New furin inhibitors based on weakly basic amidinohydrazones

Article abstract of DOI:10.1016/j.bmcl.2010.11.092

A novel series of amidinohydrazone-derived furin inhibitors was prepared; the most potent compounds 17 and 21 inhibit furin with K_i values of 0.46 and 0.59 μM, respectively. In contrast to inhibitor 17, which still contains a guanidino residue,

Full text of DOI:10.1016/j.bmcl.2010.11.092

Bioorganic & Medicinal Chemistry Letters 21 (2011) 836–840
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New furin inhibitors based on weakly basic amidinohydrazones
Frank Sielaff a, Manuel E. Than b, Dorian Bevec c, Iris Lindberg d, Torsten Steinmetzer a,
⇑
a Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
b Leibniz Institute for Age Research—Fritz Lipmann Institute (FLI), Protein Crystallography Group, Beutenbergstrasse 11, 07745 Jena, Germany
c mondoBIOTECH Holding AG, Das Kloster, Mürgstrasse 18, CH-6370 Stans, Switzerland
d Department of Anatomy and Neurobiology, University of Maryland-Baltimore, Baltimore, MD 21201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of amidinohydrazone-derived furin inhibitors was prepared; the most potent compounds
Received 5 October 2010
Revised 17 November 2010
Accepted 18 November 2010
Available online 25 November 2010
17 and 21 inhibit furin with Ki values of 0.46 and 0.59 lM, respectively. In contrast to inhibitor 17, which
still contains a guanidino residue, compound 21 possesses only weakly basic amidinohydrazone groups.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Furin
Proprotein convertase
Protease inhibitor
Amidinohydrazone
Serine protease
Furin is a type-I transmembrane protein which contains a Ca2+
-
guanabenz.11 Furthermore, CNI-1493, an anti-inflammatory and
anti-parasitic compound that contains four amidinohydrazone
groups, reached phase II clinical trials for the treatment of Crohn’s
disease.12–14 Very recently, in parallel to our work, a related amid-
inohydrazone-derived furin inhibitor 2 was identified by HTS.15
Such amidinohydrazones have a significantly decreased basicity
compared to other furin inhibitors, which often contain strongly
basic guanidino or amidino groups. For example, we have calcu-
lated16 a pKa of 8.03 for the amidinohydrazone group of inhibitor
1, which is similar to the value of 8.1 reported for the orally avail-
able guanabenz.17
After identification of 1 we prepared several analogues with one
or two amidinohydrazone groups by treatment of commercially
available carbonyl compounds with aminoguanidine (Table 1). In
addition, the known inhibitor 215 was synthesized as a reference
compound. For this inhibitor we found a similar potency (Ki =
dependent subtilisin-like serine protease domain. It is ubiquitously
distributed in human tissues and is the best characterised member
of the family of proprotein convertases (PCs), which convert
numerous precursors of secreted proteins to their active forms.
Various studies have confirmed that furin plays a crucial role in
many bacterial and viral diseases, tumorigenesis, neurodegenera-
tive disorders and diabetes.1,2 Furin possesses a strong preference
for substrates with the multibasic cleavage motif Arg-X-Arg/
Lys-Arg;-X. In addition to various types of peptidic substrate-
analogues,3–5 potent non-peptidic furin inhibitors based on
guanylated 2,5-dideoxystreptamines6 have also been described.
Recently, we have designed a series of highly potent peptidomi-
metic furin inhibitors which contain a 4-amidinobenzylamide
group as the P1 residue. Using a cell-based assay we could demon-
strate that these inhibitors are able to reduce the cleavage of the
hemagglutinin precursor HA0 in H7N1 fowl plague viruses.7
Correct cleavage of the HA0 precursor is a crucial step during an
influenza virus infection.8
25 lM) as described in the literature. In contrast, the mono-amid-
inohydrazones 3 and 4 derived from benzaldehyde and benzophe-
none, as well as the acylated analogue 5 obtained from reaction
In parallel to the design of these inhibitors we screened various
compounds available to us for furin inhibition and could identify a
bis-(amidinohydrazone)-derivative 1 with a Ki value of 1.82 lM.
This compound and several close analogues were originally
described for the treatment of trypanosomiasis9 and for inflamma-
tion.10 Interestingly, there already exists an approved amidinohyd-
razone-based drug used for the treatment of hypertension,
with benzoyl chloride showed poor inhibition (Ki >250 lM). Intro-
duction of a second amidinohydrazone group in the meta and para
position resulted in improved affinity, whereas both acylated
aminoguanidines 8 and 11 were less active. Bis-amidinohydrazone
12 derived from 1,3-indandione and 13 obtained from 4,40-diacyldi-
phenylether inhibit furin with Ki values >15 lM and were not
further modified.
From the X-ray structure of furin in complex with the irrevers-
ible inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone it is
known that furin has an unusually acidic active site explaining
⇑
Corresponding author. Tel.: +49 6421 2825900; fax: +49 6421 2825901.
E-mail address: steinmetzer@staff.uni-marburg.de (T. Steinmetzer).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.092

