Studies in natural products chemistry volume 41

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Contributors

Numbers in Parentheses indicate the pages on which the author’s contributions begin.

Daniela P.S. Alho (33), Grupo de Quı´mica Farmaceˆutica, Faculdade de Farma´cia,
Universidade de Coimbra, Po´lo das Cieˆncias da Sau´de, Azinhaga de Santa Comba,
and Centro Neurocieˆncias e Biologia Celular, Universidade de Coimbra, Coimbra,
Portugal
Dmitry L. Aminin (73), G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far
East Division, Russian Academy of Sciences, Vladivostok, Russia

Atta-ur-Rahman (257), International Center for Chemical and Biological Sciences,
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan
Sergey A. Avilov (73), G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far
East Division, Russian Academy of Sciences, Vladivostok, Russia
Francisco J. Barba (317), Department of Nutrition and Food Chemistry, Universitat
de Vale`ncia, Burjassot, Spain
Joa˜o M. Batista Jr. (379), Nucleus for Bioassays, Biosynthesis and Ecophysiology of
Natural Products (NuBBE), Institute of Chemistry, Sa˜o Paulo State University—
UNESP, Araraquara, Sa˜o Paulo, Brazil
Rachid Benhida (191), Institut de Chimie de Nice, UMR 7272 CNRS, Universite´ de
Nice-Sophia Antipolis, Nice Cedex 2, France
Khalid Bougrin (191), Laboratoire de Chimie des Plantes et de Synthe`se Organique et
Bioorganique, URAC23, Faculte´ des Sciences, Universite´ Mohammed V-Agdal,
Rabat, Morocco
Vanderlan da Silva Bolzani (379), Nucleus for Bioassays, Biosynthesis and
Ecophysiology of Natural Products (NuBBE), Institute of Chemistry, Sa˜o Paulo
State University—UNESP, Araraquara, Sa˜o Paulo, Brazil
Koen Dewettinck (343), Department of Food Safety and Food Quality, Ghent
University, Ghent, Belgium
Marı´a J. Esteve (317), Department of Nutrition and Food Chemistry, Universitat de
Vale`ncia, Burjassot, Spain
Ana. Frı´gola (317), Department of Nutrition and Food Chemistry, Universitat de
Vale`ncia, Burjassot, Spain
Takahiko Fujikawa (219), Laboratory of Molecular Prophylaxis and Pharmacology,
Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, and
Department of Biochemistry and Proteomics, Mie University Graduate School of
Medicine, Mie, Japan

xiii

xiv

Contributors

Bruno M.F. Gonc¸alves (33), Grupo de Quı´mica Farmaceˆutica, Faculdade de Farma´cia,
Universidade de Coimbra, Po´lo das Cieˆncias da Sau´de, Azinhaga de Santa Comba,
and Centro Neurocieˆncias e Biologia Celular, Universidade de Coimbra, Coimbra,
Portugal
Rajinder K. Gupta (415), School of Biotechnology, Guru Gobind Singh Indraprastha
University, Delhi, India
Tetsuya Hirata (219), R&D Center, Kobayashi Pharmaceutical Co., Ltd., Osaka,
Japan
Ingrid Hook (115), School of Pharmacy and Pharmaceutical Sciences, Trinity
Biosciences Institute (TBSI), Trinity College Dublin, Dublin, Ireland
Tsuyoshi Ikeda (219), Faculty of Pharmaceutical Sciences, Sojo University,
Kumamoto, Japan
Shabnam Javed (257), Center for Undergraduate Studies, University of Punjab,
Quaid-e-Azam Campus, Lahore-54590, Pakistan
Yongkui Jing (33), Department of Medicine, Mount Sinai School of Medicine,
New York, USA
Vladimir I. Kalinin (73), G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far
East Division, Russian Academy of Sciences, Vladivostok, Russia
Thien Trung Le (343), Faculty of Food Science and Technology, Nong Lam
University, Ho Chi Minh City, Viet Nam
Ana S. Leal (33), Grupo de Quı´mica Farmaceˆutica, Faculdade de Farma´cia,
Universidade de Coimbra, Po´lo das Cieˆncias da Sau´de, Azinhaga de Santa Comba,
and Centro Neurocieˆncias e Biologia Celular, Universidade de Coimbra, Coimbra,
Portugal
Monica Rosa Loizzo (1), Department of Pharmacy, Health Sciences and Nutrition,

University of Calabria, Rende (CS), Italy
Nadine Martinet (191), Institut de Chimie de Nice, UMR 7272 CNRS, Universite´ de
Nice-Sophia Antipolis, Nice Cedex 2, France
Hamid Marzag (191), Institut de Chimie de Nice, UMR 7272 CNRS, Universite´ de
Nice-Sophia Antipolis, Nice Cedex 2, France, and Laboratoire de Chimie des
Plantes et de Synthe`se Organique et Bioorganique, URAC23, Faculte´ des Sciences,
Universite´ Mohammed V-Agdal, Rabat, Morocco
Ekaterina S. Menchinskaya (73), G.B. Elyakov Pacific Institute of Bioorganic
Chemistry, Far East Division, Russian Academy of Sciences, Vladivostok, Russia
Vanessa I.S. Mendes (33), Grupo de Quı´mica Farmaceˆutica, Faculdade de Farma´cia,
Universidade de Coimbra, Po´lo das Cieˆncias da Sau´de, Azinhaga de Santa Comba,
and Centro Neurocieˆncias e Biologia Celular, Universidade de Coimbra, Coimbra,
Portugal
Francesco Menichini (1), Department of Pharmacy, Health Sciences and Nutrition,
University of Calabria, Rende (CS), Italy
Clive Mills (115), School of Pharmacy and Pharmaceutical Sciences, Trinity
Biosciences Institute (TBSI), Trinity College Dublin, Dublin, Ireland


Contributors

xv

Sansei Nishibe (219), Department of Pharmacognosy, Faculty of Pharmaceutical
Sciences, Health Sciences University of Hokkaido, Kanazawa, Japan
Evgeny A. Pislyagin (73), G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far
East Division, Russian Academy of Sciences, Vladivostok, Russia
Kristy M. Richards (93), U.S. FDA, Lenexa, Kansas, USA
Jorge A.R. Salvador (33), Grupo de Quı´mica Farmaceˆutica, Faculdade de Farma´cia,
Universidade de Coimbra, Po´lo das Cieˆncias da Sau´de, Azinhaga de Santa Comba,

and Centro Neurocieˆncias e Biologia Celular, Universidade de Coimbra, Coimbra,
Portugal
Helen Sheridan (115), School of Pharmacy and Pharmaceutical Sciences, Trinity
Biosciences Institute (TBSI), Trinity College Dublin, Dublin, Ireland
Alexandra S. Silchenko (73), G.B. Elyakov Pacific Institute of Bioorganic Chemistry,
Far East Division, Russian Academy of Sciences, Vladivostok, Russia
Robert E. Smith (93), U.S. FDA, Lenexa, Kansas, and Department of Science, Park
University, Parkville, Missouri, USA
Kavita Tiwari (415), School of Biotechnology, Guru Gobind Singh Indraprastha
University, Delhi, India
Joel D.W. Toh (157), Department of Pharmacy, National University of Singapore,
Singapore, Singapore
Kevin Tran (93), U.S. FDA, Lenexa, Kansas, USA
Rosa Tundis (1), Department of Pharmacy, Health Sciences and Nutrition, University
of Calabria, Rende (CS), Italy
Yoshihide Usami (283), Laboratory of Pharmaceutical Organic Chemistry, Osaka
University of Pharmaceutical Sciences, Takatsuki, Osaka, Japan
Ana S. Valdeira (33), Grupo de Quı´mica Farmaceˆutica, Faculdade de Farma´cia,
Universidade de Coimbra, Po´lo das Cieˆncias da Sau´de, Azinhaga de Santa Comba,
and Centro Neurocieˆncias e Biologia Celular, Universidade de Coimbra, Coimbra,
Portugal
John Van Camp (343), Department of Food Safety and Food Quality, Ghent
University, Ghent, Belgium
Pierre Warnault (191), Institut de Chimie de Nice, UMR 7272 CNRS, Universite´ de
Nice-Sophia Antipolis, Nice Cedex 2, France
Esther C.Y. Woon (157), Department of Pharmacy, National University of Singapore,
Singapore, Singapore


