Design and optimization of benzimidazole-containing transient receptor potential melastatin 8 (TRPM8) antagonists

Article abstract of DOI:10.1021/jm101075v

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that is thermoresponsive to cool to cold temperatures (8-28 ° C) and also may be activated by chemical agonists such as menthol and icilin. Antagonism of TRPM8 activation i

Full text of DOI:10.1021/jm101075v

J. Med. Chem. 2011, 54, 233–247 233
DOI: 10.1021/jm101075v
Design and Optimization of Benzimidazole-Containing Transient Receptor Potential Melastatin 8
(TRPM8) Antagonists
Daniel J. Parks, William H. Parsons, Raymond W. Colburn, Sanath K. Meegalla, Shelley K. Ballentine, Carl R. Illig, Ning Qin,
Yi Liu, Tasha L. Hutchinson, Mary Lou Lubin, Dennis J. Stone, Jr., Judith F. Baker, Craig R. Schneider, Jianya Ma,
Bruce P. Damiano, Christopher M. Flores, and Mark R. Player*
Janssen Research and Development, Welsh and McKean Roads, Spring House, Pennsylvania 19477-0776, United States
Received August 18, 2010
Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that is thermo-
responsive to cool to cold temperatures (8-28 °C) and also may be activated by chemical agonists such
as menthol and icilin. Antagonism of TRPM8 activation is currently under investigation for the
treatment of painful conditions related to cold, such as cold allodynia and cold hyperalgesia. The design,
synthesis, and optimization of a class of selective TRPM8 antagonists based on a benzimidazole scaffold
is described, leading to the identification of compounds that exhibited potent antagonism of TRPM8 in
cell-based functional assays for human, rat, and canine TRPM8 channels. Numerous compounds in the
series demonstrated excellent in vivo activity in the TRPM8-selective “wet-dog shakes” (WDS) pharmaco-
dynamic model and in the rat chronic constriction injury (CCI)-induced model of neuropathic pain. Taken
together, the present results suggest that the in vivo antagonism of TRPM8 constitutes a viable new
strategy for treating a variety of disorders associated with cold hypersensitivity, including certain types
of neuropathic pain.
Introduction
pathologic conditions.5 Pain hypersensitivity may manifest in
two forms: allodynia, wherein a normally innocuous stimulus
triggers a pain response, and hyperalgesia, wherein a noxious
stimulus results in a prolonged or more intense response to
pain. Allodynia to cold is prevalent in forms of neuropathic
pain such as that arising from traumatic neuropathy, or fibro-
myalgia, whereas cold hyperalgesia is a common manifestation
of chronic inflammation, such as occurs in rheumatoid ar-
thritis, although many conditions can present both types of
hypersensitivity disorder.6 Thus, TRPM8 antagonists repre-
sent a novel and potentially useful approach to the treatment
of these disorders for which there continues to be a high unmet
medical need.7 Indeed, numerous patents8 and journal articles9
have recently been disclosed in the literature purporting the
utility of TRPM8 antagonists in the treatment of various
disease states such as urological disorders, asthma, COPD,
prostate and colon cancers, and pain. A diverse range of scaf-
folds have been employed as TRPM8 antagonists, among
them are menthylamides, benzyloxybenzylamides, benzyloxy-
benzamides, alkylphosphonate esters, arylacetamides, and
fused amino-oxazoles and thiazoles.
Transient receptor potential melastatin 8 (TRPM8a) be-
longs to the melastatin subgroup of the transient receptor
potential (TRP) channel superfamily. TRP channels are non-
selective cation channels activated by a variety of chemical
and physical stimuli. A subset of the TRP channel superfamily
is thermoresponsive, each receptor being activated over a discrete
temperature range, cumulatively spanning from noxious cold to
noxious hot. Specifically, TRPM8 is stimulated by cool to cold
temperatures (8-28 °C) and by chemical agonists such as
menthol and icilin.1
TRPM8 is expressed in a range of tissues predominately in
the prostate and liver and, to a lesser degree, in brain, lung,
bladder, gastrointestinal tract, blood vessels, and immune
cells.2,3 In addition, TRPM8 has been localized in the dorsal
root and trigeminal ganglia to subsets of primary sensory
neurons (Aδ and C fibers) which are largely distinct from
TRPV1/TRPA1 expressing populations.1,4 Native and induced
expression of TRPM8 on multiple neuronal subtypes provides a
rationale for the observed cold hypersensitivity in numerous
In this paper, we describe the preparation of heteroatom-
substituted spiro[4,5]dec-2-enes substituted at the 2-position
of a benzimidazole core (Figure 1) and the lead optimization
studies that culminated in the preparation of 5, an orally
bioavailable, potent, and selective TRPM8 antagonist exhibiting
antiallodynic properties in in vivo pain models.
*To whom correspondence should be addressed. Phone: 215-628-7860.
E-mail: mplayer@its.jnj.com.
a Abbreviations: TRPM8, transient receptor potential melastatin 8;
LM, liver microsomes; MTCA, methyl trichloroacetamidate; ECHA,
ethyl chlorohydroximinoacetate; MTP, methylenetriphenylphosphorane;
DMBB, 3,3-dimethylbenzo[c][1,2]oxaborol-1(3H)-ol; pyBrOP, bromotri-
pyrrolidinophosphonium hexafluorophosphate; WDS, “wet-dog” shakes;
CCI, chronic constriction injury; FLIPR, fluorometric imaging plate reader;
FDSS, functional drug screening system; NMR, nuclear magnetic reso-
nance; TMS, tetramethylsilane; TLC, thin layer chromatography; HPLC,
high pressure liquid chromatography; CAM, ceric ammonium molybdate;
CSA, 10-camphorsulfonic acid.
Chemistry
The synthesis of target compounds 1-3 is illustrated in
Scheme 1. Reduction of the biphenylnitroaniline 6 (vide infra)
r
2010 American Chemical Society
Published on Web 12/03/2010
pubs.acs.org/jmc

234 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Parks et al.
Scheme 2a
Figure 1. Prototypical structures of the spiro-oxazolines (1 and 2),
spiro-imidazoline (3), and spiro-isoxazoline (4) substituted benzimid-
azole ring systems and the structure of optimized TRPM8 antagonist 5.
a Reagents and conditions: (a) NO2CH2CO2Et, DABCO, EtOH, 80 °C,
42 h; (b) “Ph3PdCH2”,22 Et2O, rt 16 h; (c) ECHA, DIPEA, DCM, rt,
24 h; (d) LiOH, MeOH, H2O, rt, 16 h; (e) (COCl)2, DCM, cat. DMF, rt, 1 h.
Scheme 1a
Scheme 3a
a Reagents and conditions: (a) H2, 10% Pd on C, EtOH, rt, 15 h;
(b) MTCA, AcOH, rt, 14 h; (c) (1-aminomethyl)cyclohexanol hydro-
chloride, NaOH, H2O, dioxane, rt, 14 h; (d) 1-aminomethyl-1-cyclo-
hexylamine, THF, TEA, rt, 1 h; (e) (1-aminocyclohexyl)methanol,
H2O, dioxane, rt, 14 h.
was performed by catalytic hydrogenation to yield biphenyl-
diamine 7. Reaction of 7 with methyl trichloroacetamidate
(MTCA) afforded 2-trichloromethylbenzimidazole 8 as a key
intermediate.10 Treatment of 8 with the appropriate amino
alcohol or diamine produced 1-3.
a Reagents and conditions: (a) (dppf)PdCl2 DCM, DME, 2 M aq
3
Na2CO3, 90 °C; (b) ArX, (dppf)PdCl2 DCM, DME, 2 M aq Na2CO3,
3
90 °C. Bpin = 4,4,5,5-tetramethyl-1,3,2-dioxaborolyl.
The spiro-isoxazoline-substituted benzimidazole series was
synthesized in a convergent manner through the assembly of
the benzimidazole five-membered ring in the final step. The
key intermediates for this step were the isoxazoline carboxylic
acid chlorides 18-20 (Scheme 2) and the biphenylnitro-
anilines 6, 34-47, 49 (Schemes 3 and 4).
The spiro-isoxazoline ring system was synthesized via a [3 þ 2]
dipolar cycloaddition between the in situ generated nitrile
oxide and exo-methylenecycloalkyl (Scheme 2). Isoxazoline
ester 1211 was prepared from commercially available ethyl
nitroacetate in which the reactive dipolarophile is generated
via dehydration, whereas esters 13 and 14 were prepared by
the base-induced elimination of HCl from ethyl chlorohydrox-
iminoacetate (ECHA). Saponification of the esters gave the
corresponding carboxylic acids 15-17, which were treated
with oxalyl chloride to afford the isoxazoline carboxylic acid
chlorides 18-20.
instances, the boronic acid or ester of the substituted aryl
was not commercially available, so the coupling partners were
reversed. In this case, the aminonitrophenylboronate ester 28
was used for the Suzuki coupling with the aryl halides 29 and
30toafford biphenylnitroanilines36and37, respectively. This
coupling strategy was also employed for the 2,6-disubstituted
aryl bromides and iodides 31-33 because better yields of
biphenylnitroanilines 38-40 were obtained.
Biphenylnitroaniline6washalogenatedaccordingtoScheme4
to afford 46-48. Treatment of iodide 48 with CuCN afforded
the nitrile 49. Silyl ether 52 was prepared via a Sonagashira
coupling reaction between 48 and propargyl alcohol to afford
acetylene 50. Hydrogenation of 50 produced 51, which was
subsequently protected with TBSCl to afford silyl ether 52
(Scheme 4).
The benzimidazole core was assembled via the two general
routes outlined in Scheme 5. In the first method (path A), the
biphenylnitroaniline was reduced to the biaryldiamine and used
without purification. Acylation with one of the acid chlorides
Biphenylnitroanilines 6, 34-45 were prepared by Suzuki
coupling from the 4-halo-2-nitroanilines 21-23 and 2-
substituted phenylboronic acids 24-27 (Scheme 3). In some

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 235
Scheme 6a
Scheme 4a
a Reagents and conditions: (a) 18, TEA, DCM, rt, 1 h; (b) dioxane,
a Reagents and conditions: (a) NCS, ACN, 80 °C, 72h;(b) Br2, AcOH, rt,
30 min; (c) I2, Ag2SO4, EtOH, rt, 24 h; (d) CuCN, DMA, 140 °C, 14 h;
(e) propargyl alcohol, (Ph3P)2PdCl2, CuI, THF, TEA, rt, 16 h; (f) H2,
10% Pd on C, EtOH, rt, 16 h; (g) TBSCl, imidazole, DCM, rt, 2 h.
CSA, 100 °C, 3 h; (c) DMBB or 27, (dppf)PdCl2 DCM, DME, 2 M aq
3
Na2CO3, 90 °C.
Results and Discussion
Scheme 5a
TRPM8 in vitro assays used to assess the functional
inhibitory activity of test compounds utilized either a Fluoro-
metric Imaging Plate Reader (FLIPR) manufactured by
Molecular Devices or a Functional Drug Screening System
(FDSS) manufactured by Hamamatsu. These devices mea-
sured icilin-induced increases in intracellular calcium concen-
tration by monitoring changes in fluorescent signal using a
Ca2þ-sensitive fluorescent dye in HEK293 cells that stably or
transiently express canine, human, or rat TRPM8 channels.13
Functional activity of new compounds were assessed in the
canine TRPM8 Ca2þ flux assay (cTRPM8), and the IC50
values obtained or the percent inhibition at a test concentra-
tion of 0.2 μM are shown in Tables 1-4. The metabolic
stability of the compounds were assayed in rat, human, and
dog liver microsomal (RLM, HLM, and DLM, respectively)
preparations and are reported as percent remaining after 10
min of incubation.
Compounds that were relatively potent in the cTRPM8
functional assay and stable in the liver microsomal prep-
arations were assessed in the “wet-dog” shakes (WDS) phar-
macodynamic (PD) model in rats. Rats exhibit shaking and
jumping behaviors in response to icilin, a relatively selective
and potent TRPM8 agonist.14 These behaviors are TRPM8-
mediated, as they cannot be reproduced in TRPM8-null mice.5b
Pretreatment of the rats with a TRPM8 antagonist prior to
icilin administration inhibits the observed shaking behavior,
providing a convenient PD assessment of the test compounds.
Thus, compounds were tested at a standard dose of 10 mg/kg,
and the percent inhibition of WDS relative to vehicle-treated
animals was assessed over time.
a Reagents and conditions: (a) Fe, HCl or H2, 10% Pd on C, EtOH;
(b) (i) 18-20, TEA, DCM, rt, (ii) CSA, dioxane, 100 °C; (c) (i) NaH,
THF, (ii) 18-20; (d) Fe, AcOH, 80 °C.
18-20 produced a mixture of monoamides and a minor
amount of the diacylated byproduct, which was separated
from the monoamide mixture prior to cyclization. The pro-
duction of the diacylated byproduct could be minimized through
the use of a slight excess of the biarylamine and employing a
slow addition of the acid chloride solution to the reaction
mixture. The monoamide mixture was cyclized under acid
catalysis to afford the corresponding benzimidazole.
In the alternate synthesis (Scheme 5, path B), the biphe-
nylnitroaniline was deprotonated withthe strong base NaH to
afford the sodium anilide salt, which was subsequently treated
with one of the acid chlorides 18-20 to produce the corre-
sponding biphenylnitroamide. Reduction of the nitro group
with iron in acetic acid produced the desired benzimidazole in
a one-pot reduction/acid-catalyzed cyclization cascade.
The presence of the acid-sensitive tertiary alcohol in benz-
imidazole 75 necessitated the reversal of the reaction sequence
(Scheme 6). Bromobenzimidazole 73 was prepared by acyla-
tion of 4-bromobenzene-1,2-diamine (72) with 18 followed
by acid-catalyzed cyclization. Subsequent Suzuki coupling
with 3,3-dimethylbenzo[c][1,2]oxaborol-1(3H)-ol12 (DMBB)
afforded the desired product 75. Benzimidazole 74 was also
prepared using the same synthetic sequence except using
boronic acid 27.
Compounds that displayed efficacy in the PD model were
evaluated in the rat chronic constriction injury (CCI) model of
neuropathic pain. In this model, loose ligatures are surgically
placed around the sciatic nerve, resulting in the development of
hypersensitivity in the affected limb for up to several weeks.15
Treatment of the hind paw with cold or substances that evoke
cold sensations, such as evaporating acetone, results in exagger-
ated paw lifting and licking behaviors. Test compounds were
orally administered to the rats, and the paw lifting/licking
behaviors were monitored over time. The average number of
paw lifting/licking events compared to vehicle-treated controls
was monitored over time. A reduction in the average number of
events (expressed as percent inhibition) is indicative of an
antiallodynic effect as a result of the administered compound.
Initially, four potential benzimidazole-containing scaffolds,
two oxazoline regioisomers, an imidazoline, and an isoxazoline,

