Design, Synthesis and Biological Evaluation of N

molecules Article

Design, Synthesis and Biological Evaluation of N,N-Substituted Amine Derivatives as Cholesteryl Ester Transfer Protein Inhibitors Xinran Wang 1 , Lijuan Hao 1 , Xuanqi Xu 2 , Wei Li 1 , Chunchi Liu 1 , Dongmei Zhao 1, * Maosheng Cheng 1 1

2

*

ID

and

Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China; [email protected] (X.W.); [email protected] (L.H.); [email protected] (W.L.); [email protected] (C.L.); [email protected] (M.C.) Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53715, USA; [email protected] Correspondence: [email protected]; Tel.: +86-24-4352-0219

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 µM) in vitro. Furthermore, compound 17 was selected for an in vitro metabolic stability study. 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) [1,2]. Epidemiological studies have provided compelling evidence to demonstrate an inverse association between HDL-C and cardiovascular events [3–5]. Therefore, CETP inhibitors 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 [9–13]. Accordingly, torcetrapib’s phase III trial was halted in 2006. Dalcetrapib (2, 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 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

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III, could raise HDL-C levels to 130% and decrease LDL-C levels to 40%. Researchers found that Molecules 2017, 22, 1658 2 of 15 anacetrapib demonstrated potential clinical benefits and would not produce adverse clinical effects similarthat to those observed with torcetrapib the DEFINE [20,21].not TA-8995 (5,adverse Figure clinical 1), a novel anacetrapib demonstrated potentialinclinical benefitsstudy and would produce similar thosetolerated observedand withhad torcetrapib in the DEFINE study [20,21]. Figure CETP effects inhibitor, wastowell beneficial effects on lipids, raisingTA-8995 HDL-C(5, levels by1), up to a novel CETP inhibitor, was well tolerated and had beneficial effects on lipids, raising HDL-C levels 179% and decreasing LDL-C by up to 45% [22,23]. It remains to be seen whether these potent CETP Molecules 2017, 22, 1658 2 of 15 by upwill to 179% and decreasing by up to 45% [22,23]. It remains to be seen whether these potent inhibitors proceed further in LDL-C the future. CETP inhibitors willdemonstrated proceed further in theclinical future. that anacetrapib potential benefits and would not produce adverse clinical

effects similar to those observed with torcetrapib in the DEFINE study [20,21]. TA-8995 (5, Figure 1), a novel CETP inhibitor, was well tolerated and had beneficial effects on lipids, raising HDL-C levels by up to 179% 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.

Figure 1. Representative Figure 1. RepresentativeCETP CETP inhibitors. inhibitors.

In a previous study, compound 6 (Figure 2) was identified to show weak micromolar activity

Figure 1. Representative inhibitors. to show weak micromolar activity In a previous study, compound 6 (Figure 2) wasCETP identified (IC50 = 20.97 μM) [24]. Our initial approach was to build a ring to replace the carbamate part to (IC50 = 20.97 µM) [24]. Our initial approach was to build a ringthat to tetrazole replace the carbamate part to investigate effect study, of steric hindrance on activity. is a bioisoster In athe previous compound 6 (Figure 2) was Considering identified to show weak micromolar activity of the investigate effect of steric hindrance on that tetrazole is a bioisoster of the carboxylic the of activity. compound 6 could as carboxylic acidtomethyl (ICthe 50 = acid 20.97 and μM) [24].carbamate Our initialpart approach was to Considering build a ringbetotreated replace thea carbamate part carboxylic acid and the carbamate part of compound 6 could be treated as a carboxylic acid methyl investigate the effect of steric hindrance on activity. Considering that tetrazole is a bioisoster of the ester, we hold that 2-methyltetrazole is a bioisoster of carbamate. In this paper, our group carboxylic acid cyclization and the carbamate part of compound 6 could treated as apaper, carboxylic ester, we hold that the 2-methyltetrazole a bioisoster ofusing carbamate. In this ouracid group investigated investigated of is the carbamate a be bioisosterism strategy to methyl afford a 2ester, we hold that 2-methyltetrazole is a bioisoster of carbamate. In this paper, our methyltetrazole compound. using Fortunately, the compound containing theatetrazole moietygroup (40,compound. Figure the cyclization of the carbamate a bioisosterism strategy to afford 2-methyltetrazole investigated the cyclization of the carbamate using a bioisosterism strategy to afford a 22, IC50 = the 0.81 compound ± 0.03 μM) showed increased inhibitory activity. A(40, series of novel N,N-substituted Fortunately, containing thethe tetrazole moiety IC50 (40, = 0.81 ± amine 0.03 µM) methyltetrazole compound. Fortunately, compound containing theFigure tetrazole2,moiety Figure derivatives were then synthesized. Optimization efforts on this scaffold are discussed in this study. showed increased inhibitory activity. A series of novel N,N-substituted derivatives 2, IC50 = 0.81 ± 0.03 μM) showed increased inhibitory activity. A series of novel amine N,N-substituted aminewere then derivatives were thenefforts synthesized. Optimization on this scaffold are study. discussed in this study. synthesized. Optimization on this scaffold efforts are discussed in this

Figure 2. Design of of new based on compound compound Figure Design new structures based on on compound 6. 6.6. Figure 2.2.Design newstructures structures based 2. Results and Discussion

2. Results and Discussion 2. Results and Discussion 2.1. Chemistry

2.1. Chemistry 2.1. Chemistry

Compounds 12–22 were prepared according to the procedure shown in Scheme 1. The

Compounds10 12–22 were inprepared according todescribed the procedure shownpublished in Scheme 1. The intermediate obtained a manner similar that inshown the previously paper Compounds 12–22was were prepared according totothe procedure in Scheme 1. The intermediate intermediate 10 was obtained incyanogen a mannerbromide similarunder to that described in the previously published [24]. The treatment of 10 with basic conditions furnished 11 in good yield. paper 10 was obtained in a manner similar to that described in the previously published paper [24]. the -CN ofofthe intermediate was subjected to cycloaddition with sodium to yield. [24]. Then, The treatment 10resulting with cyanogen bromide under basic conditions furnished 11 azide in good The treatment of12. 10of with cyanogen bromide under basic conditions furnished 11 in good yield. produce Compound 12 was subjected to was substitution reactions with various substituted alkyl Then, the -CN the resulting intermediate subjected to cycloaddition with sodium azide to Then, produce the bromides -CN12. of Compound the resulting intermediate was subjected to cycloaddition with sodium azide to to yield compounds 13–15. Alternatively, treatment of 12 with 2-(methylsulfonyl) ethanol 12 was subjected to substitution reactions with various substituted alkyl under Mitsunobu conditions afforded 16. Carboxylic acid derivatives 17 and 18 were prepared by produce 12. Compound 12 was subjected to substitution reactions with various substituted bromides to yield compounds 13–15. Alternatively, treatment of 12 with 2-(methylsulfonyl) ethanolalkyl hydrolysis of the ester groups of compounds 13 and 14, respectively. Subsequent reduction or bromides toMitsunobu yield compounds 13–15. Alternatively, treatment of 12 with 2-(methylsulfonyl) under conditions afforded 16. Carboxylic acid derivatives 17 and 18 were preparedethanol by aminolysis reaction of compounds 13 and 14 produced compounds 19–21. Compound 11 reacted of the ester groups of compounds 13 and 14, respectively. Subsequent reduction or by underhydrolysis Mitsunobu conditions afforded 16. Carboxylic acid derivatives 17 and 18 were prepared with hydroxylamine hydrochloride and subsequently, acetic anhydride, to obtained compound 22. aminolysis reaction of compounds 13 and 14 produced compounds 19–21. Compound 11 reacted hydrolysis of the ester groups of compounds 13 and 14, respectively. Subsequent reduction or with hydroxylamine hydrochloride anhydride, to obtained compound 22. aminolysis reaction of compounds 13 and andsubsequently, 14 producedacetic compounds 19–21. Compound 11 reacted with hydroxylamine hydrochloride and subsequently, acetic anhydride, to obtained compound 22.

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Compounds 32–37 were prepared according to the procedure in Scheme 2. The resulting 9 was reacted Compounds Molecules 2017, 32–37 22, 1658 were prepared according to the procedure in Scheme 2. The resulting 9 3was of 15 with NaBH 4 and subsequently, SOCl2 to afford intermediate 23. reacted with NaBH4 and subsequently, SOCl2 to afford intermediate 23. Compounds 32–37 were prepared according to the procedure in Scheme 2. The resulting 9 was reacted with NaBH4 and subsequently, SOCl2 to afford intermediate 23.

Scheme 1. Synthesis of target compounds 12–22. Reagents and conditions: (a) DMF, PBr3, CHCl3, rt; (b)

Scheme 1. Synthesis of target compounds 12–22. 2,Reagents and conditions: (a)80DMF, 5-isopropyl-2-methoxyphenylboronic acid, Pd(OAc) K2CO3, acetylacetone, EtOH, °C; (c)PBr 3,5-3 , CHCl3 , ◦ rt; (b) bis(trifluromethyl)benzyl 5-isopropyl-2-methoxyphenylboronic Pd(OAc) , DMF, acetylacetone, EtOH, Scheme 1. Synthesis of target compounds 12–22. Reagents and conditions: PBr3, CHCl , rt;(e) (b) 80 C; amine, NaBH(OAc) 3acid, , 1,2-dichloroethane, (d)(a) DIEA, THF, 3rt; 2 , Krt; 2 CO 3CNBr, 5-isopropyl-2-methoxyphenylboronic acid, Pd(OAc)3reflux; 2,, 1,2-dichloroethane, K2CO EtOH, 80 °C; reflux, (c) 3,5- THF, rt; NaN 3, NH4Cl, DMF, 100 °C; (f) amine, R1-X, Et3NaBH(OAc) N, acetonitrile, or3,(i)acetylacetone, Boc-R1-X, Et3N, acetonitrile, (c) 3,5-bis(trifluromethyl)benzyl rt; (d) CNBr, DIEA, ◦ C; bis(trifluromethyl)benzyl amine, NaBH(OAc) 3,acetonitrile, 1,2-dichloroethane, rt; (d)(i)CNBr, DIEA, THF, rt; (e) (ii) TFA, DCM, rt; or R 1-OH, PPh 3,R DIAD, rt; (g) NaBH 4/MeOH,reflux; THF, reflux; or NH 3-EtOH, rt; (h) (i) (e) NaN , NH Cl, DMF, 100 (f) -X, Et N, or Boc-R -X, Et N, acetonitrile, 3 4 1 3 1 3 NaN 3, NH 4Cl, DMF, 100 °C; (f) R(l) 1-X, Et23OH· N, acetonitrile, or (i) Boc-R 1-X, Et3N, acetonitrile, reflux, 1 M aq. NaOH, (ii) 1 M aq. HCl; NH HCl, Ac 2O, reflux; Et 3N, EtOH, reflux. reflux, (ii) TFA, DCM, rt; or R1 -OH, PPh3 , DIAD, rt; (g) NaBH4 /MeOH, THF, reflux; or NH3 -EtOH, rt; (ii) TFA, DCM, rt; or R1-OH, PPh3, DIAD, rt; (g) NaBH4/MeOH, THF, reflux; or NH3-EtOH, rt; (h) (i) (h) (i) 1 M aq. NaOH, (ii) 1 M aq. HCl; (l) NH2 OH·HCl, Ac2 O, Et3 N, EtOH, reflux. 1 M aq. NaOH, (ii) 1 M aq. HCl; (l) NH2OH·HCl, Ac2O, Et3N, EtOH, reflux.

Scheme 2. Synthesis of target compounds 32–37. Reagents and conditions: (a) (i) NaBH4 , MeOH, rt, (ii) SOCl2 , DMF, rt; (b) 3,5-bis(trifluromethyl)benzaldehyde, NaBH4 , MeOH, rt; (c) NaH, DMF, 0 ◦ C; (d) TFA/CH2 Cl2 (1:1), rt; (e) (i) methyl 2-bromoacetate, TEA, DMF, rt, (ii) NaBH4 /MeOH, THF, reflux; or 1 M aq. NaOH, 1 M aq. HCl; (f) TEA, DCM, rt.

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Scheme 2. Synthesis of target compounds 32–37. Reagents and conditions: (a) (i) NaBH4, MeOH, rt, (ii) SOCl2, DMF, rt; (b) 3,5-bis(trifluromethyl)benzaldehyde, NaBH4, MeOH, rt; (c) NaH, DMF, 0 °C; (d) Molecules 2017, 22, TFA/CH 2Cl1658 2 (1:1), rt; (e) (i) methyl 2-bromoacetate, TEA, DMF, rt, (ii) NaBH4/MeOH, THF, reflux; or4 of 15 1 M aq. NaOH, 1 M aq. HCl; (f) TEA, DCM, rt.

