Optimization of the first small-molecule relaxin/insulin-like family peptide receptor (RXFP1) agonists: Activation results in an antifibrotic gene expression profile

Article abstract of DOI:10.1016/j.ejmech.2018.06.008

A dose responsive quantitative high throughput screen (qHTS) of >350,000 compounds against a human relaxin/insulin-like family peptide receptor (RXFP1) transfected HEK293 cell line identified 2-acetamido-N-phenylbenzamides 1 and 3 with modest agonist acti

Full text of DOI:10.1016/j.ejmech.2018.06.008

European Journal of Medicinal Chemistry 156 (2018) 79e92
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Optimization of the first small-molecule relaxin/insulin-like family
peptide receptor (RXFP1) agonists: Activation results in an antifibrotic
gene expression profile
Kenneth J. Wilson a, Jingbo Xiao a, Catherine Z. Chen a, Zaohua Huang b,
Irina U. Agoulnik b, Marc Ferrer a, Noel Southall a, Xin Hu a, Wei Zheng a, Xin Xu a,
Amy Wang a, Courtney Myhr b, Elena Barnaeva a, Emmett R. George a,
,
, *
Alexander I. Agoulnik b **, Juan J. Marugan a
a NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville,
MD, 20850, USA
b Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, 11200 SW 8th Street, HLSI 419B,
Miami, FL, 33199, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A dose responsive quantitative high throughput screen (qHTS) of >350,000 compounds against a human
relaxin/insulin-like family peptide receptor (RXFP1) transfected HEK293 cell line identified 2-acetamido-
N-phenylbenzamides 1 and 3 with modest agonist activity. An extensive structure-activity study has
been undertaken to optimize the potency, efficacy, and physical properties of the series, resulting in the
identification of compound 65 (ML-290), which has excellent in vivo PK properties with high levels of
systemic exposure. This series, exemplified by 65, has produced first-in-class small-molecule agonists of
RXFP1 and is a potent activator of anti-fibrotic genes.
Received 27 February 2018
Received in revised form
25 May 2018
Accepted 2 June 2018
Available online 7 June 2018
Keywords:
Relaxin
© 2018 Elsevier Masson SAS. All rights reserved.
G-protein coupled receptor
Antifibrotic
2-Acetamido-N-Phenylbenzamide
1. Introduction
males and females, which suggest the pharmacological utility of
relaxin as a modulator of cardiovascular and renal functions in
The small-peptide hormone relaxin was discovered in 1926 and
is primarily associated with pregnancy, due to its effects to relax
pubic ligaments and soften the cervix to facilitate parturition. [1],
[2] Since then it has been shown that blood concentrations of
relaxin rise during the first trimester of pregnancy, stimulating
cardiovascular and renal adjustments to accommodate the
increased nutritional demands of the growing fetus and the
elevated requirements for renal clearance of metabolic waste [3].
Relaxin production increases cardiac output, arterial compliance,
renal blood flow and a decrease in systemic vascular resistance
during pregnancy [4e6]. Both clinical and non-clinical studies us-
ing this hormone reinforce these cardiovascular effects in both
humans. [7], [8].
The target of relaxin is a group of G-protein coupled receptors,
relaxin/insulin-like family peptide receptor (RXFP). [9], [10] Some
of the physiological effects of relaxin are mediated by its interaction
with RXFP1, which modulates several signal transduction path-
ways. [11], [12] Relaxin activation of RXFP1 up-regulates the
endothelin system which leads to vasodilation. Activation also
promotes extracellular matrix remodeling through regulation of
collagen deposition, proliferation, cell invasiveness, and perhaps
most importantly, overall tissue homeostasis. It also moderates
inflammation by reducing levels of inflammatory cytokines such as
TNF-a and TGF-b, as well as the induction of angiogenesis via the
activation of transcription of vascular endothelial growth factor
(VEGF) [13].
Understanding the biological effects of RXFP1 activation by
relaxin has led to the evaluation of relaxin as a pharmacologic agent
for the treatment of patients with acute heart failure (AHF),
* Corresponding author.
** Corresponding author.
E-mail address: maruganj@mail.nih.gov (J.J. Marugan).
https://doi.org/10.1016/j.ejmech.2018.06.008
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

80
K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
preeclampsia, and hypertensive disease. [6], [14] A recent phase 3
clinical trial utilizing recombinant human relaxin-2 (serelaxin) met
its primary endpoint of improving dysponea through the fifth day
in patients admitted for acute heart failure. [15], [16] We have
previously reported the identification and evaluation of a series of
small-molecule human relaxin agonists. [17], [18] Optimized
compounds from this series are potent and highly selective acti-
vators of RXFP1 with similar efficacy to the natural hormone in
functional assays. [19], [20] Using quantitative high-throughput
screening, over 350,000 compounds from our Molecular Libraries
Small Molecule Repository (MLSMR) were screened in an effort to
identify small-molecule agonists of RXFP1 [21]. This small molecule
library was created by the NIH to combine chemical resources from
both academic and private institutions to increase chemical space
diversity of compounds for a large variety of screening campaigns.
In this disclosure we present the syntheses, SAR studies, and
compound optimization which led to the identification of preclin-
ical candidate 65.
Scheme 1. Synthesis of the Western Cyclohexyl and Optimized Analogues.
Table 1
SAR of the cyclohexyl-2-acetamido-N-phenylbenzamide Series.
2. Results and discussion
The ability of these compounds to increase cyclic adenosine
monophosphate (cAMP) levels in a human RXFP1 transfected
HEK293 cell line was measured, and two compounds (1 and 3),
both having a unifying molecular motif of 2-acetamido-N-phenyl-
benzamide were discovered (Fig. 1) [17].
The activity is reported through two parameters: EC50 (con-
centration necessary to reach 50% of maximum cAMP signal) and
maximum response (efficacy indicated as the maximum observed
increase in cAMP response), which were used to perform SAR
analysis. Efficacies were normalized to forskolin, which had an EC50
of 47 nM and a defined maximum response of 100% [22].
Cmpd
R
EC50
(
m
M)a
Max. Responseb
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
3-trifluoromelhylphenyl
4-trifluoromethylphenyl
phenyl
2-methylphenyl
3-methylpheny!
4-methyphenyl
3-tert-butlyphenyl
3-nitrophenyl
3-fluorophenyl
3-chlorophenyl
1.88
94.0
94.0
inactive
37.4
187
2.66
5.93
13.3
3.34
2.65
37.4
5.29
1.88
1.06
inactive
2.65
92%
46%
57%
N/A
65%
32%
70%
93%
81%
89%
91%
74%
84%
90%
87%
N/A
74%
3-bromophenyl
Initial measurements of aqueous kinetic solubility of 1 and 3
(1.6 mg/mL, 3.7 mM and 0.9 mg/mL, 2.2 mM respectively) show poor
3-methoxy phenyl
3-thiomelhylphenyl
3-trifluoromelhylhiophenyl
3-trifluoromethylsulfonylphenyl
3-biphenyl
solubility for these compounds. However, in assessing the stability
of compounds 1 and 3 in mouse liver microsomes, compound 3
demonstrated very good stability after 60 min (70% parent
remaining) versus 1 (2% parent remaining), so these data and the
inherent problems associated with furans in mammalian meta-
bolism led us to focus on the optimization of the cyclohexyl series
based on compound 3 [23]. The general synthetic scheme to these
is represented in Scheme 1.
20
inactive
N/A
Reagents and Conditions: For 3e22, 62e67: (a) acid chloride,
DCM, TEA, 0 ꢀC to RT, 1e3 h; (b) substituted aniline or benzylamine,
2 M AlMe3 in toluene, 100 ꢀC, 16 h; (c) For 17: 16, DCM, MCPBA, RT,
12 h; (d) For 18: 13, phenylboronic acid, Pd(PPh3)4, 2 M Na2CO3,
DMF, microwave irrad., 100 οC, 1 h; For 19: 13, 2-(tri-
fluoromethylphenyl)boronic acid, Pd(PPh3)4, 2 M Na2CO3, DMF,
microwave irrad., 100 ꢀC, 1 h; For 20: 13, 3-(trifluoromethylphenyl)
21
22
inactive
9.40
N/A
45%
boronic acid, Pd(PPh3)4, 2 M Na2CO3, DMF, m
W., 100 ꢀC, 1 h; The first
series of compounds 3e22 (Table 1) were prepared in a straight-
forward manner from 2 (see Experimental Standard Procedures),
with the first amide formation performed by reaction with cyclo-
hexylcarbonyl chloride followed by subsequent AlMe3 mediated
a
The EC50 is expressed in micromolar and is the concentration necessary to reach
50% of maximum cAMP signal.
b
The maximum response is the efficacy indicated as the maximum observed
increase in cAMP response.
second amide formation with the appropriate aniline or amine. This
direct amidation using an aniline and an ester proved to be robust
and broad in scope. Compounds 18e20 were prepared from bromo-
compound 13 using standard Suzuki conditions.
Table 1 discloses the initial results of our SAR studies. It is clear
upon viewing the data that the 3-position of the pendant R-phenyl
Fig. 1. Initial RXFP1 hits from the Molecular Probe Center Network Library.

K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
81
Table 2
group provides the best agonism activity. Most of the compounds
have single digit micromolar acivity, except for 7 (37.4 M), 18 and
SAR of the western hemisphere N-3-trifluorophenylbenzamide Series.
m
20 (both inactive). Interestingly, the 3-trifluoromethylbiphenyl
derivative 19 is active (EC50 ¼ 2.65 mM), leading us to believe that
the trifluoromethyl moiety in this part of the molecule could be
important by promoting the activation of RXFP1. This is further
supported by compounds 16 and 17, the latter tri-
fluoromethylsulfonyl compound being the most potent in this set,
Cmpd
R
EC50
(
m
M)a
Max. Responseb
having an EC50 of 1.06 mM with an 87% of maximal activity. It is also
worth noting that tert-butyl compound 9 maintains activity, and
that less polar substituents may be preferred over more polar ones
such as the 3-methoxy derivative 14.
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
3-pentyl
1-butyl
cyclopropyl
cyclobutyl
cyclopentyl
cycloheptyl
benzyl
3-pyridyl
5.29
5.29
8.38
2.97
2.36
2.11
8.38
5.93
9.40
1.32
1.88
59%
94%
79%
75%
88%
75%
86%
95%
71%
64%
94%
99%
90%
92%
96%
94%
99%
42%
98%
The unsubstituted phenyl compound 5 is minimally active as is
4-trifluoromethylsulfonyl phenyl compound 4. The inactivity of
compound 6 might indicate that substituents at the ortho position
impair agonism. Thus, either no substitution or 2- and 4-phenyl
substitutions are deleterious to RXFP1 activation, with the 3-
substituted phenyl compounds generally being more active.
Drawing parallels to compounds 1 and 3, we combined the cyclo-
hexyl and furanyl chemotypes, exemplified by compound 22, which
retained some activity but did not encourage further investigation.
We then explored the replacement of the cyclohexyl group with
various saturated alkyl rings, alkyl groups and differentially
substituted aryl rings, while holding the 3-trifluoromethylphenyl
eastern hemisphere region of the molecule constant (Scheme 2).
Readily available BOC-protected anthranilic acid 23 was coupled to
3-trifluoromethylaniline and deprotected to afford 24a (which is
also commercially available), and then converted to the amides
25e44 b y reacting with the appropriate acid chloride, or coupled
with the apprpriate carboxylic acid in the case of the cycloalkyl
isosteres 27e30 (Table 2). The ethers 43e44 required an additional
synthetic step using CuI catalyzed coupling of either 2-
methoxyethan-1-ol or dimethylaminoethan-1-ol with iodo-
compound 24b. It is evident from the data in Table 2 that the ring
size of R is important. The cycloalkyl analogues 28e30 maintain
good potency while the smaller cyclopropyl adduct 27 experiences
a significant (>4-fold) reduction in activity from the parent com-
pound 3.
4-methoxyphenyl
phenyl
3-methoxyphenyl
2-melhoxyphenyl
2-methylphenyl
2-chlorophenyl
2-trifluoromethoxyphonyl
2-ethoxyphenyl
2-lsopropoxyphenyl
2,6-dimethoxyphenyl
0.334
4,20
3.74
1.18
0.265
0.471
7.47
0.747
44
3.74
92%
a
The EC50 is expressed in micromolar and is the concentration necessary to reach
50% of maximum cAMP signal.
b
The maximum response is the efficacy indicated as the maximum observed
increase in cAMP response.
manifest as seen in the activity of micromolar to submicromolar 2-
alkyl ethers 36, 39e41 and 43, with the 2-ethoxy compound 43
having the best activity at EC50 ¼ 265 nM. These data were very
encouraging as we demonstrated the additive effects of the 3-
trifluorophenyl eastern hemisphere and the 2-alkoxyphenyl west-
ern hemisphere.
Imparting more solubility to this series of compounds would be
desirable. Our next objective was to assess whether the central
phenyl ring could tolerate derivatization in the form of pyridine
analogues and retain, if not improve, potency and especially the
solubility.
Reagents and Conditions: (a) 3-trifluoromethylaniline, DCM,
DMAP, EDC, 24 h; (b) TFA, DCM, 0 ꢀC; (c) For 24b: 2-iodobenzoyl
chloride, DCM, TEA, 0 ꢀC, 1 h; (d) For 25e26, 31e42: aryl acid
chloride, DCM, TEA, RT, 2 h; For 27e30: cycloalkyl carboxylic acid,
DMF, EDC, DMAP, 12 h; (e) For 43: 24b, 2-methoxyethan-1-ol,
Cs2CO3, CuI, 1,10-phenanthroline, toluene, sealed tube, 110 ꢀC, 24 h;
For 44: 24b, 2-dimethylaminoethan-1-ol, Cs2CO3, CuI, 1,10-
phenanthroline, toluene, sealed tube, 110 ꢀC, 24 h;
Compounds 27e30 clearly demonstrated a correlation between
activity and ring size (3 < 4 < 5 < 6 > 7, EC50 ¼ 8.38, 2.97, 2.36, 1.88,
The four possible central core pyridine isomers were synthe-
sized using readily available and inexpensive aminopyridines
45e48 (Scheme 3). From our SAR data the 3-sulfonylphenyl was
chosen as a constant for the eastern region and 2-ethoxyphenyl
and 2.11 mM, respectively), with the 6-membered ring being the
optimal size for activity. The branched and linear alkanes 25 and 26
are tolerated but not ideal for agonism activity. Again, as in com-
pound 8, substitution at the 4-position substantially reduces the
activity of compound 33 relative to 2-substitution (compound 36).
The phenyl example 34 retains good activity while the 3-pyridyl 32
is an unfavorable substitution. A clear SAR profile begins to
Scheme 2. Synthesis of 3-Trifluoromethyl Analogues 25e44.
Scheme 3. Synthesis of Pyridine Core Derivatives.

