Benzo[d]imidazole transient receptor potential vanilloid 1 antagonists for the treatment of pain: Discovery of trans-2-(2-{2-[2-(4-Trifluoromethyl-phenyl)-vinyl]-1H-benzimidazol-5-yl}-phenyl)-propan-2-ol (Mavatrep)

Article

pubs.acs.org/jmc

Benzo[d]imidazole Transient Receptor Potential Vanilloid 1

Antagonists for the Treatment of Pain: Discovery of trans-2-(2-{2-[2-

(4-Triﬂuoromethyl-phenyl)-vinyl]‑1H‑benzimidazol-5-yl}-phenyl)-

propan-2-ol (Mavatrep)

William H. Parsons, Raul R. Calvo, Wing Cheung, Yu-Kai Lee, Sharmila Patel, Jian Liu,

Mark A. Youngman, Scott L. Dax, Dennis Stone, Ning Qin, Tasha Hutchinson, Mary Lou Lubin,

Sui-Po Zhang, Michael Finley, Yi Liu, Michael R. Brandt, Christopher M. Flores, and Mark R. Player*

Janssen Research & Development, Welsh and McKean Roads, Spring House, Pennsylvania 19477, United States

ABSTRACT: Reported herein is the design, synthesis, and

pharmacologic characterization of a class of TRPV1 antago-

nists constructed on a benzo[d]imidazole platform that

evolved from a biaryl amide lead. This design composes

three sections: a 2-substituted 5-phenyl headgroup attached to

the benzo[d]imidazole platform, which is tethered at the two

position to a phenyl tail group. Optimization of this design led

to the identiﬁcation of 4 (mavatrep), comprising a triﬂuoromethyl−phenyl−vinyl tail. In a TRPV1 functional assay, using cells

expressing recombinant human TRPV1 channels, 4 antagonized capsaicin-induced Ca²⁺inﬂux, with an IC₅₀value of 4.6 nM. In

the complete Freund’s adjuvant- and carrageenan-induced thermal hypersensitivity models, 4 exhibited full eﬃcacy, with ED₈₀

values of 7.8 and 0.5 mg/kg, respectively, corresponding to plasma levels of 270.8 and 9.2 ng/mL, respectively. On the basis of its

superior pharmacologic and safety proﬁle, 4 (mavatrep) was selected for clinical development for the treatment of pain.

as from a variety of visceral disorders, including inﬂammatory

bowel disease, inﬂammatory bowel syndrome, detrusor hyper-

reﬂexia, and rectal hypersensitivity.¹⁶⁻¹⁸Together, these results

suggest an important role for TRPV1 in the development and

maintenance of chronic inﬂammatory and neuropathic

conditions.

INTRODUCTION

■

Transient receptor potential vanilloid subfamily type 1

(TRPV1) is a thermosensitive, nonselective cation channel

and a member of the TRP channel superfamily. Structural

features include six transmembrane domains, with intracellular

N- and C-termini.¹The channel is selectively activated by

thermal stimuli (>43 °C), acidic pH (<6.8), and endogenous

lipidic mediators (e.g., anandamide and oxidative metabolites of

linoleic acid). TRPV1 was originally identiﬁed based on its

gating of calcium in response to the pungent vanilloid capsaicin.

In humans, application of capsaicin initially produces a burning

pain sensation as well as the development of thermal and tactile

hypersensitivity and allodynia,^2,3followed by a delayed and

prolonged period of hyposensitivity, depending on the

magnitude and duration of exposure. TRPV1 is predominately

expressed by small- and medium-sized dorsal root and

trigeminal ganglion neurons, wherein it is localized to both

peripheral and central nerve terminals.^4,5Several studies have

demonstrated that TRPV1 expression is increased in models of

inﬂammation and osteoarthritis.^4,6−9TRPV1 is also regulated in

models of neuropathic pain, such that its expression is

decreased in injured neurons and increased in neighboring

undamaged neurons.¹⁰⁻¹²This up-regulation appears to be

important in some models of neuropathic pain (e.g., chronic

constriction injury of the sciatic nerve), wherein thermal

hypersensitivity is a prominent manifestation.¹³In humans,

disease-related changes in TRPV1 expression have been

demonstrated in patients suﬀering from painful diabetic

neuropathy,¹⁴osteoarthritis, and rheumatoid arthritis¹⁵as well

In vivo studies support a role for TRPV1 in the modulation

of pain sensitivity. Mice lacking TRPV1 exhibit reduced thermal

hypersensitivity in response to incision or to the administration

of mustard oil, carrageenan, or complete Freund’s adjuvant

(CFA).¹⁹⁻²¹Importantly, these mice display normal sensitivity

to other stimuli (e.g., tactile) following inﬂammation or nerve

injury. With the discovery of selective TRPV1 antagonists,

additional support for the role of TRPV1 in pain conditions has

been demonstrated. For example, antagonists attenuate thermal

hypersensitivity in models of inﬂammatory pain, bone cancer

pain, and postsurgical pain.²²⁻²⁶Some, though not all, studies

have suggested that TRPV1 antagonists attenuate tactile

hypersensitivity in certain models of neuropathic pain, albeit

at higher doses and/or to a lesser extent compared with

inﬂammatory models.^22,25These eﬀects occur at doses that are

not associated with modiﬁcation of other types of behavior such

as locomotor activity or motor coordination. The diversity of

tissues expressing TRPV1, including the central nervous

system, as well as the wide array of preclinical disease models

in which TRPV1 has been implicated, suggests broad

Received: January 23, 2015

A

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Figure 1. Evolution of benzo[d]imidazole-based TRPV1 antagonists (IC₅₀values refer to blockade of capsaicin-induced Ca²⁺ﬂux in recombinant

HEK293 cells).

a

Scheme 1

a

Reagents and conditions: (a) pyridine, piperidine (cat.), 70 °C; (b) oxalyl chloride, DMF (cat.), CH₂Cl₂; (c) HOAc, 80 °C; (d) Pd(dppf)Cl,

Cs₂CO₃, dioxane:EtOH (5:1), 115 °C or Pd(dtbpf)Cl₂, Na₂CO₃, DME:H₂O (4:1) 90 °C; (e) Pd(dppf)Cl₂, Na₂CO₃, DME:H₂O (4:1), 100 °C; (f)

HCl; (g) POCl₃100 °C; (h) Pd(dppf)Cl₂, KOAc, DMF, 80 °C; (i) Pd(dtbpf)Cl₂, Na₂CO₃, DME:H₂O (4:1), 90−100 °C.

therapeutic applicability for TRPV1 receptor antagonists.

However, the tendency of some TRPV1 antagonists to cause

hyperthermia in humans merits caution.²⁵

Reported herein is the design, optimization, and pharmaco-

logic characterization of TRPV1 antagonists constructed on a

benzo[d]imidazole platform that evolved from a biaryl amide

design exempliﬁed by 1 (Figure 1). Compounds from this early

class exhibited relatively potent TRPV1 inhibitory activity in

functional assays but demonstrated poor oral exposure in rats.

It was proposed that the biarylamide design could be

remodeled and rigidiﬁed by elaborating the amide group into

an imidazole ring fused to the biaryl fragment, as has been

exempliﬁed by other groups.^27,28This design, which follows the

prototypical TRPV1 pharmacophore, is composed of three

sections: a 2-substituted 5-phenyl headgroup appended to a

benzo[d]imidazole platform (hydrogen bond interacting),

which in turn is tethered from the two position to a substituted

phenyl tail group. These studies demonstrated that (1) the

B

DOI: 10.1021/acs.jmedchem.5b00132

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a

Scheme 2

a

Reagents and conditions: (a) n-BuLi, THF, −78 °C; (b) CO₂, THF; (c) oxalyl chloride, DMF (cat.), CH₂Cl₂; (d) 1,4-dioxane, 90 °C; (e) Suzuki

conditions; (f) KOH, CH₃OH, 1,4-dioxane, 50 °C

substituent attached to the head phenyl group is required to be

a hydrogen bond acceptor and is appended at the ortho

position, (2) the optimal tether is trans-vinyl, and (3) the tail

phenyl group requires a hydrophobic substituent, preferably at

the meta or para position.

The ﬁrst compound (2) prepared, based on this design,

retained substantial potency in a human TRPV1 (hTRPV1)

functional assay.²⁹⁻³¹This article reports the optimization of

this design, resulting in benzo[d]imidazoles 3 and 4, both

potent, orally bioavailable TRPV1 antagonists that exhibit

eﬃcacy in animal models of pain. While both compounds were

considered for clinical development, 4 was selected because of

its superior pharmacologic proﬁle, as described herein.

Route C was utilized when phenylboronic acids were not

commercially available or not easily prepared. In this route, the

imidazole nitrogen of bromobenzo[d]imidazole 8 was

protected with a Boc group to produce bromobenzo[d]-

imidazole 12. Compound 12 was heated with bis(pinacolato)-

diboron 13 and potassium acetate in DMF to yield boronate

ester 14. The reaction times for this coupling could be

accelerated at lower temperatures by using a microwave

irradiation apparatus. Boronate ester 14 and phenyl bromide

15 were coupled under the indicated Suzuki conditions. These

conditions also served to remove the Boc group to yield ﬁnal

products. cis-Vinyl 57 was prepared from 61 by hydrogenation

in ethyl acetate with Lindlar catalyst. Compounds containing

methylene, ethylene, or n-propylene tethers were prepared

from the corresponding acids and bromophenyldiamine 7

according to route A (Scheme 1).

Scheme 2 describes the route used for the preparation of

acetylene analogues 60−63. Substituted aryl propiolic acids 17

were prepared by reaction of acetylene 16 with n-butyl lithium

in tetrahydrofuran at −78 °C. The acetylenic anion was then

transferred to a saturated solution of carbon dioxide in THF at

−78 °C. The propiolic acids were isolated in good yield after

aqueous workup and used in the next step without further

puriﬁcation. Acid 17 was treated with oxalyl choride to form

chlorocinnamic acid chloride 18 as a mixture of E/Z isomers.

The vinyl chloride conveniently served as a protecting group

for the acetylene during the Suzuki coupling. Heating acid

chloride 18 and diaminobromobenzene 7 in 1,4-dioxane gave

bromobenzo[d]imidazole 19. When boronic acid 9 was then

coupled to 19 under Suzuki conditions, the vinyl chloride was

converted to acetylenes (60−63).

RESULTS

■

Three synthetic routes were utilized to synthesize the reported

compounds (Scheme 1). In the primary route A, when

cinnamic acid 6 was not commercially available, the

corresponding benzaldehyde 5 was heated with malonic acid

in pyridine with a catalytic amount of piperidine to yield

cinnamic acid 6. The product was isolated without puriﬁcation

by precipitation upon cooling and dilution with dilute

hydrochloric acid. Acid 6 was converted to its acid chloride

with oxalyl chloride in methylene chloride and catalytic

dimethylformamide. The isolated acid chloride and 4-

bromodiaminobenzene 7 were heated in acetic acid to give

bromobenzo[d]imidazole 8. The product was isolated by

precipitation upon cooling the reaction mixture and diluting

it with an ethyl acetate/heptane mixture. The overall yield for

this sequence, for most compounds beginning with benzalde-

hyde 5, was approximately 80%. Finally, bromobenzo[d]-

imidazole 8 was reacted with phenylboronic acid 9 under the

indicated Suzuki conditions to give ﬁnal products that were

isolated by chromatography. Palladium catalyst loading of 10%

was optimal for conversion. Yields for this last step ranged

between 70% and 90%.

DISCUSSION AND CONCLUSIONS

■

In vitro TRPV1 assays designed to assess the functional

inhibitory activity of test compounds utilized FLIPR or FDSS

technology, which measured capsaicin-induced increases in

intracellular Ca²⁺using a Ca²⁺-sensitive ﬂuorescent dye.²⁹⁻³¹

These assays utilized HEK293 cells that stably express human

or rat TRPV1 (rTRPV1) channels. The IC₅₀values obtained

from eight-point concentration−response studies or the

percent inhibition at a test concentration of 1 μM are shown

in Tables 1−5.

Alternate route B was useful to more rapidly explore the SAR

of the tail region. Bis-Boc-protected bromodiaminobenzene 10

was reacted with phenylboronic acid 9 under the indicated

Suzuki conditions to give bis-Boc-biphenyldiamine 11. The Boc

groups of 11 were removed under acidic conditions (HCl), and

the diamine product as a bis-HCl salt was reacted with the acid

chloride of 6, as described for route A to give ﬁnal product.

Alternatively, the diamine can be heated with acid 6 in POCl₃

to give ﬁnal product. Yields for this last step ranged between

60% and 80%.

SAR studies demonstrated that the vinyl tether is optimal

(vide infra). The initial optimization focused on the head

phenyl group, with the t-butylphenyl tail moiety held constant,

as summarized in Table 1. Optimal potency requires a

C

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Table 1. In Vitro hTRPV1 Functional Activity of Head

Phenyl R₁Substitution

Table 2. In Vitro hTRPV1 Functional Activity of Tail Phenyl

R₂Substitution

a

compd

R₂

hTRPV1 Activity IC₅₀(nM) or %I

compd

R₁

hTRPV1 activity IC₅₀(nM) or % I

30

31

32

33

34

35

36

37

38

39

40

41

4-tBu

2

2-OH

140

40

3,4-di-F

250

190

76

20

21

22

23

24

25

26

27

28

29

30

2-CONH₂

4-CH₃

3-CONH₂

12%

10%

25

4-Cl

4-CONH₂

4-CF₃

10

2-COCH₃

3-CF₃

48%

44%

17

2-CH(OH)CH₃

2-CH₂OH

12

2-CF₃

10

4-OCF₃

2-NHCO₂t-Bu

2-NH₂

280

210

400

10%

2

4-OCH₂CF₃

4-OCH₂CF₂CF₃

4-CH(CH₃)₂

4-N(CH₃)₂

300

63%

9.2

2-NHCOCH₃

4-NHCOCH₃

2-NHSO₂CH₃

240

a

IC₅₀values or % inhibition of capsaicin-induced Ca²⁺inﬂux at a test

concentration of 1 μM averaged from four determinations (n = 4).

a

IC₅₀values or % inhibition of capsaicin-induced Ca²⁺inﬂux at a test

concentration of 1 μM averaged from four determinations (n = 4).

hydrophilic group such as hydroxyl appended to the ortho

position of the phenyl ring. Replacing the ortho hydroxy group

of 2 with hydrogen or alkyl, or migrating the hydroxy group to

the meta or para positions, eliminated activity. Amides 20−22

conﬁrm the importance of a polar ortho substituent. Acetyl

analogue 23 and related hydroxy analogues 24 and 25 were

more potent than 2, and the former two provided clues for

further optimization (vide infra). Boc-protected amine 26,

amine 27, and acetamide 28 retained modest activity. However,

when amine 27 was converted to sulfonamide 30, potency

increased 70-fold compared to 2, and 30 was signiﬁcantly more

potent than the analogous acetamide 28.

