A Bioreductive Prodrug of Cucurbitacin B Significantly Inhibits Tumor Growth in the 4T1 Xenograft Mice Model

Article abstract of DOI:10.1021/acsmedchemlett.9b00161

Cucurbitacin B (CuB), a highly cytotoxic constituent of the Cucurbitaceae plant, was identified to exhibit potent inhibitory activity against human cancer cells as well as normal cells. This disadvantage hampers the possibility of developing this compound into an anticancer drug candidate. In this work, several bioreductive prodrugs of CuB were designed to reduce toxicity to normal cells while maintaining the cytotoxic effect to cancer cells. Embedded with a bioreductive delivery and cleavable system in cancer tissues, cucurbitacin B-based prodrugs 1, 2, and 3 were synthesized and evaluated by in vitro and in vivo experiments. Compared with the parent CuB, prodrug 1 was found to significantly reduce the toxicity down to 310-fold lower against noncancerous cells. LC-MS analyses show that prodrug 1 efficiently releases the parent compound in the reductase-overexpressed MCF-7 cells. In addition, prodrug 1 shows satisfactory and comparable effectiveness in controlling tumor growth as that by tamoxifen in the 4T1 xenograft mice model.

Full text of DOI:10.1021/acsmedchemlett.9b00161

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Letter
A Bioreductive Prodrug of Cucurbitacin B Significantly
Inhibits Tumor Growth in 4T1 Xenograft Mice Model
Parichat Suebsakwong, Jie Wang, Phorntip Khetkam, Natthida Weerapreeyakul,
Jing Wu, Yun Du, Zhu-Jun Yao, Jianxin Li, and Apichart Suksamrarn
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00161 • Publication Date (Web): 10 Sep 2019
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A Bioreductive Prodrug of Cucurbitacin B Significantly Inhibits
Tumor Growth in 4T1 Xenograft Mice Model
Parichat Suebsakwong,† Jie Wang,‡ Phorntip Khetkam,† Natthida Weerapreeyakul,§ Jing Wu,‡ Yun Du,‡
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Zhu-Jun Yao,‡ Jian-Xin Li,*,‡ and Apichart Suksamrarn*,†
†Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng
University, Bangkok 10240, Thailand
‡State Key Laboratory of Coordination Chemistry, Jiangsu Key Laboratory of Advanced Organic Materials, School of
Chemistry and Chemical Engineering, Nanjing University, 163 Xianlin Avenue, Nanjing, Jiangsu 210023, China
§Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
KEYWORDS: Cucurbitacin B, Bioreductive prodrug, Drug delivery, Cytotoxicity
ABSTRACT: Cucurbitacin B (CuB), a highly cytotoxic constituent of the Cucurbitaceae plant, was identified to exhibit potent
inhibitory activity against human cancer cells as well as normal cells. This disadvantage hampers the possibility of developing this
compound into an anticancer drug candidate. In this work, several bioreductive prodrugs of CuB were designed to reduce toxicity to
normal cells while maintaining cytotoxic effect to cancer cells. Embedded with a bioreductive delivery and cleavable system in
cancer tissues, cucurbitacin B-based prodrugs 1, 2 and 3 were synthesized and evaluated by in vitro and in vivo experiments.
Compared with the parent CuB, prodrug 1 was found to significantly reduce the toxicity down to 310-fold lower against non-
cancerous cells. LC-MS analyses show that prodrug 1 efficiently releases the parent compound in the reductase-overexpressed
MCF-7 cells. In addition, prodrug 1 shows satisfactory and comparable effectiveness in controlling tumor growth as that by
tamoxifen in the 4T1 xenograft mice model.
