Dual targeting of histone deacetylase and topoisomerase II with novel bifunctional inhibitors

Article abstract of DOI:10.1021/jm200799p

Strategies to ameliorate the flaws of current chemotherapeutic agents, while maintaining potent anticancer activity, are of particular interest. Agents which can modulate multiple targets may have superior utility and fewer side effects than current single-target drugs. To explore the prospect in cancer therapy of a bivalent agent that combines two complementary chemo-active groups within a single molecular architecture, we have synthesized dual-acting histone deacetylase and topoisomerase II inhibitors. These dual-acting agents are derived from suberoylanilide hydroxamic acid (SAHA) and anthracycline daunorubicin, prototypical histone deacetylase (HDAC) and topoisomerase II (Topo II) inhibitors, respectively. We report herein that these agents present the signatures of inhibition of HDAC and Topo II in both cell-free and whole-cell assays. Moreover, these agents potently inhibit the proliferation of representative cancer cell lines.

Full text of DOI:10.1021/jm200799p

Article
pubs.acs.org/jmc
Dual Targeting of Histone Deacetylase and Topoisomerase II with
Novel Bifunctional Inhibitors
William Guerrant,§,† Vishal Patil,§,† Joshua C. Canzoneri,§,† and Adegboyega K. Oyelere*,†,‡
‡
†School of Chemistry and Biochemistry, Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of
Technology, Atlanta, Georgia 30332-0400, United States
S
* Supporting Information
ABSTRACT: Strategies to ameliorate the flaws of current
chemotherapeutic agents, while maintaining potent anticancer
activity, are of particular interest. Agents which can modulate
multiple targets may have superior utility and fewer side effects
than current single-target drugs. To explore the prospect in
cancer therapy of a bivalent agent that combines two complementary chemo-active groups within a single molecular architecture,
we have synthesized dual-acting histone deacetylase and topoisomerase II inhibitors. These dual-acting agents are derived from
suberoylanilide hydroxamic acid (SAHA) and anthracycline daunorubicin, prototypical histone deacetylase (HDAC) and
topoisomerase II (Topo II) inhibitors, respectively. We report herein that these agents present the signatures of inhibition of
HDAC and Topo II in both cell-free and whole-cell assays. Moreover, these agents potently inhibit the proliferation of
representative cancer cell lines.
themselves, including p53, E2F, tubulin, and Hsp90.18−22
HDAC inhibition has been clinically validated as a therapeutic
INTRODUCTION
Several rational pharmacological strategies, including vaccina-
■
strategy for cancer treatment with the FDA approvals of sub-
tion, gene therapy, immunotherapy, and new target identi-
eroylanilide hydroxamic acid (SAHA) and romidepsin (FK-228)
fication and validation, have emerged for the treatment of meta-
for treatment of cutaneous T cell lymphoma.23−25 However, a
static diseases. Despite these progresses, chemotherapy remains
large number of the currently known HDACi have elicited only
the primary treatment of choice for most cancer cases. How-
limited in vivo antitumor activities and have not progressed
ever, almost all chemotherapeutic agents suffer from severe
beyond preclinical characterizations.26−28 HDACi that modulate
toxicities and other undesirable side effects. To address these
the functions of additional intracellular targets, other than the
problems, the cancer medicine of the future will incorporate,
various HDAC isoforms, may be able to ameliorate many of the
within a single molecule, elements that allow for simultaneous
targeting of multiple cancer-fighting targets while maintaining
shortcomings of current inhibitors.
lower side effects.1−3 This realization has continued to spawn
Because of the presence of large hydrophobic patches at the
HDAC surface rim,29,30 it is conceivable that appropriate
immense efforts in the literature. Studies aimed at identifying
conjugation of the surface recognition group of a prototypical
multivalent ligands as promising pharmacological tools that
HDACi to other hydrophobic antitumor pharmacophores could
may be more efficacious for various human diseases than highly
furnish a new class of bifunctional agents. To date, there exist a few
selective single-target drugs are ongoing in several academic
and pharmaceutical laboratories.4−7 A subset of these studies
examples of this subtype of bifunctional HDACi derived
compounds.31−33 Expansion of the repertoire of such bifunctional
has revealed that balanced modulation of a small number of
compounds could lead to broad acting, therapeutically viable
targets may have superior efficacy and fewer side effects than
single-target treatments.1,7,8
anticancer agents.
An attractive starting point for a secondary target is the
Epigenetic control has become widely accepted as a mech-
anism for cell regulation.9−11 Specifically, histone deacetylase
topoisomerase class of enzymes (Topo I and Topo II), which
are validated targets for many small molecule inhibitors
(HDAC) is a class of epigenetic enzymes that has generated
including clinically useful anthracyclines such as doxorubicin
much interest in cancer therapeutics literature. HDACs are
(DOX) and daunomycin or daunorubicin (DAU) (Figure 1)
known to associate with many oncogenes and tumor
and camptothecins such as irinotecan and topotecan.34 Topo
suppressors, leading to altered expression patterns, and have
inhibitors elicit anticancer activities primarily by stabilizing the
consequently become attractive targets for small-molecule
inhibition.12,13 Histone deacetylase inhibitors (HDACi) have
been shown to cause growth arrest, differentiation, and apo-
ptosis in tumor cells and in animal models by inducing histone
hyperacetylation and p21waf1 expression.14−17 Additionally,
modulation of activities of HDACs alters the activity of a diverse
range of proteins, many of which are attractive therapeutic targets
DNA−enzyme cleavable complex through intercalation be-
tween DNA base pairs. However, DNA does not exist as a naked
structure in the nucleus. It is noncovalently associated with
Received: March 25, 2011
Published: January 19, 2012
© 2012 American Chemical Society
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Figure 1. Representative structures of anthracycline antibiotics.
Figure 2. Design of dual-acting Topo II−HDAC inhibitors.
histones to form the nucleosomes which make up chromatin
subunits. Agents, such as HDACi, that induce hyperacetylation of
histone proteins complexed with DNA could increase the
accessibility of DNA within chromatin and consequently potentiate
the anticancer activities of Topo inhibitors.35,36 Moreover, recent
observations have shown that HDAC1, HDAC2, and Topo II
colocalize in vivo as part of functionally coupled complexes.37,38
These evidence suggest simultaneous Topo and HDAC inhibition
could be a viable alternative approach in cancer therapy.
We disclose herein small molecules with dual acting Topo
II−HDAC inhibitory activities. We found that many of these
conjugates more potently inhibited HDAC and Topo II
activities compared to SAHA and daunomycin, standard
HDACi and Topo II inhibitors, respectively. Additionally, a
subset of these compounds exhibited potent whole cell
antiproliferative activities against representative breast, lung,
and prostate cell lines.
the anthracycline moiety could serve two other purposes: (i) as
a surface recognition cap group, allowing favorable orientation
of hydroxamic acid within the zinc binding pocket of HDAC,
and (ii) as a delivery vehicle, because the transport of the
anthracycline via proteasome could facilitate nuclear accumu-
lation of HDACi.43 On the basis of the forgoing, we designed
two classes of conjugates: a direct DAU−SAHA conjugate and
DAU−triazolylaryl hydroxamate conjugates (Figure 2). The
later conjugates were inspired by our previous studies which
revealed that triazole moiety could be incorporated in lieu of
amide bond as a surface recognition connecting group in
prototypical HDACi.48
Chemistry. Crucial to the successful synthesis of all the
conjugates described in this report is the reductive amination
reaction between DAU and appropriate aldehyde intermediates
5 and 10a−d (Schemes 1 and 2). Synthesis of aldehyde inter-
mediate 5 started with the coupling of 4-aminobenzyl alcohol 1
to suberic anhydride 2 to give benzyl alcohol 3. To assess the
suitable oxidizing agent for conversion of alcohol 3 to aldehyde
4, we employed MnO2, PCC, and Dess−Martin periodinane
(DMP) oxidation. All yielded the desired aldehyde, with DMP
giving the optimum yield among them. To synthesize the pro-
tected hydroxamate 5, we coupled 4 to O-trityl hydroxylamine
using standard EDC coupling chemistry. Interestingly, the
amide bond of compound 4 was also susceptible to nucleophilic
attack and led to a substantially lower yield under this condition.
Using TBTU as coupling reagent resulted in the same outcome.
