Pyridine C-region analogs of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists

Article abstract of DOI:10.1016/j.ejmech.2015.02.001

A series of pyridine derivatives in the C-region of N-((6-trifluoromethyl-pyridin-3-yl)methyl) 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. The SAR analysis indicated that 6-difluorochloromethyl pyridine derivatives were the best surrogates of the C-region for previous leads. Among them, compound 31 showed excellent antagonism to capsaicin as well as to multiple hTRPV1 activators. It demonstrated stronganalgesic activity in the formalin test in mice with full efficacy and it blocked capsaicin-induced hypothermia in vivo.

Full text of DOI:10.1016/j.ejmech.2015.02.001

European Journal of Medicinal Chemistry 93 (2015) 101e108
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Pyridine C-region analogs of 2-(3-fluoro-4-
methylsulfonylaminophenyl)propanamides as potent TRPV1
antagonists
HyungChul Ryu a, Sejin Seo a, Jee-Young Lee a, Tae-Hwan Ha a, Sunho Lee a, Aeran Jung a,
Jihyae Ann a, Sung-Eun Kim a, Suyoung Yoon a, Mannkyu Hong a, Peter M. Blumberg b,
Robert Frank-Foltyn c, Gregor Bahrenberg c, Klaus Schiene c, Hannelore Stockhausen c,
,
*
Thomas Christoph c, Sven Frormann c, Jeewoo Lee a
a Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742,
Republic of Korea
b Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA
c Grunenthal Innovation, Grunenthal GmbH, D-52078 Aachen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyridine derivatives in the C-region of N-((6-trifluoromethyl-pyridin-3-yl)methyl) 2-(3-
fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. The SAR
analysis indicated that 6-difluorochloromethyl pyridine derivatives were the best surrogates of the C-
region for previous leads. Among them, compound 31 showed excellent antagonism to capsaicin as well
as to multiple hTRPV1 activators. It demonstrated strong analgesic activity in the formalin test in mice
with full efficacy and it blocked capsaicin-induced hypothermia in vivo.
Received 3 December 2014
Received in revised form
16 January 2015
Accepted 1 February 2015
Available online 2 February 2015
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Vanilloid receptor 1
TRPV1 antagonists
Analgesic
1. Introduction
Using this approach, we have developed antagonists with activities
in the low nM to sub-nM range [20e24]. Here, we report our recent
The vanilloid receptor TRPV1 has emerged as a promising
therapeutic target for treatment of neuropathic and inflammatory
pain as well as for a wide range of other conditions [1e3]. TRPV1
functions as a nociceptor activated by elevated temperature, by low
pH, and by both endogenous endovanilloids and exogenous agents
such as capsaicin [1,2,4e6]. It is further subject to extensive regu-
lation through signaling pathways, such as protein kinase C or
protein kinase D, positioning TRPV1 to integrate the multitude of
influences impacting the cell [7,8]. In view of the potential of TRPV1
as a therapeutic target, intense effort by many groups has been
directed at the development of potent TRPV1 antagonists [9e17]
and at understanding of the structural basis for potent ligand
binding [18]. We have used the ultrapotent TRPV1 ligand resin-
iferatoxin as a structural lead for antagonist development [19].
efforts at further optimization of our lead structures.
Recently, we reported a series of N-{(6-trifluoromethyl-pyridin-
3-yl)methyl} 2-(3-fluoro-4-methylsulfonylaminophenyl)propana-
mides, designed by a pharmacophoric combination approach,
which proved to be potent hTRPV1 antagonists (Fig. 1) [20e24].
Structurally the antagonistic template can be divided into three
pharmacophoric parts which were designated as the A-region (3-
fluoro-4-methylsulfonylaminophenyl), the B-region (prop-
anamide), and the C-region ((6-trifluoromethyl-pyridin-3-yl)
methyl). The structure activity relationships of the 2-substituent in
the pyridine C-region have been investigated extensively by
incorporating various functional groups, including amino [20], oxy
[21], thio [22], alkyl [23] and aryl [24] groups. In these series,
multiple compounds showed highly potent and stereospecific
antagonism to hTRPV1 activators including capsaicin, pH, heat
(45 ꢀC) and NADA. In addition, selected compounds were evaluated
for antinociceptive activity in neuropathic pain models, where they
showed potent activity and were found to block capsaicin-induced
* Corresponding author.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
http://dx.doi.org/10.1016/j.ejmech.2015.02.001
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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H. Ryu et al. / European Journal of Medicinal Chemistry 93 (2015) 101e108
corresponding 2-chloropyridine 4, which was condensed with a
library of amines and thiols and then reduced to provide the C-
region amines 6. For the synthesis of 2-oxy analogs, 3-
amidopyridone 7 was synthesized instead and then converted to
the C-region amines 8 via an O-alkylation/reduction sequence. A
series of 5-methylpyridine C-regions 11 were synthesized from 9, a
b-methylated form of 2, employing the same sequences described
in Scheme 1 (Scheme 2).
