Design and Synthesis of Bioisosteres of Acylhydrazones as Stable Inhibitors of the Aspartic Protease Endothiapepsin

Article abstract of DOI:10.1002/cmdc.201800446

Acylhydrazone-based dynamic combinatorial chemistry (DCC) is a powerful strategy for the rapid identification of novel hits. Even though acylhydrazones are important structural motifs in medicinal chemistry, their further progression in development may be

Full text of DOI:10.1002/cmdc.201800446

Accepted Article
Title: Design and Synthesis of Bioisosteres of Acylhydrazones as
Stable Inhibitors of the Aspartic Protease Endothiapepsin
Authors: Anna Katharina Herta Hirsch, Varsha R Jumde, Milon Mondal,
Robin M. Gierse, M. Yagiz Unver, Francesca Magari, Roos C.
W. van Lier, Andreas Heine, and Gerhard Klebe
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To be cited as: ChemMedChem 10.1002/cmdc.201800446
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10.1002/cmdc.201800446
ChemMedChem
COMMUNICATION
Design and Synthesis of Bioisosteres of Acylhydrazones as
Stable Inhibitors of the Aspartic Protease Endothiapepsin
Varsha R. Jumde, [a,b] # Milon Mondal,[a] # Robin M. Gierse,[a,b,c] M. Yagiz Unver,[a] Francesca Magari,[d]
Roos C. W. van Lier,[a] Andreas Heine,[d] Gerhard Klebe,[d] and Anna K. H. Hirsch,[a,b,c]
*
[a]
Dr. V. R. Jumde, ORCID: 0000-0001-7677-7279, Dr. M. Mondal, ORCID: 0000-0002-1613-7269, R. M. Gierse, ORCID: 0000-0003-2418-1179, Dr. M.
Yagiz Unver, ORCID: 0000-0002-9797-7832, R.C.W. van Lier, Prof. Dr.A. K. H. Hirsch, ORCID: 0000-0001-8734-4663
Chemical Biology
Stratingh Institute for Chemistry
University of Groningen, Nijenborgh 7, 9747 AG Groningen, (The Netherlands)
E-mail: anna.hirsch@helmholtz-hips.de
[b]
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) – Helmholtz Centre for Infection Research (HZI), Department of Drug Design and
Optimization (DDOP), Campus Building E8.1, 66123 Saarbrücken (Germany)
Department of Pharmacy, Saarland University, Saarbrücken, Campus Building E8.1, 66123 Saarbrücken, Germany.
F. Magari, Prof. Dr. A. Heine, ORCID: 0000-0002-5285-4089, Prof. Dr. G. Klebe, ORCID: 0000-0002-4913-390X
Drug Design Group AG Klebe
[c]
[d]
Institute of Pharmaceutical Chemistry
Marbacher Weg 6, 35032 Marburg (Germany)
#
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the document.
disease, hypertension and HIV-1.19 Endothiapepsin is used as a
Abstract: Acylhydrazone-based dynamic combinatorial chemistry
(DCC) is a powerful strategy for the rapid identification of novel hits.
Even though acylhydrazones are important structural motifs in
medicinal chemistry, their further progression in development may be
hampered by major instability and potential toxicity under
physiological conditions. It is therefore of paramount importance to
identify stable replacements for acylhydrazone linkers. Here, we
present the first report on the design and synthesis of stable
bioisosteres of acylhydrazone-based inhibitors of the aspartic
protease endothiapepsin as a follow-up to a DCC study. The most
successful bioisostere is equipotent, bears an amide linker and we
confirmed its binding mode by X-ray crystallography. Having some
validated bioisosteres of acylhydrazones readily available might
accelerate hit-to-lead optimization in future acylhydrazone-based
DCC projects.
representative enzyme due to its robustness, immense stability
and similarity to the drug targets of the class of aspartic proteases.
Moreover, it has been used as a model enzyme for mechanistic
studies,20-22 as it is readily available in large quantity and
crystallizes easily and importantly remains active at room
temperature for more than 20 days.