Gratifyingly, the use of isobutylchloroformate (IBCF) as a
coupling reagent gave coupled requisite product 5 in 66% yield
within 2 h. Longer reaction times did not improve the yield and
led to degradation of product. Reductive amination with DAU
hydrochloride was the major hurdle in making the DAU−SAHA
RESULTS AND DISCUSSION
■
Design Rationale. Anthracyclines are one of the most
thoroughly studied classes of anticancer agents with copious
structure−activity relationship (SAR) data to aid the design and
characterization of new anthracycline-containing com-
pounds.39−44
Specifically, N-benzylated anthracyclines, such as N-benzyl
doxorubicin (AD-288)42 (Figure 1), have enhanced Topo II
inhibitory activities, reduced cardiotoxicity activity, and reduced
susceptibility to the Pgp-mediated multidrug resistance.45−47
We postulated that introduction of the HDACi via N-benzyla-
tion of the DAU amino group would be compatible with Topo
II inhibition and possibly engender the positive attributes of
N-benzylated anthracyclines to the resulting conjugates. In turn,
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Scheme 1. Synthesis of the SAHA-Based Dual-Acting Topo II−HDAC Inhibitor
Scheme 2. Synthesis of the SAHA-Like, Triazole-Based Topo II−HDACi
conjugate. Initial investigation with NaBH3CN as a reducing
agent with acetonitrile−water solvent system at room temper-
ature led to a complex reaction mixture. Employing a non-
aqueous solvent system, as well as a milder reducing agent such
as NaBH(OAc)3, did not produce any detectable amount of
product. Previous studies in the literature indicated that generation
of free amine by addition of base and elevated temperature might
promote the imine formation.49,50 Indeed, adding Hunig’s base
to reductive amination reactions between DAU and aldehyde 5
in the presence of NaBH3CN at 70 °C gave the desired coupled
product 6, albeit in low yield. Reducing the reaction tem-
perature to 50 °C and below worsened the yield, as did employ-
ing NaBH(OAc)3 as a reducing agent. The deprotection of the
acid sensitive trityl group of compound 6 was accomplished by
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treatment with the Lewis acid boron trifluoride etherate.51 This led
to the removal of the trityl group to give the desired conjugate 7 in
good yield without any need for further purification.
conjugates potently inhibit HeLa cell nuclear extract HDACs
with IC50 in the low to midnanomolar range. Among these con-
jugates, 12a is the least active, closely followed by 12d, which is
about 20-fold more potent. Compounds 12b and 12c have the
most potent anti-HDAC activity, with a slight preference for
the six methylene-linked 12c. Interestingly, the triazole-linked
compound 12b is 40-fold more potent than the amide-linked 7
despite their similar linker length. Relative to the standard
SAHA, 12c, the most potent compound in this series, is 70-fold
more potent (Table 1). The foregoing results showed that
these conjugates followed a trend similar to that which we
noted for the previously reported, structurally unrelated, triazole-
based HDACi.48
To obtain evidence for the HDAC isoform selectivity, we
tested these dual acting Topo II−HDACi conjugates against
selected recombinant HDACs−HDAC 1, HDAC 6 and HDAC
8. The pattern of the anti-HDAC activities of these compounds
against HDAC 1 and HDAC 6 is similar to what we observed
for the HeLa cell nuclear extract HDACs with few exceptions.
Specifically, compounds 7 and 12b have indistinguishable
activity against HDAC 1 and HDAC 6 (Table 1). Additionally,
12a which has midnanomolar IC50 against HeLa cell nuclear
extract HDACs is almost inactive against HDAC 1 (IC50 = 4.6 μM)
Similar chemistry was applied for the synthesis of triazole-based
conjugates. The synthesis of requisite aldehydes 10a−d started
with Cu-catalyzed cycloaddition of 4-ethynylbenzaldehyde 8 with
trityl-protected azido hydroxamates 9a−d (Scheme 2). Reductive
amination of triazole aldehydes 10a−d with DAU proceeds
facilely at room temperature in aqueous solvent system to give the
triazole-based conjugates 11a−d in slightly improved yield
compared to conjugate 6. Boron trifluoride etherate deprotection
of the trityl group of 11a−d yielded the desired triazole-based
conjugates 12a−d in good to excellent yields.
In vitro HDAC Inhibition. We first tested the HDAC
inhibition activity of compounds 7 and 12a−d against crude
HeLa cell nuclear extract HDACs using a cell free assay (Fluor
de Lys) as previously described.48 Overall, these compounds
showed inhibition activities against HeLa cell nuclear extract
HDACs, which are comparable to or exceed that of the
standard SAHA (Table 1). It is particularly interesting that 7
Table 1. In Vitro HDAC Inhibition
HDAC 1/2
a
HDAC 1
IC50 (nM)
HDAC 6
IC50 (nM)
HDAC 8
b
b
b
compd
n
IC50 (nM)
IC50 (nM)
while it maintains decent activity against HDAC 6 (IC50
=
SAHA
DAU
7
65.0
ND
64.7
89.9
1.6
38
NT
47
2
27
NT
20
2
1989 156
NT
0.6 μM). We are not exactly sure of the cause of this disparity.
In general, these compounds are weaker inhibitors of HDAC
8 with the exception of 7, whose anti-HDAC 8 activity is only
about 4-fold less than its anti-HDAC 1 activity (Table 1).
These data suggest that 7 is a more indiscriminate inhibitor of
these sets of HDACs while the rest of the conjugates are more
selective.
Molecular Docking. To obtain information on the struc-
tural basis of the observed disparity in the HDAC inhibitory
activity of these compounds, we performed molecular docking
using a validated molecular dock program (AutoDock)48,52
Docking analysis was performed on a HDAC 1 homology
model built from human HDAC 2 X-ray structure 3MAX
coordinates.53 We chose to dock the compound which had the
least HDAC inhibitory activity (12a) and the compound which
had the best HDAC inhibitory activity (12c) to delineate the
basis of the ∼600 fold activity difference between the two.
3
1
220 21
ND
12a
12b
12c
12d
1
2
3
4
4600 240
555 36
30
20 0.4
19
54
8
3
2
4,129 421
710 43
379 37
0.9
0.4
4.2
11 0.4
1
a
Inhibition was assayed using the Biomol HDAC Fluorimetric Assay/
b
Drug Discovery Kit. Data obtained through contract arrangement
with BPS Bioscience (San Diego, USA; www.bpsbioscience.com).
has identical anti-HDAC activity to SAHA. This result suggests
that the attachment of DAU does not impair the interaction
between the HDACi component of the conjugate and the
HDAC enzyme outer surface residues. It is also conceivable
that the conjugate may adopt a conformation whereby the an-
thracycline moiety can contribute positively to the interaction
with the crucial active site or surface residues. All triazole-linked
Figure 3. Docked structure of Topo II−HDACi conjugates at HDAC 1 active site. (a) Superposition of low energy conformations of 12a (teal) and
12c (pink). (b) Overlay of low energy conformations of 12c (pink) and 7 (orange).
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Additionally, we performed docking on compound 7 because of
its distinct structural feature compared to 12a−12c.
Topo II within 10 min at 37 °C resulted in an extensive DNA
decatenation (lane 3). As expected, addition of 50 μM DAU to
the decatenation experiment resulted in a severe impairment of
DNA decatenation (comparing lanes 3 and 4). Relative to
DAU, 12a and 12d have lower Topo II inhibition activity, with
the worst overall inhibition shown by 12a, the conjugate with a
four-methylene linker (lanes 7 and 8). Conjugates 12b and 7
inhibited Topo II activity at comparable levels to that of DAU
at the same drug concentration (lanes 6 and 9, respectively).
However, compound 12c had an enhanced Topo II inhibition
activity relative to DAU, resulting in a near total inhibition at
50 μM (comparing lanes 4 and 5). These results show that the
Topo II inhibition activity of DAU is tolerant of an appropriate
attachment of HDACi groups and, as in the case of 12c, such
groups could further enhance Topo II inhibition activities of
anthracycline derivatives. The molecular basis of the HDACi
linker length-dependent enhancement of Topo II inhibition of
these dual-acting conjugates is not entirely clear. It is plausible
that the placement of the HDACi group of these conjugates
within DNA minor grooves, through the daunosamine sugar,56
could further promote drug−DNA association, thereby enhanc-
ing the stability of the biologically relevant drug−DNA−Topo
II ternary complex. Interestingly, 12c also has the most potent
inhibition activity against HDAC 1 (Table 1). It is exciting to
observe that a single compound could embody optimum anti-
HDAC and Topo II inhibition activities under these cell-free
conditions.
Interestingly, compound 12a and 12c, differing only in linker
length, do not bind to the same pocket but instead localize to
two different pockets (Figure 3a). For six-methylene-linked
compound 12c, the hydroxyl group of the daunosamine sugar
could potentially make hydrogen bonding contact with the
guanidinium group of Arg270 and the linker region could facilitate
the presentation of the hydroxamic acid to the catalytic zinc by
entering through the top of the hydrophobic channel that leads to
the active site (Figure 3a and Supporting Information Figure S1c, d).
In addition, two of the hydroxyl groups from the anthracycline
ring of 12c could take part in the H-bonding interaction with
the backbone carbonyl group of Arg270 and the N−H group of
Gly272. Possibly to accommodate the shorter linker length, 12a
loses the H-bonding interaction between the hydroxyl group of
its daunosamine sugar and the enzyme’s Arg270. Although
other hydroxyl groups of the 12a anthracycline ring make poten-
tially compensatory H-bonding contacts with the phenolic group
of Tyr201 and the backbone carbonyl groups of Gly207 and
Pro206 (See Supporting Information Figure S1a, b), its binding
pocket is more solvent-exposed, compared to the binding
pocket of 12c (Figure 3a).
Compound 7 binds close to the same pocket as 12c; it
however derives its binding affinity through different sets of
interactions. Unlike 12c which enters the active site associating
with the top of the hydrophobic channel, 7 traverses the same
channel more closely associated with residues on the opposite
side of the channel (Figure 3b). Consequently, the daunos-
amine hydroxyl group of 7 could not interact with Arg270 in a
similar way as that of 12c. In lieu of this interaction, the anthra-
cycline ring of 7 may engage in other H-bonding interactions
with the backbone carbonyl groups of Gly272, Gly268, and
Thr304 on the enzyme surface rim (Figure 3b and Supporting
Information Figure S1e, f). These apparent differences in the
binding orientation at the enzyme surface rim could account for
the disparity in the potency of compounds 7, 12a, and 12c
against HDAC 1.
