Design, synthesis and biological evaluation of B-region modified diarylalkyl amide analogues as novel TRPV1 antagonists

Article abstract of DOI:10.1007/s12272-013-0228-x

Design, synthesis and biological evaluation of B-region, known to be a dipolar interacting pharmacophore, modified diarylalkyl amide analogues for novel TRPV1 (transient receptor potential channel, vanilloid subfamily member 1) antagonists was described. A variety of moieties including guanidines, heterocyclic rings, cinnamides, and α-substituted acetamides were introduced at the B-region. TRPV1 antagonistic activities of these analogues were evaluated by ⁴⁵Ca²⁺ uptake assay in rat DRG neuron. In particular, α,α-difluoroamide 53 exhibited 3-fold more potent TRPV1 antagonistic activity (IC₅₀ = 0.058 μM) than the parent amide analogue 6.

Full text of DOI:10.1007/s12272-013-0228-x

Arch. Pharm. Res.
DOI 10.1007/s12272-013-0228-x
RESEARCH ARTICLE
Design, synthesis and biological evaluation of B-region modified
diarylalkyl amide analogues as novel TRPV1 antagonists
•
Young Taek Han Shao-Mei Yang
•
•
Xiao-Yuan Wang Fu-Nan Li
Received: 9 June 2013 / Accepted: 26 July 2013
ꢀ The Pharmaceutical Society of Korea 2013
Abstract Design, synthesis and biological evaluation of
B-region, known to be a dipolar interacting pharmaco-
phore, modified diarylalkyl amide analogues for novel
TRPV1 (transient receptor potential channel, vanilloid
subfamily member 1) antagonists was described. A variety
of moieties including guanidines, heterocyclic rings, cin-
namides, and a-substituted acetamides were introduced at
the B-region. TRPV1 antagonistic activities of these ana-
logues were evaluated by 45Ca2? uptake assay in rat DRG
neuron. In particular, a,a-difluoroamide 53 exhibited
as capsaicin 1, noxious heat, protons (Tominaga et al.
1998), and endogenous inflammatory mediators such as
cannabinoid anandamide and lipoxygenase metabolites
(Smart et al. 2000; Hwang et al. 2000). Due to its crucial
role in pain signaling pathway, TRPV1 has attracted sig-
nificant attention to date (Caterina et al. 1997). Especially,
TRPV1 antagonists turned out to be therapeutically useful
in inflammatory pain models. This insight has made novel
TRPV1 antagonists developed for past decades. Capsaze-
pine 2, which was discovered by modifying the structure of
capsaicin, was reported as the first competitive TRPV1
antagonist in 1994 (Walpole et al. 1994). Since then,
numerous competitive antagonists equipped with various
scaffolds have been reported including cinnamide 3
(Doherty et al. 2005), and heterocycles such as pyrimidine
4 (Gunthrope and Chizh 2009). In particular, several potent
1,3-diarylalkyl thiourea TRPV1 antagonists including
SC0030 (IC50 of 0.037 lM, 5) have been reported along
with their structure-activity relationship (SAR) studies as
well as structural optimizations on the crucial pharmaco-
phoric parts; multiple H-bonding part (A-region), and
lipophilic part (C-region) (Wang et al. 2002; Suh et al.
2003, 2005; Suh and Oh 2005). In this connection, we
identified some B-region modified analogues such as amide
6 (IC50 of 0.17 lM, Li et al. 2009) as well as heterocycle-
linked phenylbenzylamides showing potent in vitro TRPV1
antagonistic activities (Kim et al. 2013). Some compounds
such as compound 6 exhibited more improved pharmaco-
kinetic profiles than TRPV1 antagonist equipped with
thiourea and thioamide (Li et al. 2011). Therefore, we
extended our investigation through design, and synthesis of
B-region modified diarylalkyl amide analogues incorpo-
rating various moieties such as guanidine, fluorine and
heterocyclic amine to enhance their dipolar interaction with
TRPV1 as well as rigid linkers such as cyclopropyl and
3-fold more potent TRPV1 antagonistic activity (IC50
=
0.058 lM) than the parent amide analogue 6.
Keywords TRPV1 ꢀ TRPV1 antagonist ꢀ Structural
modification ꢀ Diarylalkyl amide analogues
Introduction
TRPV1 (transient receptor potential channel, vanilloid
subfamily member 1) is a polymodal and nonselective
cation channel with a preference for calcium, and highly
expressed in sensory neurons (Szallasi and Blumberg
1999). This receptor is activated by vanilloid ligands such
Y. T. Han
College of Pharmacy, Woosuk University, Wanju 565-701,
Republic of Korea
S.-M. Yang ꢀ X.-Y. Wang ꢀ F.-N. Li (&)
School of Pharmaceutical Sciences, Xiamen University,
Xiang’an South Road, Xiamen 361102,
People’s Republic of China
e-mail: fnlee5@xmu.edu.cn
123

Y. T. Han et al.
cinnamoyl group to maintain their active conformation for
development of novel TRPV1 antagonists Figs. 1, 2.
FT-IR spectrometer. Low resolution mass spectra were
obtained on VG Trio-2 GC-MS.
Ethyl N-[4-(tert-butylbenzyl)aminocarbothioyl]carbamate
Material and method
(8)
General procedure
To a solution of ethoxycarbonyl isothiocyanate (270 lL,
1.2 mmol) in CH2Cl2 (5 mL) was added 4-tert-butylben-
zylamine 7 (640 lL, 1.2 mmol). The reaction mixture was
stirred at ambient temperature for 3 h, and diluted with
CH2Cl2. The diluted solution was washed with water and
brine, dried over MgSO4 and concentrated in vacuo.
Purification of the residue by column chromatography
(EtOAc/n-hexane =1:10) afforded 8 as a solid (610 mg,
91 %). 1H-NMR (CD3OD, 300 MHz): d 7.39 (d, 2H,
J = 6.2 Hz), 7.27 (d, 2H, J = 8.6 Hz), 4.79 (s, 2H), 4.22
(q, 2H, J = 7.1 Hz), 1.31 (s, 9H), 1.30 (t, 3H, J = 7.1 Hz).
Unless noted otherwise, all starting materials were
obtained from commercial suppliers and were used
without further purification. Air and moisture sensitive
reactions were performed under an argon atmosphere.
Flash column chromatography was performed using silica
gel 60 (230–400 mesh, Merck) with indicated solvents.
Thin-layer chromatography was performed using
0.25 mm silica gel F254 plates (Merck). Tetrahydrofuran
(THF) was distilled from sodium benzophenone ketyl.
N,N-dimethyl formamide (DMF) was distilled under
reduced pressure from calcium hydride and stored over
˚
4A molecular sieves under argon. Dichloromethane was
Ethyl N-[4-(tert-butylbenzyl)amino{3-fluoro-4-[(methyl
distilled from calcium hydride. All solvents used for
routine isolation of products and chromatography were
reagent grade and distilled. Reaction flasks were oven
sulfonyl)amino]benzylamino}methylene]carbamate (10)
To a solution of thiourea 8 (300 mg, 1.0 mmol) in DMF
(5 mL) were added EDCI (300 mg, 1.0 mmol) and Et3N
(2.9 mL, 20 mmol). The reaction mixture was stirred for
30 min, and ammonium salt 9 (260 mg, 1.0 mmol) and
Et3N (2.9 mL, 20 mmol) in DMF (11 mL) were added.
