Structure–Activity and Structure–Toxicity Relationships of Peptoid-Based Histone Deacetylase Inhibitors with Dual-Stage Antiplasmodial Activity

Article abstract of DOI:10.1002/cmdc.201800808

Novel malaria intervention strategies are of great importance, given the development of drug resistance in malaria-endemic countries. In this regard, histone deacetylases (HDACs) have emerged as new and promising malaria drug targets. In this work, we present the design, synthesis, and biological evaluation of 20 novel HDAC inhibitors with antiplasmodial activity. Based on a previously discovered peptoid-based hit compound, we modified all regions of the peptoid scaffold by using a one-pot multicomponent pathway and submonomer routes to gain a deeper understanding of the structure–activity and structure–toxicity relationships. Most compounds displayed potent activity against asexual blood-stage P. falciparum parasites, with IC₅₀ values in the range of 0.0052–0.25 μm and promising selectivity over mammalian cells (SI^Pf3D7/HepG2: 170–1483). In addition, several compounds showed encouraging sub-micromolar activity against P. berghei exo-erythrocytic forms (PbEEF). Our study led to the discovery of the hit compound N-(2-(benzylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-4-isopropylbenzamide (2 h) as a potent and parasite-specific dual-stage antiplasmodial HDAC inhibitor (IC₅₀ Pf3D7=0.0052 μm, IC₅₀ PbEEF=0.016 μm).

Full text of DOI:10.1002/cmdc.201800808

Accepted Article
Title: Structure-Activity and Structure-Toxicity Relationships of Novel
Peptoid-Based Histone Deacetylase Inhibitors with Dual-Stage
Antiplasmodial Activity
Authors: Marcel Karl Walter Mackwitz, Eva Hesping, Yevgeniya
Antonova-Koch, Daniela Diedrich, Tamirat Gebru, Tina
Skinner-Adams, Mary Clarke, Andrea Schöler, Laura
Limbach, Thomas Kurz, Elizabeth Ann Winzeler, Jana Held,
Katherine Andrews, and Finn Kristian Hansen
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
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To be cited as: ChemMedChem 10.1002/cmdc.201800808
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ChemMedChem
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FULL PAPER
Structure-Activity and Structure-Toxicity Relationships of Novel
Peptoid-Based Histone Deacetylase Inhibitors with Dual-Stage
Antiplasmodial Activity
[a]
[b]
[c]
[d]
Marcel K. W. Mackwitz, Eva Hesping, Yevgeniya Antonova-Koch, Daniela Diedrich, Tamirat
[
e]
[b]
[b]
[a]
[d]
Gebru Woldearegai, Tina Skinner-Adams, Mary Clarke, Andrea Schöler, Laura Limbach,
Thomas Kurz, Elizabeth A. Winzeler, Jana Held, Katherine T. Andrews,* and Finn K. Hansen*+[a]
[
d]
[c]
[e]
+[b]
[
[
a]
b]
M. K. W. Mackwitz, A. Schöler, Prof. Dr. F. K. Hansen
Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Medical Faculty
Leipzig University
Brüderstraße 34, 04103 Leipzig, Germany.
E-mail: finn.hansen@medizin.uni-leipzig.de
E. Hesping, Assoc. Prof. Dr. T. Skinner-Adams, M. Clarke, Prof. Dr. K. T. Andrews
Griffith Institute for Drug Discovery,
Don Young Road, Nathan Campus, Griffith University, QLD 4111, Australia
E-mail: k.andrews@griffith.edu.au
[c]
[d]
[e]
[+]
Dr. Y. Antonova-Koch, Prof. Dr. E. A. Winzeler
Department of Pediatrics, School of Medicine, University of California, San Diego, 9500 Gilman Drive 0741, La Jolla, CA 92093, United States
Dr. D. Diedrich, L. Limbach, Prof. Dr. T. Kurz
Institute of Pharmaceutical and Medicinal Chemistry,Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.
Dr. T. G.Woldearegai, Dr. J. Held
Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstraße 27, 72074 Tübingen, Germany.
These authors contributed equally to this work as senior authors.
Abstract: Novel malaria intervention strategies are of great
importance due to the development of drug resistance in malaria
endemic countries. In this regard, histone deacetylases (HDACs)
pregnant women are the most at-risk populations,[2] with
Plasmodium falciparum infection causing the highest mortality.[
Although some progress has been made in the field of malaria
2]
have emerged as new and promising malaria drug targets. In this work, vaccines,[3] vector control and antimalarial drugs continue to be
we present the design, synthesis and biological evaluation of 20 novel
HDAC inhibitors with antiplasmodial activity. Based on a previously
discovered peptoid-based hit compound, we modified all regions of
the peptoid scaffold by utilizing a one-pot multicomponent pathway
and submonomer routes to gain a deeper understanding of the
structure-activity as well as structure-toxicity relationships. Most
compounds displayed potent activity against asexual blood stage
the mainstays of malaria prevention and treatment.[3,4,5]
Unfortunately, the efficacy of antimalarial drugs is threatened by
the development of parasite resistance, including the current gold
standard artemisinin-based combination therapies (ACTs).[1,6] It is
therefore important to identify new antimalarial drug candidates in
order to prime the antimalarial drug development pipeline.
Histone deacetylase inhibitors (HDACi) are a promising new class
of potential antimalarial drugs.[2,7] HDACi are usually applied for
the epigenetic treatment of different types of cancer and four
drugs have been FDA-approved (vorinostat, belinostat,
romidepsin and panobinostat) for the treatment of T-cell
lymphoma or multiple myeloma. The scope of HDACi has been
extended and this class of compounds is currently being
intensively investigated in several non-cancer diseases such as
neurodegenative diseases, inflammation, HIV and parasitic
diseases.[9-10] Different Plasmodium species are highly sensitive
to HDACi treatment (Figure 1) and histone deacetylases (HDACs)
are therefore considered as emerging antimalarial drug
targets.[2,7] HDAC encoding genes have been discovered in all
Plasmodium species that can infect humans.[2,7] While there are
P. falciparum parasites with IC50 values ranging from 0.0052-0.25 µM
Pf3D7/HepG2
and promising selectivity over mammalian cells (SI
: 170-
1483). In addition, several compounds showed encouraging
submicromolar activity against P. berghei exo-erythrocytic forms
[
8]
(
PbEEF). Our study led to the discovery of the hit compound (N-(2-
benzylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-4-i-
(
propylbenzamide, 2h) as a potent and parasite-specific dual-stage
antiplasmodial HDAC inhibitor (IC50 (Pf3D7) = 0.0052 µM, IC50
(PbEEF) = 0.016 µM).
Introduction
1
8 HDAC isoforms in humans, a recent study re-confirmed the
Malaria is a potentially life-threatening parasitic disease that is
prevalent in many tropical and sub-tropical regions of the world.
Malaria parasite infection results in more than 200 million clinical
cases and greater than 400,000 deaths annually.[1] Children and
presence of five P. falciparum histone deacetylases (PfHDACs)
and identified one novel putative PfHDAC.[2,11b] Like the human
homologues, PfHDACs are divided into zinc dependent class I
(
PfHDAC1) and class II HDACs (PfHDAC2 and 3), and sirtuin-like
1
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Vorinostat
SAHA)
Romidepsin
(
IC (Pf): 120-190 nM
IC (cytotox.): 5,170-5,500 nM
5
0
IC (Pf): 200 nM
IC (cytotox.): 50-100 nM
5
0
5
0
class I
selective
50
SI: 27-46
pan
HDACi
SI: <1
Trichostatin A
Entinostat
IC (Pf): 8-11 nM
IC (Pf): 7,800-8,300 nM
50
5
0
IC (cytotox.): 200 nM
IC (cytotox.): >20,000 nM
5
0
50
SI: 18-25
SI: >2
Figure 1. Selected HDACi and their antiplasmodial activity (P. falciparum = Pf), cytotoxicity (against mammalian cells) and selectivity indices (SI = IC50
cytotox.))/IC50 (Pf)). Data are from references[2,14-15]
(
.
class III HDACs (PfSir2A and B).[2,11b] PfHDAC1 possess a high
sequence identity to its human class I homologues (61% to
hHDAC1 and 62% to hHDAC2), and homology modelling studies
show that the zinc-coordinating amino acids in HDACs and the
tubular entrance between the zinc atom and the surface of the
HDAC enzymes are well conserved between human class I
HDAC enzymes and PfHDAC1.[12-13] This might explain the
sensitivity of P. falciparum to pan- and class I selective HDACi.
However, pan- and class I-selective HDACi usually display
significant toxicity against mammalian cells which leads to
unsatisfactory selectivity against parasites versus human cells
previous publication, starting from a hHDAC6 preferential
scaffold,[ we reported on the multicomponent synthesis and
biological evaluation of novel peptoid-based antiplasmodial
HDACi.[17] Several of these HDACi displayed potent activities
against the 3D7 line of P. falciparum with IC50 values ranging from
4-158 nM and promising parasite-selectivity.[ Furthermore,
several compounds showed submicromolar activity against
P. berghei exo-erythrocytic stages, making this novel type of
HDACi an encouraging starting point for the development of
antiplasmodial drug leads with dual stage activity.[ The aim of
this study was to gain a better insight into the structure-activity
and structure-toxicity relationships of this new class of HDACi with
antiplasmodial activity and in particular, to explore the structural
requirements for potent dual-stage activity. Therefore, a series of
analogues with a variety of structural variations were designed
(Figure 2). In particular, we aimed for various modifications on the
cap group, as well as truncation of the linker. Herein, we describe
the synthesis and biological evaluation of a library of second-
generation peptoid-based HDACi.
20]
17]
17]
(
Figure 1).[2,14-16] Another interesting finding regarding
antiplasmodial HDACi is their activity against several malaria
parasite life cycle stages. While most antimalarial drugs target just
one specific life cycle stage (mostly asexual blood stages),
several HDACi are also active against liver stages and late stage
gametocytes.[5,17-18] This highlights the potential of HDACi as
novel antiplasmodial agents.
In this regard, we, and others, hypothesized that class II
selective hHDACi might be a better starting point for the
development of parasite-selective antiplasmodial HDACi, due to
their usually lower toxicity to mammalian cells.[5,17,19a] In addition,
knock out experiments with the class IIb isozyme hHDAC6 in mice
led to a viable phenotype with no significant defects.[19b] In a
Results and Discussion
Diversity-oriented synthesis of peptoid-based HDACi
The compounds described in this study are peptoid-based
hydroxamic acids. To synthesize a mini-library of these target
compounds, we applied two different diversity-oriented
approaches: (1) the Ugi four component reaction (U-4CR,
Scheme 1) and (2) submonomer pathways (Scheme 2). Most
recently, we reported on the synthesis of peptoid-based HDACi
by utilizing a microwave-assisted Ugi four-component reaction
(
U-4CR) in combination with
methanolate/hydroxylamine
hydroxylaminolysis in a one-pot fashion to afford the desired
a
subsequent sodium
hydrochloride based
Figure 2. Design of target compounds.
2
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Scheme 1. Microwave-assisted multicomponent approach utilizing the Ugi four-component reaction to synthesize hydroxamic acids (2a-j). Reagents and
1
2
conditions: a) (i) 4, 6, Et
3
N, MeOH, 4 Å MS, 150 W, 45°C, 30 min; (ii) R -NC, R -COOH, 150 W, 45°C, 120 min; b) aq. H
2
NOH, NaOH, DCM/MeOH (1:3), rt;
c) MeOH, 4Å MS, rt, 72 h; d) MeOH, Pd/C, H
2
, 2 h
hydroxamic acids of type 2.[17] In this study, we aimed for a more
convenient hydroxamic acid synthesis and applied an aqueous
hydroxylaminolysis using aqueous hydroxylamine with sodium
hydroxide as base, also in a one-pot multicomponent approach,
thus avoiding the fresh preparation of methanolate (Scheme 1).
Compounds 2c,e-h were synthesized using this protocol in 11-
the submonomer pathway 1, methyl 4-(aminomethyl)benzoate
hydrochloride 6 was reacted with t-butyl bromoacetate (10, 46 %
yield) and subsequently acylated with 3,5-dimethylbenzoyl
chloride to afford t-butyl ester 11 in 99 % yield. After deprotection
of the t-butyl group 11 (>99 %) and EDC-mediated amide coupling
reactions (56-82 %) to afford intermediates 12k-r, the desired
hydroxamic acids 2k-r were obtained by an aqueous
hydroxylaminolysis using hydroxylamine and sodium hydroxide in
24-91 % yield. Hydroxamic acids 2s,t were prepared via
submonomer pathway 2 starting with a reaction of bromoacetyl
bromide 13 with methylamine hydrochloride (46 %), followed by a
substitution reaction with 4-(aminomethyl)benzoate hydrochloride
6 to afford the secondary amine 14 in 50 % yield. Thereafter,
amine 14 was acylated with two different benzoyl chlorides to
provide 15s,t. Finally, the target compounds 2s,t were
8
8 % yield, while compound 2d,i-j were prepared using the
previously described protocol in 56-83 % yield.[17] The synthesis
of the truncated derivatives 2a-b was accomplished using the O-
benzyl protected hydroxamic acid 8 as amine in the U-4CR (8-
27
%
yield) and subsequent deprotection by catalytic
hydrogenolysis (23-54 yield, Scheme 1), since the
%
hydroxylaminolysis of the corresponding esters in the standard
protocol did not produce the desired hydroxamic acids. Based on
this synthetic approach, our peptoid-based hydroxamic acids can
1
2
be structurally divided into an isocyanide (R ), carboxylic acid (R ), synthesized in 40-86 % yield by hydroxylaminolysis of 15s,t as
3
4
carbonyl (R and R ) and amine (n = 0 and 1) region.
described above.
