Biarylcarboxybenzamide derivatives as potent vanilloid receptor (VR1) antagonistic ligands

Article abstract of DOI:10.1016/j.bmcl.2004.11.033

Seventeen biarylcarboxybenzamide derivatives were prepared for the study of their agonistic/antagonistic activities to the vanilloid receptor (VR1) in rat DRG neurons. The replacement of the piperazine moiety of the lead compound 1 with phenyl ring showed quite enhanced antagonistic activity. Among the prepared derivatives, N-(4-tert-butylphenyl)-4-pyridine-2-yl-benzamide (2, IC ₅₀ = 31 nM) and N-(4-tert-butylphenyl)-4-(3-methylpyridine-2-yl) benzamide (3g, IC₅₀ = 31 nM), showed 5-fold higher antagonistic activity than 1 in ⁴⁵Ca²⁺-influx assay.

Full text of DOI:10.1016/j.bmcl.2004.11.033

Bioorganic & Medicinal Chemistry Letters 15 (2005) 631–634
Biarylcarboxybenzamide derivatives as potent vanilloid
receptor (VR1) antagonistic ligands
Hyeung-geun Park,a,* Ji-yeon Choi,a Mi-hyun Kim,a Sea-hoon Choi,a
Mi-kyung Park,a Jihye Lee,a Young-Ger Suh,a Hawon Cho,a Uhtaek Oh,a
Hee-Doo Kim,b Yung Hyup Joo,c Song Seok Shin,c Jin Kwan Kim,c
Yeon Su Jeong,c Hyun-Ju Koh,c Young-Ho Parkc and Sang-sup Jewa,*
aResearch Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
bCollege of Pharmacy, Sookmyung Womenꢀs University, Seoul 140-742, South Korea
cAmorePacific R & D Center, Youngin-Si, Kyounggi-do 449-900, South Korea
Received 21 October 2004; revised 13 November 2004; accepted 15 November 2004
Available online 7 December 2004
Abstract—Seventeen biarylcarboxybenzamide derivatives were prepared for the study of their agonistic/antagonistic activities to the
vanilloid receptor (VR1) in rat DRG neurons. The replacement of the piperazine moiety of the lead compound 1 with phenyl ring
showed quite enhanced antagonistic activity. Among the prepared derivatives, N-(4-tert-butylphenyl)-4-pyridine-2-yl-benzamide
(2, IC50 = 31 nM) and N-(4-tert-butylphenyl)-4-(3-methylpyridine-2-yl)benzamide (3g, IC50 = 31 nM), showed 5-fold higher
antagonistic activity than 1 in 45Ca2+-influx assay.
Ó 2004 Elsevier Ltd. All rights reserved.
Vanilloid receptor (VR1) is a nonselective cation chan-
O
O
nel placed in the plasma membrane of peripheral sen-
sory neurons,1,2 which has been regarded as a new
target for the treatment of pain.3 The agonists desensi-
tize the peripheral sensory neurons by influx of cations,
especially Ca2+, into neuronal cell, which leads to an
analgesic effect.4 However, their initial excitatory side
effects, such as initial irritation, hypothermia, broncho-
constriction, and hypertension, derived from its inherent
mechanism, make it hard to develop them as systemic
analgesics.5 In order to avoid the side effects from ago-
nist, competitive antagonists have been pursued as novel
analgesic drugs. So far, several synthetic and semi-syn-
thetic antagonists were introduced and their pharmaco-
logical potential for pain treatment were evaluated.6,7
Recently Purdue Pharma research group disclosed
4-(2-pyridyl)piperazine-1-carboxybenzamides (1) as po-
tent VR1 antagonists (Fig. 1).8 In this letter, we report
the synthesis and functional assay on VR1 receptor of
N
N
H
N
H
N
N
N
1
2
Figure 1.
biarylcarboxybenzamide derivatives, based on molecu-
lar modeling studies.
R
R
2
O
N
4
3
R
R
1
3
Keywords: Vanilloid receptor; Antagonist; Structure–activity
relationship.
