Design, synthesis and biological evaluation of novel cholesteryl ester transfer protein inhibitors bearing a cycloalkene scaffold

Article abstract of DOI:10.1016/j.ejmech.2016.07.065

Cholesteryl ester transfer protein (CETP) is a potential target for cardiovascular disease therapy as inhibition of CETP leads to increased HDL-C in humans. Based on the structure of Merck's biphenyl CETP inhibitor, we designed novel N,N-substituted-cyclo

Full text of DOI:10.1016/j.ejmech.2016.07.065

Accepted Manuscript
Design, synthesis and biological evaluation of novel cholesteryl ester transfer protein
inhibitors bearing a cycloalkene scaffold
Chunchi Liu, Changqun Luo, Lijuan Hao, Qiong Wu, Honglei Xie, Shizhen Zhao,
Chenzhou Hao, Dongmei Zhao, Maosheng Cheng
PII:
S0223-5234(16)30628-6
10.1016/j.ejmech.2016.07.065
EJMECH 8783
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 May 2016
Revised Date: 21 July 2016
Accepted Date: 25 July 2016
Please cite this article as: C. Liu, C. Luo, L. Hao, Q. Wu, H. Xie, S. Zhao, C. Hao, D. Zhao, M. Cheng,
Design, synthesis and biological evaluation of novel cholesteryl ester transfer protein inhibitors
bearing a cycloalkene scaffold, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.07.065.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel cholesteryl ester transfer
protein inhibitors bearing a cycloalkene scaffold
Chunchi Liua, Changqun Luoa, Lijuan Haoa, Qiong Wua, Honglei Xiea, Shizhen Zhaoa, Chenzhou
Haoa, Dongmei Zhaoa,*, Maosheng Chenga
a
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang
Pharmaceutical University, Shenyang 110016, P.R. China.
*
Corresponding author: Dongmei Zhao, E-mail: medchemzhao@163.com, Fax: +86-24-23995043, Tel:
+86-24-23986413
Abstract
Cholesteryl ester transfer protein (CETP) is a potential target for cardiovascular disease
therapy as inhibition of CETP leads to increased HDL-C in humans. Based on the structure of
Merck’s biphenyl CETP inhibitor, we designed novel N,N-substituted-cycloalkenyl-methylamine
scaffold derivatives by utilizing core replacement and conformational restriction strategies.
Consequently, twenty-eight compounds were synthesized and evaluated for their inhibitory
activity against CETP. Their preliminary structure-activity relationships (SARs) studies indicate
that polar substituents were tolerated in moiety A and hydrophobic alkyl groups at the 5-position
of cyclohexene were critical for potency. Among them, compound 17a, bearing an
N-(5-pyrazolyl-pyrimidin-2-yl)-cycloalkenyl- methylamine scaffold, exhibited excellent CETP
inhibitory activity (IC50 = 0.07 ꢀM) in vitro. Furthermore, it showed an acceptable
pharmacokinetic profile in S-D rats and efficient HDL-C increase in high-fat fed hamsters.
Keywords: Synthesis; N,N-substituted-cycloalkenyl-methylamine derivatives; CETP inhibitors;
HDL-C; Cardiovascular disease
1. Introduction
Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide.
Epidemiological studies have indicated that low levels of high-density lipoprotein-cholesterol
(HDL-C) are considered to be a major risk factor for CVD, independent of high levels of
low-density lipoprotein-cholesterol (LDL-C) [1-4]. Each 1 mg/dL increase in HDL-C reduces
cardiovascular events by 2-3% on the basis of clinical studies [5]. Cholesteryl ester transfer
protein (CETP) is a 476-residue glycoprotein, secreted mainly from the liver, that facilitates the
transfer of cholesteryl esters (CEs) from high-density lipoprotein (HDL) to low-density
lipoprotein (LDL) and very low-density lipoprotein (VLDL) in exchange for triglycerides (TGs)
[6, 7]. It has been shown that CETP plays a proatherogenic role due to raising LDL-C levels and
reducing HDL-C levels [8, 9]. Therefore, the inhibition of CETP should result in increased
HDL-C levels and reduce the risk of CVD [6, 10].
Several small molecular CETP inhibitors have been reported, and some of them have been
through phase III clinical trials (Fig. 1) [11]. Torcetrapib (1) was the first CETP inhibitor to reach
phase III clinical trials, exhibiting a very potent inhibitory activity. Unfortunately, the
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ILLUMINATE study was prematurely halted due to unexpected effects that led to more mortality
in the torcetrapib/atorvastatin group than in the atorvastatin group [12-14]. Subsequently, the
dalcetrapib (2) phase III clinical trial was terminated, as the inhibitor did not decrease the risk of
cardiovascular events [15]. Recently, Lilly announced that the phase III ACCELERATE study of
evacetrapib (3) was terminated, but the precise explanation for the failure has not been published
in any journal article. Anacetrapib (4) is currently being studied in a phase III clinical trial, and the
data shows an effective increase in HDL-C and decrease in LDL-C, without the side effects of
torcetrapib [16, 17].
Figure 1. Representative CETP inhibitors
Compound 5 (IC50 = 0.11 ꢀM, Fig. 2), developed by Merck, showed significant
lipid-regulating activity in vivo [18, 19]. Its simple scaffold and favourable pharmacodynamic
properties encouraged us to explore novel and potent structures based on this compound. As
shown in Figure 2, the replacement of a substituted phenyl ring with a substituted cyclohexenyl
ring resulted in weak micromolar activity (10a, IC50 = 20.97 ꢀM), and efforts to enhance the
pharmacological activity through alternative substitutions on the carbamate were largely
unsuccessful. Next, our group investigated the cyclization of the carbamate using a conformational
constraint strategy to attempt to increase the CETP inhibitory activity. To avoid instability and the
generation of chiral centres, we focused on the introduction of aromatic rings onto moiety A.
Fortunately, the compound containing a pyrimidine ring (16, IC50 = 3.52 ꢀM) on moiety A showed
increased inhibitory activity. We chose compound 16 as the lead compound, and a series of novel
N,N-substituted-cycloalkenyl-methylamine derivatives were synthesized. Optimization efforts on
moiety A and ring B are discussed in this study.
Figure 2. Design of new structures based on compound 5
2. Results and discussion
2.1. Chemistry
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Compounds 10a-c were prepared according to the procedure in Scheme 1. The commercially
available 4,4-dimethylcyclohexanone (6) underwent a Vilsmeier-Haack-Arnold reaction to
produce 7. The treatment of 7 with 5-isopropyl-2-methoxyphenylboronic acid under Suzuki
coupling conditions achieved 8 in good yield. Then, the -CHO of the resulting
2-phenylcyclohex-1-enecarbaldehyde scaffold was subjected to reductive amination with
3,5-bis(trifluoromethyl)benzyl amine to produce 9. The obtained intermediate 9 was treated with
corresponding chloroformates to give compounds 10a-c.
Compounds 15, 16a-f, 17a-d, 18 and 19a-e were prepared according to the procedure in
Scheme 2. The resulting 8 was reacted with NaBH4 and subsequently, SOCl2 to afford
intermediate 12. The treatment of starting material 13 with 3,5-bis(trifluoromethyl)benzyl amine
provided 14. Compound 15 was obtained by the nucleophilic substitution of 9 and 11 in the
presence of NaH at 0°C. This compound was subjected to Buchwald-Hartwig coupling reactions
with corresponding amines to yield compounds 16a-f and Suzuki coupling reactions with
corresponding borate esters to furnish compounds 17a-d. In compound 15, –Br was replaced with
–OH to give compound 18 which was subjected to substitution reactions with various substituted
alkyl bromides to yield compounds 19a-e.
As illustrated in Scheme 3, compounds 26a-h were synthesized from the corresponding
starting material ketones. Intermediates 21a-h were generated by Vilsmeier-Haack-Arnold
reactions and treated with 5-isopropyl-2-methoxyphenylboronic acid to provide 22a-h. These
compounds were reacted with NaBH4 and subsequently, SOCl2 to afford intermediates 24a-h, and
key intermediates 25a-h were obtained by nucleophilic substitution. The conventional Suzuki
coupling of 25a-h with 1-methylpyrazole-4-boronic acid pinacol ester afforded compounds 26a-h.
Scheme 1. Synthesis of target compounds 10a–c. Reagents and conditions: (a) DMF, PBr3, CHCl3, rt, 36.4%; (b)
5-isopropyl-2-methoxyphenylboronic acid, Pd(OAc)2, K2CO3, acetylacetone, EtOH, 80°C, 78.3%; (c)
3,5-bis(trifluromethyl)benzyl amine, NaBH(OAc)3, 1,2-dichloroethane, rt, 48.8%; (d) DIEA, DCM, rt,
87.0%-91.2%.
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Scheme 2. Synthesis of target compounds 15, 16a-f, 17a-d, 18 and 19a-e. Reagents and conditions: (a) NaBH4,
MeOH, rt, 97.7%; (b) SOCl2, DMF, rt, 75.3%; (c) 3,5-bis(trifluromethyl)benzyl amine, DIEA, 1,4-dioxane, 105°C,
75.8%; (d) NaH, DMF, 0°C, 64.7%; (e) Pd2(dba)3, 2-(di-tert-butylphosphino)biphenyl, NaOt-Bu, toluene, reflux,
39.3-53.1%; (f) Pd(PPh3)4, Na2CO3, DME/H2O, 90°C, 58.3-66.9%; (g) (i) bis(pinacolato)diboron,
PdCl2(dppf)-CH2Cl2, AcOK, DMSO, 80°C; (ꢀ) 30% H2O2, THF, 0ꢁ, 46.4%; (h) K2CO3, DMF, rt, 56.5-84.3%.
Scheme 3. Synthesis of target compounds 26a-h. Reagents and conditions: (a) DMF, PBr3, CHCl3, rt, 36.4%; (b)
5-isopropyl-2-methoxyphenylboronic acid, Pd(OAc)2, K2CO3, acetylacetone, EtOH/H2O, 80°C, 78.3%; (c) NaBH4,
MeOH, rt, 97.7%; (d) SOCl2, DMF, rt, 75.3%; (e) NaH, DMF, 0°C, 64.7%; (f) Pd(PPh3)4, Na2CO3, DME/H2O,
90°C, 43.8-58.0%.