The 3,5-bis The starting starting materials materials 24 24 and and 2525were weresubjected subjectedto toreductive reductiveamination aminationwith with 3,5(trifluromethyl)benzaldehyde, which provided 26 and 27. Intermediates 28 and 29 were obtained bis(trifluromethyl)benzaldehyde, which provided 26 and 27. Intermediates 28 and 29 were obtained by the nucleophilic nucleophilic substitution substitutionofof2323byby2626 and presence of NaH at room temperature. by the and 2727 in in thethe presence of NaH at room temperature. The The key intermediates 30 and 31 were obtained by deprotection of 28 and 29 under the key intermediates 30 and 31 were obtained by deprotection of 28 and 29 under the condition condition of of TFA/DCM (v:v = 1:1). Intermediate 30 was reacted with methyl 2-bromoacetate, then the resultant TFA/DCM (v:v = 1:1). Intermediate 30 was reacted with methyl 2-bromoacetate, then the resultant ester ester intermediate intermediate was was subjected subjected to to reduction reduction conditions conditions to to yield yield compound compound 32 32 or or hydrolysis hydrolysis conditions to furnish compound 33. The obtained intermediates 30 and 31 were conditions to furnish compound 33. The obtained intermediates 30 and 31 were treated treated with with corresponding chloroformates to give compounds 34–37. As illustrated in Scheme 3, compounds 40–44 corresponding chloroformates to give compounds 34–37. As illustrated in Scheme 3, compounds 40– were synthesized from the corresponding starting benzaldehydes through reductive amination and 44 were synthesized from the corresponding starting benzaldehydes through reductive amination substitution reactions. and substitution reactions.

Scheme 3. Synthesis of target compounds 40–44. Reagents and conditions: (a) 2-methyl-2H-tetrazolScheme 3. Synthesis of target compounds 40–44. Reagents and conditions: (a) 2-methyl-2H-tetrazol-55-amine, NaBH4 , MeOH, rt; (b) NaH, DMF, 0 ◦ C. amine, NaBH4, MeOH, rt; (b) NaH, DMF, 0 °C.

2.2. 2.2. In In Vitro Vitro Activity Activity Against Against Cholesteryl Cholesteryl Ester Ester Transfer TransferProtein Protein The derivatives and and the reference compound anacetrapib (4) were screened The N,N-substituted N,N-substitutedamine amine derivatives the reference compound anacetrapib (4) were for their in vitro activity against CETP by a BODIPY-CE fluorescence assay with the CETP RP Activity screened for their in vitro activity against CETP by a BODIPY-CE fluorescence assay with the CETP Assay Kit (Catalogue # RB-RPAK; Roar, New York, NY, USA). The results are shown in Table in 1. RP Activity Assay Kit (Catalogue # RB-RPAK; Roar,New York , NY, USA). The results are shown As seen in the table, replacement of a carbamate (6, IC = 20.97 µM) with an N-2-methyltetrazole 50 Table 1. As seen in the table, replacement of a carbamate (6, IC50 = 20.97 µ M) with an N-2(40, IC = 0.81 ± 0.03 µM) caused a significant increase in the activity. we investigated 50 methyltetrazole (40, IC50 = 0.81 ± 0.03 µ M) caused a significant increaseSubsequently, in the activity. Subsequently, the relationship between various substituents at the 2-position of the tetrazole (Part A) and the CETP we investigated the relationship between various substituents at the 2-position of the tetrazole (Part inhibitory The replacement of –CH IC503 by = 2.84 ± 0.04 detrimental 3 by –H (12, A) and theactivity. CETP inhibitory activity. The replacement of –CH –H (12, IC50 µM) = 2.84was ± 0.04 µ M) was for activity. for Introduction of carboxylic esters (e.g.,esters compounds 13 and 14) at the of the detrimental activity. Introduction of carboxylic (eg. compounds 13 and 14)2-position at the 2-position tetrazole dramatically reduced the activity, however potency was recovered in the corresponding of the tetrazole dramatically reduced the activity, however potency was recovered in the carboxylic acidscarboxylic (17 and 18) and(17 straight chain alcohols (19 andalcohols 20), where especially two corresponding acids and 18) andsaturated straight chain saturated (19 and 20), where atoms alkyl chains were better. We observed that amine and sulfone groups (15 and 16) were tolerated especially two atoms alkyl chains were better. We observed that amine and sulfone groups (15 and and an amide group a weak decrease the activity. N-2-methyltetrazole 16) were tolerated and(21) an caused amide group (21) caused in a weak decreaseChanging in the activity. Changing N-2(40) to 3-methyoxadiazole (22, IC = 0.72 ± 0.06 µM) showed no advantage. We speculate that 50 methyltetrazole (40) to 3-methyoxadiazole (22, IC50 = 0.72 ± 0.06 µ M) showed no advantage. We carboxylic acids and saturated alcohols at the 2-position of tetrazole provide an important speculate that carboxylic acids and saturated alcohols at the 2-position of tetrazole contribution provide an to the potency. important contribution to the potency. Next, Next, we we investigated investigated the the relationship relationship between between an an aliphatic aliphatic heterocycle heterocycle (Part (Part A) A) and and the the CETP CETP inhibitory activity. Unfortunately, replacement of N-2-substituted tetrazole with azetidine/ piperidine inhibitory activity. Unfortunately, replacement of N-2-substituted tetrazole with azetidine/ derivatives (compounds(compounds 32–37) caused a dramatic in the activity. resultsThese indicate that piperidine derivatives 32–37) causeddecrease a dramatic decrease in These the activity. results aindicate flexiblethat fragment in Part A is unfavourable for inhibitory activity. a flexible fragment in Part A is unfavourable for inhibitory activity. To To further further study study the the relationship relationship between between Part Part B B and and the the CETP CETP inhibitory inhibitory activity, activity, another another five five compounds 40–44 were prepared and evaluated for their activity. Introduction of trifluoromethyl compounds 40–44 were prepared and evaluated for their activity. Introduction of trifluoromethyl groups 0.03 µ µM) groups on on the the 3-position 3-position and and the the 5-position 5-position of ofbenzene benzenering ring(40, (40,IC IC5050 == 0.81 0.81 ± ± 0.03 M) were were more more beneficial A single beneficial for for CETP CETP inhibition. inhibition. A single trifluoromethyl trifluoromethyl group group on on the the meta-position meta-position of of the the benzene benzene ring =10.05 10.05 ±±0.06 0.06µµM) causedaasevere severedecrease decreasein inthe theactivity. activity. A Atrifluoromethyl trifluoromethyl group group (43), (43), ring (41, (41, IC IC50 50 = M) caused fluorine atom (42), and trifluoromethoxy group (44) on the para-position of the benzene ring resulted fluorine atom (42), and trifluoromethoxy group (44) on the para-position of the benzene ring resulted in in no no CETP CETP inhibition. inhibition.

Molecules 2017, 22, 1658 1658 Molecules 2017,2017, 22, 1658 Molecules 22,

of 15 15 5 of 15 55 of 15 5 55ofof of15 15 55 5of 15 of of 1515 15 5 555ofof of15 155 of 15 Molecules 2017, 22, 1658 of 15 Molecules 2017, 22, 1658 5 of 15 Half of the compounds showed excellent activities, and four of them exhibited submicromolar Molecules 2017, 22, 1658 5 of 15 Molecules 2017, 22, 1658 5 of 15 Half of the compounds showed excellent activities, and four of them exhibited submicromolar Molecules 2017, 22, 1658 1515 Molecules 2017, 22, 1658 5ofof of Molecules 2017, 1658 555 of 15 Half of the compounds showed excellent activities, and four of them exhibited submicromolar Molecules 2017, 22, 1658 5 Half of the compounds showed excellent and four of them exhibited submicromolar Molecules 2017, 22, 1658 of 1515 Molecules 2017, 22, 1658 515 of 15 Half of22, the compounds showed excellentactivities, activities, and four of them exhibited submicromolar Half of the compounds showed excellent activities, and four of them exhibited submicromolar Molecules 22, 1658 of 15 Molecules 2017, 22, 1658 Molecules 2017, 22, 1658 5 5of activities, but the activity of ofshowed the mostexcellent outstanding compound 17 (IC 50 0.38 ± exhibited 0.08 μM) μM) was still 10Half of2017, the compounds activities, and four of submicromolar Half of the compounds showed excellent activities, and four them exhibited submicromolar Half of the compounds showed excellent activities, and four ofof exhibited submicromolar activities, but the activity the most outstanding compound 17 (IC 50them == them 0.38 ± 0.08 was still 10Molecules 2017, 22, 1658 Molecules Molecules2017, 2017,22, 22,1658 1658

Molecules Molecules2017, 2017,22, 22,1658 1658 Molecules 2017, 22, 1658 Molecules 2017, 22, 1658 Molecules 2017,2017, 22, 1658 Molecules 2017, 22, 1658 Molecules 22, Molecules 2017, 22,1658 1658

Half of the compounds showed excellent activities, and four of exhibited submicromolar Half of the compounds showed excellent activities, and four of exhibited submicromolar Half of the compounds showed excellent activities, and four of them exhibited submicromolar activities, but the activity of the most outstanding compound 17 (IC 50them 0.38 ± 0.08 μM) was still 10activities, but the activity of most outstanding compound 17 50them ====0.38 ± was Half of compounds showed excellent activities, and four of exhibited submicromolar Half of the compounds showed excellent activities, and four of them exhibited submicromolar activities, but the activity ofthe the most outstanding compound 17(IC (IC 50them 0.38 ±0.08 0.08μM) μM) wasstill still1010activities, but the activity of the most outstanding compound 17 (IC 50 ± 0.08 μM) was still 10Half of the compounds showed excellent activities, and of them exhibited submicromolar Half of compounds showed excellent activities, andfour four of exhibited submicromolar fold weaker than the reference compound. 50 activities, but the activity the most outstanding compound 17 (IC 50 ==0.38 0.38 0.08 μM) was still 10activities, but the activity ofof the most outstanding compound 17 (IC 50 =them 0.38 ±± 0.08 μM) was still 10Half of the compounds showed excellent activities, and four of them exhibited submicromolar Half of the compounds showed excellent activities, and four of them exhibited submicromolar activities, but the activity of the most outstanding compound 17 (IC = 0.38 ± 0.08 µM) was still fold weaker than the reference compound. activities, but the activity of the most outstanding compound 17 (IC 50 0.38 ± 0.08 μM) was still 10activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10Half of the compounds showed excellent activities, and four of them exhibited submicromolar 50 50 Half of the compounds showed excellent activities, and four of them exhibited submicromolar activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was stillactivities, 10fold weaker than the reference compound. fold weaker than the reference compound. Half of the compounds showed excellent activities, and four of them exhibited submicromolar Half of the compounds showed excellent activities, and four of them exhibited submicromolar activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10activities, but activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10fold weaker than the reference compound. Molecules 2017, 22, 1658 Molecules 2017, 22, 1658 5 of 15 Half of the compounds showed excellent activities, and four of them exhibited submicromolar Half of the compounds showed excellent activities, and Half four of them the compounds exhibited submicromolar showed excellent and four of th fold weaker than the reference compound. activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10fold weaker than the reference compound. fold weaker than the reference compound. activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10fold weaker than the reference compound. fold weaker than the reference compound. 10-fold weaker than the reference compound. but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10foldactivities, weaker than the reference compound. activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still 10foldweaker weakerthan thanthe thereference referencecompound. compound. fold 50