236 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Parks et al.
Table 1. Functional Activity of the Heteroatom-Substituted Spiro Ring
Systems
Table 2. SAR for Substitution on the 5-Phenyl Ring
a IC50 values or % inhibition at a test concentration of 0.2 μM
averaged from four determinations (n = 4). b Percent remaining after
10 min incubations in human (HLM), rat (RLM), or dog (DLM) liver
microsomal preparations.
were assessed for functional antagonism of cTRPM8. There
was a clear difference in potency between the two oxazoline
isomers: oxazoline 2 was 10-fold more potent than oxazoline1
(Table 1). In addition, the highly polar imidazoline 3 showed
poor activity, which may be a consequence of protonation of
the basic cyclic amidine functionality (calculated pKa ca. 9.3).16
The isoxazoline analogue (4) is comparable in potency to
oxazoline 2; however, a distinguishing attribute of 4 was its
greater metabolic stability in all three species. Therefore, efforts
focused on the isoxazoline tether.
Exploration of the SAR on the isoxazoline scaffold found
that substituents in the 2- and 6-positions of the 5-aryl group
on the benzimidazole were tolerated (Table 2). Addition of a
substituent in the 3-position as in 56 (91 nM) significantly
reduced potency compared to the structurally and electron-
ically similar analogues 54 and 55 (1.1 and 2.5 nM, respec-
tively). Hydrophobic substituents at the 2-position generally
resulted in single digit nanomolar antagonists of cTRPM8
(4, 53-55, and 74). Furthermore, polar groups such as the
tertiary alcohol of 75 were tolerated, however the metabolic
stability of 75 was poor.
Addition of a second substituent in the 6-position of the
5-phenyl ring has a minor positive effect on the cTRPM8
potency but has a detrimental effect on metabolic stability
when direct analogues were compared (4 to 58, 53 to 57, and
54 to 59). The requirement for an ortho substituent for
potency may suggest that the 5-phenyl ring needs to be forced
out of planarity with the benzimidazole core for effective
binding to the TRPM8 binding site.
To differentiate these potent cTRPM8 antagonists, attention
was turned to their metabolic stability. Of these compounds
with high metabolic stability across species, 4 was selected to
further characterize this emerging family of compounds.
Because the primary screen for the TRPM8 antagonists was
conducted using canine TRPM8, the activity of 4 was next
assessed in the corresponding rat and human TRPM8 func-
tional assays. The values obtained were 10 and 9 nM, respec-
tively, both similar in potency to the canine value (3.1 nM).
a Percent remaining after 10 min incubations in human (HLM), rat
(RLM), or dog (DLM) liver microsomal preparations.
This result can be rationalized on consideration of the high
percent identity of the TRPM8 protein sequences across human,
rat, and canine species.13
Compound 4 exhibited a relatively selective off-target
profile when assessed for binding against a panel of 50 GPCRs,
transporters, and ion channels (Cerep). Compound 4 showed
inhibition of reference compound binding to only two targets
by greater than 50% at a concentration of 10 μM (i.e., 98%
at 5-HT1B and 72% at 5-HT2B).
In pharmacokinetic studies in the rat, 4 exhibited relatively
high bioavailability (75%), with a modest Cmax of 230 ng/mL
and a long half-life (t1/2) of 18 h following an oral dose of
10 mg/kg. The high volume of distribution (Vdss) of 13.6 L/kg
(compared to rat total body water volume of 0.688 L/kg) is
indicative of extensive tissue distribution and may contribute
to the high clearance rate of 54 mL/min/kg, essentially
equivalent to hepatic blood flow (55 mL/min/kg).
In the rat PDmodel, 4 completely inhibited WDS behaviors
at a dose of 10 mg/kg po. In the rat CCI time-course study, a
dose of 10 mg/kg po of 4 maximally reversed paw lifting/licking
behaviors by 66%, peaking after 3 h postdose. The modest
oral exposure in rat and potent CYP 2C9 inhibition in HLM

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Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 237
Table 3. Functional Activity of Substitution of R1 and Z
cTRPM8
IC50
(nM)
stabilitya
HLM/
RLM/DLM
CYP 2C9 IC50
(μM)b
compd R1
Z
4
-CF3 -H
3.1
2.2
3.3
0.7
2.7
1.7
0.8
1.2
1.0
0.2
100/75/71
46/100/87
92/100/100
68/96/51
100/83/74
71/100/82
92/79/59
51/53/50
66/100/60
57/74/12
1.7
6.4
6.0
1.9
2.2
10
60 -CF3 -F
61 -CF3 -Cl
62 -CF3 -CN
63 -CF3 -Br
Figure 2. Time-course response in the rat CCI cold allodynia
model following oral dosing of 10 mg/kg of 5 (red down-pointing
triangle), 64 (green diamond), 65 (red up-pointing triangle), and 66
(violet circle) vs vehicle (20% HPβCD po; gray square). (* p < 0.05,
** p < 0.01; two-way ANOVA for repeated measures; Bonferroni
posthoc test; n = 7 rats/treatment).
64 -F
-CF3 -CF3
65 -OCF3-CF3
66 -Cl -CF3
67 -CF3 -(CH2)3OH
-CF3
5
10
10
10
6.8
a Percent remaining after 10 min incubations in human (HLM), rat
(RLM), or dog (DLM) liver microsomal preparations. b IC50 values
obtained from 15 min HLM incubations with tolbutamide as a substrate.
Table 4. TRPM8 Functional Activity with Cyclohexane Modifications
cTRPM8 IC50 stabilitya HLM/
compd
R1
Z
X
(nM)
RLM/DLM
68
69
70
71
-CF3 -H
-CF3 -H
-CF3 -CF3
-CF3 -Cl
O
CF2
O
6.7
0.9
1.4
4.0
98/74/83
66/ndb/34
100/96/90
100/82/81
Figure 3. Inhibition of cold-stimulated currents in HEK cells express-
ing canine and human TRPM8. Sequential concentrations of 5 were
applied following steady-state current activation achieved by cooling
perfusate to 10 °C (from 22 °C).
O
a Percent remaining after 10 min incubations in human (HLM), rat
(RLM), or dog (DLM) liver microsomal preparations. b Not deter-
mined.
(IC50 = 1.7 μM) precluded 4 from further development;
however, these liabilities were attenuated in subsequent
analogues.
In an attempt to reduce the CYP 2C9 inhibition observed in
4, modifications to the benzimidazole core were explored and
the 7-position was determined to be permissive for substitution
(Table 3). In general, hydrophobic substituents (5, 60-61,
63-66) and polar groups (62 and 67) increased or maintained
potency. Acidic functionality was also tolerated, whereas
basic moieties resulted in a loss of activity. The most potent
compound in this group (67) contained a hydroxypropyl group
at the 7-position, although it suffered from poor metabolic
stability.
In addition, many of the substituents at the 7-position led
to a reduction in the HLM CYP 2C9 inhibitory activity.
For example, the 7-CF3 substituent resulted in more than a
5-fold decrease in 2C9 inhibition (64, 5, 65-67: IC50 ca. 10 μM)
over 4 (IC50 = 1.7 μM). Other substituents that improved
CYP 2C9 activity were the 7-fluoro (60), 7-chloro (61), and
7-hydroxypropyl (67).
Another potential site for modification of the scaffold was
on the cyclohexyl portion of the spiro-isoxazoline ring system
(Table 4). Introduction of an ether oxygen at the 4-position
(68, 70, and 71) resulted in loss of TRPM8 functional activity
Figure 4. Inhibition of icilin-induced (3 mg/kg, ip) WDS in rats
following 120 min pretreatment with 5. (** p < 0.01, one-way
ANOVA; Dunnett’s posthoc test; n = 7 rats/dose).
when compared to the carbocyclic analogues (4, 5, and 61).
The fluorinated derivative 69 increased TRPM8 activity;
however, metabolic stability was reduced. Both modifications
resulted in an increase of CYP 2C9 inhibition (IC50 values of
70and71were 3 μM orless); thus, thesetypes of modifications
were not pursued further.

238 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Parks et al.
The potent TRPM8 antagonists 5 and 64-66, which dis-
played relatively high metabolic stability and the lowest CYP
2C9 inhibitory activities, were further scrutinized in the WDS
PD assay. All four compounds completely prevented icilin-
induced WDS at oral doses of 10 mg/kg; therefore, the rat
CCI cold allodynia model was used as a means to discriminate
the compounds. All compounds demonstrated efficacy in the
CCI model, albeit to different degrees. At an oral dose of
10 mg/kg, 5 gave the greatest reversal of 91%, whereas 64-66
peaked at 51%, 74%, and 74%, respectively, after 2 h postdose
(Figure 2).
Because of its superior efficacy in the CCI pain model, 5 was
selected for more detailed studies. In the canine, rat, and human
TRPM8 functional assays, 5 exhibited similar potencies, with
IC50 values of 0.8, 4.0, and 3.0 nM, respectively. In whole cell
patch clamp electrophysiology studies, 5 potently inhibited
cold-induced TRPM8 currents in HEK293 cells stably express-
ing canine or human TRPM8, with IC50 values of 0.413 and
ca. 1 nM, respectively (Figure 3). This inhibition was readily
reversed whenthe compoundwas removed from the perfusion
solution upon washout.
As previously mentioned, 5 completely prevented icilin-
induced WDSatanoraldoseof 10 mg/kg. Ina dose-response
study, 5 produced greater than 90% inhibition at 3, 5.6, and
10 mg/kg administered 2 h prior to icilin challenge (Figure 4)
and at 150 min postdose, the plasma levels of 5 were 295, 662,
and 1042 ng/mL, respectively. The corresponding brain levels
of 5at the 3, 5.6, and 10 mg/kg doses were 175, 414, and 638 ng/g,
respectively.
In a dose-response study of the rat CCI cold allodynia
model assessed at 4 h postdose, 5 produced an ED50 value of
4.5 mg/kg po, with corresponding plasma levels of 254 ng/mL
at the 4 h time point (Figure 5). When 5 was administered for 5
consecutive days at 10 mg/kg/day po, no loss of efficacy in
reversing CCI cold allodynia was observed between day 1 and
day 5, indicating that functional tolerance did not develop
under these conditions. It is particularly notable that the in vivo
pharmacological profile of 5 faithfully recapitulated the pheno-
type of TRPM8 null mutant mice in which icilin-induced WDS
behavior and CCI-induced cold allodynia are absent.5b
The cold pressor test (CPT) can be used to assess efficacy of
analgesic compounds17 as well as to assess cold hypersen-
sitivity.18 In the present context, it may also be used as a
marker of target engagement by a TRPM8 antagonist. Upon
cooling, activationofsensory afferentneuronsresults in blood
pressure elevation through sympathetically controlled vaso-
constriction. Oral administration of 5 (0.3, 1, 3, 10, or 30 mg/kg)
to anesthetized rats 2 h prior to cold pressor testing resulted in
a dose-dependent inhibition of the pressor response, suggesting
that it blocked the activation of TRPM8 by the cold stimulus
(Figure 6).
Figure 5. Dose-response relationship in the rat CCI cold allodynia
model following administration of 5 at 1, 3, 10, or 30 mg/kg, po)
assessed 4 h postdose. (* p < 0.05; *** p < 0.001; two-way ANOVA
for repeated measures; Bonferroni posthoc test; n = 7-13 rats/
treatment).
InPKassessments, benzimidazole5 hadexhibited veryhigh
oral bioavailability in the rat (96%) and moderate bioavail-
ability in the dog and monkey (45% and 57%, respectively).
Clearance rates in rat (13.9 mL/min/kg) and monkey
(15.5 mL/min/kg) were low to moderate and moderate in
the dog (14.5 mL/min/kg). Volume of distribution (Vdss) was
moderate for the rat (2.92 L/kg) and large for dog (4.43 L/kg)
and monkey (4.92 L/kg) in comparison to total body water.
Relevant PK parameters are summarized in Table 5.
Toobtain anevaluation of its safetypotential, the off-target
selectivity of 5 was assessed. In cytochrome P450 isoenzyme
studies in HLMs, benzimidazole 5 exhibited minimal effect on
the enzyme activityof CYP isoforms 3A4, 1A2, 2D6, 2C9, and
2C19 (IC50 > 10 μM). In a panel of 50 GPCRs, transporters,
Figure 6. Dose-response effects of 5 on the cold-induced increase
in mean arterial blood pressure (mmHg) when administered 2 h prior to
cold challenge (n = 7-11 rats/dose).
Table 5. Pharmacokinetic Assessment of 5 in Rat, Dog, And Cynomolgous Monkey after Intravenous and Oral Administration
intravenous administration
oral administration
species
CL (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
Cmax (ng/mL)
AUC (ng h/mL)
t1/2 (h)
F (%)
3
rata
13.9
14.5
15.4
2.92
4.43
4.92
3.17
ndc
5.66
1262
1641
510
12302
5252
6184
4.74
5.79
4.98
96
45
57
dogb
monkeya
a Rats and monkeys were administered 2 mg/kg iv of the HCl salt in 20% HPβCD or 10 mg/kg po in 20% HPβCD. b Dogs were administered 2 mg/kg
iv of the free form in 20% HPβCD or 10 mg/kg po in 20% HPβCD. c Not determined.

