Synthesis and evaluation of the HIF-1α inhibitory activities of novel ursolic acid tetrazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2019.04.028

The hypoxia-inducible factor-1α (HIF-1α) pathway has been implicated in tumor angiogenesis, growth, and metastasis. Therefore, the inhibition of this pathway is an important therapeutic target for the treatment of various types of cancers. Here, we designed and synthesized 31 ursolic acid (UA) derivatives containing a tetrazole moiety and evaluated them for their potential anti-tumor activities as HIF-1α transcriptional inhibitors. Of these, compound 14d (IC₅₀ 0.8 ± 0.2 μM) displayed the most potent activity and compounds 14a (IC₅₀ 4.7 ± 0.2 μM) exhibited the most promising biological profile. Analysis of the structure–activity relationships of these compounds with HIF-1α suggested that the presence of a tetrazole group located at C-28 of the UA derivatives was critical for their inhibitory activities.

Full text of DOI:10.1016/j.bmcl.2019.04.028

Accepted Manuscript
Synthesis and evaluation of the HIF-1α inhibitory activities of novel ursolic acid
tetrazole derivatives
Lin-Hao Zhang, Zhi-Hong Zhang, Ming-Yue Li, Zhi-Yu Wei, Xue-Jun Jin, Hu-
Ri Piao
PII:
S0960-894X(19)30247-1
https://doi.org/10.1016/j.bmcl.2019.04.028
BMCL 26395
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 March 2019
16 April 2019
16 April 2019
Please cite this article as: Zhang, L-H., Zhang, Z-H., Li, M-Y., Wei, Z-Y., Jin, X-J., Piao, H-R., Synthesis and
evaluation of the HIF-1α inhibitory activities of novel ursolic acid tetrazole derivatives, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.04.028
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Synthesis and evaluation of the HIF-1α inhibitory
activities of novel ursolic acid tetrazole derivatives
Lin-Hao Zhanga,1, Zhi-Hong Zhanga,1, Ming-Yue Lia, Zhi-Yu Weib, Xue-Jun
Jina,*, and Hu-Ri Piaoa,*
a Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules, Ministry
of Education, Yanbian University College of Pharmacy, Yanji 133002, China
b Life Science and Technology College of Dalian University, Dalian,116622, China
* Corresponding authors:
H.-R. Piao: Tel.: +86 433 2436149, Fax: + 86 433 2435026, E-mail address: piaohr@ybu.edu.cn
X.-J. Jin: Tel.: +86 433 2436943, Fax: + 86 433 2435026, E-mail address: xjjin@ybu.edu.cn
1 These authors contributed equally to this work.
Abstract
The hypoxia-inducible factor-1α (HIF-1α) pathway has been implicated in tumor
angiogenesis, growth, and metastasis. Therefore, the inhibition of this pathway is an
important therapeutic target for the treatment of various types of cancers. Here, we
designed and synthesized 31 ursolic acid (UA) derivatives containing a tetrazole
moiety and evaluated them for their potential anti-tumor activities as HIF-1α
transcriptional inhibitors. Of these, compound 14d (IC50 0.8 ± 0.2 µM) displayed the
most potent activity and compounds 14a (IC50 4.7 ± 0.2 µM) exhibited the most
promising biological profile. Analysis of the structure–activity relationships of these
compounds with HIF-1α suggested that the presence of a tetrazole group located at
C-28 of the UA derivatives was critical for their inhibitory activities.
Keywords: Ursolic acid; Tetrazole; HIF-1α inhibitor; Cytotoxicity
1

Hypoxia-inducible factor 1α (HIF-1α) has been implicated in a variety of diseases,
including cancer,1,2 infections,3,4 inflammatory diseases,5,6 and Parkinson's disease.7
HIF-1α expression levels are controlled by cellular oxygen concentrations. It is
abundantly expressed in solid tumor cells and plays a key role in tumor cell adaptation
to hypoxic environments created by rapid tumor growth.8 HIF-1α is a known regulator
of tumor cell proliferation, migration, and angiogenesis,9 and the presence of a high
concentration of HIF-1α is positively correlated with cancer severity.10 In addition, the
recurrence of cancer after resection is related to HIF-1α overexpression.11 Therefore,
HIF-1α has become an attractive target for the development of new anti-cancer
drugs.12-14
Several recent reports have investigated various structural modifications to develop
the bioactive properties of the pentacyclic triterpenoid ursolic acid (UA), with an aim
to generate potent novel anti-cancer agents. Indeed, structural modifications of the UA
C-28 carboxylic acid group or C-3 hydroxy group have been shown to significantly
enhance its anticancer activity.15-21 Wu et al reported that the oxidation of the UA C-3
hydroxyl group to a carbonyl group greatly enhanced its antitumor properties,19 while
Bai et al showed that acetylation of the UA C-3 hydroxy group and amidation of the
UA C-28 carboxyl group also resulted in an enhancement of its anticancer activity.16
However, studies by Gu et al indicated that substitutions of large functional groups at
the C-3 and C-28 positions are likely to decrease its anticancer activity.17,22 In our
previous work, we observed that the introduction of a triazolone moiety to the C-28
position of UA increased HIF-1α inhibition with low cytotoxic effects (Fig. 1,
compound A).23 Recently, we reported the design, synthesis, and screening of a series
of compounds containing an aminoguanidine moiety. We revealed that compound B
(Fig. 1, compound B) displayed the most potent inhibitory effect on HIF-1α activity.
However, some of these derivatives showed significant cytotoxicity against a Hep3B
cell line.24 As a result, we attempted to replace the triazolone moiety with other azoles
at the C-28 position of UA and to shorten the distance between UA and the azole ring.
In addition, we simultaneously introduced small changes at the C-3 position by
acetylation and oxidation to identify further potent novel HIF-1α inhibitors with low
2