In Vitro Topoisomerase II Decatenation. We performed
a cell-free DNA decatenation assay to determine the Topo II
inhibition activity of these Topo II−HDACi conjugates. We
used kinetoplast DNA (KDNA), a catenated network of mito-
chondrial DNA seen in trypanosomes, to quantify the con-
jugates’ Topo II inhibition activity according to a literature
protocol.54,55 Figure 4 illustrates the results obtained from this
In Vitro Cell Growth Inhibition. Cell viability experiments
were performed to probe for the prospect of biological activity
of these compounds in the cellular milieu. Three human cancer
cell lines were used to quantify IC50 values for these com-
pounds. Table 2 shows the IC50 values of each compound for
Table 2. Cell Viability Assay
DU-145
IC50 (μM)
SK-MES-1
MCF-7
a
a
a
compd
n
IC50 (μM)
IC50 (μM)
7
0.13 0.06
5.39 1.02
1.61 0.29
2.92 0.31
2.06 0.33
0.09 0.002
2.12 0.25
0.47 0.02
15.3 3.1
4.68 0.75
3.31 0.23
2.61 0.11
0.17 0.09
2.42 0.38
0.99 0.21
24.5 1.6
13.4 1.85
10.6 0.94
14.8 1.6
0.95 0.05
2.50 0.61
12a
12b
12c
12d
DAU
SAHA
1
2
3
4
a
Values are the average of two experiments performed in triplicate.
IC50 values were determined using the MTS assay (Promega).
all cancer cell types studied. The positive control compounds
DAU and SAHA inhibit the proliferation of these tumor cell
lines with IC50 similar to the values in the literature.57,58 DAU
displays cell line dependent cytotoxicity that varies by as much
as 10-fold among these three cell lines while SAHA shows no
such cell line dependent effect (Table 2). Bifunctional
compounds 12a−d show linker length dependent antiprolifer-
ative activities that closely matched their anti-HDAC activities.
Among the three transformed cell lines investigated, these
compounds decreased the viability of DU-145 the most, while
they are least cytotoxicity against MCF-7. Although less pro-
nounced than that seen with DAU, these compounds display
cell line-dependent cytotoxicity as well. Nevertheless, the micro-
molar IC50 values and the traction with anti-HDAC activities
suggest that HDAC inhibition is the dominating mode of anti-
proliferative activities of compounds 12a−d. Specifically, the
Figure 4. Topoisomerase II decatenation assay. Lanes 1−3: (1)
KDNA, (2) decatenated KDNA marker, (3) KDNA and Topo II.
Lanes 4−9: KDNA, Topo II, and 50 μM (4) DAU, (5) 12c, (6) 12b,
(7) 12d, (8) 12a, (9) 7.
study. KDNA and decatenated KDNA marker (lanes 1 and 2,
respectively) were used as controls. Treatment of KDNA with
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antiproliferative activities of 12c and SAHA, compounds with
similar linker length, are virtually indistinguishable against DU-
145 and SK-MES-1 cell lines. This finding is surprising because
12c displays the most potent HDAC and Topo II inhibition
activities. Interestingly, compound 7, a true hybrid between
SAHA and DAU, showed the best cytotoxicity of all the bi-
functional compounds across all cell lines, possessing sub-
micromolar activities. In fact, the cytotoxicity of 7 closely rivals
that of DAU, and they are equipotent against MCF-7. This is
contrary to the trend seen in the cell-free assays. The poten-
tiation of the activity of 7 within the cellular environment could
be due to many factors, including the predominance of the
Topo II inhibition character in dictating the bioactivity of 7, the
indiscriminate inhibition of multiple HDAC isoforms, or an
alternative mechanism(s) that is unrelated to the inhibition of
the activities of either target.
(Figure 5, second panel, lanes 2 and 3), while DAU and 7
display moderate dose-dependent change in acetylation at the
concentrations tested (2nd panel, lanes 4−7). Compound 12b
shows a strong H4 acetylation, with levels close to that of
SAHA, at both concentrations (2nd panel, lanes 8 and 9). The
trend of the drug induced perturbation of the acetylation state
of H3, in core histones purified by acid extraction of DU-145
cell nuclear extract, is similar to what obtained for H4. Relative
to the control, we observed distinct H3 hyperacetylation in
DU-145 cells exposed to the same drug concentrations used for
H4 immunoblotting (Figure 5, third panel). These results
provide evidence supporting the involvement of intracellular
HDAC inhibition as part of the mechanisms of bioactivity of
the dual acting compounds 7 and 12b.
Tubulin Acetylation. Additional data was sought in order
to clarify the mechanisms involved in the antiproliferative
activities and to delineate the disparity in enzyme inhibition
versus antiproliferative activity. Tubulin was chosen as a target
because it is acetylated by the cytoplasmic HDAC6,21,61,62 for which
7 and 12b had nearly identical inhibition. Interestingly, inhibition
of HDAC6-associated tubulin acetylation has been shown to
enhance the cytotoxicity of DNA-damaging agents.63 While
most HDACi induce p21waf1 overexpression, inhibition of tubu-
lin deacetylation is compound specific,64 potentially allowing
for differentiation between the mechanisms and potencies of 7
and 12b. Because tubulin acetylation in response to HDACi is a
relatively early event,65 we dosed DU-145 cells for 4 h with
inhibitors at either IC50 concentrations (Table 2) or a high
concentration (∼5× IC50). Immunoblotting revealed highly
varied levels of tubulin acetylation among the inhibitors. DAU
induced the lowest levels of acetylation, with IC50 concen-
tration showing levels comparable with control and only a slight
increase in response to higher concentration of drug (Figure 6,
Histone Hyperacetylation and p21waf1 Expression. To
gain a better perspective of the molecular mechanism of the
antiproliferative activities of these dual-acting inhibitors, we
probed the effect of their exposure on the intracellular status of
p21waf1 (p21) protein in DU-145 prostate cancer cells. p21 has
been shown to be upregulated in response to HDACi treatment,
as well as in a p53-independent response to DOX.59,60 We dosed
inhibitors at concentrations near the determined IC50 in DU-145
and evaluated protein expression status using Western blotting
(Figure 5). We controlled for equivalent protein loading using
Figure 5. Immunoblot detection of cellular HDACi Markers. DU-145
cells were dosed for 24 h at the indicated concentrations to probe for
acetylated histones H4 and H3, and p21waf1. Actin was also probed to
show equal loading of protein. Lane: (1) control; SAHA (2) 2.5 μM,
(3) 5 μM; DAU (4) 0.1 μM, (5) 0.5 μM; 7 (6) 0.5 μM, (7) 1 μM; 12b
(8) 2.5 μM, (9) 5 μM.
Figure 6. Tubulin acetylation in response to TopoII−HDACi. DU-
145 cells were dosed for 4 h with: (1) control (0.1% DMSO), (2)
DAU 90 nM, (3) DAU 500 nM, (4) 7 130 nM, (5) 7 500 nM, (6) 12b
1.31 μM, (7) 12b 10 μM.
antiactin antibody (Figure 5, bottom panel). As expected, SAHA
results in marked upregulation of p21, even at 2.5 μM (top panel,
lanes 2 and 3). However, neither DAU nor 7 shows noticeable
upregulation in p21 expression compared to control levels (top
panel, comparing lanes 4−7 to lane 1). This trend is reversed with
12b, as a dose-dependent upregulation of p21 expression was
observed (top panel, lanes 8 and 9). Relative to SAHA,
however, the extent of p21 upregulation by 12b is lower,
although both were dosed at the same concentrations. Because
these experiments were done at the IC50 of the respective
compounds, these results may indicate that 7 and 12b derived
their cytotoxic activity primarily through Topo II and HDAC
inhibition, respectively. Alternatively, the cytotoxic activity 7
could be due to perturbation of other intracellular HDAC
inhibition markers.
lanes 1−3). Compound 7 induced moderate levels of acetylation
(Figure 6, lanes 4 and 5), while 12b caused robust acetylation
at 5× its IC50 (Figure 6, lanes 6 and 7). p21 expression at 4 h
remained low across all compounds tested, with no discernible
induction relative to control (data not shown).
Intracellular Topoisomerase II Inhibition. To obtain
information about the intracellular fate of Topo II upon cell
exposure to these dual-acting agents, we used an immunoblot-
ting kit to assay compound-induced Topo II inhibition in an
intracellular environment (Figure 7).66 DU-145 cells were
dosed with drug concentrations corresponding to cell growth
inhibition IC50s, while the control cells were dosed with vehicle
(0.1% DMSO). The relative levels of stabilized Topo II−DNA
cleavage complexes were determined for a 30 min drug
treatment, as described by the manufacturer. Within this period,
the control cells showed no significant amounts of Topo II
inhibition, evidenced by the low levels of Topo II associated
DNA (Figure 7a, lane 1). Cells treated with DAU and 12b
contained high levels of Topo II−DNA cleavage complexes,
To further investigate into the prospect of distinct
mechanisms of action for 7 and 12b, we probed for histone
acetylation status in DU-145 cells exposed to the same drug
concentrations used for p21 immunoblotting. Intracellular
histone acetylation status is a more direct indicator of class I
HDAC inhibition. SAHA shows a strong histone H4 acetylation
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DAU, and we observed clear differences in the intracellular
distribution profiles of the tested compounds. In agreement
with previous study in the literature,67 DAU is localized within
the nuclear and perinuclear regions of DU-145 cells. Although
it shows a less nuclear localization, compound 7 is more
widely distributed within the cytosol, with evidence for
perinuclear localization in similar manner to that of DAU. In
contrast, 12b shows a highly diminished intracellular dis-
tribution, with the bulk of the compound trapped in vesicle-
like bodies within the cell (Figure 8). The relatively poor
intracellular distribution of 12b could be due to low cell
membrane penetration or an enhanced pump-induced efflux
of compound from within the cell.68 We obtained a similar
result with a lung tumor derived A549 cells (Supporting
Information Figure S2). These results show that 7 and 12b
have different intracellular residency, which may affect access
to their targets and consequently offer additional insight into
underlying factors that could contribute to the disparity in the
in vitro potency of these compounds.