2.2. In vitro activity against Cholesteryl Ester Transfer Protein
The N,N-substituted-cycloalkenyl-methylamine derivatives and reference compound
anacetrapib (4) were screened for their in vitro activity against CETP by a BODIPY-CE
fluorescence assay with the CETP RP Activity Assay Kit (Catalog # RB-RPAK; Roar). The
results are shown in Table 1 and Table 2. The IC50 values reveal that most of the compounds
exhibit potent CETP inhibitory activity.
As shown in Table 1, moiety A is critical for the pharmacological activity in vitro, and
replacement of a carbamate (10a, IC50 = 20.97 µM) with a pyrimidine ring (18, IC50 = 3.52 µM)
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caused a significant increase in the activity. Next, we investigated the relationship between various
substituents at the 5-position of the pyrimidine ring (moiety A) and the CETP inhibitory activity.
The introduction of bromine (15) dramatically reduced the activity due to the hydrophobic effect.
Interestingly, the potency of piperazine group 16a (IC50 = 0.35 µM) was nearly 10-fold stronger
than that of a free hydroxyl group (18, IC50 = 3.52 µM), and N-methyl piperazine (16b, IC50
=
2.07 µM) was 6-fold less potent than piperazine (16a). Replacing the free NH of piperazine (16a)
with an oxygen atom (morpholine, 16f, IC50 = 0.86 µM) caused a weak decrease in the activity.
Nevertheless, the nitrogen atom of piperazine (16a) moved outside to construct exocyclic amine
16c (IC50 = 0.17 µM), exhibiting 2-fold higher potency than compound 16a, while a substitution
of the amine of 16c with an acetyl group (16d, IC50 = 0.52 µM) somewhat decreased the potency.
Changing the 5-NH2 group (16c) to a 5-COOH group (16e, IC50 = 0.37 µM) showed no advantage.
Aromatic heterocycles attached at the 5-position of the pyrimidine ring were also examined, and
of these, the 1-menthylpyrazole fragment was the most potent (17a, IC50 = 0.07 µM), although the
other compounds also exhibited obvious decreases in activity. Interestingly, the potency of straight
chain saturated carboxylic acids 19b (IC50 = 0.36 µM) and 19c (IC50 = 0.30 µM) were nearly
10-fold stronger than that with a free hydroxyl group (18, IC50 = 3.52 µM), but the corresponding
ester (19a, IC50 = 0.91 µM) exhibited low potency vs 19b. Meanwhile, a straight chain saturated
alcohol (19d, IC50 = 0.19 µM) showed potent inhibitory activity, but the corresponding ether (19e,
IC50 = 0.42 µM) exhibited almost 2-fold weaker potency. According to these results, we could
speculate that the hydrophilic group at the 5-position of pyrimidine provides an important
contribution to the potency due to interaction with the hydrophilic area.
Table 1 Structures and activities of compounds 10a-c, 15, 16a-f, 17a-d, 18, 19a-e
NO.
10a
Moiety A
R
IC50 (µM)
NO.
17a
Moiety A
R
IC50 (µM)
20.97
0.07
10b
10c
15
˃50b
˃50b
˃50b
0.35
2.07
0.17
17b
17c
17d
18
˃50b
˃50b
˃50b
3.52
0.91
0.36
16a
16b
16c
19a
19b
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16d
16e
0.52
19c
0.30
0.19
N
O
N
H
0.37
19d
16f
0.86
19e
0.42
0.04
Anacetrapiba
a Used as a positive control.
b Considered with no CETP inhibition activity
To further study the relationship between ring B and the CETP inhibitory activity, another
eight compounds 26a-h were prepared and evaluated for their activity (Table 2). Compounds
containing hydrophobic alkyl groups at the 5-position of cyclohexene (26d, IC50 = 0.06 µM; 26e,
IC50 = 0.08 µM; 26f, IC50 = 0.07 µM) showed better inhibitory activity than their counterparts
lacking these groups (26b, IC50 = 0.93 µM). However, replacing the cyclohexene ring with a
cyclopentene ring (26a, IC50 = 2.76 µM) or cycloheptene ring (26c, IC50 = 1.62 µM) resulted in an
obvious decrease in activity. The incorporation of a benzene ring onto the cyclohexene to form a
1,2-dihydronaphthalene group (26g, 26h) resulted in a decrease of activity due to the steric effect.
These results indicate that hydrophobic alkyl groups at the 5-position of cyclohexene are critical
for potency.
Table 2. Structures and activities of compounds 26a-h
NO.
26a
26b
B ring
IC50 (µM)
2.76
NO.
26e
26f
B ring
IC50 (µM)
0.08
0.93
0.07
26c
26d
17a
26g
26h
1.62
0.06
0.07
0.22
6.63
0.04
anacetrapiba
a Used as a positive control.
2.3. In vivo test of compound 17a in high-fat fed hamsters
Based on the result of the in vitro CETP inhibitory assay, potent inhibitor 17a was
selected for the in vivo assay. The high-fat fed hamster model was our primary animal
model to measure the compound’s ability to elevate HDL-C by CETP inhibition. As shown
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in Figure 3, compound 17a dose-dependently elevates HDL-C. Multiple oral dosing of 17a
at 30 mg/kg for 5 days in hamsters (n=6) produced a 9.3% elevation of HDL-C at similar
levels to that observed for
4 (anacetrapib). A 50 mg/kg dose of 17a showed a statistically
significant 27.9% elevation of HDL-C. The results indicate that 17a demonstrates a potent
dose-dependent HDL-C elevation in hamsters.
Figure 3. Elevation in HDL-C for compounds
4 and 17a in moderately fat-fed hamsters. Predose blood
samples were collected by retro-orbital bleed. Compounds were formulated in ethanol/hydrogenated
castor oil/water at a 1:1:8 ratio and dosed at 30mg/kg, 50mg/kg (po, q.d.) for 5 days. Blood was drawn 2 h
after the last dose and HDL-C levels were measured in the predose and postdose samples using chemistry
analyser. (
A
) Predose and postdose plasma HDL-C levels and (
B) percent changes in HDL-C after dosing
compounds for 5 days are reported as the mean
±
SEM (n = 6), (**) p < 0.01 vs vehicle.
2.4. Pharmacokinetic characterization of 17a
The pharmacokinetic parameters of 17a were determined in Sprague-Dawley rats (n =
4) dosed at 50 mg/kg, using a solution of 10% ethanol, 10% hydrogenated castor oil, and 80%
water as the vehicle for the peros route. As shown in Table 3, observed maximal plasma
concentration following oral dosing is 697.4 ng/mL, and time to reach Cmax is 2.5 h; in rats,
terminal half-life of compound 17a is 2.5 h; area under curve (AUC) is 6994.4 ng·h/mL. On the
basis of pharmacokinetic parameters, we can know that compound 17a exhibited an acceptable
pharmacokinetic profile in S-D rats.
Table 3. Pharmacokinetic parameters for 17a in rats
Compound
17a
Species（n = 4）
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC (ng·h/mL)
6994.4
Rat (50 mpk)
697.4
2.5
11.1
3. Conclusions
In summary, an N,N-substituted-cycloalkenyl-methylamine scaffold was designed from a
reported CETP inhibitor by utilizing core replacement and conformational restriction. New
compounds were synthesized and evaluated for their inhibitory activity against CETP by a
BODIPY-CE fluorescence assay. An initial SAR revealed that polar substituents were tolerated in
moiety A and hydrophobic alkyl groups at the 5-position of cyclohexene were critical for potency.
Compound 17a was identified as a promising CETP inhibitor with good inhibitory activity (IC50 =
0.07 ꢀM), and based on its excellent in vitro property, it was selected for in vivo evaluation.
Compound 17a demonstrated a dose-dependent HDL-C elevation in hamsters and an acceptable
pharmacokinetic profile in S-D rats. Future studies of 17a are currently underway in our
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laboratory.
4. Experimental
4.1. Chemistry
All chemicals were obtained from commercial sources and were used without
purification unless otherwise specified. Solvents were distilled and dried using standard
methods. TLC was performed on silica gel plates (Indicator F-254) and visualized by
UV-light. NMR spectra were recorded on Bruker 400 MHz and 600MHz instruments, and
the chemical shifts were reported in terms of parts per million with TMS as the internal
reference. High-resolution accurate mass determinations (HRMS) for all final target
compounds were obtained on a Bruker Micromass Time of Flight mass spectrometer
equipped with electrospray ionisation (ESI). Column chromatography was performed with
silica gel (200-300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. The
purities of target compounds were ≥95%, measured by HPLC, performed on a Waters
1525-2489, eluting with 100%CH3OH or a mixture of solvents H2O (A) and MeOH (B)
(VA : VB = 5 : 95). Peaks were detected at λ 254 nm with a flow rate of 1.0 mL/min.
4.2. 2-Bromo-5,5-dimethylcyclohex-1-enecarbaldehyde (7).
PBr3 (2.8 mL, 27.0 mmol) was added slowly to a solution of DMF (2.1 mL, 30.0 mmol) in
CHCl3 (10 mL) that was cooled to 0 . After stirring for 1 h at 0 , 4,4-dimethylcyclohexanone
°
C
°C
(1.3 g, 10.0 mmol) was added to the mixture. The reaction mixture was again stirred at room
temperature for 8 h and then poured into ice water (60 mL) and neutralized slowly with solid
NaHCO3. The mixture was extracted with CH2Cl2 (20 mL×3), and the combined organic layers
were washed with water (20 mL×3) and brine (20 mL×3), dried over Na2SO4, and concentrated in
vacuo. The residue was purified by chromatography on silica gel (petroleum ether : EtOAc = 8:1)
to give 7 (0.8 g, 36.4%), which was used immediately for the next step because of its instability.
4.3. 2-(5-Isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enecarbaldehyde (8).
