Dual-acting histone deacetylase-topoisomerase i inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.03.108

Current chemotherapy regimens are comprised mostly of single-target drugs which are often plagued by toxic side effects and resistance development. A pharmacological strategy for circumventing these drawbacks could involve designing multivalent ligands th

Full text of DOI:10.1016/j.bmcl.2013.03.108

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3283–3287
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dual-acting histone deacetylase-topoisomerase I inhibitors
William Guerrant a, , Vishal Patil a, , Joshua C. Canzoneri a, , Li-Pan Yao a, Rebecca Hood a,
Adegboyega K. Oyelere a,b,
⇑
a School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA
b Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Current chemotherapy regimens are comprised mostly of single-target drugs which are often plagued by
toxic side effects and resistance development. A pharmacological strategy for circumventing these draw-
backs could involve designing multivalent ligands that can modulate multiple targets while avoiding the
toxicity of a single-targeted agent. Two attractive targets, histone deacetylase (HDAC) and topoisomerase
I (Topo I), are cellular modulators that can broadly arrest cancer proliferation through a range of down-
stream effects. Both are clinically validated targets with multiple inhibitors in therapeutic use. We
describe herein the design and synthesis of dual-acting histone deacetylase–topoisomerase I inhibitors.
We also show that these dual-acting agents retain activity against HDAC and Topo I, and potently arrest
cancer proliferation.
Received 11 December 2012
Revised 25 March 2013
Accepted 27 March 2013
Available online 4 April 2013
Keywords:
Histone deacetylase
Histone deacetylase inhibitors
Topoisomerase I
Published by Elsevier Ltd.
Camptothecin
Suberoylanilide hydroxamic acid
Current chemotherapeutic options for the treatment of cancer
are often plagued by debilitating side effects and off-target toxici-
ties. While other pharmacological options, such as gene or immu-
notherapies, are attaining increasing viability for researchers, most
clinical options still center on traditional small molecule chemo-
therapy. There is considerable interest in designing novel small
molecule agents that retain efficacy, while increasing the specific-
ity toward the target of choice, thereby reducing side effects. While
single-target drugs remain a popular design endpoint, there has
been a recent surge of interest toward multivalent ligand design.
It is thought that these drugs could possess a greater therapeutic
advantage, by modulating multiple targets and avoiding the side
effects of any single agent. Additionally, multivalent ligands are
not expected to face the inherent pharmacokinetic and pharmaco-
dynamics disadvantages of administering two or more separate
drugs, a common liability that may complicate the outcome of tra-
ditional combination therapy.1 The benefit of drugs with multiple
targets relative to the conventional combination therapies has only
begun to be elucidated, and these therapies are becoming increas-
ingly common across a variety of pharmacological applications.1–5
Cancer offers a unique opportunity for the design of a multi-
functional drug due to the multiple pathways contributing to the
disease state. One promising pathway for tumor growth inhibition
is that of epigenetic and protein acetylation state modulation by
histone deacetylases (HDACs). HDACs function within a pathway
that was originally discovered to alter the acetylation of histone
proteins, leading to a more condensed nucleosome and decreased
transcription.6,7 The counterpart enzyme, histone acetyltransferase
(HAT), has the opposite effects; acetylating histones and upregulat-
ing transcription.8 The proposed cancer-promoting mechanism of
HDAC involves transcriptional silencing of tumor suppressors via
deacetylation of nucleosomes containing tumor suppressor
genes.9,10 However, recent evidence has shown HDAC involvement
in the deacetylation of important non-histone regulatory proteins
such as p53,11 E2F,12 and tubulin.13 HDACs inhibitors (HDACi) have
been shown to cause growth arrest, differentiation, and apoptosis
in cancer cells.14–16 Two HDACi, SAHA (Vorinostat) (Fig. 1) and
FK-228 (Romidepsin), have been approved by the FDA for the
treatment of cutaneous T-cell lymphoma,17,18 thus opening the
door for HDACi as viable therapeutic agents.19,20 For these reasons,
HDACs remain an attractive target for small molecule inhibition.
Another proven anticancer target is topoisomerase I (Topo I).
The Topo I enzyme relieves the torsional strain on DNA during
DNA replication by cutting one strand of the DNA double helix
and passing one strand over the other.21,22 Due to the inherent
need for rapid replication in cancer, inhibitors of topoisomerases
result in DNA strand breaks, cell cycle arrest, and apoptosis.23–27
Many small molecule inhibitors of Topo I have proven clinically
Abbreviations: HDAC, histone deacetylase; HAT, histone acetyltransferase;
HDACi, histone deacetylase inhibitors; SAHA, suberoylanilide hydroxamic acid;
TSA, trichostatin A; Topo I, topoisomerase I class of enzymes.