Figure 7. Intracellular Topo II Inhibition. DU-145 cells were probed
for stabilized DNA−Topo II cleavage complexes upon (a) 30 min
treatment; (b) 72 h treatment with bifunctional compounds: (1)
control, (2) 90 nM DAU, (3) 130 nM 7, (4) 1.6 μM 12b.
with 12b showing a significantly higher amount (Figure 7a,
lanes 2 and 4, respectively). This result suggests that 12b could
derive its cytotoxic activity, in part, from intracellular Topo II
inhibition. Conversely, the levels of Topo II−DNA cleavage
complexes in cells exposed to 7 is indistinguishable from that of
the control cells (Figure 7, lane 3), suggesting a minimal
contribution of Topo II inhibition to the cytotoxic activity of 7
within this period. This observation is surprising in light of the
seemingly contradictory moderate effect of 7 on H4 hyper-
acetylation (Figure 5), tubulin acetylation (Figure 6), and its
potent cell growth inhibition activity (Table 2).
To elucidate any contribution Topo II inhibition could be
adding to long-term inhibition of cell proliferation, Topo II
cleavage complexes were assayed after 72 h of treatment with
compounds (Figure 7b). As expected, DAU treatment results in
significant inhibition of Topo II activity relative to control
levels (Figure 7b, comparing lanes 1 and 2). Compound 7
shows a measured increase in Topo II inhibition relative to
control levels (Figure 7b, comparing lanes 1 and 3). Interestingly,
we observed a drastic drop in the levels of stabilized Topo II−
DNA cleavage complexes upon cell exposure to 12b for 72 h
(comparing lane 4 of parts a and b of Figure 7). This result
suggests that the Topo II inhibition activity of 7 increases with
time while that of 12 decreases. The persistence of the stabilized
Topo II−DNA cleavage complexes over a longer period indicates
that Topo II inhibition may contribute significantly to the mech-
anism of the antiproliferative activity of 7.
CONCLUSION
■
There is evidence for the synergistic effect of combined Topo II
and HDAC inhibitors on cancer.38 However, this synergy is
schedule dependent, hence traditional combination therapy
involving Topo and HDAC inhibitors may be complicated by
the inherent pharmacokinetic disadvantage of two separate
drugs. To critically delineate the benefits of simultaneous Topo
and HDAC inhibition in cancer therapy, it will be of interest to
identify agents that possess Topo and HDAC inhibition
activities within a single molecule. Toward this end, we have
created dual-acting Topo II−HDAC inhibitors. A subset of
these compounds potently inhibits the proliferation of repre-
sentative cancer cell lines. When subjected to target-specific
screening, these agents present both HDAC and Topo II
inhibition signatures under cell-free conditions and in in vitro
cell cultures. This observation suggests that the cytotoxic
activities and potency of these dual-acting compounds could be
dictated by either of the two antitumor pharmacophores. Speci-
fically, results from HDAC and Topo inhibition studies, and
p21, acetyl-H4, and acetyl-tubulin immunoblots highlight com-
pound 7 as a moderate, yet sustained modulator of several
intracellular targets important in tumor etiology. This may
explain the superior antitumor activity of compound 7 relative
to the other dual acting agents disclosed in this study. It is,
however, instructive to emphasize that the target validation
experiments described herein are performed under different
conditions at different incubation periods, so parsing out the
specific contributions of the two targets to the bioactivity of
these agents may not be direct.
Cellular Localization. HDAC1 and Topo II are cell nucleus-
localized targets of these bifunctional compounds, while HDAC6
is cytoplasmic. To probe if cell penetration issues could be one of
the alternative reasons for the difference in the potencies of
compounds 7 and 12b, we used confocal microscopy to visualize
their intracellular localization (Figure 8). We exposed DU-145
cells to 1 μM of DAU, 7 and 12b. After 4 h incubation time, cells
were monitored at 488 nm, the excitation wavelength (λex) of
Figure 8. Cellular localization of dual-acting inhibitors. (a) DAU, (b) 7, (c) 12b. DU-145 cells were dosed at 1 μM for 4 h with indicated
compounds and visualized by confocal microscopy.
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Journal of Medicinal Chemistry
Article
was stirred for the next 16 h at room temperature. The reaction was
quenched by adding an aqueous solution of saturated sodium bicar-
bonate and saturated sodium thiosulfate (1:1, 40 mL) with stirring for
15 min. Methanol−CH2Cl2 1:9 (70 mL) was added after the cessation
of bubbling. The organic layer was isolated, washed subsequently with
sodium bicarbonate−sodium thiosulfate mixture (1:1, 40 mL), brine
(40 mL), and dried on Na2SO4. Solvent was evaporated under reduced
pressure, and the crude product was purified by column chromatog-
raphy using CH2Cl2−methanol (step gradient, 0−12% methanol) to
give 1.10 g of compound 4 (65%). 1H NMR (DMSO-d6, 400 MHz) δ
1.24−1.30 (4H, m), 1.47 (2H, p, J = 7.2, 15.6 Hz), 1.56 (2H, p, J = 7.2,
14.0 Hz), 2.17 (2H, t, J = 7.6 Hz), 2.33 (2H, t, J = 7.2 Hz), 7.77−7.83
(4H, m), 9.84 (1H, s), 10.28 (1H, s), 11.99 (1H, s). 13C NMR
(DMSO-d6, 100 MHz) δ 24.3, 24.7, 28.3, 28.4, 33.6, 36.4, 118.6, 130.8,
131.0, 144.8, 172.0, 174.4, 191.5. HRMS (EI) calcd for C15H19NO4
[M + H]+ 277.1314; found 277.1315.
N-(4-Formylphenyl)-N-(trityloxy)octanediamide (5). Carboxylic
acid 4 (0.30 g, 1.07 mmol) was dissolved in anhydrous THF. N-Methyl-
morpholine (0.12 mL, 1.07 mmol) was added to the solution. The
reaction mixture was then cooled down to −15 °C and stirred for 5 min.
Isobutylchloroformate (0.14 mL, 1.07 mmol) was added, and the
mixture was stirred for 10 min at −15 °C. O-Tritylhydroxylamine
(0.29 g, 1.07 mmol) was added, followed by 2 more equiv of
N-methylmorpholine. Stirring continued for 15 min at −15 °C and 2 h
at room temperature. Afterward, the mixture was poured into CH2Cl2
(50 mL) and water (50 mL). The organic layer was separated and
extracted three times in each case with water, sodium bicarbonate
solution (5%), and water. After washing with brine and drying over
Na2SO4, solvent was evaporated in vacuo. Column chromatography
with eluent system CH2Cl2−acetone (step gradient, 0−12% acetone)
gave 0.37 g of compound 5 (66%) as a white solid. 1H NMR (DMSO-
d6, 400 MHz) δ 1.10−1.21 (4H, m), 1.44−1.51 (2H, m), 1.73−1.77
(2H, m), 7.23−7.35 (15H, m), 7.78−7.84 (4H, m), 9.84 (1H, s), 10.16
(1H, s), 10.29 (1H, s). 13C NMR (DMSO-d6, 100 MHz) δ 24.7, 28.3,
31.9, 36.4, 55.1, 91.5, 118.6, 127.4, 128.9, 130.9, 142.6, 144.6, 170.4,
172.1, 191.2. HRMS (ESI) calcd for C34H34N2O4Na [M + Na]+
557.2207; found 557.2219.
Daunorubicin-N-benzyl-4-amino-8-oxo-N-(trityloxy)-
octanediamide (6). A solution of daunorubicin (0.07 g, 0.12 mmol),
aldehyde 5 (0.07 g, 0.12 mmol), and diisopropylethylamine (0.03 mL,
0.25 mmol) in DMF (2.5 mL) and MeOH (2.5 mL) was heated at
70 °C for 3 h and then allowed to cool to room temperature. Sodium
cyanoborohydride (0.03 g, 0.50 mmol) was added, and stirring was
continued at 70 °C for additional 24 h and allowed to cool at ambient
temperature. The reaction was partitioned between CH2Cl2 (30 mL)
and 5% NaHCO3 (25 mL). The two layers were separated, and the
aqueous layer was extracted with CH2Cl2 (2 × 30 mL). The combined
organic layer was washed with water (2 × 30 mL) and brine (1× 20 mL)
and dried over Na2SO4. Solvent was evaporated off and crude was
purified on preparative TLC plates, eluting with mixture of CH2Cl2−
MeOH (12:1) to give 0.05 g of compound 6 (38%). 1H NMR
(DMSO-d6, 400 MHz) δ 0.93−0.98 (3H, m), 1.04−1.21 (8H, m),
1.38−1.53 (4H, m), 1.60−1.64 (2H, m), 1.71−1.75 (2H, m), 2.06−
2.18 (4H, m), 2.24 (3H, s), 2.80−2.98 (3H, m), 3.15 (1H, d, J =
4.4 Hz), 3.57 (1H, s), 3.67−3.70 (1H, m), 4.05 (1H, q, J = 6.4, 12.8 Hz),
4.91 (1H, t, J = 4.4 Hz), 5.21 (1H, s), 5.42 (1H, s), 7.17 (2H, d,
J = 8.4 Hz), 7.25−7.34 (15H, m), 7.42 (2H, d, J = 8.0 Hz), 7.58−7.62
(1H, m), 7.85−7.87 (2H, m), 9.70 (1H, s), 10.12 (1H, s). 13C NMR
(CD3OD, 100 MHz) δ 17.0, 24.7, 25.1, 25.5, 28.4, 29.6, 30.0, 30.9,
33.1, 34.8, 37.2, 49.7, 52.5, 56.5, 66.6, 66.7, 67.9, 69.7, 100.8, 111.1,
111.3, 118.3, 119.6, 119.8, 120.7, 127.7, 128.0, 128.6, 128.9, 134.2,
134.3, 135.3, 135.6, 137.2, 141.0, 155.7, 156.3, 160.9, 171.5, 186.5,
186.8, 211.8. HRMS (MALDI) calcd for C61H64N3O13 [M + H]+
1046.4439; found 1046.4415.