Preface

Natural products continue to provide a treasure of novel materials with potential
applications in a variety of different fields. This volume of Studies in Natural
Product Chemistry presents a number of exciting articles on a wide range of
bioactive natural products.
In Chapter 1 by Tundis et al., the potential role of natural triterpenoids with
cycloartane, cucurbitane, friedelane, tirucallane, and lupane skeleton in tumor
chemoprevention and treatment is discussed. Their structures and mechanisms
of action are presented. In Chapter 2, Salvador et al. discuss the effects of natural pentacyclic triterpenoids and their semisynthetic derivatives, highlighting
their potential in anticancer drug discovery.
Chapter 3 by Aminin et al. describes the medicinal chemistry of sea
cucumber triterpene glycosides. Some of these were found to be against cancer while others showed immunomodulatory activity.
Annonaceous acetogenins constitute a large class of natural polyketides,
with over 400 representatives. They show pesticidal, anti-infective, and cytotoxic properties. In Chapter 4, Smith et al. discuss their properties, including
neurotoxicity exhibited by them.
In Chapter 5, the various classes of naphthoquinones and their biological
activities are discussed by Hook et al. Their production, biosynthesis, and
synthesis are also discussed.
Obesity has become a common health problem. Woon and Toh, in
Chapter 6, review the applications of several natural compounds to cure obesity from an epigenetic perspective. They describe their bioactivity, clinical
data, and mechanisms of action against obesity.
In Chapter 7, Marzag et al. have focused their contribution on natural polyphenols and their relationship with epigenetic modifications, particularly as potent
inhibitors of DNA methyl transferase. Chapter 8 covers different biological activities of Eucommia ulmoides Oliver leaves, including the antiobesity effects.
In Chapter 9, Javed and Atta-ur-Rahman present the increasing interest in
Aloe vera extracts not just for their use in cosmetics but in a wide variety of other
illnesses. Their chemistry and processing techniques have also been discussed.
Carbasugars are a class of carbohydrates that are known to possess various
biological activities including glycosidase inhibition, antitumor, anticancer,
antiviral, antifungal, antibacterial, and antimalarial activities. Usami, in
Chapter 10, discusses different aspects with reference to the chemical synthesis

of such compounds. In Chapter 11, Esteve et al. discuss the antioxidant and antimicrobial potential of leaf vegetable products.

xvii


xviii

Preface

Van Camp et al. have presented a comprehensive discourse in Chapter 12
on milk fat globule membrane materials, their isolation techniques, healthbeneficial properties, and applications as functional foods and nutraceuticals.
In Chapter 13, Batista and da Silva Bolzani discuss the current state of the art
of vibrational circular dichroism spectroscopy for the determination of absolute configuration of various natural products. Moreover, quantum chemical
calculations have also been discussed.
In Chapter 14, Tiwari and Gupta have presented a comprehensive review
on the bioactive secondary metabolites from rare actinomycetes. Particular
emphasis has been placed on their structures, relevant biological activities,
and source organisms.
I hope that this volume will be received with the same interest and enthusiasm as the previous volumes of this long-standing series on natural product
chemistry.
I would like to thank Ms. Taqdees Malik, Ms. Darshna Kumari, and
Ms. Humaira Hashmi for their assistance in the preparation of this volume.
I am also grateful to Mr. Mahmood Alam for his editorial assistance.
Atta-ur-Rahman, FRS
International Center for Chemical and Biological Sciences
H.E.J. Research Institute of Chemistry
University of Karachi
Karachi, Pakistan

Chapter 1

Recent Insights into the
Emerging Role of Triterpenoids
in Cancer Therapy: Part II
Rosa Tundis, Francesco Menichini and Monica Rosa Loizzo
Department of Pharmacy, Health Sciences and Nutrition, University of Calabria,
Rende (CS), Italy

Chapter Outline
Introduction
Antitumor Activity of
Triterpenoids
The Cycloartane Group
The Cucurbitane Group
The Friedelane Group

1
2
2
6
8

The Tirucallane Group
The Lupane Group
Miscellaneous Compounds
Concluding Remarks
References

10

11
25
27
30

INTRODUCTION
In this chapter, we report the recent insights into the role of triterpenoids in
cancer therapy. In the first chapter, we have focused our interest on triterpenoids with oleanane, dammarane, hopane, lanostane, and ursane skeleton
and their synthetic derivatives [1]. Herein, we report an overview of the most
recent progress in the in vitro and in vivo studies on the anticancer properties
of natural triterpenoids with a cycloartane, cucurbitane, friedelane, tirucallane,
and lupane skeleton and their synthetic derivatives with a special focus on the
structure–activity relationship (SAR).
Cancer is a complex genetic disease with hallmark traits acquired during
their multistep development including activating cell invasion and metastasis,
exhibiting genetic diversity, inflammation, reprogramming of energy metabolism, sustaining cell-proliferative signaling, evading activity of cell population
growth suppressors, enabling replicative immortality, inducing angiogenesis,
evading immune destruction, and resisting programmed cell death [2].
Studies in Natural Products Chemistry, Vol. 41. />Copyright © 2014 Elsevier B.V. All rights reserved.

1


2

Studies in Natural Products Chemistry

Natural products derived from plant sources have been a rich source of
agents of value to medicine. More than half of currently available drugs
are natural compounds or are related to them, and in the case of cancer

this proportion surpasses 60%. This situation is accompanied by
increasing interest from drug companies and institutions devoted to the search
for new drugs. Additionally, many natural compounds have been considered
leads or heads of series and their later structural modification has afforded
compounds with pharmacological activity and extraordinary therapeutic
possibilities.
Recent efforts into the research and development of anticancer drugs
derived from natural source have led to the identification of a variety of triterpenoids, characterized as possessing a wide variety of remarkable antitumor
properties, for example, induction of cell-cycle arrest, induction of apoptosis,
and differentiation, as well as inhibition of cell growth and proliferation, or a
combination of two or more of these mechanisms [3]. In our recent work, we
demonstrated the interesting antiproliferative activity on renal, prostate, and
melanoma cancer cell lines of Sarcopoterium spinosum and its major constituent tormentic acid [4]. This ursane-type triterpene demonstrated a higher
cytotoxicity than the positive control against renal cell adenocarcinoma
ACHN cell line with an inhibitory concentration 50% (IC50) value of
23.7 mM (vinblastine IC50 value of 25.0 mM).
The potential antitumor properties of triterpenoids would have broader
implications if we consider that these secondary metabolites represent the
largest group of naturally occurring phytochemicals and that are also present
in common foods.

ANTITUMOR ACTIVITY OF TRITERPENOIDS
The Cycloartane Group
Cancer chemopreventive effects and antiproliferative activity against several
cancer lines of cycloartane-type triterpenoids have been reported [5,6].
Recently, two cycloartane glycosides isolated from the aerial parts of
Thalictrum fortunei, 3-O-b-D-glucopyranosyl (1 ! 4)-b-D-fucopyranosyl(22S,24Z)-cycloart-24-en-3b,22,26,30-tetraol 26-O-b-D-glucopyranoside (1) and
3-O-b-D-glucopyranosyl (1 ! 4)-b-D-fucopyranosyl-(22S,24Z)-cycloart-24-en3b,22,26,29-tetraol 26-O-b-D-glucopyranoside (2), demonstrated IC50 values of
6.8, 24.3, 5.6 mg/ml and 3.3, 7.8, 3.1 mg/ml toward Bel-7402, LoVo, and NCIH460 human tumor cells, respectively (Table 1) [7]. In the same year, from Astragalus stereocalyx 12 cycloartane-type triterpene glycosides were isolated and
were tested for their potential cytotoxicity in vitro against HeLa (human cervical

cancer), HT-29 (human colon cancer), U937 (human leukemia), and H446 (human
lung cancer) [8]. 3-O-b-D-Glucopyranosyl-16-O-b-D-glucopyranosyl-3b,6a,16b,24(R),25-pentahydroxycycloartane was the most potent compound against


Chapter

1

3

Emerging Role of Triterpenoids in Cancer Therapy

TABLE 1 In Vitro Cytotoxicity of Cycloartane-Type and Cucurbitane-Type
Triterpenes
Compound

Cell Line

IC50, ED50

Ref.