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Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 239
and ion channel binding assays, 5 showed no inhibition of
reference compound binding to any of the targets greater than
50% when tested at 10 μM (Cerep). Similarly, in testing
against a panel of 190 kinase assays, 5 did not show inhibition
of specific binding for any kinase (Invitrogen).
The cardiovascular safety of 5 was assessed in a whole-cell
hERG binding assay, wherein it inhibited [3H]-astemizole
binding with an IC50 value of 16 μM. However, when 5 was
administered intravenously to anesthetized guinea pigs, no
notable hemodynamic or electrocardiographic effects were
induced up to a cumulative iv dose of 10 mg/kg. The corre-
sponding plasma level of 5 taken 5 min after the cumulative
dose was 18157 ng/mL. No notable effects were observed in a
CNS safety assessment study after a single oral dose of 30 or
300 mg/kg of 5 throughout the 14-day observation period.
Flash chromatography was carried out on an ISCO Combiflash
Companion or an Analogix IntelliFlash 280 system using ISCO
or Analogix prepacked silica gel columns (12-80 g sizes). High-
performance liquid chromatography (HPLC) was carried out
on a Varian Prep Star system using a Phenomenex C18 (analytical:
10 μM, 4.6 mm ꢀ 150 mm; semiprep: 5 μM, 21.2 mm ꢀ 150 mm) or
PrincetonSpher_100 phenyl (analytical: 5 μM, 4.6 mm ꢀ 150 mm;
semiprep: 5 μM, 21.2 mm ꢀ 150 mm) reverse phase columns. All
compounds used for biological assays are at least of 95% purity
based on HPLC analytical results monitored with 220 and 254 nm
wavelengths, unless otherwise noted.
Preparation of the Biphenylnitroanilines 6, 34-45: General
Suzuki Procedure. The boronic acid or ester (1.3 equiv), (dppf)-
PdCl2 DCM (0.05 equiv), and aryl halide (1 equiv) in a mixture
3
(1.5:1) of DME and 2 M Na2CO3 (3 equiv) under an argon
atmosphere was stirred at 90 °C for 18 h. After cooling to rt, the
product was extracted with EtOAc and then the organic solution
was dried over MgSO4 and filtered. The filtrate was concentrated
under reduced pressure, and then the crude product was purified
by SiO2 chromatography.
Conclusion
In summary, four spirocycle-substituted benzimidazole
series were prepared and investigated as TRPM8 antagonists.
The spiro-isoxazoline series was selected for optimization
due to its superior metabolic stability and potency over the
other series. SAR investigations demonstrated that a 2-
substituent on the phenyl ring at the 5-position of the benzimid-
azole core was essential for potency in the TRPM8 in vitro
assay, whereas substituents at the 7-position reduced CYP 2C9
inhibition.
3-Nitro-20-(trifluoromethyl)-[1,10-biphenyl]-4-amine (6). The
title compound was prepared from 4-bromo-2-nitroaniline (21)
(10.1 g, 46.7 mmol), 2-trifluoromethylphenylboronic acid (24)
(11.5 g, 60.7 mmol), and (dppf)PdCl2 DCM (1.91 g, 2.34 mmol)
3
in DME (180 mL) and 2 M aq Na2CO3 (60.0 mL, 120 mmol)
according to the general Suzuki procedure, yielding an orange
solid (12.8 g, 97%). 1H NMR (400 MHz, CDCl3) δ: 8.11 (d, J =
2.0 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.54-7.62 (m, J = 7.6 Hz,
1H), 7.44-7.53 (m, J = 7.8 Hz, 1H), 7.36 (dd, J = 8.6, 1.3 Hz,
1H), 7.33 (d, J = 7.6 Hz, 1H), 6.84 (d, J = 8.6 Hz, 1H).
20-Fluoro-3-nitro-[1,10-biphenyl]-4-amine (34). The title com-
pound was prepared from nitroaniline 21 (2.17 g, 10.0 mmol),
2-fluorophenylboronic acid (25) (1.40 g, 10.0 mmol), and Pd-
(PPh3)4 (116 mg, 0.100 mmol) in dioxane (40 mL) and 2 M aq
NaHCO3 (40.0 mL, 80.0 mmol) according to the general Suzuki
Optimization of the spiro-isoxazoline series culminated in
the discovery of5, a selective, high affinity TRPM8 antagonist
that exhibited potent in vitro functional activity and robust
oral efficacy in an inflammatory model of neuropathic pain at
relatively low plasma levels. Pharmacokinetic data indicate
that 5 exhibited moderate to high oral bioavailability across
several species, and preliminary pharmacological studies indi-
cate that 5 possesses an appreciable therapeutic window with
respect to cardiovascular and CNS safety. Taken together, the
present results suggest that the in vivo antagonism of TRPM8
constitutes a viable new strategy for treating a variety of
disorders associated with cold hypersensitivity, including certain
types of neuropathic pain.
1
procedure, yielding a yellow solid (1.80 g, 78%). H NMR
(400 MHz, CDCl3) δ: 8.36 (d, J = 1.8 Hz, 1H), 7.64 (dt, J = 8.6,
2.0 Hz, 1H), 7.45 (td, J = 7.8, 1.8 Hz, 1H), 7.30-7.38 (m, 1H),
7.13-7.27 (m, 2H), 6.91 (d, J = 8.6 Hz, 1H), 6.18 (br s, 2H).
3-Nitro-20-(trifluoromethoxy)-[1,10-biphenyl]-4-amine (35). The
title compound was prepared from nitroaniline 21 (1.20 g,
5.54 mmol), 2-trifluoromethoxyphenyl-boronic acid (26) (1.71 g,
8.31 mmol), and (dppf)PdCl2 DCM (226 mg, 0.277 mmol) in
3
DME (26 mL) and 2 M aq Na2CO3 (10.0 mL, 20.0 mmol) according
to the general Suzuki procedure, yielding an orange solid (1.59 g,
97%). 1H NMR (400 MHz, CDCl3) δ: 8.25 (d, J = 2.0 Hz, 1H),
7.53 (dd, J = 8.7, 2.1 Hz, 1H), 7.39-7.45 (m, 1H), 7.30-7.39
(m, 3H), 6.89 (d, J = 8.6 Hz, 1H), 6.21 (br s, 2H).
Experimental Section
General. Reagents were purchased from commercial sources
and used without further purification. Nuclear magnetic reso-
nance (NMR) spectra were measured in the indicated solvent
with tetramethylsilane (TMS) or the residual solvent peak as the
internal standard on a Bruker Avance or Varian (300, 400, or
500 MHz) spectrometer. Chemical shifts are reported in parts
per million (ppm) relative to the internal standard. Abbrevia-
tions for the multiplicities of the signals are: s (singlet), d (doublet),
t (triplet), q (quartet), quin (quintet), sep (septet), m (multiplet), br
(broad), and combinations thereof. Mass spectra (MS) were
determined with a Finnegan LCQ Classics or Hewlett-Packard
series 1100 mass spectrometer as (ESI) m/z (M þ Hþ) using an
electrospray technique. High-resolution mass spectral (HRMS)
measurements were obtained using an Agilent LC/MSD TOF
which consisted of an 1100 HPLC system coupled with a G1969
MSD TOF mass spectrometer operating in positive electrospray
ionization mode.
20-(Difluoromethoxy)-3-nitro-[1,10-biphenyl]-4-amine (36).Thetitle
compound was prepared from 1-bromo-2-(difluoromethoxy)-
benzene (29) (223 mg, 1.00 mmol), 4-amino-3-nitrophenylboronic
acid (273 mg, 1.50 mmol), and (dppf)PdCl2 DCM (82.0 mg,
3
0.0100 mmol) in DME (4 mL) and 2 M aq Na2CO3 (1.50 mL, 3.00
mmol) according to the general Suzuki procedure, yielding an orange
solid (291 mg, 100%). 1H NMR (400 MHz, CDCl3) δ: 8.26 (d, J =
2.0 Hz, 1H), 7.58 (dd, J =8.6, 2.0Hz, 1H), 7.42(dd, J=7.5, 1.9Hz,
1H), 7.36 (td, J = 7.7, 2.0 Hz, 1H), 7.29 (td, J = 7.5, 1.4 Hz, 1H),
7.22 (dd, J = 8.1, 1.0 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 6.39 (t, J =
73.9 Hz, 1H), 6.16 (br s, 2H).
4-(2,2-Difluorobenzo[d][1,3]dioxol-4-yl)-2-nitroaniline (37). The
title compound was prepared from 4-bromo-2,2-difluorobenzo-
[d][1,3]dioxole (30) (500 mg, 2.11 mmol), 4-amino-3-nitrophenyl-
boronic acid pinacol ester (32) (724 mg, 2.74 mmol), and (dppf)-
PdCl2 DCM (86.0 mg, 0.106 mmol) in DME (8 mL) and 2 M aq
3
Na2CO3 (3.00 mL, 6.00 mmol) according to the general Suzuki
procedure, yielding an orange solid (492 mg, 79%). 1H NMR
(400 MHz, CDCl3) δ: 8.48 (d, J = 2.3 Hz, 1H), 7.76 (dd, J = 8.8,
2.3 Hz, 1H), 7.26 (dd, J = 8.1, 1.0 Hz, 1H), 7.15 (t, J = 8.1 Hz,
1H), 7.02 (dd, J = 8.0, 1.1 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.02
(br s, 2H).
All reactions were monitored by thin layer chromatography
(TLC) carried out on EMD silica gel plates (2.5 cm ꢀ 7.5 cm,
250 μm thick, 60 F254), visualized by using UV (254 nm) or stains
such as KMnO4, p-anisaldehyde, and ceric ammonium molyb-
date (CAM). All organic solutions were dried over anhydrous
MgSO4 or Na2SO4 and concentrated on a rotary evaporator.