activities, butthan theactivity activity thecompound. most outstanding (IC =0.38 0.38 0.08 μM) wasstill still 10activities, but the of the most outstanding compound activities, 1717 (IC but 5050 =32–37, the activity ± ±0.08 of μM) thewas most outstanding 10compound 17 (IC50 = Table 1.ofStructures Structures and activities of ofcompound compounds 12–22, 32–37, 40–44. fold the reference foldweaker weaker than the reference compound. Table 1. and activities compounds 12–22, 40–44. fold weaker than the reference compound. fold weaker than the reference compound. fold weaker than the reference compound. fold weaker than the reference compound. Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. fold weaker than the reference compound. fold weaker than the reference compound. Half of 22, the compounds showed excellent activities, and Half four of them the compounds exhibited submicromolar showed excellent activities, and four of th Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Molecules 2017, 1658 Molecules 2017, 22, 1658 5 of 15 fold weaker than the reference compound. fold weaker than the reference compound. fold weaker than the reference compound. Table Structures and activities compounds 12–22, 32–37, 40–44. Table 1.1. Structures and activities ofof compounds 12–22, 32–37, 40–44. Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Table 1. Structures and activities compounds 12–22, 32–37, 40–44. Table 1.Structures Structures andactivities activitiesof ofcompounds compounds 12–22, 32–37, 40–44. Table 1.Structures Structures and activities ofof compounds 12–22, 32–37, 40–44. Table 1.of and activities of compounds 12–22, 32–37, 40–44. activities, but the activity the most outstanding compound activities, 17 (IC but 50 = the 0.38 activity ± 0.08 of μM) the was most still outstanding 10compound 17 (IC50 = Table 1. and 12–22, 32–37, 40–44. Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Table 1.1. Structures and activities ofof compounds 12–22, 32–37, 40–44. Table Structures and activities compounds 12–22, 32–37, 40–44. Table 1. and activities of 12–22, 32–37, 40–44. Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Table 1.Structures Structures and activities ofcompounds compounds 12–22, 32–37, 40–44. Table 1.Structures Structures and activities ofcompounds compounds 12–22, 32–37, 40–44. Half of 22, the1658 compounds showed excellent activities, and weaker Half four of 22, them the compounds exhibited submicromolar showed excellent activities, and four of th Molecules 2017, Molecules 2017, 1658 5 ofactivities 15 fold weaker than the reference compound. fold than the reference compound. Table Structures and activities compounds 12–22, 32–37, 40–44. Table 1.1. and activities ofof 12–22, 32–37, 40–44. Table 1. Structures and of compounds 12–22, 32 activities, but the activity of the most outstanding compound activities, 17 (IC but 50 = the 0.38 activity ± 0.08 of μM) thewas most still outstanding 10compound 17 (IC50 = Half of 22, the1658 compounds showed excellent activities, and weaker Half four of 22, them the1658 compounds exhibited submicromolar showed excellent activities, and four of th Molecules 2017, Molecules 2017, 5 ofactivities 15 Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Table 1. Structures and of compounds 12–22, 32 fold weaker than the reference compound. fold than the reference compound. activities, but the activity of the most outstanding compound activities, 17 (IC but 50 = the 0.38 activity ± 0.08 of μM) thewas most still outstanding 10compound 17 (IC50 = Molecules 2017, Molecules 2017, 5 ofactivities 15 Half of 22, the1658 compounds showed excellent activities, and weaker Half four of 22, them the1658 compounds exhibited submicromolar showed excellent activities, and four of th Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Table 1. Structures and of compounds 12–22, 32 fold weaker than the reference compound. fold than the reference compound. activities, but the activity of the most outstanding compound activities, 17 (IC but 50 = the 0.38 activity ± 0.08 of μM) thewas most still outstanding 10compound 17 (IC50 = Molecules 2017, 5 ofactivities 15 Half of 22, the1658 compounds showed excellent activities, and weaker Half four of them the compounds exhibited submicromolar showed excellent activities, and four of th Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. Table 1. Structures and of compounds 12–22, 32 fold weaker than the reference compound. fold than the reference compound. activities, but the activity of the most outstanding compound activities, 17 (IC but 50 = the 0.38 activity ± 0.08 of μM) thewas most still outstanding 10compound 17 (IC50 = NO. Part A A R (Part (Part B) B) IC50 50 (µM) NO. Part A A R (Part (Part B) B) IC50 50 (µM) NO. Part R IC (µM) NO. Part R IC (µM) Molecules 2017, 22, 1658 5(µM) of 15 Half of Part the compounds showed excellent activities,NO. and weaker four ofPart them exhibited submicromolar NO. Part A (Part IC (µM) NO. Part (Part (µM) fold weaker than reference compound. fold than reference NO. RR B)B) 50 50 (µM) AA RR B)B) ICIC 50 50 50 50 Table 1. Structures activities 12–22, 32–37, 40–44. Table 1. Structures and activities of compounds 12–22, 32 NO. PartA Athe R(Part (Part B) andIC IC (µM) of compounds NO. Part A the R(Part (Part B)compound. IC (µM) NO. Part A R (Part B) IC 5050 50 (µM) NO. Part A R (Part B) IC 5050 50 (µM) 50 50 NO. Part A R (Part B) IC 50 (µM) NO. Part A R (Part B) IC (µM) NO. Part AA Rof (Part B) ICIC 50 (µM) NO. Part A0.38 RR (Part B)B) IC 5050 (µM) b10NO. Part R (Part B) (µM) NO. Part A (Part IC (µM) NO. Part A R (Part B) IC 5050 (µM) NO. Part A R (Part B) IC 5050 (µM) NO. Part A R (Part B) IC 50 (µM) NO. Part A R (Part B) IC 50 (µM) activities, but the activity the most outstanding compound 17 (IC 50 = ± 0.08 μM) was still 12 33 2.84 ± 0.04 ˃50 b NO. Part A R (Part B) IC (µM) NO. Part A R (Part B) IC (µM) NO. Part A R (Part B) IC 50 50 (µM) NO. Part A R (Part B) IC 50 50 (µM) 12 33 2.84 ± 0.04 ˃50 b bb15 NO. A R (Part B) IC 50 ± (µM) NO. A R (Part B) IC 50˃50 (µM) Molecules 22, 1658 550 of 12 33 2.84 0.04 NO. Part A R (Part B) excellent IC 50± (µM) NO. Part A exhibited R (Part B) IC 50 (µM) 12 33 2.84 ˃50 b bb Half of Part the compounds showed activities, and ofPart them submicromolar NO. Part A RR (Part B)B) IC 50 ± (µM) NO. Part AA RR (Part B)B) ICIC 50˃50 (µM) NO. Part A (Part IC (µM) NO. Part (Part (µM) 12 2017, 33 four 2.84 ±0.04 0.04 12 33 0.04 ˃50 NO. Part A R (Part B) IC 50 50 (µM) NO. Part A R (Part B) IC 50 (µM) fold weaker than the reference compound. NO. Part R (Part IC (µM) NO. Part (Part B) IC 50 (µM) Table 1. Structures and2.84 activities of compounds 12–22, 32–37, Table 1. Structures and of compounds 12–22, 32 bb bactivities NO. Part A R (Part B)B) IC 50 50 (µM) NO. Part AA R (Part B)B) IC 50˃50 (µM) 12 33 2.84 ±±± 0.04 12 33 2.84 ± 0.04 ˃50 NO. Part A R (Part B) IC IC 50 (µM) NO. Part A R (Part IC 50 bb NO. Part AA RRR(Part B) (µM) NO. Part A A40–44. RR IC b (µM) 50 50 50 50(µM) 12 33 2.84 0.04 ˃50 12 33 2.84 ± 0.04 12 Part 33 2.84 0.04 ˃50 NO. Part R (Part B) IC (µM) NO. (Part B) 50 (µM) NO. Part AA (Part B) IC 50 (µM) NO. NO. Part Part A A RR (Part R(Part (Part B)(Part B) B) ICIC 50 ˃50 (µM) IC 50 NO. Part A NO. A (Part B) IC 50 (µM) NO. Part R IC 50 (µM) b(µM) b 12 33 2.84 ± 0.04 ˃50 12 33 2.84 ± 0.04 ˃50 activities, but the activity of the most outstanding compound 17 (IC 50 = 0.38 ± 0.08 μM) was still bb10b 12 33 2.84 ± 0.04 ˃50 b 12 33 2.84 ± 0.04 ˃50 b b 1312 2017, 22, 1658 3433 ˃50 b 0.04 12 33 2.84 ± 0.04 0.04 ˃50 2.84 ± b b Molecules 5˃50 of 15 13 34 ˃50 ˃50 12 33 2.84 ±± Half of the compounds showed excellent and four of them exhibited submicromolar 12 33 2.84 ± ˃50 bb activities,34 b 0.04 b bbb b 12 33 2.84 0.04 13 34 ˃50 ˃50 12 33 13 2.84 ± 0.04 ˃50 ˃50 ˃50 b b b fold weaker than the reference compound. b bb b bb± 0.04 b Table 1. Structures and activities of compounds 12 2.84 ± ˃50 12 3333 12 12–22, 32–37, 40–44. 33 2.84 ±0.04 0.04 ˃50 2.84 12 2.84 ± 13 34 ˃50 ˃50 >50 13 34 ˃50 12 33 2.84 ± 0.04 0.04 ˃50 b bb b bbb(µM) ˃50 ˃50 1313 3434 ˃50 ˃50 NO. Partthe A activity R (Part B) most outstanding IC 50 ˃50 (µM) NO. NO. 17 (IC Part Part (Part R (Part B) B)was IC50still (µM) IC NO. Part A b 13 34 activities, but of the compound 50 A =A0.38 ±R0.08 μM) 10˃50 ˃50 13 34 ˃50 13 34 ˃50bb bbb ˃50bb50 b ˃50 ˃50 1313 3434 ˃50 ˃50 b b b 15 b 13 34 ˃50 ˃50 Molecules 2017, 22, 1658 5 of b bactivities,35 b b 13 34 four of them exhibited submicromolar ˃50 ˃50 Half of the compounds showed excellent and 14 ˃50 ˃50 13 34 13 34 ˃50 ˃50 b b b b fold weaker than the reference compound. 14 35 13 ˃50 ˃50 13 34 ˃50 b˃50 b bbb b bbb b b Table 1. Structures and activities of compounds b b 13 3435 ˃50 ˃50 13 3413 12 3334 12 12–22, 32–37, 40–44. 33 2.84 ±˃50 0.04 ˃50 2.84 ±b0.04 13 14 >50 >50 ˃50 14 35 b b b b b ˃50 ˃50 b bb b bb b 13 34 ˃50 ˃50 13 34 34 14 35 ˃50 ˃50 14 35 ˃50 activities, but of the compound 50 A =A0.38 ±R0.08 μM) still NO. Partthe A activity R (Part B) most outstanding IC˃50 50 ˃50 (µM) NO. NO. 17 (IC Part Part (Part R (Part B) B)was IC˃50 50 (µM) IC˃50 50 NO. Part A bb(µM) bb bb b10b ˃50 ˃50 1414 3535 ˃50 13 34 ˃50 b bb b bb Molecules 2017, 22, 1658 5 of 15˃50 14 35 ˃50 ˃50 14 35 ˃50 ˃50 14 35 ˃50 ˃50 b bactivities,35 b b 14 35 ˃50 ˃50 14 ˃50 ˃50 Half of the compounds showed excellent and four of them exhibited submicromolar b b b b 35 ˃50 ˃50 fold14 weaker than the reference compound. b 14 35 ˃50 ˃50 b b b Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. 15 36 1.92 ± 0.10 0.10 ˃50 1414 3535 ˃50 14 3512 ˃50 ˃50 b b bb b 15 36 14 35 1.92 ± ˃50 ˃50 ˃50 12 33 33 2.84 ±b˃50 0.04 ˃50 2.84 ± 0.04 b b b b b 35 14 b bbb >50 >50 b b b b 13 34 13 34 1415 3536 ˃50 ˃50 14 35 ˃50 ˃50 1.92 ˃50 activities, but the activity of the outstanding compound 50 = 0.38 ± 0.08 μM) was ˃50 still 101.92 ± ˃50 b 0.10 b b± b ˃50 b b 14 35 ˃50 1415 3536 14 17 (IC 35 ˃50 ˃50 NO. Part A compounds R (Part B) mostexcellent IC 50 (µM) NO. NO. Part Part AA exhibited R (Part R (Part B)submicromolar B) IC˃50 50 ˃50 (µM) IC 50bb(µM) NO. Part A 15 36 1.92 ±0.10 0.10 ˃50 15 36 1.92 ± 0.10 ˃50 b bbb Half of the showed activities, and four of them 15 36 1.92 ± 0.10 ˃50 15 36 1.92 ± 0.10 ˃50 b b b b 15 36 1.92 0.10 ˃50 15 3636 1.92 ± ±± 0.10 ˃50 15 36 1.92 ± 0.10 ˃50b b ˃50 14 35 ˃50 fold15 weaker than the reference compound. 15 1.92 0.10 ˃50 15 36 1.92 ± 0.10 ˃50 Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. b b 36 1.92 ± 0.10 ˃50 15 3612 17 (IC50 = 0.38 ± 0.08 μM) was ˃50 1.92 ± 0.10 0.10 compound ˃50 b bb activities, but the activity of the most1.92 outstanding still b 16 3736 1.20 ±b 0.01 0.01 ˃50 1515 1.92 0.10 1.92 ± ˃50 b b b1012 33 33 2.84 ±0.10 0.04 2.84 ± 0.04 15 ± 36 >50 13 3436 13 34 15 ˃50 ˃50 ˃50 1.92 ± ˃50 16 37 1.20 15 36 1.92 ± 0.10 ˃50 b b bbb b b b 14 35 14 35 1516 3637 ˃50 1.92 ±b ±0.10 0.10 ˃50 15 36 1.92 ± 0.01 0.10 ˃50 NO. Part A R (Part B) IC 50 (µM) NO. NO. Part PartAA R (Part R (Part B) B) IC˃50 50 ˃50 (µM) IC 50bbb(µM) NO. Part A 1.20 ˃50 1.20 b b ˃50 15 36 1.92 ± 0.10 b 1516 3637 15 36 1.92 ± 0.10 ˃50 1.92 ± 0.10 16 37 1.20 ±0.01 0.01 ˃50 16 37 1.20 ± 0.01 ˃50 fold weaker than the reference compound. b bbb 16 37 1.20 ± 0.01 ˃50 16 37 1.20 ± 0.01 ˃50 b b Table 1. Structures and activities of compounds 12–22, 32–37, 40–44.