3-O-b-D-Glucopyranosyl (1 ! 4)-b-Dfucopyranosyl-(22S,24Z)cycloart-24-en-3b,22,26,30-tetraol
26-O-b-D-glucopyranoside (1)

Bel-7402, LoVo,
NCIH-460

6.8, 24.3,
5.6 mg/ml

[7]

3-O-b-D-Glucopyranosyl (1 ! 4)-b-Dfucopyranosyl-(22S,24Z)cycloart-24-en-3b,22,26,29-tetraol
26-O-b-D-glucopyranoside (2)

Bel-7402, LoVo,
NCIH-460

3.3, 7.8,
3.1 mg/ml

[7]

25-Pentahydroxycycloartane

HeLa

29.9 mM

[8]

Askendoside G

HeLa

24.4 mM

[8]

3-O-b-D-Glucopyranosyl-16-O-b-Dglucopyranosyl-3b,6a,16b,24(R),25pentahydroxycycloartane

HeLa

10 mM

[8]

Cyclotrisectoside

MCF-7

30 mM

[9]

6-O-b-D-Xylopyranosyl-3b,6a,16b,24atetrahydroxy-20(R),25-epoxycycloartane

HL-60

45 mM

[9]

Sinocalycanchinensin E (3)
(þcolchicine)

KB-C2

1.5 mg/ml

[10]

25-O-Acetylcimigenol-3-O-b-Dxylopyranoside (4)

MCF-7, R-MCF-7

4.0,
5.3 mg/ml

[11]

25-O-Acetylcimigenol-3-O-a-Larabinopyranoside (5)

MCF-7, R-MCF-7

4.3,
4.8 mg/ml

[11]

25-O-Acetylcimigenol-3-O-[40 -O-(E)-2butenoyl]-b-D-xylopyranoside (6)

HepG2

1.3 mM

[12]

25-O-Acetylcimigenol-3-O-[30 -Oacetyl]-b-D-xylopyranoside (7)

HepG2

0.7 mM

[12]

30 -O-Acetyl-23-epi-26-deoxyactein (8)

HepG2

1.4 mM

[12]

Gardenoin A

CHAGO, HepG2

1.6,
4.5 mg/ml

[13]

Gardenoin C

CHAGO, HepG2,
SW-260

4.4, 2.8,

2.5 mg/ml

[13]

G292, MG-63,
HT-161, HOS,
SAOS-2, SJSA

50 nM

[14]

Cycloartane skeleton

Cucurbitane skeleton
Cucurbitacin B (9)

Continued


4

Studies in Natural Products Chemistry

TABLE 1 In Vitro Cytotoxicity of Cycloartane-Type and Cucurbitane-Type
Triterpenes—Cont’d
Compound

Cell Line

IC50, ED50

Ref.

Cucurbitacin D (10)

HT-29

0.12 mM

[15]

3-epi-Isocucurbitacin D

HT-29

0.039 mM

[15]

Cucurbitacin I

HT-29

0.19 mM

[15]

Cucurbitacin E 2-O-b-Dglucopyranoside

BGC-823, A549

4.98,
3.2 mM

[16]

Machilusides A (12)

A2780, HCT-8,
Bel-7402,
BGC-823, A549

0.4–6.5 mM

[17]

Machilusides B (13)

A2780, HCT-8,
Bel-7402,
BGC-823, A549

0.4–6.5 mM

[17]

HeLa cells (IC50 value of 10 mM), while 3-O-{a-L-rhamnopyranosyl-(1 ! 4)[a- L -arabinopyranosyl-(1 ! 2)]-b- D -glucopyranosyl}-3b,6a,16b,24(R)25pentahydroxycycloartane, and askendoside G exhibited IC50 values of 29.9
and 24.4 mM, respectively. All the other compounds exhibited a lower cytotoxic
activity.

Previously, from A. aureus other 18 cycloartane-type triterpene glycosides
were isolated [9]. Their potential antiproliferative activity was tested against
HL-60 (human promyelocytic leukemia), MCF-7 (human breast cancer),
HT-29 (human colon carcinoma), A549 (human lung adenocarcinoma), and
PC3 (human prostate cancer) cell lines using etoposide as positive control.
Among tested compounds, cyclotrisectoside exhibited an IC50 value of
30 mM against MCF-7 cells, while 6-O-b-D-xylopyranosyl-3b,6a,16b,24atetrahydroxy-20(R),25-epoxycycloartane was cytotoxic against HL-60 cells
with an IC50 value of 45 mM (Fig. 1).
29-nor-Cycloartanes, isolated from Sinocalycanthus chinensis, were
tested against KB (human epidermoid carcinoma of nasopharynx), K562
(leukemia), and MCF-7 (breast carcinoma) cell lines, as well as multidrugresistant (MDR) human cancer cell lines, including KB-C2 (colchicineresistant KB) and K562/Adr (doxorubicin-resistant K562) [10]. Sinocalycanchinensin E (3) showed significant enhanced cytotoxicity against KB-C2
cells in the presence of colchicine with an IC50 value of 1.51 mg/ml. Since
colchicine had no effect on the growth of KB-C2 cells at this concentration
level, it was suggested that sinocalycanchinensins E might show some
MDR-reversing effects.


Chapter

1

OH

O

OH

O

OR1

H
RO
HOH2C

5

Emerging Role of Triterpenoids in Cancer Therapy

OR1

H
RO

CH2OH

1
R = β-D-Glc (1

O

H

MeO
H

2

H H
O

O

O

HO

H
H

O

O

O
O

O
5

OH

O

H

H
OAc

OR2

O

6 R = 4Ј-O-(E)-2-Butenoyl-β-D-xylose, R1 = H, R2 = Ac
7 R = 3Ј-O-Acetyl-β-D-xylose, R1 = H, R2 = Ac

O

H

OH
RO

O

OH
O

4

R1

O
O
OH

O

OH

H H

O

OH
HO
HO

3

4)-β-D-fuc; R1 = β-D-Glc

RO
8 R = 3'-O-Acetyl-β-D-xylose

FIGURE 1 Chemical structures of cycloartane-type triterpenoids 1–8.

Five cycloartane triterpenoids isolated from Cimicifuga yunnanensis,
cimigenol-3-O-b-D-xylopyranoside, 25-O-acetylcimigenol-3-O-b-D-xylopyranoside (4), 25-chlorodeoxycimigenol-3-O-b-D-xylopyranoside, 25-O-acetylcimigenol-3-O-a-L-arabinopyranoside (5) and 23-O-acetylcimigenol-3-O-b-Dxylopyranoside, have antitumor activity on the breast cancer cell line MCF-7 and
its corresponding drug-resistant cell subline R-MCF-7 [11]. Compound 4 was the
most active with IC50 values of 4.0 and 5.3 mg/ml against MCF-7 and R-MCF-7,
respectively.
Comparable effects to the positive control taxol (IC50 values of 0.03 and
4.8 mg/ml against MCF-7 and R-MCF-7, respectively) were also obtained
with 25-O-acetylcimigenol-3-O-a-L-arabinopyranoside (5) with IC50 values
of 4.3 and 4.8 mg/ml against MCF-7 and R-MCF-7, respectively. Furthermore,
in MCF-7 cells isolated compounds induced the upregulation of caspase-7
activity and increased the RNA level of p53 and pro-apoptotic gene bax.
Taken together, these compounds are potential antitumor agents on both
parental and drug-resistant breast tumors. The antiproliferative activity of
these analogs apparently increases when some hydrophobic groups are introduced to the C23 and the C25 carbon sites, such as acetyl and halogen.