The two regioisomers of the 6-CF3 pyridine C-region, 15 (5-CF3-
pyridine) and 19 (2-CF3-pyridine), were synthesized efficiently
from appropriate starting material (Scheme 3). For the synthesis of
the 5-CF3-pyridine C-region 15, 2,3-dichloropyridine 12 was sub-
jected to 2-cyano substitution followed by a BuchwaldeHartwig
amination/reduction sequence to provide 15. For the 2-CF3-pyri-
dine C-region, only one example 19 (R ¼ Ph) was prepared. Pyr-
idone 17 was synthesized from the ketal 16 in 2 conventional steps
and then converted to the corresponding 2-chloropyridine 18,
which was reduced to the amine 19, concomitant with
dehalogenation.
Fig. 1. Chemical structure of previously reported TRPV1 antagonist.
hypothermia, consistent with their in vitro mechanism of action.
Finally, modeling analysis using our established hTRPV1 homology
model [18,20] indicated that, among the receptor binding in-
teractions, the two hydrophobic interactions by the 6-
trifluoromethyl group and the 2-substituents in the C-region
were critical for their potent antagonism and were made with the
hydrophobic pockets composed of Leu547/Thr550 and Met514/
Leu515, respectively.
In continuation of our effort to further optimize the pyridine C-
region, we herein have investigated the structure activity re-
lationships for hTRPV1 antagonism of various pyridine derivatives,
including isosteres of the 6-trifluoromethyl moiety and isomers of
pyridine. In this investigation, 2-substituents in the pyridine were
fixed, using selected representative groups which had provided
high potency in previous reports. With selected potent antagonists
in the series, we have further characterized their analgesic activity
and inhibition of capsaicin-induced hypothermia in animal models.
The synthesized amines (6, 8, 11, 15, 19) were coupled with
propionic acid [20] as previously reported to afford the final com-
pounds 20e46 (Scheme 4).
2.2. In vitro activity
The synthesized TRPV1 ligands were evaluated in vitro for
antagonism as measured by inhibition of activation by capsaicin
(CAP). The assays were conducted using a fluorometric imaging
plate reader (FLIPR) with human TRPV1 heterologously expressed
in Chinese hamster ovary (CHO) cells [20]. The results are sum-
marized in Tables 1e4, together with the potencies of the previ-
ously reported antagonists IeV [20e24].
2. Result and discussion
2.1. Chemistry
First, we investigated the SAR of the 6-position (R1) in the pyr-
idine C-region by substituting the 6-trifluoromethyl moiety with
the corresponding isosteres, including the methyl, difluoromethyl,
difluorochloromethyl, cyclopropyl and 4-fluorophenyl groups
(Table 1). The 2-position (R2) in the pyridine was held fixed as
either the 4-methylpiperidinyl or the piperidinyl group, which had
provided high antagonistic potency as reference compounds I and
II from the previous report.
A series of 6-substituted pyridine C-regions (6, 8), containing
R1 ¼ CH3, CF2H, CF2Cl, cyclopropyl, and Ph-4-F, were synthesized
starting from the corresponding anhydride 1 by a modification of
the previously reported procedure (Scheme 1) [20]. For the syn-
thesis of the 2-amino (R2 ¼ NR2) and 2-thio (R2 ¼ SR) analogs, 3-
cyanopyridone 3 was prepared and then converted into the
Scheme 1. Syntheses of 6-substituted pyridine C-region analogs. Reagents and conditions: (a) CH2]CHOEt, pyridine, CHCl3, 0 ꢀC to rt; (b) 2-cyanoacetamide, NaOEt, EtOH, reflux,
3 h; (c) POCl3, sealed tube, 120 ꢀC, 6 h; (d) K2CO3, ReNH2 (or ReSH), CH3CN; (e) BH3eSMe2, THF, 6 h; (f) malonamide, NaOMe, MeOH, reflux, 2 h; (g) RX, K2CO3, 18-crown-6 ether,
CH3CN/DMF, reflux, 12 h; (h) BH3eSMe2, THF, reflux, 12 h.

H. Ryu et al. / European Journal of Medicinal Chemistry 93 (2015) 101e108
103
Scheme 2. Syntheses of 4-methyl-6-CF3 pyridine C-region analogs. Reagents and conditions: (a) 2-cyanoacetamide, NaOEt, EtOH, reflux, overnight; (b) POCl3, sealed tube, 120 ꢀC,
6 h; (c) K2CO3, ReNH2, CH3CN or K2CO3, ReOH, CH3CN; (d) Pd/C (10%), H2 (1 atm), MeOH, rt, 8 h or BH3eSMe2, THF, 6 h.
Scheme 3. Syntheses of regioisomeric pyridine C-region analogs. Reagents and conditions: (1) (a) NaCN, DMF, 120 ꢀC, 8 h; (b) PdCl2, DPPF, t-BuONa, ReNH2, toluene; (c) Pd/C
(10%), H2 (1 atm), MeOH, rt, 8 h or BH3eSMe2, THF, 6 h; (2) (a) TFAA, Py, CHCl3, rt; (b) NaOEt, EtOH, 2-cyanoacetamide; (c) POCl3, sealed tube, 120 ꢀC, 6 h; (d) Pd/C (10%), H2 (1 atm),
MeOH, rt, 8 h or BH3eSMe2, THF, 6 h.
Scheme 4. Syntheses of final compounds. Reagents and conditions: (a) HOBt, EDC, CH3CN, rt.
