Design, synthesis and biological evaluation of n,n-substituted amine derivatives as cholesteryl ester transfer protein inhibitors

Article abstract of DOI:10.3390/molecules22101658

N,N-Substituted amine derivatives were designed by utilizing a bioisosterism strategy. Consequently, twenty-two compounds were synthesized and evaluated for their inhibitory activity against CETP. Structure-activity relationship (SAR) studies indicate tha

Full text of DOI:10.3390/molecules22101658

molecules
Article
Design, Synthesis and Biological Evaluation of
N,N-Substituted Amine Derivatives as Cholesteryl
Ester Transfer Protein Inhibitors
1
1
2
1
1
1,
ID
Xinran Wang , Lijuan Hao , Xuanqi Xu , Wei Li , Chunchi Liu , Dongmei Zhao *
Maosheng Cheng 1
and
1
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China; wangxinrancai@163.com (X.W.);
8341482606@163.com (L.H.); 15542173071@163.com (W.L.); liuchunchi1990@163.com (C.L.);
mscheng@263.net (M.C.)
Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53715, USA; xxu53@wisc.edu
Correspondence: medchemzhao@163.com; Tel.: +86-24-4352-0219
1
2
*
Received: 20 August 2017; Accepted: 30 September 2017; Published: 3 October 2017
Abstract: N,N-Substituted amine derivatives were designed by utilizing a bioisosterism strategy.
Consequently, twenty-two compounds were synthesized and evaluated for their inhibitory activity
against CETP. Structure-activity relationship (SAR) studies indicate that hydrophilic groups at the
2
-position of the tetrazole and 3,5-bistrifluoromethyl groups on the benzene ring provide important
contributions to the potency. Among these compounds, compound 17 exhibited excellent CETP
inhibitory activity (IC50 = 0.38 0.08 M) in vitro. Furthermore, compound 17 was selected for an
in vitro metabolic stability study.
±
µ
Keywords: synthesis; N,N-substituted amine derivatives; CETP inhibitors; HDL-C
1
. Introduction
Cholesteryl ester transfer protein (CETP) is a hydrophobic glycoprotein secreted predominately
by the liver. CETP facilitates the movement 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) [ ]. Epidemiological studies have provided compelling evidence to demonstrate
an inverse association between HDL-C and cardiovascular events [ ]. Therefore, CETP inhibitors
(
1,2
3–5
can provide benefits to high risk coronary heart disease (CHD) patients by increasing HDL-C plasma
levels [1,6–8].
Torcetrapib (1, Figure 1) was the first CETP inhibitor to undergo advanced clinical development.
Although early studies have demonstrated that torcetrapib exhibited a significant lipid-regulating
ability, subsequent trials indicated that this inhibitor possessed off-target effects affecting blood pressure
and aldosterone levels, and failed to demonstrate any favourable impact on CHD patients [
Accordingly, torcetrapib’s phase III trial was halted in 2006. Dalcetrapib ( , Figure 1) was a modest
inhibitor without the off-targets effect seen with torcetrapib, elevating HDL-C levels by up to 30%
and with a minimal effect on LDL-C levels [14 15]. However, dal-OUTCOMES study was stopped
9–13].
2
,
due to lack of clinical benefit [16]. More recently, a Canadian study found that dalcetrapib exhibited
potentially clinical effects on atherosclerotic patients with polymorphisms in the ADCY9 gene using
a genome-wide approach [17]. Evacetrapib (3, Figure 1) possessed profound lipid-regulating effects,
raising HDL-C levels by more than 129% and decreasing LDL-C by up to 36%. Evacetrapib did
not exhibit torcetrapib-like side-effects, but its phase III ACCELERATE study was terminated due
to clinical futility [18
,
19]. Anacetrapib (
4
, Figure 1) a potent CETP inhibitor currently in phase
Molecules 2017, 22, 1658; doi:10.3390/molecules22101658
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1658
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III, could raise HDL-C levels to 130% and decrease LDL-C levels to 40%. Researchers found that
anacetrapib demonstrated potential clinical benefits and would not produce adverse clinical effects
similar to those observed with torcetrapib in the DEFINE study [20
CETP inhibitor, was well tolerated and had beneficial effects on lipids, raising HDL-C levels by up to
79% and decreasing LDL-C by up to 45% [22 23]. It remains to be seen whether these potent CETP
inhibitors will proceed further in the future.
,21]. TA-8995 (5, Figure 1), a novel
1
,
Figure 1. Representative CETP inhibitors.
In a previous study, compound
IC50 = 20.97 M) [24]. Our initial approach was to build a ring to replace the carbamate part to
investigate the effect of steric hindrance on activity. Considering that tetrazole is a bioisoster of the
carboxylic acid and the carbamate part of compound could be treated as a carboxylic acid methyl
6 (Figure 2) was identified to show weak micromolar activity
(
µ
6
ester, we hold that 2-methyltetrazole is a bioisoster of carbamate. In this paper, our group investigated
the cyclization of the carbamate using a bioisosterism strategy to afford a 2-methyltetrazole compound.
Fortunately, the compound containing the tetrazole moiety (40, Figure 2, IC50 = 0.81
showed increased inhibitory activity. A series of novel N,N-substituted amine derivatives were then
±
0.03 µM)
synthesized. Optimization efforts on this scaffold are discussed in this study.
Figure 2. Design of new structures based on compound 6.
2
. Results and Discussion
2
.1. Chemistry
Compounds 12–22 were prepared according to the procedure shown in Scheme 1. The intermediate
1
0 was obtained in a manner similar to that described in the previously published paper [24].
The treatment of 10 with cyanogen bromide under basic conditions furnished 11 in good yield.
Then, the -CN of the resulting intermediate was subjected to cycloaddition with sodium azide to
produce 12. Compound 12 was subjected to substitution reactions with various substituted alkyl
bromides to yield compounds 13–15. Alternatively, treatment of 12 with 2-(methylsulfonyl) ethanol
under Mitsunobu conditions afforded 16. Carboxylic acid derivatives 17 and 18 were prepared by
hydrolysis of the ester groups of compounds 13 and 14, respectively. Subsequent reduction or
aminolysis reaction of compounds 13 and 14 produced compounds 19
–21. Compound 11 reacted
with hydroxylamine hydrochloride and subsequently, acetic anhydride, to obtained compound 22
.

Molecules 2017, 22, 1658
3 of 15
Compounds 32
–
37 were prepared according to the procedure in Scheme 2. The resulting
9
was reacted
with NaBH and subsequently, SOCl to afford intermediate 23.
4
2
Scheme 1. Synthesis of target compounds 12
–22. Reagents and conditions: (a) DMF, PBr , CHCl ,
3 3
◦
rt; ( ) 5-isopropyl-2-methoxyphenylboronic acid, Pd(OAc) , K CO , acetylacetone, EtOH, 80 C;
b
2
2
3
(
(
c
e
) 3,5-bis(trifluromethyl)benzyl amine, NaBH(OAc) , 1,2-dichloroethane, rt; (
d
) CNBr, DIEA, THF, rt;
3
◦
) NaN , NH Cl, DMF, 100 C; (
f
) R -X, Et N, acetonitrile, reflux; or (i) Boc-R -X, Et N, acetonitrile,
3
4
1
3
1
3
reflux, (ii) TFA, DCM, rt; or R -OH, PPh , DIAD, rt; (g) NaBH /MeOH, THF, reflux; or NH -EtOH, rt;
4 3
1
3
(
h) (i) 1 M aq. NaOH, (ii) 1 M aq. HCl; (l) NH OH·HCl, Ac O, Et N, EtOH, reflux.
2
2
3
Scheme 2. Synthesis of target compounds 32
–
37. Reagents and conditions: (
a
) (i) NaBH , MeOH, rt,
4
◦
(
(
ii) SOCl , DMF, rt; (
b
) 3,5-bis(trifluromethyl)benzaldehyde, NaBH , MeOH, rt; (
c) NaH, DMF, 0 C;
2
4
d
) TFA/CH Cl (1:1), rt; (e) (i) methyl 2-bromoacetate, TEA, DMF, rt, (ii) NaBH /MeOH, THF, reflux;
2 2 4
or 1 M aq. NaOH, 1 M aq. HCl; (f) TEA, DCM, rt.

Molecules 2017, 22, 1658
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The starting materials 24 and 25 were subjected to reductive amination with 3,5-bis
trifluromethyl)benzaldehyde, which provided 26 and 27. Intermediates 28 and 29 were obtained
(
by the nucleophilic substitution of 23 by 26 and 27 in the presence of NaH at room temperature.
The key intermediates 30 and 31 were obtained by deprotection of 28 and 29 under the condition of
TFA/DCM (v:v = 1:1). Intermediate 30 was reacted with methyl 2-bromoacetate, then the resultant
ester intermediate was subjected to reduction conditions to yield compound 32 or hydrolysis
conditions to furnish compound 33. The obtained intermediates 30 and 31 were treated with
corresponding chloroformates to give compounds 34–37. As illustrated in Scheme 3, compounds 40–44
were synthesized from the corresponding starting benzaldehydes through reductive amination and
substitution reactions.
Scheme 3. Synthesis of target compounds 40–44. Reagents and conditions: (a) 2-methyl-2H-tetrazol-
◦
-amine, NaBH , MeOH, rt; (b) NaH, DMF, 0 C.
4
5
2
.2. In Vitro Activity Against Cholesteryl Ester Transfer Protein
The N,N-substituted amine derivatives and the 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 (Catalogue # RB-RPAK; Roar, New York, NY, USA). The results are shown in Table 1.
As seen in the table, replacement of a carbamate (
40, IC50 = 0.81 0.03 M) caused a significant increase in the activity. Subsequently, we investigated
the relationship between various substituents at the 2-position of the tetrazole (Part A) and the CETP
inhibitory activity. The replacement of –CH by –H (12, IC50 = 2.84 0.04 M) was detrimental
6, IC50 = 20.97 µM) with an N-2-methyltetrazole
(
±
µ
±
µ
3
for activity. Introduction of carboxylic esters (e.g., compounds 13 and 14) at the 2-position of the
tetrazole dramatically reduced the activity, however potency was recovered in the corresponding
carboxylic acids (17 and 18) and straight chain saturated alcohols (19 and 20), where especially two
atoms alkyl chains were better. We observed that amine and sulfone groups (15 and 16) were tolerated
and an amide group (21) caused a weak decrease in the activity. Changing N-2-methyltetrazole
(
40) to 3-methyoxadiazole (22, IC50 = 0.72
±
0.06 µM) showed no advantage. We speculate that
carboxylic acids and saturated alcohols at the 2-position of tetrazole provide an important contribution
to the potency.
Next, we investigated the relationship between an aliphatic heterocycle (Part A) and the CETP
inhibitory activity. Unfortunately, replacement of N-2-substituted tetrazole with azetidine/ piperidine
derivatives (compounds 32–37) caused a dramatic decrease in the activity. These results indicate that
a flexible fragment in Part A is unfavourable for inhibitory activity.
To further study the relationship between Part B and the CETP inhibitory activity, another five
compounds 40
groups on the 3-position and the 5-position of benzene ring (40, IC50 = 0.81
beneficial for CETP inhibition. A single trifluoromethyl group on the meta-position of the benzene
ring (41, IC50 = 10.05 0.06 M) caused a severe decrease in the activity. A trifluoromethyl group (43),
–
44 were prepared and evaluated for their activity. Introduction of trifluoromethyl
±
0.03 M) were more
µ
±
µ
fluorine atom (42), and trifluoromethoxy group (44) on the para-position of the benzene ring resulted
in no CETP inhibition.

