Synthesis of Peptoid-Based Class I-Selective Histone Deacetylase Inhibitors with Chemosensitizing Properties

Article abstract of DOI:10.1021/acs.jmedchem.9b01489

There is increasing evidence that histone deacetylase (HDAC) inhibitors can (re)sensitize cancer cells for chemotherapeutics via "epigenetic priming". In this work, we describe the synthesis of a series of class I-selective HDAC inhibitors with 2-aminoanilides as zinc-binding groups. Several of the synthesized compounds revealed potent inhibition of the class I HDAC isoforms HDAC1, HDAC2, and/or HDAC3 and promising antiproliferative effects in the human ovarian cancer cell line A2780 and the human squamous carcinoma cell line Cal27. Selected compounds were investigated in a cellular model of platinum resistance. In particular, compound 2a revealed potent chemosensitizing properties and full reversal of cisplatin resistance in Cal27CisR cells. This effect is related to a synergistic increase in caspase 3/7 activation and induction of apoptosis. Thus, this work demonstrates that pan-HDAC inhibition or dual class I/class IIb inhibition is not required for full reversal of cisplatin resistance.

Full text of DOI:10.1021/acs.jmedchem.9b01489

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Article
Synthesis of peptoid-based class I selective histone
deacetylase inhibitors with chemosensitizing properties
Viktoria Krieger, Alexandra Hamacher, Fangyuan Cao, Katharina Stenzel, Christoph G. W. Gertzen, Linda
Schäker-Hübner, Thomas Kurz, Holger Gohlke, Frank J. Dekker, Matthias U. Kassack, and Finn K. Hansen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01489 • Publication Date (Web): 24 Nov 2019
Downloaded from pubs.acs.org on November 25, 2019
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Synthesis of peptoid-based class I selective
histone deacetylase inhibitors with
chemosensitizing properties
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Viktoria Krieger,†,# Alexandra Hamacher,†,# Fangyuan Cao,# Katharina Stenzel,†
Christoph G. W. Gertzen,†,¶‖ Linda Schäker-Hübner,§ Thomas Kurz,† Holger Gohlke,†,‖
Frank J. Dekker,# Matthias U. Kassack,*,†,‡ and Finn K. Hansen*,§,‡
†Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität
Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
#Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of
Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands.
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‖John von Neumann Institute for Computing (NIC), Jülich Supercomputing Centre (JSC),
and Institute for Complex Systems - Structural Biochemistry (ICS-6), Forschungszentrum
Jülich GmbH, Wilhelm-Johnen-Straße, 52425 Jülich, Germany.
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¶Center for Structural Studies (CSS),Heinrich-Heine-Universität Düsseldorf,
Universitätsstr. 1, 40225 Düsseldorf, Germany.
§Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Medical Faculty, Leipzig
University, Brüderstraße 34, 04103 Leipzig, Germany.
KEYWORDS
Histone deacetylases, HDAC inhibitors, peptoids, cancer, cisplatin
ABSTRACT
There is increasing evidence that histone deacetylase (HDAC) inhibitors can
(re)sensitize cancer cells for chemotherapeutics via ‘epigenetic priming’. In this work, we
describe the synthesis of a series of class I selective HDAC inhibitors with 2-
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aminoanilides as zinc-binding groups. Several of the synthesized compounds revealed
potent inhibition of the class I HDAC isoforms HDAC1, 2, and/or 3 and promising
antiproliferative effects in the human ovarian cancer cell line A2780 and the human
squamous carcinoma cell line Cal27. Selected compounds were investigated in a cellular
model of platinum resistance. In particular compound 2a revealed potent
chemosensitizing properties and full reversal of cisplatin resistance in Cal27CisR cells.
This effect is related to a synergistic increase in caspase 3/7 activation and induction of
apoptosis. Thus, this work demonstrates that pan-HDAC inhibition or dual class I/class
IIb inhibition is not required for full reversal of cisplatin resistance.
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INTRODUCTION
Diverse cellular functions are regulated by dynamic modifications, metabolism,
transcription, and translation.1 The acetylation and deacetylation of lysine belongs to
crucial post-translational modifications in the epigenetic field. Epigenetics deals with
heritable changes in gene expression without influence of the DNA sequence itself.1 One
way to alter gene expression is the histone acetylation status, which is controlled via
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histone acetyl transferases (HATs) and histone deacetylases (HDACs). HDAC proteins
are responsible for the deacetylation of lysine residues located on the amino-terminal tails
of histone proteins. The deacetylation induces condensed and transcriptionally inactive
heterochromatin. Interference with the acetylation/deacetylation process has been linked
to cancer development. Inhibition of HDACs, which are overexpressed in several cancer
types, results in the expression of genes leading to terminal differentiation, growth arrest
and/or apoptosis in cancer cells.2 Thus, the inhibition of HDACs has become an important
target in cancer research. To date, four HDAC inhibitors (HDACi) have received FDA-
approval in cancer treatment. The first three approvals for HDACi (vorinostat, romidepsin,
and belinostat; Figure 1) have been granted for the treatment of rare types of lymphoma
(cutaneous T-cell lymphoma (CTCL) and/or peripheral T-cell lymphoma (PTCL)).