We previously discovered acylhydrazone-based inhibitors
of endothiapepsin using DCC in combination with de novo
structure-based drug design, which display a promising inhibitory
profile (IC50 = 12.8 ± 0.4 µM).23 Acylhydrazones are considered to
be important structural motifs in medicinal chemistry, as they hold
the potential to interact with a range of biological targets in
antiviral, anticancer and antibacterial drug discovery.
Neverthless, there are problems associated with acylhydrazones.
This class of compounds is considered by some as a member of
the pan-assay interference compounds (PAINS).24 They tend to
show photo-induced E/Z-isomerization.25 Along with this, it is
important to consider also the behavior of acylhydrazones in vivo.
The major setback of acylhydrazones is their lack of stability due
to hydrolysis into an aldehyde and a hydrazide under acidic pH.
Hydrazone and acylhydrazone linkages are used to develop pH-
degradable drug-delivery systems for site-specific targeting.26
Furthermore, some acylhydrazones, like PAC-1, are in clinical
trials as a treatment for cancer.26,27 Nevertheless, it is highly
desirable to replace the labile acylhydrazone linker with stable
and chemically benign analogues while maintaining the key
interactions in the active site of the protein without significant
changes in chemical structure.
Dynamic combinatorial chemistry (DCC) enables rapid
screening of functionally diverse compounds against a target,
circumventing the need for individual synthesis, purification and
characterization.1–7 Among many other prominent examples of
DCC, reversible disulfide-bond formation was first introduced in
DCC by the groups of Still,8 Sanders9 and Lehn10 in the late
1990’s. Later on, in 1997, the group of Lehn first applied DCC to
a protein target using imine formation/exchange.1 Since then, its
scope and wider applicability were demonstrated on a range of
biological targets. Replacement of the reversible disulfide bond
with thioether (-S-CH2-)11 or all carbon (olefin, -CH2-CH2-)12–16 and
of the imine moiety with amines,1 an ethyl linker17 or with an amide
linker18 provides stable bioisosteres with potentially preserved
binding mode, making DCC an enabling tool for medicinal
chemistry and drug discovery (Figure 1a).
Surprisingly, to the best of our knowledge, there are only few
examples of bioisosteres of acylhydrazones,17 but no report as a
direct follow-up of a DCC experiment. In most cases, the binding
mode of the bioisostere is not confirmed experimentally. Having
their bioisosteres in hand, will establish ‘acylhydrazone-based
DCC’ as a powerful hit/lead-identification strategy with the
potential for further optimization.
We chose the target protein endothiapepsin, belonging to
the family of pepsin-like aspartic proteases, which play a
causative role in numerous diseases such as malaria, Alzheimer’s
1
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Bioisosteres have been introduced as a fundamental
strategy to improve the biocompatibility of the parent hit or lead
compounds. As such, bioisosteres contribute to the field of
medicinal chemistry, in terms of improving potency, enhancing
selectivity, altering physicochemical properties, reducing or
redirecting metabolism, eliminating or modifying toxicophores and
acquiring novel intellectual property.29 Herein, we describe the
design, synthesis and biochemical activity of three bioisosteres of
the acylhydrazone (S)-1, the first acylhydrazone inhibitor of
endothiapepsin. Importantly, unlike the parent acylhydrazone,
bioisosteres (S)-2 and (S)-4 are not prone to hydrolysis, and all
three do not liberate potentially toxic hydrazides.
Scheme 1. Synthesis of bioisosteres. a) ClCO2Et, Et3N, dry THF, aq. NH3, b)
Lawesson's reagent, dry DCM, c) EtOH, reflux, 4 h, d) TFA, DCM, e) 2-
mesitylethanamine hydrochloride (10), 1,1'-carbonyldiimidazole, THF, rt, 15 h,
f) TFA, DCM, 0 oC to rt, 1.5 h, g) 2-mesitylethanol (11), DCC, DMAP (5%) DCM,
8 h, h) HCl/Et2O 1 M, 24 h, i) SOCl2, dry toluene, reflux, 3 h, j) a. TMS-
diazomethane, Et2O, b. 47.5% aq. HBr.