Molecules 2017, 22, 1658
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Half of the compounds showed excellent activities, and four of them exhibited submicromolar
activities, but the activity of the most outstanding compound 17 (IC50 = 0.38 0.08 M) was still
±
µ
1
0-fold weaker than the reference compound.
Table 1. Structures and activities of compounds 12–22, 32–37, 40–44.
NO.
Part A
R (Part B)
IC50 (µM)
2.84 ± 0.04
NO.
33
Part A
R (Part B)
IC50 (µM)
>50 b
12
13
14
15
16
17
18
>50 b
>50 b
>50 b
34
35
36
37
40
41
>50 b
>50 b
>50 b
1.92 ± 0.10
1.20 ± 0.01
0.38 ± 0.08
1.60 ± 0.02
0.81 ± 0.03
10.05 ± 0.06
>50 b
>50 b
>50 b
1
2
2
9
0
1
0.73 ± 0.09
5.76 ± 0.03
20.06 ± 0.12
42
43
44
2
2
2
0.72 ± 0.06
>50 b
Anacetrapib a
3
0.04 ± 0.01
a
Used as a positive control. b Considered with no CETP inhibition activity.
2
.3. In Vitro Metabolic Stability Study
Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected
for the in vitro metabolic stability study. As shown in Table 2, compound 17 showed weak stability,
with a clearance rate of 119.0 and 146.1 µL/min/mg in human and rat liver microsomes.
Table 2. In vitro DMPK profile.
HLM Stability a
RLM Stability b
(µL/min/mg)
CYPs Direct Inhibition Mean (10 µM)
Compound
(
µL/min/mg)
3
A4
2D6
2C9
1A2
2C19
1
7
119.0
146.1
17.7% No inhibition
14.3% No inhibition
3.1%
a
Human liver microsomal intrinsic clearance. b Rat liver microsomal intrinsic clearance.
In addition, five cytochrome P450 (CYP) enzymes that commonly metabolize exogenous chemicals
were used to test the direct inhibition of compound 17. Compound 17 possessed favourable metabolic

Molecules 2017, 22, 1658
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properties, as the inhibition ratios for five CYPs were less than 20% even at the compound concentration
of 10 µM.
3
. Experimental
3
.1. General Information
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 with F-254 indicator and visualized by UV-light. The purities of target compounds
(
all
≥
95%). were detected by HPLC, performed on a Waters 1525–2489 system (Waters, Milford,
MA, USA). The method conditions were as follows: 100% CH OH or a mixture of solvents H O (A)
3
2
and MeOH (B) (VA:VB = 5:95) as eluent, flow rate at 1.0 mL/min. Peaks were detected at λ = 254 nm.
NMR spectra were recorded on 400 MHz and 600 MHz instruments (Bruker, Karlsruhe, Germany)
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. (Qingdao, China)
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)
cyanamide (11): Intermediate 10 (0.3 g, 0.6 mmol) was dissolved in THF (5 mL) and cyanogen bromide
(0.2 g, 1.8 mmol) and N,N-diisopropylethylamine (0.4 mL, 2.4 mmol) were added. The reaction
mixture was stirred at room temperature for 5 h and then poured into H O (10 mL) and extracted
2
with EtOAc (10 mL
×
3). The combined organic layers were washed with H O (10 mL
×
3) and
2
brine (10 mL 3), dried over Na SO , and concentrated in vacuo. The residue was purified by
×
2
4
chromatography on silica gel (petroleum ether:EtOAc = 15:1) to give 11 (0.3 g, 92.8%) as a pale yellow
1
oil. H-NMR (400 MHz, DMSO-d )
δ
8.06 (s, 1H), 7.90 (s, 2H), 7.05 (dd, J = 8.5, 2.2 Hz, 1H), 6.91–6.77
6
(m, 2H), 4.29 (s, 2H), 3.64 (s, 3H), 3.49–3.35 (m, 2H), 2.72 (dt, J = 13.7, 6.9 Hz, 1H), 2.40–2.28 (m, 1H),
2
.11–1.99 (m, 1H), 1.90 (m, 2H), 1.40 (t, J = 6.4 Hz, 2H), 1.08 (dd, J = 6.9, 1.6 Hz, 6H), 0.98 (d, J = 2.8 Hz,
6
H). HPLC: t = 13.710 min, 99.27%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
H-tetrazol-5-amine (12): Intermediate 11 (0.1 g, 0.2 mmol) was dissolved in DMF (5 mL) and ammonium
2
chloride (0.01 g, 0.8 mmol) and sodium azide (0.05 g, 0.2 mmol) were added. After being stirred at
◦
1
00 C for 5 h, the reaction mixture was cooled to room temperature, and H O (20 mL) was added.
2
The mixture was extracted with CH Cl (20 mL
×
3) and the combined organic layers were washed
2
2
with water (20 mL
×
3) and brine (20 mL
×
3), dried over Na SO , and concentrated in vacuo.
2
4
The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 2:1) to give 12
◦
1
(
7
4
0.1 g, 96.3%) as a white solid. Mp 150.6–152.6 C. H-NMR (600 MHz, DMSO-d )
δ
14.88 (s, 1H),
.94 (s, 1H), 7.66 (s, 2H), 6.99 (dd, J = 8.5, 2.2 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H),
.55 (s, 2H), 3.91 (m, 2H), 3.61 (s, 3H), 2.61 (dt, J = 13.8, 6.9 Hz, 1H), 2.36–2.27 (m, 1H), 2.06–2.03 (m, 1H),
6
1
.77–1.69 (m, 2H), 1.38 (s, 2H), 1.36 (t, J = 6.4 Hz, 2H), 1.00 (d, J = 6.9 Hz, 6H), 0.91 (d, J = 10.3 Hz, 6H).
1
3
C-NMR (150 MHz, CDCl3)
27.27, 126.44, 123.95, 122.14, 121.79, 111.11(
δ
: 151.95, 142.58, 139.79, 136.20, 132.01(
×
2), 129.76, 128.36, 127.92( 2),
×
1
2
×
2), 55.84(
×
2), 53.23, 39.75, 34.93, 33.16, 30.00, 28.75,
+
8.44, 27.24, 24.07, 23.99. HRMS calcd for C H F N O, [M + H] , 582.2589; found 582.2668.
29
34
6
5
HPLC: t = 14.640 min, 96.47%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
H-tetrazol-5-amine-2-carboxylic acid methyl ester (13): Compound 12 (0.1 g, 0.2 mmol) and triethylamine
2
(
2
0.1 mL, 0.8 mmol) were dissolved in acetonitrile (2 mL) followed by the addition of methyl
-bromoacetate (0.03 mL, 0.4 mmol). After being stirred at 80 C for 2 h, the reaction mixture
◦
was cooled to room temperature, and H O (10 mL) was added. The aqueous layer was extracted
2

