Discovery of novel multiacting topoisomerase I/II and histone deacetylase inhibitors

Article abstract of DOI:10.1021/ml500327q

Designing multitarget drugs remains a significant challenge in current antitumor drug discovery. Because of the synergistic effect between topoisomerase and HDAC inhibitors, the present study reported the first-in-class triple inhibitors of topoisomerase I/II and HDAC. On the basis of 3-amino-10-hydroxylevodiamine and SAHA, a series of hybrid molecules was successfully designed and synthesized. In particular, compound 8c was proven to be a potent inhibitor of topoisomerase I/II and HDAC with good antiproliferative and apoptotic activities. This proof-of-concept study also validated the effectiveness of discovering triple topoisomerase I/II and HDAC inhibitors as novel antitumor agents.

Full text of DOI:10.1021/ml500327q

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Letter
Discovery of Novel Multi-acting Topoisomerase
I/II and Histone Deacetylase Inhibitors
Shipeng He, Guoqiang Dong, Zhibin Wang, Wei Chen, Yahui Huang, Zhengang Li, Yan
Jiang, Na Liu, Jianzhong Yao, Zhenyuan Miao, Wannian Zhang, and Chunquan Sheng
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml500327q • Publication Date (Web): 14 Jan 2015
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Discovery of Novel Multi-acting Topoisomerase I/II and His-
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Shipeng He,$,† Guoqiang Dong,$, ≠ Zhibin Wang,≠ Wei Chen,≠ Yahui Huang,≠ Zhengang Li,≠ Yan
Jiang≠, Na Liu,≠ Jianzhong Yao,≠ Zhenyuan Miao,≠ Wannian Zhang≠ and Chunquan Sheng†,≠,*
†School of Pharmacy, Fujian University of Traditional Chinese Medicine, 1 Qiuyang Road, Fuzhou, Fujian 350122, P.
R. China
≠School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of
China
KEYWORDS: Evodiamine derivatives, Topoisomerase I, Topoisomerase II, Histone Deacetylase, Antiproliferative ac-
tivity
ABSTRACT: Designing multi-target drugs remains a significant challenge in current antitumor drug discovery. Due to
synergistic effect between topoisomerase and HDAC inhibitors, the present study reported the first-in-class triple inhibi-
tors of topoisomerase I/II and HDAC. On the basis of 3-amino-10-hydroxylevodiamine and SAHA, a series of hybrid mole-
cules were successfully designed and synthesized. In particular, compound 8c was proven to be a potent inhibitor of
topoisomerase I/II and HDAC with good antiproliferative and apoptotic activities. This proof-of-concept study also vali-
dated the effectiveness of discovering triple topoisomerase I/II and HDAC inhibitors as novel antitumor agents.
Over the last two decades, drug discovery has been
primarily focused on the development of single-target
drugs with high potency and selectivity.1 However, not all
the diseases are amenable to this one disease-one target
approach. Also, these drugs are not as effective as ex-
pected for the treatment of complex diseases, such as
cancer. Recent evidence has shown that most single-
target drugs are plagued by toxic side effects and devel-
opment of resistance. To overcome these problems, de-
signing a single drug molecule that interacts with multi-
ple targets simultaneously and specifically is gaining con-
siderable interests in drug discovery.2-5
FK228 (romidepsin), have been approved by FDA for the
treatment of the cutaneous T-cell lymphoma (CTCL).10, 11
In many reports, HDACi can synergistically enhance the
inhibitory effect of other antitumor agents, such as tubu-
lin12, Hsp9013, EGFR14 and topoisomerase15-17 inhibitors, to
suppress proliferation and induce apoptosis in tumor cells.
The synergistic effects of HDAC inhibitors with other
antitumor agents can be used as a useful strategy to de-
sign multifunctional inhibitors to simultaneously interact
with multiple targets with high potency and low toxicity.