Acetophenone 23 and sulfonamide 30 were selected for

further examination. To assess metabolic stability, they were

incubated for 10 min with rat or human liver microsome

preparations, with the amount of 23 remaining after incubation

being 43% and 12%, respectively, and that of 30 remaining

being 29% and 9%, respectively. Metabolite identiﬁcation

studies with 23 indicated that polyoxidation of the tail region t-

butyl group was the primary metabolic liability.

To identify suitable t-butyl group replacements for the tail

phenyl ring, the sulfonamide group of 30 was held constant and

R₂was optimized, as summarized in Table 2. Small substituents

(e.g., 31) substantially decreased potency, as did a 4-methyl

group (32). The 4-chloro analogue 33 improved potency.

However, a 4-CF₃group in compound 34 restored most of the

potency of 30. Migrating the CF₃group to the meta or ortho

positions (35 and 36) reduced activity. The 4-OCF₃derivative

37 was comparable to 34. Lengthening the perﬂuoro alkyl chain

(38 and 39) was poorly tolerated. In comparing isopropyl

analogue 40 and dimethylamino analogue 41, it was apparent

that polar and basic substituent patterns were poorly tolerated.

The potency of 34 suggests that its 4-CF₃group is a suitable

isostere for a t-butyl group (30). Attempts to replace the head

or tail phenyl groups with heteroaromatic rings resulted in

inactive compounds.

50% and 85% remaining after 10 min incubations in rat and

human liver microsomes, respectively. In rat pharmacokinetic

studies, the bioavailability of 34 was 65%, the clearance was

moderate at 13 mL/min/kg, and the oral half-life was 4 h.

Intraplantar injection of complete Freund’s adjuvant (CFA)

in rats produces pronounced thermal hypersensitivity. To

examine the peak time and duration of compound eﬀects in this

model, a time course study with 34 was conducted 24 h after

the injection of CFA. Withdrawal latencies from a radiant heat

stimulus were measured 30, 60, 100, and 180 min after

compound administration. Compound 34 completely and

dose-dependently (1−10 mg/kg po) reversed thermal hyper-

sensitivity, with an ED₅₀value of 4.3 mg/kg when evaluated 60

min after administration.

Table 3 details the combination of the “best” head and tail

aromatic substituents. Potent compounds (i.e., those exhibiting

a hTRPV1 IC₅₀value less than 20 nM) were further assessed

for their metabolic stability. The compounds found to have

relatively good stability were then evaluated in rat CFA time

course studies at 10 mg/kg po. Sulfonamide 37 was equipotent

to 34 and exhibited greater stability in rat liver microsomes.

However, 37 was found to lack eﬃcacy in CFA studies. Though

less potent in vitro than other compounds, 42 was relatively

stable in rat and human liver microsomes after 10 min

incubations, with 89% and 97% remaining, respectively. In rat

pharmacokinetic studies, 42 demonstrated modest bioavail-

ability (F = 39%) and was found to be oxidized in vivo to the

ketone 43. To mitigate this oxidative metabolism, the tertiary

alcohol 4 was prepared and was found to be 10-fold more

potent in vitro than 42. In metabolic stability studies, 93% and

100% of 4 remained, respectively, in rat and human liver

microsomal incubations at 10 min. In the CFA time course

study, 4 completely reversed thermal hypersensitivity for 3 h

after administration at a dose of 10 mg/kg po. Migration of the

triﬂuoromethyl group of 4 to the meta position, as in 44,

retained in vitro potency. However, in CFA studies with 44, the

extent of reversal of thermal hypersensitivity was only 58%, and

the duration of reversal was only 60 min. Moving the CF₃

group to the ortho position, as in 45, reduced in vitro potency.

Compound 34 was further proﬁled with respect to its

pharmacokinetic and pharmacodynamic properties. Thus,

compared with 30, 34 was more metabolically stable, with

D

DOI: 10.1021/acs.jmedchem.5b00132

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Table 3. In Vitro hTRPV1 Functional Activity of Best Head

and Tail Group Combinations

Table 4. In Vitro hTRPV1 Functional Activity of Tether

Patterns

a

hTRPV1 activity

hTRPV1

compd

R₁

R₂

4-CF₃

IC₅₀(nM)

activity IC₅₀

a

compd

R₁

R₂

L

(nM)

34

37

42

43

4

−NHSO₂CH₃

−CH(OH)CH₃

−COCH₃

−C(OH)(CH₃)₂

−SO₂NH₂

10

17

45

120

4.6

4.9

13

5.9

5.7

19

22

7

2

−OH

−C(OH)(CH₃)₂

−SO₂NH₂

−t-butyl

−CF₃

trans-vinyl

−CH₂−

−(CH₂)₃−

trans-vinyl

cis-vinyl

140

6%

4.6

7

4-OCF₃

4-CF₃

55

56

4

4-CF₃

57

58

3

44

45

46

47

48

49

50

3

3-CF₃

−(CH₂)₂−

trans-vinyl

−(CH₂)₂−

2.8

7

2-CF₃

−CF₃

4-OCF₃

4-Cl

59

−SO₂NH₂

6

a

IC₅₀values or % inhibition of capsaicin-induced Ca²⁺inﬂux at a test

concentration of 1 μM averaged from four determinations (n = 4).

4-SO₂CF₃

H

−SO₂NH₂

4-F

Table 5. In Vitro hTRPV1 Functional Activity of Acetylene

Analogues

4-CF₃

7

51

52

53

54

−SO₂NH₂

4-OCF₃

3-OCF₃

4-OCH₂CH₃

4-SO₂CF₃

6

8

40

22

a

IC₅₀values or % inhibition of capsaicin-induced Ca²⁺inﬂux at a test

concentration of 1 μM averaged from four determinations (n = 4).

a

compd

R₁

R₂

hTRPV1 activity IC₅₀(nM)

60

61

62

63

−C(OH)(CH₃)₂

−SO₂NH₂

H

2.8

4.5

26

4-Triﬂuoromethoxy and 4-chloro analogues 46 and 47 retained

in vitro potency and metabolic stability but lacked in vivo

eﬃcacy in CFA studies. Triﬂuoromethylsulfone analogue 48

was less potent in vitro and was also inactive in CFA studies.

Among the sulfonamide-substituted compounds, 49 was less

potent than 50, presumably due to the lack of an aromatic

substituent (R₂). Compound 50 was equipotent to 3, although

it bears only a 4-ﬂuorine substituent. Although potent, 51 and

52 exhibited poor metabolic stability in rat liver microsomal

incubations. Larger substituents, as in 53 and 54, reduced

potency. In dose−response studies at the approximate T_maxof

100 min, 3 exhibited oral ED₅₀and ED₈₀values of 2.6 1.3

and 10.3 4.8 mg/kg, respectively. However, the correspond-

ing plasma levels to achieve these values were comparatively

high at 1757 and 62788 ng/mL, respectively. Thus, while 3 is a

potent and eﬃcacious TRPV1 antagonist, its pharmacologic

proﬁle was inferior to 4 (vide infra).

4-CF₃

H

−SO₂NH₂

4-CF₃

20

a

IC₅₀values or % inhibition of capsaicin-induced Ca²⁺inﬂux at a test

concentration of 1 μM averaged from four determinations (n = 4).

properties. Therefore, it was evaluated in more detail. In vitro, 4

exhibited high aﬃnity for the hTRPV1 channel, inhibiting the

binding of the TRPV1 agonist [³H]-resiniferatoxin (RTX) with

a K_ivalue of 6.5 nM.³²In TRPV1 functional assays using cells

expressing recombinant human or rat TRPV1 channels, 4

exhibited IC₅₀values of 4.6 and 21 nM, respectively. In whole-

cell patch clamp electrophysiology studies,²⁹⁻³¹4 blocked the

activation of hTRPV1 channels by capsaicin (1 μM) and by pH

(5.0) in a concentration-dependent fashion, with IC₅₀values of

23 and 6.8 nM, respectively. Moreover, a concentration of 0.1

The signiﬁcance of the vinyl tether is summarized in Table 4.

Compounds with tethers shorter (55) or longer (56) than two

carbons were inactive. The cis-vinyl- (57) and ethyl- (58)

tethered analogues were equipotent to 4. However, the ethyl-

linked 58 was much less stable in rat or human liver microsome

incubations with 5% or 45% remaining, respectively. Similar

liabilities were exhibited by the ethyl-tethered sulfonamide 59.

Studies of the acetylene tether are summarized in Table 5.

Promising compounds were those containing the tertiary

alcohol pattern at R₁(i.e., 60 and 61). Both compounds

were stable in rat and human liver microsomal incubations. In

CFA studies, 60 signiﬁcantly reversed thermal hypersensitivity

by 71% and 41% at the 30 min and 3 h time points,

respectively. However, pharmacokinetic assessment of 60 in

rats revealed relatively poor bioavailability (F = 13%).

μM of 4 produced a 74

8.1% inhibition of heat-evoked

currents mediated by hTRPV1. Compound 4 lacked activity at

other TRP channels evaluated, including TRPM8, TRPV2, and

TRPA1.

As previously mentioned, a 10 mg/kg po dose of 4

signiﬁcantly reversed CFA-induced thermal hypersensitivity,

beginning 30 min after administration and lasting for at least 3

h (Figure 2). Both the magnitude and duration of this reversal

was similar to an oral dose of 30 mg/kg of celecoxib. As shown

in Figure 3, in dose−response studies at the approximate T_max

of 100 min, 4 exhibited complete reversal of thermal

hypersensitivity, with ED₅₀and ED₈₀values of 1.8 and 7.8

mg/kg, respectively (left panel). The corresponding plasma

levels for these values were 41.9 and 270.8 ng/mL, respectively

(right panel). In a second inﬂammatory pain model (Figure 4),

4 completely reversed carrageenan-induced thermal hyper-

sensitivity, with ED₅₀and ED₈₀values of 0.18 and 0.48 mg/kg,

Of the compounds evaluated in the CFA model of

inﬂammatory pain, 4 exhibited superior pharmacodynamic

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Figure 2. Eﬀects of 4 in the CFA model of inﬂammatory of pain.

Abscissa: Time in min after the oral administration of 10 mg/kg of 3

(n = 5−11/time point). Ordinate: mean ( SEM) percent reversal of

CFA-induced thermal hypersensitivity.

Figure 4. Eﬀects and associated plasma levels of 4 in the carrageenan

model of inﬂammatory pain. Abscissa (left and right): vehicle (Veh) or

dose in mg/kg of 4 administered 100 min before behavioral

assessment (n = 13−22/group). Ordinate left: mean ( SEM) percent

reversal of carrageenan-induced thermal hypersensitivity. Ordinate

right: mean ( SEM) plasma concentration in ng/mL of 4 from

samples obtained immediately following behavioral assessment.

*Signiﬁcantly diﬀerent from vehicle (p < 0.05).

inhibition of activity. In a recombinant hERG channel assay, 10

μM 4 exhibited no inhibition of [H³]-astemizole binding.

Moreover, when administered intravenously to anesthetized

guinea pigs up to a cumulative dose of 10 mg/kg iv, 4 did not

induce notable hemodynamic or electrocardiographic eﬀects. In

a CNS safety assessment study, following a single oral dose of

30 or 300 mg/kg, 4 produced no substantial eﬀects across a 14

day observation period. In this general observational test

battery, there were no changes in core body temperature as

measured at 1 and 4 h postcompound administration at either

dose.

However, studies with other TRPV1 antagonists indicate that

some TRPV1 antagonists elevate core body temperature in

rodents,³²although in some cases the hyperthermia is <1 °C³³

or transient.³⁴The prevailing hypothesis is that the hyper-

thermic eﬀect of a TRPV1 antagonist is related to the extent to

which it causes blockade in proton mode.^35,36Indeed, some

TRPV1 antagonists have been identiﬁed that do not elevate

core body temperature in preclinical models while only

minimally blocking acid activation of TRPV1.³⁷

To explore this further with the present lead (4), a single oral

dose of 100 mg/kg was administered to Sprague−Dawley rats,

and core body temperature was evaluated over 3 h using a

rectal thermometer. Before dosing, the mean body temperature

in both groups was 37.5 °C. Compound 4 produced a

signiﬁcant 0.86 ( 0.12) °C increase in body temperature over

vehicle-treated rats 0.5 h after administration. The increase in

body temperature waned over 1.5 h and was not signiﬁcantly

diﬀerent from vehicle-treated rats at other time points evaluated

(Figure 5). This ﬁnding may explain the lack of an observed

eﬀect on core body temperature in the general observational

test battery, as the ﬁrst time point measured for both the 30 and

300 mg/kg dose was at 1 h (vide supra).

To evaluate the dose-dependency of increased core body

temperature, male Sprague−Dawley rats were implanted (ip)

with microchip temperature/ID telemetry probes 5 days prior

to the study start. In rats deprived of food for 18 h, a single oral

dose of 4 (0.1, 1.0, 10.0, or 100.0 mg/kg) or vehicle (20%

HPβCD) was administered, and core body temperatures were

Figure 3. Eﬀects and associated plasma levels of 4 in the CFA model

of inﬂammatory pain. Abscissa (left and right): vehicle (Veh) or dose

in mg/kg of 4 administered 100 min before behavioral assessment (n =

5−8/group). Ordinate left: mean ( SEM) percent reversal of CFA-

induced thermal hypersensitivity. Ordinate right: mean ( SEM)

plasma concentration in ng/mL of 4 from samples obtained

immediately following behavioral assessment. *Signiﬁcantly diﬀerent

from vehicle (p < 0.05).

respectively (left panel). The corresponding plasma levels were

3.8 and 9.2 ng/mL, respectively (right panel).

Table 6 provides the pharmacokinetic proﬁles of 4 in rat,

dog, Cynomolgus monkey, and mouse. Compound 4 exhibited

substantial bioavailability in the rat (51%), mouse (83%), and

monkey (70%), whereas bioavailability in the dog was lower

(25%). In the rat, the clearance rate of 4 was less than half of

liver blood ﬂow; in monkey and mouse, it was much less than

liver blood ﬂow, whereas in dog, it was roughly equal to liver

blood ﬂow. The high clearance of 4 in the dog was predicted by

poor stability in dog liver microsomes. While 100%, 93%, and

86% of 4 remained after 10 min incubations in human, rat, and

monkey liver microsomes, respectively, only 36% remained in

dog liver microsomes.

To obtain an early evaluation of the safety potential of 4, its

selectivity proﬁle was assessed. In cytochrome P450 isoenzyme

studies in human liver microsomes, 4 exhibited minimal eﬀect

on the enzymatic activity (IC₅₀> 25 μM) of CYP isoforms 3A4,

1A2, and 2D6. In a panel of 50 G protein-coupled receptor and

ion channel binding assays, 1 μM 4 did not exhibit greater than

50% inhibition of radioligand binding to any of the targets.

