Synthesis and evaluation of new pyridyl/pyrazinyl thiourea derivatives: Neuroprotection against amyloid-β-induced toxicity

Article abstract of DOI:10.1016/j.ejmech.2017.09.043

Herein, we report synthesis and evaluation of new twenty six small molecules against β amyloid (Aβ)-induced opening of mitochondrial permeability transition pore (mPTP) using JC-1 assay which measures the change of mitochondrial membrane potential (ΔΨm). The neuroprotective effect of seventeen compounds against Aβ-induced mPTP opening was superior to that of the standard Cyclosporin A (CsA). Fifteen derivatives eliciting increased green to red fluorescence percentage less than 40.0% were evaluated for their impact on ATP production, cell viability and neuroprotection against Aβ-induced neuronal cell death. Among evaluated compounds, derivatives 9w, 9r and 9k had safe profile regarding ATP production and cell viability. In addition, they exhibited significant neuroprotection (69.3, 51.8 and 48.2% respectively). Molecular modeling study using CDocker algorithm predicted plausible binding modes explaining the elicited mPTP blocking activity. Hence, this study suggests compounds 9w, 9r and 9k as leads for further development of novel therapy to Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2017.09.043

Accepted Manuscript
Synthesis and evaluation of new pyridyl/pyrazinyl thiourea derivatives:
Neuroprotection against amyloid-β-induced toxicity
Jung-eun Park, Ahmed Elkamhawy, Ahmed H.E. Hassan, Ae Nim Pae, Jiyoun Lee,
Sora Paik, Beoung-Geon Park, Eun Joo Roh
PII:
S0223-5234(17)30758-4
10.1016/j.ejmech.2017.09.043
EJMECH 9758
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 August 2017
Revised Date: 20 September 2017
Accepted Date: 21 September 2017
Please cite this article as: J.-e. Park, A. Elkamhawy, A.H.E. Hassan, A.N. Pae, J. Lee, S. Paik, B.-G.
Park, E.J. Roh, Synthesis and evaluation of new pyridyl/pyrazinyl thiourea derivatives: Neuroprotection
against amyloid-β-induced toxicity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.09.043.
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Graphical Abstract:

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Synthesis and evaluation of new pyridyl/pyrazinyl thiourea derivatives:
Neuroprotection against amyloid-β-induced toxicity
Jung-eun Parka,b,1, Ahmed Elkamhawya,c,1,*, Ahmed H. E. Hassand,e, Ae Nim Paef,g, Jiyoun Leeh,
Sora Paika,i, Beoung-Geon Parkf, Eun Joo Roha,g,*
a Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea
b Department of Chemistry, Sogang University, Seoul 04107, Republic of Korea
c Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
d Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
e Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea
f Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology (KIST), Seoul
02792, Republic of Korea
g Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Republic of Korea
h Department of Global Medical Science, Sungshin Women's University, Seoul 142-732, Republic of Korea
i Department of Fundamental Pharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul, 02447, Republic of Korea
Corresponding authors:
Ahmed Elkamhawy (e-mail: a.elkamhawy@gmail.com) & Eun Joo Roh (e-mail:
r8636@kist.re.kr
)
1 Both authors contributed equally to this work

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Abstract
Herein, we report synthesis and evaluation of new twenty six small molecules against β amyloid
(Aβ)-induced opening of mitochondrial permeability transition pore (mPTP) using JC-1 assay
which measures the change of mitochondrial membrane potential (∆Ψm). The neuroprotective
effect of seventeen compounds against Aβ-induced mPTP opening was superior to that of the
standard Cyclosporin A (CsA). Fifteen derivatives eliciting increased green to red fluorescence
percentage less than 40.0% were evaluated for their impact on ATP production, cell viability and
neuroprotection against Aβ-induced neuronal cell death. Among evaluated compounds,
derivatives 9w, 9r, and 9k had safe profile regarding ATP production and cell viability. In
addition, they exhibited significant neuroprotection (69.3, 51.8, and 48.2% respectively).
Molecular modeling study using CDocker algorithm predicted plausible binding modes
explaining the elicited mPTP blocking activity. Hence, this study suggests compounds 9w, 9r,
and 9k as leads for further development of novel therapy to Alzheimer’s disease.
Keywords: β-amyloid peptide (Aβ); Mitochondrial permeability transition pore (mPTP); Aβ-
induced neurotoxicity; Alzheimer’s disease (AD); Thiourea; Cyclophilin D; Molecular docking
.

