Synthesis and biological evaluation of lovastatin-derived aliphatic hydroxamates that induce reactive oxygen species

Article abstract of DOI:10.1016/j.bmcl.2016.10.005

Some hydroxamate compounds induce cancer cell death by intracellular reactive oxygen species (ROS). This study introduced the hydroxamate core into lovastatin, a fungus metabolite clinically used for the treatment of hypercholesterolemia. The resulting co

Full text of DOI:10.1016/j.bmcl.2016.10.005

Accepted Manuscript
Synthesis and biological evaluation of lovastatin-derived aliphatic hydroxa-
mates that induce reactive oxygen species
Ruo-Kai Lin, Yuh-Feng Lin, Ming-Jen Hsu, Chang-Lin Hsieh, Chen-Yu Wang,
Chih-Chiang Huang, Wei-Jan Huang
PII:
S0960-894X(16)31033-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.005
BMCL 24308
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
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20 September 2016
5 October 2016
Please cite this article as: Lin, R-K., Lin, Y-F., Hsu, M-J., Hsieh, C-L., Wang, C-Y., Huang, C-C., Huang, W-J.,
Synthesis and biological evaluation of lovastatin-derived aliphatic hydroxamates that induce reactive oxygen
species, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.005
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Synthesis and biological evaluation of
lovastatin-derived aliphatic hydroxamates
that induce reactive oxygen species
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Ruo-Kai Lin, Yuh-Feng Lin, Ming-Jen Hsu, Chang-Lin Hsieh, Chen-Yu Wang, Chih-Chiang Huang, Wei-Jan
Huang

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and biological evaluation of lovastatin-derived aliphatic hydroxamates
that induce reactive oxygen species
Ruo-Kai Lina,b,c,1, Yuh-Feng Lind,1, Ming-Jen Hsue, Chang-Lin Hsiehb, Chen-Yu Wangf, Chih-Chiang
Huanga, Wei-Jan Huanga,c,g*
aGraduate Institute of Pharmacognosy, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan
bProfessional Master Program in Pharmaceutics and Biotechnology, 250 Wu-Xing Street, Taipei Medical University, Taipei 110, Taiwan
cPh.D. Program for the Clinical Drug Discovery from Botanical Herbs, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan
dDivision of Nephrology, Shuang Ho Hospital, 291 Zong-Zheng Road, New Taipei City 235, Taiwan
eDepartment of Pharmacology, College of Medicine, Taipei Medical University, 250 Wu-Xing Street, Taipei 110, Taiwan
fTaiMed Biologics Inc., 607 Rui-Guang Road, Taipei 114, Taiwan
gSchool of Pharmacy, National Defense Medical Center, 161 Section 6 Min-Quan East Road, Taipei 114, Taiwan
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Some hydroxamate compounds induce cancer cell death by intracellular reactive oxygen species
(ROS). This study introduced the hydroxamate core into lovastatin, a fungus metabolite
clinically used for the treatment of hypercholesterolemia. The resulting compounds were
evaluated for the activity for inducing ROS production. Most compounds exhibited higher
activity than original lovastatin. Of these compounds, compound 3c had the most potent activity.
Test of cytotoxicity in a panel of human cancer cell lines indicated compound 3c had activities
superior to cisplatin in prostate cancer PC-3 cells and breast cancer T47D cells. In contrast, it in
amounts up to 40 µM had a much lower cytotoxic effect on normal human IMR-90 cells. Further
profiling of cell cycle progression, cell apoptosis, and DNA damage activated checkpoint
signaling pathway revealed the important role of compound 3c-mediated cytotoxicity in ROS
generation.
Keywords:
Lovastatin
Fungus metabolite
Reactive oxygen species
Aliphatic hydroxamate
2009 Elsevier Ltd. All rights reserved.
———
*Corresponding author. Tel: +886 2 2736 1661, ext. 6152; fax: +886 2 2735 5276.
E-mail address: wjhuang@tmu.edu.tw (W.-J. Huang)
1These authors contributed equally to this work.

