Synthesis and biological evaluation of Complex I inhibitor R419 and its derivatives as anticancer agents in HepG2 cells

Article abstract of DOI:10.1016/j.bmcl.2018.07.006

In this study, Complex I inhibitor R419 was firstly revealed to have significant anticancer activity against HepG2 cells (IC₅₀ = 5.2 ± 0.9 μM). Based on this finding, a series of R419 derivatives were synthesized and biologically evaluated. As results, 9 derivatives were found to have obvious anticancer activity. Among them, H20 exhibited the most potent activity (IC₅₀ = 2.8 ± 0.4 μM). Mechanism study revealed that H20 caused severe depletion of cellular ATP, dose-dependently activated AMPK, decreased Bcl-2/Bax ratio and induced necrotic cell death. Most importantly, H20 displayed definite inhibitory activity against Complex I.

Full text of DOI:10.1016/j.bmcl.2018.07.006

Accepted Manuscript
Synthesis and biological evaluation of Complex I inhibitor R419 and its deriv-
atives as anticancer agents in HepG2 cells
Yaping Huang, Geng Sun, Pengfei Wang, Rui Shi, Yanchun Zhang, Xiaoan
Wen, Hongbin Sun, Caiping Chen
PII:
S0960-894X(18)30558-4
https://doi.org/10.1016/j.bmcl.2018.07.006
BMCL 25937
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 April 2018
22 June 2018
3 July 2018
Please cite this article as: Huang, Y., Sun, G., Wang, P., Shi, R., Zhang, Y., Wen, X., Sun, H., Chen, C., Synthesis
and biological evaluation of Complex I inhibitor R419 and its derivatives as anticancer agents in HepG2 cells,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.07.006
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In this study, Complex I inhibitor R419 was
Synthesis and biological evaluation of
Complex I inhibitor R419 and its
derivatives as anticancer agents in
HepG2 cells
firstly revealed to have significant anticancer
activity against HepG2 cells (IC50 = 5.2±0.9 μM).
Based on this finding, a series of R419
derivatives were synthesized and biologically
evaluated. As results, 9 derivatives were found to
have obvious anticancer activity. Among them,
Yaping Huang, Geng Sun, Pengfei Wang, Rui Shi,
Yanchun Zhang, Xiaoan Wen, Hongbin Sun and Caiping Chen
H20 exhibited the most potent activity (IC50
=
2.8±0.4 μM). Mechanism study revealed that
H20 caused severe depletion of cellular ATP,
dose-dependently activated AMPK, decreased
Bcl-2/Bax ratio and induced necrotic cell death.
Most importantly, H20 displayed definite
inhibitory activity against Complex I.

Bioorganic & Medicinal Chemistry Letters
Synthesis and biological evaluation of Complex I inhibitor R419 and its
derivatives as anticancer agents in HepG2 cells
Yaping Huang ‡, Geng Sun‡, Pengfei Wang, Rui Shi, Yanchun Zhang, Xiaoan Wen, Hongbin Sun* and
Caiping Chen*
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia
Xiang, Nanjing 210009, China
Email: caiping.chen@cpu.edu.cn; hongbinsun@cpu.edu.cn
‡ These authors contributed equally
ARTICLE INFO
ABSTRACT
In this study, Complex I inhibitor R419 was firstly revealed to have significant anticancer
activity against HepG2 cells (IC50 = 5.2±0.9 μM). Based on this finding, a series of R419
derivatives were synthesized and biologically evaluated. As results, 9 derivatives were found to
Article history:
Received
Revised
have obvious anticancer activity. Among them, H20 exhibited the most potent activity (IC50
=
Accepted
2.8±0.4 μM). Mechanism study revealed that H20 caused severe depletion of cellular ATP,
dose-dependently activated AMPK, decreased Bcl-2/Bax ratio and induced necrotic cell death.
Most importantly, H20 displayed definite inhibitory activity against Complex I.
Available online
Keywords:
Complex I
R419
2009 Elsevier Ltd. All rights reserved.
Flavonoids derivatives
Human hepatocellular carcinoma
Cellular ATP for energy consumption in mammalian cells is
mainly produced by mitochondrial oxidative phosphorylation
(OXPHOS). NADH–ubiquinone oxidoreductase (Complex I) is
the first OXPHOS enzyme that mediates mitochondrial electron
transport1 and catalyzes the oxidization of NADH produced in
the tricarboxylic acid (TCA) cycle and in the β-oxidation of fatty
acids to regenerate NAD+ for the mitochondrial matrix.2
Although as revealed by Otto Warburg cancers tend to reprogram
metabolism to produce energy via glycolysis rather than
OXPHOS, some types of cancers still rely on OXPHOS.3-4 Thus,
it is possible to interfere with OXPHOS for treatment of specific
cancers. Complex I inhibitors, e.g. phenformin, metformin,
rotenone and AG311, have been demonstrated to possess
anticancer activity.5-8
is known, flavonoids are abundant in vegetables and fruits,10 and
some flavonoids were reported as potent anticancer active
ingredients in herbal medicines.11-12 Morever, some flavonoids
were reported to inhibit Complex I.13-14 Herein we briefly report
our study results.
Notably, compound R419 (Figure 1) was a newly identified
Complex I inhibitor that improved glucose and lipid homeostasis
in both AMPK-dependent and AMPK-independent fashions.9 To
the best of our knowledge, there was previously no report on the
anticancer activity of R419. Interestingly, we found that R419
showed relatively potent inhibitory activity against HepG2
hepatocellular cells (IC50 = 5.2±0.9 μM) in comparison to L02
normal liver cell line (IC50 = 25.9±6.9 μM) and A549 lung cancer
cell line (IC50 = 19.5±6.8 μM) (supplementary data). Encouraged
by this result, a series of piperidine or piperazine derivatives
bearing flavonoid fragments (H1-H20, Figure 1) were designed
and synthesized to search for more potent anticancer agents. As it
Figure 1. R419 and designed compounds H1-H20.