Among these targets, topoisomerase (Top1 and/or Top2)
is a good starting point for multivalent ligand design.8, 18 It
was reported that the resistance to Top2 inhibitors is of-
ten concomitant with a rise in the level of Top1 expression
and vice versa.19 In this regard, a single molecular able to
inhibit Top1/Top2/HDAC could be helpful to prevent
mechanism-based drug resistance and show more power-
ful antitumor activity. However, the discovery of a single
molecule targeting three proteins remains a significant
challenge and no HDAC1/Top1/Top2 triple inhibitors have
been reported up to date.
Cancer offers a unique opportunity for the multivalent
ligand design because of the complicated target networks
and multiple cellular pathways contributing to the disease
state. Epigenetic control, such as protein acetylation and
deacetylation state, is regulated by histone acetyl trans-
ferases (HATs) and histone deacetylases (HDACs). Tar-
geting HDACs represents one of the most popular ap-
proaches for tumor growth inhibition.6, 7 HDACs regulate
the acetylation of a diverse range of histone and nonhis-
tone proteins, controlling the transcription and regula-
tion of genes as well as cell proliferation, migration, death
and angiogenesis, and have been recognized as important
9
molecular targets for cancer treatment.8, Two HDAC
inhibitors (HDACi), SAHA (vorinostat) (Figure 1) and
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profiles.25 Thirdly, our previous study indicated that the
3
4
5
6
C-10 hydroxyl group of evodiamine was important in
maintaining antitumor potency. In order to validate the
importance of this hydroxyl group in the newly designed
hybrid molecules, a series of 10-methoxyl derivatives (10a-
d) were also designed and synthesized.
7
8
9
Scheme 1. Chemical synthesis of compound 7
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Figure 1. Chemical structure of SAHA, evodiamine and dual
Top1/Top2 inhibitor 1
Evodiamine (Figure 1) is a quinazolinocarboline alka-
loid isolated from the fruits of traditional Chinese herb
Evodiae fructus (Chinese name: Wu-Chu-Yu) with diverse
biological activities.20 In our previous study, a number of
evodiamine derivatives were designed and synthesized.21,
22
Among them, 3-amino-10-hydroxylevodiamine (1)
Reagents and conditions: (a) HBTU
(O-Benzotriazole-
showed excellent antitumor activity against a variety of
cancer cell-lines with good in vivo potency in xenograft
nude mice. Moreover, compound 1 was proven to be a
dual Top1/Top2 inhibitor by in silico target identification
in combination with biological assays. Inspired by these
results, compound 1 and SAHA can be used as good tem-
plates to design triple HDAC/Top1/Top2 inhibitors.
N,N,N,N-tetramethyl-uronium-hexafluorophosphate),
Et3N, MeCN, rt , 2 h, 76%; (b) PCC, rt, 2 h, 95%; (c) NaBH3CN,
THF, rt, 3 h, 46%; (d) KOH, CH3OH, 40 °C, 45 min, 89%.
The synthetic routes of the target compounds are
shown in Schemes 1-4. The commercially available com-
pound 2 was reacted with monomethyl suberate to give
intermediate 4,26 which was further oxidized by pyridini-
um chlorochromate (PCC) to afford aldehyde 5. Treat-
ment of compound 5 and 1 via reductive amination reac-
tion yielded the key intermediate 6, which was reacted
with freshly prepared hydroxylamine methanol solution
to give the target compound 7 (Scheme 1).
Scheme 2. Chemical synthesis of compounds 8a-c
Figure 2. Design of triple-acting Top1/Top2/HDAC inhibi-
tors
As depicted in Figure 2, a series of novel evodiamine-
SAHA hybrids were rationally designed and synthesized
as triple-targeting antitumor agents. Firstly, using a mo-
lecular hybridization strategy, compound 1 and SAHA
were merged into a new hybrid molecule (7). From struc-
ture-activity relationship studies on evodiamine deriva-
tives21-23, substitution at the 3-amino group was tolerable.