Another target-independent factor which influences the
bioactivity of the anthracycline-derived dual-acting agent is
their cell uptake and/or residency. In fact, diminished intra-
cellular residency, occasioned by multidrug resistance protein
(MRP) mediated efflux, is one of the problems of an anthra-
cycline based chemotherapy regimen.68 Compound 12b shows
a rapid onset of HDAC and Topo II inhibition activities which
may be quickly lost due to diminished intracellular residency.
The poor intracellular distribution of 12b may suggest that the
triazole-containing compounds 12a−d are prone to efflux, in a
similar manner to the anthracycline template. Alternatively, it
may be that 12a−d are not easily taken up into the cell. Either
of these limitations would compromise the bioactivity of 12a−d
and may explain their less than optimal cytotoxic activity,
despite evidence for potent inhibition of Topo II and HDACs.
Nevertheless, the amide-containing compound 7 is a lead that
merits additional study due primarily to its good intracellular
distribution and potency that rivals DAU. It will be of interest
to know how 7 fares with respect to common deleterious side
effects that have plagued anthracycline therapy.
EXPERIMENTAL SECTION
■
Materials and General Methods. Suberic acid, 4-aminobenzyl
alcohol, and 4-ethynylbenzyl alcohol were purchased from Sigma−
Aldrich. Anhydrous solvents and other reagents were purchased and
used without further purification. Analtech silica gel plates (60 F254)
were used for analytical TLC, and Analtech preparative TLC plates
(UV 254, 2000 μm) were used for purification. UV light was used to
examine the spots. Silica gel (200−400 Mesh) was used in column
chromatography. NMR spectra were recorded on a Varian-Gemini 400
1
magnetic resonance spectrometer. H NMR spectra were recorded in
parts per million (ppm) relative to the peak of CDCl3, (7.24 ppm),
CD3OD (3.31 ppm), or DMSO-d6 (2.49 ppm). 13C spectra were
recorded relative to the central peak of the CDCl3 triplet (77.0 ppm),
CD3OD (49.0 ppm), or the DMSO-d6 septet (39.7 ppm) and were
recorded with complete heterodecoupling. Multiplicities are described
using the abbreviation: s, singlet; d, doublet, t, triplet; q, quartet; m,
multiplet; and app, apparent. High-resolution mass spectra were
recorded at the Georgia Institute of Technology mass spectrometry
facility in Atlanta. The purity of all tested compounds was established
by HPLC to be >95%. HPLC analyses were performed on a Beckman
Coulter instrument using a Phenomenex RP C-18 column (250 mm ×
4.6 mm), eluting with solvent A (0.1% formic acid−water) and solvent
B (0.1% formic acid−acetonitrile) at a gradient of 5−50% over 30 min,
with detection at 498 nm and a flow rate of 1 mL/min. Sample
concentrations were 250 μM, injecting 50 μL. O-Trityl-protected
hydroxamates 9a−d,48 4-ethynylbenzaldehyde 8,69 and suberic
anhydride 270 were prepared according to literature protocol.
8-(4-(Hydroxymethyl)phenylamino)-8-oxooctanoic Acid (3) -. To
a stirring solution of suberic anhydride 2 (1.0 g, 6.40 mmol) in THF
(15 mL) was added (4-aminophenyl)methanol 1 (0.78 g, 6.34 mmol),
and resulting mixture was stirred at room temperature for 1 h. Ethyl
acetate was added (60 mL), followed by washing with water (2 ×
50 mL), brine (1 × 40 mL). Organic layer was dried on Na2SO4 and
solvent evaporated under reduced pressure to give crude compound 2,
which was purified by column chromatography (silica, CH2Cl2−
MeOH (step gradient, 0−10% methanol) to give 0.92 g of compound
1
3 (52%). H NMR (DMSO-d6, 400 MHz) δ 1.25−1.32 (4H, m),
1.49−1.61 (4H, m), 2.21 (2H, t, J = 7.2 Hz), 2.29 (2H, t, J = 8.0 Hz),
4.42 (2H, s), 5.10 (1H, s), 7.23 (2H, d, J = 8.4 Hz), 7.54 (2H, d, J =
8.8 Hz), 9.84 (1H, s), 12.0 (1H, s). 13C NMR (DMSO-d6, 100 MHz)
δ 24.4, 25.0, 28.3, 28.4, 33.6, 36.3, 62.5, 118.5, 126.6, 136.7, 137.7,
170.7, 174.1. HRMS (MALDI) calcd for C15H21NO4Na [M + Na]+
302.1363; found 302.1343.
Daunorubicin-N-benzyl-4-amino-8-oxooctahydroxamic Acid (7).
To a solution of trityl-protected compound 6 (0.025 g, 0.024 mmol) in
CH2Cl2−MeOH (1:1, 4 mL) was added BF3·OEt2 (0.05 mL) at room
temperature. The reaction mixture stirred for 30 min at room
temperature. Reaction was quenched by addition of water (25 mL)
and 10% MeOH in CH2Cl2 (30 mL). The aqueous layer was isolated,
8-((4-Formylphenyl)amino)-8-oxooctanoic Acid (4). To a stirring
solution of alcohol 3 (1.71 g, 6.13 mmol) in CH2Cl2 was added Dess−
Martin reagent (3.898 g, 9.19 mmol) at 0 °C. The reaction mixture
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Journal of Medicinal Chemistry
Article
and its pH was adjusted to 8−9 by addition of satd NaHCO3. This
aqueous layer was then extracted with 10% MeOH in CH2Cl2 (3 ×
30 mL). The combined organic layer was washed with satd brine and
dried over Na2SO4, and the solvent was evaporated under reduced
pressure. Trituration of crude product with CH2Cl2 (20 mL) gave pure
0.013 g of 7 (68%) as red solid. Retention time 6.46 min (solvent
gradient: 5−60% solvent B over 20 min). 1H NMR (CD3OD, 400 MHz)
δ 1.28−1.33 (9H, m), 1.50−1.60 (4H, m), 1.79−1.85 (1H, m), 1.93−
1.94 (1H, m), 2.04−2.12 (2H, m), 2.23−2.30 (3H, m), 2.34 (3H, s),
2.85−3.08 (5H, m), 3.69−3.83 (3H, m), 3.97 (3H, s), 4.18−4.23 (1H,
m), 5.04 (1H, s), 5.40 (1H, s), 7.22 (2H, d, J = 7.6 Hz), 7.39 (2H, d,
J = 8.4), 7.47 (1H, d, J = 8 Hz), 7.74 (1H, t, J = 8 Hz), 7.82 (1H, d, J =
7.2 Hz). 13C NMR (CDCl3 with drops of CD3OD, 100 MHz) δ 17.2,
24.7, 26.2, 26.3, 29.5, 29.6, 30.4, 33.2, 33.5, 36.4, 37.6, 53.3, 54.4, 57.0,
67.5, 68.2, 71.1, 77.2, 101.7, 111.8, 112.1, 115.3, 119.8, 120.3, 121.0,
121.2, 128.3, 130.1, 135.3, 135.4, 136.0, 136.8, 139.2, 155.9, 157.1,
162.0, 172.5, 174.1, 187.3, 187.7, 213.5. HRMS (MALDI) calcd for
C42H50N3O13 [M + H]+ 804.3343, found 804.336, and calcd for
[M + Na]+ 826.3163, found 826.3153.
127.9, 128.8, 130.2, 135.4, 136.3, 140.8, 141.7, 146.1, 176.9, 191.5.
HRMS (MALDI) calcd for C36H36N4O3Na [M + Na]+ 595.2679;
found 595.2609.
