Design, synthesis, and screening of novel ursolic acid derivatives as potential anti-cancer agents that target the HIF-1α pathway

Article abstract of DOI:10.1016/j.bmcl.2018.12.060

The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays an important role in tumor angiogenesis, growth, and metastasis and is recognized as an important potential therapeutic target for cancer. Here, we designed and synthesized three novel series of ursolic acid derivatives containing an aminoguanidine moiety and evaluated them as HIF-1α inhibitors and anti-cancer agents using human cancer cell lines. Most of the compounds exhibited significant inhibition of HIF-1α transcriptional activity, as measured using a Hep3B cell-based luciferase reporter assay. Among these compounds, 7b was the most potent inhibitor of HIF-1α expression under hypoxic conditions (IC₅₀ 4.0 μM) and did not display significant cytotoxicity against any cell lines tested. The mechanism of action of 7b was investigated, we found that 7b downregulated HIF-1α protein expression, possibly by suppressing its synthesis, reduced production of vascular endothelial growth factor, and inhibited the proliferation of cancer cells.

Full text of DOI:10.1016/j.bmcl.2018.12.060

Accepted Manuscript
Design, synthesis, and screening of novel ursolic acid derivatives as potential
anti-cancer agents that target the HIF-1α pathway
Jie Wu, Zhi-Hong Zhang, Lin-Hao Zhang, Xue-Jun Jin, Juan Ma, Hu-Ri Piao
PII:
S0960-894X(18)31017-5
https://doi.org/10.1016/j.bmcl.2018.12.060
BMCL 26229
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
19 October 2018
25 December 2018
27 December 2018
Please cite this article as: Wu, J., Zhang, Z-H., Zhang, L-H., Jin, X-J., Ma, J., Piao, H-R., Design, synthesis, and
screening of novel ursolic acid derivatives as potential anti-cancer agents that target the HIF-1α pathway, Bioorganic
&
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.12.060
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Design, synthesis, and screening of novel ursolic acid
derivatives as potential anti-cancer agents that target
the HIF-1α pathway
a,1
a,1
a
a
a,*
Jie Wu , Zhi-Hong Zhang , Lin-Hao Zhang , Xue-Jun Jin , Juan Ma , and
Hu-Ri Piao a,*
a
Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules, Ministry
of Education, Yanbian University College of Pharmacy, Yanji 133002, China
*
Corresponding authors:
H.-R. Piao: Tel.: +86 433 2436149, Fax: + 86 433 2435026, E-mail address: piaohr@ybu.edu.cn X
J. Ma: Tel.: +86 433 2436940, Fax: + 86 433 2435026, E-mail address: majuan@ybu.edu.cn
1
These authors contributed equally to this work.
Abstract
The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays an important
role in tumor angiogenesis, growth, and metastasis and is recognized as an important
potential therapeutic target for cancer. Here, we designed and synthesized three novel
series of ursolic acid derivatives containing an aminoguanidine moiety and evaluated
them as HIF-1α inhibitors and anti-cancer agents using human cancer cell lines. Most
of the compounds exhibited significant inhibition of HIF-1α transcriptional activity, as
measured using a Hep3B cell-based luciferase reporter assay. Among these
compounds, 7b was the most potent inhibitor of HIF-1α expression under hypoxic
conditions (IC50 4.0 µM) and did not display significant cytotoxicity against any cell
lines tested. The mechanism of action of 7b was investigated, we found that 7b
downregulated HIF-1α protein expression, possibly by suppressing its synthesis,
reduced production of vascular endothelial growth factor, and inhibited the
proliferation of cancer cells.
1

