Novel alkoxyamide-based histone deacetylase inhibitors reverse cisplatin resistance in chemoresistant cancer cells

Article abstract of DOI:10.1016/j.bmc.2019.115108

Although histone deacetylase inhibitors (HDACi) have shown promising antitumor effects in specific types of blood cancer, their effects on solid tumors are limited. Previously, we developed LMK235 (5), a class I and class IIb preferential HDACi with chemosensitizing effects on breast cancer, ovarian cancer and HNSCC. Based on its promising effects on solid tumor cells, we modified the cap group of 5 to improve its anticancer activity. The tri- and dimethoxy-phenyl substituted compounds 13a and 13d turned out to be the most potent HDAC inhibitors of this study. The isoform profiling revealed a dual HDAC2/HDAC6 inhibition profile, which was confirmed by the acetylation of α-tubulin and histone H3 in Cal27 and Cal27CisR. In combination with cisplatin, both compounds enhanced the cisplatin-induced cytotoxicity via caspase-3/7 activation. The effect was more pronounced in the cisplatin resistant subline Cal27CisR. The pretreatment with 13d resulted in a complete resensitisation of Cal27CisR with IC₅₀ values in the range of the parental cell line. Therefore, 13d may serve as an epigenetic tool to analyze and modulate the cisplatin resistance of solid tumors.

Full text of DOI:10.1016/j.bmc.2019.115108

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Novel alkoxyamide-based histone deacetylase inhibitors reverse
cisplatin resistance in chemoresistant cancer cells
Yodita Asfaha, Christian Schrenk, Leandro A. Alves Avelar,
Friedrich Lange, Chenyin Wang, Jan J. Bandolik, Alexandra
Hamacher, Matthias U. Kassack, Thomas Kurz
PII:
S0968-0896(19)30524-3
https://doi.org/10.1016/j.bmc.2019.115108
BMC 115108
DOI:
Reference:
To appear in:
Received date:
Revised date:
Accepted date:
29 March 2019
3 September 2019
11 September 2019
Please cite this article as: Y. Asfaha, C. Schrenk, L.A. Alves Avelar, et al., Novel
alkoxyamide-based histone deacetylase inhibitors reverse cisplatin resistance in
chemoresistant cancer cells, (2018), https://doi.org/10.1016/j.bmc.2019.115108
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Novel alkoxyamide-based histone deacetylase inhibitors
reverse cisplatin resistance in chemoresistant cancer cells
Yodita Asfaha*, Christian Schrenk*, Leandro A. Alves Avelar*, Friedrich Lange*,
Chenyin Wang*, Jan J. Bandolik*, Alexandra Hamacher*, Matthias U. Kassack*#,
Thomas Kurz*#
*Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität
#
Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany, MUK and TK share senior
and corresponding authorship
Keywords: Histone Deacetylases, cisplatin Resistance, Chemosensitizing effects
ABSTRACT
Although histone deacetylase inhibitors (HDACi) have shown promising antitumor
effects in specific types of blood cancer, their effects on solid tumors are limited.
Previously, we developed LMK235 (5), a class I and class IIb preferential HDACi with
chemosensitizing effects on breast cancer, ovarian cancer and HNSCC. Based on its
promising effects on solid tumor cells, we modified the cap group of 5 to improve its
anticancer activity. The tri- and dimethoxy-phenyl substituted compounds 13a and 13d
turned out to be the most potent HDAC inhibitors of this study. The isoform profiling
revealed a dual HDAC2/HDAC6 inhibition profile, which was confirmed by the
acetylation of α-tubulin and histone H3 in Cal27 and Cal27CisR. In combination with
cisplatin, both compounds enhanced the cisplatin-induced cytotoxicity via caspase-3/7
activation. The effect was more pronounced in the cisplatin resistant subline Cal27CisR.

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The pretreatment with 13d resulted in a complete resensitisation of Cal27CisR with IC50
values in the range of the parental cell line. Therefore, 13d may serve as an epigenetic
tool to analyze and modulate the cisplatin resistance of solid tumors.