Development of natural products to new analogues with
improved properties are highly desired in the drug discovery
process.1 Targeted prodrug design for anticancer drug is one of
the interesting strategies in drug discovery.2–5 Many anticancer
natural products are not target specific, and might also quite
toxic to normal cells. To lower the toxicity while keeping the
therapeutic property of active natural products, proper
structure modification serves as an important and useful tool
in drug discovery. Prodrug design has been proven one of the
workable strategies to improve the physicochemical properties
of a molecule and overcome unacceptable biopharmaceutical
performance. Embedment of an additional molecular device,
activities.18–20 Our previous phytochemical study on T.
cucumerina revealed that cucurbitacin B (CuB, Figure 1), the
major constituent of the plant, shows potent inhibitory
activities on human breast cancer cells (MCF-7, MDA-MB-
231 and SKBR-3)16,21 and colon cancer cells (Caco-2 and
SW620).22 Unfortunately, CuB is also highly toxic against
normal cells. This disadvantage hampers the possibility of
developing this compound into an anticancer drug. Lowering
cytotoxic effect of CuB against normal cells while maintaining
the cytotoxic potency to cancer cells keeps as a challenging
task yet.
O
which is cleavable by
a specific enzyme expressed
24
21HO
26
predominantly in tumor cells, is a commonly applied manner
in contemporary drug design and development.6 Bioreductive
prodrug can be designed to target specific tumor followed by
in situ release of the therapeutic drugs with the reductase
(NAD(P)H:quinone oxidoreductase 1, NQO1) over-expressed
in some tumor cells.7–9 Fortunately, the reductases are often
reported to be overexpressed in a variety of cancer cells, such
as breast cancer, ovarian cancer, thyroid cancer, adrenal
cancer and colon cancer.3,10–13
Trichosanthes cucumerina L. (Cucurbitaceae), distributed in
many countries of Asia and well known for its bitter taste, is a
Thai medicinal herb. Cucurbitacins, the main components of
T. cucumerina,14–17 exhibit a variety of pharmacological
effects in vitro and in vivo, such as anticancer,
hepatoprotective, cardiovascular, purgative, antiinflammatory,
antimicrobial, anthelmintic, CNS effects and antifertility
22
H
18
OAc
11
O
H
OH
27
17
16
H
14
HO
O
2
8
10
19
6
30
4
29
28
Figure 1. Structure of cucurbitacin B.
In this work, we explored the conversion of CuB into a
prodrug design to improve the toxicity/safety index between
breast cancer cells and non-cancerous cells. Such a prodrug
system is expected to be non-toxic or less toxic against normal
cells until it reaches the tumor tissue and releases CuB. We
rationally designed three prodrugs that were composed of a
variety of linkers and the general quinone delivery system that
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Figure 2. Rational design of CuB-based bioreductive prodrugs 1–3.
has been successfully used in a number of prodrug research-
es.3,12,13,23,24 Presence of the over-expressed reductase in breast
tumor cells is supposed to trigger the release of CuB and cause
the death of cancer cells. Three prodrugs 1–3 have been
synthesized and their crucial properties and biological
activities were evaluated and compared.
acetylated than the 16-hydroxy group, whereas the 20-hydroxy
group was relatively inert under normal experimental
condition. The result indicated that attachment of the
bioreductive unit at the 16-hydroxy group would need two
extra synthetic steps: protection and de-protection of the more
active 2-hydroxy group. We therefore decided to attach the
bioreductive unit at the 2-hydroxy position. Following such a
bioreductive prodrug concept, CuB was converted into
prodrug molecules 1, 2 and 3, in which the quinone system
was introduced through the carbamate and ester linkages
(Figure 2).
Cucurbitacin B (CuB, Figure 1) is the major triterpenoid
isolated from the fruit fibers of T. cucumerina. The
1
spectroscopic (IR, H NMR, 13C NMR and mass spectra) data
of CuB are consistent with those reported.25–27 CuB showed
significant cytotoxic activities against various cancer cells,
such as breast cancer,16,21 colon cancer,22 human nasopharynx
carcinoma cells,28 non-small cell lung cancer, human
hepatocellular cells,29 HeLa cells, and HepG2 cells.30 CuB
upregulated DNA methyltransferase 1 and heavy methylation
in the promoters of c-Myc, cyclin D1, and survivin, which
consequently downregulated the expression of all these
oncogenes, were observed.31 However, its non-selective
cytotoxic actions against both cancerous and normal cells
greatly limited its further development to a potential
anticancer drug. Conversion of this cytotoxic natural product
to a safe prodrug is of extreme interest for potential treatment
of cancers.