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1. Introduction
Mitochondria are double-membraned subcellular organelles responsible mainly for vital cellular
processes controlling energy production and calcium ions storage. Among subcellular organelles,
mitochondria are unique with their high transmembrane potential that could reach up to 180 mV
with an internal negative membrane potential [1, 2]. This electric potential across the membrane
is essential for the flow of protons through ATP synthase and, hence, ATP generation. It is also
the impetus for calcium uptake by mitochondria [3]. A mega channel known as mitochondrial
permeability transition pore (mPTP) capable of permeating solutes up to 1500 Da, has been
identified across mitochondrial membranes [4]. Under normal physiological conditions, mPTP
elicits a low conductance which assists in maintaining the potential across the membrane. In fact;
acute mPTP opening under these conditions participates in calcium regulation and cycling [5-7].
Nonetheless, under pathological conditions, aberrant extended opening of mPTP dissipates the
mitochondrial membrane potential, resulting in cessation of ATP production, disruption of
mitochondrial homeostasis, swelling and rupture of mitochondrial membrane with release of cell
death factors [8-11]. As mitochondria are distributed in almost every tissues’ cells within the
body, detrimental mPTP opening can be manifested in a wide variety of diseases including
neurodegenerative diseases, cardiovascular diseases and hepatic diseases [12-22]. Consequently,
development of mPTP blockers might be a promising approach for cytoprotection against
diseases whose etiology is due to loss of mitochondrial membrane potential [23-26].
Since mPTP discovery in 1979 by Haworth and Hunter [27], there has been a long-lasting debate
about its molecular components. The latest accepted model suggests mPTP as a conglomerate of

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several molecular subunits with a core formed by dimers of F0/F1 ATP synthase. This core is in a
direct interaction with cyclophilin D (CypD) in the mitochondrial matrix. Other molecular
components of this suggested model include adenine nucleotide translocator (ANT), inorganic
phosphate carrier (PiC), mitochondrial creatine kinase (CK), voltage dependent anion channel
(VDAC), Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist killer (Bak). In
addition to these structural elements, regulatory elements have been proposed in this model
including mitochondrial translocator protein (TSPO), protein kinase C epsilon (PKCε) and
glycogen synthase kinase 3-beta (GSK3-β) [28]. Whether this proposed model or other suggested
model is the exact model, it is a fact that CypD has been always an integral component of every
suggested mPTP model [29, 30]. In fact, Haworth’s discovery of mPTP [27] was confirmed
when permeability transition was blocked by the inhibitory effect of cyclosporin A (CsA) on
CypD [31, 32].
Despite being an effective mPTP inhibitor, CsA is also a powerful immunosuppressant. This
renders it unsuitable as a therapeutic agent for in vivo mPTP opening inhibition [33]. Moreover,
its polypeptidic structure imparts poor kinetic properties for crossing blood brain barrier (BBB)
which hampers its bioavailability to central nervous system neurons [34]. Apparently, the
development of non-immunosuppressant small molecule inhibitors of mPTP having good BBB-
crossing kinetics could be a viable approach to achieve neuronal cytoprotection and hence
prevention of neurodegenerative diseases progression. Actually, non-immunosuppressant
analogs of CsA have been developed exemplified by NIM811 and UNIL025. Nevertheless, these
polypeptidic molecules suffer from side effects coupled with poor BBB-crossing kinetics which
curbs their use in neurological diseases [35, 36].

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Reports of efforts to develop mPTP opening modulators are scarce in literature. Among these
efforts, Guo et al. described a micromolar activity for quinoxaline derivative (1) (Fig. 1) as
selective inhibitor of CypD over CypA [37]. Rather than this report, no further effort was
reported to develop more potent and selective mPTP modulator of quinoxaline scaffold. Also,
furamide derivatives (2) were reported to inhibit calcium induced mitochondrial swelling, albeit
at a high micromolar concentration [38]. The diarylisoxazole-3-carboxamide (3) and N-
phenylbenzamide (4) were reported to maintain mitochondrial potential in models of calcium-
induced mitochondrial permeability transition. However, they were found to target unconfirmed
biological target other than CypD [39, 40]. To the best of our knowledge, the therapeutic small
molecule inhibitor of mPTP has not been realized yet. This prompted us to pursue identification
of promising mPTP modulators able to achieve neuroprotection and stall progression of
neurodegenerative diseases. In a recent report, screening of our institute’s internal chemical
library using JC-1 assay unveiled quinuclidinyl oxime ether (5) as a promising molecule
maintaining mitochondrial membrane potential in models of amyloid beta (Aβ)-induced mPTP
opening [41]. Modifications of hit compound 5 resulted in series of pyrrolidinyl triazoles (6) and
N-alkyl-N’-pyridyl ureas (7) as mPTP inhibitors [42, 43]. In these two series, the linkers might
contribute to the elicited affinity.
<Please Insert Fig.1. Here>
CypD bears 75% sequence identity with CypA which is another member of cyclophilin family of
peptidyl-prolyl isomerases (PPI). Moreover, the contact surface amino acid residues of human