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1. Introduction
Epigenetic control plays an essential role in gene regulation through chemical
modifications of DNA and post-translational modifications of histones.1 An important
post-translational modification is the reversible histone acetylation, which is regulated by
histone acetyltransferases (HATs) and histone deacetylases (HDACs). The mammalian
zinc dependent HDACs are subdivided into three classes: class I (HDACs 1-3, 8), class
II (IIa: HDACs 4, 5, 7, 9; IIb: HDACs 6, 10) and class IV (HDAC 11).2 Recent studies
have shown that HDACs are overexpressed in a wide spectrum of cancer types.3–5
Therefore, their inhibition provides a promising tool for developing novel anticancer
drugs. The success of this approach is shown by the FDA approval of four HDAC
inhibitors (HDACi) for the treatment of specific types of blood cancer (Figure 1). The
currently used and widely accepted pharmacophore model for HDACi features the
following three elements: a zinc binding group (ZBG), a linker, that mimics the alkylic
chain of the acetyl-lysine motif and interacts with the substrate binding tunnel and a cap,
also known as surface recognition domain.6,7 According to their structural features,
HDACi can be classified into 4 main classes: hydroxamates, cyclic peptides, carboxylic
acids and o-amino anilides.7,8
Figure 1: Chemical structures of FDA approved HDAC inhibitors 1-4 and their indications. CTCL: cutaneous T-cell
lymphoma, MM: multiple myeloma, PTCL: peripheral T-cell lymphoma.9–12
Most of the currently approved HDACi inhibit a broad isoform spectrum (pan-inhibitor)
and can cause severe side effects such as diarrhea, fatigue or cardiac toxicity. It is still
under debate if inhibition of specific HDAC isoforms will improve these drawbacks.13
In a previous study, we have varied the CAP and linker region of vorinostat resulting in
compound LMK235 (5) (N-((6-(hydroxyamino)-6-oxohexyl)oxy)-3,5-dimethylbenz-
amide) with improved cytotoxic and synergistic effects against several human cancer cell

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lines compared to vorinostat.14 Reassessment of the HDAC isoform profile with improved
assays and reference compounds showed a class I / class IIb preference of LMK235
(5).15,16 Due to the promising anticancer properties of 5, we aimed to explore the effects
of further chemical cap group modifications with regard to antiproliferative and
synergistic activity in cisplatin-sensitive and cisplatin-resistant cancer cells. (Scheme 1).
Scheme 1: Modifications of 5 in its cap region.
2. Results and Discussion
2.1. Chemistry
The synthesis of the alkoxyamide-based HDACi 5 has been reported previously by Marek
et. al..14 Here, we report the synthesis of nine novel analogs (13a-h, 14) starting from the
O-protected aminoxy-hydroxamates 7, 8 and 9. Initial attempts to introduce the
alkoxyamide moiety using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI)
mediated standard protocols were unsuccessful. However, HATU mediated acylation of
7, 8 and 9 with the respective carboxylic acid provided the protected intermediates 10a-
e, 11, 12a-c. Finally, their deprotection was accomplished by hydrogenolysis and by a
Lewis acid mediated procedure17, respectively.
Scheme 2: Reagents and conditions: a) 1.00 eq R2COOH, 1.00 eq HATU, 2.00 eq DIPEA, DMF, rt, 16 h, 40-65% b)
Pd/C, H2, (1 bar), MeOH, rt, 35-40% c) 10.0 eq TFA, 10.0 eq Et3SiH, DCM, 30-40%.

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2.2. Biological evaluation
2.2.1. Cellular HDAC inhibitory activity and antiproliferative effects
The novel derivatives of compound 5, 13a - 13h and 14, were first tested for their
antiproliferative effects by MTT assay and for cellular HDAC inhibitory activity in the
ovarian cancer cell line A2780 and the head-neck cancer cell line Cal27 (Table 1). Since
the cellular HDAC assay only serves as an explorative assay and needs confirmation in
enzymatic assays, it was performed only once. Thus, no errors are reported for the cellular
HDAC assay. Results are presented in Table 1. Compound 5, vorinostat, and cisplatin
served as controls. The compounds with the highest antiproliferative activity against the
cell line A2780 and Cal27 were 13b and 13d with IC50 values ranging from 0.72 to 1.55
µM. 13b and 13d were also the most active compounds in the cellular HDAC assay with
similar IC50 values compared to the MTT assay. Interestingly 13a and 13f showed
moderate antiproliferative activity in contrast to 13b and 13d, and at the same time
increased HDAC inhibitory activity with IC50 values similar to 13b but slightly higher
than 13d. 2 Methoxy groups (13d) attached to the cap group instead of 3 (13a) seem to
play a minor role for the HDAC inhibitory activity of the compounds, while cytotoxicity
is increased (over 15-fold in A2780, approx. 4-fold in Cal27). Of note, all tested
compounds were slightly more potent in Cal27 cells (MTT assay). We could confirm the
superior potency of compound 5 (LMK235) over vorinostat. The initial evaluation
revealed comparable cytotoxicity and cellular HDAC inhibition for 13d and 5.
Interestingly, increasing the size of the phenyl substituents was tolerated (13b) whereas
introduction of halogen, -CF3, the pyridyl heterocycle or a shorter linker (14) reduced
activity.