Thus, SAHA was attached at this position. Meanwhile,
due to the presence of large hydrophobic patches at the
HDAC surface rim, conjugating SAHA with hydrophobic
Reagents and conditions: (a) propane-1,3-diol, toluene, reflux,
6 h, 95%; (b) NH2OH—HCl, NaHCO3, CH3OH, reflux, 2 h, 76%;
(c) HO2C(CH2)nCO2CH3(n = 3, 4, 6), HBTU, DIPEA, DMF,
microwave, 191 °C, 2 min; (d) Fe(HSO4)3, acetone:H2O = 1:1,
reflux, 1 h, 85-86%, over two steps; (e) 1, NaBH3CN, CH3OH,
rt, 3 h, 45%; (f) KOH, CH3OH, 45 °C, 45 min, 90-92%.
antitumor agent
1
may generate potent HDAC
inhibitors.24 Secondly, 1,2,4-oxadiazoles and 1,3,4-
oxadiazoles were introduced as a proper spacer between
the evodiamine scaffold and the zinc binding group of
SAHA (compounds 8a-c and 9a-c). Oxadiazole was cho-
sen as the spacer because it is a drug-like privileged struc-
ture in many therapeutic drugs and always used as a flat,
aromatic linker to place substituents in the appropriate
orientation for ligand binding.25 Moreover, introduction
of 1,3,4-oxadiazole ring was proven to be an effective
strategy to modulate lipophilicity and pharmacokinetic
Scheme 3. Chemical synthesis of compounds 9a-c
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O
O
O
group of 14a-c yielded the requisite aldehydes 15a-c using
a
b
c
O
O
O
O
3
O
O
O
freshly prepared Fe(HSO4)3 solution. Then, the synthesis
of target compounds 8a-c was similar to that of com-
pound 7. Synthesis of compounds 9a-c was summarized
in Scheme 3. Protection of aldehyde 17 with 1,3-
propanediol gave the intermediate 18, which was reacted
with hydrazine hydrate to yield compound 19. Acylation
of 19 provided the amides 20a-c, which were cyclized to
give key intermediates 21a-c. Then, compounds 9a-c were
synthesized using conditions similar to that of com-
pounds 8a-c. As depicted in Scheme 4, compounds 10a-d
were prepared from 3-amino-10-methoxylevodiamine (24)
by a two-step synthesis via the condensation and ammo-
nolysis reaction.
O
O
O
HN
O
HN
O
4
5
6
7
8
N
H
O
NH2
18
19
17
20a-c
O
O
d
O
O
O
O
O
O
O
O
N
O
O
f
e
N
HO
N
n
O
n
H
N
N
N
N
N
N
N
H
21a-c
22a-c
23a-c
9
10
11
12
13
14
15
HO
g
NH2OH HCl
NH
N
O
O
O
N
HO
N
H
N
N
N
H
n = 3, 4, 6
9a-c
Reagents and conditions: (a) propane-1,3-diol,toluene, reflux,
h, 93%; (b) N2H4—H2O, rt, 24 h, 73%; (c)
16
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Table 2. Inhibitory Activity of HDAC6 and HDAC8
6
HO2C(CH2)nCO2CH3(n = 3, 4, 6), HBTU, Et3N, DMF, rt, 2 h,
72%; (d) p-toluenesulfonyl chloride, Et3N, THF, reflux, 8 h,
78%; (e) Fe(HSO4)3, acetone:H2O=1:1, reflux, 1 h, 59-69%; (f) 1,
NaBH3CN, CH3OH, rt, 3 h; (g) KOH, CH3OH, 45 °C, 45min,
84-91%, over two steps.
HDAC2
IC50 (nM)
275 ± 52
174 ± 70
HDAC3
IC50 (nM)
71 ± 6.4
56 ± 4.6
HDAC6
IC50 (nM)
13 ± 1.7
23 ± 2.1
HDAC8
IC50 (µM)
2.5 ± 0.29
8.8 ± 0.70
Compd
8c
SAHA
Initially, we tested the inhibitory activity of the target
compounds against human recombinant HDAC1 enzyme
using the assay as previously described by Bradner et al. 27
Generally, most compounds showed good to excellent
HDAC1 inhibitory activity (Table 1). For example, com-
pound 7 was highly active against HDAC1 (IC50 = 30 nM).