After confirming the completion of the reaction with TLC,
the reaction solution was acidified by 2N HCl and extracted
with EtOAc. The organic layer was washed with water and
brine, dried over MgSO4 and concentrated in vacuo.
Purification of the residue by column chromatography
(EtOAc/n-hexane =1:1) afforded 10 (210 mg, 43 %) as a
1
dried at 120 ꢁC. H and 13C NMR spectra were recorded
on a JEOL LNM-LA 300 (300 MHz), Brucker FT-NMR
AVANCE 400 (400 MHz), or FT-NMR AVANCE 500
(500 MHz) spectrometer as solutions in deuteriochloro-
form (CDCl3). Chemical shifts were expressed in parts
per million (ppm, d) and tetramethylsilane (TMS) was
1
use as an internal standard. H-NMR data were reported
in order of chemical shift, multiplicity (s, singlet; brs,
broad singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet and/or multiple resonance), number of protons and
coupling constant in herz (Hz). Infrared spectra were
recorded on Jasco FT/IR-4200 or Perkin-Elmer 1710
1
solid. H-NMR (CD3OD, 300 MHz) d 7.36–7.39 (m, 3H),
7.03–7.20 (m, 4H), 4.47 (s, 2H), 4.43 (s, 2H), 4.07 (q, 2H,
Fig. 1 Capsaicin (1) known as
the TRPV1 agonist and other
selected TRPV1 antagonists
(2,3,4,5)
123

Design, synthesis and biological evaluation of B-region
Fig. 2 Design and synthesis of
B-region modified diarylalkyl
amide analogues
J = 7.1 Hz), 2.96 (s, 3H), 1.30 (s, 9H), 1.23 (t, 3H,
J = 7.1 Hz); IR (neat) cm-1: 2962, 1597, 1512, 1401,
1327, 1156; LRMS (FAB?): m/z 479 (M?H?).
9H); IR (neat) cm-1: 2959, 1642, 1558, 1511, 1457, 1327;
LRMS (FAB?): m/z 423 (M?H?).
N-{4-[(2-Chloropyrimidin-4-ylamino)methyl]-2-
fluorophenyl}methanesulfonamide (14) and N-{4-[(4-
chloropyrimidin-2-ylamino)methyl]-2-
N-[4-([4-(tert-Butylbenzyl)amino(imino)methyl]
aminomethyl)-2-fluorophenyl]methanesulfonamide (11)
fluorophenyl}methanesulfonamide (15)
To a solution of guanidine 10 (40 mg, 0.08 mmol) in DMF
(2 mL) was added bromotrimethylsilane (50 lL,
0.42 mmol). The reaction mixture was refluxed overnight,
and poured into MeOH (0.5 mL). After 30 min, the reaction
mixture was concentrated in vacuo. The residue was diluted
with 2N HCl and washed with Et2O. Filtration of the com-
bined aqueous layers afforded 11 (4.0 mg, 10 %) as a white
solid. 1H-NMR (CD3OD, 400 MHz) d 7.49 (t, 1H,
J = 6.3 Hz), 7.42 (d, 2H, J = 6.3 Hz), 7.21 (d, 2H,
J = 6.1 Hz), 7.04–7.11 (m, 2H), 4.43 (s, 2H), 4.40 (s, 2H),
2.98 (s, 3H), 1.31 (s, 9H); IR (neat) cm-1: 2961, 1643, 1513,
1398, 1334, 1156; LRMS (FAB?): m/z 407 (M?H?).
To a solution of ammonium salt 9 (310 mg, 1.2 mmol) in
THF (5 mL) was added NaH (60 % dispersion in mineral oil
49 mg, 1.2 mmol), and the reaction mixture was stirred for
2 h. To the reaction mixture was added 2,4-dichloropyrim-
idine 13 (200 mg, 1.3 mmol) at ambient temperature. After
being stirred at 50 ꢁC for overnight, the reaction mixture
was diluted with EtOAc. The organic layer was washed with
water and brine, dried over MgSO4 and concentrated in
vacuo. Purification of the residue by column chromatogra-
phy (EtOAc/n-hexane =3:2) afforded 220 mg (54 %) of
regioisomers 14 and 15 (3: 1) as a white solid. The stereo-
chemical assignment for the compounds was confirmed on
the basis of 2D-NOESY NMR analysis. 14: 1H-NMR
(CDCl3, 300 MHz) d 8.01 (d, 1H, J = 6.0 Hz), 7.51 (t, 1H,
J = 8.3 Hz), 7.11 (d, 1H, J = 3.3 Hz), 7.08 (s, 1H), 6.73 (s,
1H), 6.23 (d, 1H, J = 5.7 Hz), 4.55 (d, 2H, J = 6.3 Hz),
N-[4-([4-(tert-Butylbenzyl)amino(hydroxyimino)methyl]
aminomethyl)-2-fluorophenyl]methane sulfonamide (12)
1
To a solution of thiourea 5 (100 mg, 0.24 mmol) in DMF
(3 mL) were added HgO (56 mg, 0.26 mmol), NH2OH.HCl
(29 mg, 0.42 mmol) and Et3N (0.1 mL, 0.83 mmol). After
being stirred for 2 h at ambient temperature, the reaction
mixture was quenched by aq. NaHCO3 and diluted with
EtOAc. The combined organic layer was washed with brine,
and dried over MgSO4. Purification of the residue by column
chromatography (EtOAc/n-hexane =1:2) afforded 12
3.02 (s, 3H); 15: H-NMR (CDCl3, 300 MHz): d 8.14 (d,
1H, J = 5.3 Hz), 7.51 (m, 1H), 7.11–7.14 (m, 2H), 6.61 (d,
1H, J = 5.1 Hz), 6.50 (br s, 1H), 5.66 (br s, 1H), 4.60 (d,
2H, J = 6.2 Hz), 3.00 (s, 3H).
N-(4-{[2-(4-tert-Butylphenyl)pyrimidin-4-
ylamino]methyl}-2-fluorophenyl)methanesulfonamide (17)
1
(20 mg, 20 %) as a solid. H-NMR (CDCl3, 300 MHz) d
To a solution of pyrimidine 14 (50 mg, 0.15 mmol) in
CH3CN (2 mL) were added Pd(PPh3)4 (8.7 mg, 0.01 mmol),
potassium 4-tert-butylphenyltrifluoroborate 16 (48 mg,
0.20 mmol) and Na2CO3 (48 mg, 0.45 mmol). The
7.50 (t, 1H, J = 8.1 Hz), 7.30 (d, 2H, J = 8.3 Hz), 7.23 (d,
2H, J = 8.3 Hz), 7.01–7.08 (m, 2H), 6.47 (br s, 1H), 4.65 (br
s, 2H), 4.36 (dd, 2H, J = 5.9, 3.0 Hz), 2.98 (s, 3H), 1.29 (s,
123

Y. T. Han et al.
reaction mixture was refluxed overnight. After being
cooled to room temperature, the mixture was concentrated
in vacuo and diluted with EtOAc. The organic layer was
washed with water and brine, dried over MgSO4 and
concentrated in vacuo. Purification of the residue by col-
umn chromatography (EtOAc/n-hexane =1:1) afforded 17
(s, 1H), 7.08 (s, 1H), 6.56 (br s, 1H), 6.34 (s, 1H), 4.53 (d,
2H, J = 6.1 Hz), 3.01 (s, 3H).