82
K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
Table 3
SAR of pyridine central core Derivatives.
Cmpd
AC
50
Max. Response PBS Solubilitya RLM Stability (t1/2 min)
49 (N1)
50 (N2)
51 (N3)
52 (N4)
1.67
2.97
0.747 97%
4.71 73%
98%
99%
<1 mM
<1 mM
ND
19.85
ND
>30
>30
<1 mM
a
Pion's
mSOL assay for kinetic solubility determination was used. Saturation
shake-flask solubility method was adapted to a 96-well microtiter plate format and
a co-solvent method with n-propanol as the reference compound was utilized.
was chosen for the western hemisphere. The resultant amino-
pyridines 49e52 were then assayed and the results are shown in
Table 3. There is a substantial loss in activity of all but 3-pyridyl
compound 51, which exhibits approximately a 3-fold reduction in
activity from compound 40.
Reagents and Conditions: (a) 2-ethoxybenzoylchloride, DCM,
TEA, 0 οC-RT, 2 h, 11e94%; (b) 3-trifluoromethylsulfonylaniline, 2 M
AlMe3 in toluene, 100 οC, 12 h, 56e75%; The modification of the
central benzamide linker was evaluated next, where the western
region sulfonamide 53, N-methylated amides 54 and 55, amine 57,
thiophene surragate 59, and 1,3-benzamide 61 were prepared us-
ing the standard chemistry detailed in this manuscript (Scheme 4),
and then evaluated in the cAMP assay (Table 4). As our binding
model predicts (vide infra), the 1,3 benzamide linked compound 61
was inactive, as were the N-methylated version 54 and sulfonamide
53. Methylated amide 55 retained weak activity, and surprisingly,
so did the ethylmethoxyaniline 57. The thiophene isostere 59 had
Scheme 4. Benzamide Linker Modifications: Sulfonamide, Amine, N-Methyl and 3-
Acetamido-N-Phenylbenzamide Compounds.
in conjunction with computational modeling analysis allowed us to
develop a predictive model of our small molecule agonists' mode of
binding. (Fig. 2). Fig. 2 shows the binding model of compound 65
(ML-290) to the RXFP1 receptor. The intramolecular hydrogen
bonding interaction of the amide carbonyl to the threonine 660 is
essential for favorable conformational bias within the active site.
The inactivity of compound 54 and to a lesser extent the reduced
activity of 55 (vide supra) clearly demonstrate that the replacement
of the N-amido hydrogen with a methyl group results in the
disruption of the required active site conformation for binding to
RXFP1, as well as altering the critical T660 H-bonding interaction to
one of the sulfonyl oxygens.
The microsome stability, PBS solubility, and toxicity in both rat
and mice were investigated for compounds 62e67. Rat and mouse
microsome stability are excellent for these compounds (Table 5). An
improvement in PBS solubility was also observed for compounds 62
and 64e67. It can be argued that the sulonyl group imparts greater
solubility to these compounds as compared to the more lipophilic
thrifluoromethyl moiety.
As we previously reported, 65 was chosen as our lead compound
for further PK analysis because of favorable profiles which included
[1]: selectivity against HEK293 cells transfected with the RXFP2
receptor [2] decreased adenosine triphosphate (ATP) cytotoxicity,
and [3] especially its ability to activate the transcription of the
known relaxin target gene VEGF in human monocyte leukemia cells
(THP1). [9], [17].
The pharmacokinetics of IV and PO delivery of 65 in mice was
analyzed (Table 6). Compound 65 has an excellent half-life in
plasma by both routes of administration and in heart by IV dosing,
with the exposure in heart being about six times greater than
plasma by IV administration. This is important as organ fibrosis
the best activity (EC50 ¼ 2.35
mM).
Reagents and Conditions: (a) 2-ethoxyphenylsulfonylchloride,
DCM, TEA, 0 ꢀC-RT, 2 h; (b) 3-trifluoromethylsulfonylaniline, 2 M
AlMe3
in
toluene, 100 ꢀC, 1e16 h, 10e82%;
(c)
2-
methoxybenzoylchloride, DCM, TEA, RT; For 54: (d) NaH, DMF,
CH3I, 0 ꢀC, 1.5 h, then (b); For 55: (b), then (d); (e) 3-
trifluoromethylsulfonylaniline, DCM, TEA, 0 ꢀC-RT; (f) 2-
dimethylaminoethan-1-ol, Cs2CO3, CuI, 1,10-phenanthroline,
toluene, sealed tube, 110 ꢀC, 24 h; A clear picture had emerged of
the optimal SAR requirements for the 2-acetamido-N-phenyl-
benzamide series to effect RXFP1 activation. From the SAR data we
determined that the 2-alkoxyphenyl western hemisphere and 3-
trifluoromethylsulfonyl eastern hemisphere groups offered the
best opportunity for discovering the most potent compounds, and
gratifyingly, they did. Compounds 62e67 were synthesized ac-
cording to the procedure in Scheme 1. The assay results of these
compounds are shown in Table 5. The larger (65e67) or more hy-
drophobic alkyl groups (such as 64) confer the best activity, e.g., 62
or 63 (EC50 ¼ 0.188
(EC50 ¼ 0.047 M). That the 3-trifluoromethylsulfonyl is a superior
pharmacophore for activity to 3-trifluoromethyl on the eastern
region phenyl is confirmed comparing 36 to 62 (EC50 ¼ 0.334
versus EC50 ¼ 0.188 M), 40 to 63 (EC50 ¼ 0.265 versus
EC50 ¼ 0.188 M), and especially the obvious contrast between 41
and 65 (EC50 ¼ 0.471 M).
mM) as compared to compound 67
m
mM
m
mM
m
mM versus EC50 ¼ 0.094
m
We have previously reported the structural elucidation and
insight into the activation of the activation of human relaxin family
peptide receptor by 65 [18]. Exhaustive site-directed mutagenesis

K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
83
Table 4
SAR of benzamide linker Modifications.
Cmpd
53
R
1
R
R3
R4
Linker
EC50
(
m
M)a
Max.b Response
2
H
H
SO CF
2
inactive
N
/A
3
54
55
CH
H
CF
3
inactive
N
/
A
3
H
CH
CF
3
29.7
77%
3
57
59
H
H
H
H
OF
,
59.3
2.36
63%
95%
SO CF
2
3
61
H
H
CF
3
inactive
N/A
a
The EC50 is expressed in micromolar and is the concentration necessary to reach 50% of maximum cAMP signal.
The maximum response is the efficacy indicated as the maximum observed increase in cAMP response.
b
Table 5
SAR of optimized 2-acetamido-N-Phenylbenzamide compounds including ML-290.
Cmpd
R
EC50
(m
M)a
Max. Responseb
ATP toxicity EC50
(
m
M)c
PBS Solubility (
m
M)d
Stability(t1/2 min)
RLM
MLMe
62
63
64
65
66
67
OMe
OEt
OCH CF
2
0.188
0.188
0.067
0.094
99%
99%
97%
98%
98%
98%
29.7
18.8
29.7
18.8
9.4
6.3
<1.1
3.3
7.0
17.0
5.3
>30
>30
>30
28.2
26.8
19.5
ND
1732
100
122
133
178
3
OCH
(
CH
)
C
3
2
OCH CH
2
H
3
0.052
2
OBu
0.047
59.3
a
The EC50 is expressed in micromolar and is the concentration necessary to reach 50% of maximum cAMP signal.
The maximum response is the efficacy indicated as the maximum observed increase in cAMP response.
b
c
Cytotoxicity assay to measure the effect of compounds on cell viability by measuring ATP levels (ATPLite; Promega, Madison, WI).
d
e
Kinetic solubility analysis in PBS solution performed by Analiza (www.analiza.com).
Rodent microsomal stability analysis performed by Pharmaron (www.pharmaron.com).
(heart, liver, lung, etc) is an important indication of therapeutic
intervention. While the plasma clearance is high, the high volume
of distribution probably imparts the half-life observed. Importantly,
no toxicity or abnormal behavior were detected. We have recently
performed a maximum tolerated PO administration) dose study on
C57/Bl6 mice (5 day with 5 day post-dosing observation), with no
mortality observed at 90, 300, and 3000 mg/kg (see supplementary
material). No obvious abnormalities were noted post-necropsy in
any of the in vivo mammalian studies.
Previously described antifibrotic effects of relaxin in various
organs were associated with changes in transcriptional activity of
key genes involved in extracellular matrix production and turnover
[24]. In fibrotic liver, hepatic stellate cells (HSCs) become activated
and differentiate into profibrogenic myofibroblast-like cells.
Importantly, RXFP1 gene expression is significantly upregulated in
activated HSCs [25]. It has been shown that relaxin treatment of
activated HSCs leads to a significant decrease in profibrotic genes
such as smooth muscle actin (ACTA2), transforming growth factor

84
K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
Replacing the central phenyl ring with either a thiophene or pyri-
dine is tolerated although a decrease in activity is observed. The
western hemisphere amide is critical for the activity.
For the western region, phenyl, pyridine and cycloalkanes of
varying ring size are acceptable, but a six membered ring is optimal.
An electron-donating substituent on the aromatic ring is tolerated
at both the ortho- and meta-positions, with ortho-alkoxy having the
best activity. At the eastern phenyl position, a meta-substitution is
optimal and the trifluoromethylsulfonyl is best as
a critical
hydrogen bond interaction is formed between the sulfonyl oxygens
and threonine 660.
Thus, the first small-molecule preclinical candidate agonist for
the relaxin hormone receptor has been optimized through a me-
dicinal chemistry approach and the resulting SAR is supported by a
later-stage binding model obtained by iterative site-directed
mutagenesis.
4. Experimental section
Fig. 2. Predicted model of ML290 bound in the TM binding site of hRXFP1. The re-
ceptor hRXFP1 is shown in ribbon (white) and key residues are shown in sticks
(brown). In addition to an intramolecular H-bond, ML290 (sticks in cyan) forms a
hydrogen-bond with the backbone N atom of residue T660 in ECL3 (magenta) [18]. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the Web version of this article.)
Biology. Materials and Methods. Primary human hepatic stellate
cells (HSCs) were grown on plastic in hepatic stellate growth media
provided by the manufacturer (Zen-Bio Inc., Research Triangle Park,
NC). Cells were seeded overnight in 35 mm poly-L-lysine-coated
dishes (Corning, Durham, NC) at a density of 5 ꢁ 104 cells per dish
and treated in triplicates with either 5 mM of ML290 in DMSO
(ML290 treatment) or DMSO (control) for 24 or 72 h. Total RNA was
extracted from cells samples with RNeasy kit (Qiagen, German-
town, MD). Quality and concentration of RNA was analyzed with a
Nanovue (GE Healthcare Biosciences, Pittsburgh, PA). cDNA was
synthesized using a Verso cDNA kit (Thermo Scientific, Fair Lawn,
NJ). Quantitative PCR (qPCR) was performed using GoTaq qPCR
Master Mix (Promega, Madison, WI), and gene specific primers in a
RealPlex [2] Mastercycler (Eppendorf, Westbury, NY) according to
the manufacturer's instructions. Values were normalized to GAPDH
gene expression. The relative fold change in mRNA level was
beta-1 (TGFB1), connective tissue growth factor (CTGF), and several
collagens, but at the same time stimulates expression of matrix
metalloproteinase 1 (MMP1) and peroxisome proliferator-activated
receptor gamma (PPARG). [26], [27].
Therefore, we have tested the effect of ML290 on primary hu-
man HSC gene expression (Fig. 3). Cells were cultured on plastic,
which induces and maintains activation of HSCs [28]. Upon treat-
ment with 5 mM of ML290, we observed a clear anti-fibrotic,
relaxin-like gene expression pattern at 24 and 72 h. Similar re-
sults have been recently reported by another group in the HSC LX-
2 cell line using ML-290 [29].
DD
calculated by the comparative Ct (2e Ct) method. Results were
analyzed using GraphPad Prism software (GraphPad Software, La
Jolla, CA) for statistical significance using Student's t-test.
3. Conclusion
Measurements of cAMP concentration. cAMP assay was per-
formed using HTRF cAMP HiRange kit (CisBio, Bedford, MA, USA).
The THP1 and HEK293 cells (ATCC, Manassas, VA, USA) stably or
transiently transfected with human RXFP1, RXFP2 or AVPR1B re-
ceptor were stimulated with relaxin, the compounds or forskolin
for 30 min at 37 ꢀC, 5% CO2, after which, 8 mL per well of each HTRF
detection reagent (diluted according to assay kit directions in HTRF
lysis buffer) was added. [30], [31] The plates were incubated for
30 min at room temperature, and the signal was read on a ViewLux
(PerkinElmer, Waltham, MA, USA) or a FLUOstar Omega (BMG
Optimization of two lead compounds discovered by an exten-
sive screening campaign of over 350,000 compounds at the Na-
tional Center for the Advancement of Translational Science resulted
in a series of potent, stable, selective and well-tolerated compounds
which exhibit excellent systemic exposure in plasma and especially
heart.
To summarize the SAR of the compounds presented here, the
core aromatic ring must be ortho substituted to maintain activity.
Table 6
Pharmacokinetic parameters for compound 65 in male C57/Bl6 mice.
Dosing Level (mg/kg) CL obsa(mL/min/kg) t1/2b(h) C0
(
m
g/mL) Cmax
(m
g/L) AUClast
(
m
g-h/mL) AUCinf (m
g-h/mL) MRTlastg(h) Vss obsh(L/kg) Fi(%)
c
d
e
f
Route
IV (plasma)
PO (plasma) 30
IV (heart)
PO (heart)
3
67.2
ND
8.78
ND
6.6
5.5
6.3
1.0
0.49
ND
2.93
ND
ND
0.30
ND
0.70
0.98
5.19
2.63
0.74
1.00
5.69
2.68
4.4
3.1
5.5
2.2
24.5
ND
4.2
ND
14
ND
ND
3
30
1.03
ND
a
Total clearance after IV administration.
Half-life.
Concentration.
Maximum serum concentration.
Area under the curve.
Area under the curve.
Mean residense time.
Volume of distribution in steady state.
Bioavailability.
b
c
d
e
f
g
h
i

