Design, synthesis and in vitro evaluation of benzothiazole-based ureas as potential ABAD/17β-HSD10 modulators for Alzheimer's disease treatment

Article abstract of DOI:10.1016/j.bmcl.2016.05.087

Amyloid-beta peptide (Aβ) has been recognized to interact with numerous proteins, which may lead to pathological changes in cell metabolism of Alzheimer's disease (AD) patients. One such known metabolic enzyme is mitochondrial amyloid-binding alcohol dehydrogenase (ABAD), also known as 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10). Altered enzyme function caused by the Aβ-ABAD interaction, was previously shown to cause mitochondrial distress and a consequent cytotoxic effect, therefore providing a feasible target in AD drug development. Based on previous frentizole derivatives studies, we report two novel series of benzothiazolyl ureas along with novel insights into the structure and activity relationships for inhibition of ABAD. Two compounds (37, 39) were identified as potent ABAD inhibitors, where compound 39 exhibited comparable cytotoxicity with the frentizole standard; however, one-fold higher cytotoxicity than the parent riluzole standard. The calculated and experimental physical chemical properties of the most potent compounds showed promising features for blood–brain barrier penetration.

Full text of DOI:10.1016/j.bmcl.2016.05.087

Accepted Manuscript
Design, synthesis and in vitro evaluation of benzothiazole-based ureas as po-
tential ABAD/17β-HSD10 modulators for Alzheimer’s disease treatment
Lukas Hroch, Ondrej Benek, Patrick Guest, Laura Aitken, Ondrej Soukup, Jana
Janockova, Karel Musil, Vlastimil Dohnal, Rafael Dolezal, Kamil Kuca, Terry
K Smith, Frank Gunn-Moore, Kamil Musilek
PII:
S0960-894X(16)30597-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.087
BMCL 23946
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 March 2016
26 May 2016
28 May 2016
Please cite this article as: Hroch, L., Benek, O., Guest, P., Aitken, L., Soukup, O., Janockova, J., Musil, K., Dohnal,
V., Dolezal, R., Kuca, K., Smith, T.K., Gunn-Moore, F., Musilek, K., Design, synthesis and in vitro evaluation of
benzothiazole-based ureas as potential ABAD/17β-HSD10 modulators for Alzheimer’s disease treatment,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.087
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Design, synthesis andin vitro evaluation of benzothiazole-based ureasaspotential ABAD/17β-HSD10
modulators for Alzheimer’sdisease treatment
Lukas Hroch#a,b, Ondrej Benek#b,c, Patrick Guest#d, Laura Aitken#d, Ondrej Soukupb, Jana Janockovab,
Karel Musilb,f, Vlastimil Dohnalf, Rafael Dolezalb,e, Kamil Kucab,f, Terry K Smithg, Frank Gunn-Moored,
Kamil Musilekb,f*
a Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Pharmaceutical Chemistry
and Drug Control, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
b University Hospital, Biomedical Research Center, Sokolska 581, 500 05 Hradec Kralove, Czech Republic
c
University of Defence, Faculty of Military Health Sciences, Department of Toxicology, Trebesska 1575, 500 01
Hradec Kralove, Czech Republic
d
University of St. Andrews, School of Biology, Medical and Biological Sciences Building, North Haugh, St.
Andrews KY16 9TF, United Kingdom
e
University of Hradec Kralove, Faculty of Informatics and Management, Center for Basic and Applied Research,
Rokitanskeho 62, 500 03 Hradec Kralove, Czech Republic
f
University of Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62, 500 03 Hradec
Kralove, Czech Republic; kamil.musilek@gmail.com
g Biomedical Science Research Complex, University of St. Andrews, North Haugh, St. Andrews. KY16 9ST, United
Kingdom
# These authors contributed equally to the paper
Abstract
Amyloid-beta peptide (Aβ) has been recognized to interact with numerous proteins, which may lead
to pathological changes in cell metabolism of Alzheimer’s disease (AD) patients. One such known
metabolic enzyme is mitochondrial amyloid-binding alcohol dehydrogenase (ABAD), also known as
17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10). Altered enzyme function caused by the Aβ-
ABAD interaction, was previously shown to cause mitochondrial distress and a consequent cytotoxic
effect, therefore providing a feasible target in AD drug development. Based on previous frentizole
derivatives studies, we report two novel series of benzothiazolyl ureas along with novel insights into
the structure and activity relationships for inhibition of ABAD. Two compounds (37, 39) were
identified as potent ABAD inhibitors, where compound 39 exhibited comparable cytotoxicity with the
frentizole standard; however, one-fold higher cytotoxicity than the parent riluzole standard. The
calculated and experimental physical chemical properties of the most potent compounds showed
promising features for blood-brain barrier penetration.
1