N-(4-{[6-(4-tert-Butylphenyl)pyrimidin-4-
ylamino]methyl}-2-fluorophenyl)methanesulfonamide (24)
1
(20 mg, 48 %, brsm) as a white solid. H-NMR (CDCl3,
Titled compound 24 (24 %) was prepared from 23 by the
synthetic procedure of 17 as a white solid. 1H-NMR
(CDCl3, 300 MHz) d 8.65 (d, 1H, J = 1.1 Hz), 7.85 (d,
2H, J = 8.6 Hz), 7.54 (t, 1H, J = 8.1 Hz), 7.46 (d, 2H,
J = 8.7 Hz), 7.16 (d, 1H, J = 1.4 Hz), 7.13 (s, 1H), 6.67
(d, 1H, J = 1.1 Hz), 6.56 (br s, 1H), 5.40 (br s, 1H), 4.61
(d, 2H, J = 6.2 Hz), 2.99 (s, 3H), 1.33 (s, 9H); IR (neat)
cm-1: 3365, 2925, 1603, 1514, 1461, 1333, 1273; LRMS
(FAB?): m/z 429 (M?H?).
300 MHz) d 8.26 (s, 1H), 8.23 (d, 2H, J = 8.6 Hz),
7.24–7.53 (m, 4H), 7.16 (s, 1H), 7.13 (s, 1H), 6.23 (d, 1H,
J = 6.0 Hz), 5.49 (br s, 1H), 4.64 (d, 2H, J = 5.9 Hz),
2.98 (s, 3H), 1.32 (s, 9H); IR (neat) cm-1: 3393, 2962,
1597, 1511, 1401, 1335, 1157; LRMS (FAB?): m/z 429
(M?H?).
N-(4-{[4-(4-tert-Butylphenyl)pyrimidin-2-
ylamino]methyl}-2-fluorophenyl)methanesulfonamide (18)
(Z)-Methyl N-4-tert-butylbenzoyl-N0-[3-fluoro-4-
(methylsulfonamido)benzyl]-carbamimidothioate (27)
Titled compound 18 (54 %, brsm) was prepared from 15 by
1
the synthetic procedure of 17 as a white solid. H-NMR
To a solution of 4-tert-butylbenzoyl chloride 25 (40 mg,
0.20 mmol) in THF (2 mL) was added NaSCN (18 mg,
0.20 mmol). The reaction mixture was refluxed for 3 h, and
ammonium salt 9 (50 mg, 0.20 mmol) and Et3N (57 lL,
0.40 mmol) were added. After being refluxed overnight,
the reaction mixture was cooled to ambient temperature
and concentrated in vacuo. The residue was diluted with
EtOAc, washed with water and brine, dried over MgSO4
and concentrated in vacuo. Purification of the residue by
column chromatography (EtOAc/n-hexane =1:2) afforded
58 mg (66 % for 2 steps) of N-acylthiourea intermediate.
To the solution of N-acylthiourea (58 mg, 0.38 mmol) in
DMF (2 mL) was added CH3I (240 lL, 3.8 mmol). After
being stirred overnight, the reaction mixture was concen-
trated in vacuo, and diluted with EtOAc and water. The
organic layer was washed with brine, dried over MgSO4
and concentrated in vacuo. Purification of the residue by
column chromatography (EtOAc/n-hexane =1:2) afforded
(CDCl3, 300 MHz) d 8.30 (d, 1H, J = 5.3 Hz), 7.91 (d,
2H, J = 8.4 Hz), 7.45–7.53 (m, 3H), 7.17–7.24 (m, 2H),
7.00 (d, 1H, J = 5.3 Hz), 4.69 (d, 2H, J = 6.2 Hz), 2.97 (s,
3H), 1.33 (s, 9H); IR (neat) cm-1: 2963, 1581, 1515, 1457,
1335, 1158; LRMS (FAB?): m/z 429 (M?H?).
N-{4- [(6-Chloropyrazin-2-ylamino)methyl]-2-
fluorophenyl}methanesulfonamide (20)
Titled compound 20 (39 %) was prepared from 19 by the
synthetic procedure of 14 as a white solid. 1H-NMR
(CDCl3, 300 MHz) d 7.83 (s, 1H), 7.75 (s, 1H), 7.52 (t, 1H,
J = 8.1 Hz), 7.14 (d, 1H, J = 2.6 Hz), 7.11 (s, 1H), 5.21
(br s, 1H), 4.53 (d, 2H, J = 6.0 Hz), 3.01 (s, 3H).
N-(4-{[6-(4-tert-Butylphenyl)pyrazin-2-ylamino]methyl}-2-
fluorophenyl)methanesulfonamide (21)
1
27 (130 mg, 75 %). H-NMR (CDCl3, 400 MHz) d 11.52
Titled compound 21 (35 %) was prepared from 20 by the
synthetic procedure of 17 as a white solid. 1H-NMR
(CDCl3, 300 MHz) d 8.30 (s, 1H), 7.84–7.86 (m, 3H), 7.53
(t, 1H, J = 8.4 Hz), 7.46 (d, 2H, J = 8.4 Hz), 7.20 (s, 1H),
7.17 (s, 1H), 6.56 (s, 1H), 5.17 (br s, 1H), 4.65 (d, 2H,
(s, 1H), 8.10 (d, 2H, J = 8.4 Hz), 7.51 (t, 1H, J = 8.4 Hz),
7.38 (d, 2H, J = 8.4 Hz), 7.10 (d, 1H, J = 3.6 Hz), 6.93 (s,
1H), 6.46 (s, 1H), 4.49 (s, 2H), 2.97 (s, 3H), 2.58 (s, 3H),
1.27 (s, 9H); IR (neat) cm-1: 3232, 2962, 1594, 1554,
1513, 1375; LRMS (FAB?): m/z 452 (M?H?).
J = 5.9 Hz), 2.99 (s, 3H), 1.33 (s, 9H); IR (neat) cm-1
:
2961, 1514, 1334, 1278, 1201, 1156; LRMS (FAB?): m/z
429 (M?H?).
N-(4-{[5-(4-tert-Butylphenyl)-1H-1,2,4-triazol-3-
ylamino]methyl}-2-fluorophenyl)methanesulfonamide (28)
N-{4-[(6-Chloropyrimidin-4-ylamino)methyl]-2-
To a solution of S-methyl-N-acylthiourea 27 (32 mg,
0.07 mmol) in EtOH (1 mL) was added hydrazine hydrate
(66 lL, 2.1 mmol). The reaction mixture was refluxed for
overnight. After being cooled to room temperature, the
reaction mixture was concentrated in vacuo. Recrystalli-
zation of the solid residue using EtOAc and n-hexane
fluorophenyl}methanesulfonamide (23)
Titled compound 23 (38 %) was prepared from 22 by the
synthetic procedure of 14 as a white solid. 1H-NMR
(CDCl3, 300 MHz) d 8.37 (s, 1H), 7.51–7.56 (m, 2H), 7.11
123

Design, synthesis and biological evaluation of B-region
1
afforded 28 (11 mg, 37 %) as a colorless solid. H-NMR
(CDCl3, 400 MHz) d 7.62 (d, 2H, J = 8.4 Hz), 7.24–7.27
(m, 3H), 6.98–7.01 (m, 2H), 4.29 (s, 2H), 3.16 (s, 3H), 1.45
(s, 9H); LRMS (FAB?): m/z 418 (M?H?).
water and brine, dried over MgSO4 and concentrated in
vacuo. Purification of the residue by column chromatogra-
phy (EtOAc/n-hexane =1:6) afforded 36 (316 mg, 99 %) as
a colorless oil. 1H-NMR (CDCl3, 300 MHz) d 7.32–7.37 (m,
2H), 7.07–7.11 (m, 2H), 4.10 (d, 2H, J = 5.9 Hz), 2.12 (d,
3H, J = 4.4 Hz), 1.30 (s, 9H), 0.97 (t, 3H, J = 7.1 Hz).