Keywords: Ursolic acid; Aminoguanidine; Anticancer activity; HIF-1α inhibitor;
Cytotoxicity
Cancer is the second leading cause of death worldwide after cardiovascular disease.
The World Health Organization estimates that cancer will account for approximately
1
9
.6 million deaths globally in 2018, and its incidence is steadily increasing. The
major treatments for cancer are surgery, radiation, and chemotherapy.2,3 However,
traditional chemotherapeutic agents have a number of critical drawbacks, including
harmful side-effects, non-specific biodistribution, short circulation times, and poor
solubility, which result in poor therapeutic efficiency.4,5 Thus, there is a tremendous
need to develop new compounds for the prevention and treatment of cancer.6
Hypoxia is a common feature of many solid tumors and is generally caused by the
rapid proliferation of tumor cells, which leads to formation of solid masses and
obstruction and compression of the blood vessels surrounding them.7
Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that regulates the
expression of numerous genes involved in nutrient uptake, cell survival, angiogenesis,
invasion, and metastasis, and thus plays an important role in cancer development.8-10
In addition to hypoxia, exposure to certain hormones, cytokines, and growth factors
11
can also upregulate HIF-1α expression. Consequently, HIF-1α has gained attention
as a potential target for the development of anti-cancer agents.12 Inhibition of the
HIF-1α pathway may be a particularly useful therapy for specific types of cancers,
especially those commonly associated with hypoxia.13,14 Due to the importance of
HIF-1α in tumor development and progression, a considerable amount of effort has
been made to identify HIF-1α inhibitors for treatment of cancer.15-18
1
9
Ursolic acid (UA) is a pentacyclic triterpenoid found in most plant species, and it
2
0
21
is known to possess a number of bioactive properties such as anti-inflammatory,
anti-microbial,22 anti-oxidant,23 immunomodulatory,24 and anti-cancer activities.25
Indeed, Japanese researchers have ranked UA as one of the most promising potential
2

2
6
therapeutic compounds for tumor prevention. Over the past decade, many attempts
have been made to develop bioactive UA derivatives that more potently inhibit cancer
2
7
cell growth. These studies have indicated that the configuration at C-3 is a critical
factor for the anti-proliferative activity of UA, whereas a free hydroxyl at C-3
decreases its anti-cancer activity.27 Lin et al. showed that incorporation of an
isopropyl ester at C-28 significantly improves the anti-proliferative activity of UA,28
whereas introduction of a methyl at the same position together with an amino moiety
2
9
at C-3 significantly enhanced its activity against HeLa cells (Fig. 1A). In contrast,
Dar et al. showed that incorporation of various substituted benzene rings at the C-2
position and retention of the carboxyl group at C-28 in UA (Fig. 1B) also improved its
anti-cancer activity, as reflected by induction of cell cycle arrest in G1 and apoptosis
of HCT-116 cells.30
The medicinal properties of guanidine derivatives are also of great interest due to
their diverse anti-microbial,31 anti-inflammatory,32 anti-viral,33 and anti-cancer34,35
activities (Fig. 1C and 1D). Guanidine-containing drugs, such as
m-iodobenzylguanidine and methylglyoxal bis(guanylhydrazone), were shown several
decades ago to have anti-tumor properties and have since been subjected to intense
3
6
preclinical and clinical evaluation. In our previous work, we reported the design and
synthesis of a series of UA derivatives containing heterocyclic moieties at the C-28
position of UA, and one compound (Fig. 1E) was shown to potently inhibit HIF-1α
transcriptional activity under hypoxic conditions.37 Building on this, our ongoing
research seeks to identify HIF-1α inhibitors with the potential to be developed as
anti-cancer agents.
Here, we report on the activities of three series of UA derivatives in which we
changed the hydroxyl moiety of UA to an aminoguanidine group at C-3 and
simultaneously (i) introduced smaller moieties, such as benzene and alkyl groups, at
the C-28 position; (ii) retained the carboxyl group at C-28; or (iii) introduced different
substituted benzene rings or heterocyclic groups at the C-2 position. These novel
series, totaling 28 compounds, were evaluated for their ability to inhibit
hypoxia-induced HIF-1α transcriptional activity. The biological mechanism of action
3

of one selected compound, 7b, was investigated in detail.
Figure 1. Structures of previously reported compounds as anticancer agents. (blue: previous
work on UA derivatives; red: structure of aminoguanidine- active group).
Scheme 1. Synthetic scheme for the synthesis of compounds 3a-c, 5a-i, 7a-p. Reagents and
conditions: (a) Jones reagent, acetone, 0 °C, 5h, 90%; (b) Methyl iodide, bromoethane or
bromopentane, K
0%-90%; (d) Aldehydes, 5 % NaOH, absolute ethanol, r.t 2h, 30%-75%; (e) Concentrated HCl,
absolute ethanol, reflux, 8h, 34%-65%.
2
CO
3
, DMF, r.t., 6h, 80%-82%; (c) Benzyl chlorides, K
2
CO , acetone, reflux, 8h,
3
8
4