Even though this multicomponent one-pot synthesis is
efficient and versatile, it features some disadvantages. The
U-4CR requires the use of isocyanides, which are mostly quite
toxic, often possess a very unpleasant odor and the commercially
available isocyanides are limited regarding chemical space. The
typical synthetic strategy of isocyanides is the formamidation of
the amine followed by dehydration, with the most common
In vitro inhibition of P. falciparum asexual intraerythrocytic
parasite growth, cytotoxicity and parasite selectivity
The synthesized mini-library was tested for in vitro activity against
asexual intraerythrocytic stage parasites as well as for cytotoxicity
against human liver hepatocellular carcinoma cells (HepG2;
Table 1). All compounds, except 2a, 2b, 2d and 2i, revealed
potent activity (IC50 (Pf3D7): ≤ 0.25 µM) in combination with good
parasite selectivity (selectivity index (SI) > 100) with IC50 values
against the 3D7 line of P. falciparum ranging from 0.0052 to 0.25
µM and selectivity indices ranging from 170 to 1483. In contrast,
3
dehydration reagents (e.g. POCl ) additionally having drawbacks
due to their toxicity.[21] However, peptoids are also accessible by
so-called submonomer pathways (Scheme 2). In this approach,
three of the previously described regions (for instance the linker,
carbonyl and carboxylic acid region) are kept unchanged, while
one region can be varied in a diversity-oriented fashion. Therefore, the reference HDACi vorinostat (SAHA, IC50 (Pf3D7): 0.17 µM,
we applied two submonomer pathways in order to derivatize the
isocyanide (R ) and carboxylic acid region (R ). The synthesis of
IC50 (HepG2): 2.51 µM) showed only a low selectivity index of 15.
Interestingly, the observed structure-activity (SARs) and
1
2
the target compounds 2k-t is summarized in Scheme 2. Briefly, in
structure-toxicity
(STRs)
relationships
seem
3
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Scheme 2. Submonomer pathways for the synthesis of peptoid-based hydroxamic acids (2k-t). Reagents and conditions: a) t-Butyl bromoacetate, Et
3
N,
1
THF, rt, o.n.; b) 3,5-dimethylbenzoyl chloride, Et
DCM/MeOH (1:3), rt, o.n.; f) methylamine hydrochloride, K
Ph-COCl, Et N, DCM, rt, o.n..
3
N, DCM, rt, o.n.; c) TFA/DCM (1:1), rt; d) R -NH
2
, EDC, DMAP; Et
, DCM, 3 h; g) methyl 4-(aminomethyl)benzoate hydrochloride 6, Et
3
N, DCM, rt, o.n.; e) aq. H
2
NOH, NaOH,
CO
3
3
N, THF, rt, o.n.; h) R2-
2
3
1
to be highly dependent on the nature of the different peptoid
regions (see Figure 3). First, our aim was to investigate the impact
of structural modifications at the α-carbon of the cap group as well
as the linker lengths, hence varying the linker and the carbonyl
region. Truncation of the linker length (n = 0) reduced the
antiplasmodial activity, as seen in compound 2a,b (IC50 (Pf3D7):
to optimize the benzyl residue (R ) by introducing several
substituents (halogene, methoxy, alkyl groups) and a bioisosteric
3-picolyl group (2j-r), while keeping the carboxylic region identical
-Ph). Compound 2j with potent activity and moderate
toxicity (IC50 (Pf3D7): 0.037 µM, IC50 (HepG2): 9.57 µM, SI: 259)
2
(R : 3,5-CH
3
1
and no further substitution on the benzylic group (R ) served as a
0
.57-77.7 µM) and therefore does not seem to be a beneficial
reference. The results indicate, that substituents on the benzylic
group (R ) in general tend to decrease toxicity (IC50 (HepG2): 15-
1
structural modification. The introduction of substituents in the
carbonyl region (R , R ) provided interesting results. While
compounds 2c and 2d both displayed low toxicity (IC50 (HepG2):
3
4
26 µM) slightly, while showing no clear SAR and STR. Notably,
1
compound 2r, which possesses a 3-picolyl R residue, revealed
>
2
50 µM), the sterically demanding phenyl residue in compound
d decreased the antiplasmodial activity (IC50 (Pf3D7): 0.39 µM)
in comparison to its dimethyl substituted counterpart 2c (IC50
Pf3D7): 0.095 µM) [p = 0.0025]. Therefore, the introduction of
low toxicity (IC50 (HepG2): 41.93 µM, SI: 530), but on the other
hand also exhibited a significantly lower antiplasmodial activity
(IC50 (Pf3D7): 0.079 µM) than its structurally related compound 2j
[p = 0.0175]. Furthermore, we introduced a methyl group into the
(
1
small residues in the carbonyl region suggests improvement in
toxicity, while retaining good antiplasmodial activity. Next, we
observed high antiplasmodial activity for compounds bearing a
isocyanide region (R ) and synthesized compound 2s and 2t.
Initially, we investigated the methyl based compound 2s bearing
2
a 3,5-dimethyl group (R ) to have a comparison to the benzylic
1
benzyl residue (R ) in the isocyanide region, hence we tried to
based compounds 2j-r. Compound 2s displayed an improved
gain a better insight by introducing several structural variations.
2
Consequently, we modified the carboxylic region (R ) and
•
•
Methyl group decreases toxicity
Benzylic groups enhance activity (but
also toxicity, further substitution show
no beneficial effects)
synthesized compounds 2g-j which exhibited potent
antiplasmodial activity in the single- to double-digit nanomolar
range (IC50 (Pf3D7): 0.0052-0.037 µM). The compounds bearing
branched alkyl groups in p-position of the carboxylic region (R ),
g and 2h, showed the highest potency (IC50 (Pf3D7): 0.0052-
.0088 µM, SI: 889-1483), while having moderate toxicity against
mammalian cells (IC50 (HepG2): 5-13 µM). Compound 2h (IC50
Pf3D7): 0.0052 µM, SI: 889), bearing an i-propyl group (R ),
displayed antiplasmodial activity comparable to the reference
2
•
•
Small alkyl groups
e.g. methyl) improve
2
0
•
Shorter linker (n = 0)
decreases activity
(
toxicity
Bulky groups (e.g.
phenyl) decrease
activity
2
(
compound chloroquine (IC50 (Pf3D7): 0.0068 µM). In contrast, the
2
replacement of the i-propyl group (R ) by the bioisosteric and
more polar N,N-dimethylamino group (compound 2i) distinctly
increased toxicity against human cells, resulting in a decreased
parasitic selectivity (IC50 (HepG2): 0.62 µM, SI: 64). We next tried
Figure 3. Structure-activity and -toxicity relationship of the peptoid-based
hydroxamic acids.
4
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FULL PAPER
toxicity profile against mammalian HepG2 cells (IC50 (HepG2):
region also lowers toxicity as seen in compounds 2e,f (IC50
(HepG2): >50 µM), however, the antiplasmodial activity is
simultaneously decreased as well (IC50 (Pf3D7): 0.11-0.25 µM).
Taken together, the substitution of the carboxylic region (R2)
has a strong influence on the antiplasmodial activity and
especially branched alkyl groups, like the 4-i-propyl group, seem
47.98 µM, SI: 889) than hydroxamic acids 2j-r
(
IC50 (HepG2): 9.57-41.93 µM, SI: 170-711), while keeping
promising antiplasmodial activity (IC50 (Pf3D7): 0.054 µM).
Accordingly, the small methyl group in the isocyanide region
seems to have an impact on toxicity, which is also seen in
compound 2t (IC50 (HepG2): >50 µM, SI: >704). Notably, the
introduction of substituents in the 2-position of the carboxylic
to
have
an
enhancing
effect.
Table 1. In vitro activity against asexual intraerythrocytic stage P. falciparum parasites, cytotoxicity and selectivity indices of 2a – 2t.
Pf 3D7[a]
IC50 [µM]
HepG2
IC50 [μM]
Compound
R1
R2
95% CI for HepG2 IC50
SI[b]
2
a
c-Hex
c-Hex
c-Hex
c-Hex
t-Bu
t-Bu
Bn
4-i-Pr
0.57(±0.089)
77.70(±7.94)
35.83
47.21
>50
27.69-46.37
41.81-53.32
n/a
63
<1
2b
3,5-CH
3
2c
4-(CH
4-(CH
3
)
2
N
0.095(±0.015)
0.39(±0.075)
>526
>128
>455
>200
1483
889
64
2d
)
3 2
N
>50
n/a
2e
2-CH
2-CH
3
0.11(±0.080)
>50
n/a
2
f
3
O
0.25(±0.14)
>50
n/a
2
g
h
4-t-Bu
4-i-Pr
0.0088(±0.0032)
0.0052(±0.0036)
0.0097(±0.0029)
0.037(±0.012)
0.042(±0.011)
0.033(±0.0090)
0.087(±0.044)
0.094(±0.0073)
0.11(±0.019)
13.05
4.62
9.48-17.97
2.48-8.58
0.44-0.88
6.97-13.15
10.75-19.79
Very wide
16.79-24.74
14.25-29.47
17.59-39.08
17.65-23-09
13.16-31.53
35.22-49.93
Very wide
n/a
2
Bn
2
i
Bn
4-(CH
)
3 2
N
0.62
2
j
Bn
3,5-CH
3,5-CH
3,5-CH
3,5-CH
3,5-CH
3,5-CH
3,5-CH
3,5-CH
3,5-CH
3,5-CH
3
9.57
259
347
581
234
218
238
262
170
530
889
>704
15
2k
4-CH
4-CH
3
-Bn
3
3
3
3
3
3
3
3
3
14.59
19.18
20.38
20.49
26.22
20.19
20.37
41.93
47.98
>50
2
l
3
O-Bn
2m
4-F-Bn
4-Cl-Bn
2n
2o
2p
2q
4-t-Bu-Bn
3,5-CH
3-CH -Bn
3-Picolyl
3
-Bn
0.077(±0.022)
0.12(±0.048)
3
2
r
0.079(±0.018)
0.054(±0.0029)
0.071(±0.015)
2s
CH
CH
3
3
2
t
2-CH
3
SAHA
Chloroquine
0.17(±0.035)
2.51
1.63-3.86
0.0068(±0.0023)
[
a] Three independent assays were carried out, each carried out in triplicate wells. [b] Selectivity index (SI) = (mammalian cell IC50)/(P. falciparum IC50).
5
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In our previous study, we could not identify any clear SAR for the
isocyanide region, which is confirmed by the results in this
study.[ Nonetheless, the isocyanide region appears to play a
certain role in the STR, with methyl groups having the most
promising toxicity improvement. Additionally, modifications at the
α-carbon of the cap group have effects on the toxicity as well. In
that respect, the introduction of methyl groups in the carbonyl
region is beneficial regarding the toxicity profile.
include differences in compound uptake and possible
P. falciparum HDAC specificity. It needs to be noted that these
data are only a snapshot of treating a single developmental stage
(asexual trophozoites). There may be different effects on different
developmental forms or life stages where expression levels of
target proteins, or compound exposure dynamics, may vary and
therefore influence the level of acetylation. Future work should
include the investigation of potential acetylation changes in other
life stages within the intraerythrocytic lifecycle as well as the
investigation of acetylation alterations to other histones. The
differences in acetylation levels shown here may be related to
different target preferences and further analysis will be required,
however this is complicated by the lack of recombinant PfHDAC
proteins.
18]
Inhibition of human HDAC1 and HDAC6
The result of the evaluation of the cytotoxicity of compounds 2a-t
against mammalian HepG2 cells revealed a broad toxicity profile
from submicromolar IC50 values to no cytotoxicity (IC50 (HepG2):
>50 µM), in which the different residues of the peptoid-based
hydroxamic acids suggest certain structure-toxicity relationships.
In general, pan- and class I selective human HDAC inhibitors
seem to show increased toxicity. Thus, it has been hypothesized,
that class IIb preferential inhibitors may decrease side effects, due
to a inter alia more precise alteration of biological substrates in
the cell.[17a,18,22] In that regard, we screened selected compounds
in a biochemical assay against the class I isoform hHDAC1 and
Table 2. Activities of compounds 2c-i,s-t and the reference HDACis SAHA,
Ricolinostat, HPOB and Tubastatin A against human HDAC1 (hHDAC1) and
HDAC6 (hHDAC6).
hHDAC1
hHDAC6
Compound
SI[a]
IC50 [μM]
IC50 [μM]
the
class
IIb
isoform
hHDAC6
using
ZMAL
2
2
2
c
d
e
0.45(±0.026)
0.84(±0.095)
0.089(±0.002)
0.064(±0.007)
0.051(±0.004)
0.038(±0.004)
0.071(±0.007)
0.051(±0.003)
0.014(±0.0005)
0.044(±0.004)
0.032(±0.001)
0.055(±0.011)
0.018(±0.003)
0.0085(±0.0090)
5
(
Z-Lys(Ac)-AMC) as substrate to investigate, if the toxicity against
mammalian cells shows any correlation to the human HDAC
isoform profile. We tested the six compounds with the lowest
toxicity against HepG2 cells (2c-f, 2s-t), and the three compounds
with highest activity against the 3D7 line of P. falciparum and
moderate (2g and 2h) to high (2i) cytotoxicity (see Table 2).
Indeed, all compounds revealed potent activity against the
hHDAC6 isoform with IC50 values ranging from 0.014 to 0.089 µM.
On the contrary, we observed differences in the hHDAC1 activity.