cytotoxicity.
Tetrazole derivatives have been reported to possess a broad spectrum of therapeutic
activities including anti-microbial,25,26 anti-bacterial,27,28 antioxidant,29,30 anti-HIV,31
anti-leishmanial,32 and anti-cancer activities.26,33 Tetrazole is also a mimetic of
carboxylic acid.34 Several tetrazole-containing drugs have been developed for clinical
use, including cefazolin,35 valsartan,36 and pentetrazole,37 some of which possess a
tetrazolium group as a terminal moiety (Fig. 1). Therefore, we hypothesized that the
introduction of a terminal tetrazole functional group to the C-28 of UA is likely to
enhance its biological potency. In this study, we designed and synthesized eight novel
series of UA derivatives containing a tetrazole moiety (31 compounds in total) and
evaluated their ability to inhibit hypoxia-induced HIF-1α transcriptional activity.
Figure 1. Structures of previously reported compounds as anticancer agents and
clinical medicines containing tetrazole moiety.
The synthetic pathway of the target compounds 3a–d, 4a–d, 6a–d, and 7a–d is
presented in Scheme 1. The intermediate 1 was synthesized according to a reported
method.2 The intermediate 2 was prepared by mixing UA with an equimolar
concentration of K2CO3, a catalytic amount of KI, and more than two equivalents of
1,2-dibromoethane or 1,3-dibromopropane in DMF and stirring at 50 °C for 6h. The
3

target compounds 3a–d, 4a–d, 6a–d, and 7a–d were prepared by reacting
intermediate 2 with 5-substituted-phenyl-1H-tetrazoles (1) or amines (5), respectively,
in the presence of KI/K2CO3 in refluxing acetone. The synthetic pathway of the target
compounds 10a–d, 11a–d, 12a–b, and 14a–e is presented in Scheme 2. Jones reagent
was used to prepare intermediate 8 with UA, which was subsequently alkylated to
yield intermediate 9. The series 10 and 11 compounds were synthesized using the
same method described above to generate series 6 and 7. The intermediate 8 was
reacted with sulfoxide chloride for 4h in refluxing dichloromethane (DCM) to obtain
acyl chloride, which was dissolved in anhydrous DCM and added to a mixture of
DMF/DCM (1:1), trimethylamine, and 5-aminotetrazole in an ice bath. Following a
1h reaction, the mixture was stirred for another 2 days at room temperature to yield
compound 12a. Compound 12b was synthesized by reacting 12a with phenyl
hydrazine hydrochloride with a few drops of acetic acid in refluxing ethanol.
Intermediate 13 was prepared by mixing UA with corresponding anhydrides in the
presence of DMAP and trimethylamine in an ice bath, followed by stirring overnight
at room temperature. Compounds 14a-d were synthesized by reacting 13 with
sulfoxide chloride in refluxing dichloromethane (DCM). Compound 14e was prepared
by hydrolyzing compound 14a with 10%NaOH. The structures of all derivatives were
characterized by 1H and 13C NMR spectroscopy and high-resolution mass
spectrometry.
4

Scheme 1. Synthetic scheme for the synthesis of compounds 3a-d, 4a-d, 6a-d and
7a-d. Reagents and conditions: (a) 1,2-dibromoethane or 1,3-dibromopropane, K2CO3,
KI, DMF, 50 °C, 6h; (b) K2CO3, KI, acetone, reflux, 8h; (c) K2CO3, KI, acetone,
reflux, 10h.
Scheme 2. Synthetic scheme for the synthesis of compounds 10a-d, 11a-d, 12a-b and
14a-f. Reagents and conditions: (a) Jones’ reagent, acetone, 0 °C, 5 h, 90%; (b)
1,2-dibromoethane or 1,3-dibromopropane, K2CO3, KI, DMF, 50 °C, 6h; (c) K2CO3,
5

