Discovery of pentacyclic triterpene 3β-ester derivatives as a new class of cholesterol ester transfer protein inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.08.012

A series of pentacyclic triterpene 3β-ester derivatives were designed, synthesized and evaluated as a new class of cholesteryl ester transfer protein (CETP) inhibitors for the treatment of dyslipidemia. In vitro screening assay showed that 5 out of 30 compounds displayed moderate inhibiting human CETP activity with IC₅₀s less than 10 μM. Among them, compound 20 (IC₅₀ = 2.3 μM) had the most potent biological activity, and effectively ameliorated plasma lipid levels of human adipose tissue specific CETP transgenic (ap2-CETPTg) mice and guinea pigs. Additional safety evaluation (no blood pressure elevation in guinea pigs) and pharmacokinetics studies indicated that the potential druggability for compound 20 which is a promising lead for development of a new class of CETP inhibitors for the treatment of dyslipidemia.

Full text of DOI:10.1016/j.ejmech.2017.08.012

Accepted Manuscript
Discovery of pentacyclic triterpene 3β-ester derivatives as a new class of cholesterol
ester transfer protein inhibitors
Dongyin Chen, Xin Huang, Hongwen Zhou, Hanqiong Luo, Pengfei Wang, Yongzhi
Chang, Xinyi He, Suiying Ni, Qingqing Shen, Guoshen Cao, Hongbin Sun, Xiaoan
Wen, Jun Liu
PII:
S0223-5234(17)30615-3
10.1016/j.ejmech.2017.08.012
EJMECH 9656
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2017
Revised Date: 8 July 2017
Accepted Date: 3 August 2017
Please cite this article as: D. Chen, X. Huang, H. Zhou, H. Luo, P. Wang, Y. Chang, X. He, S. Ni, Q.
Shen, G. Cao, H. Sun, X. Wen, J. Liu, Discovery of pentacyclic triterpene 3β-ester derivatives as a new
class of cholesterol ester transfer protein inhibitors, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.08.012.
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ACCEPTED MANUSCRIPT
Graphical abstract:
Discovery of pentacyclic triterpene 3β-ester derivatives as a new class of
cholesterol ester transfer protein inhibitors
a
Dongyin Chen a, b, d, Xin Huang a, d, Hongwen Zhou c, d, Hanqiong Luo , Pengfei
Wang a, Yongzhi Chang a, Xinyi He a, Suiying Ni a, Qingqing Shen a, Guoshen Cao a,
Hongbin Sun a, Xiaoan Wen a, *, Jun Liu a, *
a Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases and State Key
Laboratory of Natural Medicines, Center of Drug Discovery, China Pharmaceutical
University, 24 Tongjia Xiang, Nanjing 210009, China
b
Department of Medicinal Chemistry, School of Pharmacy, Nanjing Medical
University, Nanjing 211166, China
c
Department of Endocrinology, the First Affiliated Hospital of Nanjing Medical
University, Nanjing 210029, China
d These authors contribute equally

ACCEPTED MANUSCRIPT
Discovery of pentacyclic triterpene 3β-ester derivatives as a new
class of cholesterol ester transfer protein inhibitors
Dongyin Chen a, b, d, Xin Huang a, d, Hongwen Zhou c, d, Hanqiong Luo a, Pengfei Wang a,
Yongzhi Chang a, Xinyi He a, Suiying Ni a, Qingqing Shen a, Guoshen Cao a, Hongbin Sun a,
Xiaoan Wen a, *, Jun Liu a, *
a Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases and State Key Laboratory
of Natural Medicines, Center of Drug Discovery, China Pharmaceutical University, 24 Tongjia
Xiang, Nanjing 210009, China
b Department of Medicinal Chemistry, School of Pharmacy, Nanjing Medical University, Nanjing
211166, China
c
Department of Endocrinology, the First Affiliated Hospital of Nanjing Medical University,
Nanjing 210029, China
d These authors contribute equally
Corresponding Author
X. Wen, E-mail address: wxagj@126.com.
J. Liu, E-mail address: junliu@cpu.edu.cn.
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Abstract
A series of pentacyclic triterpene 3β-ester derivatives were designed, synthesized and
evaluated as a new class of cholesteryl ester transfer protein (CETP) inhibitors for the treatment
of dyslipidemia. In vitro screening assay showed that 5 out of 30 compounds displayed moderate
inhibiting human CETP activity with IC50s less than 10 µM. Among them, compound 20 (IC50 =
2.3 µM) had the most potent biological activity, and effectively ameliorated plasma lipid levels
of human adipose tissue specific CETP transgenic (ap2-CETPTg) mice and guinea pigs.
Additional safety evaluation (no blood pressure elevation in guinea pigs) and pharmacokinetics
studies indicated that the potential druggability for compound 20 which is a promising lead for
development of a new class of CETP inhibitors for the treatment of dyslipidemia.
Keywords
CETP inhibitor, pentacyclic triterpenes, non-HDL-C, dyslipidemia
Abbreviations used
CVDs, cardiovascular diseases; CETP, cholesteryl ester transfer protein; HDL-C, high density
lipoprotein-cholesterol; LDL-C, low density lipoprotein-cholesterol; VLDL, very-low-density
lipoprotein; TC, total cholesterol; TG, triglyceride; CE, cholesteryl ester; ACAT, acyl-CoA:
cholesterol acyl transferase; PPARs, peroxisome proliferator-activated receptors; PLs, pancreatic
lipase; FXR, farnesoid X receptor; LXR, liver X receptor; PTs, pentacyclic triterpenes; SAR,
structure activity relationship; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride; DMAP, 4-dimethylaminopyridine; H-mont, proton-exchanged montmorillonite;
TLC, thin layer chromatography; HPLC, high performance liquid chromatography
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1. Introduction
Cardiovascular diseases (CVDs) continue to be the leading cause of mortality worldwide over
the past two decades [1, 2]. Atherosclerosis, a progressive disease characterized by the
accumulation of lipids and the formation of atheroma plaque within the arteries, which is the
primary cause of CVDs [3]. Numerous epidemiological evidences confirmed that lowering low-
density lipoprotein cholesterol (LDL-C) levels as well as raising high density lipoprotein-
cholesterol (HDL-C) levels in plasma appeared as a promising treatment strategy for
cardiovascular pathologies [4]. Additional clinical studies indicated that non-HDL-C (total
cholesterol minus HDL-C) and triglyceride (TG) could not be neglected for the prediction and
treatment of CVDs, and even had many other compelling advantages over LDL-C and other
traditional lipid parameters [5]. Currently, statin drugs are widely used in the prevention and
treatment of CVDs by significantly lowering serum LDL-C levels; however, part of patients
receiving statin therapy suffer from serious adverse events, such as muscle pain, myopathy and
even fatal rhabdomyolysis [6]. It is undeniable that intensive-dose statin therapy is associated
with an increased risk of new-onset diabetes [7]. Niacin, one of the oldest HDL-C-elevating
drugs tested in clinical trials, has less benefit in patients at high risk of vascular events, and
possesses adverse effects (cutaneous flushing and hyperglycemia) [8]. Therefore, the reasonable
therapeutic strategy for patients with cardiovascular disease should effectively accommodate the
balance between HDL-C and LDL-C, meanwhile positively ameliorate the other risk factors for
atherosclerosis including total cholesterol (TC) and TG.
