Design, synthesis and biological evaluation of phenyl urea derivatives as IDO1 inhibitors

Article abstract of DOI:10.3390/molecules25061447

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing intracellular enzyme that catalyzes the first and rate-determining step of tryptophan metabolism and is an important immunotherapeutic target for the treatment of cancer. In this study, we designed and synthesized a new series of compounds as potential IDO1 inhibitors. These compounds were then evaluated for inhibitory activity against IDO1 and tryptophan 2,3-dioxygenase (TDO). Among them, the three phenyl urea derivatives i12, i23, i24 as showed potent IDO1 inhibition, with IC50 values of 0.1–0.6 μM and no compound exhibited TDO inhibitory activity. Using molecular docking, we predicted the binding mode of compound i12 within IDO1. Compound i12 was further investigated by determining its in vivo pharmacokinetic profile and anti-tumor efficacy. The pharmacokinetic study revealed that compound i12 had satisfactory properties in mice, with moderate plasma clearance (22.45 mL/min/kg), acceptable half-life (11.2 h) and high oral bioavailability (87.4%). Compound i12 orally administered at 15 mg/kg daily showed tumor growth inhibition (TGI) of 40.5% in a B16F10 subcutaneous xenograft model and 30 mg/kg daily showed TGI of 34.3% in a PAN02 subcutaneous xenograft model. In addition, the body weight of i12-treated mice showed no obvious reduction compared with the control group. Overall, compound i12 is a potent lead compound for developing IDO1 inhibitors and anti-tumor agents.

Full text of DOI:10.3390/molecules25061447

molecules
Article
Design, Synthesis and Biological Evaluation of
Phenyl Urea Derivatives as IDO1 Inhibitors
Chuan Zhou , Fangfang Lai *, Li Sheng *, Xiaoguang Chen *, Yan Li and Zhiqiang Feng 1,*
1
2,
2,
2,
2
1
Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
zhouchuan@imm.ac.cn
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,
2
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;
yanli@imm.ac.cn
*
Correspondence: laifangfang@imm.ac.cn (F.L.); shengli@imm.ac.cn (L.S.); chxg@imm.ac.cn (X.C.);
fengzhq@imm.ac.cn (Z.F.); Tel.: +86-10-63189351 (Z.F.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Orazio Nicolotti
Received: 3 March 2020; Accepted: 19 March 2020; Published: 23 March 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing intracellular enzyme that
catalyzes the first and rate-determining step of tryptophan metabolism and is an important
immunotherapeutic target for the treatment of cancer. In this study, we designed and synthesized
a new series of compounds as potential IDO1 inhibitors. These compounds were then evaluated
for inhibitory activity against IDO1 and tryptophan 2,3-dioxygenase (TDO). Among them, the three
phenyl urea derivatives i12, i23, i24 as showed potent IDO1 inhibition, with IC50 values of 0.1–0.6 µM
and no compound exhibited TDO inhibitory activity. Using molecular docking, we predicted the
binding mode of compound i12 within IDO1. Compound i12 was further investigated by determining
its in vivo pharmacokinetic profile and anti-tumor efficacy. The pharmacokinetic study revealed that
compound i12 had satisfactory properties in mice, with moderate plasma clearance (22.45 mL/min/kg),
acceptable half-life (11.2 h) and high oral bioavailability (87.4%). Compound i12 orally administered
at 15 mg/kg daily showed tumor growth inhibition (TGI) of 40.5% in a B16F10 subcutaneous xenograft
model and 30 mg/kg daily showed TGI of 34.3% in a PAN02 subcutaneous xenograft model. In
addition, the body weight of i12-treated mice showed no obvious reduction compared with the
control group. Overall, compound i12 is a potent lead compound for developing IDO1 inhibitors and
anti-tumor agents.
Keywords: indoleamine 2,3-dioxygenase 1; tryptophan metabolism; immunotherapeutic target;
anti-tumor; IDO1 inhibitor
1
. Introduction
The tryptophan/kynurenine pathway plays an important role in cancer immunotherapy [1].
Activation of this pathway promotes the degradation of tryptophan and leads to the formation of
kynurenine and other bioactive metabolites, such as 3-hydroxykynurenine and 3-hydroxyanthranilic
acid. A local depletion of tryptophan induces T cell cycle arrest and the accumulation of tryptophan
metabolites converses naïve T cells into regulatory T cells and induces T cell apoptosis. These exert a
local immunosuppressive effect which can lead to tumor progression and recurrence [2–4].
Indoleamine 2, 3-dioxygenase 1 (IDO1, EC 1.13.11.52) catalyzes the initial and rate-limiting step in
the catabolism of tryptophan along the kynurenine pathway [
cells in the tumor microenvironment are shown to overexpress IDO1, which is often associated with
worse response to anticancer therapies and decreased survival of cancer patients [ ]. Inhibition of
5]. Cancer cells and a variety of immune
6,7
Molecules 2020, 25, 1447; doi:10.3390/molecules25061447
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1447
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IDO1 was shown to increase the therapeutic efficacy of cancer vaccines, immune checkpoint inhibitors,
or chemotherapy in multiple clinical mouse models [ 10]. On this basis, several IDO1 inhibitors have
been developed and are currently under clinical development (Figure 1) [1].
8
–
Br
F
F
F
O
NH
2
O
H
N
N
F
N
OH
H
O
H
NH
S
H
O
O
O
N
N
H
2
N
NH
H
Cl
O
HO
N
N
OH
N
N
N
H
N
H
OH
indoximod
pathway IC50 = 450 nM
navoximod
IDO1 IC50 = 13 nM
epacadostat
IDO1 IC50 = 72 nM
BMS-986205
IDO1 IC50 = 1.7nM
PF-06840003
IDO1 IC50 = 150nM
Figure 1. IDO1 inhibitors in clinical trials.
In this study, a new series of compounds were designed based on the phenyl urea scaffold in order
to search for new IDO1 inhibitors. The compounds were synthesized and their IDO1/TDO inhibitory
activities were determined. The in vivo pharmacokinetic profile and anti-tumor efficacy of a potent
IDO1 inhibitor were evaluated to explore its potential as an anti-tumor agent.
2
. Results and Discussion
2
.1. Design Strategies of the Compounds
It was disclosed that the compound BMS-E30 showed potent IDO1 inhibitory activity
(IC50 = 0.7 nM) in the IDO1 kynurenine assay with human IDO1/HEK 293 cells (Scheme 1) [11],
however, its enzyme inhibitory activity was relatively weak with an IC50 of 8.569 M (Table 1). The
µ
lack of heme-coordinating element distinguished compound BMS-E30 from other IDO1 inhibitors
in the literature. We considered that BMS-E30 effectively inhibited IDO1 by targeting its apo-form
subsequent to the disclosure of the IDO1/BMS-978587 crystal structure (6AZV) because the structures
of BMS-E30 and BMS-978587 were similar (Scheme 1) [12]. Considering the two flexible chains of
diisobutylamino group in compound BMS-E30, a new series of compounds were designed by replacing
the diisobutylamino group with a 3,5-dimethylpiperidinyl group for reducing the entropy loss of
binding IDO1. The peripheral phenyl urea group was also modified to explore the structure-activity
relationship (SAR) which was used for further optimization to obtain better IDO1 inhibitors.
F
Ar
O
X
F
O
CF3
O
NH
NH
O
NH
NH
NH
O
CF3
rigidization
HO
O
HO
N
OH
N
N
BMS-E30
X=NH or CH
2
BMS-978587
Scheme 1. Design strategies of the inhibitors and the chemical structure of BMS-978587. The flexible
disobutylamino group was replaced with the rigid 3, 5-dimethylpiperidinyl group to optimize the
space steric effect of substituent and NH of the phenyl urea group was further replaced with CH to test
whether the phenyl urea group is essential to IDO1 potency.
2
.2. Synthesis of Selected Compounds
In Scheme 2, nitration of 4-fluorobenzaldehyde was performed with nitric acid and sulfuric
acid to produce compound
nucleophilic aromatic substitution reaction with 3,5-dimethylpiperidine. An aldehyde group addition
reaction was performed in the presence of (trifluoromethyl)trimethylsilane to obtain compound 14],
a [13]. Under basic conditions, compound b was obtained through a
c
[

Molecules 2020, 25, 1447
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which was oxidized to compound
d
with Dess-Martin periodinane. Compound
e
was prepared by a
Horner-Wadsworth-Emmons reaction reaction of
and nitro group were reduced simultaneously with palladium on carbon under a hydrogen atmosphere
to give compound . Treatment of compound with a suitable isocyanate afforded compounds g1 g24
which were further subjected to hydrolysis to obtain final products i1 i24. HATU-mediated amide
coupling reaction between compound and a suitable p-substituted phenylacetic acid formed the
intermediates h1–h3, which were converted into the final products j1–j3 by hydrolysis reactions.
d with triethyl phosphonoacetate. The double bond
f
f
–
,
–
f
OH
O
O
CF3
NO
N
2
NO2
N
NO
N
2
NO2
NO
F
2
O
F
3
C
3
F C
O
i
O
ii
iii
iv
v
O
F
N
a
b
c
d
e
R
R
O
NH
O
NH
O
CF3
O
CF3
NH
N
NH
O
HO
ix
1
6
2
ii
N
v
O
CF
3
5
3
NH
2
4
O
vi
7
g1-g24
i1-i24
N
f
R
R
v
ii
i
O
O
O
CF
3
O
CF
3
NH
N
O
HO
ix
1
6
2
N
5
3
4
7
h1-h3
j1-j3
Scheme 2. Synthesis of compounds i1–i24 and j1–j3. Reagents and Conditions: (i) sulfuric acid,
nitric acid, yield = 95.4%; (ii) 3,5-dimethylpiperidine, Et N, dichloromethane, yield = 93.5%;
3
(
iii) (trifluoromethyl)trimethylsilan, K CO , DMF, yield = 86.8%; (iv) Dess-Martin periodinane, NaHCO ,
2
3
3
dichloromethane; (v) NaH, triethyl phosphonoacetate, dry tetrahydrofuran, yield = 32.5% (two steps);
vi) Pd-C, hydrogen balloon, dry tetrahydrofuran, yield = 42.3%; (vii) appropriate isocyanate, dry
tetrahydrofuran, yield = 78.4%–87.5%; (viii) p-substituted phenylacetic acid, HATU, Et N, DMF,
(
3
yield = 68.7%–71.2%; (ix) NaOH, THF/MeOH/H O; 1N HCl, yield = 70.1%–86.5%.
2
2
.3. In Vitro Biological Evaluation
We prepared the compounds using the synthetic scheme in Section 2.2 and these compounds were
screened in vitro for their IDO1 and TDO inhibitory activities. The reported IDO1 inhibitor epacadostat
was used as the reference compound in this study [15].
As shown in Table 1, compounds g1
group in compounds i1 i3 was exposed by hydrolysis, IDO1 inhibitory activity appeared. The
enzymatic results of compounds g1 g3 and i1 i3 indicated that the carboxyl group plays a critical
role in the binding activity. In addition, the replacement of phenyl ring with a cyclohexyl group
compound i4) or an n-hexyl group (compound i5) resulted in loss of inhibition. This suggested that
–g3 showed no IDO1 inhibitory activity. When the carboxyl
–
–
–
(
phenyl ring is important for the binding activity.
Modification of the benzene ring of compound i1 was performed and the results are presented in
Table 2. When the ortho-position or the meta-position was substituted by CH3 (i6, i7), Cl (i8
, i9), CN (i10,
i11) or OCH3 (i13 i14), respectively, the obtained compounds lost all IDO1 inhibitory activity. However,
,
p-substituted phenyl urea derivatives (i2, i3, i12 and i15) showed more potent IDO1 inhibitory activity
than the unsubstituted i1.

Molecules 2020, 25, 1447
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Table 1. IC50 values of compounds g1–g3 and i1–i5 against IDO1 and TDO.
H
O
N
O
CF
3
R
1
NH
2
R O
N
IC50 (µM)
Compound
R1
R2
IDO1
TDO
g1
g2
g3
i1
Et
Et
Et
H
H
H
H
>100
>100
>100
>100
34.130
8.613
5.687
>100
>100
>100
>100
>100
>100
>100
CH
3
Cl
i2
CH
3
i3
Cl
i4
i5
H
-
-
>100
8.569
0.049
>100
>100
-
BMS-E30-2
Epacadostat
-
-
The o,p-disubstituted phenyl urea derivatives, bearing o,p-difluoro (i16) or o,p-dichloro (i17
)
substituents, were also explored and had no inhibitory activities. These results demonstrated a strong
preference for para-substitution.
At the para-position, substitution with other halogens such as fluorine (i18, 5.475
i19, 4.077 M) had similar activity as chlorine (i3, 5.687 M). In addition, small para-alkyl substituents
i2, 8.613 M; i20, 9.975 M) produced inhibition against IDO1, whereas substitution with an isopropyl
µM) and bromine
(
(
µ
µ
µ
µ
group (i21) led to the loss of activity. This result suggested that the size of the para-substituent on
the benzene ring cannot be too large. Further optimization revealed that substitution with electron
withdrawing groups at the para-position was beneficial for the activity (i12, 0.331
µM; i23, 0.415 µM;
i24, 0.157 µM) and the p-NO derivative (i24) was 55-fold more potent than compound BMS-E30.
2
Finally, replacement of proximal NH of the urea with methylene was also investigated. The
observation that phenylacetamide derivatives j1, j2, j3 had no inhibitory activity was suggestive that
the proximal NH was crucial to IDO1 potency. As shown in Tables 1 and 2, none of the synthesized
compounds showed inhibitory activity against TDO, which demonstrated that the phenyl urea
derivatives were selective IDO1 inhibitors.
In summary, compound i24 showed the most potent IDO1 inhibitory activity based on the SAR
study, however, it contains a nitro group which is considered to be easily metabolized and toxic
in vivo
[16–18], so the second most potent IDO1 inhibitor i12 was used as the lead compound for
further study.

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Table 2. IC50 values of compounds i1–i3, i6–i24 and j1–j3 against IDO1 and TDO.
O
X
O
CF3
Ar
NH
HO
N
IC50 (µM)
Compound
Ar
X
IDO1
TDO
i1
i6
i7
NH
NH
NH
34.130
>100
CH
3
>100
>100
>100
>100
CH
3
i2
i8
NH
NH
8.613
>100
>100
CH
3
Cl
>100
Cl
Cl
i9
i3
NH
NH
>100
>100
>100
5.687
CN
i10
NH
>100
>100
CN
i11
i12
NH
NH
>100
>100
>100
0.331
CN
OCH
3
i13
NH
>100
>100
OCH
3
i14
i15
NH
NH
>100
>100
>100
1.617
OCH
3
F
i16
i17
NH
NH
>100
>100
>100
>100
F
Cl
Cl
F
i18
i19
NH
NH
5.475
4.077
>100
>100
Br

Molecules 2020, 25, 1447
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Table 2. Cont.
O
X
O
CF3
Ar
NH
N
HO
IC50 (µM)
Compound
Ar
X
IDO1
TDO
i20
i21
i22
i23
i24
j1
NH
NH
NH
NH
NH
CH
CH
CH
9.975
>100
2 3
CH CH
>100
1.324
0.415
0.157
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3 2
CH(CH )
OCF
3
CF
3
NO
2
CN
j2
CF
3
j3
NO
2
BMS-E30
Epacadostat
-
-
-
-
8.569
0.049
>100
-
2
.4. Predicted Binding Mode of Compound i12 with IDO1
The molecular docking study was performed to investigate the binding mode of compound i12
with IDO1 (6AZV) by using CDOCKER protocol integrated in Accelrys Discovery Studio [12 19]. As
,
shown in Figure 2, the carboxylic group in compound i12 forms hydrogen bonds with the backbone
amide of Ala-264 and with His-346, which is supposed to make critical contributions to the binding
since the carboxylic group is an essential pharmacophore as SAR demonstrated.
The phenyl urea group in compound i12 binds via edge-to-face π-interaction with Tyr126 and
hydrogen bonds with Ser167, which is also important for potency and is consistent with the SAR
results. The peripheral phenyl ring was placed into the hydrophobic pocket where it is suited to
extend a small para-substituent, which explained the loss of activity of compounds bearing bulky
substituents or substituents at the ortho- and meta-positions. Particularly, electron-withdrawing groups
at the para-position of the benzene ring were beneficial to the phenyl urea group as hydrogen bond
donors, providing a rationale for 100-fold improvement in IDO1 inhibitory activity observed when
compound i12 is compared to compound i1.