240 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Parks et al.
20,60-Difluoro-3-nitro-[1,10-biphenyl]-4-amine (38). The title
compound was prepared from 1-bromo-2,6-difluorobenzene
(31) (965 mg, 5.00 mmol), boronate ester 28 (1.72 g, 6.50 mmol), and
and (dppf)PdCl2 DCM (145 mg, 0.178 mmol) in DME (15 mL)
3
and 2 M aq Na2CO3 (5 mL, 10 mmol) according to the general
Suzuki procedure, yielding an orange solid (1.05 g, 94%). H
1
(dppf)PdCl2 DCM (204 mg, 0.250 mmol) in DME (20 mL) and
NMR (400 MHz, CDCl3) δ: 8.47 (d, J = 2.0 Hz, 1H), 7.89
(d, J = 2.0 Hz, 1H), 7.47-7.53 (m, 1H), 7.30-7.37 (m, 3H), 6.76
(br s, 2H).
3
2 M aq Na2CO3 (7.00 mL, 14.0 mmol) according to the general
Suzuki procedure, yielding an orange solid (1.14 g, 91%). 1H NMR
(400 MHz, CDCl3) δ: 8.29 (s, 1H), 7.46-7.52 (m, 1H), 7.23-7.34
(m, 1H), 6.94-7.04 (m, 2H), 6.90 (d, J = 8.6 Hz, 1H), 6.20
(br s, 2H).
20-Fluoro-3-nitro-60-(trifluoromethyl)-[1,10-biphenyl]-4-amine (39).
The title compound was prepared from 1-bromo-2-fluoro-6-tri-
fluoromethylbenzene (32) (0.850 mL, 6.09 mmol), boronate ester
20-(Trifluoromethyl)-[1,10-biphenyl]-3,4-diamine (7). Biphenyl-
nitroaniline 6 (818 mg, 2.90 mmol) was dissolved in absolute
EtOH (10 mL) then 10% Pd on carbon was added. The reaction
was stirred at rt under a H2 atmosphere at balloon pressure for 15 h.
The H2 was vented then the suspension was filtered. The solids were
washed with MeOH (3 ꢀ 10 mL), and then the combined filtrates
were concentrated under reduced pressure. The crude product was
chromatographed on a 24 g SiO2 prepacked column eluting with
0:1 to 4:1 EtOAc/hexanes, affording the title compound as an off-
white solid (570 mg, 78%). 1H NMR (400 MHz, CDCl3) δ: 7.69
(d, J = 8.1 Hz, 1H), 7.49 (t, J = 7.1 Hz, 1H), 7.39 (t, J = 7.5 Hz,
1H), 7.31 (d, J = 7.6 Hz, 1H), 6.64-6.73 (m, 3H), 3.41 (br s, 4H).
2-(Trichloromethyl)-5-(2-(trifluoromethyl)phenyl)-1H-benzimid-
azole (8). Biphenyl diamine 7 (222 mg, 0.880 mmol) was dissolved
in AcOH (4 mL) then treated with methyl 2,2,2-trichloroacetimi-
date (MTCA) (0.120 mL, 0.968 mmol) and stirred at rt for 14 h.
The reaction was poured into H2O (50 mL), and the resulting
precipitate was isolated by filtration. The solid was washed with
H2O (2 ꢀ 10 mL) and then dissolved in EtOAc (50 mL) and
dried over MgSO4. The solution was filtered and then concen-
trated under reduced pressure. The residue was triturated with
a minimum amount of DCM, affording the title compound as a
28 (2.09 g, 7.92 mmol), and (dppf)PdCl2 DCM (249 mg,
3
0.300 mmol) in DME (24 mL) and 2 M aq Na2CO3 (8.00 mL,
16.0 mmol) according to the general Suzuki procedure, yielding an
orange solid (1.62 g, 89%). 1H NMR (400 MHz, CDCl3) δ: 8.10
(s, 1H), 7.54-7.60 (m, 1H), 7.44-7.53 (m, 1H), 7.35 (t, J = 8.5 Hz,
1H), 7.30 (d, J = 7.3 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 5.44
(br s, 2H).
20-Fluoro-3-nitro-60-(trifluoromethoxy)-[1,10-biphenyl]-4-
amine (40). The title compound was prepared from 2-fluoro-1-
iodo-6-trifluoromethoxybenzene19 (33) (612 mg, 2.00 mmol),
boronate ester 28 (687 mg, 2.60 mmol), and (dppf)PdCl2 DCM
3
(81.6 mg, 0.100 mmol) in DME (10 mL) and 2 M aq Na2CO3
(4.00 mL, 8.00 mmol) according to the general Suzuki proce-
dure, yielding an orange solid (548 mg, 87%). 1H NMR (400 MHz,
CDCl3) δ: 8.21 (s, 1H), 7.30-7.47 (m, 2H), 7.18 (d, J = 8.1 Hz,
1H), 7.14 (t, J = 8.6 Hz, 1H), 6.91 (d, J = 6.6 Hz, 1H), 5.37
(br s, 2H).
1
yellow powder (326 mg, 100%). H NMR (400 MHz, CDCl3)
3-Fluoro-5-nitro-20-(trifluoromethyl)-[1,10-biphenyl]-4-amine (41).
The title compound was prepared from 4-bromo-2-fluoro-6-
nitroaniline (22) (992 mg, 4.22 mmol), boronic acid 24 (1.04 g,
δ: 10.46 (br s, 1H), 7.76 (d, J= 7.8 Hz, 1H), 7.57 (t, J = 7.6 Hz,
1H), 7.49 (t, J = 7.6 Hz, 1H), 7.38-7.93 (br s, m, 2H), 7.29-7.38
(m, 2H). Mass spectrum (LCMS, ESI pos.) calcd for
C15H8Cl3F3N2 (M þ H)þ 379.0, found 379.0.
5.49 mmol), and (dppf)PdCl2 DCM (172 mg, 0.211 mmol) in
3
2-(5-(2-(Trifluoromethyl)phenyl)-1H-benzimidazol-2-yl)-1-
oxa-3-azaspiro[4.5]dec-2-ene (1). 1-(Aminomethyl)cyclohexanol
hydrochloride (294 mg, 1.77 mmol) was dissolved in H2O
(8 mL) and treated with 2 M NaOH (0.890 mL, 1.77 mmol),
and then a dioxane (16 mL) solution of 8 (163 mg, 0.443
mmol) was added. The reaction was stirred at rt for 14 h and
then diluted with brine (30 mL) and extracted with EtOAc
(3 ꢀ 20 mL). The combined organic extracts were dried over
MgSO4 and concentrated under reduced pressure. The crude
product was chromatographed on a 24 g SiO2 prepacked
column eluting with 0:1 to 2:5 EtOAc/hexanes, affording the
title compound (114 mg, 64%). 1H NMR (400 MHz, CD3OD)
δ: 7.79 (d, J = 7.8 Hz, 1H), 7.73 (br s, 1H), 7.65 (t, J = 7.2 Hz,
1H), 7.60 (br. s, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.43 (d, J =
7.6 Hz, 1H), 7.29 (br. d, J = 6.8 Hz, 1H), 3.84 (s, 2H), 1.80-1.98
(m, 4H), 1.69-1.79 (m, 2H), 1.43-1.65 (m, 4H). HRMS calcd for
C22H20F3N3O (M þ H)þ 400.1637, found 400.1630.
DME (16 mL) and 2 M aq Na2CO3 (6.00 mL, 12.0 mmol)
according to the general Suzuki procedure, yielding an orange
solid (1.12 g, 88%). 1H NMR (400 MHz, CDCl3) δ: 7.93 (t, J =
1.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 7.2 Hz, 1H),
7.51 (t, J = 7.5 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.24-7.30
(m, 1H), 6.18 (br s, 2H).
20-Fluoro-3-nitro-5-(trifluoromethyl)-[1,10-biphenyl]-4-amine (42).
The title compound was prepared from 4-bromo-2-nitro-6-
trifluoromethyl-phenylamine (23) (285 mg, 1.00 mmol), boronic
acid 25 (182 mg, 1.30 mmol), and (dppf)PdCl2 DCM (41.0 mg,
3
0.050 mmol) in DME (4 mL) and 2 M aq Na2CO3 (1.25 mL,
2.50 mmol) according to the general Suzuki procedure, yielding an
orange solid (268 mg, 89%). 1H NMR (400 MHz, CDCl3)
δ: 8.49-8.59 (m, 1H), 7.97 (s, 1H), 7.41 (td, J = 7.8, 1.8 Hz, 1H),
7.32-7.39 (m, 1H), 7.23 (td, J = 7.5, 1.1 Hz, 1H), 7.17 (ddd, J =
10.9, 8.2, 1.1 Hz, 1H), 6.75 (br s, 2H).
3-Nitro-20,5-bis(trifluoromethyl)-[1,10-biphenyl]-4-amine (43).
The title compound was prepared from 4-bromo-2-nitro-6-
trifluoromethyl-phenylamine (23) (10.0 g, 35.2 mmol), boronic
2-(5-(2-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazol-2-yl)-3-
oxa-1-azaspiro[4.5]dec-1-ene (2). (1-Aminocyclohexyl)methanol20
(172 mg, 1.33 mmol) was dissolved in H2O (8 mL), and then a
dioxane (16 mL) solution of 8 (164 mg, 0.445 mmol) was added.
The reaction was stirred at rt for 14 h and then diluted with brine
(30 mL) and extracted with EtOAc (3 ꢀ 20 mL). The combined
organic extracts were dried over MgSO4 and concentrated under
reduced pressure. The crude product was chromatographed on a
24 g SiO2 prepacked column eluting with 0:1 to 2:5 EtOAc/hexanes,
affording the title compound (13.9 mg, 7.8%). 1H NMR (400 MHz,
CD3OD) δ: 7.79 (d, J = 7.8 Hz, 1H), 7.71 (br s, 1H), 7.65 (t, J =
7.2 Hz, 1H), 7.62 (br s, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.42 (d, J =
7.6 Hz, 1H), 7.29 (d, J = 6.6 Hz, 1H), 4.32 (s, 2H), 1.75-1.94 (m,
4H), 1.65-1.75 (m, 2H), 1.41-1.65 (m, 4H). HRMS calcd for
C22H20F3N3O (M þ H)þ 400.1637, found 400.1636.
acid 24 (8.70 g, 45.8 mmol), and (dppf)PdCl2 DCM (1.44 g,
3
1.76 mmol) in DME (150 mL) and 2 M aq Na2CO3 (50.0 mL,
100 mmol) according to the general Suzuki procedure, yielding
an orange solid (1.30 g, 92%). H NMR (400 MHz, CDCl3)
1
δ: 8.35 (d, J = 2.0 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.74 (s, 1H),
7.61 (t, J = 7.2 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.33 (d, J =
7.6 Hz, 1H), 6.75 (br s, 2H).
3-Nitro-20-(trifluoromethoxy)-5-(trifluoromethyl)-[1,10-biphenyl]-
4-amine (44). The title compound was prepared from nitroani-
line 23 (285 mg, 1.00 mmol), boronic acid 26 (268 mg, 1.30 mmol),
and (dppf)PdCl2 DCM (41.0 mg, 0.050 mmol) in DME (4 mL)
3
and 2 M aq Na2CO3 (1.25 mL, 2.50 mmol) according to the general
Suzuki procedure, yielding an orange solid (326 mg, 89%).
1H NMR (400 MHz, CDCl3) δ: 8.50 (d, J = 2.3 Hz, 1H), 7.91
(d, J = 2.0 Hz, 1H), 7.33-7.48 (m, 4H), 6.77 (br s, 2H).
20-Chloro-3-nitro-5-(trifluoromethyl)-[1,10-biphenyl]-4-amine (45).
The title compound was prepared from nitroaniline 23 (1.01 g,
3.55 mmol), 2-chlorophenyl-boronic acid (27) (833 mg, 5.33 mmol),
2-(1,3-Diazaspiro[4.5]dec-2-en-2-yl)-5-(2-(trifluoromethyl)-
phenyl)-1H-benzimidazole Hydrochloride (3). A mixture of
1-(aminomethyl)cyclohexylamine dihydrochloride21 (54.1 mg,
0.269 mmol) and 8 (102 mg, 0.269 mmol) in 8 mL of THF
was treated with TEA (0.262 mL, 1.88 mmol) and stirred for 1 h.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 241
The reaction was concentrated under reduced pressure, and
then the residue was chromatographed on a 12 g prepacked
SiO2 column using 0:1-1:4 EtOH/EtOAc afforded the title
compound as a colorless resin (116 mg, quantitative). 1H
NMR (400 MHz, CDCl3) δ: 8.22 (br s, 1H), 7.73 (d, J = 7.8 Hz,
1H), 7.48-7.65 (m, 3H), 7.40-7.48 (m, 1H), 7.33 (d, J = 7.6 Hz,
1H), 7.21 (d, J = 8.6 Hz, 1H), 3.81 (s, 2H), 1.62-1.81 (m, 6H),
1.34-1.53 (m, 4H). Mass spectrum (LCMS, ESI pos.) calcd for
The residue was dissolved in H2O and acidified to pH ca. 2 by
dropwise addition of 3 M aq HCl. The solution was extracted
thrice with DCM, and then the combined organic extracts were
dried over MgSO4 and filtered. The solvent was removed under
reduced pressure.
1-Oxa-2-azaspiro[4.5]dec-2-ene-3-carboxylic Acid (15). The
title compound was prepared from spiro-isoxazoline ester 12
(1.06 g, 5.00 mmol) and LiOH H2O (210 mg, 5.00 mmol) in
3
C
22H21F3N4 (M þ H)þ 399.2, found 399.2.
MeOH (15 mL) and H2O (5 mL) according to the general
procedure, yielding a white powder (766 mg, 92%). H NMR
1
The title compound (0.269 mmol) dissolved in Et2O (6 mL)
was treated with 1 M HCl in Et2O (269 μL, 0.269 mmol). After
stirring for 10 min, the precipitate was isolated by filtration and
washed with Et2O (2 ꢀ 5 mL). The residual solvent was removed
under reduced pressure, affording the hydrochloride salt of the
(400 MHz, CDCl3) δ: 2.93 (s, 2H), 1.72-1.89 (m, 4H), 1.60-1.72
(m, 2H), 1.37-1.54 (m, 4H).
1,8-Dioxa-2-azaspiro[4.5]dec-2-ene-3-carboxylic Acid (16). The
title compound was prepared from spiro-isoxazoline ester 13
(264 mg, 1.24 mmol) and LiOH H2O (57.0 mg, 1.36 mmol) in
1
title compound as a white powder (76.6 mg, 65%). H NMR
3
MeOH (9 mL) and H2O (3 mL) according to the general procedure,
yielding a white powder (228 mg, 99%). H NMR (400 MHz,
(400 MHz, CD3OD) δ: 7.80-7.84 (m, 2H), 7.71 (s, 1H), 7.69
(t, J = 7.3 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.39-7.47 (m, 2H),
3.99 (s, 2H), 1.76-2.03 (m, 6H), 1.45-1.69 (m, 4H). HRMS
calcd for C22H21F3N4 (M þ H)þ 399.1797, found 399.1795.
Ethyl 1-Oxa-2-aza-spiro[4.5]dec-2-ene-3-carboxylate (12). Spiro-
isoxazoline ester 12 was prepared according to the literature
procedure.11 To a 300 mL pressure vessel equipped with a magnetic
stir bar were added methylenecyclohexane (9) (5.83 g, 60.6 mmol)
and EtOH (150 mL). Ethyl nitroacetate (16.8 mL, 152 mmol) and
DABCO (680 mg, 6.06 mmol) were then added. Additional EtOH
(30 mL) was added to rinse the sides of the vessel, and then the vessel
was tightly capped. The mixture was heated to 80 °C for 42 h and
then cooled to rt. The solvent was removed under reduced pressure,
and the residue was divided into three equal portions. Each portion
was purified by column chromatography using an 80 g SiO2
prepacked column eluting with 0:1 to 1:4 EtOAc/hexanes, affording
1
DMSO-d6) δ: 3.67-3.78 (m, 2H), 3.48-3.58 (m, 2H), 3.01 (s, 2H),
1.69-1.79 (m, J = 5.4, 5.4 Hz, 4H).
8,8-Difluoro-1-oxa-2-azaspiro[4.5]dec-2-ene-3-carboxylic
Acid (17). The title compound was prepared from spiro-isoxazo-
line ester 14 (391 mg, 1.58 mmol) and LiOH H2O (73.0 mg,
3
1.74 mmol) in MeOH (12 mL) and H2O (4 mL) according to the
general procedure, yielding a white powder (284 mg, 82%). 1H
NMR (400 MHz, CDCl3 þ CD3OD) δ: 3.00 (s, 2H), 2.10-2.29
(m, 2H), 1.95-2.10 (m, 4H), 1.81-1.95 (m, 2H).
Preparation of Spiro-isoxazoline Acid Chlorides 18-20: Gen-
eral Procedure. The spiro-isoxazoline acid (1 equiv) was dis-
solved in DCM, and then a catalytic amount of DMF (10 μL)
was added. Oxalyl chloride (1.5 equiv) was added dropwise, and
then the reaction was stirred at rt for 1 h. The solvent was
removed under reduced pressure, and then the residue was taken
up in the appropriate solvent as used directly in the next step.
3-Chloro-5-nitro-20-(trifluoromethyl)-[1,10-biphenyl]-4-amine
(46). Biphenyl nitroaniline 6 (12.7 g, 45.0 mmol) was placed in a
250 mL round-bottom flask equipped with a magnetic stir bar
and a reflux condenser, and then dry acetonitrile (150 mL) was
added. The solid was allowed to dissolve, and then NCS (1.5 equiv,
9.02 g, 67.5 mmol) was added. The reaction was heated at 80 °C for
3 days, cooled to rt, diluted with EtOAc (100 mL), and then
washed twice with water (20 mL) and once with brine (30 mL).
The combined organic extracts were dried over MgSO4 and
filtered, and then the solvent was removed under reduced pressure.
The crude material was chromatographed on an 80 g SiO2 pre-
packed column eluting with 0:1 to 3:7 EtOAc/hexanes, yielding
1
the title compound as a colorless liquid (8.12 g, 64%). H NMR
(400 MHz, CDCl3) δ: 4.34 (q, J = 7.2 Hz, 2H), 2.91 (s, 2H),
1.71-1.88 (m, 4H), 1.60-1.71 (m, 2H), 1.39-1.53 (m, 4H), 1.37
(t, J = 7.1 Hz, 3H).
Preparation of Spiro-isoxazoline Esters 13-14: General Pro-
cedure. Methyl triphenyl phosphonium bromide/sodium amide
mixture22 (MTP) (1.2 equiv, 6.00 mmol) was suspended in dry
Et2O (6 mL) for 1 h at rt, and then an Et2O (4 mL) solution of the
ketone (5.00 mmol) was added. This mixture was stirred at rt for
16 h and then quenched by addition of H2O (10 mL). The
aqueous phase was extracted with Et2O (2 ꢀ 5 mL), and then the
organic extracts were combined and dried over MgSO4. DIPEA
(1.1 equiv, 5.5 mmol) was added to the dried Et2O solution
(25 mL), and then a DCM (50 mL) solution of ethyl 2-chloro-
2-(hydroxyimino)acetate (ECHA) (1 equiv, 5.00 mmol) was
added dropwise over 7 h. The mixture was stirred at rt for 60 h,
and then the solvent was removed under reduced pressure. The
residue was purified by SiO2 chromatography.
1
an orange solid (6.96 g, 49%). H NMR (400 MHz, CDCl3)
δ: 8.10 (d, J = 2.0 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.57-7.62
(m, 1H), 7.56 (d, J = 1.8 Hz, 1H), 7.44-7.54 (m, 1H), 7.32 (d,
J = 7.3 Hz, 1H), 6.64 (br s, 2H).
3-Bromo-5-nitro-20-(trifluoromethyl)-[1,10-biphenyl]-4-amine (47).
Biphenyl nitroaniline 6 (302 mg, 1.07 mmol) was placed in an 8 mL
vial equipped with a magnetic stir bar, and then glacial AcOH
(2 mL) was added. The solid was allowed to dissolve, and then Br2
(1.07 equiv, 59.0 μL, 1.14 mmol) was added dropwise. The reaction
was stirred at rt for 30 min, during which time a precipitate formed.
The reaction was poured into crushed ice, and the precipitate
was isolated by filtration. The precipitate was washed with H2O
(50 mL), and then dissolved in DCM (40 mL). The solution was
dried over anhydrous MgSO4 and filtered, and then the solvent
removed under reduced pressure. The crude material was chroma-
tographed on a 24 g SiO2 prepacked column eluting with 0:1 to
Ethyl 1,8-Dioxa-2-azaspiro[4.5]dec-2-ene-3-carboxylate (13).
The title compound was prepared from MTP (2.50 g, 6.00 mmol),
dihydro-2H-pyran-4(3H)-one (10) (0.460 mL, 5.00 mmol),
DIPEA (0.96 mL, 5.5 mmol), and ECHA (758 mg, 5.00 mmol)
according to the general procedure yielding a tan oil (264 mg, 25%).
1H NMR (400 MHz, CDCl3) δ: 4.35(q, J = 7.2 Hz, 2H), 3.89 (ddd,
J = 11.8, 8.7, 3.3 Hz, 2H), 3.65-3.75 (m, 2H), 2.99 (s, 2H),
1.89-1.97 (m, 2H), 1.76-1.86 (m, 2H), 1.37 (t, J = 7.2 Hz, 3H).
Ethyl 8,8-Difluoro-1-oxa-2-azaspiro[4.5]dec-2-ene-3-carbox-
ylate (14). The title compound was prepared from MTP (1.95 g,
4.68 mmol), 4,4-difluorocyclohexanone (11) (500 mg, 3.73 mmol),
DIPEA (0.71 mL, 4.10 mmol), and ECHA (565 mg, 3.73 mmol)
according to the general procedure yielding a colorless oil
1
1:4 EtOAc/hexanes, yielding an orange solid (341 mg, 88%). H
1
(391 mg, 42%). H NMR (400 MHz, CDCl3) δ: 4.34 (q, J =
7.2 Hz, 2H), 3.00 (s, 2H), 2.10-2.29 (m, 2H), 1.96-2.09 (m, 4H),
1.81-1.93 (m, 2H), 1.37 (t, J = 7.2 Hz, 3H).
NMR (400 MHz, CDCl3) δ: 8.14 (d, J = 2.0 Hz, 1H), 7.76 (d, J =
7.8 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.51
(t, J = 7.7 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 6.70 (br s, 2H).
3-Iodo-5-nitro-20-(trifluoromethyl)-[1,10-biphenyl]-4-amine (48).
Biphenyl nitroaniline 6 (752 mg, 2.66 mmol) was placed in a
40 mL vial equipped with a magnetic stir bar, and anhydrous
EtOH (27 mL) was added. Iodine (1.4 equiv, 945 mg, 3.72 mmol)
was added as a solid to the stirred solution. Silver sulfate (1.4 equiv,
Preparation of Spiro-isoxazoline Acids 15-17: General Pro-
cedure. The spiro-isoxazoline ester (1 equiv) was dissolved in a
mixture of MeOH and H2O (3:1), and then then LiOH H2O
3
(1.1 equiv) was added as a solid. The reaction was stirred at rt for
16 h, and then the solvent was removed under reduced pressure.