Keywords
Alzheimer's disease (AD), amyloid-beta peptide (Aβ), mitochondria, amyloid binding alcohol dehydrogenase
(ABAD), 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10), benzothiazole, riluzole
Alzheimer’s disease (AD) is the most common form of dementia in the elderly, characterized by
the slow deterioration of cognitive functions.1 The accumulation of amyloid-beta peptide (Aβ) and its
formation into plaques, along with the formation of neurofibrillary tangles, highlights the
pathological changes in affected brain regions with progressed AD. These hallmarks have become
commonly monitored markers, however the initiating events still remain unclear.2 Even though a
precise mechanism of Aβ-induced toxicity has not been fully understood, several studies have
reported synaptic and mitochondrial Aβ accumulation and dysfunction in early stages of AD
development.2–4 Furthermore, it has been described that Aβ interacts with various mitochondrial
proteins, resulting in enhanced oxidative stress, energy misbalance and overall cell toxicity.2,5–8
Amyloid-binding alcohol dehydrogenase (ABAD), also known as 17β-hydroxysteroid
dehydrogenase type 10 (17β-HSD10), is one of the proteins which was identified to interact directly
with Aβ at nanomolar concentrations.9,10 Moreover, it has been reported that the interaction
between Aβ and ABAD promotes oxidative stress and mitochondrial dysfunction, consequently
resulting in cell death.5 Cell based assays and transgenic mice experiments demonstrated that the
overexpression of both Aβ and ABAD show enhanced cell cytotoxicity, reduced levels of ATP and COX
activity along with impaired energy metabolism in mice. Conversely, the overexpression of Aβ with
inactive ABAD displayed less cytotoxicity and transgenic (ABAD) mice when compared with non-
transgenic mice do not display these changes.7,10 Lim et al. reported an ABAD inhibition study where
the compound (AG18051) appeared to reduce the levels of Aβ-induced oxidative stress and
mitochondrial respiration impairment, as well as alleviate the Aβ-induced down-regulation of ABAD
activity.11 Whereas elevated levels of ABAD have been associated with AD pathology.10,12 Reduced
levels of ABAD has been reported in brain of Parkinson’s disease patients.13 These findings suggest
that both ABAD-Aβ interaction and ABAD itself may represent a viable objective for deeper
understanding of AD pathogenesis in context of Aβ-induced toxicity. Consequently, it may also aid in
the development of novel AD therapeutics.
There has been a limited number of reported compounds acting as ABAD or ABAD-Aβ interaction
inhibitors.14,15 In 2006, Xie et al. described the marketed drug frentizole acting as a poor inhibitor of
ABAD-Aβ interaction (IC50 ~200 µM) along with a novel series of synthesised frentizole analogues
displaying a 30-fold increase in improved potency (IC50 ~6.5 µM).16 A more recent study reported two
phosphonate analogues of these previously discussed compounds, which also exhibited moderate to
2

weak ABAD inhibition (IC50 53 µM and 342 µM).17,18 Thus, our aim of the study was focused on
generating more potent inhibitors based around the benzothiazole scaffold. To this end, a first series
was designed around a 6-halogen-benzothiazole urea scaffold with various phenyl ring substitution
patterns including those previously reported (Scheme 1).16–18 Furthermore, many benzothiazole
analogues have been shown to possess various biological activities in the central nervous system.19
Riluzole, a well-known drug with neuroprotective properties, has a similar benzothiazolyl core,
however its neuroprotective mechanism of action is not still completely understood.20 Riluzole is now
an FDA approved drug to treat amyotrophic lateral sclerosis, and several other studies report riluzole
exhibiting neuroprotective properties in other neurological disorders (e.g. Parkinson’s disease,
Huntington’s disease or cerebral ischemia).21–23 Riluzole possesses a wide range of mechanisms of
action including anti-glutamate activity, Na+ and Ca2+ channel blockage, GABAergic modulation20
throughout the modulation of the excitatory cascade.24 As Aβ is known to disturb cell ionic
homeostasis on various levels including calcium balance,25 it can be hypothesized that a riluzole
moiety may partially mitigate such Aβ-induced ionic homeostasis misbalance. Thus, a second series
of compounds was designed with a riluzole core moiety and further combined with a urea linker and
substituted phenyl moiety to more closely address the effect of phenyl ring substitution variations
(Scheme 1). The urea linker was selected based on compounds that were found to be the most
potent in perturbing the ABAD-Aβ interaction.16 Additionally, the phenyl substitutions were based
upon the first series of compounds, where oxygen-based substitutions with additional halogen
substitutions were selected. The second series includes both overlapping substitutions of the first
compound series (to confirm possible lead structures and validate structure-activity relationship).
More importantly additional variations of the phenyl ring were made to more exhaustively explore
possible phenyl ring variations and structure-activity relationship necessary for ABAD inhibition.
3

