Facile preparation and characterization of novel oleanane-type triterpene functionalized β-cyclodextrin conjugates

Article abstract of DOI:10.1016/j.cclet.2018.10.009

Oleanolic acid (OA) and echinocystic acid (EA), two naturally occurring pentacyclic oleanane triterpenes, are gaining increasing attention due to their promising pharmacological activities. Conjugation with amphiphilic α(β)-cyclodextrin (CD) via “click chemistry” can improve their solubility and anti-HCV entry potency. In the present work, four water-soluble β-CD-pentacyclic triterpene conjugates were designed and synthesized, in which OA and EA was coupled to one of the primary hydroxyl groups of β-CD via ester and amide bonds. The structures of the conjugates were unambiguously determined by ¹H NMR, ¹³C NMR and HRMS or MALDI-TOF-MS. All the conjugates showed lower hydrophobicity (AlogP) than their parent compounds and no significant cytotoxicity was found to HL-60, A549, Hela and Bel-7402 cells at concentrations up to 10 μmol/L. Further anti-HCV entry activity and mechanism studies are under way in our laboratory.

Full text of DOI:10.1016/j.cclet.2018.10.009

Accepted Manuscript
Title: Facile preparation and characterization of novel
oleanane-type triterpene functionalized β-cyclodextrin
conjugates
Authors: Pingxuan Jiao, Shouxin Wang, Shuobin Liang, Man
Li, Qianqian Gao, Dezhong Ji, Yingying Chen, Haiwei Li,
Fuxiang Ran, Yongmin Zhang, Lihe Zhang, Demin Zhou,
Sulong Xiao
PII:
DOI:
Reference:
S1001-8417(18)30395-4
https://doi.org/10.1016/j.cclet.2018.10.009
CCLET 4673
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
1-6-2018
25-9-2018
10-10-2018
Please cite this article as: Jiao P, Wang S, Liang S, Li M, Gao Q, Ji D, Chen Y, Li H, Ran
F, Zhang Y, Zhang L, Zhou D, Xiao S, Facile preparation and characterization of novel
oleanane-type triterpene functionalized β-cyclodextrin conjugates, Chinese Chemical
Letters (2018), https://doi.org/10.1016/j.cclet.2018.10.009
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Communication
Facile preparation and characterization of novel oleanane-type triterpene
functionalized β-cyclodextrin conjugates
a
a,c
a
a
a
a
a
Pingxuan Jiao , Shouxin Wang , Shuobin Liang , Man Li , Qianqian Gao , Dezhong Ji , Yingying Chen ,
a
a
d
a
a,*
a,b
Haiwei Li , Fuxiang Ran , Yongmin Zhang , Lihe Zhang , Demin Zhou , Sulong Xiao
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
b
Kunming 650201, China
c
School of Pharmacy, Jining Medical University, Rizhao 276826, China
Sorbonne Université, Institut Parisien de Chimie Moléculaire, CNRS UMR 8232, 4 place Jussieu, 75005 Paris, France
d
Graphical abstract
Four water-soluble β-CD-pentacyclic triterpene conjugates were synthesized via ester and amide linkages. All the conjugates
showed lower hydrophobicity (AlogP) than their parent compounds while no significant cytotoxicity was found to HL-60, A549,
Hela and Bel-7402 cells at concentrations up to 10 μmol/L. Further anti-HCV entry activity and mechanism studies are under way
in our laboratory.
ARTICLE INFO
ABSTRACT
Article history:
Received 1 June 2018
Received in revised form 25 September 2018
Accepted 30 September 2018
Available online
Oleanolic acid (OA) and echinocystic acid (EA), two naturally occurring pentacyclic oleanane
triterpenes, are gaining increasing attention due to their promising pharmacological activities.
Conjugation with amphiphilic α(β)-cyclodextrin (CD) via “click chemistry” can improve their
solubility and anti-HCV entry potency. In the present work, four water-soluble β-CD-pentacyclic
triterpene conjugates were designed and synthesized, in which OA and EA was coupled to one
of the primary hydroxyl groups of β-CD via ester and amide bonds. The structures of the
conjugates were unambiguously determined by H NMR, C NMR and HRMS or MALDI-
TOF-MS. All the conjugates showed lower hydrophobicity (AlogP) than their parent compounds
and no significant cytotoxicity was found to HL-60, A549, Hela and Bel-7402 cells at
concentrations up to 10 μmol/L. Further anti-HCV entry activity and mechanism studies are
under way in our laboratory.
Keywords:
β-Cyclodextrin
DIBAL-H
Oleanolic acid
Echinocystic acid
Synthesis
1
13
—
——