Scheme 2
O
Reactive oxygen species (ROS), which are chemically
reactive molecules containing oxygen, have an essential role in
maintaining the homeostasis of physiological function.1 Moderate
ROS levels regulate cell growth and differentiation, control
HO
O
R
O
O
H
O
HO
N
O
O
O
O
OH
O
R
O
O
a
b
O
O
O
H
H
H
3
enzyme activity, and modulate inflammation.2, In contrast,
excess ROS cause oxidative damage leading to cell death by
reacting with intracellular biomolecules such as DNA, proteins
and lipids.4 Many tumor cell types reportedly have higher ROS
levels compared to their normal counterparts.5-7 Enhanced ROS
production in cancer cells can cause ROS levels to reach the toxic
threshold at which malignant cells death is preferentially induced.
Therefore, agents that promote ROS production may have
therapeutic applications in anticancer strategies.8, 9
1
2a: R=Me (84%)
2b: R=t-Bu (77%)
2c: R=Ph (85%)
2d: R=4-CF3OPh (64%)
2e: R=4-NO2Ph (33%)
2f: R=4-ClPh (64%)
2g: R=2-Naphth (86%)
2h: R=PhCH2 (58%)
2i: R=PhCH2CH2 (43%)
3a: R=Me (62%)
3b: R=t-Bu (62%)
3c: R=Ph (77%)
3d: R=4-CF3OPh (54%)
3e: R=4-NO2Ph (30%)
3f: R=4-ClPh (64%)
3g: R=2-Naphth (31%)
3h: R=PhCH2 (51%)
3i: R=PhCH2CH2 (42%)
c
HO
O
H
HO
N
O
OH
b
OH
O
OH
OH
H
H
Lovastatin (1), a fungal metabolite originally isolated from
Aspergillus terreus and from Monascus rubber,10-12 is widely
used to treat hypercholesterolemia. It competitively inhibits 3-
hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase,
which catalyzes the reduction of HMG-CoA into mevalonate.
This effect then lower cholesterol.13 Lovastatin (1) also blocks
the synthesis of mevalonate-related downstream isoprenoids such
as geranylpyrophosphate and farnesylpyrophosphate, both of
which bind to Ras and its related proteins, and thus perturbs the
function of G-proteins.14, 15 These studies suggest that lovastatin
(1) may have various cell physiological activities that affect cell
growth, including cell proliferation, apoptosis, invasion and
differentiation.16-19 These physiological activities are further
described below. In several tumor cell types, lovastatin (1) is
known to inhibit proliferation, induce apoptosis, and arrest the
cell cycle in G1/S phase.15, 20 Studies reveal that lovastatin (1)
combined with chemotherapeutic drugs synergistically sensitizes
cancer cells to anticancer agents in several tumor cell lines as
well as in-vivo models.21-25 Despite its many anticancer studies
reported, attempts for applying chemical synthesis to improve
lovastatin activity are few.26 Various hydroxamate-containing
compounds reportedly can not only inhibit histone deacetylase
(HDAC), but also induce intracellular ROS production. The
combinatorial effect causes cancer cell death.27-29 To enhance the
antiproliferative activity of lovastatin (1), the hydroxamate motif
that is associated with the ROS production was experimentally
incorporated into the chemical structure of lovastatin (Scheme 1).
We hypothesized that this modification would enhance the ROS
promoting activity of lovastatin (1) in tumor cells. Evaluations of
ROS-inducing ability of all synthesized compounds (3a-j)
showed that compound 3c significantly induced ROS production
in cancer cells, but had no HDAC enzyme inhibitory activity.
Notably, the cytotoxicities of compound 3c in several cancer
cells were higher than those of lovastatin (1). This study then
examined how compound 3c-mediated ROS production affects
cell cycle progression, cell apoptosis, and DNA damage in the
checkpoint signaling pathway.
3j (63%)
4 (73%)
Reagents and condition : (a) Ac2O, Pyr, RT, for 2a, ; RCOCl, Pyr, PhCH3, RT, for 2b-i; (b) 50% NH2OH, THF,
N2, RT; (c) (1) LiOH, MeOH-THF, Δ; (2) PhCH3, Δ.
Lovastatin-based aliphatic hydroxamates were synthesized as
shown in Scheme 2. Lovastatin (1) reacted with the appropriate
acyl chlorides gave 2a-i, respectively. Reaction of compounds
2a-i with NH2OH provided the corresponding hydroxamates 3a-i.
Basic saponification of lovastatin (1) in the presence of LiOH
and subsequent lactonization gave 4. Compound 4 was reacted
with NH2OH to provide 3j.
Figure 1 shows that flow cytometric analysis was performed
using DCFH-DA fluorescent probe in human breast cancer
MDAMB-231 cells to test all lovastatin-derived compounds 3a-j
synthesized in this study for activity that induced ROS
production. Additionally, two positive controls doxorubicin30 and
cisplatin31 that reported increased ROS production in cells were
included in the test. Comparisons of compounds 3a-j and
lovastatin indicated that the activities of all compounds were
improved compared to that of original lovastatin. However, these
compounds exhibited no HDAC-inhibiting activity (Figure 1S).
Most compounds, except for compound 3j, had higher ROS
production activity than did cisplatin. Notably, the activities of
compound 3c and 3g were strongest. The activities of benzoyl
substituted series 3c-g were higher than those of both compounds
3a with acetyl group and 3b with t-butanoyl group, which
suggested that the benzoyl moiety positively contributed to
activity. Comparisons of substituted benzoyl aliphatic
hydroxamates 3d-g showed that compound 3g had the highest
potency. However, its potency was comparable to that of
compound 3c, which indicated that the substituent on phenyl ring
only slightly increased ROS production. Next, compounds 3h-i
with one to two carbons chain-length of linker attached to phenyl
ring were synthesized. These compounds had equally potent
activities. Moreover, compound 3j had only weak activity. These
experimental results speculated that the weak ROS-inducing
activities of compounds 3a, 3b, and 3j were possibly caused by
their poor cell membrane permeability and the weak electron-
donating ability of the substituent on the β-position of these tree
compounds.
HO
O
H
O
HO
N
O
O
OH
O
O
O
O
O
H
H
R
The results of growth inhibition against human prostate PC-3
cells indicated that the cytotoxicities of three of these
compounds, 3c, 3h and 3i, equaled or exceeded that of lovastatin
(Table 1S). Moreover, their activities were much higher than that
of cisplatin. In particular, the antiproliferative activity of
compound 3c was highest.
Lovastatin
3a-i
Scheme 1. Design of lovastatin-derived hydroxamates 3a-i