1.20 ±± 0.01 ˃50 1616 3737 1.20 0.01 ˃50 16 37 1.20± 0.01 ˃50b b ˃50 b 36 1.92 0.10 1.20 ± 0.01 ˃50 1616 3737 1.20 ±± 0.01 0.01 ˃50 b bb b 16 37 1.20 ± ˃50 12 33 33 2.84 ±0.01 0.04 ˃50 2.84 1.20 ± 37 16 16 3712 1.20 ± 0.01 0.01 ˃50 >50 b b b ± 0.04 17 40 13 34 13 34 0.38 ±bb 0.01 0.08 0.81 0.03 ˃50 ˃50 ˃50 1616 3737 1.20 0.01 ˃50 16 1.20 ± ˃50 b b50 b 14 35 14 35 ˃50 ˃50 ˃50 16 37 NO. Part A R (Part B) IC 50 (µM) NO. NO. Part Part A A R (Part R (Part B) B) IC 50 (µM) IC (µM) NO. Part A 1.20 ± ˃50 17 40 0.38 0.08 0.81 ±±bb ±0.03 37 1.20 ± 0.01 ˃50 b b b 1517 36 15 36 1.92 ± 0.10 ˃50 1.92 ± 0.10 b 16 37 1.20 ± 0.01 ˃50 16 37 1.20 ± 0.01 ˃50 17 40 0.38 ± 0.08 0.81 40 b 0.03 0.38 ± 0.08 0.81 ± 0.03 b Table 1. Structures and activities of compounds 12–22, 32–37, 40–44. 1.20 ˃50 1616 3737 37 1.20 ±± 0.01 ˃50 1.20 ± 0.01 17 4016 0.38 ±0.01 0.08 0.81 ±0.03 0.03 17 40 0.38 ± 0.08 0.81 ± 17 40 0.38 ± 0.08 0.81 ± 0.03 17 40 0.38 ± 0.08 0.81 ± 0.03 17 40 0.38 ± 0.08 0.81 ± 0.03 1717 4040 0.38 ±± 0.08 0.81 ±b ± 0.03 0.38 ± 0.08 0.81 ± 0.03 0.38 0.08 0.81 1717 4040 0.38 ±±b 0.08 0.08 0.81 12 33 33 2.84 ±0.08 0.04 ˃50 2.84 ±0.03 0.04 17 0.38 ± 0.81 ±0.03 b b 0.03 b 16NO. 3712 1.20 ˃50 13 34 13 34 ˃50 ˃50 40 0.38 ± 0.81 ±± b 17 40 0.38 ±0.01 0.08 0.81 ±˃50 0.03 Part A R (Part B) IC 50 (µM) NO. Part A R (Part B) IC 50 (µM) 18 41 1417 35 14 35 1.60 ±b 0.08 0.02 10.05 0.06 ˃50 ˃50 ˃50 1717 4040 0.38 0.08 0.81 ±bbb±±0.03 0.03 0.38 ± 0.08 0.81 ± 0.03 15 36 15 36 1.92 ± 0.10 ˃50 1.92 ± 0.10 18 41 40 1.60 0.02 10.05 0.06 0.38 ± 0.81 ±± 0.03 17 40 0.38 ± 0.08 0.81 ± 0.03 1617 37 16 37 1.20 ± 0.01 ˃50 1.20 ± 0.01 17 40 0.38 ± 0.08 0.81 0.03 17 40 0.38 ± 0.08 0.81 ± 0.03 18 41 1.60 ± 0.02 10.05 ± 0.06 1.60 10.05 ±0.03 17 40 0.38 0.08 0.81 ± 1718 4041 40 0.38 ±± 0.08 0.81 ±0.38 0.03 ± 0.08 18 4117 1.60 ±0.02 0.02 10.05 ±0.06 0.06 18 41 1.60 ± 0.02 10.05 ± 0.06 18 41 1.60 ± 0.02 10.05 ± 0.06 18 41 1.60 ± 0.02 10.05 ± 0.06 b± ± 18 41 1.60 ± 0.02 10.05 0.06 18 1.60 ± 0.02 10.05 0.06 18 41 1.60 ± 0.02 10.05 ± 0.06 12 33 2.84 ± 0.04 ˃50 18 1.60 ± 0.02 41 10.05 ± 0.06 b b b 13 34 13 34 ˃50 ˃50 ˃50 18 41 1.60 ± 0.02 10.05 ± 0.06 18 41 1.60 ± 0.02 10.05 ± 0.06 NO. Part A R (Part B) IC 50 (µM) NO. Part A R (Part B) IC 50 (µM) b b b b± 0.06 35 35 ˃50 ˃50 18 41 1.60 ± 10.05 ±± ˃50 0.06 b 18 41 1.60 ±0.08 0.02 10.05 19 42 0.73 ± 0.09 ˃50 17 14 4014 0.38 0.81 ± 0.03 15 36 15 36 1.92 ± 0.10 ˃50 1.92 ± 0.10 1818 4141 1.60 ± 0.02 0.02 10.05 0.06 1.60 ± 0.02 10.05 ± 0.06 b 19 42 0.73 0.09 ˃50 16 37 16 37 1.20 ± 0.01 ˃50 ± 0.01 18 41 1.60 ± 0.02 10.05 ±± bb0.06 18 41 1.60 ± 0.02 10.05 ± bb0.06 0.73 ± 0.09 ˃50 17 40 17 40 1819 4142 42 0.38 ± 0.08 0.81 ±±1.20 0.38 0.03 ± 0.08 1.60 0.02 10.05 0.06 0.73 ± 0.09 ˃50 18 41 1.60 ± 0.02 10.05 ± 0.06 b b b 18 41 19 42 1.60 ± 0.02 10.05 ± 0.06 0.73 ± 0.09 ˃50 1819 41 18 41 1.60 ± 0.02 10.05 1.60 0.06 ± 0.02 19 4242 0.73 0.09 ˃50 bb b b b 19 0.73 ± 0.09 ˃50 19 0.73 ± 0.09 ˃50 12 33 2.84 ± 0.04 ˃50 19 0.73 ± 0.09 42 >50 b bb b± b 42 0.73 0.09 ˃50 1919 42 0.73 ± 0.09 NO. Part A R (Part B) IC 50 (µM) NO. Part A R (Part B) IC 50 ˃50 (µM) 19 42 0.73 ± 0.09 ˃50 13 34 ˃50 ˃50 b b b b± b ˃50 19 42 0.73 0.09 ˃50 19 42 0.73 ± 0.09 ˃50 14 35 14 35 ˃50 ˃50 b b b 0.10 42 0.73 ± 0.09 ˃50 20 43 5.76 0.03 1519 36 36 1.92 ± 0.10 ˃50 1.92 ± 19 4215 0.73 ± 0.09 0.09 ˃50 b b b b 1920 4243 0.73 0.09 20 43 5.76 ± 0.03 ˃50 19 42 0.73 ± ˃50 37 37 1.20 ± 0.01 ˃50 ± 0.01 bb bbb 19 42 0.73 0.09 19 42 0.73 ± 0.09 ˃50 18 16 4116 1.60 ±±±0.03 0.02 10.05 ± 0.06 17 40 17 40 0.38 ± 0.08 0.81 ±±1.20 0.38 0.03 ± 0.08 5.76 0.03 ˃50 20 43 5.76 ± ˃50 b 19 42 0.73 0.09 bbb 19 42 b 0.73 ± 0.09 ˃50 18 41 18 41 1.60 ± 0.02 10.05 1.60 0.06 ± 0.02 b b 20 43 5.76 0.03 ˃50 20 43 5.76 ± 0.03 ˃50 12 33 2.84 ± 0.04 ˃50 20 5.76 ± 0.03 19 0.73 0.09 19 4242 19 42 >50 0.73 0.09 0.73 b bbb± 0.09 20 43 5.76 ± 0.03 ˃50 b b 20 43 5.76 ± 0.03 ˃50 NO. Part A R (Part B) IC 50 (µM) NO. Part A R (Part B) IC 50 (µM) b b 132020 344343 ˃50 ˃50 5.76 ± 0.03 ˃50 5.76 ± 0.03 ˃50 20 43 5.76 ± 0.03 ˃50 b b b b 14 35 ˃50 ˃50 20 43 5.76 ± 0.03 ˃50 20 43 5.76 ± 0.03 ˃50 b b b b 1520 3643 15 36 1.92 ±± 0.10 ˃50 1.92 ± 0.10 5.76 0.03 ˃50 20 43 5.76 ± 0.03 ˃50 21 44 b b 20.06 ± 0.12 ˃50 b b b 20 43 5.76 ± 0.03 ˃50 20 43 5.76 ± 0.03 ˃50 16 37 16 37 1.20 ± 0.01 ˃50 1.20 ± 0.01 2121 4444 20.06 ±±±0.12 20 43 5.76 ± ˃50 20 43 5.76 0.03 ˃50 b bbb 17 40 40 0.38 0.08 0.81 ±±0.38 0.03 ± 0.08 b b 20.06 0.12 ˃50 2021 4344 21 44 5.76 ±± 0.03 20.06 ±0.03 ˃50 20 43 5.76 ±0.09 0.03 ˃50 b 12 33 2.84 0.04 21 20.06 ±±±(µM) 0.12 44 18 41 18 41 1.60 0.02 10.05 1.60 0.06 ±b˃50 0.02 >50 b bbb b 19NO. 4217 19 42 19 42 0.73 0.73 0.09 ˃50 0.73 0.09 b b±0.03 b 5.76 ± 20.06 ±0.12 0.12 ˃50 20 43 20 43 5.76 0.03 5.76 21 44 20.06 0.12 Part A R (Part B) IC 50 ± NO. Part A R (Part B) IC˃50 50 ˃50 (µM) 1320 3443 ˃50 ˃50 b bbb± 0.03 21 44 20.06 ± 0.12 ˃50 21 44 20.06 ± 0.12 ˃50 b± ± b b b 21 44 20.06 0.12 ˃50 44 20.06 0.12 ˃50 1421 35 ˃50 ˃50 21 44 20.06 ± 0.12 ˃50 b b b 21 44 20.06 ± 0.12 ˃50 21 44 20.06 ± 0.12 ˃50 15 36 1.92 ± 0.10 ˃50 b b b b 20.06 0.12 ˃50 21 4416 20.06 ± 0.12 0.12 ˃50 1621 3744 37 1.20 ±± 0.01 ˃50 1.20 ±b 0.01 22 0.72 ±±±0.06 0.06 2121 4444 20.06 0.12 ˃50 b b b 20.06 ± ˃50 22 0.72 17 40 17 40 0.38 0.08 0.81 ±(µM) 0.03 ± 0.08 12 33 2.84 ± 0.04 22 0.72 ± 0.06 21 44 20.06 ±±±0.06 0.12 NO. Part A R (Part B) IC 50 (µM) NO. Part A R (Part B) IC˃50 50 ˃50 21 20.06 ± 0.12 ˃50 b b b b 18 41 18 41 1.60 ± 0.02 10.05 ±0.38 1.60 0.06 ± 0.02 0.72 2122 4444 20.06 0.12 ˃50 0.72 21 44 20.06 ±0.06 0.12 ˃50 19 42 19 42 0.73 0.09 0.73 0.09 34 ˃50 ˃50 20 43 20 43 5.76 ± 0.03 5.76 ±±˃50 0.03 b b b 21 22 20.06 ± 0.12 ˃50 0.72 ±0.06 0.06 2122 4444 21 44 20.06 ±± 0.12 ˃50 20.06 0.12b 20 13 43 22 5.76 ± 0.03 0.72 ± b b 14 35 ˃50 ˃50 0.72 ± 0.06 22 2222 0.72 ± ±± 0.06 b 0.72 0.06 22 0.72 ± 0.06 22 0.72 0.06 1522 36 1.92 ±b0.10 ˃50 ±b 0.01 aa b 0.06 22 0.72 ± 0.72 ± 0.06 32 Anacetrapib ˃50 0.04 12 33 2.84 ±± 0.04 ˃50 0.04 ± 0.01 32 16 37 1.20 0.01 >50 a b 0.06 Anacetrapib 22 0.72 0.06 22 32 Anacetrapib 0.72 ± ˃50 0.04 ± 0.01 17 40 17 40 0.38 ± 0.08 0.81 ±±0.38 0.03 ± 0.08 bb a aa b 0.06 2222 0.72 ±b 0.06 0.06 22 0.72 ± b Anacetrapib 0.04 ± 0.01 32 Anacetrapib ˃50 0.04 ± 13 34 ˃50 ˃50 22 0.72 ± b 18 41 41 0.72 ± 1.60 ± 0.02 10.05 1.60 0.06 ± bb a aa b 19 42 19 42 0.73 ±±˃50 0.09 ˃50 0.73 ±±0.02 0.09 20 43 20 43 5.76 0.03 5.76 0.03 32 Anacetrapib ˃50 0.04 ±0.01 0.01 2232 0.72 ±b0.12 0.06 32 Anacetrapib ˃50 0.04 ± 0.01 b 22 0.72 ±bb 0.06 0.06 2122 44 18 21 44 20.06 20.06 0.12b bb a aaa 14 35 ˃50 ˃50 0.72 ± 0.06 22 0.72 ± 0.06 0.72 ± 0.06 32 Anacetrapib ˃50 0.04 ± 0.01 32 Anacetrapib ˃50 0.04 ± 0.01 bb a b a b a b a b 32 Anacetrapib ˃50 0.04 ± 0.01 32 Anacetrapib 21 22 44 ˃50 0.04 ± 0.01 20.06 ± 0.12 ˃50 32 Anacetrapib 153232 36 ˃50 0.04± ± ± 0.01 1.92 ˃50 ± ˃50 0.10 ˃50 Used asasaa apositive positive control. Considered with no CETP inhibition activity. a Used b Used positive control. with no inhibition activity. aCETPinhibition b aCETP b Considered Anacetrapib 0.04 0.01 as control. Considered with no activity. Anacetrapib 0.04 0.01 b a Used bbConsidered a aCETP inhibition activity. a Used b0.01 b 16 37 1.20 ± ˃50 32 Anacetrapib ˃50 0.04 ± 0.01 as a positive control. with no as a positive control. Considered with no CETP inhibition activity. b b 32 Anacetrapib ˃50 0.04 ± 0.01 a b b a Used as a positive control. aCETP inhibition activity. a Used bb 13 34 ˃50 ˃50 3232 Anacetrapib ˃50 0.04 ± 0.01 0.01 Anacetrapib ˃50 0.04 ± 0.01 a CETP Considered with no 17 40 0.38 ± 0.08 0.81 ±±1.60 0.03 a as aaapositive control. with no inhibition activity. b bbbConsidered aaa Used a Used b bConsidered 32 Anacetrapib ˃50 0.04 ± Anacetrapib ˃50 0.04 ± 0.01 a CETP aaCETPinhibition 18 41 18 41 1.60 ± 0.02 10.05 ± 0.02 positive control. with inhibition activity. asas positive control. Considered with no activity. a Used bbb b 3232 Anacetrapib ˃50 0.04 ±bb0.06 0.01 a b bb 32 Anacetrapib 0.04 ±˃50 0.01 19 42 19 42 0.73 ± 0.09 ˃50 0.73 ± a CETP ano a b 20 43 20 43 14 35 5.76 0.03 5.76 0.03 as positive control. with no CETP inhibition activity. ˃50 as positive control. Considered with inhibition activity. 21 44 21 44 20.06 ±˃50 ˃50 0.12 Anacetrapib ˃50 0.04 ± 32 Anacetrapib 32no Anacetrapib ˃50 0.04 ±20.06 0.01 a Used bbConsidered a Used b0.12 22 22 0.72 ± 0.72 ±±0.09 0.06 b 0.01 Used asaaa aapositive positive control. Considered with noCETP CETPinhibition inhibitionactivity. activity. as positive control. Considered with no CETP inhibition activity. 1532In Vitro Metabolic 36 1.92 ± 0.10 ˃50 a Used b0.06 a b 2.3. Stability Study as control. Considered with no b b Considered Used asa aapositive positive control. Considered withnono noCETP CETPinhibition inhibitionactivity. activity. a Used b0.06 2.3. In Vitro Metabolic Stability Study 16 37 1.20 ± 0.01 ˃50 Used as control. with aa Used b 2.3.222.3. In Vitro Metabolic Stability Study as positive control. Considered with CETP inhibition activity. 0.72 ± a b as a positive control. with no CETP inhibition activity. a Used b Considered Used as a positive control. Considered with no CETP inhibition activity. 2.3. In Vitro Metabolic Stability Study In Vitro Metabolic Stability Study a b a b as a positive control. Considered with no CETP inhibition activity. 17 40 0.38 ± 0.08 0.81 ± 0.03 Used apositive positive control. Considered with no CETP inhibition activity. a Used a Used b Considered a Used 2.3.In InVitro VitroMetabolic Metabolic Stability Study b Considered b 2.3. Stability Study 14 35 ˃50 ˃50 asaasapositive control. with CETP inhibition activity. as control. with nono CETP inhibition activity. as a positive10.05 control. with noaCETP inhib 18 41 1.60 ± b0.09 0.02 ±0.73 0.06b± Considered b 2.3. Vitro Metabolic Stability Study 2.3. InIn Vitro Metabolic Stability Study b 19 42 42 0.73 ± ˃50 0.09 b b 2.3. Vitro Metabolic Stability Study b 20 43 20 43 5.76 5.76 2.3. In Vitro Metabolic Stability Study 32 Anacetrapib 32a Anacetrapib ˃50 0.04 ±20.06 0.01 b ˃50 21 44 19 21 44 20.06 0.12 0.12 15 36 1.92 ±±0.03 0.10 ˃50 22 22 0.72 0.06 0.72 ±±0.03 0.06 2.3. In Vitro Metabolic Stability Study 2.3. InIn Vitro Metabolic Stability Study b Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected 2.3. In Vitro Metabolic Stability Study Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected 16 37 1.20 ± 0.01 ˃50 2.3. In Vitro Metabolic Stability Study a the potent inhibitor 17 was selected b inhibitory on the result ofofthe the ininvitro vitro CETP assay, 2.3. InBased Vitro Metabolic Stability Study 2.3. In Vitro Metabolic Stability Study 322.3. Anacetrapib ˃50 0.04 ± 0.01 In Vitro Metabolic Stability Study 17 40 assay, 0.38CETP ± 0.08 0.81selected ±selected 0.03 2.3. In Vitro Metabolic Stability Study Based on result the vitro inhibitory assay, the potent inhibitor 17 was on the of in inhibitory the potent inhibitor 17 2.3. InBased Vitro Metabolic Stability Study 2.3. In Vitro Metabolic Stability Study a the b CETP a Used Based on theresult result ofthe the in vitro CETP inhibitory assay, the potent inhibitor 17was was selected Based on the result of in vitro CETP inhibitory assay, the potent inhibitor 17 was selected Used as astability positive control. Considered with no CETP inhibition activity. as a showed positive control. Considered with no CETP inhib 2.3. In Vitro Metabolic Stability Study 18 41 1.60 ±As 0.02 10.05 ±selected 0.06bstability, Vitro Metabolic Stability Study 2.3. In Metabolic Stability Study for the in vitro metabolic stability study. As shown in Table 2,Vitro compound 17 showed weak stability, b Based on the result of the vitro CETP inhibitory assay, the potent inhibitor 17 was b Based on the result of the inin vitro CETP inhibitory assay, the potent inhibitor 17 was selected for2.3. the in vitro metabolic study. As shown in Table 2, compound 17 weak 15 36 1.92 0.10 19In 42 0.73 0.09 for the in vitro metabolic stability study. shown in Table 17 showed weak stability, 20 43 20 43 a 2, compound a 5.76 ± 0.03 ˃50 5.76 ±±0.03 b b Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected b ˃50 Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected 32 Anacetrapib 32 Anacetrapib ˃50 0.04 ± 0.01 21 44 21 44 20.06 ± 0.12 ˃50 20.06 0.12 for the in vitro metabolic stability study. shown in Table 2, compound 17 showed weak stability, for in vitro metabolic stability study. shown in Table 2, compound 17 showed weak stability, 22 22 0.72 ± 0.06 0.72 ± 0.06 Based on the result of the vitro CETP inhibitory assay, the potent inhibitor 17 was selected Based on the result of the inin vitro CETP inhibitory assay, the potent inhibitor 17 was selected b a Used bAs 16 37 forthe the in vitro metabolic stability study. As shown in Table 2, compound 17 showed weak stability, 1.20μL/min/mg ±As 0.01 ˃50 for the in vitro metabolic stability study. As shown in Table 2, compound 17 showed weak stability, Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected as a positive control. Considered with no CETP inhibition activity. Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected with a clearance rate of 119.0 and 146.1 in human and rat liver microsomes. for the in vitro metabolic stability study. As shown in Table 2, compound 17 showed weak stability, for the in vitro metabolic stability study. As shown Table 2, compound 17 showed weak stability, Based on the result of the in146.1 vitro CETP inhibitory assay, the potent inhibitor 17 was selected Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected 17 40 0.38µL/min/mg ±As 0.08 0.81stability, ± 0.03 with a clearance rate of 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. withwith a clearance rate of 119.0 and in human and rat liver microsomes. for the in vitro metabolic stability study. As shown in Table 2, compound 17 showed weak stability, for the in vitro metabolic stability study. shown Table 2, compound 17 showed weak Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected with clearance rate and 146.1 in human and rat liver microsomes. aBased clearance rate of 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. on the result of the incontrol. vitro CETP inhibitory assay, the potent inhibitor 17 was selected on the result of the in vitro CETP inhibitory the potent inhibitor 17 was 18 41 1.60 ±μL/min/mg 0.02 10.05 ±selected 0.06b Considered for the in vitro metabolic stability study. As shown in Table 2, compound 17 showed weak stability, a Used b a Used for the in vitro metabolic stability study. As shown Table 2, compound 17 showed weak stability, 2.3. In Vitro Metabolic Stability Study 2.3. In Vitro Metabolic Stability Study with clearance rate of 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. b CETP Based on the result of the in vitro CETP inhibitory assay, the potent inhibitor 17 was selected asof positive Considered noassay, CETP inhibition activity. as aofpositive control. withassay, no CETP Based on the result of119.0 the in vitro CETP inhibitory assay, Based the potent on the inhibitor result 17 the was in vitro selected inhibitory theinhib pot with aaaaaBased clearance rate of and 146.1 μL/min/mg in human and rat liver microsomes. for the in metabolic stability study. As Table 2, compound 17 showed weak stability, 19 42 0.73 ± 0.09 ˃50 b for the invitro vitro metabolic stability study. Asshown shown in Table 2, compound 17 showed weak stability, with clearance rate ofa119.0 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. 20 43 5.76 ± 0.03 ˃50 with clearance rate of 119.0 and 146.1 μL/min/mg inwith human and rat liver microsomes. for the in vitro metabolic stability study. As shown Table 2,2, compound 17 showed weak stability, b ˃50 for the in vitro metabolic stability study. As shown Table compound 17 showed weak a b b 21 44 21 44 20.06 ±As 0.12 20.06 0.12 16 37 1.20 ± 0.01 ˃50 with clearance rate of 119.0 and 146.1 in human and rat liver microsomes. 32 Anacetrapib 32 Anacetrapib with aaaaclearance rate of 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. ˃50 0.04 ±stability, 0.01 for the in vitro metabolic stability study. shown Table compound 17 showed weak stability, for the in vitro metabolic stability study. As shown in Table 2, compound 17 showed weak stability, 22 22a 2, 0.72 ±μL/min/mg 0.06 0.72 ±±0.06 for the in vitro metabolic stability study. As shown Table 2, compound 17 showed weak stability, for the in vitro metabolic stability study. As shown Table 2, compound 17 showed weak stability, with clearance rate of 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. with clearance rate of 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. for the in vitro metabolic stability study. As shown Table 2, compound 17 showed weak stability, the in vitro metabolic stability study. As shown in Table for the 2, compound in vitro metabolic 17 showed stability weak study. stability, As shown in Table 2, compoun Table 2. In vitro DMPK profile. with a clearance rate of 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. 17 40 0.38 ± 0.08 0.81 ± 0.03 with a clearance rate of 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. 2.3.for In Vitro Metabolic Stability Study Table 2. In vitro DMPK profile. with clearance rate ofof 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. with clearance rate 119.0and and 146.1 μL/min/mg in human and rat liver microsomes. Table 2.2.In In vitro DMPK profile. 18 41 1.60 ± 0.02 ± b0.06b with aaa aaaclearance rate of 146.1 μL/min/mg in human and rat liver microsomes. with clearance rate of and 146.1 μL/min/mg in human and rat liver microsomes. Table In vitro DMPK profile. Table 2. vitro DMPK profile. a Used b a Used with clearance rate of 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. 19 42 0.73 ± 0.09 ˃50 on the result of119.0 the in vitro CETP inhibitory assay, Based the potent on the inhibitor result 17 the was in10.05 vitro selected CETP inhibitory assay, therat pot Table 2.In In vitro DMPK profile. 2.3. InBased Metabolic Stability Study 2.3. In Vitro Metabolic Stability Study with clearance rate of119.0 119.0 and 146.1 μL/min/mg in human and rat liver microsomes. Table 2. vitro profile. as119.0 a119.0 positive control. Considered with no CETP inhibition activity. as119.0 aofpositive control. Considered no CETP inhib bμL/min/mg with clearance rate of and 146.1 μL/min/mg in human and rat liver microsomes. with a aVitro clearance rate of and 146.1 μL/min/mg inDMPK human aand clearance rat liver rate microsomes. of and 146.1 in with human and li 20 43 5.76 ±± 0.03 ˃50 b Table 2. vitro DMPK profile. Table 2. InIn vitro DMPK profile. a b b 21 44with 20.06 0.12 ˃50 Table 2. In vitro DMPK profile. Table 2. In vitro DMPK profile. 32 Anacetrapib 32 Anacetrapib a ˃50 0.04 ± 0.01 ˃50 22 22 0.72 ± 0.06 ± 0.06 40forCYPs 0.38 ± 0.08 0.81 ±0.72 0.03 Table 2. In vitro DMPK profile. Table 2. In vitro DMPK profile. Direct Inhibition Mean (10 μM) a b for17the in vitro metabolic stability study. As shown in Table the 2, compound in vitro metabolic 17 showed stability weak study. stability, As shown in Table 2, compoun Table 2. In vitro DMPK profile. CYPs Direct Inhibition Mean (10 μM) HLM Stability RLM Stability Table 2. In vitro DMPK profile. a b Table 2. In vitro DMPK profile. Table 2. In vitro DMPK profile. 18 41 1.60 ± 0.02 10.05 ± 0.06 HLM Stability RLM Stability CYPs Direct Inhibition Mean (10 μM) Based on the result of atheaa aain RLM vitro CETP inhibitory assay, theInhibition potent inhibitor 17 was selected CYPs Direct Mean (10 μM) b DMPK bvitro b Table 2. profile. CYPs Direct Inhibition Mean (10 µM) Table 2. In DMPK profile. Compound 19 42 0.73 ± 0.09 ˃50 bvitro CYPs Direct Inhibition Mean (10 μM) HLM Stability RLM Stability Stability Stability CYPs Direct Inhibition Mean (10 b CETP aHLM b a Used b Considered Table 2.In Invitro vitro DMPK profile. b in Table 2.In In DMPK profile. HLM Stability bvitro Compound 20 432.3. 5.76 ± 0.03 ˃50 Based on the result the in146.1 vitro CETP inhibitory assay, Based the potent on the inhibitor result 17 the was inμM) vitro selected inhibitory assay, therat pot Stability 2.3. In Metabolic Stability Study In Vitro Metabolic Stability Study Used as aofpositive control. Considered with no CETP inhibition activity. as119.0 aof positive control. no CETP inhib HLM Stability RLM Stability with a Vitro clearance rate of 119.0 and μL/min/mg human aand clearance rat liver rate microsomes. of and 146.1 in with human and li CYPs Direct Inhibition Mean (10 μM) HLM Stability RLM Stability CYPs Direct Inhibition Mean (10 μM) Table 2. In vitro DMPK profile. Table 2. profile. Table 2. bIn vitro DMPK profile. (μL/min/mg) (μL/min/mg) bμL/min/mg a aa RLM b bb DMPK Compound Compound Compound Direct Inhibition Mean (10 μM) CYPs Direct Inhibition (10 μM) a 21 44with b 20.06 ±As 0.12 ˃50 HLM Stability RLM Stability HLM Stability RLM Stability (μL/min/mg) (μL/min/mg) a aa b bb 3A4 2D6 2C9 17Mean 1A2 2C19 32 Anacetrapib 32CYPs Anacetrapib a ˃50 0.04 ± 0.01 ˃50 Compound Compound 22 0.72 ± 0.06 HLM Stability RLM Stability HLM Stability RLM Stability CYPs Direct Inhibition Mean (10 μM) CYPs Direct Inhibition Mean (10 μM) forfor the in vitro metabolic stability study. shown in Table 2, compound showed weak stability, 3A4 2D6 2C9 1A2 2C19 (μL/min/mg) (μL/min/mg) a b (µL/min/mg) (μL/min/mg) (μL/min/mg) a b (µL/min/mg) Compound Compound 3A4 2D6 2C9 1A2 2C19 CYPs Direct Inhibition Mean (10 μM) HLM Stability RLM Stability HLM Stability RLM Stability CYPs Direct Inhibition Mean (10 μM) a b (μL/min/mg) (μL/min/mg) 18 41 1.60 ± 0.02 10.05 ± 0.06 3A4 2D6 2C9 1A2 2C19 the in vitro metabolic stability study. As shown in Table for the 2, compound in vitro metabolic 17 showed stability weak study. stability, As shown in Table 2, compoun 3A4 2D6 2C9 1A2 2C19 (μL/min/mg) (μL/min/mg) Compound Compound a b CYPs Direct Inhibition Mean (10 μM) CYPs2D6 DirectInhibition Inhibition Mean (10μM) μM) 2C19 Stability RLM Stability b b HLM Stability RLM Stability a 3A4 2D6 2C9 Mean 1A2 (μL/min/mg) (μL/min/mg) 3A4 2C9 1A2 (μL/min/mg) (μL/min/mg) CYPs Direct (10 Compound HLM b 2C19 Compound CYPs Direct Inhibition Mean (10 μM) HLM Stability RLM Stability aa a b HLM Stability RLM Stability (μL/min/mg) (μL/min/mg) 19 42 0.73 ± 0.09 ˃50 (μL/min/mg) (μL/min/mg) CYPs Direct Inhibition Mean (10 μM) b 2C19 3A4 2D6 2C9 1A2 CYPs Direct Inhibition Mean (10 μM) 3A4 2D6 2C9 1A2 2C19 Stability RLM Stability a a b b HLM Stability RLM Stability b aHLM b a liver b Considered withCYPs 43 Compound 5.76 ± 0.03 ˃50 with aCompound clearance rate of 119.0 and 146.1 μL/min/mg in human and rat microsomes. 3A4 2D6 2C9 1A2 2C19 a control. 3A4 2D6 2C9 1A2 2C19 (μL/min/mg) (μL/min/mg) CYPs Direct Inhibition Mean (10 μM) (μL/min/mg) (μL/min/mg) CYPs Direct Inhibition Mean (10 μM) Direct In HLM Stability RLM Stability bμL/min/mg HLM Stability RLM Stability Used as aofpositive with no CETP inhibition Used activity. as aof positive control. no CETP inhib Compound Compound a in 2.3.20 In Metabolic Stability 2.3. In Vitro Metabolic Stability Based on the result the vitro CETP inhibitory assay, Based the potent on the inhibitor result 17 the was in vitro selected CETP inhibitory assay, therat pot aStudy b b b DMPK aStudy b Table 2. In vitro profile. Table 2.3.1% In vitro DMPK profile. 21 44 20.06 ±Considered 0.12 ˃50 with a Vitro clearance rate of 119.0 and 146.1 μL/min/mg in3A4 human with and clearance rat liver rate microsomes. of 119.0 and 146.1 in human and li 17 119.0 146.1 17.7% No inhibition 14.3% No inhibition (μL/min/mg) (μL/min/mg) Compound 3A4 2D6 2C9 1A2 2C19 HLM Stability RLM Stability 3A4 2D6 2C9 1A2 2C19 HLM Stability RLM Stability HLM Stability RLM Stability Compound (μL/min/mg) (μL/min/mg) a a b Compound Compound 32 Anacetrapib ˃50 0.04 ± 0.01 (μL/min/mg) (μL/min/mg) (μL/min/mg) (μL/min/mg) 22 2D6 2C9 1A2 2C19 0.72 ± 0.06 3A4 2D6 2C9 1A2 2C19 (μL/min/mg) (μL/min/mg) Compound Compound Compound (μL/min/mg) (μL/min/mg) 3A4 2D6 2C9 1A2 2C19 3A4 2D6 2C9 1A2 2C19 (μL/min/mg) (μL/min/mg) (μL/min/mg) (μL/min/mg) 3A4 2D6 2C9 1A2 2C19 3A4 2D6 2C9 1A2 b 2C19 for19the in vitro metabolic stability study. As shown in Table for the 2, compound in vitro metabolic 17 showed stability weak study. stability, As shown in Table 2, compoun a Human b (μL/min/mg) (μL/min/mg) 42 0.73 ± 0.09 ˃50 (μL/min/mg) (μL/min/mg) (μL/min/mg) (μL/min/mg) 3A4 2D6 2C9 1A2 2C19 3A4 2D6 2C9 1A2 2C19 liver microsomal intrinsic clearance. Rat liver2D6 microsomal intrinsic clearance. b 3A4 2D6 2C9 1A2 2C19 3A4 2C9 1A2 2C19 3A4 2D6 20 43 5.76 b±±0.03 ˃50 a Used as a positive control. b CYPs Direct Inhibition Mean (10 μM) CYPs Direct In 21 442.3. 20.06 0.12vitro ˃50 with noIn CETP inhibition activity. 2.3. Metabolic Stability Vitro Metabolic Stability aStudy b vitro aStudy b on the result of the inRLM vitro CETP inhibitory assay, Based therat potent on the inhibitor result of 17 the was in vitro selected CETP inhibitory assay, therat pot Table 2. Table 2.Considered DMPK profile. Table 2. In vitro DMPK profile. with a Vitro clearance rate of 119.0 and 146.1 μL/min/mg inDMPK human with aand clearance liver rate microsomes. of 119.0 and 146.1 μL/min/mg in human and li HLM Stability Stability HLM Stability RLM Stability aprofile. bInIn 22InBased 0.72 ± 0.06 32 Anacetrapib ˃50 0.04 ± 0.01 Compound Compound b As shown in Table 2, compoun for20the in vitro metabolic stability study. shown in Table for the 2, compound in vitro metabolic 17 showed stability weak study. stability, (μL/min/mg) (μL/min/mg) (μL/min/mg) (μL/min/mg) 43 5.76As ± 0.03 ˃50 3A4 2D6 2C9 1A2 2C19 2D6 b chemicals 3A4 a Used b±Considered In addition, five cytochrome (CYP) commonly metabolize exogenous 21 44that 20.06 0.12enzymes ˃50μM) as119.0 aofpositive control. noCYPs inhibition activity. CYPs Direct Inhibition (10 μM) CYPs Direct In 2.3. Metabolic Stability aCETP b on the result the inP450 vitro CETP inhibitory assay, Based therat potent on the inhibitor result of 17 the in vitro selected CETP inhibitory assay, therat pot aaStudy b bAnacetrapib a was b Direct Inhibition Mean (10 22 0.72 ± 32InBased ˃50 0.04 ± 0.01 Table 2.0.06 In vitro DMPK profile. Table 2. In vitro DMPK profile. with a Vitro clearance rate of and 146.1 μL/min/mg in with human with aand clearance liver rate microsomes. ofMean 119.0 and 146.1 μL/min/mg in human and li HLM Stability RLM Stability HLM Stability RLM Stability HLM Stability RLM Stability