However, the approval of panobinostat (Figure 1) to treat multiple myeloma is a good
evidence for the application of HDACi in more common cancer types.3 Beyond
hematologic cancers, HDACi have reached advanced stages of clinical studies in solid
cancers.3 Furthermore, HDACi have shown therapeutic potential in several other
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diseases beyond cancer such as parasitic diseases, immune disorders, HIV,
inflammation, and neurodegenerative diseases.4
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In humans, 18 different HDAC isoforms have been described and classified into four
classes based on their homology to yeast HDACs. Class I (HDAC1, 2, 3, 8), class IIa
(HDAC4, 5, 7, 9), class IIb (HDAC6, 10), and class IV (HDAC11) HDACs are Zn2+-
dependent enzymes, whereas class III HDACs are NAD+-dependent (sirtuins).5 The
aforementioned hydroxamate-based HDACi (vorinostat, belinostat, and panobinostat) do
not exert selectivity towards a specific isoform. In addition, the potential genotoxicity of
hydroxamate-containing HDACi represents a major drawback in their application and has
fueled the search for alternative ZBGs.6 In this regard, 2-aminoanilides have emerged as
promising alternatives to hydroxamates. Notably, they possess a different isoform profile
compared to most hydroxamates and do not exhibit genotoxicity.3,6 In detail, 2-
aminoanilides display selectivity for HDAC1, HDAC2, and HDAC3, which can be further
fine-tuned towards HDAC1 and HDAC2 by the introduction of bulky (hetero)aromatic
substituents in the 5-position of the 2-aminoanilide scaffold. In the latter case, the
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additional (hetero)aromatic group is binding to a hydrophobic 14 Å cavity (the so-called
“foot pocket”)7 in HDAC1 and HDAC2.
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There is increasing evidence that 2-aminoanilides can succeed in clinical trials. For
instance, chidamide (tucidinostat, Figure 1B) has been approved in China for relapsed or
refractory PTCL.8 Furthermore, entinostat (MS-275, Phase III, Figure 1B), mocetinostat
(MGCD0103, Phase II, Figure 1B), and RG2833 (RGFP109, Phase I, Figure 1B) show
selective inhibition of class I HDACs and are currently studied in different stages of clinical
trials.3,9 Interestingly, RG2833 is developed as drug against the neurodegenerative
disease Friedreich ataxia (FRDA), which highlights the therapeutic potential of
aminoanilide-based HDACi beyond oncology.9
O
A
cap
B
O
NH
O
O
zinc-binding
group (ZBG)
HN
linker
S
S
N
H
O
N
H
H
N
H
N
O
NH
O
O
F
H
N
N
N
OH
HN
N
H
O
O
O
H2N
H2N
O
O
Chidamide
Entinostat
Vorinostat
N
Romidepsin (prodrug)
O
O
O
O
O
H
N
N
N
N
H
H
N
S
OH
OH
N
H
N
H
N
H
N
H
O
HN
HN
O
H2N
H2N
Mocetinostat
RG2833 (RGFP109)
Panobinostat
Belinostat
Figure 1. FDA approved HDACi (A) and aminoanilide-based HDACi (B).
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We previously discovered hydroxamate-based HDACi utilizing peptoid-based cap
groups, which are accessible by a facile synthetic protocol based on the Ugi four-
component reaction (U-4CR).10,11 Peptoid-based hydroxamates with a benzyl linker were
identified as HDAC6 preferential inhibitors,10 whereas hydroxamic acids with an alkyl
linker displayed a preference for class I isoforms.11 Several peptoid-based hydroxamates
showed promising chemosensitizing properties and were able to revert cisplatin
resistance in cancer cells.10,11 Due to the aforementioned drawbacks of hydroxamic acids,
we aimed at the development of peptoid-based HDACi with a 2-aminoanilide ZBG. We
herein report on the rational design, diversity-oriented synthesis, and biological evaluation
of class I selective HDACi with peptoid-based cap groups.
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RESULTS AND DISCUSSION
Design and Synthesis of Peptoid-Based 2-Aminoanilides.