The compounds with low toxicity against HepG2 cells (2c-f, 2s-t)
demonstrated moderate to low hHDAC1 activity (IC50 (hHDAC1):
13
47
82
<1
<1
<1
7
2.41(±0.086)
2
f
3.11(±0.308)
2g
0.034(±0.0064)
0.019(±0.0017)
0.0077(±0.0000095)
0.29(±0.054)
2h
2
i
2s
0.29-3.11 µM), while compounds 2g-i displayed potent activity in
the double-digit nanomolar range (IC50 (hHDAC1): 0.0077-0.034
µM) with similar activity against hHDAC1 and hHDAC6. Hence,
the potent activity against the human class I hHDAC1 isoform
seems to be a possible explanation for the increased toxicity of
certain compounds against mammalian cells. Notably,
compounds 2e-f, t turned out to be promising selective hHDAC6
inhibitors (IC50 (hHDAC6): 0.032-0.051 µM, SIHDAC1/HDAC6: 47-82),
comparable to other known selective HDAC6i like ricolinostat
2
t
2.01(±0.137)
63
2
SAHA
0.082(±0.015)
0.19(±0.022)
Ricolinostat[b]
11
25
HPOB[b]
2.10(±0.23)
Tubastatin
A[b]
2.49(±0.14)
0.014(±0.00061)
178
HDAC1/HDAC6
HDAC1/HDAC6
(
(
SI
: 11), HPOB (SI
: 25) and Tubastatin A
SIHDAC1/HDAC6: 178).22-23
[
a] Selectivity index (SI) = hHDAC1/hHDAC6. [b] Data from ref.[22-23]
.
Effect of peptoid-based HDAC inhibitors on P. falciparum
histone acetylation
Hyperacetylation analysis was carried out on compounds 2c, 2g,
In vitro activity against P. berghei exo-erythrocytic forms and
stage V P. falciparum gametocytes.
2h, 2s and 2t (Figure 4). Treatment with 2s resulted in the highest
Our previous study revealed that several peptoid-based
hydroxamic acids act as antiplasmodial compounds with dual-
stage activity against asexual blood stages and liver stages
levels of hyperacetylation, causing a significant (~2-fold) relative
density increase compared to the 3h-C non-treatment control
(
p < 0.01; Figure 4) irrespective of whether the signal
P. berghei exo-erythocytic forms, Pb EEF).[18] Liver stage activity
(
corresponding to H4 (~11 kDa) or total signal including cross
reactive bands (H2B/H2Bv (~13-14 kDa); H2A.Z (~16 kDa) was
used. None other the peptoid-based HDAC inhibitors caused a
significant increase in acetylation signals (p <0.5; Figure 4). As
expected, the HDAC inhibitor control, vorinostat, also caused
significantly increased acetylation compared to the vehicle control
is an interesting additional feature, since this might prevent the
development of the disease and could be applied as
chemoprotection. Therefore, all 20 compounds were screened for
their activity against P. berghei exo-erythocytic forms. The results
are summarized in Table 3. Nine compounds (2g-m, p, q)
revealed submicromolar activity against Pb EEF with IC50 values
ranging from 0.016 to 0.58 µM (SIHepG2/Pb EEF: 23-289), while three
out of these nine compounds displayed double-digit nanomolar
(
p < 0.01; Figure 4). It is not clear why 2s, but not the other
peptoid-based HDAC inhibitors in this series, caused histone
hyperacetylation, however factors that need to be considered
activity 2h-j (IC50 (Pb EEF): 0.016-0.073 µM). Interestingly, all of
6
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ChemMedChem
10.1002/cmdc.201800808
FULL PAPER
Table 3. Activity of compounds 2a-t against P. berghei exo-erythrocytic forms.
Pb EEF[a]
IC50 [µM]
95% CI for Pb
EEF IC50
Compound
SI[b]
2
a
9.70
20.19
20.14
19.26
9.70
5.20-18.09
Very wide
5.64-71.96
Very wide
4.08-23.05
11.13-63.97
0.28-1.20
4
2
2b
2c
>3
>3
>5
>2
23
289
28
131
70
34
185
21
2
2d
2e
2
f
26.69
0.58
2
g
h
2
0.016
0.022
0.073
0.21
0.0080-0.031
0.013-0.038
0.044-0.12
0.11-0.43
2
i
2
j
2k
2
l
0.57
0.32-1.02
2m
0.11
0.045-0.28
0.33-3.00
Figure 4. Hyperacetylation analysis of P. falciparum histone H4 by selected
peptoid-based HDAC inhibitors. Western blot analysis of protein lysates
prepared from trophozoite-stage P. falciparum 3D7 infected erythrocytes
treated for 3 h with 5x IC50 of test or control compounds. Controls included a
vehicle control (0.2 % DMSO; 3h-C), the non-HDAC inhibitor control chloroquine
2n
2o
2p
2q
1.00
16.65
0.58
Very wide
0.21-1.57
35
157
3
(CQ) and the positive HDAC inhibitor control vorinostat. (A) Western Blot was
carried out using anti-(tetra)acetyl-histone H4 primary antibody and IRDye 680
0.13
0.066-0.24
Very wide
2.38-21.65
9.54-82.53
0.0055-0.011
goat anti-rabbit secondary antibody. Total protein per lane was detected using
TM
REVERT
Total Protein Stain on the same membrane. Representative
2
r
16.73
7.17
Western Blot shown. Protein molecular weight marker bands (kDa) are
indicated. (B) Graph showing mean relative density (± SD) for three independent
Western Blot experiments. Western signal densities were normalized to total
protein load in the respective lane and expressed as fold change compared to
the 3h-C DMSO vehicle control (taken as 1.0; dotted line). Data are mean (±
SD) for three independent experiments. Significant differences are shown for
H4 signal (~11 kDa; grey bars; ** p < 0.01) Total signal included H4 signal and
cross reactive bands H2B/H2Bv (~13-14 kDa) and H2A.Z (~16 kDa).
2s
7
2
t
28.05
0.0077
>2
326
SAHA
[
a] P. berghei exo-erythrocytic forms (Pb EEF). [b] Selectivity index (SI) =
the nine compounds with submicromolar activity bear a benzylic
or substituted benzylic residue in the isocyanide region. However,
the respective substitution in the carboxylic region seems to alter
the activity drastically. The most promising derivative in regard to
dual-stage activity was hydroxamic acid 2h bearing a 4-i-propyl
(mammalian cell IC50)/(Pb EEF IC50). For HepG2 values (mammalian cell IC50
see Table 1.
)
All compounds were found to have only moderate to poor
activity against mature gametocytes (IC50 > 5 µM). The best
activity was observed for compound 2p (IC50 = 5.27 µM). It is an
interesting finding that several compounds showed promising
submicromolar activity against asexual blood stages and liver
stages, but no noteworthy activity against mature gametocytes.
The reason for this is currently unknown. However, this
phenomenon could be related to an altered expression of the
three zinc-dependent PfHDACs in the different malaria parasite
life cycle stages.
2
group in the carboxylic acid region (R ), showing the highest
activity both against P. falciparum asexual blood stages (Pf 3D7:
HepG2/Pf 3D7
0
.0052 µM; SI
: 889) and against P. berghei exo-
erythocytic forms (IC50 (Pb EEF): 0.016 µM; SIHepG2/Pb EEF: 289) in
combination with encouraging parasite-selectivity.
Gametocytes are responsible for the transmission of the
parasite to another host and targeting this life stage would
contribute to the prevention of transmission, thus sooner or later
eradicating the parasite. Hence, to further investigate the potential
of these peptoid-based HDACi as multi-stage inhibitors, all
compounds were screened for their activity against mature stage
V P. falciparum gametocytes using an ATP bioluminescence
Conclusions
In conclusion, we synthesized a mini-library of 20 novel second
generation peptoid-based HDACi. To this end, we applied
efficient and diversity-oriented synthetic routes, which include a
convenient one-pot Ugi multicomponent reaction and
straightforward submonomer pathways. Based on our previous
study, we derivatized all structural regions of the peptoid scaffold
assay. First, all compounds were screened at
a fixed
concentration of 5 μM. Subsequently, compounds with inhibitory
activity in the primary screen were tested in dose response to
determine IC50 values (Table 4).
7
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ChemMedChem
10.1002/cmdc.201800808
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(
254 nm) or staining in potassium permanganate solution following heating.
Table 4. Activity of peptoid-based HDAC inhibitors against mature stage V P.
falciparum gametocytes.
Flash column chromatography was performed using prepacked silica
cartridge with the solvent mixtures specified in the corresponding
experiment. Melting points (mp) were taken in open capillaries on a Mettler
FP 5 melting-point apparatus and are uncorrected. Proton ( H) and carbon
(13C) NMR spectra were recorded on a Bruker (Avance) or Varian (Mecury-
R1
R2
Compound
Pf stage V
gametocytes[
IC50 [μM]
a]
1
6 3
plus) 300, 400, 500 or 600 using DMSO-d or CDCl as solvents. Chemical
2
2
2
2
2
c
d
l
c-Hex
4-(CH
4-(CH
3
3
)
2
N-Ph
N-Ph
16.29(±5.99)
14.81(±5.87)
10.21(±6.68)
5.27(±3.39)
16.84(±5.77)
shifts are given in parts per million (ppm), relative to residual solvent peak
for 1H and 13C. Due to the well-known phenomenon of cis/trans-amide
bond rotamers in peptoids[20c], 1H and 13C NMR-signals can occur as two
c-Hex
)
2
1
distinct sets of signals. H NMR signals marked with an asterisk (*)
4-MeO-Bn
3,5-Me-Bn
Me
3,5-Me-Ph
3,5-Me-Ph
3,5-Me-Ph
correspond to peaks assigned to the minor rotamer conformation. High
resolution mass spectra (HRMS) analysis was performed on a UHR-TOF
maXis 4G, Bruker Daltonics, Bremen by electrospray ionization (ESI).
Analytical HPLC analysis were carried out on a Knauer HPLC system
equipped with a Knauer UV detector K-2600 (254nm) using a Vertex Plus
Column (length 150 x 4 mm with precolumn, packing material of the
column was Eurospher II 100-5 C18). UV absorption was detected at 254
nm with a isocratic gradient of 10% B to 100% B in 30 min using HPLC-
grade water +0.1% TFA (solvent A) and HPLC-grade acetonitrile +0.1%
TFA (solvent B) for elution at a flow rate of 1 mL/min. The purities of all
final compounds were 95% or higher.
p
s
[
a] P. falciparum stage V gametocytes; at least two independent experiments,
each in duplicate wells.
to get a better understanding of the structure-activity (SAR) and
structure-toxicity relationships (STR). All synthesized compounds
were screened and assessed for their antiplasmodial activity and
human cell (HepG2) cytotoxicity. The majority of compounds
displayed potent activity against asexual blood stage
P. falciparum parasites (3D7 line) with IC50 values ranging from
Synthesis
of
N-(2-(cyclohexylamino)-2-oxoethyl)-N-(4-
(hydroxycarbamoyl)phenyl)-4- isopropylbenzamid 2a:
0
.0052-0.25 µM and selectivity indices over mammalian cells
4
-Amino-N-benzyloxy-benzamide was synthesized according to
(
HepG2) ranging from 170 to 1483. The results suggest that
reference[ . A mixture of 4-amino-N-benzyloxy-benzamide (436 mg, 1.8
mmol, 1 eq.), paraformaldehyde (66 mg, 2.26 mmol, 1.2 eq.) and 50 mg of
crushed molecular sieves (MS) 4 Å in dry methanol (4 mL, 0.5 M) was
stirred for 30 min at room temperature. Next, 4-i-propylbenzoic acid (5a,
296 mg, 1.8 mmol, 1 eq.) and cyclohexyl isocyanide (3a, 221 µL, 1.8 mmol,
1 eq.) was added and stirred for 72 h at room temperature. Subsequently,
the solvent was removed under reduced pressure and the residue was
dissolved in ethyl acetate (20 mL). The organic phase was washed with 1
24]
2
especially the carboxylic region (R ) seems to have the greatest
impact on SAR and STR, however, certain structural modification
on the isocyanide (R ) and carbonyl region (R ) also possess
beneficial effects regarding cytotoxicity improvement.
Furthermore, all compounds were tested against mature stage V
P. falciparum gametocytes and for activity against liver stage
P. berghei exo-erythrocytic forms. Although only poor activity was
observed against gametocytes, various compounds showed
1
3,4
3
M HCl (3 x 30 mL) and with sat. NaHCO /water (1:1, 3 x 20 mL). The
organic phase was dried over sodium sulfate, filtered, and the solvent was
removed under reduced pressure. The crude product was purified by flash
column chromatography (dichloromethane/methanol, gradient) to yield the
desired compound 9a in 8 % yield as an brown oil: 1H-NMR (CDCl
MHz): 훿 [ppm] = 1.11-1.45 (m, 5 H), 1.19 (d, J = 6.9 Hz, 6 H), 1.56-1.75
encouraging
submicromolar
activity
against
P. berghei exo-erythrocytic forms. Selected compounds were
further screened for their inhibition of two representative human
HDAC isoforms (hHDAC1 and 6), which indicated a correlation
between higher cytotoxicity against human cells and increased
activity against the human class I isoform hHDAC1. The most
promising compound 2h emerged as a potent dual-stage
antiplasmodial HDACi (IC50 (Pf 3D7) = 0.0052 µM, IC50 (Pb EEF)
3
, 300
(m, 3 H), 1.87-1.92 (m, 2 H), 2.76-2.90 (m, 1 H), 3.71-3.84 (m, 1 H), 4.45
(
2
s, 2 H), 5.00 (s, 2 H), 6.37-6.40 (m, 1 H), 7.04-7.07 (m, 2 H), 7.12-7.15 (m,
H), 7.24-7.27 (m, 2 H), 7.38-7.45 (m, 5 H), 7.51-7.54 (m, 2 H), 8.86 (s, 1
H); 13C-NMR (CDCl
, 75 MHz): 훿 [ppm] = 23.8, 24.8, 25.6, 33.1, 34.1, 48.5,
5.1, 77.4, 78.5, 126.4, 127.1, 128.4, 128.8, 129.0, 129.3, 129.4, 130.2,
3
5
1
=
0.016 µM) with parasite-specific activity. In addition, the
31.8, 135.3, 147.4, 152.1, 167.7, 171.3.; HRMS calculated for
biological evaluation led to several compounds (2c, 2e, 2s, 2t)
with promising antiplasmodial activity (IC50(Pf 3D7) = 0.054-0.12
+
C
32
H
37
N
3
O
4
: 528.2859 [M+H] , found 528.2857.