Scheme 1. Design of benzothiazole-based urea ABAD inhibitors.
While commercially available 6-flouro and 6-chloro substituted benzo[d]thiazole-2-amines were
used in the first series of compounds, 6-(trifluoromethoxy)benzo[d]thiazol-2-amine (riluzole) was
prepared from its corresponding para-substituted aniline derivative (Scheme 2). Therefore, 4-
(trifluoromethoxy)aniline was treated with potassium thiocyanate and bromine to afford 6-
(trifluoromethoxy)benzo[d]thiazol-2-amine (1) in excellent yield (94%).26
Scheme 2. Synthesis of 4-(trifluoromethoxy)aniline 1. Reagents and conditions: (a) Br2, KSCN, CH3COOH.
Subsequently, two series of benzothiazolyl ureas 5-50 were prepared in a two-step synthesis
(Scheme 3). Selected 6-subtituted benzo[d]thiazole-2-amines were activated with N,N′-
carbonyldiimidazole to obtain intermediates 2-4 in excellent yields (90-95%), followed by subsequent
treatment with a substituted aromatic amine16 to produce final benzothiazole ureas 5-50 (Table 1) in
moderate to excellent yields (36-99%).
4

Scheme 3. Synthesis of benzothiazolyl ureas 5-50. Reagents and conditions: (a) CDI, DCM, reflux; (b) Ar-NH2, MeCN, reflux.
A series of compounds (5-50) was screened at 25 µM to determine their ABAD inhibitory ability
(Table 1) and to show their structure-activity relationship. Within the first series neither frentizole
nor compounds 5, 6, 11 and 15 (as with previously reported substitutions16–18) showed any
noteworthy ABAD inhibitory ability. However, compounds 13 and 14 showed a substantial decrease
(~60%) in ABAD activity. Furthermore, compound 12 showed a notable decrease (~20%) in ABAD
activity. Therefore, selected substitutions were included in the second riluzole-based series with
additional phenyl ring substitution patterns. The para-hydroxy along meta-chlorine substitution
patterns proved to display the most pronounced inhibitory activity, where compound 37 showed a
significant decrease (~80%) in ABAD activity. Moreover, the second chlorine in close proximity of the
phenolic group (39) showed comparable inhibitory activity with compound 37. Therefore, the
presence of the free phenolic group attached to the distal phenyl ring, is shown to be essential for its
ability to inhibit ABAD, and the addition of an electron-withdrawing group (such as chlorine) in its
adjacent proximity greatly increases such an inhibitory ability. Other compounds with a single
phenolic group (12, 33-35), displacement of 3-chloro-4-hydroxy pattern (36, 38) or isosteric change
of hydroxyl to thiol group (50) showed a less pronounced drop in ABAD activity (10-30 %). However,
such a low decrease in enzymatic activity could be accounted by an experimental error. Thus these
substitution patterns did not provide sufficient SAR information, but they rather indicate the
presence of weak ABAD inhibition. Interestingly, 3,4-dihydroxy substitution displayed a similarly low
decrease in enzyme activity (~20%). On the other hand, compound 41 revealed an approximately 20%
increase in ABAD activity, which will be further investigated. Both compounds 37 and 39
demonstrated a two-fold decrease in ABAD activity when compared to compounds 13 and 14, thus
underlining that the introduction of a 6-trifluromethoxy moiety instead of a 6-halogen moiety, led to
an increased inhibitory ability towards ABAD. IC50 values were attempted to be determined for
compounds 37 and 39, however, this data was unable to be obtained due to the limited solubility of
these compounds within the enzyme activity assay, and due to the possibility that a biophysical
effect was observed rather than a true ligand-protein interaction.
Table 1. Prepared benzothiazole ureas (5-50) and their in vitro evaluation.
5