Cholesteryl ester transfer protein (CETP), a hydrophobic glycoprotein containing 476 amino
acids, plays an unfavorable physiological action by transferring cholesteryl esters (CE) from
HDL to very-low-density lipoprotein (VLDL) or LDL particles in exchange for TG, thereby
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raising proatherogenic LDL-C and lowering atheroprotective HDL-C [9]. Accordingly, inhibition
of CETP has been considered as a promising approach for the prevention and treatment of CVDs
[10]. To date, a number of structurally distinct CETP inhibitors have been developed, and four of
them entered phase III clinical trials [11]. (Fig. 1) Torcetrapib, as the first CETP inhibitor tested
in a long-term outcomes clinical trial, discontinued in 2006 due to the elevation of blood pressure
and aldosterone levels [12]. In 2015 Evacetrapib's phase III trials was terminated because of the
lack of sufficiently clinical efficacy for patients with ‘high-risk’ coronary disease [13]. However,
the Dal-GenE randomized trial is currently being conducted in patients with a recent acute
coronary syndrome to evaluate the effects of Dalcetrapib on cardiovascular risk in a genetically
defined population [14]. The large phase III clinical outcome trial of Anacetrapib is also ongoing
to determine the safety and efficacy in patients with atherosclerotic cardiovascular diseases [14].
Recent research findings showed that Anacetrapib reduced progression of atherosclerosis by
systematically reducing non-HDL-cholesterol, improved lesion stability and added to the
beneficial effects of atorvastatin [15]. Nevertheless, almost all the reported CETP inhibitors
exhibited highly lipophilic and poorly aqueous soluble characteristics [11], which might be an
unfavorable factor for their discovery and clinical development [16]. Consequently, it is in urgent
need to develop novel CETP inhibitors with different structural scaffolds to overcome the
adverse effects and improve the efficacy.
<<Fig.1 here>>
In order to find new structural CETP inhibitor candidates, a literature research was carried out
to screen active components from natural Chinese herbal medicine. Pentacyclic triterpenes (PTs),
a group of widespread natural products that synthesized by the cyclization of squalene [17],
attracted our attention, that were observed to possess hypolipidemic activity more than thirty
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years ago [18]. In China, the tablet of fine extract of hawthorn containing oleanolic acid (1) and
ursolic acid (3) as the main triterpene ingredients has been marketed as an OTC drug for the
treatment of hyperlipidemia. Many clinical studies had confirmed that there was a significant
decrease in the serum TC, TG and LDL levels, and increase in HDL levels after treatment with
the plant extracts containing various natural pentacyclic triterpene compounds [19]. However, the
mechanism of hypolipidemic activity of PTs is still poorly understood. Given the similar
structures of natural PTs with that of cholesterol and CE, it was proposed that PTs might exert
hypolipidemic action through the regulation of cholesterol transport and metabolism [19].
Based on the above findings, we believed that PTs and their derivatives should be explored as
novel CETP inhibitors for the treatment of dyslipidaemia. In fact, we have previously reported a
new class of CETP inhibitors designed by linking the PTs scaffolds and the active fragment from
Torcetrapib with the linker methylene chain [20]. In this manuscript, we first describe the design
and synthesis of pentacyclic triterpene 3β-ester derivatives as novel CETP inhibitors, as well as
structure-activity relationship (SAR) analysis, in vitro and in vivo biological testing, preliminary
safety evaluation and pharmacokinetic studies.
2. Results and discussion
2.1. Design of pentacyclic triterpene 3β-ester derivatives as novel CETP inhibitors
To design our compounds, oleanolic acid (1) was selected as a potential lead compound. As we
have observed, 1 bears a hydroxyl group at C-3 position and a rigid pentacyclic skeleton with a
large surface area of hydrophobic environment, which is quite similar with that of cholesterol.
(Fig. 2) In addition, a carboxyl group at C-17 position of 1 might be a potential hydrogen bond
site (HBS) that form interaction with CETP. In our previous studies, molecular docking of 1 into
the active site of CETP confirmed that it almost occupied the binding position of the endogenous
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ligand CE. Besides, hydrogen bond interactions between 1 and Ser191, Ser230 were observed
[20]. (Fig. 2) Based on these findings, we hypothesized that PTs might be substrate analogue
inhibitors of CETP, mimicking the protein-ligand interactions between the active site and CE
scaffold. This could provide a reasonable explanation for the hypolipidemic effect of PTs.
<<Fig.2 here>>
To better simulate the interactions between endogenous ligand CE and CETP, we proposed the
strategy for molecular design of novel CETP inhibitors by linking the pentacyclic scaffold of 1
with some hydrophobic and flexible alkyl chains at C-3 position. Besides, some hydrophilic
segments introduced into the another end of these alkyl chains could form crucial hydrogen bond
interactions with several polar residues in the active site region of CETP, such as Gln-199, Ser-
230, and His-232 [21]. (Fig. 3) In addition, the length of alkyl chains and different pentacyclic
skeletons would also be investigated to point us toward the most promising candidates.
<<Fig.3 here>>
2.2. Chemistry
Synthesis of the target compounds 7-11 is shown in Scheme 1. Esterification of the 3β-
hydroxyl group of 1 with corresponding anhydrides in anhydrous pyridine gave the target
products 7-9 in 52-90% yield. As described in our previous report [22], benzylation of 1 with
benzyl bromide or 4-methoxy benzyl chloride in the presence of K2CO3 in DMF at room
temperature, followed by condensation with n-nonylic acid or oleic acid in the present of EDCI
and DMAP afforded the corresponding benzyl esters 33 and 34 in 81% and 68% yield over two
steps, respectively. Debenzylation of 33 with hydrogen over palladium/carbon produced target
compound 10. According our previous reported method [23], compound 11 was obtained in 70%
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yield by deprotection of the p-methoxybenzyl group of 34 with proton-exchanged
montmorillonite (H-mont).
<<Scheme 1 here>>
Actually, the new drug research and development of PTs for prevention and treatment of
metabolic and vascular diseases has been an important part of our work, and a large amount of
triterpenoid derivatives have been synthesized and formed a chemical compound library.
Compounds 12-16 were prepared according to the procedures described in our previous studies
[24]. Remarkably, heterocyclic, sulfonic and carboxyl group at the end of these compounds could
form crucial hydrogen bond interactions with the active site region of CETP.