Pf
µM) and low cytotoxicity (IC50 (HepG2) = 47.98->50 µM, SI
Compound 9a (146 mg, 0.28 mmol, 1 eq.) was dissolved in methanol (20
mL) and a catalytical amount of Pd/C (10wt. %) was added. Subsequently,
the mixture was stirred for 2 h at room temperature under a hydrogen
atmosphere. Next, the solution was filtered through pad of Celite and the
solvent was removed under reduced pressure. After purification by flash
column chromatography (dichloromethane/methanol, gradient), the
desired product 2a was obtained in 23 % yield as white solid: mp = 120 °C;
3
D7/HepG2
=
>417-889). Thus, this series of compounds represent a
valuable starting point for the development of novel drug
candidates with potent dual-stage antiplasmodial activity and an
optimized toxicity profile.
1H-NMR (CDCl
3
, 600 MHz): 훿 [ppm] = 1.14 (d, J = 6.9 Hz, 6 H), 1.17-1.36
Experimental Section
(
2
m, 5 H), 1.57-1.59 (m, 1 H), 1.66-1.69 (m, 2 H), 1.84-1.86 (m, 2 H), 2.77-
.84 (m, 1 H), 3.72-3.79 (m, 1 H), 4.48 (s, 2 H), 6.75-6.76 (m, 1 H), 7.02-
Synthesis
7.08 (m, 4 H), 7.22-7.23 (m, 2 H), 7.55-7.56 (m, 2 H), 8.32 (brs, 1 H), 10.38
(
s, 1 H); 13C-NMR (CDCl
3
, 151 MHz): 훿 [ppm] = 23.7, 24.7, 25.6, 32.9, 34.1,
4
1
4
8.6, 55.1, 126.4, 127.1, 128.3, 129.1, 129.4, 131.5, 146.9, 152.3, 164.7,
All chemicals and solvents were obtained from commercial suppliers
+
31 3 4
68.4, 171.6.; HRMS calculated for C25H N O : 438.2387 [M+H] , found
(Sigma-Aldrich, Acros Organics, Carbolution Chemicals) and used as
38.2387; HPLC purity: 96.4 %.
purchased without further purification. All microwave-assisted reactions
were carried out with a CEM Focused Microwave System, Model Discover.
The progress of all reactions was monitored by thin layer chromatography
Synthesis
of
N-(2-(cyclohexylamino)-2-oxoethyl)-N-(4-
(TLC) using Merck precoated silica gel plates (with fluorescence indicator
(hydroxycarbamoyl)phenyl)-3,5- dimethylbenzamide 2b:
UV254). Components were visualized by irradiation with ultraviolet light
8
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ChemMedChem
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FULL PAPER
4
-Amino-N-benzyloxy-benzamide was synthesized according to
silicon septum and the reaction mixture was subjected to microwave
irradiation (Discover mode; power: 150W; hold time: 20 min; temperature:
45 °C; PowerMax-cooling mode) under medium speed magnetic stirring.
Next, the appropriate carboxylic acid (5a-f) (1.0 eq.) and isocyanide (3a-c)
(1.0 eq.) components were added and the reaction mixture was again
irradiated at 45 °C for 60 min (150W). Subsequently, a mixture of
hydroxylamine hydrochloride (10 eq.) in a sodium methanolate solution,
freshly prepared from dry methanol (8 mL) and sodium (7.5 mmol, 7.5 eq)
was added and the reaction mixture was subjected to microwave
irradiation at 55 °C for 30 min (150W). After completion of the reaction, the
reaction mixture was filtered, and the solvent was removed under reduced
pressure. Water (15 mL for 0.5 mmol) was added and the pH was adjusted
to a pH 7-8 using 4 M HCl. The mixture was extracted with ethyl acetate,
the combined organic layers were dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuum. The crude products were purified by
flash column chromatography (dichloromethane/methanol, gradient) to
yield the desired products 2d,i-j.
[
24]
reference . A mixture of 4-amino-N-benzyloxy-benzamide (290 mg, 1.2
mmol, 1.2 eq.), paraformaldehyde (36 mg, 1.2 mmol, 1.2 eq.) and 50 mg
of crushed molecular sieves (MS) 4 Å in dry methanol (4 mL, 0.5 M) was
stirred for 30 min at room temperature. Next, 3,5-dimethylbenzoic acid (5b,
150 mg, 1.0 mmol, 1 eq.) and cyclohexyl isocyanide (3a, 122 µL, 1.0 mmol,
1
eq.) was added and stirred for 72 h at room temperature. Subsequently,
the solvent was removed under reduced pressure and the residue was
dissolved in ethyl acetate (20 mL). The organic phase was extracted with
1
organic phase was dried over sodium sulfate, filtered, and the solvent was
removed under reduced pressure. The crude product was purified by flash
column chromatography (dichloromethane/methanol, gradient) to yield the
3
M HCl (3 x 30 mL) and with sat. NaHCO /water (1:1, 3 x 20 mL). The
1
desired compound 9b in 27 % yield as an oil: H-NMR (CDCl
3
, 300 MHz):
훿 [ppm] = 1.12-1.41 (m, 5 H), 1.64-1.92 (m, 5 H), 2.15 (s, 6 H), 3.70-3.83
m, 1 H), 4.43 (s, 2 H), 4.98 (s, 2 H), 6.27-6.30 (m, 1 H), 6.90-6.93 (m, 3
H), 7.11-7.14 (m, 2 H), 7.36-7.43 (m, 5 H), 7.50-7.53 (m, 2 H), 8.64 (s, 1
H); 13C-NMR (CDCl
, 75 MHz): 훿 [ppm] = 15.4, 21.2, 21.4, 24.8, 25.6, 33.0,
8.5, 54.9, 66.0, 77.4, 78.5, 123.8, 126.7, 127.0, 128.3, 128.8, 128.9,
(
3
4
1
1
5
N-(1-(Cyclohexylamino)-2-methyl-1-oxopropan-2-yl)-4-
29.4, 129.6, 130.1, 131.5, 132.4, 134.5, 135.4, 137.9, 138.5, 147.2, 165.6,
67.7, 171.8.; HRMS calculated for C31
14.2700.
(dimethylamino)-N-(4-(hydroxycarbamoyl)benzyl)benzamide
2c:
+
H
35
N
3
O
4
: 514.2699 [M+H] , found
Synthesized from 6, cyclohexyl isocyanide (3a), acetone (4b), 4-
(
dimethylamino)benzoic acid (5c) according to the general procedure A
1
(Method 1) in 88 % yield as a white solid: mp = 204-206 °C; H-NMR
Compound 9b (139 mg, 0.27 mmol, 1 eq.) was dissolved in methanol (20
mL) and a catalytical amount of Pd/C (10wt. %) was added. Subsequently,
the mixture was stirred for 2 h at room temperature under a hydrogen
atmosphere. Next, the solution was filtered over pad of Celite and the
solvent was removed under reduced pressure. After purification by flash
column chromatography (dichloromethane/methanol, gradient), the
desired product 2b was obtained in 54 % yield as pink solid: mp = 120 °C;
6
(DMSO-d , 400 MHz): 훿 [ppm] = 1.03-1.24 (m, 5 H), 1.27 (s, 6 H), 1.53-
1.66 (m, 5 H), 2.89 (s, 6 H), 3.41-3.54 (m, 1 H), 4.75 (s, 2 H), 6.62-6.64 (m,
2 H), 7.07-7.09 (m, 1 H), 7.38-7.41 (m, 4 H), 7.70-7.72 (m, 2 H), 9.01 (s, 1
H), 11.18 (s, 1 H); 13C-NMR (DMSO-d
6
, 101 MHz): 훿 [ppm] = 24.2, 24.9,
25.3, 32.4, 47.8, 50.1, 62.0, 111.0, 124.2, 126.7, 127.0, 128.2, 131.5,
143.3, 150.9, 163.9, 172.1, 173.0.; HRMS calculated for C27
503.2629 [M+Na] , found 503.2630; HPLC purity: 97.5 %.
36 4 4
H N O :
+
1H-NMR (DMSO-d
, 300 MHz): 훿 [ppm] = 1.00-1.34 (m, 5 H), 1.45-1.77 (m,
H), 2.13 (s, 6 H), 3.45-3.60 (m, 1 H), 4.37 (s, 2 H), 6.89-6.93 (m, 3 H),
.19-7.21 (m, 2 H), 7.59-7.62 (m, 2 H), 7.84-7.94 (m, 1 H), 9.02 (s, 1 H),
1.19 (s, 1 H); 13C-NMR (DMSO-d
, 75 MHz): 훿 [ppm] = 20.7, 24.5, 25.2,
2.4, 47.8, 52.5, 59.1, 126.0, 126.8, 127.4, 130.1, 131.2, 135.8, 136.9,
6
5
7
1
3
1
rac-N-(2-(Cyclohexylamino)-2-oxo-1-phenylethyl)-4-(dimethylamino)-
N-(4-(hydroxycarbamoyl)benzyl)benzamide 2d: Synthesized from 6,
cyclohexyl isocyanide (3a), benzaldehyde (4c), 4-(dimethylamino)benzoic
6
acid (5c) according to the general procedure A (Method 2) in 56 % yield
29 3 4
46.4, 163.3, 166.7, 169.8; HRMS calculated for C24H N O : 424.2231
1
as a white solid: mp = 154 °C; H-NMR (DMSO-d
6
, 500 MHz): 훿 [ppm] =
+
[M+H] , found 424.2224; HPLC purity: 96.2 %.
0
4
.97-1.37 (m, 5 H), 1.45-1.87 (m, 5 H), 2.94 (s, 6 H), 3.51-3.75 (m, 1 H),
.23-4.52 (m, 1 H), 4.62-4.86 (m, 1 H), 5.77 (s, 1 H), 6.61-6.83 (m, 2 H),
General procedure A for the preparation of target compounds 2c-j via
the Ugi-4CR:
6.93-7.42 (m, 9 H), 7.44-7.60 (m, 2 H), 7.91-8.07 (m, 1 H), 8.91 (s, 1 H),
11.05 (s, 1 H); 13C-NMR (DMSO-d
, 126 MHz): 훿 [ppm] = 24.3, 24.4, 25.1,
2.0, 39.6, 40.0, 47.6, 49.0, 64.0, 111.1, 122.7, 126.0, 126.4, 127.7, 128.2,
6
3
1
28.4, 128.6, 130.3, 136.5, 142.6, 151.2, 164.0, 168.1, 172.4; HRMS
Method 1: A mixture of methyl 4-(aminomethyl)benzoate hydrochloride (6)
+
calculated for C31
H
36
N
4
O
4
: 529.2809 [M+H] , found 529.2811; HPLC
(1.2 eq.), the respective carbonyl (4a-b) (1.2 eq.), triethylamine (1.2 eq.)
purity: 96.5 %.
and crushed molecular sieves (MS, 4 Å, 50 mg for 0.5 mmol) in dry
methanol (0.5 M) was added into a 10 mL glass pressure microwave tube
equipped with a magnetic stirrer bar. The tube was closed with a silicon
septum and the reaction mixture was subjected to microwave irradiation
N-(2-(tert-Butylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-
2-methylbenzamide 2e: Synthesized from 6, tert-butyl isocyanide (3b),
paraformaldehyde (4a), o-toluic acid (5d) according to the general
procedure A (Method 1) in 11 % yield as a white solid: mp = 162-164 °C;
(Discover mode; power: 150 W; hold time: 30 min; temperature: 45 °C;
PowerMax-cooling mode) under medium speed magnetic stirring. Next,
the appropriate carboxylic acid (5c, d-g) (1.0 eq.) and isocyanide (3a-c)
1H-NMR (DMSO-d
, 300 MHz): 훿 [ppm] = 1.15/1.26* (2 x s, 9 H),
6
(
1.0 eq.) components were added and the reaction mixture was again
2.23/2.33* (2 x s, 3 H), 3.43*/3.54 (2 x s, 2 H), 4.34/4.68 (s and brs, 2 H),
7.16-7.51 (m, 7 H), 7.65-7.83 (m, 2 H), 9.05 (brs, 1 H), 11.04 (brs, 1 H);
irradiated at 45 °C for 120 min (150W). Subsequently, after filtration of the
molecular sieves, methanol (3 mL for 0.5 mmol) was added and the
mixture was cooled to 0 °C. Then, aqueous hydroxylamine (50 %wt, ρ =
13C-NMR (DMSO-d
6
, 75 MHz): 훿 [ppm] = 18.6, 28.3, 28.6, 46.5, 48.3, 50.3,
50.7, 52.1, 125.5, 125.68, 125.73, 127.0, 127.15, 127.21, 127.8, 128.75,
128.78, 130.2, 130.3, 131.8, 132.0, 134.0, 134.3, 136.1, 136.1, 139.7,
1
.078 g/mL, 30 eq.) and sodium hydroxide (10 eq.) was added and the
reaction mixture was stirred for 20 min at 0 °C. The solution was allowed
to warm to room temperature and was stirred overnight. The solvent was
evaporated under reduced pressure and the residue was dissolved in
water (15 mL). The pH was adjusted to 7-8 using 1 M HCl and the formed
solid was filtered, washed with water and diethyl ether to give the pure
hydroxamic acid or was purified by flash column chromatography
140.7, 163.9, 166.6, 166.7, 171.1, 171.2; HRMS calculated for
+
22 27 3 4
C H N O : 398.2074 [M+H] , found 398.2075; HPLC purity: 97.5 %.