The synthetic pathway of the target compounds 3a–c, 5a–i, and 7a–p is presented
in Scheme 1. The intermediate 2 was synthesized by reacting UA with Jones reagent
in acetone at 0°C.38 Compounds 3a, 3b, and 3c were prepared by reacting
intermediate 2 with aminoguanidine bicarbonate, semicarbazide hydrochloride, and
thiosemicarbazide, respectively, in refluxing ethanol. Compounds 4a, 4b, and 4c were
prepared by reacting intermediate 2 with methyl iodide, ethyl bromide, and amyl
bromide, respectively. Intermediate 2 was reacted with different substituents of benzyl
chloride to provide compounds 4d–i. Compounds 6a–p were prepared by
Claisen–Schmidt condensation of intermediate 2 with different aldehydes.30
Compounds 4a–i and 6a–p were reacted with aminoguanidine bicarbonate in
refluxing ethanol to yield the target compounds 5a–i and 7a–p. The structures of the
desired compounds were characterized by H and 13C NMR spectroscopy and
high-resolution mass spectrometry.
1
The in vitro activities of the UA derivatives are summarized in Table 1. To examine
the effects on hypoxia-induced HIF-1α transcriptional activity, Hep3B cells were
transfected with a luciferase reporter construct driven by six hypoxia-response
elements and then exposed to hypoxia (1% O ). After 24 h, luciferase activity in the
2
supernatants was measured. UA was used as a positive control. We found that most of
the tested compounds inhibited HIF-1α transcriptional activity. For the substituted
guanidine derivatives 3a (IC50 > 100 µM), 3b (IC50 88.8µM), and 3c (IC50 13.3µM),
the inhibitory activity was in the order O > S > NH. It indicated that the
semicarbazide derivatives have good HIF-1α inhibitory activities and merit further
investigation as potential anti-cancer agents.
Table 1. In vitro inhibition of HIF-1α transcriptional activity in cell-based HRE
reporter assay under hypoxia conditions and cytotoxic activity.
5

IC50 (μM)
Cytotoxic activity b
HRE a
>100
>100
88.8
13.3
6.7
Compound
R
UA
－
23.8
>100
>100
69.8
3
3
3
5
5
5
5
5
5
5
5
5
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
a
b
c
d
e
f
NH
S
O
Methyl
>100
6.0
Ethyl
>100
>100
30.3
21.1
40.5
13.3
13.2
10.3
37.8
4.0
Amyl
22.0
Phenyl(3-Cl)
Phenyl(4-Cl)
Phenyl(2,4-2Cl)
Phenyl(2,6-2Cl)
Phenyl(4-F)
Phenyl(4-CH3)
Phenyl
34.1
32.1
>100
22.3
g
h
i
12.9
18.0
a
b
c
d
e
f
>100
>100
>100
>100
>100
>100
10.5
Phenyl(4-Cl)
Phenyl(2-Cl)
Phenyl(3-Cl)
Phenyl(4-F)
Phenyl(2-F)
Phenyl(3-F)
Phenyl(2-Br)
Phenyl(3-Br)
Phenyl(2,4-2Cl)
Phenyl(2,6-2Cl)
>100
>100
>100
>100
>100
>100
75.4
90.4
>100
>100
>100
>100
>100
22.8
g
h
i
>100
>100
>100
>100
>100
>100
>100
>100
>100
j
k
l
Phenyl(4-CH )
3
m
n
o
p
Phenyl(4-NO2)
Naphthyl
Furyl
7
a
Thienyl
The inhibitory effects of all the derivatives on HIF-1α transcriptional activity were tested by
HRE luciferase reporter assay after 24 h treatment of HCT116 cells under hypoxic conditions.
Values were shown as mean, n=3.
b
The cytotoxic activity was evaluated by MTT assay after 24 h treatment of compounds under
normoxic conditions. Values were shown as mean, n=3.
For the compounds in series 5, alkyl groups of different carbon chain lengths were
introduced at C-28 of UA. Of these, only compound 5a showed potent anti-cancer
activity (IC50 6.7 µM), indicating that a methyl group at C-28 improves the
anti-cancer activity UA, whereas extending the carbon chain may have the reverse
6