K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
85
Table 7
Primer sequences used in qPCR.
Gene
Forward primer
Reverse primer
GAPDHa
ACTA2b
COL1A1c
CTGFd
AGCCACATCGCTCAGACAC
AAGCACAGAGCAAAAGAGGAAT
AGACAGTGATTGAATACAAAACCA GGAGTTTACAGGAAGCAGACA
CCCAGACCCAACTATGATTAGAG
GCACTGAGAAAGAAGACAAAGG
CATACAGGATCATTGGCTACAC
GCCCAATACGACCAAATCCc
ATGTCGTCCCAGTTGGTGAT
AGGCGTTGTCATTGGTAACC
CTAAGTCCACATCTTGCTCTTG
TCACATCGCTCCAGACTTG
TGAGAGGGCCAAGCAAAG
GTCCCCTGTGCCTTGATG
MMP1e
MMP2f
PPARGC1Ag ATAAATCACACGGCGCTCTT
TGFB1h
CACTCCCACTCCCTCTCTC
a
Glyceraldehyde-3-phosphate dehydrogenase.
Alpha-acti-2.
Collagen.
Connective tissue growth factor.
Matrix metallopeptidase 1.
Matrix metallopeptidase2.
Peroxisome proliferative activated receptor, gamma, coactivator 1 alpha.
Volume of distribution in steady state i Transforming growth factor beta-1.
b
c
d
e
f
g
h
preparative HPLC system (Waters Corp., Milford, Mass.). The col-
umn used was a Phenomenex Luna C18 (5
m, 30 ꢁ 75 mm; Phe-
m
nomenex, Inc., Torrance, Calif.) at a flow rate of 45.0 mL/min. The
mobile phase consisted of acetonitrile and water (each containing
0.1% trifluoroacetic acid). A gradient of 10%e50% acetonitrile over
8 min was used during the purification. Fraction collection was
triggered by UV detection at 220 nM. Analytical analysis was per-
formed on an Agilent LC/MS (Agilent Technologies, Santa Clara,
Calif.). Method 1 (t1): A 7-min gradient of 4%e100% acetonitrile
(containing 0.025% trifluoroacetic acid) in water (containing 0.05%
trifluoroacetic acid) was used with an 8-min run time at a flow rate
of 1.0 mL/min. Method 2 (t2): A 3-min gradient of 4%e100%
acetonitrile (containing 0.025% trifluoroacetic acid) in water (con-
taining 0.05% trifluoroacetic acid) was used with a 4.5-min run time
Fig. 3. ML290 activates an antifibrotic gene expression profile in activated human
hepatic stellate cells. HSCs were treated with 5 M of ML290 or DMSO for 24 and 72 h.
Gene expression was evaluated by quantitative RT-PCR, normalized to GAPDH gene
expression and presented as the fold change vs DMSO-treated control. N ¼ 3, *p < 0.05,
**p < 0.01.
m
at a flow rate of 1.0 mL/min. A Phenomenex Luna C18 column (3 mm,
3 ꢁ 75 mm) was used at a temperature of 50 ꢀC. Purity determi-
nation was performed using an Agilent diode array detector for
both Method 1 and Method 2. Mass determination was performed
using an Agilent 6130 mass spectrometer with electrospray ioni-
zation in positive detection mode. 1H NMR spectra were recorded
on Varian 400 MHz spectrometers (Agilent Technologies, Santa
Clara, Calif.). Chemical shifts are reported in ppm with undeu-
terated solvent (DMSO‑d6 at 2.49 ppm) as internal standard for
DMSO‑d6 solutions. All of the analogues tested in the biological
assays have a purity of greater than 95% based on both analytical
methods. High resolution mass spectrometry was recorded on
Agilent 6210 Time-of-Flight (TOP) LC/MS system. Confirmation of
molecular formula was accomplished using electrospray ionization
in the positive mode with Agilent Masshunter software (Version
B.02).
Labtech, Cary, NC, USA) plate readers. Nonlinear regressions to the
Hill equation were performed using Prism software (GraphPad
Software, San Diego, CA, USA).
Modulation of HSC gene expression. Primary human hepatic
stellate cells (HSCs) were grown on plastic in hepatic stellate
growth media provided by the manufacturer (Zen-Bio Inc.,
Research Triangle Park, NC). Cells were seeded overnight in 35 mm
poly-
L-lysine-coated dishes (Corning, Durham, NC) at a density of
5ꢁ104 cells per dish and treated in triplicates with either 5
mM of
ML290 in DMSO (ML290 treatment) or DMSO (control) for 24 or
72 h. Total RNA was extracted from cells samples with RNeasy kit
(Qiagen, Germantown, MD). Quality and concentration of RNA was
analyzed with a Nanovue (GE Healthcare Biosciences, Pittsburgh,
PA). cDNA was synthesized using a Verso cDNA kit (Thermo Sci-
entific, Fair Lawn, NJ). Quantitative PCR (qPCR) was performed us-
ing GoTaq qPCR Master Mix (Promega, Madison, WI), and gene
specific primers in a RealPlex2 Mastercycler (Eppendorf, Westbury,
NY) according to the manufacturer's instructions. Values were
normalized to GAPDH gene expression. The relative fold change in
mRNA level was calculated by the comparative Ct (2eDDCt)
method. Results were analyzed using GraphPad Prism software
(GraphPad Software, La Jolla, CA) for statistical significance using
Student's t-test. The sequences of the primers are shown in Table 7.
Chemistry. General Methods. All air or moisture sensitive re-
actions were performed under positive pressure of nitrogen with
oven-dried glassware. Anhydrous solvents such as dichloro-
methane, N,N-dimethylformamide (DMF), acetonitrile, methanol
and tetrahydrofuran were purchased from Sigma-Aldrich (St. Louis,
Mo.). Preparative purification was performed on a Waters semi-
General Protocol A. A solution of methyl or ethyl benzoate
(0.191 mmol) and amine (0.383 mmol) in toluene (2.00 mL) was
treated at room temperature with AlMe3 (0.192 mL, 2.0 M in
toluene, 0.384 mmol). The reaction mixture was stirred overnight
at 100 ꢀC and then quenched with 100
mL of water. The mixture was
concentrated, redissolved in 2.00 mL of DMSO, filtered and purified
via C18 reverse phase HPLC to give the final product.
General Protocol B. A solution of carboxylic acid (0.178 mmol) in
DMF (2.00 mL) was treated at room temperature with 2-amino-N-
(3-(trifluoromethyl)pheny1)benzamide (25.0 mg, 0.089 mmol) fol-
lowed by EDC (17. l mg 0.089 mmol) and DMAP (10.9 mg, 0.089
mnol). The reaction mixture was stirred overnight at room tem-
perature. The mixture was purified via C18 reverse phase HPLC to
give the final product.
General Protocol C.
fluoromethyl)phenyl)benzamide
A
solution of 2-amino-N-(3- (tri-
(50.0 mg, 0.178 mmol) in

86
K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
dichloromethane (2.00 mL) and TEA (0.075 mL, 0.535 mmol) was
treated at room temperature with carbonyl chloride (0.357 mmol).
The reaction mixture was stirred at room temperature for 2 h. The
mixture was concentrated, re-dissolved in 2.00 mL of DMSO,
filtered and purified via C18 reverse phase HPLC to give the final
product.
General Protocol D. A solution of carboxylic acid (0.357 mmol) in
DMF (2.00 mL) was added DIPEA (0.093 mL, 0.535 mmol) and HATU
(136 mg, 0.357 mmol). The reaction mixture was stirred at room
temperature for 5 min, followed by 2-amino-N-(3-(tri-
fluoromethyl)-phenyl)benzamide (50.0 mg, 0.178 mmol). The re-
action mixture was stirred overnight at room temperature, filtered
and purified via C18 reverse phase HPLC to give the final product.
7.3, 1.6 Hz, lH), 7.21 (td, J ¼ 7.6, 1.2 Hz, lH), 2.18e2.35 (m, lH), 1.80
(dd, J ¼ l2.7, 2.9 Hz, 2H), 1.68 (dt, J ¼ l2.2, 3.4 Hz, 2H), 1.59 (d,
19
J ¼ 11.9 Hz, lH), 1.01e1.43 (m, 5H);
FNMR (376 MHz, DMSO‑d6)
d
ppm ꢂ60.26 (s, 3 F); 13C NMR (151 MHz, DMSO‑d6)
d 173.94,
167.32, 142.64, 141.12, 137.79, 134.12, 131.86, 131.13, 128.90, 125.96,
123.64, 123.51, 122.03, 120.34, 45.10, 29.02, 25.41, 25.13. HRMS (ESI)
m/z (M þ H) calcd. For C21H22F3N20, 391.1628; found 391.1628.
2-(Cyclohexanecarboxamido)-N-phenylbenzamide (5). The
title compound was prepared according to general protocol A.
Yield: 68% LC-MS Retention Time: t1 (Method 1) 6.326 min; 1H
NMR (400 MHz, DMSO‑d6)
d ppm 10.54 (s, lH), 10.37 (s, lH), 8.23
(dd, J ¼ 8.2, l.0 Hz, lH), 7.77 (dd, J ¼ 7.9, l.5 Hz, lH), 7.63e7.71 (m, 2H),
7.49 (dd, J ¼ 8.4, 7.2, 1.6 Hz, lH), 7.28e7.41 (m, 2H), 7.19 (td, J ¼ 7.6,
1.3 Hz, lH), 7.07e7.14 (m, lH), 2.17e2.34 (m, lH), 1.76e1.88 (m, 2H),
1.69 (dd, J ¼ l2.5, 3.4, 3.2 Hz, 2H), l.59 (d, J ¼ 12. l Hz, lH), 1.03e1.43
General Protocol E.
fluoromethyl)phenyl)benzamide (50.0 mg, 0.145 mmol, 1.0 equiv.)
or N-(3-bromophenyl)-2-(cyclohexanecarboxamido)benzamide
A mixture of 2-bromo-N-(3- (tri-
(m, 5H); 13C NMR (151 MHz, DMSO‑d6)
d 176.98, 170.13, 141.77,
(100 mg, 0.249 mmol, 1.0 equiv.), boronic acid or pinacol ester (2.0
equiv.) and Pd(PPh3)4 (0.05 eq) in DMF (1.50 mL) and 2.0 N Na2CO3
141.35, 134.88, 131.88, 131.73, 127.14, 126.60, 125.99, 124.48, 123.91,
48.42, 43.00, 42.86, 42.72, 42.58, 42.44, 42.30, 42.16, 32.14, 28.48,
28.20; HRMS (ESI) m/z (M þ H) calcd. For C20H23N2O2, 323. l754;
found 323. l758.
(0.50 mL) aqueous solution was heated in m
W at 100 ꢀC for 30 min
to 1 h. The reaction was cooled to room temperature, and a small
portion of Si-THIOL was added to remove palladium. The reaction
mixture was filtered and purified via C18 reverse phase HPLC to
give the final product.
General Protocol F. 2-Iodo-N-(3-(trifluoromethyl) phenyl)ben-
zamide (100 mg, 0.256 mmol), amine (0.767 mmol), and CuCl
(7.59 mg, 0.077 mmol) in DMF (1.00 mL) was stirred at room tem-
perature for 15e30 min. The reaction was treated with a small
portion of Si-THIOL to remove palladium, filtered and purified via
C18 reverse phase HPLC to give the final product.
General Protocol G. A solution of thio-compound (0.255 mmol)
in dichloromethane (3.00 mL) was treated at room temperature
with MCPBA (220 mg, 1.27 mmol). The reaction mixture was stirred
overnight at room temperature. 10% Aqueous NaHSO3 solution was
added to quench excess MCPBA and the mixture was stirred at
room temperature for 15 min. The reaction mixture was partitioned
between dichloromethane and water. The organic layer was sepa-
rated, dried (Na2SO4), filtered, concentrated, and purified via C18
reverse phase HPLC to give the final product.
General Protocol H. A tube was charged with CuI (0.1 equiv.),
1,10-phenanthroline (0.2 equiv.), Cs2CO3 (2.0 equiv.), iodo substrate
(1.0 equiv.) and alcohol (2.0 equiv.) in toluene (2.00 mL) under N2.
The tube was sealed and the reaction mixture was stirred at 110 ꢀC
for 24 h. The resulting mixture was cooled to room temperature
and treated with a small portion of Si-THIOL to remove copper. The
mixture was concentrated, dissolved in 2.00 mL of DMSO, filtered
and purified via C18 reverse phase HPLC to give the final product.
3-(Cyclohexanecarboxamido)-N-(3-(trifluoromethyl)
2-(Cyclohexanecarboxamido)-N-o-tolylbenzamide (6). The
title compound was prepared according to general protocol A.
Yield: 38%; LC-MS Retention Time: t1 (Method 1) ¼ 6.401 min; 1H
NMR (400 MHz, DMSO‑d6) d ppm 11.00 (s, lH),10.09 (s, lH), 8.38 (dd,
J ¼ 8.4, 1.2 Hz, lH), 7.91 (d, J ¼ 7.8 Hz, lH), 7.51 (dd, J ¼ 8.6, 7.2, 1.7 Hz,
lH), 7.25-7.35 (m, 2H), 7.10e7.25 (m, 3H), 2.21 (s, 3H), 2.16e2.28 (m,
lH), 1.77e1.91 (m, 2H), 1.69 (dt, J ¼ l2.4, 3.5 Hz, 2H), l.53-l.65 (m, lH),
0.99e1.44 (m, 5H); 13C NMR (101 MHz, DMSO‑d6)
d 173.86, 167.36,
139.15, 135.79, 134.00, 132.11, 130.36, 128.60, 126.87, 126.43, 126.11,
122.63, 121.31, 120.78, 45.61, 29.06, 25.36, 25.06, 17.81; HRMS (ESI)
m/z (M þ H) calcd. for C21H25N2O2, 337.1911; found 337.1912.
2-(Cyclohexanecarboxamido)-N-m-tolylbenzamide (7). The
title compound was prepared according to general protocol A.
Yield: 55%; LC-MS Retention Time: t1 (Method 1) ¼ 6.632 min; 1H
NMR (400 MHz, DMSO‑d6)
d ppm 10.54 (s, lH), 10.30 (s, lH), 8.24
(dd, J ¼ 8.3, 1.1 Hz, lH), 7.76 (dd, J ¼ 7.8, 1.4 Hz, lH), 7.38e7.54 (m,
3H), 7.10e7.31 (m, 2H), 6.93 (ddd, J ¼ 7.5, l.5, l.l, 0.8 Hz, lH), 2.29 (s,
3H), 2.19e2.3 S (m, lH), 1.82 (dd, J ¼ l2.8, 2.6 Hz, 2H), 1.69 (dt,
J ¼ l2.4, 3.4 Hz, 2H), l.S9 (d, J ¼ l2.3 Hz, lH), 1.04e1.42 (m, 5H); 13C
NMR (101 MHz, DMSO‑d6)
d 173.88, 167.00, 138.53, 138.30, 137.75,
131.75, 128.74, 128.45, 124.76, 123.40, 122.83, 121.39, 121.32, 118.09,
45.33, 29.05, 25.38, 25.07, 21.19; HRMS (ESI) m/z (M þ H) calcd. for
C21H25N2O2, 337.1911; found 337.1908.
2-(Cyclohexanecarboxamido)-N-p-tolylbenzamide (8). The
title compound was prepared according to general protocol A.
Yield: 49%; LC-MS Retention Time: t1 (Method 1) ¼ 6.636 min; 1H
NMR (400 MHz, DMSO‑d6)
d ppm 10.61 (s, 1H), 10.30 (s, 1H), 8.26
phenyl)-benzamide (3). The title compound was prepared ac-
cording to general protocol A. Yield: 35%; LC-MS Retention Time: t1
(dd, J ¼ 8.2, 0.8 Hz, 1H), 7.77 (dd, J ¼ 7.8, 1.4 Hz, 1H), 7.52e7.59 (m,
2H), 7.48 (dd, J ¼ 8.4, 7.2, 1.6 Hz, 1H), 7.18(dd, J ¼ 7.8, 1.2 Hz, 1H),
7.11e7.17 (m, 2H), 2.26 (s, 3H), 2.16e2.36 (m, 1H), 1.82 (dd, J ¼ 13.0,
2.1 Hz, 2H), 1.69 (dd, J ¼ 12.2, 3.3, 3.1 Hz, 2H), l.51e1.64 (m, 1H),
(Method 1) ¼ 6.516 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.53
(s, lH), 10.00 (s, lH), 8.21 (t, J ¼ 2. l Hz, lH), 8.14 (t, J ¼ 2.0 Hz, lH), 8.03
(dd, J ¼ 8.2, 1.2, 1.0 Hz, lH), 7.81 (dd, J ¼ 8. l, 2.2, 1.1 Hz, lH), 7.55e7.64
(m, 2H), 7.37e7.49 (m, 2H), 2.24e2.41 (m, lH), 1.69e1.92 (m, 4H),
1.57e1.69 (m, lH),1.08e1.50 (m, 5H); 19F NMR (376 MHz, DMSO- d6)
1.02e1.47 (m, 5H); 13C NMR (101 MHz, DMSO‑d6)
d 174.29, 167.36,
138.85, 136.50, 133.57, 132.20, 129.45, 129.14, 123.60, 123.23, 121.67,
121.37, 45.84, 29.50, 25.83, 25.54, 20.98; HRMS (ESI) m/z (M þ H)
calcd. for C21H25N2O2, 337.1911; found 337.1916.
N-(3-(tert-Butyl)phenyl)-2-(cyclohexanecarboxamido)ben-
zamide (9). The title compound was prepared according to general
protocol A. Yield: 80%; LC-MS Retention Time: t1 (Method
d
ppm ꢂ61.29 (s, 3 F); 13C NMR (101 MHz, DMSO‑d6)
d 174.40,
167.65, 140.12, 138.19, 132.20, 130.29, 129.90, 129.58, 129.19, 125.92,
125.07, 124.52, 123.62, 122.48, 120.58, 120.54, 117.14, 117.10, 45.47,
29.45, 25.82, 25.50; HRMS (ESI) m/z (M
21H22F3N2O2, 391.1628; found 391.1637.
2-(Cyclohexanecarboxamido)-N-(4-(trifluoromethyl)phenyl-
þ
H) calcd. for
C
1) ¼ 7.324 min; 1H NMR (400 MHz, DMSO‑d6)
d 10.51 (s, 1H), 10.30
(s, 1H), 8.20 (d, J ¼ 8.3 Hz, 1H), 7.77 (d, J ¼ 7.7 Hz, 1H), 7.61 (d,
J ¼ 8.4 Hz, 2H), 7.54e7.45 (m, 1H), 7.27 (dd, J ¼ 8.3, 7.6 Hz, 1H),
7.24e7.11 (m, 2H), 2.30e2.20 (m, 1H), 1.91e1.79 (m, 2H), 1.69 (d,
J ¼ 12.7 Hz, 3H), 1.60 (d, J ¼ 13.3 Hz, 1H), 1.43e1.32 (m, 4H), 1.27 (s,
benzamide (4). The title compound was prepared according to
general protocol A. Yield; 62%; LC-MS Retention Time: t1 (Method
1) ¼ 6.943 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.65 (s, lH),
10.29 (s, lH), 8.09 (dd, J ¼ . 8.4, 1.0 Hz, lH), 7.91 (d, J ¼ 8.6 Hz, 2H),
9H); 13C NMR (101 MHz, DMSO‑d6)
d 173.82, 167.22, 140.35, 137.77,
7.74 (dd, J ¼ 7.8, 1.4 Hz, lH), 7.71 (d, J ¼ 8.4 Hz, 2H), 7.5 l (dd, J ¼ 8.4,
132.75, 131.81, 130.31, 128.73, 124.21, 123.55, 123.04, 121.80, 120.03,

