Fused piperidines as a novel class of potent and orally available transient receptor potential melastatin type 8 (TRPM8) antagonists

Article

pubs.acs.org/jmc

Fused Piperidines as a Novel Class of Potent and Orally Available

Transient Receptor Potential Melastatin Type 8 (TRPM8) Antagonists

Nuria A. Tamayo,*^,†Yunxin Bo,^†Vijay Gore,^†Vu Ma,^†Nobuko Nishimura,^†Phi Tang,^†Hong Deng,^§

Lana Klionsky,^§Sonya G. Lehto,^§Weiya Wang,^§Brad Youngblood,^§Jiyun Chen,^∥Tiffany L. Correll,^‡

Michael D. Bartberger,^†Narender R. Gavva,^§and Mark H. Norman^†

Departments of ^†Chemistry Research and Discovery, ^§Neuroscience, ^‡Analytical Research and Development, and ^∥Pharmacokinetics

and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States

S

* Supporting Information

ABSTRACT: The transient receptor potential melastatin type 8

(TRPM8) is a nonselective cation channel primarily expressed in

a subpopulation of sensory neurons that can be activated by a

wide range of stimuli, including menthol, icilin, and cold

temperatures (<25 °C). Antagonism of TRPM8 is currently

under investigation as a new approach for the treatment of pain.

As a result of our screening efforts, we identified tetrahydro-

thienopyridine 4 as an inhibitor of icilin-induced calcium influx in CHO cells expressing recombinant rat TRPM8. Exploration of

the structure−activity relationships of 4 led to the identification of a potent and orally bioavailable TRPM8 antagonist,

tetrahydroisoquinoline 87. Compound 87 demonstrated target coverage in vivo after oral administration in a rat

pharmacodynamic model measuring the prevention of icilin-induced wet-dog shakes (WDS).

INTRODUCTION

■

The transient receptor potential melastatin type 8 (TRPM8) is

a member of the transient receptor potential (TRP) super-

family of ion channels. Based on amino acid homology, the

mammalian members of this family have been classified into six

subfamilies: TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML.

The TRP channels have six transmembrane polypeptide

subdomains flanked by intracellular C- and N-terminal regions.

In addition, the TRP proteins assemble as homo- or

heterotetramers to form cation-permeable pores.¹These

channels are nonselective cation channels activated by a variety

of chemical and physical stimuli. For example, TRPM8 is a

ligand-gated, Ca²⁺-permeable, nonspecific cation channel

primarily expressed in a subset of small diameter sensory

neurons.²TRPM8 is activated by cold temperatures (<25 °C),

Figure 1. TRPM8 agonists, menthol (1), eucalyptol (2), and icilin (3),

as well as by natural cooling compounds such as menthol (1)

and eucalyptol (2).³The TRPM8 channel is also activated by

the synthetic cooling agent icilin (3) (Figure 1).⁴The cloning

and characterization of the TRPM8 receptor, as well as several

studies with knockout mice, revealed its role in cold sensation

as well as cold allodynia (pain induced by normally innocuous

cold) and its potential utility as a therapeutic target.^5,6Cold

allodynia is one of the predominant clinical symptoms in

patients going through chemotherapy,⁷as well as in diseases

such as diabetic neuropathy,⁸fibromyalgia,⁹and traumatic

neuropathy.¹⁰Furthermore, correlation of pain with increased

expression of TRPM8 in nerve fibers of overactive and painful

bladders suggests TRPM8 involvement in bladder pain.¹¹

Preclinically, an antagonist of TRPM8 has been shown to

decrease the frequency of volume-induced bladder contractions,

without reducing the amplitude of contraction in rats.¹²

the initial high-throughput TRPM8 hit 4, tetrahydrothienopyridine 5,

and the generic structure 6 used for SAR investigations.

Together, the above studies suggest a potential role for

TRPM8 antagonists in chronic pain conditions as well as in

painful bladder syndromes.¹³Small molecule antagonists of the

TRPM8 channel would provide important pharmacological

tools for fully assessing the therapeutic potential of inhibiting

this novel target.

At the onset of our efforts to validate TRPM8 as a novel

target for pain, there were very few reports of TRPM8

antagonists in the public domain.^14,15We therefore set out to

Received: October 10, 2011

Published: February 13, 2012

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a

Scheme 1

a

Reagents and conditions: (a) R¹COCl, Et₃N, CH₂Cl₂, room temperature, 2 h; (b) POCl₃, CH₃CN, 60 °C, 18 h; (c) NaBH₄, MeOH, room

temperature, 2 h; (d) benzaldehyde, toluene, reflux, 16 h; (e) trifluoroacetic acid, room temperature, 72 h; (f) phenyl isocyanate, CH₂Cl₂, room

temperature, 1 h.

discover a novel small molecule antagonist by performing a

high-throughput screen to identify inhibitors of icilin activation

of the rTRPM8 channel. Tetrahydrothienopyridine 4 was

identified as a hit from this screening campaign (Figure 1).

Compound 4 inhibited the Ca²⁺influx evoked by 3 in CHO

cells transfected with rTRPM8 with an IC₅₀value of 1.39 μM.

However, stability data from rat liver microsomal (RLM)

preparations predicted that compound 4 would be extensively

metabolized (CL_int> 399 μL/(min·mg))¹⁶and therefore, it

would not have adequate pharmacokinetic (PK) characteristics

necessary for in vivo target validation. An initial substructure

search of the corporate database identified several other

tetrahyhydrothienopyridines as antagonists of rTRPM8. From

this set, compound 5 (Figure 1) was found to be 5-fold more

potent than the initial hit, albeit displaying similar instability in

RLM (IC₅₀= 293 nM; CL_int> 399 μL/(min·mg)).¹⁶Therefore,

we undertook a structure−activity relationship (SAR) inves-

tigation to identify analogues of 5 with the goal of improving

both the potency and the pharmacokinetic properties of this

series.

7 with benzaldehyde formed the imine 9, which was cylized in

the presence of trifluoroacetic acid. The targeted ureas (5, 11−

23), were prepared by reacting phenyl isocyanate with

tetrahydrothienopyridines 10a−10n.

To study the role of the urea substituent in compound 5 (R²

at the piperidine nitrogen), several analogues having amines

(24−26), amides (27−31), or ureas (32−39) substituents were

prepared from 4-(4-(trifluoromethyl)phenyl)-4,5,6,7-

tetrahydrothieno[3,2-c]pyridine (10g) (Scheme 2). Reductive

a

Scheme 2

a

Reagents and conditions: (a) ketone/aldehyde, trifluoroacetic acid,

To investigate the SAR around compound 5, we decided to

systematically vary three segments of the molecule: the

heterocyclic core, the R¹group on the 2-position of the

piperidine ring, and the R²substituent on the piperidine

nitrogen (general structure 6; Figure 1). Toward this goal,

novel analogues of 5 were synthesized and tested for their

ability to antagonize rTRPM8. The compounds were also

evaluated for in vitro metabolic stability in rat liver microsomes

(RLM). This investigation provided an understanding of the

SAR of this class of compounds and resulted in the discovery of

a novel series of rTRPM8 antagonists, exemplified by

tetrahydroisoquinoline 87. This compound potently inhibited

rTRPM8 in vitro and demonstrated adequate target coverage in

vivo.

NaB(OAc)₃H, DCE, room temperature, 1 h; (b) acid chloride, Et₃N,

CH₂Cl₂, room temperature, 2 h; (c) isocyanate, CH₂Cl₂, room

temperature, 1 h.

amination of 10g with acetone, isobutyraldehyde, or

benzaldehyde led to amines 24−26. Amides (27−31) were

prepared by the reaction of 4-(4-(trifluoromethyl)phenyl)-

4,5,6,7-tetrahydrothieno[3,2-c]pyridine (10g) with the corre-

sponding acid chlorides in the presence of triethylamine.

Finally, the urea derivatives 32−39 were prepared by

condensation of 10g with various isocyanates (Scheme 2).

To extend our SAR study, we also investigated a variety of

replacements of the tetrahydrothienopyridine core. Initially, we

examined the nature of the five-membered heterocyclic ring

fused to the piperidine. For example, tetrahydrofuropyridine 41

was synthesized by N-acylation of commercially available 2-

(furan-2-yl)ethanamine (40) with 4-(trifluoromethyl)benzoyl

chloride, followed by cyclization with POCl₃and reduction of

the cyclic imine intermediate (Scheme 3). The regiosomeric

analogues, tetrahydrofuropyridine 42 and tetrahydrothienopyr-

idine 43, were obtained by previously described proce-

dures,^17,18and the tetrahydropyrrolo[1,2-a]pyrazine 44 was

commercially available. The requisite urea analogues 45−48

were synthesized in good yields by treatment of fused

CHEMISTRY

■

Tetrahydrothienopyridine analogues where the R¹group was

varied were prepared via “amide” or “imine” synthetic routes as

depicted in Scheme 1. Acylation of 2-(thiophen-2-yl)-

ethanamine (7) with various acid chlorides led to amides of

general structure 8. Cyclization of the amides with POCl₃and

subsequent reduction of the resulting imines with NaBH₄led to

the desired tetrahydrothienopyridines 10a−10m. Alternatively,

4-phenyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (10n) was

prepared via a Pictet−Spengler reaction. Treatment of amine

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a

Scheme 3

a

Reagents and conditions: (a) 4-CF₃COCl, Et₃N, CH₂Cl₂, room temperature, 2 h; (b) POCl₃, benzene, reflux, 2 h; (c) NaB(OAc)₃H, CH₂Cl₂, room

temperature, 18 h; di-tert-butyl dicarbonate, Et₃N, CH₂Cl₂, room temperature, 18 h; (d) HCl/dioxane, room temperature, 18 h; (e) 4-F-phenyl

isocyanate, Et₃N, CH₂Cl₂, room temperature, 1 h.

a

Scheme 4

a

Reagents and conditions: (a) MeONHMe·HCl, EDC, DIEA, CH₂Cl₂, room temperature, 16 h; (b) DIBAL, THF, −78 °C, 1 h; (c) 4-CF₃PhMgBr,

THF, 0 °C; (d) N-vinylphtalimide, Pd₂(dba)₃, X-Phos, Et₃N, DMF, 80 °C, 24 h; (e) Pd/C, H₂, MeOH; (f) MnO₂, CH₂Cl₂, room temperature, 4 h;

(g) NH₂NH₂, EtOH, room temperature, 72 h; (h) NaBH₄, MeOH, room temperature, 30 min; (i) 4-F-phenyl isocyanate, CH₂Cl₂, room

temperature, 1 h.

piperidines 41−44 with 4-fluorophenyl isocyanate at room

temperature.

yield 53. Catalytic hydrogenation of the vinyl group of

compound 53, followed by oxidation with manganese oxide

led to ketone 55. Removal of the phthalimide protecting group

with hydrazine in ethanol and reduction of the resulting

dihydrothiazolopyridine intermediate 56 gave tetrahydrothia-

zolopyridine 57. Finally, compound 57 was treated with 4-

fluorophenyl isocyanate to give the desired analogue, 58.

Analogues having a fused imidazole ring were readily

accessible by thermal cyclization of the Schiff base obtained

from histamine (59) and 4-trifluoromethylbenzaldehyde

The synthesis of the fused 4,5-thiazole derivative 58 needed

for this investigation required a longer synthetic route (Scheme

4). Intermediate 51 was synthesized following a two-step

procedure starting from commercially available acid 49 via the

Weinreb amide 50. Reaction of 5-bromothiazole-4-carbalde-

hyde (51) with 4-(trifluoromethyl)phenyl Grignard gave the

benzylic alcohol 52, which was then coupled with N-

vinylphthalimide under palladium-catalyzed conditions to

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a

Scheme 5

a

Reagents and conditions: (a) 4-CF₃PhCHO, KOH, EtOH/H₂O, 100 °C, 4 h; (b) 4-F-phenyl isocyanate, CH₂Cl₂, room temperature, 1 h; (c) di-

tert-butyl dicarbonate, NaHCO₃, MeOH, room temperature, 2 h; (d) MeI, NaHCO₃, DMF, room temperature, 2 h; (e) trifluoroacetic acid/CH₂Cl₂,

room temperature, 2 h.

a

Scheme 6

a

Reagents and conditions: (a) 4-CF₃PhCOCl, DIEA, CH₂Cl₂, room temperature, 16 h; (b) P₂O₅, polyphosphoric acid, 165 °C, 2 h; (c) NaBH₄,

MeOH, room temperature, 2 h; (d) 4-F-phenyl isocyanate, CH₂Cl₂, room temperature, 1 h.

(Scheme 5). The tetrahydroimidazopyridine 60 was converted

to the 4-fluorophenyl urea 61 by reaction with 4-fluorophenyl

isocyanate. To prepare analogue 63, the piperidine amino

group of compound 60 was first protected using di-tert-butyl

carbonate to give intermediate 62. Methylation of the imidazole

ring with methyl iodide in the presence of sodium bicarbonate

gave the 1-methyl regioisomer as the major product.¹⁹The t-

butoxycarbonyl protecting group was removed by treatment

with trifluoroacetic acid in dichloromethane. Further funtion-

alization with 4-fluorophenyl isocyanate and purification gave

the dihydroimidazopyridine, 63.

We next examined a set of compounds containing either a

substituted or an unsubstituted fused six-membered aromatic

ring. Compounds having a tetrahydroisoquinoline or benzaze-

pine core substituted with a 4-trifluoromethylphenyl group as

R¹were prepared by standard methods from 4-

(trifluoromethyl)benzoyl chloride and the appropriate 2-

arylethylamines (64a−64d) or 2-phenylpropylamine (64e) as

shown in Scheme 6. The intermediate amides 65a−e

underwent a Bischler−Napieralski reaction upon treatment

with polyphosporic acid and phosphorus pentoxide. The

resulting imines 66a−e were reduced with sodium borohydride.

Treatment of tetrahydroisoquinolines 67a−d or benzazepine

67e with 4-fluorophenyl isocyanate at room temperature gave

the corresponding ureas 68−72 in good yields.

The synthesis of the final fluorotetrahydroisoquinoline

derivative 78 is shown in Scheme 7. In this case, the 4-

(trifluoromethyl)phenyl group was introduced at the C-1

position of the isoquinoline ring via activation of 8-

fluoroisoquinoline (73) with benzyl bromide followed by

reaction with (4-(trifluoromethyl)phenyl)magnesium bromide.

Reduction of the tetrahydroisoquinoline core, deprotection of

the N-benzyl amine, and subsequent urea formation led to the

8-fluoro analogue, 78.

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a

Scheme 7

a

Reagents and conditions: (a) PhCH₂Br, CH₃CN, 90 °C, 3 h; (b) 4-CF₃PhMgBr, THF, 0 °C to room temperature, 4 h; (c) NaBH₄, AcOH, THF,

room temperature, 2 h; (d) Pd(OH)₂, H₂, EtOH, 50 psi, 3 h; (e) 4-F-phenyl isocyanate, CH₂Cl₂, room temperature, 1 h.

a

Scheme 8

a

Reagents and conditions: (a) 4-CF₃PhMgBr, THF, 0 °C, 2 h; (b) trifluoroacetic acid, Et₃SiH, CH₂Cl₂, room temperature, 18 h; (c) LiAlH₄, THF,

75 °C, 2 h; (d) 4-F-phenyl isocyanate, CH₂Cl₂, room temperature, 2 h.

a

Scheme 9

a

Reagents and conditions: (a) 4-CF₃PhMgBr, THF, −78 °C to room temperature, 2.5 h; (b) trifluoroacetic acid, room temperature, 4 h; (c) NaBH₄,

MeOH, room temperature, 18 h; (d) 4-F-phenyl isocyanate, CH₂Cl₂, room temperature, 3 h.

The isoindoline analogue 82 (Scheme 8) was prepared by

the addition of 4-trifluoromethylphenyl Grignard to phthali-

mide (79), followed by deoxygenation of the intermediate with

triethylsilane and trifluoroacetic acid to give 3-(4-

(trifluoromethyl)phenyl)isoindolin-1-one (80). Isoindolinone

80 was reduced to isoindoline 81 by treatment with lithium

aluminum hydride. The requisite urea analogue, 82, was

obtained by derivatization of 81 with 4-fluorophenyl isocyanate.