3-Bromo-5-(4-tert-butylphenyl)-4,5-dihydroisozazole (32)
To a solution of acid 29 (2.0 g, 22 mmol) in 75 % aq.
DME (32 mL) was added N-bromosuccinimide (7.2 g,
40 mmol) at 0 ꢁC. The reaction mixture was stirred for 3 h,
and 1-tert-butyl-4-vinylbenzene 31 (7.2 g, 45 mmol),
KHCO3 (9.0 g, 22 mmol) and DME (25 mL) were added.
After confirming the completion of the reaction with TLC,
the reaction mixture was concentrated in vacuo and diluted
with EtOAc and water. The organic layer was washed with
brine, dried over MgSO4 and concentrated in vacuo.
Purification of the residue by column chromatography
(EtOAc/n-hexane =1:30) afforded 32 (3.2 g, 50 % for 2
steps). 1H-NMR (CDCl3, 300 MHz) d 7.39 (d, 2H,
J = 8.4 Hz), 7.26 (d, 2H, J = 8.4 Hz), 5.63 (dd, 1H,
J = 9.2, 10.6 Hz), 3.56 (dd, 1H, J = 10.6, 17.2 Hz), 3.21
(dd, 1H, J = 9.2, 17.2 Hz), 1.30 (s, 9H).
(Z)-3-(4-tert-Butylphenyl)-2-fluoro-N-[3-fluoro-4-
(methylsulfonamido)benzyl]but-2-enamide (37)
To a solution of ester 36 (316 mg, 1.2 mmol) in 50 %
aq.THF (6 mL) was added LiOH•H2O (201 mg,
4.8 mmol). After being stirred for 1 h at ambient temper-
ature for 1 h, the reaction mixture was acidified with
1N HCl, and extracted with EtOAc. The combined organic
layer were washed with water and brine, dried over
MgSO4, and concentrated in vacuo. Crude acid interme-
diate (256 mg, 91 %) was obtained as a white solid, and
used for next step without further purification. To a solu-
tion of the acid intermediate (100 mg, 0.42 mmol) in THF
(6 mL) were added DMTMM (4-(4,6-dimethoxy-1,3,5-
triazin-2-yl)-4-methylmorpholinium chloride) (173 mg,
0.63 mmol), NMM (185 lL, 0.42 mmol) and Et3N
(140 lL, 1.26 mmol). After being stirred for 4 h, the
reaction mixture was added N-[4-(aminomethyl)-2-fluoro-
phenyl]methane sulfonamide ammonium salt 9 (128 mg,
0.50 mmol). After additional 10 h, the reaction mixture
was diluted with CH2Cl2, washed with water and brine,
dried over MgSO4, and concentrated in vacuo. Purification
of the residue by column chromatography (EtOAc/n-hex-
ane =2:1) afforded 37 as a white solid (146 mg, 79 %). 1H-
NMR (CDCl3, 300 MHz) d 7.45 (t, 1H, J = 9.3 Hz), 7.38
(d, 2H, J = 8.4 Hz), 7.20 (d, 2H, J = 8.4 Hz), 6.91–7.00
(m, 1H), 6.77 (d, 1H, J = 1.8 Hz), 6.41 (br s, 1H), 4.37 (d,
2H, J = 5.9 Hz), 2.99 (s, 3H), 2.13 (d, 3H, J = 4.6 Hz),
1.33 (s, 9H); IR (neat) cm-1: 2962, 1651, 1516, 1334,
1274, 1158, 1111, 974, 758; LRMS (EI?): m/z 436 (M).
N-(4-{[5-(4-tert-Butylphenyl)-4,5-dihydroisozazol-3-
ylamino]methyl}-2-fluorophenyl)methanesulfonamide (33)
To
a
solution of bromoisoxazole 32 (150 mg,
0.53 mmol) in toluene (5 mL) were added tert-BuONa
(120 mg, 1.3 mmol), Pd2(dba)3 (10 mg, 0.01 mmol),
P(i-BuNCH2CH2)3N (7 lL, 0.02 mmol) and ammonium salt
9 (160 mg, 0.64 mmol). The reaction mixture was refluxed
overnight. After being cooled to ambient temperature, the
mixture was concentrated in vacuo and diluted with EtOAc
and water. The organic layer was washed with brine and
dried over MgSO4. Purification of the residue by column
chromatography (EtOAc/n-hexane =1:1) afforded 33 as a
solid (22 mg, 10 %). 1H-NMR (CDCl3, 300 MHz) d 7.44 (t,
1H, J = 8.5 Hz), 7.32 (d, 2H, J = 8.4 Hz), 7.25 (d, 2H,
J = 8.6 Hz), 7.04–7.12 (m, 2H), 6.55 (s, 1H), 5.43 (t, 1H,
J = 18.0 Hz), 4.29 (d, 2H, J = 5.3 Hz), 4.08 (br s, 1H), 3.24
(dd, 1H, J = 9.5, 15.0 Hz), 2.95 (dd, 1H, J = 8.8, 15.0 Hz),
2.95 (s, 3H), 1.24 (s, 9H); IR (neat) cm-1: 3363, 2959, 1620,
1513, 1330; LRMS (FAB?): m/z 420 (M?H?).
(4-tert-Butylphenyl)(phenyl)methanone (38)
To a solution of phenylmagnesium chloride (3 M in ether,
1 mL, 3.0 mmol) in THF (15 mL) was added 4-tert-
butylbenzaldehyde 35 (500 lL, 3.0 mmol) at -78 ꢁC.
After being stirred for 2 h at same temperature, the reaction
mixture was quenched with sat. NH4Cl, and diluted with
Et2O. The organic layer was washed with water and brine,
dried over MgSO4, and concentrated in vacuo. Purification
of the residue by silica gel column chromategraphy
(EtOAc/n-hexane =1:5) afforded (4-tert-butylphenyl)
phenyl methanol (275 mg, 38 %) as a white solid. 1H-NMR
(CDCl3, 400 MHz) d 7.44 (d, 2H, J = 7.3 Hz), 7.37–7.39
(m, 4H), 7.35 (d, 2H, J = 3.7 Hz), 7.30 (m, 1H), 5.86 (s, 1H),
1.32 (s, 9H).