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CypD and CypA with CsA are conserved in both of them [44]. This might be an indication of the
usefulness of CypA inhibitors as hits for designing potential promising CypD inhibitors.
Compound 8 (Fig. 1) was designed as a CypA inhibitor by linking two hydrophobic aromatic
fragments via an acylurea linker [45]. In addition, triazole, urea and acylurea linkers of
compounds 6, 7 and 8 respectively might donate and/or accept hydrogen bonds. In fact, the
crystal structure of CsA bound to CypD (pdb ID = 2Z6W) discloses favorable hydrogen bonding
interactions within the binding cavity between amide groups of CsA and Arg55, Asn102, Trp121
and His126 [44]. Therefore, we envisioned that replacing oxime ether, triazole, urea or acylurea
linkers with lipophilic thiourea linker which has stronger tendency to form hydrogen bonding
[46], would enable more efficient simulation of some hydrogen bonding interactions pattern of
CsA, while improving the biological activity and BBB penetration at the same time. In addition,
exploration of the structure activity relationship through replacing the eastern aliphatic moieties
of compounds 5 and 6 with different aromatic moieties or aliphatic chains was planned.
Moreover, investigations of the impact of retaining the pyridine ring or isosteric replacement
with pyrazine ring, in addition to, incorporation of non-halogenated or different halogenated
benzyloxy moieties were considered.
2. Results and discussion
2.1. Chemistry
Achieving conciseness of the target molecules’ synthetic pathway is one of the ultimate goals in
the science of synthesis. The more synthetic steps involved the more attrition of the yield and
higher cost of the final compounds. Thereby, ideal synthesis should involve only one high
yielding synthetic step. While achieving ideal synthesis remains far from practice in many cases,

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it should be an inspiring aim for planning near ideal synthesis. Keeping up these goals, a concise
preparation of the target compounds (9a−z) was achieved in two synthetic steps starting from
commercially available 2-amino-3-hydroxypyridine (10a) or 2-amino-3-chloropyrazine (10b).
While 3-(benzyloxy)pyridine-2-amine (11a) is commercially available, other 3-benzyloxy-2-
pyridinamines (11b−d) were prepared via O-alkylation of the starting pyridine derivative 10a
with the appropriate benzyl bromide derivative in biphasic medium employing a phase transfer
catalyst under basic conditions. For pyrazine derivatives, nucleophilic aromatic substitution of
10b with the appropriate phenylmethoxide derivative generated by treatment of the
corresponding benzyl alcohol with sodium hydride afforded 3-(substituted-benzyloxy)pyrazin-2-
amine derivatives (11e−f). Nucleophilic addition of intermediates 11 to aromatic isothiocyanate
afforded the corresponding 1,3-diarylthioureas. However, for reaction with aliphatic thiourea
derivatives, the intermediates 11 were first converted in situ to sodium amide derivatives which
underwent reaction with the appropriate aliphatic isothiocyanate derivative to afford the targeted
1-alkyl-3-arylthioureas.
<Please Insert Scheme 1. Here>
2.2. Biological evaluation
2.2.1. Protection against loss of mitochondrial membrane potential assay (JC-1 assay)
As highlighted previously, mitochondrial membrane potential (∆ΨM) is crucial for maintaining
integrity of mitochondria. Prolonged opening of the mega-channel mPTP results in decay of
mitochondrial membrane potential. Mitochondria with maintained membrane potential are
featured with an inside negative matrix potential. Consequently, the decline in mitochondrial
membrane potential might be quantified using fluorescent cationic dyes. For accurate

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measurements, an ideal cationic dye to be used should be affected only by mitochondrial
membrane potential without probability to be affected by other factors including the plasma
membrane potential (∆Ψp) which would interfere with these measurements. JC-1 (5,5’,6,6’-
tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocarbocyanine iodide) is a lipophilic cationic cyanine
dye; offers many advantages over other cationic dyes as its performance is specific for the
mitochondrial membrane potential. JC-1 is not affected by other components including plasma
membrane potential (∆Ψp), mitochondrial size or shape [47, 48]. It self-assembles into red
fluorescing aggregates in the matrix of mitochondria having physiologically normal membrane
potential. Upon loss of membrane potential, these aggregates break up into green fluorescing
monomers. Induction of mPTP opening in presence of compounds to be evaluated as mPTP
blockers and measuring the ratio of the red to green fluorescence enables quantification of
protective effects of these compounds. Amyloid beta (Aβ) is a neurotoxin which plays a crucial
role in progression of neurodegeneration. Upon interaction with mitochondria, Aβ triggers mPTP
opening with a concomitant loss of membrane potential. Consequently, Aβ-induced mPTP
opening model was selected for initial biological evaluation of prepared compounds. In this
model, Aβ produced fluorescence shift from red to green as the membrane potential is lost. The
protective effect of synthesized target compounds against Aβ-induced potential loss was
quantified by measuring the decrement of green to red fluorescence ratio in hippocampal
neuronal cells. The results of evaluation are presented in Table 1. The standard CsA lowered the
green to red ratio to 46.0%, thus recovering 54.0% of mitochondrial membrane potential.
<Please Insert Table 1. Here>

