Repurposing N,N'-bis-(arylamidino)-1,4-piperazinedicarboxamidines: An unexpected class of potent inhibitors of cholinesterases

Article abstract of DOI:10.1016/j.ejmech.2016.09.051

Drug repurposing (=drug repositioning) is an effective way to cut costs for the development of new therapeutics and to reduce the time-to-market time-span. Following this concept a small library of compounds was screened for their ability to act as inhibitors of acetyl- and butyrylcholinesterase. Picloxydine, an established antiseptic, was shown to be an inhibitor for both enzymes. Systematic variation of the aryl substituents led to analogs possessing almost the same good properties as gold standard galantamine hydrobromide.

Full text of DOI:10.1016/j.ejmech.2016.09.051

Accepted Manuscript
Repurposing N,N'-bis-(arylamidino)-1,4-piperazinedicarboxamidines: An unexpected
class of potent inhibitors of cholinesterases
Anne Loesche, Jana Wiese, Sven Sommerwerk, Vivienne Simon, Wolfgang Brandt,
René Csuk
PII:
S0223-5234(16)30787-5
10.1016/j.ejmech.2016.09.051
EJMECH 8917
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 August 2016
Revised Date: 15 September 2016
Accepted Date: 17 September 2016
Please cite this article as: A. Loesche, J. Wiese, S. Sommerwerk, V. Simon, W. Brandt, R. Csuk,
Repurposing N,N'-bis-(arylamidino)-1,4-piperazinedicarboxamidines: An unexpected class of
potent inhibitors of cholinesterases, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.09.051.
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ACCEPTED MANUSCRIPT
Repurposing N,N‘-bis-(arylamidino)-1,4-piperazinedicarboxamidines: an unexpected
class of potent inhibitors of cholinesterases
Anne Loesche a,#, Jana Wiese a,#, Sven Sommerwerk a, Vivienne Simon a, Wolfgang Brandt b,
René Csuk*a
# contributed equally
* corresponding author
a
Martin-Luther University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str. 2, D-
06120 Halle (Saale), Germany
b
Leibniz Institute of Plant Biochemistry, Bioorganic Chemistry, Weinberg 3, 06120 Halle
(Saale), Germany
Corresponding author:
Prof. Dr. René Csuk
Bereich Organische Chemie
Martin-Luther Universität Halle-Wittenberg
Kurt-Mothes-Str. 2
D-06120 Halle (Saale)
Germany
Tel.: +49 (0) 345 5525660
Fax: +49 (0) 345 5527030
Email: rene.csuk@chemie.uni-halle.de
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Graphical Abstract:
Repurposing N,N‘-bis-(arylamidino)-1,4-piperazinedicarboxamidines: an unexpected
class of potent inhibitors of cholinesterases
Anne Loesche, Jana Wiese, Sven Sommerwerk, Vivienne Simon, Wolfgang Brandt, René
Csuk*
2

ACCEPTED MANUSCRIPT
Abstract
Drug repurposing (= drug repositioning) is an effective way to cut costs for the development
of new therapeutics and to reduce the time-to-market time-span. Following this concept a
small library of compounds was screened for their ability to act as inhibitors of acetyl- and
butyrylcholinesterase. Picloxydine, an established antiseptic, was shown to be an inhibitor for
both enzymes. Systematic variation of the aryl substituents led to analogs possessing almost
the same good properties as gold standard galantamine hydrobromide.
Keywords: picloxydine; acetylcholinesterase; butyrylcholinesterase; bisbiguanides
1. Introduction
The development of a new drug is costly, and on average it takes about US$ 2-3 billion or
more to develop a new therapeutic. This process is also time-consuming, and the time-span
from target discovery to approval is about 12-16 years. The development of a new therapeutic
is complex since the failure rate during this process is about 95%.[1]
Several years ago, the concept of “repurposing” (also known as “repositioning”) has been
invented. This concept extends the “off-label-prescription” [2] already being in use for many
years. The concept of repurposing improves the development of new treatments by creating
new uses for “older” drugs that are already in use for another disease or have passed several
key-steps along the development paths.[1, 3] A successful example for drug repurposing
comprises the use of thalidomide for treatment of leprosy [4] or several malignant tumors [5,
6]. Last but not least it is worth to mention the more or less serendipitous discovery of
sildenafil for treating men suffering from erectile dysfunction.[7, 8]
The number of people with age-related diseases such as Alzheimer’s disease (AD) grows
continuously. AD causes progressive loss of memory, and worldwide nearly 44 million to
46.8 million [9,10] persons are affected. Applying a repurposing concept early in 2015 (from
a cooperation of the US based National Center for Advancing Translational Science, NCATS,
and Yale University School of Medicine) antitumor active saracatinib was found and
successfully tested in mice for treating AD.[11] Presently AD cannot be cured but the quality
of life for AD patients (or patients suffering from Myasthenia gravis pseudoparalytica) is
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significantly improved upon treatment with inhibitors of cholinesterases (ChE) such as
donezepil (reversible ChE inhibitor), galantamine (reversible ChE inhibitor), or rivastigmine
(irreversible ChE inhibitor).[12-16]
2. Results and discussion
2.1. Synthesis
Following this concept we screened a small library of established drugs for their ability to
inhibit enzymes of therapeutic interest, among them acetyl- and butyrylcholinesterase (AChE,
BChE).