Scheme 1. Reagent and conditions: (a) K2CO3, DMF, 70 oC, 44-86%; (b) NaOH (1N), MeOH/DCM; (c) amines, CDI, DMF, 70 oC, 13-68%; (d) TFA, DCM; (e)
R4X, K2CO3, acetone, 70 oC, 31-88%.
H
H
Bn
Bn
16.7±0.1
14.0±0.9
7.2±1.0
>20
H1
H2
R419 was synthesized according to the previously reported
method.15 The derivatives H1-H20 were synthesized following
the routes shown in Scheme 1. Treatment of hydroxyl flavonoids
(1)16-18 with 2-bromoacetates (2) in the presence of potassium
carbonate provided flavonoid esters (3), which were hydrolyzed
in 1 N sodium hydroxide methanol solution to afford carboxylic
acids (4). Direct coupling of the carboxylic acids (4) with
appropriate 1-benzylpiperidin-4-amines or 1-benzylpiperazine
amines by CDI in DMF gave piperidine amides (H1, H2, H9,
H11, H14, H16, H19) or piperazine amides (H6, H13, H16).
Alternatively, coupling of 4 with tert-butyl 4-aminopiperidine-1-
carboxylate was followed by de-Boc reaction in TFA to give
piperidines (6), which were further alkylated or acylated by
diverse benzyl halides or benzoyl chloride to achieve the final
targets compounds (H3, H4, H5, H7, H8, H10, H12, H15, H17,
H18, H20).
Me Me
H
H
4-CF3-Bn
4-CF3-Bn
4-OMe-Bn
Bn
H3
Me Me
H4
H
H
H
H
H
H
H
H
H
H
H
H
8.7±0.8
>20
H5
H6
4-CN-Bn
Bz
9.3±0.1
>20
H7
H8
Bn
>20
H9
4-OMe-Bn
Bn
>20
H10
H11
H12
H13
H14
H15
H16
H17
H18
H19
H20
R419
Me Me
>20
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-CN-Bn
Bn
>20
>20
Bn
15.5±0.5
>20
Compounds R419 and H1-H20 were evaluated for their
anticancer activity against HepG2 cells. The IC50 values are
summarized in Table 1. R419 and its 9 derivatives exhibited
significant anticancer activity (IC50 < 20 μM), among which H20
displayed the best activity with IC50 2.8±0.4 μM. SAR analysis
revealed the following features. Firstly, compounds with 4-
substitued benzyl groups were generally more active than those
with 4-unsubstitued benzyl (e.g. H3 vs H1, H17 vs H14).
Secondly, all the compounds with isoflavone fragment (H9-H13)
were all inactive. Thirdly, all the compounds with a piperazine
ring (H6, H13, H16) were also inactive, suggesting that the
piperidine ring is probably an important pharmacophore.
Moreover, benzoyl as the R4 group resulted in complete loss of
activity (H8, H15). Interestingly, electrical change did not take
obvious effect on anticancer activity, as evidenced by that
compounds with EWG (electron withdrawing group) and EDG
(electron donating group) on the benzyl showed similar activity
(e.g. H5 vs H7, H17 vs H18).
Bz
Bn
>20
4-CN-Bn
4-OMe-Bn
Bn
6.7±1.2
6.8±0.6
>20
Me Me
Me Me 4-OMe-Bn
2.8±0.4
5.2±0.9
a HepG2 cells were incubated with vehicle (0.1% DMSO) or compounds for
72 h and cell viability was measured by MTT assay;
b The data represented as means ± SD of three separate experiments.
Interestingly, the most potent compound H20 exhibited good
selectivity for HepG2 cell line over L02 normal liver cell line
(IC50 = 17.6±1.6 μM) and A549 lung cancer cell line (IC50
=
40.0±2.7 μM) (Figure 2). We next selected H20 for further
mechanism study. Cell death analysis was performed through
flow cytometry by Annexin V-FITC/PI assay to determine
whether necrosis or apoptosis dominated the anticancer activity
of H20. As shown in Figure 3, treatment of HepG2 cells with 0.3
μM and 3 μM of compound H20 for 40 hours resulted in 11.0%
and 46.4% cellular necrosis, respectively, remarkably more than
7.0% in DMSO control group. Through flow cytometry, no
apoptosis was observed in cells exposed to H20. This was
further confirmed by western blot assay of the apoptotic markers,
since H20 did not induce caspase 3 and PARP cleavage in
HepG2 cells (supplementary data).
Table 1. Cytotoxicity of R419 and H1-H20 in HepG2 cell
line. a
Cpd
R1
R2
R3
R4
IC50 (μM) b