242 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Parks et al.
1.16 g, 3.72 mmol) was added in one portion as a solid, and the
reaction was stirred at rt for 24 h. The reaction was filtered, and
then the solvent was removed under reduced pressure. The residue
was dissolved in DCM (30 mL), washed with 10% aq Na2S2O3
(10 mL), dried over anhydrous MgSO4 and filtered, and then the
solvent was removed under reduced pressure. The crude product
was purified by column chromatography using a 40 g SiO2
prepacked column eluting with 0:1 to 1:4 EtOAc/hexanes, yielding
7.49 (t, J = 7.5 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.32 (d, J =
7.6 Hz, 1H), 6.61 (s, 1H), 6.59 (s, 1H), 3.66 (t, J = 5.9 Hz, 2H),
3.52 (br s, 4H), 2.65 (t, J = 7.3 Hz, 2H), 1.75-1.87 (m, 2H), 0.91
(s, 9H), 0.07 (s, 6H).
Preparation of the Spiro-isoxazoline Benzimidazoles 4, 5,
53-69: General Procedure. The biphenylnitroaniline (1 equiv)
was dissolved in a mixture of EtOH and 3 M HCl (5:1), and then
Fe powder (5 equiv) was added and the reaction was stirred at
80 °C for 4 h. The reaction was cooled to rt and filtered. The
solids were washed with MeOH, and then the combined filtrates
were concentrated under reduced pressure. To the residue was
added 2 M NaOH (10 mL), and then the aqueous mixture was
extracted with EtOAc (3 ꢀ 15 mL). The organic extracts were
combined, dried over MgSO4, and filtered. The solvent was
removed under reduced pressure to afford the biphenyldiamine,
which was used without purification in the next step.
The biphenyldiamine (1.12 equiv) was dissolved in DCM, and
then TEA (3 equiv) was added. A DCM solution of the spiro-
isoxazoline acid chloride was added dropwise over 10 min, and
then the reaction was stirred at rt for 2 h. The solvent was removed
under reduced pressure, and then the residue was purified by column
chromatography to afford a mixture of the two monoamides.
The monoamide mixture was dissolved in dry dioxane, and
then CSA (0.2 equiv) was added and the reaction was stirred at
100 °C for 4 h. The reaction was cooled to rt and then poured
into a saturated aqueous NaHCO3 solution and extracted with
EtOAc (3ꢀ). The combined organic extracts were dried over
MgSO4 and filtered, and then the solvent was removed under
reduced pressure. The residue was purified by chromatography
to afford the desired benzimidazole.
Optionally, a salt form of the benzimidazole was prepared
using the following general procedure: The benzimidazole was
dissolved in EtOAc, and then 1 M HCl in Et2O (1 equiv) was
added. A precipitate immediately formed, which was isolated by
filtration and rinsed once with EtOAc. The residual solvent was
removed under high vacuum to afford the benzimidazole hydro-
chloride salt.
3-[5-(2-Trifluoromethyl-phenyl)-1H-benzimidazol-2-yl]-1-oxa-2-
aza-spiro[4.5]dec-2-ene (4). The title compound was prepared
according to the general procedure from biphenyldiamine 7
(157 mg, 0.626 mmol) and TEA (1.00 mL, 7.17 mmol) dissolved
in DCM (20 mL) by slow addition of acid chloride 18 (0.796 mmol)
in DCM (30 mL), affording a mixture of the monoamides (172 mg,
79%). The monoamide mixture (172 mg, 0.413 mmol) in dioxane
1
an orange solid (902 mg, 83%). H NMR (400 MHz, CDCl3)
δ: 8.17 (d, J = 2.0 Hz, 1H), 7.94 (d, J = 2.0 Hz, 1H), 7.76 (d, J =
7.3 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H),
7.32 (d, J = 7.6 Hz, 1H), 6.74 (br s, 2H).
4-Amino-5-nitro-20-(trifluoromethyl)-[1,10-biphenyl]-3-carbonitrile
(49). Biphenyl nitroaniline 48 (230 mg, 0.564 mmol) and Cu-
(I)CN (1.5 equiv, 7.57 mg, 0.845 mmol) were placed in an 8 mL vial
equipped with a magnetic stir bar. Dry dimethylacetamide (2.5 mL)
was added, and the vial was tightly capped and was stirred at 140 °C
for 14 h. The reaction was cooled to rt and then poured into
water. The precipitate was isolated by filtration and washed with
water (10 mL). The precipitate was dissolved in EtOAc (25 mL),
dried over anhydrous MgSO4, and filtered, and then the solvent
was removed under reduced pressure. The crude product was
purified by preparative TLC on a 2000 μm SiO2 plate developed
with 1:9 EtOAc/hexanes, yielding a yellow solid (86 mg, 50%).
1H NMR (400 MHz, CDCl3) δ: 8.37 (d, J = 1.8 Hz, 1H), 7.79
(d, J = 7.8 Hz, 1H), 7.71 (d, J = 1.5 Hz, 1H), 7.62 (t, J = 7.3 Hz,
1H), 7.55 (t, J = 7.7 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 6.84
(br s, 2H).
3-(4-Amino-5-nitro-20-(trifluoromethyl)-[1,10-biphenyl]-3-yl)-
prop-2-yn-1-ol (50). Biphenyl nitroaniline 48 (466 mg, 1.14 mmol),
(Ph3P)2PdCl2 (0.05 equiv, 40.1 mg, 0.057 mmol), and CuI
(0.05 equiv, 10.2 mg, 0.054 mmol) were placed in a 40 mL vial
equipped with a magnetic stir bar. The vial was evacuated and
backflushed with argon, and then anhydrous THF (6 mL) and TEA
(4.0 equiv, 0.64 mL, 4.56 mmol) were added. Propargyl alcohol
(4 equiv, 0.270 mL, 4.56 mmol) was added, and then the reaction
was stirred at rt for 16 h. The solution was diluted with EtOAc
(20 mL) and filtered. The solvent was removed under reduced
pressure, and the crude product was purified by column chroma-
tography on a 24 g SiO2 prepacked column eluting with 0:1 to
3:2 EtOAc/hexanes, yielding the title compound (279 mg, 73%). 1H
NMR (400 MHz, CDCl3) δ: 8.10 (d, J = 2.0 Hz, 1H), 7.73 (d, J =
7.6 Hz, 1H), 7.53-7.59 (m, 1H), 7.53 (d, J = 1.8 Hz, 1H),
7.43-7.51 (m, 1H), 7.29 (d, J = 7.6 Hz, 1H), 6.82 (br s, 2H), 4.59
(s, 2H).
3-(4,5-Diamino-20-(trifluoromethyl)-[1,10-biphenyl]-3-yl)-
propan-1-ol (51). Biphenyl nitroaniline 50 (148 mg, 0.440 mmol)
was placed in an 8 mL vial equipped with a magnetic stir bar, and
then dry EtOH (2 mL) was added. To the ethanol solution was
added 10% Pd on activated carbon (27 mg), and then the reaction
was stirred under an atmosphere of H2 at balloonpressure for 16 h.
The H2 was released, and then the reaction was filtered. The filter
cake was washed three times with MeOH (5 mL). The filtrates were
combined then the solvent was removed under reduced pressure to
afford the title compound (101 mg, 73%). 1H NMR (400 MHz,
CDCl3) δ: 7.68 (d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.3 Hz, 1H), 7.38
(t, J = 7.6 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 6.61 (s, 1H), 6.60
(s, 1H), 3.65 (t, J = 5.9 Hz, 2H), 3.48 (br s, 4H), 2.66 (t, J =
7.3 Hz, 2H), 1.86 (quin, J = 6.6 Hz, 2H).
(6 mL) was treated with TsOH H2O (78.5 mg, 0.413 mmol) at
3
80 °C for 14 h, yielding a white foam (67.8 mg, 41%). 1H NMR
(400 MHz, CD3OD) δ: 7.79 (d, J = 7.8 Hz, 1H), 7.65 (td, J =
7.6, 0.8 Hz, 1H), 7.64 (br s, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.54
(br s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 8.1 Hz, 1H), 3.29
(s, 2H), 1.71-1.91 (m, 6H), 1.48-1.64 (m, 4H). HRMS calcd for
C22H20F3N3O (M þ H)þ 400.1637, found 400.1629.
3-[7-Trifluoromethyl-5-(2-trifluoromethyl-phenyl)-1H-benzimid-
azol-2-yl]-1-oxa-2-aza-spiro[4.5]dec-2-ene Hydrochloride (5).The title
compound was prepared according to the general procedure from
nitroaniline 43 (330 mg, 0.942 mmol) and Fe powder (263 mg,
4.71 mmol) in EtOH (5 mL) and 3 M HCl (1 mL) in the first step,
and then the resulting biphenyldiamine (0.942 mmol) and TEA
(0.330 mL, 2.39 mmol) dissolved in DCM (50 mL) was treated with
acid chloride 18 (0.796 mmol) in DCM (30 mL), affording a mixture
of the monoamides (272 mg, 70%). The monoamide mixture
(272 mg, 0.560 mmol) in dioxane (10 mL) was treated with CSA
(26.0 mg, 0.112 mmol) at 100 °C for 3 h, yielding a white foam
(255 mg, 97%). Treatment of the benzimidazole (255 mg,
0.545 mmol) in EtOAc (3 mL) with 1 M HCl in Et2O (0.55 mL,
0.550 mmol) afforded the title compound as a white powder
(241 mg, 88%). 1H NMR (400 MHz, DMSO-d6) δ: 7.89 (d, J =
7.6 Hz, 1H), 7.74-7.81 (m, 1H), 7.71 (s, 1H), 7.65-7.70 (m, 1H),
7.56 (d, J = 7.6 Hz, 1H), 7.48 (s, 1H), 3.33 (s, 2H), 1.63-1.82
(m, 6H), 1.35-1.55 (m, 4H). HRMS calcd for C23H19F6N3O (M þ
H)þ 468.1511, found 468.1515.
5-(3-((tert-Butyldimethylsilyl)oxy)propyl)-20-(trifluoromethyl)-
[1,10-biphenyl]-3,4-diamine (52). Biphenyldiamine 51 (101 mg,
0.324 mmol) was dissolved in DCM (2 mL), and then imidazole
(1.1 equiv, 24.5 mg, 0.356 mmol) and TBSCl (1.1 equiv, 53.7 mg,
0.356 mmol) were added sequentially. The reaction was stirred
at rt for 2 h. The reaction was filtered, the precipitate was washed
once with DCM (5 mL), and the filtrate was concentrated under
reduced pressure. The crude product was purified by column
chromatography on a 24 g SiO2 prepacked column eluting with
0:1 to 1:1 EtOAc/hexanes, yielding the title compound (93.2 mg,
68%). 1H NMR (400 MHz, CDCl3) δ: 7.69 (d, J = 7.8 Hz, 1H),