Daunorubicin-N-benzyl-4-triazolyl-O-tritylpentahydroxamate
(11a). A solution of daunorubicin hydrochloride (0.08 g, 0.14 mmol)
and 10a (0.15 g, 0.28 mmol) in acetonitrile (9 mL) and water (3 mL)
was stirred at room temperature for 30 min. A suspension of sodium
cyanoborohydride in THF was added within 1 min, and stirring con-
tinued overnight at room temperature during which all of the starting
material got consumed. The reaction mixture was partitioned between
CH2Cl2 (30 mL) and water (30 mL), and the two layers were sepa-
rated. The aqueous layer was extracted with CH2Cl2 (30 mL), and the
combined organic layer was dried over Na2SO4. Solvent was removed
under reduced pressure, and the crude was purified by preparative
TLC, eluting with 12:1 CH2Cl2−MeOH to give 0.065 g of 11a (44%)
as red solid. 1H NMR (DMSO-d6, 400 MHz) 1.23−1.27 (5H, m), 1.36
(3H, d, J = 6.4 Hz), 1.60−1.68 (5H, m), 1.76−1.83 (2H, m), 2.10−
2.15 (1H, m), 2.34−2.39 (1H, m), 2.88−2.93 (1H, m), 2.99−3.03
(1H, m), 3.21−3.26 (1H, m), 3.64 (1H, s), 3.67−3.70 (1H, m), 3.79−
3.83 (1H, m), 3.97 (1H, q, J = 6.4 Hz), 4.06 (3H, s), 4.16−4.19 (2H,
m), 4.73 (1H, s), 4.76 (1H, s), 5.30 (1H, s), 5.51 (1H, d, J = 3.2 Hz),
7.26−7.33 (15H, m), 7.36−7.42 (3H, m), 7.64 (1H, s), 7.71 (2H, d,
J = 8.0 Hz), 7.76 (1H, t, J = 8.4 Hz), 8.01 (1H, d, J = 7.2 Hz). 13C
NMR (CDCl3, 100 MHz) δ 17.4, 20.4, 29.7, 29.9, 30.4, 34.2, 35.7,
50.2, 50.3, 52.5, 56.9, 65.7, 67.0, 67.1, 69.5, 101.0, 111.6, 111.7, 118.6,
119.6, 120.0, 121.1, 126.0, 127.4, 128.1, 128.4, 128.7, 129.2, 129.9,
133.8, 134.0, 135.7, 136.0, 139.6, 141.1, 155.9, 156.4, 161.3, 186.9,
187.3, 213.9. HRMS (ESI) calcd for C60H60N5O12 [M + H]+ 1042.4233;
found 1042.4189.
Representative Procedure for Cu(I)-Catalyzed Cycloaddition
Reaction: O-Trityl-4-formylphenyltriazolylpentahydroxamate
(10a). 5-Azido-O-tritylpentahydroxamate 9a (0.15 g, 0.37 mmol)
and 4-ethynylbenzaldehyde 8 (0.06 g, 0.49 mmol) were dissolved in
anhydrous THF (10 mL) and stirred under argon at room tempera-
ture. Copper(I) iodide (9 mg, 0.05 mmol) and Hunig’s base (0.1 mL)
were then added to the reaction mixture, and stirring continued for 24 h.
The reaction mixture was diluted with CH2Cl2 (30 mL) and washed
with 1:4 NH4OH−satd NH4Cl (3 × 30 mL) and satd NH4Cl (30 mL).
The organic layer was dried over Na2SO4 and concentrated in vacuo.
The crude product was purified by flash chromatography (CH2Cl2−
acetone, gradient 12:1, 10:1, 8:1) to give 0.16 g of 10a (82%) as a
Daunorubicin-N-benzyl-4-triazolyl-O-tritylhexahydroxamate
(11b). Reaction of daunorubicin hydrochloride (0.055 g, 0.097 mmol)
with 10b (0.159 g, 0.29 mmol) and sodium cyanoborohydride (0.018 g,
0.29 mmol) in acetonitrile−water solvent system, as described in the
1
white solid. H NMR (DMSO-d6, 400 MHz) δ 1.13−1.20 (2H, m),
1.49−1.56 (2H, m), 1.82 (2H, t, J = 7.2), 4.27 (2H, t, J = 6.8 Hz),
7.24−7.28 (15H, m), 7.97 (2H, d, J = 8.8 Hz), 8.05−8.07 (2H, d, J =
8.4 Hz), 8.68 (1H, s), 9.99 (1H, s), 10.23 (1H, s). 13C NMR (CDCl3,
100 MHz) δ 20.0, 29.2, 30.1, 50.0, 93.4, 120.8, 125.8, 127.7, 128.0,
128.8, 130.2, 135.5, 136.3, 140.8, 146.1, 176.2, 191.6. HRMS (MALDI)
calcd for C33H30N4O3Na [M + Na]+ 553.2210; found 553.2191.
1
synthesis of 11a, gave 0.05 g of 11b (49%) as red solid. H NMR
(DMSO-d6, 400 MHz) 1.23−1.28 (5H, m), 1.38 (3H, d, J = 6.4 Hz),
1.56−1.60 (3H, m), 1.66−1.85 (6H, m), 2.12−2.17 (1H, m), 2.35−
2.41 (1H, m), 2.91−2.94 (1H, m), 2.99−3.04 (1H, m), 3.23−3.28
(1H, m), 3.66−3.72 (2H, m), 3.81−3.84 (1H, m), 3.99 (1H, q, J = 6.8
Hz), 4.07 (3H, s), 4.28 (2H, t, J = 7.6 Hz), 4.74 (1H, s), 4.78 (1H, s),
5.29−5.32 (1H, m), 5.53 (1H, d, J = 2.8 Hz), 7.30−7.35 (15H, m),
7.37−7.45 (3H, m), 7.67 (1H, s), 7.73−7.80 (3H, m), 8.02 (1H, d, J =
7.2 Hz). 13C NMR (CDCl3, 100 MHz) δ 17.1, 22.5, 25.7, 29.6, 29.8,
30.2, 30.7, 33.9, 35.4, 49.9, 50.0, 52.3, 56.6, 65.4, 66.7, 66.8, 69.3,
100.8, 111.3, 111.5, 118.4, 119.4, 119.8, 120.8, 125.8, 127.8, 128.1,
128.4, 128.9, 129.7, 133.5, 133.8, 135.4, 135.7, 139.5, 141.1, 147.3,
155.6, 156.2, 161.0, 186.7, 187.0, 213.7. HRMS (ESI) calcd for
C61H62N5O12 [M + H]+ 1056.4389; found 1056.4440.
O-Trityl-4-formylphenyltriazolylhexahydroxamate (10b). Reac-
tion of 6-azido-O-tritylhexahydroxamate 9b (0.24 g, 0.58 mmol) and
4-ethynylbenzaldehyde 8 (0.09 g, 0.69 mmol) as described for the
synthesis of 10a, followed by purification using column chromatog-
raphy (CH2Cl2−acetone, gradient 12:1, 10:1, 8:1) gave 0.19 g of 10b
1
(61%) as a white solid. H NMR (DMSO-d6, 400 MHz) δ 0.92−0.99
(2H, m), 1.19−1.26 (2H, m), 1.70−1.78 (4H, m), 4.30 (2H, t, J =
6.8), 7.26−7.29 (15H, m), 7.96 (2H, d, J = 8.0 Hz), 8.05 (2H, d, J =
8.4 Hz), 8.72 (1H, s), 9.99 (1H, s), 10.15 (1H, s). 13C NMR (CDCl3,
100 MHz) 22.2, 25.5, 29.6, 30.7, 49.9, 93.1, 120.8, 125.7, 127.6, 127.9,
128.8, 130.1, 135.4, 136.3, 140.7, 141.6, 146.0, 176.5, 191.5.
Daunorubicin-N-benzyl-4-triazolyl-O-tritylheptahydroxamate
(11c). Reaction of daunorubicin hydrochloride (0.055 g, 0.097 mmol)
with 10c (0.162 g, 0.29 mmol) and sodium cyanoborohydride (0.018 g,
0.29 mmol) in acetonitrile−water solvent system, as described in the
O-Trityl-4-formylphenyltriazolylheptahydroxamate (10c). Reac-
tion of 7-azido-O-tritylheptahydroxamate 9c (0.30 g, 0.70 mmol) and
4-ethynylbenzaldehyde 8 (0.09 g, 0.70 mmol) as described for the
synthesis of 10a, followed by purification using column chromatog-
raphy (CH2Cl2−acetone, gradient 12:1, 10:1, 8:1) gave 0.25 g of 10c
1
synthesis of 11a, gave 0.05 g of 11c (48%) as a red solid. H NMR
(DMSO-d6, 400 MHz) δ 0.99−1.07 (2H, m), 1.13−1.25 (6H, m), 1.36
(3H, d, J = 6.4 Hz), 1.52−1.57 (2H, m), 1.70−1.83 (5H, m), 2.02−
2.10 (2H, m), 2.14 (1H, s), 2.33 (1H, s), 2.85−2.88 (1H, m), 2.93
(1H, s), 2.97−3.00 (1H, s), 3.14−3.19 (1H, m), 3.67−3.70 (2H, m),
3.80−3.83 (1H, m), 4.04 (3H, s), 4.28 (3H, t, J = 6.8 Hz), 4.66−4.69
(1H, m), 5.24 (1H, s), 5.50 (1H, d, J = 3.2 Hz), 7.26−7.30 (15H, m),
7.34−7.43 (3H, m), 7.63 (1H, s), 7.66−7.79 (3H, m), 7.97 (1H, d, J =
7.6 Hz). 13C NMR (CDCl3, 100 MHz) δ 17.1, 23.1, 24.8, 25.9, 28.2,
29.7, 30.0, 30.9, 30.9, 33.2, 34.9, 49.9, 50.2, 52.3, 56.6, 66.6, 66.7, 69.9,
100.6, 111.3, 111.5, 118.4, 119.4, 119.9, 120.9, 125.7, 127.8, 128.1,
128.4, 128.9, 129.9, 134.2, 134.2, 135.4, 135.7, 139.5, 141.1, 147.3,
155.6, 156.2, 161.0, 186.7, 187.1, 212.0. HRMS (ESI) calcd for
C62H64N5O12 [M + H]+ 1070.4546; found 1070.4508.
