Synthesis of a new class of bis(thiourea)hydrazide pseudopeptides as potential inhibitors of β-sheet aggregation

Article abstract of DOI:10.1021/ol203074p

The modular synthesis of a novel pseudopeptide scaffold based on a bis(thiourea)hydrazide motif is reported. This compound class is designed to display "amphifinity", i.e. association with a peptide strand on one but not the other face of the scaffold, and hence could potentially inhibit β-sheet aggregation.

Full text of DOI:10.1021/ol203074p

ORGANIC
LETTERS
2012
Vol. 14, No. 1
330–333
Synthesis of a New Class of
Bis(thiourea)hydrazide Pseudopeptides
as Potential Inhibitors of β-Sheet
Aggregation
Jan J. Klein and Stefan Hecht*
€
Department of Chemistry, Humboldt-Universitat zu Berlin, 12489 Berlin, Germany
sh@chemie.hu-berlin.de
Received November 16, 2011
ABSTRACT
The modular synthesis of a novel pseudopeptide scaffold based on a bis(thiourea)hydrazide motif is reported. This compound class is designed to
display “amphifinity”, i.e. association with a peptide strand on one but not the other face of the scaffold, and hence could potentially inhibit β-sheet
aggregation.
The conversion of peptides or proteins from their soluble
forms into highly organized fibrillar aggregates is relevant
to many neurogenerative diseases, such as Alzheimer’s,
Parkinson’s, Creutzfeldt-Jakob, and Huntington’s.1
These aggregates are generally described as amyloid
fibrils or plaques when they accumulate extracellulary.
For example in Alzheimer’s disease, proteolytic cleavage
of the amyloid precursor protein (APP) generates the
natively unfolded 39À43 residue amyloid β-peptide (Aβ),
which assembles into a characteristic cross-β-structure
via the association of β-strands. Finding molecules that
prevent Aβ peptide aggregation is the goal for many
approaches to develop therapeutics for Alzheimer’s and
related diseases.2 There are various approaches to reduce
or inhibit Aβ peptide production by the modulation of the
proteases, which are involved in the processing of APP,3
or the development of antibodies,4 which directly target
monomeric Aβ or oligomeric Aβ. In addition, the direct
inhibition of oligomerization (Aβ self-assembly) appears
to be a promising way to reduce neurotoxicity, mainly
since the toxic forms of Aβ are soluble oligomers, as small
as dimers and trimers.5 Therefore, several classes of
peptidic and pseudopeptidic aggregation inhibitors have
(1) For reviews, see: (a) Miller, Y.; Ma, B.; Nussinov, R. Chem. Rev.
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Selkoe, D. J. Nature 2003, 426, 900–904. (e) Hamley, I. W. Angew.
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Natl. Acad. Sci. U.S.A 2007, 104, 15659–15664. (b) Bard, F.; Cannon,
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Zheng, H.; Li, Y.-M. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 20228–
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10.1021/ol203074p
Published on Web 12/14/2011
2011 American Chemical Society

