Natural Product Evodiamine with Borate Trigger Unit: Discovery of Potent Antitumor Agents against Colon Cancer

Article abstract of DOI:10.1021/acsmedchemlett.9b00513

In order to improve the antitumor potency of the natural product evodiamine, novel boron-containing evodiamine derivatives were designed by incorporating boronic acid and boronate as trigger units. Boronate derivative 13a could be triggered by reactive oxygen species (ROS) in the HCT116 colon cancer cell line and showed excellent antitumor activity in vitro and in vivo. It induced apoptosis in HCT116 cancer cells in a dose-dependent manner and cell growth arrest at the G2 phase.

Full text of DOI:10.1021/acsmedchemlett.9b00513

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Letter
Natural Product Evodiamine with Borate Trigger Unit:
Discovery of Po-tent Antitumor Agents against Colon Cancer
Xinglin Li, Shanchao Wu, Guoqiang Dong, Shuqiang Chen, Zonglin Ma, Dan Liu, and Chunquan Sheng
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00513 • Publication Date (Web): 28 Feb 2020
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ACS Medicinal Chemistry Letters
Natural Product Evodiamine with Borate Trigger Unit:
Discovery of Potent Antitumor Agents against Colon Cancer
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Xinglin Li,$, † Shanchao Wu,$, ‡ Guoqiang Dong,$, ‡ Shuqiang Chen,‡ Zonglin Ma,† Dan Liu,*, † and Chunquan
Sheng*, ‡
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† Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, People’s Republic of China
‡ Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
People’s Republic of China
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KEYWORDS: evodiamine, borate unit, ROS, antitumor, Top1, Top2
ABSTRACT: In order to improve the antitumor potency of natural product evodiamine, novel boron-containing evodiamine derivatives were
designed by incorporating boronic acid and boronate as trigger units. Boronate derivative 13a could be triggered by ROS in the HCT116
colon cancer cell line and showed excellent antitumor activity in vitro and in vivo. It induced apoptosis in HCT116 cancer cell in a dose-
dependent manner and cell growth arrest at the G2 phase.
Boron is an essential element of diverse cells, being
ubiquitous in nature.1 It has an empty p-orbital with
electrophilic property, which can form covalent bonds with
biological nucleophiles including amine and hydroxyl groups
in nucleic acids. Moreover, the sp2 boron center can be easily
changed to sp3 hybridization under certain physiological
conditions.2-6 Given the special characteristic of boron atom,
boron-containing compounds (BCCs) have attracted great
interests in medicinal chemistry and several of them have
been approved (e.g. bortezomib, tavaborole, Figure 1)7,8 or
under clinical trials (e.g. crisaborole, GSK8175, Figure 1).9,10
In recent years, evodiamine (Figure 1) has been
investigated as an antitumor lead compound that possesses
multi-targeting profiles.23-26 Previously, systemic structural
optimizations of evodiamine was performed by our group
and several highly active evodiamine derivatives (1a and 1b,
Figure 1) were identified.27-29 Unfortunately, further
development of evodiamine derivatives was hindered by the
unsatisfied in vivo antitumor potency. In light of the success
of borate in drug discovery,17-22 herein a series of novel
boron-containing evodiamine analogues were reported by
incorporating borates as trigger units at C10 position of
compounds 1a and 1b through various cleavable linkers with
an aim to improve their in vivo antitumor efficacy (Figure 2).
OH
B
O
OH
B
O
O
OH
B
H
NC
N
N
N
N
OH
H
O
F
O
O
R
R
1 = F or H, 2 = Boc or H
HO
OH
N
O
O
or
HO
B
B
O
Bortezomib
Tavaborole
Unit: A =
Crisaborole
B
=
R
R1
N
O
N
H
OH
Cl
N
B
F
Borate
Trigger
Units
1a
1b
R = H
R = F,
N
Linkers
O
N
R2
O
O
H
N
HO
N
Linkers
N
SO2Me
Me
R
O
O
O
O
N
N
N
N
Linker: M =
N =
H
H
Borate Trigger Units
O
GSK8175
1a R = F, 1b R = H
Evodiamine
ROS Triggering Evodiamine Borate Analogues
Figure 1. Chemical structures of bortezomib, tavaborole,
crisaborole, GSK8175, evodiamine and evodiamine derivatives.
Figure 2. Design of ROS triggering evodiamine borate
analogues.