Similarly, in a panel of 190 kinase assays, 4 did not exhibit any

F

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Table 6. Pharmacokinetic Assessment of 4 in Rat, Dog, Monkey, and Mouse after Intravenous and Oral Administration

a

b

intravenous administration

oral administration

species

Cl (mL/min/kg)

V_ss(L/kg)

T_1/2(h)

C_max(ng/mL)

AUC (ng·h/mL)

T_1/2(h)

F (%)

rat

33

29

7

10

3.4

1.1

421

1607

969

4203

4186

3.8

1.4

51

dog

2.7

3.8

2.5

25

monkey

mouse

12.9

2.75

13747

13178

12.9

5.87

70

10.5

944

83.3

a

b

2 mg/kg of solution of sodium salt in 20% HPβCD, n = 4 animals per group. 10 mg/kg solution of sodium salt in 20% HPβCD, n = 4 animals per

group.

HPβCD) was administered QD for ﬁve consecutive days, and

core body temperature was assessed prior to and 0.5, 1, 1.66,

and 3 h after administration of 4. On day 1, a dose of 10 mg/kg

4 produced a transient increase in body temperature that

peaked 0.5 h after administration (Figure 7, left panel). The

right panel of Figure 7 shows that the eﬀects on day 5 were

similar to those observed on day 1. Similar results were

observed on days 2−4 (data not shown), with signiﬁcant

treatment eﬀects observed on days 3 and 4, illustrating that

tolerance to this eﬀect does not develop through the treatment

period studied. Throughout the 5-day study, the average

maximal increase in core body temperature was 1.18 0.21 °C.

Together, these results indicate that 4 produces a moderate

(≤1.18 °C) hyperthermic response in rats immediately

following administration. This response was transient (i.e.,

signiﬁcant changes were observed only 30 min after

administration) and did not parallel other physiologic or

behavioral eﬀects observed following 4. For example, reversal of

thermal hypersensitivity and antagonism of capsaicin-induced

ﬂinching indicates a much longer duration of action (i.e., over 7

h depending on the assay), which is more consistent with the

pharmacokinetics of the compound. These results suggest that

there may be compensatory mechanisms that rapidly restore

core body temperature to baseline.

This work summarizes the transformation of an early

biarylamide motif into a benzo[d]imidazole-based design and

its optimization to produce 4. In summary, 4 is a selective, high-

aﬃnity TRPV1 antagonist that exhibits potent in vitro

functional activity and robust oral eﬃcacy in multiple models

of inﬂammatory pain at relatively low plasma levels.

Pharmacokinetic and safety data in a number of species

indicate that 4 possesses an acceptable oral bioavailability and

therapeutic window for development. Taken together, the

results generated to date suggest that 4 may be a safe and

eﬀective pharmacotherapy, possessing the potential to provide

signiﬁcant relief of patient pain and suﬀering. Compound 4 has

been advanced to phase 2 clinical trials, data from which will be

published elsewhere.

Figure 5. Eﬀects of 4 on core body temperature in rats. Abscissa: time

in min after oral administration of 100 mg/kg of 4 (n = 8) or vehicle

(HPβCD) (n = 8). Ordinate: mean ( SEM) of core body temperature

of 4. *Signiﬁcantly diﬀerent from core body temperature at time = 0 (p

< 0.05).

obtained prior to and at 0.5, 1, 1.66, 3, and 5 h after dosing. In

general, 4 elevated core body temperature above that of vehicle-

treated rats in a manner that was not clearly dose-dependent

(Figure 6). Doses of 0.1 and 100 mg/kg produced signiﬁcant

Figure 6. Dose response of 4 for elevating core body temperature in

rats. Abcissa: time in min after oral administration of 0.1, 1, 10, or 100

mg/kg of 4 (n = 5−6) or vehicle (HPβCD) (n = 5−6). Ordinate:

change in core body temperature.

EXPERIMENTAL SECTION

■

Chemistry. Reagents and solvents were obtained from commercial

suppliers and used without further puriﬁcation. Flash chromatography

was performed using Fisher Chemical Silica Gel Sorbent (230−400

Mesh, grade 60). Preparative thin-layer chromatography was

performed on Analtech Silica Gel GF plates (1000 or 2000 μm, 20

cm × 20 cm). Preparative HPLC was performed on a Gilson 215

HPLC system using a C-18 YMC ODS-A 5m 30 mm × 100 mm, 120

A column with a ﬂow rate of 32 mL/min. The solvents, both

acetonitrile (MeCN) and water, contain 0.1% triﬂuoroacetic acid

(TFA, v/v). Hydrogenation was performed in an H-Cube

(ThalesNano, Inc., Budapest, Hungary). ¹H NMR spectra were

recorded on a Bruker B-ACS-120 (400 MHz) spectrophotometer at rt.

Chemical shifts are given in ppm (δ), coupling constants (J) are in

treatment eﬀects, although intermediate doses of 1 and 10 mg/

kg did not. Over all times and doses of 4, the average change of

core body temperature was not signiﬁcantly above vehicle and

did not increase more than 1.04 °C.

To determine whether tolerance to this eﬀect would develop

following repeated administration, 4 was administered daily for

5 days. Male Sprague−Dawley rats were implanted (ip) with

microchip temperature/ID telemetry probes 3 weeks prior to

the study start. Compound 4 (10 mg/kg) or vehicle (20%

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Figure 7. Eﬀects of daily 10 mg/kg 4 administration on core body temperature in rats. Abcissa: time in min after oral administration of 10 or 100

mg/kg of 4 (n = 5−6) or vehicle (HPβCD) (n = 3−5) on Day 1 (left panel) and Day 5 (right panel). Ordinate: change in core body temperature.

hertz (Hz), and signals are designated as follows: (s) singlet, (d)

doublet, (t) triplet, (q) quartet, (m) multiplet, (br s) broad singlet,

(dd) doublet of doublets, (dt) doublet of triplets, and (ddd) triplet of

doublets. Elemental analysis was performed by Quantitative

Technologies, Inc., Whitehouse, NJ. LC-MS was performed on a

system consisting of an atmospheric pressure ionization (API) source

on a Finnigan LCQ ion trap mass spectrometer, an Agilent 1100 DAD

UV detector (214 nm wavelength), an Agilent 1100-LC binary

gradient pumping system, a Gilson 215 conﬁgured as an autosampler,

and a Zorbax SB-C18 HPLC column (5 μm, 50 mm × 2.1 mm). The

purities of the key target compounds were determined on the Finnigan

LCQ instrument, which gave both the MS and LC trace of the

compound in an 11 min run and were ≥95% in all cases. The HPLC

method used was as follows: column, Zorbax SB-C18 HPLC column

(5 μm, 50 mm × 2.1 mm); column temperature, ambient; ﬂow rate,

0.5 mL/min; gradient, 5% acetonitrile in water to 100% acetonitrile in

water over 10 min. Both MeCN and water contain 0.1% of TFA, v/v.

(trans)-2-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-phenol (2). To a solution of 3-(4-tert-butyl-phenyl)-acrylic acid (12

g, 59 mmol) in POCl₃(200 mL) was slowly added 4-bromo-benzene-

1,2-diamine (10 g, 53 mmol). The solution was heated at reﬂux for 18

h. The solution was concentrated, and the residual POCl₃was

azeotropically removed with toluene. The residue was partitioned

between EtOAc and 10% aq Na₂CO₃. The organic layer was washed

with brine, dried over Na₂SO₄, and ﬁltered, and the ﬁltrate was

concentrated. The residue was puriﬁed by chromatography (silica,

EtOAc:hexanes, 3:7) to give (E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-

N-tert-butyl-2-{2-[2-(4-triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimida-

zol-5-yl}-benzenesulfonamide as yellow oil. ¹H NMR (400 MHz,

CD₃OD) δ (ppm): 8.14 (dd, 1H, J = 1.6 Hz, 7.6 Hz), 7.84 (d, 2H, J =

8.4 Hz), 7.73 (d, 2H, J = 8.4 Hz), 7.70−7.62 (m, 4H), 7.54 (dt, 1H, J =

1.6 Hz, 8.6 Hz), 7.41 (dd, 1H, J = 1.2 Hz, 7.6 Hz), 7.37−7.31 (m, 2H),

1.00 (s, 9H). MS (ESI, pos. ion) m/z: 490.3 (M + 1).

A solution of N-tert-butyl-2-{2-[2-(4-triﬂuoromethyl-phenyl)-vinyl]-

1H-benzoimidazol-5-yl}-benzenesulfonamide in 60 mL of TFA was

heated at 70 °C for 2 h. The reaction was concentrated, and the

residue was dissolved in EtOAc and washed with saturated aqueous

NaHCO₃. The organic layer was dried over Na₂SO₄and ﬁltered, and

the residue was concentrated. The residue was puriﬁed by

chromatography (silica gel, hexanes:EtOAc, 1:2) to give 11 g (68%)

3 as a light-brown solid; mp 168−170 °C. ¹H NMR (400 MHz,

CD₃OD) δ (ppm): 8.16 (dd, 1H, J = 1.2 Hz, 8.0 Hz), 7.95−7.91 (m,

3H), 7.82−7.76 (m, 4H), 7.70−7.56 (m, 3H), 7.44 (s, 1H), 7.16 (d,

1H, J = 9.6 Hz). MS (ESI, pos. ion) m/z: 444.3 (M + 1).

(trans)-2-(2-{2-[2-(4-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzimi-

dazol-5-yl}-phenyl)-propan-2-ol (4). A solution of 4-triﬂuoromethyl-

benzaldehyde (7.7 mL, 58 mmol), malonic acid (12.0 g, 115 mmol),

and piperidine (0.57 μL, 5.8 mmol) in 30 mL of pyridine was stirred at

70 °C for 18 h. The reaction solution was cooled to room temperature.

Water (300 mL) was added, and the resulting mixture was acidiﬁed to

pH 4 (litmus) using concentrated hydrochloric acid. The solution was

ﬁltered. The solid was washed with water until the ﬁltrate was neutral

and was dried in air then in vacuo to give 3-(4-triﬂuoromethyl-

1

phenyl)-acrylic acid as a white powder (11.2 g, 90%). H NMR (400

1

MHz, DMSO-d₆) δ (ppm): 12.60 (br s, 1H), 7.92 (d, 2H, J = 8.2 Hz),

7.77 (d, 2H, J = 8.2 Hz), 7.66 (d, 1H, J = 16.0 Hz), 6.70 (d, 1H, J =

16.0 Hz).

vinyl]-1H-benzoimidazole (7.1 g, 31% yield). H NMR (400 MHz,

CDCl₃) δ (ppm): 7.64 (d, 1H, J = 13.9 Hz), 7.62 (s, 1H), 7.41 (d, 2H,

J = 8.4 Hz), 7.36 (d, 1H, J = 8.5 Hz), 7.33 (d, 2H, J = 8.4 Hz), 7.28 (s,

1H), 7.22 (dd, 1H, J = 1.8 Hz, 8.5 Hz), 7.02 (d, 1H, J = 16.5 Hz), 1.25

(s, 9H). A solution of (E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-vinyl]-

1H-benzoimidazole (0.029 g, 0.10 mmol), 2-hydroxybenzeneboronic

acid (0.034 g, 0.25 mmol), Pd(dppf)Cl₂(0.016 g, 0.020 mmol), and

Cs₂CO₃(0.081 g, 0.25 mmol) in 1,4-dioxane:EtOH 5:1 (2.5 mL) was

heated to 115 °C. After 18 h, the solution was cooled and

concentrated. The residue was puriﬁed using preparative TLC plates

(2000 μm, EtOAc:hexanes, 3:7) to give the title compound (0.0048

A solution of 3-(4-triﬂuoromethylphenyl)-acrylic acid (21 g, 95

mmol) in anhydrous methylene chloride (200 mL) was treated with

oxalyl chloride (17 mL, 190 mmol) and 3 drops of anhydrous

dimethylformamide. The resulting solution was stirred at room

temperature under an argon atmosphere for 18 h. The solvent was

concentrated to give 3-(4-triﬂuoromethyl-phenyl)-acryloyl chloride as

a solid, which was used without further puriﬁcation in the next step.

To a solution of 4-bromo-benzene-1,2-diamine (16.1 g, 86.7 mmol)

in acetic acid (100 mL) was added dropwise a solution of 3-(4-

triﬂuoromethyl-phenyl)-acryloyl chloride (assumed 95.4 mmol) in

acetic acid (100 mL). The solution was stirred at 100 °C for 18 h. The

solution was cooled to room temperature. A mixture of ethyl

acetate:hexanes 3:7 (500 mL) was added to the reaction solution.

The mixture was triturated at room temperature for 3 h. The solution

was ﬁltered, and the solid was dried and concentrated in vacuo to give

5-bromo-2-[2-(4-triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimidazole

1

g,16% yield). H NMR (400 MHz, CDCl₃) δ (ppm): 7.56 (s, 1H),

7.31 (dd, 2H, J = 12.2 Hz, 24.1 Hz), 7.10 (m, 8H), 6.86 (t, 1H, J = 7.3

Hz), 6.78 (d, 1H, J = 16.4 Hz), 1.19 (s, 9H). MS (ESI, pos. ion) m/z:

368.3 (M + 1).

(trans)-2-{2-[2-(4-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimida-

zol-5-yl}-benzenesulfonamide (3). A mixture of 2-(tert-butylamino)-

sulfonylphenylboronic acid (12 g, 0.048 mol), (E)-5-bromo-2-[2-(4-

triﬂuoromethyl-phenyl)-vinyl]-1H-benzo[d]imidazole (14 g, 0.037

mol), Pd(dppf)Cl₂·CH₂Cl₂(3.0 g, 3.7 mmol), tetrabutylammonium

bromide (TBAB, 12 g, 0.037 mmol), and Na₂CO₃(31.4 g, 0.30 mol)

in 750 mL of mixed solvent (DME:water, 4:1) was heated at 90 °C for

12 h. The reaction mixture was concentrated, and the residue was

puriﬁed by chromatography (silica gel, hexanes:EtOAc, 1:1) to aﬀord

1

(23 g, 73%). H NMR (400 MHz, DMSO-d₆/CDCl₃) δ (ppm): 8.45

(d, 1H, J = 16.7 Hz), 7.84−7.90 (m, 1H), 7.74 (d, 2H, J = 8.3 Hz),

7.56−7.62 (m, 3H), 7.50−7.52 (m, 1H), 7.34 (d, 1H, J = 16.7 Hz).

To a solution of methyl 2-bromobenzoate (20.76 g, 96 mmol) in

120 mL of anhydrous ether under argon at 0 °C was slowly added

H

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methylmagnesium bromide (77 mL, 3.26 M in ether) at a rate such

that the internal temperature of the mixture remained below 20 °C. A

white suspension resulted, and the mixture was stirred at room

temperature for 2 h. The mixture was cooled with an ice/water bath.