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Twenty six derivatives (9a−z) bearing halogenated or non-halogenated benzyloxy moieties of
pyridine or pyrazine scaffolds linked to aliphatic or aromatic moieties through a thiourea spacer
were synthesized and biologically assessed for their mPTP blocking activity using JC-1 assay.
The results of the evaluation are illustrated in Fig. 2. The dashed line across the figure represents
the level of green to red ratio elicited by CsA in the course of the employed test. Compounds
having values of percentage increase in the fluorescence ratio (green/red) lower than the dashed
line are eliciting superior mPTP blocking activity than that of standard CsA, while those
compounds whose values of percentage increase in the fluorescence ratio (green/red) above the
dashed line are less effective than CsA.
In the beginning, compounds having pyridine scaffold were prepared and evaluated. As shown in
Table 1 and illustrated in Fig. 2, all ethyl, tert-butyl and benzyl derivatives (9a, 9c, 9d, 9h, 9j, 9l
and 9q) which bear unsubstituted, 3-fluoro, 2-chloro or 3-chlorobenzyloxy substitution in the
western part, were ineffective or having weak efficiency in reducing the green to red
fluorescence ratio. The isopropyl derivatives elicited mixed results; while isopropyl derivative 9i
which bears 3-fluorobenzyloxy substitution in the western part was ineffective, the isopropyl
derivative 9b elicited efficacy almost as CsA (45.2% and 46.0% respectively), whereas, the
isopropyl derivative 9m showed better reduction of green to red ratio (31.7%). In contrast to the
aliphatic derivatives, all aromatic derivatives having unsubstituted, 3-fluoro, 2-chloro or 3-
chlorobenzyloxy substitution in the western part exhibited superior activity than that of CsA in
retaining mitochondrial potential. The best performing aromatic derivative 9o showed increase in
the green to red fluorescence ratio around fourth of CsA value (13.0% and 46.0% respectively),
while aromatic derivative 9r which is the least effective among prepared aromatic derivatives is

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still more efficient than CsA (38.0% and 46.0% respectively). Out of ten prepared aromatic
derivatives, five derivatives (9g, 9k, 9n, 9o and 9p) showed excellent reductions of the increased
green to red fluorescence ratio within range of (13.0%~17.5%), while other four derivatives (9e,
9f, 9s and 9t) were less efficient, however, still also excellent reductions of the increased green
to red fluorescence ratio within range of (23.3%~26.0%).
Next, we explored the performance of derivatives having pyrazine scaffold in JC-1 assay. Six
derivatives having isopropyl, tert-butyl, benzyl, 4-methylphenyl and 3-chlorophenyl
substitutions in eastern part were prepared and evaluated. In paradox to ineffective analogous
pyridine derivatives, compounds 9u and 9v having pyrazine scaffold, bearing isopropyl and tert-
butyl substituents in eastern part and 3-fluorobenzyloxy in the western part showed appreciable
activities (reduced the percent of increased green to red fluorescence ratio to 43.1 and 28.1%
respectively). However, compounds 9x and 9z having pyrazine scaffold, bearing isopropyl and
3-chlorophenyl substituents in eastern part and 2-chlorobenzyloxy in the western part were less
effective than the analogous pyridine derivatives. The most active compounds prepared among
both pyridine and pyrazine series were pyrazine derivatives 9w and 9y (reduced the percent of
increased green to red fluorescence ratio to 4.6 and 5.3% respectively).
<Please Insert Fig.2. Here>
2.2.2. Assessment of compounds toxic effect on ATP production (Luciferase-based assay)
and neurocytotoxicity (MTT assay)