Figure 2. Cytotoxicity assay of H20 in HepG2 cells, L02 cells and A549 cells.
Figure 3. Cell death analysis in HepG2 cells exposed to compound H20 by Annexin V-FITC/PI assay.
Figure 4. (A) Effect of compound H20 on intracellular ATP levels. (B) Effects of compounds H20 and R419 on AMPK activation and Bcl-2/Bax ratio.
In contrast to apoptosis that ATP is required, necrotic cell
death is a consequence of ATP depletion.19 Therefore, effect of
H20 on intracellular ATP levels was measured. It was found that
exposure to 3 μM of H20 for 44 h caused severe depletion of
cellular ATP levels, while on the contrary low dose 0.3 μM led to
a slight increase despite without statistical significance (Figure
4A). Effects of compound H20 on AMPK phosphorylation and
Bcl-2 family proteins were further studied, as it is known that
increase of AMP/ATP ratio can activate AMPK unconditionally20
and Bcl-2 family proteins play an important role in regulating
cell response to lethal signals by governing the mitochondrial
membrane permeabilization.21-22 As expected, H20 triggered
AMPK activation in HepG2 cell in a dose-dependent manner,
which at 10 μM seemed as potent as R419 (Figure 4B). Bcl-2
protein level was obviously decreased by H20, but Bax not, thus
leading to an increase in mitochondrial membrane
permeabilization and necrotic cell death at low ATP level.
Together, these results indicate that H20 may cause destruction
of mitochondrial energy metabolism.
Mitochondrial oxidative phosphorylation (OXPHOS) not only
produces the majority of ATP, but also participates in regulation
of cell death.23 We therefore sought to verify whether H20-
induced ATP depletion was through Complex I inhibition. H20,
R419 and compound H10 were selected to assess the inhibitory
effect on Complex I. As shown in Figure 5, both H20 and R419
exhibited dose-dependent and moderate inhibitory activity of
Complex I, while H10 with weak anticancer activity had weak
inhibitory activity on Complex I merely at 50 μM. Inconsistent
with the in vitro cytotoxicity against HepG2 cells, the inhibitory
potency of H20 against Complex I was somewhat lower than that
of R419, suggesting that H20-induced dysfunction of OXPHOS
may be through both Complex I-dependent and independent
pathways.

References and notes
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Figure 5. Effects of R419, H20 and H10 on Complex I activity. The
Complex I activity values are represented as percentages of the vehicle group.
The data are analyzed with Student’s t test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001
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In summary, we synthesized and biologically evaluated
Complex I inhibitor R419 and a series of its derivatives. R419
and 9 of its derivatives displayed significant inhibitory activity
against HepG2 cell. Among them, compound H20 was the most
potent one with IC50 value of 2.8±0.4 μM. Flow cytometric
analysis revealed that H20 induced necrotic cell death but not
apoptosis. Further mechanism study showed that H20 caused
ATP depletion, AMPK activation and Bcl-2/Bax ratio decrease in
HepG2 cells. Enzyme activity assay demonstrated that H20,
similar to R419, had moderate potency of Complex I inhibition.
Taken together, H20 may induce destruction of mitochondrial
energy metabolism, at least in part through Complex I-dependent
pathway.
18. Sagrera G, Seoane G. Synthesis, 2010, 2010(16): 2776-2786.
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Acknowledgments
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Financial support from the National Natural Science Foundation
of China (31501182, 81730094, 81473262 and 81573299) is
gratefully acknowledged. This project was also supported by the
“111 Project” from the Ministry of Education of China, the State
Administration of Foreign Expert Affairs of China (No. 111-2-
07) and the Fundamental Research Funds for the Central
Universities (2632017ZD05).
Supplementary Material
Supplementary material is prepared and provided as a separate
electronic file.

1. First report on anticancer activity of Complex I inhibitor
R419
2. Twenty R419 derivatives were synthesized
3. H20 is the most potent compound against HepG2 cells
4. H20 caused cellular ATP depletion and necrosis
5. H20 displayed inhibitory activity against Complex I