15

Compound Compound were used testmetabolic the directstability inhibition of compound Compound possessed favourable metabolic Compound for the intovitro study. As shown in17. Table for the 2, compound in vitro17 metabolic 17 showed stability weakstudy. stability, (μL/min/mg) (μL/min/mg) (μL/min/mg) (μL/min/mg) b As shown in Table 2, compoun

21 44 no CETP 20.06b±Considered 0.12 ˃50 a Used as a positive control. (μL/min/mg) (μL/min/mg) 3A4 2D6 2D6 2C9activity. 1A2 1A2 2C19 2C19 3A4 2D6 with inhibition a Direct 3A4 2C9 b 2.3. Metabolic Stability Study 22 0.72 ± on the result of the vitro CETP inhibitory assay, therat potent inhibitor 17 selected CYPs Inhibition Mean (10 μM) CYPs Direct 32InBased Anacetrapib ˃50 0.04 ± 0.01 a inRLM b a was b with a Vitro clearance rate of 119.0 and 146.1 μL/min/mg in human with aand clearance liver rate microsomes. of 119.0 and 146.1 μL/min/mg in human and rat In li Table 2.0.06 In vitro DMPK profile. Table 2. In vitro DMPK profile. HLM Stability Stability HLM Stability RLM Stability Compound Compound for the in vitro metabolic stability study. As shown in Table 2, compound(μL/min/mg) 17 showed weak stability, (μL/min/mg) (μL/min/mg) a(μL/min/mg) b UsedStability as a positive control. with noaCETP inhibition activity. 1A2 0.04 ±2C19 3A4 2D6 2C9 3A4 2D6 0.72 ± Considered 0.06 2.3.22 Vitro on Metabolic Study b 32InBased Anacetrapib ˃50 0.01 the result of the in146.1 vitro CETP inhibitory assay, therat potent inhibitor was selected CYPs Direct Inhibition Mean17 (10 Direct In a b a μM) b with a clearance rate of 119.0 and μL/min/mg in human and liver microsomes. Table 2. In vitro DMPK profile. Table 2. In vitro DMPK CYPs profile. HLM Stability RLM Stability HLM Stability RLM Stability Compound Compound for the in vitro metabolic stability study. As shown in Table 2, compound 17 showed weak stability, a(μL/min/mg) b Considered (μL/min/mg) (μL/min/mg) (μL/min/mg) b Used as aofpositive control. withassay, noaCETP inhibition activity. 1A2 Anacetrapib ˃50 ±2C19 0.01 2.3.32InBased Vitro on Metabolic Stability Study 3A4 2D6 2C9 3A4 2D6 the result the in146.1 vitro CETP inhibitory therat potent inhibitor was0.04 selected CYPs Direct Inhibition Mean17 (10 CYPs Direct In with a clearance rate of 119.0 and μL/min/mg in human and liver microsomes. a b a μM) Table 2. In vitro DMPK profile. HLM Stability RLM Stability HLM Stability RLM Stability b for the in vitro metabolic stability study. As shown in Table 2, compound 17 showed weak stability, a Used as a b Compound Compound positive control. Considered with no CETP inhibition activity. (μL/min/mg) (μL/min/mg) (μL/min/mg) 2.3. InBased Vitro on Metabolic Stability Study the result of the in146.1 vitro CETP inhibitory assay, therat potent inhibitor 1A2 17 was (μL/min/mg) selected 3A4 2D6 2C19 3A4 2D6 with a clearance rate of 119.0 and μL/min/mg in human and liver2C9 microsomes. CYPs Direct Inhibition Mean (10 μM) Table 2. In vitro DMPK profile. a b