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In drug discovery process, it is critical to implement assessment of toxicity early in workflow of
the discovery program. These front toxicity assays would filter out likely toxic compounds and
thus minimize the probability of developing molecules that would fail later because of their
toxicities [49]. As mentioned before, mitochondria are cells’ indispensable power-plants
generating most of the ATP produced in various tissues of the body. Compounds inducing
mitochondrial toxicity would seriously affect normal functioning of cells, and thereby, it is a
pivotal issue to be considered for development of safe drugs [50, 51]. In addition to these general
concerns in drug development, a special concern arises from the assumed model of
mitochondrial permeability transition pore (mPTP) which suggests dimers of ATP synthase as
the core element forming the pore. Consequently, it is important to confirm that molecules
designed as mPTP blockers do not interfere with normal physiological production of ATP
because if ATP synthases were inhibited as a part of mPTP inhibition process, this would deprive
cells from the vital process of mitochondrial ATP production. Hence, the most active fifteen
derivatives which elicited increased green to red fluorescence percentage less than 40.0% were
evaluated for their effect on cellular ATP production in hippocampal neuronal cells. After 7
hours incubation of cells with 5 µM solution of the tested compound, a luciferase based assay
was employed for measurement of ATP in the cells’ lysate. As presented in Table 2 and
illustrated in Fig. 3, compounds 9e, 9f, and 9g with unsubstituted benzyloxy moiety significantly
decreased the levels of ATP. Other tested compounds (9k, 9m, 9n, 9o, 9p, 9r, 9s, 9t, 9v, 9w, and
9z) showed more or less insignificant impact on cellular ATP production. Despite thiourea
derivative 9y which elicited high efficacy in lowering the g/r ratio showed some reduction of
ATP production, this reduction is still comparable to that of the standard piracetam (85.1 and
88.0% for 9y and piracetam respectively).

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After excluding the unsubstituted benzyloxy derivatives turned out to possess significant
detrimental effects on mitochondrial ATP production, the remaining twelve derivatives were
subjected to a subsequent filter to exclude compound(s) possessing cytotoxic effects.
Accordingly, the promising twelve derivatives (9k, 9m, 9n, 9o, 9p, 9r, 9s, 9t, 9v, 9w, 9y, and
9z) were submitted to MTT cell viability assay to assess the level of hippocampal neuronal cells
survival after incubation for 24 hours with 5 µM solutions of tested compounds. The results of
MTT assay are presented in Table 2. With exception of compound 9p whose MTT assay result
was approximately similar to reference CsA, the other tested eleven compounds showed superior
results than that of CsA. Almost no significant reductions in neuronal cells viability were exerted
by these compounds. From the combined excellent performance of compounds passed through
the applied two consecutive toxicity filters, it can be assumed that these derivatives are devoid of
direct toxic effects on mitochondrial ATP production and cytotoxicity on hippocampal neuronal
cells.
<Please Insert Table 2 Here>
<Please Insert Fig. 3. Here>
2.2.3. Protection against Aβ-induced neuronal cell death
Neurodegenerative diseases are progressive in nature which is a major obstacle in developing
effective therapies thereof. The neurodegenerative disease tends to worsen by time as of death of
more neuronal cells takes place. The death of neurons in neurodegenerative diseases such as
Alzheimer’s disease is a consequence of multiple complex mechanisms [52]. However,
achieving protection of neurons from Aβ-induced cells death is one of the ultimate goals in

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developing new effective treatments for Alzheimer’s disease. At sub-micromolar concentrations
Aβ is highly neurocytotoxic. It has been reported that 500 nM concentrations induced substantial
neuronal necrosis of cerebellar granule cells within 72-168 hours, while at 1 µM, substantial
cerebellar granule cells death took place rapidly within only 24 hours [53]. Moreover, incubation
of hippocampal neurons cell line with 2.5 µM concentrations of Aβ reduced its viability to
around 50.0% [54]. Considering these values of elicited cytotoxicity of Aβ, we decided to test
the ability of each candidate compound to protect hippocampal neuronal cells, over 24 hours
incubation time, from cytotoxic effects of 5 µM concentrations of Aβ which is double the
concentration that produced reduction of hippocampal cells viability to around 50.0%. This
highly elevated concentration of Aβ was applied hoping that high selection criteria would reveal
potentially effective lead compounds protecting neurons against Aβ-induced cell death even in
the presence of high concentrations of toxic Aβ. The results of protective effects elicited by
evaluated compounds are presented in Table 2 and illustrated in Fig. 3. Under these strict criteria
of selection, three compounds (9w, 9r, and 9k) elicited a decent protection of hippocampal
neuronal cells against Aβ-induced neurocytotoxicity (69.3, 51.8, and 48.2% for 9w, 9r, and 9k
respectively). These values translate into, for compound 9w for example, that approximately
70.0% of hippocampal neuronal cells died upon incubation with 5 µM concentration Aβ are still
viable after 24 hours of exposure to this highly toxic concentration of Aβ when compound 9w is
present. Compounds tested other than 9w, 9r, and 9k were less or not effective in protecting cells
against Aβ-induced death.
2.2.4. Molecular Modeling Study
To gain insights into the possible causes triggering variations in the activity of tested compounds
as mPTP modulators, molecular modeling study was conducted using the reported crystal