Scheme 1. Synthetic Procedures for Compounds 5−8a
O
HO
HO
O
OH
b
a
OH
O
O
O
4
5
6
(90%)
(80%)
O
c
HN
R
NH
R
HN
N
O
R
R
7a
7b
, R = CH3
, R = H
8a
8b
, R = CH3 (69%)
, R = H (64%)
For bioreductive prodrug design, quinone delivery systems
is capable to deliver drugs to target specific tumor and can be
activated by overexpressed reductases in cancer cells.3,23,32 The
steric hindrance imparted by the three methyl groups on the
quinone moiety (the “trimethyl lock”) has been shown to
induce intramolecular cyclization to release the active drugs
from the prodrugs in the target sites (Figure 2).24,33 At the
beginning of the design of the prodrug(s), we found that upon
acetylation of CuB, the 2-hydroxy group was more readily
aReagents and conditions: (a) 3,3-dimethylacrylic acid, CH3SO3H,
70 °C; (b) NBS, acetone, water; (c) Boc2O, DCM.
The synthesis of prodrugs 1, 2 and 3 is depicted in Schemes
1–3. First of all, reaction between trimethylhydroquinone (4)
and 3,3-dimethylacrylic acid in the presence of dry
methanesulfonic acid provided 5, which was further treated
with N-bromosuccinimide (NBS) in aqueous acetone to afford
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6. The 1,3-diaminopropanes 7a and 7b were treated with di-
tert-butyldicarbonate (Boc2O) in DCM, yielding the mono-
Boc-protected linkers 8a and 8b (Scheme 1).
1
2
3
4
Scheme 2. Synthesis of Prodrugs 1 and 2a
5
6
7
8
O
H
O
O
H
HO
HO
HO
OAc
H
OAc
O2N
OAc
O
H
OH
O
H
OH
O
O
H
OH
O
O
H
H
H
a
b
N
R
N
R
O
O
HO
O
O
O
O
O
9
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10a
10b
, R = CH3 (96%)
, R = H (74%)
CuB
9
(78%)
O
O
H
HO
HO
H
OAc
OAc
O
H
OH
O
H
OH
O
O
O
H
H
O
c
d
N
R
N
R
O
O
HN
R
N
R
O
O
O
11a
11b
, R = CH3 (93%)
, R = H (96%)
1
2
, R = CH3 (72%)
, R = H (74%)
aReagents and conditions: (a) 4-nitrophenyl chloroformate, Et3N, DCM, 0 °C; (b) compounds 8a and 8b, Et3N, DCM; (c) 10% TFA, DCM;
(d) compound 6, EDCI, DMAP, DCM.
Scheme 3. Synthesis of Prodrug 3a
functionality of prodrug 3 to bind CuB with the quinone
moiety. The experiment confirmed that prodrugs 1 and 2 were
more stable than prodrug 3 (see Figure S2). In addition, it was
also observed that the 1,3-di-N-methylaminopropyl linkage of
showed better stability than the unsubstituted 1,3-
diaminopropyl linker employed in compound 2.