effect. Compounds 5d–i which contained different substituents on the phenyl ring,
exhibited significant HIF-1α inhibition, with IC50 values between 10.3 and 40.5 µM.
For the chlorinated compounds 5d (IC50 30.3µM), 5e (IC50 21.1µM), 5f (IC50
4
4
0.5µM), and 5g (IC50 13.3µM), the inhibitory activity was in the order 2,6-Cl
2
>
-Cl > 3-Cl > 2,4-Cl . For compounds 5e (IC50 21.1µM), 5h (IC50 13.2µM), and 5i
2
(
IC50 10.3µM), electron-donating groups showed beneficial effects compared with
electron-withdrawing groups (4-CH > 4-F > 4-Cl). No clear structure-activity
relationship could be found in series 7. Compounds 7a (H), 7b (4-Cl), 7i (3-Br), and
j (2,4-Cl ) exhibited potent anti-cancer activities when a benzene ring was
3
7
2
introduced at the C-2 position of UA. Among these compounds, 7b was the most
potent inhibitor of HIF-1α transcriptional activity (IC50 4.0 µM). Compound 7p also
potently inhibited HIF-1α, suggesting that the thiophene moiety is more favorable
than furan for inhibition.
The cytotoxicity of these compounds against Hep3B cells was assessed using the
MTT assay. As shown in Table 1, compounds in series 3 and 7 (except 7g), which
retained the carboxyl at C-17, showed no appreciable cytotoxic activity (IC50 >100
μM), whereas compounds in series 5 (except 5a,f) were modestly cytotoxic,
indicating that introduction of a phenyl ring or long carbon chain (C > 2) into the
carboxyl group could increase the cytotoxicity. Interestingly, we previously showed
that esterification of the carboxyl group of UA greatly decreased its cytotoxicity and
enhanced its HIF-1α inhibitory activity. However, introduction of an aminoguanidine
group at C-3 but retention of the free carboxyl group also reduced the cytotoxicity and
increased the inhibitory activity of UA, indicating that a free carboxyl group is not
critical for the compounds’ cytotoxicity or HIF-1α inhibitory activity. Importantly, the
cytotoxicity of UA derivatives were weaker than their HIF-1α transcription inhibitory
activities, suggesting that ursolic acid derivatives suppressed HIF-1α transcriptional
activity without cytotoxicity.
Compound 7b, with the best HIF-1α inhibitory effect (IC50 4.0μM), was selected
for further biological evaluation. As shown in Figure 2, 7b dose-dependently inhibited
the luciferase activity in Hep3B cells (Fig. 2A) and concentrations up to 30 µM did
7

not adversely affect cell viability (Fig. 2B).
Figure 2. Effect of compound 7b on HRE-mediated reporter gene expression.
(
A) Hep3B cells were transiently co-transfected with a pGL3-HRE-Luciferase and pRL-CMV
vectors. Following 24 h incubation, the cells were incubated under hypoxia in the absence or
presence of the indicated concentrations of compound 7b. Luciferase activities were determined
as described in “Materials and Methods”. Data are represented as the mean ± standard deviation
of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, significant with respect to
the control. (B) Hep3B cells were treated with the indicated concentrations of compound 7b. After
24 h incubation, cell viability was determined by MTT assays.
To understand the mechanism underlying the ability of 7b to suppress HIF-1α
transcriptional activity, we first measured HIF-1α protein levels by western blot
analysis. Under normoxic conditions, HIF-1α protein is normally rapidly turned over
and is virtually undetectable in cells. In contrast, hypoxic conditions or exposure to
CoCl leads to stabilization of HIF-1α, and its expression becomes readily detectable.
2
Notably, HCT116, Hep3B, A549, and HeLa cells treated with 7b under hypoxic
conditions for 12 h showed a dose-dependent inhibition of HIF-1α protein levels
compared with untreated cells (Fig. 3A), whereas 7b had virtually no effect on levels
of the control protein topoisomerase-I. To confirm these results, we performed
8