To explore the effect of eliminating the fused aromatic ring,

the 2-(4-(trifluoromethyl)phenyl)piperidine analogue 86 was

prepared (Scheme 9). Nucleophilic addition of 4-trifluorome-

thylphenyl Grignard to the protected γ-lactam 83 provided

ketone 84.²⁰Removal of the t-butoxycarbonyl protecting group

with trifluoroacetic acid gave the intermediate amine that

cyclized readily under the reaction conditions. Reduction of the

resulting Schiff base with sodium borohydride gave the

piperidine derivative, 85, which was treated with 4-fluorophenyl

isocyanate to provide the piperidine derivative, 86.

RESULTS AND DISCUSSION

■

The fused piperidine analogues prepared in this study were

evaluated for their ability to block the icilin-induced calcium

influx in CHO cells expressing the rTRPM8 channel. The

results are shown in Tables 1−4. All compounds reported

herein behaved as full antagonists. Additionally, the metabolic

stability of the compounds was assayed in rat liver microsomal

(RLM) preparations for initial assessment of metabolism, and

the data is reported as intrinsic clearance (CL_int).¹⁶

To establish a thorough understanding of the SAR of this

series, each of the three variables of general structure 6 were

systematically modified including the R¹and R²substituents

and the central heterocyclic core. We began our investigation

by examining the effect of altering the R¹group at the 2-

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19); however, 2- and 4-trifluoromethyl or chloro substituted

phenyl analogues (15, 17, 18, and 20) had similar activities as

the parent compound 5. By contrast, no benefit was realized by

the addition of the smaller fluoro group at any of the three

positions on the phenyl ring (14 vs 21−23). Although the 2-

substituted derivatives 15 and 18 showed good activities, the 4-

trifluoromethyl group gave slightly better stability in rat liver

microsomes. From this set of analogues, we determined that

substituting the n-propyl group of compound 5 by a 4-

trifluoromethyl phenyl group (to give compound 17) resulted

in an analogue that retained rTRPM8 antagonistic activity but

Table 1. In Vitro rTRPM8 Activities and Rat Microsomal

Stabilities for Tetrahydrothienopyridines (R¹Group

Modifications)

a

had an improved stability in the rat microsomal assay (CL_int

=

211 μL/(min·mg)).

Since compound 17, which contains a 4-trifluoromethyl

phenyl group (R¹substituent), had the best combination of

potency and microsomal stability, this group was maintained at

the 2-position in the next phase of our SAR investigations

whereby we examined the effect of varying the R²substituent

on the piperidine nitrogen (Table 2). Removal of the phenyl

urea group to give the unsubstituted piperidine derivative 10g

(R²= H) completely abolished functional activity against the

rTRPM8 channel (IC₅₀> 20 μM). We then explored other

functional groups on the piperidyl nitrogen such as alkyl

groups, amides, or ureas. The isopropyl, isobutyl, and benzyl

derivatives (24, 25, and 26) did not show any antagonistic

activity against the rTRPM8 channel. In contrast, adding a

carbonyl group adjacent to the piperidyl nitrogen, by replacing

the R²isobutyl group (derivative 25) with an isopropyl amide

group (analogue 27), increased the potency significantly (27

IC₅₀= 1260 nM), indicating that a carbonyl group in this

position was beneficial. Next, we examined analogues bearing

aromatic amides at the piperidinyl nitrogen, 2-fluoro (28), 3-

fluoro (29), and 4-fluorophenyl amide (30). Interestingly, this

substitution was only tolerated when the fluoro was at the 2-

position on the phenyl group (28, IC₅₀= 510 nM). The 3- and

4-fluorophenyl analogues (29 and 30, respectively) had IC₅₀

values greater than 20 μM, indicating that the substitution at

the phenyl group was very sensitive to substituent’s position.

Although amide 28 was moderately potent, it had extremely

poor rat microsomal stability. Therefore, we turned our

attention to ureas and explored the substitution of the phenyl

urea of compound 17 by alkyl ureas. Introduction of an

isopropyl urea (32) or a cyclopropyl urea (33) at the

piperidinyl nitrogen led to compounds that were 2−5-fold

weaker than the parent phenyl urea 17, indicating that a phenyl

group was preferred for activity. Also, the antagonistic activity

against rTRPM8 of 17 was abolished when a morpholino urea

group was introduced at R²(compound 34). We then explored

substitution at the 4-position on the N-phenyl urea group with

electron-donating (35, R²= 4-methoxyphenyl urea) and

electron-withdrawing groups (36, R²= 4-chlorophenyl urea).

We postulated that introduction of substituents at this position,

if tolerated, may improve the in vitro microsomal stability

because the 4-position in a phenyl group is often a site for

oxidative metabolism. Unfortunately, these substitutions

a

IC₅₀values based on inhibition of icilin (1 μM) induced influx of

Ca²⁺into rTRPM8-expressing CHO cells. Each IC₅₀value reported

represents an average of at least two independent experiments with

three replicates at each concentration. In vitro microsomal stability

measured in a high-throughput automated format.¹⁶

position of the tetrahydrothienopyridine core structure.

Derivatives of lead compound 5 with modifications at R¹are

shown in Table 1. The unsubstituted tetrahydrothienopyridine

11 (R¹= H) was devoid of activity against the rTRPM8

channel indicating that substitution at the 2-position on the

heterocyclic core was required. Decreasing the size of the alkyl

group from n-propyl (compound 5) to methyl and cyclopropyl

(compounds 12 and 13) also proved to be detrimental (IC₅₀

values of 6800 and 5570 nM, respectively). A loss in potency

was also observed for the phenyl, the 3-trifluoromethyl phenyl,

and the 3-chlorophenyl substituted derivatives (14, 16, and

resulted in a loss of activity (35, IC₅₀= 2320 nM; 36, IC₅₀

=

1630 nM). In contrast, the 2-, 3-, and 4-fluoro substituted

phenyl derivatives were more encouraging, as illustrated by

compounds 37−39. Of these analogues, the 4-fluoro derivative,

39, proved to be both the most potent and the most

metabolically stable. As a direct comparison of the 4-

fluorophenyl urea, and to interrogate the contribution of the

urea NH functionality to potency and microsomal stability, we

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a

Table 2. In Vitro rTRPM8 Activities and Rat Microsomal Stabilities for Tetrahydrothienopyridines (R²Group Modifications)

a

IC₅₀values based on inhibition of icilin (1 μM) induced influx of Ca²⁺into rTRPM8-expressing CHO cells. Each IC₅₀value reported represents an

average of at least two independent experiments with three replicates at each concentration (SEM). In vitro microsomal stability measured in a high-

throughput automated format.¹⁶

also prepared the 4-fluorophenylacetamide derivative 31. This

analogue was slightly less potent than urea 39 but was

significantly less stable in rat liver microsomes demonstrating

the added stability provided by the urea functionality. We

therefore continued our investigations using analogue 39 for

further optimization studies.

The final SAR examined in this work resulted from

modifications of the heterocyclic core of compound 39

(Table 3). Replacement of the fused thiophene ring in 39

with its regioisomeric five-membered ring heterocycle gave

4,5,6,7-tetrahydrothieno[2,3-c]pyridine 45. This small modifi-

cation gave only a 2-fold improvement in potency against

rTRPM8; however, it did not improve the microsomal stability.

Similarly, substitution of the fused thiophene ring by a furan

ring did not result in a significant change in activity with the

regioisomeric 4,5,6,7-tetrahydrofuro[3,2-c]pyridine 46 and

4,5,6,7-tetrahydrofuro[3,2-c]pyridine 47 having IC₅₀values of

260 and 230 nM, respectively. This suggested that the sulfur

atom did not play a critical role in the antagonistic activity

against the rTRPM8 channel. Other fused five-membered ring

heterocycles such as pyrrole 48, thiazole 58, and imidazoles 61

and 63 were also assessed for functional antagonism of

rTRPM8. There was not a significant difference in potency

between the parent thiophene analogue 39 and compounds 48

and 58. However, introduction of an additional H-bond

donating group such as in imidazole 61 proved detrimental

for activity (IC₅₀= 1430 nM). Masking the NH in the

imidazole ring by methylation led to analogue 63, which had

improved potency and RLM values. We then examined six-

membered rings and a significant improvement in activity was

observed when the (5,6)-bicyclic core was replaced with a

(6,6)-core. For example, tetrahydroisoquinoline 68 was not

only 6-fold more potent than the parent compound, but it was

also significantly more metabolically stable with a CL_int= 92

μL/(min·mg). This result demonstrated that the tetrahydro-

thienopyridine moiety, a highly electron-rich ring system and

potentially the main source of metabolic instability, could be

replaced while enhancing rTRPM8 antagonistic activity.

With the tetrahydroisoquinoline core identified as a superior

structural motif, four isomeric monofluoro tetrahydroisoquino-

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Table 3. In Vitro rTRPM8 Activities and Rat Microsomal Stabilities for Substituted Piperidines (Heterocyclic Core

a

Modifications)

a

IC₅₀values based on inhibition of icilin (1 μM) induced influx of Ca²⁺into rTRPM8-expressing CHO cells. Each IC₅₀value reported represents an

average of at least two independent experiments with three replicates at each concentration (SEM). In vitro microsomal stability measured in a high-

throughput automated format.¹⁶

line analogues were prepared (i.e., 69−71, 78) in an effort to

further improve in vitro stability by blocking possible sites of

metabolism on this new series of compounds (Table 3).

Introduction of a fluoro substituent at the 6- or 8-position (70,

78) was not well tolerated for rTRPM8 activity. However, the

5- and 7-fluoro analogues (69, 71) were potent antagonists of

rTRPM8, and a moderate improvement in metabolic stability

was obtained.

The role of the aromatic ring fused to the piperidine was also

investigated with the piperidine analogue 86. This compound

was significantly less potent than the parent tetrahydrothieno-

pyridine 39, indicating that a fused bicyclic core was preferred

for activity.

contrast, the indoline analogue 82 was only 6-fold less potent

than the parent tetrahydroisoquinoline 68.

Due to its improved in vitro metabolic clearance, as well as its

intrinsic potency at the target, tetrahydroisoquinoline 68 was

selected for in vivo evaluation. Separation of the racemic

mixture 68 using preparative chiral chromatography provided

the two enantiomers (87 and 88). The absolute stereo-

chemistries of tetrahydroisoquinolines 87 and 88 were

determined by two different methods that compare theoretical

and experimental measurements: vibrational circular dichroism

(VCD)²¹and optical rotation.²²In the first method, VCD

spectra for each enantiomer were acquired on a chiral infrared

(IR) instrument (Figure 2a), and the corresponding theoretical

VCD spectra were determined quantum mechanically (B3LYP/

6-31G*)^22a−c(Figure 2b). Experimental and theoretical achiral

IR spectra were also obtained and are illustrated in Figure 2c.

Alignment of the experimental and theoretical achiral IR

spectra allowed for the major absorption transitions (i−iv in

Next we turned our attention to the fused piperidine ring and

examined the effect of the ring size on potency and microsomal

stability. Homologation of the piperidine ring to the seven-

membered ring analogue resulted in benzazepine 72, which was

not active against rTRPM8 following activation by 3. In

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+379°, respectively; at 589 nm) were in qualitative agreement

with the measured optical rotations for 87 and 88 ([α]²⁶

D

−106.6° and +106.7° [c 0.1, chloroform], respectively), thereby

corroborating the VCD-based assignments.

Having determined the absolute configuration of compounds

87 and 88, we analyzed both enantiomers in vitro. The R-

isomer 87 was a significantly more potent rTRPM8 antagonist

and was more stable in rat liver microsomes preparations (IC₅₀

= 56 nM, CL_int= 42 μL/(min·mg); Table 4). Following

Table 4. In Vitro rTRPM8 Activities and Rat Microsomal

Stabilities for Tetrahydroisoquinoline Enantiomers 87 and

a

88

a

IC₅₀values based on inhibition of icilin (1 μM) induced influx of

Ca²⁺into rTRPM8-expressing CHO cells. Each IC₅₀value reported

represents an average of at least two independent experiments with

three replicates at each concentration. In vitro microsomal stability

measured in a high-throughput automated format.¹⁶For selectivity

information on other TRP channels as well as potency for the human

channel and human liver microsomal data, see Supporting

Information.

intravenous administration in Sprague−Dawley rats (Table 5),

87 displayed a relatively high total systemic clearance (2.9 L/

(h·kg), ∼88% of hepatic blood flow), a high volume of

distribution (15.3 L/kg), and a moderate terminal half-life of

6.7 h. In contrast, an even higher rate of clearance (CL) was

observed for compound 88 (5.8 L/(h·kg)). The pharmacoki-

netic profile of compound 87 after single oral administration

(po) is summarized in Table 5. Tetrahydroisoquinoline 87 was

well-absorbed, with a bioavailability of 57%. The maximum

plasma concentration (C_max) following oral administration of

compound 87 was 265 ng/mL at 2.7 h. Rat plasma protein

binding for compound 87 was determined to be 90.1%.

Although pharmacokinetic properties of tetrahydroisoquino-

line 87 were not optimal, its potency and overall profile made it

a suitable candidate for in vivo evaluation in an on-target

biochemical challenge model. In this model, prevention of

icilin-induced wet-dog shakes (WDS) in rats by tetrahydroiso-

quinolines 87 and 88 was measured (Figure 3).²³Vehicle or

test compounds (87 and 88) were administered orally to

Sprague−Dawley rats 90 min prior to challenge with 3 (0.5

mg/kg, ip). In the vehicle/3 group, over 150 shakes were

counted during a 30 min period. Pretreatment with compound

87 significantly reduced icilin-induced WDS in a dose-

dependent manner. A reduction significantly different from

vehicle was observed at 3 mg/kg (30%; p < 0.01) and more

than 80% reduction was obtained at 30 mg/kg (p < 0.001). A

Figure 2. IR and VCD spectra for compounds 87 and 88: (a)

experimental VCD spectra; (b) theoretical VCD spectra; (c)

experimental and theoretical achiral IR spectra, superimposed. See

Supporting Information for computational details.

Figure 2) to be mapped to their corresponding peaks in the

chiral VCD spectra. Comparison of these unambiguous regions

of the corresponding experimental VCD traces of 87 and 88 to

those computed for the (R)- and (S)-forms, led to the

assignments of compounds 87 and 88 as the (R)- and (S)-

enantiomers, respectively. The second method used to

determine the absolute stereochemistries of 87 and 88 utilized

optical rotation measurements. In this method, the predicted

optical rotation values for the Boltzmann-weighted, conforma-

tional average of the (R)- and (S)-enantiomers (−379° and

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a

Table 5. Mean (SD) Pharmacokinetic (PK) Parameters of Compounds 87 and 88 in Male Sprague−Dawley Rats

route

dose (mg/kg)

AUC_inf(ng·h/mL)

CL (L/(h·kg))

Vss (mL/kg)

t_1/2(h)

C_max(ng/mL)

F (%)

b

87

88

87

iv

2

763(306)

347(19.1)

2.9(1.0)

15.3(2.0)

15.8(3.2)

6.7(2.1)

4.4(1.8)

b

iv

5.8(0.31)

c

po

10

1747(548)

265(55.7)

57

a

b

All PK parameters were reported as mean(SD). Study in fed male Sprague−Dawley rats dosed at 2 mg/kg in DMSO, n = 3 animals per study.

c

Study in fasted male Sprague−Dawley rats dosed at 10 mg/kg as a suspension in 5% Tween 80/Oraplus, n = 3 animals per study.

EXPERIMENTAL SECTION

■

Chemistry. Unless otherwise noted, all materials were obtained

from commercial suppliers and used without further purification.

Anhydrous solvents were obtained from Aldrich or EM Science and

used directly. All reactions involving air- or moisture-sensitive reagents

were performed under a nitrogen or argon atmosphere. All microwave-

assisted reactions were conducted with a Smith synthesizer from

Personal Chemistry, Uppsala, Sweden. All final compounds were

purified to >95% purity, as determined by LC/MS obtained on Agilent

1100 and HP 1100 spectrometers. Silica gel chromatography was

performed using either glass columns packed with silica gel (200−400

mesh, Aldrich Chemical) or prepacked silica gel cartridges (Biotage or

1

RediSep). H NMR spectra were determined with a Bruker 300 MHz

or DRX 400 MHz spectrometer. Chemical shifts are reported in parts

Figure 3. Inhibition of icilin-induced wet-dog shakes (WDS) in rats

per million (ppm, δ units).

and corresponding plasma levels (bar graph) of compounds 87 and 88,

The following 4,5,6,7-tetrahydrothieno[3,2-c]pyridine derivatives

dosed po in 5% Tween 80/Oraplus 90 min prior to challenge by 3; n =

were commercially available: 4,5,6,7-tetrahydrothieno[3,2-c]pyridine

8 per group of Sprague−Dawley male rats. Plasma samples were

(10b), 4-cyclopropyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (10d), 4-

collected 2 h after oral dosing.

