Design, synthesis, and biofunctional evaluation of novel pentacyclic triterpenes bearing O-[4-(1-piperazinyl)-4-oxo-butyryl moiety as antiproliferative agents

Article abstract of DOI:10.1016/j.bmcl.2015.08.076

A series of pentacyclic triterpenoids derivatives bearing O-[4-(1-piperazinyl)-4-oxo-butyryl moiety has been synthesized and investigated for their potential antiproliferative activities. Pentacyclic triterpenoids derivative compounds were synthesized by a four or six step synthetic procedure. The activity studies were evaluated using Cell Counting Kit-8 method, and Western blotting analysis on A549 cells, MCF-7 cells and Hela cells. Compounds methyl 3-O-[4-(1-piperazinyl)-4-oxo-butyryl]olean-12-ene-28-oate (OA-4) and compound 2-O-[4-(1-piperazinyl)-4-oxo-butyryl]-3,23-dihydroxyurs-12-ene-28-oate (AA-5) were found to be promising antiproliferative agents. These compounds show potentiality for further optimization as antitumor drugs.

Full text of DOI:10.1016/j.bmcl.2015.08.076

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biofunctional evaluation of novel pentacyclic
triterpenes bearing O-[4-(1-piperazinyl)-4-oxo-butyryl moiety as
antiproliferative agents
a,d,
Chun-hui Zhao a, Cui-li Zhang b, Jin-jie Shi a, Xi-yan Hou c, Bin Feng b, , Long-xuan Zhao
⇑
⇑
a Liaoning Normal University, School of Life Sciences, Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Dalian 116029, China
b Dalian Medical University, Department of Biotechnology, Dalian 116044, China
c Dalian Nationalities University, College of Life Science, Dalian 116600, China
d Liaoning Normal University, School of Chemistry and Chemical Engineering, Dalian 116029, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pentacyclic triterpenoids derivatives bearing O-[4-(1-piperazinyl)-4-oxo-butyryl moiety has
been synthesized and investigated for their potential antiproliferative activities. Pentacyclic triterpenoids
derivative compounds were synthesized by a four or six step synthetic procedure. The activity studies
were evaluated using Cell Counting Kit-8 method, and Western blotting analysis on A549 cells, MCF-7
cells and Hela cells. Compounds methyl 3-O-[4-(1-piperazinyl)-4-oxo-butyryl]olean-12-ene-28-oate
(OA-4) and compound 2-O-[4-(1-piperazinyl)-4-oxo-butyryl]-3,23-dihydroxyurs-12-ene-28-oate
(AA-5) were found to be promising antiproliferative agents. These compounds show potentiality for fur-
ther optimization as antitumor drugs.
Received 20 March 2015
Revised 6 August 2015
Accepted 28 August 2015
Available online xxxx
Keywords:
Pentacyclic triterpenoid
Antiproliferative agent
Apoptosis
Ó 2015 Published by Elsevier Ltd.
Cell cycle
Modern medical science is constantly searching for new and
more powerful agents to prevent, treat, or retard cancer. Therefore,
a worldwide search for new anticancer drugs of natural origin is
underway. An important part of this research field is the applica-
tion of biosynthetic knowledge to synthesize interesting, natural
products (i.e., biomimetic synthesis). To date, approximately
20,000 triterpenoids have been identified from various parts of
medicinal plants.1 The naturally occurring pentacyclic triter-
penoids 18-b-glycyrrhetinic acid (GA), asiatic acid (AA), oleanolic
acid (OA), and ursolic acid (UA) can inhibit tumor initiation and
growth, and induce apoptosis in various cancer cells.2–13 They all
contain hydroxyl and carboxylic groups, as shown in Figure 1.
Numerous attempts have been made to obtain potent synthetically
feasible analogs of pentacyclic triterpenoids with a heteroatom
incorporated in positions on the triterpenoid skeleton. Previous
Letters have suggested that the activity of these pentacyclic triter-
penoids is related to their basic triterpenoid skeletal structure, and
the attached functional groups offer opportunities for chemical
modification and improvement of activity.13–16 Dicarboxylic acid
hemiesters of pentacyclic triterpenoid such as UA, OA, and betuli-
nic acid showed more potent inhibitory activity on the human
immunodeficiency virus (HIV)-protease.17
Previous studies in our group on AA and related analogs showed
that structurally modified compounds could inhibit the growth of
human non-small cell lung cancer (NSCLC) A549 and PC9/G
(acquired resistance to gefitinib) cell lines in vitro, when the car-
boxylic group at C-28 of AA was converted to methyl ester, and
butanedioic acid was introduced into the hydroxyl group at the
C-2 position.18 These results encouraged us to select GA, AA, OA,
and UA as templates, and to design a series of pentacyclic triter-
penoids analogs.
Piperazine derivatives possess pharmacological properties.
Many currently notable anticancer agents, including imatinib
(STI571),19 dasatinib (BMS-354825),20 bosutinib (SKI-606),21 danu-
sertib (PHA-739358),22 and VX-680,23 contain a piperazine ring as
part of their molecular structure.
In the present study, functional groups attached to pentacyclic
triterpene molecules were modified to generate derivatives, while
maintaining the pentacyclic triterpene skeletal structure. As a
result of these modifications, the carboxylic group was converted
to a methyl ester with retention of stereochemistry, which may
significantly influence its physical properties or receptor interac-
tions. Piperazine and butanedioic acid were introduced into the
⇑
Corresponding authors. Tel./fax: +86 411 86110350 (B.F.); tel.: +86 411
82158329 (L.-x.Z.).
E-mail addresses: binfeng@dmu.edu.cn (B. Feng), lxzhao@lnnu.edu.cn
(L.-x. Zhao).
http://dx.doi.org/10.1016/j.bmcl.2015.08.076
0960-894X/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Zhao, C.-h.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.076