Reactive oxygen species (ROS) including hydrogen
peroxide (H2O2) are natural byproducts of normal oxygen
metabolism, which act a pivotal part in cell signaling and
homeostasis.11 In particular, cancer cells exhibit exclusive
feature of increased ROS (mainly H2O2)12-14, which can be
used to design drugs for targeted cancer therapy.15,16
Moreover, borates, including boronate and boronic acid, are
ROS responsive functional groups.11,17-22 Borates attached to
carbon are easily cleavable by ROS.11,17-22 Therefore, it is
advantageous to attach borates as “trigger unit” to improve
the pharmacological activity and druggability of antitumor
agents.17-22
The synthesis of evodiamine borates were depicted in
Schemes 1-3. First, starting from evodiamine derivatives 1,
triflates 2 were prepared according to the literature.22 Then,
Miyaura reaction was performed with 2 to afford boronoates
3. Deprotection of 3 in the presence of potassium
difluorohydride (KHF2) and methanol at room temperature
gave potassium trifluoroborates 4, followed by hydrolysis
under acid conditions to yield target compound 5 (Scheme
1).
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Scheme 1. Chemical synthesis of compounds 3 and 5a
As depicted in Scheme 3, evodiamine boronates
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1
2
3
4
5
6
7
8
derivatives with carbonate linker (13 and 15) were prepared.
Boronate pinacol ester 11 was reacted with 4-nitrophenyl
chloroformate and trimethylamine (TFA) in tetrahydrofuran
(THF) to afford intermediate 12, which was reacted with
compounds 1 to give target compounds 13 with high yields.
Then, target compounds 15 were prepared using the similar
synthetic methods as described for derivatives 5.
O
B
O
O
O
OH
OTf
O
N
N
N
N
N
N
a
b
N
N
N
R
R
R
H
H
H
1a
R = F
R = H
3a
3b
R = F
R = H
2
1b
OH
B
O
O
BF3K
OH
N
N
N
N
d
c
N
N
H
R
R
H
4
5a
5b
R = F
R = H
9
Table 1. In vitro antitumor activity of borate
evodiamines (IC50, μM)
aReagents and Conditions: (a) Tf2O, Pyridine, DCM, 0 °C, 3 h, yield
87%; (b) Bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc,1,4-dioxane;
100 C, 8 h , yield 90-93%; (c) KHF2, MeOH, rt, 30 min; yield 63-68%;
(d) TMSCl, H2O, MeCN, 2 h, yield 95-98%.
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Compds
3a
HCT116
0.17 ± 0.004
0.22 ± 0.03
MCF-7
0.96 ± 0.2
0.39 ± 0.075
A549
0.11 ± 0.14
0.17 ± 0.022
Target compounds 7-10 were synthesized as shown in
Scheme 2. The NH groups of compounds 1 were protected
with Boc group via three steps to afford intermediates 6. In
the presence of NaH and 4-bromomethylphenylboronic
acid, phenylboronic acids 7 and phenylboronates 9 were
obtained, respectively. Finally, Boc deprotection was
conducted to give target compounds 8 and 10.
3b
5a
0.063 ± 0.003 0.094 ± 0.006 0.040 ± 0.006
5b
0.069 ± 0.005 0.16 ± 0.031
0.10 ± 0.008
2.13 ± 0.15
7.20 ± 1.4
7a
4.91 ± 0.97
0.52 ± 0.36
2.39 ± 0.48
1.58 ± 0.32
7b
8a
0.083 ± 0.001 0.059 ± 0.010 0.073 ± 0.003
Scheme 2. Chemical synthesis of compounds 7-10a
8b
0.12 ± 0.04
2.17 ± 0.47
4.18 ± 0.79
0.11 ± 0.021 0.074 ± 0.014
b
O
O
9a
7.85 ± 0.92
4.13 ± 0.83
2.23 ± 0.43
3.44 ± 0.65
N
OH
N
OH
a
R
R
9b
N
N
N
N
c
H
Boc
10a
10b
13a
13b
15a
15b
1a
0.15 ± 0.014 0.097 ± 0.016 0.12 ± 0.001
0.10 ± 0.06 0.16 ± 0.031 0.39 ± 0.075
0.016 ± 0.003 0.033 ± 0.008 0.037 ±0.002
0.065 ± 0.022 0.086 ± 0.017 0.10 ±0.006
0.041 ± 0.005 0.032 ±0.006 0.044 ± 0.002
0.052 ±.007 0.14 ± 0.028 0.13 ± 0.05
1a
1b
R = F
R = H
6a
6b
R = F
R = H
OH
B
OH
B
O
OH
O
OH
N
O
O
7a
N
N
d
R
R
N
N
N
H
8a
8b
R = F
R = H
R = F
R = H
Boc
7b
O
B
O
B
O
O
O
O
N
N
N
O
O
e
R
R
N
N
Boc
N
H
9a
9b
10a
10b
R = F
R = H
R = F
R = H
0.027 ± 0.006 0.031 ± 0.008 0.029 ± 0.004
0.055 ± 0.005 0.27 ± 0.006 0.084 ± 0.013
0.009 ± 0.003 0.084 ± 0.005 0.039 ± 0.008
aReagents and Conditions: (a) 1) DMAP, (Boc)2O, THF, 2 h; 2)
K2CO3, MeOH, 4 h; 3) AcOH, 1 h, yield 62-65%; (b) NaH, 4-
bromomethylphenylboronic acid, DMF, 0 C, 8 h, 1 N HCl, pH = 2,
yield 80-83%; (c) NaH, 4-bromomethylphenylboronic acid pinacol
ester, DMF, 0 C, 8 h, yield 82-83%; (d) TFA, DCM, rt, 2 h, yield 63-
66%; (e) TFA, DCM, rt, 2 h, yield 45-48%.