The 6-methyl derivative 20 showed a ca. 240-fold reduction in
activity compared to the parent I. However, the 6-difluoromethyl
21 and the 6-difluorochloromethyl 22 derivatives retained high
Table 1
potency, with 22 showing the same potency as I. To investigate
In vitro hTRPV1 antagonistic activities for 2,6-substituted pyridine C-region de-
the halogen effect of the above two substituents, a cyclopropyl
rivatives.
group, which has a lipophilicity similar to that of trifluoromethyl
group, was incorporated into the 6-position. The 6-cyclopropyl
derivative 23 still exhibited potent antagonism comparable to
21. We conclude that the lipophilicity of the 6-substituent was
more important for potent antagonism than was its electronic
effect. This result is likewise predicted from our molecular
modeling, in which the 6-trifluoromethyl group in the pyridine
R1
R2
Ki [CAP] (nM)
0.3a
C-region made
a hydrophobic interaction with a pocket
composed of Leu547 and Thr550. To confirm the relative potency
of the 6-difluorochloromethyl and cyclopropyl groups, their 2-
piperidinyl derivatives 25 and 26 were also prepared and
compared to their parent II. As was the case with 22 and 23,
compounds 25 and 26 showed potent and comparable antago-
nism to their parent compound (II), indicating that the lip-
ophilicity of the substituent at the 6-position was critical for
potent antagonism.
Since the 6-difluorochloromethyl group proved to be a prom-
ising bioisostere of the 6-trifluoromethyl group above, we explored
the SAR of 2-substituents in the 6-difluorochloromethyl pyridine C-
region to further evaluate its activity. The various 2-substituents
including amino, oxy and thio groups were selected based on
their potencies in previous reports [20e24] and activities were
compared to those for the corresponding 6-trifluoromethyl surro-
gates (Table 2). As expected, most 6-difluorochloromethyl pyridine
I
20
21
22
23
24
72.1
1
0.3
0.9
59
II
0.43a
25
26
1.3
1.1
a
Values from Ref. [20].

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H. Ryu et al. / European Journal of Medicinal Chemistry 93 (2015) 101e108
Table 2
Table 3
In vitro hTRPV1 antagonistic activities for 6-difluorochloropyridine derivatives.
In vitro hTRPV1 antagonistic activities for 4-methylpyridine derivatives.
R2
R1 ¼ CH3
Ki [CAP] (nM)
17.5
R1 ¼ H
R2
R1 ¼ CF2C1
Ki [CAP] (nM)
0.6
R1 ¼ CF3
Ki [CAP] (nM)a
0.2
Ki [CAP] (nM)a
40
41
42
0.3
27
9.2
0.2
0.4
28b
29
1
0.7
0.8
100
0.5
43
44
0.9
30
31
0.8
0.3
0.7
0.3
a
Values from Refs. [20,21].
parents (Table 3). The incorporation of a 4-methyl group to the
pyridine led to ca. 50e250 fold reductions in potency compared to
those of the corresponding parent antagonists, indicating that the
4-methyl group was poorly tolerated probably by inducing steric
repulsion with the receptor or unfavorable conformation of B/C-
regions.
Finally, the SAR of pyridine isomers in the C-region was inves-
tigated. Due to limitations of synthetic accessibility, only a few
representative compounds were prepared and compared to the
parents (Table 4). Relative to the parent compounds III and IV
32
0.8
0.4
33
34
0.6
0.5
0.5
0.9
35
0.9
1.3
Table 4
In vitro hTRPV1 antagonistic activities for 2 (or 5)-trifluoromethylpyridine de-
rivatives.
36
37
38
39
0.9
14.6
4.2
0.5
142
29.5
0.9
3.0
R
X
N
Y
Z
Ki [CAP] (nM)
0.6a
III
44
IV
CH
CH
CH
N
N
CH
CH
WE
a
Values from Refs. [20e22].
Mixture of cis/trans.
b
CH
0.43a
derivatives exhibited very potent antagonism in the low nanomolar
range comparable with those of the corresponding 6-
trifluoromethyl surrogates. Among them, compounds 31 and 34
with 2-butyloxy and 2-cyclopentyloxy groups were selected as the
most promising antagonists for further study.
Next, we sought to evaluate 4-methyl derivatives of selected 6-
trifluoromethyl pyridine antagonists to investigate their steric
tolerance by the receptor. The four representative derivatives
40e43 were synthesized and compared to their corresponding
45
V
CH
N
N
CH
CH
N
28.6
6.4a
32.7
CH
CH
46
CH
a
Values from Refs. [20,24].

H. Ryu et al. / European Journal of Medicinal Chemistry 93 (2015) 101e108
105
Table 5
nociceptive response by 29% and 15% at the doses of 0.3 and 1.0 mg/
kg, respectively.
In vitro hTRPV1 antagonistic activities of 31 and 34 for multiple activators.
Activators, parameter
I
31
34
0.5
18.5
19.3
3. Conclusion
CAP (f)Ki (nM)
0.3
15.8
2.56
0.02
0.3
8.3
7.0
0.02
pH, IC50 (nM)
Heat 45 ꢀC, IC50 (nM)
NADA (f)Ki (nM)
The structure activity relationship of pyridine derivatives in the
C-region of N-(6-trifluoromethyl-pyridin-3-ylmethyl) 2-(3-fluoro-
4-methylsulfonylaminophenyl)propanamides was investigated for
hTRPV1 antagonism. The analysis indicated that among the current
series the 6-difluorochloromethyl pyridine C-region was the most
adequate surrogate of the C-region for the leads described previ-
ously. The two selected antagonists 31 and 34 showed excellent
antagonism to multiple hTRPV1 activators and blocked capsaicin-
induced hypothermia, consistent with their actions in vitro being
through TRPV1. Compound 31 demonstrated strong analgesic ac-
tivity in the formalin test in mice with full TRPV1 efficacy.