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Table 1. Antiproliferative effects and HDAC inhibitory activity of 13a-13h, 14, 5, vorinostat and cisplatin in
A2780 and Cal27
A2780 (µM)
Cal27 (µM)
Compound
13a
R2
HDACb
MTT
HDACb
MTT
1.18
13.8 ± 0.84
1.48
3.07 ± 0.15
13b
0.93
3.79
1.55 ± 0.12
13.7 ± 1.38
1.30
3.77
1.0 ± 0.001
7.98 ± 1.10
13c
13d
13e
13f
0.83
11.0
0.45
0.89 ± 0.006
30.5 ± 4.16
0.72 ± 0.09
15.8 ± 0.57
10.56
1.65
7.20
10.2 ± 0.22
64.0 ± 6.68
1.80
9.40
3.63 ± 0.16
45.0 ± 11.3
13g

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68.4 ± 6.50
28.1 ± 1.67
8.67 ± 0.40
14.5
4.24
12.7
3.36
13h
14
9.34 ± 0.14
0.97 ± 0.01
2.04 ± 0.20
0.76 ± 0.04
1.50 ± 0.10
0.17
0.91
0.55
0.59
5
vorinostat
cisplatina)
1.88 ± 0.06
2.27 ± 0.20
n.d.
n.d.
a)Data taken from18 b)Cellular HDAC assay was only performed once with 3 replicates due to its explorative character.
Values are the mean ± SEM of three independent experiments conducted in triplicate. n.d. = not determined.
2.2.2. Inhibitory activity on HDAC2, HDAC4, HDAC6 and HDAC8
For further evaluation, compounds were tested against human recombinant HDAC2,
HDAC4, HDAC6 and HDAC8. Compound 5, vorinostat, and CHDI-00390576-0000-004
served as reference compounds (Table 2.). Cellular HDAC assay revealed compounds
13a, 13b, 13d and 13f as the most promising inhibitors. The two compounds with
methoxy substituents attached to the cap group showed the strongest inhibition of
HDAC2 and HDAC6. 13a and 13d displayed Ki values of 0.03 µM to 0.09 µM for
HDAC2 and HDAC6, with low inhibition of HDAC4 (Ki: 93.1 µM and 49.2 µM) and
moderate inhibition of HDAC8 (Ki: 6.40 µM and 5.69 µM).

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Table 2: Inhibitory activity of 13a – 13h, 14, vorinostat and CHDI-00390576-0000-004 on recombinant human
HDAC2, HDAC4, HDAC6 and HDAC8.
Ki (µM)
R2
Compound
HDAC2
HDAC4
HDAC6
HDAC8
13a
13b
0.09
93.1
0.06
6.40
1.57
0.58
>100
>100
2.15
0.10
28.1
14.7
13c
13d
13e
13f
0.06
0.55
49.2
83.7
0.03
0.56
5.69
4.90
0.21
1.33
31.8
0.12
0.33
4.44
10.0
13g
>100

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1.36
0.37
>100
0.35
0.42
18.9
22.3
13h
14
>100
0.08
0.09
>100
28.4
0.05
0.02
4.81
5.33
5
vorinostat
n.d.
0.17
n.d.
n.d.
CHDI-00390576-0000-004
Data shown are the mean of pooled data from at least three experiments each carried out in triplicates. The standard deviations are <
10% of the mean. nd = not determined
2.2.3. Acetylation of α-tubulin and histone H3
In order to prove HDAC inhibition at a cellular level, acetylation of α-tubulin and histone-
H3 was tested by western blot analysis in Cal27 and Cal27CisR (Figure 2). Incubation
with vorinostat, compound 5, 13a, or 13d induced accumulation of acetyl α-tubulin
compared to control indicating that all four compounds inhibit HDAC6. Further, the four
compounds increased acetylation of histone-H3 proving cellular inhibition of class I
HDACs.

