Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1 (plasminogen activator inhibitor-1)

Article abstract of DOI:10.1016/j.bmcl.2014.03.045

The natural product embelin was found to have PAI-1 inhibitory activity with the IC₅₀ value of 4.94 μM. Based on the structure of embelin, a series of analogues were designed, synthesized, and evaluated for their ability to inhibit PAI-1. The SAR study on these compounds disclosed that the inhibitory potency largely depended on the hydroxyl groups at C2 and C5, and the length of the alkyl chains at C3 and C6. Compound 11 displayed the best PAI-1 inhibitory potency with the IC₅₀ value of 0.18 μM.

Full text of DOI:10.1016/j.bmcl.2014.03.045

Accepted Manuscript
Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1
(Plasminogen Activator Inhibitor-1)
Fanglei Chen, Guiping Zhang, Zebin Hong, Zhonghui Lin, Min Lei, Mingdong
Huang, Lihong Hu
PII:
S0960-894X(14)00272-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.03.045
BMCL 21438
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 January 2014
12 March 2014
15 March 2014
Please cite this article as: Chen, F., Zhang, G., Hong, Z., Lin, Z., Lei, M., Huang, M., Hu, L., Design, synthesis, and
SAR of embelin analogues as the inhibitors of PAI-1 (Plasminogen Activator Inhibitor-1), Bioorganic & Medicinal
Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.03.045
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Graphical Abstract
Design, synthesis, and SAR of embelin
analogues as the inhibitors of PAI-1
(Plasminogen Activator Inhibitor-1)
Leave this area blank for abstract info.
Fanglei Chen, Guiping Zhang, Zhonghui Lin,
Zebin Hong, Min Lei, Mingdong Huang, Lihong Hu

1
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1
(Plasminogen Activator Inhibitor-1)
Fanglei Chena, Guiping Zhanga, Zebin Hongb, Zhonghui Linb, Min Leia, Mingdong Huangb,∗ and Lihong
Hua,∗
a Shanghai Research Center for the Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, P.R. China.
b State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, P.R.
China.
ARTICLE INFO
ABSTRACT
Article history:
Received
The natural product embelin was found to have PAI-1 inhibitory activity with the IC50 value of
4.94 µM. Based on the structure of embelin, a series of analogues were designed, synthesized,
and evaluated for their ability to inhibit PAI-1. The SAR study on these compounds disclosed
that the inhibitory potency largely depended on the hydroxyl groups at C2 and C5, and the
length of the alkyl chains at C3 and C6. Compound 11 displayed the best PAI-1 inhibitory
potency with the IC50 value of 0.18 µM.
Revised
Accepted
Available online
Keywords:
Embelin
2009 Elsevier Ltd. All rights reserved.
Natural product
PAI-1
Inhibitory activity
SAR study
As the primary physiological inhibitor of urokinase-type
plasminogen activator (uPA) and tissue-type plasminogen
activator (tPA), plasminogen activator inhibitor-1 (PAI-1) has
been participated in a wide range of metabolic responses, such as
blood coagulation, wound healing, angiogenesis, cell attachment
and detachment, cell migration, and tumor-cell invasion.1–7 In
human malignant tumors, the levels of uPA and PAI-1 are
significantly higher than those in the corresponding normal
tissues. Over-expressed uPA and PAI-1 can promote
angiogenesis in tumor, facilitating the progression, dissemination
and metastasis of tumors.8,9 In breast cancer, uPA and PAI-1 have
been identified as the most extensively validated biological
prognostic factors.10–12 Furthermore, increased levels of PAI-1
have been associated with increased risk of cardiovascular
diseases.13–20 Therefore, PAI-1 is a potential target for treatment
of cancer and cardiovascular diseases. Although many PAI-1
inhibitors have been reported,21–28 most of them have a certain
degree of limitations, such as low affinity to bind with PAI-1,
poor physicochemical properties, or lack of potency to inhibit
PAI-1 in the presence of its cofactor. Therefore, searching for
novel PAI-1 inhibitors with improved potencies is quite
necessary and meaningful.
Embelin (Figure 1) comes from the fruit of Embelia ribes
Burm plant (Myrsinaceae), which has been used for the treatment
of fever, inflammatory and gastrointestinal diseases for thousands
of years.29 As the active component of this plant, embelin has
been shown to have analgesic,30 anti-inflammatory,30
antidiabetes,31 and antitumor properties.30,32–35 In our previous
drug screening test, we found that embelin was able to inhibit
PAI-1 activity with the IC50 value of 4.94 µM.36 Comparing with
other small molecule inhibitors of PAI-1, embelin is distinctive.
Specifically, the source is natural and rich, the structure is simple
and stable, the potency is prominent, and the binding site is clear.
To explore its structure and PAI-1 inhibitory activity
relationship, we focused on our crystal structure of PAI-1 in
complex with embelin (Figure 2).36
Figure 1. The structure of embelin.
———
∗ Corresponding author. Tel.: +86-591-8370-4996; fax: +86-591-8370-4996; e-mail: mhuang@fjirsm.ac.cn (M. Huang)
∗ Corresponding author. Tel.: +86-21-2023-1965; fax: +86-21-2023-1965; e-mail: lhhu@simm.ac.cn (L. Hu)