As part of our program to develop novel VR1 antago-
nists as potent analgesics, we attempted to replace the
piperazine moiety of 1 with phenyl ring based on the
*
Corresponding authors. Tel.: +82 28807871; fax: +82 28729129
(H.P.); e-mail: hgpk@plaza.snu.ac.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.033

632
H. Park et al. / Bioorg. Med. Chem. Lett. 15 (2005) 631–634
Figure 2. Stereoviews of the preferred three-dimensional conformations. (a) Energy-minimized conformation of 1 (5.113 kcal/mol); (b) energy-
minimized conformation of 2 (6.786 kcal/mol).
molecular modeling studies of 1 and 2. As shown in
Figure 2, the energy-minimized conformation of 1
(5.113 kcal/mol) is very similar to that of biarylcarb-
oxybenzamide 2 (6.876 kcal/mol).9 The chair-like con-
CHO
CHO
i
(HO) B
R
2
5
4
formation of the piperazine in
1 maintains the
molecular linear form, which is quite overlapped with
that of 2. Based on the similarity of the two conforma-
tions, we presumed that the biarylcarboxybenzamide
analogues (3) could retain the antagonistic activity on
VR1. Seventeen biarylcarboxybenzamide derivatives
were easily prepared in three steps (2, 3a–o) or four steps
(3p) from 4 (Scheme 1). The Suzuki coupling of 4 with
various aryl halides gave the corresponding 4-arylbenz-
aldehydes.10 Oxidation of the aldehydes 5 with NaClO2,
followed by the coupling with the corresponding aniline
derivatives in the presence of EDC afforded 2 and
3a–o.11 Compound 3p was prepared by N-methylation
of 2 in basic condition. The agonistic or antagonistic
activities of the prepared derivatives12 on VR1 receptor
were evaluated by the 45Ca2+-influx assay, previously re-
ported by using neonatal rat cultured spinal sensory
neurons.13 As shown in Table 1, the phenyl-substituted
analogue 3a still retained antagonistic activity
(IC50 = 3.6 lM), but less than 1 (IC50 = 0.15 lM).
Among the pyridine derivatives, 2-pyridine analogue 2
(IC50 = 0.031 lM) showed the highest antagonistic
activity, which is five times higher than that of the lead
compound 1. However, 3-pyridine analogue (3b,
ii, iii
R
R
R
R
2
2
O
O
iv
N
N
H
3
3
CH
3
R
1
R
R
1
3p
3a-o
= H
R
= benzene
2-pyridine
3-pyridine
4-pyridine
2-pyrimidine
5-pyrimidine
2-pyrazine
2
1
CH
3
3
CF
iso-propyl
tert-butyl
R
3
= H
CH
3-CH -2-pyridine
3
3
3-CF -2-pyridine
3
3-Cl-2-pyridine
3-NO -2-pyridine
2
Scheme 1. Reagents and conditions: (a) (i) R1X, Pd(PPh3)4, MeCN,
0.4 M NaHCO3, reflux, 5 h, 40–70%; (ii) NaClO2, NaH2PO4Æ2H2O,
CH3CH@C(CH3)2, tert-BuOH, H2O, rt, 2 h, 80–90%; (iii) 4-R2PhNH2,
EDC, DMAP, Et3N, CH2Cl2, rt, 2 h, 70–80%; (iv) CH3I, NaH, THF,
0 °C, 92%.
IC50 = 1.3 lM), and 4-pyridine analogue (3c, IC50
=
0.28 lM) showed lower activities, implying the ortho-N
of pyridine is quite important for the binding with recep-
tor, which is in accordance with the previous reports.8 In
the case of two-nitrogen possessing six-membered
heterocycle series, 2-pyrimidine analogue (3d,
IC50 = 0.06 lM), exhibited superior potency to 5-pyrim-

H. Park et al. / Bioorg. Med. Chem. Lett. 15 (2005) 631–634
Table 1. In vitro biological activity of the derivatives by 45Ca2+-influx
assay in rat DRG (Dorsal root ganglion) neurons
633
O
N
H
3
R
No.
R
45Ca2+-influx activity (lM)a
Agonist (IC50) Antagonist (IC50
)
1
—
NE
NE
0.15
3.6
3a
Figure 3. Reversible antagonistic effects of
capsaicin receptor.