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Figure 2: Compound-induced α-tubulin and histone H3 acetylation in Cal27 and Cal27CisR. Representative
immunoblot analysis of α- tubulin (α-tub), acetylated α-tubulin (Ac-α-tub), and acetylated histone H3 (Ac-histone-H3)
in Cal27 and Cal27CisR after 24 h incubation with vehicle (C) or 3 µM vorinostat (V), 1.5 µM 5, 5 µM 13a, or 1.5 µM
13d, respectively.
2.2.4 Enhancement of cisplatin-induced cytotoxicity
Next, the effect of the most active compounds 13a and 13d on cisplatin-induced
cytotoxicity was tested in comparison to compound 5 at Cal27 and the cisplatin-resistant
subline Cal27CisR. Cal27CisR is about 10-fold resistant compared to Cal27 and was
established by intermittent treatment of Cal27 with cisplatin over several weeks.19 Results
are displayed in Table 3. Overall, both new HDACi 13a and 13d induce an increase in
cisplatin potency with 13d being more potent than 13a. Interestingly, 13d gave a higher
shift factor than compound 5, thus indicating a higher potency to reverse cisplatin
resistance than compound 5. Figure 3 displays these results: 13d is able to shift the
cisplatin concentration-effect curve of Cal27CisR back to the one of Cal27. Furthermore,
the effect of increased cisplatin potency by HDACi was higher in the cisplatin-resistant
cell line Cal27CisR than it was in cisplatin-sensitive Cal27 as evident by higher shift
factors obtained in Cal27CisR (Table 3). The question remains whether class I HDAC
inhibition or HDAC6 inhibition is responsible for the observed effect. In a previous study,
we could demonstrate that inhibition of class I HDACs is responsible for the
chemosensitizing effect and not HDAC6 inhibition.18 However, dual inhibitors (class I
and HDAC6) are not detrimental for chemosensitization as shown in our previous study
18
and in this study with compound 13d. This is also in accordance with literature data:
Caponigro et al. have highlighted the effectiveness of class I HDAC inhibition in
sensitizing oral squamous cell carcinomas to cisplatin.20 In addition, HDAC1 and
HDAC2 are known as critical regulators of the DNA damage response by modulating the
ATM pathway.21 We thus conclude that inhibition of class I HDACs is mainly responsible

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for the chemosensitizing effects of the inhibitors 13a and 13d seen in this study in
Cal27CisR.
Table 3. IC50 values of cisplatin in the absence and presence of 0.4 µM / 0.8 µM 5, 1.4 µM / 6 µM 13a, 0.2 µM / 0.8
µM 13d in Cal27 / Cal27CisR cells.
Cell line
Compound
Cal27
Cal27CisR
IC50
4.36
SF
IC50
42.8
5.54
3.35
5.12
SF
---
cisplatin
---
cisplatin + 13a
cisplatin + 13d
cisplatin + 5
2.24 1.95
1.15 3.79
1.47 3.00
7.73
12.8
8.36
Data shown are the mean of pooled data from at least three experiments
each carried out in triplicates. The standard deviations are < 10% of the
mean. All shift factors are significant (t-test, p < 0.05).
Figure 3: 48 h preincubation with 13d reversed cisplatin resistance of Cal27CisR cells. Cal27 (white dots) and Cal27CisR (black
dots) were incubated with cisplatin for 72 h. Preincubation of Cal27CisR with 0.8 µM 13d 72 h prior to cisplatin incubation (blue
dots) reduced the IC50 value even below the IC50 of the parental cell line Cal27. SF were calculated as the ratio of the IC50 of cisplatin
alone and the IC50 of the combination of cisplatin with 13d.
2.2.5 Enhancement of cisplatin-induced caspase 3/7 activation
Cancer cells can develop resistance to platinum-based anticancer agents by DNA repair
mechanisms or DNA damage tolerance such as translesion synthesis (TLS). The