Inspection of the soaked crystal structure of endothiapepsin
with acylhydrazone (S)-1 in the active site, shows that the
aromatic parts of the compound such as indolyl and/or mesityl
moieties are able to form π–π-stacking interactions with the
amino acid residues of the protein backbone. In all of the
structures (Figure 1b), the binding modes of the indolyl- and
mesityl- moieties are preserved. It was computationally observed
that the a-amino groups of all bioisosteres (S)-2–(S)-4 form
charge-assisted H bonds to the catalytic dyad (Asp35 and Asp
219) as well as additional H-bonding interactions with Asp81, and
Gly221. The indolyl –NH forms H bonds either with Asp81 or
Asp33, the NH group of the amide donates an H-bond to Gly221
in (S)-2. In addition to these, the thiazolyl ring of (S)-4 is involved
in several hydrophobic interactions with the protein backbone.
The main building blocks required for the synthesis of bioisosteres
(S)-2–(S)-4, are N-α-Boc-L-tryptophan (5) and the 2-mesitylene-
derived compounds (S)-5, 8 and 11 (see Schemes S1 to S4 in the
ESI and Scheme 1).
Figure 1. a) Previous examples of bioisosteres and b) proposed bioisosteres
(2–4) of the acylhydrazone 1 as stable inhibitors of endothiapepsin.
We chose the X-ray crystal structure of endothiapepsin in
complex with acylhydrazone (S)-1 (PDB: 4KUP)23 as a starting
point for the design of stable bioisosteres of the labile
acylhydrazone moiety. Hit (S)-1 displays an IC50 value of 12.8 μM
and ligand efficiency (LE) of 0.27. It interacts with the catalytic
dyad using H-bonding interactions (Asp35 (2.8 Å, 3.2 Å) and
Asp219 (2.9 Å)) through its a-amino group.
Very mild peptide-coupling conditions afforded the bioisostere
(S)-2 with the amide linker, followed by deprotection of the Boc-
group. Starting from N-Boc-L-tryptophan (5) and 2-
mesitylethanamine hydrochloride (10) in presence of the weak
base carbonyldiimidazole, we obtained the corresponding amide
(S)-14 in 80% yield, and after deprotection with TFA, the test
compound (S)-2 in quantitative yield. The ester (S)-3 was
accessible through the Steglich esterification.32 We synthesized
the bioisostere (S)-4 from the building blocks thioamide (S)-7 and
ketobromide 9, which can be both accessed in two steps from N-
α-Boc-L-tryptophan (5) and mesitylacetic acid (8),33,34 respectively.
We designed bioisosteres using two different design
approaches, namely Recore in the LeadIT suite30 and the
molecular modeling software Moloc31 for molecular modeling and
computation of the dipole moments. In Recore, a defined moiety
of a molecule (the core) is replaced by fragments from a 3D
database whilst keeping the rest of the molecule intact. To restrict
the number of solutions, defined ligand-based pharmacophore
constraints can be assigned. This modeling and docking resulted
in various compounds displaying heterocyclic, ester or amide
linkages. Among the various heterocycles (e.g., triazole, tetrazole,
oxazole (ESI Figure S8)), we chose the best three compounds
(Figure 1b) based on their dipole moments, their calculated DG
values and predicted binding modes, which are comparable with
the parent acylhydrazone (S)-1 and synthesized them as a proof-
of-concept study. The predicted binding modes of three
representative bioisosteres in the active pocket of endothiapepsin
are shown in Figure S4 (See ESI).