2
C. -h. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
butanedioic anhydride in the presence of 4-dimethylaminopy-
ridine (DMAP) in dichloromethane (CH2Cl2) to generate inter-
mediates (GA-2, OA-2, and UA-2). Coupling reactions of the
intermediates with N-tert-butoxycarbonylpiperazine and DMAP
were performed in the presence of 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDCI) in CH2Cl2 to obtain compounds GA-3,
OA-3, and UA-3. The protecting group was then removed by a
treatment with
a trifluoroacetic acid/dichloromethane (TFA/
CH2Cl2) mixture to result in the deprotected analogs (GA-4, OA-4,
and UA-4). AA preparation and synthesis of its derivatives (AA-1,
AA-2, and AA-3) were performed as previously described.18 As
shown in Scheme 2, the dihydroxyl groups at C-3 and C-23 of AA
were treated with 2, 2-dimethoxypropane ((CH3)2C(OCH3)2) in
the presence of p-toluenesulfonic acid (p-TsOH) in DMF to generate
acetonide. The carboxylic group at C-28 was converted to a methyl
ester, and the hydroxyl group at C-2 was treated with succinic
anhydride using the same synthetic pathways as used for other
pentacyclic triterpenoids to obtain compounds AA-4 and AA-5.
All of the compounds were dissolved in dimethyl sulfoxide (DMSO)
prior to use in biological activity assays.
We compared the inhibitory effects of the four pentacyclic
triterpenoids (GA, AA, OA, and UA) and 17 analogs against three
cancer cell lines: MCF-7 (human breast cancer cells), Hela (human
cervical carcinoma cells), and A549 (human NSCLC cells). The
screening procedure was performed using the Cell Counting Kit-8
(CCK-8) (Dojindo, Kumamoto, Japan), and the results are summa-
rized in Table 1.
The pentacyclic triterpenoids derivatives with a piperazine
group (GA-4, AA-5, OA-4, and UA-4) showed much stronger
growth inhibitory effects than their corresponding analogs with
Figure 1. Chemical structures of pentacyclic triterpenoids.
hydroxyl group at the C-2 position of AA, and at the C-3 positions
of GA, OA, and UA. The dihydroxyl groups at C-3 and C-23 of AA
were protected with acetonide using 2,2-dimethoxy propane. In
addition, we described the synthesis and cytotoxicity evaluation
of the derivatives. The effect of the novel compounds on cell cycle
blockage was also analyzed.
Pentacyclic triterpenoids GA, AA, OA, and UA were used as lead
compounds, and the structural modifications were made at
positions C-3, C-30 of GA, C-2, C-3, C-23, C-28 of AA, and C-3 and
C-28 of OA and UA, respectively. Similar procedures were used to
synthesize derivatives of GA, OA, and UA, and the synthetic
pathways are presented in Schemes 1–4. The carboxylic groups
(C-30 of GA, and C-28 of OA and UA) were subjected to methyl
iodide treatment in the presence of potassium carbonate in
dimethyl formamide (DMF) to create methyl ester moieties with
retention of stereochemistry (GA-1, OA-1, and UA-1). The hydroxyl
groups at C-3 of these pentacyclic triterpenoids were treated with
IC50 values from 7.05 to 13.13 lM in the three cancer cells. How-
ever, GA-2, AA-3, OA-2, and UA-2, which contain a substituted
butanedioic acid, displayed weak cytotoxicity against the cancer
cell lines with IC50 values from 26.03 to more than 100 lM. The
pronounced influence of the incorporation of piperazine on
antiproliferative effects in the series of synthesized compounds
could be explained on the capacity for the formation of hydrogen
bonds, improvement of water-solubility, and adjustment of molec-
ular physicochemical properties.
To evaluate the cytotoxic mechanism of AA-5 and OA-4 against
MCF-7, Hela, and A549 cells and to gain further insight into the
Scheme 1. Synthesis of glycyrrhetinic acid derivatives. Reagent and conditions: (i) CH3I, K2CO3, DMF, rt; (ii) butanedioic anhydride, DMAP, CH2Cl2, reflux; (iii) N-tert-
butoxycarbonylpiperazine, EDCI, DMAP, CH2Cl2, rt; (iv) TFA, CH2Cl2, rt.
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C. -h. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Scheme 2. Synthesis of asiatic acid derivatives. Reagent and conditions: (i) 5N-NaOH, MeOH, reflux; (ii) (CH3)2C(OCH3)2, p-TsOH, DMF, rt; (iii) CH3I, K2CO3, DMF, rt; (iv)
butanedioic anhydride, DMAP, CH2Cl2, reflux; (v) N-tert-butoxycarbonylpiperazine, EDCI, DMAP, CH2Cl2, rt; (vi) TFA, CH2Cl2, 1 N HCl, CH3OH, rt.
Scheme 3. Synthesis of oleanolic acid derivatives. Reagent and conditions: (i) CH3I, K2CO3, DMF, rt; (ii) butanedioic anhydride, DMAP, CH2Cl2, reflux; (iii) N-tert-
butoxycarbonylpiperazine, EDCI, DMAP, CH2Cl2, rt; (iv) TFA, CH2Cl2, rt.
Please cite this article in press as: Zhao, C.-h.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.076