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R2
HN
O
5
6
7
8
O
O
O
N
H2N
H
H
O
N
OH
O
N
N
H
N
N
H
R1
cap
N
O
H2N
H
linker
I
ZBG
O
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Entinostat
RG2833 (RGFP109)
R2
HN
R2
O
O
N
HN
H2N
O
H
N
O
N
R3
R3
N
R1
O
H
R1
O
H2N
2a-f
1a-k
Figure 2. Design of target compounds 1a-k and 2a-f.
The synthesis of our HDACi library is summarized in Schemes 1-3. The series of HDACi
1a-k was designed as hybrid compounds derived from our peptoid-based hydroxamate
scaffold I and RG2833 (Figure 2). We retained the aliphatic linker and substituted the
hydroxamate-based ZBG by an aminoanilide-based ZBG in order to improve the
selectivity for the class I isoforms HDAC1-3. The synthesis of the compounds 1a-h was
achieved through straightforward U-4CRs differing in the use of the carboxylic acid and
the isocyanide components (Scheme 1). In detail, for the synthesis of compounds 3a-h
the amine methyl 6-aminohexanoate hydrochloride, trimethylamine, and
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paraformaldehyde were stirred in dry methanol in the presence of 4 Å molecular sieves
(4 Å MS) to provide the imine intermediate. The subsequent addition of the carboxylic
acid and isocyanide component provided the desired Ugi products 3a-h after purification
by flash column chromatography (Scheme 1).
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Scheme 1. Synthesis of benzamide HDACi 1a-h.a
R2
HN
O
O
O
H
O
O
OH
HCl
a)
NC
R2
+
+
+
O
N
H2N
O
H
O
R1
R1
3a-h
R2
R2
O
HN
HN
H2N
O
BocHN
O
O
b)
c)
3a-h
O
N
O
N
R3
R3
N
H
N
H
R1
R1
4a-h
1a-h
aReagents and conditions: a) (i) Methyl 6-aminohexanoate hydrochloride (1.2 eq),
paraformaldehyde (1.2 eq), MeOH, 4 Å MS, RT, 30 min; (ii) R1COOH (1 eq), RT, 10 min;
(iii) R2NC (1 eq), RT, 16h; b) (i) LiOH.H2O (2 eq), MeOH, RT, 16h; (ii) acid (1.2 eq), amine
(1 eq), pyridine (2 eq), DIC (1.2 eq), HOAt (1.2 eq), CH2Cl2/DMF (1:1, v/v), RT, 16h; c)
TFA/CH2Cl2 (1:5, v/v), RT, 30 min.
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The target compounds 1a-h were subsequently prepared by performing suitable post-Ugi
transformations. The methyl esters of 3a-h were cleaved via hydrolysis with LiOH.H2O in
methanol overnight. Afterwards, the resulting carboxylic acids were coupled with the
respective mono-Boc-protected phenylenediamine derivative using the coupling reagents
DIC and HOAt in the presence of pyridine in CH2Cl2/DMF to afford the Boc-protected
compounds 4a-h. Finally, the acidic deprotection yielded the desired peptoid-based
HDACi 1a-h.
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Scheme 2. Submonomer synthesis of compound 1i.a
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O
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O
O
HCl
O
O
HN
a)
b)
H2N
Br
+
+
Br
O
O
O
O
HN
N
H2N
H
O
O
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Br
5
O
O
HN
HN
HN
BocHN
O
H2N
O
O
O
O
O
c)
d)
e)
5
O
N
O
N
O
N
N
H
N
O
H
6
7
1i
aReagents and conditions: a) (4-Methoxyphenyl)methanamine (1 eq), 2-bromoacetyl
bromide (1 eq), DIPEA (1 eq), CH2Cl2, 0 °CRT, 16 h; b) methyl 6-aminohexanoate
hydrochloride (1 eq), 2-bromo-N-(4-methoxybenzyl)acetamide (1 eq), Et3N (2 eq),
CH2Cl2, RT, 16 h; c) methyl 6-({2-[(4-methoxybenzyl)amino]-2-oxoethyl}amino)hexanoate
(1.2 eq), pyridine (1.2 eq), 3,5-dimethylbenzoyl chloride (1 eq), CH2Cl2, 0 °CRT, 16 h;
d) (i) LiOH.H2O (2 eq), MeOH, RT, 16 h; (ii) acid (1.2 eq), tert-butyl (2-
aminophenyl)carbamate (1 eq), pyridine (2 eq), DIC (1.2 eq), HOAt (1.2 eq), CH2Cl2/DMF
(1:1, v/v), RT, 24 h; e) TFA/CH2Cl2 (15%, v/v), RT, 30 min.