5-Isopropyl-2-methoxyphenylboronic acid (1.6 g, 8.0 mmol) was dissolved in water (20 mL)
and ethanol (20 mL), and intermediate 7 (1.7 g, 8.0 mmol), potassium carbonate (2.2 g, 16.0
mmol), acetylacetone (0.2 mL, 2.0 mmol) and Pd(OAc)2 (0.02 g, 0.08 mmol) were added. The
reaction mixture was heated to reflux for 4 h and then cooled to room temperature. After
concentration, the residue was dissolved in EtOAc (20 mL), washed with water (20 mL×3) and
brine (20 mL×3), dried over Na2SO4, and concentrated in vacuo. The residue was purified by
chromatography on silica gel (petroleum ether : EtOAc = 8:1) to give 8 (1.8 g, 78.3%) as a pale
1
yellow oil. H NMR (400 MHz, DMSO-d6) δ: 9.21 (s, 1H), 7.13 (dd, J = 8.5Hz, 2.2Hz, 1H),
6.92-6.90 (m, 2H), 3.64 (s, 3H), 2.80-2.73 (m, 1H), 2.56 (m, 1H), 2.29-2.28 (m, 1H), 1.94-1.91 (m,
2H), 1.38 (t, J = 6.4 Hz, 2H), 1.09 (d, J = 6.9 Hz, 6H), 0.89 (s, 6H). MS (ESI) m/z 309.4 [M+Na]+.
4.4. N-(3,5-Bis(trifluoromethyl)benzyl)-1-(2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex
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-1-enyl)methanamine (9).
Under an argon atmosphere, intermediate 8 (0.2 g, 0.8 mmol), 3,5-bis(trifluromethyl)benzyl
amine (0.2 g, 0.8 mmol) and Na2SO4 (0.2 g) were dissolved in 1,2-dichloroethane (2 mL). After
stirring for 1 h at room temperature, NaBH(OAc)3 (0.2 g, 1.0 mmol) was added to the mixture.
The reaction mixture was stirred at room temperature overnight and then poured into saturated
sodium bicarbonate solution (10 mL). The mixture was extracted with CH2Cl2 (20 mL×3) and the
combined organic layers were washed with water (20 mL×3) and brine (20 mL×3), dried over
Na2SO4, and concentrated in vacuo. The residue was purified by chromatography on silica gel
(petroleum ether : EtOAc = 4:1) to give 9 (0.2 g, 48.8%) as a pale yellow oil. 1H NMR (400 MHz,
DMSO-d6) δ: 7.86 (s, 3H), 6.98 (dd, J = 8.4 Hz, 2.3 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.75 (d, J =
2.3 Hz, 1H), 3.65 (s, 3H), 3.64 (s, 2H), 2.87-2.79 (m, 2H), 2.73-2.66 (m, 1H), 2.32-2.28 (m, 2H),
1.95 (s, 3H), 1.37 (t, J = 6.3 Hz, 2H), 1.07 (d, J = 6.9 Hz, 6H), 0.96 (s, 6H). MS (ESI) m/z 514.2
[M+H]+.
4.5. N-(3,5-Bis(trifluoromethyl)benzyl)-N-methoxycarbonyl-(2-(5-isopropyl-2-methoxyphenyl)-5,5
-dimethylcyclohex-1-enyl)methanamine (10a).
Intermediate 9 (0.2 g, 0.4 mmol) was dissolved in CH2Cl2 (5 mL) and methyl chloroformate
(0.1 mL, 0.6 mmol) and N,N-diisopropylethylamine (0.2 mL, 1.4 mmol) were added. The reaction
mixture was stirred at room temperature for 30 min and then poured into H2O (10 mL), and
extracted with CH2Cl2 (10 mL×3). The combined organic layers were washed with H2O (10 mL×3)
and brine (10 mL×3), dried over Na2SO4, and concentrated in vacuo. The residue was purified by
chromatography on silica gel (petroleum ether : EtOAc = 8:1) to give 10a (0.2 g, 87.0%) as a
1
white solid. mp 77.4-83.2°C. H NMR (400 MHz, CDCl3) δ: 7.67 (s, 1H), 7.46 (s, 1H), 7.35 (s,
1H), 7.04-7.00 (m, 1H), 6.74-6.67 (m, 2H), 4.46-4.33 (m, 1H), 4.24-4.17 (m, 1H), 4.10-3.90 (m,
1H), 3.73-3.65 (m, 7H), 2.73 (s, 1H), 2.47-2.41 (m, 1H), 2.09-2.05 (m, 1H), 1.83-1.77 (m, 2H),
1.46-1.40 (m, 2H), 1.12 (d, J = 3.6 Hz, 6H), 0.98 (s, 6H). 13C NMR (100 MHz, CDCl3) δ:
154.15(×2), 141.12, 140.97(×2), 130.44(×2), 128.01(×2), 127.34(×2), 125.82(×2), 124.63, 121.91,
120.85, 110.84, 55.33, 52.85, 40.21, 35.47, 33.09(×2), 29.04, 28.17(×2), 24.05(×2), 24.00(×2).
HRMS calcd for C30H36F6NO3, [M+H]+, 572.2594; found 572.2592. HPLC: tR = 6.500 min,
95.23%.
4.6. N-(3,5-Bis(trifluoromethyl)benzyl)-N-isopropoxycarbonyl-(2-(5-isopropyl-2-methoxyphenyl)-
5,5-dimethylcyclohex-1-enyl)methanamine (10b).
Colourless oil; yield 89.3%; 1H NMR (600 MHz, CDCl3) δ: 7.67 (d, J = 4.7 Hz, 1H), 7.43 (m,
2H), 7.02 (d, J = 6.9 Hz, 1H), 6.75-6.68 (m, 2H), 4.44-4.19 (m, 2H), 3.95-3.87 (m, 3H), 3.74-3.64
(m, 4H), 2.74 (d, J = 5.3 Hz, 1H), 2.44-2.42 (m, 1H), 2.09-2.07 (m, 1H), 1.85-1.82 (m, 3H), 1.42
(t, J = 6.5 Hz, 2H), 1.13-1.11 (m, 6H), 0.98-0.97 (m, 9H), 0.80-0.79 (m, 3H). 13C NMR (150 MHz,
CDCl3) δ: 157.14, 154.17(×2), 141.54, 141.00(×2), 135.41, 131.29, 130.51, 128.05, 127.96,
127.33, 125.79, 124.18, 122.37, 120.78, 110.93, 71.96(×2), 55.31, 48.83, 47.27, 40.36, 35.49,
33.08(×2), 29.02, 28.20, 28.02(×2), 24.01(×2), 18.87. HRMS calcd for C33H42F6NO3, [M+H]+,
614.3063; found 614.3072. HPLC: tR = 7.913 min, 95.46%.
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4.7. N-(3,5-Bis(trifluoromethyl)benzyl)-N-benzyloxycarbonyl-(2-(5-isopropyl-2-methoxyphenyl)-5
,5- dimethylcyclohex-1-enyl)methanamine (10c).
Colourless oil; yield 91.2%; 1H NMR (400 MHz, CDCl3) δ: 7.67 (d, J = 5.5 Hz, 1H), 7.46 (s,
1H), 7.39-7.33 (m, 5H), 7.22-7.20 (m, 1H), 7.04-7.01 (m, 1H), 6.75-6.69 (m, 2H), 5.20-5.12 (m,
2H), 4.46-4.21 (m, 1H), 4.17-4.16 (m, 1H), 4.14-3.98 (m, 1H), 3.93-3.64 (m, 4H), 2.74 (t, J = 6.8
Hz, 1H), 2.52-2.50 (m, 1H), 2.06-2.05 (m, 1H), 1.86-1.74 (m, 2H), 1.44-1.40 (m, 2H), 1.12 (d, J =
6.9 Hz, 6H), 0.99-0.93 (m, 6H). 13C NMR (100 MHz, CDCl3) δ: 161.25, 154.99(×2), 154.24,
141.66, 140.90, 138.34, 135.06, 131.30, 130.98, 130.73, 128.18, 128.08(×2), 127.77, 125.66(×2),
124.23(×2), 122.21(×2), 117.80, 110.71, 55.34, 48.75, 47.51, 40.38, 35.57, 33.12, 29.16,
29.11(×2), 28.26, 28.10, 24.06(×2). HRMS calcd for C36H39F6NO3Na, [M+Na]+, 670.2726; found
670.2750. HPLC: tR = 7.774 min, 97.13%.
4.8. (2-(5-Isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methanol (11).
Intermediate 8 (114.5 mg, 0.4 mmol) was dissolved in ethanol (5 mL), and thereto was added
sodium borohydride (19.0 mg, 0.5 mmol). After being stirred at room temperature for 30 min, the
reaction mixture was poured into H2O (20 mL) and extracted with EtOAc (10 mL×3). The
combined organic layers were washed with H2O (10 mL×3) and brine (10 mL×3), dried over
Na2SO4, and concentrated in vacuo. The residue was purified by chromatography on silica gel
(petroleum ether : EtOAc = 2:1) to give 11 (112.7 mg, 97.7%) as a colourless oil. 1H NMR (400
MHz, DMSO-d6) δ: 7.06 (dd, J = 8.4 Hz, 2.2 Hz, 1H), 6.88-6.85 (m, 2H), 4.25 (t, J = 5.3 Hz, 1H),
3.67 (s, 3H), 3.64-3.53 (m, 2H), 2.85-2.75 (m,1H), 2.35-2.31 (m, 1H), 2.04-1.86 (m, 3H), 1.38 (t,
J = 6.5 Hz, 2H), 1.16 (d, J = 6.9 Hz, 6H), 0.96 (d, J = 3.0 Hz, 6H). MS (ESI) m/z 311.4[M+Na]+.
4.9. 2-(2-(Chloromethyl)-4,4-dimethylcyclohex-1-enyl)-4-isopropyl-1-methoxybenzene (12).
SOCl2 (0.1 mL, 1.4 mmol) was added to a solution of intermediate 11 (115.3 mg, 0.4 mmol)
in DMF (2 mL) cooled to 0°C. After being stirred at room temperature for 1 h, the reaction
mixture was poured into H2O (10 mL) and was extracted with EtOAc (5 mL×3). The combined
organic layers were washed with H2O (10 mL×3) and brine (10 mL×3), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether :
EtOAc = 10:1) to give 12 (92.5 mg, 75.3%), which was used immediately for the next step
because of its instability.