F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 836–840
837
NH
HN
NH2
N
H2N
N
HN
NH2
N
H2
N
N
NH
HN
NH
H2N
N
NH
H
N
H
N
HN
NH
NH
H
C
Cl
H2
O
6
O
N
N
Cl
NH2
H2N
NH
HN
NH2
NH
HN
NH
1
guanabenz
CNI-1493
NH
H
H2
N
N
NH
NH
H
N
N
H2
N
NH2
O
O
H
O
HN
NH
HN
NH
NH2
Cl
NH2
compound 1e
from Jiao et al.6
2
its preference for substrates with basic P6-P1 residues.19 Based on
preliminary modelling we assumed that one amidinohydrazone
group of 1 should occupy the S1 pocket, whereas the second one
might bind into the S2 pocket. Therefore, we used the easily
accessible aniline group of 1 for further modifications with basic
residues to address additional acidic binding sites of furin
(Table 2).
The arginine derivative 15 exhibits slightly enhanced affinity; a
similar potency was found for its des-amino analogue 16. There-
fore, we also introduced the shorter 4-(guanidino)butyryl- and
the homologous 6-(guanidino)caproyl residue (17–18). The most
potent inhibitor 17 (preparation see Scheme 1) possesses a Ki value
of 0.46 M, which is approximately 4-fold improved compared to
1. The -aminobutyric acid analogue 19, an intermediate from syn-
thesis of compound 17, has slightly reduced inhibitory activity.
The most efficient non-peptidic furin inhibitor is a 2,5-dideoxy-
streptamine derivative with four guanidine residues (compound 1e
from Jiao et al.6), therefore, we prepared compound 20 by dimer-
ization of 1 via a malonyl spacer to obtain a first analogue contain-
ing four amidinohydrazone groups (Table 3).
l
c
However, inhibitor 20 had only marginally improved potency
compared to analogue 1. Alternatively, we used chloroacetyl
O
O
O
O
a, b
c
HN
NH2
NH2
O
26
27
NH
NH
NH
NH
HN
HN
HN
HN
NH2
H2N
N
NH2
N
H2N
N
N
d
NH
NH2
HN
HN
NH2
N
H
O
O
19
17
Scheme 1. Reagents and conditions: (a) Boc-
c-aminobutyric acid, N-methylmorpholine, isobutylchloroformate, ꢀ15 °C, 10 min in DMF, followed by addition of 26, 1 h at
ꢀ15 °C and overnight at room temperature, (b) 1 N HCl in acetic acid, 1 h room temperature, (c) 2.6 equiv aminoguanidine hydrochloride, 5 mol % HCl, in 50% EtOH reflux for
6 h, (d) 3 equiv 1H-pyrazole-1-carboxamidine hydrochloride, 6 equiv diisopropylethylamine in DMF, 16 h. Final inhibitors 19 and 17 were purified by preparative reversed
phase HPLC to a purity of >95% according to HPLC at 220 nm.

838
F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 836–840
O
O
OH
O
O
H2N
O
O
O
O
O
O
O
26
29
Cl
N
H
Cl
Cl
O
N
a
b
H
O
O
28
30
NH
HN
N
NH2
HN
HN
NH2
N
O
O
N
H
N
c
NH
NH
N
H2N
HN
NH2
NH
21
Scheme 2. Reagents and conditions: (a) 1.2 equiv 26, 1.2 equiv K2CO3 in dry dichloromethane, reflux 5 h, (b) 2 equiv 29, 2 equiv Cs2CO3 in acetonitrile, reflux 5 h, (c) (i)
5.2 equiv aminoguanidine hydrochloride, 5 mol % HCl, in 50% EtOH reflux for 6 h; (ii) purification by preparative reversed phase HPLC.
Table 1
Table 1 (continued)
Amidinohydrazone and acylated aminoguanidine-derived furin inhibitors
Compound
Structure
Ki (l
M)18
H
N
H
N
NH2
NH
NH2
NH
R1
=
O
R
=
N
N
H
R1
R1
O
8
27.9
Compound
Structure
Ki (l
M)18
R
R
R
R
9
10
11
1.47
1
1.82
H
R
H
R
NH2
O
1.84
H
R
R1
O
R1
O
173
2
25.3a
R
R
Cl
12
13
17.3
R
3
4
5
>500
H
R
R
R
273
376
44.0
R1
O
O
R
H
R
O
S
O
H
N
14
1.55
R
6
7
11.5
H
R
R
Komiyama et al. have reported a Ki value of 11.8
M for this compound.15
R
a
l
4.78