K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
87
118.95, 45.10, 29.00, 25.37, 25.05, 45.46, 33.89, 30.55, 29.44, 25.82,
25.51; HRMS (ESI) m/z (M þ H) calcd. For C24H31N2O2, 379.2380;
found 379.2387.
2-(Cyclohexanecarboxamido)-N-(3-nitrophenyl)benzamide
(10). The title compound was prepared according to general pro-
tocol A. Yield: 30%; LC-MS Retention Time: t1 (Method
2-(Cyclohexanecarboxamido)-N-(3-(methylthio)phenyl)ben-
zamide (15). The title compound was prepared according to gen-
eral protocol A. Yield: 36%; LC-MS Retention Time: t11 (Method
1) ¼ 6.687 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.44 (s, lH),
10.37 (s, lH), 8.18 (d, J ¼ 8.2 Hz, lH), 7.75 (dd, J ¼ 7.8, 1.6 Hz, lH), 7.63
(t, J ¼ 2. l Hz, lH), 7.44e7.55 (m, 2H), 7.29 (t, J ¼ 7.9 Hz, lH), 7.20 (td,
J ¼ 7.6, 1.3 Hz, lH), 7.01 (dd, J ¼ 7.8, 2.0, 1.0 Hz, lH), 2.46 (s, 3H),
2.19e2.35 (m, lH), 1.76e1.90 (m, 2H), 1.65e1.76 (m, 2H), 1.52e1.65
1) ¼ 6.392 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.76 (s, lH),
10.26 (s, lH), 8.69 (t, J ¼ 2.2 Hz, lH), 8.01e8. l5 (m, 2H), 7.95 (dd,
J ¼ 8.3, 2.3, 1.0 Hz, lH), 7.75 (dd, J ¼ 7.8, 1.6 Hz, lH), 7.64 (t, J ¼ 8.2 Hz,
lH), 7.46e7.56 (m, lH), 7.22 (td, J ¼ 7.6, 1.2 Hz, lH), 2.19e2.35 (m, lH),
1.79 (dd, J ¼ 12.5, 2.5 Hz, 2H), 1.68 (dt, J ¼ 3.1 Hz, 2H), 1.58 (d,
(m, lH), 1.03e1.47 (m, 5H); 13C NMR (101 MHz, DMSO‑d6)
d 173.89,
167.05, 139.25, 138.41, 138.08, 131.74, 129.12, 128.70, 123.86, 122.95,
121.60, 121.35, 117.88, 117.23, 45.23, 29.04, 25.38, 25.08, 14.67;
HRMS (ESI) m/z (M þ H) calcd. for C21H25N2O2S, 369.1631; found
369.1625.
J ¼ 12.5 Hz, lH), 1.03e1.43 (m, 5H); 13C NMR (101 MHz, DMSO-d6
)
d
174.40, 167.74, 148.30, 140.58, 138.17, 132.30, 130.49, 129.23,
126.87, 125.04, 123.66, 122.55, 118.72, 115.03, 45.46, 29.44, 25.82,
25.51; HRMS (ESI) m/z (M þ H) calcd. for C20H22N3O4, 368.1605;
found 368.1616.
2-(Cyclohexanecarboxamido)-N-(3-fluorophenyl)benzamide
(11). The title compound was prepared according to general pro-
tocol A. Yield: 73%; LC-MS Retention Time: t1 (Method
2-(Cyclohexanecarboxamido)-N-(3-(trifluoromethyl)phenyl)
benzamide (16). The title compound was prepared according to
general protocol A. Yield: 50%; LC-MS Retention Time: t1 (Method
1) ¼ 7.349 min; 1H NMR (600 MHz, DMSO‑d6)
d 10.58 (s, 1H), 10.28
(s, 1H), 8.12 (d, J ¼ 2.0 Hz, 1H), 8.04 (d, J ¼ 8.2 Hz, 1H), 7.90 (dd,
J ¼ 8.3, 2.2, 1.1 Hz, 1H), 7.72 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.50 (q, J ¼ 7.2,
6.5 Hz, 2H), 7.43 (dt, J ¼ 7.9, 1.3 Hz, 1H), 7.21 (td, J ¼ 7.6, 1.2 Hz, 1H),
2.26 (tt, J ¼ 11.5, 3.6 Hz, 1H), 1.82e1.75 (m, 2H), 1.67 (dt, J ¼ 12.9,
3.5 Hz, 2H), 1.58 (dt, J ¼ 13.0, 3.4 Hz, 1H), 1.37e1.17 (m, 5H), 1.11
(ddd, J ¼ 16.1, 12.4, 7.9, 3.5 Hz, 1H), 1.02 (s, 2H); 13C NMR (151 MHz,
1) ¼ 6.529 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.51 (s, lH),
10.33 (s, lH), 8.12 (dd, J ¼ 8.3, 1.1 Hz, lH), 7.73 (dd, J ¼ 7.7, l.5 Hz, lH),
7.64 (dt, J ¼ ll.7, 2.3 Hz, lH), 7.43e7.55 (m, 2H). 7.37 (td,
J ¼ 8.2.6.7 Hz, lH), 7.20 (td, 7.5, 1.2 Hz, lH), 6.82e6.98 (m, lH),
2.21e2.35 (m, lH), 1.81 (d, J ¼ l1.5 Hz, 2H), 1.69 (dt, J ¼ 12.4, 3.3 Hz,
2H), 1.59 (d, J ¼ 11.7 Hz, lH), 1.02e1.43 (m, 5H); 19F NMR (376 MHz,
DMSO‑d6)
133.33, 131.88, 131.75, 130.35, 127.94, 126.33, 126.18, 125.19, 48.41,
32.09, 28.48, 28.18; HRMS (ESI) m/z (M H) calcd. for
21H22F3N2O2S, 423.1349; found 423.1360.
2-(Cyclohexanecarboxamido)-N-(3((trifluoromethyl)sulfo-
d 177.05, 170.21, 143.28, 140.76, 134.83, 134.25, 133.79,
DMSO‑d6)
d
ppm ꢂ112.20-112.29 (m, 1 F); HRMS (ESI) m/z (M þ H)
þ
calcd. for C20H22FN2O2, 341.1660; found 341.1660.
C
N-(3-Chlorophenyl)-2-(cyclohexanecarboxamido)benzamide
(12). The title compound was prepared according to general pro-
tocol A. Yield: 40%; LC-Retention Time: t1 (Method 1) ¼ 6.898 min;
nyl)phenyl)benzamide (17). The title compound was prepared
according to general protocol G. Yield: 80%; LC-MS Retention Time:
1H NMR (400 MHz, DMSO‑d6)
d
ppm 10.48 (s, lH), 10.31 (s, lH), 8.11
t1 (Method 1) ¼ 6.964 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm
(d, J ¼ 7.8 Hz, lH), 7.85 (t, J ¼ 2.0 Hz, lH), 7.71(dd, J ¼ 7.7, 1.3 Hz, lH),
7.59 (dd, J ¼ 8.0, 1.8 Hz, lH), 7.43e7.54 (m, lH), 7.35 (t, J ¼ 8. l Hz, lH),
7.06e7.24 (m, 2H), 2.26 (tt, J ¼ 11.3, 3.6 Hz, lH), 1.79 (d, J ¼ 14. l Hz,
2H), 1.68 (dd, J ¼ 12.7, 3.0, 2.7 Hz, 2H), 1.52e1.62 (m, lH), 1.03e1.41
10.83 (s, lH), 10.16 (s, lH), 8.57 (t, J ¼ l.6 Hz, lH), 8.26 (dd, J ¼ 5.7, 3.7,
2.2 Hz, lH), 7.93 (dd, J ¼ 8.3, 1.3 Hz, lH), 7.76e7.86 (m, 2H), 7.72 (dd,
J ¼ 7.8, 1.6 Hz, lH), 7.52 (dd, J ¼ 8.5, 7.2, 1.6 Hz, lH), 7.23 (td, J ¼ 7.6,
1.2 Hz, lH), 2.16e2.36 (m, lH), 1.73e1.87 (m, 2H), 1.62e1.73 (m, 2H),
1.50e1.62 (m, lH), 0.82e1.46 (m, 5H); 19F NMR (376 MHz, DMSO‑d6)
(m, 5H); 13C NMR (101 MHz, DMSO‑d6)
d 173.90, 167.10, 140.30,
137.91, 132.85, 131.77, 130.27, 128.73, 124.17, 123.55, 123.04, 121.80,
120.03, 118.95, 45.10, 29.00, 25.37, 25.05; HRMS (ESI) m/z (M þ H)
calcd. for C20H22ClN2O2, 357.1364; found 357.1366.
d
ppm ꢂ78.42 (s, 3 F); 13C NMR (151 MHz, DMSO‑d6)
d 174.04,
167.31, 141.09, 137.28, 131.77, 131.30, 129.82, 128.79, 128.47, 125.80,
125.24, 123.52, 122.71, 122.61, 120.99, 120.55, 118.38, 116.22, 44.85,
N-(3-Bromophenyl)-2-(cyclohexanecarboxamido)benzamide
(13). The title compound was prepared according to general pro-
tocol A. Yield: 90%; LC-MS Retention Time: t1 (Method
29.00, 25.42, 25.11; HRMS (ESI) m/z (M
21H22F3N2O4S, 455.1247; found 455.1253.
N-([1,1′-Biphenyl]-3-yl)-2-(cyclohexanecarboxamido) benza-
þ
H) calcd. for
C
1) ¼ 6.904 min; 1 H NMR (400 MHz, DMSO‑d6)
d
ppm 10.47 (s, lH),
mide (18). The title compound was prepared according to general
10.31 (s, lH), 8.11 (dd, J ¼ 8.4, 1.0 Hz, lH), 7.94e8.04 (m, lH), 7.72 (dd,
J ¼ 7.8, 1.6 Hz, lH), 7.64 (dd, J ¼ 6.7, 2.3, 2.2 Hz, lH), 7.49(dd, J ¼ 8.4,
7.2, 1.6 Hz, lH), 7.24e7.35 (m, 2H), 7.19 (td, J ¼ 7.6, 1.2 Hz, lH),
2.21e2.37 (m, lH), 1.80 (d, J ¼ 11.5 Hz, 2H), 1.69 (dt, J ¼ l2.2, 3.2 Hz,
2H). l.59 (d, J ¼ 12.5 Hz, lH), 1.07e1.42 (m, 5H); 13C NMR (151 MHz,
protocol A. Yield: 39%; LC-MS Retention Time: t1 (Method
1) ¼ 7.181 min; 1H NMR (600 MHz, DMSO‑d6)
10.51 (d, J ¼ 13.2 Hz,
d
2H), 8.22 (dd, J ¼ 8.4, 1.1 Hz, 1H), 8.00 (t, J ¼ 2.0 Hz, 1H), 7.82 (dd,
J ¼ 7.9, 1.6 Hz, 1H), 7.73 (dt, J ¼ 8.0, 1.6 Hz, 1H), 7.67e7.61 (m, 2H),
7.56e7.36 (m, 6H), 7.23 (td, J ¼ 7.6, 1.2 Hz, 1H), 2.29 (tt, J ¼ 11.5,
3.6 Hz, 1H), 1.88e1.81 (m, 2H), 1.71 (dp, J ¼ 10.7, 3.5 Hz, 2H),
1.64e1.57 (m, 1H), 1.37 (qd, J ¼ 12.5, 3.3 Hz, 2H), 1.26 (qt, J ¼ 12.6,
3.3 Hz, 2H), 1.15 (qt, J ¼ 12.5, 3.5 Hz, 1H); 13C NMR (151 MHz,
DMSO‑d6)
d 177.00, 170.14, 143.70, 141.05, 134.85, 133.69, 131.84,
129.67, 129.53, 127.55, 126.17, 125.07, 124.93, 122.38, 48.30, 32.24,
28.61, 28.29; HRMS (ESI) m/z (M þ H) calcd. for C20H22BrN2O2,
401.0859; found 401.0859.
DMSO‑d6)
d 177.02, 170.21, 143.70, 143.15, 142.36, 141.23, 134.87,
2-(Cyclohexanecarboxamido)-N-(3-methoxyphenyl)benza-
mide (14). The title compound was prepared according to general
protocol A. Yield: 47%; LC-MS Retention Time: t1 (Method
132.36, 132.11, 131.85, 130.73, 129.72, 126.97, 126.08, 125.49, 124.68,
122.92, 122.25, 48.36, 32.16, 28.48, 28.18; HRMS (ESI) m/z (M þ H)
calcd. for C26H27N2O2, 399.2067; found 399.2071.
2-(Cyclohexanecarboxamido)-N-(2'-(trifluoromethyl)-[1,l'-
biphenyl]-3- yl)benzamide (19). The title compound was prepared
according to general protocol E. Yield: 25%; LC-MS Retention Time:
1) ¼ 6.344 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 10.47 (s, lH),
10.33 (s, lH), 8.20 (dd, J ¼ 8.4, 1.0 Hz, lH), 7.74 (dd, J ¼ 7.8, 1.4 Hz, lH),
7.49 (dd, J ¼ 8.4, 7.2, 1.6 Hz, lH), 7.33 (t, J ¼ 2.2 Hz, lH), 7.13e7.30 (m,
3H), 6.69 (dd, J ¼ 7.8, 2.5, 1.4 Hz, lH), 3.73 (s, 3H), 2.26 (tt, J ¼ 11.3,
3.6 Hz, lH), 1.82 (dd, J ¼ 12.5, 2.7 Hz, 2H), 1.69 (dd, J ¼ 12.4, 3.1,
2.8 Hz, 2H), l.5 l ꢂ1.64 (m, 1H), 1.02e1.41 (m, 5H); 13C NMR
t1 (Method 1) ¼ 7.417 min; 1H NMR (600 MHz, DMSO‑d6)
d 10.50 (s,
1H),10.41 (s,1H), 8.16 (dd, J ¼ 8.2,1.1 Hz,1H), 7.85 (dd, J ¼ 7.9,1.2 Hz,
1H), 7.82e7.71 (m, 4H), 7.63 (td, J ¼ 7.6, 1.1 Hz, 1H), 7.51 (dd, J ¼ 8.6,
7.4, 1.6 Hz, 1H), 7.43 (t, J ¼ 7.9 Hz, 2H), 7.22 (td, J ¼ 7.6, 1.2 Hz, 1H),
7.07 (dd, J ¼ 7.5, 1.6 Hz, 1H), 2.28 (tt, J ¼ 11.4, 3.5 Hz, 1H), 1.82 (dd,
J ¼ 12.9, 3.5 Hz, 2H), 1.69 (dp, J ¼ 10.6, 3.2 Hz, 2H), 1.60 (dtt, J ¼ 12.7,
3.4, 1.5 Hz, 1H), 1.35 (qd, J ¼ 12.5, 3.2 Hz, 2H), 1.25 (qt, J ¼ 12.6,
(101 MHz, DMSO‑d6)
d 174.31, 167.47, 159.82, 140.28, 138.58, 132.17,
129.84, 129.16, 124.20, 123.34, 121.95, 113.48, 109.81, 107.12, 55.49,
45.72, 29.48, 25.83, 25.53; HRMS (ESI) m/z (M þ H) calcd. for
C
21H25N2O3, 353.1860; found 353.1856.