4.10. N-(3,5-Bis(trifluoromethyl)benzyl)-5-bromopyrimidin-2-amine (14).
3,5-Bis(trifluromethyl)benzyl amine (5.0 g, 20.6 mmol) and 5-bromo-2-chloropyrimidine
(3.9 g, 20.3 mmol) were dissolved in 1,4-dioxane (25 mL) and DIEA (5.2 mL, 30.5 mmol) was
added. The reaction mixture was heated to reflux for 10 h and then cooled to room temperature.
After concentration, the residue was dissolved in EtOAc (50 mL), washed with H2O (20 mL×3)
and brine (20 mL×3), dried over Na2SO4, and concentrated in vacuo. The residue was purified by
chromatography on silica gel (petroleum ether : EtOAc = 10:1) to give 14 (6.2 g, 75.8%) as a
10

ACCEPTED MANUSCRIPT
white solid. mp 127.1-131.1°C. 1H NMR (600 MHz, DMSO-d6) δ: 8.41 (s, 2H), 8.18 (t, J = 6.3
Hz, 1H), 7.98 (d, J = 3.8 Hz, 3H), 4.64 (d, J = 6.3 Hz, 2H). MS (ESI) m/z 398.0[M-H]-.
4.11. N-(3,5-Bis(trifluoromethyl)benzyl)-5-bromo-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-di
methylcyclohex-1-enyl)methyl)pyrimidin-2-amine (15).
NaH (20 mg, 0.5 mmol, 60% in oil) was added to a solution of intermediate 14 (120.0 mg,
0.3 mmol) in DMF (2 mL) cooled to 0°C. After stirring at 0°C for 30 min, a solution of
intermediate 12 (92.1 mg, 0.3 mmol) in DMF (2 mL) was added. The reaction mixture was
allowed to warm to room temperature and stirred for 30 min, and then it was poured onto crushed
ice. The mixture was diluted with EtOAc (15 mL), and the layers were separated. The aqueous
layer was extracted with EtOAc (5 mL×3), and the combined organic layers were washed with
H2O (10 mL×3) and brine (10 mL×3), dried over Na2SO4, and concentrated in vacuo. The residue
was purified by chromatography on silica gel (petroleum ether : EtOAc = 20:1) to give 15 (129.9
mg, 64.7%) as a white solid. mp 120.1-123.7°C. 1H NMR (600 MHz, CDCl3) δ: 8.26 (s, 2H), 7.64
(s, 1H), 7.47 (s, 2H), 7.01 (dd, J = 8.5 Hz, 2.3 Hz, 1H), 6.75 (d, J = 2.2 Hz, 1H), 6.69 (d, J = 8.4
Hz, 1H), 4.76-4.73 (m, 1H), 4.56-4.50 (m, 2H), 4.01-3.99 (m, 1H), 3.67 (s, 3H), 2.76-2.70 (m, 1H),
2.48-2.45 (m, 1H), 2.11-2.08 (m, 1H), 1.80-1.71 (m, 2H), 1.45-1.40 (m, 2H), 1.12 (d, J = 6.8 Hz,
6H), 0.95 (d, J = 10.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ: 160.43, 157.86(×4), 154.23,
141.38, 140.93, 135.16, 131.35, 131.01, 130.68, 128.25(×2), 128.05(×2), 127.81, 125.71(×2),
110.74, 55.33, 48.96, 47.62, 40.39, 35.54, 33.12, 29.17, 29.09, 28.23, 28.12, 24.05(×2). HRMS
calcd for C32H35BrF6N3O, [M+H]+, 670.1862; found 670.1847. HPLC: tR = 13.921 min, 99.30%.
4.12. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)-5-(piperazin-1-yl)pyrimidin-2-amine (16a).
Under an argon atmosphere, intermediate 15 (133.8 mg, 0.2 mmol) was dissolved in toluene
(2 mL), and N-Boc-piperazine (55.9 mg, 0.3 mmol), NaOt-Bu (28.8 mg, 0.3 mmol), Pd2(dba)3 (9.2
mg, 0.01 mmol), and 2-(di-tert-butylphosphino)biphenyl (6.0 mg, 0.02 mmol) were added. The
reaction mixture was heated to reflux for 4 h and then cooled to room temperature and poured into
saturated sodium bicarbonate solution. The mixture was diluted with EtOAc (5 mL), and the
layers were separated. The aqueous layer was extracted with EtOAc (5 mL×3), and the combined
organic layers were washed with H2O (5 mL×3) and brine (5 mL×3), dried over Na2SO4, and
concentrated in vacuo. The residue was immediately dissolved in saturated trifluoroacetic acid–
CH2Cl2 (1:1) solution (2 mL) and stirred at room temperature overnight. After concentration, the
residue was dissolved in EtOAc (5 mL), washed with H2O (5 mL×3) and brine (5 mL×3), dried
over Na2SO4, and concentrated in vacuo. The residue was purified by chromatography on silica
gel (petroleum ether : EtOAc = 2:1) to give 16a (65.3 mg, 48.3%) as a yellow solid. mp
61.8-64.2°C. 1H NMR (400 MHz, DMSO-d6) δ: 9.45 (brs, 1H), 8.22 (s, 2H), 7.87 (s, 1H), 7.59 (s,
2H), 6.97 (dd, J = 8.4 Hz, 1.9 Hz, 1H), 6.74-6.73 (m, 2H), 4.75-4.62 (m, 2H), 4.30-4.16 (m, 2H),
3.63 (s, 3H), 3.25 (t, J = 3.1 Hz, 4H), 3.19 (t, J = 2.2 Hz, 4H), 2.66-2.60 (m, 1H), 2.37-2.33 (m,
1H), 2.04-2.00 (m, 1H), 1.75 (s, 2H), 1.37 (t, J = 6.1 Hz, 2H), 1.00 (d, J = 6.9 Hz, 6H), 0.91 (d, J
= 3.9 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ: 157.88, 154.53, 148.29(×2), 143.40, 140.51,
136.41, 133.97, 130.61(×2), 130.30, 128.57, 127.75, 127.59(×2), 125.73, 125.08, 122.37, 120.61,
11

ACCEPTED MANUSCRIPT
111.33, 55.75, 52.51, 46.82(×2), 42.97(×2), 35.52, 32.81, 29.27, 29.13, 28.62, 28.17, 24.28(×2),
24.21(×2). HRMS calcd for C36H44F6N5O, [M+H]+, 676.3445; found 676.3436. HPLC: tR =
25.670 min, 95.99%.
4.13. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)-5-(4-methylpiperazin-1-yl)pyrimidin-2-amine (16b).
Colourless oil; yield 43.5%; 1H NMR (600 MHz, CDCl3) δ: 8.07 (s, 2H), 7.62 (s, 1H), 7.49 (s,
2H), 6.98 (dd, J = 8.4 Hz, 2.1 Hz, 1H), 6.75 (d, J = 2.1 Hz, 1H), 6.66 (d, J = 8.5 Hz, 1H),
4.77-4.74 (m, 1H), 4.55-4.52 (m, 2H), 4.02-4.00 (m, 1H), 3.65 (s, 3H), 3.06 (t, J = 4.6 Hz, 4H),
2.71 (t, J = 6.9 Hz, 1H), 2.60 (d, J = 4.0 Hz, 4H), 2.47-2.44 (m, 1H), 2.35 (s, 3H), 2.09-2.06 (m,
1H), 1.79 (d, J = 7.5 Hz, 2H), 1.44-1.38 (m, 2H), 1.11 (d, J = 6.9 Hz, 6H), 0.93 (d, J = 12.2 Hz,
6H). 13C NMR (150 MHz, CDCl3) δ: 158.19, 154.27, 148.41(×2), 142.43, 140.78, 136.48, 134.47,
130.88, 128.68, 128.18(×2), 127.62(×2), 125.48(×2), 124.32, 122.51, 120.25, 110.64, 55.28,
54.83(×2), 50.25(×2), 48.76, 47.61, 45.77, 40.36, 35.61, 33.08, 29.12, 29.07, 28.28, 28.03,
24.03(×2). HRMS calcd for C37H46F6N5O, [M+H]+, 690.3601; found 690.3632. HPLC: tR =
28.113 min, 97.71%.
4.14. 5-(4-Aminopiperidin-1-yl)-N-(3,5-bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxy
phenyl)-5,5-dimethylcyclohex-1-enyl)methyl)pyrimidin-2-amine (16c).
1
Red oil; yield 53.1%; H NMR (400 MHz, DMSO-d6) δ: 8.35 (s, 2H), 8.14 (s, 2H), 7.85 (s,
1H), 7.57 (s, 2H), 6.94 (dd, J = 6.4 Hz, 1.2 Hz, 1H), 6.73 (d, J = 6.4 Hz, 1H), 6.71 (d, J = 1.2 Hz,
1H), 4.70-4.60 (m, 2H), 4.25-4.13 (m, 2H), 3.60 (s, 3H), 3.40 (d, J = 8.0 Hz, 2H), 2.90-2.84 (m,
1H), 2.62-2.60 (m, 2H), 2.59 (s, 1H), 2.34-2.31 (m, 1H), 2.01-1.98 (m, 1H), 1.86 (d, J = 7.2 Hz,
2H), 1.72 (s, 2H), 1.51-1.49 (m, 2H), 1.34 (t, J = 4.3 Hz, 2H), 0.98 (d, J = 4.6 Hz, 6H), 0.88 (d, J =
4.6 Hz, 6H).13C NMR (150 MHz, DMSO-d6) δ: 157.47, 154.54, 148.27(×2), 143.56, 140.53,
137.17, 133.85, 130.68, 130.56, 130.34, 128.68, 127.75, 127.58(×2), 125.72, 124.63, 122.82,
120.54, 111.38, 55.77, 48.48(×2), 47.74(×2), 45.80(×2), 35.54, 32.79, 29.71(×2), 29.11(×2), 28.62,
28.17, 24.27, 24.20. HRMS calcd for C37H46F6N5O, [M+H]+, 690.3601; found 690.3611. HPLC:
tR = 26.614 min, 96.55%.