(4-chlorophenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (10j), 1-(4-

(trifluoromethyl)phenyl)-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine

(44), and 4-methyl-N-phenyl-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-

carboxamide (12).

nonlinear regression model (log inhibitor versus response with

variable slope) of plasma concentrations of compound 87

versus WDS yielded an EC₅₀of 209 ng/mL. In the same study,

animals were also predosed with 100 mg/kg of compound 88,

the enantiomer of 87 (rTRPM8 IC₅₀> 20 μM). Plasma levels

of compound 88 at 100 mg/kg (521 ng/mL) were comparable

to 30 mg/kg of 87 (460 ng/mL); however, no inhibition of

icilin-induced WDS was observed. These results demonstrated

that 87 was a potent antagonist of TRPM8 in vivo and that the

inhibition of WDS in rats induced by 3 was due to TRPM8

antagonism.

(a). General Procedure for the Preparation of Tetrahydro-

thienopyridines. Amide Route. (Step 1) To a solution of 2-

(thiophen-2-yl)ethanamine (7, 20 g, 0.16 mol) and triethyl-

amine (17.8 g, 0.18 mol, 1.1 equiv) in CH₂Cl₂(120 mL) was

added the acid chlorides (1.05 equiv) dropwise at 0 °C. The

mixture was stirred until completion of the reaction was

determined by TLC (∼2 h). The reaction mixture was diluted

with EtOAc and washed with 10% aq HCl, saturated aqueous

NaHCO₃, and saturated aqueous NaCl. The EtOAc solution

was dried over Na₂SO₄, and the solvent was eliminated under

vacuum to afford the target amides, 8.

(Step 2) To a stirred solution of the amide (8) in CH₃CN was

added POCl₃(4 equiv) dropwise, and the reaction mixture was stirred

at 60 °C overnight. The reaction mixture was cooled to room

temperature, and the solvent was removed under vacuum. The residue

obtained was dissolved in EtOAc, and the mixture was washed with

saturated aqueous NaHCO₃. The organic layer was separated, washed

with water and saturated aqueous NaCl, and dried over Na₂SO₄, and

the solvent was removed under reduced pressure. The crude product

was purified by silica gel column chromatography eluting with EtOAc/

hexanes mixtures.

(Step 3) To a stirred solution of the tetrahydrothienopyridines (1.5

mmol) in dry MeOH (10 mL) was added NaBH₄(2 equiv) at room

temperature. Stirring was continued until the completion of the

reaction as determined by TLC, ∼2 h. The reaction mixture was

concentrated under vacuum, and the residue was dissolved in EtOAc.

The organic layer was washed with water and saturated aqueous NaCl

and dried over Na₂SO₄, and the solvent was eliminated under vacuum.

The crude product was purified by silica gel column chromatography

eluting with EtOAc/hexanes mixtures.

SUMMARY

■

Following the identification of tetrahydrothienopyridine 4 as a

TRPM8 antagonist hit from a high-throughput screening

campaign, we undertook a detailed SAR investigation aimed

at improving potency as well as increasing metabolic stability of

this compound. SAR studies aimed at improving potency and

microsomal stability established a 4-trifluorophenyl group as

the preferred R¹substituent and a 4-fluorophenyl urea as the R²

substituent of choice on the piperidine nitrogen, leading to

tetrahydrothienopyridine 39. Subsequent modifications of the

central heterocyclic core led to of a new series of

tetrahydroisoquinolines as potent TRPM8 antagonists with

increased metabolic stability, exemplified by compound 87. The

overall profile of this TRPM8 antagonist (87), potency and

pharmacokinetic properties, made it a suitable candidate for in

vivo studies. Compound 87 showed a dose-dependent

reduction of icilin-induced WDS in rats, an on-target

biochemical challenge model. Although optimization of

potency and overall PKDM profile is still required in this

new series, these data represent an encouraging starting point

for the discovery of TRPM8 antagonists as novel therapies for

pain.

The following compounds were prepared according to this general

procedure (amide route).

1

4-Propyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (10a). H NMR

(400 MHz, CDCl₃): 1.03 (t, J = 7.34 Hz, 3 H), 1.70 (dq, J = 15.28,

7.56 Hz, 2 H), 1.98−2.19 (m, 2 H), 3.07−3.19 (m, 1 H), 3.26−3.43

(m, 2 H), 3.64−3.73 (m, 1 H), 4.43 (t, J = 6.16 Hz, 1 H), 6.81 (d, J =

5.28 Hz, 1 H), 7.20 (d, J = 5.28 Hz, 1 H). MS (ESI pos. ion) m/z:

calcd for C₁₀H₁₅NS 182.1; found 182.1.

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4-(2-(Trifluoromethyl)phenyl)-4,5,6,7-tetrahydrothieno[3,2-c]-

with EtOAc and washed with 10% aqueous HCl, saturated aqueous

NaHCO₃, and saturated aqueous NaCl and dried over Na₂SO₄, and

the solvent was removed under reduced pressure. Purification by

column chromatography using a mixture of 1:5 to 1:4 EtOAc/hexanes

afforded N-(2-(furan-2-yl)ethyl)-4-(trifluoromethyl)benzamide as a

yellow solid (3.5 g, 22%).

(Step 2) To a stirred solution of phosphorus oxide (5.0 g, 35 mmol)

and phosphorus trichloride (5.4 mL, 59 mmol) in benzene (100 mL)

at reflux was added N-(2-(furan-2-yl)ethyl)-4-(trifluoromethyl)-

benzamide (3.4 g, 12 mmol). The reaction mixture was heated at

reflux for 2 h and then allowed to cool to room temperature. The

solution was poured into crushed ice, basified with potassium

carbonate to pH = 10−11, and extracted with benzene. The organic

layer was separated, washed with water, and dried over Na₂SO₄, and

the solvent was eliminated under vacuum. Purification by column

chromatography using a mixture of 1:5 to 1:4 EtOAc/hexanes afforded

4-(4-(trifluoromethyl)phenyl)-6,7-dihydrofuro[3,2-c]pyridine as a red

oil (0.94 g, 30%).

1

pyridine (10e). H NMR (400 MHz, CDCl₃): 2.79−2.92 (m, 1 H),

2.99−3.22 (m, 2 H), 3.29−3.41 (m, 1 H), 5.45 (s, 1 H), 6.33 (d, J =

5.09 Hz, 1 H), 6.99 (d, J = 5.28 Hz, 1 H), 7.29−7.39 (m, 2 H), 7.40−

7.49 (m, 1 H), 7.68 (d, J = 7.63 Hz, 1 H), 10.20 (br s, 1 H). MS (ESI

pos. ion) m/z: calcd for C₁₄H₁₂F₃NS 284.1; found 284.0.

4-(3-(Trifluoromethyl)phenyl)-4,5,6,7-tetrahydrothieno[3,2-c]-

1

pyridine (10f). H NMR (400 MHz, CDCl₃): 2.82−2.91 (m, 1 H),

2.96−3.06 (m, 1 H), 3.09−3.18 (m, 1 H), 3.26−3.35 (m, 1 H), 5.08

(s, 1 H), 6.43 (d, J = 5.09 Hz, 1 H), 7.03 (d, J = 5.09 Hz, 1 H), 7.40−

7.50 (m, 2 H), 7.54 (d, J = 7.63 Hz, 1 H), 7.58 (br s, 1 H). MS (ESI

pos. ion) m/z: calcd for C₁₄H₁₂F₃NS 284.1; found 284.0.

4-(4-(Trifluoromethyl)phenyl)-4,5,6,7-tetrahydrothieno[3,2-c]-

pyridine (10g). ¹H NMR (400 MHz, DMSO-d₆): 2.70−2.80 (m, 1 H),

2.81−2.99 (m, 2 H), 3.04−3.16 (m, 1 H), 5.01 (s, 1 H), 6.41 (d, J =

5.28 Hz, 1 H), 7.19 (d, J = 5.09 Hz, 1 H), 7.51 (d, J = 8.02 Hz, 2 H),

7.67 (d, J = 8.02 Hz, 2 H). MS (ESI pos. ion) m/z: calcd for

C₁₄H₁₂F₃NS 284.1; found 284.0.

4-(2-Chlorophenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine

(10h). ¹H NMR (400 MHz, CDCl₃): 2.82−3.00 (m, 2 H), 3.06−3.14

(m, 1 H), 3.15−3.24 (m, 1 H), 5.55 (s, 1 H), 6.50 (d, J = 5.09 Hz, 1

H), 7.05 (d, J = 1.00 Hz, 1 H), 7.18 (dtd, J = 18.34, 7.41, 7.41, 1.47

Hz, 2 H), 7.40 (dd, J = 7.73, 1.47 Hz, 2 H). MS (ESI pos. ion) m/z:

calcd for C₁₃H₁₂ClNS 249.0; found 249.0.

(Step 3) To a stirred solution of the 4-(4-(trifluoromethyl)phenyl)-

6,7-dihydrofuro[3,2-c]pyridine (0.61 g, 2.3 mmol) in dry CH₂Cl₂(15

mL) was added NaB(OAc)₃H (1.63 g, 7.7 mmol) at room

temperature. The reaction mixture was stirred at room temperature

overnight, diluted with CH₂Cl₂, and cooled to 0 °C. An aqueous

solution of NaOH (10%) was added slowly to this solution. The

organic layer was separated and dried over Na₂SO₄, and the solvent

was eliminated under vacuum. This material was used in the next step

without further purification. To a solution of 4-(4-(trifluoromethyl)-

phenyl)-4,5,6,7-tetrahydrofuro[3,2-c]pyridine (0.61 g, 2.3 mmol) in

CH₂Cl₂(10 mL) at 0 °C were added triethylamine (0.47 mL, 3.4

mmol) and di-tert-butyl dicarbonate (0.55 g, 2.5 mmol). The reaction

mixture was stirred at room temperature overnight. The solvent was

removed under reduced pressure, and the residue was purified by

column chromatography using a mixture of 1:9 EtOAc/hexanes as

eluent to give tert-butyl 4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrofuro[3,2-c]pyridine-5(4H)-carboxylate as a yellow oil (0.7 g,

83%).

4-(3-Chlorophenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (10i).

¹H NMR (400 MHz, CDCl₃): 2.75−2.90 (m, 1 H), 2.92−3.05 (m, 1

H), 3.06−3.17 (m, 1 H), 3.29 (dt, J = 12.13, 4.79 Hz, 1 H), 5.00 (s, 1

H), 6.46 (d, J = 5.09 Hz, 1 H), 7.02 (d, J = 5.09 Hz, 1 H), 7.13−7.21

(m, 1 H), 7.23−7.30 (m, 3 H). MS (ESI pos. ion) m/z: calcd for

C₁₃H₁₂ClNS 249.0; found 249.0.

4-(2-Fluorophenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (10k).

¹H NMR (400 MHz, CDCl₃): 2.78−3.02 (m, 2 H), 3.07−3.17 (m, 1

H), 3.20−3.32 (m, 1 H), 5.44 (s, 1 H), 6.51 (d, J = 5.09 Hz, 1 H),

6.99−7.13 (m, 4 H), 7.19−7.30 (m, 1 H). MS (ESI pos. ion) m/z:

calcd for C₁₃H₁₂FNS 234.1; found 234.1.

4-(3-Fluorophenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (10l).

¹H NMR (400 MHz, CDCl₃): 2.79−2.89 (m, 1 H), 2.93−3.04 (m, 1

(Step 4) To a solution of tert-butyl 4-(4-(trifluoromethyl)phenyl)-

6,7-dihydrofuro[3,2-c]pyridine-5(4H)-carboxylate (0.65 g, 1.8 mmol)

in Et₂O (10 mL) was added HCl/dioxane (4.85 M, 5 mL). The

reaction mixture was stirred at room temperature overnight, and the

resulting white precipitate was filtered, washed with Et₂O, and dried

under vacuum to give the title compound as a white solid (0.52 g,

H), 3.06−3.16 (m, 1 H), 3.29 (dt, J = 12.18, 4.87 Hz, 1 H), 5.02 (s, 1

H), 6.47 (d, J = 5.28 Hz, 1 H), 6.93−7.04 (m, 3 H), 7.08 (d, J = 7.63

Hz, 1 H), 7.27−7.32 (m, 1 H). MS (ESI pos. ion) m/z: calcd for

C₁₃H₁₂FNS 234.1; found 234.1.

4-(4-Fluorophenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine

(10m). ¹H NMR (400 MHz, CDCl₃): 2.78−2.90 (m, 1 H), 2.93−3.03

(m, 1 H), 3.06−3.16 (m, 1 H), 3.23−3.36 (m, 1 H), 5.01 (s, 1 H), 6.44

(d, J = 5.09 Hz, 1 H), 6.95−7.06 (m, 3 H), 7.16−7.29 (m, 2 H). MS

(ESI pos. ion) m/z: calcd for C₁₃H₁₂FNS 234.1; found 234.1.

(b). Amine Route. 4-Phenyl-4,5,6,7-tetrahydrothieno[3,2-c]-

pyridine (10n). A solution of 2-(thiophen-2-yl)ethanamine (2.2

mL, 17.4 mmol) and benzaldehyde (1.8 g, 17.4 mmol) in

toluene (50 mL) was heated at reflux (Dean−Stark trap, H₂O

removed) for 16 h. The reaction mixture was then concentrated

in vacuo, and trifluoroacetic acid (30 mL) was cautiously added

to the residue. The mixture was stirred at room temperature for

3 d. The reaction mixture was concentrated in vacuo. The

residue was dissolved in EtOAc (50 mL), and the solution was

washed with 2 M aqueous NaOH solution (50 mL). The

organic phase was dried over Na₂SO₄, filtered, and concen-

trated. The residue was purified by column chromatography

using a mixture of 2% to 4% MeOH in CH₂Cl₂to give the title

1

96%). H NMR (400 MHz, DMSO-d₆): 2.92−3.04 (m, 1 H), 3.07−

3.21 (m, 1 H), 3.37−3.53 (m, 2 H), 5.76 (br s, 1 H), 6.17 (s, 1 H),

7.68 (d, J = 8.53 Hz, 2 H), 7.71 (s, 1 H), 7.86 (d, J = 8.53 Hz, 2 H),

9.73 (br s, 1 H), 10.47 (br s, 2 H). MS (ESI pos. ion) m/z: calcd for

C₁₄H₁₂F₃NO 268.1; found 268.1.

General Procedure for the Preparation of Ureas (4−5, 11−

23, 32−39, and 45−48). To a solution of the tetrahydrothieno/

furanopyridine (1.9 mmol) in CH₂Cl₂(4 mL) at room temperature

was added 1 equiv of the appropriate isocyanate. The reaction mixture

was stirred at room temperature for 1 h and then concentrated in

vacuo. The compounds were purified by silica gel chromatography.

N-(4-Chlorophenyl)-4-propyl-6,7-dihydrothieno[3,2-c]pyridine-

1

5(4H)-carboxamide (4). H NMR (400 MHz, CDCl₃): 1.00 (t, J =

7.34 Hz, 3 H), 1.47−1.62 (m, 2 H), 1.75−1.87 (m, 2 H), 2.79 (dd, J =

16.14, 2.84 Hz, 1 H), 2.93−3.05 (m, 1 H), 3.21−3.35 (m, 1 H), 4.33

(dd, J = 13.79, 5.18 Hz, 1 H), 5.03 (t, J = 6.75 Hz, 1 H), 6.38 (s, 1 H),

6.81 (d, J = 5.09 Hz, 1 H), 7.12 (d, J = 5.09 Hz, 1 H), 7.23 (d, J = 9.00

Hz, 2 H), 7.27−7.33 (m, 2 H). HRMS calcd for C₁₇H₁₉ClN₂OS (M +

H): 335.0976; found 335.0970.