Hydrochloric acid (400 mL, 0.5 M) was added VERY SLOWLY to the

mixture. The pH of the ﬁnal mixture was adjusted to <6 with a few

drops of 2 M hydrochloric acid. The two layers were separated, and

the aqueous layer was extracted twice with ether. The organic layers

were combined and dried with anhydrous magnesium sulfate. The

mixture was then ﬁltered, and the ﬁltrate was removed under reduced

pressure to yield a pale-yellow liquid. The liquid was then distilled

under vacuum to aﬀord 2-(2-bromo-phenyl)-propan-2-ol as a colorless

DMSO-d₆) δ (ppm): 12.73 (m, 1H), 7.90 (d, 2H, J = 8.2 Hz), 7.85

(dd, 1H, J = 0.6 Hz, 8.0 Hz), 7.78 (d, 2H, J = 8.4 Hz), 7.74 (d, 1H, J =

16.8 Hz), 7.59−7.47 (m, 1H), 7.41 (s, 1H), 7.37−7.32 (m, 2H), 7.21

(dt, 1H, J = 1.2 Hz, 7.4 Hz), 7.06 (s, 1H), 7.02 (d, 1H, J = 7.4 Hz),

4.85 (s, 1H), 1.21 (s, 6H). Mass spectrum (LCMS, APCI pos.) calcd

For C₂₅H₂₁F₃N₂O: 423.2 (M + H), found 423.3; mp (uncorr.) 250−

251 °C.

(trans)-2-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-benzamide (20). The title compound (0.0010 g) was prepared

from 2-aminocarbonyl benzeneboronic acid (0.14 g, 0.76 mmol) and

(E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-vinyl]-1H-benzo[d]imidazole

1

(0.11 g, 0.30 mmol) according to procedures used to prepare 2. H

1

liquid (16.9 g, 82%, bp ca. 65−70 °C/0.3 mmHg). H NMR (400

NMR (400 MHz, CD₃OD) δ (ppm): 7.48 (m, 6H), 7.37 (m, 5H),

7.26 (dd, 1H, J = 1.6 Hz, 8.3 Hz), 7.04 (d, 1H, J = 16.5 Hz), 1.26 (s,

9H). MS (ESI, pos. ion) m/z: 396.1 (M + 1).

MHz, CDCl₃) δ (ppm): 7.67 (dd, 1H, J = 1.7 Hz, 7.9 Hz), 7.58 (dd,

1H, J = 1.3 Hz, 7.9 Hz), 7.30 (ddd, 1H, J = 1.4 Hz, 7.4 Hz, 7.9 Hz),

7.10 (ddd, 1H, J= 1.7 Hz, 7.4 Hz, 7.8 Hz), 2.77 (br s, 1H), 1.76 (s,

6H).

(trans)-3-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-benzamide (21). The title compound (0.0010 g) was prepared

from 3-aminocarbonylbenzene boronic acid (0.14 g, 0.76 mmol) and

(E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-vinyl]-1H-benzo[d]imidazole

n-BuLi (166 mL, 2.6 M in hexane, 430 mmol) was added to 200 mL

of anhydrous THF at −78 °C under argon. When the internal

temperature of the solution reached about −75 °C, a solution of 2-(2-

bromo-phenyl)-propan-2-ol (42.2 g, 196 mmol) in 60 mL of

anhydrous THF was slowly added at −78 °C over 60−90 min at a

rate such that the internal temperature remained below −70 °C. After

the addition, the mixture was stirred at −75 °C for 2 h.

Triisopropylborate (58.8 mL, 255 mmol) was then added to the

mixture in three portions at −78 °C. The mixture was allowed to

slowly warm to room temperature overnight. The mixture was then

cooled to 0 °C and carefully quenched with dilute hydrochloric acid

(250 mL, 2N). The mixture was then stirred at room temperature for

1 h. The pH of the mixture was checked and adjusted to acidic using

additional 2 N HCl if needed. The two layers were separated. The

aqueous layer was extracted twice with ether. The organic layers were

combined and dried with anhydrous magnesium sulfate. The ﬁltrate

was concentrated under reduced pressure to yield a pale-yellow oil.

The residue was then diluted with ethyl acetate (400 mL) and washed

with 1N sodium hydroxide solution (3 × 150 mL). The basic aqueous

layers were combined and acidiﬁed with 2 N HCl. The clear solution

turned cloudy when the acid was added. The mixture was then

extracted with ether (3 × 150 mL). The organic layers were combined

and dried with magnesium sulfate. The solution was ﬁltered, and the

ﬁltrate was concentrated under reduced pressure to yield 3,3-dimethyl-

3H-benzo[c][1,2]oxaborol-1-ol as a colorless oil (26.2 g, 82%), which

was used without further puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆)

δ (ppm): 9.00 (s, 1H), 7.66 (dm, 1H, J = 7.3 Hz), 7.45 (dt, 1H, J = 1.1

Hz, 7.7 Hz), 7.40 (dm, 1H, J = 7.6 Hz), 7.31 (dt, 1H, J = 1.2 Hz, 7.1

Hz), 1.44 (s, 6H).

1

(0.11 g, 0.30 mmol) according to procedures used to prepare 2. H

NMR (400 MHz, CD₃OD) δ (ppm): 8.22 (t, 1H, J = 1.7 Hz), 7.88

(m, 3H), 7.62 (m, 6H), 7.51 (m, 2H), 7.17 (d, 1H, J = 16.5 Hz), 1.38

(s, 9H). MS (ESI, pos. ion) m/z: 396.1 (M + 1).

(trans)-4-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-benzamide (22). The title compound (0.0022 g) was prepared

from 4-aminocarbonylbenzene boronic acid (0.14 g, 0.76 mmol) and

(E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-vinyl]-1H-benzo[d]imidazole

1

(0.11 g, 0.30 mmol) according to procedures used to prepare 2. H

NMR (400 MHz, CD₃OD) δ (ppm): 8.98−9.04 (m, 2H), 8.76−8.85

(m, 3H), 8.57−8.70 (m, 5H), 8.48−8.52 (m, 2H), 8.13−8.20 (d, 1H, J

= 16 Hz), 2.40 (s, 9H). MS (ESI, pos. ion) m/z: 396.1 (M + 1).

(trans)-1-(2-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-

5-yl}-phenyl)-ethanone (23). The title compound (0.0015 g) was

prepared from 2-acetylbenzene boronic acid (0.14 g, 0.76 mmol) and

(E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-vinyl]-1H-benzo[d]imidazole

1

(0.24 g, 0.75 mmol) according to procedures used to prepare 2. H

NMR (400 MHz, CD₃OD) δ (ppm): 7.54 (m, 11H), 7.23 (dd, 1H, J =

1.6 Hz, 8.3 Hz), 7.15 (d, 1H, J = 16.5 Hz), 2.02 (s, 3H), 1.36 (s, 9H).

MS (ESI, pos. ion) m/z: 395.3 (M + 1).

(E)-1-(2-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-phenyl)-ethanol (24). A solution of (E)-1-(2-{2-[2-(4-tert-butyl-

phenyl)-vinyl]-1H-benzimidazol-5-yl}-phenyl)-ethanone (22) (0.0050

g, 0.010 mmol) in ethanol (0.5 mL) was treated with NaBH₄(0.0030

g, 0.080 mmol). After 3 h, the reaction mixture was applied to a

preparative TLC plate (2000 μm) and developed using EtOAc:hex-

anes, 1:1. The desired band was extracted and concentrated to give the

1

title compound (0.002 g). H NMR (400 MHz, CD₃OD) δ (ppm):

To a mixture of 3,3-dimethyl-3H-benzo[c][1,2]oxaborol-1-ol (12 g,

71 mmol), 5-bromo-2-[2-(4-triﬂuoromethyl-phenyl)-vinyl]-1H-ben-

zoimidazole (20 g, 54 mmol), sodium carbonate (46.1 g, 435

mmol), and Pd(dppf)Cl₂·CH₂Cl₂(8.9 g, 11 mmol) in a 1 L round-

bottom ﬂask equipped with water condenser was added 400 mL of

anhydrous DME and 200 mL of water. The mixture was evacuated and

ﬁlled with argon three times. The mixture was heated to 100 °C for 20

h. The mixture was then cooled to room temperature. The biphasic

system was transferred to a 1 L separatory funnel, and the two layers

were separated. The organic layer was washed with brine (2 × ∼300

mL). The aqueous layers were combined and extracted with ethyl

acetate (∼300 mL). The organic layers were combined, dried with

anhydrous sodium sulfate for 2 h, and ﬁltered. The volume of the

ﬁltrate was reduced to ∼170 mL under reduced pressure. The mixture

was then ﬁltered through a pad of silica gel and washed with ethyl

acetate until the wash did not contain any product. A light-orange

solution was obtained. After removal of solvents, a light-pink/biege

solid was obtained. The solid was triturated with 50 mL of ethyl

acetate and heated to 85 °C for 5 min. The mixture was slowly cooled

to rt, then placed into a freezer for 0.5 h. The mixture was then ﬁltered,

and the residual solid in the ﬂask was rinsed oﬀ with the ﬁltrate. The

solid cake was then washed with a minimal amount of fresh cold ethyl

acetate twice and dried under vacuum at 40 °C to yield 4 as a light-

7.69 (m, 1H), 7.62 (m, 3H), 7.50 (m, 3H), 7.41 (ddd, 2H, J = 2.5 Hz,

10.1 Hz, 14.1 Hz), 7.32 (dt, 1H, J = 1.4 Hz, 7.5 Hz), 7.23 (m, 2H),

7.17 (d, 1H, J = 16.5 Hz), 4.99 (q, 1H, J = 6.3 Hz), 1.38 (s, 9H), 1.35

(d, 1.5H, J = 6.1 Hz), 1.33 (d, 1.5H, J = 6.1 Hz). MS (ESI, pos. ion)

m/z: 397.2 (M + 1).

(trans)-(2-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-phenyl)-methanol (25). The title compound (0.0046 g) was

prepared from 2-hydroxymethylbenzene boronic acid (0.11 g, 0.76

mmol) and (E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-vinyl]-1H-benzo-

[d]imidazole according to procedures used to prepare 2 (0.11 g, 0.30

1

mmol). H NMR (400 MHz, CD₃OD) δ (ppm): 7.59 (m, 5H), 7.49

(d, 2H, J = 8.4 Hz), 7.40 (m, 2H), 7.34 (m, 2H), 7.27 (dd, 1H, J = 1.6

Hz, 8.3 Hz), 7.16 (d, 1H, J = 16.6 Hz), 4.58 (s, 2H), 1.37 (s, 9H). MS

(ESI, pos. ion) m/z: 383.2 (M + 1).

(trans)-(2-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-phenyl)-carbamic Acid tert-Butyl Ester (26). The title compound

(0.0016 g) was prepared from 2-N-Boc-benzeneboronic acid (0.18 g,

0.76 mmol) and (E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-vinyl]-1H-

aminobenzo[d]imidazole (0.11 g, 0.30 mmol) according to procedures

1

used to prepare 2. H NMR (400 MHz, CD₃OD) δ (ppm): 7.62 (m,

5H), 7.50 (m, 2H), 7.33 (dd, 1H, J = 1.6 Hz, 8.3 Hz), 7.14 (m, 3H),

6.83 (m, 2H), 3.37 (s, 9H), 1.37 (s, 9H). MS (ESI, pos. ion) m/z:

468.1 (M + 1).

1

beige solid (7.6 g, 33%). RP-HPLC 95% pure. H NMR (400 MHz,

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Article

(trans)-2-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-phenylamine (27). A solution of (trans)-(2-{2-[2-(4-tert-butyl-

phenyl)-vinyl]-1H-benzimidazol-5-yl}-phenyl)-carbamic acid tert-butyl

ester (25, 0.0050 g, 0.010 mmol) in methylene chloride (1 mL) was

treated with triﬂuoroacetic acid (0.5 mL). The solution was stirred at

room temperature for 4 h. The solution was concentrated to give the

(m, 6H), 8.10−8.16 (d, 1H, J = 16 Hz), 3.69 (s, 3H). MS (ESI, pos.

ion) m/z: 424.2 (M + 1).

(trans)-N-(2-{2-[2-(4-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimi-

dazol-5-yl}-phenyl)-methanesulfonamide (34). The title compound

(0.094 g) was prepared from 2-methylsulfonylamino benzeneboronic

acid (0.16 g, 0.76 mmol) and (E)-5-bromo-2-[2-(4-triﬂuoromethyl-

phenyl)-vinyl]-1H-benzo[d]imidazole (0.11 g, 0.30 mmol) according

to procedures used to prepare 2. (E)-5-Bromo-2-[2-(4-triﬂuorometh-

yl-phenyl)-vinyl]-1H-benzo[d]imidazole was prepared from 3-(4-

triﬂuoromethylphenyl)-acrylic acid and 4-bromo-benzene-1,2-diamine

according to procedures used to prepare 2. ¹H NMR (400 MHz,

DMSO-d₆) δ (ppm): 12.82 (s, 1H), 8.89 (s, 1H), 7.91 (d, 2H, J = 8.1

Hz), 7.80 (m, 2H), 7.75 (s, 1H), 7.70 (m, 1H), 7.58 (d, 1H, J = 6.9

Hz), 7.37 (m, 6H), 2.73 (d, 3H, J = 28.8 Hz). MS (ESI, pos. ion) m/z:

458.2 (M + 1).

1

title compound (0.003 g). H NMR (400 MHz, CD₃OD) δ (ppm):

8.03 (d, 1H, J = 16.5 Hz), 7.90 (d, 1H, J = 8.5 Hz), 7.85 (s, 1H), 7.74

(d, 2H, J = 8.4 Hz), 7.65 (dd, 1H, J = 1.5 Hz, 8.5 Hz), 7.59 (d, 2H, J =

8.4 Hz), 7.43 (m, 5H), 1.39 (s, 9H). MS (ESI, pos. ion) m/z: 368.1

(M + 1).

(trans)-N-(2-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-

5-yl}-phenyl)-acetamide (28). The title compound (0.0010 g) was

prepared from 2-N-acetyl-benzeneboronic acid (0.14 g, 0.76 mmol)

and (E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-vinyl]-1H-benzo[d]-

imidazole (0.11 g, 0.30 mmol) according to procedures used to

(E)-N-(2-{2-[2-(3-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimida-

zol-5-yl}-phenyl)-methanesulfonamide (35). 4-Bromo-phenylenedi-

amine (25 g, 0.13 mol) was added portionwise to di-tert-butyl

dicarbonate (175 g, 0.80 mol) at 25 °C, and the reaction mixture was

stirred for 10 h. The mixture was puriﬁed by chromatography (silica

gel, hexanes to hexanes:EtOAc, 1:1) to give (5-bromo-2-tert-

butoxycarbonylamino-phenyl)-carbamic acid tert-butyl ester as a

1

prepare 2. H NMR (400 MHz, CD₃OD) δ (ppm): 7.44 (m, 4H),

7.24 (m, 6H), 6.91 (d, 1H, J = 12.4 Hz), 6.47 (d, 1H, J = 12.4 Hz),

1.86 (s, 1H), 1.20 (s, 9H). MS (ESI, pos. ion) m/z: 410.2 (M + 1).