1
(65%) as a white solid. H NMR (DMSO-d6, 400 MHz) δ 0.96−1.17
(6H, m), 1.71−1.78 (4H, m), 4.34 (2H, t, J = 7.2 Hz), 7.24−7.34
(15H, m), 7.96 (2H, d, J = 7.6 Hz), 8.06 (2H, d, J = 8.0 Hz), 8.75 (1H,
s), 9.99 (1H, s), 10.14 (1H, s). 13C NMR (CDCl3, 100 MHz) δ 22.8,
25.8, 28.0, 29.7, 30.7, 50.1, 93.2, 120.7, 125.7, 127.9, 128.8, 130.1,
135.4, 136.3, 140.8, 141.7, 146.1, 176.8 191.5.
O-Trityl-4-formylphenyltriazolyloctahydroxamate (10d). Reaction
of 8-azido-O-trityloctahydroxamate 9d (0.25 g, 0.57 mmol) and 4-
ethynylbenzaldehyde 8 (0.07 g, 0.57 mmol) as described for the
synthesis of 10a, followed by purification using column chromatog-
raphy (CH2Cl2−acetone, gradient 12:1, 10:1, 8:1) gave 0.19 g of 10d
1
(59%) as a white solid. H NMR (DMSO-d6, 400 MHz) δ 0.85−0.93
Daunorubicin-N-benzyl-4-triazolyl-O-trityloctahydroxamate
(11d). Reaction of daunorubicin hydrochloride (0.055 g, 0.097 mmol)
with 10d (0.166 g, 0.29 mmol) and sodium cyanoborohydride (0.018 g,
0.29 mmol) in acetonitrile−water solvent system, as described in the
(2H, m), 1.08−1.17 (6H, m), 1.72−1.80 (4H, m), 4.38 (2H, t, J =
7.2), 7.24−7.28 (15H, m), 7.96 (2H, d, J = 7.6 Hz), 8.06 (2H, d, J =
8.0 Hz), 8.77 (1H, s), 9.99 (1H, s), 10.13 (1H, s). 13C NMR (CDCl3,
100 MHz) δ 23.0, 26.0, 28.3, 28.5, 30.0, 30.8, 50.3, 93.1, 120.7, 125.8,
1
synthesis of 11a, gave 0.048 g of 11d (46%) as a red solid. H NMR
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(DMSO-d6, 400 MHz) δ 0.98−1.06 (2H, m), 1.19−1.24 (8H, m), 1.37
(3H, d, J = 6.4 Hz), 1.53−1.58 (2H, m), 1.69−1.85 (5H, m), 2.07−
2.10 (2H, m), 2.35 (1H, s), 2.88−2.92 (1H, m), 2.96−2.99 (1H, m),
3.15−3.20 (1H, m), 3.67−3.70 (2H, m), 3.79−3.83 (1H, m), 4.05
(3H, s), 4.31 (3H, t, J = 6.8 Hz), 4.68 (1H, s), 5.25 (1H, s), 5.50 (1H,
s), 7.27−7.31 (15H, m), 7.35−7.47 (3H, m), 7.66 (1H, s), 7.69−7.77
(3H, m), 7.98 (1H, d, J = 7.6 Hz). 13C NMR (CDCl3, 100 MHz) δ
17.0, 23.1, 24.8, 26.1, 28.5, 29.6, 30.1, 30.2, 31.0, 33.2, 34.8, 50.0, 50.2,
52.5, 56.6, 66.5, 66.7, 69.8, 100.9, 111.1, 111.2, 118.3, 119.2, 119.7,
120.7, 125.7, 127.8, 128.0, 128.4, 128.9, 129.6, 134.2, 134.3, 135.4,
135.6, 139.5, 141.0, 147.3, 155.7, 156.3, 160.9, 186.5, 186.8, 211.8.
HRMS (ESI) calcd for C63H66N5O12 [M + H]+ 1084.4702; found
1084.4657.
Daunorubicin-N-benzyl-4-triazolylpentahydroxamic Acid (12a).
Reaction of 11a (0.05 g, 0.048 mmol) and BF3·OEt2 (0.1 mL in
CH2Cl2−MeOH (4 mL/4 mL) within 2 h, as described for the
synthesis of 7, followed by preparative TLC (eluent, CH2Cl2−MeOH
7:1) to give 0.028 g of 12a (73%) as red solid. Retention time 15.06 min
(solvent gradient: 0−100% solvent B over 30 min). 1H NMR (DMSO-
d6, 400 MHz) δ 1.11−1.21 (7H, m), 1.42−1.50 (4H, m), 1.74−1.80
(2H, m), 1.96 (2H, t, J = 7.6 Hz), 2.06−2.22 (5H, m), 2.84−2.97 (3H,
m), 3.63−3.80 (1H, m), 3.96 (3H, s), 4.07−4.09 (1H, m), 4.33 (2H, t,
J = 6.8 Hz), 4.91 (1H, m), 5.22 (1H, s). 5.47 (1H, s), 7.37 (2H, d, J =
8.0 Hz), 7.61−7.63 (1H, m), 7.69−7.71 (2H, d, J = 8.0 Hz), 7.87−7.92
(2H, m), 8.46 (1H, s), 10.37 (1H, s). 13C NMR (CDCl3 with drops of
CD3OD, 125 MHz) δ 15.1, 22.5, 24.1, 24.2, 27.3, 27.4, 28.3, 31.0, 31.3,
34.2, 35.4, 51.1, 52.2, 54.8, 65.4, 66.1, 68.9, 75.1 99.5, 109.7, 109.9,
113.1, 117.6, 118.2, 118.8, 119.0, 126.3, 127.9, 133.1, 133.2, 133.8,
134.7, 137.0, 153.7, 154.9, 159.8, 170.4, 172.0, 185.1, 185.5, 211.1.
HRMS (ESI) calcd for C41H46N5O12 [M + H]+ 800.3137; found
800.3088.
Daunorubicin-N-benzyl-4-triazolylhexahydroxamic Acid (12b).
Reaction of 11b (0.05 g, 0.047 mmol) and BF3·OEt2 (0.1 mL) in
CH2Cl2−MeOH (4 mL/4 mL) within 2 h, as described for the
synthesis of 7, followed by preparative TLC (eluent, CH2Cl2−MeOH
7:1) gave 0.028 g of 12b (76%) as red solid. Retention time 15.20 min
(solvent gradient: 0−100% solvent B over 30 min). 1H NMR (CDCl3 +
CD3OD, 400 MHz) δ 0.74−0.84 (2H, m), 1.25−1.31 (7H, m),
1.54−1.62 (3H, m), 1.72−1.73 (2H, m), 1.83−1.93 (2H, m), 1.99
(1H, t, J = 7.6 Hz), 2.03−2.08 (1H, m), 2.19−2.33 (2H, m), 3.57−
3.60 (5H, m), 3.68−3.73 (2H, m), 4.00 (3H, s), 4.30 (2H, q, J = 7.2
Hz), 4.65−4.66 (1H, m), 5.22−5.23 (1H, m), 5.44 (1H, s), 7.24−7.25
(1H, m), 7.34 (2H, d, J = 8.4 Hz), 1.61−1.65 (2H, m), 7.70−7.72
(1H, m), 7.74−7.75 (1H, m), 7.95 (1H, d, J = 7.6 Hz). 13C NMR
(CDCl3 with drops of CD3OD, 100 MHz) δ 16.8, 22.5, 24.3, 25.4,
29.5, 30.7, 32.2, 33.6, 35.3, 50.0, 50.1, 51.9, 56.5, 65.0, 67.0, 67.2, 69.0,
100.6, 111.3, 111.4, 118.4, 119.7, 119.9, 125.7, 127.8, 128.6, 129.1,
133.7, 133.8, 135.3, 135.7, 147.3, 160.9, 170.2, 186.7, 187.1, 213.5.
HRMS (ESI) calcd for C42H48N5O12 [M + H]+ 814.3294; found
814.3323.
Daunorubicin-N-benzyl-4-triazolylheptahydroxamic Acid (12c).
Reaction of 11c (0.05 g, 0.047 mmol) and BF3·OEt2 (0.1 mL) in
CH2Cl2−MeOH (4 mL/4 mL) within 2 h, as described for the syn-
thesis of 7, followed by preparative TLC (eluent, CH2Cl2−MeOH
7:1) gave 0.030 g of 12c (78%) as red solid. Retention time 16.98 min
(solvent gradient: 5−60% solvent B over 20 min). 1H NMR (DMSO-
d6, 400 MHz) δ 1.17−1.26 (8H, m), 1.37−1.49 (3H, m), 1.70−1.83
(5H, m), 1.89−1.92 (1H, m), 1.99 (1H, t, J = 7.2 Hz), 2.09−2.23 (5H,
m), 2.83−2.99 (4H, m), 3.63−3.84 (2H, m), 3.92−3.96 (3H, m), 4.08
(1H, t, J = 6.8 Hz), 4.31 (2H, t, J = 6.8 Hz), 4.93−4.96 (1H, m), 5.25
(1H, s). 5.34 (1H, s), 7.36 (2H, d, J = 8.0 Hz), 7.53−7.60 (1H, m),
7.70 (2H, d, J = 8.0 Hz), 7.79−7.80 (1H, m), 7.85−7.86 (1H, m), 8.41
(1H, s), 10.21 (1H, s). 13C NMR (DMSO-d6, 100 MHz) δ 17.7, 24.6,
25.5, 25.8, 26.1, 26.2, 27.2, 28.4, 28.6, 28.6, 30.1, 32.4, 32.8, 37.0, 50.1,
57.3, 67.4, 70.7, 76.1, 101.2, 111.5, 119.8, 120.6, 120.9, 121.8, 125.6,
128.6, 129.0, 129.5, 135.6, 136.4, 136.9, 146.8, 155.2, 155.9, 161.7,
186.8, 212.0. HRMS (ESI) calcd for C43H50N5O12 [M + H]+ 828.3450;
found 828.3407.