(Z)-Ethyl 3-(4-tert-butylphenyl)-2-fluorobut-2-enoate (36)
To a solution of triethyl 2-fluoro-2-phosphonoacetate
(218 lL, 1.2 mmol) in THF (10 mL) was added NaH (60 %
dispersion in mineral oil 72 mg, 1.8 mmol) at 0 ꢁC. After
30 min, 1-(4-tert-butylphenyl)ethanone 34 (218 lL, 1.2
mmol) was added to the reaction mixture. After additional
12 h, the reaction mixture was acidified by 2N HCl and
extracted with Et2O. The organic layer was washed with
123

Y. T. Han et al.
To a solution of the (4-tert-butylphenyl)phenyl metha-
stirred for 3 h at -78 ꢁC. The reaction mixture was
quenched by saturated sodium potassium tartrate aqueous
solution, and diluted with EtOAc. The organic layer was
washed with brine, dried over MgSO4, and concentrated in
vacuo. Purification of the residue by column chromatog-
raphy (EtOAc/n-hexane =1:2) afforded (E)-3-[4-(tert-
butyl)phenyl]-2-propen-1-ol (72 mg, 88 %). 1H-NMR
(CD3OD, 300 MHz) d 7.24–7.38 (m, 4H), 6.55 (m, 1H),
6.30 (m, 1H), 4.21 (dd, 2H, J = 5.7, 1.5 Hz), 1.30 (s, 9H).
To a solution of the (E)-3-[4-(tert-butyl)phenyl]-2-pro-
pen-1-ol (34 mg, 0.18 mmol) in CH2Cl2 (3 mL) was added
diethyl zinc (1.0 M soln in n-hexane, 0.54 mL, 0.54 mmol)
at 0 ꢁC. After being stirred for 5 min, diiodomethane
(70 lL, 0.89 mmol) added dropwise to the reaction mix-
ture over 10 min. After additional 4 h, the reaction mixture
was quenched by aq. NH4Cl and diluted with EtOAc. The
organic layer was washed with brine and dried over
MgSO4, and concentrated in vacuo. Purification of the
residue by column chromatography (EtOAc/n-hexane
=1:2) afforded 42 (18 mg, 50 %). 1H-NMR (CDCl3,
300 MHz) d 7.30 (d, 2H, J = 8.4 Hz), 7.02 (d, 2H,
J = 8.4 Hz), 3.53–3.65 (m, 2H), 1.81 (m, 1H), 1.51 (m,
1H), 1.28 (s, 9H).
nol (273 mg, 1.1 mmol) in CH2Cl2 (10 mL) were added
˚
PDC (855 mg, 2.3 mmol) and 4 A molecular sieve. After
being stirred for 12 h at ambient temperature, the reaction
mixture was diluted with Et2O and water. The organic layer
was washed with brine, dried over MgSO4, and concen-
trated in vacuo. Purification of the residue by column
chromatography (EtOAc/n-hexane =1:5) afforded 38 as a
solid (265 mg, 26 %). 1H-NMR (CDCl3, 300 MHz) d
7.72–7.80 (m, 5H), 7.43–7.60 (m, 4H), 1.35 (s, 9H).
(E)-Ethyl 3-(4-tert-butylphenyl)-3-phenylacrylate (39)
Titled compound 39 (54 %) was prepared from 38 by the
synthetic procedure of 36 as a colorless oil. 1H-NMR
(CDCl3, 300 MHz) d 7.29–7.39 (m, 5H), 7.12–7.25 (m,
4H), 6.36 (d, 1H, J = 18.9 Hz), 3.99–4.08 (m, 2H), 1.29 (s,
9H), 1.09 (t, 3H, J = 6.2 Hz).
(E)-3-(4-tert-Butylphenyl)-N-[3-fluoro-4-
(methylsulfonamido)benzyl]-3-phenylacrylamide (40)
Titled compound 40 (52 % for 2 steps) was prepared from
1
39 by the synthetic procedure of 37 as a white solid. H-
NMR (CDCl3, 300 MHz) d 7.35–7.44 (m, 5H), 7.25–7.32
(m, 3H), 7.15–7.18 (m, 2H), 6.71–6.82 (m, 2H), 6.52 (s,
1H), 6.42 (d, 1H, J = 13.4 Hz), 5.46 (br s, 1H), 4.22 (q,
2H, J = 3.1 Hz), 2.98 (s, 3H), 1.28 (s, 9H); IR (neat)
cm-1: 2961, 1646, 1594, 1515, 1366, 1335, 1158, 972, 758;
LRMS (FAB?): m/z 481 (M?H?).
2-[4-(tert-Butyl)phenyl]-N-3-fluoro-4-
[(methylsulfonyl)amino]benzylcyclopanecarboxamide (43)
To a solution of alcohol 42 (100 mg, 0.49 mmol) in DMF
˚
(3 mL) were added PDC (220 mg, 0.59 mmol) and 4 A
molecular sieve. After being stirred for 12 h at ambient
temperature, the reaction mixture was diluted with Et2O
and water. The organic layer was washed with brine, dried
over MgSO4, and concentrated in vacuo. Purification of the
residue by column chromatography (EtOAc/n-hexane
=1:10) afforded 2-[4-(tert-butyl)phenyl]cyclopropanecar-
Ethyl (E)-3-[4-(tert-butylphenyl)]-2-propenoate (41)
To a solution of 4-tert-butylbenzaldehyde 35 (1.0 g,
6.2 mmol) in CH3CN (15 mL) were added triethyl phos-
phonoacetate (1.5 mL, 7.4 mmol), diisopropyl ethylamine
(1.6 mL, 9.2 mmol) and LiCl (520 mg, 12 mmol). The
reaction mixture was stirred at ambient temperature for
overnight. The reaction mixture was concentrated in vacuo,
and diluted with EtOAc and water. The organic layer was
washed with brine, dried over MgSO4, and concentrated in
vacuo. Purification of the residue by column chromatog-
raphy (EtOAc/n-hexane =1:1) afforded 41 as a solid
1
boxylic acid (77 mg, 72 %). H-NMR (CDCl3, 300 MHz)
d 9.30 (d, 1H, J = 4.8 Hz), 7.33 (d, 2H, J = 8.4 Hz), 7.06
(d, 2H, J = 8.4 Hz), 2.58 (m, 1H), 1.50 (m, 1H), 1.28 (s,
9H).
To a solution of the 2-[4-(tert-butyl)phenyl]cyclopro-
panecarboxylic acid (70 mg, 0.32 mmol) in CH2Cl2
(5 mL) were added two drops of DMF and oxalyl chloride
(56 lL, 0.64 mmol) at 0 ꢁC. In a seperate flask, Et3N
(140 lL, 1.0 mmol) was added to a solution of ammonium
salt 9 (98 mg, 0.38 mmol) in CH2Cl2 (5 mL) at room
temperature, and the reaction mixture was stirred for
10 min. To the solution of ammonium salt was added the
solution of acid chloride at ambient temperature. After
being stirred for 2 h at same temperature, the reaction
mixture was diluted with CH2Cl2 and water. The organic
layer was washed with brine, dried over MgSO4, and
concentrated in vacuo. Purification of the residue by col-
umn chromatography (EtOAc/n-hexane =1:1) afforded 43
1
(940 mg, 66 %). H-NMR (CDCl3, 300 MHz) d 7.68 (d,
1H, J = 16.0 Hz), 7.47 (d, 2H, J = 8.4 Hz), 7.40 (d, 2H,
J = 8.6 Hz), 6.41 (d, 1H, J = 16.0 Hz), 4.28 (q, 2H,
J = 7.1 Hz), 1.34 (t, 3H, J = 7.1 Hz), 1.31(s, 9H).