Corresponding authors.
E-mail addresses: deminzhou@bjmu.edu.cn (D. Zhou); slxiao@bjmu.edu.cn (S. Xiao)

Pentacyclic triterpenes are secondary plant metabolites, widespread in fruit peels, leaves, and stem bark [1], with a few species
containing up to 30% of their dry weight [2]. Oleanolic acid (OA, Fig. 1), an oleanane-type pentacyclic triterpene, has been isolated from
more than 1,600 plant species, including many dietary and medicinal plants [3]. Numerous studies indicate natural OA and its saponins
exhibit a wide range of pharmacological activities, such as antiviral, antitumor, anti-inflammation, and anti-microbial activities [4]. Due
to its inherent bioactivities, availability and low cost, OA has been used as a starting molecule for the synthesis of oleanane triterpenes.
A series of novel synthetic oleanane triterpenes have been prepared by chemical modifications of OA at three sites, including C-3
1
2-13
hydroxyl, Δ
double bond and C-28 carboxylic acid [5]. Several synthetic derivative of OA, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic
acid (CDDO) and its C-28 methyl ester (CDDO-Me) and C-28 imidazole (CDDO-Im) derivatives, have been tested in clinical trials [6].
Recently, we found that echinocystic acid (EA), an analog of OA with an extra hydroxyl group at C-16, displays substantial inhibitory
activity on hepatitis C virus (HCV) entry with one derivative Q8 (Fig. 1) showing EC50 at nanomolar level [7]. Despite their promising
bioactivity, pentacyclic triterpenes are bulky, non-polar, and poorly soluble in water, which are major drawbacks for various applications,
in particular, the development of drugs. For example, the solubility of OA in water is only about 0.05 µg/mL [8]. Hence, the synthesis
of more water-soluble triterpene derivatives is needed. Some water-soluble derivatives have been synthesized by introducing polar sugar
moieties at C-3 and/or C-28 [9].
Fig. 1. The chemical structures of OA and its derivatives.
β-cyclodextrin (CD) is a macrocyclic oligomers of D-glucose with the secondary C-2 and C-3 hydroxyl groups on the secondary face
and the primary C-6 hydroxyl group on the first face. This unique structure enables β-CD to possess high aqueous solubility. Due to the
ability to form inclusion complex with a large range of hydrophobic drug molecules, the physicochemical properties of drugs are
significantly modified after forming complexes with β-CD, such as the increased stability and water solubility, enhanced dissolution rate
and bioavailability [10]. Several CD inclusion complexes with triterpenes have been studied [11]. Usually, the drugs are released quickly
from the inclusion complex under physiological conditions. Alternatively, direct covalent linkage with CD has been suggested to improve
the physicochemical properties of triterpines, which has been widely used for other water insoluble bioactive molecules, such as
fluorouracil (5-FU) and folic acid [12].
In our previous studies, a series of water-soluble triazole-bridged α- and β-CD-pentacyclic triterpene conjugates have been synthesized
via “click chemistry” (Fig. 2) [13]. As a continuation towards the elucidation of the relationship between the structure and anti-HCV
entry activity of triterpene derivatives, we have synthesized conjugates a-d in which the triazolyl linker was replaced by ester or amide
bond. Herein, we describe a simple method to prepare those β-CD derivatives and their lipophilicity (AlogP) along with their cytotoxicity
to three human cancer cell lines.
Fig. 2. Structure of novel 1:1 CD-pentacyclic triterpene conjugates. (Top: linked by triazol bond [13], bottom: linked by ester or amide bond).
A
As shown in Scheme 1, conjugation of β-CD with two oleanane-type triterpenes OA and EA via ester bond started from 6 -hydroxy-
per-O-benzyl-β-CD (3), a regioselective mono-debenzylated intermediate. 3 was prepared from the per-O-benzylated β-CD using
diisobutylaluminium hydride (DIBAL-H) (30 equiv.) as a chemical “scalpel” as first introduced by P. Sinaӱ group [14]. In addition to
the mono-debenzylated β-CD (3), the di-de-O-benzylated product 5 was also obtained in 20% yield after chromatography separation.
The structures of 3 and 5 were further confirmed by their acetylated derivatives 4 and 6, respectively. Initial attempts using classical
methods to conjugate triterpenes OA and EA with 3 directly via ester bond yielded no desired product. Therefore, the compound 3 was
4 3
converted to its bromide derivative 7 using the commercially available carbon tetrabromide (CBr ) and triphenylphosphine (PPh ) in