Subsequent deprotection of the Boc-group of compound (S)-
12 afforded bioisostere (S)-4 in quantitative yield. The first step to
obtain thioamide (S)-7 consists of the synthesis of amide (S)-6
followed by thionation using Lawesson’s reagent. On the other
hand, using modified Arndt-Eistert reaction conditions, starting
from mesitylacetic acid (8), afforded intermediate 9. To
investigate the biochemical activity of the designed bioisosteres
(S)-2–(S)-4, we performed a fluorescence-based assay adapted
from the HIV-protease assay (see Figures S1—S3 for the IC50
curves, in the ESI).35
2
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COMMUNICATION
Table 1. Biochemical evaluation of acylhydrazone (S)-1 and its bioisosteres (S)-
2–(S)-4. Each experiment was carried out in duplicate.
molecule is well coordinated and seems to displace several water
molecules. This may be important for the stabilization of the ligand
bound to the protein. A similar DMSO molecule can be observed
in previous crystal structures (e.g., PDB code: 4KUP).23
Inhibitor
IC50 [μM][a]
Ki [μM][b]
ΔGEXPT
[kJ.mol-1][b]
ΔGHYDE
[kJ.mol-1][c]
(S)-1
(S)-2
(S)-3
(S)-4
12.8 ± 0.4
12.9 ± 0.7
28.7 ± 4.1
193.7 ± 11.4
6.0 ± 0.2
6.1 ± 0.4
13.5 ± 1.9
91.2 ± 5.4
–30
–30
–28
–23
–32
̶
̶
̶
27
28
31
[a] 11 different concentrations of inhibitor were used; the errors are given in
standard deviations (SD). [b] Values indicate the inhibition constant (Ki) and the
Gibbs free energy of binding (ΔG) derived from IC50 values using the Cheng-
Prusoff equation.36 [c] Values indicate the calculated Gibbs free energy of
binding (ΔGHYDE; calculated by the HYDE scoring function in the LeadIT suite).
The three designed bioisosteres inhibit the activity of
endothiapepsin to a different extent. The most potent inhibitor, the
amide bioisostere (S)-2, displays a Ki value of 6.1 µM, very similar
to the parent acylhydrazone (S)-1 (Ki = 6.0 µM, Table 1). We
calculated the Ki values from experimental IC50 values using the
Cheng–Prusoff equation.36 To verify the predicted binding mode
of the bioisosteres, we soaked crystals of endothiapepsin with the
most potent bioisostere (S)-2 and determined the crystal structure
of (S)-2 in complex with endothiapepsin at 1.58 Å resolution (PDB
code: 5OJE). The structure features clear electron density for the
ligand, as shown in Figure 2b.
Figure 3. Superimposition of the acylhydrazone inhibitor (S)-1 (cyan) and the
amide bioisostere (S)-2 (green). H bonds below 3.0 Å are shown as black,
dashed lines (Color code: protein backbone: C: gray, O: red, N: blue, (S)-1: C:
cyan and (S)-2: C: green).
The single bond connecting the mesityl unit to the rest of the
acylhydrazone (S)-1 is part of a conjugated system and prefers a
planar orientation. It is twisted out of planarity to an unfavorable
angle of +50° compared to the ideal angle of 90° as in bioisostere
(S)-2 (ESI Figure S6).
Upon closer examination, the location of the ligand is similar
to the docked pose shown in Figure S4 (See ESI). The amino
group of the ligand forms two H bonds with Asp35 (2.8 Å) and
Asp219 (2.8 Å). The indolyl nitrogen atom forms a H-bond with
Asp81 (3.0 Å). The hydrophobic part of the indolyl moiety is
engaged in hydrophobic interactions with Phe116, Leu125, Tyr79
and Gly221. The mesityl substituent is involved in hydrophobic
interactions with Ile300, Ile304, Tyr226, Gly80 and Asp81. The
oxygen atom of the amide linkage donates two H-bonds to a
network of two water molecules with the backbone of Gly37 and
Gly80, which is conserved between the crystal structures in
complex with (S)-1 and (S)-2 (4KUP and 5OJE, respectively, ESI
Figure S7).