As shown in Scheme 2, esterification of the 3β-hydroxyl group of 1 separately with succinic
anhydride, glutaric anhydride, and phthalic anhydride to furnish the corresponding target
compounds 17, 18 and 20 in 68-80% yield. Alternatively, benzylation of 1 with benzyl bromide,
followed by condensation with adipic acid in presence of EDCI and DMAP, and then
debenzylation with hydrogen over palladium/carbon in THF generated target product 19 in 65%
yield over three steps. According to the above procedures, benzylation of 1 with benzyl bromide,
followed by esterification separately with succinic anhydride and glutaric anhydride to give key
intermediates 35 and 36. Treatment of 35 and 36 with sulfinyl chloride in CH2Cl2, followed by
amidation with separately ammonia, morpholidine, and 2-hydroxyethylamine, and further
debenzylation over palladium/carbon gave the corresponding target products 21-26 in 32-94%
yield over three steps.
<<Scheme 2 here>>
Additionally, target compounds 27-32 were synthesized to investigate the roles of carboxyl
group at C-17 position and different pentacyclic skeletons. As outlined in Scheme 3, compounds
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27 and 28 were synthesized by esterification of the 3β-hydroxyl group of β-amyrin (5) separately
with succinic anhydride and glutaric anhydride. In similar fashions, target product 29 was
synthesized from carboxylic acid 6, whose preparation was described in our previous report [25].
As shown in Scheme 4, acylation of the 3β-hydroxyl groups of δ-oleanolic acid (2) [26], ursolic
acid (3) and betulinic acid (4) using glutaric anhydride provided target compounds 30-32
containing three different pentacyclic skeletons. The details of the synthetic procedures and
structural characterizations of target compounds are described in the Experimental Section.
<<Scheme 3 here>>
<<Scheme 4 here>>
2.3. Screening assay of natural PTs and their 3β-ester derivatives as CETP inhibitors and SAR
analysis.
The inhibition of CETP mediated CE transfer for natural PTs 1-4 and all target compounds 7-
32 were characterized in vitro using a fluorescence transfer assay with human recombinant
CETP. The initial screening was carried out at a concentration of 10 µΜ for each compound, and
compounds that displayed >30% inhibition at 10 µΜ were further evaluated for their IC50s. The
results are summarized in Table 1, and the details of the bioassay procedures are described in the
Experimental Section. As shown in Table 1, thirteen compounds demonstrated moderate
inhibitory activities, with >30% inhibition at a concentration of 10 µΜ for each compound.
Three natural PTs such as oleanolic acid (1), δ-oleanolic acid (2) and betulinic acid (4) failed to
show their inhibitory activity in vitro, but ursolic acid (3) displayed a moderate inhibitory
potency against CETP with an IC50 of 46.6 µM. It gives us great encouragement to explore a
series of pentacyclic triterpene 3β-ester derivatives (see compounds 7-11, as shown in Table 1
and Scheme 1), which better mimicking the structure of endogenous ligand CE. Unfortunately,
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the introduction of these hydrophobic alkyl groups at C-3 position caused a loss in inhibitory
activity, which suggested that these compounds with high lipid solubility might not be a
potentially strong contender to CE in the active site region of CETP. However, the introduction
of heterocyclic groups into the end of the alkyl chain of compound 8 afforded the new
compounds 12-14, which brought us some exciting surprises. Among them, compound 12
displayed a moderate inhibitory effect toward CETP with an IC50 of 68.5 µM, but compounds 13
and 14 have hardly any inhibitory potency. Additionally, compound 15 with sulfonic group in
the end exhibited IC50 of 23.1 µM, while succinic acid ester 17 displayed an excellent inhibitory
effect toward CETP (IC50 = 4.3 µM), which was nearly 6-fold better than that of compound 15.
However, malic acid ester 16 showed no inhibitory activity against CETP, since the stable
intramolecular hydrogen bond between the carboxyl group and hydroxyl group disturbed its
binding to the active site region. This unexpected result indicates that the significant impact of
hydrophilic segments in the end of these alkyl chains on the potency.
On the basis of above results, we held the carboxyl groups at opposite ends on the molecular
scaffolds of compounds 18-20, which all displayed an excellent CETP inhibitory activity.
Among them, phthalic acid ester 20 exhibited the most potent CETP inhibitory activity; its IC50
reached 2.3 µM. Remarkably, glutaric acid ester 18 exhibited IC50 of 3.5 µM, while adipic acid
ester 19 showed a faint loss in inhibitory potency against CETP, with an IC50 of 7.0 µM. This
indicates the significant impact of the carbon chain length on the potency. To further investigate
the importance of carboxyl group, we attempted to introduce several amides to the end of the
carbon chain of compounds 17 and 18, which caused a complete loss in inhibitory activity (see
compounds 21-26, as shown in Table 1 and Scheme 2). Besides, we further substituted the
carboxyl group from C-17 position of compounds 17 and 18 with methyl group to form
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compounds 27 and 28, which almost completely ablated their activities. However, compound 29
with a carboxylmethoxycarbonyl group at C-17 position had a moderate inhibitory potency, and
its IC50 value was 38.9 µM. The above results suggest that carboxyl groups at opposite ends on
the molecular scaffold are both important to maintain the inhibitory potency of these compounds
toward CETP.
Based on the above findings, we also synthesized compounds 30-32 with different pentacyclic
skeletons, and further evaluated their biological activities toward CETP. As shown in Table 1, a
decrease in CETP inhibitory activity was observed with the bioassay of olean-13(18)-ene-type
derivative 30 and lupane-type derivative 32, which exhibited IC50 of 10.3 µM and 38.0 µM,
respectively. However, ursane-type derivative 31 exhibited excellent CETP inhibitory activity,
with an IC50 of 3.4 µM, which was as well as that of the corresponding oleanane-type derivative
18. These data show that the different pentacyclic scaffolds have an important influence on the
CETP inhibitory activities of these compounds; oleanane-type and ursane-type scaffolds might
be more suitable to fit the active pocket of CETP than other pentacyclic scaffolds. Consequently,
according to the preliminarily SAR analysis, compound 20 was identified as a potent CETP
inhibitor for an in-depth study in vivo.
<<Table 1 here>>
2.4. In vivo studies of compound 20.
Compound 20 was first tested in human adipose tissue specific CETP transgenic (ap2-
CETPTg) mice pharmacodynamic model. Adipose tissue of ap2-CETPTg mice has a high level
of CETP, which secrets into the blood circulation, reduces HDL-C, and increases non-HDL-C
significantly; the lipid profile is very similar with lipid metabolism of type 2 diabetic [27].