4.15. N-(1-(2-((3,5-Bis(trifluoromethyl)benzyl)((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethyl
cyclohex-1-enyl)methyl)amino)pyrimidin-5-yl)piperidin-4-yl)acetamide (16d).
1
Brown solid; yield 46.1%; mp 143.5-147.3°C. H NMR (600 MHz, CDCl3) δ: 8.06 (s, 2H),
7.61 (s, 1H), 7.48 (s, 2H), 6.97 (dd, J = 8.5 Hz, 2.2 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 6.66 (d, J =
8.4 Hz, 1H), 5.71 (d, J = 7.8 Hz, 1H), 4.76-4.73 (m, 1H), 4.54-4.51 (m, 2H), 4.01-3.99 (m, 1H),
3.90-3.84 (m, 1H), 3.65 (s, 3H), 3.31-3.29 (m, 2H), 2.78 (t, J = 11.8 Hz, 2H), 2.70 (t, J = 6.9 Hz,
1H), 2.47-2.44 (m, 1H), 2.08-2.06 (m, 1H), 2.03 (d, J = 13.0 Hz, 2H), 1.97 (s, 3H), 1.78 (d, J = 8.2
Hz, 2H), 1.58-1.56 (m, 2H), 1.42-1.39 (m, 2H), 1.10 (d, J = 7.0 Hz, 6H), 0.92 (d, J = 12.0 Hz, 6H).
13C NMR (150 MHz, CDCl3) δ: 169.52, 158.21, 154.26, 148.85(×2), 142.33, 140.79, 134.57,
131.10, 130.85, 128.58, 128.17(×2), 127.62(×2), 125.50(×2), 124.31, 122.50, 120.28, 110.65,
55.28(×2), 50.55, 48.79, 47.60, 46.00, 40.35, 35.59, 33.08, 31.98, 29.11, 29.06, 28.27, 28.02,
12

ACCEPTED MANUSCRIPT
24.02(×2), 23.44. HRMS calcd for C39H48F6N5O2, [M+H]+, 732.3707; found 732.3714. HPLC: tR
= 29.532 min, 97.18%.
4.16. 1-(2-((3,5-Bis(trifluoromethyl)benzyl)((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)amino)pyrimidin-5-yl)piperidine-4-carboxylic acid (16e).
Brown solid; yield 39.3%; mp 72.0-75.5°C. 1H NMR (600 MHz, DMSO-d6) δ: 8.14 (s, 2H),
7.87 (s, 1H), 7.56 (s, 2H), 6.95 (dd, J = 8.5 Hz, 2.2 Hz, 1H), 6.74-6.71 (m, 2H), 4.67-4.63 (m, 2H),
4.21-4.13 (m, 2H), 3.61 (s, 3H), 2.64-2.59 (m, 3H), 2.33-2.28 (m, 2H), 2.01-1.98 (m, 1H),
1.88-1.86 (m, 2H), 1.72 (s, 2H), 1.65-1.62 (m, 2H), 1.34 (t, J = 6.5 Hz, 2H), 1.22 (s, 2H), 0.99 (d,
J = 6.9 Hz, 6H), 0.89 (d, J = 8.0 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ: 179.68, 158.02, 154.26,
148.83(×2), 142.39, 140.78, 137.08, 134.53, 131.14, 130.86, 130.81, 128.64, 128.19(×2), 127.57,
125.49, 124.78, 122.07, 120.63, 110.62, 55.29(×2), 50.79, 50.71, 48.76, 47.57, 40.31, 35.61,
33.09(×2), 29.08(×2), 28.29, 28.05(×2), 24.06(×2). HRMS calcd for C38H43F6N4O3, [M-H]-,
717.3239; found 717.3205. HPLC: tR = 22.183 min, 95.13%.
4.17. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)-5-morpholinopyrimidin-2-amine (16f).
1
Colourless oil; yield 46.0%; H NMR (600 MHz, DMSO-d6) δ: 8.10 (s, 2H), 7.63 (s, 1H),
7.48 (s, 2H), 6.98 (dd, J = 8.4 Hz, 2.3 Hz, 1H), 6.75 (d, J = 2.3 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H),
4.82-4.76 (m, 1H), 4.57-4.53 (m, 2H), 4.05-4.02 (m, 1H), 3.86 (t, J = 4.6 Hz, 4H), 3.66 (s, 3H),
3.02 (t, J = 4.6 Hz, 4H), 2.76-2.69 (m, 1H), 2.50-2.42 (m, 1H), 2.10-2.04 (m, 1H), 1.80-1.78 (m,
2H), 1.43 (s, 2H), 1.12 (d, J = 6.9 Hz, 6H), 0.94 (d, J = 8.0 Hz, 6H). 13C NMR (100 MHz, CDCl3)
δ: 154.25(×2), 140.82(×2), 134.72, 131.19, 130.86, 130.80, 128.48, 128.19(×2), 127.60(×2),
125.53(×2), 124.76, 122.05, 120.35, 110.64(×2), 66.76(×2), 55.31, 50.76, 48.85, 47.60, 40.35,
35.59, 33.10(×2), 29.12, 29.09, 28.29, 28.05, 24.07, 24.03. HRMS calcd for C36H43F6N4O2,
[M+H]+, 677.3285; found 677.3272. HPLC: tR = 23.472 min, 97.80%.
4.18. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (17a).
Under an argon atmosphere, intermediate 15 (0.2 g, 0.3 mmol), 1-methylpyrazole-4-boronic
acid pinacol ester (0.1 g, 0.5 mmol), Pd(PPh3)4 (34.7 mg, 0.03 mmol), Na2CO3 (63.6 mg, 0.6
mmol), DME (5 mL), and H2O (0.5 mL) were added to a three-necked bottle containing a stirring
bar. After being stirred at 90°C for 12 h, the reaction mixture was cooled to room temperature, and
H2O (20 mL) was added. The aqueous layer was extracted with EtOAc (10 mL×3), and the
combined organic layers were washed with H2O (10 mL×3) and brine (10 mL×3), dried over
Na2SO4, and concentrated in vacuo. The residue was purified by chromatography on silica gel
(petroleum ether : EtOAc = 2:1) to give 17a (125.6 mg, 62.8%) as a white solid. mp 45.4-49.1°C.
1H NMR (600 MHz, DMSO-d6) δ: 8.58 (s, 2H), 8.06 (s, 1H), 7.89 (s, 1H), 7.80 (s, 1H), 7.60 (s,
2H), 6.96 (dd, J = 8.4 Hz, 1.8 Hz, 1H), 6.76-6.74 (m, 2H), 4.78-4.66 (m, 2H), 4.32-4.20 (m, 2H),
3.84 (s, 3H), 3.62 (s, 3H), 2.64-2.59 (m, 1H), 2.35-2.32 (m, 1H), 2.03-2.00 (m, 1H), 1.74 (s, 2H),
1.36 (t, J = 6.4 Hz, 2H), 0.99-0.98 (m, 6H), 0.91 (d, J = 2.9 Hz, 6H). 13C NMR (150 MHz,
13

ACCEPTED MANUSCRIPT
DMSO-d6) δ: 160.37, 154.50(×2), 154.41, 142.63, 140.20, 135.40(×2), 133.76, 130.20, 130.02,
127.98, 127.35, 127.23(×2), 126.89, 125.40, 124.21, 122.41, 120.35, 116.05, 115.96, 111.03,
55.42, 48.18, 47.28, 38.69, 35.12, 32.41(×2), 28.92, 28.74, 28.23, 27.80, 23.89, 23.82. HRMS
calcd for C36H40F6N5O, [M+H]+, 672.3132; found 672.3164. HPLC: tR = 8.010 min, 98.72%.
4.19. N-(3,5-bis(trifluoromethyl)benzyl)-5-(3,5-dimethylisoxazol-4-yl)-N-((2-(5-isopropyl-2-
methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)pyrimidin-2-amine (17b).
Pale yellow oil; yield 58.3%; 1H NMR (600 MHz, CDCl3) δ: 8.21 (s, 2H), 7.65 (s, 1H), 7.51
(s, 2H), 7.01 (dd, J = 8.5 Hz, 2.2 Hz, 1H), 6.78 (d, J = 2.2 Hz, 1H), 6.70 (d, J = 8.5 Hz, 1H),
4.85-4.82 (m, 1H), 4.67-4.64 (m, 1H), 4.61-4.58 (m, 1H), 4.08-4.06 (m, 1H), 3.67 (s, 3H),
2.76-2.72 (m, 1H), 2.50-2.47 (m, 1H), 2.39 (s, 3H), 2.25 (s, 3H), 2.13-2.10 (m, 1H), 1.88-1.79 (m,
2H), 1.47-1.42 (m, 2H), 1.13 (d, J = 6.9 Hz, 6H), 0.97 (d, J = 10.6 Hz, 6H). 13C NMR (150 MHz,
CDCl3) δ: 154.85(×2), 154.23, 141.45, 140.95, 138.01, 135.25, 131.30, 131.08, 130.70,
128.15(×2), 128.09(×2), 127.74(×2), 125.68(×2), 124.39, 122.38, 120.60, 117.85, 110.76,
55.35(×2), 48.95, 47.59, 40.43, 35.55, 33.09(×2), 29.18(×2), 28.25(×2), 24.04(×2). HRMS calcd
for C37H41F6N4O2, [M+H]+, 687.3128; found 687.3128. HPLC: tR = 8.026 min, 99.84%.
4.20. N-(3,5-bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)-5-(1-methyl-1H-pyrazol-5-yl)pyrimidin-2-amine (17c).