Molecules 2020, 25, 1447
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Figure 2. Predicated binding mode of compound i12 with IDO1 using CDOCKER. (A) The binding
mode of compound i12 with IDO1 (6AZV), the enzyme is shown in yellowish brown, compound i12 is
shown as sticks with cyan carbon atoms. The residues that interact with compound i12 are shown as
sticks with pink carbon atoms, and hydrogen bonds are indicated by yellow dashed lines. The images
were generated by using Chimera 1.12. (B) Schematic 2D diagram of the key interactions between
compound i12 with IDO1 (6AZV).
2
.5. Pharmacokinetic Study of Compound i12
Compound i12 was further evaluated in in vivo pharmacokinetic study using male C57BL/6 mice.
The plasma concentration-time profiles are shown in Figure 3 and the pharmacokinetic parameters
were determined by non-compartmental analysis (Table 3).

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Figure 3. Mean plasma concentration-time profile in male C57BL/6 mice after administration of a single
oral (30 mg/kg) and intravenous (3 mg/kg) dose of compound i12. PO stands for oral administration of
drug; IV stands for intravenous administration of drug.
Table 3. Pharmacokinetic parameters of compound i12 after a single oral or intravenous dose in male
C57BL/6 mice.
PO
0 mg/kg
IV
3 mg/kg
Parameters
Units
3
t1/2β
Tmax
h
h
11.2
2
7.17
-
Cmax
ng/mL
h·ng/mL
h·ng/mL
h
L/kg
mL/min/kg
2702
11,523
12,849
8.18
-
6958
1318
2159
8.60
15.80
22.45
-
AUC(0–t)
AUC(0–∞)
MRT(0–∞)
Vss
Cl
F%
-
87.4
When intravenously administered at a dose of 3 mg/kg, compound i12 had moderate clearance of
2.45 mL/min/kg and an extensive distribution in tissues with Vss value of 15.80 L/kg. After an oral
2
administration of 30 mg/kg, compound i12 was absorbed with Tmax value of 2 h and Cmax value of
702 ng/mL. Compound i12 was eliminated slowly with a half-time of 11.2 h and showed reasonable
oral exposure with AUC(0– ) value of 11,523 h ng/mL. The oral bioavailability of compound i12 was
determined to be 87.4%, supporting a further evaluation of its in vivo efficacy.
2
∞
·
2
.6. In Vivo Anti-Tumor Efficacy Study of Compound i12
Compound i12 was tested for its in vivo antitumor activities in melanoma and pancreatic cancer
xenograft models (Figures 4 and 5). In a mouse B16F10 subcutaneous xenograft model, compound
i12 orally administered at 15 mg/kg daily demonstrated obvious in vivo anti-tumor activity and
showed tumor growth inhibition (TGI) of 40.5%. In addition, the body weight of i12-treated mice
showed no significant changes compared with the control group. The classic first line anti-cancer drug
cyclophosphamide (CTX) was used as the reference drug.

Molecules 2020, 25, 1447
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Figure 4. In vivo anti-tumor activity of i12 in B16F10 xenograft mice. (A) Tumor weights of each group
after 17 days of treatment. The control group mice bearing B16F10 xenografts were dosed orally with
vehicle (0.5% sodium salt of carboxymethyl cellulose, CMC-Na); the CTX group were administered
CTX intraperitoneally at the dose of 100 mg/kg; the treated group were administered i12 orally at the
dose of 15, 30 or 100 mg/kg. * p < 0.05, ** p < 0.01 and **** p < 0.0001 versus vehicle. (B) The body
weight of each group after the treatment. There is no obvious body weight difference among all of the
i12-treated groups.
Figure 5. In vivo anti-tumor activity of i12 in PAN02 pancreatic cancer xenograft mice. (A) Tumor
weights of each group after 15 days of treatment. The control group mice bearing PAN02 pancreatic
cancer xenografts were dosed orally with vehicle (0.5% sodium salt of carboxymethyl cellulose,
CMC-Na); the CTX group were administered CTX intraperitoneally at the dose of 60 mg/kg; the treated
group were administered i12 orally at the dose of 10, 30 or 100 mg/kg. ** p < 0.01 and *** p < 0.001
versus vehicle. (
B) The body weight of each group after the treatment. There is no obvious body weight
difference among any of the i12-treated groups.

Molecules 2020, 25, 1447
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We also evaluated compound i12 in a PAN02 pancreatic cancer xenograft model, in which
compound i12 oral treatment resulted in a 34.3% decrease in tumor weight at a dose of 30 mg/kg daily
compared with the control group.
3
. Experimental Section
3
.1. General Information
1
Reagents and solvents were obtained from commercial suppliers and used as received. H-NMR
spectra were obtained on a 400 MHz Mercury NMR spectrometer (Varian, San Diego, CA, USA).
Electrospray ionization (ESI) mass spectra and high-resolution mass spectroscopy (HRMS) were
performed with a liquid chromatograph/mass selective detector time-of-flight mass spectrometer
(
LC/MSD TOF, Agilent Technologies, Santa Clara, CA, USA). Silica gel column chromatography was
performed with silica gel 60G (Qingdao Haiyang Chemical, Qingdao, China). Purity was determined
using HPLC, LC/MS and NMR spectroscopy (Supplementary Materials). All of the synthesized
compounds have purities over 95%.
3
.1.1. Preparation of 4-Fluoro-3-nitro-benzaldehyde (a)
To a solution of sulfuric acid (24 mL) and nitric acid (3 mL) was slowly added 4-fluoro-benzaldehyde
◦
(
6 g, 48.3 mmol) at
−
5 C. The reaction mixture was stirred at room temperature for 2 h, poured onto
ice and extracted with ethyl acetate (200 mL). The organic extract was washed with brine (60 mL
dried over anhydrous Na SO and concentrated. The residue was purified by column chromatography
silica gel, PE/EA = 30:1, v/v) to afford compound a as a light yellow solid (7.8 g, 95.4% yield). H-NMR
CDCl3): 10.05 (s, 1H, CHO), 8.60 (dd, J = 7.0, 1.7 Hz, 1H, H-phenyl), 8.21 (ddd, J = 8.6, 4.2, 2.1 Hz,
H, H-phenyl), 7.51 (dd, J = 9.9, 8.7 Hz, 1H, H-phenyl). HRMS (ESI) m/z: [M + H] calculated for
C H O NF, 170.02480; found, 170.02446, ∆ −1.99 ppm.
×
2),
2
4
1
(
(
1
δ
+
7
5
3
3
.1.2. Preparation of 4-(3,5-Dimethylpiperidin-1-yl)-3-nitro-benzaldehyde (b)
To a solution of 4-fluoro-3-nitrobenzaldehyde (
was added 3,5-dimethylpiperidine (5.9 g, 52.1 mmol) and ethylamine (5.3 g, 52.4 mmol) at 0 C. The
resulting mixture was stirred at room temperature for 0.5 h, poured into water (200 mL) and extracted
a
, 5.9 g, 34.9 mmol) in dichloromethane (100 mL)
◦
with dichloromethane (200 mL
×
2). The combined organic layers were washed with 1N HCl aqueous
2), dried over anhydrous Na SO
solution (150 mL 2), saturated NaCl aqueous solution (150 mL
×
×
2
4
and concentrated. The residue was purified by column chromatography (silica gel, PE/EA = 10:1, v/v)
1
to afford the product b as a yellow solid (8.6 g, 93.5% yield). H-NMR (DMSO-d ):
δ 9.82 (s, 1H, CHO),
6
8
.31 (s, 1H, H-phenyl), 7.90 (t, J = 9.8 Hz, 1H, H-phenyl), 7.39 (d, J = 8.6 Hz, 1H, 1H-phenyl), 3.30 (d,
J = 12.9 Hz, 2H, CH -1 and CH -5), 2.58 (t, J = 12.0 Hz, 2H, CH -1 and CH -5), 1.84–1.65 (m, 3H, CH -3,
CH-2, CH-4), 0.92–0.74 (m, 8H, CH -6, CH -7 and CH -3). HRMS (ESI) m/z: [M + H] calculated for
a
a
b
b
a
+
3
3
b
C H O N , 263.13902; found, 263.13876, ∆ −0.98 ppm.
14
19
3
2
3
.1.3. Preparation of 1-[4-(3,5-Dimethylpiperidin-1-yl)-3-nitro-phenyl]-2,2,2-trifluoroethanol (c)
To a solution of 4-(3,5-dimethylpiperidin-1-yl)-3-nitrobenzaldehyde ( , 4.2 g, 16.0 mmol) and
b
trimethyl(trifluoromethyl)silane (4.6 g, 32.0 mmol) in dimethylformamide (40 mL) was added potassium
◦
carbonate (4.4 g, 32.0 mmol) at 0 C. The resulting mixture was warmed to room temperature and
stirred for 12 h. The mixture was then treated with 40 mL of 1 mol/L HCl aqueous solution, stirred for
another 30 min, poured into water (200 mL) and extracted with ethyl acetate (200 mL
×
2). The organic
extract was washed with saturated NaCl aqueous solution (150 mL 3), dried over anhydrous Na SO
×
2
4
and concentrated. The residue was purified by column chromatography (silica gel, PE/EA = 8:1,
1
v/v) to afford the product c as a brown red oil (4.6 g, 86.8% yield). H-NMR (DMSO-d ):
δ 7.91 (d,
6
J = 1.9 Hz, 1H, H-phenyl), 7.64 (dd, J = 8.6, 1.7 Hz, 1H, H-phenyl), 7.31 (dd, J = 8.7, 2.5 Hz, 1H,
H-phenyl), 7.00–6.94 (m, 1H, CH), 5.24 (d, J = 7.0 Hz, 1H, OH), 3.13 (dd, J = 12.0, 1.5 Hz, 2H, CH -1
a

Molecules 2020, 25, 1447
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and CH
a
-5), 2.38 (t, J = 11.5 Hz, 2H, CH -1 and CH -5), 1.81–1.65 (m, 3H, CH
a
-3, CH-2, CH-4), 0.85 (d,
b
b
+
J = 6.5 Hz, 6H, CH -6, CH -7), 0.69 (q, J = 9.6 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated
3
3
b
for C H O N F , 333.14205; found, 333.14246, ∆ 1.22 ppm.
15
20
3
2 3
3
.1.4. Preparation of 3-[4-(3,5-Dimethylpiperidin-1-yl)-3-nitrophenyl]-4,4,4-trifluorobut-2-enoic Acid
Ethyl Ester (e)
To a solution of 1-[4-(3,5-dimethylpiperidin-1-yl)-3-nitrophenyl]-2,2,2-trifluoroethanol (
2.6 mmol) in dichloromethane (200 mL) at 0 C was added sodium bicarbonate (3.2 g, 37.8 mmol)
c
, 4.2 g,
◦
1
followed by Dess-Martin periodinane (8.0 g, 18.9 mmol). The resulting solution was warmed to room
temperature and stirred for 2 h. The reaction mixture was then diluted with saturated NaHCO3
aqueous solution (100 mL) and stirred for another 30 min. The organic layer was separated and washed
with saturated NaCl aqueous solution (100 mL
The residue was preliminarily purified by column chromatography (silica gel, PE/EA = 20:1, v/v) to
afford crude product as a yellow solid. The crude product was used directly without further
×
2), dried over anhydrous Na SO and concentrated.
2 4
d
d
purification. Next ethyl 2-(diethoxyphosphoryl)acetate (1.1 g, 4.9 mmol) was added to a solution of
◦
NaH (2.28 mg, 5.7 mmol) in dry tetrahydrofuran at 0 C. The reaction mixture became a clear solution
after stirring for 30 min. The crude product
d in dry tetrahydrofuran was then added. The resulting
mixture was warmed to room temperature, stirred for 2 h, quenched with saturated NH Cl aqueous
4
solution (10 mL). The aqueous layer was further extracted with ethyl acetate (50 mL
combined organic extracts were washed with saturated NaCl aqueous solution (50 mL
anhydrous Na SO and concentrated. The residue was purified by column chromatography (silica
×
2) and the
2), dried over
×
2
4
1
gel, PE/EA = 40:1, v/v) to afford the product e as an orange yellow oil (1.64 g, 32.5% yield). H-NMR
DMSO-d6): 7.73 (d, J = 2.2 Hz, 1H, H-phenyl), 7.44 (dd, J = 8.7, 2.0 Hz, 1H, H-phenyl), 7.32 (dd,
J = 8.8, 2.8 Hz, 1H, H-phenyl), 6.86 (s, 1H, =CH), 4.04 (q, J = 7.2 Hz, 2H, CH ), 3.21–3.13 (m, 2H, CH -1
-3, CH-2, CH-4), 1.04 (t,
J = 7.2 Hz, 3H, CH ), 0.85 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.73 (q, J = 11.8 Hz, 1H, CH -3). HRMS
(
δ
2
a
b b
a a
and CH -5), 2.44 (t, J = 11.7 Hz, 2H, CH -1 and CH -5), 1.83–1.64 (m, 3H, CH
3
+
3
3
b
(
ESI) m/z: [M + H] calculated for C H O N F , 401.16827; found, 401.16788, ∆ −0.97 ppm.
15
20
3
2 3
3
.1.5. Preparation of 3-[3-Amino-4-(3,5-dimethylpiperidin-1-yl)-phenyl]-4,4,4-trifluorobutyric Acid
Ethyl Ester (f)
To a solution of 3-[4-(3,5-dimethylpiperidin-1-yl)-3-nitrophenyl]-4,4,4-trifluorobut-2-enoic acid
ethyl ester ( , 2.4 g, 6.0 mmol) in dry tetrahydrofuran (60 mL) was added palladium on carbon (640 mg,
0% Pd/C, 0.6 mmol) and the suspension was hydrogenated (1 atm, balloon) for 2 h. Thin layer
chromatography (TLC) indicated completion. The suspension was filtered through a pad of Celite
and the filter cake was rinsed with ethyl acetate (50 mL 3). The combined filtrate and rinses were
e
1
×
concentrated and the residue was purified by column chromatography (silica gel, PE/EA = 30:1, v/v) to
1
afford the product f as a colorless oil (945 mg, 42.3% yield). H-NMR (DMSO-d ):
δ 6.82 (d, J = 8.1 Hz,
6
1
H, H-phenyl), 6.67 (d, J = 1.7 Hz, 1H, H-phenyl), 6.54 (dd, J = 8.0, 1.8 Hz, 1H, H-phenyl), 4.76 (s, 2H,
NH ), 4.07–3.93 (m, 2H, CH ), 3.85–3.70 (m, 1H, CH), 3.02–2.76 (m, 4H, CH , CH -1 and CH -5), 2.00
-3, CH-2, CH-4), 1.08 (t, J = 7.1 Hz,
2
2
2
a
a
(
td, J = 10.9, 4.4 Hz, 2H, CH -1 and CH -5), 1.86–1.70 (m, 3H, CH
a
b b
+
3
H, CH ), 0.85 (d, J = 6.4 Hz, 6H, CH -6, CH -7), 0.68–0.56 (m, 1H, CH -3). HRMS (ESI) m/z: [M + H]
3 3 3 b
calculated for C15H20O3N2F3, 373.20974; found, 373.20999, ∆ 0.67 ppm.
3
.1.6. General Procedure A for the Synthesis of g1–g24
To a solution of 3-[3-amino-4-(3,5-dimethylpiperidin-1-yl)-phenyl]-4,4,4-trifluorobutyric acid ethyl
ester (f, 1 equiv.) in tetrahydrofuran was added a suitable isocyanate (1 equiv.). The reaction mixture
was stirred at room temperature until the starting material disappeared in TLC. The reaction mixture
was concentrated and the residue was purified by column chromatography (silica gel, PE/EA = 8:1, v/v)
to afford the products g1–g24.