⇑
Corresponding author. Tel.: +1 404 894 4047; fax: +1 404 894 2291.
E-mail address: aoyelere@gatech.edu (A.K. Oyelere).
These authors contributed equally to this work.
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.03.108

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W. Guerrant et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3283–3287
O
O
O
OH
H
N
N
H
OH
N
H
N
O
Trichlostatin A (TSA)
Suberoylanilide Hydroxamic
Acid (SAHA)
O
O
O
HO
HO
N
N
N
N
N
N
O
O
O
O
O
O
OH
OH
OH
7-Ethyl-10-hydroxycamptothecin
(SN-38)
10-Hydroxycamptothecin
Camptothecin
Figure 1. Representative HDAC and Topo I inhibitors.
effective and are currently FDA-approved for cancer chemother-
apy.25 Since both HDAC and Topo I enzymes are localized to the
nucleus, the opportunity for dual inhibition from a single agent is
a promising possibility. Creating a dual-acting HDAC–Topo I inhib-
itor could prove beneficial for many reasons. First, HDACi have
been shown to act synergistically with Topo I inhibitors, resulting
in enhanced apoptosis in cancer.28 Also, since both enzymes are
nuclear-localized, dual-acting agents may have better therapeutic
indices.
HDAC Inhibition Moiety
O
R1
N
N
N
N
OH
O
N
H
O
n
N
1 = H, Et
n = 1 - 4
O
R
Using fused-frameworks design approach,1 we have previously,
described dual-acting agents derived from an anthracycline, a
topoisomerase II (Topo II) inhibitor and SAHA analogs, prototypical
HDACi. A subset of these dual-acting HDAC–Topo II inhibitors
inhibited Topo II and HDAC activities more potently compared to
parent anthracycline and SAHA, respectively.29 Furthermore, a lead
compound from this series was equipotent to daunorubin against
selected breast, lung and prostate cancer cell lines. As a follow-
up to our work on dual-acting HDAC–Topo II inhibitors, we have
designed and synthesized dual-acting HDAC–Topo I inhibitors de-
rived from the camptothecin ring system and the linker region of
SAHA-like HDACi. We show here that an alternative designed mul-
tiple ligand approach, merged-frameworks strategy,1b proved suc-
cessful in the design of HDAC–Topo I inhibitors. We present
evidence here that these compounds retain inhibitory activities
against both target enzymes and inhibit the proliferation of se-
lected cancer cell lines.
The camptothecin family of Topo I inhibitors are potent antican-
cer drugs that form a ternary complex at the interface of the cleav-
age complex, inhibiting dissociation of Topo I from DNA. We chose
10-hydroxycamptothecin and 7-ethyl-10-hydroxycamptothecin
(SN-38) (Fig. 1) as the Topo I inhibiting templates for the design
of the proposed dual-acting HDAC–Topo I inhibitors due to their
promising activity against a range of tumor types and the presence
of a functionalizable phenolic group at their C-10 position. Also,
both templates have demonstrated more potency and less toxicity
than camptothecin.30–32 From structure–activity relationship (SAR)
studies on camptothecins, substitution at the 10-hydroxy group
has been found to be tolerable,33 so we used this position as the
point of attachment for the HDACi moiety. We have already re-
ported the suitability of 1,2,3-triazole ring as a surface recognition
cap group-linking moiety in SAHA-like HDAC inhibitors.34 These
studies showed cap group-dependent preference for five to six
methylene linkers. In the designed dual-acting compounds, the lin-
ker region of SAHA-like HDACi is coupled through a triazole moiety
to the camptothecin template, which in turn is anticipated to act as
an aromatic surface recognition cap group essential for HDAC
O
OH
Topoisomerase I
Inhibition Moiety
Figure 2. Designed dual-acting HDAC–Topo I inhibitors.
inhibition while also retaining its Topo
I inhibition activity
(Fig. 2). We introduced variations into the linker region to test
the linker length-dependent potency of the resulting dual-acting
agents. Additionally, incorporation of the triazole ring into com-
pound design helped to simplify synthesis and SAR studies.
The reaction route to all the designed compounds is shown in
Scheme 1. The phenolic OH-group of 7-ethyl-10-hydroxycampto-
thecin 1a and 10-hydroxycamptothecin 1b was alkylated with
propargyl bromide to yield the corresponding alkyne intermedi-
ates 2a and 2b, respectively. Cu-catalyzed Huisgen cycloaddition35
with known azido intermediates 3a–e34,36 afforded trityl-protected
compounds 4a–h. Subsequent TFA deprotection of 4a–h yielded
the desired compounds 5a–h in good yields with minimal purifica-
tion required.