To our surprise picloxydine (PD, Fig. 1), an antiseptic, was shown to be a good inhibitor for
BChE. PD is a bisbiguanide, and these compounds are known as useful microbiocides,
insecticides or disinfectants.[17] Except for a very early report [18] about the inhibitory effect
of paludrine (PA, a prophylactic antimalarial drug usually taken together with another
antimalarial drug, Fig. 1) on cholinesterases, to our knowledge, the cholinesterase inhibiting
effect of these compounds has not been investigated so far. Recently, cyclic acyl guanidines
bearing extra carbamate moieties [19] and aryl-urea bnzofuranylthiazole hybrids [20] have
been found to inhibit cholinesterases, too
Fig. 1. Structure of antiseptic picloxydine (PD) and anti-malarial paludrine (PA)
As a consequence we decided to investigate bisbiguanides more detail. The synthetic route to
bisbiguanides is depicted in Scheme 1.
Thus, commercially available (substituted) anilines were allowed to react at 60 °C with
sodium dicyanamide in the presence of aq. hydrochloric acid for three hours,[21] and the
corresponding N-aryl-N’-cyanoguanides 1-21 were obtained in good to excellent yields. From
the microwaves assisted reaction (120 °C, 2 hours) of 1-21 with piperazine the
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bis(arylamidino)-1,4-piperazine-dicarboxamids 22-43 were obtained. As exemplified for 22,
these compounds are characterized in their 13C NMR spectra by the presence of two signals at
δ = 159.7 ppm (assigned to the NHC(=NH)-piperazine fragment) and δ = 157.6 ppm [NH-
C(=NH)] while the 4 carbons of the piperazine moiety were detected at δ = 44.9 ppm. In their
ESI-MS spectra always a quasimolecular ion [M+2H]2+ was detected.
Scheme 1. Synthesis of the target compounds 22-43 starting from substituted amines and
dicyanamide: a) 1 eq. HCl (aq.), then 1 eq. NaN(CN)2, 60 °C, 3 hours; b) 2 eq. HCl (aq.),
piperazine (1 eq.), MeOH, then 4 eq. 1-20, 120 °C, 2 h (microwave-assisted).
2.2. Biological screening and structure activity relationships
Compounds 22-43 were subjected to Ellman’s test [22] to determine their ability to inhibit the
enzymes AChE and BCh; the inhibition constants Ki (for competitive inhibition) and Ki’ (for
non-competitive inhibition) as well as the type of inhibition was determined by using
Lineweaver-Burk plots, Dixon plots and Cornish-Bowden plots. The results from these assays
are compiled in Table 1.
Table 1. Inhibitory constants for galantamine hydrobromide (GH) and compounds 22-43 (Ki
(competitive inhibition) and Ki’ (non-competitive inhibition) in µM), determined using
Ellman`s assay employing acetylcholinesterase with galantamine hydrobromide (GH) as
standard.
Compound
X
Type of
Ki (µM)
Ki' (µM)
inhibition
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0.54±0.01
competitive
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
GH
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
H
13.53±0.14
14.92±0.25
14.86±0.32
7.77±0.24
6.11±0.03
4.45±0.29
22.00±0.09
9.34±0.79
4.19±1.74
6.17±0.51
3.43±0.92
1.56±0.25
2.79±0.26
5.08±0.87
4.34±0.47
10.41±0.66
25.55±1.37
7.74±0.98
19.83±1.83
9.25±0.04
1.41±0.06
0.52±0.06
69.16±2.56
58.58±1.97
104.27±9.61
16.19±0.17
66.68±11.5
> 30
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
28.31±0.24
75.30±5.69
40.53±22.01
25.40±2.22
35.55±1.63
> 100
2,4'-Cl
2-Br
3-Br
4-Br
2-I
3-I
19.94±2.43
49.10±5.62
13.68±0.82
45.23±2.71
88.86±2.79
41.21±0.56
62.24±8.95
> 100
4-I
2-OMe
3-OMe
4-OMe
3,4,5-OMe
4-OCF3
4-Me
4-tButyl
4-NO2
21.77±1.06
2.96±0.40
Table 2. Inhibitory constants for galantamine hydrobromide (GH) and compounds 22-43 (Ki
(competitive inhibition) and Ki’ (non-competitive inhibition) in µM), determined using
Ellman`s assay employing butyrylcholinesterase with galantamine hydrobromide (GH) as
standard.