Molecules 2020, 25, 1447
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3
.1.7. General Procedure B for the Synthesis of i1–i24 and j1–j3
To a solution of compound g1–g24 or h1–h3 (1 equiv.) in tetrahydrofuran (4 volumes), methanol
1 volume) and water (1 volume) was added sodium hydroxide (3 equiv.). The resulting mixture was
(
stirred at room temperature until the starting material disappeared in TLC. Part of the tetrahydrofuran
and methanol was removed in vacuo and the crude was diluted with water (2 volumes) and the pH
was adjusted to ca. 4 using 1 N HCl solution. The aqueous phase was then extracted with ethyl acetate
(
15 v
×
3) and the combined organic extracts were washed with saturated NaCl solution, dried over
anhydrous Na SO and concentrated. The residue was purified by column chromatography (silica gel,
2
4
DCM/MeOH = 30:1, v/v) to afford the products i1–i24 or j1–j3.
3
4
.1.8. Preparation of Racemic 3-[4-(3,5-dimethylpiperidin-1-yl)-3-(3-phenylureido)-phenyl]-4,4,
-trifluorobutyric Acid Ethyl Ester (g1)
Reaction of compound
compound g1 (white solid, 85.3% yield). H-NMR (DMSO-d ):
f
and phenyl isocyanate following the general procedure A afforded
1
δ 9.54 (s, 1H, NH), 8.14 (d, J = 1.6 Hz,
6
1
H, H-phenyl), 8.07 (s, 1H, NH), 7.49 (d, J = 7.9 Hz, 2H, H-phenyl), 7.29 (t, J = 7.8 Hz, 2H, H-phenyl),
7
.13 (d, J = 8.2 Hz, 1H, H-phenyl), 7.02–6.95 (m, 2H, H-phenyl), 4.07–3.87 (m, 3H, CH , CH), 3.07–2.79
2
(
m, 4H, CH , CH
a
-1 and CH
CH-4), 1.80 (d, J = 12.7 Hz, 1H, CH
CH -7), 0.67 (q, J = 12.0 Hz, 1H, CH -3). C-NMR (DMSO-d6):
a
b b
-5), 2.14 (t, J = 10.3 Hz, 2H, CH -1 and CH -5), 2.06–1.91 (m, 2H, CH-2,
2
a
-3), 1.08 (t, J = 7.1 Hz, 3H, CH ), 0.87 (d, J = 6.6 Hz, 6H, CH -6,
3 3
13
δ
169.61, 152.54, 141.92, 139.82, 134.19,
3
b
1
3
29.17, 128.87 (2C), 128.14, 122.53, 122.04, 120.38, 119.34, 118.56 (2C), 60.48, 59.72 (2C), 45.02, 41.62,
3.81, 30.87 (2C), 19.33 (2C), 13.92. HRMS (ESI) m/z: [M + H] calculated for C H O N F , 492.24685;
26 33 3 3 3
+
found, 492.24582, ∆ −2.10 ppm.
3
4
.1.9. Preparation of Racemic 3-[4-(3,5-dimethylpiperidin-1-yl)-3-(3-phenylureido)-phenyl]-4,4,
-trifluorobutyric Acid (i1)
Hydrolysis of compound g1 (80 mg, 0.16 mmol) following the general procedure B afforded
◦
1
compound h1 (white solid, 61 mg, 80.7% yield). mp: 94.1–95.7 C. H-NMR (DMSO-d ):
1
1
δ
12.61 (s,
H, COOH), 9.58 (s, 1H, NH), 8.14 (d, J = 1.9 Hz, 1H, H-phenyl), 8.09 (s, 1H, NH), 7.49 (dd, J = 8.6,
.0 Hz, 2H, H-phenyl), 7.33–7.26 (m, 2H, H-phenyl), 7.13 (d, J = 8.2 Hz, 1H, H-phenyl), 7.02–6.95 (m,
H, H-phenyl), 3.95–3.83 (m, 1H, CH), 2.97–2.71 (m, 4H, CH , CH -1 and CH
6
2
3
a
a
-5), 2.14 (td, J = 11.2,
2
.7 Hz, 2H, CH -1 and CH -5), 2.05–1.91 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.8 Hz, 1H, CH
J = 6.6 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.4 Hz, 1H, CH -3). HRMS(ESI) m/z: [M + H] calculated
a
+
-3), 0.87 (d,
b
b
3
3
b
for C H O N F , 464.21555; found, 464.21466, ∆ −1.92 ppm.
24
29
3
3 3
3
.1.10. Preparation of Racemic 3-[4-(3,5-dimethylpiperidin-1-yl)-3-(3-p-tolylureido)phenyl]-4,4,
-trifluorobutyric Acid Ethyl Ester (g2)
4
Reaction of compound
compound g2 (white solid, 86.1% yield). H-NMR (DMSO-d ):
f
and p-tolyl isocyanate following the general procedure A afforded
1
δ 9.42 (s, 1H, NH), 8.14 (d, J = 2.0 Hz,
6
1
H, H-phenyl), 8.02 (s, 1H, NH), 7.38–7.35 (m, 2H, H-phenyl), 7.15–7.07 (m, 3H, H-phenyl), 6.97 (dd,
J = 8.2, 2.0 Hz, 1H, H-phenyl), 4.07–3.88 (m, 3H, CH , CH), 3.06–2.79 (m, 4H, CH , CH -1 and CH -5),
.25 (s, 3H, CH -phenyl), 2.13 (td, J = 11.2, 2.0 Hz, 2H, CH -1 and CH -5), 2.02–1.90 (m, 2H, CH-2,
2
2
a
a
2
3
b
b
CH-4), 1.79 (d, J = 13.1 Hz, 1H, CH
a
3 3
-3), 1.07 (t, J = 6.8 Hz, 3H, CH ), 0.86 (d, J = 6.6 Hz, 6H, CH -6,
13
CH -7), 0.66 (q, J = 12.4 Hz, 1H, CH -3). C-NMR (DMSO-d6):
δ
169.61, 152.59, 141.81, 137.19, 134.32,
3
b
1
3
5
30.95, 129.28 (2C), 129.20, 122.40, 120.39, 119.19, 118.79 (2C), 118.27, 60.48, 59.71 (2C), 44.90, 41.62,
3.81, 30.87 (2C), 20.42, 19.33 (2C), 13.92. HRMS (ESI) m/z: [M + H] calculated for C H O N F ,
27 35 3 3 3
06.26250; found, 506.26242, ∆ −0.16 ppm.
+

Molecules 2020, 25, 1447
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3
.1.11. Preparation of Racemic 3-[4-(3,5-dimethylpiperidin-1-yl)-3-(3-p-tolylureido)phenyl]-4,4,
-trifluorobutyric Acid (i2)
4
Hydrolysis of compound g2 (140 mg, 0.28 mmol) following the general procedure B afforded
◦
1
compound i2 (white solid, 113 mg, 85.4% yield). mp: 134.3–136.1 C. H-NMR (DMSO-d ):
δ
12.50 (s,
6
1
2
H, COOH), 9.45 (s, 1H, NH), 8.15 (d, J = 1.8 Hz, 1H, H-phenyl), 8.04 (s, 1H, NH), 7.37 (d, J = 8.4 Hz,
H, ArH-phenyl), 7.15–7.06 (m, 3H, H-phenyl), 6.97 (dd, J = 8.2, 1.8 Hz, 1H, H-phenyl), 3.96–3.82 (m,
H, CH), 2.99–2.75 (m, 4H, CH , CH -1 and CH -5), 2.25 (s, 3H, -CH , CH -phenyl), 2.13 (td, J = 11.1,
.3 Hz, 2H, CH -1 and CH -5), 2.03–1.89 (m, 2H, CH-2, CH-4), 1.79 (d, J = 12.7 Hz, 1H, CH
1
3
a
a
2
3
3
b
b
a
-3), 0.86
171.05,
52.62, 141.76, 137.22, 134.36, 130.95, 129.48, 129.28 (2C), 126.88, 122.49, 120.39, 119.17, 118.82 (2C),
13
(d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.0 Hz, 1H, CH -3). C-NMR (DMSO-d6): δ
3 3 b
1
+
5
9.75 (2C), 45.14, 41.65, 33.80, 30.87 (2C), 20.42, 19.33 (2C). HRMS (ESI) m/z: [M + H] calculated for
C H O N F , 478.23120; found, 478.23230, ∆ 2.29 ppm.
25
31
3
3 3
3
.1.12. Preparation of Racemic 3-[3-[3-(4-chlorophenyl)-ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (g3)
Reaction of compound
afforded compound g3 (white solid, 83.4% yield). H-NMR (DMSO-d6):
f
and 4-chlorophenyl isocyanate following the general procedure A
1
δ 9.69 (s, 1H, NH), 8.14 (d,
J = 1.9 Hz, 1H, H-phenyl), 8.09 (s, 1H, NH), 7.55–7.49 (m, 2H, H-phenyl), 7.37–7.31 (m, 2H, H-phenyl),
7
3
2
.14 (d, J = 8.2 Hz, 1H, H-phenyl), 7.00 (dd, J = 8.2, 1.9 Hz, 1H, H-phenyl), 4.07–3.88 (m, 3H, CH , CH),
2
.07–2.76 (m, 4H, CH , CH
a
a
b b
-1 and CH -5), 2.14 (t, J = 10.6 Hz, 2H, CH -1 and CH -5), 2.06–1.91 (m,
2
H, CH-2, CH-4), 1.80 (d, J = 12.9 Hz, 1H, CH
a
-3), 1.07 (t, J = 7.2 Hz, 3H, CH ), 0.87 (d, J = 6.6 Hz, 6H,
3
13
CH -6, CH -7), 0.67 (q, J = 12.4 Hz, 1H, CH -3). C-NMR (DMSO-d6):
δ
169.59, 152.40, 141.96, 138.84,
3
3
b
1
4
5
34.02, 129.22, 128.72 (2C), 125.51, 122.73, 120.44, 120.21, 119.96 (2C), 119.32, 60.48, 59.72 (2C), 45.00,
1.61, 33.79, 30.88 (2C), 19.32 (2C), 13.92. HRMS (ESI) m/z: [M + H] calculated for C H O N ClF ,
26 32 3 3 3
26.20788; found, 526.20862, ∆ 1.41 ppm.
+
3
.1.13. Preparation of Racemic 3-[3-[3-(4-chlorophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (i3)
Hydrolysis of compound g3 (134 mg, 0.26 mmol) following the general procedure B afforded
◦
1
compound i3 (light yellow solid, 102 mg, 80.3% yield). mp: 123.5–124.7 C. H-NMR (DMSO-d6):
δ
1
2.51 (s, 1H, COOH), 9.70 (s, 1H, NH), 8.13 (d, J = 1.8 Hz, 1H, H-phenyl), 8.10 (s, 1H, NH), 7.55–7.49 (m,
2
1
2
H, H-phenyl), 7.36–7.31 (m, 2H, H-phenyl), 7.14 (d, J = 8.2 Hz, 1H, H-phenyl), 6.99 (dd, J = 8.2, 1.9 Hz,
H, H-phenyl), 3.95–3.83 (m, 1H, CH), 2.98–2.76 (m, 4H, CH , CH -1 and CH -5), 2.15 (td, J = 11.2,
.9 Hz, 2H, CH -1 and CH -5), 2.05–1.92 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.9 Hz, 1H, CH -3), 0.87 (d,
171.08, 152.47,
2
a
a
b
b
a
13
J = 6.6 Hz, 6H, CH -6, CH -7), 0.67 (q, J = 12.4 Hz, 1H, CH -3). C-NMR (DMSO-d6):
δ
3
3
b
1
4
4
41.93, 138.91, 134.13, 129.58, 128.73 (2C), 126.94, 125.52, 122.83, 120.46, 120.01 (2C), 119.34, 59.80 (2C),
+
5.20, 41.65, 33.86, 30.92 (2C), 19.32 (2C). HRMS (ESI) m/z: [M + H] calculated for C H O N ClF ,
24
28
3
3
3
98.17658; found, 498.17761, ∆ 2.07 ppm.
3
4
.1.14. Preparation of Racemic 3-[3-(3-cyclohexylureido)-4-(3-methylpiperidin-1-yl)-phenyl]-4,4,
-trifluorobutyric Acid Ethyl Ester (g4)
Reaction of compound
compound g4 (white solid, 86.3% yield). H-NMR (DMSO-d ):
f
and cyclohexyl isocyanate following the general procedure A afforded
1
δ 8.08 (s, 1H, NH), 7.63 (s, 1H, NH),
6
7
.12–7.02 (m, 2H, H-phenyl), 6.89 (d, J = 8.2 Hz, 1H, H-phenyl), 4.06–3.93 (m, 2H, CH ), 3.92–3.80
2
(
m, 1H, CH), 3.52–3.40 (m, 1H, CH-cyclohexyl), 3.04–2.75 (m, 4H, CH , CH
a
-1 and CH
a
-5), 2.09 (dt,
-3,
CH-cyclohexyl), 1.36–0.99 (m, 8H, CH-cyclohexyl, CH ), 0.86 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.65 (q,
2
J = 11.0, 5.5 Hz, 2H, CH -1 and CH -5), 1.96 (d, J = 6.7 Hz, 2H, CH-2, CH-4), 1.87–1.49 (m, 6H, CH
a
b
b
3
3
3
+
J = 12.0 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 498.29380; found,
b
26 39
3
3 3
4
98.29480, ∆ 2.00 ppm.