Building on our previous observations about the linker length-
dependent potency of aryltriazolyl HDACi,34,36 we first synthesized
and evaluated the anti-HDAC activity of 7-ethylcamptothecin-de-
rived compounds 5a–e against HeLa cell nuclear extract HDACs
as described previously with a slight modification.34 Briefly, cam-
ptothecin has a fluorescence emission (excitation k = 370 nm,
emission k = 434 nm) close to the wavelength (460 nm) of the fluo-
rescence generated by the HDAC enzyme cleavage of its fluoro-
genic substrate. To circumvent this potential interference,
controls containing the same concentration of the test compound
without the enzyme were used, and the background fluorescence
of these controls were subtracted from the experimental fluores-
cence readings. Compound 5a, an analog with a three methylene
linker separating the triazole ring and the hydroxamate moiety,
has no measurable anti-HDAC activity at concentrations as high
as 10
lM (Table 1). The inactivity of 5a may be due to the fact that
its linker region is too short to effectively position its hydroxamate

W. Guerrant et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3283–3287
3285
O
R1
n = 1; 3a
n = 2; 3b
n = 3; 3c
Ph
Ph
R1
N
N3
O
Ph
N
H
O
O
n
O
HO
N
N
3d
3e
n = 4;
n = 5;
i
N
O
O
O
O
ii
OH
R1 = Et; 2a
OH
R1 = Et; 1a
1 = H; 1b
R
1 = H; 2b
R
Ph
O
Ph
Ph
O
HO
R1
N
H
N
R1
N
N
H
N
N
N
N
N
N
O
O
n
O
O
O
n
N
N
iii
O
O
O
R1 = Et; n = 1; 5a
R1 = Et; n = 2; 5b
R1 = Et; n = 1; 4a
4b
R1 = Et; n = 2;
R1 = Et; n = 3; 4c
1 = Et; n = 4; 4d
R1 = Et; n = 5; 4e
O
OH
OH
R1 = Et; n = 3;
R1 = Et; n = 4;
5c
5d
R
R1 = Et; n = 5; 5e
R1 = H; n = 2; 5f
R1 = H; n = 3; 5g
R1 = H; n = 2;
4f
R1 = H; n = 3; 4g
R1 = H; n = 4; 4h
R1 = H; n = 4;
5h
Scheme 1. Synthesis of dual-acting HDAC–Topo
I inhibitors. Reagents and conditions: (i) propargyl bromide, K2CO3, DMSO, rt, 48 h; (ii) compounds 3a–e, CuI,
THF:DMSO:Hunig’s base 10:1:0.1; (iii) TFA, thioanisole, CH2Cl2, 0 °C.
Table 1
In vitro HDAC inhibition activity of novel HDAC–Topo I inhibitors
O
R1
N
N
N
N
OH
O
N
H
O
N
n
O
O
OH
Compound
n
R1
HeLaa IC50 (nM)
HDAC 1b IC50 (nM)
HDAC 6b IC50 (nM)
HDAC 8b IC50 (nM)
5a
5b
5c
5d
5e
5f
5g
5h
1
2
3
4
5
2
3
4
—
—
–CH2CH3
–CH2CH3
–CH2CH3
–CH2CH3
–CH2CH3
–H
–H
–H
—
—
ND
ND
NT
129 33
50
369 111
116 40
NT
37
NT
38
85 34
NT
1726 577
NT
ND
ND
2599 475
ND
NT
1046 316
NT
155.4
120.7
64.65
212.3
144.5
112.2
56.2
42
36
6
5
7
75 34
260 40
NT
81 26
NT
7
SN-38
SAHA
ND
65.0
2
27
2
1989 156
ND—Nondeterminable within tested range, 1 nM–10
l
M; NT—not tested.
a
HeLa nuclear extract. Each value is obtained from three independent experiments.