Compound 20 was dosed in ap2-CETPTg mice at 30 mg/kg, once-a-day (q.d.); plasma samples
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were obtained at 2 h after dosing on day 14, and then HDL-C, LDL-C, TG, and TC levels were
measured. As shown in Fig. 4, compound 20 increased HDL-C level by 11.1% and decrease
LDL-C level by 34.2% versus control, while an increase of 51.0% in HDL-C and a decrease of
40.4% in LDL-C were observed after dosing with Anacetrapib. Although compound 20 showed
a slightly weaker activity in raising HDL-C and lowering LDL-C than Anacetrapib, it could
effectively reduce TG and TC levels by 29.3% and 5.8%, respectively. Quite unexpectedly, 4.7%
increase in TG level and 17.8% increase in TC level were observed after dosing of Anacetrapib
for two weeks in ap2-CETPTg mice, which is worthy of further investigation.
<<Fig.4 here>>
In view of reasonably regulation of lipid profile in CETP transgenic mice, we further evaluated
compound 20 in a species that naturally expresses CETP. Since guinea pigs (unlike mice and rats)
express CETP, and the lipid metabolism of which has some similarities to that of human,
compound 20 was next tested in the normal fed and high-fat fed guinea pig models. As depicted
in Fig. 5, compound 20 produced elevation in HDL-C of approximately 32.3% (P < 0.05,
compared with control) and reduction in LDL-C of 19.8% in normal fed guinea pigs after dosing
30 mg/kg (q. d.) for two weeks. In our further studies, high-fat fed guinea pig models have a
higher level of LDL-C, TG and TC, and a lower level of HDL-C than the control guinea pigs.
(Fig. 6) An increase of 72.8% in HDL-C and a decrease of 40.8% in LDL-C versus model were
observed after dosing with compound 20, while Anacetrapib increased HDL-C level by 82.2%
and decreased LDL-C level by 30.2%. (Fig. 6A and 6B) Much to our satisfaction, compound 20
could also reduce TG and TC levels by 27.3% and 20.3% compared with model, respectively.
(Fig. 6C and 6D)
<<Fig.5 here>>
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<<Fig.6 here>>
According the above results, compound 20 showed similar effects on modulating lipid profile
as Anacetrapib in the normal fed and high-fat fed guinea pig models, in spite of its moderate
inhibiting human CETP activity in vitro. However, one thing that needs to be addressed is that,
natural PTs are often mild and sometimes even weak modulators for their targets, which can
interact with multiple target proteins in lipid metabolism pathways [19]. For example, oleanolic
acid (1) showed mild ACAT inhibitory activity (IC50 = 77.9 µM) [28] and weak PL inhibitory
activity (IC50 = 83 µg/mL) [29]. Therefore, compound 20 with a natural product scaffold may be
a multi-site modulator of lipid metabolism at a holistic level. To better explain its hypolipidemic
effects, we would investigate the interaction of compound 20 with some other target proteins in
the lipid metabolism pathways, such as ACAT, PPARs, PLs, FXR and LXR, etc. In our opinion,
the mild interaction of compound 20 with these related target proteins may be the key to
systematically modulate lipid metabolism.
2.5. Safety evaluation of compound 20.
In view of excellent biological activities of compound 20, we further evaluated its effects on
the systolic blood pressure in normal fed and high-fat fed guinea pig models. As shown in Fig.
7A and 7B, the systolic blood pressure of normal fed and high-fat fed guinea pigs had hardly any
change versus control and model after dosing with 30 mg/kg (q. d.) compound 20 for two weeks.
In addition, a comparison of physiochemical property profiles of compound 20 and four typical
CETP inhibitors are shown in Table 2. To our satisfaction, compound 20 with two carboxyl
groups exhibited better aqueous solubility, with a partition coefficient of 0.49, which might be an
advantage for drug absorption and drug safety over the existing CETP inhibitors.
<<Fig.7 here>>
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<<Table 2 here>>
2.6. Pharmacokinetic evaluation of compound 20.
Compound 20 was further evaluated for its preliminary pharmacokinetic profile in normal fed
guinea pig models, compared with 1 at the same oral dose, and the relevant pharmacokinetic
parameters are listed in Table 3. The results showed that compound 20 had a good
pharmacokinetic profile, with an AUC0-48 (area under the plasma concentration-time curve from
0 to 48 hours) of 9337.83 ± 2152.23 ng/mL*h and a good half-life (t1/2 = 18.75 ± 5.15 h). After
oral administration of compound 20 at 30 mg/kg dose, plasma concentrations reached its peak
(Cmax = 1266.46 ± 406.61 ng/mL) at 1.46 ± 0.40 h. It is noteworthy that only a very small amount
of compound 20 metabolized into 1 in normal fed guinea pigs. (Fig. 8) On the basis of its
favorable in vitro and in vivo properties, compound 20 was selected as a candidate for further
development.
<<Table 3 here>>
<<Fig.8 here>>
3. Conclusions
In the present study, a series of pentacyclic triterpene 3β-ester derivatives were designed and
synthesized, and their inhibitory activities in vitro were evaluated using a fluorescence transfer
assay with human recombinant CETP. Among them, five compounds displayed moderate
inhibitory activities against CETP, with IC50s < 10 µM. Compound 20 showed the best biological
activity, with an IC50 of 2.3 µM. The preliminary SAR analysis indicated that hydrophilic
segments at opposite ends on the molecular scaffold are indispensable to maintain the CETP
inhibitory potency of these compounds, and different pentacyclic skeletons play a key role for
the inhibitory activities. Further studies in vivo showed that compound 20 provided an 11.1%
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increase in HDL-C levels and a 34.2% decrease in LDL-C levels versus control in ap2-CETPTg
mice. Remarkably, compound 20 could effectively reduce TG and TC levels by 29.3% and 5.8%,
which was superior to the positive control drug Anacetrapib. Furthermore, compound 20 also
showed robust potential to regulate the lipid profile both in the normal fed and high-fat fed
guinea pig models, which is very comparable to Anacetrapib. Additionally, no significant
changes on the systolic blood pressure in normal fed and high-fat fed guinea pigs when
compound 20 was administered orally for two weeks. Preliminary pharmacokinetics studies in
guinea pigs indicated that compound 20 has a good pharmacokinetic profile. In summary, the
studies described herein demonstrate that these pentacyclic triterpene 3β-ester derivatives with
hydrophilic segments at opposite ends on the molecular scaffold, especially compound 20, have
promising potential as novel generation of CETP inhibitors for the treatment of dyslipidemia.
4. Experiments
4.1. Chemistry
All commercially available solvents and reagents were used without further purification.
Reactions were monitored by TLC on Silica Gel 60 F254 plates (Qingdao Ocean Chemical
Company, China). Column chromatography was carried out on silica gel (200-300 mesh,
1
Qingdao Ocean Chemical Company, China). H and 13C NMR spectra were recorded on an
ACF* 300 Q Bruker or ACF* 500 Q Bruker spectrometer in CDCl3, or in DMSO-d6 with Me4Si
as the internal reference. Low- and high- resolution mass spectra (LRMS and HRMS) were
recorded in electron impact mode. Chemical shifts were reported in parts per million (ppm).
Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), multiplet (m), and
broad (br). In addition, compound 20 was confirmed with over 98% purity, which was
determined by Agilent 1260 with binary pump, photodiode array detector, using Agilent Extend-
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C18 column (0.46 cm × 15 cm, 5 µm), CH3OH/H2O = 80/20 (v/v) at 1.0 mL/min, and calculated
the peak areas at 254 nM.
4.1.1. General procedure for the synthesis of compounds 7-9, 17, 18 and 20.
To a solution of 1 (100 mg, 0.219 mmol) dissolved in dry pyridine (3 mL) was added
anhydrides (0.876 mmol) and DMAP (27 mg, 0.219 mmol). The reaction mixture was stirred at
room temperature (or heated to reflux) until the TLC indicated the consumption of starting
material, and then concentrated in vacuo to dryness. Then water (10 mL) was added to the
reaction mixture and the aqueous layer was extracted with EtOAc (3 × 5 mL). The combined
organic layers were washed with brine and dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by flash chromatography (SiO2, petroleum
ether/ethyl acetate 30 : 1 to 5 : 1) to give the desirable product.
4.1.1.1. 3
β
-Acetyloxy-olean-12-en-28-oic acid (7) [30]. Compound 7 (98 mg, 90%) was obtained
1
as a white solid. H NMR (300 MHz, CDCl3) δ 5.28 (s, 1H), 4.56-4.42 (m, 1H), 2.82 (dd, J =
13.3, 2.9 Hz, 1H), 2.04 (s, 3H), 1.99-0.65 (m, 22H), 1.13 (s, 3H), 0.94 (s, 3H), 0.93 (s, 3H), 0.91
(s, 3H), 0.86 (s, 3H), 0.85(s, 3H), 0.75 (s, 3H); ESI-MS m/z: 521.3 [M + Na]+.
4.1.1.2. 3β-Propionyloxy-olean-12-en-28-oic acid (8) [30]. Compound 8 (92 mg, 79%) was
obtained as a white solid. 1H NMR (300 MHz, CDCl3) δ 5.27 (s, 1H), 4.58-4.40 (m, 1H), 2.81 (d,
J = 13.5 Hz, 1H), 2.32 (q, J = 7.5 Hz, 2H), 2.06-0.70 (m, 25H), 1.13 (s, 3H), 0.94 (s, 3H), 0.93
(s, 3H), 0.90 (s, 3H), 0.85 (s, 6H), 0.75 (s, 3H); ESI-MS m/z: 511.4 [M - H]-.
4.1.1.3. 3β-Butyryloxy-olean-12-en-28-oic acid (9) [30]. Compound 9 (60 mg, 52%) was
obtained as a white solid. 1H NMR (300 MHz, CDCl3) δ 5.28 (s, 1H), 4.57-4.43 (m, 1H), 2.81 (d,
J = 12.9 Hz, 1H), 2.28 (t, J = 7.3 Hz, 2H), 2.06-0.65 (m, 27H), 1.13 (s, 3H), 0.93 (s, 6H), 0.90 (s,
3H), 0.85 (s, 6H), 0.75 (s, 3H); ESI-MS m/z: 525.4 [M - H]-.
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4.1.1.4. 3
β
-[3-(Carboxyl)propionyloxy]-olean-12-en-28-oic acid (17) [30]. Compound 17 (98 mg,
80%) was obtained as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 5.29 (s, 1H), 4.56 (t, J =
7.8 Hz, 1H), 2.90-2.79 (m, 1H), 2.78-2.53 (m, 4H), 2.00-0.70 (m, 22H), 1.13 (s, 3H), 0.96 (s,
3H), 0.94 (s, 3H), 0.92 (s, 3H), 0.87 (s, 3H), 0.82 (s, 3H), 0.75 (s, 3H); ESI-MS m/z: 579.3 [M +
Na]+.
4.1.1.5. 3
β
-[(4-Carboxyl)butyryloxy]-olean-12-en-28-oic acid (18) [30]. Compound 18 (85 mg,
1
68%) was obtained as a white solid. H NMR (300 MHz, DMSO-d6) δ 12.09 (brs, 1H), 5.13 (s,
1H), 4.39-4.36 (m, 1H), 2.73-2.69 (m, 1H), 2.29-2.27 (m, 2H), 2.21 (t, J = 6.93, 2H), 2.00-0.70
(m, 24H), 1.08 (s, 3H), 0.84 (s, 9H), 0.78 (s, 6H), 0.69 (s, 3H); ESI-MS m/z: 569.4 [M - H]-.
4.1.1.6. 3
β
-[(2-Carboxyphenyl)carbonyloxy]-olean-12-en-28-oic acid (20) [30]. Compound 20
1
(99 mg, 75%) was obtained as a white solid. H NMR (300 MHz, DMSO-d6) δ 7.70-7.67 (m,
1H), 7.60-7.57 (m, 3H), 5.15 (s, 1H), 4.63-4.57 (m, 1H), 2.74-2.71 (m, 1H), 2.00-0.70 (m, 22H),
1.10 (s, 3H), 0.88 (s, 6H), 0.85 (s, 6H), 0.80 (s, 3H), 0.71 (s, 3H); ESI-MS m/z: 627.3 [M + Na]+.
4.1.2. 3-[(2-Carboxyethyl)carbonyloxy]-β-amyrin (27) [31].
Compound 27 (98 mg, 79%) was prepared from 5 as a white solid following the similar
1
procedure carried out for compound 17. H NMR (300 MHz, CDCl3) δ 5.17 (s, 1H), 4.53-4.51
(m, 1H), 2.68-2.62 (m, 4H), 1.97-0.70 (m, 23H), 1.12 (s, 3H), 0.95 (s, 6H), 0.86 (s, 9H), 0.82 (s,
6H); ESI-MS m/z: 549.4 [M + Na]+.
4.1.3. 3-[(3-Carboxypropyl)carbonyloxy]-β-amyrin (28) [31].
Compound 28 (102 mg, 81%) was prepared from 5 as a white solid following the similar
1
procedure carried out for compound 18. H NMR (300 MHz, CDCl3) δ 5.20 (s, 1H), 4.66-4.45
(m, 1H), 2.48-2.38 (m, 4H), 2.09-0.80 (m, 25H), 1.15 (s, 3H), 0.99 (s, 6H), 0.89 (s, 12H), 0.85 (s,
3H); ESI-MS m/z: 539.4 [M - H]-.
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4.1.4. Carboxymethyl 3
β
-[(4-carboxy)butyryloxy]-olean-12-en-28-oate (29).
Compound 29 (14 mg, 11%) was prepared from 6 as a white solid following the similar
1
procedure carried out for compound 18. H NMR (300 MHz, CDCl3) δ 5.26 (s, 1H), 4.62-4.47
(m, 2H), 4.47-4.45 (m, 1H), 2.91-2.87 (m, 1H), 2.41-2.32 (m, 4H), 2.15-0.70 (m, 24H), 1.18 (s,
3H), 0.98 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.77 (s, 3H); ESI-MS m/z:
674.4 [M + 2Na]2+.