1

compound as an off-white solid (1.46 g, 39%). H NMR (400

MHz, DMSO-d₆): 2.63−2.97 (m, 4 H), 3.12 (dt, J = 11.54, 4.50

Hz, 1 H), 4.90 (s, 1 H), 6.38 (d, J = 5.09 Hz, 1 H), 7.16 (d, J =

5.09 Hz, 1 H), 7.20−7.36 (m, 5 H). MS (ESI pos. ion) m/z:

calcd for C₁₃H₁₃NS 216.1; found 216.1.

4-(4-(Trifluoromethyl)phenyl)-4,5,6,7-tetrahydrofuro[3,2-c]-

pyridine hydrochloride (41). (Step 1) To a solution of 2-(furan-2-

yl)ethanamine (40, 6.39 g, 0.06 mol) and triethylamine (12 mL, 0.08

mol) in CH₂Cl₂(100 mL) at 0 °C was added 4-(trifluoromethyl)-

benzoyl chloride (8.5 mL, 0.06 mol) dropwise. The solution was

stirred at room temperature for 2 h. The reaction mixture was diluted

N-Phenyl-4-propyl-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-car-

1

boxamide (5). H NMR (400 MHz, CDCl₃): 1.00 (t, J = 7.34 Hz, 3

H), 1.55 (sxt, J = 7.43 Hz, 2 H), 1.75−1.92 (m, 2 H), 2.78 (dd, J =

16.04, 2.93 Hz, 1 H), 2.90−3.09 (m, 1 H), 3.27 (ddd, J = 13.64, 12.08,

3.81 Hz, 1 H), 4.35 (dd, J = 13.69, 5.09 Hz, 1 H), 5.06 (t, J = 6.85 Hz,

1 H), 6.40 (br s, 1 H), 6.81 (d, J = 5.28 Hz, 1 H), 6.99−7.06 (m, 1 H),

7.11 (d, J = 5.28 Hz, 1 H), 7.29 (d, J = 7.43 Hz, 2 H), 7.32−7.39 (m, 2

H). HRMS calcd for C₁₇H₂₀N₂OS (M + H): 301.1360; found

301.1365.

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N-Phenyl-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-carboxamide

(11). ¹H NMR (400 MHz, CDCl₃): 2.95 (t, J = 5.58 Hz, 2 H), 3.84 (t,

J = 5.67 Hz, 2 H), 4.60 (s, 2 H), 6.40 (br s, 1 H), 6.82 (d, J = 5.09 Hz,

1 H), 7.05 (t, J = 7.34 Hz, 1 H), 7.16 (d, J = 5.09 Hz, 1 H), 7.30 (t, J =

7.92 Hz, 2 H), 7.37 (d, J = 7.61 Hz, 2 H). HRMS calcd for

C₁₄H₁₄N₂OS (M + H): 259.0897; found 259.0900.

4-(3-Fluorophenyl)-N-phenyl-6,7-dihydrothieno[3,2-c]pyridine-

1

5(4H)-carboxamide (22). H NMR (300 MHz, DMSO-d₆): 2.79−

3.22 (m, 3 H), 4.20−4.35 (m, 1 H), 6.55 (s, 1 H), 6.87 (d, J = 5.12 Hz,

1 H), 6.92−7.05 (m, 2 H), 7.06−7.18 (m, 2 H), 7.25 (t, J = 7.89 Hz, 2

H), 7.34−7.44 (m, 2 H), 7.48 (dd, J = 8.55, 0.95 Hz, 2 H), 8.71 (s, 1

H). MS (ESI pos. ion) m/z: calcd for C₂₀H₁₇FN₂OS 353.1; found

353.0.

4-Cyclopropyl-N-phenyl-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-

1

carboxamide (13). H NMR (300 MHz, CDCl₃): 0.57−0.78 (m, 4

4-(4-Fluorophenyl)-N-phenyl-6,7-dihydrothieno[3,2-c]pyridine-

1

H), 1.15−1.34 (m, 1 H), 2.77−2.89 (m, 1 H), 2.91−3.09 (m, 1 H),

3.40−3.56 (m, 1 H), 4.29 (dd, J = 13.96, 5.33 Hz, 1 H), 4.67 (d, J =

8.33 Hz, 1 H), 6.39 (s, 1 H), 6.92 (d, J = 5.12 Hz, 1 H), 6.98−7.07 (m,

1 H), 7.12 (d, J = 5.12 Hz, 1 H), 7.27−7.39 (m, 4 H). HRMS calcd for

C₁₇H₁₈N₂OS (M + H): 299.1209; found 299.1210.

5(4H)-carboxamide (23). H NMR (300 MHz, DMSO-d₆): 2.81−

3.18 (m, 3 H), 4.19−4.36 (m, 1 H), 6.55 (s, 1 H), 6.82 (d, J = 5.26 Hz,

1 H), 6.91−7.01 (m, 1 H), 7.11−7.32 (m, 6 H), 7.40 (d, J = 5.12 Hz, 1

H), 7.47 (dd, J = 8.55, 0.95 Hz, 2 H), 8.69 (s, 1 H). MS (ESI pos. ion)

m/z: calcd for C₂₀H₁₇FN₂OS 353.1; found 353.0.

N,4-Diphenyl-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-carboxa-

N-Isopropyl-4-(4-(trifluoromethyl)phenyl)-6,7-dihydrothieno[3,2-

1

mide (14). H NMR (400 MHz, DMSO-d₆): 2.84−3.02 (m, 2 H),

c]pyridine-5(4H)-carboxamide (32). H NMR (300 MHz, DMSO-

3.03−3.15 (m, 1 H), 4.28 (dd, J = 13.89, 4.50 Hz, 1 H), 6.56 (s, 1 H),

6.83 (d, J = 5.28 Hz, 1 H), 6.95 (t, J = 7.34 Hz, 1 H), 7.19−7.30 (m, 5

H), 7.30−7.36 (m, 2 H), 7.39 (d, J = 5.09 Hz, 1 H), 7.48 (d, J = 7.63

Hz, 2 H), 8.67 (s, 1 H). HRMS calcd for C₂₀H₁₈N₂OS (M + H):

335.1209; found 335.1220.

d₆): 1.07 (dd, J = 6.50, 2.56 Hz, 6 H), 2.72−3.03 (m, 3 H), 3.38 − 3.59

(m, 1 H), 3.82 (d, J = 6.87 Hz, 1 H), 4.08 (d, J = 9.50 Hz, 1 H), 6.43

(d, J = 7.60 Hz, 1 H), 6.49 (s, 1 H), 6.83 (d, J = 5.26 Hz, 1 H), 7.37−

7.44 (m, 3 H), 7.70 (d, J = 8.18 Hz, 2 H). HRMS calcd for

C₁₈H₁₉F₃N₂OS (M + H): 369.1239; found 369.1230.

N-Phenyl-4-(2-(trifluoromethyl)phenyl)-6,7-dihydrothieno[3,2-c]-

N-Cyclopropyl-4-(4-(trifluoromethyl)phenyl)-6,7-dihydrothieno-

[3,2-c]pyridine-5(4H)-carboxamide (33). ¹H NMR (300 MHz,

DMSO-d₆): 0.33−0.43 (m, 2 H), 0.50−0.61 (m, 2 H), 2.53−2.62

(m, 1 H), 2.68−3.01 (m, 3 H), 3.99 (d, J = 8.92 Hz, 1 H), 6.46 (s, 1

H), 6.83 (d, J = 5.26 Hz, 2 H), 7.35−7.44 (m, 3 H), 7.70 (d, J = 8.04

Hz, 2 H). HRMS calcd for C₁₈H₁₇F₃N₂OS (M + H): 367.1083; found

367.1090.

1

pyridine-5(4H)-carboxamide (15). H NMR (300 MHz, DMSO-d₆):

2.79−3.01 (m, 2 H), 3.33−3.45 (m, 1 H), 4.11−4.25 (m, 1 H), 6.63

(d, J = 5.12 Hz, 1 H), 6.82 (s, 1 H), 6.88−6.98 (m, 1 H), 7.16 (s, 1 H),

7.23 (d, J = 7.45 Hz, 2 H), 7.36 (d, J = 5.26 Hz, 1 H), 7.41 (dd, J =

8.55, 1.10 Hz, 2 H), 7.52 (t, J = 7.50 Hz, 1 H), 7.56−7.64 (m, 1 H),

7.79 (d, J = 6.72 Hz, 1 H), 8.87 (s, 1 H). HRMS calcd for

C₂₁H₁₇F₃N₂OS (M + H): 403.1083; found 403.1090.

Morpholino(4-(4-(trifluoromethyl)phenyl)-6,7-dihydrothieno[3,2-

1

c]pyridin-5(4H)-yl)methanone (34). H NMR (300 MHz, DMSO-

N-Phenyl-4-(3-(trifluoromethyl)phenyl)-6,7-dihydrothieno[3,2-c]-

1

pyridine-5(4H)-carboxamide (16). H NMR (300 MHz, DMSO-d₆):

d₆): 2.95 (d, J = 4.68 Hz, 2 H), 3.03−3.39 (m, 5 H), 3.46−3.74 (m, 5

H), 6.03 (s, 1 H), 6.75 (d, J = 5.12 Hz, 1 H), 7.35 (d, J = 5.12 Hz, 1

H), 7.46 (d, J = 8.18 Hz, 2 H), 7.69 (d, J = 8.18 Hz, 2 H). HRMS calcd

for C₁₉H₁₉F₃N₂O₂S (M + H): 397.1188; found 397.1190.

N-(4-Methoxyphenyl)-4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrothieno[3,2-c]pyridine-5(4H)-carboxamide (35). ¹H NMR

(300 MHz, DMSO-d₆): 2.81−3.15 (m, 3 H), 3.71 (s, 3 H), 4.27 (d,

J = 10.82 Hz, 1 H), 6.60 (s, 1 H), 6.79−6.91 (m, 3 H), 7.31−7.39 (m,

2 H), 7.42 (d, J = 5.26 Hz, 1 H), 7.47 (d, J = 8.18 Hz, 2 H), 7.72 (d, J

= 8.18 Hz, 2 H), 8.58 (s, 1 H). HRMS calcd for C₂₂H₁₉F₃N₂O₂S (M +

H): 433.1188; found 433.1198.

N-(4-Chlorophenyl)-4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrothieno[3,2-c]pyridine-5(4H)-carboxamide (36). ¹H NMR

(300 MHz, DMSO-d₆): 2.84−3.18 (m, 3 H), 4.20−4.37 (m, 1 H),

6.61 (s, 1 H), 6.88 (d, J = 5.12 Hz, 1 H), 7.01 (dd, J = 7.89, 1.32 Hz, 1

H), 7.27 (t, J = 8.11 Hz, 1 H), 7.40−7.53 (m, 4 H), 7.65−7.69 (m, 1

H), 7.73 (d, J = 8.18 Hz, 2 H), 8.91 (s, 1 H). HRMS calcd for

C₂₁H₁₆ClF₃N₂OS (M + H): 437.0694; found 437.0700.

N-(2-Fluorophenyl)-4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrothieno[3,2-c]pyridine-5(4H)-carboxamide (37). ¹H NMR

(300 MHz, DMSO-d₆): 2.83−3.22 (m, 3 H), 4.16−4.33 (m, 1 H),

6.58 (s, 1 H), 6.87 (d, J = 5.26 Hz, 1 H), 7.07−7.27 (m, 3 H), 7.36−

7.45 (m, 2 H), 7.49 (s, 2 H), 7.73 (d, J = 8.18 Hz, 2 H), 8.57 (s, 1 H).

HRMS calcd for C₂₁H₁₇F₄N₂OS (M + H): 421.0989; found 421.0990.

N-(3-Fluorophenyl)-4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrothieno[3,2-c]pyridine-5(4H)-carboxamide (38). ¹H NMR

(300 MHz, DMSO-d₆): 2.82−3.21 (m, 3 H), 4.19−4.39 (m, 1 H),

6.62 (s, 1 H), 6.69−6.83 (m, 1 H), 6.88 (d, J = 5.12 Hz, 1 H), 7.21−

7.34 (m, 2 H), 7.38−7.55 (m, 4 H), 7.73 (d, J = 8.18 Hz, 2 H), 8.93 (s,

1 H). HRMS calcd for C₂₁H₁₇F₄N₂OS (M + H): 421.0989; found

421.0980.

2.82−3.19 (m, 3 H), 4.20−4.41 (m, 1 H), 6.63 (s, 1 H), 6.88 (d, J =

5.26 Hz, 1 H), 6.92−7.02 (m, 1 H), 7.25 (t, J = 7.89 Hz, 2 H), 7.43 (d,

J = 5.1 Hz, 1 H), 7.44 − 7. 48 (m, 2 H), 7.51 − 7.64 (m, 3 H), 7.68 (d,

J = 8.90 Hz, 1 H), 8.75 (s, 1 H). HRMS calcd for C₂₁H₁₇F₃N₂OS (M +

H): 403.1083; found 403.1080.

N-Phenyl-4-(4-(trifluoromethyl)phenyl)-6,7-dihydrothieno[3,2-c]-

1

pyridine-5(4H)-carboxamide (17). H NMR (300 MHz, DMSO-d₆):

2.81−3.18 (m, 3 H), 4.24−4.35 (m, 1 H), 6.63 (s, 1 H), 6.88 (d, J =

5.26 Hz, 1 H), 6.92−7.01 (m, 1 H), 7.19−7.29 (m, 2 H), 7.43 (d, J =

5.26 Hz, 1 H), 7.48 (d, J = 7.60 Hz, 3 H), 7.72 (d, J = 8.18 Hz, 2 H),

8.73 (s, 1 H). HRMS calcd for C₂₁H₁₇F₃N₂OS (M + H): 403.1083;

found 403.1088.

4-(2-Chlorophenyl)-N-phenyl-6,7-dihydrothieno[3,2-c]pyridine-

1

5(4H)-carboxamide (18). H NMR (400 MHz, DMSO-d₆): 2.84−

2.97 (m, 2 H), 3.17−3.26 (m, 1 H), 4.15−4.29 (m, 1 H), 6.73−6.77

(m, 2 H), 6.90−6.96 (m, 2 H), 7.22 (t, J = 7.92 Hz, 2 H), 7.27 (dd, J =

7.53, 1.47 Hz, 1 H), 7.33 (dd, J = 7.5, 1.9 Hz, 1 H), 7.38 (d, J = 5.28

Hz, 1 H), 7.41−7.46 (m, 2 H), 7.49 (dd, J = 7.83, 1.37 Hz, 1 H), 8.82

(s, 1 H). HRMS calcd for C₂₀H₁₇ClN₂OS (M + H): 369.0820; found

369.0830.

4-(3-Chlorophenyl)-N-phenyl-6,7-dihydrothieno[3,2-c]pyridine-

1

5(4H)-carboxamide (19). H NMR (400 MHz, CDCl₃): 2.87−2.98

(m, 1 H), 3.02−3.17 (m, 1 H), 3.31−3.45 (m, 1 H), 4.08 (dd, J =

14.38, 4.21 Hz, 1 H), 6.38 (s, 1 H), 6.44 (s, 1 H), 6.73 (d, J = 5.28 Hz,

1 H), 7.01−7.08 (m, 1 H), 7.17 (d, J = 5.28 Hz, 1 H), 7.27−7.37 (m, 8

H). HRMS calcd for C₂₀H₁₇ClN₂OS (M + H): 369.0820; found

369.0820.

4-(4-Chlorophenyl)-N-phenyl-6,7-dihydrothieno[3,2-c]pyridine-

1

5(4H)-carboxamide (20). H NMR (300 MHz, CDCl₃): 2.85−2.99

(m, 1 H), 3.01−3.17 (m, 1 H), 3.36 (ddd, J = 14.03, 11.69, 3.95 Hz, 1

H), 4.07 (dd, J = 13.96, 4.17 Hz, 1 H), 6.39 (s, 1 H), 6.44 (s, 1 H),

6.70 (d, J = 5.26 Hz, 1 H), 7.05 (tt, J = 6.36, 2.19 Hz, 1 H), 7.16 (d, J =

5.26 Hz, 1 H), 7.27−7.35 (m, 8 H). HRMS calcd for C₂₀H₁₇ClN₂OS

(M + H): 369.0820; found 369.0829.