KI, acetone, reflux, 10h; (d) SOCl2, DMF/DCM (1:1), trimethylamine; (e) Phenyl
hydrazine hydrochloride, acetic acid, ethanol, reflux,8h; (f) anhydride, DMAP, DCM,
trimethylamine, r.t., overnight; (g) 10%NaOH, 4N HCl, 50% methanol in water.
The in vitro activities of the UA derivatives are summarized in Table 1 and Table 2.
To examine the effect of the UA derivatives on hypoxia-induced HIF-1α
transcriptional activity, Hep3B cells were transfected with a luciferase reporter
construct containing six hypoxia-response elements, and then exposed to hypoxia (1%
O2) for 24h. Luciferase activity in the cell culture supernatants was subsequently
measured.2 The cytotoxicity of each of the derivatives against Hep3B cells was
evaluated using the MTT assay. UA and compounds A and B were used as positive
controls.
We found the majority of the synthesized compounds inhibited HIF-1α
transcriptional activity. While six of the eight series 3 and 4 compounds exhibited
moderate to good inhibitory activities, compound 4b exhibited a potent inhibitory
activity (IC50 7.5 ± 0.2 µM). The substituents on the benzene ring attached to the
tetrazole moiety were found to affect potency in an arbitrary manner, which meant
that a meaningful structure–activity relationship could not be determined from the
data. Similarly, we did not observe any significant correlation between inhibitory
potency and the relative distance between UA and the tetrazole moiety. Most of the
series 6 and 7 compounds containing a terminal free tetrazole moiety showed
moderate inhibitory activities in the following order: meta position > para position >
ortho position. Compounds with two carbon linkages between UA and the tetrazole
moiety exhibited higher inhibitory activities compared with compounds with three
carbon linkages. A comparison of the activities of compounds 6a or 7a with
compounds containing a benzene group connected to the tetrazole ring suggested that
HIF-1 inhibitory activity was enhanced in the absence of a benzene ring. Next, we
compared the inhibitory activities of the compounds in series 10 and 11 to their series
6 and 7 counterparts. The only difference between these compounds is that the
hydroxy group at the C-3 position in the series 6 and 7 compounds was oxidized by
Jones’ reagent to create the series 10 and 11 compounds. All other substituents were
equivalent. We concluded that the series 10 and 11 compounds were slightly less
6

potent than their series 6 and 7 counterparts. Furthermore, the structure–activity
relationships of the series 10 and 11 compounds were comparable to the compounds
from series 6 and 7.
Almost all of the series 12 and 14 compounds exhibited higher activities
compared with the compounds in the other series. In these series, the tetrazole group
was directly linked to a nitrogen atom of the amide group at the C-28 position, and the
C-3 hydroxy group was either unmodified, oxidized, esterified, or changed to
hydrazine. The unique feature of this series of compounds was that the tetrazole
moiety was directly introduced to the C-28 position by means of an amide bond
without any additional linkages. Compound 14d showed the most potent inhibition of
HIF-1α transcriptional activity with an IC50 value of 0.8 ± 0.2 µM. However, it did
exert a moderate cytotoxic effect (IC50 18.5 ± 0.2 µM). In contrast, we recorded a
reasonable inhibitory activity without an enhancement of cytotoxicity when the C-3
hydroxy group was oxidized to a ketone, as shown for compound 12a (IC50 18.1 ± 0.2
µM). However, further modification of the C-3 hydroxy group to a phenylhydrazine
derivative (12b) resulted in increases in both the HIF-1α transcriptional inhibitory
activity (IC50 2.2 ± 0.1 µM) and cytotoxicity (IC50 11.9 ± 0.2 µM). The results
obtained for compounds 14a–e (IC50 4.7 ± 0.2 µM, 1.6 ± 0.2 µM, 1.4 ± 0.2 µM, 0.8 ±
0.2 µM, and 13.3 ± 0.2 µM respectively) strongly suggested that the esterification of
the 14e C-3 hydroxy group could not only increase the HIF-1α inhibitory activity but
also enhanced the cytotoxicity except for 14a, indicating that the presence of a larger
group in this position is detrimental for the development of a suitable HIF-1α
inhibitor.
Table 1. In vitro inhibition of HIF-1α transcriptional activity and cytotoxicity of the
series 3, 4, 6 and 7 compounds.
IC50(µM)
Compound n
R1
R2
HRE a
Cytotoxicity b
7