N-(2-(tert-Butylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-
-methoxybenzamide 2f: Synthesized from 6, tert-butyl isocyanide (3b),
2
paraformaldehyde (4a), 2-methoxybenzoic acid (5e) according to the
(dichloromethane/methanol, gradient) to yield the desired products 2c,e-h.
general procedure A (Method 1) in 20 % yield as a white solid: mp = 139-
1
1
41 °C; H-NMR (DMSO-d
6
, 400 MHz): 훿 [ppm] = 1.13/1.24* (2 x s, 9 H),
Method 2: A mixture of methyl 4-(aminomethyl)benzoate hydrochloride (6)
1.2 eq.), the respective carbonyl (4a, c) (1.2 eq), triethylamine (1.2 eq.),
3.50-5.31 (m, 4 H), 3.74*/3.84 (2 x s, 3 H), 6.95-7.41 (m, 7 H), 7.69-7.78
(m, 2 H), 9.03 (brs, 1 H), 11.19 (brs, 1 H); 13C-NMR (DMSO-d
, 101 MHz):
(
6
and 50 mg (50 mg for 0.5 mmol) of crushed molecular sieves (MS) 4 Å in
dry methanol (0.5 M) was added into a 10mL glass pressure microwave
tube equipped with a magnetic stirrer bar. The tube was closed with a
훿 [ppm] = 28.4, 28.5, 47.2, 48.2, 50.1, 50.6, 52.4, 55.4, 55.6, 111.3, 111.6,
120.5, 120.7, 125.3, 125.6, 127.0, 127.1, 127.3, 127.6, 127.7, 130.3, 130.5,
131.5, 139.8, 140.6, 154.5, 154.8, 163.7, 163.8, 166.5, 166.7, 169.0,
9
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201800808
FULL PAPER
1
4
69.2; HRMS calculated for
14.2025; HPLC purity: >99 %.
22 27 3 5
C H N O :
414.2023 [M+H]+, found
훿 [ppm] = 1.47 (s, 9 H), 1.82 (brs, 1 H), 3.30 (s, 2 H), 3.85 (s, 2 H), 3.91 (s,
3 H), 7.40-7.42 (m, 2 H), 7.98-8.01 (m, 2 H); 13C-NMR (CDCl
3
, 151 MHz):
훿 [ppm] = 28.3, 51.1, 52.2, 53.1, 81.5, 128.2, 129.2, 129.9, 145.2, 167.2,
1
71.7; Elemental analysis calculated for C15
4
H21NO : C 64.50, H 7.58, N
N-(2-(Benzylamino)-2-oxoethyl)-4-(tert-butyl)-N-(4-
hydroxycarbamoyl)benzyl)benzamide 2g: Synthesized from 6, benzyl
5.01, found: C 64.66, H 7.51, N 5.07.
(
isocyanide (3c), paraformaldehyde (4a), 4-tert-butylbenzoic acid (5f)
according to the general procedure A (Method 1) in 38 % yield as a white
3
To a solution of 10 (2.700 g, 0.0097 mol, 0.01 eq.) and Et N (1.68 mL,
1
6
solid: mp = 157-158 °C; H-NMR (DMSO-d , 600 MHz): 훿 [ppm] = 1.26-
0.012 mol, 1.25 eq.) in dichloromethane (50 mL) was added 3,5-
dimethylbenzoyl chloride (1.57 mL, 0.011 mol, 1.1 eq.). The mixture was
stirred over night at room temperature. The reaction was quenched with
water (175 mL) and diluted with dichloromethane (200 mL). The organic
layer was washed with a HCl-solution (1 M, 100 mL) and dried over
1.29 (m, 9 H), 3.81/3.99* (2 x s, 2 H), 4.27/4.33* (2 x d, J = 5.4, 5.0 Hz, 2
H), 4.61*/4.68 (2 x s, 2 H), 7.20-7.21 (m, 1 H), 7.24-7.27 (m, 2 H), 7.32-
7
9
=
1
1
.33 (m, 3 H), 7.37-7.44 (m, 5 H), 7.74-7.76 (m, 2 H), 8.44-8.47 (m, 1 H)
.41 (brs, 1 H), 10.38 (brs, 1 H); 13C-NMR (DMSO-d
6
, 126 MHz): 훿 [ppm]
30.9, 34.4, 42.1, 47.5, 48.7, 51.1, 53.1, 125.0, 126.4, 126.7, 127.0, 127.2,
27.5, 128.1, 131.7, 131.9, 132.8, 133.0, 139.0, 139.2, 139.9, 140.3, 152.1,
Na
2 4
SO . After filtration and removal of the solvent under reduced pressure,
column chromatographic purification (n-hexane/EtOAc
afforded the desired product 11 in 99 % yield as a white wax (3.919 g): H-
=
gradient)
: 474.2387 [M+H]+,
1
63.8, 167.7, 171.3; HRMS calculated for C28
H N O
31 3 4
found 474.2390; HPLC purity: 98.1 %.
NMR (CDCl
s, 6 H), 3.92-3.93 (m, 3 H), 3.75*/4.04 (2 x s, 2 H), 4.64/4.85* (s, 2 H),
.03-7.08 (m, 3 H), 7.26-7.27 (m, 1 H), 7.40-7.41 (m, 1 H) 8.02-8.03 (m, 2
H); 13C-NMR (CDCl
, 151 MHz): 훿 [ppm] = 21.4, 28.1, 28.3, 47.2, 49.2,
1.0, 52.29, 52.35, 53.7, 82.2, 82.5, 124.3, 124.5, 127.1, 128.5, 129.7 ,
29.9, 130.2, 130.3, 131.6, 131.7, 135.4, 135.7, 142.0, 142.1, 166.8, 167.0,
3
, 600 MHz): 훿 [ppm] = 1.43*/1.49 (2 x s, 9 H), 2.28*/2.31 (2 x
7
N-(2-(Benzylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-4-
isopropylbenzamide 2h: Synthesized from 6, benzyl isocyanide (3c),
paraformaldehyde (4a), 4-i-propylbenzoic acid (5a) according to the
3
5
1
general procedure A (Method 1) in 45 % yield as a white solid: mp = 189-
1
168.1, 168.5, 172.9, 173.1; Elemental analysis calculated for C24
5
H29NO :
1
2
2
90 °C; H-NMR (DMSO-d
6
, 400 MHz): 훿 [ppm] = 1.18-1.22 (m, 6 H), 2.85-
C 70.05, H 7.10, N 3.40, found: C 70.04, H 7.36, N 3.29.
.95 (m, 1 H), 3.81/3.99* (2 x s, 2 H), 4.27-4.33 (m, 2 H), 4.60*/4.68 (2 x s,
H), 7.18-7.40 (12 H), 7.74-7.76 (m, 1 H), 8.43-8.44 (m, 1 H), 9.03 (s, 1
H), 11.21 (s, 1 H); 13C-NMR (DMSO-d
, 101 MHz): 훿 [ppm] = 23.7, 33.3,
2.1, 42.2, 47.6, 48.8, 51.1, 53.2, 126.3, 126.8, 126.9, 127.1, 127.3, 127.7,
28.3, 131.7, 131.9, 133.2, 133.5, 139.0, 139.3, 140.2, 140.5, 150.0, 150.2,
Compound 11 (731 mg, 1.78 mmol, 1 eq.) was stirred in trifluoroacetic
acid/dichloromethane (1:1, 10 mL) at room temperature until complete
consumption of the starting material. Afterwards, the solvent was
evaporated under reduced pressure. Further trifluoroacetic acid was
subsequently removed by codestillation with toluene, which afforded the
corresponding N-(3,5-dimethylbenzoyl)-N-(4-(methoxycarbonyl)benzyl)-
glycine in >99 % yield as a yellow solid (664 mg): mp = 141-146 °C; 1H-
6
4
1
1
4
63.9, 164.0, 167.7, 167.9, 171.5; HRMS calculated for C27
60.2231 [M+H] , found 460.2234; HPLC purity: >99 %.
H
29
N
3
O
4
:
+
N-(2-(Benzylamino)-2-oxoethyl)-4-(dimethylamino)-N-(4-
hydroxycarbamoyl)benzyl)benzamide 2i: Synthesized from 6, benzyl
isocyanide (3c), paraformaldehyde (4a), 4-(dimethylamino)benzoic acid
NMR (300 MHz, DMSO-d
3
7
DMSO-d
6
5
1
6
): 훿 [ppm] = 2.21-2.32 (m, 6 H), 3.85 (s, 3 H),
.89*/4.02 (2 x s, 2 H), 4.58*/4.73 (2 x s, 2 H), 6.94-7.25 (m, 3 H), 7.36-
.64 (m, 2 H), 7.91-8.04 (m, 2 H), 12.82 (brs, 1 H); 13C NMR (151 MHz,
): 훿 [ppm] = 20.72, 20.75, 20.8, 46.7, 47.1, 48.9, 49.5, 50.7,
2.11, 52.12, 52.3, 53.1, 123.9, 124.0, 126.4, 127.0, 127.2, 127.9, 128.5,
28.7, 129.4, 129.46, 129.49, 130.4, 130.7, 131.0, 131.1, 134.2, 135.5,
(
(
5c) according to the general procedure A (Method 2) in 83 % yield as a
1
white solid: mp = 193 °C; H-NMR (DMSO-d
6
, 300 MHz): 훿 [ppm] = 2.93
(s, 6 H), 3.90 (s, 2 H), 4.30 (d, J = 5.8 Hz, 2 H), 4.67 (s, 2 H), 6.55-6.75 (m,
2
H), 7.14-7.46 (m, 9 H), 7.66-7.83 (m, 2 H), 8.32-8.60 (m, 1 H), 9.03 (s, 1
H), 11.20 (s, 1 H); 3C-NMR (DMSO-d
1
135.8, 136.7, 137.7, 137.8, 142.8, 143.0, 165.8, 166.0, 166.1, 167.5, 168.0,
70.1, 170.7, 171.6.; HRMS calculated for C20
: 356.1492 [M+H]+,
found 356.1493.
6
, 75 MHz): 훿 [ppm] = 39.7, 42.1,
11.0, 121.9, 126.86, 126.92, 127.0, 127.2, 127.3, 128.3, 128.6, 131.7,
39.2, 140.8, 151.2, 164.0, 168.15, 171.7; HRMS calculated for
1
H21NO
5
1
1
+
C
H N O
26 28 4 4
: 461.2183 [M+H] , found 461.2182; HPLC purity: 98.2 %.
To a solution of N-(3,5-dimethylbenzoyl)-N-(4-(methoxycarbonyl)benzyl)-
glycine (1 eq.), EDC hydrochloride (1.2 eq.) and DMAP (0.3 eq.) in
dichloromethane (5 mL for 0.5 mmol) was added the appropriate amine (1
eq.). The mixture was stirred over night at room temperature. The solution
was diluted with dichloromethane (25 mL) and washed with distilled water
(15 mL) and 1 N HCl-solution (2 x 15 mL). The organic layer was dried
N-(2-(Benzylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-3,5-
dimethylbenzamide 2j: Synthesized from 6, benzyl isocyanide (3c),
paraformaldehyde (4a), 3,5-dimethylbenzoic acid (5b) according to the
general procedure A (Method 2) in 77 % yield as a white solid: mp =
1
1
94 °C; H-NMR (DMSO-d
6
, 300 MHz): 훿 [ppm] = 2.24 (s, 6 H), 3.79/3.99*
4
over MgSO and the solvent was evaporated under reduced pressure. The
(2 x s, 2 H), 4.21-4.41 (m, 2 H), 4.55*/4.68 (2 x s, 2 H), 6.96-7.13 (m, 3 H),
residue was purified by column chromatography (n-hexane/ethyl acetate,
gradient) or by recrystallization (ethyl acetate/n-hexane) to give the
desired products 12k-r.
7
1
4
1
.12-7.48 (m, 7 H), 7.69-7.87 (m, 2 H), 8.33-8.52 (m ,1 H), 9.03 (s, 1 H),
1.21 (s, 1 H); 13C-NMR (DMSO-d
6
, 126 MHz): 훿 [ppm] = 20.7, 42.1, 47.4,
8.6, 51.0, 53.1, 124.0, 126.8, 127.1, 127.6, 128.2, 130.7, 131.7, 135.9,
37.5, 139.0, 140.4, 163.95, 167.7, 171.5; HRMS calculated for
+
C
26
H
27
N
3
O
4
: 446.2074 [M+H] , found 446.2077; HPLC purity: >99 %.
Methyl
4-((N-(2-((3,5-dimethylbenzyl)amino)-2-oxoethyl)-4-
methylbenzamido)methyl)-benzoate 12k: The compound was obtained
as described in the general procedure B using 4-methylbenzylamine in
General Procedure
(
(
B
for the synthesis of intermediates 12k-r
submonomer pathway 1): To solution of methyl-4-
aminomethyl)benzoate hydrochloride (6) (5.000 g, 0.025 mol, 1 eq.) in
1
80 % yield as a white solid: mp = 160-168 °C; H-NMR (CDCl
3
, 300 MHz):
a
훿 [ppm] = 2.26 (s, 6 H), 2.33 (s, 3 H), 3.83*/4.07 (2 x s, 2 H), 3.92 (s, 3 H),
.36-4.41 (m, 2 H), 4.69/4.82* (2 x s, 2 H), 6.12-6.19*/6.76 (m and brs, 1
H), 6.98-7.03 (m, 3 H), 7.08-7.15 (m, 4 H) 7.23-7.36 (m, 2 H), 7.99-8.02
m, 2 H); 13C-NMR (CDCl
, 151 MHz): 훿 [ppm] = 21.2, 21.3, 43.4, 49.5,
2.3, 54.0, 124.4, 127.2, 127.8, 128.0, 129.5, 129.8, 130.3, 132.0, 134.9,
4
THF (50 mL) was added triethylamine (10.31 mL, 0.075 mol, 3 eq.),
followed by the addition of tert-butyl-bromoacetate (4.40 mL, 0.030, 1.2
eq.). The suspension was stirred over night at room temperature. Further
tert-butyl-bromoacetate (0.74 mL, 0.005 mol, 0.2 eq.) was added, because
of incomplete consumption of the starting material and the solution was
stirred at room temperature for additional 4 h. After filtration, the solvent
was evaporated, and the residue was dissolved in ethyl acetate (75 mL)
and washed with water (2 x 30 mL). The organic layer was dried over
(
3
5
1
35.2, 137.3, 138.5, 141.5, 141.8, 166.8, 168.5, 173.6; HRMS calculated
+
for C28
30 2 4
H N O : 459.2278 [M+H] , found 459.2284.