0.04
(X ¼ N, Y ¼ Z ¼ H), the shift of a nitrogen to the 4-position led to a
dramatic reduction in antagonism, with 44 and 45 (Y ¼ N,
X ¼ Z ¼ H) proving to be weak and moderate antagonists, respec-
tively. Likewise, relative to the parent compound V, the shift of a
nitrogen to the 5-position to provide 46 (Z ¼ N, X ¼ Y ¼ H) caused a
5-fold reduction in potency. These results indicate that among the
pyridine isomers the 6-trifluoromethyl pyridine was optimal for
antagonism.
Detailed in vitro activities of 31 and 34, the most potent antag-
onists in this study, were investigated for multiple TRPV1 activa-
tors, namely capsaicin, pH, heat (45 ꢀC) and N-arachidonoyl
dopamine (NADA), and were compared to the activity of lead
compound I (Table 5). Compound 31 showed excellent antagonism
toward all activators comparable to I, but compound 34 exhibited
2e3 fold less potency than 31.
4. Experimental
4.1. Chemistry
4.1.1. General
All chemical reagents were commercially available. Melting
points were determined on a Büchi Melting Point B-540 apparatus
and are uncorrected. Silica gel column chromatography was per-
formed on silica gel 60, 230e400 mesh, Merck. Nuclear magnetic
resonance (1H NMR and 13C NMR) spectra were recorded on JEOL
JNM-LA 300 [300 MHz (1H), 75 MHz (13C)] and Bruker Avance
400 MHz FT-NMR [400 MHz (1H), 100 MHz (13C)] spectrometers.
Chemical shifts are reported in ppm units with Me4Si as a reference
standard. Infrared (IR) spectra were recorded on a JASCO FT/IR-
4200 spectrometer. Mass spectra were recorded on a VG Trio-2
GC-MS and 6460 Triple Quad LC/MS. All final compounds were
purified to >95% purity, as determined by high-performance liquid
chromatography (HPLC). HPLC was performed on an Agilent 1120
Compact LC (G4288A) instrument using an Agilent Eclipse Plus C18
2.3. In vivo activity
Consistent with its in vitro mechanism of action as an hTRPV1
antagonist, in vivo 31 and 34 likewise blocked response to capsaicin
(Table 6). Compounds 31 and 34 were administered orally at a dose
0.3 mg/kg 15 min before intraperitoneal injection of 3 mg/kg
capsaicin, following the procedure described previously [20]. This
dose of 31 and 34 inhibited the hypothermic response to capsaicin,
assayed 30 min after capsaicin injection, by 43% and 41%, respec-
tively. Compound 34 showed a dose-dependent inhibition in
capsaicin-induced hypothermia and almost completely blocked the
response at a dose of 10 mg/kg.
We evaluated the in vivo analgesic activities of the two selected
antagonists, 31 and 34, in the formalin test [25] in mice upon oral
administration (Table 7). Compound 31 showed a significant anti-
nociceptive effect, with 56% and 73% inhibition of response at the
doses of 0.1 and 0.3 mg/kg, respectively. Since we had observed that
TRPV1 knock-out mice showed approximately 50% of the magni-
tude of response in the formalin test as was seen in wild-type mice
(unpublished observations), the inhibition of the formalin response
that we found for this compound would correspond to the expected
result for full TRPV1 blockade. Compound 34 showed weaker ac-
tivity than that of 31 in the formalin assay, inhibiting the
column (4.6 ꢁ 250 mm, 5
mm) and a Daicel Chiralcel OD-H column
(4.6 ꢁ 250 mm, 5 m).
m
4.1.2. General procedure for chemistry
The general synthetic procedure for the syntheses of the final
compounds was described in previous reports [20e22].
4.1.2.1. N-((6-Methyl-2-(4-methylpiperidin-1-yl)pyridin-3-yl)
methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(20). 75% yield, white solid, mp ¼ 78e80 ꢀC, 1H NMR (300 MHz,
CDCl3)
d
7.51 (t, 1H, J ¼ 8.2 Hz), 7.13 (d, 1H, J ¼ 8.6 Hz), 7.07 (d, 2H,
J ¼ 8.0 Hz), 6.76 (s, 1H), 6.74 (s, 1H), 6.46 (brs, 1H), 4.41 (t, 2H,
J ¼ 4.4 Hz), 3.51 (q, 1H), 3.18 (m, 2H), 3.02 (s, 3H), 2.78 (m, 2H), 2.42
(s, 3H), 1.73 (m, 2H), 1.50 (d, 3H, J ¼ 7.1 Hz), 1.26 (m, 2H), 0.97 (d, 3H,
J ¼ 6.4 Hz); MS (FAB) m/z 463 (MþH).
Table 6
Effect of compounds 31 and 34 on capsaicin-induced hypothermia in mice.
