffer
at a concentration of 5 mg of tissue/mL and 50 µL aliquots were
rapidly distributed to tubes with 0.5 mL of room-temperature buffer
containing 25pM [35S] 2-{(2)-1-[(3,4-dichlorophenyl)sulfonyl]-3-
oxo-1,2,3,4-tetrahydroquinoxalin-2-yl}-N-{2-[4-(4,5-dihydro-1-imi-
dazol-2-yl)phenyl]ethyl}acetamide. At 2, 4, 6, 8, and 10 min
following membrane addition, incubations were terminated by
filtration of three tubes over glass fiber filters. A parallel set of
incubations performed in the presence of 100 nM unlabeled
compound X was used to determine nonspecific radioligand binding
at each time point. Radioactivity on the filters was determined by
liquid scintillation counting, and [35S] 2-{(2)-1-[(3,4-dichlorophe-
nyl)sulfonyl]-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl}-N-{2-[4-
(4,5-dihydro-1-imidazol-2-yl)phenyl]ethyl}acetamide rates of as-
sociation for vehicle and test compound were determined by linear
regression. Receptor occupancy in a drug treated animal was
calculated as: % occupancy ) (1 - (slopedrug/slopevehicle)) × 100.
The brain and spinal cord concentrations of the test compound
required to achieve 90% receptor occupancy (Occ 90%) were
derived by nonlinear curve fitting using Prism software.
CFA Model of Inflammatory Pain. Male and female humanized
B1 mice and wild-type mice (8-13 weeks) were obtained from
Charles River Labs. All animals were maintained in a climate-
controlled room on a 12 h/12 h light/dark cycle with free access to
food and water. All procedures were approved by the Institutional
Animal Care and Use Committee (IACUC) at Merck and Co., Inc.,
West Point, PA. Baseline tactile thresholds to von Frey filaments
were measured by applying a series of calibrated von Frey filaments
(Stoelting, Wood Dale, IL) to the plantar aspect of the left hind
paw and determining the median withdrawal threshold (grams) using
the Dixon up-down method.28 Inflammation was induced by
injection of 30 µL of 50% complete Freund’s adjuvant (CFA) (1:1
solution of complete and incomplete Freund’s adjuvant, Sigma, St.
Louis, MO) subcutaneously into the plantar surface of the hind paw.
Twenty-four hours following CFA injection, mice were tested for
tactile hypersensitivity using von Frey filaments. Mice were then
administered either drug (6, 20, or 60 mg/kg, po) or vehicle (0.5%
methyl cellulose with 10 mg/mL of malic acid, pH ) 4, 10 mL/
kg, po), and paw withdrawal thresholds were recorded at 60 min
post-administration. Following testing at 60 min, brain and plasma
samples were collected for analysis. Dose-response curves were
compared by two-way repeated measures ANOVA (time × dose)
with post-hoc Tukey’s test (SigmaStat). Significance was defined
as p < 0.05. CFA-induced inflammation produced a significant
decrease in the paw withdrawal thresholds of both wild-type and
hB1 knock-in mice when measured 24 h after CFA injection (Figure
2). The paw withdrawal thresholds of CFA mice were unaffected
by vehicle treatment. Compound 13b dose dependently reversed
the hypersensitive paw withdrawal thresholds in hB1 knock-in mice
(ED50 ) 9.76 mg/kg; 95% CL 6.05-15.74) but not wild-type mice
(see Supporting Information).
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log P measurements and Joan S. Murphy for determination of
high resolution mass spectra.

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