Molecules 2017, 22, 1658
7 of 15
with EtOAc (5 mL
×
3) and the combined organic layers were washed with H O (5 mL
×
3)
2
and brine (5 mL
×
3), dried over Na SO , and concentrated in vacuo. The residue was purified
2
4
by chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 13 (0.09 g, 68.4%) as
1
a colourless oil. H-NMR (400 MHz, CDCl )
δ
7.67 (s, 1H), 7.54 (s, 2H), 7.03 (dd, J = 8.4, 2.3 Hz, 1H),
3
6
1
2
.76 (d, J = 2.3 Hz, 1H), 6.72 (d, J = 8.5 Hz, 1H), 5.18 (s, 2H), 4.58–4.38 (m, 2H), 4.19 (d, J = 14.5 Hz,
H), 4.00 (d, J = 14.4 Hz, 1H), 3.76 (s, 3H), 3.68 (s, 3H), 2.76 (dt, J = 13.8, 6.9 Hz, 1H), 2.54–2.37 (m, 1H),
.16–2.00 (m, 1H), 1.83 (s, 2H), 1.50–1.33 (m, 2H), 1.15 (d, J = 6.9 Hz, 6H), 0.94 (d, J = 11.9 Hz, 6H).
1
3
C-NMR (150 MHz, CDCl3)
28.06( 2), 127.76( 2), 127.63, 125.66(
3.03, 29.10, 28.99, 28.03( 2), 23.99(
54.2877. HPLC: t = 8.753 min, 95.8%.
δ
: 169.64, 165.70, 154.12, 141.11, 140.87, 135.34, 131.28( 2), 130.52,
×
1
3
6
×
×
×
2), 110.64(
×
2), 55.24, 53.01, 52.91, 51.69, 49.38, 40.57, 35.42,
+
×
×
2). HRMS calcd for C H F N O , [M + H] , 654.2801; found
32
38
6
5
3
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
1
2
H-tetrazol-5-amine-2-butyric acid ethyl ester (14): Colourless oil; yield 71.3%; H-NMR (400 MHz,
CDCl3)
δ
7.66 (s, 1H), 7.54 (s, 2H), 7.03 (d, J = 7.6 Hz, 1H), 6.82–6.65 (m, 2H), 4.46 (d, J = 3.5 Hz,
H), 4.18–4.11(m, 3H), 3.98 (d, J = 14.5 Hz, 1H), 3.67 (s, 3H), 2.76 (s, 1H), 2.46 (d, J = 19.3 Hz, 1H),
.30 (d, J = 4.5 Hz, 2H), 2.23 (s, 2H), 2.12–2.03 (m, 1H), 1.82 (s, 2H), 1.42 (s, 2H), 1.25–1.24 (m, 3H),
4
2
1
13
.14 (s, 6H), 0.94 (d, J = 11.1 Hz, 6H). C-NMR (150 MHz, CDCl3)
40.85, 135.18, 131.24( 2), 130.57, 128.08, 127.77( 2), 125.63, 124.16, 122.35, 120.60, 110.62(
5.22, 51.77, 51.65, 49.36, 40.61, 35.43, 33.03, 30.60, 29.10, 29.00, 28.04, 28.02, 24.12, 23.99, 23.98, 14.04.
δ: 172.11, 169.37, 154.14, 141.34,
1
×
×
×
2), 60.54,
5
+
HRMS calcd for C H F N O , [M + H] , 696.3270; found 656.3361. HPLC: t = 7.653 min, 96.4%.
35
44
6
5
3
R
2-(2-Aminoethyl)-N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethyl-
cyclohex-1-enyl)methyl)-2H-tetrazol-5-amine (15): Compound 12 (0.5 g, 0.9 mmol) and triethylamine
(
2
1.8 mL, 13.0 mmol) were dissolved in acetonitrile (10 mL), followed by the addition of tert-butyl
-bromoethylcarbamate (0.6 mL, 2.6 mmol). After being stirred at 80 C for 2 h, the reaction mixture
◦
was cooled to room temperature, and H O (10 mL) was added. The aqueous layer was extracted with
2
EtOAc (5 mL
5 mL 3), dried over Na SO , and concentrated in vacuo. The residue was immediately dissolved in
a trifluoroacetic acid–dichloromethane (1:1) solution (2 mL) and stirred at room temperature overnight.
After concentration, the residue was dissolved in EtOAc (5 mL), washed with H O (5 mL 3)
3), dried over Na SO , and concentrated in vacuo. The residue was purified by
×
3) and the combined organic layers were washed with H O (5 mL
×
3) and brine
2
(
×
2
4
×
2
and brine (5 mL
×
2
4
chromatography on silica gel (petroleum ether:EtOAc = 2:1) to give 15 (0.35 g, 62.2%) as a colourless
1
oil. H-NMR (400 MHz, DMSO-d )
δ
7.93 (s, 1H), 7.66 (s, 2H), 7.00 (dd, J = 8.4, 2.2 Hz, 1H), 6.79–6.76
6
(m, 2H), 4.49 (s, 2H), 4.35 (t, J = 6.1 Hz, 2H), 4.02–3.89 (m, 2H), 3.62 (s, 3H), 2.94 (t, J = 6.2 Hz, 2H),
2
.67 (dt, J = 13.8, 6.9 Hz, 1H), 2.54–2.37 (m, 1H), 2.16–2.00 (m, 1H), 1.76 (s, 2H), 1.35 (t, J = 6.3 Hz, 2H),
13
1.04 (d, J = 6.9 Hz, 6H), 0.89 (d, J = 10.2 Hz, 6H). C-NMR (150 MHz, CDCl3)
δ
: 169.41, 154.13, 141.31,
2), 55.81,
1
40.86, 135.29, 131.25( 2), 130.55, 128.07, 127.80, 127.70, 125.65, 124.14, 122.34, 120.63, 110.62(
×
×
5
5.22, 51.72, 49.39, 40.93, 40.64, 35.42, 33.03, 29.11, 29.01, 28.06, 28.03, 24.00, 23.98. HRMS calcd for
+
C H F N O, [M + H] , 625.3011; found 625.3070. HPLC: t = 18.895 min, 96.6%.
31
39
6
6
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
2
-(2-(methylsulfonyl)ethyl)-2H-tetrazol-5-amine (16): 2-(methylsulfonyl)ethanol (0.04 mL, 0.4 mmol) was
◦
added to a solution of compound 12 (0.1 g, 0.2 mmol) in THF (2 mL) and cooled to 0 C, followed by
the addition of Ph P (0.07 g, 0.3 mmol) and DIAD (0.05 mL, 0.3 mmol). After being stirred at room
3
temperature for 4 h, the reaction mixture was poured into H O (10 mL) and extracted with EtOAc
2
(
5 mL
×
3), and the combined organic layers were washed with H O (5 mL
×
3) and brine (5 mL
×
3),
2
dried over Na SO , and concentrated in vacuo. The residue was purified by chromatography on
2
4
1
silica gel (petroleum ether:EtOAc = 1:1) to give 16 (0.09 g, 65.3%) as a colourless oil. H-NMR
400 MHz, CDCl3) 7.68 (s, 1H), 7.50 (s, 2H), 7.03 (dd, J = 8.4, 2.2 Hz, 1H), 6.76 (d, J = 2.3 Hz, 1H),
.71 (d, J = 8.5 Hz, 1H), 4.90 (t, J = 6.9 Hz, 2H), 4.48 (s, 2H), 4.17 (d, J = 15.4 Hz, 1H), 3.99 (d, J = 14.4 Hz,
H), 3.70–3.60 (m, 5H), 2.82–2.71 (m, 4H), 2.46 (d, J = 18.1 Hz, 1H), 2.10 (d, J = 18.4 Hz, 1H), 1.79 (s, 2H),
(
6
1
δ