2
(MK-180) on the
2
NE
0.031
N
N
binding site, and the electron-donating groups seem
more favorable for binding, compared to electron-with-
drawing groups. In a series of aniline amide analogues
(2, 3k–o) in Table 2, the bulky and hydrophobic substi-
tuted analogues, 2 (tert-butyl, IC50 = 0.031 lM) and 3n
(iso-propyl, IC50 = 0.038 lM) gave higher antagonistic
activities than relatively smaller or polar group ana-
3b
3c
3d
NE
NE
NE
1.3
N
0.28
0.06
N
N
N
logues, 3k (H, IC50 > 10 lM), 3l (4-CH3, IC50
=
N
N
3e
3f
NE
NE
NE
1.0
2.3 lM), 3m (3,4-di-CH3, IC50 = 2.0 lM), and 3n (4-
CF3, IC50 = 7.9 lM), implying that the hydrophobic
interaction contributes to efficient binding. In particular,
N–CH3 analogue 3p (IC50 > 10 lM), exhibited dramatic
loss of activity. This might support the hypothesis that
the hydrogen bonding of N–H is essential for the favor-
able binding with VR1. One of the best antagonistic
analogues, 2 (MK-180) was chosen for the confirmation
of its VR1 antagonism via the inhibition of the capsai-
cin-induced agonist action on patch-clamped rat DRG
neurons. As shown in Figure 3, the application of capsa-
icin (1 lM) greatly activated the capsaicin receptors in
inside-out membrane patches. However, 2 (0.3 lM) al-
most inhibited the channel activity, evoked by capsaicin
(1 lM). After 2 was removed by washing, capsaicin
(1 lM) reactivated the channel activity, suggesting that
2 clearly antagonizes the capsaicin at the VR1 in a
reversible manner. Figure 4 shows the magnitude of
inhibition by 2 (0.3 lM) in the presence of capsaicin
(1 lM).
10>
N
CH
3
3g
0.031
0.14
N
CF
3
3h
3i
NE
NE
N
Cl
0.055
N
NO
2
3j
NE
0.06
N
NE: not effective at 30 lM.
a EC50 (the concentration of derivative necessary to produce 50% of the
maximal response) and IC50 values (the concentration of derivative
necessary to reduce the response to 0.5 lM capsaicin by 50%) were
estimated with at least three replicates at each concentration. Each
compound was tested in two independent experiments. Antagonist
data were fitted with a sigmoidal function.
idine analogue (3f, IC50 > 10 lM) and 2-pyrazine ana-
logue (3e, IC50 = 1.0 lM). Most of 3-substituted-2-pyri-
dine analogues (3g–j) showed higher antagonistic
activities than 1 except 3h. The electron-donating group
(CH3, 3g, IC50 = 0.031 lM) exhibited higher antagonis-
tic activity than that of the electron-withdrawing groups
(3h, CF3, IC50 = 0.14 lM; 3i, Cl, IC50 = 0.055 lM; 3j,
NO2, IC50 = 0.06 lM). Based on the cumulative in vitro
antagonistic activity results, the general tendency could
be summarized as follows: (1) The ortho-N plays an inte-
gral role in antagonistic activity, but di-ortho-N2 cannot
give additive effect; (2) the antagonistic potency is in the
order of ortho-N > para-N > meta-N; (3) There is some
space at the 3-position of 2-pyridine group around the
Figure 4. Comparison of the channel activity of capsaicin (1lM) to 2
(0.3lM) in the presence of capsaicin (1lM). CTL is control activity
before the application of capsaicin.

634
H. Park et al. / Bioorg. Med. Chem. Lett. 15 (2005) 631–634
Table 2. In vitro biological activity of the derivatives by 45Ca2+-influx
assay in rat DRG neurons
Brough, S.; Stevens, A. J.; Randall, A. D.; Smart, D.;
Gunthorpe, M. J.; Davis, J. B. Bioorg. Med. Chem. Lett.
2004, 14, 3631.
R
R
1
O
7. (a) Park, H.-g.; Park, M.-k.; Choi, J.-y.; Choi, S.-h.; Lee,
J.; Suh, Y.-g.; Oh, U.; Lee, J.; Kim, H.-D.; Park, Y.-H.;
Jeong, Y. S.; Choi, J. K.; Jew, S.-s. Bioorg. Med. Chem.