The bioisostere (S)-2, however, contains a peptidic bond in
the linker, which also prefers planarity. This forces the C–N bond,
its third bond, counting from the mesityl substituent, into an
unfavorable torsional angle of 124° compared to the preferred
170° of the acylhydrazone (Figure S6). In conclusion, both ligands
have to adopt a slightly unfavorable conformation to bind in the
pocket of the enzyme, which is reflected in their binding affinities.
Based on our observations, it might be difficult to design a linker
with improved binding affinity, which would need to be more
flexible with respect to the torsional angles, while the H-bond
donor and –acceptor functions of the peptidic nitrogen and oxygen
atoms should ideally be preserved.
We report the successful replacement of the acid-sensitive
and hydrolyzable acylhydrazone linker of parent hit (S)-1,
affording stable and equipotent inhibitors of endothiapepsin. We
designed and synthesized three bioisosteres and evaluated them
for their inhibitory potency against endothiapepsin. Compounds
(S)-2 and (S)-3, possessing amide and ester linkers, respectively
display comparable Ki values as the parent hit (S)-1, while
compound (S)-4 is an order of magnitude weaker than the parent
hit. The crystal structure of amide (S)-2 (Ki = 6.1 µM) in complex
with endothiapepsin validates predicted binding modes. In this
proof-of-concept study, we identified molecular interactions that
should be taken into consideration if further modifications are
done to achieve
a more drug-like replacement for the
acylhydrazone linker. Taken together, we demonstrate that
acylhydrazones can be replaced without affecting the binding
mode and whilst preserving the activity, demonstrating that
acylhydrazone-based DCC is a powerful tool to identify hits, which
Figure 2. a) Zoomed-out view of the protein shown as surface; b) Electron
density omit-map of the crystal structure of endothiapepsin in complex with
compound (S)-2 and a coordinated DMSO molecule. Fo-Fc map contoured at
3.3 σ (color code: protein cartoon: light blue; C: green; O: red; N: blue; S: yellow).
can then be optimized to stable lead compounds in
straightforward manner.
a
The only difference compared to the docked pose is at the
amide linkage. In contradiction to the computational modeling, the
nitrogen atom of the amide does not form a H bond with the
oxygen atom of Gly221, the distance is 4.3 Å. Instead, the
hydroxyl group of Thr222 acts as a H-bond acceptor and forms a
H bond (2.8 Å) with the amide nitrogen atom of the ligand, which
is also shown in Figure 3.
Experimental Section
Full experimental details are provided in Supporting Information.
Due to the slightly bent shape of the coordinated ligand, both
aromatic groups are able to form hydrophobic interactions (4.2 Å)
with one DMSO molecule, shown in Figure 2. This DMSO
3
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Acknowledgments
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A.K.H.H. gratefully acknowledges funding from the Netherlands
Organisation for Scientific Research (ECHO-STIP and LIFT
grants), the Dutch Ministry of Education, Culture and Science
(Gravitation Program 024.001.035), the European Research
Council (ERC starting grant 757913) and the Helmholtz-
Association’s Initiative and Networking Fund.
[21] L. Coates, P. T. Erskine, S. P. Wood, D. A. A. Myles, J. B. Cooper,
Biochemistry 2001, 40, 13149–13157.
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Keywords: Dynamic combinatorial chemistry • bioisosteres •
aspartic proteases • acylhydrazones • drug design
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4
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Entry for the Table of Contents
Acylhydrazone-based dynamic combinatorial chemistry (DCC) can be used for the rapid identification of novel hits. To identify
replacements of the unstable acylhydrazones, we performed a computer-guided search for biosiosteres on a previously found hit
inhibiting the aspartic protease endothiapepsin. A peptidic linker emerged as an equipotent bioisostere and its predicted binding
mode was verified by determination of the crystal structure with the bound bioisostere. This readily available biosiosteric linker might
accelerate future lead development from hits found using acylhydrazone-based DCC.
5
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