Molecules 2017, 22, 1658
8 of 15
1
.43 (dd, J = 9.7, 6.4 Hz, 2H), 1.15 (d, J = 6.9 Hz, 6H), 0.94 (d, J = 11.3 Hz, 6H). 13C-NMR (150 MHz,
CDCl3) : 169.58, 154.08,140.96, 135.68, 131.36( 2), 130.40, 128.01, 127.69, 127.33, 125.74( 2), 124.10,
δ
×
×
122.29, 120.77, 110.66(
×
2), 55.25, 52.90, 51.66, 49.20, 46.20, 41.37, 40.66, 35.36, 33.03, 29.10, 29.02,
+
2
8.08, 28.03, 24.00, 23.98. HRMS calcd for C H F N O S, [M + H] , 688.2678; found 688.2754.
32
40
6
5
3
HPLC: t = 13.667 min, 95.6%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
H-tetrazol-5-amine-2-acetic acid (17): Compound 13 (0.2 g, 0.3 mmol) was dissolved in MeOH (5 mL)
and 1 mol/L NaOH (5 mL) was added and the mixture was stirred at room temperature for 2 h.
After concentration, the residue was dissolved in H O (10 mL). Then, 1 mol/L HCl (5 mL) was added
2
2
to the mixture, the mixture was extracted with EtOAc (10 mL
×
3), and the combined organic layers
were washed with H O (10 mL 3) and brine (10 mL 3), dried over Na SO , and concentrated in
×
×
2
2
4
vacuo. The residue was purified by chromatography on silica gel (DCM:MeOH = 5:1) to give 17 (0.17 g,
1
8
2
3
6.4%) as a colourless oil. H-NMR (600 MHz, DMSO-d )
.2 Hz, 1H), 6.77 (dd, J = 7.9, 5.4 Hz, 2H), 5.35 (s, 2H), 4.57–4.45 (m, 2H), 3.97 (t, J = 10.4 Hz, 2H),
.63 (s, 3H), 2.67 (dt, J = 13.7, 6.9 Hz, 1H), 2.33 (d, J = 17.9 Hz, 1H), 2.02 (d, J = 18.0 Hz, 1H), 1.76 (s, 2H),
δ
7.92 (s, 1H), 7.65 (s, 2H), 7.00 (dd, J = 8.5,
6
13
1.36 (t, J = 6.4 Hz, 2H), 1.04 (d, J = 6.9 Hz, 6H), 0.90 (d, J = 14.3 Hz, 6H). C-NMR (150 MHz,
DMSO-d6) : 169.21, 167.87, 154.37, 142.28, 140.54, 134.58, 130.63( 2), 130.29, 127.81, 127.74, 127.62,
δ
×
1
2
25.85, 124.50, 122.69, 120.98, 111.32(
8.46, 28.09, 24.27, 24.21. HRMS calcd for C H F N O , [M + H] , 640.2644; found 640.2709.
×
2), 55.70, 53.85, 51.65, 49.55, 40.57, 35.36, 32.75, 29.24, 29.07,
+
31
36
6
5
3
HPLC: t = 19.013 min, 97.3%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
1
2
H-tetrazol-5-amine-2-butyric acid (18): Colourless oil; yield 81.3%; H-NMR (400 MHz, DMSO-d )
δ
6
1
4
1
0
2.18 (s, 1H), 7.90 (s, 1H), 7.65 (s, 2H), 6.99 (d, J = 8.5 Hz, 1H), 6.78 (d, J = 8.5 Hz, 2H), 4.50 (s, 2H),
.44 (t, J = 6.8 Hz, 2H), 4.00–3.90 (m, 2H), 3.62 (s, 3H), 2.66 (dt, J = 13.7, 6.8 Hz, 1H), 2.32 (d, J = 17.7 Hz,
H), 2.19–2.15(m, 2H), 2.07–1.98 (m, 3H), 1.75 (s, 2H), 1.35 (t, J = 5.9 Hz, 2H), 1.03 (d, J = 6.9 Hz, 6H),
.88 (d, J = 7.5 Hz, 6H). 13C-NMR (150 MHz, DMSO-d6)
: 173.76, 169.19, 154.39, 142.35, 140.54, 134.46,
2), 127.88, 127.74, 127.72( 2), 125.84, 124.51, 122.70, 120.94, 111.33( 2), 55.69, 51.88, 51.70, 49.66,
0.63, 35.36, 32.76, 30.40, 29.25, 29.05, 28.40, 28.13, 24.44, 24.25, 24.21. HRMS calcd for C H F N O ,
δ
130.61(
×
×
×
4
33
40
6
5
3
+
[M + H] , 668.2957; found 668.3050. HPLC: t = 18.767 min, 97.1%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
H-tetrazol-5-amine-2-ethyl alcohol (19): Compound 13 (0.4 g, 0.6 mmol) was dissolved in THF (5 mL)
2
and NaBH (0.05 g, 1.2 mmol) and 2 drops of MeOH were added and the mixture was stirred at 70
4
◦
C for 2 h and then cooled to room temperature. After concentration, the residue was dissolved
in EtOAc (10 mL), washed with H O (10 mL
and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum
ether:EtOAc = 4:1) to give 19 (0.3 g, 79.8%) as a colourless oil. H-NMR (600 MHz, DMSO-d )
×
3) and brine (10 mL
×
3), dried over Na SO ,
2 4
2
1
δ
7.92
6
(s, 1H), 7.67 (s, 2H), 7.00 (dd, J = 8.5, 2.2 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 2.3 Hz, 1H),
4
5
.95 (t, J = 5.6 Hz, 1H), 4.49 (s, 2H), 4.42 (t, J = 5.4 Hz, 2H), 3.97 (q, J = 14.4 Hz, 2H), 3.78 (dd, J = 10.8,
.5 Hz, 2H), 3.62 (s, 3H), 2.67 (dt, J = 13.8, 6.9 Hz, 1H), 2.33 (d, J = 18.0 Hz, 1H), 2.02 (d, J = 18.0 Hz,
13
1H), 1.76 (s, 2H), 1.35 (t, J = 6.4 Hz, 2H), 1.04 (d, J = 6.9 Hz, 6H), 0.89 (d, J = 16.3 Hz, 6H). C-NMR
(
150 MHz, CDCl3) : 169.30, 154.11, 141.18, 140.89, 135.43, 131.32( 2), 130.51, 128.04, 127.72, 127.55,
δ
×
1
25.67, 124.13, 122.32, 120.71, 110.63(
9.02, 28.04(
HPLC: t = 16.776 min, 95.8%.
×
2), 60.30, 55.23, 54.95, 51.80, 49.41, 40.68, 35.40, 33.03, 29.12,
+
2
×
2), 23.99, 23.98. HRMS calcd for C H F N O , [M + H] , 626.2851; found 626.2920.
31
38
6
5
2
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
1
2
H-tetrazol-5-amine-2-butanol (20): Colourless oil; yield 73.8%; H-NMR (400 MHz, DMSO-d )
δ
7.92
6
(s, 1H), 7.67 (s, 2H), 7.02 (dd, J = 8.5, 2.1 Hz, 1H), 6.83–6.76 (m, 2H), 4.52 (s, 2H), 4.48–4.39 (m, 3H),
4.03–3.92 (m, 2H), 3.64 (s, 3H), 3.37 (d, J = 5.4 Hz, 2H), 2.68 (dt, J = 13.7, 6.8 Hz, 1H), 2.40–2.29

Molecules 2017, 22, 1658
9 of 15
(
0
1
4
m, 1H), 2.06–2.00 (m, 1H), 1.89–1.80 (m, 2H), 1.77 (s, 2H), 1.40–1.28 (m, 4H), 1.05 (d, J = 6.9 Hz, 6H),
.90 (d, J = 7.5 Hz, 6H). 13C-NMR (150 MHz, CDCl3)
: 169.27, 154.14, 141.37, 140.85, 135.19, 131.22( 2),
2), 125.63, 124.16, 122.36, 120.59, 110.62( 2), 61.78, 55.22, 52.52, 51.64, 49.35,
2), 25.53, 23.99, 23.98. HRMS calcd for C H F N O ,
δ
×
30.58, 128.09, 127.77(
×
×
0.60, 35.43, 33.03, 29.12, 29.10, 29.00, 28.04(
×
33
42
6
5
2
+
[
M + H] , 654.3164; found 654.3257. HPLC: t = 15.755 min, 96.4%.
R
2
-(5-((3,5-Bis(trifluoromethyl)benzyl)((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)
amino)-2H-tetrazol-2-yl)acetamide (21): Compound 13 (0.2 g, 0.3 mmol) was dissolved in a saturated
NH –EtOH solution (5 mL). After being stirred at room temperature for 12 h, the reaction mixture
3
was concentrated in vacuo. The residue was dissolved in EtOAc (10 mL), washed with H O (10 mL
2
×
3) and brine (10 mL
×
3), dried over Na SO , and concentrated in vacuo. The residue was
2 4
purified by chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 21 (0.12 g, 62.6%) as
1
a colourless oil. H-NMR (400 MHz, DMSO-d )
δ 7.91 (s, 1H), 7.70 (s, 1H), 7.66 (s, 2H), 7.42 (s, 1H),
6
6
3
.99 (dd, J = 8.5, 2.2 Hz, 1H), 6.78–6.75(m, 2H), 5.12 (s, 2H), 4.50 (s, 2H), 3.97 (d, J = 4.6 Hz, 2H),
.62 (s, 3H), 2.66 (dt, J = 13.7, 6.8 Hz, 1H), 2.33 (d, J = 18.0 Hz, 1H), 2.01 (d, J = 17.7 Hz, 1H), 1.76 (s, 2H),
.35 (t, J = 6.2 Hz, 2H), 1.04 (d, J = 6.9 Hz, 6H), 0.89 (d, J = 10.9 Hz, 6H). 1 C-NMR (150 MHz, CDCl3)
3
δ:
1
1
1
69.77, 154.08, 140.96, 140.88, 131.43(
20.84, 110.68(
×
2), 130.42, 127.99(
×
2), 127.73, 127.29, 125.77( 2), 124.07, 122.26,
×
×
2), 55.24, 54.83, 51.87, 49.43, 40.79, 35.35, 33.04, 29.13, 29.03, 28.10, 28.00, 24.01, 23.98.
+
HRMS calcd for C H F N O , [M + H] , 639.2804; found 639.2909. HPLC: t = 12.757 min, 98.2%.
31
37
6
6
2
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
-methyl-1,2,4-oxadiazol-3-amine (22): Intermediate 11 (0.3 g, 0.6 mmol) was dissolved in EtOH (5 mL)
5
and triethylamine (0.08 mL, 0.6 mmol) and hydroxylamine hydrochloride (0.04 g, 0.6 mmol) were
◦
added. After being stirred at 80 C for 2 h, the reaction mixture was cooled to room temperature
and concentrated in vacuo. Then, the residue was added pyridine (5 mL) and acetic anhydride
◦
(
0.07 mL, 0.7 mmol). After being stirred at 80 C for 12 h, the reaction mixture was cooled to room
temperature and concentrated in vacuo. The residue was dissolved in EtOAc (10 mL), washed with
H O (10 mL 3) and brine (10 mL 3), dried over Na SO , and concentrated in vacuo. The residue
×
×
2
2
4
was purified by chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 22 (0.15 g, 41.9%)
1
as a colourless oil. H-NMR (600 MHz, CDCl )
δ 7.66 (s, 1H), 7.50 (s, 2H), 7.00 (dd, J = 8.4, 2.1 Hz,
3
1
H), 6.72 (d, J = 2.0 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 4.43 (q, J = 16.2 Hz, 2H), 4.06 (d, J = 14.5 Hz, 1H),
3
1
.90 (d, J = 14.5 Hz, 1H), 3.65 (s, 3H), 2.73 (dt, J = 13.8, 6.9 Hz, 1H), 2.47–2.43 (m, 4H), 2.08 (d, J = 18.2 Hz,
H), 1.81 (s, 2H), 1.42 (dq, J = 13.0, 6.5 Hz, 2H), 1.13 (d, J = 6.9 Hz, 6H), 0.95 (d, J = 13.1 Hz, 6H).
1
3
C-NMR (150 MHz, CDCl3)
28.00, 127.54, 127.35, 125.66, 124.13, 122.32, 120.70, 110.62(
9.04, 29.01, 28.10, 27.97, 23.96(
found 596.2716. HPLC: t = 9.953 min, 95.8%.
δ
: 175.39, 170.36, 154.07, 140.92, 140.85, 135.59, 131.31( 2), 130.37,
×
1
2
×
2), 55.22, 51.08, 48.61, 40.40, 35.41, 33.01,
+
×
2), 12.63. HRMS calcd for C H F N O , [M + H] , 596.2633;
31
36
6
3
2
R
2
0
-(2-(Chloromethyl)-4,4-dimethylcyclohex-1-enyl)-4-isopropyl-1-methoxybenzene (23): Intermediate
.4 mmol) was dissolved in ethanol (5 mL). Sodium borohydride (19.0 mg, 0.5 mmol) was added to the
mixture. After being stirred at room temperature for 30 min, the reaction mixture was poured into H O
9 (114.5 mg,
2
(
(
1
20 mL) and extracted with EtOAc (10 mL
10 mL 3) and brine (10 mL 3), dried over Na SO , and concentrated in vacuo. SOCl (0.1 mL,
.4 mmol) was added to a solution of the above residue in DMF (2 mL) cooled to 0 C. After being
stirred at room temperature for 1 h, the reaction mixture was poured into H O (10 mL) and was
×
3). The combined organic layers were washed with H O
2
×
×
2
4
2
◦
2
extracted with EtOAc (5 mL
×
3). The combined organic layers were washed with H O (10 mL
×
3)
2
and brine (10 mL 3), dried over Na SO , and concentrated in vacuo. The residue was purified by
×
2
4
chromatography on silica gel (petroleum ether:EtOAc = 10:1) to give 23 (92.5 mg, 75.3% in two steps),
which was used immediately for the next step because of its instability. HPLC: t = 7.706 min, 96.8%.
R
tert-Butyl 4-(3,5-bis(trifluoromethyl)benzylamino)piperidine-1-carboxylate (26): tert-butyl 4-amino-
piperidine-1-carboxylate (1.0 g, 5.0 mmol) and 3,5-bis(trifluoromethyl) benzaldehyde (1.0 g, 4.1 mmol)