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structure of CypD-CsA complex (pdb ID = 2Z6W). Analysis of CypD-CsA complex revealed the
pivotal role of hydrophobic binding interactions. CsA's methylated valine residue (Mva11) docks
into a critical binding pocket 1. Juxtaposed to binding pocket 1, a less hydrophobic pocket 2 is
partially filled with the CsA's α-amino-butyric acid residue (Aba2). A saddle separates pocket 1
and pocket 2. On the opposite side, there is a flat hydrophobic surface adjoined to pocket 1
which interacts with CsA's methylated leucine residue side chain (Mle9). Two hydrogen bonds
contribute to binding of CsA in the saddle region, another two to the edge of pocket 1, and one to
the flattened hydrophobic surface.
CDocker [55, 56] was used to perform flexible docking of the synthesized twenty six derivatives
(9a−z) followed by refining of the best twenty poses via in situ minimizations and calculation of
binding and complex energies. The analysis of the results of molecular docking simulations
revealed two general patterns for binding of docked compounds which might underlay the
elicited biological activity. In the first binding pattern, the hydrophobic pocket 1 of CypD is
filled by a hydrophobic moiety of the ligand. The ligands effectively maintained mitochondrial
membrane potential elicited strong binding interaction of modes belonging to this pattern. The
second binding pattern is characterized by hydrophobic pocket 1 being unoccupied by any
moiety of the ligand while the ligand docks above this pocket. The molecular simulation study
revealed that ineffective compounds have much less calculated binding interaction for the first
binding pattern and/or preference towards the second binding pattern.
The distinctive binding patterns of the thiourea derivative 9w with the highest efficiency in
maintaining mitochondrial membrane potential among tested compounds are illustrated in Fig. 4.

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There are three discernible subtypes belonging to the first binding pattern. In binding subtype 1
(Fig. 4A), the aromatic ring of the benzyloxy moiety docks into the hydrophobic pocket 1. For
compound 9w, the best pose within this predicted binding mode, was ranked the second best
pose among all retrieved twenty poses in terms of calculated binding energies (-53.34 kcal/mol
for binding energy, -50.89 kcal/mol for total binding energy and -3053.63 kcal/mol for ligand-
receptor complex energy). In this pose, the phenyl ring of benzyloxy moiety docked into the
hydrophobic pocket 1. The pyrazine ring and the thiourea linker are directed towards the saddle
region.
The second subtype of binding modes belonging to binding pattern 1 is illustrated in Fig. 4B.
This subtype is marked by the central pyrazine ring (or pyridine ring in case of pyridine
derivatives) being docked within the hydrophobic pocket 1. For compound 9w, the pyrazine ring
is docked in hydrophobic pocket 1 and the thiourea linker is in interaction with the saddle. By a
slight difference in calculated binding energies, the best pose of this subtype of binding mode is
ranked over the best pose for subtype 1 (-54.53 kcal/mol for binding energy and -51.48 kcal/mol
for total binding energy for subtype 2, while, -53.34 kcal/mol for binding energy and -50.89
kcal/mol for total binding energy for subtype 1). However, the ligand-receptor complex energy
of the best pose belonging to subtype 1 is more energetically favored than that of subtype 2 (-
3030.32 kcal/mol for complex energy of subtype 2, while, -3053.63 kcal/mol for complex energy
of subtype 1). These opposing differences in binding and complex energies may indicate that
both poses could contribute to the elicited biological activity of compound 9w.
<Please Insert Fig. 4. Here>

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As illustrated in Fig. 4C, in subtype 3 of binding pattern 1, the eastern substitution on the
thiourea linker is docked within the hydrophobic pocket 1. In case of compound 9w. The
thiourea spacer is in favorable interactions with the saddle region. The calculated low energy
terms for the best pose belonging to this subtype among calculated poses of compound 9w (-
45.964 kcal/mol for binding energy, -44.2041 kcal/mol for total binding energy and -3053.18
kcal/mol for complex energy) indicates low probability of this pose for its contribution to the
elicited biological activity of this derivative.
The second general binding pattern for compound 9w is depicted in Fig. 4D. In this binding
pattern, the hydrophobic pocket 1 remains vacant while the ligand is docked above this pocket.
The calculated low binding energies (-48.58 kcal/mol for binding energy, -45.53 kcal/mol for
total binding energy and -3056.86 kcal/mol for complex energy) indicate little effect of this
binding mode on the activity of compound 9w. Scrutinizing the different anticipated binding
patterns combined with calculated energy terms would attribute the high efficiency of compound
9w to high contribution of subtypes 1 and 2 of the first general binding pattern.
In contrast, inefficient or compounds with low efficiency to maintain the mitochondrial
membrane potential, have propensity for the second general binding pattern and/or less
energetically favored poses of the first general binding pattern. This is illustrated by predicted
binding poses of compound 9c which was inefficient in maintaining the mitochondrial membrane
potential eliciting 91% of green to red fluorescence. The best calculated poses for binding of
compound 9c with CypD are illustrated in Fig. 5. The best energetically favored pose was of the