Molecules 2020, 25, 1447
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3
4
.1.15. Preparation of Racemic 3-[3-(3-cyclohexylureido)-4-(3,5-dimethylpiperidin-1-yl)phenyl]-4,4,
-trifluorobutyric Acid (i4)
Hydrolysis of compound g4 (80 mg, 0.16 mmol) following the general procedure B afforded
◦
1
compound i4 (white solid, 53 mg, 70.1% yield). mp: 134.2–135.7 C. H-NMR (DMSO-d ):
(
δ
7.91–7.76
m, 1H, H-phenyl), 7.45–7.21 (m, 2H, NH, H-phenyl), 7.15–6.98 (m, 1H, H-phenyl), 4.00–3.85 (m,
H, CH), 3.54–3.42 (m, 1H, CH-cyclohexyl), 3.14–2.75 (m, 4H, CH -1 and CH -5, CH ), 2.06–1.90 (m,
H, CH -1 and CH -5), 1.85–1.76 (m, 3H, CH-2, CH-4, CH-cyclohexyl), 1.75–1.62 (m, 3H, CH
6
1
a
a
2
2
a
-3,
b
b
CH-cyclohexyl), 1.60–1.48 (m, 1H, CH-cyclohexyl), 1.37–1.11 (m, 6H, CH-cyclohexyl), 0.89 (d, J = 6.6 Hz,
+
6
H, CH -6, CH -7), 0.75 (q, J = 12.6 Hz, 1H, CH -3). HRMS(ESI) m/z: [M + H] calculated for
3
3
b
C H O N F , 470.26250; found, 470.26343, ∆ 1.97 ppm.
24
35
3
3 3
3
4
.1.16. Preparation of Racemic 3-[3-(3-butylureido)-4-(3,5-dimethylpiperidin-1-yl)-phenyl]-4,4,
-trifluorobutyric Acid Ethyl Ester (g5)
Reaction of compound
compound g5 (white solid, 84.1% yield). H-NMR (DMSO-d ):
f
and butyl isocyanate following the general procedure A afforded
1
δ 8.08 (d, J = 1.2 Hz, 1H, H-phenyl),
6
7.65 (s, 1H, NH), 7.15 (t, J = 5.4 Hz, 1H, NH), 7.06 (d, J = 8.2 Hz, 1H, H-phenyl), 6.92–6.87 (m, 1H,
H-phenyl), 4.06–3.93 (m, 2H, CH ), 3.93–3.81 (m, 1H, CH), 3.12–3.05 (m, 2H, CH-butyl), 3.04–2.76
2
(
m, 4H, CH , CH
a
-1 and CH
CH-2, CH-4), 1.78 (d, J = 12.7 Hz, 1H, CH
CH ), 0.93–0.82 (m, 9H, CH- butyl, CH -6, CH -7), 0.65 (q, J = 12.0 Hz, 1H, CH -3). HRMS (ESI) m/z:
a
b b
-5), 2.10 (td, J = 11.0, 2.9 Hz, 2H, CH -1 and CH -5), 2.03–1.88 (m, 2H,
2
a
-3), 1.47–1.26 (m, 4H, CH-butyl), 1.07 (t, J = 7.1 Hz, 3H,
3
3
3
b
+
[
M + H] calculated for C H O N F , 472.27815; found, 472.27792, ∆ −0.49 ppm.
24
37
3
3 3
3
4
.1.17. Preparation of Racemic 3-[3-(3-butylureido)-4-(3,5-dimethylpiperidin-1-yl)-phenyl]-4,4,
-trifluorobutyric Acid (i5)
Hydrolysis of compound g5 (80 mg, 0.17 mmol) following the general procedure B afforded
◦
1
compound i5 (white solid, 56 mg, 74.3% yield). mp: 131.0–132.9 C. H-NMR (DMSO-d ):
δ
8.00–7.80
6
(m, 1H, -phenyl), 7.36–7.15 (m, 2H, NH, H-phenyl), 7.09–6.96 (m, 1H, H-phenyl), 3.98–3.83 (m, 1H,
H-phenyl), 3.11 (t, J = 6.6 Hz, 2H, CH- butyl), 3.04–2.76 (m, 4H, CH , CH
a
-1 and CH
a
-5), 2.46–2.21 (m,
a
-3), 1.49–1.29 (m,
2
2
4
H, CH -1 and CH -5), 2.05–1.89 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.6 Hz, 1H, CH
b
b
H, CH-butyl), 0.95–0.83 (m, 9H, CH- butyl, CH -6, CH -7), 0.73 (q, J = 11.7 Hz, 1H, CH -3). HRMS
3
3
b
+
(
ESI) m/z: [M + H] calculated for C H O N F , 444.24685; found, 444.24750, ∆ 1.46 ppm.
22 33 3 3 3
3
4
.1.18. Preparation of Racemic 3-[4-(3,5-dimethylpiperidin-1-yl)-3-(3-o-tolylureido)phenyl]-4,4,
-trifluorobutyric Acid Ethyl Ester (g6)
Reaction of compound
compound g6 (white solid, 78.4% yield). H-NMR (DMSO-d ):
f
and o-tolyl isocyanate following the general procedure A afforded
1
δ 8.62 (s, 1H, NH), 8.07 (s, 1H, NH),
6
8
3
.04–8.01 (m, 1H, H-phenyl) 7.58–7.54 (m, 1H, H-phenyl), 7.20–7.09 (m, 2H, H-phenyl), 7.06–6.90 (m,
H, H-phenyl), 4.03–3.80 (m, 3H, CH , CH), 3.01–2.75 (m, 4H, CH , CH -1 and CH -5), 2.20 (s, 3H,
-3), 1.03 (t,
J = 7.0 Hz, 3H, CH ), 0.79 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.57 (q, J = 12.0 Hz, 1H, CH -3). HRMS
2
2
a
a
CH -phenyl), 2.05 (t, J = 10.4 Hz, 2H, CH -1 and CH -5), 1.84–1.66 (m, 3H, CH-2, CH-4, CH
a
3
b
b
3
+
3
3
b
(ESI) m/z: [M + H] calculated for C H O N F , 506.26250; found, 506.26297, ∆ 0.92 ppm.
27 35 3 3 3
3
4
.1.19. Preparation of Racemic 3-[4-(3,5-dimethylpiperidin-1-yl)-3-(3-o-tolylureido)phenyl]-4,4,
-trifluorobutyric Acid (i6)
Hydrolysis of compound g6 (203 mg, 0.40 mmol) following the general procedure B afforded
◦
1
compound i6 (white solid, 156 mg, 81.3% yield). mp: 109.3–111.2 C. H-NMR (DMSO-d ):
1
1
δ
12.53 (s,
H, COOH), 8.67 (s, 1H, NH), 8.08 (s, 1H, NH), 8.04 (d, J = 1.0 Hz, 1H, H-phenyl), 7.56 (d, J = 7.7 Hz,
H, H-phenyl), 7.25–7.14 (m, 2H, H-phenyl), 7.11–6.94 (m, 3H, H-phenyl), 3.96–3.80 (m, 1H, CH),
6
2
.98–2.72 (m, 4H, CH
a
-1 and CH
a
-5, CH ), 2.25 (s, 3H, CH -phenyl), 2.10 (td, J = 11.1, 3.7 Hz, 2H,
2
3
CH -1 and CH -5), 1.86–1.68 (m, 3H, CH-2, CH-4, CH
a
-3), 0.84 (d, J = 6.4 Hz, 6H, CH -6, CH -7),
b
b
3
3

Molecules 2020, 25, 1447
15 of 27
0
.62 (q, J = 12.2 Hz, 1H, CH -3). 13C-NMR (DMSO-d6):
δ
171.12, 153.19, 142.24, 136.94, 133.95, 130.91,
b
1
30.47, 129.16, 126.93, 126.34, 124.53, 124.37, 122.80, 120.12, 119.96, 59.50 (2C), 45.13, 41.62, 33.87, 30.88
+
(2C), 19.31 (2C), 18.02. HRMS (ESI) m/z: [M + H] calculated for C H O N F , 478.23120; found,
25
31
3
3 3
4
78.23215, ∆ 1.98 ppm.
3
4
.1.20. Preparation of Racemic 3-[4-(3,5-dimethylpiperidin-1-yl)-3-(3-m-tolylureido)phenyl]-4,4,
-trifluorobutyric Acid Ethyl Ester (g7)
Reaction of compound
compound g7 (white solid, 86.1% yield). H-NMR (DMSO-d ):
f
and m-tolyl isocyanate following the general procedure A afforded
1
δ 9.46 (s, 1H, NH), 8.14 (d, J = 1.9 Hz,
6
1
(
(
3
H, H-phenyl), 8.05 (s, 1H, NH), 7.37–7.33 (m, 1H, H-phenyl), 7.28–7.23 (m, 1H, H-phenyl), 7.20–7.11
m, 2H, H-phenyl), 6.98 (dd, J = 8.2, 1.9 Hz, 1H, H-phenyl), 6.81 (d, J = 7.4 Hz, 1H, H-phenyl), 4.07–3.87
m, 3H, CH , CH), 3.07–2.80 (m, 4H, CH , CH -1 and CH -5), 2.29 (s, 3H, CH -phenyl), 2.14 (td, J = 11.1,
.0 Hz, 2H, CH -1 and CH -5), 2.04–1.89 (m, 2H, CH-2, CH-4), 1.80 (d, J = 13.2 Hz, 1H, CH
2
2
a
a
3
a
-3), 1.08 (t,
b
b
J = 7.1, 3H, CH ), 0.87 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.4 Hz, 1H, CH -3). HRMS (ESI)
3
3
3
b
+
m/z: [M + H] calculated for C H O N F , 506.26250; found, 506.26297, ∆ 0.92 ppm.
27
35
3
3 3
3
4
.1.21. Preparation of Racemic 3-[4-(3,5-dimethylpiperidin-1-yl)-3-(3-m-tolylureido)phenyl]-4,4,
-trifluorobutyric Acid (i7)
Hydrolysis of compound g7 (209 mg, 0.41 mmol) following the general procedure B afforded
◦
1
compound i7 (white solid, 148 mg, 75.0% yield). mp: 121.2–122.4 C. H-NMR (DMSO-d ):
1
δ
9.48 (s,
H, NH), 8.14 (d, J = 1.5 Hz, 1H, H-phenyl), 8.06 (s, 1H, NH), 7.37–7.34 (m, 1H, H-phenyl), 7.26 (d,
J = 8.6 Hz, 1H, H-phenyl), 7.20–7.10 (m, 2H, H-phenyl), 6.97 (dd, J = 8.2, 1.6 Hz, 1H, H-phenyl), 6.81 (d,
J = 7.4 Hz, 1H, H-phenyl), 3.96–3.82 (m, 1H, CH), 3.00–2.74 (m, 4H, CH , CH -1 and CH -5), 2.29 (s,
H, CH -phenyl), 2.14 (td, J = 11.0, 4.2 Hz, 2H, CH -1 and CH -5), 2.05–1.91 (m, 2H, CH-2, CH-4), 1.80
6
2
a
a
3
3
b
b
(
d, J = 12.8 Hz, 1H, CH
a
-3), 0.87 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.0 Hz, 1H, CH -3).
171.16, 152.57, 141.84, 139.79, 138.09, 134.28, 129.56, 128.75, 126.94, 122.82,
3
3
b
1
3
C-NMR (DMSO-d6):
δ
1
22.60, 120.39, 119.30, 119.17, 115.78, 59.77 (2C), 45.21, 41.67, 33.93, 30.90 (2C), 21.30, 19.35 (2C). HRMS
+
(ESI) m/z: [M + H] calculated for C H O N F , 478.23120; found, 478.23227, ∆ 2.23 ppm.
25
31
3
3 3
3
.1.22. Preparation of Racemic 3-[3-[3-(2-chlorophenyl)-ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (g8)
Reaction of compound
afforded compound g8 (white solid, 81.7% yield). H-NMR (DMSO-d ):
f
and 2-chlorophenyl isocyanate following the general procedure A
1
δ 9.03 (s, 1H, NH), 8.43 (s, 1H,
6
NH), 7.97–7.90 (m, 2H, H-phenyl), 7.47 (dd, J = 8.0, 1.2 Hz, 1H, H-phenyl), 7.34–7.27 (m, 1H, H-phenyl),
7
.13–6.99 (m, 3H, H-phenyl), 4.07–3.87 (m, 3H, CH , CH), 3.07–2.82 (m, 4H, CH , CH
a
-1 and CH
a
-5),
2
2
2
.12 (t, J = 11.0 Hz, 2H, CH -1 and CH -5), 2.00–1.86 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.8 Hz, 1H,
b
b
a
3 3 3
CH -3), 1.08 (t, J = 7.2 Hz, 3H, CH ), 0.86 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.65 (q, J = 12.0 Hz, 1H,
+
CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N ClF , 526.20788; found, 526.20917,
b
26 32
3
3
3
∆
2.45 ppm.
3
.1.23. Preparation of Racemic 3-[3-[3-(2-chlorophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (i8)
Hydrolysis of compound g8 (60 mg, 0.11 mmol) following the general procedure B afforded
◦
1
compound i8 (white solid, 46 mg, 80.7% yield). mp: 111.2–113.7 C. H-NMR (DMSO-d ):
1
δ
12.43 (s,
H, COOH), 9.01 (s, 1H, NH), 8.40 (s, 1H, NH), 7.95–7.85 (m, 2H, H-phenyl), 7.43 (d, J = 7.9 Hz, 1H,
H-phenyl), 7.31–7.22 (m, 1H, H-phenyl), 7.09–6.93 (m, 3H, H-phenyl), 3.89–3.79 (m, 1H, CH), 2.93–2.71
m, 4H, CH , CH -1 and CH -5), 2.08 (td, J = 11.0, 2.6 Hz, 2H, CH -1 and CH -5), 1.96–1.87 (m, 2H,
6
(
a
a
2
b
b
CH-2, CH-4), 1.74 (d, J = 12.7 Hz, 1H, CH
a
-3), 0.82 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.61 (q, J = 12.0 Hz,
3
3
+
1
H, CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N ClF , 498.17658; found, 498.17786,
b
24 28
3
3
3
∆
2.57 ppm.

Molecules 2020, 25, 1447
16 of 27
3
.1.24. Preparation of Racemic 3-[3-[3-(3-chlorophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (g9)
Reaction of compound
compound g9 (white solid, 79.7% yield). H-NMR (DMSO-d ):
f
and 3-chlorophenyl isocyanate following the general procedure A afforded
1
δ 9.76 (s, 1H, NH), 8.15–8.09 (m, 2H,
6
H-phenyl, NH), 7.79–7.75 (m, 1H, H-phenyl), 7.35–7.25 (m, 2H, H-phenyl), 7.15 (d, J = 8.2 Hz, 1H,
H-phenyl), 7.05–6.98 (m, 2H, H-phenyl), 4.06–3.92 (m, 3H, CH , CH), 3.08–2.79 (m, 4H, CH , CH -1 and
-5), 2.15 (t, J = 11.0 Hz, 2H, CH -1 and CH -5), 2.05–1.90 (m, 2H, CH-2, CH-4), 1.78 (d, J = 12.0 Hz,
2
2
a
CH
a
3 3 3
H, CH -3), 1.08 (t, J = 7.1 Hz, 3H, CH ), 0.87 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.67 (q, J = 12.4 Hz, 1H,
a
b
b
1
+
CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N ClF , 526.20788; found, 526.20776,
∆
b
26 32
3
3
3
−
0.23 ppm.
3
.1.25. Preparation of Racemic 3-[3-[3-(3-chlorophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (i9)
Hydrolysis of compound g9 (300 mg, 0.57 mmol) following the general procedure B afforded
◦
1
compound i9 (white solid, 234 mg, 82.5% yield). mp: 129.3–131.7 C. H-NMR (DMSO-d )
δ
12.50
6
(
s, 1H, COOH), 9.77 (s, 1H, NH), 8.14–8.09 (m, 2H, NH, H-phenyl), 7.77 (t, J = 1.9 Hz, 1H, H-phenyl),
7
.35–7.25 (m, 2H, H-phenyl), 7.15 (d, J = 8.1 Hz, 1H, H-phenyl), 7.07–6.97 (m, 2H, H-phenyl), 3.98–3.84
-1 and CH -5), 2.15 (td, J = 11.1, 2.9 Hz, 2H, CH -1 and
CH -5), 2.06–1.91 (m, 2H, CH-2, CH-4), 1.81 (d, J = 12.9 Hz, 1H, CH
(m, 1H, CH), 3.01–2.75 (m, 4H, CH , CH
a
a
2
b
b
a
-3), 0.87 (d, J = 6.6 Hz, 6H, CH -6,
3
1
3
CH -7), 0.67 (q, J = 12.1 Hz, 1H, CH -3). C-NMR (DMSO-d6):
δ 171.03, 152.36, 141.97, 141.42, 133.92,
3
b
1
3
4
33.33, 130.49, 129.55, 126.86, 122.91, 121.60, 120.48, 119.38, 117.78, 116.74, 59.75 (2C), 45.10, 41.61, 33.78,
+
0.90 (2C), 19.32 (2C). HRMS (ESI) m/z: [M + H] calculated for C H O N ClF , 498.17658; found,
24
28
3
3
3
98.17770, ∆ 2.25 ppm.
3
.1.26. Preparation of Racemic 3-[3-[3-(2-cyanophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (g10)
Reaction of compound
compound g10 (white solid, 79.1% yield). H-NMR (DMSO-d ):
f
and 2-cyanophenyl isocyanate following the general procedure A afforded
1
δ 10.17 (s, 1H, NH), 8.28 (s, 1H, NH),
6
8
7
2
.17–8.11 (m, 2H, H-phenyl), 8.03 (dd, J = 8.0, 3.8 Hz, 1H, H-phenyl), 7.63–7.57 (m, 1H, H-phenyl),
.22–7.01 (m, 3H, H-phenyl), 4.07–3.87 (m, 3H, CH , CH), 3.08–2.82 (m, 4H, CH , CH -1 and CH -5),
.13 (t, J = 11.0 Hz, 2H, CH -1 and CH -5), 2.03–1.88 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.8 Hz, 1H,
2
2
a
a
b
b
a
3 3 3
CH -3), 1.07 (t, J = 7.1 Hz, 3H, CH ), 0.86 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.65 (q, J = 12.4 Hz, 1H,
+
CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 517.24210; found, 517.24335,
∆
b
27 32
3
4 3
2
.41 ppm.
3
.1.27. Preparation of Racemic 3-[3-[3-(2-cyanophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (i10)
Hydrolysis of compound g10 (40 mg, 0.08 mmol) following the general procedure B afforded
◦
1
compound i10 (white solid, 30 mg, 79.8% yield). mp: 178.2–179.9 C. H-NMR (DMSO-d ):
δ
12.51
6
(s, 1H, COOH), 10.18 (s, 1H, NH), 8.28 (s, 1H, NH), 8.16–8.10 (m, 2H, H-phenyl), 8.02 (dd, J = 8.1,
3
.6 Hz, 1H, H-phenyl), 7.62–7.56 (m, 1H, H-phenyl), 7.22–7.01 (m, 3H, H-phenyl), 3.99–3.84 (m, 1H,
-1 and CH -5), 2.14 (t, J = 10.6 Hz, 2H, CH -1 and CH -5), 2.04–1.89
m, 2H, CH-2, CH-4), 1.80 (d, J = 12.8 Hz, 1H, CH
CH), 3.02–2.76 (m, 4H, CH , CH
(
J = 12.4 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 489.20987; found,
a
a
2
b
b
a
-3), 0.87 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.66 (q,
3
3
+
b
25 28
3
4 3
4
89.21080, ∆ −1.90 ppm.
3
.1.28. Preparation of Racemic 3-[3-[3-(3-cyanophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (g11)
Reaction of compound
compound g11 (white solid, 87.5% yield). H-NMR (DMSO-d ):
f
and 3-cyanophenyl isocyanate following the general procedure A afforded
1
δ 9.91 (s, 1H, NH), 8.17 (s, 1H, NH),
6