Molecules 2017, 22, 1658
10 of 15
were dissolved in MeOH (15 mL). After stirring for 3 h at room temperature, NaBH (0.3 g, 7.0
4
mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 30 min
and then poured into a saturated sodium bicarbonate solution (20 mL). The mixture was extracted
with CH Cl (20 mL
×
3) and the combined organic layers were washed with water (20 mL
×
3)
2
2
and brine (20 mL
×
3), dried over Na SO , and concentrated in vacuo. The residue was purified by
2
4
chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 26 (1.4 g, 80.0%) as a pale yellow oil.
1
H-NMR (600 MHz, CDCl3) δ 7.83 (s, 2H), 7.76 (s, 1H), 4.04 (s, 2H), 3.96 (s, 2H), 2.82 (s, 2H), 2.71–2.63
(
m, 1H), 1.88 (d, J = 11.2 Hz, 2H), 1.46 (s, 9H), 1.35–1.26 (m, 2H). HPLC: t = 13.657 min, 95.7%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)
piperidine-4-amine-1-carboxylic acid tert-butyl ester (28): NaH (30.0 mg, 0.7 mmol, 60% in oil) was added
◦
to a solution of intermediate 26 (213.1 mg, 0.5 mmol) in DMF (5 mL) cooled to 0 C. After stirring
◦
at 0 C for 30 min, a solution of intermediate 23 (153.4 mg, 0.5 mmol) in DMF (5 mL) was added.
The reaction mixture was allowed to warm to room temperature and stirred for 30 min, and then
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 H O (10 mL 3) and brine (10 mL 3), dried over Na SO , and concentrated in
×
×
2
2
4
vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 20:1) to
1
give 28 (235.9 mg, 67.7%) as a colourless oil. H-NMR (400 MHz, CDCl )
δ
7.83 (s, 2H), 7.73 (s, 1H),
3
7
3
.08 (dd, J = 8.4, 2.2 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 2.3 Hz, 1H), 4.11 (s, 2H), 3.70 (s, 3H),
.50 (q, J = 15.0 Hz, 2H), 2.90–2.78 (m, 2H), 2.74–2.48 (m, 4H), 2.44–2.31 (m, 1H), 2.09–2.00 (m, 1H),
1
1
.99–1.83 (m, 2H), 1.52 (d, J = 12.0 Hz, 1H), 1.45–1.34 (m, 12H), 1.34–1.25 (m, 2H), 1.22 (dd, J = 6.9,
.1 Hz, 6H), 0.96 (s, 6H). HPLC: t = 7.023 min, 96.6%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)
piperidin-4-amine (30): Intermediate 28 (0.2 g, 0.3 mmol) was dissolved in a trifluoroacetic acid–
dichloromethane (1:1) solution (20 mL) and stirred at room temperature overnight. After concentration,
the residue was dissolved in EtOAc (10 mL), washed with H O (10 mL
×
3) and brine (10 mL
×
3),
2
dried over Na SO , and concentrated in vacuo. The residue was purified by chromatography on silica
2
4
1
gel (petroleum ether:EtOAc = 2:1) to give 30 (172.1 mg, 96.1%) as a colourless oil. H-NMR (400 MHz,
DMSO-d6) 8.81 (s, 1H), 8.10–7.88 (m, 3H), 7.11 (dd, J = 8.4, 2.1 Hz, 1H), 6.91 (d, J = 8.5 Hz, 1H),
.83 (d, J = 2.0 Hz, 1H), 3.66 (s, 3H), 3.57 (d, J = 2.4 Hz, 2H), 3.29 (d, J = 11.8 Hz, 2H), 2.89–2.68 (m, 6H),
.26 (d, J = 17.6 Hz, 1H), 2.01 (s, 1H), 1.87 (d, J = 7.9 Hz, 2H), 1.67–1.48 (m, 4H), 1.35 (t, J = 6.2 Hz, 2H),
.17 (d, J = 6.9 Hz, 6H), 0.92 (d, J = 10.5 Hz, 6H). HPLC: t = 13.003 min, 95.9%.
δ
6
2
1
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)
piperidin-4-amine-1-ethyl alcohol (32): Intermediate 30 (0.7 g, 1.2 mmol) was dissolved in acetonitrile
(
10 mL) and methyl 2-bromoacetate (0.4 g, 2.4 mmol) and triethylamine (0.6 mL, 4.7 mmol) were added.
The reaction mixture was stirred at room temperature for 16 h and then poured into H O (10 mL) and
2
extracted with EtOAc (10 mL × 3). The combined organic layers were washed with H O (10 mL × 3)
2
and brine (10 mL
×
3), dried over Na SO , and concentrated in vacuo. The residue was dissolved
2 4
in THF (10 mL), and sodium borohydride (0.1 g, 2.6 mmol) and 2 drops of MeOH were added.
The mixture was stirred at 70 C for 2 h and then cooled to room temperature. After concentration,
◦
the residue was dissolved in EtOAc (10 mL), washed with H O (10 mL
×
3) and brine (10 mL
×
3),
2
dried over Na SO , and concentrated in vacuo. The residue was purified by chromatography
2
4
1
on silica gel (petroleum ether:EtOAc = 1:1) to give 32 (0.4 g, 52.0%) as a colourless oil. H-NMR
400 MHz, DMSO-d6) 8.01 (s, 2H), 7.93 (s, 1H), 7.08 (dd, J = 8.4, 2.0 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H),
.74 (d, J = 2.0 Hz, 1H), 4.39 (s, 1H), 3.64 (s, 3H), 3.55 (d, J = 18.4 Hz, 2H), 3.44 (d, J = 5.9 Hz, 2H),
.84 (d, J = 13.3 Hz, 2H), 2.78 (dd, J = 13.7, 6.8 Hz, 1H), 2.69 (s, 2H), 2.43–2.37 (m, 3H), 2.26 (d, J = 17.8 Hz,
(
δ
6
2
13
1
(
1
H), 1.96–1.77 (m, 4H), 1.44–1.42 (m, 7H), 1.16 (d, J = 6.9 Hz, 6H), 0.91 (d, J = 7.1 Hz, 6H). C-NMR
150 MHz, CDCl3) : 154.49, 144.61, 140.59, 132.83, 131.67, 131.25( 2), 129.57, 128.19, 128.00, 125.01,
24.38, 122.57, 120.40, 110.56( 2), 59.29, 57.73, 55.43, 55.13, 53.47, 53.39, 52.55, 52.25, 40.99, 35.60,
δ
×
×