Article
3-[5-(2-Fluorophenyl)-1H-benzimidazol-2-yl]-1-oxa-2-aza-spiro-
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 243
1.0 Hz, 1H), 7.76 (d, J= 8.6 Hz, 1H), 7.67 (dd, J=8.5, 1.6Hz, 1H),
7.55 (dd, J = 8.0, 1.1 Hz, 1H), 7.41 (dd, J = 8.1, 1.3 Hz, 1H), 7.34
(t, J = 8.1 Hz, 1H), 1.63-1.82 (m, 6H), 1.37-1.55 (m, 4H). HRMS
calcd for C22H19F2N3O3 (M þ H)þ 412.1473, found 412.1486.
3-[5-(2,6-Difluorophenyl)-1H-benzimidazol-2-yl]-1-oxa-2-aza-
spiro[4.5]dec-2-ene Trifluoroacetic Acid Salt (57). The title com-
pound was prepared according to the general procedure from
nitroaniline 38 (204 mg, 0.815 mmol) and Fe powder (228 mg,
4.08 mmol) in EtOH (5 mL) and 3 M HCl (1.5 mL) in the first
step, and then the resulting biphenyldiamine (0.762 mmol) and
TEA (0.290 mL, 2.08 mmol) dissolved in DCM (30 mL) was
treated with acid chloride 18 (0.508 mmol) in DCM (20 mL),
affording a mixture of the monoamides (154 mg, 79%). The
monoamide mixture (154 mg, 0.400 mmol) in dioxane (4 mL)
was treated with CSA (19.0 mg, 0.080 mmol) at 100 °C for 4 h.
The product was further purified by HPLC, affording the title
[4.5]dec-2-ene Trifluoroacetic Acid Salt (53). The title compound
was prepared according to the general procedure from nitroaniline
34 (175 mg, 0.753 mmol) and Fe powder (210 mg, 3.76 mmol) in
EtOH (5 mL) and 3 M HCl (1.5 mL) in the first step, and then the
resulting biphenyldiamine (0.942 mmol) and TEA (0.280 mL,
2.03 mmol) dissolved in DCM (30 mL) was treated with acid
chloride 18 (0.676 mmol) in DCM (20 mL), affording a mixture of
the monoamides (199 mg, 80%). The monoamide mixture (199 mg,
0.543 mmol) in dioxane (6 mL) was treated with CSA (25.0 mg,
0.109 mmol) at 100 °C for 4 h. The product was further purified by
HPLC, affording the titlecompound as the trifluoroacetic acid salt
(122 mg, 48%). 1H NMR (400 MHz, DMSO-d6) δ: 7.76 (s, 1H),
7.71 (d, J = 8.3 Hz, 1H), 7.59 (td, J = 8.0, 1.5 Hz, 1H), 7.47-7.51
(m, 1H), 7.38-7.47 (m, 1H), 7.27-7.38 (m, 2H), 3.30 (s, 2H),
1.63-1.80 (m, 6H), 1.34-1.56 (m, 4H). HRMS calcd for
C21H20FN3O (M þ H)þ 350.1669, found 350.1672.
1
compound as the trifluoroacetic acid salt (121 mg, 63%). H
3-[5-(2-Trifluoromethoxyphenyl)-1H-benzimidazol-2-yl]-1-oxa-2-
aza-spiro[4.5]dec-2-ene Hydrochloride (54). The title compound was
prepared according to the general procedure from nitroaniline 35
(299 mg, 1.00 mmol) and Fe powder (279 mg, 5.00 mmol) in EtOH
(5 mL) and 3 M HCl (1 mL) in the first step, and then the resulting
biphenyldiamine (0.932 mmol) and TEA (0.350 mL, 2.50 mmol)
dissolved in DCM (50 mL) was treated with acid chloride 18 (0.832
mmol) in DCM (10 mL), affording a mixture of the monoamides
(339 mg, 94%). The monoamide mixture (339 mg, 0.783 mmol) in
dioxane (6 mL) was treated with CSA (36.0 mg, 0.157 mmol) at
100 °C for 4 h, yielding a white foam (146 mg, 45%). Treatment of
the benzimidazole (125 mg, 0.301 mmol) in EtOAc (2 mL) with 1 M
HCl in Et2O (0.30 mL, 0.300 mmol) afforded the title compound as
a white powder (124 mg, 91%). 1H NMR (400 MHz, DMSO-d6)
δ: 7.72 (d, J = 8.6 Hz, 1H), 7.70 (s, 1H), 7.57-7.63 (m, 1H),
7.47-7.56 (m, 3H), 7.42 (dd, J = 8.3, 1.3 Hz, 1H), 3.31 (s, 2H),
1.62-1.80 (m, 6H), 1.34-1.57 (m, 4H). HRMS calcd for
C22H20F3N3O2 (M þ H)þ 416.1586, found 416.1586.
NMR (400 MHz, DMSO-d6) δ: 7.70 (d, J = 8.3 Hz, 1H), 7.66
(s, 1H), 7.43-7.54 (m, 1H), 7.33 (dd, J = 8.6, 1.3 Hz, 1H),
7.19-7.29 (m, 2H), 3.30 (s, 2H), 1.63-1.79 (m, 6H), 1.35-1.55
(m, 4H). HRMS calcd for C21H19F2N3O (M þ H)þ 368.1574,
found 368.1590.
3-[5-(2-Fluoro-6-trifluoromethylphenyl)-1H-benzimidazol-2-yl]-1-
oxa-2-aza-spiro[4.5]dec-2-ene (58). The title compound was pre-
pared according to a modified general procedure from nitroaniline
39 (754mg, 2.51mmol), NH4Cl (1.34 g, 25.1 mmol), and Fe powder
(701 mg, 12.6 mmol) in EtOH (20 mL) and H2O (5 mL) stirred at
80 °C for 14 h, affording the biphenyldiamine (662 mg, 98%) in
the first step, and then the resulting biphenyldiamine (258 mg,
0.953 mmol) and TEA (0.360 mL, 2.55 mmol) dissolved in DCM
(50 mL) was treated with acid chloride 18 (0.851 mmol) in DCM
(10 mL), affording a mixture of the monoamides (315 mg, 85%).
The monoamide mixture (315 mg, 0.723 mmol) in dioxane (6 mL)
was treated with CSA (34.0 mg, 0.145 mmol) at 100 °C for
1
4 h, yielding a white foam (151 mg, 50%). H NMR (400 MHz,
DMSO-d6 þ TFA-d1) δ: 7.63-7.77 (m, 4H), 7.57 (s, 1H), 7.23
(d, J = 8.3 Hz, 1H), 3.31 (s, 2H), 1.63-1.85 (m, 6H), 1.34-1.59
(m, 4H). HRMS calculated for C22H19F4N3O (M þ H)þ 418.1543,
found 418.1552.
3-[5-(2-Difluoromethoxyphenyl)-1H-benzimidazol-2-yl]-1-oxa-
2-aza-spiro[4.5]dec-2-ene Hydrochloride (55). The title compound
was prepared according to the general procedure from nitroani-
line 36 (163 mg, 0.583 mmol) and Fe powder (163 mg, 2.92 mmol)
in EtOH (3 mL) and 3 M HCl (0.6 mL) in the first step, and then
the resulting biphenyldiamine (0.561 mmol) and TEA (0.210 mL,
1.53 mmol) dissolved in DCM (30 mL) was treated with acid
chloride18(0.510 mmol) inDCM (20mL), affording a mixture of
the monoamides (163 mg, 77%). The monoamide mixture (163 mg,
0.392 mmol) in dioxane (2 mL) was treated with CSA (18.0 mg,
0.078 mmol) at 100 °C for 3 h, yielding a white foam (80.4 mg,
52%). Treatment of the benzimidazole (80.4 mg, 0.202 mmol) in
EtOAc (2 mL) with 1 M HCl in Et2O (0.200 mL, 0.200 mmol)
afforded the title compound as a white powder (78.7 mg, 89%).
1H NMR (400 MHz, DMSO-d6) δ: 7.72 (d, J = 1.0 Hz, 1H), 7.71
(d, J = 5.3 Hz, 1H), 7.48-7.55 (m, 1H), 7.47 (dd, J = 5.6, 1.8 Hz,
1H), 7.45 (dd, J = 6.1, 1.5 Hz, 1H), 7.37-7.41 (m, 1H), 7.33 (d,
J = 8.3 Hz, 1H), 7.16 (t, J = 74 Hz, 1H), 3.32 (s, 2H), 1.63-1.83
(m, 6H), 1.37-1.56 (m, 4H). HRMS calcd for C22H21F2N3O2
(M þ H)þ 398.1680, found 398.1679.
3-[5-(2-Fluoro-6-trifluoromethoxyphenyl)-1H-benzimidazol-2-yl]-
1-oxa-2-aza-spiro[4.5]dec-2-ene Hydrochloride (59). The title com-
pound was prepared according to the general procedure from nitro-
aniline 40 (285 mg, 0.901 mmol) and Fe powder (252 mg,
4.51 mmol) in EtOH (5 mL) and 3 M HCl (1 mL) in the first step,
and then the resulting biphenyldiamine (0.831 mmol) and TEA
(0.310 mL, 2.23 mmol) dissolved in DCM (50 mL) was treated with
acid chloride 18 (0.742 mmol) in DCM (10 mL), affording a mixture
of the monoamides (269 mg, 80%). The monoamide mixture
(269 mg, 0.595 mmol) in dioxane (6 mL) was treated with CSA
(28.0 mg, 0.119 mmol) at 100 °C for 4 h, yielding a white
foam (121 mg, 47%). Treatment of the benzimidazole (113 mg,
0.261 mmol) in EtOAc (2 mL) with 1 M HCl in Et2O (0.260 mL,
0.260 mmol) afforded the title compound as a white powder
(112 mg, 91%). 1H NMR (400 MHz, DMSO-d6) δ: 7.72 (d, J =
8.3 Hz, 1H), 7.63 (s, 1H), 7.55-7.62 (m, 1H), 7.45 (t, J = 8.8 Hz,
1H), 7.40 (d, J = 8.3 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 3.31 (s, 2H),
1.63-1.80 (m, 6H), 1.33-1.57 (m, 4H). HRMS calcd for
C22H19F4N3O2 (M þ H)þ 434.1492, found 434.1494.
3-[5-(2,2-Difluorobenzo[1,3]dioxol-4-yl)-1H-benzimidazol-2-yl]-1-
oxa-2-aza-spiro[4.5]dec-2-ene Hydrochloride (56). The title com-
pound was prepared according to the general procedure from
nitroaniline 37 (217 mg, 0.738 mmol) and Fe powder (206 mg,
3.69 mmol) in EtOH (4 mL) and 3 M HCl (0.8 mL) in the first step,
and then the resulting biphenyldiamine (0.561 mmol) and TEA
(0.210 mL, 1.53 mmol) dissolved in DCM (30 mL) was treated with
acid chloride 18 (0.510 mmol) in DCM (20 mL), affording a mixture
of the monoamides (172 mg, 79%). The monoamide mixture
(172 mg, 0.399 mmol) in dioxane (2 mL) was treated with CSA
(19.0 mg, 0.080 mmol) at 100 °C for 3 h, yielding a white foam
(120 mg, 73%). Treatment of the benzimidazole (120 mg,
0.291 mmol) in EtOAc (2 mL) with 1 M HCl in Et2O (0.290 mL,
0.290 mmol) afforded the title compound as a white powder
(111 mg, 85%). 1H NMR (400 MHz, DMSO-d6) δ: 7.95 (d, J =
3-[7-Fluoro-5-(2-trifluoromethylphenyl)-1H-benzimidazol-
2-yl]-1-oxa-2-aza-spiro[4.5]dec-2-ene Hydrochloride (60). The title
compound was prepared according to the general procedure from
nitroaniline 41 (300 mg, 1.00 mmol) and Fe powder (279 mg,
5.00 mmol) in EtOH (5 mL) and 3 M HCl (1 mL) in the first step,
and then the resulting biphenyldiamine (0.561 mmol) and TEA
(0.210 mL, 1.53 mmol) dissolved in DCM (30 mL) was treated with
acid chloride 18 (0.510 mmol) in DCM (20 mL), affording a mixture
of the monoamides (140 mg, 63%). The monoamide mixture
(140 mg, 0.322 mmol) in dioxane (2 mL) was treated with CSA
(15.0 mg, 0.064 mmol) at 100 °C for 3 h, yielding a white foam
(116 mg, 86%). Treatment of the benzimidazole (116 mg,