(trans)-N-(4-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-

5-yl}-phenyl)-acetamide (29). The title compound (0.0038 g) was

prepared from 4-N-acetylbenzeneboronic acid (0.14 g, 0.76 mmol)

and (E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-vinyl]-1H-benzo[d]-

imidazole (0.11 g, 0.30 mmol) according to procedures used to

1

light-brown solid. H NMR (400 MHz, CDCl₃) δ (ppm): 7.76 (br

s, 1H), 7.32 (br s, 1H), 7.22 (dd, 1H, J = 2.0 Hz, 8.8 Hz), 6.72 (br s,

1H), 6.53 (br s, 1H).

1

prepare 2. H NMR (400 MHz, CD₃OD) δ (ppm): 7.73 (m, 2H),

To a solution of (5-bromo-2-tert-butoxycarbonylamino-phenyl)-

carbamic acid tert-butyl ester (2.0 g, 5.1 mmol), 2-methylsulfonylami-

nophenyl boronic acid (1.1 g, 5.1 mmol), and K₂CO₃(2.1 g, 15 mmol)

in 1,4-dioxane:water (4:1, 120 mL) was added Pd(dppf)Cl₂·CH₂Cl₂

(0.42 g, 0.51 mmol) under an argon atmosphere. The mixture was

heated at 95 °C for 12 h. The reaction mixture was cooled to room

temperature and concentrated under reduced pressure. The residue

was puriﬁed by chromatography (silica, EtOAc:hexanes, 3:7) to aﬀord

(3-tert-butoxycarbonylamino-2′-methanesulfonylamino-biphenyl-4-yl)-

7.61 (m, 7H), 7.49 (m, 3H), 7.12 (d, 1H, J = 16.5 Hz), 2.17 (s, 3H),

1.36 (s, 9H). MS (ESI, pos. ion) m/z: 410.2 (M + 1).

(trans)-N-(2-{2-[2-(4-tert-Butyl-phenyl)-vinyl]-1H-benzoimidazol-

5-yl}-phenyl)-methanesulfonamide (30). The title compound

(0.0045 g) was prepared from 2-methanesulfonamide benzeneboronic

acid (0.16 g, 0.76 mmol) and (E)-5-bromo-2-[2-(4-tert-butyl-phenyl)-

vinyl]-1H-benzo[d]imidazole (0.11 g, 0.30 mmol) according to

procedures used to prepare 2. ¹H NMR (400 MHz, CD₃OD) δ

(ppm): 7.62 (m, 6H), 7.50 (d, 2H, J = 8.5 Hz), 7.41 (m, 2H), 7.34 (m,

2H), 7.17 (d, 1H, J = 16.5 Hz), 2.75 (s, 3H), 1.37 (s, 9H). MS (ESI,

pos. ion) m/z: 446.2 (M + 1).

1

carbamic acid tert-butyl ester as a yellow solid (1.7 g, 70% yield). H

NMR (400 MHz, CDCl₃) δ (ppm): 7.64 (d, 1H, J = 8.4 Hz), 7.57 (d,

1H, J = 14.4 Hz), 7.34 (t, 1H, J = 8.4 Hz), 7.26−7.15 (m, 3H), 7.07 (s,

1H), 7.04 (d, 1H, J = 8 Hz), 6.87 (s, 1H), 6.63 (s, 1H), 2.90 (s, 3H),

1.51 (s, 9H), 1.49 (s, 9H). Mass spectrum (LCMS, ESI pos.) calcd For

C₂₃H₃₁N₃O₆S: 478.2 (M + H), found 478.0.

(trans)-N-(2-{2-[2-(3,4-Diﬂuoro-phenyl)-vinyl]-1H-benzoimidazol-

5-yl}-phenyl)-methanesulfonamide (31). The title compound

(0.0021 g) was prepared from 2-methylsulfonylamino benzeneboronic

acid (0.16 g, 0.76 mmol) and (E)-5-bromo-2-[2-(3,4-diﬂuoro-phenyl)-

vinyl]-1H-benzo[d]imidazole (0.10 g, 0.30 mmol) according to

procedures used to prepare 2. (E)-5-Bromo-2-[2-(3,4-diﬂuoro-

phenyl)-vinyl]-1H-benzo[d]imidazole was prepared from 3-(3,4-

diﬂuorophenyl)-acrylic acid and 4-bromo-benzene-1,2-diamine accord-

A solution of (3-tert-butoxycarbonylamino-2′-methanesulfonylami-

no-biphenyl-4-yl)-carbamic acid tert-butyl ester (1.0 g, 2.1 mmol) in a

mixture of 4 M HCl in 1,4-dioxane (10 mL) was heated at 70 °C for 2

h. After concentration of the reaction, the residue was dissolved in

dichloromethane (20 mL), and to the solution was added K₂CO₃

(0.50 g) and the mixture was stirred at room temperature for 20 min.

After ﬁltration and removal of solvents, the residue was dried to aﬀord

N-(3′,4′-diamino-biphenyl-2-yl)-methanesulfonamide as a brown solid

1

ing to procedures used to prepare 20. H NMR (400 MHz, CD₃OD)

δ (ppm): 7.50 (m, 5H), 7.36 (m, 1H), 7.26 (m, 5H), 7.07 (d, 1H, J =

16.5 Hz), 2.64 (s, 3H). MS (ESI, pos. ion) m/z: 426.1 (M + 1).

(trans)-N-{2-[2-(2-p-Tolyl-vinyl)-1H-benzoimidazol-5-yl]-phenyl}-

methanesulfonamide (32). The title compound (0.011 g) was

prepared from 2-methylsulfonylamino benzeneboronic acid (0.050 g,

0.18 mmol) and (E)-5-bromo-2-[2-(4-tolyl)-vinyl]-1H-benzo[d]-

imidazole (0.029 g, 0.18 mmol) according to procedures used to

prepare 2. (E)-5-Bromo-2-[2-(4-tolyl)-vinyl]-1H-benzo[d]imidazole

was prepared from 3-(4-methylphenyl)-acrylic acid and 4-bromo-

1

(0.58 g, quantitative yield). H NMR (400 MHz, CD₃OD) δ (ppm):

7.47 (d, 1H, J = 8.4 Hz), 7.40−7.35 (m, 1H), 7.32−7.29 (m, 2H),

7.16−7.09 (m, 3H), 2.80 (s, 3H). Mass spectrum (LCMS, ESI pos.)

calcd For C₁₃H₁₅N₃O₂S: 278.09 (M + H), found 278.1.

A solution of N-(3′,4′-diamino-biphenyl-2-yl)-methanesulfonamide

(0.050 g, 0.18 mmol) and 3-(3-triﬂuoromethylphenyl)-acrylic acid

(0.039 g, 0.18 mmol) in POCl₃(1.5 mL) was heated to 100 °C for 12

h. The reaction was cooled, the excess POCl₃was removed under

reduced pressure, and the residue was puriﬁed by preparative HPLC

(gradient of 5%−80% CH₃CN in water) to aﬀord the title compound

as an oﬀ-white solid (0.026 g, 32% yield). ¹H NMR (400 MHz,

CD₃OD) δ (ppm): 7.90−7.96 (m, 2H), 7.54−7.76 (m, 6H), 7.26−

7.42 (m, 5H), 2.76 (s, 3H). MS (ESI, pos. ion) m/z: 458.1 (M + 1).

(E)-N-(2-{2-[2-(2-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimida-

zol-5-yl}-phenyl)-methanesulfonamide (36). The title compound

(0.014 g) was prepared from 2-methylsulfonyl aminobenzeneboronic

acid (0.37 g, 1.7 mmol) and (E)-5-bromo-2-[2-(2-triﬂuoromethyl-

phenyl)-vinyl]-1H-benzo[d]imidazole (0.19 g, 0.51 mmol) according

1

benzene-1,2-diamine according to procedures used to prepare 2. H

NMR (400 MHz, CD₃OD) δ (ppm): 7.65−7.60 (m, 3H), 7.41−7.36

(m, 3H), 7.41−7.36 (m, 2H), 7.33−7.30 (m, 2H), 7.26 (d, 2H, J = 10

Hz), 7.18 (d, 1H, J = 20 Hz), 2.74 (s, 3H), 2.39 (s, 3H). Mass

spectrum (LCMS, ESI pos.) calcd For C₂₃H₂₁N₃O₂S: 404.1 (M + H),

found 404.3.

(trans)-N-(2-{2-[2-(4-Chloro-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-phenyl)-methanesulfonamide (33). The title compound (0.0080

g) was prepared from 2-methylsulfonylamino benzeneboronic acid

(0.37 g, 1.7 mmol) and 5-bromo-2-[2-(4-chloro-phenyl)-vinyl]-1H-

benzoimidazole (0.17 g, 0.51 mmol) according to procedures used to

prepare 2. 5-Bromo-2-[2-(4-chloro-phenyl)-vinyl]-1H-benzoimidazole

was prepared from 3-(4-chlorophenyl)-acrylic acid and 4-bromo-

1

to procedures used to prepare 4. H NMR (400 MHz, CD₃OD) δ

1

benzene-1,2-diamine according to procedures used to prepare 2. H

(ppm): 8.92−9.04 (m, 2H), 8.72−8.78 (d, J = 7.8 Hz, 1H), 8.60−8.70

(m, 3H), 8.50−8.55 (m, 2H), 8.28−8.40 (m, 4H), 8.16−8.22 (d, J =

NMR (400 MHz, DMSO-d₆) δ (ppm): 8.47−8.64 (m, 6H), 8.24−8.40

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16 Hz, 1H), 3.71 (s, 3H). Mass spectrum (LCMS, ESI pos.) calcd For

C₂₃H₁₈F₃N₃O₂S: 458.1 (M + H), found 458.2.

pound (0.0086 g) was prepared from N-(3′,4′-diamino-biphenyl-2-yl)-

methanesulfonamide (0.050 g, 0.18 mmol) and 3-(4-N,N-dimethyla-

minophenyl)-acrylic acid (0.034 g, 0.18 mmol) according to

procedures used to prepare 35. ¹H NMR (400 MHz, CD₃OD) δ

(ppm): 7.69−7.61 (m, 2H), 7.57−7.51 (m, 2H), 7.46−7.39 (m, 3H),

7.29 (d, 2H, J = 16.2 Hz), 7.05 (d, 1H, J = 12 Hz), 6.67 (d, 2H, J =

16.2 Hz), 6.33 (d, 1H, J = 12 Hz), 3.01 (s, 3H), 2.97 (s, 6H). Mass

spectrum (LCMS, ESI pos.) calcd For C₂₄H₂₄N₄O₂S: 433.2 (M + H),

found 433.3.

(trans)-1-(2-{2-[2-(4-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimi-

dazol-5-yl}-phenyl)-ethanol (42). A solution of (E)-1-(2-{2-[2-(4-

triﬂuoromethyl-phenyl)-vinyl]-1H-benzimidazol-5-yl}-phenyl)-etha-

none (43) (0.0050 g, 0.010 mmol) in ethanol (0.5 mL) was treated

with NaBH₄(0.0030 g, 0.08 mmol). After 3 h, the solution was applied

to a preparative TLC plate (2000 μm) and developed using

EtOAc:hexanes, 1:1. The desired band was extracted and concentrated

to give the title compound (0.0010 g). ¹H NMR (400 MHz, CD₃OD)

δ (ppm): 7.87 (d, 1H, J = 8.2 Hz), 7.76 (d, 1H, J = 8.0 Hz), 7.67 (m,

1H), 7.51 (s, 1H), 7.42 (m, 1H), 7.32 (m, 1H), 7.25 (m, 1H), 4.98 (q,

1H, J = 6.4 Hz), 1.35 (d, 1H, J = 6.4 Hz). MS (ESI, pos. ion) m/z:

409.2 (M + 1).

(trans)-1-(2-{2-[2-(4-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimi-

dazol-5-yl}-phenyl)-ethanone (43). The title compound (0.0010 g)

was prepared from 2-acetylbenzeneboronic acid (0.13 g, 0.76 mmol)

and (E)-5-bromo-2-[2-(4-triﬂuoromethyl-phenyl)-vinyl]-1H-benzo-

[d]imidazole (0.11 g, 0.30 mmol) according to procedures used to

make 2. ¹H NMR (400 MHz, CD₃OD) δ (ppm): 7.75 (d, 2H, J = 8.2

Hz), 7.60 (m, 4H), 7.48 (m, 2H), 7.38 (m, 3H), 7.23 (d, 1H, J = 16.6

Hz), 7.16 (dd, 1H, J = 1.7 Hz, 8.3 Hz), 1.92 (s, 3H). MS (ESI, pos.

ion) m/z: 407.3 (M + 1).

(trans)-2-(2-{2-[2-(3-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimi-

dazol-5-yl}-phenyl)-propan-2-ol (44). The title compound was

prepared from 3,3-dimethyl-3H-benzo[c][1,2]oxaborol-1-ol (0.033 g,

0.20 mmol) and 5-bromo-2-[2-(3-triﬂuoromethyl-phenyl)-vinyl]-1H-

benzoimidazole (0.050 g, 0.14 mmol) according to procedures used to

make 4. ¹H NMR (400 MHz, CD₃OD) δ (ppm): 7.89−7.87 (m, 2H),

7.79 (dd, 1H, J = 1.1 Hz, 8.1 Hz), 7.67−7.56 (m, 3H), 7.52 (m, 1H),

7.40 (br s, 1H), 7.31 (m, 1H), 7.27 (d, 1H, J = 16.6 Hz), 7.18 (dt, 1H,

J = 1.3 Hz, 7.4 Hz), 7.14 (dd, 1H, J = 1.4 Hz, 8.3 Hz), 7.04 (dd, 1H, J

= 1.38 Hz, 7.52 Hz), 1.31 (s, 6H). Mass spectrum (LCMS, APCI pos.)

calcd For C₂₅H₂₁F₃N₂O: 423.2 (M + H), found 423.3.

(trans)-2-(2-{2-[2-(2-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimi-

dazol-5-yl}-phenyl)-propan-2-ol (45). The title compound was

prepared from 3,3-dimethyl-3H-benzo[c][1,2]oxaborol-1-ol (0.033 g,

0.20 mmol) and 5-bromo-2-[2-(2-triﬂuoromethyl-phenyl)-vinyl]-1H-

benzoimidazole (0.050g, 0.14 mmol) according to procedures used to

make 4. ¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.00−7.94 (m, 2H),

7.79 (dd, 1H, J = 1.1 Hz, 8.1 Hz), 7.73 (d, 1H, J = 7.9 Hz), 7.66 (t, 1H,

J = 7.7 Hz), 7.53 (br s, 1H), 7.50 (t, 1H, J = 7.67 Hz), 7.41 (br s, 1H),

7.31 (m, 1H), 7.21−7.13 (m, 3H), 7.04 (dd, 1H, J = 1.4 Hz, 7.5 Hz),

1.31 (s, 6H). Mass spectrum (LCMS, APCI pos.) calcd For

C₂₅H₂₁F₃N₂O: 423.2 (M + H), found 423.3.