Interestingly, all the 1,2,4-oxadiazole derivatives (8a-c)
potently inhibited HDAC1 with IC50 values in the nano-
molar range. Moreover, it was observed that the HDAC1
inhibitory activity was enhanced with the increasing of
the carbon chain length (8c > 8b > 8a, 9c > 9b > 9a, 10d >
10b > 10a)with the exception of compound 10c. Com-
Scheme 4. Chemical synthesis of compounds 10a-d
Reagents and conditions: (a) HBTU, Et3N, DMF, rt, 2 h, 63-
75%; (b) KOH, CH3OH, 45 °C, 45 min, 83-90%.
Table 1. In Vitro HDAC1 Inhibition and Antitumor Activity of Target Compounds
HDAC1
MDA-MB-231
IC50 (µM)
HCT116
IC50 (µM)
HLF
IC50 (µM)
Compd
IC50 (nM)
NTa
0.49 ± 0.02
7.4 ± 0.65
14 ± 1.0
16 ± 1.9
5.9 ± 0.43
2.3 ± 0.14
>168
30 ± 3.4
5.7 ± 0.61
191 ± 20
36 ± 3.1
58 ± 4.5
54 ± 5.7
0.43 ± 0.03
3.7 ± 0.30
2.1 ± 0.26
3.5 ± 0.39
3.7 ± 0.28
0.41 ± 0.03
11 ± 2.4
4.9 ± 0.32
9.7 ± 1.2
31 ± 3.1
0.47 ± 0.03
3.0 ± 0.33
13 ± 1.5
6.7 ± 0.48
8.2 ± 0.75
1.3 ± 0.10
>168
26 ± 3.0
6.1 ± 0.55
97 ± 10
1
23 ± 2.1
30 ± 4.5
321 ± 21
190 ± 13
24 ± 3.2
>10000
93 ± 5.7
89 ± 7.9
527 ± 45
275 ± 23
>10000
64 ± 5.5
SAHA
7
8a
8b
8c
9a
9b
9c
10a
10b
22 ± 2.1
35 ± 4.1
20 ± 2.3
62 ± 5.6
68 ± 6.7
30 ± 3.8
10c
10d
aNT = not tested.
Similar strategy was applied for the synthesis of 1,2,4-
oxadiazole-based conjugates (8a-c) (Scheme 2). Acetali-
zation of the 4-formylbenzonitrile 11 afforded the pro-
panediol-protected compound 12, which was treated by
hydroxylamine hydrochloride in the presence of NaHCO3
and methanol to give compound 13 in good yield. Con-
densation of compound 13 with one of three carboxylic
acid monomethyl esters under microwave conditions af-
forded compounds 14a-c. Deprotection of 1,3-propanediol
pounds 9c and 10d exhibited good anti-HDAC1 activity
with the IC50 values of 89 nM and 64 nM, respectively. In
particular, compound 8c, the best HDAC1 inhibitor (IC50
= 24 nM), showed comparable activity to SAHA (IC50 = 23
nM). To obtain evidence for the HDAC isoform selectivity,
the inhibitory activity of compound 8c was examined
against selected recombinant HDAC2, HDAC3, HDAC6
and HDAC8 (Table 2). Compound 8c displayed nanomo-
lar activity against HDAC2 (IC50 = 275 nM), HDAC3 (IC50
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= 71 nM)and HDAC6 (IC50 = 13 nM). In contrast, its activi-
ty against HDAC8 (IC50 = 2.5 µM) was significantly de-
creased. As compared with SAHA, compound 8c was
more potent against HDAC6 and HDAC8.