N-(4-Fluorophenyl)-4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrothieno[3,2-c]pyridine-5(4H)-carboxamide (39). ¹H NMR

(300 MHz, DMSO-d₆): 2.81−3.22 (m, 3 H), 4.18−4.37 (m, 1 H),

6.61 (s, 1 H), 6.88 (d, J = 5.26 Hz, 1 H), 7.01−7.16 (m, 2 H), 7.36−

7.58 (m, 5 H), 7.72 (d, J = 8.18 Hz, 2 H), 8.77 (s, 1 H). HRMS calcd

for C₂₁H₁₇F₄N₂OS (M + H): 421.0989; found 421.0990.

N-(4-Fluorophenyl)-7-(4-(trifluoromethyl)phenyl)-4,5-

dihydrothieno[2,3-c]pyridine-6(7H)-carboxamide (45). ¹H NMR

(400 MHz, DMSO-d₆): 2.81−3.17 (m, 3 H), 4.23−4.32 (m, 1 H),

6.61 (s, 1 H), 6.88 (d, J = 5.52 Hz, 1 H), 7.09 (t, J = 8.78 Hz, 2 H),

7.42 (d, J = 5.02 Hz, 1 H), 7.43 (s, 1 H), 7.45−7.53 (m, 4 H), 7.72 (d,

4-(2-Fluorophenyl)-N-phenyl-6,7-dihydrothieno[3,2-c]pyridine-

5(4H)-carboxamide (21). ¹H NMR (300 MHz, DMSO-d₆): 2.92 (d, J

= 4.38 Hz, 2 H), 3.13−3.31 (m, 1 H), 4.24−4.36 (m, 1 H), 6.73 (d, J =

5.1 Hz, 1 H), 6.77 (s, 1 H), 6.90−6.98 (m, 1 H), 7.01 (dd, J = 7.51,

1.43 Hz, 1 H), 7.09−7.19 (m, 1 H), 7.19−7.28 (m, 3 H), 7.30−7.40

(m, 2 H), 7.41−7.50 (m, 2 H) 8.73 (s, 1 H). HRMS calcd for

C₂₀H₁₇FN₂OS (M + H): 353.1115; found 353.1120.

1604

dx.doi.org/10.1021/jm2013634 | J. Med. Chem. 2012, 55, 1593−1611

Journal of Medicinal Chemistry

Article

J = 8.03 Hz, 2 H), 8.77 (s, 1 H). HRMS calcd for C₂₁H₁₇F₄N₂OS (M +

H): 421.0989; found 421.1000.

(m, 1 H), 4.13 (d, J = 13.45 Hz, 1 H), 6.77 (s, 1 H), 6.87 (d, J = 5.41

Hz, 1 H), 7.30−7.47 (m, 3 H), 7.71 (d, J = 8.04 Hz, 2 H). HRMS

calcd for C₁₈H₁₈F₃NOS (M + H): 354.1130; found 354.1130.

(2-Fluorophenyl)(4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanone (28). ¹H NMR

(400 MHz, DMSO-d₆): 2.87−2.96 (m, 1 H), 3.02−3.14 (m, 1 H),

3.20−3.28 (m, 1 H), 4.07 (br s, 1 H), 6.54 (br s, 1 H), 6.85 (d, J = 5.28

Hz, 1 H), 7.43 (d, J = 5.09 Hz, 1 H), 7.46−7.57 (m, 3 H), 7.57−7.64

(m, 1 H), 7.75 (d, J = 8.22 Hz, 2 H), 7.86 (d, J = 7.43 Hz, 2 H).

HRMS calcd for C₂₁H₁₅F₄NOS (M + H): 406.0880; found 406.0870.

(3-Fluorophenyl)(4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanone (29). ¹H NMR

(400 MHz, DMSO-d₆): 2.80−2.92 (m, 1 H), 2.95−3.08 (m, 1 H),

3.19−3.31 (m, 1 H), 3.66−3.69 (m, 1 H), 6.84 (br s, 1 H), 6.91 (d, J =

5.28 Hz, 1 H), 7.25 (d, J = 7.04 Hz, 1 H), 7.30−7.38 (m, 2 H), 7.41−

7.59 (m, 4 H), 7.75 (d, J = 8.02 Hz, 2 H). HRMS calcd for

C₂₁H₁₅F₄NOS (M + H): 406.0880; found 406.0870.

N-(4-Fluorophenyl)-4-(4-(trifluoromethyl)phenyl)-6,7-

1

dihydrofuro[3,2-c]pyridine-5(4H)-carboxamide (46). H NMR (400

MHz, DMSO-d₆): 2.69 (dd, J = 16.31, 3.26 Hz, 1 H), 2.81−2.97 (m, 1

H), 2.98−3.14 (m, 1 H), 4.31 (dd, J = 14.05, 5.02 Hz, 1 H), 6.50 (d, J

= 14.56 Hz, 2 H), 7.09 (t, J = 9.03 Hz, 2 H), 7.48 (dd, J = 8.53, 5.02

Hz, 2 H), 7.56 (d, J = 8.03 Hz, 2 H), 7.64 (s, 1 H), 7.73 (d, J = 8.03

Hz, 2 H), 8.78 (s, 1 H). HRMS calcd for C₂₁H₁₆F₄N₂O₂(M + H):

405.1217; found 405.1210.

N-(4-Fluorophenyl)-7-(4-(trifluoromethyl)phenyl)-4,5-

1

dihydrofuro[2,3-c]pyridine-6(7H)-carboxamide (47). H NMR (400

MHz, DMSO-d₆): 2.53 (dd, J = 13.7, 3.01 Hz, 1 H), 2.63−2.79 (m, 1

H), 2.93−3.09 (m, 1 H), 4.28 (dd, J = 14.31, 4.77 Hz, 1 H), 6.51 (d, J

= 11.04 Hz, 2 H), 7.09 (t, J = 8.78 Hz, 2 H), 7.43−7.54 (m, 4 H), 7.67

(s, 1 H), 7.76 (d, J = 8.03 Hz, 2 H), 8.81 (s, 1 H). HRMS calcd for

C₂₁H₁₆F₄N₂O₂(M + H): 405.1217; found 405.1234.

N-(4-Fluorophenyl)-1-(4-(trifluoromethyl)phenyl)-3,4-

dihydropyrrolo[1,2-a]pyrazine-2(1H)-carboxamide (48). ¹H NMR

(300 MHz, CDCl₃): 3.62 (ddd, J = 12.79, 8.92, 3.87 Hz, 1 H), 3.95−

4.29 (m, 3 H), 5.89−6.07 (m, 1 H), 6.20 (dd, J = 3.51, 2.78 Hz, 1 H),

6.30 (s, 1 H), 6.41 (s, 1 H), 6.68 (dd, J = 2.63, 1.61 Hz, 1 H), 6.92−

7.04 (m, 2 H), 7.17−7.25 (m, 2 H), 7.45−7.54 (m, 2 H), 7.56−7.67

(m, 2 H). HRMS calcd for C₂₁H₁₇F₄N₃O (M + H): 404.1377; found

404.1370.

General Procedure for the Preparation of Amines (24−26).

To a stirred solution of 4-(4-(trifluoromethyl)phenyl)-4,5,6,7-

tetrahydrothieno[3,2-c]pyridine (10g, 0.7 mmol) in dichloroethane

(DCE) (5 mL) at room temperature was added 1 equiv of

trifluoroacetic acid (TFA) followed by 1 equiv of the corresponding

ketone/aldehyde and then 3 equiv of NaB(OAc)₃H. The reaction

mixture was stirred at room temperature for 3 h and quenched by the

addition of a saturated aqueous solution of NaHCO₃. The reaction

mixture was extracted with CH₂Cl₂. The combined organic extracts

were dried over Na₂SO₄, concentrated, and evaporated under reduced

pressure vacuum to give the crude compound. Purifications were

carried out by chromatography on silica gel eluting with EtOAc/

hexanes mixtures.

(4-Fluorophenyl)(4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanone (30). ¹H NMR

(400 MHz, DMSO-d₆): 2.82−2.94 (m, 1 H), 2.95−3.09 (m, 1 H),

3.18 − 3.33 (m, 1 H), 3.67 (br s, 1 H), 6.84 (br s, 1 H), 6.90 (d, J =

5.09 Hz, 1 H), 7.29 (t, J = 8.90 Hz, 2 H), 7.42−7.58 (m, 5 H), 7.75 (d,

J = 8.22 Hz, 2 H). HRMS calcd for C₂₁H₁₅F₄NOS (M + H): 406.0880;

found 406.0890.

2-(4-Fluorophenyl)-1-(4-(4-(trifluoromethyl)phenyl)-6,7-

1

dihydrothieno[3,2-c]pyridin-5(4H)-yl)ethanone (31). H NMR (300

MHz, DMSO-d₆): 2.76−2.94 (m, 2 H), 3.08−3.22 (m, 1 H), 3.32 (s, 3

H), 4.13 (dd, J = 13.88, 3.65 Hz, 1 H), 6.76 (s, 1 H), 6.85 (d, J = 5.26

Hz, 1 H), 7.06−7.16 (m, 2 H), 7.22−7.31 (m, 2 H), 7.34−7.45 (m, 3

H), 7.70 (d, J = 8.18 Hz, 2 H). HRMS calcd for C₂₂H₁₇F₄NOS (M +

H): 420.1036; found 420.1030.

5-Bromo-N-methoxy-N-methylthiazole-4-carboxamide (50). To

a 100 mL round-bottomed flask were added 5-bromothiazole-4-

carboxylic acid (49; 0.98 g, 4.7 mmol), CH₂Cl₂(20 mL), and N-(3-

dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (11.08 g,

5.6 mmol). The reaction mixture was stirred at room temperature for

20 min. To this mixture, N,O-dimethylhydroxylamine hydrochloride

(0.55 g, 5.6 mmol) and N,N-diisopropylethylamine (0.98 mL, 5.6

mmol) were added. The reaction mixture was stirred at room

temperature for 16 h, diluted with water (40 mL), and extracted with

EtOAc (50 mL). The organic extract was washed with water (20 mL)

and saturated aqueous NaCl (20 mL), dried over Na₂SO₄, filtered, and

concentrated in vacuo. The crude product was purified by silica gel

chromatography eluting with 50% EtOAc/hexanes to give the title

compound (0.68 g, 58%). ¹H NMR (300 MHz, CDCl₃): 3.37 (s, 3 H),

3.73 (s, 3 H), 8.76 (s, 1 H). MS (ESI pos. ion) m/z: calcd for

C₆H₇BrN₂O₂S 250.9; found 250.9.

5-Bromothiazole-4-carbaldehyde (51). To a 100 mL round-

bottomed flask were added 5-bromo-N-methoxy-N-methylthiazole-4-

carboxamide (50, 0.63 g, 2.5 mmol) and THF (10 mL). The reaction

mixture was cooled to −78 °C, and diisobutylaluminum hydride (2.7

mL, 2.7 mmol, 1 M in THF) was added. The reaction mixture was

stirred at −78 °C for 1 h and then quenched by the addition of MeOH

(1 mL). The reaction mixture was diluted with water (20 mL) and

extracted with EtOAc (2 × 50 mL). The combined organic extracts

were washed with water (10 mL), saturated aqueous NaCl (10 mL),

dried over Na₂SO₄, filtered, and concentrated in vacuo to give the title

compound (0.42 g, 84%). ¹H NMR (300 MHz, CDCl₃): 8.85 (s, 1 H),

10.12 (s, 1 H). MS (ESI pos. ion) m/z: calcd for C₄H₂BrNOS 192.9;

found 192.9.

5-Isopropyl-4-(4-(trifluoromethyl)phenyl)-4,5,6,7-

1

tetrahydrothieno[3,2-c]pyridine (24). H NMR (300 MHz, DMSO-

d₆): 0.97 (dd, J = 12.20, 6.65 Hz, 6 H), 2.55−2.75 (m, 2 H), 2.82−2.94

(m, 2 H), 3.10−3.22 (m, 1 H), 4.84 (s, 1 H), 6.25 (d, J = 5.12 Hz, 1

H), 7.13 (d, J = 5.12 Hz, 1 H), 7.58 (dd, J = 8.04, 1.00 Hz, 2 H), 7.68

(d, J = 8.48 Hz, 2 H). HRMS calcd for C₁₇H₁₈F₃NS (M + H):

326.1181; found 326.1180.

5-Isobutyl-4-(4-(trifluoromethyl)phenyl)-4, 5, 6, 7-

1

tetrahydrothieno[3,2-c]pyridine (25). H NMR (300 MHz, DMSO-

d₆): 0.74 (dd, J = 9.43, 6.50 Hz, 6 H), 1.73−1.90 (m, 1 H), 1.95−2.08

(m, 1 H), 2.18 (dd, J = 12.28, 5.26 Hz, 1 H), 2.53−2.57 (m, 1 H),

2.79−3.01 (m, 2 H), 3.18 (dt, J = 11.73, 4.51 Hz, 1 H), 4.52 (s, 1 H),

6.28 (d, J = 5.12 Hz, 1 H), 7.18 (d, J = 5.12 Hz, 1 H), 7.51 (d, J = 8.04

Hz, 2 H), 7.68 (d, J = 8.04 Hz, 2 H). MS (ESI pos. ion) m/z: calcd for

C₁₈H₂₀F₃NS 340.1; found 340.1.

5-Benzyl-4-(4-(trifluoromethyl)phenyl)-4,5,6,7-tetrahydrothieno-

1

[3,2-c]pyridine (26). H NMR (300 MHz, DMSO-d₆): 2.56 (dt, J =

11.40, 4.60 Hz, 1 H), 2.75−2.92 (m, 2 H), 2.96−3.06 (m, 1 H), 3.39

(d, J = 12.30 Hz, 1 H), 3.64 (d, J = 13.74 Hz, 1 H), 4.70 (s, 1 H), 6.33

(d, J = 5.12 Hz, 1 H), 7.20 (d, J = 5.26 Hz, 1 H), 7.22−7.36 (m, 5 H),

7.64 (dd, J = 8.33, 1.00 Hz, 2 H), 7.73 (dd, J = 8.18, 1.00 Hz, 2 H).

HRMS calcd for C₂₁H₁₈F₃NS (M + H): 374.1181; found 374.1190.

General Procedure for the Preparation of Amides (27−31).

(5-Bromothiazol-4-yl)(4-(trifluoromethyl)phenyl)methanol (52).

To a suspension of magnesium turnings (0.11 g, 4.4 mmol) in THF

(10 mL) was added 1-bromo-4-(trifluoromethyl)benzene (0.62 mL,

4.4 mmol). The reaction mixture was stirred at 60 °C for 2 h and

cooled to 0 °C. To this solution, 5-bromothiazole-4-carbaldehyde (51,

0.42 g, 2.2 mmol) in THF (10 mL) was added dropwise. After the

addition was completed, the reaction mixture was stirred at 0 °C for 30

min, then the reaction was quenched by the addition of saturated

aqueous NH₄Cl (20 mL) and extracted with EtOAc (40 mL). The

organic extract was washed with water and saturated aqueous NaCl,

To

a solution of 4-(4-(trifluoromethyl)phenyl)-4,5,6,7-

tetrahydrothieno[3,2-c]pyridine (10g, 100 mg, 0.35 mmol) in

CH₂Cl₂(4 mL) at room temperature were added 1.1 equiv of

triethylamine and 1.05 equiv of the corresponding acid chloride. The

reaction mixture was stirred at room temperature for 2 h and then

concentrated in vacuo. Purifications were carried out by chromatog-

raphy on silica gel eluting with 50% EtOAc/hexanes.

2-Methyl-1-(4-(4-(trifluoromethyl)phenyl)-6,7-dihydrothieno[3,2-

1

c]pyridin-5(4H)-yl)propan-1-one (27). H NMR (300 MHz, DMSO-

d₆): 1.03 (dd, J = 15.27, 6.65 Hz, 6 H), 2.87−3.05 (m, 3 H), 3.09−3.26

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dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue

was purified by silica gel chromatography, eluting with 40% EtOAc/

hexanes to give the title compound (0.51 g, 69%). H NMR (300

4 H), 5.27 (s, 1 H), 7.44 (d, J = 8.33 Hz, 2 H), 7.59 (d, J = 8.18 Hz, 2

H), 8.60 (s, 1 H). MS (ESI pos. ion) m/z: calcd for C₁₃H₁₁F₃N₂S

285.0; found 285.0.