Compound
X
Type of
inhibition
competitive
Ki (µM)
Ki' (µM)
2.07±0.29
0.95±0.04
6.82±1.32
1.70±0.01
1.18±0.16
1.85±0.34
0.74±0.08
0.62±0.03
0.41±0.15
GH
22
23
24
25
26
27
28
29
H
> 40
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
2-F
25.49±10.06
32.85±2.80
15.18±1.59
12.70±3.82
12.24±4.04
9.37±0.09
7.51±3.08
3-F
4-F
2-Cl
3-Cl
4-Cl
2,4'-Cl
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2-Br
1.25±0.05
0.23±0.11
0.61±0.09
1.96±0.09
0.21±0.02
0.31±0.12
5.20±0.17
3.02±0.01
1.78±0.13
9.03±0.51
3.34±0.23
0.45±0.05
0.12±0.02
10.78±0.81
12.34±3.83
10.32±3.02
3.84±0.32
11.19±0.13
3.04±1.37
2.74±0.69
33.96±0.93
54.84±3.43
35.13±4.97
---
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
Mixed type
competitive
Mixed type
Mixed type
Mixed type
Mixed type
30
31
32
33
34
35
36
37
38
39
40
41
42
43
3-Br
4-Br
2-I
3-I
4-I
2-OMe
3-OMe
4-OMe
3,4,5-OMe
4-OCF3
4-Me
4-tButyl
4-NO2
> 20
11.80±4.97
4.41±1.42
22.87±4.79
To get a deeper insight into the mode of action of these compounds, some molecular
modelling calculations were performed. The ligands contain protonable nitrogen atoms at
three different moieties in the compounds. To decide which one are most likely protonated
when binding to the enzymes, the proton affinities (for a single protonation only) were
calculated. The results clearly predict that protonation at the piperazine ring is disfavored by
26.8 kcal/mol. Thus protonation at this site can be ruled out under physiological conditions in
the active site of the enzymes. Despite a favored protonation at the second guanidinyl group
by 9.7 kcal/mol (as compared to a protonation of the guanidinyl moiety close to the piperazine
ring) both protonation options were taken into consideration during docking. Altogether three
forms of each ligand were used for docking, the neutral unprotonated one and both
symmetrically protonated at the guanidine moieties at both sides, i.e. double protonation.
The results from the calculations showed for all docking results, that the protonated forms of
the ligands are clearly favored over the neutral form. This is due to the fact that in both
enzymes there are two negatively charged amino acid residues (AchE: D74, E202;
BChE:D70, E197) allowing the formation of salt bridges with the ligands. However, for AchE
no docking arrangement could be observed where one of the protonated guanidino groups
interacts with E202, whereas in BChE this appears quite often. There is an essential difference
between the active sites of AChE and BChE. The gorge in AchE is much narrower than the
one in BChE (Figure 2).
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Fig. 2: Comparison of the active sites of AChE with bound 43 (p-NO2, left) and BChE with
bound 34 (m-I, right) from the top.
This striking difference is also reflected when comparing the most favored docking
arrangements of the most active compounds displayed in Figure 3. Whereas for AChE in all
poses the ligands adopt an almost extended conformation, for BChE the ligands are folded or
bend because of the available space for optimal interactions. The most active compound for
AchE inhibition is 43 which gave a significantly higher Ki value for BChE. In AChE one
guanidino moiety forms a strong salt bridge with D74 and the other one a hydrogen bond with
the backbone carbonyl of S293. Furthermore, a hydrogen bond is formed between the p-NO2
group and the hydroxyl group of Y133. Molecular dynamics simulations over 5 ns in a water
box applying periodic boundary condition proved the stability of the entire complex with only
minor movements of the ligand in the active site. The presence of some water molecules
(having been automatically placed in the active site) led to a further stabilization of the
complex by bridging water hydrogen bonds between the guanidino groups and E202, E292
and N87.
All other compounds except the p-NO2 derivative 43 were not able to form such a hydrogen
bond with Y133; this obviously led to the reduction of their affinity to the enzyme. In BChE
one guanidino group forms a salt bridge with E197, and W82 may form π−π interactions with
the same moiety. In comparison to the binding pose with AchE there is only a (not perfect)
salt bridge of one of the guanidino groups with E74 and a hydrogen bond with the carbonyl
backbone of S293. Corresponding molecular dynamics simulations (performed as for 43 and
AchE) resulted in only one new bridging water hydrogen bond with of one nitro-group with
E197. There were no further stabilizing interactions as they have been discussed for the the p-
NO2 substituted ligand.