Dose (mpk)
0.1
0.3
1
10
92
4.1.2.2. N-((6-(Difluoromethyl)-2-(4-methylpiperidin-1-yl)pyridin-3-
yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(21). 48% yield, white solid, mp ¼ 105e107 ꢀC, 1H NMR (300 MHz,
% Inhibition
31
34
43
41
23
60
CDCl3)
d
7.50 (t, 2H, J ¼ 4.7 Hz), 7.17 (d, 1H, J ¼ 8.1 Hz), 7.09 (d, 1H,
J ¼ 6.0 Hz), 6.48 (s, 1H), 6.42 (brs, 1H), 4.46 (brs, 1H), 3.55 (q, 1H),
3.26 (t, 2H, J ¼ 13.2 Hz), 3.02 (s, 3H), 2.79 (t, 2H, J ¼ 11.9 Hz), 1.71
(brs, 2H), 1.52 (d, 3H, J ¼ 6.6 Hz), 1.19 (m, 2H), 0.97 (d, 3H,
J ¼ 6.0 Hz); MS (FAB) m/z 499 (MþH).
Table 7
Analgesic activity of compound 31 and 34 on formalin model after oral adminis-
tration in mice.
Dose (mpk)
0.1
0.3
1
4.1.2.3. N-((6-(Chlorodifluoromethyl)-2-(4-methylpiperidin-1-yl)
pyridin-3-yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)
propanamide (22). 77% yield, white solid, mp ¼ 175e176 ꢀC, 1H
MPE
56
31
34
73
29
15
NMR (300 MHz, CDCl3) d 7.45e7.53 (m, 2H), 7.07e7.18 (m, 3H), 6.72

106
H. Ryu et al. / European Journal of Medicinal Chemistry 93 (2015) 101e108
(bs,1H), 6.37 (bt,1H), 4.46 (d, 2H, J ¼ 5.7 Hz), 3.56 (q,1H, J ¼ 6.9 Hz),
3.32 (m, 2H), 3.02 (s, 3H), 2.82 (m, 2H), 1.71 (m, 2H), 1.53 (d, 3H,
J ¼ 7.5 Hz), 1.23 (m, 3H), 0.97 (d, 3H, J ¼ 6.9 Hz); MS (FAB) m/z 534
(MþH).
1.56 (m, 4H), 1.52 (d, 3H, J ¼ 6.9 Hz); MS (FAB) m/z 534 (MþH).
4.1.2.11. N-((6-(Chlorodifluoromethyl)-2-(4-phenylpiperazin-1-yl)
pyridin-3-yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)
propanamide (30). 65% yield, white solid, mp ¼ 108 ꢀC, 1H NMR
4.1.2.4. N-((6-Cyclopropyl-2-(4-methylpiperidin-1-yl)pyridin-3-yl)
methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(23). 58% yield, white solid, mp ¼ 84 ꢀC, 1H NMR (300 MHz, CDCl3)
(300 MHz, CD3OD)
d
8.60 (m, 1H), 7.58 (d, 1H, J ¼ 7.5 Hz), 7.47e7.25
(m, 4H), 7.25e7.00 (m, 4H), 4.57e4.35 (m, 2H), 3.73 (d, 1H,
J ¼ 7.1 Hz), 3.45e3.32 (m, 8H), 2.95 (bs, 3H), 1.47 (d, 3H, J ¼ 7.1 Hz);
MS (FAB) m/z 597 (MþH).
d
7.50 (t, 1H, J ¼ 8.3 Hz), 7.23e7.06 (m, 3H), 6.74 (d, 2H, J ¼ 7.5 Hz),
6.47 (bs, 1H), 4.40 (t, 2H, J ¼ 5.9 Hz), 3.50 (q, 1H, J ¼ 7.1 Hz), 3.18 (m,
2H), 3.01 (s, 3H), 2.72 (m, 2H), 1.90 (m, 1H), 1.69 (m, 2H), 1.50 (d, 3H,
J ¼ 7.1 Hz), 1.26e1.13 (m, 3H), 0.95 (d, 3H, J ¼ 6.4 Hz); MS (FAB) m/z
489 (MþH).
4.1.2.12. N-((2-Butoxy-6-(chlorodifluoromethyl)pyridin-3-yl)
methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(31). 70% yield, white solid, mp ¼ 75e77 ꢀC, 1H NMR (300 MHz,
CDCl3)
d
7.49e7.54 (m, 2H), 7.14 (d, 1H, J ¼ 7.5 Hz), 7.05e7.09 (m,
4.1.2.5. N-((6-(4-Fluorophenyl)-2-(4-methylpiperidin-1-yl)pyridin-
3-yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)prop-
anamide (24). 52% yield, white solid, mp ¼ 154e156 ꢀC, 1H NMR
2H), 6.46 (bs, 1H), 6.02 (bt, 1H), 4.36 (m, 4H), 3.52 (q, 1H, J ¼ 6.9 Hz),
3.03 (s, 2H), 1.71 (m, 2H), 1.48 (m, 5H), 0.97 (t, 3H, J ¼ 7.2 Hz); MS
(FAB) m/z 508 (MþH).
(300 MHz, CDCl3)
d
7.49e7.54 (m, 2H), 7.51 (dd, 1H, J ¼ 8.1 and
8.1 Hz), 7.43 (d, 1H, 7.8 Hz), 7.29 (d, 1H, J ¼ 7.8 Hz), 7.09e7.17 (m,
4H), 6.64 (bt, 1H), 4.48 (d, 2H, J ¼ 5.7 Hz), 3.52 (q, 1H, J ¼ 6.9 Hz),
3.30 (m, 2H), 3.03 (s, 3H), 2.88 (m. 2H), 1.76 (m, 2H), 1.51 (d, 3H,
J ¼ 6.9 Hz), 1.24 (m, 3H), 0.99 (d, 3H, J ¼ 6.6 Hz); MS (FAB) m/z 543
(MþH).