6

Studies in Natural Products Chemistry

A significant cytotoxicity against HepG2 cells (IC50 values of 1.3, 0.7, and
1.4 mM, respectively) demonstrated 25-O-acetylcimigenol-3-O-[40 -O-(E)-2butenoyl]-b-D-xylopyranoside (6), 25-O-acetylcimigenol-3-O-[30 -O-acetyl]-b0
D-xylopyranoside (7) and 3 -O-acetyl-23-epi-26-deoxyactein (8), isolated from
the roots of Cimicifuga fetida [12]. An IC50 value of 1.7 mM against HepG2 cell
line was found for cisplatin used in this study as positive control.
Four new 3,4-seco-cycloartanes, gardenoins A–D, were isolated from the
exudate of Gardenia tubifera and were tested for their cytotoxicity against
BT474, CHAGO, HepG2, KATO-3, and SW-620 [13]. Among these compounds,
gardenoin A demonstrated a significant antiproliferative activity against CHAGO
and HepG2 cell lines with IC50 values of 1.6 and 4.5 mg/ml, respectively.
Gardenoin C was also active against the CHAGO, HepG2, and SW-260
cancer cell lines, with IC50 values of 4.4, 2.8, and 2.5 mg/ml, respectively,
whereas gardenoin B and D showed IC50 values >5 mg/ml against all tested cell
lines. As previously reported, these results support the role of an exomethylene
g-lactone ring system for the cytotoxicity of these compounds [18,19].

The Cucurbitane Group
Cucurbitacin B (9), a component of plant-derived tetracyclic triterpenoids
originally found in the plant family of Cucurbitaceae, was studied as a single
agent or in combination with methotrexate (MTX) for human osteosarcoma
(OS) treatment [14]. Cucurbitacin B showed antiproliferative activity against
seven human OS cell lines in vitro (U2OS, G292, MG-63, HT-161, HOS,
SAOS-2, and SJSA) accompanying G2/M cell-cycle arrest, apoptosis, and
inhibition of extracellular signal-regulated kinase (ERK), Akt, and mammalian target of rapamycin (mTOR) proteins. Cucurbitacin B (9) in combination
with MTX synergistically inhibited OS cell growth in vitro. Low-dose cucurbitacin B (LD-CuB, 0.5 mg/kg body weight) or low-dose MTX (LD-MTX,

150 mg/kg) failed to decrease the size of human OS xenografts in nude mice.
However, combined therapy at identical concentrations inhibited tumor
growth by 62% versus LD-CuB and 81% versus LD-MTX. Strikingly, the
effect persisted even when the dose of MTX was decreased by two-thirds.
Accumulating evidences indicate that cucurbitacin B inhibits the growth of
numerous human cancer cell lines and tumor xenografts including breast,
prostate, lung, uterine cervix, liver, skin, and brain cancers. Cucurbitacin
B inhibits tumor growth and Stat (signal transducer and activator of transcription) 3 signaling pathway in numerous human cancer cells and induces
apoptosis [20–25]. Liu et al. [26] demonstrated that Hep-2 cells treated with
a combination of cucurbitacin B/cisplatin display synergistic effects on growth
inhibition, cell-cycle arrest, and apoptosis induction. Xenograft models containing Hep-2 cells in mice also demonstrated that this cucurbitacin B/cisplatin
combination led to the synergistic inhibition of tumor growth (Fig. 2).


Chapter

1

HO

O

O
O

O
H

HO

OH

H

HO

O

HO
OH

O
H

HO

O
H

OH

H

O

O

10
HO
HO

HO
O
H

HO
HO

OH
GlcO

O
12

O

OH

OH

HO

OH

O
H

H

11

OH

O

O

H

H

HO

9

GlcO

7

Emerging Role of Triterpenoids in Cancer Therapy

O
H

O

O

OH

O

H

OH

O
13

FIGURE 2 Chemical structures of cucurbitane-type triterpenoids 9–13.

Combined therapy with cucurbitacin B and gemcitabine (at doses 0.5 and
25 mg/kg, respectively) resulted in significant tumor growth inhibition of pancreatic cancer xenografts (up to 79%) without any significant signs of toxicity [27]. Western blot analysis of the tumor samples of mice who received
both cucurbitacin B and gemcitabine revealed stronger inhibition of Bcl-XL,
Bcl-2, and c-myc, and higher activation of the caspase cascades, than mice
treated with either agent alone. Eight new 16,23-epoxycucurbitacin derivatives, elaeocarpucins A–H, and five known cucurbitacin were isolated from
the chloroform-soluble partitions of separate methanol extracts of the fruits
and stem bark of Elaeocarpus chinensis [15]. Cucurbitacin D (10), 3-epiisocucurbitacin D, 25-O-acetylcucurbitacin F, and cucurbitacin I were found
to be the most active in inhibiting the proliferation of HT-29 cancer cells, with
IC50 values ranging from 0.039 to 0.54 mM.
Elaeocarpucin C (11) showed an IC50 value of 0.41 mM, while elaeocarpucins D, G, and H were less active against the same cell line. Probably, a
24(25)-en-27-ol functionality on the side chain of these compounds seems
to be required for potent cytotoxicity. In general, when the C-17–C-23 unit
is contained in an epoxide ring, the resultant cytotoxicity is reduced when
compared with known compounds such as 3-epi-isocucurbitacin D. Previously, cucurbitacin D (10) was found to inhibit proliferation and to induce
apoptosis of T-cell leukemia cells [28]. Constitutively activated NF-kB was
inhibited by cucurbitacin D in the nucleus, which resulted in the accumulation
of NF-kB in the cytoplasm, leading to downregulation of the expression
of antiapoptotic proteins Bcl-xL and Bcl-2. Furthermore, cucurbitacin


8

Studies in Natural Products Chemistry

D induced the accumulation of inhibitor of NF-kB (IkB)a by inhibition of
proteasome activity. Low doses of cucurbitacin D (10) synergistically potentiated the antiproliferative effects of the histone deacetylase inhibitor valproic
acid. The pro-apoptotic and proteasome inhibitory activity of cucurbitacin
D was also demonstrated in an in vivo study using SCID mice.
Cucurbitacin E 2-O-b-D-glucopyranoside showed selective cytotoxic
activity against the BGC-823 and A549 cell lines (IC50 4.98 and 3.20 mM,
respectively) [16]. IC50 values >10 mM were found for the A2780, HCT-8,
and Bel-7402 cells. The positive control camptothecin gave IC50 values of
0.26–11.8 mM. These results confirmed the role of acetylation of the OH-25
in enhancing the cytotoxicity of cucurbitacins. Machilusides A (12) and
B (13), novel homocucurbitane triterpenoid glycosides, isolated from the stem
bark of Machilus yaoshansis, were tested against the ovary (A2780), colon
(HCT-8), hepatoma (Bel-7402), stomach (BGC-823), and lung (A549) human
cancer cell lines, and showed nonselective cytotoxic activities with IC50
values of 0.40–6.52 mM [17]. The positive control camptothecin gave IC50
values of 0.28–12.5 mM.

The Friedelane Group
From Garcia parviflora friedelane-type triterpenes, namely, 1,2dehydro-2,3-secofriedelan-3-oic acid, friedelin-3,4-lactone, acetyl aleuritolic
acid (14), 1b-hydroxyfriedelin, and 3b-hydroxyfriedelan-23-oic acid, were
isolated and were investigated for their cytotoxic activity against breast
(MCF-7), leukemia (K562), central nervous system (U251, Glia), prostate
(PC-3), colon (HCT-15), and lung (SKLU-1) cancer cell lines [29]. Moreover,
some derivatives of 1b-hydroxyfriedelin were obtained via oxidation, reduction, and esterification in order to explore the cytotoxic potential of the
resulting semisynthetic friedelane derivatives.
As reported in Table 2, acetyl aleuritolic acid (14) exhibited the highest
cytotoxic activity against U251 cell line with an IC50 value of 8.4 mM.