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C. -h. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Scheme 4. Synthesis of ursolic acid derivatives. Reagent and conditions: (i) CH3I, K2CO3, DMF, rt; (ii) butanedioic anhydride, DMAP, CH2Cl2, reflux; (iii) N-tert-
butoxycarbonylpiperazine, EDCI, DMAP, CH2Cl2, rt; (iv) TFA, CH2Cl2, rt.
Table 1
Cytotoxicity of compounds
a
Compounds
IC50
Hela
(lM)
MCF-7
A549
GA
>100
>100
>100
>100
17.74 2.33
67.58 6.21
59.85 7.84
>100
45.55 5.97
21.12 3.29
7.58 1.25
>100
>100
>100
>100
>100
18.72 2.54
91.07 10.14
60.04 5.87
>100
55.63 7.63
23.75 2.87
8.13 1.69
>100
>100
>100
>100
>100
15.14 3.02
>100
GA-1
GA-2
GA-3
GA-4
AA
AA-1
AA-2
AA-3
AA-4
AA-5
OA
34.73 1.74b
48.86 2.93b
26.03 2.47b
33.61 4.12
13.13 4.37
>100
OA-1
OA-2
OA-3
OA-4
UA
UA-1
UA-2
UA-3
UA-4
Gefitinib
>100
55.71 4.81
>100
7.05 1.45
44.84 5.41
>100
36.04 3.25
50.16 4.39
10.72 2.24
17.83 7.85
>100
57.81 6.62
>100
7.75 1.09
64.48 6.75
>100
46.68 3.68
>100
>100
51.84 4.94
>100
9.91 2.11
74.65 9.42
>100
74.24 8.77
>100
13.07 1.98
15.40 4.63
17.63 2.54
11.02 3.27
Human tumor cells were treated with different concentrations of samples for 48 h (n = 3 independent experiments).
a
Data are presented as IC50
Cited from Ref. 18.
(lM, the concentration of 50% proliferation-inhibitory effect).
b
mode of action, we systematically assessed cell cycle signal check-
point proteins, including p16, p21, CDK1, Cyclin D1, and Cyclin B in
MCF-7, Hela, and A549 cells after treating the cells with or without
the analogs. p16 and p21 play an important role in cell cycle regu-
lation by decelerating the progression of cells from G1 to S phase,
and therefore act as a tumor suppressor that is implicated in the
prevention of cancers. As shown in Figure 2, AA-5 and OA-4 at
synthesized and investigated for their cytotoxic potential against
three human cancer cell lines. Most of these compounds showed
significant antiproliferative effects compared to their original
compounds when tested in MCF-7, Hela, and A549 cells using
the CCK-8 assay. In particular, compounds methyl 3-O-[4-(1-
piperazinyl)-4-oxo-butyryl]olean-12-ene-28-oate (OA-4) and
2-O-[4-(1-piperazinyl)-4-oxo-butyryl]-3,23-dihydroxyurs-12-
ene-28-oate (AA-5) showed the highest inhibitory activity against
three human cancer cell lines. Furthermore, OA-4 and AA-5 can
induce cell cycle arrest in all of the three cells. Therefore, as indi-
cated by our results, compounds OA-4 and AA-5 were identified
as promising candidate drugs.
15 lM induced the significant expression of p16 and p21 com-
pared with the control in the three cell lines. In contrast, AA-5
and OA-4 inhibited the expression of Cyclin D1, Cyclin B, and CDK1.
In conclusion, a series of pentacyclic triterpenoid derivatives
bearing the O-[4-(1-piperazinyl)-4-oxo-butyryl moiety were
Please cite this article in press as: Zhao, C.-h.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.076

C. -h. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Figure 2. Effects of AA-5 and OA-4 on cell cycle related proteins in MCF-7 (human breast cancer cells), Hela (human cervical carcinoma cells), and A549 (human NSCLC cells).
Cells were treated for 48 h with 15 M of AA-5 and OA-4 and the expressions of cell cycle signal checkpoint proteins, including p16, p21, Cyclin D1, Cyclin B, and CDK1 were
l
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