Lett. 2003, 13, 197; (b) Park, H.-g.; Park, M.-k.; Choi,
J.-y.; Choi, S.-h.; Lee, J.; Suh, Y.-g.; Cho, H.; Oh, U.; Lee,
J.; Kim, H.-D.; Park, Y.-H.; Koh, H.-J.; Lim, K. M.;
Moh, J.-H.; Jew, S.-s. Bioorg. Med. Chem. Lett. 2003, 13,
601; (c) Yoon, J. W.; Choi, H. Y.; Lee, J. J.; Ryu, C. H.;
Park, H.-g.; Suh, Y.-g.; Oh, U.; Jeong, Y. S.; Choi, J. K.;
Park, Y.-H.; Kim, H.-D. Bioorg. Med. Chem. Lett. 2003,
13, 1549; (d) Lee, J.; Lee, J.; Kang, M.; Shin, M.-Y.; Kim,
J.-M.; Kang, S.-U.; Lim, J.-O.; Choi, H.-K.; Suh, Y.-G.;
Park, H.-g.; Oh, U.; Kim, H.-D.; Park, Y.-H.; Ha, H.-J.;
Kim, Y.-H.; Toth, A.; Wang, Y.; Tran, R.; Pearce, L. V.;
Lundberg, D. J.; Blumberg, P. M. J. Med. Chem. 2003, 46,
3116; (e) Suh, Y.-G.; Lee, Y.-S.; Min, K.-H.; Park, O.-H.;
Seung, H.-S.; Kim, H.-D.; Park, H.-g.; Choi, J.-y.; Lee, J.;
Kang, S.-W.; Oh, U.; Koo, J.-y.; Joo, Y.-H.; Kim, S.-Y.;
Kim, J.-K.; Park, Y.-H. Bioorg. Med. Chem. Lett. 2003,
13, 4389; (f) Park, H.-g.; Choi, J.-y.; Choi, S.-h.; Park,
M.-k.; Lee, J.; Suh, Y.-g.; Cho, H.; Oh, U.; Lee, J.; Kang,
S.-U.; Lee, J.; Kim, H.-D.; Park, Y.-H.; Jeong, Y. S.;
Chou, J. K.; Jew, S.-s. Bioorg. Med. Chem. Lett. 2004, 14,
787; (g) Park, H.-g.; Choi, J.-y.; Choi, S.-h.; Park, M.-k.;
Lee, J.; Suh, Y.-g.; Cho, H.; Oh, U.; Kim, H.-D.; Joo, Y.
H.; Kim, S.-Y.; Park, Y.-H.; Jeong, Y. S.; Choi, J. K.;
Kim, J. K.; Jew, S.-s. Bioorg. Med. Chem. Lett. 2004, 14,
1693; (h) Ryu, C. H.; Jang, M. J.; Jung, J. W.; Park, J.-H.;
Choi, H. Y.; Suh, Y.-g.; Oh, U.; Park, H.-g.; Lee, J.; Koh,
H.-J.; Mo, J.-H.; Joo, Y. H.; Park, Y.-H.; Kim, H.-D.
Bioorg. Med. Chem. Lett. 2004, 14, 1751.
N
3
2
R
3
N
No. R1
R2
R3
45Ca2+-influx activity (lM)a
Agonist (IC50) Antagonist (IC50
)
1
—
—
H
H
—
H
H
H
H
H
H
NE
NE
NE
NE
NE
NE
NE
0.15
10>
2.3
3k
3l
H
CH3
3m CH3
CH3
H
2.0
7.9
3n
3o
2
CF3
iso-propyl
tert-butyl
tert-butyl
H
0.038
0.031
H
3p
H
CH3 NE
10>
NE: not effective at 30 lM.
a EC50 and IC50 values were estimated by the same method described in
Table 1.
In conclusion, 17 diarylcarboxybenzamides were pre-
pared and their biological activities were evaluated.
Quite highly enhanced antagonistic activities were ob-
served by the replacement of piperazine moiety of the
lead compound 1 with phenyl ring. Among them,
N-(4-tert-butylphenyl)-4-pyridine-2-yl-benzamide
(2,
MK-180) and N-(4-tert-butylphenyl)-4-(3-methylpyri-
dine-2-yl)benzamide (3g) showed the best antagonistic
activities. We believe this pharmacophore information
would be very useful to design more potent antagonistic
scaffolds for the development of potential analgesics.
8. (a) Sun, Q.; Tafesse, L.; Islam, K.; Zhou, X.; Victory, S.
F.; Zhang, C.; Hachicha, M.; Schmid, L. A.; Patel, A.;
Rotshteyn, Y.; Valenzano, K. J.; Kyle, D. J. Bioorg. Med.
Chem. Lett. 2004, 13, 3621; (b) Sun, Q.; Schmid, L.;
Valenzano, K. J.; Rotshteyn, Y.; Su, X.; Kyle, D. J.