UA
3a
3b
3c
3d
4a
4b
4c
4d
–
1
1
1
1
2
2
2
2
–
–
–
–
–
>100
23.8 ± 0.2
H
18.1 ± 0.2 >100
76.4 ± 0.2 >100
4-Cl
4-CH3O
>100
>100
>100
>100
3,4-(OCH2O) –
H
–
–
–
40.0 ± 0.2 >100
7.5 ± 0.2 >100
27.4 ± 0.2 >100
83.0 ± 0.2 >100
4-Cl
4-CH3O
3,4-(OCH2O) –
6a
6b
1
1
–
>100
>100
–
8.9 ± 0.2 >100
6c
6d
7a
1
1
2
–
–
–
26.2 ± 0.2 >100
8.5 ± 0.2 >100
>100
>100
7b
7c
2
2
–
–
13.2 ± 0.2 >100
>100
>100
8

7d
2
–
–
–
–
–
10.3 ± 0.2 >100
36.9 ± 0.3 >100
4.0 ± 0.2 >100
Comp. A
–
–
Comp. B
a The inhibitory effects of all the derivatives on HIF-1α transcriptional activity were
tested by HRE luciferase reporter assay after 24 h treatment of Hep3B cells under
hypoxic conditions. Values were shown as mean ± SE, 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 ± SE, n=3.
Table 2. In vitro inhibition of HIF-1α transcriptional activity and cytotoxicity of the
series 10, 11, 12 and 14 compounds.
IC50(µM)
Compound n
R2
R3
–
R4
–
HRE a
Cytotoxicity b
>100
10a
10b
10c
10d
11a
11b
1
1
1
1
2
2
>100
–
–
>100
>100
–
–
18.1 ± 0.2 >100
14.2 ± 0.2 >100
–
–
–
–
>100
>100
>100
>100
–
–
9

11c
11d
12a
12b
14a
14b
14c
14d
14e
2
2
–
–
–
–
–
–
–
–
–
–
–
–
–
>100
>100
16.7 ± 0.2 >100
–
–
–
–
–
–
–
18.1 ± 0.2 >100
2.2 ± 0.1 11.9 ± 0.2
4.7 ± 0.2 >100
–
–
–
–
–
1.6 ± 0.2 6.4 ± 0.2
1.4 ± 0.2 4.7 ± 0.2
0.8 ± 0.2 18.5 ± 0.2
13.3 ± 0.2 >100
–
a The inhibitory effects of all the derivatives on HIF-1α transcriptional activity were
tested by HRE luciferase reporter assay after 24 h treatment of Hep3B cells under
hypoxic conditions. Values were shown as mean ± SE, 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 ± SE, n=3.
Compound 14a with the most desirable inhibitory activity against HIF-1α out of
all the derivatives tested, was selected for further biological evaluation. As shown in
Fig. 2, compound 14a dose-dependently inhibited the luciferase activity in Hep3B
cells (Figs. 2A) and concentration up to 30 µM did not adversely affect cell viability
(Fig. 2B).
10

Figure 2. Effect of compound 14a 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 14a.
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 14a. After
24 h incubation, cell viability was determined by MTT assays.
In summary, several series of ursolic acid tetrazole derivatives were designed,
synthesized, and evaluated for their HIF-1α inhibitory activities. Of the compounds
tested, compound 14a showed the most promising HIF-1α inhibitory activity with an
IC50 of 4.7 ± 0.2 µM and did not show any significant cytotoxicity at a concentration
of 30µM against a Hep3B cell line. Furthermore, its inhibitory activity was
comparable to compound B and eight times more potent than compound A. Analysis
of the structure–activity relationships of the compounds inhibiting HIF-1α suggested
that introduction of the tetrazole moiety at the C-28 position of UA is critical for
increasing the HIF-1α inhibitory activity and a larger group at the C-3 position is
detrimental for the development of a suitable HIF-1α inhibitor.
Acknowledgement
The work was supported by the National Science Foundation of China (No. 81660608)
11

and the Natural Science Foundation of Jilin Province (No. 20160101218JC). 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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Graphical abstract
Synthesis and evaluation of the HIF-1α inhibitory
activities of novel ursolic acid tetrazole derivatives
Lin-Hao Zhanga,1, Zhi-Hong Zhanga,1, Ming-Yue Lia, Zhi-Yu Weib,Xue-Jun Jina,*, and Hu-Ri Piaoa,*
13

14

Highlights:
Eight series of ursolic acid-tetrazole derivatives were designed and synthesized.
The inhibitory activity on HIF-1α pathway and cytotoxicity of these compounds
were evaluated.
Compound 14a is a promising lead for searching effective HIF-1α inhibitors.
 The investigation of dose-dependently inhibiting the luciferase activity in Hep3B
cells of compound 14a was performed.
15