Methyl
4-((N-(2-((4-methoxybenzyl)amino)-2-oxoethyl)-3,5-
Na
2
SO
4
and the solvent was removed under reduced pressure. Column
dimethylbenzamido)methyl)-benzoate 12l: The compound was
obtained as described in the general procedure from 4-
methoxybenzylamine in 82 % yield as a yellowish solid: mp = 154-161 °C;
chromatographic purification (n-hexane/EtOAc, gradient) afforded product
B
1
10 in 46 % yield as a yellowish wax (3.191 g). H-NMR (CDCl
3
, 600 MHz):
1
0
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201800808
FULL PAPER
1H-NMR (CDCl
, 600 MHz): 훿 [ppm] = 2.25 (s, 6 H), 3.79 (s, 3 H),
.82*/4.06 (2 x s, 2 H), 3.91 (s, 3 H), 4.33-4.36 (m, 2 H), 4.69/4.82* (2 x s,
H), 6.14*/6.73 (2 x s, 1 H), 6.85-6.86 (m, 2 H), 6.98-7.03 (m, 3 H), 7.13-
.24 (m, 3 H), 7.31-7.41 (m, 1 H), 7.99-8.01 (m, 2 H); 13C-NMR (CDCl
141.6, 166.8, 168.5, 173.5; HRMS calculated for C28
[M+H] , found 459.2282.
H
N
O
: 459.2278
3
30
2
4
+
3
2
7
1
1
1
4
3
,
Methy
4-((3,5-dimethyl-N-(2-oxo-2-((pyridin-3-
12r: The
51 MHz): 훿 [ppm] = 21.3, 43.1, 49.5, 52.3, 54.1, 55.4, 114.2, 124.4, 127.2,
28.4, 129.1, 129.4, 129.9, 130.2, 134.9, 138.6, 141.5, 159.1, 166.8, 166.8,
68.4, 173.5; HRMS calculated for C28
75.2227.
ylmethyl)amino)ethyl)benzamido)methyl)-benzoate
compound was obtained as described in the general procedure B using 3-
picolylamine in 77 % yield as a white solid: mp = 175-178 °C; 1H-NMR
+
H
30
N
2
O
5
: 475.2227 [M+H] , found
(
3
CDCl
H), 4.43-4.45 (m, 2 H), 4.70-4.83 (m, 2 H), 6.51-7.04 (m, 4 H), 7.23-7.25
(m, 2 H), 7.31-7.62 (m, 2 H), 7.99-8.02 (m, 2 H,), 8.51-8.53 (m, 2 H); 13C-
NMR (CDCl , 126 MHz): δ [ppm] = 21.3, 41.1, 43.1, 49.6, 52.3, 54.3, 77.2,
3
, 300 MHz): δ [ppm] = 2.26 (s, 6 H), 3.85*/4.08 (2 x s, 2 H), 3.92 (s,
Methyl
4-((N-(2-((4-fluorobenzyl)amino)-2-oxoethyl)-3,5-
dimethylbenzamido)methyl)benzoate 12m: The compound was
3
obtained as described in the general procedure
fluorobenzylamine in 62 % yield as a white solid: mp = 182-184 °C; H-
B
using 4-
123.7, 124.5, 124.9, 126.6, 127.2, 128.5, 130.1, 130.3, 132.1, 133.4, 134.0,
1
134.9, 135.6, 138.6, 141.5, 149.0, 149.3, 166.7, 168.9, 173.7; HRMS
+
NMR (CDCl
.95 (s, 3 H), 4.39-4.42 (m, 2 H), 4.72-4.85 (m, 2 H), 6.20-6.25*/6.87 (m
and s, 1 H), 7.00-7.39 (m, 9 H), 8.03-8.04 (m, 2 H); 13C-NMR (CDCl
, 126
MHz): 훿 [ppm] = 21.3, 43.0, 49.8, 52.3, 54.2, 76.9, 77.2, 77.4, 115.6, 115.8,
3
, 600 MHz): 훿 [ppm] = 2.29 (s, 6 H), 3.87*/4.10 (2 x s, 2 H),
calculated for C26
H
27
N
3
O
4
: 446.2074 [M+H] , found 446.2075.
3
3
General Procedure
C
for the synthesis of intermediates 15s-t
(
submonomer pathyway 2): A mixture of methylamine hydrochloride
124.5, 127.3, 129.5, 130.0, 130.3, 132.0, 134.1, 135.0, 138.6, 141.5, 161.4,
(
1.674 g, 0.025 mol, 1 eq.) and K CO (6.855 g, 0.050 mol, 2 eq.) in
2
3
1
63.4, 166.7, 168.6, 173.6; HRMS calculated for C27
H
27FN
2
O
4
: 463.2028
dichloromethane (75 mL) was cooled to 0 °C under nitrogen atmosphere.
Bromoacetyl bromide (2.15 mL, 0.025 mol, 1 eq.) was then added
dropwise under vigorous stirring. The solution was stirred for 1 h at 0 °C
followed by 2 h at room temperature. Subsequently, water (25 mL) was
added to the solution followed by phase separation and drying of the
+
[M+H] , found 463.2032.
Methyl
4-((N-(2-((4-chlorobenzyl)amino)-2-oxoethyl)-3,5-
dimethylbenzamido)methyl)-benzoate 12n: The compound was
obtained as described in the general procedure
chlorobenzylamine in 66 % yield as a white solid: mp = 177-180 °C; H-
B
using 4-
2 4
organic phase over Na SO overnight. After filtration, the solvent was
removed under reduced pressure and 2-bromo-N-methylacetamide
1
NMR (CDCl
s, 3 H), 4.39-4.41 (m, 2 H), 4.70-4.85 (m, 2 H), 6.05*/6.87 (brs and s, 1
H), 6.98-7.05 (m, 3 H), 7.12-7.24 (m, 3 H), 7.28-7.42 (m, 3 H), 8.00-8.03
m, 2 H); 13C-NMR (CDCl
, 126 MHz): 훿 [ppm] = 21.3, 35.8, 42.9, 49.8,
2.3, 54.3, 77.1, 77.2, 77.36, 77.41, 124.4, 127.3, 129.0, 129.2, 130.0,
3
, 300 MHz): 훿 [ppm] = 2.27 (s, 6 H), 3.86-4.08 (m, 2 H), 3.93
(CAUTION: This substance is an irritant and must be handled with care)
1
(
was obtained as a white solid (1.730 g) in 46 % yield: H-NMR (CDCl
3
, 300
MHz): 훿 [ppm] = 2.85 (d, 3 H), 3.87 (s, 2 H), 6.60 (brs, 1 H); 13C-NMR
CDCl , 75 MHz): 훿 [ppm] = 27.1, 29.3, 166.1.
(
5
1
3
3
30.3, 132.0, 133.5, 134.9, 136.9, 138.6, 141.5, 166.7, 168.6, 173.6;
To a solution of methyl 4-(aminomethyl)benzoate hydrochloride (6) (2.335
g, 0.012 mol, 1.1 eq.) and triethylamine (4.52 mL, 0.033 mol, 3.1 eq.) in
THF (20 mL) was added 2-bromo-N-methylacetamide (1.600 g, 0.011 mol,
1 eq.) in three portions. The mixture was then stirred at room temperature
overnight. The mixture was filtered, and the solvent was removed under
reduced pressure. The residue was dissolved in ethyl acetate (50 mL),
+
HRMS calculated for C27
H
27ClN
2
O
4
: 479.1732 [M+H] , found 479.1735.
Methyl
4-((N-(2-((4-(tert-butyl)benzyl)amino)-2-oxoethyl)-3,5-
dimethylbenzamido)methyl)benzoate 12o: The compound was
obtained as described in the general procedure
butylbenzylamine in 57 % yield as a white solid: mp = 99-100 °C; H-NMR
B
using 4-tert-
1
2 4
washed with water (2 x 20 mL), dried over Na SO and the solvent was
(
2
(
1
CDCl
H), 3.95 (s, 3 H), 4.40-4.45 (m, 2 H), 4.74/4.88* (2 x s, 2 H), 6.00*/6.71
2 x s, 1 H), 7.04-7.06 (m, 3 H), 7.19-7.39 (m, 6 H), 8.03-8.05 (m, 2 H);
3
, 600 MHz): 훿 [ppm] = 1.34 (s, 9 H), 2.29 (s, 6 H), 3.86*/4.11 (2 x s,
removed under reduced pressure. The product (14) was obtained after
flash column chromatography (n-hexane/ethyl acetate, gradient) as a
1
white solid in 50 % yield (1.235 g): mp = 76-78 °C; H-NMR (DMSO-d
6
,
3C-NMR (CDCl
, 126 MHz): 훿 [ppm] = 21.34, 31.49, 34.69, 43.40, 49.70,
2.27, 54.14, 76.90, 77.16, 77.41, 124.51, 125.84, 127.26, 127.58, 128.46,
30.00, 130.31, 131.95, 135.08, 138.59, 141.66, 150.73, 166.77, 168.47,
600 MHz): 훿 [ppm] = 2.60 (d, J = 4.7 Hz, 3 H), 2.71 (brs, 1 H), 3.05 (s, 2
3
5
1
1
5
H), 3.74 (s, 2 H), 3.84 (s, 3 H), 7.48-7.50 (m, 2 H, ), 7.76-7.77 (m, 1 H),
7.90-7.91 (m, 2 H); 13C-NMR (DMSO-d
6
, 125 MHz): 훿 [ppm] = 25.2, 51.4,
73.52; HRMS calculated for
01.2752.
C
31 36 2 4
H N O :
501.2748 [M+H]+, found
51.9, 52.3, 128.0, 128.0, 128.8, 129.0, 146.2, 166.1, 171.2; HRMS
+
calculated for C12
H
16
N
2
O
3
: 237.1234 [M+H] , found 237.1237.
Methyl
4-((N-(2-((3,5-dimethylbenzyl)amino)-2-oxoethyl)-3,5-
The amine (14) (1 eq.) was dissolved in dichloromethane (5 mL for 1.1
mmol amine) followed by the addition of triethylamine (1.25 eq.).
Subsequently, the respective acyl chloride (1.1 eq.) was added and the
solution was stirred at room temperature overnight. The reaction mixture
was quenched by the addition of water (15 mL) followed by dilution with
dichloromethane (25 mL). The organic phase was separated, washed with
dimethylbenzamido)-methyl)benzoate 12p: The compound was
obtained as described in the general procedure
dimethylbenzylamine in 56 % yield as a white solid: mp = 177-181 °C; H-
B
using 3,5-
1
NMR (CDCl
x s, 2 H), 3.92 (s, 3 H), 4.32-4.36 (m, 2 H), 4.71/4.84* (2 x s, 2 H),
.01*/6.76 (2 x s, 1 H), 6.83-7.03 (m, 6 H), 7.23-7.37 (m, 2 H), 8.00-8.01
m, 2 H); 13C-NMR (CDCl
, 151 MHz): 훿 [ppm] = 21.3, 21.4, 43.5, 49.5,
3
, 600 MHz): 훿 [ppm] = 2.26*/2.29 (2 x s, 12 H), 3.83*/4.09 (2
6
(
2 4
1 M HCl (15 mL) and dried over Na SO . After filtration the solvent was
3
evaporated under reduced pressure and the residue was purified either by
recrystallization from dichloromethane and n-hexane or by flash column
chromatography (n-hexane/ethylacetate, gradient) to give the respective
ester (15s-t).
52.3, 54.0, 124.4, 125.5, 127.1, 128.5, 129.2, 129.9, 132.0, 134.9, 138.0,
138.4, 138.5, 141.5, 166.8, 168.5, 173.6; HRMS calculated for
+
29 32
C H N
O
2 4
: 473.2435 [M+H] , found 473.2437.
Methyl
4-((3,5-dimethyl-N-(2-((3-methylbenzyl)amino)-2-
Methyl
oxoethyl)benzamido)methyl)benzoate 15s: The compound was
obtained as described in the general procedure using 3,5-
dimethylbenzoyl chloride in 88 % yield as a white solid: mp = 218-219 °C;
1H-NMR (CDCl
, 600 MHz): 훿 [ppm] = 2.27-2.30 (m, 6 H), 2.80 (s, 3 H),
4-((3,5-dimethyl-N-(2-(methylamino)-2-
oxoethyl)benzamido)methyl)benzoate 12q: The compound was
obtained as described in the general procedure
methylbenzylamine in 63 % yield as a white solid: mp = 156-158 °C; H-
NMR (CDCl
B
using 3-
C
1
3
, 600 MHz): 훿 [ppm] = 2.29 (s, 6 H), 2.36 (s, 3 H), 3.86*/4.11
3
(
6
2 x s, 2 H), 3.94 (s, 3 H), 4.40-4.44 (m, 2 H), 4.73/4.88* (2 x s, 2 H), 5.92-
3.80*/4.04 (2 x s, 2 H), 3.92 (s, 3 H), 4.70/4.84* (2 x s, 2 H), 5.79*/6.43 (2
x s, 1 H), 7.03-7.05 (m, 3 H), 7.24-7.40 (m, 2 H), 8.02-8.03 (m, 2 H); 13C-
.00*/6.79 (m and s, 1 H), 6.99-7.41 (m, 9 H), 8.03-8.04 (m, 2 H); 13C-NMR
, 126 MHz): 훿 [ppm] = 21.3, 21.5, 43.7, 49.7, 52.3, 54.1, 124.5,
24.9, 127.2, 128.4, 128.6, 128.8, 130.0, 130.3, 132.0, 135.0, 138.2, 138.6,
(CDCl
3
NMR (CDCl
3
, 151 MHz): 훿 [ppm] = 21.4, 26.4, 49.6, 52.3, 54.2, 124.4,
1
124.6, 127.2, 128.5, 129.9, 130.3, 132.1, 134.8, 138.6, 141.6, 166.8, 169.4,
1
1
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201800808
FULL PAPER
1
3
73.6; HRMS calculated for
69.1815.