Molecules 2017, 22, 1658
11 of 15
+
33.21(
×
2), 29.30, 28.93, 28.28, 27.72. 27.25, 24.23, 24.12. HRMS calcd for C H F N O , [M + H] ,
35
47
6
2
2
6
41.3463; found 641.3525. HPLC: t = 16.772 min, 97.9%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)
piperidin-4-amine-1-acetic acid (33): Intermediate 30 (0.5 g, 0.9 mmol) was dissolved in acetonitrile
(10 mL), and methyl 2-bromoacetate (0.3 g, 1.8 mmol) and triethylamine (0.4 mL, 3.4 mmol) were
added. The reaction mixture was stirred at room temperature for 16 h and then poured into H O
2
(
(
10 mL) and extracted with EtOAc (10 mL
×
3). The combined organic layers were washed with H O
2
10 mL 3) and brine (10 mL 3), dried over Na SO , and concentrated in vacuo. The residue was
×
×
2
4
dissolved in MeOH (5 mL) and 1 mol/L NaOH (5 mL) was added and the mixture was stirred at room
temperature for 2 h. After concentration, the residue was dissolved in H O (10 mL). Then, 1 mol/L
2
HCl (5 mL) was added to the mixture, the mixture was extracted with EtOAc (10 mL
combined organic layers were washed with H O (10 mL 3) and brine (10 mL 3), dried over
Na SO , and concentrated in vacuo. The residue was purified by chromatography on silica gel
×
3), and the
×
×
2
2
4
1
(
DCM:MeOH = 5:1) to give 33 (0.4 g, 67.8%) as a colourless oil. H-NMR (400 MHz, DMSO-d ) δ 8.02
6
(s, 2H), 7.95 (s, 1H), 7.09 (dd, J = 8.5, 2.1 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 2.1 Hz, 1H),
3
2
.65 (s, 3H), 3.63–3.51 (m, 2H), 3.20 (s, 2H), 3.14 (d, J = 10.4 Hz, 2H), 2.79 (dt, J = 13.7, 6.9 Hz, 1H),
.70 (s, 2H), 2.62–2.51 (m, 3H), 2.26 (d, J = 17.7 Hz, 1H), 1.96 (s, 1H), 1.91–1.75 (m, 2H), 1.70–1.51
13
(
(
m, 2H), 1.40 (s, 2H), 1.35 (t, J = 6.3 Hz, 2H), 1.16 (d, J = 6.8 Hz, 6H), 0.91 (d, J = 8.7 Hz, 6H). C-NMR
150 MHz, DMSO-d6) : 168.60, 154.62( 2), 145.76, 140.43, 132.53, 131.21, 130.22( 2), 129.42, 128.93,
δ
×
×
1
27.74, 125.54, 124.78, 122.97, 111.48(
×
2), 59.03, 55.81, 54.09, 52.70, 52.43, 52.37, 51.58, 41.02, 35.50, 32.94,
+
2
9.40, 29.00, 28.53, 27.95, 24.55(
×
2), 24.44(
×
2). HRMS calcd for C H F N O , [M + H] , 655.3256;
35
45
6
2
3
found 655.3345. HPLC: t = 19.973 min, 98.1%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)
piperidin-4-amine-1- methanoic acid tetrahydro-2H-pyran-4-ol ester (34): Intermediate 30 (0.2 g, 0.3 mmol)
was dissolved in CH Cl (5 mL), and tetrahydro-2H-pyran-4-yl carbonochloridate (0.1 g, 0.5 mmol)
2
2
and triethylamine (0.2 mL, 1.4 mmol) were added. The reaction mixture was stirred at room
temperature for 30 min and then poured into H O (10 mL) and extracted with CH Cl (10 mL
×
3).
2
2
2
The combined organic layers were washed with H O (10 mL
×
3) and brine (10 mL
×
3), dried over
2
Na SO , and concentrated in vacuo. The residue was purified by chromatography on silica gel
2
4
1
(
petroleum ether:EtOAc = 4:1) to give 34 (0.1 g, 45.9%) as a colourless oil. H-NMR (600 MHz, CDCl )
3
δ
7.82 (s, 2H), 7.73 (s, 1H), 7.08 (dd, J = 8.3, 2.0 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.73 (d, J = 2.0 Hz,
H), 4.86–4.80 (m, 1H), 4.13 (d, J = 7.1 Hz, 2H), 3.90–3.85 (m, 2H), 3.71 (s, 3H), 3.56–3.53 (m, 3H),
.49–3.41 (m, 1H), 2.84–2.81 (m, 2H), 2.75–2.53 (m, 4H), 2.37 (d, J = 17.7 Hz, 1H), 2.02 (s, 1H), 1.93–1.90
m, 3H), 1.87 (s, 1H), 1.65–1.63 (m, 3H), 1.55 (d, J = 12.5 Hz, 1H), 1.46–1.37 (m, 3H), 1.35–1.29
1
3
(
(
13
m, 1H), 1.22 (dd, J = 6.9, 2.4 Hz, 6H), 0.96 (s, 6H). C-NMR (150 MHz, CDCl3)
δ
: 154.47( 2), 144.35,
×
140.64, 131.57, 131.32, 131.10, 129.35, 128.18( 2), 125.09( 2), 124.35, 122.54, 120.51, 110.61(
×
×
×
2), 69.63,
6
2
5.30(
×
2), 55.44(
×
2), 55.35, 52.46, 52.23, 43.82, 43.66, 41.07, 35.57, 33.19, 32.08(
×
2), 29.24, 28.94,
+
8.33, 27.64, 24.24, 24.11(
×
2). HRMS calcd for C H F N O , [M + H] , 725.3675; found 725.3751.
39
51
6
2
4
HPLC: t = 15.553 min, 96.0%.
R
0
0
0
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((5 -isopropyl-2 -methoxy-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1 -biphenyl]
-2-yl)methyl)azetidin-3-amine-1-methanoic acid ethyl ester (35): Intermediate 31 (0.2 g, 0.3 mmol) was
dissolved in CH2Cl2 (5 mL), and ethyl chloroformate (0.1 g, 0.7 mmol) and triethylamine (0.2 mL,
1.4 mmol) were added. The reaction mixture was stirred at room temperature for 30 min and then
poured into H O (10 mL) and extracted with CH Cl (10 mL
×
3). The combined organic layers were
2
2
2
washed with H O (10 mL
The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 35
×
3) and brine (10 mL
×
3), dried over Na SO , and concentrated in vacuo.
2
2
4
1
(
0.2 g, 89.2%) as a colourless oil. H-NMR (400 MHz, CDCl )
δ
7.78 (s, 2H), 7.76 (s, 1H), 7.09 (dd, J = 8.4,
3
2
3
.1 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.88–3.81 (m, 1H),
.80–3.72 (m, 3H), 3.71 (s, 3H), 3.68–3.56 (m, 2H), 3.55–3.47 (m, 1H), 2.90–2.77 (m, 2H), 2.77–2.65

Molecules 2017, 22, 1658
12 of 15
(
0
1
2
6
m, 1H), 2.43–2.29 (m, 1H), 2.10–2.04 (m, 1H), 2.01–1.82 (m, 2H), 1.45–1.35 (m, 2H), 1.24–1.19 (m, 9H),
.96 (s, 6H). 13C-NMR (150 MHz, CDCl3)
: 156.60( 2), 154.30( 2), 140.75, 131.29( 2), 131.14, 128.37,
2), 125.39, 124.21, 122.41, 120.87, 110.55( 2), 60.92, 55.32, 54.44, 53.43, 41.59, 35.49, 33.14(
2), 14.60(
δ
×
×
×
27.94(
×
×
×
2),
+
9.38, 28.97, 28.22, 27.80, 24.18, 24.12(
×
×
2). HRMS calcd for C H F N O , [M + H] ,
34
43
6
2
3
41.3100; found 641.3180. HPLC: t = 11.763 min, 96.7%.
R
0
0
0
1
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((5 -isopropyl-2 -methoxy-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1 -biphenyl]
-
2-yl)methyl)azetidin-3-amine-1-methanoic acid isopropyl ester (36): Colourless oil; yield 69.3%; H-NMR
(
6
1
2
1
δ
400 MHz, CDCl3)
.74 (d, J = 2.2 Hz, 1H), 4.84 (dt, J = 12.5, 6.2 Hz, 1H), 3.83 (t, J = 8.4 Hz, 1H), 3.77 (d, J = 7.9 Hz,
H), 3.73 (d, J = 5.4 Hz, 2H), 3.70 (s, 3H), 3.67–3.56 (m, 2H), 3.55–3.47 (m, 1H), 2.89–2.77 (m, 2H),
.76–2.64 (m, 1H), 2.40–2.28 (m, 1H), 2.09–2.04 (m, 1H), 2.00–1.83 (m, 2H), 1.44–1.35 (m, 2H),
.22 (d, J = 6.9 Hz, 6H), 1.19 (d, J = 6.2 Hz, 6H), 0.96 (d, J = 2.8 Hz, 6H). 13C-NMR (150 MHz, CDCl3)
: 156.38( 2), 154.32( 2), 140.72, 131.48, 131.26, 131.19, 128.34, 127.93( 2), 125.35, 124.23, 122.43,
20.83, 110.53( 2), 68.17, 55.31( 2), 54.44, 53.43, 51.10, 41.56, 35.50, 33.14, 29.38, 28.96( 2), 28.23,
7.79, 24.19, 24.13, 22.11( 2). HRMS calcd for C H F N O , [M + H] , 655.3256; found 655.3344.
HPLC: t = 10.757 min, 96.8%.
δ 7.77 (s, 2H), 7.76 (s, 1H), 7.09 (dd, J = 8.4, 2.2 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H),
×
×
×
1
2
×
×
×
+
×
35
45
6
2
3
R
0
0
0
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((5 -isopropyl-2 -methoxy-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1 -biphenyl]
-
2-yl)methyl)azetidin-3-amine-1-methanoic acid tetrahydro-2H-pyran-4-ol ester (37): Colourless oil;
1
yield 73.6%; H NMR (600 MHz, CDCl )
δ 7.78 (s, 2H), 7.76 (s, 1H), 7.09 (dd, J = 8.4, 2.1 Hz, 1H),
3
6
3
2
.80 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 2.2 Hz, 1H), 4.79 (tt, J = 8.4, 4.0 Hz, 1H), 3.89–3.83 (m, 3H),
.82–3.78 (m, 1H), 3.75 (s, 2H), 3.71 (s, 3H), 3.62 (s, 2H), 3.55–3.49 (m, 3H), 2.90–2.77 (m, 2H),
.73 (s, 1H), 2.39–2.30 (m, 1H), 2.09–2.02 (m, 1H), 2.00–1.94 (m, 1H), 1.92–1.85 (m, 3H), 1.64–1.59
13
(
δ
m, 2H), 1.45–1.34 (m, 2H), 1.22 (d, J = 6.9 Hz, 6H), 0.97 (d, J = 4.2 Hz, 6H). C-NMR (150 MHz, CDCl3)
: 155.77( 2), 154.30, 140.76, 131.53, 131.31, 131.14, 128.36, 127.90( 3), 125.40, 124.20, 122.40, 120.92,
2), 69.44, 65.26( 2), 55.33( 2), 54.49, 53.46, 41.59, 35.48, 33.14(
×
×
1
10.57(
×
×
×
×
2), 32.07(
×
2), 29.40, 28.98( 2),
×
+
2
8.23, 27.82, 24.20, 24.13. HRMS calcd for C H F N O , [M + H] , 697.3362; found 697.3459.
37
47
6
2
4
HPLC: t = 16.003 min, 95.8%.
R
N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)-
2
-methyl-2H-tetrazol-5-amine (40): The title compound was obtained in a manner similar to that
1
described for the preparation of intermediate 28. Colourless oil; yield 70.5%; H-NMR (600 MHz,
DMSO-d6)
.10 (s, 3H), 3.99–3.93 (m, 2H), 3.60 (s, 3H), 2.65 (dt, J = 13.7, 6.9 Hz, 1H), 2.32 (d, J = 18.0 Hz,
H), 2.01 (d, J = 18.0 Hz, 1H), 1.79–1.72 (m, 2H), 1.35 (t, J = 6.4 Hz, 2H), 1.02 (d, J = 6.9 Hz, 6H),
.89 (d, J = 13.2 Hz, 6H). 13C-NMR (150 MHz, CDCl3)
: 169.46, 154.12, 141.34, 140.83, 135.26,
δ 7.92 (s, 1H), 7.64 (s, 2H), 6.98 (dd, J = 8.4, 2.2 Hz, 1H), 6.77–6.73 (m, 2H), 4.50 (s, 2H),
4
1
0
δ
1
4
5
31.26(
×
2), 130.54, 128.05, 127.70, 127.65, 125.61, 124.16, 122.35, 120.60, 110.59(
×
2), 55.21, 51.59, 49.22,
+
0.56, 39.20, 35.44, 33.02, 29.09, 29.02, 28.08, 27.99, 23.98(
96.2746; found 596.2847. HPLC: t = 8.776 min, 96.1%.
×
2). HRMS calcd for C H F N O, [M + H] ,
3
0
36
6
5
R
N-((2-(5-Isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)-2-methyl-N-(3-(trifluoromethyl)-
1
benzyl)-2H-tetrazol-5-amine (41): Colourless oil; yield 78.4%; H-NMR (600 MHz, DMSO-d )
δ 7.52
6
(
d, J = 7.7 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.34 (s, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.02 (dd, J = 8.5, 2.3 Hz,
1
3
1
H), 6.80 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 2.3 Hz, 1H), 4.41 (s, 2H), 4.10 (s, 3H), 3.92 (q, J = 14.7 Hz, 2H),
.63 (s, 3H), 2.68 (dt, J = 13.8, 6.9 Hz, 1H), 2.32 (d, J = 18.0 Hz, 1H), 2.04 (d, J = 18.1 Hz, 1H), 1.76 (s, 2H),
.38 (t, J = 6.5 Hz, 2H), 1.05 (dd, J = 6.9, 0.8 Hz, 6H), 0.91 (d, J = 5.1 Hz, 6H). C-NMR (150 MHz,
13
CDCl3) : 169.66, 154.26, 140.64, 139.40, 134.35, 130.80, 130.73, 128.32, 128.03, 125.36, 124.31, 124.29,
δ
1
2
23.43, 123.40, 110.45(
×
2), 55.17, 51.16, 49.31, 40.42, 39.15, 35.54, 33.05, 29.19, 29.03, 28.20, 27.98, 24.09,
+
4.01. HRMS calcd for C H F N O, [M + H] , 528.2872; found 528.2960. HPLC: t = 8.335 min, 96.2%.
29
37
3
5
R
N-(4-Fluorobenzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)-2-methyl-2H-
1
tetrazol-5-amine (42): Colourless oil; yield 80.1%; H-NMR (600 MHz, DMSO-d )
δ
7.07–7.05 (m, 1H),
6