1
White solid; yield 66.9%; mp 97.8-102.8°C. H NMR (600 MHz, CDCl3) δ: 8.35 (s, 2H),
7.66 (s, 1H), 7.53 (d, J = 1.9 Hz, 1H), 7.51 (s, 2H), 7.02 (dd, J = 8.3 Hz, 2.2 Hz, 1H), 6.78 (d, J =
2.2 Hz, 1H), 6.71 (d, J = 8.5 Hz, 1H), 6.29 (d, J = 1.8 Hz, 1H), 4.87-4.84 (m, 1H), 4.68-4.66 (m,
1H), 4.62-4.59 (m, 1H), 4.09-4.07 (m, 1H), 3.88 (s, 3H), 3.68 (s, 3H), 2.75-2.72 (m, 1H),
2.50-2.47 (m, 1H), 2.13-2.10 (m, 1H), 1.86-1.78 (m, 2H), 1.47-1.42 (m, 2H), 1.13 (d, J = 6.9 Hz,
6H), 0.97 (d, J = 9.8 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ: 161.56, 157.07(×2), 154.23, 140.96,
138.70, 138.21, 135.35, 131.08, 130.68, 128.12(×2), 127.82(×2), 125.73(×2), 124.24, 122.43,
120.62, 113.43, 110.79, 106.00(×2), 55.34, 48.84, 47.53, 40.46, 37.31, 35.55, 33.10, 29.18, 29.10,
28.23, 28.08, 24.02(×2). HRMS calcd for C36H40F6N5O, [M+H]+, 672.3132; found 672.3130.
HPLC: tR = 7.777 min, 99.74%.
4.21. N-(3,5-bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)-5-(thiophen-2-yl)pyrimidin-2-amine (17d).
White solid; yield 62.1%; mp 53.2-57.3°C. 1H NMR (600 MHz, CDCl3) δ: 8.53 (s, 2H), 7.65
(s, 1H), 7.50 (s, 2H), 7.27 (s, 1H), 7.17 (d, J = 3.5 Hz, 1H), 7.09-7.07 (m, 1H), 7.00 (dd, J = 8.4
Hz, 2.2 Hz, 1H), 6.77 (d, J = 2.2 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 4.88-4.86 (m, 1H), 4.67-4.65
(m, 1H), 4.61-4.59 (m, 1H), 4.10-4.07 (m, 1H), 3.68 (s, 3H), 2.76-2.72 (m, 1H), 2.49-2.46 (m, 1H),
2.12-2.09 (m, 1H), 1.86-1.75 (m, 2H), 1.45-1.41 (m, 2H), 1.13 (d, J = 6.9 Hz, 6H), 0.96 (d, J =
10.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ: 161.25, 154.99(×2), 154.24, 141.66, 140.90, 138.34,
135.06, 131.30, 130.98, 130.73, 128.18(×2), 128.08(×2), 127.77, 125.66(×2), 124.23(×2),
122.21(×2), 117.80, 110.71, 55.34, 48,75, 47.51, 40.38, 35.57, 33.12, 29.16, 29.10, 28.26, 28.10,
24.06(×2). HRMS calcd for C36H38F6N3OS, [M+H]+, 674.2634; found 674.2657. HPLC: tR =
14

ACCEPTED MANUSCRIPT
14.710 min, 99.27%.
4.22. 2-((3,5-Bis(trifluoromethyl)benzyl)((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-
1-enyl)methyl)amino)pyrimidin-5-ol (18).
Under an argon atmosphere, intermediate 15 (3.6 g, 5.4 mmol), PdCl2(dppf)-CH2Cl2 (0.9 g,
1.1 mmol), AcOK (1.6 g, 16.2 mmol), bis(pinacolato) diboron (2.7 g, 10.8 mmol), and DMSO (40
mL) were added to a three-necked bottle containing a stirring bar. After being stirred at 80°C for 1
h, the reaction mixture was cooled to room temperature, and H2O (200 mL) was added. The
aqueous layer was extracted with EtOAc (50 mL×3), and the combined organic layers were
washed with H2O (50 mL×3) and brine (50 mL×3), dried over NaSO4, and concentrated in vacuo.
30% H2O2 (15 mL) was added to a solution of the residue in THF (100 mL) cooled to 0°C, and the
resultant mixture was stirred at 0ꢁ for 1 h prior to the addition of saturated sodium thiosulfate
solution (10 mL). The mixture was extracted with EtOAc (20 mL×3), and the combined organic
layers were washed with H2O (20 mL×3) and brine (20 mL×3), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether :
EtOAc = 2:1) to give 18 (1.5 g, 46.4%) as a white solid. mp 43.8-47.4°C. 1H NMR (600 MHz,
DMSO-d6) δ: 9.18 (s, 1H), 7.98 (s, 2H), 7.86 (s, 1H), 7.56 (s, 2H), 6.96 (dd, J = 8.3 Hz, 2.0 Hz,
1H), 6.75-6.72 (m, 2H), 4.68-4.58 (m, 2H), 4.21-4.11 (m, 2H), 3.62 (s, 3H), 2.64-2.60 (m, 1H),
2.34-2.31 (m, 1H), 2.01-1.98 (m, 1H), 1.72 (s, 2H), 1.35 (t, J = 6.4 Hz, 2H), 1.00 (d, J = 6.9 Hz,
6H), 0.89 (d, J = 7.4 Hz, 6H). 13C NMR (150 MHz, DMSO) δ: 156.61, 154.18, 145.47(×2), 143.72,
143.24, 140.15, 133.25, 130.35, 130.14, 129.93, 128.44, 127.39, 127.23(×2), 125.33, 124.27,
122.46, 120.15, 111.01, 55.41(×2), 48.47, 47.55, 35.17, 32.42, 28.91, 28.74, 28.25, 27.80, 23.92,
23.85. HRMS calcd for C32H36F6N3O2, [M+H]+, 608.2706; found 608.2707. HPLC: tR = 6.600min,
97.53%.
4.23. Methyl2-(2-((3,5-bis(trifluoromethyl)benzyl)((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethyl
cyclohex-1-enyl)methyl)amino)pyrimidin-5-yloxy)acetate (19a).
Intermediate 18 (0.1 g, 0.2 mmol) and methyl bromoacetate (45.9 mg, 0.3 mmol) were
dissolved in DMF (2 mL), followed by the addition of K2CO3 (41.5 mg, 0.3 mmol). After being
stirred at room temperature for 2 h, the reaction mixture was poured into H2O (10 mL) and
extracted with EtOAc (5 mL×3), and the combined organic layers were washed with H2O (5
mL×3) and brine (5 mL×3), dried over NaSO4, and concentrated in vacuo. The residue was
purified by chromatography on silica gel (petroleum ether : EtOAc = 8:1) to give 19a (101.8 mg,
74.9%) as a colourless oil. 1H NMR (600 MHz, DMSO-d6) δ: 8.13 (s, 2H), 7.82 (s, 1H), 7.51 (s,
2H), 6.90 (dd, J = 8.5 Hz, 2.2 Hz, 1H), 6.69-6.66 (m, 2H), 4.72 (s, 2H), 4.65-4.55 (m, 2H),
4.20-4.09 (m, 2H), 3.61 (s, 3H), 3.55 (s, 3H), 2.57-2.53 (m, 1H), 2.28-2.25 (m, 1H), 1.96-1.93 (m,
1H), 1.67 (s, 2H), 1.30 (t, J = 6.4 Hz, 2H), 0.93 (d, J = 6.9 Hz, 6H), 0.84 (d, J = 6.9 Hz, 6H). 13C
NMR (150 MHz, DMSO-d6) δ: 169.15, 157.71, 154.13, 146.15(×2), 144.84, 142.86, 140.15,
133.59, 130.19, 128.11, 127.34(×2), 127.16(×2), 125.36(×2), 124.22, 122.41, 120.25, 110.98,
66.20, 55.38, 51.82(×2), 48.55, 47.55, 35.13, 32.40, 28.90, 28.74, 28.23, 27.78, 23.88, 23.82.
HRMS calcd for C35H40F6N3O4, [M+H]+, 680.2918; found 680.2919. HPLC: tR = 6.696 min,
98.50%.
15

ACCEPTED MANUSCRIPT
4.24. 2-(2-((3,5-Bis(trifluoromethyl)benzyl)((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)amino)pyrimidin-5-yloxy)acetic acid (19b).
1
Colourless oil; yield 84.3%; H NMR (600 MHz, DMSO-d6) δ: 12.95 (s, 1H), 8.18 (s, 2H),
7.87 (s, 1H), 7.58 (s, 2H), 6.96 (dd, J = 8.4 Hz, 2.2 Hz, 1H), 6.76-6.73 (m, 2H), 4.72-4.62 (m, 1H),
4.26-4.24 (m, 1H), 4.17-4.15 (m, 1H), 3.62 (s, 3H), 2.65-2.60 (m, 1H), 2.35-2.32 (m, 1H),
2.03-2.01 (m, 1H), 1.74 (s, 2H), 1.36 (t, J = 6.2 Hz, 2H), 1.00 (d, J = 6.9 Hz, 6H), 0.91 (d, J = 7.1
Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ: 170.50, 158.00, 154.54, 146.40(×2), 145.35, 143.30,
140.57, 133.96, 130.65, 128.55, 127.75(×2), 127.58(×2), 125.77(×2), 124.63, 122.82, 120.64,
111.40, 66.45, 55.80(×2), 48.95, 47.96, 35.54, 32.80, 29.31, 29.15, 28.63, 28.19, 24.29, 24.23.
HRMS calcd for C34H36F6N3O4, [M-H]+, 664.2610; found 664.2579. HPLC: tR = 17.043 min,
99.35%.
4.25. 4-(2-((3,5-Bis(trifluoromethyl)benzyl)((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)amino)pyrimidin-5-yloxy)butanoic acid (19c).
1
Colourless oil; yield 72.1%; H NMR (600 MHz, DMSO-d6) δ: 12.11 (s, 1H), 8.16 (s, 2H),
7.87 (s, 1H), 7.58 (s, 2H), 6.96 (dd, J = 8.5 Hz, 2.1 Hz, 1H), 6.76-6.73 (m, 2H), 4.72-4.62 (m, 2H),
4.25-4.15 (m, 2H), 3.96 (t, J = 6.4 Hz, 2H), 3.62 (s, 3H), 2.63-2.60 (m, 1H), 2.37-2.34 (m, 2H),
2.32 (s, 1H), 2.03 (s, 1H), 2.01-1.86 (m, 2H), 1.73 (s, 2H), 1.36 (t, J = 6.4 Hz, 2H), 1.00 (d, J = 7.0
Hz, 6H), 0.91 (d, J = 6.7 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ: 178.34, 154.25, 145.66, 145.35,
142.26, 140.80, 134.59, 131.17, 130.84(×2), 128.58, 128.19(×2), 127.56, 125.51(×2), 124.76,
122.06, 120.27, 110.63(×2), 68.59, 55.30, 49.00, 47.73, 40.35, 35.59, 33.10, 30.22, 29.13, 29.08,
28.28, 28.05, 24.45, 24.06, 24.02. HRMS calcd for C36H40F6N3O4, [M-H]+, 692.2923; found
692.2943. HPLC: tR = 17.508 min, 99.67%.
4.26. 2-(2-((3,5-Bis(trifluoromethyl)benzyl)((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)amino)pyrimidin-5-yloxy)ethanol (19d).