been developed.6 It has been shown that short peptide
sequences based on the central hydrophobic core, Aβ
(16À20) (KLVFF), act as β-sheet blockers.7 Other ap-
proaches deal with the modification of the amide back-
bone. For example, peptides with incorporated N-methyl
amino acids,8 ester linkages,9 or proline residues10 have
been designed and show inhibition of β-sheet propagation
or even disassembly of preformed fibrils.
bond acceptor as compared to the amide carbonyl
oxygen.11 On the other hand, hydrogen-bonding to the
opposite face is strongly enhanced due to the bidentate
binding mode of the more acidic thiourea protons.11c,12
These N-methyl and thiourea motifs are connected via
hydrazide linkage to maintain the direction of the peptide
backbone, allowing the use of amino acid building blocks
for the ease of synthesis and diversification. The resulting
target scaffolds should hence present a complementary
and a blocking face, which offer the possibility of stronger
hydrogen-bonding to the β-sheet on the one face and
ensure inhibition of further association on the opposite
face, respectively (Figure 1). As our scaffold displays
orthogonal binding affinity it can be described as
“amphifinic”.
To the best of our knowledge, the targeted compound
class of bis(thiourea)hydrazides has not been reported
thus far. Merely, structure-related peptidyl thiosemicar-
bazides have been reported recently as intermediates
of oxadiazol synthesis.13 However, these structures
lack both multiple thioureahydrazide units as well as
N-methylation.
A modular synthesis was devised, which in principle
allows for the rapid variation of side chain functionality
as well as stereochemistry for optimizing the interaction
with specific peptide sequences or proteins. A key step in
our synthesis is the formation of the two thioureas from
an ambident R-N-methyl hydrazide and two isothiocya-
nates, all of which are readily derived from commercially
available amino acid building blocks.
Figure 1. Inhibition of β-sheet aggregation by a multivalent
scaffold, its structure, and its retrosynthesis (SG = solubilizing
group).
Here, we report a novel scaffold based on bis(thiourea)-
hydrazide pseudopeptides (Figure 1), which due to its
rational design could potentially inhibit β-sheet aggrega-
tion and therefore stabilize a given peptide in its native
folded state. Our scaffold is based on several design
considerations: On the one hand we take advantage of
N-methylated amides,8 thus hydrogen-bonding to one
face is blocked and further association of β-sheets as well
as homoassociation of the scaffold itself can be prevented.
Furthermore, blocking is enhanced by the employment of
two thiourea units since the sulfur is a weaker hydrogen-
Scheme 1. Synthesis of Ambident R-N-Methyl Hydrazide Core
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Perez, K.; Masters, C. L.; Cappai, R.; Wade, J. D.; Barnham, K. J.
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2001, 40, 8237–8245. (b) Chatterjee, J.; Gilon, C.; Hofman, A.; Kessler,
H. Acc. Chem. Res. 2008, 41, 1331–1342.
The first target for synthesis was the R-N-methyl
hydrazide core 6 (Scheme 1) from commercially available
(9) Gordon, D. J.; Meredith, S. C. Biochemistry 2003, 42, 475–485.
(10) Soto, C.; Sigurdsson, E. M.; Morelli, L.; Kumar, R. A.; Castano,
E. M.; Frangione, B. Nat. Med. 1998, 4, 822–826.
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Org. Lett., Vol. 14, No. 1, 2012
331