244 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Parks et al.
0.278 mmol) in EtOAc (2 mL) with 1 M HCl in Et2O (0.280 mL,
0.280 mmol) afforded the title compound as a white powder
(110 mg, 87%). 1H NMR (400 MHz, DMSO-d6) δ: 7.86 (d, J =
7.8 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.50
(d, J = 7.6 Hz, 1H), 7.26 (s, 1H), 7.03 (d, J = 11.4 Hz, 1H), 3.31
(s, 2H), 1.60-1.81 (m, 6H), 1.29-1.58 (m, 4H). HRMS calcd for
C22H19F4N3O (M þ H)þ 418.1543, found 418.1559.
4.45 mmol) in EtOH (5 mL) and 3 M HCl (1 mL) in the first step,
and then the resulting biphenyldiamine (0.892 mmol) and TEA
(0.330 mL, 2.39 mmol) dissolved in DCM (50 mL) was treated with
acid chloride 18 (0.796 mmol) in DCM (30 mL), affording a mixture
of the monoamides (256 mg, 74%). The monoamide mixture
(256 mg, 0.589 mmol) in dioxane (10 mL) was treated with CSA
(27.3 mg, 0.118 mmol) at 100 °C for 3 h, yielding a white foam
(242 mg, 98%). Treatment of the benzimidazole (242 mg,
0.579 mmol) in EtOAc (3 mL) with 1 M HCl in Et2O (0.580 mL,
0.580 mmol) afforded the title compound as a white powder
(211 mg, 80%). 1H NMR (400 MHz, DMSO-d6) δ: 7.92 (s, 1H),
7.71 (s, 1H), 7.62-7.70 (m, 1H), 7.44-7.52 (m, 1H), 7.32-7.41 (m,
2H), 3.33 (s, 2H), 1.61-1.83 (m, 6H), 1.35-1.59 (m, 4H). HRMS
calcd for C22H19F4N3O (M þ H)þ 418.1543, found 418.1542.
3-[5-(2-Trifluoromethoxyphenyl)-7-trifluoromethyl-1H-benz-
imidazol-2-yl]-1-oxa-2-aza-spiro[4.5]dec-2-ene Hydrochloride (65).
The title compound was prepared according to the general pro-
cedure from nitroaniline 44 (326 mg, 0.890 mmol) and Fe powder
(249 mg, 4.45 mmol) in EtOH (5 mL) and 3 M HCl (1 mL) in
the first step, and then the resulting biphenyldiamine (294 mg,
0.876 mmol) and TEA (0.330 mL, 2.39 mmol) dissolved in DCM
(50 mL) was treated with acid chloride 18 (0.796 mmol) in DCM
(30 mL), affording a mixture of the monoamides (276 mg, 69%).
The monoamide mixture (276 mg, 0.550 mmol) in dioxane (10 mL)
was treated with CSA (25.6 mg, 0.110 mmol) at 100 °C for 3 h,
yielding a white foam (252 mg, 95%). Treatment of the benzimid-
azole (252 mg, 0.521 mmol) in EtOAc (3 mL) with 1 M HCl in
Et2O (0.520 mL, 0.520 mmol) afforded the title compound as a
white powder (232 mg, 86%). 1H NMR (400 MHz, DMSO-d6)
δ: 7.86 (s, 1H), 7.66-7.73 (m, 1H), 7.64 (s, 1H), 7.50-7.62 (m,
3H), 3.33 (s, 2H), 1.62-1.83 (m, 6H), 1.34-1.61 (m, 4H). HRMS
calcd for C23H19F6N3O2 (M þ H)þ 484.1460, found 484.1458.
3-[5-(2-Chlorophenyl)-7-trifluoromethyl-1H-benzimidazol-2-yl]-1-
oxa-2-aza-spiro[4.5]dec-2-ene Hydrochloride (66). The title com-
pound was prepared according to the general procedure from
nitroaniline 45 (291 mg, 0.918 mmol) and Fe powder (256 mg,
4.59 mmol) in EtOH (5 mL) and 3 M HCl (1 mL) in the first step,
and then the resulting biphenyldiamine (266 mg, 0.929 mmol) and
TEA (0.330 mL, 2.39 mmol) dissolved in DCM (50 mL) was treated
with acid chloride 18 (0.796 mmol) in DCM (30 mL), affording a
mixture of the monoamides (233 mg, 65%). The monoamide
mixture (233 mg, 0.517 mmol) in dioxane (10 mL) was treated with
CSA (24.0 mg, 0.103 mmol) at 100 °C for 3 h, yielding a white
foam (156 mg, 70%). Treatment of the benzimidazole (156 mg,
0.359 mmol) in EtOAc (2 mL) with 1 M HCl in Et2O (0.360 mL,
0.360 mmol) afforded the title compound as a white powder
3-[7-Chloro-5-(2-trifluoromethylphenyl)-1H-benzimidazol-2-yl]-1-
oxa-2-aza-spiro[4.5]dec-2-ene Hydrochloride (61). The title com-
pound was prepared according to the general procedure from nitro-
aniline 46 (161 mg, 0.508 mmol) and Fe powder (142 mg,
2.54 mmol) in EtOH (3 mL) and 3 M HCl (0.6 mL) in the first step,
and then the resulting biphenyldiamine (140 mg, 0.490 mmol) and
TEA (0.190 mL, 1.34 mmol) dissolved in DCM (30 mL) was treated
with acid chloride 18 (0.445 mmol) in DCM (20 mL), affording a
mixture of the monoamides (145 mg, 72%). The monoamide
mixture (145 mg, 0.321 mmol) in dioxane (3 mL) was treated with
CSA (15.0 mg, 0.064 mmol) at 100 °C for 3 h, yielding a white foam
(116 mg, 84%). Treatment of the benzimidazole (116 mg, 0.268
mmol) in EtOAc (2 mL) with 1 M HCl in Et2O (0.270 mL, 0.270
mmol) afforded the title compound as a white powder (103 mg,
1
82%). H NMR (400 MHz, DMSO-d6) δ: 13.52 (br s, 1H), 7.86
(d, J = 7.6 Hz, 1H), 7.75 (t, J = 7.5 Hz, 1H), 7.65 (t, J = 7.6 Hz,
1H), 7.51 (d, J = 7.6 Hz, 1H), 7.35 (s, 1H), 7.25 (s, 1H), 1.63-1.82
(m, 6H), 1.34-1.60 (m, 4H). HRMS calcd for C22H19ClF3N3O
(M þ H)þ 434.1247, found 434.1258.
2-(1-Oxa-2-aza-spiro[4.5]dec-2-en-3-yl)-6-(2-trifluoromethyl-
phenyl)-3H-benzimidazole-4-carbonitrile Hydrochloride (62). The
title compound was prepared according to a modified general pro-
cedure from nitroaniline 49 (86.1 mg, 0.280 mmol), NH4Cl (150 mg,
2.80 mmol), and Fe powder (78.0 mg, 1.40 mmol) in EtOH (3 mL)
and H2O (1 mL) stirred at 80 °C for 1 h, affording the biphenyldi-
amine (76.0 mg, 98%) in the first step, and then the resulting
biphenyldiamine (76.0 mg, 0.274 mmol) and TEA (0.100 mL,
0.747 mmol) dissolved in DCM (25 mL) was treated with acid
chloride 18 (0.249 mmol) in DCM (15 mL), affording a mixture
of the monoamides (77.7 mg, 71%). The monoamide mixture
(77.7 mg, 0.176 mmol) in dioxane (2 mL) was treated with CSA
(18.0 mg, 0.077 mmol) at 100 °C for 3 h, yielding a white
foam (40.8 mg, 54%). Treatment of the benzimidazole (40.8 mg,
0.096 mmol) in EtOAc (2 mL) with 1 M HCl in Et2O (0.096 mL,
0.096 mmol) afforded the title compound as a white powder
(23.7 mg, 54%). 1H NMR (400 MHz, DMSO-d6) δ:7.88(d, J=7.6
Hz, 1H), 7.74-7.81 (m, 1H), 7.71 (s, 2H), 7.64-7.70 (m, 1H), 7.53
(d, J = 7.6 Hz, 1H), 3.35 (s, 2H), 1.63-1.82 (m, 6H), 1.31-1.59
(m, 4H). HRMS calcd for C23H19F3N4O (M þ H)þ 425.1589,
found 425.1589.
1
(115 mg, 68%). H NMR (400 MHz, DMSO-d6) δ: 13.52 (br s,
1H), 7.86 (d, J = 7.6 Hz, 1H), 7.75 (t, J = 7.5 Hz, 1H), 7.65 (t, J =
7.6 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.35 (s, 1H), 7.25 (s, 1H), 3.33
(s, 2H), 1.63-1.82 (m, 6H), 1.33-1.59 (m, 4H). HRMS calcd for
C22H19ClF3N3O (M þ H)þ 434.1247, found 434.1245.
3-[7-Bromo-5-(2-trifluoromethylphenyl)-1H-benzimidazol-2-yl]-1-
oxa-2-aza-spiro[4.5]dec-2-ene Hydrochloride (63). The title com-
pound was prepared according to the general procedure from nitro-
aniline 47 (98.2 mg, 0.272 mmol) and Fe powder (76.0 mg,
1.36 mmol) in EtOH (2 mL) and 3 M HCl (0.4 mL) in the first step,
and then the resulting biphenyldiamine (89.1 mg, 0.269 mmol) and
TEA (0.100 mL, 0.735 mmol) dissolved in DCM (15 mL) was
treated with acid chloride 18 (0.245 mmol) in DCM (10 mL),
affording a mixture of the monoamides (87.9 mg, 72%). The mono-
amide mixture (87.9 mg, 0.177 mmol) in dioxane (2 mL) was treated
with CSA (8.0 mg, 0.035 mmol) at 100 °C for 3 h, yielding a white
foam (69.2 mg, 82%). Treatment of the benzimidazole (69.2 mg,
0.145 mmol) in EtOAc (2 mL) with 1 M HCl in Et2O (0.150 mL,
0.150 mmol) afforded the title compound as a white powder
(49.5 mg, 66%). 1H NMR (400 MHz, DMSO-d6) δ: 7.86 (d, J =
7.1 Hz, 1H), 7.75 (t, J = 7.3 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.51
(d, J = 7.6 Hz, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 3.33 (s, 2H),
1.62-1.81 (m, 6H), 1.32-1.59 (m, 4H). HRMS calcd for
C22H19BrF3N3O (M þ H)þ 478.0742, found 478.0740.
3-[2-(1-Oxa-2-aza-spiro[4.5]dec-2-en-3-yl)-6-(2-trifluoromethyl-
phenyl)-3H-benzimidazol-4-yl]-propan-1-ol Hydrochloride (67).
The title compound was prepared according to the general pro-
cedure from biphenyldiamine 52 (93.2 mg, 0.220 mmol) and
TEA (0.0840 mL, 0.600 mmol) dissolved in DCM (20 mL) by
slow addition of acid chloride 18 (0.200 mmol) in DCM (10 mL),
affording a mixture of the monoamides, which was used as crude
material in the next step. The monoamide mixture (0.200 mmol)
in dioxane (6 mL) was treated with CSA (9.0 mg, 0.040 mmol) at
100 °C for 3 h, during which time the silyl ether was cleaved
concurrently with the cyclization, affording the hydroxypropyl-
benzimidazole (51.3 mg, 56%). Treatment of the benzimidazole
(51.3 mg, 0.112 mmol) in EtOAc (2 mL) with 1 M HCl in Et2O
(0.110 mL, 0.110 mmol) afforded the title compound as a white
powder (48.6 mg, 88%). 1H NMR (400 MHz, DMSO-d6) δ: 7.85
(d, J = 7.6 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.63 (t, J = 7.6 Hz,
1H), 7.48 (d, J = 7.3 Hz, 1H), 7.37 (s, 1H), 7.08 (s, 1H), 3.45
(t, J = 6.6 Hz, 2H), 3.33 (s, 2H), 3.01 (t, J = 7.5 Hz, 2H),
1.79-1.90 (m, 2H), 1.61-1.79 (m, 6H), 1.31-1.57 (m, 4H).
3-[5-(2-Fluorophenyl)-7-trifluoromethyl-1H-benzimidazol-2-yl]-1-
oxa-2-aza-spiro[4.5]dec-2-ene Hydrochloride (64). The title com-
pound was prepared according to the general procedure from nitro-
aniline 42 (268 mg, 0.892 mmol) and Fe powder (249 mg,