Molecules 2017, 22, 1658
13 of 15
6
3
1
0
.97–6.93 (m, 4H), 6.86–6.85 (m, 1H), 6.80 (d, J = 2.3 Hz, 1H), 4.30 (q, J = 15.8 Hz, 2H), 4.10 (s, 3H),
.85 (dd, J = 44.8, 14.8 Hz, 2H), 3.66 (s, 3H), 2.72 (dt, J = 13.8, 6.9 Hz, 1H), 2.32 (d, J = 18.0 Hz,
H), 2.08 (d, J = 18.0 Hz, 1H), 1.76 (s, 2H), 1.40 (t, J = 6.5 Hz, 2H), 1.09 (dd, J = 6.9, 0.6 Hz, 6H),
.93 (s, 6H). 13C- NMR (150 MHz, CDCl3)
: 169.67, 154.42, 140.66, 133.58, 130.93, 129.47, 129.41, 128.30,
28.14, 127.90, 125.25, 114.72, 114.58, 110.44(
δ
1
×
2), 55.23, 50.53, 48.79, 40.35, 39.12, 35.64, 33.12, 29.28,
+
29.04, 28.25, 28.01, 24.20, 24.06. HRMS calcd for C H FN O, [M + H] , 478.2904; found 478.2999.
28
37
5
HPLC: t = 7.750 min, 96.7%.
R
N-((2-(5-Isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)-2-methyl-N-(4-(trifluoromethyl)-
1
benzyl)-2H-tetrazol-5-amine (43): Colourless oil; yield 67.5%; H-NMR (600 MHz, CDCl )
δ 7.37
3
(d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 7.02 (dd, J = 8.4, 2.3 Hz, 1H), 6.76 (d, J = 2.3 Hz, 1H),
6.70 (d, J = 8.4 Hz, 1H), 4.45 (s, 2H), 4.11 (s, 3H), 4.02 (s, 2H), 3.65 (s, 3H), 2.73 (dt, J = 13.8, 6.9 Hz,
1
1
1
3
5
H), 2.44 (d, J = 18.2 Hz, 1H), 2.12 (d, J = 18.1 Hz, 1H), 1.84 (d, J = 1.6 Hz, 2H), 1.48–1.42 (m, 2H),
1
3
.14 (dd, J = 6.9, 1.8 Hz, 6H), 0.96 (s, 6H). C-NMR (150 MHz, CDCl3)
δ: 169.62, 154.32, 142.35, 140.67,
34.24, 130.74, 128.03, 127.98, 127.70( 3), 125.30, 124.87, 124.85, 110.43(
9.16, 35.60, 33.06, 29.22, 29.05, 28.30, 27.93, 24.15, 23.98. HRMS calcd for C H F N O, [M + H] ,
×
×
2), 55.20, 50.82, 49.03, 40.41,
+
29
37
3
5
28.2872; found 528.2953. HPLC: t = 8.985 min, 96.7%.
R
N-((2-(5-Isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)-2-methyl-N-(4-(trifluoro-methoxy)
1
benzyl)-2H-tetrazol-5-amine (44): Colourless oil; yield 66.3%; H-NMR (600 MHz, DMSO-d )
δ 7.14
6
(d, J = 8.1 Hz, 2H), 7.06–7.01 (m, 3H), 6.82 (d, J = 8.5 Hz, 1H), 6.78 (d, J = 2.2 Hz, 1H), 4.35 (s, 2H),
4
2
0
.11 (s, 3H), 3.88 (m, 2H), 3.66 (s, 3H), 2.70 (dt, J = 13.8, 6.9 Hz, 1H), 2.33 (d, J = 18.4 Hz, 1H),
.06 (d, J = 18.0 Hz, 1H), 1.76 (s, 2H), 1.39 (t, J = 6.4 Hz, 2H), 1.06 (dd, J = 6.9, 1.0 Hz, 6H),
.92 (d, J = 1.6 Hz, 6H). 13C-NMR (150 MHz, CDCl3)
δ: 169.66, 154.37, 140.66, 136.94, 133.96, 130.81,
1
3
29.00(
×
3), 128.15, 128.11, 125.29, 120.46(
×
2), 110.43(
×
2), 55.19, 50.66, 48.76, 40.39, 39.13, 35.61,
+
3.10, 29.21, 29.03, 28.25, 27.97, 24.14, 24.01. HRMS calcd for C H F N O , [M + H] , 544.2821;
29
37
3
5
2
found 544.2898. HPLC: t = 8.302 min, 97.3%.
R
3
.2. In Vitro Test for CETP Inhibitory Activity
All tested compounds were dissolved in 100% DMSO, making sure the compound was dissolved
totally. The solution was vibrated hard on an oscillator for more than 30 s 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 activity was estimated in accordance with
the instruction for the CETP inhibitor screening kit and recombinant CETP. Compounds were
tested at eight concentrations, and the fluorescence intensity were measured using a fluorometer
(
ExEm = 465/535 nm). The IC50 was determined from a curve fit of the data with each concentration
tested three times.
3
.3. Cytochrome P450 Inhibition Assay
Five specific probe substrates (CYP3A4, 2
CYP2C9, 5 µM diclofenac; CYP1A2, 10 µM phenacetin; CYP2C19, 30 µ
µ
M midazolam; CYP2D6, 5
M S-mephenytoin) were used
to evaluated cytochrome P450 inhibition in human liver microsomes (0.25 mg/mL). A mixture of
µM dextromethorphan;
20
µL specific probe substrate solution and 20
µ
L buffer solution (100 mM K PO , 33 mM MgCl ,
3
4
2
pH = 7.4) was added compound (2
The mixture were incubated at 37 C for 10 min and another 10 min underwent after 20
solution added. Consecutively, 400
µ
L) and 158
µ
L human liver microsomes solution (0.25 mg/mL).
L NADPH
L cold stop solution (200 ng/mL tolbutamide and 200 ng/mL
◦
µ
µ
labetalol in acetonitrile) was added to terminated the reaction. After the reactions were terminated,
The plates were centrifuged (4000 rpm) at room temperature for 20 min, and the supernatants were
analysed by LC/MS/MS.