immunofluorescence staining of HIF-1α protein in Hep3B cells. As expected,
exposure of the cells to 7b (10 µM) under hypoxic conditions for 12 h virtually
abolished the expression of HIF-1α protein in the nuclei (Fig. 3B).
Figure 3. Compound 7b inhibits hypoxia-induced the expression of HIF-1α protein.
(
A) Cancer cells lines (HCT116, A549, Hep3B, and HeLa) were incubated under normoxia or
hypoxia for 12 h, in the absence or presence of indicated concentration of 7b, the nuclear extract
for HIF-1α was analyzed by Western bloting. The same blot was reprobed with an anti-Topo-I
antibody as a loading control. (B) Hep3B cells were incubated with 10 μM 7b for 12 h under
hypoxic conditions. After fixation, the cells were stained with anti-HIF-1α (1:100) antibody and
Alexa fluor® 488 goat anti-mouse lgG (H+L) and examined by fluorescence microscopy. DAPI
staining shows the location and size of nuclei. Scale bars: 20 μm. Images were acquired for each
fluorescence channel, using suitable filters with 40× objective. The green and blue images were
merged using J software.
To understand the potential mechanism underlying HIF-1α inhibition by 7b, we
explored the effects of 7b on HIF-1α translational regulation. To examine whether the
down-regulation of HIF-1α protein was caused by proteasomal degradation, we
9

blocked proteolytic activity of the 26S proteasome using the proteasome inhibitor
MG132. Even in the presence of MG132, 7b treatment decreased HIF-1α protein
levels (Fig. 4A), suggesting that HIF-1α protein synthesis in Hep-3B cells is markedly
impaired in the presence of 7b. To address the effect of 7b on HIF-1α protein stability,
the protein translation inhibitor cycloheximide (CHX) was used to prevent de novo
HIF-1α protein synthesis, and the half-life (t1/2) of HIF-1α in the presence of 7b was
then calculated. We found that 7b did not significantly modify the degradation rate of
HIF-1α (Fig. 4B). To determine whether 7b inhibited HIF-1α expression at the
transcriptional level, the mRNA levels of HIF-1α were evaluated using RT-PCR. No
significant effects of 7b treatment on HIF-1α mRNA expression were observed under
either normoxic or hypoxic conditions (Fig. 5A). Taken together, these findings
suggest that 7b does not facilitate or speed up the degradation of HIF-1α.
Figure 4. Compound 7b inhibits the protein synthesis of HIF-1α but not by enhancing its
degradation.
(
A) Proteasome inhibitor MG-132 (10 μM) was added to Hep3B cells for 30 min prior to the
treatment of compound 7b (10 μM) and then the cells were incubated under hypoxia conditions
for 12 h. The nuclear extract for HIF-1α was detected by western blotting. (B) Hep3B cells were
incubated under normoxic or hypoxic conditions for 4 h. Cycloheximide (CHX) (10 μM) and
compound 7b (10 μM) were then mixed with culture media. After 0, 15, 30, or 45 min following
the addition of cycloheximide, HIF-1α protein level was detected by western blotting.
Expression of VEGF, a crucial growth factor involved in tumor cell proliferation,
angiogenesis, invasion, and metastasis, is known to be regulated by HIF-1α.39,40 To
determine whether 7b suppresses VEGF gene expression, we performed RT-PCR
10

analysis in Hep3B cells. Indeed, treatment with 7b resulted in a dose-dependent
decrease in VEGF mRNA (Fig. 5B), and the effective concentrations were
comparable to those inhibiting HIF-1α protein expression.
Figure 5. Effect of compound 7b on the expression of HIF-1α target genes.
Hep3B cells were incubated under normoxic or hypoxic conditions for 12 h in the absence or
presence of indicated concentration of compound 7b. RNA was isolated from cells,
reverse-transcribed, and analyzed by RT-PCR for HIF-1α (A) and VEGF (B). GAPDH was used to
show equal loading of total RNA. Data are respresented as the mean ± standard deviation of three
independent experiments. *p < 0.05, significant with respect to the control.
EdU is a thymidine analog that is incorporated into replicated chromosomal DNA
4
1
during the S phase of the cell cycle. DAPI is a fluorescent dye that binds strongly to
A-T-rich regions in DNA and is commonly used for cell nucleus staining. We used the
EdU incorporation assay, which is a highly sensitive and specific method, to
investigate the inhibitory effects of 7b. As shown in Figure 6, treatment of Hep3B
cells with 7b (10 µM) for 12 h reduced the percentage of EdU-positive cells
compared with the vehicle-treated cells (Fig. 6A), indicating that 7b inhibits Hep3B
11