O
O
H
HO
HO
H
OAc
OAc
O
H
OH
a
O
O
H
OH
1
H
O
H
HO
O
O
O
CuB and the prodrugs 1, 2 and 3 were evaluated for their
cytotoxicity against breast cancer (MCF-7) and non-cancerous
Vero (African green monkey kidney) cells using the MTT
assay. Tamoxifen (TAM), a well established breast cancer
drug,34 was used as a positive control. As shown in Table 1,
CuB exhibited potent activity toward MCF-7 cells (IC50 12.0
µM) and it was highly toxic toward the noncancerous Vero cells
(IC50 0.04 µM). The prodrugs 1, 2 and 3 exhibited better
cytotoxicity over TAM (IC50 22.6 µM) against MCF-7 cells with
IC50 values of 18.1, 15.4 and 16.6 μM, respectively. Compared
with CuB, the prodrugs 1, 2 and 3 showed lower cytotoxic
activity towards the Vero cells, with an IC50 values of 12.4,
1.87 and 0.07 M, respectively. They are approximately
310-, 47- and 2-fold less toxic than CuB, respectively. Based
on the above, significant decrease of the cytotoxic action
against the normal Vero cells while keeping its cytotoxic
effect against the cancerous MCF-7 cells mentions that the
bioreductive prodrug design of CuB works by aid of the
reductase overexpressed in the cancer cells. To further verify
the role of NQO1 in the drug release process in the cells, we
also performed MTT assays for MCF-7 cells pretreated with
dicoumarol (DIC), an inhibitor of NQO1.35 Upon pretreatment
with DIC, the cell inhibition decreased significantly (Table 1),
suggesting that the prodrugs exerted its cytotoxicity depending
on NQO1 bioreductive activation. We also evaluated whether
the prodrugs could be efficiently reduced by NQO1 and could
specifically release CuB3 (see Figure S3).
O
CuB
3
(70%)
aReagents and condition: (a) 6, EDCI, cat. DMAP, DCM.
Connection of the quinone bioreductive unit 6 to CuB is
summarized in Scheme 2. CuB was treated with 4-nitrophenyl
chloroformate in DCM in the presence of Et3N to furnish
compound 9. The attachment of the leaving group at the 2-
position was evident from large down-field shifts of the NMR
chemical shifts from CuB (H 4.39 and C 71.6) to 9 (H 5.39
and C 77.7) and was further confirmed by HMBC
experiments (see Supporting Information). Parallel
condensation of 9 with the linkers 8a and 8b, respectively,
provided 10a and 10b. Treatment of 10a and 10b with 10%
TFA gave amine intermediates 11a and 11b, in parallel.
Finally, quinone unit 6 was coupled to amines 11a and 11b
using EDCI and a catalytic amount of DMAP to yield the
prodrugs 1 and 2, respectively (Scheme 2). Following similar
procedure, quinone 6 was directly conjugated to CuB to afford
prodrug 3 (Scheme 3). The purity of the prodrugs 1–3 was
checked by HPLC (see Figure S1).
Stability of the prodrug is an important parameter for further
bioassays in vitro and in vivo. The stability of prodrugs 1, 2
and 3 was examined in the cell culture medium, DMEM with
10% FBS for a period of 72 h by using HPLC. The results
show that all prodrugs were reasonably stable under the
experiment conditions, and more than 90% of the tested
molecules remained after 24 h. Chemically, the carbamate
functionality applied in the prodrug linkage of 1 and 2 is
expected to be more stable than the corresponding ester
To get an insight into how the prodrug releases drug (CuB)
or other active species with the reductases in cancer cells,
cellular uptake study was performed using MCF-7 cells, a
typical reductase-overexpressing cancer cells.12 First, MCF-7
cells were treated with 25 µM prodrugs, and the cells were
lysed after 24 h incubation. The concentrations of prodrug in
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the culture medium and cell lysate were determined by HPLC
concentration of 2 in the culture medium was not detected.
This might be caused by better cell permeability of prodrug 2
compared with those of prodrugs 1 and 3.
with a calibration curve.2 As shown in Figure 3A, the concen
trations of 1 and 3 in culture medium decreased to 8.39 and
6.21 μM after 24 h incubation, respectively. However, the
1
2
3
4
5
Table 1. In Vitro Cytotoxicity Activity of CuB and Prodrugs
6
7
8
9
IC50a (µM)
Verob
Compound
CIc
MCF-7
MCF-7+DIC
CuB
12.0 ± 0.4
18.1 ± 0.4
15.4 ± 0.2
16.6 ± 0.9
22.6 ± 0.3
–
0.04 ± 0.02
12.4 ± 0.5
1.87 ± 0.24
0.07 ± 0.02
–
9.72 ± 1.08
–
310
46.8
1.75
–
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1
>100
>100
>100
–
2
3
TAM
Ellipticine
4.06 ± 0.58
–
–
aEach value was reproduced in three experiments. bAfrican green monkey kidney. cCytotoxicity index, IC50 of prodrug in
Vero compared with IC50 of CuB parent in Vero.