88
K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
3.3 Hz, 2H), 1.14 (qt, J ¼ 12.5, 3.5 Hz, 1H); 19F NMR (376 MHz,
128.93, 128.24, 128.00, 125.64, 124.16, 123.99, 122.75, 122.45,
DMSO‑d6)
d
ppm ꢂ55.34 (s, 3 F); 13C NMR (151 MHz, DMSO‑d6)
120.09, 116.59, 116.55, 93.28; HRMS (ESI) m/z (M þ H) calcd. for
d
173.96, 167.09, 140.54, 139.69, 138.49, 137.97, 132.37, 132.08,
C21H15F3N2O2, 511.0125; found 511.0122.
131.72, 128.81, 128.29, 128.20, 126.92, 126.73, 126.10, 125.10, 124.27,
123.29, 123.09, 121.78, 120.66, 119.82, 45.20, 29.05, 25.40, 25.10;
HRMS (ESI) m/z (M þ H) calcd. for C27H26F3N2O2, 467.1941; found
467.1931.
2-(Cyclohexanecarboxamido)-N-(3'-(trifluoromethyl)-[1,1′-
biphenyl]-3-yl)benzamide (20). A mixture of N-(3-bromophenyl)-
2-(cyclohexanecarboxamido)benzamide (50.0 mg, 0.125 mmol), 3-
(trifluoromethyl)phenylboronic acid (35.5 mg, 0.187 mmol) and
2-(2-Ethylbutanamido)-N-(3-(trifluoromethyl)phenyl)ben-
zamide (25). The title compound was prepared according to gen-
eral protocol C. Yield: 12%; LC-MS Retention Time: t1 (Method
1) ¼ 6.751 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.60 (s, lH),
10.25 (s, lH), 8.15 (t, J ¼ 2.2 Hz, lH), 7.98 (dd, J ¼ 8.2, 1.4 Hz, lH), 7.93
(dd, J ¼ 8.2, 1.2, 1.0 Hz, 1H), 7.72 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.57 (t,
J ¼ 7.9 Hz, lH), 7.50 (dd, J ¼ 8.4, 7.1,1.7 Hz, lH), 7.38e7.47 (m, lH), 7.23
(td, J ¼ 7.6, 1.3 Hz, 1H), 2.15 (tt, J ¼ 8.8, 5.4 Hz, lH), 1.30e1.62 (m, 4H),
Pd(PPh3)4 (7.2 mg, 6.23
(0.50 mL) aqueous solution was heated in
m
mol) in DMF (l.50 mL) and 2.0 N Na2CO3
0.74e0.86 (m, 6H); 19F NMR (376 MHz, DMSO‑d6)
d
ppm ꢂ61.23 (s,
m
W at 100 ꢀC for 30 min.
3 F); 13C NMR (151 MHz, DMSO‑d6)
d 176.94, 170.24, 142.91, 140.15,
The reaction was cooled to room temperature, a small portion of Si-
THIOL was added to remove palladium. The mixture was filtered
and purified via C18 reverse phase HPLC to give the 35 mg (70%
yield) of product. LC-MS Retention Time: t1 (Method
134.62, 132.92, 132.47, 132.26, 131.68, 129.09, 128.15, 126.93, 126.67,
126.34, 125.82, 123.11, 123.09, 123.06, 119.56, 119.53, 119.50, 53.43,
28.26, 14.82; HRMS (ESI) m/z (M þ H) calcd. for C20H22F3N2O2,
379.1628; found 379.1634.
2-Pentanamido-N-(3-(trifluoromethyl)phenyl)benzamide
(26). The title compound was prepared according to general pro-
tocol C. Yield: 22%; LC-MS Retention Time: t1 (Method
1) ¼ 7.438 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 10.50 (br s,
1H), 10.49 (s, 1H), 8.14e8.24 (m, 1H), 8.01e8.08 (m, 1H), 7.94e8.01
(m, 1H), 7.91 (s, 1H), 7.78e7.84 (m, 2H), 7.70e7.77 (m, 2H),
7.41e7.56 (m, 3H), 7.22 (td, J ¼ 7.6,1.3 Hz, lH), 2.21e2.35 (m, lH),1.83
(d, J ¼ l5.8 Hz, 2H), 1.64e1.74 (m, 2H), 1.52e1 .63 (m, 1H), 1.08e1.46
1) ¼ 6.546 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.61 (s, lH),
10.19 (s, lH), 8.17 (t, J ¼ 2.2 Hz, lH), 7.98 (dd, J ¼ 8. l, 1.1 Hz, lH), 7.92
(dd, J ¼ 8.2, 2.0, 0.8 Hz, lH), 7.71 (dd, J ¼ 7.7, 1.7 Hz, lH), 7.57 (t, J ¼ 8. l
Hz, 1H), 7.47e7.54 (m, lH), 7.39e7.47 (m, 1H), 7.21 (td, J ¼ 7.6, 1.3 Hz,
lH), 2.28 (t, J ¼ 7.4 Hz, 2H), 1.43e1.60 (m, 2H), 1.18e1.34 (m, 2H),
0.80 (t, J ¼ 7.6 Hz, 3H); 19F NMR (376 MHz, DMSO‑d6) d ppm ꢂ61.21
(m, 5H); 19F NMR (376 MHz, DMSO‑d6)
d
ppm ꢂ61.11 (s, 3 F); 13C
NMR (151 MHz, DMSO‑d6)
d 173.12, 167.66, 142.23, 139.78, 139.54,
137.09, 135.78, 132.21, 131.29, 129.51, 129.48, 127.76, 127.72, 126.89,
126.84, 124.47, 124.22, 123.65, 122.98, 120.51, 119.86, 43.09, 29.23,
25.11, 24.84; HRMS (ESI) m/z (M þ H) calcd. for C27H26F3N2O2,
467.1941; found 467.1943.
2-(Cyclohexanecarboxamido)-N-(3-(trifluoromethyl)benzyl)
benzamide (21). The title compound was prepared according to
general protocol A. Yield: 71%; LC-MS Retention Time: t1 (Method
(s, 3 F); 3C NMR (101 MHz, DMSO‑d6)
d 171.63,167.56,140.24,137.76,
132.01, 130.23, 129.90, 129.59, 129.08, 125.93, 124.35, 123.79,
122.82, 120.49, 116.95, 116.91, 36.83, 27.59, 22.10, 14.08; HRMS (ESI)
m/z (M þ H) calcd. for C19H20F3N2O2, 365.1471; found 365.1476.
2-(Cyclopropanecarboxamido)-N-(3-(trifluoromethyl)
phenyl)benzamide (27). The title compound was prepared ac-
cording to general protocol B. Yield: 51%; LC-MS Retention Time: t1
1) ¼ 6.781 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 11.13 (s, lH),
9.34 (t, J ¼ 6. l Hz, lH), 8.37 (dd, J ¼ 8.4, 1.4 Hz, lH), 7.76 (dd, J ¼ 7.9,
1.7 Hz, lH), 7.66e7.70 (m. lH), 7.58e7.66 (m. 2H), 7.53e7.58 (m, lH),
7.47 (dd, J ¼ 8.5, 7.2, 1.7 Hz, lH), 7.13 (td, J ¼ 7.6, 1.3 Hz, lH), 4.54 (d,
J ¼ 5.9 Hz, 2H), 2.20 (tt, J ¼ l.12, 3.5 l H), 1.75e1.87 (m, 2H), 1.68 (dt,
J ¼ 12.3, 3.2 Hz, 2H), 1.53e1.64 (m, lH), 1.02e1.42 (m, 5H); 19F NMR
(Method 1) ¼ 6.131 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.61
(s, lH), 10.46 (s, lH), 8.17 (s, lH), 7.95e8.08 (m, lH), 7.91 (dd, J ¼ 7.9,
1.3 Hz, lH), 7.71 (dd, J ¼ 7.9, 1.5 Hz, lH), 7.57 (t, J ¼ 7.9 Hz, lH),
7.46e7.53 (m, lH), 7.40e7.46 (m, lH), 7.20 (tt, J ¼ 7.6, 0.8 Hz, lH),
1.61e1.81 (m, lH), 0.65e0.79 (m, 4H); 19F NMR (376 MHz, DMSO‑d6)
(376 MHz, DMSO‑d6)
DMSO‑d6)
d
ppm ꢂ61.02 (s, 3 F); 13C NMR (101 MHz,
d
173.76, 168.56, 140.60, 139.05, 132.02, 131.43, 129.40,
d
ppm ꢂ61.28 (s, 3 F); 13C NMR (101 MHz, DMSO‑d6)
d 172.12,167.57,
129.17, 128.86, 128.01, 123.82, 123.78, 123.64, 123.60, 122.54, 120.71,
120.59, 45.69, 42.25, 29.03, 25.35, 25.07; HRMS (ESI) m/z (M þ H)
calcd. for C22H24F3N2O2, 405.1784; found 405.1790.
140.23, 137.84, 132.08, 130.26, 129.90, 129.13, 125.54, 124.42, 123.69,
122.71, 120.53, 117.02, 116.98, 15.48, 7.85; HRMS (ESI) m/z (M þ H)
calcd. for C18H16F3N2O2, 349.1158; found 349.1160.
2-(Cyclohexanecarboxamido)-N-(furan-2-yl-methyl)benza-
mide (22). The title compound was prepared according to general
protocol A. Yield: 42%; LC-MS Retention Time: t1 (Method
2-(Cyclobutanecarboxamido)-N-(3-(trifiuoromethyl)phenyl)
benzamide (28). The title compound was prepared according to
general protocol B. Yield: 53%; LC-MS Retention Time: t1 (Method
1) ¼ 6.799 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 12.07 (s, 1H),
1) ¼ 6.412 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.61 (s, lH),
10.69 (s, 1H), 9.29 (t, J ¼ 5.8 Hz, 1H), 8.41 (dd, J ¼ 8.3, 1.3 Hz, 1H),
8.22 (dd, J ¼ 8.3, 1.3 Hz, 1H), 7.80 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.73 (dd,
J ¼ 7.8, 1.6 Hz, 1H), 7.45e7.64 (m, 3H), 7.13e7.29 (m, 2H), 6.36 (dd,
J ¼ 3.2, 1.9 Hz, 1H), 6.22e6.33 (m, 1H), 4.45 (d, J ¼ 5.5 Hz, 2H),
2.18e2.36 (m, 1H), 1.76e1.91 (m, 2H), 1.64e1.76 (m, 2H), 1.50e1.64
10.22 (s, lH), 8.15 (t, J ¼ 2.2 Hz, lH), 8.09 (dt, J ¼ 8. l, 0.6 Hz, lH), 7.93
(dd, J ¼ 8.2, 1.2, 1.0 Hz, lH), 7.75 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.58 (t,
J ¼ 7.8 Hz, lH), 7.51 (dd, J ¼ 8.5, 7.2, 1.6 Hz, 1H), 7.40e7.48 (m, 1H),
7.21 (td, J ¼ 7.6, 1.3 Hz, lH), 3.15e3.26 (m, lH), 2.01e2.24 (m, 4H),
1.80e1.97 (m, 1H), 1.64e1.80 (m, 1H); 19F NMR (376 MHz, DMSO‑d6)
(m, 1H), 1.05e1.44 (m, 5H); 13C NMR (101 MHz, DMSO‑d6)
d
174.40,
d
ppm ꢂ61.18 (s, 3 F); 13C NMR (101 MHz, DMSO‑d6)
d 172.88,
168.70, 166.89, 152.14, 142.57, 139.06, 138.97, 132.64, 132.60, 128.73,
128.13, 123.82, 122.10, 121.79, 121.73, 110.92, 107.51, 45.81, 36.52,
29.41, 25.82, 25.53; HRMS (ESI) m/z (M þ H) calcd. for C26H28N3O4,
446.2074; found 446.2080.
167.20, 139.68, 137.72, 131.81, 129.83, 129.45, 128.76, 124.03, 123.15,
121.87, 116.61, 39.73, 24.62, 17.51; HRMS (ESI) m/z (M þ H) calcd. for
C19H18F3N2O2, 363.1315; found 363.1317.
2-(Cyclopentanecarboxamido)-N-(3-(trifluoromcthyl)
2-Iodo-N-(2-((3-(trifluoromethy1)phenyl)carbamoyl)
pheny1)benzamide (24b). The title compound was prepared ac-
cording to general protocol C. Yield: 89%; LC-MS Retention Time: t1
phenyl)benzamide (29). The title compound was prepared ac-
cording to general protocol B. Yield: 76%; LC-MS Retention Time: t1
(Method 1) ¼ 6.780 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.60
(Method 1) ¼ 6.879 min; 1H NMR (400 MHz, DMSO‑d6)
d
10.79 (s,
(s, lH),10.29 (s, lH), 8.15 (t, J ¼ 2.2 Hz, lH), 8.04 (d, J ¼ 7.8 Hz, lH), 7.93
(d, J ¼ 8.4 Hz, lH), 7.73 (dd, J ¼ 7.8, 1.6 Hz, lH), 7.57 (t, J ¼ 7.9 Hz, lH),
7.50 (dd, J ¼ 8.5, 7.2, 1.6 Hz, lH), 7.44 (d, J ¼ 7.6 Hz, lH), 7.21 (td,
J ¼ 7.6, 1.3 Hz, lH), 2.75 (quin, J ¼ 8.0 Hz, lH), 1.74e1.86 (m, 2H),
1.63e1.74 (m, 2H), 1.41e1.63 (m, 4H); 19F NMR (376 MHz, DMSO‑d6)
1H), 10.74 (s, 1H), 8.19e8.07 (m, 2H), 8.02e7.90 (m, 2H), 7.83 (dd,
J ¼ 7.8, 1.5 Hz, 1H), 7.67e7.41 (m, 5H), 7.34 (td, J ¼ 7.6, 1.2 Hz, 1H),
7.23 (dd, J ¼ 7.9, 7.1, 2.0 Hz, 1H); 19F NMR (376 MHz, DMSO‑d6)
d
ꢂ61.29 (s, 3 F); 13C NMR (101 MHz, DMSO‑d6)
d 167.10, 166.99,
142.26, 139.72, 139.42, 136.99, 131.84, 131.43, 129.83, 129.44, 129.13,
d
ppm ꢂ61.21 (s, 3 F); 13C NMR (151 MHz, DMSO‑d6)
d 174.82, 174.17,