cell proliferation in vitro. We also performed clonogenic assays to determine the
long-term anti-proliferative activity of 7b. We observed a dose-dependent reduction in
colony formation by 7b-treated compared with control Hep3B cells (Fig. 6B).
Figure 6. Effect of compound 7b on the tumor cell proliferation.
(
A) Hep3B cells were incubated for 12 h in the presence or absence of compound 7b (10 μM). The
EdU-labeled replicating cells were examined under a fluorescence microscope. DNA was stained
using DAPI (blue). (B) Colony formation capability assay with treatment of compound 7b (0, 1, 3
and 10 μM) for 12 h in Hep3B cells. Then the medium was replaced by fresh medium. Cells were
allowed to grow for 10 d. The images were captured with a Nikon camera (Japan).
A series of UA derivatives containing an aminoguanidine moiety were designed,
synthesized, and evaluated for their biological activity as HIF-1α inhibitors. Among
the compounds tested, 7b was the most active inhibitor of HIF-1α (IC50 4.0 μM) and
did not show significant cytotoxic activity against any of the tested cell lines. We
12

investigated the mechanism of action of compound 7b and found that it reduced
HIF-1α protein levels without affecting its transcription or degradation. Moreover, 7b
inhibited hypoxia-induced expression of VEGF at both the mRNA and protein levels.
Compound 7b was confirmed to inhibit the proliferation of cancer cells in vitro. These
results provide initial support for the development of 7b as a potential anti-cancer
agent.
Acknowledgement
The work was supported by the National Science Foundation of China (No. 81260468,
8
1660608, 81760657) and the Natural Science Foundation of Jilin Province (No.
2
0160101218JC). We thank Anne M. O’Rourke, PhD, from Liwen Bianji, Edanz
Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this
manuscript.
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Table 1. In vitro inhibition of HIF-1α transcriptional activity in cell-based HRE reporter
assay under hypoxia conditions and cytotoxic activity.
IC50 (μM)
15

Compound
R
HRE a
>100
>100
88.8
13.3
6.7
Cytotoxic activity b
23.8
UA
－
3
3
3
5
5
5
5
5
5
5
5
5
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
NH
>100
>100
69.8
S
O
a
b
c
Methyl
>100
6.0
Ethyl
>100
>100
30.3
21.1
40.5
13.3
13.2
10.3
37.8
4.0
Amyl
22.0
d
e
f
Phenyl(3-Cl)
Phenyl(4-Cl)
Phenyl(2,4-2Cl)
Phenyl(2,6-2Cl)
Phenyl(4-F)
Phenyl(4-CH3)
Phenyl
34.1
32.1
>100
22.3
g
h
i
12.9
18.0
a
b
c
>100
>100
>100
>100
>100
>100
10.5
Phenyl(4-Cl)
Phenyl(2-Cl)
Phenyl(3-Cl)
Phenyl(4-F)
Phenyl(2-F)
Phenyl(3-F)
Phenyl(2-Br)
Phenyl(3-Br)
Phenyl(2,4-2Cl)
Phenyl(2,6-2Cl)
>100
>100
>100
>100
>100
>100
75.4
90.4
>100
>100
>100
>100
>100
22.8
d
e
f
g
h
i
>100
>100
>100
>100
>100
>100
>100
>100
>100
j
k
l
Phenyl(4-CH
3
)
m
n
o
p
Phenyl(4-NO2)
Naphthyl
Furyl
7
a
Thienyl
The inhibitory effects of all the derivatives on HIF-1α transcriptional activity were tested by HRE
luciferase reporter assay after 24 h treatment of HCT116 cells under hypoxic conditions. Values
were shown as mean, n=3.
b
The cytotoxic activity was evaluated by MTT assay after 24 h treatment of compounds under
normoxic conditions. Values were shown as mean, n=3.
Highlights:


Three series of ursolic acid derivatives were designed and synthesized.
The inhibitory effects on HIF-1α transcriptional activity and cytotoxicity of
compounds were evaluated.

Compound 7b is a promising HIF-1α inhibitory hit as anticancer agent.
16


The investigation of the mechanism on the mode of action of 7b was performed.
Design, synthesis, and screening of novel ursolic acid derivatives as
potential anti-cancer agents that target the HIF-1α pathway
Jie Wu a,1, Zhi-Hong Zhang a,1, Lin-Hao Zhang , Xue-Jun Jin , Juan Ma a,*, and Hu-Ri Piao a,*
a
a
Three novel series of ursolic acid derivatives containing an aminoguanidine moietiy
were designed, synthesized, and evaluated for anti-tumor activity as HIF-1α
inhibitors.
17