these compounds showed better activity against HCT116
than the other two cell-lines. Increasing the length of
methylene chain of 1,2,4-oxadiazole derivatives led to the
improvement of antiproliferative activities, which was
closely correlated to their anti-HDAC activities. This
trend was also observed in 1,3,4-oxadiazole and 10-
methoxyl derivatives, respectively. In particular, com-
pound 8c showed the most potent antitumor activity with
IC50 values in the range of 0.41 μM to 2.29 μM. For the
HCT116 cell line, compound 8c was significantly more
active than the reference drug SAHA and comparable to
lead compound 1. Thus, it was subjected to an apoptotic
assay for further evaluation. Apoptosis was evaluated by
annexin V test.22 As shown in Figure 4, compound 8c
exhibited strong pro-apoptotic activity in the HCT116 cell
line. After treating with 5 μM of the compound for 24 h
and 48 h, the percentage of apoptotic cells for compound
8c was 39.32% and 79.1% (Q2 + Q4), respectively. It dis-
played much higher apoptosis level than compound 1 and
SAHA (11.09% and 16.39% for 1 and 25.54% and 46.81% for
SAHA, respectively). This result demonstrated that com-
bination topoisomerase and HDAC inhibitor in one single
molecular led to synergy at the cellular level. On the basis
of the above results, compound 8c was subjected for fur-
ther evaluation of cellular HDAC inhibition. The effect of
the compound 8c on the acetylation level of histone 3 is
shown in Figure 5. Exposure to compound 8c for 24 h
and 48 h induced a hyperacetylation of histone 3 in the
HCT116 cell line.
Previously, compound 1 and other evodiamine deriva-
tives were identified as dual Top1/Top2 inhibitors.22 Top1-
and Top2α-mediated pBR322 DNA relaxation assays with
purified Top1 and Top2α were used to evaluate the inhibi-
tory activity of the target compounds. Camptothecin
(CPT, Top1 inhibitor) and etoposide (Eto, Top2 inhibitor)
were employed as positive controls. As shown in Figure
3A, all the tested compounds were found to be active
against Top1-mediated DNA relaxation. Most of them
exhibited comparable or superior Top1 inhibitory activity
to CPT. The 1,2,4-oxadiazole derivatives (8a-c) showed
higher Top1 inhibitory activity than CPT. The best inhibi-
tion was observed for compound 8c containing the six
methylene alkyl chain. In contrast, the 10-methoxyl deriv-
atives (10a-c) showed weak inhibitory activity and com-
pound 10c was almost inactive. Similarly, all the tested
compounds were active against Top2 (Figure 3B). As
compared to Eto, compounds 7, 8b-c, 10a and 10c showed
stronger Top2 inhibitory activity. Among them, com-
pound 7 was the most active one. Interestingly, the activi-
ty of 1,2,4-oxadiazole derivatives was dependent on the
length of methylene chain. Reducing the alkyl chain to
three carbon atoms (8a) resulted in decrease of Top2 in-
hibitory activity.
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Figure 3. Top1 and Top2 inhibitory activity of target com-
pounds. (A) Inhibition of Top1 relaxation activity at 50 µM.
Lane 1, supercoiled plasmid DNA; Lane 2, DNA + Top1; Lanes
3, DNA + Top1 + CPT (50 µM); Lanes 4-14, DNA + Top1 +
compounds (1, 7, 8a-c, 9a-c, 10a-c at 50 µM). (B) Inhibition
of Top2 relaxation activity at 50 µM. Lane 1, supercoiled
plasmid DNA; Lane 2, DNA + Top2; Lanes 3, DNA + Top2 +
Eto (50 µM); Lanes 4-14, DNA + Top2 + compounds (1, 7, 8a-c,
9a-c, 10a-c at 50 µM). The assay was repeated 3 times.
Figure 4. Cell apoptosis induced by compound 1, 8c and
SAHA. HCT116 cells were treated with DMSO and 5 μM of
compounds for 24 and 48 h, respectively. Apoptosis was ex-
amined by flow cytometer (n = 3).
On the basis of the above results, the hybrid com-
pounds derived from 1 and SAHA were proven to be tri-
ple-targeting inhibitors of HDAC/Top1/Top2. Particularly,
compound 8c generally exhibited the highest activity.
Furthermore, we investigated the antiproliferative activi-
ties of the target compounds against MDA-MB-231 (breast
cancer), HCT116 (colon cancer) and HLF (liver cancer) cell
lines using parent compounds 1 and SAHA as reference
drugs. The antitumor activity was determined using the
standard MTT assay.21 As shown in Table 1, most of the
target compounds showed moderate to good antiprolifer-
ative activity against all the three cell lines. Moreover,
Figure 5. Western blot probing for acetylated histones H3
in the HCT116 cell line after 24 h treatment with com-
pounds. Lanes: (1) control, (2) SAHA, 2.5 μM, (3) SAHA
5.0 μM, (4) 8c, 2.5 μM, (5) 8c, 5 μM.