2-[4-(tert-Butyl)phenyl]cyclopropylmethanol (42)
To a solution of ester 41 (100 mg, 0.43 mmol) in CH2Cl2
(3 mL) was added DIBAL-H (1 M in toluene, 1.3 mL,
1.3 mmol) dropwise at -78 ꢁC. The reaction mixture was
123

Design, synthesis and biological evaluation of B-region
(72 mg, 54 %) as a white solid. 1H-NMR (CD3OD,
300 MHz) d 7.46 (t, 1H, J = 8.3 Hz), 7.30 (d, 2H,
J = 8.4 Hz), 7.09–7.14 (m, 2H), 7.05 (d, 2H, J = 8.4 Hz),
4.44 (d, 1H, J = 15.0 Hz), 4.36 (d, 1H, J = 15.0 Hz), 2.37
(m, 1H), 1.86 (m, 1H), 1.47 (m, 1H), 1.28 (s, 9H); IR (neat)
cm-1: 2960, 1646, 1514, 1455, 1333, 1157; LRMS
(FAB?): m/z 419 (M?H?).
aqueous layer was acidified by c-HCl, and the extracted
with Et2O. The organic layer was washed with water and
brine, dried over MgSO4, and concentrated in vacuo to give
63 mg (20 %) of the crude 2-(4-tert-butylphenylami-
no)acetic acid 49, which was used for the next step without
further purification. 1H-NMR (CDCl3, 300 MHz)
d
7.23–7.28 (m, 2H), 6.70 (d, 2H, J = 8.3 Hz), 4.06 (s, 2H),
1.26 (s, 9H).
3-[4-(tert-Butyl)phenyl]-N-3-fluoro-4-
[(methylsulfonyl)amino]benzyl-2-oxiranecarboxamide (45)
2-(4-tert-Butylphenylamino)-N-[3-fluoro-4-
(methylsulfonamido)benzyl]acetamide (50)
A solution of m-CPBA (68 mg, 0.39 mmol) in CH2Cl2
(5 mL) was added to a solution of amide 44 (50 mg,
0.12 mmol) in CH2Cl2 (3 mL) at 0 ꢁC. The reaction mix-
ture was stirred at room temperature overnight, and washed
with 10 % aq. Na2SO3 (100 mL), 5 % aq. NaHCO3, water,
and brine. The organic layer was dried over MgSO4, and
concentrated in vacuo. Purification of the residue by col-
umn chromatography (EtOAc/n-hexane =1:1) afforded
epoxide 45 (32 mg, 58 %). 1H-NMR (CDCl3, 300 MHz) d
7.59 (t, 1H, J = 8.3 Hz), 7.42 (d, 2H, J = 8.4 Hz), 7.24 (d,
2H, J = 8.4 Hz), 7.10–7.15 (m, 2H), 6.66 (br s, 1H), 6.64
(br s, 1H), 4.37–4.55 (m, 2H), 3.91 (d, 1H, J = 2.0 Hz),
3.62 (d, 1H, J = 2.0 Hz), 3.05 (s, 3H), 1.33 (s, 9H); IR
(neat) cm-1: 2962, 1666, 1515, 1453, 1332, 1157; LRMS
(FAB?): m/z 443 (M?Na?).
Titled compound 50 (24 %) was prepared from 49 by the
synthetic procedure of 37 as a white solid. 1H-NMR
(CDCl3, 300 MHz) d 8.27 (dd, 1H, J = 8.8, 9.0 Hz), 8.11
(dd, 2H, J = 8.8, 9.0 Hz), 6.98–7.06 (m, 2H), 6.63 (br s,
1H), 6.48 (br s, 1H), 4.51 (d, 2H, J = 5.1 Hz), 2.98 (s, 3H),
2.10 (d, 2H, J = 4.2 Hz), 1.29 (s, 9H); IR (neat) cm-1
:
3424, 2956, 1659, 1520, 1326, 1156, 984, 575; LRMS
(FAB?): m/z 408 (M?H?).
Ethyl 2-[4-(tert-butyl)phenoxy]-2,2-difluoroacetate (51)
To
a solution of 4-tert-buthylphenol 48 (100 mg,
0.67 mmol) in DMF (3 mL) were added K2CO3 (138 mg,
1.0 mmol), KI (22 mg, 0.13 mmol) and ethyl bromodi-
fluoroacetate (129 lL, 1.0 mmol). The reaction mixture
was stirred at 110 ꢁC for 24 h. After being acidified by
2N HCl at ambient temperature, the reaction mixture was
diluted with EtOAc and water. The organic layer was
washed with brine, dried over MgSO4, and concentrated in
vacuo. Purification of the residue by column chromatog-
raphy (EtOAc/n-hexane =1:5) afforded 51 (46 mg, 25 %)
as a yellow oil. 1H NMR (CDCl3, 300 MHz) d 7.35 (d, 2H,
J = 9.0 Hz), 7.12 (d, 2H, J = 9.0 Hz), 4.36 (q, 2H,
J = 7.1 Hz), 1.34 (t, 3H, J = 7.1 Hz), 1.29 (s, 9H).
3-[4-(tert-Butyl)phenyl]-N-3-fluoro-4-
[(methylsulfonyl)amino]benzyl-2-hydroxypropanamide
(46)
To a solution of epoxide 45 (30 mg, 0.07 mmol) in MeOH
(5 mL) was added catalytic amount of 10 % palladium on
activated carbon, and the reaction mixture was stirred for
10 min under H2 atmosphere at ambient temperature. The
reaction mixture was diluted with Et2O, filtered through
Celite pad, and concentrated in vacuo to give 46 (30 mg,
98 %). 1H-NMR (CDCl3, 300 MHz) d 7.51 (t, 1H,
J = 8.3 Hz), 7.35 (d, 2H, J = 8.2 Hz), 7.17 (d, 2H,
J = 8.2 Hz), 7.00–7.04 (m, 2H), 6.90 (br s, 1H), 4.33–4.48
(m, 3H), 3.27 (dd, 1H, J = 14.0, 3.9 Hz), 2.99 (s, 3H), 2.92
2-(4-tert-Butylphenoxy)-2,2-difluoroacetic acid (52)
Titled compound 52 (95 %) was prepared from 51 by the
synthetic procedure of 37 as a white solid. 1H NMR
(300 MHz, CDCl3) d 7.37 (d, 2H, J = 8.8 Hz), 7.14 (d,
2H, J = 8.8 Hz), 1.30 (s, 9H).
(dd, 1H, J = 14.0, 8.4 Hz), 1.29 (s, 9H); IR (neat) cm-1
:
3371, 2961, 1652, 1514, 1451, 1331; LRMS (FAB?): m/z
423 (M?H?).
2-(4-tert-Butylphenoxy)-2,2-difluoro-N-[3-fluoro-4-
2-(4-tert-Butylphenylamino)acetic acid (49)
(methylsulfonamido)benzyl]acetamide (53)
To a solution of 2-bromoacetic acid (215 mg, 1.5 mmol) in
MeOH (5 mL) were added KOH (191 mg, 3.4 mmol) and
4-tert-butylaniline 47 (317 lL, 2.0 mmol). The reaction
mixture was refluxed for 2 h. After being cooled to ambient
temperature, the reaction mixture was added 5 % aq.