1

MHz, CDCl₃): 3.28 (d, J = 7.75 Hz, 1 H), 6.03 (d, J = 7.75 Hz, 1 H),

7.50−7.70 (m, 4 H), 8.78 (s, 1 H). MS (ESI pos. ion) m/z: calcd for

C₁₁H₇BrF₃NOS 338.9; found 338.9.

N-(4-Fluorophenyl)-4-(4-(trifluoromethyl)phenyl)-6,7-

dihydrothiazolo[4,5-c]pyridine-5(4H)-carboxamide (58). To a 25

mL round-bottomed flask were added 4-(4-(trifluoromethyl)phenyl)-

4,5,6,7-tetrahydrothiazolo[4,5-c]pyridine (57, 5 mg, 18 μmol), CH₂Cl₂

(1 mL), and 4-fluorophenyl isocyanate (2 mg, 18 μmol). The solution

was stirred at room temperature for 1 h, and the solvent was removed

in vacuo. The residue was purified by silica gel chromatography,

eluting with 40% EtOAc/hexanes to give the title compound (5 mg,

(E)-2-(2-(4-(Hydroxy(4-(trifluoromethyl)phenyl)methyl)thiazol-5-

yl)vinyl)isoindoline-1,3-dione (53). To a 50 mL round-bottomed flask

were added (5-bromothiazol-4-yl)(4-(trifluoromethyl)phenyl)-

methanol (52, 0.25 g, 0.75 mmol), N-vinylphthalimide (0.14 g, 0.82

mmol), acetato(2′-di-t-butylphosphino-1,1′-biphenyl-2-yl)palladium-

(II) (35 mg, 75 μmol), triethylamine (0.10 mL, 0.75 mmol), and

DMF (2 mL). The reaction mixture was stirred at 80 °C for 24 h and

cooled to room temperature. The reaction mixture was diluted with

water (10 mL) and extracted with EtOAc (2 × 40 mL). The combined

organic extracts were washed with water and saturated aqueous NaCl,

dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue

was purified by silica gel chromatography, eluting with 40% EtOAc/

1

67%). H NMR (300 MHz, CDCl₃): 2.92−3.06 (m, 1 H), 3.08−3.24

(m, 1 H), 3.28−3.44 (m, 1 H), 4.36 (dd, J = 13.88, 4.68 Hz, 1 H), 6.36

(s, 1 H), 6.53 (s, 1 H), 6.93−7.07 (m, 2 H), 7.19−7.36 (m, 2 H),

7.52−7.70 (m, 4 H), 8.73 (s, 1 H). MS (ESI pos. ion) m/z: calcd for

C₂₀H₁₅F₄N₃OS 422.0942; found 422.0951.

4-(4-(Trifluoromethyl)phenyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-

c]pyridine (60). To a 100 mL round-bottomed flask were added

histamine dihydrochloride (59, 0.19 g, 1.03 mmol), potassium

hydroxide (0.18 g, 3.2 mmol), and water (1 mL). The reaction

mixture was treated with 4-(trifluoromethyl)benzaldehyde (0.14 mL,

1.03 mmol) in EtOH (2.5 mL) followed by the addition of water (12.5

mL). The reaction mixture was heated at 100 °C for 4 h. The mixture

was allowed to cool to room temperature, and the resulting precipitate

was collected by filtration. The crude product was dissolved into

MeOH/H₂O and filtered. The filtrate was concentrated in vacuo to

give the title compound as a white solid (94 mg, 34%). ¹H NMR (300

MHz, CD₃OD): 2.59−2.88 (m, 2 H), 3.02 (ddd, J = 12.61, 7.20, 5.19

Hz, 1 H), 3.08−3.23 (m, 1 H), 5.07 (s, 1 H), 7.38−7.55 (m, 3 H),

7.56−7.68 (m, 2 H). MS (ESI pos. ion) m/z: calcd for C₁₃H₁₂F₃N₃

268.1; found 268.0.

1

hexanes to give the title compound (56 mg, 17%). H NMR (300

MHz, CDCl₃): 3.19 (d, J = 4.24 Hz, 1 H), 3.55 (d, J = 7.02 Hz, 1 H),

6.00−6.16 (m, 2 H), 7.14 (dd, J = 1.97, 0.80 Hz, 1 H), 7.20 (d, J = 1

4.76 Hz, 1 H), 7.52−7.68 (m, 6 H), 7.79 (dd, J = 5.48, 3.14 Hz, 2 H),

7.89−7.96 (m, 2 H), 8.63 (s, 1 H), 8.80 (d, J = 1.90 Hz, 1 H). MS (ESI

pos. ion) m/z: calcd for C₂₁H₁₃F₃N₂O₃S 431.1; found 431.0.

2-(2-(4-(Hydroxy(4-(trifluoromethyl)phenyl)methyl)thiazol-5-yl)-

ethyl)isoindoline-1,3-dione (54). To a 50 mL round-bottomed flask

were added (E)-2-(2-(4-(hydroxy(4-(trifluoromethyl)phenyl)methyl)-

thiazol-5-yl)vinyl)isoindoline-1,3-dione (53, 0.12 g, 0.27 mmol), 10%

palladium on carbon (2.9 mg, 27 μmol), and MeOH (2 mL). The

reaction mixture was hydrogenated under 1 atm of hydrogen for 6

days. The solution was filtered to remove the catalyst through a pad of

Celite, and the solvent was removed in vacuo. The residue was purified

by silica gel chromatography, eluting with 50% EtOAc/hexanes to give

the title compound (28 mg, 24%). ¹H NMR (300 MHz, CDCl₃): 3.29

(t, J = 7.09 Hz, 2 H), 3.49 (d, J = 6.87 Hz, 1 H), 3.88−4.00 (m, 2 H),

6.01 (d, J = 6.72 Hz, 1 H), 7.52 (s, 4 H), 7.55−7.66 (m, 1 H), 7.67−

7.76 (m, 2 H), 7.76−7.87 (m, 2 H). MS (ESI pos. ion) m/z: calcd for

C₂₁H₁₅F₃N₂O₃S 433.1; found 433.0.

N-(4-Fluorophenyl)-4-(4-(trifluoromethyl)phenyl)-6,7-dihydro-

1H-imidazo[4,5-c]pyridine-5(4H)-carboxamide (61). To a 5 mL

microwave vial, 4-(4-(trifluoromethyl)phenyl)-4,5,6,7-tetrahydro-1H-

imidazo[4,5-c]pyridine (60, 94 mg, 0.35 mmol), 4-fluorophenyl

isocyanate (0.040 mL, 0.36 mmol), and CH₂Cl₂(3 mL) were

added. The reaction mixture was stirred at room temperature for 30

min. The resulting precipitate was collected via filtration and washed

with CH₂Cl₂to yield the title compound as a white solid (87 mg,

2-(2-(4-(4-(Trifluoromethyl)benzoyl)thiazol-5-yl)ethyl)isoindoline-

1,3-dione (55). To a 50 mL round-bottomed flask were added 2-(2-(4-

(hydroxy(4-(trifluoromethyl)phenyl)methyl)thiazol-5-yl)ethyl)-

isoindoline-1,3-dione (54, 26 mg, 60 μmol), CH₂Cl₂(2 mL), and

manganese(IV) oxide (104 mg, 1.2 mmol). The reaction mixture was

stirred at room temperature for 4 h. The solid was removed by

filtration, washed with EtOAc, and the solvent was removed in vacuo.

The residue was purified by silica gel chromatography, eluting with

1

61%). H NMR (300 MHz, DMSO-d₆): 2.54−2.66 (m, 1 H), 2.70−

3.10 (m, 2 H), 4.29−4.44 (m, 1 H), 6.40 (s, 1 H), 7.08 (t, J = 8.92 Hz,

2 H), 7.41−7.55 (m, 2 H), 7.58−7.80 (m, 5 H), 8.78 (s, 1 H), 12.02

(br s, 1 H). MS (ESI pos. ion) m/z: calcd for C₂₀H₁₆F₄N₄O 405.1;

found 405.0.

tert-Butyl 4-(4-(Trifluoromethyl)phenyl)-6,7-dihydro-1H-imidazo-

[4,5-c]pyridine-5(4H)-carboxylate (62). To a 100 mL round-

bottomed flask, 4-(4-(trifluoromethyl)phenyl)-4,5,6,7-tetrahydro-1H-

imidazo[4,5-c]pyridine (60, 0.58 g, 2.2 mmol), di-tert-butyl

dicarbonate (0.54 g, 2.5 mmol), NaHCO₃(0.28 g, 3.2 mmol), and

MeOH (10 mL) were added. The mixture was stirred at room

temperature for 2 h. The solvent was evaporated, and the residue was

dissolved in EtOAc (20 mL). The organic phase was washed with

water (20 mL) and saturated aqueous NaCl (20 mL), dried over

Na₂SO₄, filtered, and concentrated in vacuo. The crude product was

purified by silica gel chromatography (iPrOH (w/10% NH₄OH) in

CHCl₃0−10%) to afford the title compound as a pale-yellow solid

(0.59 g, 74%). ¹H NMR (300 MHz, DMSO-d₆): 1.43 (s, 9 H), 2.54−

2.80 (m, 2 H), 2.84−2.99 (m, 1 H), 4.34 (d, J = 4.09 Hz, 1 H), 6.03

(br s, 1 H), 7.52−7.60 (m, 3 H), 7.71 (d, J = 8.18 Hz, 2 H), 12.02 (br

s, 1 H). MS (ESI pos. ion) m/z: calcd for C₁₈H₂₀F₃N₃O₂368.2; found

368.0.

N-(4-Fluorophenyl)-1-methyl-4-(4-(trifluoromethyl)phenyl)-6,7-

dihydro-1H-imidazo[4,5-c]pyridine-5(4H)-carboxamide (63). To a 5

mL microwave vial were added iodomethane (0.18 mL, 2.91 mmol),

NaHCO₃(0.16 g, 1.95 mmol), tert-butyl 4-(4-(trifluoromethyl)-

phenyl)-6,7-dihydro-1H-imidazo[4,5-c]pyridine-5(4H)-carboxylate

(62, 0.36 g, 0.97 mmol), and DMF (4 mL). The mixture was stirred at

room temperature for 2 h. The reaction mixture was partitioned

between water (20 mL) and EtOAc (20 mL). The organic phase was

taken and washed with saturated aqueous NaHCO₃(2 × 20 mL). The

1

30% EtOAc/hexanes to give the title compound (22 mg, 85%). H

NMR (300 MHz, CDCl₃): 3.71 (t, J = 6.43 Hz, 2 H), 4.11 (t, J = 6.43

Hz, 2 H), 7.60−7.71 (m, 4 H), 7.72−7.83 (m, 2 H), 8.06 (d, J = 8.04

Hz, 2 H), 8.64 (s, 1 H). MS (ESI pos. ion) m/z: calcd for

C₂₁H₁₃F₃N₂O₃S 431.1; found 431.0.

4-(4-(Trifluoromethyl)phenyl)-6,7-dihydrothiazolo[4,5-c]pyridine

(56). To a 50 mL round-bottomed flask were added 2-(2-(4-(4-

(trifluoromethyl)benzoyl)thiazol-5-yl)ethyl)isoindoline-1,3-dione (55,

22 mg, 51 μmol), EtOH (1 mL), and anhydrous hydrazine (8 μL, 0.26

mmol). The reaction mixture was stirred at room temperature for 3

days, and the solvent was removed in vacuo. The residue was purified

by silica gel chromatography, eluting with 80% EtOAc/hexanes to give

the title compound (6 mg, 42% yield). ¹H NMR (300 MHz, CDCl₃):

3.03−3.19 (m, 2 H), 3.99−4.15 (m, 2 H), 7.70 (d, J = 8.18 Hz, 2 H),

8.11 (d, J = 8.18 Hz, 2 H), 8.68 (s, 1 H). MS (ESI pos. ion) m/z: calcd

for C₁₃H₉F₃N₂S 283.0; found 283.0.

4-(4-(Trifluoromethyl)phenyl)-4,5,6,7-tetrahydrothiazolo[4,5-c]-

pyridine (57). To a 50 mL round-bottomed flask were added 4-(4-

(trifluoromethyl)phenyl)-6,7-dihydrothiazolo[4,5-c]pyridine (56, 6

mg, 21 μmol), MeOH (1 mL), and NaBH₄(1 mg, 21 μmol). The

reaction mixture was stirred at room temperature for 30 min, and the

solvent was removed in vacuo. The residue was purified by silica gel

chromatography, eluting with 10% MeOH/EtOAc to give the title

compound (5 mg, 83%). ¹H NMR (300 MHz, CDCl₃): 2.85−3.36 (m,

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organic phase was dried over Na₂SO₄, filtered, and concentrated in

vacuo. The crude product was taken into CH₂Cl₂(5 mL), and TFA (2

mL) was added. The yellow solution was stirred at room temperature

for 2 h. The reaction mixture was partitioned between saturated

aqueous NaHCO₃(30 mL) and CH₂Cl₂(50 mL). The aqueous phase

was taken and extracted with CH₂Cl₂(20 mL). The combined organic

phases were dried over Na₂SO_4,filtered, and concentrated in vacuo.

The residue was dissolved in CH₂Cl₂(2 mL), and 4-fluorophenyl

isocyanate (0.05 mL, 0.13 mmol) was added. The resulting yellow

solution was stirred at room temperature for 3 h. The reaction mixture

was directly purified by silica gel chromatography (eluent iPrOH (w/

10% NH₄OH) in CHCl₃0−10%) to afford the title compound as a

white solid (19 mg, 5%). ¹H NMR (300 MHz, DMSO-d₆) 2.59 (dd, J

= 15.35, 3.22 Hz, 1 H) 2.68−3.05 (m, 2 H) 3.56 (s, 3 H) 4.32−4.51

(m, 1 H) 6.38 (s, 1 H) 7.02−7.14 (m, 2 H) 7.43−7.55 (m, 2 H) 7.57−

7.76 (m, 5 H) 8.80 (s, 1 H). MS (ESI pos. ion) m/z: calcd for

C₂₁H₁₈F₄N₄O 419.1486; found 419.1490.

7-Fluoro-1-(4-(trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoqui-

noline (67d). MS (ESI pos. ion) m/z: calcd for C₁₆H₁₃F₄N 296.1;

found 296.1

1-(4-(Trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1H-benzo[c]-

azepine (67e). MS (ESI pos. ion) m/z: calcd for C₁₇H₁₆F₃N 292.1;

found 292.1

General Procedure for the Preparation of N-(4-Fluorophen-

yl)-1-(4-(trifluoromethyl)phenyl)-3,4-dihydroisoquinolines

(68−72). To a solution of the tetrahydroisoquinoline/benzoazepine,

67a−e (1.9 mmol) in CH₂Cl₂(4 mL) at room temperature was added

1 equiv of 4-fluorophenyl isocyanate. The reaction mixture was stirred

at room temperature for 1 h and then concentrated in vacuo.

Purification was carried out by chromatography on silica gel to give

compounds 68−72.

N-(4-Fluorophenyl)-1-(4-(trifluoromethyl)phenyl)-3,4-dihydroiso-

1

quinoline-2(1H)-carboxamide (68). H NMR (400 MHz, CD₃OD):

2.82 (dt, J = 16.24, 5.58 Hz, 1 H), 2.96−3.10 (m, 1 H), 3.60 (ddd, J =

12.96, 8.17, 5.09 Hz, 1 H), 3.88 (dt, J = 12.62, 6.02 Hz, 1 H), 6.64 (s, 1

H), 6.93−7.07 (m, 2 H), 7.19−7.33 (m, 4 H), 7.35−7.48 (m, 4 H),

7.61 (d, J = 8.22 Hz, 2 H). MS (ESI pos. ion) m/z: calcd for

C₂₃H₁₈F₄N₂O 415.1424; found 415.1420.

The mixture was resolved using chiral SFC (OJH column, 21 × 250

mm², 5 μM) using 70% supercritical CO₂/30% iPrOH (0.2%

diethylamine) at a flow rate of 65 mL/min) to give two products

with enantiomeric excesses greater than 99%.