In summary, a series of novel evodiamine/SAHA hy-
brids were rationally designed and synthesized on the
basis of synergistic effect observed between topoisomer-
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gether, the present study provided a proof-of-concept
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ASSOCIATED CONTENT
Supporting Information
Chemical synthesis and structural characterization of the
target compounds; Protocols of biological assays. This mate-
rial is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*
(C.S.)
Phone/fax:
86-21-81871239;
E-mail:
shengcq@hotmail.com.
Author Contributions
$ S.H. and G.D. contributed equally to this work.
Funding Sources
(14) Lai, C. J.; Bao, R.; Tao, X.; Wang, J.; Atoyan, R.; Qu, H.;
Wang, D. G.; Yin, L.; Samson, M.; Forrester, J.; Zifcak, B.; Xu,
G. X.; DellaRocca, S.; Zhai, H. X.; Cai, X.; Munger, W. E.;
This work was supported by National Natural Science
Foundation of China (Grants 81222044, 81373278), Key
Project of Science and Technology of Shanghai (Grant
11431920402, 14YF1405400), the 863 Hi-Tech Program of
China (Grant 2014AA020525) for financial support.
Notes
Keegan, M.; Pepicelli, C. V.; Qian, C. CUDC-101,
a
multitargeted inhibitor of histone deacetylase, epidermal
growth factor receptor, and human epidermal growth factor
receptor 2, exerts potent anticancer activity. Cancer. Res.
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increases cytotoxicity to anticancer drugs targeting DNA.
Cancer. Res. 2003, 63, 7291-7300.
(16) Catalano, M. G.; Fortunati, N.; Pugliese, M.; Poli, R.;
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acid, a histone deacetylase inhibitor, enhances sensitivity to
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(17) Bevins, R. L.; Zimmer, S. G. It's about time: scheduling
alters effect of histone deacetylase inhibitors on
camptothecin-treated cells. Cancer. Res. 2005, 65, 6957-6966.
(18) Guerrant, W.; Patil, V.; Canzoneri, J. C.; Oyelere, A. K.
Dual targeting of histone deacetylase and topoisomerase II
with novel bifunctional inhibitors. J. Med. Chem. 2012, 55,
1465-1477.
(19) Salerno, S.; Da Settimo, F.; Taliani, S.; Simorini, F.; La
Motta, C.; Fornaciari, G.; Marini, A. M. Recent advances in
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anticancer drugs. Curr. Med. Chem. 2010, 17, 4270-4290.
(20) Jiang, J.; Hu, C. Evodiamine: a novel anti-cancer alkaloid
from Evodia rutaecarpa. Molecules 2009, 14, 1852-1859.
(21) Dong, G.; Sheng, C.; Wang, S.; Miao, Z.; Yao, J.; Zhang,
W. Selection of evodiamine as a novel topoisomerase I
inhibitor by structure-based virtual screening and hit
optimization of evodiamine derivatives as antitumor agents.
J. Med. Chem. 2010, 53, 7521-7531.
The authors declare no competing financial interest.
ABBREVIATIONS
Top1, topoisomerase I; Top2, topoisomerase II; HDAC, his-
tone deacetylase; HDACi, HDAC inhibitors; PCC, pyridinium
chlorochromate;
HBTU,
O-Benzotriazole-N,N,N,N-
tetramethyl-uronium-hexafluorophosphate; CPT, Camptoth-
ecin; Eto, etoposide.
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Lay Summary
According to the synergistic effect observed between topoisomerase and HDAC inhibitors, a se-
ries of novel evodiamine/SAHA hybrids were rationally designed and synthesized. They were
identified as the first-in-class triple inhibitors of Top1/Top2/HDAC. Notably, compound 8c was
proven to be a potent inhibitor of Top1/Top2/HDAC, which also showed good antiproliferative
activities and high apoptosis level, and induced a hyperacetylation of histone 3 in HCT116 cell
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line. This study provided
a
proof-of-concept study for discovering triple-acting
Top1/Top2/HDAC inhibitors as novel antiproliferative agents.
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