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Scheme 3. Submonomer synthesis of compounds 1j,k.a
9
OtBu
O
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OtBu
HCl
O
O
O
a)
b)
O
O
O
N
H2N
Br
+
OtBu
O
O
HN
O
9
8
R1
R1
O
R1
O
HN
HN
HN
BocHN
O
H2N
O
O
O
c)
d)
e)
9
O
N
O
N
O
N
N
H
N
O
H
10a,b
1j,k
11a,b
aReagents and conditions: a) Methyl 6-aminohexanoate hydrochloride (1 eq), tert-butyl
bromoacetate (1 eq), Et3N (2 eq), THF, RT, 16 h; b) methyl 6-{[2-(tert-butoxy)-2-oxoethyl]-
amino}hexanoate (1.2 eq), pyridine (1.2 eq), 3,5-dimethylbenzoyl chloride (1 eq), CH2Cl2,
RT, 18 h; c) (i) TFA/CH2Cl2 (1:5, v/v), RT, 2 h; (ii) acid (1 eq), amine (1 eq), EDC.HCl (2
eq), DMAP (0.4 eq), DMF/CH2Cl2, RT, 16 h; d) (i) LiOH.H2O (2 eq), MeOH, RT, 16 h; (ii)
acid (1.2 eq), tert-butyl (2-aminophenyl)carbamate (1 eq), pyridine (2 eq), DIC (1.2 eq),
HOAt (1.2 eq), CH2Cl2/DMF (1:1, v/v), RT, 24 h; e) TFA/CH2Cl2 (15%, v/v), RT, 30 min.
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Since the number of purchasable isocyanides is limited, we switched to a peptoid
submonomer synthesis for further modifications of the isocyanide region (Scheme 2 and
3). The synthesis of compound 1i is illustrated in Scheme 2. The reaction of 4-
methoxybenzylamine with bromoacetyl bromide in the presence of DIPEA afforded the
corresponding bromoacetyl amide. The subsequent alkylation of methyl 6-
aminohexanoate hydrochloride in the presence of triethylamine in dichloromethane
yielded the secondary amine 5. Intermediate 5 was directly treated with 3,5-
dimethylbenzoyl chloride using pyridine as a base to generate the peptoid 6. The methyl
ester in 6 was hydrolyzed and the resulting carboxylic acid derivative was subjected to an
amide coupling reaction with mono-Boc-protected phenylenediamine to yield 7. Removal
of the Boc-protecting group furnished the desired aminoanilide 1i.
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The synthesis of the HDACi 1j,k was performed as highlighted in Scheme 3. The
alkylation of methyl 6-aminohexanoate hydrochloride with tert-butyl bromoacetate in the
presence of triethylamine in THF provided the secondary amine 8. Without further
purification, the intermediate 8 was directly acylated with 3,5-dimethylbenzoyl chloride
using pyridine as a base to furnish the key building block 9. Subsequently, 9 was used as
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starting point for the variation of the isocyanide region. To this end, we performed the
deprotection of the tert-butyl group followed by amide coupling reactions of the free
carboxylic acid with the respective amine using DIC and HOAt as coupling agents to
furnish 10a,b. The HDACi 1j,k were prepared via the saponification of the methyl ester in
10a,b and a subsequent amide coupling with tert-butyl (2-aminophenyl)carbamate to yield
11a,b. Next, the Boc-protecting group was removed by acidolysis to afford the target
compounds 1j,k.
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The second type of HDACi 2a-f (Figure 2) is derived from our peptoid-based hydroxamate
scaffold I and entinostat (MS-275), a selective inhibitor of HDAC1, HDAC2, and HDAC3.
Compared to the first type of HDACi 1a-k they differ in their linker. The synthesis of
compounds 2a-f was performed essentially as described for compounds 1a-h (Scheme
4). Briefly, in the case of compounds 2a-f we used methyl 4-(aminomethyl)benzoate as
amine component in the U-4CR to provide the intermediates 12a-f bearing a benzyl linker.
The Boc-protected aminoanilides 13a-f were then prepared by hydrolysis of the methyl
ester followed by amide coupling reactions with the respective mono-Boc-protected
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phenylenediamine derivative. Finally, deprotection of the Boc group was achieved by
treatment of 13a-f with trifluoroacetic acid in dichloromethane to provide the peptoid-
based HDACi 2a-f.
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Scheme 4. Synthesis of HDACi 2a-f.a
R2
HCl
HN
O
N
O
H
O
OH
H2N
a)
NC
R2
+
+
+
R1
O
H
O
R1
O
O
O
12a-f
R2
HN
R2
HN
O
N
O
b)
c)
N
9a-f
O
O
R1
O
NH2
R1
O
NHBoc
HN
HN
R3
R3
13a-f
2a-f
aReagents and conditions: a) (i) Methyl 4-(aminomethyl)benzoate hydrochloride (1.2 eq),
paraformaldehyde (1.2 eq), MeOH, 4 Å MS, RT, 30 min; (ii) R1COOH (1 eq), RT, 10 min;
(iii) R2NC (1 eq), RT, 16h; b) (i) LiOH.H2O (2 eq), MeOH, RT, 16h; (ii) acid (1.2 eq), amine
(1 eq), EDC.HCl (2 eq), DMAP (0.4 eq), RT, 16h; c) TFA/CH2Cl2 (1:5, v/v), RT, 30 min.