(R)-N-(4-Fluorophenyl)-1-(4-(trifluoromethyl)phenyl)-3,4-dihy-

droisoquinoline-2(1H)-carboxamide (87). First eluting peak. ¹H

NMR (400 MHz, CD₃OD): 2.82 (dt, J = 16.04, 5.58 Hz, 1 H),

2.94−3.09 (m, 1 H), 3.60 (ddd, J = 12.91, 8.12, 5.18 Hz, 1 H), 3.81−

3.93 (m, 1 H), 6.64 (s, 1 H), 6.96−7.07 (m, 2 H), 7.20−7.32 (m, 4 H),

7.34−7.47 (m, 4 H), 7.61 (d, J = 8.22 Hz, 2 H). Anal. (C₂₃H₁₈F₄N₂O):

C, H, N.

N-Phenethyl-4-(trifluoromethyl)benzamide (65a). To a solution

of phenethylamine (64a, 14.6 mL, 0.12 mol) and N-ethyl-N-

isopropylpropan-2-amine (20.1 mL, 0.12 mol) in CH₂Cl₂(500 mL)

at 0 °C was added 4-(trifluoromethyl)benzoyl chloride (17.2 mL, 0.12

mol) dropwise. The reaction was allowed to warm to room

temperature and stirred for 16 h. The reaction mixture was diluted

with water, and the organic phase was separated. The aqueous layer

was extracted with CH₂Cl₂. The combined organic extracts were dried

over Na₂SO₄, and the solvent was eliminated under vacuum to give the

1

title compound as an off-white solid (31.8 g, 94%). H NMR (400

MHz, CD₃OD): 2.94 (t, J = 7.34 Hz, 2 H), 3.63 (t, J = 7.34 Hz, 2 H),

7.15−7.35 (m, 5 H), 7.76 (d, J = 8.22 Hz, 2 H), 7.93 (d, J = 8.22 Hz, 2

H). MS (ESI pos. ion) m/z: calcd for C₁₆H₁₄F₃NO 294.1; found

294.1.

1-(4-(Trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoquinoline

(67a). To a round-bottomed flask were added N-phenethyl-4-

(trifluoromethyl)benzamide (65a, 5.0 g, 17.0 mmol), phosphorus(V)

oxide (1.21 g, 8.5 mmol), and polyphosphoric acid (60 g). The

reaction mixture was heated at 165 °C for 2 h and allowed to cool to

room temperature. The reaction mixture was carefully poured into an

ice-cold solution of 20% KOH, adjusting the pH to 7. The reaction

mixture was extracted with EtOAc (2×), the combined organic

extracts were dried over Na₂SO₄, and the solvent was eliminated under

vacuum to give 66a, which was used without further purification. To a

crude solution of 1-(4-(trifluoromethyl)phenyl)-3,4-dihydroisoquino-

line (66a, 4.69 g, 17.0 mmol) in MeOH (25 mL) at 0 °C was added

NaBH₄(1.93 g, 51.1 mmol). The reaction mixture was stirred at 0 °C

for 15 min and at room temperature for 2 h. The solvent was removed

under vacuum, and the reaction mixture was diluted with saturated

aqueous NaHCO₃and extracted with EtOAc (2×). The combined

organic extracts were dried over MgSO₄, filtered, and concentrated in

vacuo to give the crude material. Purification by silica gel

chromatography with 20−100% EtOAc/hexanes as eluent provided

(S)-N-(4-Fluorophenyl)-1-(4-(trifluoromethyl)phenyl)-3,4-dihy-

1

droisoquinoline-2(1H)-carboxamide (88). Second eluting peak. H

NMR (400 MHz, CD₃OD): 2.82 (dt, J = 16.19, 5.60 Hz, 1 H), 3.03

(ddd, J = 16.09, 8.17, 5.67 Hz, 1 H), 3.60 (ddd, J = 13.01, 8.12, 5.09

Hz, 1 H), 3.88 (dt, J = 12.47, 5.99 Hz, 1 H), 6.64 (s, 1 H), 6.96−7.08

(m, 2 H), 7.20−7.33 (m, 4 H), 7.36−7.46 (m, 4 H), 7.61 (d, J = 8.41

Hz, 2 H). Anal. (C₂₃H₁₈F₄N₂O): C, H, N.

5-Fluoro-N-(4-fluorophenyl)-1-(4-(trifluoromethyl)phenyl)-3,4-di-

hydroisoquinoline-2(1H)-carboxamide (69). ¹H NMR (400 MHz,

DMSO-d₆): 2.74−2.86 (m, 1 H), 2.88−3.01 (m, 1 H), 3.23−3.40 (m,

1 H), 3.95−4.13 (m, 1 H), 6.66 (s, 1 H), 7.02−7.20 (m, 4 H), 7.25−

7.36 (m, 1 H), 7.39−7.52 (m, 4 H), 7.71 (d, J = 8.53 Hz, 2 H). MS

(ESI pos. ion) m/z: calcd for C₂₃H₁₇F₅N₂O 433.1330; found

433.1320.

6-Fluoro-N-(4-fluorophenyl)-1-(4-(trifluoromethyl)phenyl)-3,4-di-

hydroisoquinoline-2(1H)-carboxamide (70). ¹H NMR (400 MHz,

CD₃OD): 2.83 (dt, J = 16.38, 5.40 Hz, 1 H), 2.98−3.10 (m, 1 H), 3.55

(ddd, J = 13.20, 8.41, 4.99 Hz, 1 H), 3.84−3.95 (m, 1 H), 6.65 (s, 1

H), 6.96−7.09 (m, 4 H), 7.26 (dd, J = 8.31, 5.58 Hz, 1 H), 7.34−7.47

(m, 4 H), 7.62 (d, J = 8.22 Hz, 2 H). MS (ESI pos. ion) m/z: calcd for

C₂₃H₁₇F₅N₂O 433.1330; found 433.1340.

7-Fluoro-N-(4-fluorophenyl)-1-(4-(trifluoromethyl)phenyl)-3,4-di-

hydroisoquinoline-2(1H)-carboxamide (71). ¹H NMR (400 MHz,

DMSO-d₆): 2.70−2.82 (m, 1 H), 2.89−3.01 (m, 1 H), 3.45 (ddd, J =

13.16, 8.46, 4.99 Hz, 1 H), 3.86−3.96 (m, 1 H), 6.65 (s, 1 H), 7.05−

7.16 (m, 3 H), 7.20 (dd, J = 9.59, 2.54 Hz, 1 H), 7.33 (dd, J = 8.41,

5.87 Hz, 1 H), 7.43 (d, J = 8.22 Hz, 2 H), 7.46−7.54 (m, 2 H), 7.70

(d, J = 8.22 Hz, 2 H), 8.68 (s, 1 H). MS (ESI pos. ion) m/z: calcd for

C₂₃H₁₇F₅N₂O 433.1330; found 433.1320.

1

the title compound as a white solid (1.99 g, 42%). H NMR (400

MHz, CD₃OD): 2.84−2.95 (m, 1 H), 2.99−3.10 (m, 2 H), 3.16−3.25

(m, 1 H), 5.19 (s, 1 H), 6.69 (d, J = 7.82 Hz, 1 H), 7.05 (dd, J = 8.02,

2.15 Hz, 1 H), 7.13−7.23 (m, 2 H), 7.44 (d, J = 8.22 Hz, 2 H), 7.64

(d, J = 8.02 Hz, 2 H). MS (ESI pos. ion) m/z: calcd for C₁₆H₁₄F₃N

278.1; found 278.1

Compounds 67b−e were prepared in an analogous manner to that

described for 67a.

5-Fluoro-1-(4-(trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoqui-

1

noline (67b). H NMR (400 MHz, DMSO-d₆): 2.66−2.82 (m, 2 H),

2.85−2.97 (m, 1 H), 3.00−3.11 (m, 2 H), 5.11 (s, 1 H), 6.49 (d, J =

7.63 Hz, 1 H), 6.99 (t, J = 9.00 Hz, 1 H), 7.04−7.12 (m, 1 H), 7.49 (d,

J = 8.22 Hz, 2 H), 7.68 (d, J = 8.22 Hz, 2 H). MS (ESI pos. ion) m/z:

calcd for C₁₆H₁₃F₄N 296.1; found 296.1

N-(4-Fluorophenyl)-1-(4-(trifluoromethyl)phenyl)-4,5-dihydro-

1

1H-benzo[c]azepine-2(3H)-carboxamide (72). H NMR (400 MHz,

CD₃OD): 1.71−1.82 (m, 1 H), 1.83−1.96 (m, 1 H), 2.80−2.88 (m, 2

H), 3.21−3.29 (m, 1 H), 4.04 (dt, J = 15.36, 3.77 Hz, 1 H), 6.78 (s, 1

H), 6.96−7.02 (m, 2 H), 7.12 (d, J = 7.43 Hz, 1 H), 7.22 (td, J = 6.75,

2.74 Hz, 1 H), 7.26−7.35 (m, 5 H), 7.37−7.42 (m, 1 H), 7.68 (d, J =

8.22 Hz, 2 H). MS (ESI pos. ion) m/z: calcd for C₂₄H₂₀F₄N₂O

429.1580; found 429.1605.

6-Fluoro-1-(4-(trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoqui-

1

noline (67c). H NMR (400 MHz, CD₃OD): 2.82−2.94 (m, 1 H),

2.96−3.11 (m, 2 H), 3.13−3.23 (m, 1 H), 5.16 (s, 1 H), 6.70 (dd, J =

8.51, 5.77 Hz, 1 H), 6.80 (dt, J = 8.40, 2.70 Hz, 1 H), 6.94 (dd, J =

9.59, 0.39 Hz, 1 H), 7.44 (d, J = 8.02 Hz, 2 H), 7.65 (d, J = 8.02 Hz, 2

H). MS (ESI pos. ion) m/z: calcd for C₁₆H₁₃F₄N 296.1; found 296.1

2-Benzyl-8-fluoro-1-(4-(trifluoromethyl)phenyl)-1,2-dihydroiso-

quinoline (75). To a 25 mL round-bottomed flask were added 8-

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fluoroisoquinoline (73, 0.40 g, 2.7 mmol), CH₃CN (5 mL), and

benzyl bromide (0.37 mL, 3.0 mmol). The reaction mixture was

heated at reflux for 3 h and cooled to room temperature, and the

solvent was removed under vacuum to give 2-benzyl-8-fluoroisoqui-

nolinium bromide (74) as a yellow solid that was used without further

purification. To a 100 mL round-bottomed flask were added

magnesium turnings (0.28 g, 11 mmol), THF (10 mL), and a crystal

of iodine. The reaction mixture was placed under an argon

atmosphere, and 4-bromobenzotrifluoride (1.16 mL, 8.3 mmol) was

added. The reaction mixture was stirred at room temperature for 3 h

during which (4-(trifluoromethyl)phenyl)magnesium bromide was

formed. A separate 100 mL round-bottomed flask was charged with 2-

benzyl-8-fluoroisoquinolinium bromide (74, 0.87 g, 2.7 mmol) and

THF (15 mL). The solution was cooled to 0 °C. To this flask was

added (4-(trifluoromethyl)phenyl)magnesium bromide over a period

of 5 min, and the solution was allowed to stir at 0 °C for 1 h and at

room temperature for 16 h. The reaction mixture was quenched with

saturated aqueous NH₄Cl (35 mL) and extracted with EtOAc (2 × 25

mL). The combined organic extracts were washed with water and

saturated aqueous NaCl and dried over Na₂SO₄, and the solvent was

removed under vacuum. The title compound was obtained as brown

oil and used without further purification. MS (ESI pos. ion) m/z: calcd

for C₂₃H₁₇F₄N 384.2; found 384.1.

Polymer Lab-HCO₃macroporous resin cartridge. The filtrate was

concentrated under vacuum to give the title compound as a white film

(0.42 g, 70%). H NMR (400 MHz, DMSO-d₆) 2.79−2.89 (m, 1 H)

2.96−3.09 (m, 1 H) 3.20−3.26 (m, 1 H) 4.00−4.06 (m, 1 H) 6.82 (s,

1 H) 7.11 (q, J = 8.80 Hz, 3 H) 7.18 (d, J = 7.63 Hz, 1 H) 7.35 − 7.42

(m, 3 H) 7.50 (dd, J = 9.00, 5.09 Hz, 2 H) 7.72 (d, J = 8.22 Hz, 2 H)

8.81 (s, 1 H). MS (ESI pos. ion) m/z: calcd for C₂₃H₁₇F₅N₂O

433.1330; found 433.1358.

1

3-(4-(Trifluoromethyl)phenyl)isoindolin-1-one (80). To a solution

of phthalimide (79, 22.4 g, 16.3 mmol) in THF (20 mL) under N₂

atmosphere at 0 °C was added a solution of (4-(trifluoromethyl)-

phenyl)magnesium bromide in THF (1M, 60 mL), followed by 1,3-

dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (2 mL). The reaction

mixture was stirred at 0 °C for 2 h and quenched by the addition of an

aqueous solution of sodium phosphate. The resulting mixture was

diluted with EtOAc and stirred at room temperature for 15 min. The

organic layer was separated, washed with water and saturated aqueous

NaCl, and dried over Na₂SO₄. Evaporation of the solvent under

vacuum gave 3-hydroxy-3-(4-(trifluoromethyl)phenyl)isoindolin-1-one

(5.1 g) as an orange solid that was used without further purification.

To a solution of 3-hydroxy-3-(4-(trifluoromethyl)phenyl)isoindolin-1-

one (3.0 g, 10.2 mmol) in CH₂Cl₂(60 mL) was added TFA (5.3 mL,

71.6 mmol), and the reaction mixture was stirred at room temperature

for 5 min. Triethylsilane (3.3 mL, 20.5 mmol) was added, and the

solution was stirred at room temperature for 16 h. The reaction was

quenched by the addition of water (50 mL), and CH₂Cl₂(20 mL) was

added. The mixture was stirred at room temperature for 5 min, the

organic layer was collected, and the aqueous layer was extracted with

CH₂Cl₂(30 mL). The combined organic extracts were dried over

MgSO₄and partially concentrated in vacuo. A white precipitate was

obtained, filtered, and dried in vacuo to give the title compound as a

2-Benzyl-8-fluoro-1-(4-(trifluoromethyl)phenyl)-1,2,3,4-tetrahy-

droisoquinoline (76). To a 250 mL round-bottomed flask were added

2-benzyl-8-fluoro-1-(4-(trifluoromethyl)phenyl)-1,2-dihydroisoquino-

line (75, 1.2 g, 3.1 mmol), THF (20 mL), and NaBH₄(0.32 g, 8.4

mmol) at room temperature. After 20 min, acetic acid (3.3 mL) was

added, and the reaction mixture was stirred at room temperature for

2.5 h. The reaction mixture was concentrated and dissolved in EtOAc

(150 mL), and water (25 mL) was added. The organic layer was

separated, dried over anhydrous Na₂SO₄, and concentrated to yield the

crude product. Purification by flash silica gel chromatography (eluent

EtOAc/hexanes gradient) gave the title compound as a colorless oil

1

white solid (1.15 g, 53%). H NMR (400 MHz, CD₃OD): 5.85 (s, 1

H), 7.35 (d, J = 7.83 Hz, 1 H), 7.52 (d, J = 8.41 Hz, 2 H), 7.56 (d, J =

7.04 Hz, 1 H), 7.60 (dt, J = 6.30, 1.40 Hz, 1 H), 7.70 (d, J = 8.22 Hz, 2

H), 7.86 (d, J = 7.24 Hz, 1 H). MS (ESI pos. ion) m/z: calcd for

C₁₅H₁₀F₃NO 278.1; found 278.1.

1

(0.78 g, 65%). H NMR (400 MHz, DMSO-d₆): 2.61 (dt, J = 12.55,

5.27 Hz, 1 H), 2.72−2.87 (m, 2 H), 2.89−3.02 (m, 1 H), 3.55 (d, J =

13.55 Hz, 1 H), 3.72 (d, J = 13.55 Hz, 1 H), 5.06 (s, 1 H), 6.89−6.98

(m, 1 H), 7.11 (d, J = 7.53 Hz, 1 H), 7.21−7.42 (m, 8 H), 7.68 (d, J =

8.03 Hz, 2 H). MS (ESI pos. ion) m/z: calcd for C₂₃H₁₉F₄N 386.2;

found 386.1.