1b
CPT
The in vitro antitumor activities of evodiamine borates
(Table 1) were assayed against HCT116, MCF-7 and A549
cell lines using the CCK8 assay.30 Evodiamine derivatives 1a,
1b and camptothecin (CPT) were selected as positive
controls. As illustrated in Table 1, boronic acids derivatives
(5a, 5b, 8a, 8b and 15b) generally showed better antitumor
activity than the corresponding boronates (3a, 3b, 10a, 10b
and 13b). Most N13-Boc protected intermediates (7a, 7b, 9a
and 9b) showed decreased antitumor activity against the
three cell lines. Interestingly, excellent antitumor activity was
retained for evodiamine borates with a carbonate linker (13
and 15). Most boronic acid derivatives showed comparable
antitumor activity to lead compounds 1a and 1b. In
Scheme 3. Chemical synthesis of compounds 13 and 15a
O
O
B
O
B
B
O
O
O
O
O
O
N
O
O
O
a
b
HO
R
O
N
N
H
13a
13b
R = F
R = H
O
O2
N
11
12
OH
B
BF3
K
OH
O
O
O
N
O
O
N
d
c
R
O
R
N
O
N
H
N
N
15a
15b
R = F
R = H
H
14
aReagents and Conditions: (a) 4-Nitrophenyl chloroformate, TEA,
THF, rt, 6 h, yield 58%; (b) 1, K2CO3, THF, 70 C, 10 h, yield 76-80%;
(c) KHF2, MeOH, rt, 30 min; yield 74-76%; (d) TMSCl, H2O, MeCN,
2 h, yield 59-62%.
particular,
compound
13a
exhibited
excellent
antiproliferative activity (IC50 range: 16 to 37 nM), whose
efficacy against HCT116 cells (IC50 = 16 nM) was superior
to that of lead compound 1a (IC50 = 27 nM).
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Information). On the basis of the above results, compounds
1
2
3
4
8b, 13a, 13b and 15a were proven to be Top1/Top2 dual
inhibitors, indicating that these borate compounds might act
by similar antitumor mechanism to that of lead compounds
1a and 1b.29
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7
8
9
To better illuminate the binding mode with Top1 and
Top2, compounds 1a, 1b, 13a and 15a were subjected to
molecule docking. Similar to compounds 1a and 1b (Figure
S1A, S1B in Supporting Information), A-ring of compound
13a formed π-π interactions with TGP11 base pair
enhancing the base stacking at the active site of Top1 (Figure
4A). Carbonyl group and carbonate carbonyl group formed
hydrogen bonding interactions with Try426 and Asn722,
respectively. Boric acid group of compound 15a formed
hydrogen bond with DA11 and DG12 additionally (Figure
4B). Additional hydrogen bonds were also found for
compounds 13a and 15a in the active site of Top1.
Moreover, the four compounds fitted well into the ATPase
of Top2α. For compounds 1, hydrogen bonds between
hydroxy group and Top2 was the predominant interactions
(1a, DC8 and His758; 1b, DC8 and Ser763, Figure S1C,
S1D in Supporting Information). However, compounds 13a
and 15a bound to the active domain of Top2 through
different ways. For compound 13a, D-ring carbonyl group
participated in the formation of hydrogen bond with Ser464
(Figure 4C). For compound 15a, D-ring carbonyl group
formed a hydrogen bond with Ser763 and boric acid group
formed hydrogen bonds with His758 and DC8, respectively
(Figure 4D).