Relative % activity
remaining @25 µM1
Comp.
R1
R2
Control
Frentizole
5
-
OCH3
F
-
100 ± 1.2
97.9 ± 3.6
97.2 ± 3.8
95.6 ± 4.5
94.4 ± 1.1
91.9 ± 1.9
94.8 ± 1.0
93.6 ± 1.9
91.5 ± 1.5
77.1 ± 1.0
39.8 ± 0.5
38.6 ± 0.7
101.3 ± 1.6
100.5 ± 1.1
110.9 ± 1.8
104.6 ± 1.2
103.6 ± 1.9
104.4 ± 1.5
105.5 ± 1.5
107.2 ± 2.5
108.8 ± 2.2
107.4 ± 1.9
107.8 ± 1.7
105.5 ± 1.7
110.3 ± 1.7
108.6 ± 2.2
114.7 ± 5.3
99.3 ± 3.2
94.7 ± 3.3
97.9 ± 3.4
92.0 ± 0.1
78.8 ± 0.1
69.6 ± 0.1
92.0 ± 1.6
17.3 ± 0.8
65.5 ± 3.0
20.3 ± 1.1
104.8 ± 2.6
119.8 ± 2.1
94.8 ± 5.6
-
3-COOMe, 4-OH
3-COOMe, 4-OH
4-OH
6
Cl
7
F
8
Cl
4-OH
9
F
3-OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Cl
3-OH
F
2-OH
Cl
2-OH
F
3-Cl, 4-OH
3-Cl, 4-OH
3-COOH, 4-OH
3-COOH, 4-OH
4-OMe
Cl
F
Cl
F
Cl
4-OMe
F
3,4-OMe
3,4-OMe
3-COOH, 4-OMe
3-COOH, 4-OMe
4-OPh
Cl
F
Cl
F
Cl
4-OPh
F
4-COOH
Cl
4-COOH
F
4-COOEt
4-COOEt
4-COOMe
4-COOMe
4-NHCOMe
4-NHCOMe
2-OH
Cl
F
Cl
F
Cl
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
3-OH
4-OH
2-Cl, 4-OH
3-Cl, 4-OH
2-OH, 4-Cl
3,5-Cl, 4-OH
3-Cl, 4-COOH
2-OH, 4-COOH
3-OH, 4-COOH
6

43
44
45
46
47
48
49
50
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
3-COOH, 4-OH
4-OMe
96.6 ± 5.5
100.7 ± 2.4
91.0 ± 2.6
98.3 ± 6.1
78.9 ± 5.9
86.8 ± 3.0
98.2 ± 1.8
80.6 ± 5.0
3-Cl, 4-OMe
3-OH, 4-OMe
3,4-OMe
3-COOH, 4-OMe
4-COOMe
2-SH
1 Relative remaining activity is displayed in percentage of 3 independent measurements ± SEM.
A key aspect in the design of any potential ABAD inhibitors is that they should be CNS
penetrable. Thus, essential physical chemical properties (HBA, HBD, TPSA, ClogP, ClogD) for efficient
blood-brain barrier (BBB) penetration were calculated for the two most promising inhibitors 37 and
39 using ACDLabs PhysChem Suite 2014 (Table 2). The calculated values of the selected physical
chemical properties either oscillate close to the upper threshold or were found to be slightly higher
than optimal values proposing CNS bioavailability. Additionally, logP and logD values (further referred
as ElogP and ElogD) were experimentally determined using adapted OECD guideline.27 Both
calculated values of ClogP and ClogD were found to be in very good agreement with experimental
data. The presented physical chemical properties of 37 and 39 showed reasonable conformity with
the requirements for centrally acting compounds28 (based on earlier defined Lipinski rule of five for
orally administrated drugs).29 Experimental prediction of BBB penetration is further shown in Table 2.
Data obtained for 37 and 39 are correlated to standard drugs (e.g. donepezil) , where CNS availability
is known and also reported using the PAMPA assay (Supporting information, Table S1).30 The
acquired data from the PAMPA assay predicted a possible high BBB penetration for compound 39,
whereas experimental data for compound 37 estimated a rather uncertain BBB penetration.
Table 2. Physicochemical properties of 37 and 39.
TPSA
(Å2)
1
Comp.
Mw
HBA/HBD
ClogP ± SD1 ELogP ± SD2 ClogD7.4 ELogD7.4 ± SD2 Pe ± SEM3
Optimal
LR5 28,29
≤ 450
≤ 7 / ≤ 3
4/0
≤ 90
≤ 5
≤ 5
---
0-3
2.79
3.35
0-3
---
-
donepezil 379.49
38.8
111.7
4.23 ± 0.40
4.24 ± 0.41
7.3 ± 0.9
2.2 ± 0.4
4.07 ±
0.11‰
4.65 ±
0.43‰
37
403.76
6/3
4.04 ± 2.16‰
39
438.20
6/3
111.7
5.18 ± 0.43
3.79
4.40 ± 2.17‰
7.0 ± 1.4
1 The values were calculated by ACDLabs PhysChem Suite 2014.
2 The values refer to experimentally acquired data.
Prediction of blood-brain barrier penetration of drugs expressed as Pe ± SEM (n = 2-4). High BBB permeation
3
predicted for Pe > 4; BBB permeation uncertain for Pe between 2.0 and 4.0; low BBB permeation predicted for
< 2.0
7