1
White solid; yield 56.5%; mp 85.5-89.9°C. H NMR (600 MHz, DMSO-d6) δ: 8.18 (s, 2H),
7.88 (s, 1H), 7.57 (s, 2H), 6.96 (dd, J = 8.5 Hz, 2.1 Hz, 1H), 6.76-6.73 (m, 2H), 4.88 (t, J = 5.5 Hz,
1H), 4.72-4.62 (m, 2H), 4.25-4.15 (m, 2H), 3.98 (t, J = 4.8 Hz, 2H), 3.66 (t, J = 4.9 Hz, 2H), 3.62
(s, 3H), 2.64-2.59 (m, 1H), 2.35-2.32 (m, 1H), 2.03-2.00 (m, 1H), 1.74 (s, 2H), 1.36 (t, J = 6.4 Hz,
2H), 1.00 (d, J = 6.9 Hz, 6H), 0.91 (d, J = 6.7 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ: 158.22,
154.27, 145.86(×2), 145.34, 142.28, 140.81, 134.56, 130.85(×2), 128.60, 128.19(×2), 127.63,
125.53(×2), 124.77, 122.06, 120.32, 110.64, 71.34, 61.43, 55.30, 48.97, 47.74, 40.36, 35.59, 33.10,
29.13, 29.08, 28.28, 28.06, 24.06, 24.03. HRMS calcd for C34H40F6N3O3, [M+H]+, 652.2968;
found 652.2968. HPLC: tR = 14.536 min, 98.18%.
4.27. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclo
hex-1-enyl)methyl)-5-(2-methoxyethoxy)pyrimidin-2-amine (19e).
1
Colourless oil; yield 68.7%; H NMR (600 MHz, DMSO-d6) δ: 8.18 (s, 2H), 7.88 (s, 1H),
16

ACCEPTED MANUSCRIPT
7.58 (s, 2H), 6.96 (dd, J = 8.5 Hz, 2.2 Hz, 1H), 6.75-6.73 (m, 2H), 4.72-4.62 (m, 2H), 4.25-4.15
(m, 2H), 4.09-4.07 (m, 2H), 3.62 (s, 3H), 3.61-3.59 (m, 2H), 3.27 (s, 3H), 2.63-2.61 (m, 1H),
2.35-2.32 (m, 1H), 2.03-1.99 (m, 1H), 1.73 (s, 2H), 1.36 (t, J = 6.4 Hz, 2H), 1.00 (d, J = 6.9 Hz,
6H), 0.91 (d, J = 7.1 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ: 157.82, 154.54, 146.17(×2),
145.90, 143.37, 140.55, 133.88, 130.65, 130.36, 128.60, 127.75(×2), 127.55(×2), 125.75, 124.63,
122.83, 120.62, 111.39, 70.91, 69.04, 58.54(×2), 55.80, 48.88, 47.92, 35.54, 32.80, 29.30, 29.14,
28.65, 28.18, 24.30, 24.23. HRMS calcd for C35H42F6N3O3, [M+H]+, 666.3125; found 666.3136.
HPLC: tR = 8.043 min, 96.90%.
4.28. N-(3,5-bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)cyclopent-1-enyl)
methyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (26a).
The title compound was obtained in a manner similar to that described for the preparation of
1
17a. Colourless oil; yield 43.8%; H NMR (600 MHz, CDCl3) δ: 8.43 (s, 2H), 7.67 (s, 2H),
7.54-7.53 (m, 3H), 7.07 (dd, J = 8.3 Hz, 1.6 Hz, 1H), 6.85 (d, J = 1.7 Hz, 1H), 6.81 (d, J = 8.4 Hz,
1H), 4.69 (s, 2H), 4.41 (s, 2H), 3.95 (s, 3H), 3.75 (s, 3H), 2.78-2.76 (m, 1H), 2.74-2.71 (m, 2H),
2.39 (t, J = 7.1 Hz, 2H), 1.89-1.84 (m, 2H), 1.13 (d, J = 6.9 Hz, 6H). 13C NMR (150 MHz, CDCl3)
δ: 160.93, 155.15, 154.88(×2), 141.21, 136.07(×2), 135.33, 133.63, 131.33, 129.80, 127.65,
127.11, 126.61, 126.57, 126.06, 125.42, 124.25, 122.44, 120.57, 117.23, 115.91, 111.00, 55.50(×2),
48.73, 48.17, 39.12, 33.09, 28.08, 25.07, 24.04(×2). HRMS calcd for C33H34F6N5O, [M+H]+,
630.2662; found 630.2658. HPLC: tR = 6.493 min, 97.66%.
4.29. N-(3,5-bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)cyclohex-1-enyl)
methyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (26b).
Colourless oil; yield 51.3%; 1H NMR (600 MHz, CDCl3) δ: 8.39 (s, 2H), 7.66-7.64 (m, 2H),
7.59 (s, 2H), 7.51 (s, 1H), 7.02 (dd, J = 8.4 Hz, 2.2 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 6.73 (d, J =
8.4 Hz, 1H), 4.80-4.77 (m, 1H), 4.63-4.58 (m, 2H), 4.07-4.05 (m, 1H), 3.94 (s, 3H), 3.70 (s, 3H),
2.76-2.73 (m, 1H), 2.44-2.41 (m, 1H), 2.09-2.00 (m, 3H), 1.69-1.67 (m, 2H), 1.63-1.61 (m, 2H),
1.13 (d, J = 6.9 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ: 161.00, 154.75(×2), 154.23, 141.98,
140.84, 136.04(×2), 135.84, 131.07(×2), 129.63, 128.22, 127.97(×2), 125.99, 125.62, 124.32,
122.50, 120.50, 117.34, 115.54, 110.78, 55.28, 48.93, 48.06, 39.07, 33.10, 31.26, 26.70, 24.03(×2),
22.98, 22.58. HRMS calcd for C34H36F6N5O, [M+H]+, 644.2819; found 644.2830. HPLC: tR =
6.735 min, 95.77%.
4.30. N-(3,5-bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)cyclohept-1-enyl)
methyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (26c).
Colourless oil; yield 48.5%; 1H NMR (600 MHz, CDCl3) δ: 8.40 (s, 2H), 7.65-7.64 (m, 2H),
7.54 (s, 2H), 7.51 (s, 1H), 6.99 (dd, J = 8.4 Hz, 2.2 Hz, 1H), 6.74 (d, J = 2.2 Hz, 1H), 6.71 (d, J =
8.4 Hz, 1H), 4.86-4.84 (m, 1H), 4.81-4.78 (m, 1H), 4.59-4.56 (m, 1H), 3.94 (d, J = 3.6 Hz, 3H),
3.91-3.89 (m, 1H), 3.72 (s, 3H), 2.75-2.73 (m, 1H), 2.50-2.46 (m, 1H), 2.34-2.30 (m, 1H),
2.15-2.14 (m, 2H), 1.74-1.71 (m, 3H), 1.51-1.49 (m, 3H), 1.13 (dd, J = 6.8 Hz, 2.6 Hz, 6H). 13C
NMR (150 MHz, CDCl3) δ: 160.98, 154.82(×2), 154.11, 142.91, 141.88, 140.53, 136.03(×2),
17

ACCEPTED MANUSCRIPT
135.48, 132.47, 131.08, 130.87, 128.33, 128.01, 126.01, 125.42, 124.32, 122.51, 120.39, 117.33,
115.57, 110.69, 55.22, 49.21, 47.59, 39.10, 35.97, 33.11, 32.69, 30.76, 26.43, 24.85, 24.05(×2).
HRMS calcd for C35H38F6N5O, [M+H]+, 658.2975; found 658.2970. HPLC: tR = 7.483 min,
96.73%.
4.31. N-(3,5-bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5-methylcyclohex-
1-enyl)methyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (26d).
Colourless oil; yield 46.8%; 1H NMR (600 MHz, CDCl3) δ: 8.40 (s, 2H), 7.65 (s, 2H), 7.58 (s,
2H), 7.52 (s, 1H), 7.01 (t, J = 8.6 Hz, 1H), 6.78 (s, 1H), 6.73 (dd, J = 8.4 Hz, 3.8 Hz, 1H),
4.85-4.64 (m, 2H), 4.56-4.54 (m, 1H), 4.15-3.99 (m, 1H), 3.94 (s, 3H), 3.69 (s, 3H), 2.77-2.72 (m,
1H), 2.54-2.40 (m, 1H), 2.12-2.05 (m, 2H), 1.72-1.62 (m, 4H), 1.13 (t, J = 7.5 Hz, 6H), 0.93 (d, J
= 5.5 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ: 160.93, 154.83(×2), 154.31, 141.99, 140.80,
136.04, 135.70, 131.19, 130.79, 129.07, 128.19, 127.95(×2), 126.00(×2), 125.67, 124.31, 122.50,
120.46, 117.32, 115.56, 110.78, 55.28, 49.04, 48.00, 39.09, 35.32, 33.10, 31.18, 31.11, 28.70,
24.02(×2), 21.80. HRMS calcd for C35H38F6N5O, [M+H]+, 658.2975; found 658.2982. HPLC: tR =
7.098 min, 98.23%.
4.32. N-(3,5-bis(trifluoromethyl)benzyl)-N-((5-ethyl-2-(5-isopropyl-2-methoxyphenyl)cyclohex-1-
enyl)methyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (26e).