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cotreatment of cancer cells with an HDACi can overcome these resistance mechanisms
and may therefore obtain fundamental importance for the resensitization of resistant cell
lines.22
13a and 13d increased the cisplatin-induced cytotoxicity significantly. This prompted us
to test if the increased cytotoxic activity of the combination treatment (13a or 13d with
cisplatin) was mediated by apoptosis. Estimation of caspase 3/7 activation was used as
marker for apoptosis induction. Because cisplatin is only incubated for 24 h in the cell-
based caspase 3/7 activation assay, 2-fold higher concentrations of 13a, 13d and 5 were
used than in the MTT shift experiments above. Results are presented in Figure 4 and
Figure 5. 13a and 13d markedly enhanced caspase 3/7 activation in the combination
treatment in comparison to HDACi or cisplatin alone in both, Cal27 and Cal27CisR. Both
compounds are similarly effective as compound 5. Notably, the increase in cisplatin-
induced caspase activation upon combination treatment was more pronounced in the
cisplatin-resistant cell line Cal27CisR than in Cal27. Since caspase activation is
downstream of DNA damage and the HDACi effect is significantly larger in resistant
Cal27CisR (resistant – i.e. reduced DNA damage signaling) than in sensitive Cal27
(sensitive – i.e. intact DNA damage signaling), we have a hint of enhanced DNA damage
signaling induced by HDACi. This is also in accordance with data from Table 3 showing
higher shift factors for Cal27CisR than for Cal27 cells. In Cal27CisR, 13a, 13d, and
compound 5 gave even more caspase-positive cells than 100 µM cisplatin which is an
approx. 2-fold IC50 of cisplatin. Furthermore, the combined effects of the HDACi and
cisplatin is synergistic, as the sum of HDACi- and cisplatin-induced caspase activation is
significantly lower than the caspase activation by combination treatment. This is
illustrated in Figure 5 for compound 13d: all samples (control, 13d only, cisplatin only,
13d plus cisplatin) contained approx. the same number of cells stained in blue
(Hoechst33342). Caspase activation only slightly increased after mono-compound
treatment whereas the combination of 13d plus cisplatin dramatically increased caspase
activation shown in green.
A synergistic apoptotic effect of HDACi and cisplatin is in agreement with
Zacharioudakis and coworkers.22 They suggested that HDACi increase the accessibility
of platinum-based anticancer agents and showed that this resulted in an accumulation of
DNA-Pt adducts at particular sites in the genome. DNA-Pt adducts colocalized with the
E3 ubiquitin ligase RAD18 triggering accumulation and colocalization of the polymerase
Polƞ which in turn colocalized with the proliferating cell nuclear antigen (PCNA).

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Cotreatment of HDACi and cisplatin then resulted in the ubiquitinylation of PCNA and
subsequent apoptosis. The synergistic apoptotic response is either due to the inability of
polymerases to efficiently bypass DNA-Pt clusters,23 or HDAC inhibition caused a lesion
24
bypass at the expense of DNA repair mechanisms.
Due to the structural similarity
between SAHA and the HDACi reported in this study, a similar mode of action can be
anticipated, but remains to be further investigated.
Figure 4: Compounds 13a and 13d enhanced caspase-dependent cell death in Cal27 and Cal27CisR.
Shown are the mean values of caspase 3/7 positive cells in percent ± SEM (n= 3). Cal27 cells were
incubated with 2.8 µM 13a, 0.4 µM 13d or 0.8 µM 5 prior to an additional 24 h incubation with
3 µM Cisplatin (cDDP). Cal27CisR cells were incubated with 8 µM 13a, 0.8 µM 13d or 0.8 µM 5
prior to an additional 24 h incubation with 25 µM Cisplatin. 100 µM Cisplatin served as positive
control.

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Figure 5: Preincubation with 0.8 µM 13d led to synergistic enhancement of caspase 3/7 activation when combined
with cisplatin. Exemplary pictures of nuclear stain Hoechst (blue) and active caspase 3/7 (green) of Cal27CisR
incubated with 0.8 µM 13d or 25 µM cisplatin alone or in combination. Scale bar, 200 µm (applies to all images).