2
Bioorganic & Medicinal Chemistry
In this crystal structure, embelin binds to the groove
intermediates 1a, 1b and 1c were prepared successively. After
the reaction of 1c with cerium (IV) ammonium nitrate (CAN),
compounds 2 and 3 could be obtained.39,40
constituted by α helixes D and F and β strand 2A. The polar, six-
member ring of embelin sits in the middle of the groove forming
the direct strong contacts with Tyr-79 (α helix D), Asp-95 and
Thr-93 (β strand 2A), and His-143 (α helix F). The aliphatic
carbon chain of embelin stretches out of the cavity involving in
the interactions with Ser-119 (hE-s1A connecting loop) and Trp-
139 (α helix F).36 This interaction mode was proposed to fix β
strand 2A to the neighboring α helixes D and F, delaying the
insertion of the reaction center loop into β strand 2A that is the
perquisite for translocation of the protease to the opposite pole of
PAI-1 and subsequent covalent complex formation.36–38 From the
inhibitory mechanism of embelin, it is clear that both the nucleus
and the alkyl chain played an important role in the process of
fixing β strand 2A to the neighboring α helixes D and F.
Therefore, to explore the importance of the 2,5-dihydroxyl-p-
benzoquinone nucleus and the optimal length of the alkyl chain,
we designed and synthesized series of embelin analogues (Figure
3).
In order to evaluate the PAI-1 inhibitory activity of embelin
analogues, a chromogenic assay was adopted, in which the
activity of PAI-1 was evaluated by its inhibition towards uPA-
mediated hydrolysis of chromogenic substrate.36 The PAI-1
inhibitory activities of compounds 2 and 3 were tested and
exhibited in Table 1. From this table, we found that methylation
of 5-hydroxyl (2) resulted in a sharp decrease of potency, while
methylation of both 2-hydroxyl and 5-hydroxyl (3) led to a
complete loss of potency even at 100 µM. Hence, retaining
hydroxyl groups at C2 and C5 were crucial for the compounds to
keep PAI-1 inhibitory activity.
O
OH
OMe
OH
OMe
OMe
a, b
c
HO
MeO
MeO
O
OH
OMe
1
1a
1b
OMe
O
OMe
OR2
C11H23
d, e
MeO
f
1 = Me, R2 = H
2:
R
C11H23
R1O
: R1 = Me, R2 = Me
3
OMe
O
1c
Scheme 1. Reagents and conditions: (a) con. HCl, MeOH; (b)
Na2S2O4, H2O; (c) KOH, MeI, DMSO; (d) n-BuLi, THF, –10
oC; (e) 1-Bromo-undecane, –10 oC~rt; (f) CAN, CH3CN, –10
oC~rt.
Table 1. IC50 Values of compounds 2 and 3 against PAI-1a
Compd.
IC50 value (µM)
embelin
4.94 0.41
Figure 2. Close-up view of the embelin binding site with direct contacts
shown as yellow dashed lines. Selected residues of PAI-1 binding to embelin
are given in stick. Tyr-79, located on top of embelin, is not shown for clarity
(PDB code: 3UT3).
2
3
90.19 4.04
b
–
a IC50 values are based on at least three independent experiments.
bNo activity at 100 µM.
O
O
O
OH
R3
O
O
a, b
c
O
O
HO
R3
O
O
O
a-2
R3 =
4a-8a
4-8
4
7
5
6
: n = 3; : n = 4; : n = 5;
n
8
: n = 6; : n = 7.
Scheme 2. Reagents and conditions: (a) n-BuLi, THF, –10oC;
(b) R3Br, –10oC~rt; (c) 1,4-dioxane, 6 mol/L HCl, air, reflux.
Then, to explore the relationship between the length of the
side chain at C3 and PAI-1 inhibitory activity, compounds 4–8
containing different length of alkyl chains at C3 were
synthesized. As shown in Scheme 2, compound a-2 reacted with
n-BuLi, and alkyl bromide in THF to generate intermediates 4a–
8a which deprotected with 6 mol/L HCl in 1,4-dioxane to obtain
compounds 4–8.
Figure 3. Design of embelin analogues.
Firstly, we prepared two etherification derivatives (2, 3)
(Scheme 1). Using 2,5-dihydroxycyclohexa-2,5-diene-1,4-dione
(1) as the starting material, via four steps of reactions,