Figure 3. Cellular uptake experiments of prodrugs 1, 2 and 3. (A) Concentration of prodrugs 1 and 3 in cell culture media after 24 h
incubation. Not detected by HPLC. ** P < 0.01 (n = 3). (B), (C), (D) HRMS analyses for prodrugs 1, 2 and 3 in cell lysate after 24 h
a
incubation, respectively.
Next, we examined whether the prodrugs could release ther-
apeutic drug CuB in the cells. As shown in Figure 3, prodrugs
1, 2 and 3 efficiently released CuB in the cell lysate, while the
intermediates 11a and 11b were also detected in the cell lysate
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of 1 and 2, respectively. Very interestingly, 1, 2 and 3 in the
indicated the presence of intermediate 11b in the cell lysate of
2 (Figure 3C). It is noteworthy that the byproducts cyclic ureas
X and Y (see Figure 2) were undetectable in the cell lysate
under the HPLC conditions, because they are not UV-
detectable. However, both compounds showed low-intensity
ion peaks [M + H]+ ion at m/z 129.1025 and [M + H]+ ion at
m/z 101.0695 in the HRMS analysis (see Figures 3B and 3C,
respectively). It should also be noted that the ion peaks of the
prodrugs 1, 2 and 3 at the extended m/z range to 1000 were not
detected (see Figure S4). The cytotoxicity of the lactone 5 and
cyclic ureas X and Y against MCF-7 and Vero cells using the
MTT assay was evaluated and they were found to be nontoxic
1
2
3
4
5
6
7
8
cell lysate were not detected by HPLC. This highly mentions
that the transformation of prodrugs to CuB is very efficient in
the cancer cells by aid of the corresponding reductases.
Furthermore, observation of the anticipated compounds or
intermediates released from the prodrugs by high resolution
ESI-TOFMS analysis confirmed such a conclusion. For the
prodrug 1, the corresponding ions including CuB ([M + Na]+
at m/z = 581.3102), 11a ([M + H]+ at m/z = 687.4216) and the
lactone 5 ([M + H]+ at m/z = 235.1334) were detected (Figure
3B). In addition, the ions of CuB and the lactone 5 were also
detected by HRMS for prodrugs 2 and 3 (Figure 3C and 3D,
respectively). Moreover, the [M + H]+ ion at m/z 659.3896
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(see
Table
S1).
Figure 4. In vivo antitumor activity of prodrug 1 on tumor growth against 4T1 xenograft in BALB/c mice. (A) Growth curves of tumor
volume and days were plotted. (B) xenografted mice after being sacrificed. (C) Mean tumor weight, and (D) tumor photographs after mice
a
were sacrificed. All mice died, and the tumor weight of the CuB (3 mg/kg/d) treated group was not determinable. ** P < 0.01, *** P <
0.001 vs Control group; Student’s t test (n = 6).
The possibility of deactivation of prodrugs by thiol-
containing species36 was also investigated. The prodrugs 1−3
was treated with glutathione (GSH), but no reaction was
observed (Supporting Information).