The two most potent compounds (37, 39) were assayed for acute cytotoxicity evaluation
(Table 3). The compounds’ acute cytotoxicity was determined via a combined MTT and LDH assay
using CHO-K1 cell lines. A combined cytotoxicity assay was chosen due to the MTT test being partially
dependent on the mitochondrial oxidoreductases,31 whose activity may be influenced by compounds
targeted to mitochondria. Both MTT and LDH assays for compound 37 showed a greater cytotoxicity
when compared to frentizole, whereas compound 39 displayed comparable cytotoxicity level with
frentizole. All evaluated benzothiazolyl ureas (frentizole and compounds 37, 39) showed one order of
magnitude higher acute cytotoxicity compared to riluzole. In general, the MTT assay data showed a
trend of a slightly lower IC50 value compared to the LDH data. However, this fact may be caused by
the compounds’ involvement in the mitochondrial electron transport chain and requires to be
further elucidated.
Table 3. Acute cytotoxicity evaluation of compound 37 and 39.
IC50 ± SEM1(µM)
Comp.
MTT CHO-K1
LDH CHO-K1
frentizole
riluzole
37
31 ± 3
310 ± 33
7.7 ± 0.7
31 ± 5
46 ± 6
480 ± 45
9.0 ± 1.7
41 ± 12
39
1 The IC50 value refers to 3 independent measurements ± SEM.
In summary, two novel series of benzothiazolyl ureas were designed and synthesised. All
compounds were evaluated for ABAD inhibitory ability in vitro, where two riluzole-based compounds
(37, 39) showed the most promising ABAD inhibitory activity. In terms of the structure-activity
relationship for the benzothiazole-urea-phenyl scaffold, the combination of a 4-phenolic moiety with
a chlorine in its close proximity was confirmed to be essential for compounds ABAD inhibitory ability
(13, 14, 37, 39). Possible good BBB permeation was predicted for compound 39 by experimental
determination, and the cytotoxicity of compound 39 was also found to be similar to the frentizole
standard, but was one order of magnitude higher to its parent compound riluzole. Overall the most
promising compounds (37 and 39) exhibited solubility issues within the activity assay and were
predicted with at least a borderline BBB penetration.
8

Acknowledgement
This work was supported by the Ministry of Health of the Czech Republic (no. NV15-28967A),
Charles University in Prague (no. GAUK B-CH/992214, SVV 260 291) and the Alzheimer’s Society
(specifically The Barcopel Foundation). This research is part-funded by the MSD Scottish Life Sciences
fund. As part of an on-going contribution to Scottish life sciences, Merck Sharp & Dohme Limited
(MSD), a global healthcare leader, has given substantial monetary funding to the Scottish Funding
Council (SFC) for distribution via the Scottish Universities Life Science Alliance (SULSA) to develop and
deliver a high quality drug discovery research and training programme. All aspects of the programme
have been geared towards attaining the highest value in terms of scientific discovery, training and
impact. The opinions expressed in this research are those of the authors and do not necessarily
represent those of MSD, nor its Affiliates.
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Graphical abstract
Design, synthesis and in vitro evaluation of benzothiazole-based ureas as potential ABAD/17β-HSD10 modulators for
Alzheimer’sdiseasetreatment
Lukas Hroch, Ondrej Benek, Patrick Guest, Laura Aitken, Ondrej Soukup, Jana Janockova, Karel Musil,
Vlastimil Dohnal, Rafael Dolezal, Kamil Kuca, Patrick Guest, Terry K Smith, Frank Gunn-Moore, Kamil
Musilek*
11