(trans)-2-(2-{2-[2-(4-Triﬂuoromethoxy-phenyl)-vinyl]-1H-benzoi-

midazol-5-yl}-phenyl)-propan-2-ol (46). The title compound was

prepared from 3,3-dimethyl-3H-benzo[c][1,2]oxaborol-1-ol (0.33 g,

2.0 mmol) and 5-bromo-2-[2-(4-triﬂuoromethoxy-phenyl)-vinyl]-1H-

benzoimidazole (0.38 g, 1.0 mmol) according to procedures used to

make 4. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 7.84 (dd, 1H, J =

1.26 Hz, 8.08 Hz), 7.75−7.85 (m, 2H), 7.66 (d, 1H, J = 16.4 Hz),

7.53−7.58 (m, 1H), 7.45 (br s, 1H), 7.34−7.38 (m, 3H), 7.17−7.26

(m, 3H), 7.09 (dd, 1H, J = 1.58 Hz, 7.58 Hz), 1.36 (s, 6H). MS (ESI,

pos. ion) m/z: 439.2 (M + 1).

(trans)-N-(2-{2-[2-(4-Triﬂuoromethoxy-phenyl)-vinyl]-1H-benzoi-

midazol-5-yl}-phenyl)-methanesulfonamide (37). A solution of 4-

triﬂuoromethoxybenzaldehyde (5.0 g, 26 mmol), malonic acid (5.6 g,

54 mmol), and piperidine (0.27 mL, 2.7 mmol) in pyridine (15 mL)

was heated to 70 °C for 18 h. Water (200 mL) was added to the

reaction solution. The mixture was acidiﬁed to pH 4 using

concentrated hydrochloric acid. The solution was ﬁltered. The solid

was washed with water. The solid was dried and concentrated in vacuo

to give 3-(4-triﬂuoromethoxy-phenyl)-acrylic acid (1.2 g, 20% yield).

¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 6.80 (d, 1H, J = 16.02 Hz),

6.72 (m, 2H), 6.38 (m, 2H), 5.55 (d, 1H, J = 16.00 Hz).

A solution of 3-(4-triﬂuoromethoxy-phenyl)-acrylic acid (1.2 g, 5.2

mmol) in methylene chloride (20 mL) was treated with oxalyl chloride

(7.8 mL, 3.6 mmol). To the solution was added DMF (0.02 mL). The

reaction solution was stirred at room temperature for 2 h. The reaction

solution was concentrated. The residue was dried and concentrated in

vacuo to give 3-(4-triﬂuoromethoxy-phenyl)-acryloyl chloride.

A solution of 3-(4-triﬂuoromethoxy-phenyl)-acryloyl chloride (1.0

g, 4.0 mmol) in acetic acid (10 mL) was added slowly to a solution of

4-bromo-benzene-1,2-diamine (0.74 g, 4.0 mmol) in acetic acid (10

mL). The solution was heated to 100 °C for 18 h. The solution was

cooled to room temperature. To the solution was added ethyl

acetate:hexanes 3:7 (100 mL). The solution was ﬁltered. The solid was

dried and concentrated in vacuo to give (E)-5-bromo-2-[2-(4-

triﬂuoromethoxy-phenyl)-vinyl]-1H-benzoimidazole (1.1 g, 72%

1

yield). H NMR (400 MHz, DMSO-d₆) δ (ppm): 8.12 (d, 1H, J =

16.60 Hz), 7.98 (m, 1H), 7.87 (m, 2H), 7.82−7.68 (m, 1H), 7.67−

7.37 (m, 4H), 7.34 (d, 1H, J = 16.58 Hz). MS (ESI pos. ion) m/z:

383.2 and 385.2.

The title compound (0.05 g) was prepared from 2-methylsulfonyl

aminobenzeneboronic acid (0.52 g, 2.4 mmol) and (E)-5-bromo-2-[2-

(4-triﬂuoromethoxy-phenyl)-vinyl]-1H-benzo[d]imidazole (0.46 g, 1.2

1

mmol) according to procedures used to prepare 2. H NMR (400

MHz, DMSO-d₆) δ (ppm): 12.76 (d, 1H, J = 5.43 Hz), 8.90 (d, 1H, J

= 4.68 Hz), 7.83 (d, 2H, J = 8.72 Hz), 7.65 (m, 1H), 7.50−7.22 (m,

8H), 2.76 (s, 1.5H), 2.69 (s, 1.5H). MS (ESI, pos. ion) m/z: 474.2 (M

+ 1).

(E)-N-[2-(2-{2-[4-(2,2,2-Triﬂuoroethoxy)-phenyl]-vinyl}-1H-ben-

zoimidazol-5-yl)-phenyl]-methanesulfonamide (38). The title com-

pound (0.015 g) was prepared from N-(3′,4′-diamino-biphenyl-2-yl)-

methanesulfonamide (0.050 g, 0.18 mmol) and 3-(4-triﬂuoroethox-

yphenyl)-acrylic acid (0.044 g, 0.18 mmol) according to procedures

1

used to prepare 34. H NMR (400 MHz, CD₃OD) δ (ppm): 7.67−

7.62 (m, 5H), 7.54−7.52, (m,1H), 7.42−7.33 (m, 4H), 7.09 (d, 2H, J

= 16.4 Hz), 7.08 (d, 1H, J = 12 Hz), 4.60 (q, 2H, J = 8.4 Hz), 2.74 (s,

3H). Mass spectrum (LCMS, ESI pos.) calcd For C₂₄H₂₀F₃N₃O₃S:

488.1 (M + H), found 488.3.

(trans)-N-[2-(2-{2-[4-(2,2,3,3,3-Pentaﬂuoro-propoxy)-phenyl]-

vinyl}-1H-benzoimidazol-5-yl)-phenyl]-methanesulfonamide (39).

The title compound (0.0044 g) was prepared from N-(3′,4′-

diamino-biphenyl-2-yl)-methanesulfonamide (0.070 g, 0.25 mmol)

and 3-(4-(2,2,3,3,3-pentaﬂuoro-propoxy)-phenyl)-acrylic acid (0.067

1

g, 0.23 mmol) according to procedures used to prepare 35. H NMR

(400 MHz, CD₃OD) δ (ppm): 7.65−7.60 (m, 5H), 7.54−7.52 (m,

1H), 7.41−7.37 (m, 2H), 7.34−7.30 (m, 2H), 7.10−7.06 (m, 3H),

4.68 (t, 2H, J = 12.8 Hz), 2.74 (s, 3H). Mass spectrum (LCMS, ESI

pos.) calcd For C₂₅H₂₀F₅N₃O₃S: 538.1 (M + H), found 538.2.

(trans)-N-(2-{2-[2-(4-Isopropyl-phenyl)-vinyl]-1H-benzoimidazol-

5-yl}-phenyl)-methanesulfonamide (40). The title compound

(0.0026 g) was prepared from N-(3′,4′-diamino-biphenyl-2-yl)-

methanesulfonamide (0.050 g, 0.18 mmol) and 3-(4-isopropyl-

phenyl)-acrylic acid (0.034 g, 0.18 mmol) according to procedures

(E)-2-(2-{2-[2-(4-Chloro-phenyl)-vinyl]-1H-benzimidazol-5-yl}-

phenyl)-propan-2-ol (47). The title compound was prepared from

3,3-dimethyl-3H-benzo[c][1,2]oxaborol-1-ol (0.15 g, 0.90 mmol) and

5-bromo-2-[2-(4-chloro-phenyl)-vinyl]-1H-benzoimidazole (0.20 g,

0.60 mmol) according to procedures used to make 4. ¹H NMR

(400 MHz, CD₃OD) δ (ppm): 7.79 (dd, 1H, J = 1.1 Hz, 8.1 Hz),

7.63−7.54 (m, 3H), 7.51 (br d, 1H, J = 7.4 Hz, 9.7 Hz), 7.40−7.38 (m,

3H), 7.31 (m, 1H), 7.19 (dt, 1H, J = 1.4 Hz, 7.4 Hz), 7.15 (d, 1H, J =

1

used to prepare 35. H NMR (400 MHz, CD₃OD) δ (ppm): 7.67−

7.52 (m, 6H), 7.14 (d, 1H, J = 16.4 Hz), 3.13−3.12 (m, 1H), 2.72 (s,

3H), 1.27 (d, 6H, J = 6.8 Hz). Mass spectrum (LCMS, ESI pos.) calcd

For C₂₅H₂₅N₃O₂S: 432.2 (M + H), found 432.2.

(trans)-N-(2-{2-[2-(4-Dimethylamino-phenyl)-vinyl]-1H-benzoi-

midazol-5-yl}-phenyl)-methanesulfonamide (41). The title com-

K

DOI: 10.1021/acs.jmedchem.5b00132

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Journal of Medicinal Chemistry

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1

16.5 Hz), 7.13 (dd, 1H, J = 1.8 Hz, 7.9 Hz), 7.04 (dd, 1H, J = 1.4 Hz,

7.5 Hz), 1.30 (s, 6H). Mass spectrum (LCMS, APCI pos.) calcd For

C₂₄H₂₁ClN₂O: 389.1 (M + H), found 389.3.

mmol) according to procedures used to prepare 51. H NMR (400

MHz, CD₃OD) δ (ppm): 8.14 (dd, 1H, J = 1.2 Hz, 7.6 Hz), 7.37−7.51

(m, 8H), 7.41 (dd, 1H, J = 1.2 Hz, 8.0 Hz), 7.35 (dd, 1H, J = 1.6 Hz,

8.0 Hz), 7.29 (d, 1H, J = 8.8 Hz), 7.25 (d, 1H, J = 16.8 Hz), 7.34 (dd,

1H, J = 1.6 Hz, 8.4 Hz). MS (ESI, pos. ion) m/z: 460.4 (M + 1).

2-{2-[2-(4-Ethoxy-phenyl)-vinyl]-1H-benzoimidazol-5-yl}-benze-

nesulfonamide (53). The title compound was prepared from 3′,4′-

diamino-biphenyl-2-sulfonic acid tert-butylamide (0.12 g, 0.38 mmol)

and trans-4-ethoxy-cinnamic acid (0.11 g, 0.45 mmol) according to

procedures used to prepare 51. ¹H NMR (400 MHz, CD₃OD) δ

(ppm): 8.13 (dd, 1H, J = 1.6 Hz, 8 Hz), 7.94 (dd, 1H, J = 1.6 Hz, 8

Hz), 7.68−7.62 (m, 6H), 7.41 (dd, 1H, J = 1.6 Hz, 8 Hz), 7.32 (d, 1H,

J = 1.6 Hz, 8 Hz), 7.28 (t, 1H, J = 8 Hz), 7.16 (d, 1H, J = 16.8 Hz).

Mass spectrum (LCMS, ESI pos.) calcd For C₂₁H₁₅BrFN₃O₂S: 473.3

(M + H), found 473.2.

(trans)-2-(2-{2-[2-(4-Triﬂuoromethanesulfonyl-phenyl)-vinyl]-1H-

benzoimidazol-5-yl}-phenyl)-propan-2-ol (48). The title compound

was prepared from 3,3-dimethyl-3H-benzo[c][1,2]oxaborol-1-ol

(0.068 g, 0.40 mmol) and 5-bromo-2-[2-(4-triﬂuoromethanesulfonyl-

phenyl)-vinyl]-1H-benzoimidazole (0.090 g, 0.21 mmol) according to

1

procedures used to make 4. H NMR (400 MHz, CD₃OD) δ (ppm):

8.07 (d, 2H, J = 8.6 Hz), 7.98 (d, 2H, J = 8.6 Hz), 7.78 (dd, 1H, J = 1.1

Hz, 8.1 Hz), 7.69 (d, 1H, J = 16.5 Hz), 7.54 (d, 1H, J = 8.2 Hz), 7.44

(d, 1H, J = 16.5 Hz), 7.42 (br s, 1H), 7.31 (ddd, 1H, J = 1.5 Hz, 7.8

Hz, 8.1 Hz), 7.18 (dt, 1H, J = 1.3, 7.4 Hz), 7.16 (dd, 1H, J = 1.5 Hz,

8.3 Hz), 7.03 (dd, 1H, J = 1.4 Hz, 7.5 Hz), 1.31 (s, 6H). Mass

spectrum (LCMS, APCI pos.) calcd For C₂₅H₂₁F₃N₂O₃S: 487.1 (M +

H), found 487.1.

2-(2-Styryl-1H-benzoimidazol-5-yl)-benzenesulfonamide (49).

The title compound was prepared from 3′,4′-diamino-biphenyl-2-

sulfonic acid tert-butylamide (0.12 g, 0.38 mmol) and trans-cinnamic

acid (0.11 g, 0.45 mmol) according to procedures used to prepare 51.

¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.13 (dd, 1H, J = 1.6 Hz, 7.8

(trans)-2-{2-[2-(4-Triﬂuoromethanesulfonyl-phenyl)-vinyl]-1H-

benzoimidazol-5-yl}-benzenesulfonamide (54). The title compound

was prepared from 2-(tert-butylamino)sulfonylphenylboronic acid (12

g, 0.048 mol) and 5-bromo-2-[2-(4-triﬂuoromethanesulfonyl-phenyl)-

vinyl]-1H-benzo[d]imidazole (14 g, 0.037 mol) according to

procedures used to prepare 3. ¹H NMR (400 MHz, CD₃OD) δ

(ppm): 8.14 (dd, 1H, J = 1.2 Hz, 7.6 Hz), 8.10 (d, 2H, J = 8.4 Hz),

8.01 (d, 2H, J = 8.4 Hz), 7.71−7.53 (m, 5H), 7.44 (d, 1H, J = 16.4

Hz), 7.41 (dd, 1H, J = 1.2 Hz, 7.6 Hz), 7.35 (dd, 1H, J = 1.2 Hz, 8.4

Hz). MS (ESI, pos. ion) m/z: 508.3 (M + 1).

Hz), 7.66−7.58 (m, 6H), 7.54 (dt, 1H, J = 1.6 Hz, 7.6 Hz), 7.44−7.40

(m, 3H), 7.38−7.34 (m, 1H), 7.31 (dd, 1H, J = 1.6 Hz, 8 Hz), 7.18 (d,

1H, J = 16.4 Hz). Mass spectrum (LCMS, ESI pos.) calcd For

C₂₁H₁₇N₃O₂S: 376.1 (M + H), found 376.3.

2-[2-(4-tert-Butyl-benzyl)-1H-benzoimidazol-5-yl]-phenol (55).