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 245
1
HRMS calcd for C25H26F3N3O2 (M þ H)þ 458.2055, found
458.2055.
foam (107 mg, 69%). H NMR (400 MHz, CD3OD) δ: 7.86
(d, J = 7.6 Hz, 1H), 7.83 (s, 1H), 7.69-7.76 (m, 1H), 7.59-7.67
(m, 2H), 7.49 (d, J = 7.6 Hz, 1H), 3.88-3.97 (m, 2H), 3.72-3.81
(m, 2H), 3.47 (s, 2H), 1.94-2.00 (m, 4H). HRMS calcd for
C22H17F6N3O2 (M þ H)þ 470.1303, found 470.1301.
3-[5-(2-Trifluoromethylphenyl)-1H-benzimidazol-2-yl]-1,8-dioxa-
2-aza-spiro[4.5]dec-2-ene (68). The title compound was prepared
according to the general procedure from biphenyldiamine 7
(131 mg, 0.520 mmol) and TEA (0.19 mL, 1.39 mmol) dissolved
in DCM (50 mL) by slow addition of acid chloride 19
(0.464 mmol) in DCM (15 mL), affording a mixture of the
monoamides (133 mg, 68%). The monoamide mixture (133 mg,
0.317 mmol) in dioxane (6 mL) was treated with CSA (15.0 mg,
0.063 mmol) at 100 °C for 4 h, yielding a white foam (27.6 mg,
3-[7-Chloro-5-(2-trifluoromethyl-phenyl)-1H-benzimidazol-2-
yl]-1,8-dioxa-2-aza-spiro[4.5]dec-2-ene (71). The title compound
was prepared according to the general procedure from nitroani-
line 46 (174 mg, 0.549 mmol) and NaH (65.8 mg of 60% mineral
oil dispersion, 1.65 mmol) in THF (10 mL) at 0 °C and acid
chloride 19 (0.604 mmol) in THF (5 mL), affording the nitro-
amide (126 mg, 48%). The nitroamide (126 mg, 0.260 mmol) in a
mixture of AcOH (8 mL) and MeOH (2 mL) was treated with Fe
powder (72.7 mg, 1.30 mmol) at 100 °C for 4 h, yielding a
colorless glass (103 mg, 91%). Treatment of the benzimidazole
(103 mg, 0.235 mmol)) in EtOH (2 mL) with 5 M HCl in IPA
(47.0 μL, 0.235 mmol) afforded the title compound as a colorless
1
22%). H NMR (400 MHz, DMSO-d6 þ TFA-d1) δ: 7.86 (d,
J = 7.6 Hz, 1H), 7.71-7.78 (m, 1H), 7.68 (d, J = 8.3 Hz, 1H),
7.60-7.67 (m, 1H), 7.55 (s, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.26
(d, J = 9.1 Hz, 1H), 3.75-3.87 (m, 2H), 3.54-3.68 (m, 2H), 3.42
(s, 2H), 1.79-1.94 (m, 4H). HRMS calcd for C22H18F5N3O
(M þ H)þ 402.1429, found 402.1432.
1
foam (50.0 mg, 45%). H NMR (400 MHz, CD3OD) δ: 7.84
8,8-Difluoro-3-[5-(2-trifluoromethylphenyl)-1H-benzimidazol-2-
yl]-1-oxa-2-aza-spiro[4.5]dec-2-ene (69). The title compound was
prepared according to the general procedure from biphenyldi-
amine 7 (97.5 mg, 0.386 mmol) and TEA (0.14 mL, 1.04 mmol)
dissolved in DCM (50 mL) by slow addition of acid chloride 20
(0.345 mmol) in DCM (15 mL), affording a mixture of the
monoamides (103 mg, 66%). The monoamide mixture (103 mg,
0.228 mmol) in dioxane (1 mL) was treated with CSA (11.0 mg,
0.046 mmol) at 100 °C for 4 h, yielding a white foam (53.5 mg,
54%). 1H NMR (400 MHz, DMSO-d6 þ TFA-d1) δ: 7.85 (d, J =
7.8 Hz, 1H), 7.71-7.78 (m, 1H), 7.69 (d, J = 8.3 Hz, 1H),
7.59-7.67 (m, 1H), 7.56 (s, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.27
(d, J = 8.3 Hz, 1H), 3.45 (s, 2H), 1.85-2.23 (m, 8H). HRMScalcd
for C22H18F5N3O (M þ H)þ 436.1448, found 436.1462.
(d, J = 7.6 Hz, 1H), 7.71 (t, J = 7.3 Hz, 1H), 7.63 (t, J = 7.6 Hz,
1H), 7.57 (br s, 1H), 7.47 (d, J = 6.8 Hz, 2H), 3.87-3.97 (m, 2H),
3.73-3.82 (m, 2H), 3.45 (s, 2H), 1.95-2.01 (m, 4H). HRMS
calcd for C21H17ClF3N3O2 (M þ H)þ 436.1040, found 436.1037.
3-(5-Bromo-1H-benzo[d]imidazol-2-yl)-1-oxa-2-azaspiro[4.5]dec-
2-ene (73). 4-Bromobenzene-1,2-diamine (72) (377 mg, 2.02 mmol)
was dissolved in DCM (100 mL) then TEA (0.65 mL, 4.65 mmol)
was added. A DCM (50 mL) solution of acid chloride 18
(1.55 mmol) was added dropwise over 10 min, and then the
reaction was stirred at rt for 1 h. The solvent was removed
under reduced pressure, and then the residue was purified by
column chromatography using an 80 g SiO2 prepacked col-
umn eluting with 0:1-2:3 EtOAc/hexanes to afford a mixture
of the two monoamides (460 mg, 84%). The monoamide
mixture (460 mg, 1.31 mmol) was dissolved in anhydrous
dioxane (10 mL), and then CSA (61.0 mg, 0.261 mmol) was
added and the reaction was stirred at 100 °C for 3 h. The reaction
was cooled to rt, the acid was quenched with TEA (0.1 mL), and
then the reaction mixture was purified by chromatography on a
40 g SiO2 prepacked column eluting with 0:1-2:3 EtOAc/hexanes
to afford the title compound (299 mg, 68%). 1H NMR (400 MHz,
CD3OD) δ:7.74(s, 1H), 7.51(d, J= 8.6 Hz, 1H), 7.40 (dd, J= 8.6,
1.8 Hz, 1H), 3.25 (s, 2H), 1.69-1.88 (m, 6H), 1.43-1.63 (m, 4H).
3-[5-(2-Chlorophenyl)-1H-benzimidazol-2-yl]-1-oxa-2-aza-spiro-
[4.5]dec-2-ene Hydrochloride (74). The title compound was pre-
pared from 73 (148 mg, 0.442 mmol), 2-chlorophenylboronic acid
Preparation of the Spiro-isoxazoline Benzimidazoles 70, 71:
General Procedure. The biphenylnitroaniline (1 equiv) in dry
THF (50 mL) was cooled to 0 °C in an ice bath, and then NaH
(3 equiv, 60% dispersion in oil) was added in small portions.
Spiro-isoxazoline acid chloride 19 (1.1 equiv) in dry THF
(20 mL) was added dropwise to the reaction mixture over
10 min, and then the mixture was stirred for 30 min at 0 °C,
warmed to rt, and stirred for an additional 16 h. The mixture
was quenched with satd aq NH4Cl solution and extracted
three times with EtOAc. The combined organic extracts were
dried over MgSO4 and filtered. The solvent was removed
under reduced pressure, and then the residue was purified by
column chromatography to afford the nitroamide. The ni-
troamide was dissolved in a mixture of AcOH and MeOH
(4:1) and treated with Fe powder (5 equiv). The mixture was
heated to 100 °C for 4 h, and then the solvent was removed
under reduced pressure. The residue was dissolved in EtOAc
and washed with satd aq NaHCO3 solution. The aqueous
layer was extracted twice with EtOAc, and then the combined
organic extracts were dried over MgSO4 and filtered. The
solvent was removed under reduced pressure, and then the
crude material was chromatographed on an 80 g SiO2 prepacked
column eluting with 0:1 to 2:3 EtOAc/hexanes, affording the
benzimidazole. The benzimidazole was dissolved in EtOH, and then
5 M HCl in IPA (1 equiv) was added. The reaction was stirred at rt
for 30 min, and then the solvent was removed under high vacuum
to afford the benzimidazole hydrochloride salt.
3-[7-Trifluoromethyl-5-(2-trifluoromethylphenyl)-1H-benzimid-
azol-2-yl]-1,8-dioxa-2-aza-spiro[4.5]dec-2-ene (70). The title com-
pound was prepared according to the general procedure from nitro-
aniline 43 (200 mg, 0.571 mmol) and NaH (68.5 mg of 60%
mineral oil dispersion, 1.71 mmol) in THF (10 mL) at 0 °C and
acid chloride 19 (0.628 mmol) in THF (5 mL), affording the
nitroamide (178 mg, 60%). The nitroamide (178 mg, 0.344 mmol)
in a mixture of AcOH (8 mL) and MeOH (2 mL) was treated with
Fe powder (96.1 mg, 1.72 mmol) at 100 °C for 4 h, yielding a
colorless glass (144 mg, 89%). Treatment of the benzimidazole
(144 mg, 0.307 mmol)) in EtOH (2 mL) with 5 M HCl in IPA
(61.4 μL, 0.307 mmol) afforded the title compound as a colorless
(27) (90.0 mg, 0.575 mmol), and (dppf)PdCl2 DCM (18.0 mg,
3
0.0220 mmol) in DME (2 mL) and 2 M aq Na2CO3 (0.500 mL,
1.00 mmol) according to the general Suzuki procedure, yielding
a white solid (75.7 mg, 47%). Treatment of the benzimidazole
(75.7 mg, 0.207 mmol) in EtOAc (1 mL) with 1 M HCl in Et2O
(0.200 mL, 0.200 mmol) afforded the title compound as a white
powder (74.3 mg, 89%). 1H NMR (400 MHz, DMSO-d6) δ: 7.71
(d, J = 8.6 Hz, 1H), 7.66 (s, 1H), 7.58-7.62 (m, 1H), 7.41-7.51
(m, 3H), 7.39 (dd, J = 8.5, 1.6 Hz, 1H), 3.31 (s, 2H), 1.63-1.80
(m, 6H), 1.36-1.55 (m, 4H). HRMS calcd for C21H20ClN3O
(M þ H)þ 366.1373, found 366.1372.
2-{2-[2-(1-Oxa-2-aza-spiro[4.5]dec-2-en-3-yl)-1H-benzimid-
azol-5-yl]-phenyl}-propan-2-ol (75). The title compound was
prepared from 73 (150 mg, 0.448 mmol), 3,3-dimethyl-3H-
benzo[c][1,2]oxaborol-1-ol12 (DMBB) (145 mg, 0.898 mmol),
and (dppf)PdCl2 DCM (37.0 mg, 0.0450 mmol) in DME
3
(2 mL) and 2 M aq Na2CO3 (0.75 mL, 1.50 mmol) according
to the general Suzuki procedure, yielding a white solid (16.4 mg,
9.4%). 1H NMR (400 MHz, CD3OD) δ: 7.82 (dd, J = 8.1, 1.3 Hz,
1H), 7.53-7.68 (m, 1H), 7.47 (br s, 1H), 7.35 (td, J = 7.6, 1.6 Hz,
1H), 7.19 - 7.24 (m, 2H), 7.05 (dd, J = 7.6, 1.3 Hz, 1H), 3.28
(s, 2H), 1.69-1.90(m, 6H), 1.46-1.65 (m, 4H), 1.33 (s, 6H). HRMS
calcd for C24H27N3O2 (M þ H)þ 390.2182, found 390.2174.
Biological Assays. In Vitro Canine TRPM8 Functional Assay.
TRPM8 functional activity was initially determined by measur-
ing changes in intracellular calcium concentration using a Ca2þ
-

246 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Parks et al.
sensitive fluorescent dye. The changes in fluorescent signal were
monitored by a fluorescence plate reader, either FLIPR manu-
factured by Molecular Devices or FDSS manufactured by
Hamamatsu. Increases in intracellular Ca2þ concentration were
readily detected upon activation with icilin.
of an abrupt or protracted withdrawal or lifting of the paw.
Rats were acclimated to this test procedure over the course of
several practices sessions. Only rats exhibiting a 100% positive
response rate (5 of 5) at the time of the predose baseline were
included in the study. The inhibition of positive responses
following administration of test compound (n = 7-13 rats/
treatment) was taken as a percentage of the number of positive
responses prior to test compound treatment according to the
following formula: % iInhibition = [1 - (treatment responses/
baseline responses)] ꢀ 100. Time course data were analyzed by
ANOVA, followed by a Bonferroni posthoc test. ED50 values
and associated statistics were calculated using PharmTools
Plus software (The McCary Group, Schnecksville, PA).
Cold Pressor Test. Fasted, male, Sprague-Dawley rats (300-
450 g) were orally administered 5 (0.3, 1, 3, 10, or 30 mg/kg) or
vehicle (20% HPβCD) 120 min prior to cold pressor testing.
Then 75 min following oral dose administration, rats were
anesthetized with 65 mg/kg ip sodium pentobarbital, followed
by 10 mg/kg/h supplemental doses, with continuous intravenous
infusion (jugular cannulation) until the end of the study. A sur-
gically placed carotid artery catheter was connected to a pressure
transducer to continuously record blood pressure. Cold stimulation
of the fore paws was accomplished by placing the paws in ice
water for 3-5 min, followed by active rewarming with rt water.
The percent change in mean arterial pressure in response to this
cold stimulus was calculated for vehicle and test compound
pretreatments. Percent inhibition attributed to treatment with
test compound was then determined using the following formula:
At 24 h prior to the assay, HEK293 cells stably expressing
canine TRPM8 were seeded in culture medium in black wall,
clear-base poly-D-lysine coated 384-well plates (BD Biosciences,
NJ, USA) and grown overnight in 5% CO2 at 37 °C. On assay
day, the growth media was removed and cells were loaded with
Calcium 3 Dye (Molecular Devices) for 35 min at 37 °C under
5% CO2 and then for 25 min at rt. Subsequently, cells were
tested for agonist-induced increases in intracellular Ca2þ levels
using FLIPR or FDSS. Cells were then exposed to test com-
pound (at varying concentrations), and intracellular Ca2þ was
measured for 5 min prior to the addition of icilin to all wells to
achieve a final concentration that produces approximately an
80% maximal response. EC50 or IC50 values were determined from
eight-point concentration-response studies. Curves were generated
using the average of quadruplicate wells for each data point.
In Vitro Canine TRPM8 Electrophysiology. HEK293 cells
stably expressing cTRPM8 were cultured in DMEM supple-
mented with 10% fetal bovine serum, 100 units/mL penicillin,
100 μg/mL streptomycin, and 400 μg/mL G418. Cells were
plated on glass coverslips, maintained at 37 °C and in 5% CO2,
and used 1-2 days after plating.
Recordings were performed using the conventional whole-
cell patch clamp technique. Currents were continuously sampled
(at 100 Hz)/recorded using Digidata 1322A/pClamp 9.0 and
amplified and filtered (at 2 kHz) using Axopatch 200B. The
holding potential was -60 mV. The extracellular solution
contained (in mM): NaCl (132), KCl (5.4), MgCl2 (0.8), EGTA
(1), HEPES (10), and glucose (10), pH = 7.4. The intracellular
solution used to fill recording pipettes contained (in mM): CsCl
(145), EGTA (5), HEPES (10), and glucose (5), pH = 7.4.
The temperature of the solution perfusing the cell was cooled
to 10 °C from 22 °C to activate the channel using an in-line
cooler. After steady-state current activation was achieved, one
or several concentrations (sequentially applied with increasing
concentration) of test compound were applied to the cell at 10 °C.
This was followed by the application of a saturating concentra-
tion of a reference antagonist (also at 10 °C) to establish a
baseline current from which the other currents were subtracted.
Percent inhibition at a given concentration of test compound
was calculated as the ratio between the amplitudes of the current
in the presence and absence of that concentration of test com-
pound. The concentration-response data were fitted to a logistic
function as follows: R = 100/(1 þ c/IC50)p, where, R is the
percentage response, p is the Hill coefficient, and c is the concentra-
tion of test compound.
%inhibition
¼ ½1 - ðcold evoked%change in BP post - test compoundÞ=
ðcold evoked%change in BP post - vehicleÞꢁ ꢀ 100
Blood sampling was performed at the end of the experiment to
assess compound exposure levels.
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