Against the same cell line, the natural compounds 1,2-dehydro-2,3secofriedelan-3-oic acid and friedelin-3,4-lactone and the semisynthetic derivative 1b,3b-dihydroxyfriedelane exhibited a lower activity with IC50 values of
36.8, 17.1, and 22.8 mM, respectively, than the positive control adriamycin
(IC50 value of 0.3 mM). A similar trend was observed also with the derivative
4a-hydroxyfriedel-1-en-3-one that showed weak cytotoxicity against the
HCT-15 cell line with an IC50 value of 41.8 mM compared with adriamycin
(IC50 value of 0.2 mM). Friedelin-3,4-lactone and acetyl aleuritolic acid were
more cytotoxic than 1,2-dehydro-2,3-secofriedelan-3-oic acid. From the stem
of Calophyllum inophyllum 3b,23-epoxy-friedelan-28-oic acid, friedelin, epifriedelanol, canophyllal, canophyllol, canophyllic acid (15), and 3-oxofriedelan-28-oic acid (16) were isolated and tested against human leukemia
HL-60 cells. Compounds 15 and 16 exhibited a significant activity with


TABLE 2 In Vitro Cytotoxicity of Friedelane- and Tiricullane-Type
Triterpenes
Compound

Cell Line

IC50 (mM)

Ref.

1,2-Dehydro-2,3-secofriedelan-3-oic
acid

U251

36.8

[29]

Friedelin-3,4-lactone

U251

17.1

[29]

Acetyl aleuritolic acid (14)

U251

8.4

[29]

1b,3b-Dihydroxyfriedelane

U251

22.8

[29]

4a-Hydroxyfriedel-1-en-3-one

HCT-15

41.8

[29]

3b,23-epoxy-Friedelan-28-oic acid

HL-60

10.7

[30]

Canophyllic acid (15)

HL-60

4.6

[30]

3-oxo-Friedelan-28-oic acid (16)

HL-60

2.7

[30]

3a-Hydroxy-21amethoxy-24,25,26,27tetranortirucall-7-ene-23(21)-lactone
(17)

MCF-7, HeLa,

HepG2, SGC-7901,
BGC-823

24.2–42.2

[31]

3a-Hydroxy-21bmethoxy-24,25,26,27tetranortirucall-7-ene-23(21)-lactone
(18)

MCF-7, HeLa,
HepG2, SGC-7901,
BGC-823

21.3–67.1

[31]

3-oxo-21a-Methoxy-24,25,26,27tetranortirucall-7-ene-23(21)-lactone

MCF-7, HeLa,
HepG2, SGC-7901,
BGC-823

70.9–166.5

[31]

3-oxo-21b-Methoxy-24,25,26,27tetranortirucall-7-ene-23(21)-lactone

MCF-7, HeLa,
HepG2, SGC-7901,
BGC-823

41.7–76.2

[31]

3-oxo-21a-Ethoxy-24,25,26,27tetranortirucall-7-ene-23(21)-lactone

MCF-7, HeLa,
HepG2, SGC-7901,
BGC-823

50.2–126.6

[31]

3a-Hydroxy-24,25,26,27tetranortirucall-7-ene-23(21)-lactone
(19)

MCF-7, HeLa,
HepG2, SGC-7901,
BGC-823

34.2–64.1

[31]

3-oxo-24,25,26,27Tetranortirucall-7-ene-23(21)-lactone

(20)

MCF-7, HeLa,
HepG2, SGC-7901,
BGC-823

15.7–97.6

[31]

Cornusalterin L (21)

A549, SK-OV-3,
SK-MEL-2, XF498

3.8–5.8

[32]

Deoxyflindissone (22)

A549, SK-OV-3,
SK-MEL-2, XF498

3.6–6.1

[32]

Friedelane skeleton

Tiricullane skeleton


10

Studies in Natural Products Chemistry

IG50 values of 4.6 and 2.7 mM, respectively, compared to the positive control
5-fluorouracile (IG50 value of 4.1 mM) [30]. A lower cytotoxicity was found
for the triterpene 3b,23-epoxy-friedelan-28-oic acid with an IG50 value of
10.6 mM.

The Tirucallane Group
Tirucallic acids isolated from Boswellia carterii exerted cytotoxic effects in
human prostate cancer cell lines both in vitro and in vivo. In particular, the
activation of the Akt is associated with aggressive clinical behavior of prostate cancer. LNCaP and PC-3 cell lines express predominantly Akt1 and
Akt2. Selective downregulation of Akt1, but not Akt2, was observed with tetracyclic triterpenoids 3-oxo-tirucallic acid, 3-a-acetoxy-tirucallic acid, and
3-b-acetoxy-tirucallic acid while nuclear factor-kB kinases remained unaffected. Docking analysis suggested that these triterpenoids could establish
hydrogen bonds within the phosphatidylinositol binding pocket of the Akt
pleckstrin homology domain. In fact, the 3-b-acetoxy-tirucallic acid did not
inhibit the activity of Akt1 because it is lacking the pleckstrin homology
domain. The tirucallic acid derivatives inhibited proliferation and induced
apoptosis in tumors xenografted onto chick chorioallantoic membranes and
decreased the growth of pre-established prostate tumors in nude mice without
overt systemic toxicity [33].
Tirucallane-type C26 triterpenoids, namely, 3a-hydroxy-21amethoxy-24,25,26,27-tetranortirucall-7-ene-23(21)-lactone (17), 3a-hydroxy-21b-methoxy-24,25,26,27-tetranortirucall-7-ene-23(21)-lactone (18), 3oxo-21a-methoxy-24,25,26,27-tetranortirucall-7-ene-23(21)-lactone, 3-oxo21b-methoxy-24,25,26,27-tetranortirucall-7-ene-23(21)-lactone, 3-oxo-21aethoxy-24,25,26,27-tetranortirucall-7-ene-23(21)-lactone, 3a-hydroxy-24,
25,26,27-tetranortirucall-7-ene-23(21)-lactone (19), and 3-oxo-24,25,26,27tetranortirucall-7-ene-23(21)-lactone (20), were isolated from the stem barks
of Aphanamixis grandifolia. These compounds were evaluated for their
cytotoxic potential using MCF-7 cells (human breast cancer), HeLa cells
(human cervical cancer), HepG2 cells (human hepatocellular carcinoma),

SGC-7901 cells (human gastric adenocarcinoma), and BGC-823 cells
(human gastric carcinoma) using taxol as positive control.
Compound 18 showed the highest cytotoxic activity with IC50 average
value of 31.6 mM, followed by 17 (IC50 average value of 32.3 mM), 19, and
20 (IC50 average value of 46.5 and 47.3 mM, respectively) while lower bioactivity was observed with the other isolated constituents. The PCA analysis
clearly evidenced the key role for the inhibitory activity in 3-hydroxyl group
and the superiority of the b-methoxyl to a-methoxyl substitution at C-21 [31]
(Fig. 3).
Twelve new tirucallane triterpenoids, named cornusalterins A–L, and two
known tirucallane triterpenoids, deoxyflindissone and (À)-leucophyllone,


Chapter

1

11

Emerging Role of Triterpenoids in Cancer Therapy

O

O

R
COOH

H

COOH

H
AcO

R

H
14
O

O

15 R = β-OH
16 R = O

HO

17 R = α-OMe
18 R = β-OMe
O

R
H
H

R1
R

19 R = α-OH, R1 = H
20 R = R1 = O

O

21 R = β-H
22 R = α-H

FIGURE 3 Chemical structures of friedelane- and tiricullane-type triterpenoids 14–22.

were isolated from a methanol extract of stems and stem bark of Cornus
walteri [32]. Cornusalterin L(21) and deoxyflindissone (22), characterized by
a tetrahydrofuran ring in the side chain, exhibited significant cytotoxic activity
against the A549, SK-OV-3, SK-MEL-2, and XF498 cell lines (IC50 values of
4.29, 3.82, 4.73, and 5.81 mM for 21, and 4.02, 3.64, 6.07, and 5.10 mM, for
22, respectively). The other compounds were essentially noncytotoxic.