K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
89
167.21, 139.81, 139.79, 137.64, 131.74, 129.88, 129.63, 129.42, 129.21,
129.00,128.77, 126.90, 125.09,125.03, 123.98, 123.28,122.15,121.48,
120.14, 120.12, 120.09, 120.06, 116.61, 116.59, 116.56, 116.53, 45.67,
29.76, 25.53; HRMS (ESI) m/z (M þ H) calcd. for C20H20F3N2O2,
377.1471; found 377.1481.
N-(2-((3(Trifiuoromethyl)phenyl)carbamoyl)phenyl)cyclo-
heptanecarboxamide (30). The title compound was prepared ac-
cording to general protocol B. Yield: 70%; LC-MS Retention Time: t1
2-Methoxy-N-(2-((3-(trifluoromethyl)phenyl)carbamoyl)
phenyl)benzamide (36). The title compound was prepared ac-
cording to general protocol C. Yield: 64%. LC-MS RetentionTime: t1
(Method 1) ¼ 6.734 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 11.54
(s, lH), 10.84 (s, lH), 8.52e8.63 (m, lH), 8.32 (t, J ¼ 2.2 Hz, lH),
7.94e8.06 (m, 2H), 7.79 (dd, J ¼ 7.7, 1.7 Hz, lH), 7.51e7.68 (m, 3H),
7.43e7.51 (m, lH), 7.24e7.30 (m, lH), 7.17e7.23 (m, lH), 6.99e7.13
(m, lH), 3.97 (s, 3H); 19 F NMR (376 MHz, DMSO‑d6)
d
ppm ꢂ61.40
(Method 1) ¼ 7.093 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 10.58
(s, 3 F); 13C NMR (101 MHz, DMSO‑d6) 167.35, 163.02, 157.16,
d
(s, lH), 10.18 (s, lH), 8.13 (s, lH), 7.98 (dd, J ¼ 8. l, 0.9 Hz, lH), 7.93 (d,
J ¼ 8.0 Hz, lH), 7.71 (dd, J ¼ 7.8, 1.6 Hz, lH), 7.57 (t, J ¼ 7.8 Hz, lH), 7.49
(dd, J ¼ 8.4, 7.2, 1.6 Hz, lH), 7.39e7.46 (m, lH), 7.21 (td, J ¼ 7.6, 1.2 Hz,
lH), 2.40e2.51 (m, lH), 1.75e1.88 (m, 2H), 1.31e1.71 (m, lH); 19F
139.69, 137.66, 133.68, 131.80, 131.52, 129.99, 129.57, 129.26, 128.57,
124.41, 123.72, 123.13, 121.99, 121.32, 120.86, 120.78, 116.30, 116.25,
112.26, 55.59; HRMS (ESI) m/z (M þ H) calcd. for C22H13F3N2O3,
415.1264; found 415.1266.
NMR (376 MHz, DMSO‑d6)
(101 MHz, DMSO‑d6)
d
ppm ꢂ61.20 (s, 3 F); 13C NMR
2
-Methyl-N-(2-((3-(trifluoromethyl)phenyl)carbamoyl)
d
174.95, 167.10, 139.75, 137.55, 131.58, 129.80,
phenyl)benzamide (37). The title compound was prepared ac-
129.43, 128.66, 125.33, 123.94, 123.25, 122.31, 120.02, 46.58, 40.15,
cording to general protocol C. Yield: 46%; LC-MS Retention Time: t1
27.92, 25.83; HRMS (ESI) m/z (M þ H) calcd. for C22H24
F
3N2O2,
(Method 1) ¼ 6.769 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.74
405.1784; found 405.1794.
(s, 1H), 10.70 (s, 1H), 8.08e8.19 (m, 2H), 7.95 (dt, J ¼ 8.2, 1.2 Hz, lH),
7.79 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.48e7.65 (m, 3H), 7.33e7.47 (m, 2H),
7.22e7.33 (m, 3H), 2.37 (s, 3H); 19 F NMR (376 MHz, DMSO‑d6)
2-(2-Phenylacetamido)-N-(3-(trifluoromethyl)phenyl)ben-
zamide (31). The title compound was prepared according to gen-
eral protocol C. Yield: 85%; LC-MS Retention Time: t1 (Method
d
ppm ꢂ61.30 (s, 3 F); 13C NMR (151 MHz, DMSO‑d6)
d 168.70,
1) ¼ 6.443 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 10.61 (s, lH),
167.24, 139.85, 139.02, 137.32, 136.46, 135.88, 131.81, 131.74, 130.91,
130.60, 130.20, 130.10, 129.62, 129.42, 129.21, 129.05, 128.88, 127.06,
125.86, 123.94, 122.57, 116.50, 21.30; HRMS (ESI) m/z (M þ H) calcd.
for C22H18F3N2O2, 399.1315; found 399.1324.
10.30 (s, lH), 8.17 (t, J ¼ 2. l Hz, lH), 8.00e8.07 (m, lH), 7.83e7.91 (m,
lH), 7.71 (dd, J ¼ 7.8, 1.6 Hz, lH), 7.57 (t, J ¼ 7.9 Hz, lH), 7.50 (dd,
J ¼ 8.5, 7.2, 1.6 Hz, lH), 7.41e7.47 (m, lH), 7.11e7.34 (m, 6H), 3.66 (s,
2H); 19F NMR (376 MHz, DMSO‑d6)
d
ppm ꢂ61.15 (s, 3 F); 13C NMR
2
-Chloro-N(2-((3-(trifluoromethyl)phenyl)carbamoyl)
(101 MHz, DMSO‑d6)
d
169.27, 166.93, 139.70, 137.18, 135.28, 131.61,
phenyl)benzamide (38). The title compound was prepared ac-
129.77, 129.45, 129.35,129.14, 128.55, 128.28, 126.61, 125.52, 125.26,
123.89, 123.51, 122.81, 122.21, 120.09, 116.42, 43.61; HRMS (ESI) m/z
(M þ H) calcd. for C22H18F3N2O2, 399.1315; found 399.1312.
N-(2-((3-(Trifiuoromethyl)phenyl)carbamoyl)-phenyl)-nico-
tinamide (32). The title compound was prepared according to
general protocol C. Yield: 55%; LC-MS Retention Time: t1 (Method
cording to general protocol C. Yield: 81%; LC-MS Retention Time: t1
(Method 1) ¼ 6.765 min; 1H NMR (600 MHz, DMSO‑d6)
d 10.82 (s,
1H), 10.71 (s, 1H), 8.12 (d, J ¼ 2.0 Hz, 1H), 8.06 (d, J ¼ 8.2 Hz, 1H),
7.95e7.92 (m, 1H), 7.78 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.62e7.51 (m, 3H),
7.48 (td, J ¼ 7.7,1.7 Hz,1H), 7.45e7.39 (m,1H), 7.32 (td, J ¼ 7.6,1.2 Hz,
1H); 19 F NMR (376 MHz, DMSO‑d6)
d
ppm ꢂ61.28 (s, 3 F); 13C NMR
1) ¼ 5.342 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 11.27 (s, lH),
(151 MHz, DMSO‑d6) d 166.97, 164.62, 139.81, 136.68, 136.22, 131.80,
10.74 (s, lH), 8.95e9.12 (m, lH), 8.67e8.91 (m, lH), 8.19e8.29 (m, lH),
8.17 (dd, J ¼ 8.3, 1.3 Hz, lH), 8.10 (t, J ¼ 2. l Hz, lH), 7.92e8.02 (m, lH),
7.86 (dd, J ¼ 7.8, 1.6 Hz, lH), 7.52e7.69 (m, 3H), 7.38e7.48 (m, lH),
7.33 (td, J ¼ 7.6, 1.4 Hz, lH); 19F NMR (376 MHz, DMSO‑d6)
131.58, 130.01, 129.98, 129.89, 129.40, 129.19, 129.03, 128.94, 127.40,
126.26, 125.05, 124.38, 123.93, 122.76, 120.10, 120.07, 116.50, 116.47;
HRMS (ESI) m/z (M þ H) calcd. for C21H15ClF3N2O2, 419.0769;
found 419.0769.
d
ppm ꢂ61.20 (s, 3 F); 13C NMR (101 MHz DMSO‑d6)
d
167.23,163.41,
2-(Trifluoromethoxy)-N-(2-((3-(trifluoromethy1)phenyl)car-
bamoyl)phenyl)benzamide (39). The title compound was pre-
pared according to general protocol C. Yield: 91%; LC-MS Retention
Time: t1 (Method 1) ¼ 6.983 min; 1H NMR (400 MHz, DMSO‑d6)
152.11, 148.03, 139.63, 137.31, 135.43, 132.04, 130.28, 129.88, 129.47,
129.16, 129.03, 125.35, 124.19, 123.94, 122.71, 120.21, 116.75, 116.71;
HRMS (ESI) m/z (M þ H) calcd. for C20H15F3N3O2, 386.1111; found
386.1116.
d
ppm 10.97 (s, lH), 10.74 (s, lH), 8.09e8.26 (m, 2H), 7.91e8.03 (m,
2-(4-Methoxybenzamido)-N-(3-((trifluoromethyl)phenyl)
benzamide (33). The title compound was prepared according to
general protocol C. Yield: 64%; LC-MS RetentionTime: t1 (Method
lH), 7.83 (dd, 1 ¼7.8, 1.6 Hz, lH), 7.77 (dd, 1 ¼7.6, 2.0 Hz, lH),
7.39e7.70 (m, 6H), 7.33 (td, J ¼ 7.6, 1.3 Hz, lH); 19F NMR (376 MHz,
DMSO‑d6)
DMSO‑d6)
d
ppm ꢂ56.63 (s, 3 F), ꢂ61.33 (s, 3 F); 13C NMR (101 MHz,
1) ¼ 6.679 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 11.31 (s, lH),
d
166.96, 162.80, 144.79, 139.64, 137.01, 132.53, 131.98,
10.75 (s, lH), 8.36 (dd, J ¼ 8.3, 1.3 Hz, lH), 8.08 (t, J ¼ 2.2 Hz, lH), 8.01
(dd, J ¼ 8.2, 1.2, 1.0 Hz, lH), 7.78e7.92 (m, 3H), 7.54e7.64 (m, 2H),
7.42e7.49 (m, lH), 7.27 (td, J ¼ 7.6, 1.2 Hz, lH), 7.00e7.12 (m, 2H),
130.14, 130.00, 129.83, 129.46, 128.90, 127.89, 125.23, 124.15, 124.00,
122.35, 121.83, 120.18, 116.60; HRMS (ESI) m/z (M þ H) calcd. for
C22H15F6N2O3, 468.0981; found 468.0982.
3.81 (s, 3H); 19F NMR (376 MHz, DMSO‑d6)
d
ppm ꢂ61.25 (s, 3 F); 13
C
2-Ethoxy-N(2-((3-(trifluoromethyl)pheny1)carbamoyl)
phenyl) benzamide (40). The title compound was prepared ac-
NMR (101 MHz, DMSO‑d6)
d
167.69, 164.23, 162.19, 139.43, 138.62,
132.31, 129.89, 129.49, 129.17, 129.01, 126.52, 124.48, 123.17, 121.66,
120.41, 117.06, 117.02, 114.07, 55.46, 11.88; HRMS (ESI) m/z (M þ H)
calcd. for C22H18F3N2O3, 415.1264; found 415.1272.
2-(3-Methoxybenzamido)-N-(3-(trifluoromethyl)phenyl)
benzamide (35). The title compound was prepared according to
general protocol C. Yield: 60%; LC-MS Retention Time: t1 (Method
cording to general protocol C. Yield: 47%; LC-MS Retention Time: t1
(Method 1) ¼ 6.998 min; 1H NMR (400 MHz, DMSO‑d6)
d 11.33 (s,
lH), 10.82 (s, lH), 8.51 (dd, J ¼ 8.4, 1.2 Hz, lH), 8.28(t, J ¼ 2.1 Hz, lH),
7.89e8.07 (m, 2H), 7.80 (dd, J ¼ 7.8, 1.6 Hz, lH), 7.55e7.65 (m, 2H),
7.38e7.55 (m, 2H), 7.27 (td, J ¼ 7.6, 1.3 Hz, lH), 7.19 (dd, J ¼ 8.4,
1.0 Hz, lH), 7.01e7.12 (m, lH), 4.30 (q, J ¼ 6.9 Hz, 2H), 1.30 (t,
1) ¼ 6.778 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 11.33 (s, 1H),
J ¼ 6.9 Hz, 3H); 19F NMR (376 MHz, DMSO‑d6)
ppm ꢂ61.40 (s, 3 F);
d
10.75 (s, 1H), 8.32 (dd, J ¼ 8.3, 1.3 Hz, 1H), 8.14 (t, J ¼ 2. l Hz, 1H), 7.98
(dt, J ¼ 8. l, 1.2 Hz, 1H), 7.89 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.52e7.66 (m,
2H), 7.39e7.52 (m, 4H), 7.30 (td, 1.4 Hz, lH), 7.13e7.25 (m, 1H), 3.80
13C NMR (101 MHz, DMSO‑d6)
d 167.66, 163.81, 156.43, 140.11,
138.08, 133.89, 132.28, 131.95, 130.39, 129.98, 129.66, 129.11, 124.75,
124.19, 123.65, 122.64, 122.57, 121.13, 120.74, 120.70, 116.73, 116.69,
113.48, 64.55, 40.60, 40.40, 40.19, 39.98, 39.77, 39.56, 39.35, 14.27;
HRMS (ESI) m/z (M þ H) calcd. for C23H20F3N2O3, 429.1421; found
429.1425.
(s, 3H); 19F NMR (376 MHz, DMSO‑d6)
(ESI) m/z (M þ H) calcd. for C22H18F3N2O3, 415.1264; found
d
ppm ꢂ61.31 (s, 3 F); HRMS
415.1270.