Colourless oil; yield 50.5%; 1H NMR (600 MHz, CDCl3) δ: 8.40 (s, 2H), 7.66-7.65 (m, 2H),
7.61 (d, J = 8.9 Hz, 2H), 7.52 (s, 1H), 7.03-7.00 (m, 1H), 6.79 (s, 1H), 6.73 (d, J = 8.4 Hz, 1H),
4.85-4.67 (m, 2H), 4.57-4.55 (m, 1H), 4.16-3.99 (m, 1H), 3.94 (s, 3H), 3.70 (d, J = 7.3 Hz, 3H),
2.76-2.73 (m, 1H), 2.54-2.40 (m, 1H), 2.16-2.09 (m, 2H), 1.80-1.78 (m, 1H), 1.63-1.58 (m, 1H),
1.46-1.37 (m, 1H), 1.27-1.24 (m, 3H), 1.14 (t, J = 7.2 Hz, 6H), 0.87-0.82 (m, 3H). 13C NMR (150
MHz, CDCl3) δ: 161.07, 154.77(×2), 154.32, 142.10, 140.77, 136.02(×2), 131.17, 130.96, 129.18,
128.23, 127.99(×2), 125.99(×2), 125.68, 124.33, 122.53, 120.47, 117.37, 115.53, 110.75, 55.26,
49.09, 48.00, 39.08, 35.52, 33.12(×2), 31.51, 29.07, 28.65, 24.04(×2), 11.43. HRMS calcd for
C36H40F6N5O, [M+H]+, 672.3132; found 672.3137. HPLC: tR = 7.096 min, 95.18%.
4.33. N-(3,5-bis(trifluoromethyl)benzyl)-N-((5-tert-butyl-2-(5-isopropyl-2-methoxyphenyl)cyclohex
-1-enyl)methyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (26f).
Colourless oil; yield 51.7%; 1H NMR (600 MHz, CDCl3) δ: 8.41 (s, 2H), 7.67-7.66 (m, 4H),
7.53 (s, 1H), 7.02 (t, J = 9.1 Hz, 1H), 6.80-6.79 (m, 1H), 6.74 (t, J = 7.7 Hz, 1H), 4.76-4.55 (m,
3H), 4.16-3.97 (m, 1H), 3.95 (s, 3H), 3.71 (d, J = 2.9 Hz, 3H), 2.77-2.74 (m, 1H), 2.58-2.45 (m,
1H), 2.15-1.95 (m, 2H), 1.84-1.80 (m, 1H), 1.73-1.71 (m, 1H), 1.66 (s, 1H), 1.45-1.31 (m, 1H),
1.14 (t, J = 5.6 Hz, 6H), 0.77 (d, J = 12.6 Hz, 9H). 13C NMR (150 MHz, CDCl3) δ: 161.02,
154.67(×2), 154.37, 142.21, 140.78, 136.03(×2), 131.21, 130.99, 129.78, 128.31, 128.12(×2),
126.02(×2), 125.70, 124.33, 122.52, 120.55, 117.32, 115.54, 110.75, 55.23(×2), 49.77, 48.31,
44.29, 43.81, 39.10, 33.10, 32.80, 32.12, 28.60, 27.13(×2), 24.02(×2). HRMS calcd for
C38H44F6N5O, [M+H]+, 700.3445; found 700.3443. HPLC: tR = 8.819 min, 96.65%.
18

ACCEPTED MANUSCRIPT
4.34. N-(3,5-bis(trifluoromethyl)benzyl)-N-((1-(5-isopropyl-2-methoxyphenyl)-3,4-dihydronaphth
alen-2-yl)methyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (26g).
White solid; yield 53.8%; mp 56.5-60.6°C. 1H NMR (600 MHz, CDCl3) δ: 8.42 (s, 2H), 7.66
(s, 2H), 7.54-7.53 (m, 3H), 7.14-7.09 (m, 3H), 7.04-7.02 (m, 1H), 6.87 (d, J = 1.7 Hz, 1H), 6.81 (d,
J = 8.5 Hz, 1H), 6.59 (d, J = 7.7 Hz, 1H), 4.88-4.86 (m, 1H), 4.79-4.77 (m, 1H), 4.70-4.68 (m, 1H),
4.28-4.25 (m, 1H), 3.95 (s, 3H), 3.59 (s, 3H), 2.81-2.76 (m, 3H), 2.39-2.37 (m, 2H), 1.13 (d, J =
6.8 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ: 160.71, 154.86(×4), 141.79, 140.73, 139.95,
136.05(×2), 135.24, 131.30, 131.08, 128.17(×2), 127.94(×2), 126.48, 126.03(×4), 124.27, 122.46,
120.69, 117.33, 115.69, 110.77(×2), 55.32, 48.49, 44.54, 39.10, 37.71, 34.82, 33.13, 24.03, 22.14.
HRMS calcd for C38H36F6N5O, [M+H]+, 692.2819; found 692.2821. HPLC: tR = 6.323 min,
95.44%.
4.35. N-(3,5-bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-3,4-dihydronaphth
alen-1-yl)methyl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (26h).
White solid; yield 58.0%; mp 70.1-73.3°C. 1H NMR (600 MHz, CDCl3) δ: 8.45 (s, 2H), 7.68
(s, 1H), 7.54 (s, 1H), 7.51 (s, 1H), 7.46-7.45 (m, 1H), 7.27 (s, 2H), 7.11 (dd, J = 8.5 Hz, 2.1 Hz,
1H), 7.06-7.05 (m, 2H), 6.97-6.96 (m, 1H), 6.84 (d, J = 2.1 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H),
5.55-5.52 (m, 1H), 4.83-4.80 (m, 1H), 4.52-4.49 (m, 1H), 4.45-4.42 (m, 1H), 3.95 (s, 3H), 3.80 (s,
3H), 2.83-2.81 (m, 1H), 2.49-2.44 (m, 3H), 2.33-2.31 (m, 1H), 1.21 (d, J = 7.0 Hz, 6H). 13C NMR
(150 MHz, CDCl3) δ: 160.52, 154.83(×2), 154.52, 142.09, 140.98, 140.88, 136.15, 136.04(×2),
133.84, 130.69, 130.47, 130.08, 128.89, 128.33, 127.27, 126.95(×2), 126.33(×2), 126.07, 124.25,
123.85, 122.44, 120.02, 117.22, 115.87, 110.84, 55.37, 48.70, 44.80, 39.11, 33.16, 30.44, 28.07,
24.86, 24.00. HRMS calcd for C38H36F6N5O, [M+H]+, 692.2819; found 692.2813. HPLC: tR =
6.586 min, 97.78%.
4.2. Biology
4.2.1. In vitro test for CETP inhibitory activity
The CETP RP Activity Assay Kit (Catalog #RB-RPAK; Roar) used a donor molecule containing a
fluorescent self-quenched neutral lipid that was transferred to an acceptor by CETP (Catalog
#R8899; Roar). The CETP-mediated transfer of the fluorescence neutral lipid to the acceptor
molecule resulted in an increase in fluorescence (Ex/Em = 465/535 nm). The inhibitor of CETP
inhibited the lipid transfer and thereby decreased the fluorescence intensity. Tested compounds
were dissolved in DMSO. The solution was vibrated on an oscillator for more than 30 seconds and
then stored in a nitrogen cabinet. The stock solutions (10 mM) were diluted with DMSO for an 8
point titration (1:5 serial dilutions) in a 96-well dilution plate. The assay was performed according
to the instruction for the CETP inhibitor screening kit and recombinant CETP. Compounds were
tested at eight concentrations, and the concentration required to inhibit 50% of the activity (IC50)
was determined from a curve fit of the data with each concentration tested for one time.
4.2.2. In vivo test of compound 17a in high-fat fed hamsters
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Male golden (Syrian) hamsters were placed on a high-fat diet containing 10% lard oil and 3%
cholesterol for 4 weeks. Source of the animals: Beijing Vital River. Age and weight of the animals:
8 weeks old. Weight: approximately 110−120 g. The test animals were randomly divided into four
groups, each consisting of 6 animals. On the first day of dosing, the hamsters were bled to
determine the baseline HDL-C. Compound 17a (30 mg/kg, 50 mg/kg) and compound 4
(anacetrapib, 30 mg/kg) were administered orally to the animals. Each test compound was
suspended in a solvent vehicle that was a solution of 10% ethanol, 10% hydrogenated castor oil,
and 80% water. To the control group, the solvent vehicle alone was administered. The animals
were dosed once a day for 5 days according to the groups (n = 5 for each group), and they were
fasted for 16 hours prior to bleeding. Two hours after the final dose, blood specimens were
retrieved from all hamsters (retro-orbital puncture) and collected in 500 ꢀL EP tubes containing
heparin sodium. The collected blood was centrifuged at 3000 rpm for 15 min, and the plasma lipid
value in the separated serum (HDL-C) was determined using a MINDRAY BS-120 chemistry
analyser.
4.2.3. Pharmacokinetic study of compound 17a
Four adult male Sprague-Dawley rats were fasted for 16 hours before the study. Compound
17a (50 mg/kg) was administered orally to the animals. Test compound was suspended in a
solvent vehicle that was a solution of 10% ethanol, 10% hydrogenated castor oil, and 80% water.
Blood samples (~ 0.5 mL) were collected into tubes containing heparin sodium at the following
time points: 0.5 h, 1 h, 2 h, 3 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 7 h, 8 h, 10 h, 12 h, 24 h, 36 h. The
collected blood was centrifuged at 3000 rpm for 15 min, plasma was stored at -70oC until analysis.
Acknowledgements
We gratefully acknowledge the financial support from the National Natural Science
Foundation of China (Grant 81373324), and Program for Innovative Research Team of the
Ministry of Education, and Program for Liaoning Innovative Research Team in University.
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ꢀ
ꢀ
ꢀ
ꢀ
A series of N,N-substituted-cycloalkenyl-methylamine scaffold derivatives were designed and synthesized.
Most compounds showed potent CETP inhibitory activity.
Compound 17a demonstrated a dose-dependent HDL-C elevation in hamsters.
Compound 17a exhibited an acceptable pharmacokinetic profile in S-D rats.