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One of the most active compounds for binding to BChE is 35 holding p-I substituents. The
attractive interaction is favored by the formation of an almost perfect salt bridge between one
guanidino group with E197 and hydrophobic interactions of the piperazine moiety with W82.
Furthermore, one of the p-iodine substituents may form attractive interactions via the positive
electrostatic potential of the sigma hole with the carbonyl group of side chain of N83 (red
dashed line in Figure 3b). It is known, that the smaller the halogen atoms, the smaller is the
sigma hole and thus the ability to form attractive interactions with lone pairs. This indeed
correlates with the stepwise reduced affinity of the p-Br, p-Cl and p-F which has almost no
positive sigma hole. The moderate activity of p-t-but substituted 42 will be discussed (vide
infra) in comparison with it surprisingly high affinity to BChE. The docking arrangements in
both AchE and BChE for the meta substituted iodine derivative 34 are very similar to the ones
shown for the p-I substituted compound 35. Thus, these calculations nicely explain the
experimental findings.
Another very interesting compound is 42 holding two tert-butyl groups in the para positions.
This compound is a good inhibitor of BChE but displays only weak inhibition for AChE.
Calculations showed the formation of a salt bridge of one guanidino group with E197 but also
the intra-molecular hydrophobic interaction between one of the tert-butyl moieties with the
other aromatic ring system. This interaction highly supports the formation of a bend
conformation resulting in binding almost perfectly into the active site. The preferred bend
conformation for binding into the active site is also reflected by the much lower
conformational force field energy (~ -80 kcal/mol) of the isolated bend conformation (like
bound in BChE) in comparison to the extended one formed in AChE. For AChE the formation
of a salt bridge of one guanidino group with D74 is observed and hydrophobic interactions of
one tert-butyl group with the side chain of W86 support binding in the extended
conformation.
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Fig. 3: Docking of the most favored arrangements of a) 43 (para-NO2), b) 35 (para-I), and c)
42 (para- tert-butyl), to the acetylcholinesterase (left) and to butyrylcholinesterase (right)
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3. Conclusion
Following the concept of drug repurposing a small library of compounds was screened for
their ability to act as inhibitor of enzymes being of interest in the treatment of diseases. Thus,
picloxydine, an established antiseptic, was shown to be an inhibitor for acetyl- and
butyrylcholinesterase. Systematic variation of the aryl substituents led to analogs possessing
almost the same enzyme inhibiting properties as gold standard galantamine hydrobromide.
While p-nitro substituted compound 43 was a good inhibitor for AChE, p-tert-butyl
substituted bisbiguanide 42 inhibited BChE in the low µM range.
4. Experimental part
4.1.General
Detailed procedures for the synthesis, screening and modelling as well as a description of the
equipment is given in the Supplementary material.
4.2. General procedure for the synthesis of N-Cyano-N’-aryl- guanidines (1-21)
To a solution of substituted aniline (22.49 mmol) in water (40 mL), hydrochloric acid (aq., 37
%, 1.9 mL, 22.49 mmol) and sodium dicyanamide (2.01 g, 22.49 mmol) were added. The
mixture was stirred at 60 °C for three hours. After cooling to 0 °C for several hours, the crude
product was filtered off and washed with ether.
4.3.General
procedure
for
the
synthesis
of
N,N’-Bis-(arylamidino)-1,4-
piperazinedicarboxamidines (22-43)
To a solution of piperazine (100 mg, 1.16 mmol) in methanol (4 mL), hydrochloric acid (aq.,
37%, 229 mg, 2.32 mmol) and the N-cyano-N’-aryl-guanidine (1-21) (4.64 mmol) were
added. The reaction mixture was heated to 120 °C for two hours in a microwave reactor. After
addition of methanol (4 mL) and aq. sodium hydroxide solution (1.16 mL, 2 M), the mixture
was cooled to 0 °C, and the crude product 22-43 was collected by filtration and re-crystallized
from methanol to yield pure 22-43 each as a colorless solid.
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Acknowledgments
We like to thank Dr. R. Kluge for measuring the ESI-MS spectra and Dr. D. Ströhl and his
team for recording the NMR spectra. Many thanks are due to Ms. J. Pech for skillful help in
the lab and for obtaining the IR-, UV-Vis spectra and the microanalyses.
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Abbreviations
Acetylcholinesterase (AChE), butyrylcholinesterase (BChE), galantanine hydrobromide
(GH), picloxydine (PD), paludrine (PA).
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•
•
•
•
Twenty one analogs of the antiseptic agent picloxydine were synthesized
They were screened for their ability to inhibit cholinesterases AChE and BChE
A p-nitro substituted bisbiguanide 43 was a good inhibitor for AChE
A p-tert-butyl substituted analog 42, however, inhibited BChE in the low µM range