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Inhibition of HDAC1-3 and HDAC6. All synthesized compounds and the reference HDACi
vorinostat were first screened for inhibitory activity against their intended targets HDAC1-
3. Furthermore, HDAC6 was selected as control isoform. The results are summarized in
Table 1. As expected, all aminoanilides were inactive against HDAC6 (IC50 > 10 µM). The
screening against HDAC1-3 provided some interesting differences depending on the type
of the linker and ZBG. In general, the 2-aminoanilides with a benzyl linker exhibited
increased activity against HDAC1 and HDAC2 compared to their corresponding alkyl
linker counterparts, whereas the alkyl-based compounds showed higher activity against
HDAC3 (see e.g. 1c vs 2a). Compound 1h bearing a phenyl ring in R3 position, which is
intended to target the foot pocket in HDAC1 and HDAC2, represents one noteworthy
exemption. 1h revealed clearly the highest activity against HDAC1 (IC50: 0.057 µM)
among the alkyl-based compounds and is almost inactive against HDAC3 (20% inhibition
at 10 µM). Interestingly, compound 1d utilizes the same cap and linker as compound 1h
and the only difference in the structure is the additional phenyl ring at the R3 position. The
introduction of this foot pocket targeting moiety leads to an ~20-fold improved potency of
1h towards HDAC1 and a >40-fold reduction in activity against HDAC3 compared to 1d.
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The hydroxamic acid-based HDACi Ia (Table 1), one of the hit compounds from our
previous study,11 represents the corresponding hydroxamate analogue of 1d and 1h. This
compound showed potent inhibition of HDAC1-3 and 6 with IC50 values ranging from
0.025 to 0.135 µM. Interestingly, the HDAC selectivity profile of this compound can be
altered towards HDAC3 preferential inhibition by replacing the hydroxamate with an
unsubstituted aminoanilide or towards HDAC1 preferential inhibition via the introduction
of a foot pocket targeting moiety.
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In the case of the benzyl-based compounds, no noteworthy change in potency against
HDAC1 was observed after introduction of an additional phenyl ring at the aminoanilide
group (see 2a (IC50: 0.038 µM) vs 2e (IC50: 0.039 µM)). As expected, compound 2e was
inactive against HDAC3. Notably, the introduction of a fluorine atom in R3 position, as
realized in the approved aminoanilide chidamide, resulted in a significant decrease of the
HDAC1 and 2 inhibition (see 2b vs 2f) and hence this substitution appears to be
detrimental for our peptoid-based aminoanilides. Compared to the well-known
aminoanilide entinostat (HDAC1 IC50: 0.519 µM, HDAC2 IC50: 0.505 µM, and HDAC3
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IC50: 2.85 µM,), our benzyl-based compounds 2a-f displayed an improved inhibition of
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HDAC1 and a clear preference for HDAC1 over HDAC2 and 3 (Table 1).
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Table 1. Inhibitory activities (IC50 [µM]) of compounds 1a-k and 2a-f against HDACs 1-3,
and 6.
R2
R2
O
HN
O
N
HN
R2
O
HN
HN
H2N
O
H2N
O
O
O
O
O
N
R1
O
NH2
O
N
N
OH
O
N
R3
N
H
HN
N
H
H
R1
1a-h
1i-k
2a-f
Ia
R3
IC50 [µM]
HDAC2
compd
R1
R2
R3
HDAC1
HDAC3
HDAC6
>10b
>10b
>10b
>10b
>10b
>10b
>10b
>10b
1a
1b
1c
1d
1e
1f
Ph
c-Hex
c-Hex
Bn
H
H
H
H
H
H
F
5.46±0.32
5.55±0.30
1.58±0.15
1.16±0.21
6.56±0.84
1.67±0.17
5.95±0.67
6.66±0.44
1.24±0.078
1.60±0.38
5.38±0.69
1.90±0.030
3.06±0.16
1.81±0.12
0.858±0.066
0.481±0.088
0.312±0.021
0.250±0.021
0.646±0.066
0.163±0.013
0.369±0.009
20% @ 10 µMa
49% @ 10 µMa
0.478±0.026
0.583±0.027
0.586±0.055
3,5-Me-Ph
4-Me2N-Ph
3,5-Me-Ph
1-Naphthyl
1-Naphthyl
4-Me2N-Ph
3,5-Me-Ph
3,5-Me-Ph
Bn
c-Hex
Bn
1g
1h
Bn
1.98±0.028
Bn
Ph
H
0.057±0.005
4-MeO-
Bn
1i
1j
2.08±0.21
1.10±0.05
30% @ 10 µMa
5.03±0.69
>10b
>10b
>10b
>10b
3,5-Me-Ph
3,5-Me-Ph
4-Me-Bn
3,5-Me-
Bn
H
H
1k
2a
0.484±0.051
0.038±0.012
4.10±0.29
4-Me2N-Ph
Bn
H
0.283±0.028
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2b
2c
3,5-Me-Ph
3-Pyridyl
Bn
Bn
Bn
H
H
0.062±0.012
0.103±0.016
0.485±0.018
0.786±0.017
34% @ 3.33
µMa
32% @ 10 µMa
0.878±0.117
>10b
>10b
3,5-Me-Ph
Ph
2d
2e
0.051±0.011
0.039±0.011
>10000b
>10000b
>10b
>10b
4-Me2N-Ph
Bn
Ph
36% @ 3.33
µMa
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2f
3,5-Me-Ph
Bn
---
---
---
Bn
F
0.277±0.045
0.111±0.015
0.519±0.063
Ki = 0.032c
25% @ 10 µMa
0.146±0.014
0.505±0.028
n.d.