Molecules 2020, 25, 1447
17 of 27
8.14–8.11 (m, 1H, H-phenyl), 8.06–8.03 (m, 1H, H-phenyl), 7.66 (d, J = 8.2 Hz, 1H, H-phenyl), 7.54–7.41
(m, 2H, H-phenyl), 7.16 (d, J = 8.1 Hz, 1H, H-phenyl), 7.02 (d, J = 8.4 Hz, 1H, H-phenyl), 4.08–3.88 (m,
3
H, CH , CH), 3.07–2.81 (m, 4H, CH , CH
a
-1 and CH
a
b b
-5), 2.15 (t, J = 10.8 Hz, 2H, CH -1 and CH -5),
2
2
2
.06–1.92 (m, 2H, CH-2, CH-4), 1.81 (d, J = 12.8 Hz, 1H, CH
a
-3), 1.08 (t, J = 7.2 Hz, 3H, CH ), 0.87 (d,
3
+
J = 6.5 Hz, 6H, CH -6, CH -7), 0.67 (q, J = 12.1 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated
3
3
b
for C H O N F , 517.24210; found, 517.24121, ∆ −1.72 ppm.
27
32
3
4 3
3
.1.29. Preparation of Racemic 3-[3-[3-(3-cyanophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (i11)
Hydrolysis of compound g11 (180 mg, 0.35 mmol) following the general procedure B afforded
◦
1
compound i11 (white solid, 121 mg, 71.2% yield). mp: 128.4–130.2 C. H-NMR (DMSO-d ):
δ
12.52 (s,
6
1
7
3
H, COOH), 9.94 (s, 1H, NH), 8.01–8.23 (m, 3H, NH, H-phenyl), 7.66 (d, J = 7.3 Hz, 1H, H-phenyl),
.57–7.39 (m, 2H, H-phenyl), 7.16 (d, J = 8.2 Hz, 1H, H-phenyl), 7.01 (d, J = 8.2 Hz, 1H, H-phenyl),
.98–3.83 (m, 1H, CH), 3.04–2.74 (m, 4H, CH , CH -1 and CH -5), 2.16 (t, J = 9.9 Hz, 2H, CH -1 and
2
a
a
b
a
3
CH -5), 2.06–1.92 (m, 2H, CH-2, CH-4), 1.81 (d, J = 11.3 Hz, 1H, CH -3), 0.87 (d, J = 6.1 Hz, 6H, CH -6,
b
13
CH -7), 0.67 (q, J = 11.6 Hz, 1H, CH -3). C-NMR (DMSO-d6):
δ
171.03, 152.38, 142.03, 140.76, 133.79,
3
b
1
4
30.31, 129.58, 126.90, 125.47, 123.06, 122.96, 120.92, 120.52, 119.40, 118.91, 111.72, 59.75 (2C), 45.10,
1.60, 33.78, 30.90 (2C), 19.33 (2C). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 489.21080;
25 28 3 4 3
+
found, 489.21274, ∆ 1.94 ppm.
3
.1.30. Preparation of Racemic 3-[3-[3-(4-cyanophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (g12)
Reaction of compound
compound g12 (white solid, 84.1% yield). H-NMR (DMSO-d ):
f
and 4-cyanophenyl isocyanate following the general procedure A afforded
1
δ 10.05 (s, 1H, NH), 8.21 (s, 1H, NH),
6
8.12 (d, J = 1.8 Hz, 1H, H-phenyl), 7.77–7.72 (m, 2H, H-phenyl), 7.70–7.66 (m, 2H, H-phenyl), 7.16
(
d, J = 8.3 Hz, 1H, H-phenyl), 7.03 (dd, J = 8.1, 2.0 Hz, 1H, H-phenyl), 4.07–3.88 (m, 3H, CH , CH),
2
3
.08–2.82 (m, 4H, CH , CH
a
-1 and CH
CH-2, CH-4), 1.81 (d, J = 12.7 Hz, 1H, CH
CH -7), 0.67 (q, J = 12.8 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N F ,
a
b b
-5), 2.15 (t, J = 10.9 Hz, 2H, CH -1 and CH -5), 2.05–1.90 (m, 2H,
2
a
-3), 1.07 (t, J = 7.1 Hz, 3H, CH ), 0.90–0.81 (m, 6H, CH -6,
3 3
+
3
b
27 32
3
4 3
5
17.24210; found, 517.24298, ∆ 1.70 ppm.
3
.1.31. Preparation of Racemic 3-[3-[3-(4-cyanophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (i12)
Hydrolysis of compound g12 (331 mg, 0.64 mmol) following the general procedure B afforded
◦
1
compound i12 (white solid, 271 mg, 86.5% yield). mp: 172.3–172.9 C. H-NMR (DMSO-d ):
1
δ
10.18 (s,
H, NH), 8.27 (s, 1H, NH), 8.12 (d, J = 1.4 Hz, 1H, H-phenyl), 7.76–7.67 (m, 4H, H-phenyl), 7.16 (dd,
J = 8.0, 4.2 Hz, 1H, H-phenyl), 7.02 (dd, J = 8.2, 1.8 Hz, 1H, H-phenyl), 3.98–3.83 (m, 1H, CH), 3.03–2.75
m, 4H, CH , CH -1 and CH -5), 2.16 (td, J = 11.1, 1.9 Hz, 2H, CH -1 and CH -5), 2.06–1.94 (m, 2H,
CH-2, CH-4), 1.80 (d, J = 12.7 Hz, 1H, CH
6
(
a
a
2
b
b
a
-3), 0.86 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.67 (q, J = 12.4 Hz,
3 3
170.92, 152.17, 144.35, 141.88, 133.53, 133.25 (2C), 129.64, 126.77
13
1
H, CH -3). C-NMR (DMSO-d6):
δ
b
(
4
q, Jc-f = 280.8 Hz), 123.29, 120.49, 119.75, 119.31, 118.11 (2C), 103.25, 59.73 (2C), 44.97 (q, J = 27.3 Hz),
1.45, 33.67, 30.62 (2C), 19.21 (2C). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 489.21080;
25 28 3 4 3
+
found, 489.20990, ∆ −1.84 ppm.
3
.1.32. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(2-methoxyphenyl)ureido]-
phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (g13)
Reaction of compound
afforded compound g13 (white solid, 85.9% yield). H-NMR (DMSO-d ):
f
and 2-methoxyphenyl isocyanate following the general procedure A
1
δ 8.91 (s, 1H, NH), 8.42 (s, 1H,
6
NH), 8.00–7.93 (m, 2H, H-phenyl), 7.10–6.96 (m, 4H, H-phenyl), 6.93–6.86 (m, 1H, H-phenyl), 4.07–3.87
m, 3H, CH , CH), 3.85 (s, 3H, OCH -phenyl), 3.05–2.83 (m, 4H, CH , CH -1 and CH -5), 2.10 (td,
(
a
a
2
3
2

Molecules 2020, 25, 1447
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J = 11.1, 2.1 Hz, 2H, CH -1 and CH -5), 2.02–1.87 (m, 2H, CH-2, CH-4), 1.77 (d, J = 12.8 Hz, 1H, CH
a
-3),
b
b
1
.07 (t, J = 7.1 Hz, 3H, CH ), 0.85 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.64 (q, J = 12.4 Hz, 1H, CH -3).
3
+
3
3
b
HRMS (ESI) m/z: [M + H] calculated for C H O N F , 522.25742; found, 522.25598, ∆ −2.75 ppm.
27
35
4
3 3
3
.1.33. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(2-methoxyphenyl)ureido]-
phenyl}-4,4,4-trifluorobutyric Acid (i13)
Hydrolysis of compound g13 (80 mg, 0.15 mmol) following the general procedure B afforded
◦
1
compound i13 (white solid, 61 mg, 80.8% yield). mp: 119.3–120.2 C. H-NMR (DMSO-d ):
(
δ
12.64
s, 1H, COOH), 8.91 (s, 1H, NH), 8.42 (s, 1H, NH), 7.99–7.93 (m, 2H, H-phenyl), 7.10–6.86 (m, 5H,
H-phenyl), 3.94–3.81 (m, 4H, CH, OCH -phenyl), 2.93–2.65 (m, 4H, CH -1 and CH -5, CH ), 2.10 (td,
J = 11.2, 4.6 Hz, 2H, CH -1 and CH -5), 2.01–1.86 (m, 2H, CH-2, CH-4), 1.77 (d, J = 13.3 Hz, 1H, CH
6
3
a
a
2
a
-3),
b
b
+
0
.85 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.63 (q, J = 12.4 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H]
3 3 b
calculated for C H O N F , 494.22612; found, 494.22455, ∆ −3.17 ppm.
25
31
4
3 3
3
.1.34. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(3-methoxyphenyl)ureido]-
phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (g14)
Reaction of compound
afforded compound g14 (white solid, 83.9% yield). H-NMR (DMSO-d ):
f
and 3-methoxyphenyl isocyanate following the general procedure A
1
δ 9.55 (s, 1H, NH), 8.13
6
(d, J = 1.9 Hz, 1H, H-phenyl), 8.07 (s, 1H, NH), 7.22–7.17 (m, 2H, H-phenyl), 7.13 (d, J = 8.2 Hz, 1H,
H-phenyl), 7.03–6.96 (m, 2H, H-phenyl), 6.59–6.55 (m, 1H, H-phenyl), 4.07–3.88 (m, 3H, CH , CH), 3.75
2
(
s, 3H, OCH -phenyl), 3.06–2.75 (m, 4H, CH , CH
a
a
b
-1 and CH -5), 2.13 (td, J = 11.2, 1.6 Hz, 2H, CH -1
3
2
and CH -5), 2.04–1.91 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.7 Hz, 1H, CH
a
-3), 1.10-1.06 (t, J = 7.2 Hz,
H, CH ), 0.87 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.4 Hz, 1H, CH -3). HRMS (ESI) m/z:
b
3
3
+
3
3
b
[
M + H] calculated for C H O N F , 522.25742; found, 522.25769, ∆ 0.52 ppm.
27 35 4 3 3
3
.1.35. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(3-methoxyphenyl)ureido]-
phenyl}-4,4,4-trifluorobutyric Acid (i14)
Hydrolysis of compound g14 (245 mg, 0.47 mmol) following the general procedure B afforded
◦
1
compound i14 (white solid, 178 mg, 76.7% yield). mp: 112.3–113.7 C. H-NMR (DMSO-d ):
δ
12.52
6
(s, 1H, COOH), 9.56 (s, 1H, NH), 8.14 (d, J = 1.6 Hz, 1H, H-phenyl), 8.08 (s, 1H, NH), 7.22–7.11 (m,
3
H, H-phenyl), 7.03–6.96 (m, 2H, H-phenyl), 6.57 (dd, J = 8.2, 2.5 Hz, 1H, H-phenyl), 3.96–3.81 (m,
-1 and CH -5), 2.14 (td, J = 11.0,
.1 Hz, 2H, CH -1 and CH -5), 2.05–1.92 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.9 Hz, 1H, CH -3), 0.87
171.08,
1H, CH), 3.75 (s, 3H, OCH -phenyl), 3.00–2.77 (m, 4H, CH , CH
a
a
3
2
2
a
b
b
13
(d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.67 (q, J = 12.0 Hz, 1H, CH -3). C-NMR (DMSO-d6):
δ
3
3
b
1
5
59.82, 152.50, 141.89, 141.09, 134.19, 129.66, 129.52, 126.90, 122.58, 120.44, 119.38, 110.87, 107.65, 104.23,
9.74 (2C), 55.06, 45.15, 41.64, 33.83, 30.88 (2C), 19.34 (2C). HRMS (ESI) m/z: [M + H] calculated for
+
C H O N F , 494.22612; found, 494.22699, ∆ 1.77 ppm.
25
31
4
3 3
3
.1.36. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-methoxyphenyl)ureido]-
phenyl}-4,4 4-trifluorobutyric Acid Ethyl Ester (g15)
Reaction of compound
afforded compound g15 (light yellow solid, 81.3% yield). H-NMR (DMSO-d ):
f
and 4-methoxyphenyl isocyanate following the general procedure A
1
δ 9.34 (s, 1H, NH),
6
8
1
.16 (d, J = 1.9 Hz, 1H, H-phenyl), 7.99 (s, 1H, NH), 7.41–7.36 (m, 2H, H-phenyl), 7.12 (d, J = 8.2 Hz,
H, H-phenyl), 6.96 (dd, J = 8.2, 1.9 Hz, 1H, H-phenyl), 6.91–6.87 (m, 2H, H-phenyl), 4.05–3.88 (m, 3H,
CH , CH), 3.73 (s, 3H, OCH -phenyl), 3.06–2.80 (m, 4H, CH , CH -1 and CH -5), 2.12 (dt, J = 11.2, 5.6
Hz, 2H, CH -1 and CH -5), 1.99–1.86 (m, 2H, CH-2, CH-4), 1.78 (d, J = 13.0 Hz, 1H, CH -3), 1.08 (t,
J = 7.2 Hz, 3H, CH ), 0.86 (d, J = 6.8 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.0 Hz, 1H, CH -3). HRMS
2
3
2
a
a
b
b
a
3
+
3
3
b
(ESI) m/z: [M + H] calculated for C H O N F , 522.25665; found, 522.25742, ∆ −1.47 ppm.
27
35
4
3 3