Figure 2. Cell viability of the breast cancer cell lines MCF7, T47D, MDA-
MB-231, and the normal cell line IMR-90 treated with compound 3c
lovastatin, and cisplatin. The MTT assay was used to measure growth in the
T47D (A), MCF7 (B), and MDA-MB-231 (C) cancer cell lines and in the
IMR-90 normal lung cell line (D) after the cells had been treated with drugs
for 48 hr. The data were presented as the means s.d. All experiments were
performed with three technical replicates.
In further experiments to identify potential anti-breast cancer
agents, compound 3c with significant growth inhibition was
selected for the evaluation of antiproliferative activity against a
panel of human breast cancer cell lines such as MDA-MB-231,
MCF7 and T47D cells using cisplatin as a positive control. In
T47D and MCF7cancer cell lines, compound 3c exhibited higher
cytotoxicities (IC50=30-40 μM) than did lovastatin (IC50>40 μM)
(Figure 2A-C). Compared to cisplatin, the antiproliferative
activities of compound 3c against MDA-MB-231 and MCF7
were comparable, but its activity against T47D cells was higher.
In contrast, compound 3c even in amounts up to 40 μM exhibited
lower cytotoxicity for normal IMR-90 cells compared to cisplatin
(Figure 2D). These experimental results indicated that compound
3c may have the therapeutic index superior to cisplatin.
Reactive oxygen species are a major source of DNA damage
because they can modify DNA, indirectly induce lesions, and
affect cell viability. Therefore, we investigated whether
compound 3c promotes DNA damage in MDA-MB-231 cells by
using phosphorylation of γH2AX as a DNA damage marker.32
Figure 3A shows that compared to lovastatin, compound 3c
significantly increased the severity of DNA damage for 48 h
(Figure 3A). This further confirmed that compound 3c markedly
enhanced phosphorylation for chk1 as well as chk2, two protein
kinases in the checkpoint signaling pathways associated with
DNA damage.33 Figure 3B shows that the effects are superior to
those of doxorubicin, a known topoisomerase II inhibitor used to
induce DNA damage.34 However, lovastatin revealed no activity
in these two proteins.
Figure 1. Effects of compounds 3a-j on intracellular ROS concentrations.
MDAMB-231 cells were treated with vehicle, lovastatin (30 µM),
compounds 3a-j (10 µM), cispaltin (10 µM) or doxorubicin (5 µM) for 24
h. The intracellular ROS level was then analyzed by flow cytometry
performed with a DCFH-DA fluorescent probe. Results shown in the upper
panel are representative of at least four independent experiments. Complied
results are show in the bottom panel of the chart. Each column represents
the mean S.E.M. of at least four independent experiments (*p < 0.05,
compared with the control group).
Figure 3. Compound 3c induced DNA damage without DNA binding. (A)
The DNA damage, (B) protein levels of cell signaling pathways for DNA
damage and cell cycle, and (C) drug-DNA binding were analyzed in breast
cancer MDA-MB-231 cells treated with compound 3c (10 µM), lovastatin
(10
µ
M) or doxorubicin (5 µM). The Notr was the control with no treatment
medium, and DMSO was the solvent control.