4.1.2.13. N-((6-(Chlorodifluoromethyl)-2-(pentyloxy)pyridin-3-yl)
methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(32). 48% yield, white solid, mp ¼ 86 ꢀC, 1H NMR (400 MHz, CDCl3)
d
7.58e7.47 (m, 2H), 7.17e7.03 (m, 3H), 6.50 (bs, 1H), 5.98 (bt, 1H),
4.43e4.25 (m, 4H), 3.51 (q, 1H, J ¼ 8.6 Hz), 3.03 (s, 3H), 1.78e1.67
(m, 2H), 1.49 (d, 3H, J ¼ 6.8 Hz), 1.46e1.27 (m, 6H), 0.94e0.87 (m,
3H); MS (FAB) m/z 522 (MþH).
4.1.2.6. N-((6-(Chlorodifluoromethyl)-2-(piperidin-1-yl)pyridin-3-yl)
methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(25). 70% yield, pale yellow solid, mp ¼ 63e65 ꢀC, 1H NMR
4.1.2.14. N-((6-(Chlorodifluoromethyl)-2-isobutoxypyridin-3-yl)
methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(33). 57% yield, white solid, mp ¼ 105 ꢀC, 1H NMR (300 MHz, CDCl3)
(300 MHz, CDCl3)
d 7.45e7.52 (m, 2H), 7.06e7.18 (m, 3H), 6.46 (bt,
1H), 4.47 (d, 2H, J ¼ 5.1 Hz), 3.58 (q, 1H, J ¼ 6.9 Hz), 3.05 (m, 4H),
3.00 (s, 3H), 1.61 (m, 6H), 1.53 (d, 3H, J ¼ 7.1 Hz); MS (FAB) m/z 520
(MþH).
d
7.58e7.48 (m, 2H), 7.16 (d, 1H, J ¼ 7.5 Hz), 7.11 (m, 1H), 7.06 (m,
1H), 6.46 (m, 1H), 5.95 (bt, 1H), 4.44e4.32 (m, 2H), 4.19e4.06 (m,
2H), 3.51 (q, 1H, J ¼ 7.1 Hz), 3.04 (s, 3H), 2.05 (m, 1H), 1.49 (d, 3H,
J ¼ 7.1 Hz), 0.99 (d, 6H, J ¼ 6.8 Hz); MS (FAB) m/z 509 (MþH).
4.1.2.7. N-((6-Cyclopropyl-2-(piperidin-1-yl)pyridin-3-yl)methyl)-2-
(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(26). 57%
7.51
yield, white solid, mp ¼ 65e75 ꢀC, 1H NMR (300 MHz, CDCl3)
d
4.1.2.15. N-((6-(Chlorodifluoromethyl)-2-(cyclopentyloxy)pyridin-3-
yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(34). 71% yield, white solid, mp ¼ 106 ꢀC, 1H NMR (300 MHz, CDCl3)
(t,1H, J ¼ 8.4 Hz), 7.24e7.06 (m, 3H), 6.74 (d, 2H, J ¼ 7.5 Hz), 6.47 (bs,
1H), 4.39 (t, 2H, J ¼ 5.3 Hz), 3.50 (q, 1H, J ¼ 7.0 Hz), 3.01 (s, 3H), 2.92
(m, 4H), 1.90 (m, 1H), 1.55 (m, 2H), 1.50 (d, 3H, J ¼ 7.1 Hz), 1.01e0.86
(m, 3H); MS (FAB) m/z 475 (MþH).
d
7.57e7.48 (m, 2H), 7.15e7.03 (m, 3H), 6.56 (bs, 1H), 6.01e5.90 (bt,
1H), 5.46 (m, 1H), 4.42e4.27 (m, 2H), 3.52 (q, 1H, J ¼ 7.1 Hz), 3.03 (s,
3H), 2.08e1.91 (m, 2H), 1.78e1.56 (m, 6H), 1.49 (d, 3H, J ¼ 7.1 Hz);
MS (FAB) m/z 520 (MþH).
4.1.2.8. N-((6-(Chlorodifluoromethyl)-2-(dipropylamino)pyridin-3-
yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(27). 58% yield, white solid, mp ¼ 101e103 ꢀC, 1H NMR (300 MHz,
4.1.2.16. N-((6-(Chlorodifluoromethyl)-2-(cyclohexyloxy)pyridin-3-
yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(35). 58% yield, white solid, mp ¼ 97 ꢀC, 1H NMR (300 MHz, CDCl3)
CDCl3)
d
7.53 (dd, 1H, J ¼ 8.1 and 8.1 Hz), 7.43 (d, 1H, J ¼ 7.8 Hz), 7.16
(dd, 1H, J ¼ 2.1 and 10.8 Hz), 7.08e7.12 (m, 2H), 6.46 (bs, 1H), 6.15
(bt, 1H), 4.44 (d, 2H, J ¼ 5.7 Hz), 3.53 (q, 1H, J ¼ 6.9 Hz), 3.10 (m, 4H),
3.02 (s, 3H), 1.44e1.54 (m, 4H), 0.83 (t, 6H, J ¼ 7.2 Hz), MS (FAB) m/z
535 (MþH).
d
7.59e7.48 (m, 2H), 7.14e7.02 (m, 3H), 6.49 (bs, 1H), 6.01 (bt, 1H),
5.13 (m, 1H), 4.47e4.29 (m, 2H), 3.52 (q, 1H, J ¼ 7.3 Hz), 3.03 (s, 3H),
1.99e1.85 (m, 2H), 1.77e1.62 (m, 2H), 1.52e1.38 (m, 9H); MS (FAB)
m/z 534 (MþH).