4.1.5. 3β-[(4-Carboxyl)butyryloxy]-olean-13(18)-en-28-oic acid (30).
Compound 30 (107 mg, 86%) was prepared from 2 as a white solid following the similar
procedure carried out for compound 18. 1H NMR (300 MHz, DMSO-d6) δ 12.08 (brs, 1H), 4.43
(dd, J = 11.8, 5.1 Hz, 1H), 2.70 (d, J = 15.2 Hz, 1H), 2.37-2.29 (m, 2H), 2.30-2.17 (m, 4H), 2.08-
2.03 (m, 1H), 1.84-0.7 (m, 22H), 1.14 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.82 (s,
3H), 0.80 (s, 3H), 0.71 (s, 3H); ESI-MS m/z: 569.3 [M - H]-.
4.1.6. 3β-[(4-Carboxyl)butyryloxy]-urs-12-en-28-oic acid (31) [32].
Compound 31 (106 mg, 85%) was prepared from 3 as a white solid following the similar
procedure carried out for compound 18. 1H NMR (300 MHz, DMSO-d6) δ 12.02 (brs, 1H), 5.13
(s, 1H), 4.45-4.39 (m, 1H), 2.35-2.30 (m, 1H), 2.26-2.21 (m, 4H), 2.11 (d, J = 11.1 Hz, 1H),
1.98-0.68 (m, 23H), 1.06 (s, 3H), 0.91 (s, 6H), 0.82 (s, 9H), 0.76 (s, 3H); ESI-MS m/z: 569.4 [M
- H]-.
4.1.7. 3β-[(4-Carboxyl)butyryloxy]-lup-20(29)-en-28-oic acid (32) [33].
Compound 32 (101 mg, 82%) was prepared from 4 as a white solid following the similar
procedure carried out for compound 18. 1H NMR (300 MHz, CDCl3) δ 4.73 (s, 1H), 4.61 (s, 1H),
4.51-4.46 (m, 1H), 3.03-2.96 (m, 1H), 2.46-2.39 (m, 4H), 2.29-0.78 (m, 26H), 1.69 (s, 3H), 0.97
(s, 3H), 0.92 (s, 3H), 0.85 (s, 6H), 0.82 (s, 3H); ESI-MS m/z: 569.3 [M - H]-.
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4.1.8. 3β-Nonanoyloxy-olean-12-en-28-oic acid (10).
As described in our previous report [22], benzylation of 1 with benzyl bromide afforded the
key intermediate benzyl ester derivative. To a solution of this benzyl ester (200 mg, 0.366 mmol)
dissolved in CH2Cl2 (10 mL), was added n-nonylic acid (70 mg, 0.439 mmol), EDCI (140 mg,
o
0.732 mmol) and DMAP (45 mg, 0.366 mmol). The reaction mixture was stirred at 40 C until
the TLC indicated the consumption of starting material. Then water (20 mL) was added to the
reaction mixture and the aqueous layer was extracted with CH2Cl2 (3 × 5 mL). The combined
organic layers were washed with brine and dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by flash chromatography (SiO2, petroleum
ether/ethyl acetate 100 : 1) to give compound 33 (203 mg, 81% for two steps) as a colorless oil.
To a solution of 33 (200 mg, 0.291 mmol) was dissolved in THF (5 mL) and treated with 10%
Pd/C (20 mg). The mixture was stirred at room temperature under H2 atmospheric pressure until
the TLC indicated the consumption of starting material. The reaction mixture was filtered
through celite and the insoluble substance was washed with THF. The filtrate was concentrated
in vacuo to give 10 (121 mg, 70%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 5.27 (s, 1H),
4.59-4.41 (m, 1H), 2.82 (d, J = 10.9 Hz, 1H), 2.29 (t, J = 7.2 Hz, 2H), 2.01-0.65 (m, 37H), 1.13
(s, 3H), 0.94 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.85 (s, 6H), 0.74 (s, 3H); ESI-MS m/z: 595.5 [M
- H]-.
4.1.9. 3β-Oleoyloxy-olean-12-en-28-oic acid (11).
Following the similar procedure carried out for compound 33, benzylation of 1 with 4-
methoxybenzylchloride, and then condensation with oleic acid in the presence of EDCI and
DMAP, gave key intermediate 34 (208 mg, 68% for two steps) as a colorless oil. To a solution of
34 (90 mg, 0.107 mmol) dissolved in CH2Cl2 (3 mL) was added H-mont (40 mg) and anisol (1
18

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mL). The mixture was stirred at room temperature until the TLC indicated the consumption of
starting material. The reaction mixture was filtered to remove H-mont. The filtrate was
concentrated in vacuo and the residue was purified by flash chromatography (SiO2, petroleum
ether/ethyl acetate 100 : 1) to give 11 (50 mg, 70%) as a white solid. 1H NMR (300 MHz,
CDCl3) δ 5.37 (s, 2H), 5.31 (s, 1H), 4.58-4.44 (m, 1H), 2.85 (d, J = 13.4 Hz, 1H), 2.32 (t, J = 7.3
Hz, 2H), 2.12-0.70 (m, 51H), 1.16 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.91 (s. 3H), 0.89 (s, 6H),
0.79 (s, 3H); ESI-MS m/z: 719.6 [M - H]-.
4.1.10. 3β-[(5-Carboxy)valeryloxy]-olean-12-en-28-oic acid (19) [34].
Following the similar procedure carried out for compound 33, benzylation of 1 with benzyl
bromide, and then condensation with adipic acid in the presence of EDCI and DMAP, gave key
intermediate 37 (247 mg, 72% for two steps) as a colorless oil. Following the procedure
described for preparation of 10, compound 19 (156 mg, 90%) was prepared from 37 as a white
1
solid. H NMR (300 MHz, DMSO-d6) δ 12.01 (brs, 1H), 5.16 (s, 1H), 4.51-4.33 (m, 1H), 2.77-
2.72 (m, 1H), 2.31-2.27 (m, 2H), 2.23-2.19 (m, 2H), 1.99-0.70 (m, 26H), 1.11 (s, 3H), 0.89 (s,
3H), 0.87 (s, 6H), 0.81 (s, 6H), 0.72 (s, 3H); ESI-MS m/z: 583.4 [M - H]-.