The Lupane Group
Lupeol
Lupeol (23) is a triterpene that has showed anticarcinogenic and antimutagenic activity both in vitro and in vivo [34]. Lupeol treatment to Mel 928
and Mel 1241 and Mel 1011 cells resulted in a dose-dependent decrease in
cell viability with IC50 values of 75, 72, and 135 mM, respectively. This triterpene is able to induce apoptosis and decrease the colonogenic potential, the
b-catenin transcriptional activity and finally the expression of Wingless target
genes that regulates a variety of cellular processes including proliferation, differentiation, survival, apoptosis and cell motility. Aberrant activation of these
pathways has been observed in approximately one-third of melanomas and
this subset has very poor prognosis.
Moreover, lupeol (23) restricted the translocation of b-catenin from the
cytoplasm to the nucleus. In vivo study demonstrated that lupeol (23) also
decreased the growth of Mel 928 but not Mel 1011-derived tumors implanted
in athymic nude mice. The effect in Mel 928-derived tumor growth was associated with a decrease in the expression of Wingless target genes c-myc,

12

Studies in Natural Products Chemistry

cyclin D1, proliferation markers proliferating cell nuclear antigen and Ki-67
and invasion marker osteopontin. These results suggest that this compound
may be useful alone or as an adjuvant to currently used therapies for the treatment of human melanomas harboring constitutive Wingless/b-catenin signaling considering that lupeol lack of effect on normal human melanocytes [35].
In the preclinical study, this triterpene exhibited also an interesting activity
against prostate cancer cell live androgen-dependent phenotype (ADPC) and
castration-resistant phenotype (CRPC). In fact, it significantly inhibited the
androgen analog (R1881) in the induction of (i) transcriptional activity of
androgen receptor and (ii) expression of PSA. Its action appears to be competed antagonistically with androgen for receptor consequently blocking the
binding of receptor to its responsive genes including PSA, TIPARP, SGK,
and IL-6. Moreover, lupeol (23) inhibited the recruitment of RNAPol II to target genes. In CRPC, lupeol sensitized cells to antihormone therapy. In vivo
study confirmed the inhibition of tumorigenicity of both ADPC and CRPC
cells and a significant serum and tumor tissues reduction of PSA levels [36].
The analysis of obtained data highlights that lupeol (23) inhibited androgen
receptor in prostate cancer cells by adopting several approaches irrespective
of androgen receptor and androgen-sensitive status and showed the potential
of this triterpene as a more effective disruptor of androgen receptor signaling
than currently used antiandrogens (e.g., bicalutamide).

Betulin and Betulinic Derivatives
Betulin (24) and betulinic acid (25) are common plant-derived lupane-type triterpenes. Both compounds demonstrated antitumor activity and overcome
resistance by inducing apoptosis in a variety of human cancers [37]. Eichenmuller et al. [38] demonstrated the ability of betulinic acid (2) to induce apoptosis in rhabdomyosarcoma cell cultures (RMS-13, RH-30, and RD) and
in vivo using female NMRI nude mice by measuring cell viability, survival,
apoptosis, hedgehog signaling activity, and neovascularization. Betulinic acid
(25) had a strong cytotoxic effect on rhabdomyosarcoma cells in a dosedependent manner with IC50 values of 5.0, 3.9, and 9.5 mg/ml for RMS-13,
RH-30, and RD, respectively (Fig. 4).
Moreover treatment with 25 caused a massive induction of apoptosis

mediated by the intrinsic mitochondrial pathway, which could be inhibited
by the broad-range caspase inhibitor zVAD.fmk. Exposure of hedgehogactivated RMS-13 cells to this triterpene resulted in a strong decrease in
GLI1, GLI2, PTCH1, and IGF2 expression as well as hedgehog-responsive
luciferase activity. Intraperitoneal injection of 20 mg of 25 per kg per day significantly retarded growth of RMS-13 xenografts in association with markedly
higher counts of apoptotic cells and downregulation of GLI1 expression compared with control tumors, while leaving microvascular density, cell proliferation, and myogenic differentiation unaffected.


Chapter

1

13

Emerging Role of Triterpenoids in Cancer Therapy

H

H

R

R
H

H

H

O

H

HO

23

O
HO

OH

30 R =

N

31 R =

NH

32 R =

N

O

N
NH2

R

OAc
27 R =

O

H
N

R

HO

AcO

R1
24 R = CH2OH, R1 = H
25 R = COOH, R1 = H
26 R = COOH, R1 = OH

N

N

R1
N
H

N

OMe

O

OH

28 R =

NH

29 R =

NH

N
N
N N
NH2

33 R = -H (Gly), R1 = Me (Ala)
34 R = -H (Gly), R1 = Me (Pro)

NH

FIGURE 4 Chemical structures of lupane-type triterpenoids 23–34.

Recently, Chintharlapalli et al. [39] demonstrated that betulinic acid (25)
inhibited growth at concentrations of ! 5 mM after 48 and 72 h exposure
and induced apoptosis in RKO and SW-480 colon cancer cells and inhibited
tumor growth in athymic nude mice bearing RKO cells as xenograft. Betulinic
acid also decreased expression of Sp1, Sp3, and Sp4 transcription factors
which are overexpressed in colon cancer cells and decreased levels of several

Sp-regulated genes including survivin, vascular endothelial growth factor, p65
subunit of NF-kB, epidermal growth factor receptor, cyclin D1, and pituitary
tumor transforming gene-1.
The mechanism of action of betulinic acid (25) was dependent on cell context, since (25) induced proteasome-dependent and proteasome-independent
downregulation of Sp1, Sp3, and Sp4 in SW-480 and RKO cells, respectively.
In RKO cells, the mechanism of betulinic acid-induced repression of Sp1,
Sp3, and Sp4 pro-oncogenic gene products was due to induction of reactive
oxygen species (ROS), ROS-mediated repression of microRNA-27a, and
induction of the Sp repressor gene ZBTB10. These results coupled with several recent reports showed potential clinical applications for betulinic acid
(25) and related compounds alone or in combination with other anticancer
agents such as ginsenoside Rh2 [40–42].
23-Hydroxybetulinic acid is a pentacyclic triterpene, isolated from Pulsatilla chinensis, largely used in Chinese medicine. This compound mediated
induced apoptosis in HL-60 cells by decreases in bcl-2 expression and telomerase activity [43].


14

Studies in Natural Products Chemistry

Zheng et al. [44] investigated the ability of 23-hydroxybetulinic acid alone
or in association with doxorubin against a series of cancer cell lines including
23-HBA in NCI-H460, SGC7901, HepG2, and sarcoma 180 cells. Both compounds inhibited the growth of all cell lines in a dose-dependent manner. In
particular, 23-hydroxybetulinic acid (26) exert its cytotoxic activity with
IC50 values of 49.2, 49.1, 306.4, and 28.0 mM, respectively while doxorubicin
showed IC50 values of 0.33, 0.66, 1.53, and 0.04 mM, respectively. Moreover,
the combination of a no toxic dose of 23-hydroxybetulinic acid (26) significantly enhanced the inhibitory effects of doxorubicin on tumor cell growth
in a concentration-dependent manner. It is possible that both compounds
could act in synergy and these results were confirmed in vivo. Interestingly,
the triterpene enhanced the intra-tumor accumulation of doxorubicin without
affecting the plasma pharmacokinetics. Nevertheless, the naturally occurring