F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 836–840
839
Table 2
Table 3
Furin inhibitors with the general formula
Inhibition of furin by amidinohydrazone inhibitors
H
N
H
H
N
NH2
NH
H2N
N
NH2
NH
R
=
N
N
N
O
NH
Compound
Structure
Ki (l
M)18
NH
R
R
R
Compound
R
Ki (l
M)18
O
O
NH
20
1.13
N
H
N
H
15
1.13
H2N
N
H
R
R
R
NH2
O
R
NH
16
17
1.04
0.46
O
H2N
H2N
N
H
O
O
21
0.58
N
H
O
H
N
R
NH
R
O
R
O
H
N
H2N
H2N
18
19
1.42
1.2
22
23
5.61
1.46
NH
O
N
H
O
R
R
R
O
chloride for the synthesis of inhibitor 21 (preparation see Scheme 2),
which also allows access to bi- and trifunctional analogues
22–25. Compound 21 has a slightly improved Ki value of 0.58 lM,
N
H
R
R
which is similar to the potency of the guanidine derivative 17,
whereas the other analogues were less potent. The marginal differ-
ences in the inhibition constants of the compounds summarised in
Table 3 suggest that amidinohydrazone groups from only one
phenyl ring are able to make specific interactions with furin.
Selected compounds were also tested towards several trypsin
like serine proteases, such as thrombin, trypsin, plasmin, factor
O
24
25
1.94
1.35
O
N
H
R
R
R
O
Xa and subtilisin. Most Ki values were >10
21 also inhibits thrombin (Ki = 0.86 M), whereas compound 17
has a Ki value of 2.8 M towards trypsin (see Supplementary data).
lM; however, inhibitor
O
N
l
H
l
Although we could obtain only slightly improved inhibitors
compared to our original screening hit, we assume that these
amidinohydrazones might represent a suitable starting point for
the design of non-peptidic furin inhibitors with relatively high
selectivity against trypsin-like serine proteases. Despite several at-
tempts, we failed to obtain a crystal structure of furin in complex
with this type of inhibitors; therefore, we presently have no infor-
mation regarding their binding mode. However, their reduced
basicity may lead to improved pharmacokinetic properties com-
pared to guanidine and amidine-derived furin inhibitors.
In general, all carbonyl compounds were converted to final
inhibitors by treatment with aminoguanidine hydrochloride in
ethanol and catalytic amounts of hydrochloric acid.9 The synthesis
of amidinohydrazone inhibitors is exemplarily described for inhib-
itors 17 and 19 (Scheme 1) and 21 (Scheme 2). Protected amino
acids were coupled to intermediate 269 using the mixed anhydride
procedure, followed by removal of the protecting group and con-
version to the amidinohydrazone (Scheme 1). Reaction of 19 with
1H-pyrazole-1-carboxamidine20 provided inhibitor 17.
R
Acylated compounds 5, 8 and 11 were synthesized from reac-
tion of corresponding acid chlorides with aminoguanidine ꢁ HCO3ꢀ
in pyridine.21
Acknowledgements
The authors are grateful to Prof. Wolfram Bode and to Prof. Jörg
Stürzebecher for initial contributions to this project.
Supplementary data
Supplementary data (a scheme with additional calculated pKa
values and a table with inhibition constants towards selected tryp-
sin-like serine proteases are provided as Supplementary data)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.11.092.
Inhibitor 21 was prepared according to Scheme 2. Reaction of
26 with chloroacetyl chloride provided intermediate 28, which
was converted with the phenol derivative 299 to compound 30.
Final conversion to the amidinohydrazones was performed as de-
scribed in Scheme 1.
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