Molecules 2020, 25, 1447
19 of 27
3
.1.37. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-methoxyphenyl)ureido]-
phenyl}-4,4,4-trifluorobutyric Acid (i15)
Hydrolysis of compound g15 (230 mg, 0.44 mmol) following the general procedure B afforded
◦
1
compound i15 (white solid, 181 mg, 81.8% yield). mp: 108.9–120.1 C. H-NMR (DMSO-d ):
δ
12.50
6
(s, 1H, COOH), 9.32 (s, 1H, NH), 8.15 (d, J = 1.9 Hz, 1H, H-phenyl), 7.99 (s, 1H, NH), 7.40–7.36 (m,
2
2
CH
H, H-phenyl), 7.12 (d, J = 8.3, 1H, H-phenyl), 6.95 (dd, J = 8.2, 1.9 Hz, 1H, H-phenyl), 6.90–6.86 (m,
H, H-phenyl), 3.94–3.83 (m, 1H, CH), 3.73 (s, 3H, OCH -phenyl), 2.98–2.75 (m, 4H, CH , CH -1 and
-5), 2.13 (td, J = 11.1, 3.1 Hz, 2H, CH -1 and CH -5), 1.98-1.85 (d, J = 6.6 Hz, 2H, CH-2, CH-4), 1.78
3
2
a
a
b
b
(
a
3 3 b
d, J = 12.8 Hz, 1H, CH -3), 0.86 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.4 Hz, 1H, CH -3).
+
HRMS (ESI) m/z: [M + H] calculated for C H O N F , 494.22612; found, 494.22815, ∆ 4.11 ppm.
25
31
4
3 3
3
.1.38. Preparation of Racemic 3-[3-[3-(2,4-difluorophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (g16)
Reaction of compound
afforded compound g16 (white solid, 80.8% yield). H-NMR (DMSO-d ):
f
and 2,4-difluorophenyl isocyanate following the general procedure A
1
δ 9.36 (s, 1H, NH), 8.34
6
(
(
s, 1H, NH), 8.09–7.98 (m, 2H, H-phenyl), 7.31 (ddd, J = 11.6, 8.9, 2.9 Hz, 1H, H-phenyl), 7.14–6.97
m, 3H, H-phenyl), 4.07–3.85 (m, 3H, CH , CH), 3.06–2.82 (m, 4H, CH , CH -1 and CH -5), 2.13 (t,
-3),
.07 (t, J = 7.1 Hz, 3H, CH ), 0.86 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.4 Hz, 1H, CH -3).
2
2
a
a
J = 10.8 Hz, 2H, CH -1 and CH -5), 2.04–1.89 (m, 2H, CH-2, CH-4), 1.79 (d, J = 13.0 Hz, 1H, CH
1
HRMS (ESI) m/z: [M + H] calculated for C H O N F , 528.22801; found, 528.22894, ∆ 1.76 ppm.
a
b
b
3
+
3
3
b
26
31
3
3 5
3
.1.39. Preparation of Racemic 3-[3-[3-(2,4-difluorophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (i16)
Hydrolysis of compound g16 (190 mg, 0.36 mmol) following the general procedure B afforded
◦
1
compound i16 (white solid, 149 mg, 82.0% yield). mp: 119.6–120.6 C. H-NMR (DMSO-d ):
(
δ
9.38
s, 1H, -NH-), 8.36 (s, 1H, -NH-), 8.08–7.97 (m, 2H, H-phenyl), 7.31 (ddd, J = 11.6, 8.9, 2.9 Hz, 1H,
H-phenyl), 7.14–6.95 (m, 3H, H-phenyl), 4.07–3.86 (m, 1H, CH), 2.94–2.70 (m, 4H, CH , CH -1 and
-5), 2.13 (td, J = 11.0, 3.8 Hz, 2H, CH -1 and CH -5), 2.04–1.88 (m, 2H, CH-2, CH-4), 1.79 (d,
6
2
a
CH
a
b
b
J = 12.6 Hz, 1H, CH
C-NMR (DMSO-d6):
a
-3), 0.86 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.65 (q, J = 12.0 Hz, 1H, CH -3).
δ 171.31, 155.16 (2C), 152.71, 142.28, 133.78, 129.47, 126.97, 123.85, 123.82, 123.10,
3
3
b
13
1
20.20, 120.08, 111.07, 103.92, 59.65 (2C), 45.23, 41.65, 34.18, 30.77 (2C), 19.33 (2C). HRMS (ESI) m/z:
+
[M + H] calculated for C H O N F , 500.19671; found, 500.19440, ∆ −4.62 ppm.
24
27
3
3 5
3
.1.40. Preparation of Racemic 3-[3-[3-(2,4-dichlorophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (g17)
Reaction of compound
afforded compound g17 (white solid, 81.3% yield). H-NMR (DMSO-d ):
f
and 2,4-dichlorophenyl isocyanate following the general procedure A
1
δ 9.12 (s, 1H, NH), 8.50 (s,
6
1
H, NH), 8.00 (d, J = 8.9 Hz, 1H, H-phenyl), 7.93 (d, J = 1.8 Hz, 1H, H-phenyl), 7.63 (d, J = 2.5 Hz, 1H,
H-phenyl), 7.39 (dd, J = 8.9, 2.5 Hz, 1H, H-phenyl), 7.10 (d, J = 8.3 Hz, 1H, H-phenyl), 7.03 (dd, J = 8.2,
.9 Hz, 1H, H-phenyl), 4.07–3.86 (m, 3H, CH , CH), 3.06–2.82 (m, 4H, CH , CH -1 and CH -5), 2.12 (t,
J = 11.1 Hz, 2H, CH -1 and CH -5), 2.02–1.88 (m, 2H, CH-2, CH-4), 1.79 (d, J = 12.7 Hz, 1H, CH -3),
1
a
a
2
2
b
b
a
1
.07 (t, J = 7.1 Hz, 3H, CH ), 0.86 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.65 (q, J = 12.4 Hz, 1H, CH -3).
3
+
3
3
b
HRMS (ESI) m/z: [M + H] calculated for C H O N Cl F , 560.16891; found, 560.17065,
∆
3.11 ppm.
26
31
3
3
2 3
3
.1.41. Preparation of Racemic 3-[3-[3-(2,4-dichlorophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (i17)
Hydrolysis of compound g17 (80 mg, 0.14 mmol) following the general procedure B afforded
◦
1
compound i17 (white solid, 61 mg, 80.1% yield). mp: 119.3–110.5 C. H-NMR (DMSO-d ):
δ
9.14
6
(s, 1H, NH), 8.50 (s, 1H, NH), 8.00 (d, J = 8.9 Hz, 1H, H-phenyl), 7.92 (d, J = 1.7 Hz, 1H, H-phenyl),
7.63 (d, J = 2.4 Hz, 1H, H-phenyl), 7.38 (dd, J = 8.9, 2.5 Hz, 1H, H-phenyl), 7.09 (d, J = 8.3 Hz, 1H,

Molecules 2020, 25, 1447
20 of 27
H-phenyl), 7.01 (dd, J = 8.2, 1.8 Hz, 1H, H-phenyl), 3.96–3.82 (m, 1H, CH), 2.96–1.71 (m, 4H, CH
a
-1 and
a
2 b b
CH -5, CH ), 2.12 (td, J = 11.1, 3.9 Hz, 2H, CH -1 and CH -5), 2.03–1.87 (m, 2H, CH-2, CH-4), 1.83–1.75
13
(
m, 1H, CH
a
3 3 b
-3), 0.86 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.65 (q, J = 12.4 Hz, 1H, CH -3). C-NMR
(
1
DMSO-d6):
δ
171.29, 152.61, 143.00, 135.17, 133.12, 129.07, 128.72, 127.56, 127.24, 126.97, 125.07, 124.91,
+
23.65, 121.31, 119.86, 59.44 (2C), 45.12, 41.66, 34.15, 30.75 (2C), 19.33 (2C). HRMS (ESI) m/z: [M + H]
calculated for C H O N Cl F , 532.13761; found, 532.13892, ∆ 2.47 ppm.
24
27
3
3
2 3
3
.1.42. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-fluorophenyl)ureido]-
phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (g18)
Reaction of compound
compound g18 (white solid, 81.3% yield). H-NMR (DMSO-d ):
f
and 4-fluorophenyl isocyanate following the general procedure A afforded
1
δ 9.57 (s, 1H, NH), 8.13 (d, J = 1.8 Hz,
6
1
H, H-phenyl), 8.04 (s, 1H, NH), 7.54–7.46 (m, 2H, H-phenyl), 7.18–7.09 (m, 3H, H-phenyl), 6.98 (dd,
J = 8.2, 1.9 Hz, 1H, H-phenyl), 4.08–3.85 (m, 3H, CH , CH), 3.07–2.79 (m, 4H, CH , CH -1 and CH -5),
.14 (t, J = 10.6 Hz, 2H, CH -1 and CH -5), 2.05–1.88 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.8 Hz, 1H,
2
2
a
a
2
b
b
a
3 3 3
CH -3), 1.07 (t, J = 7.1 Hz, 3H, CH ), 0.87 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.67 (q, J = 12.4 Hz, 1H,
13
CH -3). C-NMR (DMSO-d6):
δ
169.60, 158.68, 156.31, 152.59, 141.88, 136.13, 134.17, 129.20, 122.57,
b
1
20.34 (2C), 120.33, 119.25, 115.38 (2C), 60.47, 59.71 (2C), 45.02, 41.61, 33.79, 30.90 (2C), 19.32 (2C), 13.92.
+
HRMS (ESI) m/z: [M + H] calculated for C H O N F , 510.23743; found, 510.23688, ∆ −1.08 ppm.
26
32
3
3 4
3
.1.43. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-fluorophenyl)ureido]-
phenyl}-4,4,4-trifluorobutyric Acid (i18)
Hydrolysis of compound g18 (456 mg, 0.89 mmol) following the general procedure B afforded
◦
1
compound i18 (white solid, 371 mg, 86.3% yield). mp: 125.1–126.4 C. H-NMR (DMSO-d ):
1
δ
12.50 (s,
H, COOH), 9.76 (s, 1H, NH), 8.14 (d, J = 1.8 Hz, 1H, H-phenyl), 8.11 (s, 1H, NH), 7.55–7.48 (m, 2H,
H-phenyl), 7.16–7.09 (m, 3H, H-phenyl), 6.98 (dd, J = 8.2, 1.9 Hz, 1H, H-phenyl), 3.96–3.79 (m, 1H, CH),
.99–2.74 (m, 4H, CH , CH -1 and CH -5), 2.14 (td, J = 11.0, 2.8 Hz, 2H, CH -1 and CH -5), 2.08–1.93
m, 2H, CH-2, CH-4), 1.79 (d, J = 12.8 Hz, 1H, CH
6
2
a
a
2
b
b
(
a
3 3
-3), 0.86 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.66 (q,
J = 12.4 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 482.20613; found,
+
b
24 28
3
3 4
4
82.20630, ∆ 0.35 ppm.
3
.1.44. Preparation of Racemic 3-[3-[3-(4-bromophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (g19)
Reaction of compound
afforded compound g19 (white solid, 100 mg, 81.5% yield). H-NMR (DMSO-d ):
f
and 4-bromophenyl isocyanate following the general procedure A
1
δ 9.69 (s, 1H, NH),
6
8
.13 (d, J = 1.7 Hz, 1H, H-phenyl), 8.09 (s, 1H, NH), 7.50–7.44 (m, 4H, H-phenyl), 7.14 (d, J = 8.2 Hz, 1H,
H-phenyl), 7.00 (dd, J = 8.2, 1.8 Hz, 1H, H-phenyl), 4.07–3.87 (m, 3H, CH , CH), 3.07–2.80 (m, 4H, CH ,
2
2
CH
a a
b b
-1 and CH -5), 2.14 (t, J = 10.7 Hz, 2H, CH -1 and CH -5), 2.05–1.91 (m, 2H, CH-2, CH-4), 1.80 (d,
J = 12.6 Hz, 1H, CH
a
-3), 1.07 (t, J = 7.1 Hz, 3H, CH ), 0.87 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.67 (q,
3 3 3
+
J = 12.0 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N BrF , 570.15737; found,
b
26 33
3
3
3
5
70.15735, ∆ −0.03 ppm.
3
.1.45. Preparation of Racemic 3-[3-[3-(4-bromophenyl)ureido]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (i19)
Hydrolysis of compound g19 (100 mg, 0.18 mmol) following the general procedure B afforded
◦
1
compound i19 (white solid, 71 mg, 74.8% yield). mp: 136.9–137.7 C. H-NMR (DMSO-d ):
1
δ
12.58 (s,
H, COOH), 9.75 (s, 1H, -NH-), 8.15–8.09 (m, 2H, H-phenyl, NH), 7.51–7.42 (m, 4H, H-phenyl), 7.14 (d,
J = 8.2 Hz, 1H, H-phenyl), 6.98 (dd, J = 8.1, 1.6 Hz, 1H, H-phenyl), 3.96–3.82 (m, 1H, CH), 2.98–2.71 (m,
H, CH , CH -1 and CH -5), 2.14 (td, J = 11.0, 3.3 Hz, 2H, CH -1 and CH -5), 2.06–1.91 (m, 2H, CH-2,
CH-4), 1.80 (d, J = 13.7 Hz, 1H, CH
6
4
a
a
2
b
b
a
-3), 0.86 (d, J = 6.5 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.4 Hz,
3 3
171.14, 152.47, 141.95, 139.41, 134.10, 131.57 (2C), 129.57, 126.92,
13
1
H, CH -3). C-NMR (DMSO-d6):
δ
b