Data obtained through contract arrangement with BPS Bioscience (San Diego, USA; www.bpsbioscience.com). Assays were performed in duplicates at each concentration
b
and data reported with standard error.29,34c
moiety within the active site while maintaining the crucial surface
residue contacts. Conversely, compounds 5b–e displayed linker
length-dependent HDAC inhibitory activities with compound 5d,
analog with six methylene linkers, having inhibition activities
comparable to SAHA (Table 1). The anti-HDAC activities of these
compounds followed linker length-dependence similar to what
we observed for other aryltriazolyl HDACi.34,36 This result suggests
that the camptothecin ring could function as a cap group, facilitat-
ing HDAC inhibition perhaps through interactions with the enzyme
surface residues.
four, five and six methylene linkers, respectively. Our choice of this
linker range is based on the foregoing observation that this range
conferred the optimum activity to the 7-ethylcamptothecin de-
rived compounds 5b–d. A comparison of the anti-HDAC activities
of 5b–d and 5f–h, against the HeLa cell nuclear extract HDACs, re-
veals that pairs with the same linker length have nearly identical
HDAC inhibition activity (Table 1). These results suggest that the
presence or lack thereof of the ethyl group at the C-7 of camptothe-
cin ring system has no significant effect on the inhibition of HeLa
cell nuclear extract HDACs. As expected, the standard Topo I inhib-
itor—SN-38—had no measurable HDAC inhibition activity.
In order to elucidate the contribution of the ethyl group at the
C-7 of 7-ethylcamptothecin on HDAC inhibition activity, we syn-
thesized camptothecin-derived compounds 5f–h, analogs with
To obtain additional evidence for the specific mode of HDAC
inhibition, isoform selectivity was investigated by testing selected

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W. Guerrant et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3283–3287
against HDAC 8. The preference for HDAC6 over HDAC1 could
further explain the inactivity of 5a against HeLa nuclear extract,
which is a rich source of HDACs 1 and 2.37
We performed a cell-free DNA plasmid relaxation assay, accord-
ing to a literature protocol, in order to determine the Topo I inhibi-
tion activity of these HDAC–Topo I inhibitors.38,39 In this assay, a
supercoiled plasmid is incubated with Topo I in the presence or ab-
sence of Topo I inhibitors. Reactions are terminated by addition of
SDS, which denatures Topo I. Reaction mixtures are then electro-
phoresed in an agarose gel and DNA is visualized using a nucleic
acid dye. Stabilized cleavage complexes that are covalently bound
to DNA will inhibit migration of DNA in the gel significantly more,
relative to unbound, relaxed DNA. SN-38 was used as a positive
control for Topo I inhibition and drugs were dosed at 50 lM. The
7-ethylcamptothecin compounds 5a–e inhibited Topo I, evidenced
by both the reduction of relaxed plasmid and increase in nicked
plasmid compared to uninhibited Topo I, with no apparent drop
in activity compared to SN-38 (Fig. 3a). Similarly, camptothecin
compounds 5f–h inhibited Topo I with similar activities to each
other, but are less active relative to SN-38 (Fig. 3b). The enhanced
Topo I inhibitory activities of 5a–e relative to 5f–h is not unex-
pected as SN-38, the template for 5a–e, is a more potent Topo I
inhibitor compared to 10-hydroxycamptothecin, the template for
5f–h.40,41 These results, taken together with the HDAC inhibition
data, showed that these dual-acting HDAC–Topo I inhibitors could
function to inhibit either target enzyme and merging of the two
inhibiting moieties does not preclude the activities of either parent
compound significantly.
Figure 3. Topoisomerase I-induced plasmid relaxation assay: (a) (lane 1) pBR322
plasmid DNA, (lane 2) DNA and Topo I, (lanes 3–8) DNA, Topo I, and 50
lM (3) SN-
38, (4) 5a, (5) 5b, (6) 5c, (7) 5d, (8) 5e; (b) (lane 1) pBR322 plasmid DNA, (lane 2)
DNA and Topo I, (lanes 3–6) DNA, Topo I, and 50 lM: (3) SN-38, (4) 5f, (5) 5g, (6) 5h.
To examine the effects of these dual-acting inhibitors on cancer
cell proliferation, they were screened against the DU-145 prostate
cancer cell line and the inhibition of cell viability was measured.
SN-38 and SAHA were used as positive controls with SN-38 po-
tently inhibiting DU-145 viability in the mid-nanomolar range,
while SAHA’s IC50 was higher, in the low micromolar range. The
bifunctional compounds 5a–h showed linker length dependent
anti-proliferative activities with a five methylene linker proving
to be optimum for cytotoxicity among the 7-ethylcamptothecin
compounds (Table 2, comparing 5a–e). Compound 5a, with the
shortest linker of three methylenes, possessed the least potent
activity against DU-145. Conversely, the camptothecin compounds
5f–h displayed indistinguishable cytotoxic activity. Comparatively,
most of the 7-ethylcamptothecin compounds are generally less ac-
tive than their camptothecin congeners. One exception is com-
pound 5c which showed cytotoxicity activity that is identical to
that of 5g, its direct camptothecin analog. More importantly, the
micromolar IC50 values suggest that HDAC inhibition may be the
dominating mode of antiproliferative activities of compounds
5a–h.