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2.3. Molecular docking
To rationalize the observed preference of compounds 13a and 13d towards HDAC2 and
HDAC6 and their lack of inhibitory activity towards HDAC4, both inhibitors were
docked in the crystal structures of HDAC225, HDAC426 and HDAC627 using
Autodock4.228 (Figure 6). The docking of 13a and 13d in HDAC4 resulted in no proper
binding modes of the hydroxamic acid group with the catalytic zinc ion, which is in
agreement with the enzymatic assays. In the docking performed with HDAC2 and
HDAC6, the hydroxamate group of 13a and 13d showed bidentate interactions with the
zinc metal, showing additional hydrogen bond with the catalytic Tyr782 in HDAC6 and
Tyr308 in HDAC2. Additionally, the position of the alkoxyamide connecting unit of 13a
in both docked confirmations suggests hydrogen bonds with Ser568 (HDAC6, 2.7 Å) and
Asp104 (HDAC2, 2.3 Å) (Figure 6A and 6B). In the docking of 13d this interaction was
restricted to Asp104 (2.1 Å) of HDAC2 (Figure 6C). These residues have been previously
demonstrated to be essential for the anchoring of peptide substrates27,29 and in case of
Asp104 to interact with the amide group, which serves as connecting unit of vorinostat.25
Figure 6: A) Docking of 13a (green) in HDAC2 (PDB 4LXZ)25 with the interactions highlighted in orange, hydrogen
bond with Asp104 shown as a dashed line. B) Docking of 13a (green) in HDAC6 (PDB 5EDU)19 with the interactions

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highlighted in orange, hydrogen bond interaction with Ser568 shown as dashed line. C) Docking of 13d (blue) in
HDAC2 (PDB 4LXZ)25 with the interactions highlighted in orange, hydrogen bond with Asp104 shown as a dashed
line. D) Docking of 13d (blue) in HDAC6 (PDB 5EDU)19 with the interactions highlighted in orange.
The di- and trimethoxyphenyl cap groups occupy a hydrophobic region of the recognition
surface of HDAC2 (Figure 6A and 6D orange region). These interactions are very similar
to those observed in the crystal structure of HDAC2 and vorinostat25 suggesting that the
proposed binding modes are akin to vorinostat. The dockings in HDAC6 showed that in
this isoform, the cap group of 13a interacts with a basin present at the side of HDAC6
catalytic site (Figure 6 B orange region). Despite this region has been shown to be
essential to interaction of HDAC6 substrate recognition30, the major part of the clinically-
approved HDACi including vorinostat, were shown not to interact with this region, but
with the loop H25 (Figure 6B)27. This interaction, however, could only be observed in the
docking of 13d (Figure 6D) and could be one possible explanation for the lower activity
of 13a towards HDAC6 compared to vorinostat and 13d.

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3. Conclusions
In this study, we evaluated structural variations of the CAP and linker group of compound
5 (LMK235), a previously introduced class I and class IIb preferential HDACi. We
obtained two derivatives, 13a and 13d which displayed similar potency in HDAC2 and
HDAC6 inhibition as compound 5. Furthermore, 13a and 13d increased cisplatin potency
in cisplatin-resistant cancer cells, and 13d was slightly more effective in reversing
cisplatin resistance than compound 5. Thus, another epigenetic tool, 13d, for evaluation
in chemoresistant cancer has been developed through this study.
Acknowledgements
YA, CS, MUK, and TK gratefully acknowledge support from the Deutsche
Forschungsgemeinschaft (DFG) for grant number KA 1942/1-1 to MUK and grant
number KU 1577/2-1 to TK, and also the support from the DFG research training group
GRK2158. We further acknowledge funding by the DFG for the Thermofisher Arrayscan
XTI (Grant: INST 208/690-1 FUGG to MUK) and the CHDI Foundation Inc. for
providing the compound CHDI-00390576-0000-004.
Supplementary information
All experimental procedures and analytical data for all synthesized compounds are
provided as supplementary information.
Conflicts of interest
The authors declare that they have no conflict of interest.
Abbreviations
Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium, HATU; 3-(4,5-
Dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide, MTT;
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