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Figure 3. (A) Top1 inhibitory activity assay of evodiamine borate
derivatives at 500, 250 , 125 μM, respectively. (B) Top1 inhibitory
activity assay of compounds 13a and 15a ranging from 27.9 to 250 μM.
(C) Top2 inhibitory activity assay of evodiamine borate derivatives at
200, 100, 50 μM, respectively. (D) Top2 inhibitory activity assay of
compounds 13a and 15a ranging from 25 to 200 μM.
Previously, topoisomerase I (Top1) and topoisomerase
II (Top2) were identified as targets of compounds 1a and 1b.
Herein, evodiamine borates with good cytotoxicity were
selected to investigate their Top inhibitory activities. As
depicted in Figure 3A, all the compounds showed strong
inhibitory effect against Top1 at 500 μM. At a lower
concentration of 125 μM, six compounds (8a, 8b, 13a, 13b,
15a and 15b) were still effective. Particularly, the activity of
compounds 13a and 15a were active at 83.3 μM (Figure 3B).
Moreover, five compounds, namely 8b, 13a, 13b, 15a and
15b, retained Top2 inhibitory potency at 50 μM (Figure 3C,
3D) and three of them (8a, 13a, 15a) exhibited comparable
potency to the reference drug etoposide (ETO). Given that
3-chloro-10-hydroxy thio-evodiamine was identified as a
Top1/Top2/tubulin inhibitor, the tubulin inhibitory
activities of compounds 13a, 13b, 15a and 15b were further
determined.28 However, only compound 15b was active with
an IC50 value of 25.7 μM (Table S3 in Supporting
Figure 4. Binding modes of compounds 13a (A, C) and 15a (B, D)
with Top1-DNA complex (PDB code: 1T8I) and ATPase domain of
Top2α (PDB code: 5GWK).
On the basis of in vitro antitumor activities, two
different types of evodiamine borates 13a and 15a were
selected to test the ability in inducing the apoptosis and cell-
cycle arrest in HCT116 cell line. As shown in Figure 5A,
compounds 13a and 15a demonstrated significantly
apoptosis-inducing activity dose-dependently in HCT116
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cells. The percentages of apoptotic cells were increased
be activated by H2O2 (Figure S1, S2 in Supporting
Information). Furthermore, ultra performance liquid
1
2
3
4
5
6
7
8
significantly (13a: from 42.28% to 61.73%; 15a: from
44.40% to 64.47%) as compared to the control population
(6.25%). The cell-cycle arrest of compounds 13a and 15a in
HCT116 cells were evaluated by the flow cytometric method
(Figure 5B). After exposure to compound 13a at 0.05, 0.2
and 0.4 μM, the percentage of cells in G2 fraction were
26.88%, 45.49% and 56.25%, respectively. Similarly, the
percentage of cells exposed to compound 15a at G2 phase
were changed dramatically (29.47%, 43.45% and 51.23%,
respectively). As compared with the ratio (10.8%) of the
untreated cells at the G2 phase, compounds 13a and 15a
were confirmed to induce cell-cycle arrest in HCT116 cells
at the G2 phase, which was consistent with that of lead
compound 1a.29
chromatography
quadrupole
time-of-flight
mass
spectrometry (UPLC-QTOF/MS) was used to assess the
cleavage of borate derivatives undergoing intracellular ROS
in HCT116 cancer cells. The concentrations of compounds
13a, 15a and 1a in cell culture media were analyzed after
incubation with HCT116 cancer cells for 8, 48 and 72 hours.
Relative drug release rates were measured by relative peak
areas. Compound 15a (retention time: 8.6 min) was directly
converted into compound 1a (retention time: 7.3 min) with
the relative drug release rate of 18.5%, 24.5% and 35.5%,
respectively (Figure S4-S6 in Supporting Information). In
contrast, compound 13a (retention time: 11.4 min) was first
converted to compound 15a (78.6%, 43.7% and 36.3%,
respectively) and then transformed to compound 1a (13.6%,
39.9% and 53.1%, respectively, Figure S4-S6 in Supporting
Information). Thus, compounds 13a and 15a could be
triggered by ROS in HCT116 cells (Scheme 4). The relative
drug release rates were increased in a time dependent
manner. Reactive quinone methide (QM) precursor 4-
(chloromethyl)phenyl acetate was also used to tested its
antiproliferative effect for cancer cells, and it was almost
inactive against cancer cells (Table S1 in Supporting
Information), suggesting the released QM has little effect on
cancer cells. Furthermore, in vitro metabolic stabilities of
compounds 13a, 15a and 1a were evaluated through liver
microsome assay. Compound 13a had a terminal half-life
(t1/2) of 8.36 min, which was much longer than that of
compounds 15a (t1/2 = 0.74 min) and 1a (t1/2 = 3.69 min,
Table S2 in Supporting Information). The cLogP of
compounds 13, 15 and 1 were also predicted (Table S4 in
Supporting Information). Compounds 13 had better in vitro
antitumor potency possibly due to its tumor penetration
with increased lipophilicity of the boronate-units.