21 24 2 4
C H N O :
369.1809 [M+H]+, found
163.8, 167.7, 171.48, 171.48; HRMS calculated for
464.1980 [M+H] , found 464.1981; HPLC purity: 95.1 %.
C
26 3 4
H26FN O :
+
Methyl
4-((2-methyl-N-(2-(methylamino)-2-
N-(2-((4-Chlorobenzyl)amino)-2-oxoethyl)-N-(4-
oxoethyl)benzamido)methyl)benzoate 15t: The compound was
(hydroxycarbamoyl)benzyl)-3,5-dimethylbenzamide
2n:
The
obtained as described in the general procedure C using 2-methylbenzoyl
hydroxamic acid was obtained as described in the general procedure D
using ester 12n in 70 % yield as a white solid: mp = 149-150 °C; H-NMR
1
1
chloride in 46 % yield as a white solid: mp = 92-94 °C; H-NMR (CDCl
3
,
3
00 MHz): 훿 [ppm] = 2.30*/2.36 (2 x s, 3 H), 2.73*/2.84 (2 x d, J = 4.8, 4.9
Hz, 3 H), 3.62-4.09 (m, 2 H), 3.91/3.92* (2 x s, 3 H), 4.55/4.90* (s and brs,
H), 5.44*/6.26 (2 x s, 1 H), 7.15-7.46 (m, 6 H), 7.97-8.05 (m, 2 H); 13C-
NMR (CDCl , 75 MHz): 훿 [ppm] = 19.1, 19.2, 26.3, 43.7, 46.0, 48.4, 48.6,
1.1, 52.3, 53.3, 125.7, 125.8, 126.0, 126.1, 126.8, 127.5, 127.7, 128.7,
6
(DMSO-d , 300 MHz): 훿 [ppm] = 2.23 (s, 6 H), 3.79/3.98* (2 x s, 2 H),
4.24/4.29* (2 x d, J = 5.6, 5.7 Hz, 2 H), 4.55*/4.67 (2 x s, 2 H), 7.00-7.07
2
(m, 3 H), 7.18-7.45 (m, 6 H), 7.73-7.94 (m, 2 H), 8.42-8.49 (m, 1 H), 9.04
(s, 1 H), 11.22 (s, 1 H); 13C-NMR (DMSO-d
3
6
, 126 MHz): 훿 [ppm] = 20.6,
5
1
1
41.4, 47.4, 48.6, 50.9, 53.1, 124.0, 125.7, 126.6, 126.9, 127.4, 128.1,
128.9, 129.0, 129.21, 130.7, 131.3, 131.8, 132.4, 135.9, 137.4, 138.1,
29.5, 129.6, 129.7, 129.99, 130.08, 130.2, 130.3, 130.9, 131.3, 134.6,
34.9, 135.1, 141.1, 141.9, 166.7, 168.2, 169.1, 173.1.; HRMS calculated
139.3, 139.7, 163.7, 167.8, 171.5; HRMS calculated for C26
480.1685 [M+H] , found 480.1687; HPLC purity: 98.0 %.
3 4
H26ClN O :
+
+
for C20
H N O
22 2 4
: 355.1652 [M+H] , found 355.1656.
General procedure D for the preparation of hydroxamic acids
synthesized by the submonomer pathways (2k-t): The appropriate
ester (1 eq.) was dissolved in dichloromethane/methanol (1:3, 6 mL for 0.3
mmol) and the solution was cooled to 0 °C. This was followed by the
addition of aq. hydroxylamine solution (50 %wt, 30 eq.) and sodium
hydroxide (10 eq.). The mixture was allowed to warm to room temperature
and was stirred overnight. Next, the solvent was removed under reduced
pressure. The residue was dissolved in distilled water (20 mL) and the
solution was acidified with 1 M HCl-solution to pH 7-8. The precipitate was
washed with diethyl ether several times, recrystallized from
dichloromethane and diethyl ether or purified by column chromatography
N-(2-((4-(tert-Butyl)benzyl)amino)-2-oxoethyl)-N-(4-
(hydroxycarbamoyl)benzyl)-3,5-dimethylbenzamide
hydroxamic acid was obtained as described in the general procedure D
using ester 12o in 80 % yield as a reddish solid: mp = 180-183 °C; H-NMR
2o:
The
1
6
(DMSO-d , 600 MHz): 훿 [ppm] = 1.26 (s, 9 H), 2.23 (s, 6 H), 3.77/3.97* (2
x s, 2 H), 4.22/4.28* (2 x d, J = 5.5, 5.2 Hz, 2 H), 4.55*/4.68 (2 x s, 2 H),
7.01-7.07 (m, 3 H), 7.10-7.19 (m, 2 H), 7.27-7.40 (m, 4 H), 7.74-7.89 (m, 2
H), 8.39-8.36 (m, 1 H), 9.05 (brs, 1 H), 11.17 (brs, 1 H); 13C-NMR (DMSO-
6
d , 126 MHz): 훿 [ppm] = 20.6, 31.0, 34.0, 41.8, 47.4, 48.6, 50.9, 53.1,
124.0, 124.9, 126.8, 127.0, 127.5, 130.7, 131.7, 131.8, 135.9, 136.1, 137.4,
140.3, 149.2, 163.8, 167.6, 171.5; HRMS calculated for C30
H
35
N
3 4
O :
+
(dichloromethane/methanol, gradient) to give the desired product. In some
502.2700 [M+H] , found 502.2701; HPLC purity: 95.3 %.
cases, the respective carboxylic acid has formed as a side product, which
was removed by using the anion exchange sorbent Isolute PE-AX® (with
methanol as the eluent).
N-(2-((3,5-Dimethylbenzyl)amino)-2-oxoethyl)-N-(4-
(hydroxycarbamoyl)benzyl)-3,5-dimethylbenzamide
2p:
The
hydroxamic acid was obtained as described in the general procedure D
1
N-(4-(Hydroxycarbamoyl)benzyl)-3,5-dimethyl-N-(2-((4-
using ester 12p in 58 % yield as a white solid: mp = 116-118 °C; H-NMR
methylbenzyl)amino)-2-oxoethyl)benzamide 2k: The hydroxamic acid
6
(DMSO-d , 300 MHz): 훿 [ppm] = 2.23 (s, 12 H), 3.77/3.97* (2 x s, 2 H),
was obtained as described in the general procedure D using ester 12k in
4.19/4.25* (2 x d, J = 5.5, 5.3 Hz, 2 H), 4.55*/4.67 (2 x s, 2 H), 6.80-6.87
1
8
2 % yield as a white solid: mp = 147-149 °C; H-NMR (DMSO-d
6
, 300
(m, 3 H), 7.02-7.06 (m, 3 H), 7.25-7.39 (m, 2 H), 7.73-7.76 (m, 2 H), 8.29-
MHz): 훿 [ppm] = 2.24 (s, 6 H), 2.27 (s, 3 H), 3.77/3.97* (2 x s, 2 H),
.21/4.26* (2 x d, J = 5.6, 5.7 Hz, 2 H), 4.54*/4.67 (2 x s, 2 H); 7.02-7.40
m, 9 H), 7.73-7.87 (m, 2 H), 8.35-8.41 (m, 1 H), 9.16 (brs, 1 H), 11.24 (brs,
8.41 (m, 1 H), 10.14 (brs, 1 H); 13C-NMR (DMSO-d
6
, 126 MHz): 훿 [ppm] =
4
20.6, 20.8, 42.1, 47.2, 48.4, 50.7, 53.1, 113.9, 124.1, 124.9, 125.0, 126.5,
126.7, 127.3, 128.2, 129.0, 129.2, 130.7, 133.5, 136.0, 137.1, 137.4, 138.9,
(
H); 13C-NMR (DMSO-d
8.6, 51.0, 53.1, 124.0, 125.9, 126.8, 127.1, 127.6, 128.7, 129.3, 130.7,
31.7, 135.9, 136.1, 137.5, 140.3, 163.8, 167.6, 171.5; HRMS calculated
, 126 MHz): 훿 [ppm] = 20.5, 20.7, 41.9, 47.4,
163.6, 167.5, 171.4; HRMS calculated for C28
found 474.2391; HPLC purity: 97.1 %.
31 3 4
H N O
: 474.2387 [M+H]+,
1
4
1
6
+
for C27
29 3 4
H N O : 460.2231 [M+H] , found 460.2237; HPLC purity: 95.4 %.
N-(4-(Hydroxycarbamoyl)benzyl)-3,5-dimethyl-N-(2-((3-
methylbenzyl)amino)-2-oxoethyl)benzamide 2q: The hydroxamic acid
N-(4-(Hydroxycarbamoyl)benzyl)-N-(2-((4-methoxybenzyl)amino)-2-
oxoethyl)-3,5-dimethylbenzamide 2l: The hydroxamic acid was obtained
as described in the general procedure D using ester 12l in 38 % yield as a
was obtained as described in the general procedure D using ester 12q in
1
24 % yield as a white solid: mp = 114-118 °C; H-NMR (DMSO-d
6
, 300
MHz): 훿 [ppm] = 2.24 (s, 6 H), 2.28 (s, 3 H), 3.76/3.97* (2 x s, 2 H), 4.23-
4.28 (m, 2 H), 4.51*/4.65 (2 x s, 2 H), 6.93-7.06 (m, 6 H), 7.17-7.33 (m, 3
H), 7.72-7.74 (m, 2 H), 8.36-8.45 (m, 1 H), 10.05 (brs, 1 H); 13C-NMR
(DMSO-d , 12 MHz): 훿 [ppm] = 20.6, 20.9, 42.1, 47.4, 48.5, 50.9, 53.1,
6
124.0, 124.2, 126.8, 127.0, 127.4, 127.5, 127.8, 128.1, 129.2, 130.7, 131.8,
white solid: mp = 123-130 °C 1H-NMR (DMSO-d
, 300 MHz): 훿 [ppm] =
.22 (s, 6 H), 3.70 (s, 3 H), 3.72/3.93* (2 x s, 2 H), 4.16-4.22 (m, 2 H),
.50*/4.63 (2 x s, 2 H), 6.83-6.85 (m, 2 H), 6.99-7.33 (m, 7 H), 7.70-7.72
, 101 MHz): 훿 [ppm] =
6
2
4
(
m, 2 H), 8.30-8.37 (m, 1 H); 13C-NMR (DMSO-d
6
2
1
1
1
4
0.8, 25.0, 41.6, 41.6, 47.2, 48.5, 50.8, 53.2, 55.1, 113.7, 124.1, 126.6,
26.7, 127.5, 128.5, 128.6, 130.8, 131.1, 131.2, 133.5, 133.9, 135.9, 136.1,
135.9, 137.2, 137.4, 138.9, 139.0, 140.0, 140.3, 163.7, 167.6, 171.5;
+
29 3 4
HRMS calculated for C27H N O : 460.2231 [M+H] , found 460.2225;
37.56, 137.59, 138.6, 139.0, 158.2, 158.3, 162.9, 163.0, 167.5, 167.6,
HPLC purity: 97.2 %.
+
71.5, 175.0; HRMS calculated for C27
H
29
N
3
O
5
: 476.2180 [M+H] , found
76.2182; HPLC purity: 95.6 %.
N-(4-(Hydroxycarbamoyl)benzyl)-3,5-dimethyl-N-(2-oxo-2-((pyridin-3-
ylmethyl)amino)ethyl)benzamide 2r: The hydroxamic acid was obtained
N-(2-((4-Fluorobenzyl)amino)-2-oxoethyl)-N-(4-
hydroxycarbamoyl)benzyl)-3,5-dimethylbenzamide
as described in the general procedure D using ester 12r in 69 % yield as
1
(
2m:
The
6
a reddish solid: mp = 138-139 °C; H-NMR (DMSO-d , 600 MHz): 훿 [ppm]
hydroxamic acid was obtained as described in the general procedure D
using ester 12m in 91 % yield as a white solid: mp = 185-187 °C; H-NMR
= 2.23 (s, 6 H), 3.79/3.99* (2 x s, 2 H), 4.29/4.35* (2 x d, J = 5.7, 5.4 Hz, 2
H), 4.56*/4.68 (2 x s, 2 H), 7.00-7.06 (m, 3 H), 7.27-7.40 (m, 3 H), 7.56-
7.67 (m, 1 H), 7.73-7.75 (m, 2 H), 8.43-8.52 (m, 3 H), 9.04 (s, 1 H), 11.21
1
(
4
(
7
DMSO-d
.25/4.30* (2 x d, J = 5.7, 5.5 Hz, 2 H), 4.55*/4.68 (2 x s, 2 H), 7.01-7.08
m, 3 H), 7.12-7.16 (m, 2 H), 7.21-7.35 (m, 3 H), 7.38-7.40 (m, 1 H), 7.74-
.92 (m, 2 H), 8.43-8.47 (m, 1 H), 9.10 (brs, 1 H), 11.23 (brs, 1 H); 13C-
NMR (DMSO-d , 126 MHz): 훿 [ppm] = 20.6, 41.4, 47.5, 48.6, 51.0, 53.1,
14.8, 114.9, 124.0, 126.8, 127.0, 127.6, 129.0, 129.1, 129.2, 129.4, 130.7,
31.7, 131.8, 135.2, 135.4, 135.8, 135.9, 137.5, 140.1, 140.3, 160.2, 162.1,
6
, 600 MHz): 훿 [ppm] = 2.23 (s, 6 H), 3.79/3.98* (2 x s, 2 H),
(s, 1 H); 13C-NMR (DMSO-d
, 151 MHz): 훿 [ppm] = 20.8, 47.7, 48.7, 51.1,
6
53.3, 123.5, 124.0, 124.1, 126.9, 127.1, 127.2, 127.7, 130.9, 131.7, 134.6,
135.1, 136.0, 137.6, 140.5, 148.1, 148.2, 148.7, 148.8, 164.0, 168.0,
6
171.6; HRMS calculated for
447.2030; HPLC purity: 97.1 %.