90
K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
2-Isopropoxy-N-(2-((3-(trifluoromethyl)phenyl)carbamoyl)
phenyl)-benzamide (41). The title compound was prepared ac-
cording to general protocol H. Yield: 9%; LC-MS Retention Time: t1
according to general protocol A. Yield: 38%; LC-MS Retention Time:
t1 (Method 1) ¼ 5.766 min; HRMS (ESI) m/z (M þ H) calcd. for
C22H19F3N3O5S, 494.0992; found 494.1000.
(Method 1) ¼ 7.126 min; 1H NMR (600 MHz, DMSO‑d6)
d
11.18 (s,
4
-(2-Ethoxybenzamido)-N-(3-((trifluoromethyl)sulfonyl)
1H), 10.87 (s, 1H), 8.50 (dd, J ¼ 8.3, 1.1 Hz, 1H), 8.36 (t, J ¼ 1.9 Hz, 1H),
7.91 (dd, J ¼ 11.7, 7.5, 1.7 Hz, 2H), 7.82 (dd, J ¼ 7.8, 1.6 Hz, 1H),
7.63e7.57 (m, 2H), 7.54e7.44 (m, 2H), 7.29 (td, J ¼ 7.6, 1.2 Hz, 1H),
7.22e7.17 (m,1H), 7.05 (dd, J ¼ 7.9, 7.3,1.0 Hz,1H), 4.82 (p, J ¼ 6.1 Hz,
phenyl)nicotinamide (51). The title compound was prepared ac-
cording to general protocol A. Yield: 26%; LC-MS Retention Time: t1
(Method 1) ¼ 5.507 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 11.71
(s, lH), 11.25 (s, lH), 9.00 (s, lH), 8.77 (t, J ¼ 2.2 Hz, lH), 8.65e8.71 (m,
lH), 8.54e8.64 (m, lH), 8.15e8.26 (m, lH), 7.96 (dd.J ¼ 7.8, 2.3 Hz,
lH), 7.78e7.93 (m, 2H), 7.56 (dd, J ¼ 8.6, 7.0, 2.0 Hz, lH), 7.24 (d,
J ¼ 8.6 Hz, lH), 7.09 (t, J ¼ 7.4 Hz, lH), 4.35 (q, J ¼ 6.9 Hz, 2H), 1.29 (t,
1H), 1.34 (d, J ¼ 6.0 Hz, 6H); 13C NMR (151 MHz, DMSO‑d6)
d 170.14,
166.71,158.40,142.76,140.69, 136.44,134.96,134.68,133.00,132.55,
132.34, 131.84, 127.27, 126.83, 126.37, 125.83, 125.56, 123.54, 123.37,
123.35, 119.39, 119.36, 117.02, 74.24, 24.10; HRMS (ESI) m/z (M þ H)
calcd. For C24H22F3N2O3, 443.1577; found 443.1599.
J ¼ 6.8 Hz, 3H); 19 F NMR (376 MHz, DMSO‑d6)
d
ppm ꢂ78.37 (s,
3 F); 13C NMR (151 MHz, DMSO‑d6)
d 169.07, 167.39, 159.21, 156.20,
2,6-Dimethoxy-N-(2-((3-(trifluoromethyl)phenyl)carbamoyl)
phenyl)benzamide (42). The title compound was prepared ac-
cording to general protocol C. Yield: 88%; LC-MS Retention Time: t1
153.04, 147.91, 143.56, 137.33, 134.89, 134.57, 133.02, 131.82, 129.06,
124.40, 124.05, 123.94, 123.60, 121.44, 120.83, 117.83, 116.25, 67.26,
16.85; HRMS (ESI) m/z (M þ H) calcd. for C22H19F3N3O5S,
494.0992; found 494.1004.
(Method 1) ¼ 6.400 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.68
(s, lH), 10.62 (s, lH), 8.43 (d, J ¼ 8.2 Hz, lH), 8.08 (s, lH), 7.93 (d,
J ¼ 8.4 Hz, lH), 7.85 (dd, J ¼ 7.7,1.1 Hz, lH), 7.52e7.64 (m, 2H), 7.45 (d,
J ¼ 7.6 Hz, lH), 7.34 (t, J ¼ 8.4 Hz, lH), 7.27 (td, J ¼ 7.6, 1.1 Hz, lH), 6.71
(d, J ¼ 8.4 Hz, 2H), 3.69 (s, 6H); 19F NMR (376 MHz, DMSO‑d6)
2
-(2-Ethoxybenzamido)-N-(3-((trifluoromethyl)sulfonyl)
phenyl)-nicotinamide (52). The title compound was prepared
according to general protocol A. yield: 51%; LC-MS Retention Time:
t1 (Method 1) ¼ 7.402 min; HRMS (ESI) m/z (M þ Na)þ calcd. for
C22H18F3N3NaO5S, 516.0811; found 516.0837.
d
ppm ꢂ61.29 (s, 3 F); 1HRMS (ESI) m/z (M þ H) calcd. for
23H20F3N2O4, 445.1370; found 445.1380.
C
2
-(2-Ethoxyphenylsulfonamido)-N-(3-((trifluoromcthy1)
2-(2-Methoxyethoxy)-N-(2-((3-(trifluoromethyl)phenyl)car-
sulfonyl)phenyl)-benzamide (53). The title compound was pre-
pared according to general protocol A. Yield: 29%; LC-MS Retention
Time: t1 (Method 1) ¼ 6.631 min; 1H NMR (400 MHz, DMSO‑d6)
bamoyl)phenyl)benzamide (43). The title compound was pre-
pared according to general protocol H. Yield: 16%; LC-MS Retention
Time: t1 (Method 1) ¼ 6.625 min; 1H NMR (600 MHz, DMSO‑d6)
d
11.00 (s, 1H), 10.37 (s, 1H), 8.64 (t, J ¼ 1.9 Hz, 1H), 8.24 (dt, J ¼ 7.8,
d
11.25 (s, 1H), 10.84 (s, 1H), 8.45 (dd, J ¼ 8.4, 1.1 Hz, 1H), 8.23 (d,
1.8 Hz, 1H), 7.96e7.84 (m, 2H), 7.82 (dd, J ¼ 7.8, 1.7 Hz, 2H),
7.60e7.50 (m, 2H), 7.48 (dd, J ¼ 8.5, 7.3, 1.5 Hz, 1H), 7.18 (td, J ¼ 7.6,
1.2 Hz, 1H), 7.12e7.00 (m, 2H), 3.95 (q, J ¼ 7.0 Hz, 2H), 1.18 (t, J ¼ 7.0,
J ¼ 1.9 Hz, 1H), 7.94 (dd, J ¼ 22.5, 8.1, 2.1 Hz, 2H), 7.82 (dd, J ¼ 7.8,
1.6 Hz, 1H), 7.65e7.57 (m, 2H), 7.53 (dd, J ¼ 8.8, 7.2, 1.8 Hz, 1H),
7.51e7.46 (m, 1H), 7.29 (td, J ¼ 7.5, 1.2 Hz, 1H), 7.25 (dd, J ¼ 8.4,
1.0 Hz, 1H), 7.09 (td, J ¼ 7.6, 1.0 Hz, 1H), 4.37e4.31 (m, 2H),
3.69e3.63 (m, 2H), 3.12 (s, 3H). 19F NMR (376 MHz, DMSO‑d6)
3H); 19 F NMR (376 MHz, DMSO‑d6)
d
ꢂ78.37; 13C NMR (101 MHz,
DMSO‑d6)
d 167.47, 155.62, 140.29, 137.78, 135.61, 133.03, 131.39,
130.49, 129.95, 129.46, 128.91, 125.92, 125.37, 123.06, 121.39, 121.31,
121.05, 120.03, 118.75, 117.80, 113.56, 64.53, 13.68; HRMS (ESI) m/z
(M þ H) calcd. for C22H20F3N2O6S2, 529.0709; found 529.0716.
d
ppm ꢂ61.36 (s, 3 F); 13C NMR (151 MHz, DMSO‑d6)
d 167.18,
163.39, 156.25, 139.72, 138.97, 137.58, 133.39, 131.90, 131.37, 129.99,
129.49,129.28,128.83,124.55,123.85,123.39,122.40,122.35,121.07,
120.29, 116.39, 113.62, 69.79, 67.95, 58.06; HRMS (ESI) m/z (M þ H)
calcd. for C24H22F3N2O4, 459.1526; found 459.1527.
2
-Methoxy-N-methy1-N-(2-((3-(trifluoromethyl)pheny1)
carbamoy1)phenyl)benzamide (54). The title compound was
prepared according to general protocol A. Yield: 47%; LC-MS
Retention Time: t1 (Method 1) ¼ 6.046 min; HRMS (ESI) m/z
(M þ H) calcd. for C23H20F3N2O3, 429.1421; found 429.1438.
2-(2-Methoxybenzamido)-N-methyl-N-(3-(trifluoromethyl)
phenyl) benzamide (55). The title compound was prepared ac-
cording to general protocol A. Yield: 60%; LC-MS Retention Time: t1
2-(2-(Dimethylamino)ethoxy)-N-(2-((trifluoromethyl)
phenyl)carbamoyl)-phenyl)benzamide (44). The title compound
was prepared according to general protocol H. Yield: 4%; LC-MS
Retention Time: t1 (Method 1) ¼ 4.705 min; 1H NMR (400 MHz,
DMSO‑d6)
d
ppm 11.11 (s, 1H), 10.84 (s, 1H), 8.41 (dd, J ¼ 8.5, 1.3 Hz,
1H), 8.07e8.20 (m, 1H), 7.96 (dd. J ¼ 8.0.1.2 Hz, 1H), 7.87 (dd,
J ¼ 7.7.1.9 Hz, 1H), 7.84 (dd, J ¼ 7.8, 1.8 Hz, 1H). 7.57e7.65 (m, 2H),
7.52e7.57 (m, 1H), 7.45e7.52 (m, 1H), 7.24e7.33 (m, 2H), 7.13 (td,
J ¼ 7.5, 1.0 Hz, 1H), 4.55 (t, J ¼ 5. l Hz, 2H), 3.49e3.74 (m, 2H), 2.78 (s,
6H); HRMS (ESI) m/z (M þ H) calcd. for C25H25F3N3O3, 472.1843;
found 472.1851.
(Method 1) ¼ 6.442 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.65
(d, J ¼ 0.8 Hz, lH), 8.21 (dt, J ¼ 8.4, 0.6 Hz, lH), 8.03 (dd, J ¼ 7.8,1.8 Hz,
lH), 7.54e7.66 (m, 2H), 7.37e7.54 (m, 3H), 7.20e7.33 (m, 2H), 7.13
(dd, J ¼ 7.9, 7.2, 1.0 Hz, lH), 7.04e7.11 (m, lH), 6.86e6.96 (m, lH), 4.02
(s, 3H), 3.44 (s, 3H); HRMS (ESI) m/z (M
þ
H) calcd. for
C23H20F3N2O3, 429.1241; found 429.1418.
2-(2-Ethoxybenzamido)-N-(3-((trifluoromethyl)sulfonyl)
phenyl)nicotinamide (49). The title compound was prepared ac-
cording to general protocol A. Yield: 45%; LC-MS Retention Time: t1
2-((2-Methoxyethyl)amino)-N-(3-(trifiuoromethyl)phenyl)-
benzamide (57). The title compound was prepared according to
general protocol F. Yield: 19%; LC-MS Retention Time: t1 (Method
(Method 1) ¼ 5.472 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 10.98
1) ¼ 6.254 min; 1H NMR (400 MHz, DMSO‑d6)
d ppm 10.33 (s, lH),
(s, lH), 10.81 (s, lH), 8.47e8.66 (m, 2H), 8.05e8.24 (m, 2H),
7.75e7.87 (m, 2H), 7.72 (dd, J ¼ 7.8, 2.0 Hz, lH), 7.51 (dd, J ¼ 8.5, 7.1,
2.0 Hz, lH), 7.39 (dd, J ¼ 7.6, 4.9 Hz, lH), 7.20 (d, J ¼ 8.2 Hz, lH), 7.00
(t, J ¼ 7.4 Hz, lH), 4.27 (q, J ¼ 7.0 Hz, 2H), 1.42 (t, J ¼ 7.0 Hz, 3H); 19F
8.14 (t, J ¼ 2.2 Hz, lH), 7.88e8.05 (m, lH), 7.70 (dd, J ¼ 7.8, 1.6 Hz, lH),
7.54e7.63 (m, lH), 7.44e7.52 (m, lH), 7.37e7.44 (m, lH), 7.34 (dd,
J ¼ 8.6, 7.0, 1.7 Hz, lH), 6.72e6.84 (m, lH), 6.65 (dd, J ¼ 8.0, 7.1, 1.2 Hz,
lH), 3.53 (t, J ¼ 5.5 Hz, 2H), 3.28 (s, 3H), 3.24e3.33 (m, 2H); 19F NMR
NMR (376 MHz, DMSO‑d6)
(151 MHz, DMSO‑d6)
d
ppm ꢂ78.44 (s, 3 F); 13C NMR
(376 MHz, DMSO‑d6)
d
ppm ꢂ61.23 (s, 3 F); 13C NMR (101 MHz,
d
165.91, 163.44, 156.44, 150.26, 148.04, 141.24,
DMSO‑d6) d 168.36, 149.33, 139.94, 133.15, 129.75, 129.19, 129.08,
137.87, 133.55, 131.38, 130.94, 129.86, 128.00, 125.15, 124.02, 121.63,
120.76, 120.51, 120.43, 120.27, 118.35, 113.33, 64.71, 14.44; HRMS
(ESI) m/z (M þ H) calcd. for C22H19F3N3O5S, 494.0992; found
494.1004.
124.02, 119.73, 116.55, 116.51, 114.97, 114.37, 111.30, 70.34, 58.03,
41.90; HRMS (ESI) m/z (M þ H) calcd. for C17H18F3NO2, 339.1315;
found 339.1326.
2-(2-Ethoxybenzamido)-N-(3-((trifluoromethyl)sulfonyl)
phenyl)thiophene-3-carboxamide (59). The title compound was
prepared according to general protocol A. Yield: 33%; LC-MS
3
-(2-Ethoxybenzamido)-N-(3-((trifluoromethyl)sulfonyl)
phenyl)isonicotinamide (50). The title compound was prepared