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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% CH3 OH or a mixture of solvents H2 O (A) 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 H2 O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 15:1) to give 11 (0.3 g, 92.8%) as a pale yellow oil. 1 H-NMR (400 MHz, DMSO-d6 ) δ 8.06 (s, 1H), 7.90 (s, 2H), 7.05 (dd, J = 8.5, 2.2 Hz, 1H), 6.91–6.77 (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, 6H). HPLC: tR = 13.710 min, 99.27%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)2H-tetrazol-5-amine (12): Intermediate 11 (0.1 g, 0.2 mmol) was dissolved in DMF (5 mL) and ammonium chloride (0.01 g, 0.8 mmol) and sodium azide (0.05 g, 0.2 mmol) were added. After being stirred at 100 ◦ C for 5 h, the reaction mixture was cooled to room temperature, and H2 O (20 mL) was added. The mixture was extracted with CH2 Cl2 (20 mL × 3) and the combined organic layers were washed with water (20 mL × 3) and brine (20 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 2:1) to give 12 (0.1 g, 96.3%) as a white solid. Mp 150.6–152.6 ◦ C. 1 H-NMR (600 MHz, DMSO-d6 ) δ 14.88 (s, 1H), 7.94 (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), 4.55 (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), 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). 13 C-NMR (150 MHz, CDCl ) δ: 151.95, 142.58, 139.79, 136.20, 132.01(×2), 129.76, 128.36, 127.92(×2), 3 127.27, 126.44, 123.95, 122.14, 121.79, 111.11(×2), 55.84(×2), 53.23, 39.75, 34.93, 33.16, 30.00, 28.75, 28.44, 27.24, 24.07, 23.99. HRMS calcd for C29 H34 F6 N5 O, [M + H]+ , 582.2589; found 582.2668. HPLC: tR = 14.640 min, 96.47%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)2H-tetrazol-5-amine-2-carboxylic acid methyl ester (13): Compound 12 (0.1 g, 0.2 mmol) and triethylamine (0.1 mL, 0.8 mmol) were dissolved in acetonitrile (2 mL) followed by the addition of methyl 2-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 H2 O (10 mL) was added. The aqueous layer was extracted

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with EtOAc (5 mL × 3) and the combined organic layers were washed with H2 O (5 mL × 3) and brine (5 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 13 (0.09 g, 68.4%) as a colourless oil. 1 H-NMR (400 MHz, CDCl3 ) δ 7.67 (s, 1H), 7.54 (s, 2H), 7.03 (dd, J = 8.4, 2.3 Hz, 1H), 6.76 (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, 1H), 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), 2.16–2.00 (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). 13 C-NMR (150 MHz, CDCl ) δ: 169.64, 165.70, 154.12, 141.11, 140.87, 135.34, 131.28(×2), 130.52, 3 128.06(×2), 127.76(×2), 127.63, 125.66(×2), 110.64(×2), 55.24, 53.01, 52.91, 51.69, 49.38, 40.57, 35.42, 33.03, 29.10, 28.99, 28.03(×2), 23.99(×2). HRMS calcd for C32 H38 F6 N5 O3 , [M + H]+ , 654.2801; found 654.2877. HPLC: tR = 8.753 min, 95.8%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)2H-tetrazol-5-amine-2-butyric acid ethyl ester (14): Colourless oil; yield 71.3%; 1 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, 4H), 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), 2.30 (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), 1.14 (s, 6H), 0.94 (d, J = 11.1 Hz, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 172.11, 169.37, 154.14, 141.34, 140.85, 135.18, 131.24(×2), 130.57, 128.08, 127.77(×2), 125.63, 124.16, 122.35, 120.60, 110.62(×2), 60.54, 55.22, 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. HRMS calcd for C35 H44 F6 N5 O3 , [M + H]+ , 696.3270; found 656.3361. HPLC: tR = 7.653 min, 96.4%. 2-(2-Aminoethyl)-N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)-2H-tetrazol-5-amine (15): Compound 12 (0.5 g, 0.9 mmol) and triethylamine (1.8 mL, 13.0 mmol) were dissolved in acetonitrile (10 mL), followed by the addition of tert-butyl 2-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 H2 O (10 mL) was added. The aqueous layer was extracted with EtOAc (5 mL × 3) and the combined organic layers were washed with H2 O (5 mL × 3) and brine (5 mL × 3), dried over Na2 SO4 , 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 H2 O (5 mL × 3) and brine (5 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 2:1) to give 15 (0.35 g, 62.2%) as a colourless oil. 1 H-NMR (400 MHz, DMSO-d6 ) δ 7.93 (s, 1H), 7.66 (s, 2H), 7.00 (dd, J = 8.4, 2.2 Hz, 1H), 6.79–6.76 (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), 1.04 (d, J = 6.9 Hz, 6H), 0.89 (d, J = 10.2 Hz, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 169.41, 154.13, 141.31, 140.86, 135.29, 131.25(×2), 130.55, 128.07, 127.80, 127.70, 125.65, 124.14, 122.34, 120.63, 110.62(×2), 55.81, 55.22, 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 C31 H39 F6 N6 O, [M + H]+ , 625.3011; found 625.3070. HPLC: tR = 18.895 min, 96.6%. 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 Ph3 P (0.07 g, 0.3 mmol) and DIAD (0.05 mL, 0.3 mmol). After being stirred at room temperature for 4 h, the reaction mixture was poured into H2 O (10 mL) and extracted with EtOAc (5 mL × 3), and the combined organic layers were washed with H2 O (5 mL × 3) and brine (5 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 1:1) to give 16 (0.09 g, 65.3%) as a colourless oil. 1 H-NMR (400 MHz, CDCl3 ) δ 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), 6.71 (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, 1H), 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),

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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). 13 C-NMR (150 MHz, CDCl3 ) δ: 169.58, 154.08,140.96, 135.68, 131.36(×2), 130.40, 128.01, 127.69, 127.33, 125.74(×2), 124.10, 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, 28.08, 28.03, 24.00, 23.98. HRMS calcd for C32 H40 F6 N5 O3 S, [M + H]+ , 688.2678; found 688.2754. HPLC: tR = 13.667 min, 95.6%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)2H-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 H2 O (10 mL). Then, 1 mol/L HCl (5 mL) was added to the mixture, the mixture was extracted with EtOAc (10 mL × 3), and the combined organic layers were washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (DCM:MeOH = 5:1) to give 17 (0.17 g, 86.4%) as a colourless oil. 1 H-NMR (600 MHz, DMSO-d6 ) δ 7.92 (s, 1H), 7.65 (s, 2H), 7.00 (dd, J = 8.5, 2.2 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), 3.63 (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), 1.36 (t, J = 6.4 Hz, 2H), 1.04 (d, J = 6.9 Hz, 6H), 0.90 (d, J = 14.3 Hz, 6H). 13 C-NMR (150 MHz, DMSO-d6 ) δ: 169.21, 167.87, 154.37, 142.28, 140.54, 134.58, 130.63(×2), 130.29, 127.81, 127.74, 127.62, 125.85, 124.50, 122.69, 120.98, 111.32(×2), 55.70, 53.85, 51.65, 49.55, 40.57, 35.36, 32.75, 29.24, 29.07, 28.46, 28.09, 24.27, 24.21. HRMS calcd for C31 H36 F6 N5 O3 , [M + H]+ , 640.2644; found 640.2709. HPLC: tR = 19.013 min, 97.3%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)2H-tetrazol-5-amine-2-butyric acid (18): Colourless oil; yield 81.3%; 1 H-NMR (400 MHz, DMSO-d6 ) δ 12.18 (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), 4.44 (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, 1H), 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), 0.88 (d, J = 7.5 Hz, 6H). 13 C-NMR (150 MHz, DMSO-d6 ) δ: 173.76, 169.19, 154.39, 142.35, 140.54, 134.46, 130.61(×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, 40.63, 35.36, 32.76, 30.40, 29.25, 29.05, 28.40, 28.13, 24.44, 24.25, 24.21. HRMS calcd for C33 H40 F6 N5 O3 , [M + H]+ , 668.2957; found 668.3050. HPLC: tR = 18.767 min, 97.1%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)2H-tetrazol-5-amine-2-ethyl alcohol (19): Compound 13 (0.4 g, 0.6 mmol) was dissolved in THF (5 mL) and NaBH4 (0.05 g, 1.2 mmol) and 2 drops of MeOH were added and 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 H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , 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. 1 H-NMR (600 MHz, DMSO-d6 ) δ 7.92 (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), 4.95 (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, 5.5 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, 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). 13 C-NMR (150 MHz, CDCl3 ) δ: 169.30, 154.11, 141.18, 140.89, 135.43, 131.32(×2), 130.51, 128.04, 127.72, 127.55, 125.67, 124.13, 122.32, 120.71, 110.63(×2), 60.30, 55.23, 54.95, 51.80, 49.41, 40.68, 35.40, 33.03, 29.12, 29.02, 28.04(×2), 23.99, 23.98. HRMS calcd for C31 H38 F6 N5 O2 , [M + H]+ , 626.2851; found 626.2920. HPLC: tR = 16.776 min, 95.8%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)2H-tetrazol-5-amine-2-butanol (20): Colourless oil; yield 73.8%; 1 H-NMR (400 MHz, DMSO-d6 ) δ 7.92 (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

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(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), 0.90 (d, J = 7.5 Hz, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 169.27, 154.14, 141.37, 140.85, 135.19, 131.22(×2), 130.58, 128.09, 127.77(×2), 125.63, 124.16, 122.36, 120.59, 110.62(×2), 61.78, 55.22, 52.52, 51.64, 49.35, 40.60, 35.43, 33.03, 29.12, 29.10, 29.00, 28.04(×2), 25.53, 23.99, 23.98. HRMS calcd for C33 H42 F6 N5 O2 , [M + H]+ , 654.3164; found 654.3257. HPLC: tR = 15.755 min, 96.4%. 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 NH3 –EtOH solution (5 mL). After being stirred at room temperature for 12 h, the reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAc (10 mL), washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 21 (0.12 g, 62.6%) as a colourless oil. 1 H-NMR (400 MHz, DMSO-d6 ) δ 7.91 (s, 1H), 7.70 (s, 1H), 7.66 (s, 2H), 7.42 (s, 1H), 6.99 (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), 3.62 (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), 1.35 (t, J = 6.2 Hz, 2H), 1.04 (d, J = 6.9 Hz, 6H), 0.89 (d, J = 10.9 Hz, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 169.77, 154.08, 140.96, 140.88, 131.43(×2), 130.42, 127.99(×2), 127.73, 127.29, 125.77(×2), 124.07, 122.26, 120.84, 110.68(×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 C31 H37 F6 N6 O2 , [M + H]+ , 639.2804; found 639.2909. HPLC: tR = 12.757 min, 98.2%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)-methyl)5-methyl-1,2,4-oxadiazol-3-amine (22): Intermediate 11 (0.3 g, 0.6 mmol) was dissolved in EtOH (5 mL) 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 H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 22 (0.15 g, 41.9%) as a colourless oil. 1 H-NMR (600 MHz, CDCl3 ) δ 7.66 (s, 1H), 7.50 (s, 2H), 7.00 (dd, J = 8.4, 2.1 Hz, 1H), 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), 3.90 (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, 1H), 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). 13 C-NMR (150 MHz, CDCl ) δ: 175.39, 170.36, 154.07, 140.92, 140.85, 135.59, 131.31(×2), 130.37, 3 128.00, 127.54, 127.35, 125.66, 124.13, 122.32, 120.70, 110.62(×2), 55.22, 51.08, 48.61, 40.40, 35.41, 33.01, 29.04, 29.01, 28.10, 27.97, 23.96(×2), 12.63. HRMS calcd for C31 H36 F6 N3 O2 , [M + H]+ , 596.2633; found 596.2716. HPLC: tR = 9.953 min, 95.8%. 2-(2-(Chloromethyl)-4,4-dimethylcyclohex-1-enyl)-4-isopropyl-1-methoxybenzene (23): Intermediate 9 (114.5 mg, 0.4 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 H2 O (20 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. SOCl2 (0.1 mL, 1.4 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 H2 O (10 mL) and was extracted with EtOAc (5 mL × 3). The combined organic layers were washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by 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: tR = 7.706 min, 96.8%. tert-Butyl 4-(3,5-bis(trifluoromethyl)benzylamino)piperidine-1-carboxylate (26): tert-butyl 4-aminopiperidine-1-carboxylate (1.0 g, 5.0 mmol) and 3,5-bis(trifluoromethyl) benzaldehyde (1.0 g, 4.1 mmol)