25 26 4 4
C H N O :
447.2027 [M+H]+, found
1
1
1
2
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201800808
FULL PAPER
N-(4-(Hydroxycarbamoyl)benzyl)-3,5-dimethyl-N-(2-(methylamino)-2-
oxoethyl)benzamide 2s: The hydroxamic acid was obtained as described
and the results were expressed as the fold change in relative density
compared to the 3h-C DMSO vehicle control (set to one). Data are mean
(±) SD for three independent experiments.
in the general procedure D using ester 15s in 40 % yield as a white solid:
1
mp = 187-188 °C; H-NMR (DMSO-d
6
, 600 MHz): 훿 [ppm] = 2.23*/2.27 (2
x s, 6 H), 2.57/2.62* (2 x d, J = 4.3, 4.1 Hz, 3 H), 3.69/3.88* (2 x s, 2 H),
.53*/4.64 (2 x s, 2 H), 7.02-7.07 (m, 3 H), 7.25-7.39 (m, 2 H), 7.72-7.74
m, 2 H), 7.83-7.90 (m, 1 H), 9.04 (s, 1 H), 11.21 (s, 1 H); 13C-NMR (DMSO-
Gametocytocidal activity (ATP bioluminescence assay)
4
(
Activity against mature stage V gametocytes of the P. falciparum strain
d
1
1
6
, 151 MHz): 훿 [ppm] = 20.8, 25.5, 47.4, 48.5, 51.1, 53.1, 124.2, 126.9,
27.1, 127.3, 127.6, 130.8, 131.7, 131.9, 135.9, 136.0, 137.6, 140.3, 140.5,
NF54 was evaluated by an ATP bioluminescence assay as described
previously[26a] with minor modifications[26b]
. Gametocyte culture was
64.0, 168.0, 168.2, 171.6; HRMS calculated for C20
23 3 4
H N O : 370.1761
initiated from synchronized parasites kept in complete culture medium
supplemented with 5% human serum, starting at a hematocrit of 6% and
parasitemia of 0.3%. Culture medium was changed daily without parasite
dilution throughout the entire experiment. When the parasitemia reached
+
[M+H] , found 370.1761, HPLC purity: 96.4 %.
N-(4-(Hydroxycarbamoyl)benzyl)-2-methyl-N-(2-(methylamino)-2-
oxoethyl)benzamide 2t: The hydroxamic acid was obtained as described
5
%, the volume of the medium was doubled. From days 12 to 15,
gametocyte cultures were treated with 50 mM N-acetyl-D-glucosamine
MP Biomedicals GmbH) to remove asexual stages of the parasite, and on
in the general procedure D using 15t in 86 % yield as a white solid: mp =
1
1
47-149 °C; H-NMR (DMSO-d
6
, 400 MHz): 훿 [ppm] = 2.23/2.31* (2 x s, 3
(
H), 2.52/2.63* (2 x d, J = 4.5, 4.5 Hz, 3 H), 3.59/3.91* (s and brs, 2 H),
day 15 or 16, the culture was purified by a NycoPrep 1.077 cushion density
gradient and magnetic column separation to remove uninfected
erythrocytes and enrich the gametocyte population. All tested compounds
were dissolved in DMSO before further dilutions with complete culture
medium was done. After purification, mature gametocytes (50,000 per
well) were incubated for 48 hours with the respective compound before
performance of the ATP based luminescence assay. To evaluate the
gametocytocidal activity of the compounds a pre-screening based on 2
concentrations (5 µM and 500 nM) together with the control compounds
4
7
.38/4.68* (s and brs, 2 H), 7.16-7.31 (m, 5 H), 7.41-7.43 (m, 1 H), 7.69-
.86 (m, 3 H), 9.03 (s, 1 H), 11.07 (s, 1 H); 13C-NMR (DMSO-d
, 101 MHz):
6
훿 [ppm] = 18.58, 18.60, 25.5, 46.3, 47.9, 50.3, 52.2, 125.5, 125.60, 125.63,
1
1
1
3
25.66, 127.0, 127.15, 127.19, 127.8, 128.8, 130.2, 130.3, 131.8, 131.9,
34.1, 134.3, 135.9, 136.0, 139.8, 140.5, 163.9, 167.7, 167.9, 171.09,
19 21 3 4
C H N O :
356.1605 [M+H]+, found
71.12; HRMS calculated for
56.1608, HPLC purity: 97.4 %.
Biological evaluation
epoxomycin, methylene blue and chlorotonil
A
with known
gametocytocidal activity was performed. Of the most promising
compounds a 2-fold serial dilution was done to determine the 50%
inhibitory concentration (IC50) by analysing the nonlinear regression of log
P. falciparum asexual intraerythrocytic culture and growth inhibition
assays
concentration–response curves using the drc-package v0.9.0 of
v2.6.1[26c]
R
.
Asexual drug-sensitive P. falciparum parasites (3D7)[25a] were cultured in
vitro in O+ human erythrocytes and RPMI 1640 media (Gibco, USA)
Activity against P. berghei-Luc EFF and HepG2 viability (P. berghei-
Luc Liver stage and HepG2 cytotoxicity assays)
containing 10% heat inactivated human serum and 5 μg/ml gentamycin
o
(
Sigma Aldrich, USA). Cultures were maintained at 37 C in 5% CO
2
, 5%
3
O
2,
N
2
gas mixture. The [ H]-hypoxanthine incorporation method was used
to define in vitro growth inhibition, essentially as previously described [12a].
Briefly, ring-stage P. falciparum infected erythrocytes (0.25% final
parasitemia, 2.5% haematocrit) were incubated in 96 well plates with serial
dilutions of test compounds (dissolved in DMSO vehicle; ≤0.5% final
The liver stage activity against P. berghei-Luc (Pb-Luc) EEFs and toxicity
to HepG2 cells were evaluated in Pb-Luc and HepG2 cytotoxicity
bioluminescence assays as described previously[27a]
.
HepG2-A16-
CD81EGFP cells, stably transformed to express a GFP-tetraspanin
receptor CD81 fusion protein, were cultured at 37 °C and 5% CO in culture
3
DMSO concentration) or controls. After 48h incubation, [ H]-hypoxanthine
2
3
(
0.5 μCi/well) was added and [ H]-hypoxanthine incorporation assessed
media (DMEM with phenol red (Life Technology, CA), 10% FBS and 1x
Pen Strep Glutamine (Life Technologies, CA)). For both, P. berghei-Luc
and HepG2 cytotoxicity assays, 20-26 hour prior to Pb-Luc sporozoites
infection, 5 µL HepG2-A16-CD81EGFP cells (~6x105 cells/mL in assay
medium (DMEM without Phenol Red (Life Technologies, CA), 5% FBS,
and 5x Pen Strep Glutamine (Life Technologies, CA)) were seeded into
white, solid-bottom, 1536-well plates (custom GNF mold ref# 789173-F,
Greiner Bio-One). Next 50 nL of compounds in 1:3 serial dilutions in DMSO
after a further 24h incubation. The 50% inhibitory concentration (IC50) was
calculated using linear interpolation of inhibition curves[25b]. A minimum of
three independent assays were carried out, each in triplicate wells, and
data presented as mean IC50 ± SD (standard deviation).
Protein hyperacetylation assays
(
final DMSO concentration per well 0.5%) were transferred with an
Protein hyperacetylation assays were carried out essentially as previously
described[14]. Briefly, synchronised trophozoite-stage P. falciparum 3D7
Acoustic Transfer System (ATS) (Biosero) instrument into the assay plates.
Atovaquone (0.5 µM) and puromycin (10 μM) in 1:3 serial dilutions in
DMSO were used as positive controls for Pb-Luc and HepG2 cytotoxicity
assays, respectively. Wells containing 0.5% DMSO were used as negative
controls for both assays. For Pb-Luc infection, P. berghei-ANKA-GFP-
Luc-SMCON (Pb-Luc)[27b] sporozoites were freshly isolated from
infected Anopheles stephensi mosquitoes (received from Insectary Core
Facility at New York University). Dissected salivary glands were
homogenized in DMEM media (Life Technology, CA) using a glass tissue
grinder and filtered twice through a 20 M nylon net filter (Steriflip,
infected erythrocytes (3-5% parasitemia, 5% haematocrit, 3ml culture per
well) were incubated with 5x IC50 of test compounds or controls. Controls
included parasites treated with 5x IC50 vorinostat and DMSO vehicle
control. Protein lysates were prepared via saponin lysis and resuspension
of parasite pellets in 1x SDS-PAGE loading dye. Following heat
denaturation (95ºC; 5min), proteins were analysed by SDS-PAGE and
Western blot using PVDF membrane, anti-(tetra)acetyl histone H4 primary
antibody (1:2,000 dilution; Merck Millipore, USA; known to cross-react with
acetylated histone H2 forms; Millipore product manual) and IRDye 680
Millipore). The sporozoites were counted using
a
Neubauer
goat anti-rabbit as the secondary antibody (1:10,000 dilution; Li-Cor
Biosciences). REVERT total Protein Stain® (Li-Cor Biosciences, USA) was
hemocytometer (C-Chip, InCyto, Republic of Korea), adjusted to final
concentration of 200 sporozoites per 1 μL in assay media, and placed on
ice until needed. Next ~1,000 sporozoites (5 µL) were added to each well
with a single tip Bottle Valve liquid handler (GNF). The assay plates were
spun down at 37 °C for 3 minutes with a centrifugal force of 330xg on
normal acceleration and brake setting. For the HepG2 cytotoxicity assay,
used prior to immunoblotting to control for loading, as per the
manufacturer’s instructions. Reactions were carried out in Odyssey
Blocking Buffer (Li-Cor Biosciences), as per manufacturer’s instructions,
and membranes imaged using an Odyssey Fc (Li-Cor Biosciences, USA)
at 700 nm. The hyperacetylation profile was analysed by densitometry
analysis using Image Studio Lite Version 5.2 software. Each Western
density signal was normalised to its respective total protein loading control
5
µL per well of additional assay media (but not sporozoites) was added to
the HepG2-A16-CD81EGFP cells to maintain equal concentrations of
1
3
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ChemMedChem
10.1002/cmdc.201800808
FULL PAPER
compounds relative to the Pb-Luc infected plates. Both sets of plates were
incubated at 37 °C for 48 h in 5% CO with high humidity to minimize media
2
evaporation and edge effects. After incubation media was removed by
spinning the inverted plates at 150xg for 30 seconds. Next, 2 μL per well
of BrightGlo (Promega) for quantification of Pb-Luc EEFs or CellTiterGlo
RTS,S/AS01 malaria vaccine with or without a booster dose in infants
and children in Africa: final results of a phase 3, individually randomised,
controlled trial, Lancet 2015, 386, 31-45.
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January 2016. The Weekly Epidemiological Record (WER) 2016, 91, 33-
52.
(
Promega) reagent (diluted 1:2 with deionized water) for quantification of
HepG2-A16-CD81EGFP cell viability were dispensed with the MicroFlo
BioTek) liquid handler. Luminescence was immediately measured using
K. Stenzel, M. J. Chua, S. Duffy, Y. Antonova-Koch, S. Meister, A.
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an Envision Multilabel Reader (PerkinElmer). For data analysis EEF
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atovaquone concentrations (20 wells), and the background for the HepG2
cytotoxicity was defined as the average of the 3 highest puromycin
concentrations (12 wells). IC50 values were determined using the average
normalized bioluminescence intensity of 4 wells per concentration and
plate (96 wells in total for each compound) and a nonlinear variable slope
four-parameter regression curve fitting model in Prism 6 (GraphPad
Software Inc.).
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an assay volume of 50 µL. 5 µL test compound or control, diluted in assay
2
buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl ,
0
.1 mg/mL BSA), were incubated with 35 µL of the fluorogenic substrate
ZMAL (Z-(Ac)Lys-AMC)[28] (21.43 µM in assay buffer) and 10 µL of human
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50 µL of 0.4 mg/mL trypsin in trypsin buffer (50 mM Tris-HCl, pH 8.0, 100
mM NaCl) were added, followed by further incubation at 37 °C for 30 min.
Fluorescence was measured with an excitation wavelength of 355 nm and
an emission wavelength of 460 nm using a Fluoroskan Ascent microplate
reader (Thermo Scientific). All compounds were evaluated in duplicate in
at least two independent experiments.
2
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Acknowledgements
[
17] D. Diedrich, K. Stenzel, E. Hesping, Y. Antonova-Koch, T. Gebru, S.
Duffy, G. Fisher, A. Schöler, S. Meister, T. Kurz, V. M. Avery, E. A.
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This
Forschungsgemeinschaft (DFG) (HA 7783/1-1 to FKH and HE
607/1-1 to JH), E.A.W. is supported by grants from the NIH
5R01AI090141 and R01AI103058). E. H. is supported by Griffith
University postgraduate scholarships. The Deutsche
Forschungsgemeinschaft (DFG) is acknowledged for funds used
to purchase the UHR-TOF maXis 4G, Bruker Daltonics, Bremen
HRMS instrument used in this research. We acknowledge the
Australian Red Cross Blood Service for the provision of human
blood and sera for P. falciparum culture.
work
was
supported
by
the
Deutsche
7
(
158, 801-813.
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Entry for the Table of Contents
The synthesis and biological evaluation of a series of novel peptoid-based HDACi is presented. All compounds were screened for
activity against three different malaria parasite life cycle stages as well as for mammalian cell toxicity to investigate the structure-activity
and structure-toxicity relationships of this class of antiplasmodial HDACi. Compound 2h was identified as a parasite-selective HDACi
with potent dual-stage antiplasmodial activity.
1
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