Bioorg. Med. Chem. Lett. 2004, 14, 5513.
Acknowledgements
9. The calculations were done using the program SYBYL 6.5
from Tripos Software Inc. St. Louis, MO, USA. 1 and
2 were minimized using Tripos forcefield parameters and the
conjugate gradient algorithm with a gradient convergence
This research was supported by a grant of the Korea
Health 21 R&D Project, Ministry of Health & Welfare,
Republic of Korea: 02-PJ2-PG4-PT01-0014.
˚
value of 0.005 kcal/mol A. Partial atomic charges were
calculated using the Gasteiger–Huckel method. Low energy
¨
References and notes
conformation was searched by grid search method.
10. (a) Arvanitis, A. G.; Arrold, C. R.; Fitz, L. W.; Frietze, W.
E.; Olson, R. E.; Gilligan, P. J.; Robertson, D. W. Bioorg.
Med. Chem. Lett. 2003, 13, 289; (b) Gong, Y.; Pauls, H.
W. Synlett 2000, 829.
1. Szallasi, A.; Blumberg, P. M. Pharmacol. Rev. 1999, 51, 159.
2. Fitzgerald, M. Pain 1983, 15, 109.
3. (a) Virus, R. M.; Gebhart, G. F. Life Sci. 1979, 25, 1275;
(b) Wood, J. N.; Winter, J.; James, I. F.; Rang, H. P.;
Yeats, J.; Bevan, S. J. Neurosci. 1988, 8, 3208; (c)
Capsaicin In the Study of Pain; Wood, J. N., Ed.;
Academic: San Diego, CA, 1993.
11. Kraus, G. A.; Roth, B. J. Org. Chem. 1980, 45, 4825.
12. All compounds gave satisfactory spectroscopic data con-
sistent with the proposed structures. Selected spectral data
1
for 2: mp 177 °C. H NMR (CDCl3, 300 MHz), d 8.72 (d,
4. Appendino, G.; Szallasi, A. Life Sci. 1997, 60, 681–696.
5. (a) Wrigglesworth, R.; Walpole, C. S. J. Drugs Future
1998, 23, 531; (b) Jancso, N.; Jancso-Gaber, A.; Szolcs-
anyi, J. J. Br. Pharmacol. 1967, 31, 138; (c) Petsche, U.;
Fleischer, E.; Lembeck, F.; Handwerker, H. O. Brain Res.
1983, 265, 233; (d) Dray, A.; Bettany, J.; Forster, P.
J. Pharmacol. 1990, 101, 727; (e) Dray, A.; Bettany, J.;
Reuff, A.; Walpole, C. S. J.; Wrigglesworth, R. Eur. J.
Pharmacol. 1990, 181, 289.
6. For the review on antagonists: (a) Szallasi, A.; Appendino,
G. J. Med. Chem. 2004, 47, 2712; (b) McDonnell, M. E.;
Zhang, S.-P.; Nasser, N.; Dubin, A. E.; Dax, S. L. Bioorg.
Med. Chem. Lett. 2004, 14, 531; (c) Rami, H. K.;
Thompson, M.; Wyman, P.; Jerman, J. C.; Egerton, J.;
J = 4.77 Hz, 1H), 8.11 (d, J = 8.43 Hz, 2H), 7.96 (d, J =
8.43 Hz, 2H), 7.84 (br, 1H), 7.78 (m, 2H), 7.57 (d,
J = 8.61 Hz, 2H), 7.38 (d, J = 8.79 Hz, 2H), 7.28 (m, 1H),
1.31 (s, 9H). 13C NMR (CDCl3, 125 MHz), d 165.36, 156.05,
149.77, 147.54, 142.29, 136.96, 135.25, 135.15, 127.51,
127.10, 125.86, 122.80, 120.90, 120.08, 34.38, 31.32. IR
(KBr) 3291, 2960, 1650, 1593, 1526 cmÀ1. MS (ESI) m/z, 331
[M+H]+. HRMS (EI) calcd for [C22H22N2O]: 330.4230,
found: 330.1709 [M]+. Anal. Calcd for C22H22N2O: C,
79.97; H, 6.71; N, 8.48. Found: C, 79.39; H, 6.69; N, 8.35.
13. The uptake and the accumulation of 45Ca2+ by the
biarylcarboxybenzamide derivatives were studied in neo-
natal rat cultured spinal sensory neurons, by the method
described in detail in Ref. 3b.