Molecules 2020, 25, 1447
21 of 27
1
22.81, 120.42, 120.34 (2C), 119.36, 113.30, 59.79 (2C), 45.20, 41.68, 33.98, 30.79 (2C), 19.34 (2C). HRMS
+
(ESI) m/z: [M + H] calculated for C H O N BrF , 542.12607; found, 542.12616, ∆ 0.17 ppm.
24 28 3 3 3
3
.1.46. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-ethylphenyl)ureido]
phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (g20)
Reaction of compound
afforded compound g20 (white solid, 83.7% yield). H-NMR (DMSO-d ):
f
and 4-ethylphenethyl isocyanate following the general procedure A
1
δ 9.42 (s, 1H, NH), 8.15 (d,
6
J = 1.9 Hz, 1H, H-phenyl), 8.03 (s, 1H, NH), 7.41–7.37 (m, 2H, H-phenyl), 7.16–7.10 (m, 3H, H-phenyl),
6
.97 (dd, J = 8.2, 2.0 Hz, 1H, H-phenyl), 4.08–3.86 (m, 3H, CH , CH), 3.06–2.78 (m, 4H, CH , CH
a
-1
2
2
and CH
a
-5), 2.55 (q, J = 7.6 Hz, 2H, CH CH -phenyl), 2.13 (td, J = 11.2, 2.3 Hz, 2H, CH -1 and CH -5),
3
2
b
b
2
.01–1.87 (m, 2H, CH-2, CH-4), 1.78 (d, J = 12.8 Hz, 1H, CH
a
3
-3), 1.16 (t, J = 7.6 Hz, 3H, CH CH2-phenyl),
1
.07 (t, J = 7.6 Hz, 3H, CH ), 0.86 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.66 (q, J = 12.4 Hz, 1H, CH -3).
3
+
3
3
b
HRMS (ESI) m/z: [M + H] calculated for C H O N F , 520.27815; found, 520.27795, ∆ −0.39 ppm.
28
37
3
3 3
3
.1.47. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-ethylphenyl)ureido]
phenyl}-4,4,4-trifluorobutyric Acid (i20)
Hydrolysis of compound g20 (134 mg, 0.26 mmol) following the general procedure B afforded
◦
1
compound i20 (white solid, 103 mg, 81.2% yield). mp: 122.1–122.9 C. H-NMR (DMSO-d ):
δ
9.44
6
(s, 1H, NH), 8.14 (d, J = 1.6 Hz, 1H, H-phenyl), 8.04 (s, 1H, NH), 7.39 (d, J = 8.4 Hz, 2H, H-phenyl),
7
.16–7.10 (m, 3H, H-phenyl), 6.96 (dd, J = 8.2, 1.5 Hz, 1H, H-phenyl), 3.95–3.81 (m, 1H, CH), 2.96–2.73
-1 and CH -5), 2.55 (q, J = 7.6 Hz, 2H, CH CH -phenyl), 2.13 (td, J = 11.0, 3.8 Hz, 2H,
CH -1 and CH -5), 2.00–1.88 (m, 2H, CH-2, CH-4), 1.78 (d, J = 12.7 Hz, 1H, CH
(
m, 4H, CH , CH
a
a
2
3
2
b
b
a
-3), 1.16 (t, J = 7.6 Hz,
13
3
H, CH CH2-phenyl), 0.86 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.65 (q, J = 12.4 Hz, 1H, CH -3). C-NMR
3 3 3 b
(
DMSO-d6):
δ 171.21, 152.63, 141.72, 137.54, 137.40, 134.33, 129.60, 128.10 (2C), 126.93, 122.49, 120.36,
1
19.19, 118.97 (2C), 59.74 (2C), 45.18, 41.64, 34.00, 30.86 (2C), 27.60, 19.32 (2C), 15.85. HRMS (ESI) m/z:
+
[M + H] calculated for C H O N F , 492.24685; found, 492.24774, ∆ 1.80 ppm.
26 33 3 3 3
3
.1.48. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-isopropylphenyl)ureido]-
phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (g21)
Reaction of compound
afforded compound g21 (white solid, 80.7% yield). H-NMR (DMSO-d ):
f
and 4-isopropylphenyl isocyanate following the general procedure A
1
δ 9.42 (s, 1H, NH), 8.15 (d,
6
J = 1.9 Hz, 1H, H-phenyl), 8.03 (s, 1H, NH), 7.42–7.37 (m, 2H, H-phenyl), 7.14–7.20 (m, 2H, H-phenyl),
.12 (d, J = 8.4 Hz, 1H, H-phenyl), 6.97 (dd, J = 8.2, 1.9 Hz, 1H, H-phenyl), 4.08–3.86 (m, 3H, CH2,
CH), 3.07–2.79 (m, 5H, CH , CH -1 and CH -5, (CH ) CH-phenyl), 2.13 (td, J = 11.1, 2.5 Hz, 2H,
CH -1 and CH -5), 2.00–1.85 (m, 2H, CH-2, CH-4), 1.78 (d, J = 12.9 Hz, 1H, CH
7
2
a
a
3 2
b
b
a
-3), 1.18 (d, J = 6.8 Hz,
6
H, (CH ) CH-phenyl), 1.08 (t, J = 7.2 Hz, 3H, CH ), 0.86 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.65 (q,
3
2
3
+
3
3
J = 12.4 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 534.29380; found,
b
29 39
3
3 3
5
34.29602, ∆ 4.15 ppm.
3
.1.49. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-isopropylphenyl)ureido]-
phenyl}-4,4,4-trifluorobutyric Acid (i21)
Hydrolysis of compound g21 (200 mg, 0.35 mmol) following the general procedure B afforded
◦
1
compound i21 (white solid, 142 mg, 81.4% yield). mp: 121.7–122.7 C. H-NMR (DMSO-d ):
1
δ
9.43 (s,
H, NH), 8.15 (d, J = 1.6 Hz, 1H, H-phenyl), 8.04 (s, 1H, NH), 7.39 (d, J = 8.4 Hz, 2H, H-phenyl), 7.16 (d,
J = 8.5 Hz, 2H, H-phenyl), 7.12 (d, J = 8 Hz, 1H, H-phenyl), 6.96 (dd, J = 8.3, 1.7 Hz, 1H, H-phenyl),
6
3
.95–3.82 (m, 1H, CH), 2.98–2.73 (m, 5H, CH , CH
a
a
3 2
-1 and CH -5, (CH ) CH-phenyl), 2.13 (td, J = 11.0,
2
3
.9 Hz, 2H, CH -1 and CH -5), 2.00–1.85 (m, 2H, CH-2, CH-4), 1.77 (d, J = 12.7 Hz, 1H, CH
a
-3), 1.18 (t,
b
b
J = 7.1 Hz, 6H, (CH ) CH-phenyl), 0.85 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.65 (q, J = 12.4 Hz, 1H,
3
2
3
3
13
CH -3). C-NMR (DMSO-d6):
δ 171.13, 152.64, 142.30, 141.72, 137.44, 134.34, 129.56, 126.92, 126.62 (2C),
b

Molecules 2020, 25, 1447
22 of 27
1
22.47, 120.37, 119.18, 119.09 (2C), 59.73 (2C), 45.19, 41.63, 33.92, 32.87, 30.87 (2C), 24.09 (3C), 19.32 (2C).
+
HRMS (ESI) m/z: [M + H] calculated for C H O N F , 506.26250; found, 506.26373, ∆ 2.42 ppm.
27
35
3
3 3
3
.1.50. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-trifluoromethoxyphenyl)-
ureido]phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (g22)
Reaction of compound
afforded compound g22 (white solid, 81.5% yield). H-NMR (DMSO-d ):
f
and 4-fluorophenyl isocyanate following the general procedure A
1
δ 9.75 (s, 1H, NH), 8.14 (d,
6
J = 1.9 Hz, 1H, H-phenyl), 8.10 (s, 1H, NH), 7.62–7.58 (m, 2H, H-phenyl), 7.30 (d, J = 8.5 Hz, 2H,
H-phenyl), 7.14 (d, J = 8.2 Hz, 1H, H-phenyl), 7.00 (dd, J = 8.2, 1.9 Hz, 1H, H-phenyl), 4.07–3.88 (m,
3
H, CH , CH), 3.07–2.81 (m, 4H, CH , CH
a
-1 and CH
a
b b
-5), 2.15 (t, J = 10.4 Hz, 2H, CH -1 and CH -5),
2
2
2
.05–1.90 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.8 Hz, 1H, CH
a
-3), 1.07 (t, J = 7.1 Hz, 3H, CH ), 0.87 (d,
3
+
J = 6.6 Hz, 6H, CH -6, CH -7), 0.67 (q, J = 12.8 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated
3
3
b
for C H O N F , 576.22915; found, 576.22772, ∆ −2.49 ppm.
27
32
4
3 6
3
.1.51. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-trifluoromethoxyphenyl)-
ureido]phenyl}-4,4,4-trifluorobutyric Acid (i22)
Hydrolysis of compound g22 (586 mg, 1.02 mmol) following the general procedure B afforded
◦
1
compound i22 (white solid, 455 mg, 81.5% yield). mp: 196.2–197.8 C. H-NMR (DMSO-d ):
1
δ
12.37 (s,
H, COOH), 9.78 (s, 1H, NH), 8.17–8.08 (m, 2H, H-phenyl, NH), 7.64–7.56 (m, 2H, H-phenyl), 7.30 (d,
J = 8.7 Hz, 2H, H-phenyl), 7.15 (d, J = 8.2 Hz, 1H, H-phenyl), 7.00 (dd, J = 8.2, 1.5 Hz, 1H, H-phenyl),
.97–3.82 (m, 1H, -CH-), 3.01–2.74 (m, 4H, CH , CH -1 and CH -5), 2.15 (td, J = 11.0, 2.8 Hz, 2H, CH -1
and CH -5), 2.07–1.91 (m, 2H, CH-2, CH-4), 1.80 (d, J = 12.6 Hz, 1H, CH
6
3
a
a
2
b
b
a
-3), 0.87 (d, J = 6.5 Hz, 6H,
171.02, 152.47, 142.73, 141.93,
39.14, 134.00, 129.51, 126.86, 122.87, 121.79 (2C), 120.44, 120.28, 119.65 (2C), 119.32, 59.74 (2C), 45.09,
13
CH -6, CH -7), 0.67 (q, J = 12.4 Hz, 1H, CH -3). C-NMR (DMSO-d6):
δ
3
3
b
1
4
+
1.62, 33.77, 30.89 (2C), 19.32 (2C). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 548.19785;
25 28 4 3 6
found, 548.19781, ∆ −0.08 ppm.
3
.1.52. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-trifluoromethylphenyl)-
ureido]phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (g23)
Reaction of compound
A afforded compound g23 (white solid, 80.6% yield). H-NMR (DMSO-d ):
f
and 4-(trifluoromethyl)phenyl isocyanate following the general procedure
1
δ 9.96 (s, 1H, NH), 8.26–8.12
6
(
m, 2H, NH, H-phenyl), 7.81–7.61 (m, 4H, H-phenyl), 7.16 (d, J = 8.2 Hz, 1H, H-phenyl), 7.02 (d,
J = 8.2 Hz, 1H, H-phenyl), 4.12–3.89 (m, 3H, CH , CH), 3.13–2.79 (m, 4H, CH , CH -1 and CH -5), 2.15
t, J = 10.6 Hz, 2H, CH -1 and CH -5), 2.08–1.92 (m, 2H, CH-2, CH-4), 1.81 (d, J = 12.1 Hz, 1H, CH -3),
.14–1.03 (m, 3H, CH ), 0.87 (d, J = 6.4 Hz, 6H, CH -6, CH -7), 0.67 (q, J = 12.0 Hz, 1H, CH -3). HRMS
2
2
a
a
(
1
a
b
b
3
3
3
b
+
(ESI) m/z: [M + H] calculated for C H O N F , 560.23424; found, 560.23425, ∆ 0.02 ppm.
27 32 3 3 6
3
.1.53. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-trifluoromethylphenyl)-
ureido]phenyl}-4,4,4-trifluorobutyric Acid (i23)
Hydrolysis of compound g23 (350 mg, 0.63 mmol) following the general procedure B afforded
◦
1
compound i23 (white solid, 270 mg, 80.6% yield). mp: 179.7–180.5 C. H-NMR (DMSO-d ):
1
δ
12.50 (s,
H, COOH), 9.97 (s, 1H, NH), 8.19 (s, 1H, NH), 8.14 (d, J = 1.9 Hz, 1H, H-phenyl), 7.74–7.62 (m, 4H,
H-phenyl), 7.16 (d, J = 8.2 Hz, 1H, H-phenyl), 7.01 (dd, J = 8.3, 2.0 Hz, 1H, H-phenyl), 3.98–3.82 (m,
H, CH), 3.00–2.76 (m, 4H, CH , CH -1 and CH -5), 2.16 (td, J = 11.1, 3.1 Hz, 2H, CH -1 and CH -5),
.07–1.92 (m, 2H, CH-2, CH-4), 1.81 (d, J = 12.8 Hz, 1H, CH
6
1
a
a
2
b
b
2
0
a
+
-3), 0.87 (d, J = 6.6 Hz, 6H, CH -6, CH -7),
3 3
.67 (q, J = 12.4 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 532.20294;
b
25 28
3
3 6
found, 532.20508, ∆ 4.03 ppm.

Molecules 2020, 25, 1447
23 of 27
3
.1.54. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-nitrophenyl)ureido]
phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (g24)
Reaction of compound
compound g24 (yellow solid, 86.5% yield). H-NMR (DMSO-d ):
f
and 4-nitrophenyl isocyanate following the general procedure A afforded
1
δ 10.28 (s, 1H, NH), 8.29 (s, 1H, NH),
6
8
.24–8.19 (m, 2H, H-phenyl), 8.15 (d, J = 1.8 Hz, 1H, H-phenyl), 7.78–7.73 (m, 2H, H-phenyl), 7.17 (d,
J = 8.2 Hz, 1H, H-phenyl), 7.04 (dd, emphJ = 8.2, 1.9 Hz, 1H, H-phenyl), 4.07–3.91 (m, 3H, CH2,
CH), 3.08–2.82 (m, 4H, CH , CH -1 and CH -5), 2.15 (dd, J = 16.1, 6.1 Hz, 2H, CH -1 and CH -5),
.07–1.92 (m, 2H, CH-2, CH-4), 1.81 (d, J = 12.8 Hz, 1H, CH
2
a
a
b
b
2
a
-3), 1.08 (t, J = 7.1 Hz, 3H, CH ), 0.87 (d,
3
+
J = 6.6 Hz, 6H, CH -6, CH -7), 0.67 (q, J = 12.4 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated
3
3
b
for C H O N F , 537.23193; found, 537.23126, ∆ −1.25 ppm.
26
32
5
4 3
3
.1.55. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[3-(4-nitrophenyl)ureido]
phenyl}-4,4,4-trifluorobutyric Acid (i24)
Hydrolysis of compound g24 (160 mg, 0.30 mmol) following the general procedure B afforded
◦
1
compound i24 (yellow solid, 121 mg, 79.3% yield). mp: 151.3–153.0 C. H-NMR (DMSO-d ): δ 12.51
6
(
s, 1H, COOH), 10.29 (s, 1H, NH), 8.29 (s, 1H, NH), 8.23–8.19 (m, 2H, H-phenyl), 8.14 (d, J = 1.6 Hz, 1H,
H-phenyl), 7.77–7.72 (m, 2H, H-phenyl), 7.17 (d, J = 8.3 Hz, 1H, H-phenyl), 7.04 (dd, J = 8.2, 1.7 Hz,
H, H-phenyl), 3.98–3.85 (m, 1H, CH), 3.01–2.77 (m, 4H, CH , CH -1 and CH -5), 2.16 (td, J = 11.0,
.8 Hz, 2H, CH -1 and CH -5), 2.08-1.93 (m, 2H, CH–2, CH-4), 1.81 (d, J = 12.8 Hz, 1H, CH -3), 0.86 (t,
171.00, 152.05,
1
2
a
a
2
b
b
a
13
J = 8.4 Hz, 6H, CH -6, CH -7), 0.68 (q, J = 12.4 Hz, 1H, CH -3). C-NMR (DMSO-d6):
δ
3
3
b
1
4
5
46.58, 142.19, 141.09, 133.52, 129.62, 126.84, 125.24 (2C), 123.49, 120.59, 119.65, 117.62 (2C), 59.79 (2C),
+
5.04, 41.59, 33.74, 30.80 (2C), 19.32 (2C). HRMS (ESI) m/z: [M + H] calculated for C H O N F ,
24
28
5
4 3
09.20063; found, 509.20267, ∆ 4.00 ppm.
3
.1.56. Preparation of Racemic 3-[3-[2-(4-cyanophenyl)acetylamino]-4-(3,5-dimethylpiperidin-1-
yl)-phenyl]-4,4,4-trifluorobutyric Acid Ethyl Ester (h1)
To a solution of racemic 3-[3-amino-4-(3,5-dimethylpiperidin-1-yl)-phenyl]-4,4,4-trifluoro-butyric
acid ethyl ester ( , 120 mg, 0.32 mmol) in N,N-dimethylformamide (4 mL) was added
-cyanophenylacetic acid (52 mg, 0.32 mmol), 1-hydroxybenzotriazole (43 mg, 0.32 mmol),
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (61 mg, 0.32 mmol) and
N,N-diisopropylethylamine (106 L, 0.64 mmol). The reaction mixture was stirred at room temperature
until the starting material disappeared in TLC. The reaction mixture was then diluted with ethyl
acetate (100 mL), washed with 1 N HCl aqueous solution (40 mL 2), saturated NaCl aqueous solution
40 mL 2), dried over anhydrous Na SO and concentrated. The residue was purified by column
f
4
µ
×
(
×
2
4
chromatography (silica gel, PE/EA = 10:1, v/v) to afford the product as a colorless oil (117 mg, 70.5%
1
yield). H-NMR (DMSO-d6):
δ 8.85 (s, 1H, NH), 8.04 (s, 1H, H-phenyl), 7.84 (d, J = 8.1 Hz, 2H,
H-phenyl), 7.59 (d, J = 8.1 Hz, 2H, H-phenyl), 7.11 (d, J = 8.0 Hz, 2H, H-phenyl), 4.05–3.86 (m, 5H, CH2,
CH, CH -phenyl), 3.06–2.82 (m, 2H, CH ), 2.73–2.63 (m, 2H, CH -1 and CH -5), 2.04 (t, J = 11.1 Hz, 2H,
-3), 1.61–1.45 (m, 2H, CH-2, CH-4), 1.05 (t, J = 7.1 Hz,
H, CH ), 0.77 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.57 (q, J = 12.0 Hz, 1H, CH -3). HRMS (ESI) m/z:
2
2
a
a
CH -1 and CH -5), 1.67 (d, J = 12.0 Hz, 1H, CH
a
b
b
3
3
+
3
3
b
[M + H] calculated for C H O N F , 516.24685; found, 516.24878, ∆ 3.73 ppm.
28 33 3 3 3
3
.1.57. Preparation of Racemic 3-[3-[2-(4-cyanophenyl)acetylamino]-4-(3,5-dimethylpiperidin-1-yl)-
phenyl]-4,4,4-trifluorobutyric Acid (j1)
Hydrolysis of compound h1 (70 mg, 0.14 mmol) following the general procedure B afforded
◦
1
compound j1 (white solid, 49 mg, 73.9% yield). mp: 108.2–108.9 C. H-NMR (DMSO-d ):
δ
8.83 (s,
6
1H), 8.07 (s, 1H, H-phenyl), 7.84 (d, J = 6.1 Hz, 2H, H-phenyl), 7.59 (d, J = 6.6 Hz, 2H, H-phenyl),
7
.13–7.03 (m, 2H, H-phenyl), 3.98–3.82 (m, 3H, CH, CH -phenyl), 2.91–2.61 (m, 4H, CH , CH
a
-1 and
2
2
CH
a
-5), 2.03 (t, J = 11.0 Hz, 2H, CH -1 and CH -5), 1.68 (d, J = 12.4 Hz, 1H, CH
a
1
-3), 1.59–1.43 (m, 2H,
b
b
3
CH-2, CH-4), 0.79–0.68 (m, 6H, CH -6, CH -7), 0.57 (q, J = 12.0 Hz, 1H, CH -3). C-NMR (DMSO-d6):
3
3
b