Molecules 2017, 22, 1658
14 of 15
3
.4. Metabolic Stability Study
Ten L (100 M/L) of compound and 80
7 C for 10 min, and then 10 µL NADPH regenerating system was added. Samples were obtained
µ
µ
µ
L liver microsomes were mixture and incubated at
◦
3
at 0 min, 5 min, 10 min, 20 min, 30 min and 60 min respectively, and 300
µ
L stop solution (cold in
◦
4
C, including 100 ng/mL tolbutamide and 100 ng/mL labetalol) was added to terminate the reaction.
After oscillating for 10 min, the plates were centrifuged (4000 rpm) at room temperature for 20 min,
and the supernatants were used for analyzation.
4
. Conclusions
A series of novel N,N-substituted amine derivatives were designed by utilizing bioisosterism.
New compounds were synthesized and evaluated for their inhibitory activity against CETP by
a BODIPY-CE fluorescence assay. Compound 17 was identified as a promising CETP inhibitor with
good inhibitory activity (IC50 = 0.38
liver microsomes stability and low CYP inhibition.
±
0.08 µM), and compound 17 demonstrated weak human/rat
Acknowledgments: 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.
Author Contributions: Xinran Wang and Lijuan Hao designed and carried out the experimental and wrote
the paper; Xuanqi Xu, Wei Li and Chunchi Liu assisted in experiment; Dongmei Zhao and Maosheng Cheng
supervised the whole experiment and provided technical guidance. All authors have read and approved the
final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
2
3
.
.
.
Le Goff, W.; Guerin, M.; Chapman, M.J. Pharmacological modulation of cholesteryl ester transfer protein,
a new therapeutic target in atherogenic dyslipidemia. Pharmacol. Ther. 2004, 101, 17–38. [CrossRef] [PubMed]
Barter, P.J. Hugh Sinclair Lecture: The regulation and remodelling of HDL by plasma factors. Atheroscler. Suppl.
2
002, 3, 39–47. [CrossRef]
Curb, J.D.; Abbott, R.D.; Rodriguez, B.L.; Masaki, K.; Chen, R.; Sharp, D.S.; Tall, A.R. A prospective study of
HDL-C and cholesteryl ester transfer protein gene mutations and the risk of coronary heart disease in the
elderly. J. Lipid Res. 2004, 45, 948–953. [CrossRef] [PubMed]
4
5
.
.
Barter, P.; Gotto, A.M.; LaRosa, J.C.; Maroni, J.; Szarek, M.; Grundy, S.M.; Kastelein, J.J.P.; Bittner, V.;
Fruchart, J.C. HDL cholesterol, very low levels of LDL cholesterol, and cardiovascular events. N. Engl. J. Med.
2
007, 357, 1301–1310. [CrossRef] [PubMed]
Di Angelantonio, E.; Sarwar, N.; Perry, P.; Kaptoge, S.; Ray, K.K.; Thompson, A.; Wood, A.M.; Lewington, S.;
Sattar, N.; Packard, C.J.; et al. Major Lipids, Apolipoproteins, and Risk of Vascular Disease. JAMA 2009, 302,
1
993–2000. [PubMed]
6
.
.
De Grooth, G.J.; Klerkx, A.H.E.M.; Stroes, E.S.G.; Stalenhoef, A.F.H.; Kastelein, J.J.P.; Kuivenhoven, J.A.
A review of CETP and its relation to atherosclerosis. J. Lipid Res. 2004, 45, 1967–1974. [CrossRef] [PubMed]
Chapman, M.J.; Le Goff, W.; Guerin, M.; Kontush, A. Cholesteryl ester transfer protein: at the heart of
the action of lipid-modulating therapy with statins, fibrates, niacin, and cholesteryl ester transfer protein
inhibitors. Eur. Heart J. 2010, 31, 149–164. [CrossRef] [PubMed]
7
8
.
.
Mabuchi, H.; Nohara, A.; Inazu, A. Cholesteryl ester transfer protein (CETP) deficiency and CETP Inhibitors.
Mol. Cells 2014, 37, 777–784. [CrossRef] [PubMed]
Brousseau, M.E.; Schaefer, E.J.; Wolfe, M.L.; Bloedon, L.T.; Digenio, A.G.; Clark, R.W.; Mancuso, J.P.;
Rader, D.J. Effects of an inhibitor of cholesteryl ester transfer protein on HDL cholesterol. N. Engl. J. Med.
9
2
004, 350, 1505–1515. [CrossRef] [PubMed]
1
0. Barter, P.J.; Caulfield, M.; Eriksson, M.; Grundy, S.M.; Kastelein, J.J.P.; Komajda, M.; Lopez-Sendon, J.;
Mosca, L.; Tardif, J.; Waters, D.D.; et al. Effects of torcetrapib in patients at high risk for coronary events.
N. Engl. J. Med. 2007, 357, 2109–2122. [CrossRef] [PubMed]

Molecules 2017, 22, 1658
15 of 15
1
1
1
1
1
1. Nissen, S.E.; Tardif, J.; Nicholls, S.J.; Revkin, J.H.; Shear, C.L.; Duggan, W.T.; Ruzyllo, W.; Bachinsky, W.B.;
Lasala, G.P.; Tuzcu, E.M. Effect of torcetrapib on the progression of coronary atherosclerosis. N. Engl. J. Med.
007, 356, 1304–1316. [CrossRef] [PubMed]
2. Bots, M.L.; Visseren, F.L.; Evans, G.W.; Riley, W.A.; Revkin, J.H.; Tegeler, C.H.; Shear, C.L.; Duggan, W.T.;
Vicari, R.M.; Grobbee, D.E.; et al. Torcetrapib and carotid intima-media thickness in mixed dyslipidaemia
2
(RADIANCE 2 study): a randomised, double-blind trial. Lancet 2007, 370, 153–160. [CrossRef]
3. Kastelein, J.J.P.; van Leuven, S.I.; Burgess, L.; Evans, G.W.; Kuivenhoven, J.A.; Barter, P.J.; Revkin, J.H.;
Grobbee, D.E.; Riley, W.A.; Shear, C.L.; et al. Effect of torcetrapib on carotid atherosclerosis in familial
hypercholesterolemia. N. Engl. J. Med. 2007, 356, 1620–1630. [CrossRef] [PubMed]
4. Luscher, T.F.; Taddei, S.; Kaski, J.C.; Jukema, J.W.; Kallend, D.; Munzel, T.; Kastelein, J.J.P.; Deanfield, J.E.;
Dal-VESSEL Investigators. Vascular effects and safety of dalcetrapib in patients with or at risk of coronary
heart disease: the dal-VESSEL randomized clinical trial. Eur. Heart J. 2012, 33, 857–865. [CrossRef] [PubMed]
5. Fayad, Z.A.; Mani, V.; Woodward, M.; Kallend, D.; Abt, M.; Burgess, T.; Fuster, V.; Ballantyne, C.M.;
Stein, E.A.; Tardif, J.C.; et al. Safety and efficacy of dalcetrapib on atherosclerotic disease using novel
non-invasive multimodality imaging (dal-PLAQUE): A randomised clinical trial. Lancet 2011, 378, 1547–1559.
[CrossRef]
1
1
1
6. Schwartz, G.G.; Olsson, A.G.; Abt, M.; Ballantyne, C.M.; Barter, P.J.; Brumm, J.; Chaitman, B.R.; Holme, I.M.;
Kallend, D.; Leiter, L.A.; et al. Effects of Dalcetrapib in Patients with a Recent Acute Coronary Syndrome.
N. Engl. J. Med. 2012, 367, 2089–2099. [CrossRef] [PubMed]
7. Tardif, J.C.; Rheaume, E.; Perreault, L.P.L.; Gregoire, J.C.; Zada, Y.F.; Asselin, G.; Provost, S.; Barhdadi, A.;
Rhainds, D.; L’Allier, P.L.; et al. Pharmacogenomic Determinants of the Cardiovascular Effects of Dalcetrapib.
Circ. Cardiovasc. Genet. 2015, 8, 372–382. [CrossRef] [PubMed]
8. Nicholls, S.J.; Brewer, H.B.; Kastelein, J.J.P.; Krueger, K.A.; Wang, M.D.; Shao, M.Y.; Hu, B.; McErlean, E.;
Nissen, S.E. Effects of the CETP Inhibitor Evacetrapib Administered as Monotherapy or in Combination
With Statins on HDL and LDL Cholesterol A Randomized Controlled Trial. JAMA 2011, 306, 2099–2109.
[CrossRef] [PubMed]
1
9. Nicholls, S.J.; Lincoff, A.M.; Barter, P.J.; Brewer, H.B.; Fox, K.A.A.; Gibson, C.M.; Grainger, C.; Menon, V.;
Montalescot, G.; Rader, D.; et al. Assessment of the clinical effects of cholesteryl ester transfer protein
inhibition with evacetrapib in patients at high-risk for vascular outcomes: Rationale and design of the
ACCELERATE trial. Am. Heart J. 2015, 170, 1061–1069. [CrossRef] [PubMed]
2
2
0. Cannon, C.P.; Shah, S.; Dansky, H.M.; Davidson, M.; Brinton, E.A.; Gotto, A.M.; Stepanavage, M.; Liu, S.X.;
Gibbons, P.; Ashraf, T.B.; et al. Safety of Anacetrapib in Patients with or at High Risk for Coronary Heart
Disease. N. Engl. J. Med. 2010, 363, 2406–2415. [CrossRef] [PubMed]
1. Brinton, E.A.; Kher, U.; Shah, S.; Cannon, C.P.; Davidson, M.; Gotto, A.M.; Ashraf, T.B.; Sisk, C.M.; Dansky, H.;
Mitchel, Y.; et al. Effects of anacetrapib on plasma lipids in specific patient subgroups in the DEFINE
(
Determining the Efficacy and Tolerability of CETP INhibition with AnacEtrapib) trial. J. Clin. Lipidol. 2015
,
9
, 65–71. [CrossRef] [PubMed]
2
2. Ford, J.; Lawson, M.; Fowler, D.; Maruyama, N.; Mito, S.; Tomiyasu, K.; Kinoshita, S.; Suzuki, C.;
Kawaguchi, A.; Round, P.; et al. Tolerability, pharmacokinetics and pharmacodynamics of TA-8995, a selective
cholesteryl ester transfer protein (CETP) inhibitor, in healthy subjects. Br. J. Clin. Pharmacol. 2014, 78, 498–508.
[CrossRef] [PubMed]
2
2
3. Hovingh, G.K.; Kastelein, J.J.P.; van Deventer, S.J.H.; Round, P.; Ford, J.; Saleheen, D.; Rader, D.J.; Brewer, H.B.;
Barter, P.J. Cholesterol ester transfer protein inhibition by TA-8995 in patients with mild dyslipidaemia
(TULIP): A randomised, double-blind, placebo-controlled phase 2 trial. Lancet 2015, 386, 452–460. [CrossRef]
4. Liu, C.C.; Luo, C.Q.; Hao, L.J.; Wu, Q.; Xie, H.L.; Zhao, S.Z.; Hao, C.Z.; Zhao, D.M.; Cheng, M.S. Design,
synthesis and biological evaluation of novel cholesteryl ester transfer protein inhibitors bearing a cycloalkene
scaffold. Eur. J. Med. Chem. 2016, 123, 419–430. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 12–22, 32–37, 40–44 are available from the authors.
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