1-(4-(Trifluoromethyl)phenyl)isoindoline (81). To a solution of 3-

(4-(trifluoromethyl)phenyl)isoindolin-1-one (80, 0.50 g, 1.80 mmol)

in THF (7 mL) was added lithium aluminum hydride (1 M in THF,

5.41 mL, 5.41 mmol). The reaction mixture was heated at 75 °C for 2

h. The mixture was allowed to cool to room temperature, and the

reaction was quenched by the addition of Na₂SO₄·H₂O until no

bubbling was observed. The mixture was stirred at room temperature

overnight and filtered, and the solid was washed with EtOAc. The

combined filtrates were concentrated, and the residue was purified first

by silica gel column chromatography (0−20% MeOH in CH₂Cl₂),

then by prep HPLC (0−100% CH₃CN/water, 0.1% TFA). The

CH₃CN was removed in vacuo, saturated aqueous NaHCO₃(15 mL)

was added, and the solution was extracted with EtOAc. The combined

organic extracts were dried over MgSO₄, concentrated, and dried

under vacuum to give the title compound as a white solid (63 mg,

8-Fluoro-1-(4-(trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoqui-

noline (77). To a 25 mL reaction vial were added 2-benzyl-8-fluoro-1-

(4-(trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoquinoline (76, 0.20

g, 0.52 mmol), palladium hydroxide and carbon (20%, 0.14 g, 0.19

mmol), and EtOH (10 mL). The reaction mixture was stirred at room

temperature under a 50 psi hydrogen atmosphere for 3 h. The catalyst

was removed by filtration through a Celite pad, and the solvent was

eliminated under vacuum. The crude product was purified by prep-

HPLC using a gradient of CH₃CN/water (0.1% TFA) and a

PHENOMENEX Gemini Axia-5 C-18 column (100 × 21.2 mm²).

The solvent was removed under vacuum, and the resulting product

was dissolved in MeOH (5 mL) and neutralized by passing the

solution through a Polymer Lab-HCO₃macroporous resin cartridge.

The filtrate was concentrated under vacuum to give the title

compound as a colorless oil (0.13 g, 86%). ¹H NMR (400 MHz,

DMSO-d₆): 2.82−2.95 (m, 3 H), 2.96−3.03 (m, 1 H), 5.53 (s, 1 H),

6.99 (t, J = 9.10 Hz, 1 H), 7.13 (d, J = 7.63 Hz, 1 H), 7.28−7.36 (m, 1

H), 7.40 (d, J = 8.02 Hz, 2 H), 7.72 (d, J = 8.22 Hz, 2 H). MS (ESI

pos. ion) m/z: calcd for C₁₆H₁₃F₄N 296.1; found 296.1.

8-Fluoro-N-(4-fluorophenyl)-1-(4-(trifluoromethyl)phenyl)-3,4-di-

hydroisoquinoline-2(1H)-carboxamide (78). To a 25 mL round-

bottomed flask were added 8-fluoro-1-(4-(trifluoromethyl)phenyl)-

1,2,3,4-tetrahydroisoquinoline (77, 0.41 g, 1.4 mmol), DCE (10 mL),

and 4-fluorophenyl isocyanate (0.18 mL, 1.6 mmol). The reaction was

stirred at room temperature for 16 h, and the solvent was eliminated

under vacuum. The product was purified by prep-HPLC using a

gradient of CH₃CN/water (0.1% TFA) and a PHENOMENEX

Gemini Axia-5 C-18 column (100 × 21.2 mm²). The solvent was

removed under vacuum, and the resulting product was dissolved in

MeOH (5 mL) and neutralized by passing the solution through a

1

13%). H NMR (400 MHz, CD₃OD): 4.30 (d, J = 15.06 Hz, 1 H),

4.42 (d, J = 14.28 Hz, 1 H), 5.53 (s, 1 H), 7.00 (d, J = 7.63 Hz, 1 H),

7.19−7.34 (m, 2 H), 7.39 (d, J = 7.63 Hz, 1 H), 7.50 (d, J = 8.22 Hz, 2

H), 7.66 (d, J = 8.02 Hz, 2 H). MS (ESI pos. ion) m/z: calcd for

C₁₅H₁₂F₃N 264.1; found 264.1.

N-(4-Fluorophenyl)-1-(4-(trifluoromethyl)phenyl)isoindoline-2-

carboxamide (82). To a solution of 1-(4-(trifluoromethyl)phenyl)-

isoindoline (81, 58 mg, 0.22 mmol) and diisopropylethylamine (38

μL, 0.22 mmol) in CH₂Cl₂(1 mL) was added 4-fluorophenyl

isocyanate (25 μL, 0.22 mmol). The reaction mixture was stirred at

room temperature for 2 h. The reaction mixture was diluted with water

(2 mL) and extracted with CH₂Cl₂(2 × 2 mL). The combined organic

extracts were dried over MgSO₄, filtered, and concentrated in vacuo to

give the crude material. The product was purified by prep-HPLC (0−

100% CH₃CN/water, 0.1% TFA). The solvents were removed, and a

saturated aqueous solution of NaHCO₃was added. The aqueous layer

was extracted with EtOAc (2 × 25 mL). The combined organic

extracts were dried over MgSO₄, concentrated, and dried under

vacuum to give the title compound as a white solid (36 mg, 41%). ¹H

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NMR (400 MHz, CD₃OD): 5.01 (d, J = 13.89 Hz, 1 H), 5.18 (dd, J =

13.79, 2.45 Hz, 1 H), 6.30 (d, J = 2.15 Hz, 1 H), 6.94−7.03 (m, 2 H),

7.12 (d, J = 7.63 Hz, 1 H), 7.28 (t, J = 7.43 Hz, 1 H), 7.31−7.39 (m, 3

H), 7.44 (d, J = 7.43 Hz, 1 H), 7.54 (d, J = 8.22 Hz, 2 H), 7.64 (d, J =

8.22 Hz, 2 H). MS (ESI pos. ion) m/z: calcd for C₂₂H₁₆F₄N₂O 401.1;

found 401.1.

luminescence was measured by a charged-couple device

(CCD) camera-based FLASH-luminometer built by Amgen,

Inc. Compound activity was calculated using GraphPad Prism

4.01 (GraphPad Software Inc., San Diego, CA).

Rat Liver Microsomal Stability. Test compounds (1 μM) were

incubated with male CD1 rat liver microsomes (1 mg/mL) in the

presence of NADPH (1 mM) in phosphate-buffered saline (66.7 mM)

at 37 °C. Incubations were conducted for 30 min. Control incubations

were generated by the omission of NADPH from the incubation

reaction. Under these conditions, a cut off of <100 μL/(min·mg) was

considered desirable.

Rat PK Study. Nine male Sprague−Dawley rats were randomized to

dose groups as shown in Table 5. Animals in groups 1 and 2 were

dosed via intravenous injection, and animals in group 3 were dosed via

oral gavage. Plasma samples were collected for PK analysis predose

and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 16 h post-iv dose and predose

and 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 16 h post-po dose. Plasma

concentrations for compounds 87 and 88 were measurement using

sensitive LC/MS/MS methods. Noncompartmental analysis was

conducted on compound 87 and 88 plasma concentrations using

WinNonlin Enterprise v.5.1.1 (Pharsight Corporation, Mountain View,

CA).

In Vivo Assay. Icilin-Induced “Wet-Dog” Shaking in Rats. Male

Sprague−Dawley rats (220−300 g, n = 5−13/treatment) were

first habituated to the testing room for 30 min and then to a

transparent Plexiglas observation cylinder for 20 min. The

cylinders were placed on a custom opaque plastic apparatus

such that one rat could not view any other rats.²⁵A TRPM8

antagonist or vehicle control was administered po 90 min prior

to administration of 3 (0.5 mg/kg, ip, 100% PEG 400), and

wet-dog shakes (WDS) were counted for a duration of 30 min

postadministration. Antagonists were tested to assess the ability

to block the spontaneous wet-dog shake phenomena induced

by 3.

Absolute Sterochemical Determination. Vibrational circular

dichroism (VCD) spectra of 87 and 88 were recorded on a

BioTools/Bomem Dual PEM ChiralIR spectrometer (Biotools, Inc.,

Jupiter, FL) as CDCl₃solutions (50 mg/mL, BaF₂cell, 100 μM path

length, 8 cm⁻¹resolution) and are solvent subtracted. Optical rotation

measurements were performed at 26 °C in CHCl₃at 589 nm.

Quantum mechanical geometry optimizations and harmonic fre-

quency/VCD analysis were performed with the B3LYP hybrid density

functional and the 6-31G* basis set. Optical rotation calculations were

performed with the B3LYP functional and the 6-311++G(2d,2p) basis

upon the B3LYP/6-31G* geometries, utilizing the frequency-depend-

ent polarizability at 589.0 nm. All quantum mechanical calculations

were performed with the Gaussian 03 program system. Please see

Supporting Information for full details.

tert-Butyl 5-Oxo-5-(4-(trifluoromethyl)phenyl)pentylcarbamate

(84). To a solution of tert-butyl 2-oxopiperidine-1-carboxylate (83,

2.11 g, 10.6 mmol) in THF (20 mL) under N₂atmosphere at −78 °C

was added a solution of (4-(trifluoromethyl)phenyl)magnesium

bromide in THF (1 M, 11.7 mL). The reaction mixture was stirred

at −78 °C for 30 min, allowed to warm to room temperature, and

stirred at room temperature for an additional hour. The reaction

mixture was quenched by the addition of a 10% aqueous solution of

HCl, and the resulting mixture was extracted with CH₂Cl₂(2×). The

combined organic extracts were dried over Na₂SO₄, and the solvent

was eliminated under vacuum. The crude product was purified by silica

gel column chromatography (eluent EtOAc/hexanes 20%) to afford

the title compound as an off-white solid (2.41 g, 66%). ¹H NMR (400

MHz, CDCl₃): 1.44 (s, 9 H), 1.53−1.64 (m, 4 H), 1.70−1.89 (m, 2

H), 3.03 (t, J = 7.24 Hz, 2 H), 3.17 (q, J = 6.46 Hz, 2 H), 4.59 (br s, 1

H), 7.73 (d, J = 8.22 Hz, 2 H), 8.06 (d, J = 8.22 Hz, 2 H). MS (ESI

pos. ion) m/z: calcd for C₁₇H₂₂F₃NO₃345.1; found 246.1.

2-(4-(Trifluoromethyl)phenyl)piperidine (85). tert-Butyl 5-oxo-5-

(4-(trifluoromethyl)phenyl)pentylcarbamate (84, 2.31 g, 6.7 mmol)

was dissolved in TFA (15 mL), and the reaction mixture was stirred at

room temperature for 4 h. To the reaction mixture was added 50%

NaOH until the pH was 12−13, and then the mixture was extracted

with CH₂Cl₂(3×). The combined organic extracts were dried over

Na₂SO₄, and the solvent was eliminated under vacuum. The residue

obtained was dissolved in a 4:1 MeOH/H₂O mixture (10 mL), and

NaBH₄(0.31 g, 8.0 mmol) was added. The reaction mixture was

stirred at room temperature overnight. The mixture was acidified with

10% HCl and stirred at room temperature for 30 min. To the reaction

mixture was added 5 N NaOH until the pH was 12−13, and the

solution was extracted with CH₂Cl₂(3×). The combined organic

extracts were dried over Na₂SO₄, and the solvent was eliminated under

vacuum to give the title compound as a yellow solid (1.44 g, 94%). ¹H

NMR (400 MHz, CDCl₃): 1.39−1.60 (m, 3 H), 1.65−1.72 (m, 2 H),

1.75−1.82 (m, 1 H), 1.86−1.94 (m, 1 H), 2.80 (td, J = 11.54, 2.93 Hz,

1 H), 3.17−3.26 (m, 1 H), 3.62−3.69 (m, 1 H), 7.48 (dd, J = 8.22,

1.00 Hz, 2 H), 7.57 (d, J = 8.22 Hz, 2 H). MS (ESI pos. ion) m/z:

calcd for C₁₂H₁₄F₃N 230.1; found 230.1.

N-(4-Fluorophenyl)-2-(4-(trifluoromethyl)phenyl)piperidine-1-

carboxamide (86). To a solution of 2-(4-(trifluoromethyl)phenyl)-

piperidine (85, 0.37 g, 1.64 mmol) in DCE (4 mL) was added 4-

fluorophenyl isocyanante (0.20 mL, 1.80 mmol). The reaction mixture

was stirred at room temperature for 3 h, and the solvents were

evaporated under vacuum. The crude material was absorbed onto a

plug of silica gel and purified by silica gel chromatography eluting with

CH₂Cl₂. The title compound was obtained as a white solid (0.59 g,

ASSOCIATED CONTENT

■

S

* Supporting Information

1

99%). H NMR (400 MHz, CDCl₃): 1.40−1.54 (m, 1 H), 1.63−1.78

Computed geometric parameters, total and free energies,

Boltzmann populations, and predicted optical rotations for

the conformational ensemble of (R)-87; complete Gaussian 03

reference; TRP selectivity data, human TRPM8 activity, and

human liver microsomal clearance for compounds 87 and 88.

This material is available free of charge via the Internet at

http://pubs.acs.org.

(m, 3 H), 1.99−2.12 (m, 1 H), 2.33 (dd, J = 14.18, 3.42 Hz, 1 H),

2.97−3.14 (m, 1 H), 3.94 (br d, J = 13.10 Hz, 1 H), 5.49 (br s, 1 H),

6.32 (br s, 1 H), 6.93−7.02 (m, 2 H), 7.26−7.32 (m, 2 H), 7.42 (d, J =

8.22 Hz, 2 H), 7.63 (d, J = 8.41 Hz, 2 H). MS (ESI pos. ion) m/z:

calcd for C₁₉H₁₈F₄N₂O 367.1424; found 367.1430.

Biological Assays. In Vitro Rat TRPM8 Functional Assay.

Chinese Hamster Ovary (CHO) cells stably expressing rat

TRPM8 were generated using the tetracycline-inducible T-REx

expression system from Invitrogen, Inc. (Carlsbad, CA). To

enable a luminescence readout based on intracellular increase in

calcium, each cell line was also cotransfected with pcDNA3.1

plasmid containing jellyfish aequorin cDNA.²⁴Cells were

seeded in 96-well plates 24 h before the assay, and TRPM8

channel expression was induced with 0.5 μg/mL of tetracycline.

On assay day, the growth medium was removed, and cells were

incubated with assay buffer for 2 h. Cells were then exposed to

test compounds (at varying concentrations) and incubated for

2.5 min prior to adding the agonist (3, 1 μM). The

AUTHOR INFORMATION

■

Corresponding Author

*Phone: 805-447-8814. Fax: 805-480-1337. E-mail: ntamayo@

amgen.com.

ACKNOWLEDGMENTS

■

The authors thank Randall Hungate and Ken Wild for their

support of this research program. Thanks also go to Wesley

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Article

neuropathic pain. PLoS ONE 2009, 4, e7383 DOI: 10.1371/

journal.pone.0007383.

Barnhart for his excellent technical assistance in conducting the

HPLC chiral separation and to Xiping Zhang and Jian Jiang for

their support of the LC-MS/MS bioanalysis. Finally, we

acknowledge all of our colleagues on the TRPM8 research

team (Pharmaceutics, Toxicology, and PKDM).

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R. W.; Meegalla, S. K.; Ballentine, S. K.; Illig, C. R.; Qin, N.; Liu, Y.;

Hutchinson, T. L.; Lubin, M. L.; Stone, D. J. Jr.; Baker, J. F.;

Schneider, C. R.; Ma, J.; Damiano, B. P.; Flores, C. M.; Player, M. R.

Design and optimization of benzimidazole-containing Transient

Receptor Potential Melastatin 8 (TRPM8) antagonists. J. Med.

Chem. 2011, 54, 233−247.

ABBREVIATIONS

■

TRPM8, transient receptor potential melastatin type 8; WDS,

wet-dog shakes; TRP, transient receptor potential; TRP

subtype C, canonical; TRP subtype V, vanilloid; TRP subtype

A, ankyrin; TRP subtype P, polycystin; ML, mucolipin; CHO,

chinese hamster ovary; SAR, structure−activity relationship;

RLM, rat liver microsomes; ND, not determined; VCD,

vibrational circular dichroism; AUC_inf, area under the serum

concentration time curve from time 0 to infinity; CL, total body

clearance; V_ss, volume of distribution at steady state; t_1/2

,

terminal half-life; C_max, maximum observed serum concen-

tration

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Fused piperidines as a novel class of potent and orally available transient receptor potential melastatin type 8 (TRPM8) antagonists

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