1
8
H
5% yield. It was characterized using H and 13C NMR, which gave the distinctive bromine methyl signals at 3.55 and 3.80 ppm (2 ×
A
A
6
) and 34.59 ppm (C
6
), respectively.
Conjugation of the key intermediate 7 with triterpenes EA and OA in the presence of K
2
CO
3
provided compound 8 or 9 smoothly in
1
13
8
8%-92% yield, respectively. Fig. S1 in Supporting information presents the H and C NMR spectra of compound 9. The triplet at 5.33
1
1
1
13
ppm (J = 3.0 Hz) refers to 1H, which, according to its H- H and H- C correlation spectra, should be assigned to H12. The high-field
doublets of doublet at 3.16 ppm (J = 4.4 and 11.1 Hz) and 3.05 ppm (J = 3.5 and 14.5 Hz), each referring to 1H, are assigned to H and
] ion at m/z
19: 1712.8606), indicating that it was indeed connected via an ester bond. Similar
3
+
H
1
18, respectively. The ESI-HRMS mass spectrum of compound 9 recorded in the positive mode shows clearly an [M/2+NH
4
1
2
1
14
16
712.8676 (calcd. isotopic mass for
C
106
H
122
13
1
N O
1
patterns were also observed in H NMR and C NMR spectra of compound 8 of this series. Finally, de-O-benzylation of 8 or 9 was
accomplished by hydrogenolysis to yield 10 or 11 in high yields (78%-91%).
Scheme 1. Synthesis of β-CD-oleanane-type triterpene conjugates 10 and 11 via ester bond. Reagents and conditions: (a) BnBr, NaH, DMF,
o
8
6%; (b) DIBAL-H (30 equiv.), toluene, 50 C, 2 h 42.1% for compound 3 and 20.0% for compound 5; (c) Ac
2
O, pyridine, DMAP, 81%-85%;
(d) CBr
4 3 2 2 2 3 2 3
, PPh , CH Cl , 85%; (e) EA or OA, K CO , DMF, 62-82%; (f) Pd/C, H , CH OH, 78-91%.
A parallel experiment was carried out to conjugate pentacyclic triterpene with β-CD moiety through an amide bond rather than an
ester bond. Such alteration might enhance the stability of pentacyclic triterpene-β-CD conjugates. As shown in Scheme 2, selective
monotosylation of β-CD (1) according to the procedure described by Vizitiu et al. [15] with minor modifications afforded compound 12,
which was used without further purification for the nucleophilic substitution with NaN
3
in DMF to provide the mono-azide substituted
derivative 13 in 8% yield over two steps. Reduction of the azide group of 13 with triphenylphospine followed by hydrolysis gave the key
A
A
intermediate 6 -amino-6 -deoxy-β-CD 14. Finally, conjugation of 14 with triterpenes OA and EA in the presence of N-ethyl-N'-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC•HCl) afforded compounds 15 and 16 in moderate yields (43%-47%). Structural
1
13
elucidation of the new derivatives was also done using 1D-NMR ( H and C), 2D-NMR and MALDI-TOF-MS techniques (Supporting
information).
2
Scheme 2. Synthesis of β-CD-pentacyclic triterpene conjugates 15 and 16 via amide bond. Reagents and conditions: (a) H O, NaOH,
o
3 3 3
TsCl, 9.4%; (b) NaN , DMF, 80 C, 91%; (c) Ph P, DMF, NH (aq), 63%; (d) EDC, HOBt, DMF, 43-47%.
The 1-octanol/water partition coefficient (logP) is a well-known measure of molecular lipophilicity. In a recent study, a negative
correlation was found between the logP value of terpenes and their bioaccessibility (r = − 0.77, P < 0.001) [16]. Therefore, decrease in
the lipophilicity of pentacyclic triterpenes may increase the bioaccessibility. In this study, the calculated AlogP values based on Ghose
and Crippen’s method were determined using Pipeline Pilot software, Vers. 7.5 (Accelrys Corporation, San Diego, USA) [17]. We found
that the targeting conjugates 10/11 and 15/16 showed increased hydrophilicity comparing with their parent compounds due to the
introduction of β-CD (Table S1 in Supporting information). Compared with their analogs b and d [13a], the ALogP of conjugates 10 and
1
1 increased by 0.66 log units (triazol linker vs. ester linker) while the ALogP of conjugates 15 and 16 remained almost the same (trizolyl