L-Ala-OH as a starting point. Benzylation of L-Ala-OH and
subsequent selective deprotection of the C-terminus by sapo-
nification provided N-protected alanine 2 in very good over-
all yield (88%). Subsequent regioselective coupling of 2
with hydrazone 3, masking the primary amino function of
N-methyl hydrazine by using a ethyl chloroformate protocol14
furnished 4. A moderate yield of 72% results from the high
instability of the hydrazone functionality. Subsequent hydra-
zone cleavage followed by quantitative debenzylation by
hydrogenolysis provided the R-N-methyl hydrazide core 6.
can be carried out chemoselectively by carefully control-
ling the reaction temperature (see Supporting Information).
The more reactive primary amine adds rapidly to the
isothiocyanate even at low temperatures while the less
nucleophilic hydrazide reacts only at elevated tempera-
tures and rather slowly. Consequently, (R,R,R)-thiourea
16c as well as (S,R,S)-thiourea 16d can easily be obtained
directly by the reaction of nucleophile 6 with 2 equiv of
isothiocyanates 14a and 14b, respectively, at higher tem-
peratures, i.e. 50À60 °C.
Scheme 2. Synthesis of Isothiocyanate Termini
Scheme 3. Chemoselective Synthesis of Bis(thiourea)hydrazides
The synthetic route was demonstrated using alanine-
derived building blocks; however, in principle it allows
any desired (side-chain protected) amino acid to be
employed. Importantly, epimerization can be avoided
during synthesis therefore granting access to enantiopure
bis(thiourea)hydrazide pseudopeptides with all possible
stereochemical variations. Polar triethylene glycol resi-
dues were introduced as terminal groups to provide
sufficient solubility in aqueous (buffer) solutions, neces-
sary in the context of biological studies. Note that the
The first step in the formation of the isothiocyanate
terminal building blocks 14a and 14b involved the syn-
thesis of the polar triethylenegycol chain 11 (Scheme 2).
Benzylation of 2-(methylamino)ethanol via formation of
an iminium salt with benzaldehyde and titanium isoprop-
oxide as well as its subsequent reduction with NaBH4
provided 8 (86%). Nucleophilic substitution of 8 with
monotosylated diethyleneglycol monomethyl ether 9
gave the benzyl-protected polar triethyleneglycol precur-
sor 10, which subsequently was deprotected by hydro-
genation to obtain the amine 11 in 94% yield. The
terminal triethyleneglycol groups of the isothiocyantes
were introduced by the reaction of amine 11 with Cbz-
protected amino acids (here: L- and D-alanine) using an
ethyl chloroformate coupling protocol followed by al-
most quantitative N-deprotection by hydrogenation with
Pd/C to obtain both stereoisomers 12a and 12b. Finally
conversion into the reactive isothiocyanates 14a and 14b
was carried out in yields of 79À84% using thiophosgene.
Initial coupling of the ambident nucleophile 6 with 1
equiv of isothiocyanates 14a and 14b, respectively, yields
thioureas 15a and 15b, respectively, which can be subse-
quently coupled with another equivalent of the oppositely
configured isothiocyanate (14a or 14b) to obtain the
desired target 16a or 16b, respectively (Scheme 3). Ob-
viously, the coupling of the two building blocks 6 and 14
Figure 2. CD spectra of bis(thiourea)hydrazides 16aÀd (c = 4 Â
10À4 M) in phosphate buffer (10 mM, pH 7.4) at 25 °C.
(14) Verado, G.; Geatti, P.; Lesa, B. Synthesis 2005, 4, 559–564.
332
Org. Lett., Vol. 14, No. 1, 2012

terminal residues can be varied freely, thereby offering the
possibility for introducing labels or anchors to solid
supports, necessary for future automated synthesis. In-
itial CD spectroscopic studies demonstrate and verify
that all bis(thiourea)hydrazides 16aÀd display different
stereochemistry (Figure 2) as well as the absence of
homoassociation (see Supporting Information).
We have described a totally new class of thioureahy-
drazide pseudopeptides, which due to their “amphifinity”
could potentially be able to stabilize peptide sequences,
and perhaps even proteins, in their native folded state,
thereby preventing misfolding and subsequent aggrega-
tion into amyloid-type fibrils. Work, currently ongoing in
our laboratories, is concerned with the investigation of
the inhibition potential of these scaffolds. Furthermore,
detailed conformational analysis of the scaffold and its
peptide complexes in solution via NOE-measurements as
well as the determination of the association constants for
formation of the involved homo- and heteroduplexes will
be investigated. In order to explore the full potential of
this substance class, a respective solid phase synthesis is
being developed to generate libraries for investigating
structureÀactivity relationships to eventually target spe-
cific peptides and proteins. This approach should provide
a rational means to develop molecules based on our
scaffold to sense and potentially cure amyloid and other
protein-misfolding related diseases.
Acknowledgment. We are grateful to the German Re-
search Foundation for support (DFG via SFB 765).
Wacker Chemie AG, BASF AG, Bayer Industry Services,
and Sasol Germany are thanked for generous donations of
chemicals.
Supporting Information Available. Experimental pro-
cedures and compound characterization data. This ma-
terial is available free of charge via the Internet at http://
pubs.acs.org.
Org. Lett., Vol. 14, No. 1, 2012
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