4.1.2.9. N-((6-(Chlorodifluoromethyl)-2-(3,5-(cis/trans)-dimethylpi-
peridin-1-yl)pyridin-3-yl)methyl)-2-(3-fluoro-4-(methyl-
sulfonamido)phenyl)propanamide (28). 58% yield, colorless oil, 1H
4.1.2.17. N-((2-(Benzyloxy)-6-(chlorodifluoromethyl)pyridin-3-yl)
methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(36). 52% yield, white solid, mp ¼ 62 ꢀC, 1H NMR (300 MHz, CDCl3)
NMR (300 MHz, CDCl3) d 7.45e7.54 (m, 2H), 7.06e7.15 (m, 3H), 6.62
(bs, 1H), 6.31 (bt, 1H), 4.46 (d, 2H, J ¼ 5.7 Hz), 3.54 (q, 1H, J ¼ 7.2 Hz),
3.25 (m, 2H), 3.02 (s, 3H), 2.36 (m, 2H), 2.03 (m, 1H), 1.53e1.65 (m,
3H), 1.52 (d, 3H, J ¼ 7.2 Hz) 0.92 (d, 3H, J ¼ 6.6 Hz), 0.88 (d, 3H,
J ¼ 6.6 Hz); MS (FAB) m/z 548 (MþH).
d
7.59 (d, 1H, J ¼ 7.3 Hz), 7.50e7.30 (m, 6H), 7.20 (d, 1H, J ¼ 7.8 Hz),
7.05 (dd, 1H, J ¼ 11.2 and 2.0 Hz), 6.97 (d, 1H, J ¼ 7.9 Hz), 6.52 (bs,
1H), 6.00 (bt, 1H), 5.49e5.36 (m, 2H), 4.46e4.30 (m, 2H), 3.42 (q,
1H, J ¼ 7.1 Hz), 3.00 (bs, 3H), 1.43 (d, 3H, J ¼ 7.1 Hz); MS (FAB) m/z
542 (MþH).
4.1.2.10. N-((2-(Azepan-1-yl)-6-(chlorodifluoromethyl)pyridin-3-yl)
methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(29). 58% yield, white solid, mp ¼ 59e61 ꢀC, 1H NMR (300 MHz,
4.1.2.18. N-((6-(Chlorodifluoromethyl)-2-(pyridin-3-ylmethoxy)pyr-
idin-3-yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)prop-
anamide (37). 61% yield, white solid, mp ¼ 78 ꢀC, 1H NMR
CDCl3)
d
7.52 (dd, 1H, J ¼ 8.1 and 8.1 Hz), 7.38 (d, 1H, J ¼ 7.5 Hz), 7.17
(dd, 1H, J ¼ 1.8 and 11.1 Hz), 7.08 (d, 1H, J ¼ 8.1 Hz), 6.99 (d, 1H,
J ¼ 7.5 Hz), 6.57 (bs, 1H), 5.87 (bt, 1H), 4.42 (d, 2H, J ¼ 5.7 Hz), 3.56
(q, 1H, J ¼ 6.9 Hz), 3.39 (t, 4H, J ¼ 6.0 Hz), 3.02 (s, 3H), 1.75 (m, 4H),
(400 MHz, CDCl3) d 8.61e8.56 (m, 2H), 7.77 (m, 1H), 7.62 (d, 1H,
J ¼ 7.6 Hz), 7.48 (dd, 1H, J ¼ 8.0 and 8.0 Hz), 7.31 (m, 1H), 7.21 (d, 1H,
J ¼ 7.6 Hz), 7.09e6.92 (m, 2H), 5.88 (bt, 1H), 5.47e5.37 (m, 2H),

H. Ryu et al. / European Journal of Medicinal Chemistry 93 (2015) 101e108
107
4.43e4.30 (m, 2H), 3.49 (q, 1H, J ¼ 6.8 Hz), 3.03 (s, 3H), 1.28 (d, 3H,
J ¼ 6.8 Hz); MS (FAB) m/z 543 (MþH).
4.1.2.26. N-((2-(4-Methylbenzylthio)-6-(trifluoromethyl)pyridin-3-
yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(45). 81% yield, white solid, 1H NMR (300 MHz, CDCl3)
d 8.43 (s,
1H), 7.54e7.49 (m, 2H), 7.23e7.15 (m, 2H), 6.63 (bs, 1H), 4.67e4.52
(dq, 2H, J ¼ 13.5 and 4.4 Hz), 3.68 (q, 1H, J ¼ 7.5 Hz), 3.01 (s, 3H),
2.96e2.83 (m, 4H), 1.74e1.60 (m, 4H), 1.59 (m, 2H), 1.55 (d, 2H,
J ¼ 7.1 Hz); MS (FAB) m/z 503 (MþH).