23% @ 10 µMa
0.0097±0.0008
2.85±0.22
Ki = 0.005c
0.026±0.002d
>10b
0.102±0.009
>10b
vorinostat
entinostat
RG2833
Ia
---
---
---
---
---
---
---
n.d.
0.135±0.013d
3,5-Me-Ph
0.025±0.007d
0.061±0.003d
a% inhibition at the concentration stated (µM). b<15% inhibition at 10 µM. cdata taken from
9b. ddata taken from ref. 11.
Interestingly, compounds 1h (alkyl linker) and 2d (benzyl linker) bearing the same cap
and ZBG revealed similar inhibition profiles with potent inhibition of HDAC1 (1h (IC50:
0.057 µM) vs 2d (IC50: 0.051 µM)) and weak inhibition of HDAC2 and 3. In contrast,
compounds 1c and 2a, which also only differ in the linker, showed a completely different
inhibition profile with 1c being an HDAC3 preferential inhibitor, whereas 2a displayed
HDAC1 preference. Based on these results, it can be assumed that, in the absence of a
foot pocket group, the linker dictates the selectivity profile. However, as soon as a foot
pocket targeting moiety is added to the aminoanilide ZBG, the ability of the substituted
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aminoanilide ZBG to occupy the foot pocket is responsible for the preferential inhibition
of HDAC1.
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The most important structure–activity and structure–selectivity relationships are
summarized in Figure 3. Overall, this screening revealed the alkyl-based compound 1f as
the most potent preferential HDAC3 inhibitor and the benzyl-based compounds 2d,e as
preferential HDAC1 inhibitors. Furthermore, 1h and 2a were identified as the most potent
HDAC1 inhibitors from the alkyl and benzyl series, respectively.
Benzyl linker:
• increases HDAC1
and 2 inhibition
Cap-effects:
• Strongest HDAC1 inhibition
R2
Pentyl linker:
• enchances HDAC3
inhibition and selectivity
1h
in the alkyl series ( ):
HN
O
N
R1 = 3,5-Me-Ph, R2 = 3,5-Me-Bn;
R3 = Ph.
Linke
r
O
• Strongest HDAC1 inhibition
R1
O
NH2
2a
in the benzyl series ( ):
HN
R1 = 4-Me2N-Ph, R2 = Bn; R3 = H.
R3
Fluorine substitution:
• decreased HDAC
inhibition
Phenyl substituent:
• improves HDAC1
inhibition in combination
with a pentyl linker
Phenyl substituent:
• improves HDAC1 selectivity
in combination with a benzyl
linker
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Figure 3. Structure-activity and structure-selectivity relationships of the peptoid-based
aminoanilides 1a-k and 2a-f.
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Docking Studies. Compounds 1h and 2a, which showed the highest activity against
HDAC1 within each molecule series, respectively, and 1f, which showed preferential
inhibition of HDAC3, were docked into energetically relaxed X-ray crystal structures of
HDAC1, HDAC2, HDAC3, and HDAC6. We docked the three compounds in both their
cis- and trans-rotamers, considering the orientation of the amide bond connecting R1.