NaOH and washed with n-hexane (93). The combined
Titled compound 53 (39 %) was prepared from 52 by the
synthetic procedure of 37 as a white solid. 1H-NMR
(CDCl3, 300 MHz) d 7.53 (t, 1H, J = 8.4 Hz), 7.35 (d, 2H,
J = 8.8 Hz), 7.06–7.12 (m, 4H), 6.87 (br s, 1H), 6.55 (br s,
1H), 4.50 (d, 2H, J = 6.1 Hz), 3.01 (s, 3H), 1.29 (s, 9H);
13C-NMR (CDCl3, 125 MHz): d 160.0, 155.0, 153.0,
123

Y. T. Han et al.
149.5, 146.8, 135.7, 135.6, 126.5, 124.3, 123.6, 121.2,
115.4, 115.2, 114.6, 42.7, 39.9, 34.5, 31.3; LRMS
(FAB?): m/z 445 (M?H?); HRMS (FAB?) calcd for
C20H24F3N2O4S (M ? H?): 445.1409, found 445.1412.
compound was added to each well. The neurons were
incubated at room temperature for 10 min and the Terasaki
plates were washed six times in HEPES (10 mM, pH 7.4)-
buffered calcium and magnesium-free Hank’s balanced salt
solution and dried in an oven. Sodium dodecyl sulfate
(0.3 %, 10 lL) was then added to dissolve the cells and
extract the 45Ca2?. The contents of each well were trans-
ferred to scintillation vials and counted in 3 mL of Aqua-
sol-2 scintillant. Antagonistic activities of the test
compounds were given as IC50 (the concentration of the
compound necessary to reduce the response to 0.5 lM
capsaicin by 50 %). IC50 values were estimated at least
three replicates at each concentration. Each compound was
tested at least in two independent experiments.
In vitro assay
Culture of DRG neurons
DRG neurons were prepared from neonatal Sprague–
Dawley rats. DRGs of all spinal levels were dissected
aseptically and collected. Ganglia were incubated sequen-
tially for 30 min at 37 ꢁC in 200 U/mL collagenase and
2.5 mg/mL trypsin. The digestion was halted by an addi-
tion of an equal volume of DME/F12 medium supple-
mented with 10 % horse serum. The ganglia were then
triturated through a fire-polished Pasteur pipette, filtered
through nylon membrane and spun down. Dissociated cells
were plated onto Terasaki plates previously coated with
10 lg/mL of poly-D-ornithine at a density of 1500–1700
neurons/well. The cells were then cultured for 3 days in
DME/F12 medium containing 1.2 g/L sodium bicarbonate,
15 mM HEPES, 50 mg/L gentamycin and 10 % horse
serum diluted 1:1 with identical medium conditioned by C6
glioma cells (2 days on a confluent monolayer) under a
humidified atmosphere at 37 ꢁC containing 5 % CO2.
Medium was supplemented with 200 ng/mL nerve growth
factor. Cytosine arabinoside (100 lM) was added for the
first 2 days to kill dividing nonneuronal cells.
Results and discussion
Chemistry
At first, guanidine (11) and hydroxyguanidine (12) ana-
logues were prepared as shown in Scheme 1. Nucleophilic
addition of benzylamine 7 to ethyl isothiocyanate afforded
thiourea 8, which was subject to substitution reaction with
benzylammonium salt 9 (Li et al. 2009) in the presence of
EDCl to afford carbamate 10, and then it was converted
into desired guanidine analogue 11 by deprotection with
Me3SiBr (Manimala and Anslyn 2002). Hydroxyguanidine
analogue 12 was prepared from the thiourea analogue 5
(Suh et al. 2003) according to the reported procedure (Li
et al. 2002).
Uptake assay
We extended our investigation to synthesis of the ana-
logues having 6-membered heterocyclic amines in
B-region. The synthetic routes for the pyrimidine ana-
logues 17, 18 and 21, and a pyrazine analogue 24 were
outlined in Scheme 2. Aromatic substitution reaction of
benzylammonium salt 9 with commercially available 2,4-
dichloropyrimidine 13 afforded separable regioisomers
14 and 15 which were converted to the desired 2,4-
Terasaki plates containing DRG neurons grown for 3 days
were equilibrated with four washes of HEPES (10 mM,
pH 7.4)-buffered calcium and magnesium-free Hank’s
balanced salt solution. The solution in each well was
removed from the individual wells. For antagonistic stud-
ies, medium (10 lL) containing 10 lCi/mL 45Ca2? and
0.5 M capsaicin together with the test concentration of the
Scheme 1 Reagents and conditions: (a) ethoxycarbonyl isothiocyanate, CH2Cl2, 91 %; (b) 9, EDCI, Et3N, DMF, 43 %; (c) TMSBr, DMF,
reflux, then MeOH, 10 %; (d) HgO, NH2OH, Et3N, DMF, 20 %
123

Design, synthesis and biological evaluation of B-region
Scheme 2 Reagents and conditions: (a) 60 % NaH, THF, rt or reflux, 41 % for 14, 14 % for 15, 39 % for 20 and 38 % for 23; (b) potassium
4-tert-butylphenyltrifluoroborate (16), Pd(OAc)2, Na2CO3, CH3CN, reflux, 48 % for 17, 54 % for 18, 35 % for 21 and 24 % for 24
disubstituted pyrimidine analogues 17 and 18 by Suzuki
coupling with potassium 4-tert-butylphenyltrifluoroborate
16, respectively (Molander and Biolatto 2003). Using a
similar method, 4,6-disubstituted pyrimidine analogue 21
and 2,6-disubstituted pyrazine analogue 24 were synthe-
sized from commercially available 4,6-dichloropyrimidine
19 and 2,6-dichloropyrazine 22, respectively.
with [2 ? 3] cycloaddition of alkene 31 to dib-
romoformaldoxime 30 prepared from acetic acid 29.
Resulting isoxazoline 32 was transformed into desired
analogue 33 by subsequent Buchwald-Hartwig amination
with benzylammonium salt 9 in the presence of P(i-
BuNCH2CH2)3N and 0.5 mol% of Pd2(dba)3 (Urgaonkar
and Verkade 2004).
Five-membered heterocyclic amine analogues such as
triazole 28 and isoxazoline 33 were also prepared as shown
in Scheme 3. Treatment of 4-tert-butylbenzoyl chloride 25
with sodium thiocyanate afforded 26, which was converted
to S-methyl isothiourea 27 in good yields by addition of
the benzyl ammonium salt 9, followed by methylation.
Subsequent cyclization of the S-methyl isothiourea 27
using hydrazine gave the triazole analogue 28 (Lowe et al.
2005). Synthesis of isoxazoline analogue 33 commenced
In addition, a, b-substituted 4-tert-butylcinnamide ana-
logues (37 and 40), and cyclopropane analogue (43) in
which rigid linkers were introduced as B-region element
were prepared as shown in Scheme 4. Horner-Emmons
olefination reaction of acetophenone 34, followed by sub-
sequent hydrolysis and amide coupling (Kunishima et al.
1999) readily afforded the cinnamide 37. The cinnamide 40
was also synthesized by similar method from benzophe-
none 38 which was prepared by Grignard addition of
Scheme 3 Reagents and conditions: (a) NaNCS, THF, reflux; (b)
9, Et3N, THF, 66 % for
(f) 31, KHCO3, DME, 50 % for 2 steps; (g) 9, 0.5 % mol Pd2(dba)3,
NaO-t-Bu, P(i-BuNCH2CH2)3N, toluene, reflux, 10 %
2 steps; (c) MeI, DMF, 75 %;
(d) NH2NH2•H2O, EtOH, reflux, 37 %; (e) NBS, DME:H2O (3:1);
123

Y. T. Han et al.
Scheme 4 Reagents and conditions: (a) 60 % NaH, THF, triethyl
2-fluoro-2-phosphonoacetate, 0 ꢁC to rt, 99 % (from 34);
(b) LiOH•H2O, THF:H2O = 1:1, rt, 67–91 %; (c) 9, DMTMM,
NMM, Et3N, THF, 24–79 %; (d) PhMgCl, THF, -78 ꢁC, 38 % (from
phosphonoacetate, 0 ꢁC to rt, 54 %; (g) triethyl phosphonoacetate,
i-Pr2NEt, CH3CN, LiCl, 66 % (from 35); (h) DIBAL-H, CH2Cl2,
-78 ꢁC, 88 %; (i) Et2Zn, CH2I2, CH2Cl2, 50 %; (j) PDC, DMF, 0 ꢁC,
72 %; (k) (COCl)2, DMF, CH2Cl2, then 9, Et3N, CH2Cl2, 54 %
˚
35); (e) PDC, CH2Cl2, 4 A MS, 26 %; (f) 60 % NaH, THF, triethyl
PhMgCl to aldehyde 35, followed by oxidation reaction.