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second general binding pattern (-45.55 kcal/mol for binding energy, -42.19 kcal/mol for total
binding energy and -3047.46 kcal/mol for complex energy) which is, as illustrated in Fig. 5A,
characterized by vacant hydrophobic pocket 1.
The next best pose of compound 9c (Fig. 5B) was of the first general binding pattern subtype 1;
in which the aromatic ring of the benzyloxy moiety docks into the hydrophobic pocket 1. For this
pose of compound 9c, in addition to being less favored than the pose of second general binding
type, the calculated energy terms (-42.69 kcal/mol for binding energy, -38.39 kcal/mol for total
binding energy and -3062.26 kcal/mol for complex energy) are considerably much lower than
calculated energy terms for the corresponding poses of the same binding pattern of the effective
compounds such as 9w (-53.34 kcal/mol for binding energy, -50.89 kcal/mol for total binding
energy and -3053.63 kcal/mol for complex energy). Consequently, the inefficiency of compound
9c may be attributed to its tendency to the second general binding mode in addition to its lower
calculated binding energies in comparison with that of effective derivatives.
Other subtypes of the first general binding pattern were also detected in calculated poses of
compound 9c, however, with less favorable energetic terms. The pose visualized in Fig. 5C,
shows compound 9c binding mode 1 subtype 2; in which the central pyridine ring docks into the
hydrophobic pocket 1. The calculated energetic terms were -41.62 kcal/mol for binding energy, -
38.26 kcal/mol for total binding energy and -3047.03 kcal/mol for complex energy. Also binding
mode 1 subtype 3; in which the eastern hydrophobic tert-butyl moiety docks into the
hydrophobic pocket 1, was detected in calculated poses of compound 9c and is visualized in Fig.

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5D. The calculated energy terms for this pose was not high (-40.09 kcal/mol for binding energy,
-37.26 kcal/mol for total binding energy and -3043.58 kcal/mol for complex energy).
<Please Insert Fig. 5. Here>
3. Conclusion
In this study, three compounds, 9w, 9r and 9k, have been discovered as new mPTP modulators
maintaining mitochondrial membrane potential in models of Aβ-induced mitochondrial
membrane depolarization, and furthermore, protecting neuronal cells effectively against Aβ-
induced cytotoxicity. In vitro evaluation of mitochondrial and cellular toxicity indicated absence
of both of detrimental effects on the mitochondrial vital process of ATP production, and
cytotoxicity for these compounds. Furthermore, the conducted in silico molecular docking study
provided plausible explanation of their observed mPTP blocking activity. Consequently, this
study suggests that compounds 9w, 9r and 9k might be used as a leads for development of new
effective therapies to Alzheimer’s disease.
4. Experimental
4.1. Chemistry
General: All reactions and manipulations were performed in nitrogen atmosphere using standard
Schlenk techniques. The reaction solvents purchased from Aldrich Co., TCI and Alfa and used
without any other purification. 3-(Benzyloxy)pyridin-2-amine 11a has been purchased from Alfa
Aesar Co. The NMR spectra were obtained on Bruker Avance 300 or 400. 1H NMR spectra were
referenced to tetramethylsilane (δ = 0.00 ppm) as an internal standard and are reported as

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follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, dd = doublet
of doublet, m = multiplet). Column chromatography was performed on Merck Silica Gel 60
(230−400 mesh) and eluting solvents for all of these chromatographic methods are noted as
appropriated-mixed solvent with given volume-to-volume ratios. TLC was carried out using
glass sheets pre-coated with silica gel 60 F254 purchased by Merk. The purity of samples was
determined by analytical HPLC using a Water ACQUITY UPLC (CORTECSTM) with C18
column (2.1 mm x 100 mm; 1.6 µm) at temperature 40 °C. HPLC data were recorded using
parameters as follows: 0.1% formic acid in water and 0.1% formic acid in methanol and flow
rate of 0.3 mL/min. For more details, see supplementary file. High-resolution spectra were
performed on Waters ACQUITY UPLC BEH C18 1.7µ−Q-TOF SYNAPT G2-Si High
Definition Mass Spectrometry.
4.1.1. 3-(Benzyloxy)pyridin-2-amine derivatives (11b−d)
Compounds 11b−d have been synthesized as reported by our group in a previous report [43].
4.1.2. General procedure of 3-(benzyloxy)pyrazin-2-amine derivatives (11e and 11f)
Sodium hydride (60% in mineral oil, 0.04 g, 1 mmol) was added to a stirred solution of benzyl
alcohol derivative (1 mmol) in anhydrous N,N-dimethylformamide (3 mL of DMF) at room
temperature and stirring was continued for 1 h. 2-Amino-3-chloropyrazine (10b, 0.13 g, 1 mmol)
was added to the reaction mixture and the reaction mixture was stirred at 100 °C for 15 h. After
cooling, the solvent was evaporated and the residue was partitioned between water and
dichloromethane. The organic layer was dried over sodium sulfate anhydrous, filtered, and
concentrated. The residue was purified by column chromatography (SiO2, EA/n-Hex = 1/5).