Since the prodrug 1 exhibited significantly decreased
toxicity against normal cells, it was therefore selected for in
vivo antitumor study using the BALB/c mice model with
breast cancer cells (4T1),37,38 and CuB and TAM were used as
comparison controls. The antitumor efficacy of different
dosages of the prodrug 1 (3, 5 and 10 mg/kg/d), CuB (3
mg/kg/d) and TAM (5 mg/kg/d) were tested in the animal
model. As shown in Figure 4A, all concentrations of 1 showed
significant inhibitory effects on tumor growth. The tumor
growth inhibition (TGI) rate of 1 at dose of 5 mg/kg/d was
53.8%, which was comparable to that of TAM (55.0%, 5
mg/kg/d) (Table S2). The excised tumors from the control
animals ranged from 500 to 700 mg, while all concentrations
of the prodrug 1-treated tumors weighed less than 400 mg
(Figure 4B, 4C and 4D). In addition, both dosages of 1 (3 and
5 mg/kg/d) showed increase in body weight (6.46 and 5.27%,
respectively) compared with the control group (−1.48%) and
TAM (4.99%). The prodrug 1 at 10 mg/kg/d group exhibited
significant body weight loss −2.49% (Figure S5 and Table
S2), which could mainly ascribe to the tumor weight being
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Excellence for Innovation in Chemistry, Office of the Higher
inhibited. It is noteworthy that, when a dose of 3.0 mg/kg/d of
CuB was used in vivo, all treated mice died after one day of
CuB administration for its high in vitro cytotoxicity. These
data clearly mention that the prodrug 1 is a successful design
to reduce the in vivo toxicity of CuB, and it might be a
therapeutically promising and less toxic agent for potential
cancer treatment.
1
2
3
4
Education Commission (to AS), National Natural Science
Foundation of China (Nos. 21761142001 and 21532002, to ZJY),
and the Royal Golden Jubilee (RGJ) Ph.D. Program of TRF (No.
PHD 57K0084, to PS).
5
ABBREVIATIONS
6
7
8
9
CuB, cucurbitacin B; TAM, tamoxifen; NBS, N-
bromosuccinimide; Boc2O, di-tert-butyldicarbonate; DCM,
dichloromethane; TFA, trifluoroacetic acid; EDCI, 1-ethyl-3-(3-
In summary, we present a new successful example to
convert a highly toxic natural product into potentially useful
and less toxic anticancer compounds using cellular degradable
prodrug design. Three bioreductive prodrugs (1, 2 and 3) were
synthesized from CuB, the major constituent of T.
cucumerina. Our study showed that these prodrugs
significantly reduced toxicity against non-cancerous cells than
CuB and maintained the original actions against cancer cells.
The experiments also confirmed that the prodrugs could
efficiently release CuB in the reductase-overexpressing MCF-
7 cells. Among them, the prodrug 1 exhibited significant
toxicity reduction in both in vitro and in vivo studies, and
showed a comparable tumor growth inhibition in the 4T1
xenograft mice model at a dose of 5 mg/kg/d by comparison
with tamoxifen (TAM).
dimethylaminopropyl)carbodiimide;
DMAP,
N,N-dimethyl-4-
aminopyridine; DIC, dicoumarol; DMEM, Dulbecco’s Modified
Eagle Medium; FBS, fetal bovine serum; PBS, phosphate-buffered
saline
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental section, HPLC purity analyses of prodrugs 1, 2 and
3; Stability examination of prodrugs 1, 2 and 3 in culture medium;
In vitro cytotoxic activity of lactone 5, cyclic ureas X and Y;
HPLC assay for drug release studies; Drug release studies as a
function of time in the presence of NQO1 and NADPH in PBS;
Stability of prodrugs under the GSH activation; The original
HRMS analysis of prodrugs 1, 2 and 3 in cell lysate after 24 h
incubation; In vivo antitumor activity of prodrug 1 on mice body
weight; In vivo effects of prodrug 1 and CuB. General; Isolation
of cucurbitacin B; Synthesis of compounds 5, 6, 8a, 8b, 9, 10a,
10b, 11a and 11b and spectroscopic data; Synthesis of prodrugs
1, 2 and 3 and spectroscopic data; and NMR spectral copies of all
the compounds shown in Schemes 1–3 (PDF).
AUTHOR INFORMATION
Corresponding Author
* E-mail: s_apichart@ru.ac.th (A. Suksamrarn)
* E-mail: lijxnju@nju.edu.cn (J. Li).
ORCID
Natthida Weerapreeyakul: 0000-0002-0810-8425
Zhu-Jun Yao: 0000-0002-6716-4232
Jian-Xin Li: 0000-0002-9414-3242
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Apichart Suksamrarn: 0000-0001-8919-3555
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by The Thailand Research Fund (TRF,
Nos. DBG 5980003 and DBG 6180030, to AS), Center of
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