Horner–Emmons reaction of the benzaldehyde 35 gave
cinnamate 41, which was readily converted to alcohol 42
via subsequent reduction and cyclopropanation. Compound
42 was oxidized to carboxylic acid, which was coupled
with compound 9 to provide desired cyclopropane ana-
logue 43 using acyl halide formation, followed by amida-
tion reaction.
the amide were described in Scheme 5. Treatment of cin-
namide 44 (Li et al. 2009) with 3-chloroperoxybenzoic acid
(m-CPBA) afforded epoxide 45, subject to palladium-
catalyzed hydrogenolysis to afford the a-hydroxyamide
analogue 46. The b-aminoacetamide analogue 50 was
synthesized from aniline 47 by N-alkylation, and
DMTMM-mediated coupling with the benzyl ammonium
salt 9. Finally, difluoroamide analogue 53 was synthesized
from acid 52 which was prepared by O-alkylation of phe-
nol 48, followed by hydrolysis.
Synthetic procedures for amide analogues possessing
polar moiety such as alcohol and difluorine at a-position of
Scheme 5 Reagents and conditions: (a) m-CPBA, NaHCO3, CH2Cl2,
58 %; (b) 10 % Pd/C, H2, MeOH, 98 %; (c) 2-bromoacetic acid,
MeOH, reflux, 20 % (from 47); (d) 9, DMTMM, NMM, Et3N, THF,
50: 24 %, 53: 39 %; (e) ethyl bromodifluoroacetate, K2CO3, KI,
DMF, 110 ꢁC, 25 % (from 48); (f) LiOH•H2O, THF:H2O = 1:1, rt,
95 %
123

Design, synthesis and biological evaluation of B-region
Table 1 45Ca2? Uptake inhibition by the synthesis of compounds
Table 1 continued
Compounds
B-region
IC50 (lM)
50
53
1.8
0.058
Compounds
B-region
IC50 (lM)
6
0.17
11
12
18.2
0.84
In vitro activity
The TRPV1 antagonistic activities of the synthesized ana-
logues were evaluated by in vitro 45Ca2? uptake assay in rat
DRG neuron, and results are summarized in Table 1. We
initially investigated the analogues possessing guanidine
(11) and hydroxyguanidine (12) as the isostere of amide in
B-region. And disappointingly, these analogues exhibited
less potent antagonistic activities than the parent amide 6, to
a greater or lesser extent. Next, we introduced several het-
erocycles into the B-region considering heterocyclic core
structures of TRPV1 antagonists for clinical trial (Gunthrope
and Chizh 2009) such as AMG 517 (4). The series of het-
erocyclic core analogues including not only 6-membered
ring (pyrimidines 17, 18 and 21, and pyrazine 24) but also
5-membered ring (triazole 28 and isoxazoline 33) exhibited
significant decrease in TRPV1 antagonistic activities. These
results would support that the heterocyclic amines in
B-region were not beneficial as much as amide or (thio)urea
moieties to TRPV1 antagonist scaffolds we have reported.
Next, we turned our attention to introduction of constraint to
dipolar amide moiety in B-region. Considering beneficial
effect of conformational restriction for the TPRV1 antago-
nistic activity (Kim et al. 2013), less flexible amide ana-
logues such as cinnamides (37 and 40) and cyclopropane
(43) were estimated to have potent TRPV1 antagonistic
activities. These analogues exhibited potent TRPV1 antag-
onistic activity as much as the parent amide 6. Interestingly,
epoxide 45 (IC50 = 0.14 lM) exhibited higher activity than
the cyclopropane (IC50 = 0.33 lM of cinnamides 40).
Finally, we designed and synthesized analogues equipped
with additional moieties on a-position of dipolar amide
moiety in B-region. Alcohol 46 showed equipotent activity
to the parent amide 6. Especially, difluoroamide 53 was
identified to exhibit 3-fold more potent TRPV1 antagonistic
activity (IC50 = 0.058 lM) than the parent amide analogue
6 (IC50 = 0.17 lM). This result indicated that additional
difluorine was estimated to make compound 53 have more
17
18
21
24
[10.0
9.3
5.4
9.5
28
33
37
8.9
[10.0
3.0
40
43
45
46
0.24
0.33
0.14
1.6
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Y. T. Han et al.
oxide synthase provides mechanistic insights into NO biosyn-
thesis. Biochemistry 41: 13868–13875.
increased dipolar interaction than the corresponding parent
amide 6. In addition, it has been known that fluorine sub-
stituent sometimes makes the parent compound have a
suitable pharmacokinetic stability enough to become a drug
candidate. (Banks et al. 1994) Considering its potent in vitro
activity as well as estimated stability, compound 53 might
be developed as a TRPV1 antagonist for further progress.
In summary, we designed and synthesized B-region
modified diarylalkyl amide analogues as novel TRPV1
antagonists based on the structure of amide 6, and identi-
fied potent difluoroamide 53. This analogue possessing
difluorine moiety estimated to make the parent compound
physiochemically stable, exhibited approximately 3-fold
increased TRPV1 antagonistic activity (IC50 = 0.058 lM)
than the parent amide 6 (IC50 = 0.17 lM). Further studies
on the identified TRPV1 antagonist is in progress well.
Li, F.N., N.J. Kim, S.M. Paek, D.Y. Kwon, K.H. Min, Y.S. Jeong,
S.Y. Kim, Y.H. Park, H.D. Kim, H.G. Park, and Y.G. Suh. 2009.
Design, synthesis, and biological evaluation of novel diarylalkyl
amides as TRPV1 antagonists. Bioorganic and Medicinal
Chemistry 17: 3557–3567.
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Suh, Y.G., Y.S. Lee, K.H. Min, O.H. Park, H.S. Seung, H.D. Kim,
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Suh, Y.G., Y.S. Lee, K.H. Min, O.H. Park, J.K. Kim, H.S. Seung,
S.Y. Seo, B.Y. Lee, Y.H. Nam, K.O. Lee, H.D. Kim, H.G. Park,
J. Lee, U. Oh, J.O. Lim, S.U. Kang, M.J. Kil, J. Koo, S.S. Shin,
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Novel potent antagonists of transient receptor potential channel,
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1,3-diarylalkyl thioureas possessing new vanilloid equivalents.
Journal of Medicinal Chemistry 48: 5823–5836.
Acknowledgments This research work was supported by grant
from the Basic Research Program of the Korea Science, and sup-
ported in part by the grant from the Natural Science Foundation of
Fujian Province of China (Grant No. 2011J01257).
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