K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
91
Retention Time: t1 (Method 1) ¼ 7.341 min; 1H NMR (400 MHz,
2-Isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenyl-
DMSO‑d6)
d
ppm 13.25 (s, 1H), 10.45 (s, 1H), 8.90 (d, J ¼ 2.2 Hz, 1H),
carbamoyl)phenyl)benzamide (65). The title compound was
prepared according to general protocol A. Yield: 57%; LC-MS
Retention Time: t1 (Method 1) ¼ 6.974 min; t2 (Method
8.30e8.20 (m, 1H), 8.08 (dd, J ¼ 7.9, 1.9 Hz, 1H), 7.90e7.80 (m, 2H),
7.75 (d, J ¼ 6.0 Hz, 1H), 7.60 (dd, J ¼ 8.9, 7.3, 1.9 Hz, 1H), 7.31 (dd,
J ¼ 8.6, 0.9 Hz, 1H), 7.18e7.08 (m, 2H), 4.51 (q, J ¼ 7.0 Hz, 2H), 1.47 (t,
2) ¼ 3.881 min; 1H NMR (400 MHz, DMSO‑d6):
d 11.11 (s, 1H), 11.09
J ¼ 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
d
164.07, 162.19,
(s, 1H), 8.75 (s, 1H), 8.48 (d, J ¼ 8.2 Hz, 1H), 8.20 (dt, J ¼ 7.4, 2.2 Hz,
1H), 7.91 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.77e7.89 (m, 3H), 7.57e7.66 (m,
1H), 7.51 (dd, J ¼ 8.6, 7.0, 2.0 Hz,1H), 7.30 (td, J ¼ 7.5,1.0 Hz,1H), 7.20
(d, J ¼ 8.6 Hz, 1H), 7.05 (t, J ¼ 7.4 Hz, 1H), 4.75e4.85 (m, 1H), 1.32 (d,
156.99, 147.77, 141.20, 134.92, 132.43, 131.69, 130.33, 129.06, 125.79,
123.30, 121.47, 121.39, 119.79, 117.58, 115.83, 113.79, 64.98, 14.46; 19
NMR (376 MHz, DMSO‑d6)
F
d
ꢂ78.43 (s, 3 F); HRMS (ESI) m/z (M þ H)
calcd. for C21H18F3N2O5S2, 499.0604; found 499.0608.
J ¼ 5.9 Hz, 6H); 13C NMR (101 MHz, DMSO‑d6):
d 167.24, 163.63,
2-Methoxy-N-(3-((3-(trifluoromethyl)phenyl)carbamoyl)
phenyl)benzamide (61). The title compound was prepared ac-
cording to general protocol A. Yield: 68%; LC-MS Retention Time: t1
155.29, 140.75, 137.61, 133.30, 132.06, 131.52, 131.28, 129.97 (br.),
128.89, 128.51, 125.58, 123.93, 123.29, 122.79, 122.56, 120.82,
120.44, 119.40 (q, J ¼ 328 Hz), 113.98, 71.19, 21.05; 19F NMR
(Method 1) ¼ 6.471 min; 1H NMR (400 MHz, DMSO‑d6)
d
ppm 10.58
(376 MHz, DMSO‑d6):
for C24H22F3N2O5S, 507.1196; found 507.1221.
d
ꢂ78.38 (s, 3 F); HRMS (m/z): [MþH] calcd.
(s, lH), 10.30 (s, lH), 8.27e8.35 (m, lH), 8.23 (t, J ¼ 2. l Hz, lH), 8.04
(dt, J ¼ 8.2, 1.1 Hz, lH), 7.88e7.99 (m, lH), 7.67 (dd, J ¼ 8.0, 1.4, 1.2 Hz,
lH), 7.63 (dd, J ¼ 7.6, 1.8 Hz, lH), 7.56e7.62 (m, lH), 7.47e7.56 (m,
2H), 7.41e7.47 (m, lH), 7.14e7.23 (m, lH), 7.06 (td, J ¼ 7.4, l.0 Hz, lH),
2-Propoxy-N-(2-((3-((trifluoromethyl)sulfonyl)phenyl)car-
bamoyl)phenyl)benzamide (66). The title compound was pre-
pared according to general protocol A. Yield: 41%; LC-MS Retention
Time: t1 (Method 1) ¼ 7.147 min; 1H NMR (400 MHz, DMSO‑d6):
3.90 (s, 3H); 19F NMR (376 MHz, DMSO‑d6)
d
ppm ꢂ61.28 (s, 3 F); 13
C
NMR (101 MHz, DMSO‑d6)
d
166.43, 165.23, 156.95, 140.40, 139.69,
d
11.26 (s, 1H), 11.10 (s, 1H), 8.72 (s, 1H), 8.54 (d, J ¼ 8.6 Hz, 1H), 8.24
135.69, 132.59, 130.32, 130.08, 129.95, 129.24, 125.95, 125.21, 124.18,
123.45, 122.99, 120.93, 119.66, 116.76, 116.72, 112.46; HRMS (ESI) m/
z (M þ H) calcd. for C22H18F3N2O3, 415.1264; found 415.1275.
2-Methoxy-N-(2-((3-((trifluoromethyl)sulfonyl)phenyl)car-
bamoy1)phenyl)benzamide (62). The title compound was pre-
pared according to general protocol G. Yield: 25%; LC-MS Retention
Time: t1 (Method 1) ¼ 6.810 min; 1H NMR (400 MHz, DMSO‑d6)
(dt, J ¼ 7.1, 2.3 Hz, 1H), 7.94 (dd, J ¼ 7.8, 2.0 Hz, 1H), 7.78e7.91 (m,
3H), 7.56e7.67 (m, 1H), 7.52 (dd, J ¼ 8.5, 7.1, 2.0 Hz, 1H), 7.25e7.33
(m, 1H), 7.20 (d, J ¼ 8.2 Hz, 1H), 7.07 (t, J ¼ 7.4 Hz, 1H), 4.16 (t,
J ¼ 7.0 Hz, 2H), 1.71 (sxt, J ¼ 7.2 Hz, 2H), 0.77 (t, J ¼ 7.4 Hz, 3H); 13C
NMR (101 MHz, DMSO‑d6):
d 167.42, 163.49, 156.14, 140.78, 137.75,
133.37, 132.14, 131.39, 131.33, 129.95 (br.), 128.90, 128.48, 125.54,
123.80, 123.21, 122.25, 122.15, 120.88, 120.68, 119.43 (q, J ¼ 328 Hz),
d
ppm 11.58 (s, 1H), 11.09 (s, 1H), 8.82 (t, J ¼ 2. l Hz, 1H), 8.58 (dd,
113.09, 69.82, 21.24, 9.74; 19F NMR (376 MHz, DMSO‑d6):
d
ꢂ78.43
1 ¼8.5, 1.3 Hz, lH), 8.23 (dt, J ¼ 7.0, 2.2 Hz, lH), 8.02 (dd, J ¼ 7.7,
1.9 Hz, lH), 7.76e7.99 (m, 3H), 7.51e7.63 (m, 2H), 7.27 (td, J ¼ 7.5,
1.2 Hz, 1H), 7.22 (dd, J ¼ 8.4, 1.2 Hz, 1H), 7.09 (dd, J ¼ 7.2, 1.2 Hz, lH),
(s, 3 F); HRMS (m/z): [MþH] calcd. for C24H22F3N2O5S, 507.1196;
found 507.1219.
2-Butoxy-N-(2-((3-((trifluoromethyl)sulfonyl)phenyl)carba-
moyl)phenyl)benzamide (67). The title compound was prepared
according to general protocol A. Yield: 49%; LC-MS Retention Time:
t1 (Method 1) ¼ 7.325 min; t2 (Method 2) ¼ 3.971 min; 1H NMR
3.99 (s, 3H); 19F NMR (376 MHz, DMSO‑d6)
d
ppm ꢂ78.39 (s, 3 F);
13C NMR (101 MHz, DMSO‑d6)
d 167.57, 163.01, 157.16, 140.75,
137.78, 133.69, 132.05, 131.51, 131.37, 129.94, 128.69, 128.43, 125.58,
123.90, 123.10, 122.05, 121.24, 120.84, 120.77, 112.26, 55.59; HRMS
(ESI) m/z (M þ H) calcd. for C22H18F3N2O5S, 479.0883; found
479.0886.
2-Ethoxy-N-(2-((3-((trifluoromethyl)sulfonyl)phenyl)carba-
moyl)phenyl)benzamide (63). The title compound was prepared
according to general protocol A. Yield: 53%; LC-MS Retention Time:
(400 MHz, DMSO‑d6):
d 11.27 (s, 1H), 11.10 (s, 1H), 8.71 (s, 1H), 8.55
(d, J ¼ 8.2 Hz, 1H), 8.26 (dt, J ¼ 7.3, 2.0 Hz, 1H), 7.93 (dd, J ¼ 7.8,
2.0 Hz, 1H), 7.81e7.91 (m, 3H), 7.58e7.68 (m, 1H), 7.47e7.57 (m, 1H),
7.29 (t, J ¼ 7.4 Hz, 1H), 7.22 (d, J ¼ 8.2 Hz, 1H), 7.08 (t, J ¼ 7.4 Hz, 1H),
4.19 (t, J ¼ 6.8 Hz, 2H), 1.57e1.73 (m, 2H), 1.23 (sxt, J ¼ 7.5 Hz, 2H),
0.70 (t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO‑d6):
d 167.41,
t1 (Method 1) ¼ 6.963 min; 1H NMR (400 MHz, DMSO‑d6):
d
11.35
163.52, 156.13, 140.81, 137.81, 133.37, 132.20, 131.36, 131.34, 129.96
(br.), 128.94, 128.41, 125.54, 123.59, 123.18, 122.30, 122.06, 120.85,
120.69, 113.09, 68.19, 29.93, 18.34, 13.37; 19F NMR (376 MHz,
(s, 1H), 11.10 (s, 1H), 8.76 (t, J ¼ 2.2 Hz, 1H), 8.53 (d, J ¼ 8.2 Hz, 1H),
8.23 (dt, J ¼ 7.1, 2.3 Hz, 1H), 7.96 (dd, J ¼ 7.8, 2.0 Hz, 1H), 7.80e7.92
(m, 3H), 7.57e7.65 (m, 1H), 7.53 (dd, J ¼ 8.6, 7.0, 2.0 Hz, 1H), 7.30 (td,
J ¼ 7.6, 1.2 Hz, 1H), 7.22 (d, J ¼ 8.2 Hz, 1H), 7.04e7.10 (m, 1H), 4.31 (q,
J ¼ 6.8 Hz, 2H), 1.31 (t, J ¼ 6.8 Hz, 3H); 13C NMR (101 MHz,
DMSO‑d6):
d
ꢂ78.42 (s, 3 F); HRMS (m/z): [MþH] calcd. for
C25H24F3N2O5S, 521.1353; found 521.1359.
DMSO‑d6):
d
167.44, 163.39, 156.00, 140.75, 137.73, 133.44, 132.10,
Acknowledgments
131.48, 131.34, 129.93, 128.81, 128.49, 125.58, 123.86, 123.21, 122.27,
122.08, 120.81, 120.67, 119.41 (q, J ¼ 328 Hz), 113.00, 64.09, 13.80;
This research was supported by the Molecular Libraries Initia-
tive of the National Institutes of Health Roadmap for Medical
Research Grant U54MH084681 and the Intramural Research Pro-
gram of the National Center for Advancing Translational Sciences at
the National Institutes of Health. AIA acknowledges grants
R01DK110167 and R03MH085705 for funding. Dr. Daniel C. Talley
and Dr. Scott B. Hoyt and are acknowledged for critical editing of
this manuscript.
19F NMR (376 MHz, DMSO‑d6):
d
ꢂ78.40 (s, 3 F); HRMS (m/z):
[MþH] calcd. for C23H20F3N2O5S, 493.1040; found 493.1044.
2-Isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenyl-
carbamoyl)phenyl)benzamide (64). The title compound was
prepared according to general protocol A. Yield: 44%; LC-MS
Retention Time: t1 (Method 1) ¼ 6.804 min; 1H NMR (400 MHz,
DMSO‑d6):
d 11.10 (s, 1H), 11.05 (s, 1H), 8.64 (s, 1H), 8.44 (d,
J ¼ 8.2 Hz, 1H), 8.27 (dt, J ¼ 7.1, 2.3 Hz, 1H), 7.79e7.92 (m, 4H),
7.60e7.69 (m, 1H), 7.52e7.60 (m, 1H), 7.37 (d, J ¼ 8.2 Hz, 1H), 7.31 (t,
J ¼ 7.6 Hz, 1H), 7.19 (t, J ¼ 7.4 Hz, 1H), 4.94 (q, J ¼ 9.0 Hz, 2H); 13C
Abbreviations
NMR (101 MHz, DMSO‑d6):
d
167.46, 163.36, 154.24, 140.76, 137.68,
AcOH
Aq
acetic acid
aqueous
132.87, 132.27, 131.25, 130.85, 129.87, 128.99, 128.60, 125.45, 124.28,
123.83, 123.63 (q, J ¼ 280 Hz), 123.49, 122.52, 121.97, 121.05, 119.42
(q, J ¼ 329 Hz), 114.03, 65.00 (q, J ¼ 35.0 Hz); 19F NMR (376 MHz,
CH2Cl2
Conc
dichloromethane
concentrated;
DMSO‑d6):
d
72.46 (t, J ¼ 9.4 Hz, 3 F), ꢂ78.48 (s, 3 F); HRMS (m/z):
DIPEA
DMAP
diisopropylethylamine
dimethylaminopyridine
[MþH] calcd. for C23H17F6N2O5S, 547.0757; found 547.0762.

92
K.J. Wilson et al. / European Journal of Medicinal Chemistry 156 (2018) 79e92
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Et2O
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1,2-dimethoxyethane
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dimethyl sulfoxide
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TNF
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mW
Appendix A. Supplementary data
Mouse PK data, crystal structure of compound 65 and X-ray
data, carbon, proton and fluorine NMR spectra of selected com-
pounds including compound 65, maximum tolerated dose study for
compound 65.
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.06.008.
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