Table 2
Whole cell cytotoxicity activity against DU-145 prostate cancer cells, as determined
by MTS assay after 72 h
Compound
n
R1
IC50 (lM)
5a
5b
5c
5d
5e
5f
5g
5h
1
2
3
4
5
2
3
4
—
—
–CH2CH3
–CH2CH3
–CH2CH3
–CH2CH3
–CH2CH3
–H
–H
–H
—
—
6.27
4.25
2.05
3.11
3.51
2.50
1.95
2.03
0.11
2.12
SN-38
SAHA
All values are mean of two experiments performed in triplicate as measured by the
MTS assay (Promega).
compounds against purified HDAC1, HDAC6, and HDAC8. The pat-
tern of the anti-HDAC activities of these compounds against HDAC
1 and HDAC 6 mirrored what was observed for the HeLa cell nucle-
ar extract HDACs with one exception. Specifically, the three meth-
ylene-linked compound 5a is inactive against HDAC 1 while it
maintains low nanomolar and micromolar IC50’s against HDAC 6
and HDAC 8, respectively (Table 1). In general, these dual-acting
agents are more selective for HDAC 6 with modest or no activity
Since the anticancer activities displayed by these dual-acting
HDAC–Topo I inhibitors against DU-145 appeared to be largely dri-
ven by HDACi-based mechanisms, we profiled the contribution of
intracellular HDAC inhibition through the level of p21waf1 expres-
sion.34b Compounds 5c and 5g were used, premised on the fact that
they are representative examples from each of the two Topo I
1
2
3
4
5
6
7
8
9
Actin
p21waf1
Fig. 4. Western blot probing for actin and p21 in the DU-145 cell line. Lanes: (1) control, (2) SAHA, 2.5
2.5 M, (7) 5c, 5.0 M, (8) 5g, 2.5 M, (9) 5g, 5.0 M.
l
M, (3) SAHA 5.0 lM, (4) SN-38, 0.1 lM, (5) SN-38 0.5 lM, (6) 5c,
l
l
l
l

W. Guerrant et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3283–3287
3287
inhibiting templates with identical linker lengths and anticancer
References and notes
activities. Inhibitors were dosed at concentrations near the deter-
mined IC50’s in DU-145 (Table 2) and p21waf1 expression was
probed via immunoblot (Fig. 4). Equivalent protein loading was
demonstrated using an anti-actin antibody (Fig. 4, top). Both SAHA
and SN-38 resulted in marked upregulation of p21waf1 expression
levels with 24 hour treatment (Fig. 4, bottom, lanes 2–5). Gratify-
ingly, we observed that the dual-acting compounds 5c and 5g re-
sulted in substantial upregulation of p21waf1 expression in a
concentration-dependent manner with 5g causing upregulation
at levels comparable to SN-38 (Fig. 4, bottom panel, lanes 6–9).
These results suggest that compounds 5c and 5g derived their
cytotoxic activity, in part, through HDAC inhibition. It is unclear
at present how much of the p21waf1-dependent anticancer activity
is contributed by each inhibiting moiety as both SAHA and SN-38
significantly increased p21waf1 expression. Subsequent investiga-
tion into the expression levels of other cellular markers could clar-
ify the driving force behind the cellular effects observed.
A new class of dual-acting HDAC–Topo I inhibitors has been cre-
ated from camptothecin and SAHA-like templates. Two types of
camptothecin templates were used and both were connected
through their 10-hydroxy moieties to alklyltriazolyl hydroxamates
that we have shown possess enhanced HDAC inhibition activity.34
Results from cell-free and whole cell studies showed that these
compounds possess inhibition activities against both target en-
zymes and inhibit the growth of DU-145 prostate carcinoma cells.
Relative to the camptothecin standard SN-38, the functionalization
of the 10-hydroxy moiety presented no observable deleterious
effect on the Topo I inhibition by 7-ethyl-10-hydroxycamptothe-
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inhibitory activities of 10-hydroxycamptothecin-derived conju-
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Acknowledgments
This work was financially supported by NIH Grant
R01CA131217. W.G. and J.C.C. are recipients of the GAANN predoc-
toral fellowship from the Georgia Tech Center for Drug Design,
Development, and Delivery. R.H. is a Beckman Undergraduate
Research Fellow.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
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