The title compound (0.0014 g) was prepared from 2-hydroxybenze-

neboronic acid (0.034 g, 0.25 mmol) and 5-bromo-2-[3-(4-tert-butyl-

phenyl)-methyl]-1H-benzo[d]imidazole (0.034 g, 0.10 mmol) accord-

2-{2-[2-(4-Fluoro-phenyl)-vinyl]-1H-benzoimidazol-5-yl}-benze-

nesulfonamide (50). The title compound was prepared from 3′,4′-

diamino-biphenyl-2-sulfonic acid tert-butylamide (0.12 g, 0.38 mmol)

and trans-4-ﬂuoro-cinnamic acid (0.11 g, 0.45 mmol) according to

procedures used to prepare 51. ¹H NMR (400 MHz, CD₃OD) δ

(ppm): 8.13 (dd, 1H, J = 1.6 Hz, 8 Hz), 7.69−7.66 (m, 2H), 7.65−

7.52 (m, 5H), 7.41 (dd, 1H, J = 1.6 Hz, 8 Hz), 7.31 (dd, 1H, J = 1.6

Hz, 8 Hz), 7.18−7.10 (m, 3H). Mass spectrum (LCMS, ESI pos.)

calcd For C₂₁H₁₆FN₃O₂S: 394.4 (M + H), found 394.3.

(trans)-2-{2-[2-(4-Triﬂuoromethoxy-phenyl)-vinyl]-1H-benzoimi-

dazol-5-yl}-benzenesulfonamide (51). A mixture of 2-(tert-

butylamino)sulfonylphenylboronic acid (4.7 g, 0.018 mmol), (5-

bromo-2-tert-butoxycarbonylamino-phenyl)-carbamic acid tert-butyl

ester (4.7 g, 0.012 mmol), Pd(dppf)Cl₂·CH₂Cl₂(0.99 g, 0.0012

mmol), tetrabutylammomnium bromide (3.9 g, 0.012 mmol), and 20

mL of 1 M Na₂CO₃(aq) in 100 mL of DME was heated at 100 °C for

12 h. The reaction mixture was concentrated, and the residue was

puriﬁed by chromatography (silica gel, hexanes:EtOAc, 2:1) to give (3-

tert-butoxycarbonylamino-2′-tert-butylsulfamoyl-biphenyl-4-yl)-carba-

mic acid tert-butyl ester as a yellow oil.

A mixture of (3-tert-butoxycarbonylamino-2′-tert-butylsulfamoyl-

biphenyl-4-yl)-carbamic acid tert-butyl ester in 20 mL of 4 M HCl

in dioxane was stirred for 3 h. The reaction mixture was concentrated,

diluted with EtOAc, washed with saturated aqueous NaHCO₃, dried

over Na₂SO₄, and ﬁltered, and the ﬁltrate was concentrated to provide

3′,4′-diamino-biphenyl-2-sulfonic acid tert-butylamide as a brown oil.

¹H NMR (400 MHz, CD₃OD) δ (ppm): 8.04 (dd, 1H, J = 1.2 Hz, 7.6

Hz), 7.56 (dt, 1H, J = 1.2 Hz, 8.2 Hz), 7.44 (dt, 1H, J = 1.2 Hz, 8.4

Hz), 7.33 (dd, 1H, J = 1.2 Hz, 7.6 Hz), 6.86 (d, 1H, J = 1.6 Hz), 6.77

(d, 1H, J = 7.6 Hz), 6.73 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 0.97 (s, 9H).

MS (ESI, pos. ion) m/z: 319.9 (M + 1).

1

ing to procedures used to prepare 2. H NMR (400 MHz, CDCl₃) δ

(ppm): 7.44−7.56 (m, 4H), 7.25−7.36 (m, 3H), 7.14−7.24 (m, 2H),

7.02−7.08 (m, 1H), 6.90−6.96 (m, 1H). MS (ESI, pos. ion) m/z:

357.2 (M + 1).

2-{2-[3-(4-tert-Butyl-phenyl)-propyl]-1H-benzoimidazol-5-yl}-

phenol (56). The title compound (0.0083 g) was prepared from 2-

hydroxybenzeneboronic acid (0.070 g, 0.50 mmol) and 5-bromo-2-[3-

(4-tert-butyl-phenyl)-propyl]-1H-benzo[d]imidazole (0.074 g, 0.20

1

mmol) according to procedures used to prepare 2. H NMR (400

MHz, CDCl₃) δ (ppm): 7.51 (s, 1H), 7.39 (d, 1H, J = 8.1 Hz), 7.16

(m, 6H), 6.95 (d, 3H, J = 8.2 Hz), 6.88 (t, 1H, J = 7.4 Hz), 2.73 (t, 2H,

J = 7.5 Hz), 2.52 (t, 2H, J = 7.3 Hz), 2.00 (m, 2H), 1.20 (s, 9H). MS

(ESI, pos. ion) m/z: 385.3 (M + 1).

(cis)-2-(2-{2-[2-(4-Triﬂuoromethyl-phenyl)-vinyl]-1H-benzoimida-

zol-5-yl}-phenyl)-propan-2-ol (57). To a solution of acetylene 61

(0.025 g, 0.059 mmol) in 5 mL of ethyl acetate was added Lindlar’s

catalyst (0.017 g). The mixture was hydrogenated under 1 atm at rt for

18 h. The mixture was ﬁltered through a pad of Celite. The solution

was concentrated under reduced pressure. The residue was puriﬁed on

preparative TLC (2000 μm, ethyl acetate:hexanes, 1:1 v/v) to yield the

1

desired product (0.0052 g). H NMR (400 MHz, CD₃OD) δ (ppm):

7.77 (d, 1H, J = 8.02 Hz), 7.59 (s, 4H), 7.53−7.30 (m, 2H), 7.30 (t,

1H, J = 7.60 Hz), 7.17 (t, 1H, J = 7.36 Hz), 7.11 (d, 1H, J = 8.22 Hz,

7.07−7.01 (m, 2H), 6.74 (d, 1H, J = 12.42 Hz), 1.30 (s, 6H). MS (ESI,

pos. ion) m/z: 423.3 (M + 1).

2-(2-{2-[2-(4-Triﬂuoromethyl-phenyl)-ethyl]-1H-benzoimidazol-5-

yl}-phenyl)-propan-2-ol (58). The title compound (0.013 g) was

prepared from 5-bromo-2-[2-(4-triﬂuoromethyl-phenyl)-ethyl]-1H-

benzoimidazole (0.048 g, 0.13 mmol) and 3,3-dimethyl-3H-benzo[c]-

[1,2]oxaborol-1-ol (0.032 g, 0.20 mmol) according to procedures used

A mixture of 3′,4′-diamino-biphenyl-2-sulfonic acid tert-butylamide

(0.12 g, 0.38 mmol), trans-4-(triﬂuoromethoxy)-cinnamic acid (0.11 g,

0.45 mmol), and 0.4 mL of 4 N HCl (aq) in 4 mL of ethylene glycol

was heated at 180 °C for 1 h. The reaction mixture was puriﬁed

directly by HPLC (YMC ODS-A, H₂O:MeCN, 90:10 to 40:60, over

10 min) to aﬀord 51 as an oﬀ-white solid. ¹H NMR (400 MHz,

CD₃OD) δ (ppm): 8.14 (dd, 1H, J = 1.2 Hz, 8.0 Hz), 7.75 (d, 2H, J =

8.4 Hz), 7.68−7.53 (m, 5H), 7.41 (dd, 1H, J = 1.2 Hz, 7.6 Hz), 7.37−

7.33 (m, 3H), 7.19 (d, 1H, J = 16.8 Hz). MS (ESI, pos. ion) m/z:

460.3 (M + 1).

1

to prepared 2. H NMR (400 MHz, DMSO-d₆) δ (ppm): 7.71 (dd,

1H, J = 1.26 Hz, 8.08 Hz), 7.45 (d, 2H, J = 7.8 Hz), 7.37 (d, 1H, J =

8.34 Hz), 7.31 (d, 2H, J = 8.08 Hz), 7.20−7.26 (m, 2H), 7.10 (ddd,

1H, J = 1.51 Hz, 7.33 Hz, 7.58 Hz), 6.93−7.20 (m, 2H), 3.14 (m, 4H),

1.21 (s, 6H). MS (ESI, pos. ion) m/z: 425.1 (M + 1).

2-{2-[2-(4-Triﬂuoromethyl-phenyl)-ethyl]-1H-benzoimidazol-5-

yl}-benzenesulfonamide (59). A mixture of 3 (0.020 g, 0.045 mmol)

and 10% palladium on charcoal (0.0050 g, 0.0045 mmol) in 2 mL of

MeOH was hydrogenated under H₂gas (1 atm) for 1 h. The catalyst

was removed by ﬁltration, the ﬁltrate was concentrated to dryness, and

the residue was puriﬁed by chromatography (silica gel,

2-{2-[2-(3-Triﬂuoromethoxy-phenyl)-vinyl]-1H-benzoimidazol-5-

yl}-benzenesulfonamide (52). The title compound was prepared from

3′,4′-diamino-biphenyl-2-sulfonic acid tert-butylamide (0.12 g, 0.38

mmol) and trans-3-triﬂuoromethoxy-cinnamic acid (0.11 g, 0.45

L

DOI: 10.1021/acs.jmedchem.5b00132

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MeOH:CH₂Cl₂, 10:1) to give the title compound as a white solid. H

NMR (400 MHz, CD₃OD) δ (ppm): 8.12 (dd, 1H, J = 1.2 Hz, 8.4

Hz), 7.64−7.51 (m, 6H), 7.42 (d, 2H, J = 8.0 Hz), 7.39 (dd, 1H, J =

1.2 Hz, 7.6 Hz), 7.27 (dd, 1H, J = 1.6 Hz, 8.4 Hz), 3.24 (m, 4H). MS

(ESI, pos. ion) m/z: 446.4 (M + 1).

spectrum (LCMS, ESI pos.) calcd For C₂₁H₁₅N₃O₂S: 374.1 (M + H),

found 374.2.

2-[2-(4-Triﬂuoromethyl-phenylethynyl)-1H-benzoimidazol-5-yl]-

benzenesulfonamide (63). The title compound (0.0045 g) was

1

prepared according to procedures used to prepare 62. H NMR (400

MHz, CD₃OD) δ (ppm): 8.04 (dd, 1H, J = 1.2 Hz, 8.0 Hz), 7.76 (d,

2H, J = 8.2 Hz), 7.69 (d, 2H, J = 8.2 Hz), 7.58−7.53 (m, 2H), 7.54 (dt,

1H, J = 1.4 Hz, 7.5 Hz), 7.46 (dt, 1H, J = 1.4 Hz, 7.7 Hz), 7.31 (dd,

1H, J = 1.3 Hz, 7.5 Hz), 7.30 (dd, 1H, J = 0.9 Hz, 8.4 Hz). Mass

spectrum (LCMS, ESI pos.) calcd For C₂₂H₁₄F₃N₃O₂S: 442.1 (M +

H), found 442.2.

2-[2-(2-Phenylethynyl-1H-benzoimidazol-5-yl)-phenyl]-propan-

2-ol (60). A mixture of 4-bromo-benzene-1,2-diamine dihydrochloride

(1.3 g, 5.0 mmol) and phenylpropiolic acid (0.73 g, 5.0 mmol) in 4 mL

of ethylene glycol was heated to reﬂux for 5 h. The mixture was cooled

to rt and poured into water. The mixture was neutralized with 2 N

sodium hydroxide and ﬁltered. The solid was suspended in water and

extracted with ethyl acetate. The organic layers were combined, dried

with anhydrous sodium sulfate, and ﬁltered. The ﬁltrate was

concentrated to yield a red oil. The residue was puriﬁed using

preparative TLC plates twice (2000 μm, EtOAc:hexanes 3:7 and

EtOAc:dichloromethane 3:97) to give 5-bromo-2-(2-chloro-2-phenyl-

vinyl)-1H-benzoimidazole as a mixture of cis- and trans-isomers (0.35

Human and Rat TRPV1 Cell-Based Functional Assays.

HEK293 cells stably expressing human or rat TRPV1 were maintained

at 37 °C and 5% CO₂in Dulbecco’s Modiﬁed Eagle’s Medium

supplemented with 10% FBS, 2 mM ^L-glutamine, 100 IU/mL

penicillin, 100 μg/mL streptomycin (Mediatech), and 400 μg/mL

G418 sulfate in tissue culture treated ﬂasks. For assaying, cells were

grown on poly-^D-lysine coated 384-well black-walled plates (BD

354663) and one day later loaded with Calcium 3 Dye for 35 min at

37 °C in 5% CO₂. Following incubation for 25 min at room

temperature, cells were tested for capsaicin-induced increases in

intracellular Ca²⁺levels using FLIPR (Molecular Devices) or FDSS

(Hamamatsu) technology. Cells were exposed to test compounds at

varying concentrations, and intracellular Ca²⁺was measured for 5 min

prior to the addition of capsaicin to all wells to achieve a ﬁnal

concentration (typically 0.020−0.030 μM), eliciting an approximately

80% maximal response. All data points are the average value obtained

from quadruplicate wells in the same assay plate. EC₅₀or IC₅₀values

were obtained by eight-point nonlinear regression of sigmoidal

concentration−response modeled by GraphPad Prism 6.02 (Graph

Pad Software Inc., CA, USA).

1

g, 21% yield). H NMR (400 MHz, DMSO-d₆) δ (ppm): 12.59 (m,

1H), 7.86−7.78 (m, 3H), 7.64−7.56 (m, 1H), 7.54−7.49 (m, 3H),

7.46 (s, 1H), 7.40−7.34 (m, 1H). Mass spectrum (LCMS, APCI pos.)

calcd For C₁₅H₁₀BrClN₂: 333.0 (M + H), found 333.1.

A mixture of 5-bromo-2-(2-chloro-2-phenyl-vinyl)-1H-benzoimida-

zole (0.025 g, 0.075 mmol), 3,3-dimethyl-3H-benzo[c][1,2]oxaborol-

1-ol (0.018 g, 0.11 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(0.012 g, 0.015

mmol) in 3 mL of DME, and sodium carbonate solution (1.0 M, 0.6

mL) was heated at 150 °C for 1 h in a Biotage Initiator microwave

synthesizer. The mixture was ﬁltered through a pad of silica gel. The

reaction was repeated a total of three times. The residues were

combined and puriﬁed using preparative TLC plates twice (2000 μm,

EtOAc:hexanes 3:7 and EtOAc:hexanes:methanol 2:8:1) to give 60

1

(8.4 mg). H NMR (400 MHz, CD₃OD) δ (ppm): 7.78 (dd, 1H, J =

1.1 Hz, 8.1 Hz), 7.62−7.60 (m, 2H), 7.52 (d, 1H, J = 8.3 Hz), 7.46−

7.38 (m, 4H), 7.31 (dt, 1H, J = 1.5 Hz, 8.0 Hz), 7.21−7.17 (m, 2H),

7.02 (dd, 1H, J = 1.3 Hz, 7.5 Hz), 1.30 (s, 6H). Mass spectrum

(LCMS, APCI pos.) calcd For C₂₄H₂₀N₂O: 353.2 (M + H), found

353.3.