4.1.11. General Procedure for the Synthesis of compounds 21-26.
As described in our previous report [24], benzylation of 1 with benzyl bromide, followed by
acylation with succinic anhydride and glutaric anhydride respectively, gave key intermediate 35
and 36 in a good yield. To a solution of 35 (100 mg, 0.155 mmol) dissolved in CH2Cl2 (5 mL)
was added thionyl chloride (1 mL). The reaction mixture was refluxed for 4 h, and then
concentrated in vacuo to dryness. The residue was dissolved in CH2Cl2 (3 mL). Amine (0.155
mmol) and Et3N (0.186 mmol) were added into the reaction mixture, and then stirred at room
temperature until the TLC indicated the consumption of starting material. Water (20 mL) was
19

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added to the reaction mixture and the aqueous layer was extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were washed with brine and dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was dissolved in THF (5 mL) and treated with 10% Pd/C (20
mg). The mixture was stirred at room temperature under H2 atmospheric pressure until the TLC
indicated the consumption of the starting material. The reaction mixture was filtered through
celite and the insoluble substance was washed with THF. The filtrate was concentrated in vacuo
and the residue was purified by flash chromatography (SiO2, petroleum ether/ethyl acetate 3 : 1)
to give desirable products 21-23. Following the similar procedure carried out for 21-23,
Compounds 24-26 were prepared from key intermediate 36.
4.1.11.1. 3
β
-(4-Amino-4-oxobutyryloxy)-olean-12-en-28-oic acid (21). Compound 21 (93 mg,
1
94% for two steps) was obtained as a white solid. H NMR (300 MHz, CDCl3) δ 6.25 (s, 1H),
5.73 (s, 1H), 5.26 (s, 1H), 4.55-4.49 (m, 1H), 2.84-2.78 (m, 1H), 2.68-2.64 (m, 2H), 2.54-2.49
(m, 2H), 2.06-0.70 (m, 22H), 1.12 (s, 3H), 0.92 (s, 6H), 0.90 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H),
0.75 (s, 3H); ESI-MS m/z: 668.5 [M + Na]+.
4.1.11.2. 3
β
-(4-Morpholino-4-oxobutyryloxy)-olean-12-en-28-oic acid (22). Compound 22 (57
1
mg, 59% for two steps) was obtained as a white solid. H NMR (300 MHz, CDCl3) δ 5.27 (s,
1H), 4.53-4.48 (m, 1H), 3.70-3.65 (s, 4H), 3.63-3.60 (s, 2H), 3.50-3.48 (s, 2H), 2.85-2.79 (m,
1H), 2.68-2.61 (m, 4H), 2.04-0.70 (m, 22H), 1.13 (s, 3H), 0.93 (s, 6H), 0.90 (s, 3H), 0.87 (s, 3H),
0.85 (s, 3H), 0.75 (s, 3H); ESI-MS m/z: 624.5 [M - H]-.
4.1.11.3.
3β
-{4-[(2-Hydroxyethyl)amino]-4-oxobutyryloxy}-olean-12-en-28-oic acid (23).
1
Compound 23 (40 mg, 43% for two steps) was obtained as a white solid. H NMR (300 MHz,
CDCl3) δ 6.18 (s, 1H), 5.27 (s, 1H), 4.54-4.48 (m, 1H), 3.72-3.68 (m, 2H), 3.43-3.39 (m, 2H),
20

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2.85-2.78 (m, 1H), 2.69 (t, J = 6.2 Hz, 2H), 2.49 (t, J = 6.6 Hz, 2H), 2.04-0.70 (m, 22H), 1.12 (s,
3H), 0.93 (s, 6H), 0.90 (s, 3H), 0.84 (s, 6H), 0.74 (s, 3H); ESI-MS m/z: 622.4 [M + Na]+.
4.1.11.4. 3β-(5-Amino-5-oxovaleryloxy)-olean-12-en-28-oic acid (24). Compound 24 (33 mg,
38% for two steps) was obtained as a light yellow solid. 1H NMR (300 MHz, CDCl3) δ 6.18 (brs,
1H), 5.53 (brs, 1H), 5.28 (s, 1H), 4.54-4.49 (m, 1H), 2.86-2.80 (m, 1H), 2.43-2.38 (m, 2H), 2.32-
2.27 (m, 2H), 2.31-0.70 (m, 24H), 1.14 (s, 3H), 0.94 (s, 6H), 0.91 (s, 3H), 0.87 (s, 3H), 0.86 (s,
13
3H), 0.76 (s, 3H); C NMR (75 MHz, CDCl3) δ 183.1, 175.7, 172.8, 143.8, 122.3, 81.0, 55.2,
47.5, 46.5, 45.9, 41.6, 41.0, 39.3, 38.0, 37.7, 37.0, 34.8, 33.8, 33.7, 33.1, 32.5, 32.4, 30.7, 29.7,
28.1, 27.7, 25.9, 23.6, 23.4, 22.9, 20.8, 18.2, 17.0, 16.8, 15.4; ESI-MS m/z: 568.4 [M - H]-;
HRMS calcd for C35H54NO5 [M - H]-: 568.4002, found: 568.4015.
4.1.11.5. 3β-(5-Morpholino-5-oxovaleryloxy)-olean-12-en-28-oic acid (25). Compound 25 (35
mg, 32% for two steps) was obtained as a light yellow solid. 1H NMR (300 MHz, CDCl3) δ 5.28
(s, 1H), 4.57-4.40 (m, 1H), 3.72-3.64 (m, 4H), 3.63-3.56 (m, 2H), 3.52-3.42 (m, 2H), 2.92-2.77
(m, 1H), 2.43-2.36 (m, 4H), 2.20-0.70 (m, 24H), 1.14 (s, 3H), 0.94 (s, 3H), 0.93 (s, 3H), 0.91 (s,
13
3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.76 (s, 3H); C NMR (75 MHz, CDCl3) δ 183.1, 173.1, 171.0,
143.6, 122.5, 81.0, 66.9, 66.7, 55.3, 47.5, 46.5, 46.0, 45.8, 41.9, 41.6, 41.0, 39.3, 38.0, 37.7, 37.0,
33.8, 33.0, 32.5, 32.4, 32.1, 30.6, 29.7, 28.1, 27.7, 25.9, 23.6, 23.4, 22.9, 20.6, 18.2, 17.1, 16.7,
15.4; ESI-MS m/z: 638.4 [M - H]-; HRMS calcd for C39H60NO6 [M - H]-: 638.4421, found:
638.4438.
4.1.11.6.
3β
-{5-[(2-Hydroxyethyl)amino]-5-oxovaleryloxy}-olean-12-en-28-oic acid (26).