Molecules 2020, 25, 1447
24 of 27
δ
168.08, 143.22, 141.42, 132.58 (2C), 132.40, 130.58 (2C), 129.58, 127.02, 125.04, 121.43, 120.35, 118.84,
+
1
09.88, 59.35 (2C), 45.16, 43.55, 41.42, 34.36, 31.00 (2C), 19.16 (2C). HRMS (ESI) m/z: [M + H] calculated
for C H O N F , 488.21555; found, 488.21490, ∆ −1.34 ppm.
26
29
3
3 3
3
.1.58. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[2-(4-trifluoromethylphenyl)-
acetylamino]phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (h2)
Reaction of compound
f
and 4-(trifluoromethyl)phenylacetic acid following the similar procedure
1
described for the preparation of h1 afforded h2 as a colorless oil (128 mg, 71.2%). H-NMR (DMSO-d ):
6
δ
8.78 (s, 1H, NH), 8.12 (s, 1H, H-phenyl), 7.74 (d, J = 8.2 Hz, 2H, H-phenyl), 7.63 (d, J = 8.2 Hz, 2H,
H-phenyl), 7.10 (s, 2H, H-phenyl), 4.05–3.92 (m, 3H, CH, CH ), 3.90 (s, 2H, CH -phenyl), 3.06–2.82 (m,
2
2
2 b b
a a
2H, CH ), 2.59–2.69 (m, 2H, CH -1 and CH -5), 2.01 (t, J = 11.1 Hz, 2H, CH -1 and CH -5), 1.62 (d,
J = 13.0 Hz, 1H, CH
a
-3), 1.47-1.34 (dt, J = 22.8, 11.6 Hz, 2H, CH-2, CH-4), 1.06 (t, J = 7.1 Hz, 3H, CH ),
3
+
0.73 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.53 (q, J = 12.4 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H]
3 3 b
calculated for C H O N F , 559.23899; found, 559.24072, ∆ 3.10 ppm.
28
33
3
2 6
3
.1.59. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[2-(4-trifluoromethylphenyl)-
acetylamino]phenyl}-4,4,4-trifluorobutyric Acid (j2)
Hydrolysis of compound h2 (80 mg, 0.14 mmol) following the general procedure B afforded
◦
1
compound j2 (white solid, 49 mg, 77.8% yield). mp: 93.2–93.6 C. H-NMR (DMSO-d ): δ 8.77 (s, 1H,
6
NH), 8.13 (s, 1H, H-phenyl), 7.74 (d, J = 8.1 Hz, 2H, H-phenyl), 7.63 (d, J = 8.0 Hz, 2H, H-phenyl),
7
2
.13–7.02 (m, 2H, H-phenyl), 3.98–3.82 (m, 3H, CH, CH ), 2.90–2.58 (m, 4H, CH , CH
a
-1 and CH
a
-5),
2
2
.01 (td, J = 11.1, 4.2 Hz, 2H, CH -1 and CH -5), 1.61 (d, J = 12.6 Hz, 1H, CH
a
-3), 1.46–1.30 (m, 2H, CH-2,
b
b
13
CH-4), 0.73 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.53 (q, J = 12.0 Hz, 1H, CH -3). C-NMR (DMSO-d6):
3
3
b
δ
171.22, 168.28, 143.04, 140.23, 132.59, 130.44 (2C), 129.50, 127.98-125.48 (4C), 124.89, 123.05, 120.92,
+
1
20.49, 59.36 (2C), 45.02, 43.47, 41.29, 33.96, 30.91 (2C), 19.07 (2C). HRMS (ESI) m/z: [M + H] calculated
for C H O N F , 531.20769; found, 531.20703, ∆ −1.24 ppm.
26
29
3
2 6
3
.1.60. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[2-(4-nitrophenyl)acetylamino]-
phenyl}-4,4,4-trifluorobutyric Acid Ethyl Ester (h3)
Reaction of compound
f
and 4-nitrophenylacetic acid following the similar procedure described
1
for the preparation of h1 afforded h3 as a light yellow oil (99 mg, 68.7%). H-NMR (DMSO-d ):
δ
6
8.91 (s, 1H, NH), 8.24 (d, J = 8.6 Hz, 2H, H-phenyl), 8.03 (s, 1H, H-phenyl), 7.67 (d, J = 8.5 Hz, 2H,
H-phenyl), 7.11 (s, 2H, H-phenyl), 4.05–3.88 (m, 5H, CH , CH, CH -phenyl), 3.06–1.99 (m, 2H, CH ),
2
2
2
2.75–2.65 (m, 2H, CH
a a
b b
-1 and CH -5), 2.04 (t, J = 11.1 Hz, 2H, CH -1 and CH -5), 1.70–1.50 (m, 3H,
CH -3, CH-2, CH-4), 1.05 (t, J = 7.1 Hz, 3H, CH ), 0.75 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.56 (q,
a
3
3
3
+
J = 12.0 Hz, 1H, CH -3). HRMS (ESI) m/z: [M + H] calculated for C H O N F , 536.23668; found,
b
27 33
5
3 3
5
36.23749, ∆ 1.51 ppm.
3
.1.61. Preparation of Racemic 3-{4-(3,5-dimethylpiperidin-1-yl)-3-[2-(4-nitrophenyl)acetylamino]-
phenyl}-4,4,4-trifluorobutyric Acid (j3)
Hydrolysis of compound h3 (70 mg, 0.13 mmol) following the general procedure B afforded
◦
1
compound j3 (yellow solid, 52 mg, 78.2% yield). mp: 92.1–92.7 C. H-NMR (DMSO-d ):
δ
12.51 (s, 1H,
6
COOH), 8.90 (s, 1H, NH), 8.24 (d, J = 8.7 Hz, 2H, H-phenyl), 8.04 (s, 1H, H-phenyl), 7.67 (d, J = 8.6 Hz,
2
H, H-phenyl), 7.11 (s, 2H, H-phenyl), 3.99–3.84 (m, 3H, CH -phenyl, CH), 2.94–2.64 (m, 4H, CH ,
2
2
CH
0
a
-1 and CH
a
-5), 2.04 (t, J = 9.9 Hz, 2H, CH -1 and CH -5), 1.69–1.48 (m, 3H, CH
a
-3, CH-2, CH-4),
b
b
.75 (d, J = 6.6 Hz, 6H, CH -6, CH -7), 0.56 (q, J = 12.4 Hz, 1H, CH -3). 3C-NMR (DMSO-d6):
1
δ
170.95,
3
3
b
1
5
68.00, 146.64, 143.69, 132.37, 130.95, 130.83 (2C), 129.17, 126.78, 125.12, 123.73 (2C), 121.62, 120.45,
9.34 (2C), 45.85, 43.20, 41.37, 33.66, 30.96 (2C), 19.09 (2C). HRMS (ESI) m/z: [M + H] calculated for
+
C H O N F , 508.20538; found, 508.20618, ∆ 1.57 ppm.
25
29
5
3 3

Molecules 2020, 25, 1447
25 of 27
3
.2. Pharmacological Methods
3
.2.1. The Enzyme Assay for IDO1 and TDO Inhibition
Recombinant human IDO1 and TDO were expressed and purified according to the reported
procedures [20]. The assay was performed according to the literature: A standard reaction mixture
100 L) containing 100 mM potassium phosphate buffer (pH 6.5), 40 mM ascorbic acid (neutralized
with NaOH), 200 g/mL catalase, 20 M methylene blue and 0.05 M rhIDO1 or rhTDO was added to
(
µ
µ
µ
µ
the solution containing the substrate L-tryptophan and the test sample at a determined concentration.
◦
The reaction was carried out at 37 C for 45 min and stopped by adding 20
µ
L of 30% (w/v) trichloroacetic
◦
acid. After heating at 65 C for 15 min, 100
µL of 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid
was added to each well. The yellow pigment derived from kynurenine was measured at 492 nm using
a SYNERGY-H1 microplate reader (Biotek Instruments, Inc., Winooski, VT, USA). IC50 was analyzed
using the GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA) [21].
3
.2.2. Mice
C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.
Beijing, China). Studies involving mice were approved by the Experimental Animal Management
(
and Welfare Committee at the Institute of Materia Medica, Peking Union Medical College (Approval
No. 00000522).
3
.2.3. Pharmacokinetic Studies
The animal Care and Welfare Committee of Institute of Materia Medica, Chinese Academy of
Medical Sciences approved all animal care, housing, and laboratory procedures. Male C57BL/6 mice
were used in the single dose pharmacokinetic studies. Compound i12 was prepared as a 3 mg/mL
suspension with 0.5% CMC for oral use and was formulated as a 3 mg/mL solution with 10% DMSO in
2
0% HP-
group and six in the intravenous group. After fasting 12 h with free access to water, mice were treated
with a 3 mg/kg i.v. or 30 mg/kg oral dose of compound i12. Blood samples (50 L) were collected at 5,
5, 30 min, 1, 2, 4, 6, 8, 12 and 24 h after oral administration and 2, 5, 15, 30 min, 1, 2, 4, 6, 8, 12 and
4 h after intravenous injection. After centrifugation, the plasma samples (20 L) were precipitated
by four volumes of acetonitrile. The supernatant were analyzed by liquid chromatography/tandem
mass spectrometry with a Zorbax C18 column (50 mm 2.1 mm, 3.5 m). Compound detection
β-CD for intravenous injection. Sixteen mice were divided into two groups, 10 in the oral
µ
1
2
µ
×
µ
was performed with the mass spectrometer in positive ionization mode by t-SIM: m/z 489.209 for
compound i12. The pharmacokinetic parameters were calculated with WinNonlin software V6.3 using
non-compartmental analysis (Pharsight Corporation, Mountain View, CA, USA).
3
.2.4. In Vivo Studies
6
The mouse melanoma cells B16F10 were cultured and harvested in saline at 6
×
10 cells/0.2 mL
volume. Cells (0.2 mL) were injected subcutaneously into male C57BL/6 mice at day 0 of the experiment,
and treatment was initiated at day 1 following the mice enrolled randomly in control and experimental
groups. For control group, 0.5% CMCNa was orally administered every day. The CTX group were
administered CTX intraperitoneally at the dose of 100 mg/kg. Compound i12 was dissolved in 0.5%
CMC-Na for oral treatment. After 17 days, the mice were sacrificed and the tumors were stripped and
weighted. The tumor growth inhibition (TGI) was calculated as TGI = (1
weight vehicle 100%. The statistical analysis was performed with GraphPad Prism 8.0 software and
the significance level was evaluated with one-way ANOVA model [22].
−
tumor weight treatment/tumor
)
×
The mouse pancreatic cancer cells PAN02 were cultured and harvested in saline at
6
6
×
10 cells/0.2 mL volume. Cells (0.2 mL) were injected subcutaneously into male C57BL/6 mice at
day 0 of the experiment, and treatment was initiated at day 1 following the mice enrolled randomly in
control and experimental groups. For control group, 0.5% CMCNa was orally administered every day.

Molecules 2020, 25, 1447
26 of 27
The CTX group were administered CTX intraperitoneally at the dose of 60 mg/kg Compound i12 was
dissolved in 0.5% CMC-Na for oral treatment. After 15 days, the mice were sacrificed and the tumors
were stripped and weighted. The tumor growth inhibition (TGI) was calculated as TGI = (1
weight treatment/tumor weight vehicle 100%. The statistical analysis was performed with GraphPad
Prism 8.0 software and the significance level was evaluated with one-way ANOVA model [22].
−
tumor
)
×
4
. Conclusions
In summary, we designed a new series of phenyl urea derivatives as IDO1 inhibitors through a
ring formation strategy. Systematic SAR led to the discovery of the promising anticancer compound i12
with favourable drug-like properties. Compound i12 had potent IDO1 inhibitory activity with the IC50
of 0.331 µM and exhibited a satisfactory PK profile with moderate plasma clearance (22.45 mL/min/kg)
and high oral bioavailability (87.4%). In addition, Compound i12 orally administered at 15 mg/kg
daily showed a TGI of 40.5% in a B16F10 subcutaneous xenograft model and at 30 mg/kg daily showed
a TGI of 34.3% in a PAN02 subcutaneous xenograft model. Overall, compound i12 is a promising
anti-tumor agent with the potent in vitro enzymatic activity, good pharmacokinetic properties and
satisfied in vivo anti-tumor efficacy.
Supplementary Materials: The following are available online, Figures are H NMR, 13C NMR and MS spectra of
1
the synthesized compounds.
Author Contributions: Conceptualization, Z.F.; Data curation, C.Z., F.L. and L.S.; Formal analysis, C.Z.; Funding
acquisition, Z.F.; Investigation, C.Z., F.L. and L.S.; Methodology, F.L., L.S., X.C. and Y.L.; Project administration,
C.Z.; Resources, X.C., Y.L. and Z.F.; Supervision, Z.F.; Validation, X.C. and Y.L.; Visualization, C.Z.; Writing–original
draft, C.Z.; Writing–review and editing, F.L., L.S. and Z.F. All authors have read and agreed to the published
version of the manuscript.
Funding: The research was funded by Beijing Municipal Natural Science Foundation (7192133) and the Non-profit
Central Research Institute Fund of Chinese Academy of Medical Sciences (2018PT35003).
Acknowledgments: We would like to thank Lu Zhang for her support in the molecular docking study.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).