3
As shown in Table 2, 4 and 5 kept the activity, and 6–8
showed slightly increased activity. Among them, compound 8
with the n-nonyl group substituted at C3 exhibited the best
potency.
O
O
R5
O
O
R5
OH
R5
O
O
a, b
c
O
Table 2. IC50 values of compounds 4–8 against PAI-1a
HO
R5
O
O
Compd.
IC50 value (µM)
4.94 0.41
4.56 0.12
4.18 0.25
3.87 0.33
1.87 0.15
1.78 0.18
O
embelin
a-2
15a-20a
15-20
4
5
6
7
8
15
18
16
17
R5 =
: m = 1; : m = 2; : m = 3;
m
19 20
: m = 4; : m = 5; : m = 6.
Scheme 4. Reagents and conditions: (a) n-BuLi, THF, –10oC;
(b) R5Br, –10oC~r.t; (c) 1,4-dioxane, 6 mol/L HCl, air, reflux.
a IC50 values are based on at least three independent experiments.
Table 4. IC50 values of compounds 15–20 against PAI-1a
Compd.
embelin
15
IC50 value (µM)
4.94 0.41
5.68 0.15
7.11 0.19
0.92 0.13
0.58 0.14
0.51 0.05
0.89 0.08
On the basis of 8, we further introduced carbon chains at C6
(Scheme 3). The PAI-1 inhibitory activities of compounds 9–14
revealed that introducing carbon chains at C6 could significantly
improve the potencies of compounds. Specifically, 9, 12 and 13
showed 13-fold, 8-fold and 5-fold improved potency respectively,
10 and 14 exhibited 6.5-fold improved potency. Compound 11
afforded the best potency in this series of compounds with the
IC50 value of 0.18 µM, which was 27-fold more potent than
embelin (Table 3).
16
17
18
19
20
a IC50 values are based on at least three independent experiments.
To further explore the optimal length of carbon chains at C3
and C6, we designed and synthesized compounds 15–20 (Scheme
4). The synthetic methods of these compounds were similar to
that of 4–8. As shown in Table 4, 15 and 16 were slightly less
potent than embelin. 17 and 20 showed 5-fold improved potency
relative to embelin. 18 and 19 exhibited 9-fold and 10-fold
improved potency, respectively.
According to IC50 values of compounds 9–20, it could be
concluded that the lengths of alkyl chains at C3 and C6 have
significant effect on the potencies of compounds. Introducing
long carbon chain at C3 and short carbon chain at C6 is optimal,
such as compounds 9 and 11.
In conclusion, we have found embelin which was able to
inhibit PAI-1 with the IC50 value of 4.94 µM. Based on the
structure of PAI-1/embelin complex, a series of embelin analogs
were designed and synthesized for the study of SAR. From the
IC50 values of these compounds, the SAR could be summarized
as follows: (1) the hydroxyl groups at C2 and C5 were
indispensable for the inhibitory potency, because both of them
have been participated in the hydrogen-bonding interaction with
the binding pocket; (2) suitably shortening the length of the alkyl
chain at C3 could slightly increase activity. Introducing alkyl
chains at C6 could significantly improve potency. In sum, several
compounds have exhibited much better potencies than that of
embelin. Among them, Compound 11 showed the best activity
with the IC50 value of 0.18 µM. In addition, as our initial study,
these analogues have retained the structural character of embelin.
For the purpose to obtain more potent PAI-1 inhibitors, we are
exploring embelin analogues with more diverse and complex
substituents at C3 and C6, and the corresponding work will be
disclosed in due course.
Scheme 3. Reagents and conditions: (a) n-BuLi, THF, –10oC;
R4Br, –10oC~rt; (b) 1,4-dioxane, 6 mol/L HCl, reflux.
Table 3. IC50 values of compounds 9–14 against PAI-1a
Compd.
IC50 value (µM)
4.94 0.41
0.38 0.19
0.75 0.06
0.18 0.05
0.63 0.09
0.95 0.05
0.77 0.16
embelin
9
10
11
12
13
14
a IC50 values are based on at least three independent experiments.
Acknowledgments

4
Bioorganic & Medicinal Chemistry
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This work was supported by the National Natural Science
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the Chinese National Science & Technology Major Project “Key
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