23-hydroxybetulinic acid selectively killed cancer cells and was nontoxic to
normal tissue in animal models (Table 3).
A series of 23-hydroxybetulinic acid derivatives as possible antitumor agents
were synthesized and tested against HeLa, MCF-7, HepG2, B16, and A375 cancer cells using doxorubicin as a reference [45]. Compounds 3,23-O-diacetyl-17-1,40 -bipiperidinyl betulinic amide (27), 3,23-O-diacetyl-17-[4-(5(pyridin-2-yl)-2H-tetrazol-2-yl)-n-butyl] betulinic amide (28), 3,23-O-diacetyl-17-(2-aminoethyl)betulinic amide (29), 3,23-dihydroxy-17-1,40 -bipiperidinyl
betulinic amide (30), 3,23-dihydroxy-17-(2-aminoethyl)betulinic amide
(31), 3,23-dihydroxy-17-piperizinyl betulinic amide (32), N-[N-(3,23dihydroxy-17-betulinic acyl)-L-glycine acyl]-L-alanine methyl ester (33), and N[N-(3,23-dihydroxy-17-betulinic acyl)-L-glycine acyl]-L-proline methyl ester
(34) exhibited IC50 values lower than 10 mM on all tested cell lines. In particular,
the highest IC50 values of 6.86, 3.93, 2.93, and 2.27 mM was found for 27, 29, 31,
and 32 against MCF-7, respectively; 1.91, 5.01, and 3.34 mM for 28, 33, and 34
against A375, respectively; 5.71 mM for 30 against B16. SAR analysis revealed
that the acetyl groups at both C-3 and C-23 positions would be favorable; a bulky,
electron-donating, and hydrophilic moiety at C-23 site may benefit the potency
and modification at the C-17 site with suitable carboxylate or amide can produce
potent derivatives.
Starting from betulin (24) and betulinic acid (25), carbamate and
N-acylheterocyclic derivatives were obtained [46]. Most of the compounds
have shown a better cytotoxic profile than their parent compound against
a panel of cancer cell lines (HepG2, Jurkat, HeLa, HT-29, PC-3, BJ). In
particular, the N-acyltriazole derivatives 28-(1H-triazol-1-yl)-28-oxo-lup-20
(29)-en-3b-yl-1H-triazole-1-carboxylate and 2-hydroxy-28-(1H-triazol-1-yl)lup-1,20(29)-dien-3,28-dione were the most promising derivatives, being up
to 19- and 12-fold more potent than betulinic acid against human PC-3 cell
lines (IC50 values of 1.1 and 1.8 mM, respectively). In general, results
suggested that more than the size limitation of the moiety used to create derivatives, for the cytotoxic effect there is great significance in the electronic


Chapter

1

15

Emerging Role of Triterpenoids in Cancer Therapy

TABLE 3 In Vitro Cytotoxicity of Lupane-Type Triterpenes
Compound

Cell Line

IC50

Ref.

Lupeol (23)

Mel 928, Mel 1241, Mel
1011

75, 72,
135 mM

[35]

Lupeol (23)

LAPC4, LNCaP, 22Rn1
(ADPC cells), C4-2b
(CRPC cells)

15.9, 17.3,
19.1,

25 mmol/l

[36]

Betulinic acid (25)

RMS-13, RH-30, RD

3.9–9.5 mg/ml

[38]

Betulinic acid (25)

RKO, SW-480

! 5 mM

[39]

23-Hydroxybetulinic
acid (26)

23-HBA, NCI-H460,
SGC7901, HepG2,
Sarcoma 180

49.2, 49.1,
306.4,
28.0 mM

[44]

3,23-O-Diacetyl-17-1,40 bipiperidinyl betulinic
amide (27)

HeLa, MCF-7, HepG2, B16,
A375

6.9–10.8 mM

[45]

3,23-ODiacetyl-17-[4-(5-(pyridin-2yl)-2H-tetrazol-2-yl)-n-butyl]
betulinic amide (28)

HeLa, MCF-7, HepG2, B16,
A375

1.8–18.9 mM

[45]

3,23-ODiacetyl-17-(2-aminoethyl)
betulinic amide (29)

HeLa, MCF-7, HepG2, B16,
A375

3.9–9.99 mM

[45]

3,23-Dihydroxy-17-1,40 bipiperidinyl betulinic
amide (30)

HeLa, MCF-7, HepG2, B16,
A375

5.7–12.7 mM

[45]

3,23-Dihydroxy-17-(2aminoethyl)betulinic
amide (31)

HeLa, MCF-7, HepG2, B16,
A375

2.9–9.6 mM

[45]

3,23Dihydroxy-17-piperizinyl
betulinic amide (32)

HeLa, MCF-7, HepG2, B16,
A375

2.3–8.8 mM

[45]

N-[N-(3,23dihydroxy-17-betulinic
acyl)-L-glycine acyl]-Lalanine methyl ester (33)

HeLa, MCF-7, HepG2, B16,
A375

5.0–12.1 mM

[45]

N-[N-(3,23dihydroxy-17-betulinic
acyl)-L-glycine acyl]-Lproline methyl ester (34)

HeLa, MCF-7, HepG2, B16,
A375

3.3–9.3 mM

[45]

28-(1H-triazol-1-yl)-28-oxolup-20(29)-en-3b-yl-1Htriazole-1-carboxylate

PC-3

1.1 mM

[46]

Continued


16

Studies in Natural Products Chemistry

TABLE 3 In Vitro Cytotoxicity of Lupane-Type Triterpenes—Cont’d
Compound

Cell Line

IC50

Ref.

2-Hydroxy-28-(1Htriazol-1-yl)-lup-1,20(29)dien-3,28-dione

PC-3

1.8 mM

[46]

8-Hydroxy-lup-20(29)en-3b-yl-1Htriazole-1-carboxylate

HepG2, HeLa, HT-29, PC-3

2.1–7.3 mM

[46]

3-O-Chloroacetylbetulinic
acid

518A2, A549, FADU,
HT-29, MCF-7

3.0–8.4 mM

[47]

28-O-Chloroacetylbetulin

518A2, A549, FADU,
HT-29, MCF-7

8.1–27.6 mM

[47]

a-28-OAcetylbetulin-3-yl-Dglucopyranoside

8505C, SW-1736, A253,
FADU, A431, A2780,
DLD-1, HCT-8, HCT-116,
HT-29 SW-480, MCF-7,
518A2, A549, Lipo

4.4–10.2 mM

[48]

b-28-OAcetylbetulin-3-yl-Dglucopyranoside

8505C, SW-1736, A253,
FADU, A431, A2780,
DLD-1, HCT-8, HCT-116,
HT-29 SW-480, MCF-7,
518A2, A549, Lipo

5.3–10.1 mM

[48]

(R)-4-[3b-tertButyloxy-28-norlup-20(29)en-17b-yl]-2-methylene-gbutyrolactone

518A2, A431, A2780,
DLD-1, 8505C, SW-1736

14.4–29.5 mM

[49]

(R)-4-[3bMethoxy-28-norlup-20(29)en-17b-yl]-2-methylene-gbutyrolactone

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,

SW-480, 8505C, SW-1736,
MCF-7, Lipo

6.4–13.4 mM

[49]

(R)-4-[3bAcetoxy-28-norlup-20(29)en-17b-yl]-2-methylene-gbutyrolactone

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

3.6–9.5 mM

[49]

(R)-4-[3bAcetoxy-28-norlup-20(29)en-17b-yl]-g-butyrolactone

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

2.6–10.6 mM

[49]

Chapter

1

17

Emerging Role of Triterpenoids in Cancer Therapy

TABLE 3 In Vitro Cytotoxicity of Lupane-Type Triterpenes—Cont’d
Compound

Cell Line

IC50

Ref.