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were dissolved in MeOH (15 mL). After stirring for 3 h at room temperature, NaBH4 (0.3 g, 7.0 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 CH2 Cl2 (20 mL × 3) and the combined organic layers were washed with water (20 mL × 3) and brine (20 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by 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, CDCl ) 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 3 (m, 1H), 1.88 (d, J = 11.2 Hz, 2H), 1.46 (s, 9H), 1.35–1.26 (m, 2H). HPLC: tR = 13.657 min, 95.7%. 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 H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 20:1) to give 28 (235.9 mg, 67.7%) as a colourless oil. 1 H-NMR (400 MHz, CDCl3 ) δ 7.83 (s, 2H), 7.73 (s, 1H), 7.08 (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), 3.50 (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), 1.99–1.83 (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, 1.1 Hz, 6H), 0.96 (s, 6H). HPLC: tR = 7.023 min, 96.6%. 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 H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 2:1) to give 30 (172.1 mg, 96.1%) as a colourless oil. 1 H-NMR (400 MHz, DMSO-d6 ) δ 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), 6.83 (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), 2.26 (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), 1.17 (d, J = 6.9 Hz, 6H), 0.92 (d, J = 10.5 Hz, 6H). HPLC: tR = 13.003 min, 95.9%. 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 H2 O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was dissolved 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 H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 1:1) to give 32 (0.4 g, 52.0%) as a colourless oil. 1 H-NMR (400 MHz, DMSO-d6 ) δ 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), 6.74 (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), 2.84 (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, 1H), 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). 13 C-NMR (150 MHz, CDCl3 ) δ: 154.49, 144.61, 140.59, 132.83, 131.67, 131.25(×2), 129.57, 128.19, 128.00, 125.01, 124.38, 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,

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33.21(×2), 29.30, 28.93, 28.28, 27.72. 27.25, 24.23, 24.12. HRMS calcd for C35 H47 F6 N2 O2 , [M + H]+ , 641.3463; found 641.3525. HPLC: tR = 16.772 min, 97.9%. 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 H2 O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue 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 H2 O (10 mL). Then, 1 mol/L HCl (5 mL) was added to the mixture, the mixture was extracted with EtOAc (10 mL × 3), and the combined organic layers were washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (DCM:MeOH = 5:1) to give 33 (0.4 g, 67.8%) as a colourless oil. 1 H-NMR (400 MHz, DMSO-d6 ) δ 8.02 (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), 3.65 (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), 2.70 (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 (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). 13 C-NMR (150 MHz, DMSO-d6 ) δ: 168.60, 154.62(×2), 145.76, 140.43, 132.53, 131.21, 130.22(×2), 129.42, 128.93, 127.74, 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, 29.40, 29.00, 28.53, 27.95, 24.55(×2), 24.44(×2). HRMS calcd for C35 H45 F6 N2 O3 , [M + H]+ , 655.3256; found 655.3345. HPLC: tR = 19.973 min, 98.1%. 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 CH2 Cl2 (5 mL), and tetrahydro-2H-pyran-4-yl carbonochloridate (0.1 g, 0.5 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 H2 O (10 mL) and extracted with CH2 Cl2 (10 mL × 3). The combined organic layers were washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 34 (0.1 g, 45.9%) as a colourless oil. 1 H-NMR (600 MHz, CDCl3 ) 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, 1H), 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), 3.49–3.41 (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 (m, 1H), 1.22 (dd, J = 6.9, 2.4 Hz, 6H), 0.96 (s, 6H). 13 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, 65.30(×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, 28.33, 27.64, 24.24, 24.11(×2). HRMS calcd for C39 H51 F6 N2 O4 , [M + H]+ , 725.3675; found 725.3751. HPLC: tR = 15.553 min, 96.0%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((50 -isopropyl-20 -methoxy-4,4-dimethyl-3,4,5,6-tetrahydro-[1,10 -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 H2 O (10 mL) and extracted with CH2 Cl2 (10 mL × 3). The combined organic layers were washed with H2 O (10 mL × 3) and brine (10 mL × 3), dried over Na2 SO4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel (petroleum ether:EtOAc = 4:1) to give 35 (0.2 g, 89.2%) as a colourless oil. 1 H-NMR (400 MHz, CDCl3 ) δ 7.78 (s, 2H), 7.76 (s, 1H), 7.09 (dd, J = 8.4, 2.1 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), 3.80–3.72 (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

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(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), 0.96 (s, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 156.60(×2), 154.30(×2), 140.75, 131.29(×2), 131.14, 128.37, 127.94(×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), 29.38, 28.97, 28.22, 27.80, 24.18, 24.12(×2), 14.60(×2). HRMS calcd for C34 H43 F6 N2 O3 , [M + H]+ , 641.3100; found 641.3180. HPLC: tR = 11.763 min, 96.7%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((50 -isopropyl-20 -methoxy-4,4-dimethyl-3,4,5,6-tetrahydro-[1,10 -biphenyl] -2-yl)methyl)azetidin-3-amine-1-methanoic acid isopropyl ester (36): Colourless oil; yield 69.3%; 1 H-NMR (400 MHz, CDCl3 ) 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), 6.74 (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, 1H), 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), 2.76–2.64 (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), 1.22 (d, J = 6.9 Hz, 6H), 1.19 (d, J = 6.2 Hz, 6H), 0.96 (d, J = 2.8 Hz, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 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, 120.83, 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, 27.79, 24.19, 24.13, 22.11(×2). HRMS calcd for C35 H45 F6 N2 O3 , [M + H]+ , 655.3256; found 655.3344. HPLC: tR = 10.757 min, 96.8%. N-(3,5-Bis(trifluoromethyl)benzyl)-N-((50 -isopropyl-20 -methoxy-4,4-dimethyl-3,4,5,6-tetrahydro-[1,10 -biphenyl] -2-yl)methyl)azetidin-3-amine-1-methanoic acid tetrahydro-2H-pyran-4-ol ester (37): Colourless oil; yield 73.6%; 1 H NMR (600 MHz, CDCl3 ) δ 7.78 (s, 2H), 7.76 (s, 1H), 7.09 (dd, J = 8.4, 2.1 Hz, 1H), 6.80 (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), 3.82–3.78 (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), 2.73 (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 (m, 2H), 1.45–1.34 (m, 2H), 1.22 (d, J = 6.9 Hz, 6H), 0.97 (d, J = 4.2 Hz, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 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, 110.57(×2), 69.44, 65.26(×2), 55.33(×2), 54.49, 53.46, 41.59, 35.48, 33.14(×2), 32.07(×2), 29.40, 28.98(×2), 28.23, 27.82, 24.20, 24.13. HRMS calcd for C37 H47 F6 N2 O4 , [M + H]+ , 697.3362; found 697.3459. HPLC: tR = 16.003 min, 95.8%. 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 described for the preparation of intermediate 28. Colourless oil; yield 70.5%; 1 H-NMR (600 MHz, DMSO-d6 ) 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), 4.10 (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, 1H), 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), 0.89 (d, J = 13.2 Hz, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 169.46, 154.12, 141.34, 140.83, 135.26, 131.26(×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, 40.56, 39.20, 35.44, 33.02, 29.09, 29.02, 28.08, 27.99, 23.98(×2). HRMS calcd for C30 H36 F6 N5 O, [M + H]+ , 596.2746; found 596.2847. HPLC: tR = 8.776 min, 96.1%. N-((2-(5-Isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)-2-methyl-N-(3-(trifluoromethyl)benzyl)-2H-tetrazol-5-amine (41): Colourless oil; yield 78.4%; 1 H-NMR (600 MHz, DMSO-d6 ) δ 7.52 (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, 1H), 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), 3.63 (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), 1.38 (t, J = 6.5 Hz, 2H), 1.05 (dd, J = 6.9, 0.8 Hz, 6H), 0.91 (d, J = 5.1 Hz, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 169.66, 154.26, 140.64, 139.40, 134.35, 130.80, 130.73, 128.32, 128.03, 125.36, 124.31, 124.29, 123.43, 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, 24.01. HRMS calcd for C29 H37 F3 N5 O, [M + H]+ , 528.2872; found 528.2960. HPLC: tR = 8.335 min, 96.2%. N-(4-Fluorobenzyl)-N-((2-(5-isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)-2-methyl-2Htetrazol-5-amine (42): Colourless oil; yield 80.1%; 1 H-NMR (600 MHz, DMSO-d6 ) δ 7.07–7.05 (m, 1H),

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6.97–6.93 (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), 3.85 (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, 1H), 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), 0.93 (s, 6H). 13 C- NMR (150 MHz, CDCl3 ) δ: 169.67, 154.42, 140.66, 133.58, 130.93, 129.47, 129.41, 128.30, 128.14, 127.90, 125.25, 114.72, 114.58, 110.44(×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 C28 H37 FN5 O, [M + H]+ , 478.2904; found 478.2999. HPLC: tR = 7.750 min, 96.7%. N-((2-(5-Isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)-2-methyl-N-(4-(trifluoromethyl)benzyl)-2H-tetrazol-5-amine (43): Colourless oil; yield 67.5%; 1 H-NMR (600 MHz, CDCl3 ) δ 7.37 (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, 1H), 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), 1.14 (dd, J = 6.9, 1.8 Hz, 6H), 0.96 (s, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 169.62, 154.32, 142.35, 140.67, 134.24, 130.74, 128.03, 127.98, 127.70(×3), 125.30, 124.87, 124.85, 110.43(×2), 55.20, 50.82, 49.03, 40.41, 39.16, 35.60, 33.06, 29.22, 29.05, 28.30, 27.93, 24.15, 23.98. HRMS calcd for C29 H37 F3 N5 O, [M + H]+ , 528.2872; found 528.2953. HPLC: tR = 8.985 min, 96.7%. N-((2-(5-Isopropyl-2-methoxyphenyl)-5,5-dimethylcyclohex-1-enyl)methyl)-2-methyl-N-(4-(trifluoro-methoxy) benzyl)-2H-tetrazol-5-amine (44): Colourless oil; yield 66.3%; 1 H-NMR (600 MHz, DMSO-d6 ) δ 7.14 (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), 4.11 (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), 2.06 (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), 0.92 (d, J = 1.6 Hz, 6H). 13 C-NMR (150 MHz, CDCl3 ) δ: 169.66, 154.37, 140.66, 136.94, 133.96, 130.81, 129.00(×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, 33.10, 29.21, 29.03, 28.25, 27.97, 24.14, 24.01. HRMS calcd for C29 H37 F3 N5 O2 , [M + H]+ , 544.2821; found 544.2898. HPLC: tR = 8.302 min, 97.3%. 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 µM midazolam; CYP2D6, 5 µM dextromethorphan; CYP2C9, 5 µM diclofenac; CYP1A2, 10 µM phenacetin; CYP2C19, 30 µM S-mephenytoin) were used to evaluated cytochrome P450 inhibition in human liver microsomes (0.25 mg/mL). A mixture of 20 µL specific probe substrate solution and 20 µL buffer solution (100 mM K3 PO4 , 33 mM MgCl2 , pH = 7.4) was added compound (2 µL) and 158 µL human liver microsomes solution (0.25 mg/mL). The mixture were incubated at 37 ◦ C for 10 min and another 10 min underwent after 20 µL NADPH solution added. Consecutively, 400 µL cold stop solution (200 ng/mL tolbutamide and 200 ng/mL 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.

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3.4. Metabolic Stability Study Ten µL (100 µM/L) of compound and 80 µL liver microsomes were mixture and incubated at 37 ◦ C for 10 min, and then 10 µL NADPH regenerating system was added. Samples were obtained at 0 min, 5 min, 10 min, 20 min, 30 min and 60 min respectively, and 300 µL 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 ± 0.08 µM), and compound 17 demonstrated weak human/rat liver microsomes stability and low CYP inhibition. 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.

4.

5.

6. 7.

8. 9.

10.

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. 2002, 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] 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. 2007, 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, 1993–2000. [PubMed] 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] 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. 2004, 350, 1505–1515. [CrossRef] [PubMed] 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

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

15 of 15

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. 2007, 356, 1304–1316. [CrossRef] [PubMed] 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 (RADIANCE 2 study): a randomised, double-blind trial. Lancet 2007, 370, 153–160. [CrossRef] 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] 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] 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] 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] 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] 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] 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] 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] 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] 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] 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] 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. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).