F
igur
e 3
B s
hows
that compound 3
c was furth
er eval
uated
for
the phosphorylation of upstream ATR and ATM as well as of
downstream p53 associated with the activation of chk1 and chk2
signaling. The experimental results indicated that the
phosphorylation of ATM and p53 induced by compound 3c was
significantly increased compared to that of lovastatin. Figure 3B
shows that compound 3c was comparable to lovastatin in terms
of promoting p53 phosphorylation. Compound 3c also enhanced
the expression of p21 protein that is regulated by its counterpart
gene p21WAF1 serving as a downstream of p53. To investigate
whether compound 3c-induced DNA damage by directly binding
to DNA, this study further analyzed DNA minor groove-binding
in compound 3c. Figure 3C shows that compound 3c revealed no
DNA binding activity even at 100 µM, which indicated that its
DNA damage effect was independent of its interaction with
DNA.
Different concentrations of compound 3c were analyzed by
flow cytometry and by cleaved PARP detection to investigate
whether it induces cancer cell apoptosis. Cell apoptosis caused by
compound 3c was higher than that caused by lovastatin, and the
effect was concentration-dependent (Figure 4A). Compound 3c at
32 µM was then examined by flow cytometry combined with PI
staining at various time courses (6, 12, 24, 48h) in MDA-MB-
231 cells. Figure 4B shows that compound 3c caused cell arrest
in the S phase of the cell cycle progression at 6h and 12h.
In conclusion, this study synthesized a series of novel
aliphatic hydroxamates derived from lovastatin and disclosed
their SAR in ROS-inducing activity. Of these, the cytotoxicities
of compound 3c were improved compared to those of lovastatin
in several human cancer cells. The effects of compound 3c
mediated-ROS on cell cycle arrest, cell apoptosis, and DNA
damage activated by checkpoint signaling pathway indicated that
compound 3c induced significant DNA damage in MDA-MB-
231 cell, which then caused cell death. Further studies of
structural modifications that enhance antiproliferative activity are
now underway.
Acknowledgments
We gratefully acknowledge the financial support from the
Ministry of Science and Technology (NSC101-2320-B-038-026-
MY3 and NSC102-2320-B-038-019-MY3) and the Taipei
Medical University-Shuang Ho Hospital, Ministry of Health and
Welfare (100TMU-SHH-01-1) in Taiwan.
Figure 4. Compound 3c induced apoptosis annd cell cyycle
a
a
rrest. (A) Cell
apoptosis and (B) cell cycle distribution were analyzed in bbreast cancer
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Supplementary Material
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