linker vs amide linker). In addition, due to the 16-hydroxyl group of EA, the ALogP of conjugates 11 and 16 decreased by 1.1 log units
(10 vs. 11 and 15 vs. 16), which means that their solubility in water are increased by ~ 12.5–fold.
Oleanane-type triterpenes are widely found in the plant kingdom as free acids or triterpene saponins linked with one or more sugar
chains. Some natural pentacyclic triterpenes are shown weak cytotoxicity [18]. In this study, the cytotoxicity of the four conjugates (10,
1, 15 and 16) against human promyelocytic leukemia (HL-60), human cervical cancer (Hela), human lung cancer (A549), and human
1
liver cancer (Bel-7402) cell lines was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at
concentrations of 0.1-10 μmol/L [19]. Except conjugates 10, 11 and 15 showed weak cytotoxicity to Hela cell at concentration of 1
μmol/L and 10 μmol/L (~ 23%-26%), no significant cytotoxicity was observed at concentrations up to 10 μmol/L for other three cancer
cell (Table S2 in Supporting information). These results indicated that the synthesized β-CD-pentacyclic triterpene derivatives had no
significant toxic effect to cells in vitro.
In summary, starting from natural β-CD, four water-soluble oleanane-type triterpene-β-CD conjugates 10/11 and 15/16 were
1
13
synthesized via ester and amide linkage by a facile method. The new products were unambiguously characterized by H NMR, C NMR
and HRMS or MALDI-TOF-MS. Compared with their parent compounds, they showed higher hydrophobicity (AlogP) with no
significant cytotoxicity in vitro. This study supports that these conjugates are potential candidates for further evaluation as anti-HCV
entry agents.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 81573269, 21572015, 21877007, 91753202 and
2
1702007) and the open funding of the State Key Laboratory of Phytochemistry and Plant Resources in West China. The authors
gratefully acknowledge Dr. Hongwei Jin at State Key Laboratory of Natural and Biomimetic Drugs, Peking University for carrying out
the ALogP determinations and Dr. Lijun Zhong at the Centre of Medical and Health Analysis, Peking University for MALDI-TOF
experiments.
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