1
Compound 26 (46 mg, 43% for two steps) was obtained as a light yellow solid. H NMR (300
MHz, CDCl3) δ 6.08 (brs, 1H), 5.29 (s, 1H), 4.55-4.50 (m, 1H), 3.75-3.72 (m, 2H), 3.46-3.39 (m,
2H), 2.89-2.79 (m, 1H), 2.40 (t, J = 6.9 Hz, 2H), 2.32-2.23 (m, 2H), 2.12-0.70 (m, 24H), 1.15 (s,
21

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13
3H), 0.95 (s, 6H), 0.92 (s, 3H), 0.87 (s, 6H), 0.77 (s, 3H); C NMR (75 MHz, CDCl3) δ 183.5,
173.5, 173.2, 143.7, 122.5, 81.1, 62.2, 55.3, 47.5, 46.5, 45.8, 42.4, 41.5, 40.9, 39.3, 38.0, 37.7,
37.0, 35.5, 33.8, 33.0, 32.5, 30.6, 29.7, 28.1, 27.7, 25.9, 23.6, 23.4, 22.8, 21.1, 18.2, 17.2, 16.7,
15.4; ESI-MS m/z: 612.4 [M - H]-; HRMS calcd for C37H58NO6 [M - H]-: 612.4264, found:
612.4278.
4.2. CETP inhibitory assay in vitro.
The CETP inhibitory bioactivities of natural PTs and their 3β-ester derivatives were
determined by using a standard fluorescent-CE transfer assay with CETP activity assay kit (Roar
Biomedical Inc.) and human recombinant CETP (Roar Biomedical Inc.). Assay system was
prepared according the instruction for the CETP activity assay kit and human recombinant
CETP. The assay uses synthetic HDL donor particles that contain a fluorescent CE with an
excitation maximum at 465 nm and emission maximum at 535 nm. As the fluorescent CE is
transferred from a donor molecule to an acceptor molecule by human recombinant CETP,
fluorescence is observed and quantified. Inhibition of CETP mediated CE transfer is
characterized by a decrease in levels of fluorescence observed relative to control. Anacetrapib
was used as the positive control drug.
The assay procedure can be described briefly as follows. All testing compounds were totally
dissolved in 100% DMSO and stored at nitrogen cabinet. Reconstitute the 80 µg/mL human
recombinant CETP in total protein with assay buffer and dilute the stocking compounds with
DMSO. A solution that contains no human recombinant CETP was as background and that
contains human recombinant CETP but no testing compounds as the positive control. Donor
molecule (4 mL), acceptor molecule (4 mL) and testing compound (1 mL) with human
recombinant CETP (30 ng) were mixed in assay buffer (200 mL). After incubation at 37 oC for 3
22

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h, fluorescence intensity was read in a fluorimeter (Flex Station III) and the inhibition ratio was
also calculated. The initial screening was carried out at a concentration of 10 µΜ for each testing
compound. Compounds that displayed >30% inhibition at 10 µΜ were diluted with DMSO for 8
points titration (1 : 5 serial dilutions) in 96-well dilution plate, and the inhibition ratios were also
calculated according to the above methods. IC50 values of these compounds were determined
from a curve fit of the data with each concentration tested. The curve was fitted using "Sigmoidal
dose-response (variable slope)" in GraphPad Prism software.
4.3. In vivo sudies of compound 20 in animal models.
For the transgenic mice pharmacodynamic assay, ap2-CETPTg mice expressing human CETP
were used. Blood samples were collected by retro-orbital bleed. Compound 20 was formulated in
DMSO/cremophor/saline at a 3 : 4 : 93 ratio and oral dosing at 30 mg/kg, once-a-day, for two
weeks. Blood was drawn 2 h postdose on day 14. HDL-C, LDL-C, TG and TC levels in ap2-
CETPTg mice were measured using an automated lipid analyzer (COBAS), which were reported
as the mean ± SD (n ≥ 6), and compared to the values measured in control group. Anacetrapib
was used as the positive control drug.
For the normal fed guinea pig study, the male guinea pigs (12 weeks of age) were used. Blood
samples were collected by retro-orbital bleed. The male guinea pigs were dosed with compound
20 by gavage, 30 mg/kg, once-a-day, for two weeks, with normal diet simultaneously. At 2 h
after dosing on day 14, plasma samples were collected. HDL-C and LDL-C levels in normal fed
guinea pigs were measured, which were reported as the mean ± SD (n ≥ 6) and compared to the
values measured in control group. Anacetrapib was used as the positive control drug.
For the high-fat fed guinea pig study, the male guinea pigs (12 weeks of age) were used and
initially fed a high fat diet (2.5% coconut oil and 0.25% cholesterol) for two weeks. Blood
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samples were collected by retro-orbital bleed. Then, the high-fat fed guinea pigs were dosed with
compound 20 by gavage, 30 mg/kg, once-a-day, for two weeks, with high-fat diet
simultaneously. At 2 h after dosing on day 14, plasma samples were obtained. HDL-C, LDL-C,
TG and TC levels in high-fat fed guinea pigs were measured, which were reported as the mean ±
SD (n ≥ 6), and compared to the values measured in model group. Anacetrapib was used as the
positive control drug.
For the pharmacokinetic evaluation of compound 20, the male guinea pigs (12 weeks of age)
were used. Compound 20 dissolved in DMSO/cremophor/saline at a 3 : 4 : 93 ratio was
administered to normal fed guinea pigs at dose of 30 mg/kg (n = 6) by oral gavages. Blood
samples (300 µL) were collected before and after administration by retro-orbital bleed at 0.25 (at
the end of oral gavage), 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 24, 36 and 48 h after drug administration.
After centrifugation of blood samples, 100 µL aliquots of plasma samples were collected. After
adding 10 µL internal standard (I.S.) glycyrrhetic acid (100 ng/mL), the samples were extracted
with 1 mL of ethyl acetate and centrifuged at 4000 r/min for 10 minutes. The organic layer (900
µL) was transferred and evaporated to the dryness by SPD 2010 vacuum concentrator (Thermo
Electron Corporation, MA, USA). The residues were dissolved by methanol and then injected
into the LC-MS/MS system. The LC-MS/MS was performed by triple quadrupole system TSQ
Quantum Ultra (Thermo Scientific, MA, USA). Analyses were carried out using electrospray
ionization (ESI) in negative-ion mode using selective reactions monitoring (SRM): m/z
603.3→453.2 for compound 20, m/z 455.4→407.3 for 1 and m/z 469.3→425.3 for I.S..
Separation of the test compounds was achieved using a 150 mm × 2.1 mm i.d., 3.5 µm, Agilent
ZORBAX Elipse Plus C8 column. The mobile phase composition was (A) water with 1 mM
ammonium acetate and (B) methanol. The column temperature was maintained at 35 °C. Mobile
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phase B maintained to 15% for 1 minute and then increased to 95% in half a minute. Over next
6.5 minutes, mobile phase B went to 100% smoothly. At 8.01 minute, mobile phase B was
decreased to 15% rapidly and final equilibration held for 2 minutes.
Acknowledgment
We gratefully acknowledge financial support from the National Natural Science Foundation of
China (81373303, 81473080, 81573299, 81673443, 81602957 and 81673684), the Natural
Science Foundation of Jiangsu Province, China (BK20161035), the “111 Project” from the
Minisry of Education of China, the State Administration of Foreign Expert Affairs of China
(111-2-07), and the Program for Changjiang Scholars and Innovative Research Team in
University (IRT1193).
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