CH2Cl2−MeOH (4 mL/4 mL) within 2 h, as described for the
synthesis of 7, followed by preparative TLC (eluent, CH2Cl2−MeOH
7:1), gave 0.028 g of 12d (73%) as red solid. Retention time 16.93 min
(solvent gradient: 5−60% solvent B over 20 min). 1H NMR (DMSO-
d6, 400 MHz) δ 1.15−1.20 (8H, m), 1.41−1.44 (2H, m), 1.64−1.67
(2H, m), 1.77−1.80 (2H, m), 1.88 (2H, t, J = 7.6), 1.95−1.20 (2H,
m), 2.23−2.24 (3H, m), 2.80−2.94 (3H, m), 3.60−3.67 (2H, m),
3.76−3.79 (1H, m), 3.89−3.94 (3H, m), 4.05−4.10 (1H, m), 4.30
(2H, t, J = 7.2), 4.88−4.91 (1H, m), 5.21 (1H, m), 5.42 (1H, s), 7.33−
7.35 (2H, m), 7.57−7.59 (1H, m), 7.65−7.73 (2H, m), 7.83−7.86
(2H, m), 8.44 (1H, s), 10.29 (1H, m). 13C NMR (DMSO-d6, 125
MHz) δ 17.2, 24.0, 24.8, 25.5, 27.9, 29.4, 30.8, 31.5, 32.1, 36.2, 48.4,
49.3, 51.8, 54.8, 56.5, 66.8, 70.0, 75.1, 75.2, 100.6, 110.5, 110.6, 118.8,
119.5, 119.9, 120.8, 124.9, 128.3, 129.1, 134.4, 134.6, 135.5, 136.1,
146.2, 154.1, 155.8, 160.6, 169.0, 186.3, 211.5. HRMS (ESI) calcd for
C44H50N5O12 [M + H]+ 842.3607; found 842.3626.
In Vitro HDAC Inhibition. In vitro HDAC inhibition was assayed
using the HDAC Fluorimetric Assay/Drug Discovery Kit as previously
described.48 Briefly, 15 μL of HeLa nuclear extract was mixed with
5 μL of 10× compound and 5 μL of assay buffer. Fluorogenic substrate
(25 μL) was added, and reaction was allowed to proceed for 15 min at
room temperature and then stopped by addition of a developer con-
taining TSA. Fluorescence was monitored after 15 min at excitation
and emission wavelengths of 360 and 460 nm, respectively. IC50 values
were determined using logit plots.
KDNA Decatenation Assay. The decatenation of KDNA was
assayed according to TopoGen protocol in order to determine
topoisomerase II activity. The substrate KDNA (200 ng) and 50 μM
drug were combined in assay buffer (50 mM Tris−HCl, pH 8, 120 mM
KCl, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM dithiothreitol, 300ug/mL
bovine serum albumin (BSA)) and incubated for 10 min on ice. Next,
1 U of topoisomerase II was added and the reaction was allowed to
proceed for 10 min at 37 °C. The reaction was quenched via the
addition of loading buffer (1% sarkosyl, 0.025% bromophenol blue,
and 5% glycerol) and was then analyzed by electrophoresis on a 1%
agarose gel in TBE buffer (89 mM Tris, 89 mM borate, and 2 mM
Na−EDTA, pH 8.3) for 3.5 h at 40 V. The gel was stained with SYBR
Green I (Molecular Probes) for 30 min and was visualized under UV
illumination and photographed on an AlphaImager.
Cellular Topo II Inhibition. DU-145 cells were probed for Topo
II inhibition with an in vitro blotting kit designed to show relative
amounts of stabilized Topo II−DNA cleavage complexes (Top-
oisomerase II In Vivo Link Kit, Topogen). Briefly, cells were dosed
with Topo II−HDACi’s at concentrations pertaining to their respec-
tive IC50 values for cell viability inhibition. Control cells were dosed
with 0.1% DMSO to take into account DMSO from stock solutions of
drug. As recommended in the protocol instructions, cells were dosed
for 30 min, counted, and lysed with 1% sarkosyl. Alternately, DU-145
cells were dosed for 72 h, counted, and equalized with the cell count
from the 30 min incubation before subsequent lysis. Lysate was
collected, loaded on a CsCl gradient, and subjected to centrifugation at
31000 rpm at room temperature for 12 h. Aliquots of the gradient
separations were then taken and the Topo II−DNA cleavage com-
plexes were identified via absorbance at 260 nm. Aliquots were then
loaded into a slot blotting device and subjected to vacuum to load
proteins onto a nitrocellulose membrane. Immunoblotting using the
Odyssey Imaging System (LiCor Biosciences) revealed Topo II levels
associated with the stabilized DNA complexes.
Cell Culture and Viability. DU-145 prostate carcinoma and SK-
MES-1 nonsmall cell lung carcinoma was obtained from ATCC
(Manassass, VA) and maintained in the recommended growth
mediums. MCF-7 breast cancer cells were a generous gift from Dr.
Donald Doyle. All cell lines were maintained in a 37 °C incubator with
a 5% CO2 environment. All compounds to be tested were dissolved to
a concentration of 10 mM in DMSO and stored at −80 °C. Cells were
passaged 24 h prior to cell viability experiments. For cancer cell
viability experiments, cells were dosed for 72 h and viability was
determined through the use of the MTS assay (Promega) according
to manufacturer’s instructions. Control wells were dosed with fresh
media containing 0.1% DMSO.
Daunorubicin-N-benzyl-4-triazolyloctahydroxamic Acid (12d).
Reaction of 11d (0.05 g, 0.046 mmol) and BF3·OEt2 (0.1 mL) in
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Western Blot of p21waf1 Expression and Histone Hyper-
acetylation. DU-145 prostate cancer cells were passaged 24 h prior
to the experiment. Compounds to be tested were diluted in the growth
medium so that the final concentration of DMSO was 0.1% and
control cells were dosed with fresh media containing 0.1% DMSO.
Cells were dosed for 24 h then washed twice with ice-cold PBS and
lysed on the culture plate at 4 °C for 5 min with RIPA buffer con-
taining protease inhibitors. Lysates were mixed repeatedly by pipetting
and centrifuged at 14000g for 15 min at 4 °C. Supernatant was saved
and protein concentration was quantified using the Bio-Rad Protein
Assay (Bio-Rad) with BSA as the standard. Loading buffer was added,
and protein samples were incubated at 100 °C for 10 min before
electrophoresis. Proteins were then transferred to a nitrocellulose
membrane for 1 h, followed by blocking overnight in a 1:1 mixture of
Odyssey Blocking Buffer (LiCor Biosciences) and PBS. Membranes
were incubated with primary and secondary antibodies, both diluted in
1:1 Odyssey Blocking Buffer/PBS. Membranes were scanned on the
Odyssey infrared imaging system (LiCor Biosciences) using both 700
and 800 nm channels simultaneously at 169 μm resolution and
analyzed on the imaging software.
Western Blot of Tubulin Acetylation. DU-145 cells were
passaged 24 h prior to dosing. Compounds were diluted such that
cells, including controls, were exposed to no more than 0.1% DMSO.
Cells were dosed for 4 h before lysis in RIPA buffer with protease
inhibitors. Lysates were vortexed and centrifuged at 14000 rpm at 4 °C
for 15 min. Supernatants were removed, and protein concentration
was assayed using the Bio Rad Protein Assay with BSA as a standard.
Electrophoresis and immunoblotting were performed as described
above. Rabbit antitubulin (Sigma) and mouse antiacetyl tubulin
(Invitrogen) were used to probe the membrane after blocking with
Odyssey blocking buffer (Li Cor). Li Cor near-infrared secondary anti-
bodies were used to image the blot with the Odyssey infrared imaging
system at 700 and 800 nm.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR and 13C NMR spectral information, HPLC traces,
and molecular modeling outputs. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 404-894-4047. Fax: 404-894-2291. E-mail: aoyelere@
gatech.edu.
Author Contributions
§These authors contributed equally to the manuscript.
ACKNOWLEDGMENTS
■
We are grateful to Professor Olaf Wiest for providing us with
the HDAC 1 homology model. This work was financially
supported by NIH grant R01CA131217.
ABBREVIATIONS USED
■
HDAC, histone deacetylase; HAT, histone acetyltransferase;
HDACi, histone deacetylase inhibitors; HDLP, histone
deacetylase-like protein; SAHA, suberoylanilide hydroxamic
acid; TSA, trichostatin A; DOX, doxorubicin; DAU, daunomycin;
Topo II, topoisomerase II class of enzymes; KDNA, kinetoplast
DNA
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