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OH
O
B
B
OH
O
Hydrolysis
O
O
O
O
R
R
Figure 5. Apoptosis and cell-cycle arrest induced by compounds 13a
and 15a. (A) HCT116 cells were incubated with 0.05, 0.2 and 0.4 μM
of compounds 13a or 15a for 48 h. Apoptosis was assayed by flow
cytometry (n = 3). (B) Cell-cycle effect of compounds 13a and 15a.
HCT116 cells were incubated with 0.05, 0.2 and 0.4 μM of compounds
13a or 15a for 24 h. At least three independent experiments were done
for each condition
O
O
O
N
Compound 13a
Compound 15a
ROS
ROS
N
H
R =
F
N
O
O
OH
B
O
B
O
O
O
O
+
+
R
R
HO
OH
O
HO
F
O
O
O
OH
N
Compounds 13a and 15a were selected to investigate
the ability to release compound 1a by treating different
concentrations of H2O2 in PBS (pH = 7.4) using high
performance liquid chromatography (HPLC). At 0.05 mM
of H2O2, compound 1a could be detected with relative drug
release rate for compound 13a and 15a of 11.6% and 20.8%,
respectively. Compound 1a could be released in a
concentration dependent manner, indicating that they can
O
O
N
N
H
Compound 1a
Scheme 4. Proposed reaction mechanism of evodiamine borate
analogues with ROS.
Finally, compound 13a was administrated
intraperitoneally (IP) at 10 mg/kg to evaluate its in vivo
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$
X.L., S.W. and G.D. contributed equally to this work. D.L. and C.S.
antitumor activity in HCT116 xenograft model in mice.
After 21 consecutive days, compound 13a achieved the
tumor growth inhibition (TGI) of 64.4% (Figure 6A), which
was superior to compound 1a (TGI = 30.3% at 2 mg/kg29,
highly toxic at 10 mg/kg, Figure S7 in Supporting
Information) and comparable to the control TPT (TGI =
68.8%). Meanwhile, compound 13a had no significant effects
on the decrease of the body weight (P > 0.05, Figure 6B). In
contrast, TPT had significant toxicity on the treated mice (P
< 0.01), suggesting that toxicity of compound 13a might be
lower than that of TPT.
1
2
3
4
5
6
7
8
designed the experiments and revised the manuscript. X.L., S.W. and
G.D. synthesized evodiamine borate derivatives and carried out the
biological experiments. S.C., and Z.M. assisted in the biological
experiments and preparing the manuscript. All authors discussed the
results and commented on the manuscript. All authors read and
approved the final manuscript.
Funding Sources
This work was supported by National Natural Science Foundation of
China (grants 21807113 to S.W., 81872742 to G. D. and 81725020 to
C. S.), the National Key R&D Program of China (grant
2017YFA0506000 to C. S.), Shanghai Sailing Program (18YF1429100
to S.W.) and the Innovation Program of Shanghai Municipal
Education Commission (Grant 2019-01-07-00-07-E00073 to C.S.).
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Notes
The authors declare no competing financial interest.
ABBREVIATIONS
BCCs, boron-containing compounds; ROS, reactive oxygen species;
CPT, camptothecin; ETO, etoposide; Top1, topoisomerase I; Top2,
topoisomerase II; HPLC, high performance liquid chromatography;
UPLC-QTOF/MS, ultra performance liquid chromatography
quadrupole time-of-flight mass spectrometry; IP, intraperitoneally;
TGI, tumor growth inhibition.
Figure 6. Antitumor activity of compound 13a and TPT in HCT116
tumor xenograft model. (A) Tumor growth inhibiton. (B) The change
of body weight. Data are presented as the mean ± SEM: *P < 0.05, **P
< 0.01, ***P < 0.001 vs vehicle group, determined with Student’s t test.
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Corresponding Author
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N
H
H
Compound 1a
Compound 13a
HCT116: IC50 = 16 nM, in vivo TGI = 64.4%
HCT116: IC50 = 27 nM, in vivo TGI = 30.3%
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