Methyl (28S)-3-[3bmethoxy-28-hydroxy-lup-20
(29)-en-28-yl]-propiolate

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

2.3–6.0 mM

[49]

(R)-4-[3bMethoxy-28-norlup-20(29)en-17b-yl]-2-butenolide (41)

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

3.5–12.7 mM

[49]

3b-Hydroxy-17b-[(2R)-2oxiranyl]-28-norlup-20
(29)-en

518A2, 8505C, A253,
A2780, A431, HCT-116,
HCT-8, HT-29, Lipo, MCF-7,
SW-480

15.4–28.2 mM

[50]

(2S,3S)-2-[3bAcetoxy-28-norlup-20(29)en-17b-yl]-1,5-dioxa-spiro
[2.4]heptan-4-one

518A2, 8505C, A253,

A2780, A431, A549, DLD-1,
FADU, HCT-116, HCT-8,
HT-29, Lipo, MCF-7,
SW-1736, SW-480

2.3–9.6 mM

[50]

Ethyl-(5R)-5-[3bacetoxy-28-norlup-20(29)en-17byl]-5-hydroxy-3-oxo-valerate

518A2, 8505C, A253,
A2780, A431, A549, DLD-1,
FADU, HCT-116, HCT-8,
HT-29, Lipo, MCF-7,
SW-1736, SW-480

9.0–18.5 mM

[50]

Ethyl-(5R)-5-[3bmethoxy-28-norlup-20(29)en-17byl]-5-hydroxy-3-oxo-valerate

518A2, 8505C, A253,
A2780, A431, A549, DLD-1,
FADU, HCT-116, HCT-8,
HT-29, Lipo, MCF-7,
SW-1736, SW-480

12.1–20.7 mM

[50]

2-((RS)-[3bacetoxy-28-norlup-20(29)en-17b-yl]-3-oxomethyl)-gbutyrolactone

518A2, 8505C, A253,
A2780, A431, A549, DLD-1,
FADU, HCT-116, HCT-8,
HT-29, Lipo, MCF-7,
SW-1736, SW-480

5.5–14.8 mM

[50]

Ethyl-3-[3bacetoxy-28-norlup-20(29)en-17b-yl]-3-oxopropionate

518A2, 8505C, A253,
A2780, A431, A549, DLD-1,
FADU, HCT-116, HCT-8,
HT-29, Lipo, MCF-7,
SW-1736, SW-480

2.2–12.0 mM

[50]

(28R)-3-Acetyl-28-(2ethoxy-2-oxoethyl)
allobetulin

A253, A2780, A431, A549,
FADU, HCT-116, HCT-8,
HT-29, MCF-7, SW-1736,
SW-480

9.3–27.1 mM

[50]

Continued


18

Studies in Natural Products Chemistry

TABLE 3 In Vitro Cytotoxicity of Lupane-Type Triterpenes—Cont’d
Compound

Cell Line

IC50

Ref.

(28R)-3-Acetyl-28-(4ethoxy-2,4-dioxobut-1-yl)
allobetulin

518A2, 8505C, A253,
A2780, A431, A549, DLD-1,

FADU, HCT-116, HCT-8,
HT-29, Lipo, MCF-7,
SW-1736, SW-480

4.7–13.2 mM

[50]

(28S)-3-[3b-Acetoxy-28hydroxy-lup-20(29)en-28-yl]-propiolate

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

2.3–6.0 mM

[51]

(28S) 3-O-Acetyl-28-(3hydroxyprop-1-ynyl)-lup-20
(29)-en-3,28-diol

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

6.9–11.9 mM

[51]

28-[3-(Ethylcarboxy)-4(methylcarboxy)pyrazol-5-yl]-3,28dioxo-28-ethinyllup-20(29)ene

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

7.6–21.5 mM

[51]

28-[3-(Ethylcarboxy)-5(methylcarboxy)pyrazol-4-yl]-3,28dioxo-28-ethinyllup-20
(29)-ene

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

11.3–16.4 mM

[51]

28-[Nmethyl-3-(methylcarboxy)pyrazol-4-yl]-3,28dioxo-28-ethinyllup-20(29)ene

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

7.0–24.5 mM

[51]

28-(4-(Methylcarboxy)-1H1,2,3-triazol-5-yl)-3,28dioxo-28-ethinyllup-20
(29)-ene

518A2, A431, A253, FADU,
A549, A2780, DLD-1,
HCT-8, HCT-116, HT-29,
SW-480, 8505C, SW-1736,
MCF-7, Lipo

11.8–18.2 mM

[51]

density at C-3 position. Moreover, no cytotoxic activity was found with
acetyl esters at the C-28 and C-3 positions of 20-methylimidazole and triazole
carbamate derivatives, even at concentrations up to 30 mM. The C-3 lupane
carbamate derivative 8-hydroxy-lup-20(29)-en-3b-yl-1H-triazole-1-carboxylate


Chapter

1

Emerging Role of Triterpenoids in Cancer Therapy

19

exerted the highest cytotoxic activity against HepG2, HeLa, HT-29, and PC-3
with IC50 7.3, 6.9, 4.9, and 2.1 mM. SAR analysis demonstrated that the free
C-28 carboxylic acid function is important for the cytotoxicity. In order to clarify the mechanism of cytotoxic activity, authors analyzed the topoisomerase
inhibitory property of 28-(1H-triazol-1-yl)-28-oxo-lup-20(29)-en-3b-yl-1Htriazole-1-carboxylate
and
2-hydroxy-28-(1H-triazol-1-yl)-lup-1,20(29)dien-3,28-dione that displayed the best cytotoxicity profile, however, they were
not the most potent inhibitors of topoisomerase I. This lack of correlation
between the topoisomerase I inhibitory effect and the cytotoxicity of the different tested compounds suggested that there could be multiple mechanisms
responsible for the cytostatic activity of these compounds, one of them being
the inhibition of the herein assayed enzyme.
Successively, the same research group investigated the way in which
28-(1H-imidazol-1-yl)-lup-20(29)-en-3,28-dione,
28-(1H-imidazol-1-yl)lup-1,20(29)-dien-3,28-dione,
and
28-hydroxy-lup-20(29)-en-3b-yl-1Himidazole-1-carboxylate exerted their cytotoxic activity against HepG2,
HeLa, and Jurkat cells. All the compounds were found to be potent inducers
of apoptosis, suggesting that the introduction of the imidazolyl moiety is crucial for enhancing the induction of apoptosis and the cell-cycle arrest. The
mechanism of apoptosis induction has been studied in HepG2 cells and found
to be mediated by activation of the post-mitochondrial caspases-9 and -3 cascade and possibly by mitochondrial amplification loop involving caspase-8.
These facts were corroborated by detection of mitochondrial cytochrome c
release and DNA fragmentation [52].
Kommera et al. [47] investigated the antiproliferative activity of 13 derivatives of betulinic acid and betulin against a panel of cancer cell lines including
518A2, A549, FADU, HT-29, and MCF-7. Among them the most active compounds, 3-O-chloroacetylbetulinic acid and 28-O-chloroacetylbetulin exerted

a dose-dependent antiproliferative activity especially against HT-29 and
518A2 cells with IC50 values of 3.6 and 9.91 mM, respectively. Interestingly,
3-O-chloroacetylbetulinic acid was four times more cytotoxic than 25 on
HT-29 colon cancer cells. Successively, authors demonstrated that 3-Ochloroacetylbetulinic acid exert its cytotoxicity in colon cancer cell toward
mechanism that involves the induction of apoptosis, as observed by the
appearance of DNA fragmentation. Derivative 28-O-chloroacetylbetulin
showed better activity than betulinic acid (25) on the breast (MCF-7) cancer
cell line (IC50 value of 8.14 mM). Moreover, both derivatives were found to
be selective to tumor cells (Table 4).
The a- and b-anomers of derivative of betulin 28-O-acetylbetulin-3-yl-Dglucopyranoside exerted a dose-dependent antiproliferative action toward a
panel of tumor cell lines [48]. In particular, treatment of HCT-116 cells
for 24 h induced apoptosis, which was confirmed by the appearance of a typical ladder pattern in the DNA fragmentation assay and cell-cycle analysis.
The a- and b-anomers of 28-O-acetylbetulin-3-yl-D-glucopyranoside seem to