While valid docking poses of all three compounds were identified in HDAC1 and HDAC2,
molecular docking failed in the case of HDAC6: Here, no docking pose could be identified
in which the ZBG of the respective compound was interacting with the Zn2+ ion at the
catalytic center of HDAC6 (Table S1). The reason for this is the shape of the catalytic
center in HDAC6, which is too narrow to accommodate the sterically demanding ZBG of
the compounds. As the interaction of the ZBG with the zinc ion is known to be essential
for the activity of HDACi, these results are in perfect agreement with the compounds’
inability to inhibit HDAC6 (Table 1). Both 1f and 2a furthermore produce valid docking
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poses in HDAC3 (Table 1). Thus, the docking results neither replicate the HDAC3
preference of 1f nor the HDAC1 preference of 2a; however, given the small differences
in the respective IC50 values (Table 1), such a discrimination cannot be expected from a
scoring function.12 Yet, the docking results do reflect the compounds’ general ability to
inhibit the three HDAC isoforms (Table 1). The validity of the docking is further
corroborated by the results of compound 1h, for which valid docking poses were obtained
in HDAC1 and HDAC2, but not in HDAC3 (Table S1), in line with inhibition studies (Table
1).
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Judging from the docking results of the three HDACi, the binding affinity to HDAC1 seems
to be dominated by the aromatic character and size of the ZBG and linker. As such, 1h
and 2a, which show a high affinity towards HDAC1, bear an additional phenyl ring in their
structure: In 1h, the phenyl ring is in position R3, with which it occupies the foot pocket
adjacent to the zinc ion of HDAC1 (Figure 4A); in 2a, the phenyl ring is in the linker forming
π-stacking interactions to H178 (Figure 4B). In contrast, 1f, which shows a lower affinity
towards HDAC1 than the other two compounds, bears neither of the two aforementioned
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features. For the substituents in R1 and R2, no common interaction pattern can be found
for all three tested HDACi. While 1f and 1h both interact with Y204, 1h forms hydrophobic
contacts to L271, and 1f forms π-stacking interactions with H28 (Figure 4A+C). H28 is
also addressed by π-stacking interactions by 2a, yet R1 of this compound forms π-
stacking interactions with F205. The inability of 1h to inhibit HDAC3 is caused by a
spacious phenyl ring in R3, which does not fit the binding pocket of HDAC3. As the
residues lining the binding pocket of HDAC1 and HDAC2 are highly identical (96%,
considering residues 8 Å around the co-crystallized ligand of the HDAC2 complex
structure as part of the binding pocket), similar docking results for both isoforms could be
expected. However, the energetic relaxation of the two structures widened the brim of the
HDAC2 binding pocket by ~1 Å more than that of HDAC1, which allows HDAC2 to also
accommodate the more spacious trans-rotamers of the HDACi.
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To conclude, while the docking cannot reproduce subtle selectivity differences, it is able
to correctly predict and rationalize the ligands’ general capabilities to inhibit the HDAC
isoforms 1, 2, and 3.
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Figure 4. Predicted binding modes of compounds cis-1h (A, magenta), cis-2a (B, light
blue), and cis-1f (C, orange) in the energetically relaxed X-ray crystal structure of HDAC1
(white, cartoon and surface, PDB ID 4BKX). Prominent interacting residues of HDAC1
are shown in navy sticks. A Compound cis-1h utilizes its spacious ZBG to fill the
hydrophobic foot pocket in HDAC1 and engages in π-stacking interactions with Y204. B
Compound cis-2a engages in π-stacking interactions with H28 and F205. C Compound
cis-1f engages in π-stacking interactions with H28 and Y204.
Anticancer Activity and Inhibition of Cellular HDAC Activity. All aminoanilide HDACi 1a-k
and 2a-f were assessed for their cytotoxicity and HDAC inhibitory activity in the human
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ovarian cancer cell line A2780 and the human tongue squamous cell carcinoma cell line
Cal27. Results are summarized in Tables 2 and 3.
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Table 2. Cytotoxic activity of compounds 1a-k and 2a-f.a
6
7
8
9
R2
HN
O
N
R2
HN
R2
H2N
O
O
HN
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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28
29
30
31
32
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60
O
O
N
R1
O
NH2
H2N
N
O
O
H
HN
O
N
R3
N
H
R1
1a-h
1i-k
2a-f
R3
MTT IC50 [µM]
compd
R1
R2
R3
A2780
181
Cal27
30.9
17.5
16.0
1a
1b
1c
Ph
c-Hex
c-Hex
Bn
H
H
H
3,5-Me-Ph
4-Me2N-
Ph
42.0
18.5
1d
1e
3,5-Me-Ph
1-
Bn
H
H
20.9
18.1
13.7
24.9
c-Hex
Naphthyl
1-
1f
Bn
Bn
Bn
H
F
22.8
25.9
11.0
20.2
Naphthyl
4-Me2N-
Ph
1g
1h
1i
3,5-Me-Ph
3,5-Me-Ph
3,5-Me-Ph
3,5-Me-Ph
4-Me2N-
Ph
Ph
H
17.1
24.6
9.80
5.10
2.40
18.9
17.4
18.2
19.3
1.60
4-MeO-Bn
4-Me-Bn
3,5-Me-Bn
Bn
1j
H
1k
2a
H
H
2b
2c
2d
2e
3,5-Me-Ph
3-Pyridyl
3,5-Me-Ph
4-Me2N-
Ph
Bn
Bn
Bn
Bn
H
H
2.80
8.90
n.e.
n.e.