4.1.2.19. N-((6-(Chlorodifluoromethyl)-2-(pyridin-2-ylmethoxy)pyr-
idin-3-yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)prop-
anamide (38). 52% yield, white solid, mp ¼ 67 ꢀC, 1H NMR
(400 MHz, CDCl3)
d
8.62 (d, 1H, J ¼ 4.4 Hz), 7.75 (dd, 1H, J ¼ 7.6 and
7.6 Hz), 7.67 (d, 1H, J ¼ 7.2 Hz), 7.51e7.41 (m, 2H), 7.27e7.19 (m, 2H),
7.13 (dd, 1H, J ¼ 11.2 and 1.6 Hz), 7.04 (d, 1H, J ¼ 8.4 Hz), 6.50 (bs,
1H), 5.63e5.48 (m, 2H), 4.61e4.40 (m, 2H), 3.60 (q, 1H, J ¼ 7.2 Hz),
3.05 (s, 3H), 1.49 (d, 3H, J ¼ 7.2 Hz); MS (FAB) m/z 543 (MþH).
4.1.2.27. N-((4-Phenyl-6-(trifluoromethyl)pyridin-3-yl)methyl)-2-(3-
fluoro-4-(methylsulfonamido)phenyl)propanamide (46). 54% yield,
colorless oil, 1H NMR (300 MHz, CD3OD)
d 8.55 (s, 1H), 7.46e7.53
(m, 5H), 7.24e7.27 (m, 2H), 7.05 (dd, 1H, J ¼ 11.1 and 1.8 Hz), 6.99 (d,
1H, J ¼ 8.1 Hz), 6.62 (bs 1H), 5.67 (bt, 1H), 4.49 (d, 2H, J ¼ 5.7 Hz),
3.45 (q,1H, J ¼ 6.9 Hz), 3.04 (s, 3H), 1.45 (d, 3H, J ¼ 6.9 Hz); MS (FAB)
m/z 496 (MþH).
4.1.2.20. N-((6-(Chlorodifluoromethyl)-2-(cyclohexylthio)pyridin-3-
yl)methyl)-2-(3-fluoro-4-(methylsulfonamido)phenyl)propanamide
(39). 66% yield, white solid, mp ¼ 70e73 ꢀC, 1H NMR (300 MHz,
CD3OD)
d
7.35e7.48 (m, 3H), 7.26 (d, 1H, J ¼ 7.8 Hz), 7.16 (dd, 1H,
4.2. Biological assay
J ¼ 1.8 and 11.1 Hz), 7.10 (d, 1H, J ¼ 8.4 Hz), 6.13 (bs, 1H), 4.35 (d, 2H,
J ¼ 5.7 Hz), 3.82 (m,1H), 3.56 (q,1H, J ¼ 7.2 Hz), 3.02 (s, 3H), 2.06 (m,
2H), 1.75 (m, 2H), 1.49 (d, 3H, J ¼ 7.2 Hz), 1.26e1.33 (m, 6H); MS
(FAB) m/z 550 (MþH).
The methods for in vitro and in vivo assays were reported in the
previous literature [16,17]. All animal protocols were approved by
the institutional review committee at Grunenthal Innovations.
4.1.2.21. N-((4-Methyl-2-(4-methylpiperidin-1-yl)-6-(tri-
fluoromethyl)pyridin-3-yl)methyl)-2-(3-fluoro-4-(methyl-
sulfonamido)phenyl)propanamide (40). 66% yield, white solid, 1H
Acknowledgments
This research was supported by Research Grants from Grunen-
thal in Germany, Grants from the National Research Foundation of
Korea (NRF) (R11-2007-107-02001-0) and the Korea Science and
Engineering Foundation (KOSEF) (2014M3A9B5073755) in South
Korea, and in part by the Intramural Research Program of NIH,
Center for Cancer Research, NCI (Project Z1A BC 005270) in USA.
NMR (300 MHz, CDCl3)
d
7.51e7.46 (t, 1H, J ¼ 8.0 Hz), 7.14 (s, 1H),
7.07e7.02 (m, 2H), 6.80 (brs, 1H), 4.52 (d, 2H, J ¼ 5.3 Hz), 3.48 (q,
1H), 3.17 (d, 1H, J ¼ 11.9 Hz), 3.02 (s, 3H), 2.78 (tt, 2H, J ¼ 11.0 and
2.6 Hz), 2.38 (s, 3H), 1.71 (m, 2H), 1.47 (d, 3H, J ¼ 7.0 Hz), 1.26e1.13
(m, 2H), 0.97 (d, 3H, J ¼ 6.6 Hz), 0.88 (m, 1H); MS (FAB) m/z 531
(MþH).
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d
7.51 (t, 1H, J ¼ 8.3 Hz), 7.03 (m, 3H), 5.96 (bs,
1H), 5.43 (m, 1H), 4.45e4.33 (m, 2H), 3.46 (q, 1H, J ¼ 7.1 Hz), 3.02 (s,
3H), 2.48 (s, 3H), 1.99e1.96 (m, 2H), 1.65e1.55 (m, 6H), 1.46 (d, 3H,
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(bs, 1H), 4.59e4.54 (dq, 2H, J ¼ 12.1 and 4.2 Hz), 3.68 (q, 1H,
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