Maximal ﬂuorescence intensity (FI) achieved upon addition of

capsaicin was exported from the FDSS software and further analyzed

using GraphPad Prism. Basal FI was subtracted prior to normalizing

data to percent of maximal response. For concentration−response

relationship analysis, data were converted to % maximal capsaicin

response based on the maximal ﬂuorescence observed at either 90 or

100 μM capsaicin (i.e., 100%) and the minimal ﬂuorescence observed

at 0.6 pM capsaicin (0%). Curves were generated using the average of

quadruplicate wells for each data point and were analyzed using

nonlinear regression of sigmoidal concentration−response with

variable slope. Finally, the EC₅₀and IC₅₀values were calculated with

the best-ﬁt concentration curve determined by GraphPad Prism.

Human TRPV1 Binding Assay. Compounds were evaluated for

their ability to inhibit the binding of [³H]-RTX to hTRPV1. HEK293

cells were transfected with hTRPV1 and washed with Hank’s Balanced

Salt Solution. Cells were dissociated with cell dissociation buﬀer

(Sigma) and centrifuged at 1000g for 5 min. Cell pellets were

homogenized in cold 20 mM HEPES buﬀer (pH = 7.4), containing 5.8

mM NaCl, 320 mM sucrose, 2 mM MgCl₂, 0.75 CaCl₂, and 5 mM KCl

and centrifuged at 1000g for 15 min. The resultant supernatant was

centrifuged at 40000g for 15 min, and the pelleted membranes were

stored in a −80 °C freezer until use. Approximately 120 μg/mL of

protein from membranes were incubated with the indicated

concentrations of [³H]-RTX in 0.5 mL of the HEPES buﬀer (pH =

7.4), containing 0.25 mg/mL fatty acid-free bovine serum albumin at

37 °C for 60 min. The reaction mixture was cooled to 4 °C, 0.1 mg of

α₁-acid glycoprotein was added, and samples were incubated at 4 °C

for 15 min. The samples were centrifuged at 18500g for 15 min. The

tip of the microcentrifuge tube containing the pellet was cut oﬀ. Bound

radioactivity was quantiﬁed by scintillation spectroscopy, and non-

speciﬁc binding was determined in the presence of 200 nM unlabeled

RTX. K_ivalues were calculated using Prism according to the equation:

K_i= IC₅₀/(1 + L/K_d), wherein L = ligand concentration.

2-[2-(2-(4-Triﬂuoromethyl)-phenylethynyl-1H-benzoimidazol-5-

yl)-phenyl]-propan-2-ol (61). The title compound (0.0053 g) was

prepared from 3-[4-(triﬂuoromethyl)phenyl]-2-propynoic acid accord-

1

ing to procedures used to prepare 60. H NMR (400 MHz, CD₃OD)

δ (ppm): 7.78 (dd, 1H, J = 1.1 Hz, 8.1 Hz), 7.62−7.60 (m, 2H), 7.52

(d, 1H, J = 8.3 Hz), 7.46−7.38 (m, 4H), 7.31 (dt, 1H, J = 1.5 Hz, 8.0

Hz), 7.21−7.17 (m, 2H), 7.02 (dd, 1H, J = 1.3 Hz, 7.5 Hz), 1.30 (s,

6H). Mass spectrum (LCMS, APCI pos.) calcd For C₂₅H₁₉F₃N₂O:

421.1 (M + H), found 422.1.

2-(2-Phenylethynyl-1H-benzoimidazol-5-yl)-benzenesulfonamide

(62). A mixture of 5-bromo-2-(2-chloro-2-phenyl-vinyl)-1H-benzoimi-

dazole from the preparation of 60 (0.025 g, 0.075 mmol), 2-(tert-

butylamino)sulfonylphenylboronic acid (0.029 g, 0.11 mmol), Pd-

(dppf)Cl₂·CH₂Cl₂(0.012 g, 0.015 mmol) in 3 mL of DME, and

sodium carbonate solution (1.0 M, 0.6 mL) was heated at 150 °C for 1

h in a Biotage Initiator microwave synthesizer. The mixture was

ﬁltered through a pad of silica gel. The residue was puriﬁed by

preparative TLC (2000 μm, EtOAc:hexanes 3:7) to give N-tert-butyl-

2-(2-phenylethynyl-1H-benzoimidazol-5-yl)-benzenesulfonamide

(0.013 g, 40% yield). Mass spectrum (LCMS, APCI pos.) calcd For

C₂₅H₂₃N₃O₂S: 430.2 (M + H), found 430.3.

A mixture of N-tert-butyl-2-(2-phenylethynyl-1H-benzoimidazol-5-

yl)-benzenesulfonamide (0.015 g, 0.034 mmol) in triﬂuoroacetic acid

and 1,2-dichloroethane (2 mL, 1:1) was heated at 90 °C for 3 h. The

reaction was cooled to rt and concentrated under reduced pressure.

The residue was dissolved in dichloromethane and washed with

saturated sodium bicarbonate solution. The aqueous layer was

extracted twice with ethyl acetate. The organic layers were combined,

dried with anhydrous magnesium sulfate, and ﬁltered, and the ﬁltrate

was removed under reduced pressure. The residue was puriﬁed using

preparative TLC plates (2000 μm, EtOAc:hexanes:methanol 5:5:1) to

give 62 (12.1 mg, 93% yield). ¹H NMR (400 MHz, CD₃OD) δ

(ppm): 8.11 (dd, 1H, J = 1.2 Hz, 8.0 Hz), 7.67−7.56 (m, 5H), 7.52

(ddd, 1H, J = 1.4 Hz, 6.4 Hz, 7.6 Hz), 7.48−7.33 (m, 5H). Mass

TRPV1 Electrophysiology: Whole-Cell Patch Clamp Record-

ing of Capsaicin- and pH-Evoked Currents from hTRPV1-HEK

Cells. Compounds were evaluated for their ability to inhibit capsaicin-

induced currents using whole cell patch clamp recording. A Dynaﬂow

8 channel “proII” (DF8PROII) perfusion chip was prepared per

manufacturer’s speciﬁcations. HEK293 cells stably expressing hTRPV1

M

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

were removed by pipet trituration from a 35 mm dish with Earl’s

Balanced Salt Solution (EBSS), containing 132 mM NaCl, 1.8 mM

CaCl₂, 5.4 mM KCl, 0.8 mM MgCl₂, 10 mM HEPES, and 10 mM

glucose (pH = 7.4). However, all subsequent drug solutions were

prepared in EBSS lacking CaCl₂and containing 0.1% BSA (0Ca-EBSS

+ BSA) to avoid desensitization of the capsaicin-evoked current and to

reduce nonspeciﬁc binding of compound to the perfusion chip,

respectively. The internal pipet electrode solution contained 140 mM

CsCl, 0.6 mM MgCl₂, 1 mM EGTA, and 10 mM HEPES (pH = 7.4).

After achieving whole cell conﬁguration with the patch clamp pipet,

the cell was positioned at the channel opening containing 0Ca-EBSS +

BSA. After establishing a stable baseline, the solution was changed to

0Ca-EBSS + BSA with 1 μM capsaicin by moving the cell to the

adjacent well opening. Once the capsaicin current reached a stable

level (typically after 1−2 min), the cell was exposed to solution

containing the indicated concentration of compound in the presence

of 1 μM capsaicin. The resulting current was measured for 150 s, by

which time it typically reached a stable level. The cell was subsequently

exposed to a higher concentration of compound in the same manner.

After exposure to 1 μM of compound (the highest concentration

used), the cell was moved into 0Ca-EBSS + BSA for a ﬁnal wash. No

single cell was exposed to more than three ascending concentrations of

compound.

was unaware of the treatment administered to individual animals. Heat

hypersensitivity was evaluated following intraplantar injection of 100

μL (1 μg/μL) of CFA (1:1 CFA:saline) or 200 μL of carrageenan-λ

(10 mg/mL) using a radiant heat device (University of California San

Diego, San Diego, CA).

To assess the ability of test compounds to reverse thermal

hypersensitivity, baseline response latencies were obtained prior to

injection of CFA or carrageenan-λ. Only withdrawal responses that

were quick hind paw movements (with or without licking of the hind

paw) were recorded. Paw movements associated with locomotion or a

shifting of weight were not considered a withdrawal response. The

stimulus intensity that produced 10−15 s baseline withdrawal latencies

was used, and a maximum cutoﬀ of 20 s was imposed to protect the

animals from thermal injury. Hypersensitivity was evaluated 24 h after

CFA or 3 h after carrageenan. Only rats that exhibited at least a 25%

reduction in response latency from baseline were included in further

analysis. Following the postinﬂammogen latency assessment, rats were

orally administered test compound, reference compound, or vehicle

(2.5 mL/kg). To determine the time of peak eﬀects in the CFA

studies, latencies were redetermined 30, 60, 100, 180, and 300 min

after compound administration. To determine the potency of

compounds, withdrawal latencies were re-evaluated after compound

administration at the approximate T_maxof the compound. For CFA

studies, plasma samples were obtained immediately following

assessment of thermal hypersensitivity. For carrageenan studies,

plasma samples were obtained from a separate group of rats

administered compound under identical conditions as those in the

behavioral assessment. Blood was placed into tubes containing lithium

heparin and centrifuged at 4000 rpm for 5 min. Plasma was stored in a

−80 °C freezer until analyzed for compound concentrations.

Data were calculated as mean SEM absolute latency or percent

reversal of hypersensitivity, which was calculated for each animal as

[treatment response − postinﬂammogen response/preinﬂammogen

response − postinﬂammogen response] × 100. For time course

results, eﬀects were analyzed using a two-way, repeated-measure

analysis of variance (ANOVA) followed by the Bonferroni posthoc

test. For dose−response results, eﬀects were analyzed using a one-way

ANOVA followed by Dunnett’s posthoc test. The 50 or 80% eﬀective

dose (ED₅₀or ED₈₀) values and the relative plasma concentrations

(EC₅₀or EC₈₀) were calculated using PharmTools Plus software (The

McCary Group).

Core Body Temperature Time Course Model. A dose of 100

mg/kg 4 (n = 8) or vehicle (HPβCD; n = 8) was orally administered

to male Sprague−Dawley rats (food deprived for 18 h before dosing),

and core body temperature was evaluated over 3 h using a rectal

thermometer. Signiﬁcant main eﬀects were analyzed by two-way,

repeated measure ANOVA (vehicle vs compound) with signiﬁcant

main eﬀects further analyzed by Bonferroni post-test comparisons.

Core Body Temperature Dose-Dependency Model. Male

Sprague−Dawley rats (Charles River; Portage, ME) were implanted

(ip) with microchip temperature/ID telemetry probes (Bio Medic

Data Systems, Inc., Seaford, DE) 5 days prior to the study start. Rats

were habituated to handling and to the oral gavage 3 days prior to the

study start. In rats (n = 5−6/group) deprived of food for 18 h, a single

oral dose of 4 (0.1, 1.0, 10.0, or 100.0 mg/kg) or vehicle (20%

HPβCD) was administered and core body temperatures were obtained

prior to and 30, 60, 100, 180, and 300 min after dosing. Signiﬁcant

main eﬀects were analyzed by two-way ANOVA (vehicle vs

compound) with signiﬁcant main eﬀects further analyzed by

Bonferroni post-test comparisons.

Core Body Temperature Subchronic Model. Compound 4 was

administered daily for 5 days. Male Sprague−Dawley rats (Charles

River, Portage, ME) were implanted (ip) with microchip temperature/

ID telemetry probes (Bio Medic Data Systems, Inc., Seaford, DE) 3

weeks prior to the study start. One week prior to the study start, rats

were habituated to handling and to oral gavage dosing. Rats were

maintained in the same room throughout the 5 day temperature study.

Compound 4 (10 mg/kg; n = 5−6) or vehicle (20% HPβCD; n = 3−

5) was administered QD for 5 consecutive days, and core body

Compounds were also evaluated for their ability to block pH-

induced hTRPV1 currents. Under similar methodology as that

described above, a pH 5 solution was added, and compound was

tested at concentrations of 10, 30, 100, and 300. IC₅₀values were

calculated by a logistic equation ﬁt to the data using Origin statistical

software.

TRPV1 Electrophysiology: Whole-Cell Patch Clamp Record-

ing of Heat-Evoked Currents from hTRPV1-HEK Cells. Record-

ings from HEK293 cells stably expressing hTRPV1 were performed

using the conventional whole-cell patch clamp technique. Solutions

were applied onto the cell via a gravity-fed perfusion system. To

activate the channel, the temperature of solutions perfusing the cell

was increased from 22 to 40 °C using an in-line heater. After steady-

state current activation was achieved, compound (100 nM) was

applied for 80 s at this temperature, followed at the end of an

experiment by the additional application of 10 μM capsazepine for 55

s. The temperature in the vicinity of the recorded cell and the currents

concurrently measured in the whole-cell patch clamp mode were

measured with a custom-made miniature thermomicroprobe con-

nected to a monitoring thermometer and sampled using Digidata

1322A and pClamp 9.0. Currents were ampliﬁed by a patch clamp

ampliﬁer and ﬁltered at 2 kHz. The cell was held at −80 mV, and the

current was continuously sampled (at 100 Hz) at this holding

potential. The average current for the last 2 s of each treatment

condition was used to calculate the percent inhibition by 100 nM

compound, using the following equation:

%inhibition = current^{(capsazepine+heat)}− current^{(compound+heat)}

(

/current^{(capsazepine+heat)}− current^(heat)× 100

)

Chemically-Induced Inﬂammatory Pain Models. Male

Sprague−Dawley rats from Charles Rivers Laboratories (Portage,

ME), weighing between 195 and 350 g at the time of study, were

subjected to a one-week quarantine/acclimation period before being

transferred to a general stockroom. Animals were housed in

microisolator cages in groups of four rats per cage with corncob

bedding and free access to food and water. The environment was

maintained at an average temperature of 21 °C, with a 12 h light/dark

cycle. Rats in the CFA study were moved to a testing room the day of

the CFA injection, where they remained for the duration of the study.

Rats in the carrageenan study were moved to the testing room on the

day before the study.

Animals from several cages were randomly assigned across

treatment groups (i.e., rodents in a given cage were pseudorandomly

assigned to diﬀerent treatment groups). Treatments were coded and

randomized so that the investigator performing the behavioral analysis

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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AUTHOR INFORMATION

■

Corresponding Author

*Phone: 215-628-7860. E-mail: mplayer@its.jnj.com.

Notes

The authors declare no competing ﬁnancial interest.

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Article Doi

Benzo[d]imidazole transient receptor potential vanilloid 1 antagonists for the treatment of pain: Discovery of trans-2-(2-{2-[2-(4-Trifluoromethyl-phenyl)-vinyl]-1H-benzimidazol-5-yl}-phenyl)-propan-2-ol (Mavatrep)

DOI: 10.1021/acs.jmedchem.5b00132

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