1.60
11.2
8.90
11.1
Ph
Ph
2f
3,5-Me-Ph
---
Bn
---
---
F
5.40
2.42
2.25
5.72
2.64
2.50
vorinostat
cisplatin
---
---
---
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aValues are the mean of three experiments. Standard deviations
are <10% of the mean. n.e. = no effect up to 100 µM.
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Table 3. HDAC inhibition of compounds 1a-k and 2a-f.a
R2
HN
O
N
R1
O
HN
R2
O
H2N
HN
O
O
O
N
R1
O
NH2
H2N
N
O
H
HN
O
N
R3
N
H
R1
1a-h
2a-f
R3
1i-k
HDACi IC50 [µM]
compd
R1
R2
R3
A2780
43.6
Cal27
21.9
12.1
4.33
1a
1b
1c
Ph
c-Hex
c-Hex
Bn
H
H
H
3,5-Me-Ph
4-Me2N-
Ph
22.8
9.25
1d
3,5-Me-Ph
Bn
H
8.71
3.93
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1e
1f
1-
c-Hex
Bn
H
H
F
33.1
10.2
13.4
13.0
4.13
6.82
Naphthyl
1-
Naphthyl
4-Me2N-
Ph
1g
Bn
10
11
12
13
14
15
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1h
1i
3,5-Me-Ph
3,5-Me-Ph
3,5-Me-Ph
3,5-Me-Ph
4-Me2N-
Ph
Bn
Ph
H
1.43
3.53
4.48
3.17
0.47
4.81
4.57
3.15
4-MeO-Bn
4-Me-Bn
3,5-Me-Bn
Bn
1j
H
1k
2a
H
H
0.48
0.11
2b
2c
2d
2e
3,5-Me-Ph
3-Pyridyl
3,5-Me-Ph
4-Me2N-
Ph
Bn
Bn
Bn
Bn
H
H
0.81
1.06
1.72
0.18
1.01
0.63
Ph
Ph
2.62
0.85
2f
3,5-Me-Ph
---
Bn
---
F
1.43
0.96
0.47
0.86
vorinostat
---
aValues are the mean of three experiments. Standard deviations
are <10% of the mean. n.e. = no effect up to 100 µM.
The compounds revealed cytotoxic activity with IC50 values in the range of 1.60 µM up to
181 µM (and two compounds, 2d and 2e, with no effect up to 100 µM) depending on their
substitution pattern and the cell line investigated. In the whole cell HDAC assay, the
compounds showed HDAC inhibitory activity from lower nM range (0.11 µM) up to 43.6
µM. Overall, data from both assays (pIC50 values MTT and cellular HDAC assays) show
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significant correlation (A2780: r2: 0.7236; Cal27: r2: 0.6816). 2a and 2b were the most
potent compounds in both MTT and cellular HDAC assays. 2a and 2b were nearly equally
cytotoxic in the respective cell lines (IC50 of 1.60 µM in Cal27 and IC50 of 2.40 and 2.80
µM in A2780). In Cal27, 2a and 2b were even more potent than the reference pan-HDACi
vorinostat in the MTT (1.7fold) and cellular HDAC assay (approx. 5fold). Whereas 2a and
2b inhibit HDAC1 and HDAC2 with similar potency (Table 1), only 2a, but not 2b, potently
inhibits HDAC3. Thus, for potent cytotoxicity (MTT assay), inhibition of HDAC1 and
HDAC2 seems to be sufficient in the cellular model used in this study. However, HDAC1
inhibition only (no HDAC2 inhibition) reduces cytotoxic potency significantly, as can be
seen by comparison of 2b (HDAC1 and HDAC2 inhibitor) with 2d or 2e (both selective
HDAC1 inhibitors): in MTT assays at Cal27 cells, 2b has an IC50 of 1.6 µM whereas 2d
and 2e show IC50 values of 8.90 and 11.1 µM, respectively. These findings are in
agreement with literature reports on redundant functions of HDAC1 and HDAC2 in gene
ablation experiments: combined ablation of HDAC1 and HDAC2 resulted in severe
proliferation defects.13 However, in other cancer cells, knockdown of HDAC3 led to growth
inhibition and apoptosis.20 Thus, even though our results suggest a major role for HDAC1
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