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4.1.2.1.1. 3-(3-Fluorobenzyloxy)pyrazin-2-amine (11e)
1
Light orange solid, yield: 75.6%, H NMR (400 MHz, DMSO-d6) δ = 5.39 (2H, s, OCH2Ph),
6.36 (2H, br, NH2), 7.14 (1H, td, J = 2.6 Hz, 9.0 Hz, ArH), 7.25 (1H, d, J = 3.1 Hz, ArH), 7.32
(1H, d, J = 7.6 Hz, ArH), 7.37-7.44 (2H, m, ArH), 7.49 (1H, d, J = 3.1 Hz, ArH).
4.1.2.1.2.
3-(2-Chlorobenzyloxy)pyrazin-2-amine (11f)
1
Yellow solid, yield: 87.7%, H NMR (400 MHz, CDCl3) δ = 4.79 (2H, br, NH2), 5.51 (2H, s,
OCH2Ph), 7.27-7.30 (2H, m, ArH), 7.41-7.42 (1H, m, ArH), 7.43 (1H, d, J = 3.1 Hz, ArH), 7.47-
7.49 (1H, m, ArH), 7.58 (1H, d, J = 3.1 Hz, ArH).
4.1.3. General procedure of thiourea derivatives (9a−z)
For aliphatic thiourea derivatives (R2 = alkyl group); 2-Amino-3-benzyloxy pyridine or pyrazine
derivative (0.85 mmol) and sodium hydride (60% in mineral oil, 68 mg, 1.71 mmol) was
dissolved in dry THF (5 mL). The isothiocyanate derivative (1.02 mmol) was added and the
reaction mixture was refluxed for 5−18 h. After cooling, the reaction mixture was evaporated and
the residue was purified by flash column chromatography (SiO2, EA/n-Hex = 1/4). For aromatic
thiourea derivatives (R2 = aryl group); 2-Amino-3-benzyloxy pyridine or pyrazine derivative (2.5
mmol) and isothiocyanate derivative (3 mmol) were dissolved in dry THF (10 mL) and the
reaction mixture was refluxed for 3‒6 h. After cooling, the reaction mixture was evaporated and
the residue was purified by solidification with cold methanol and filtered to give target
compounds.
1-(3-(Benzyloxy)pyridin-2-yl)-3-ethylthiourea (9a)

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1
White solid, yield: 49%, mp: 90.6-91.0 °C, HPLC purity: 6.79 min, 100%, H NMR (400 MHz,
CDCl3) δ = 1.34 (3H, t, J = 7.3 Hz, CH3), 3.75-3.82 (2H, m, CH2), 5.15 (2H, s, OCH2Ph), 6.85
(1H, dd, J = 5.1 Hz, 8.1 Hz, ArH), 7.11 (1H, dd, J = 1.3 Hz, 8.1 Hz, ArH), 7.35-7.42 (5H, m,
13
ArH), 7.75 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH), 8.58 (1H, s, NH), 11.59 (1H, s, NH). C NMR
(100.6 MHz, CDCl3) δ = 14.09, 40.53, 70.83, 117.47, 118.27, 127.43, 128.63, 128.92, 135.05,
136.71, 141.09, 144.39, 179.24. HRMS (ES+): m/z calculated for C15H17N3OS: 288.1170
[M+H]+. Found 288.1179.
1-(3-(Benzyloxy)pyridin-2-yl)-3-isopropylthiourea (9b)
1
White solid, yield: 14%, mp: 89.4-95.0 °C, HPLC purity: 7.02 min, 98.31%, H NMR (400
MHz, CDCl3) δ = 1.35 (6H, d, J = 6.6 Hz, 2CH3), 4.59-4.60 (1H, m, CH), 5.15 (2H, s, OCH2Ph),
6.85 (1H, dd, J = 5.1 Hz, 8.1 Hz, ArH), 7.11 (1H, dd, J = 1.3 Hz, 8.1 Hz, ArH), 7.35-7.42 (5H,
m, ArH), 7.75 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH), 8.52 (1H, s, NH), 11.55 (1H, s, NH). 13C NMR
(100.6 MHz, CDCl3) δ = 22.24, 47.63, 70.79, 117.43, 118.25, 127.41, 128.61, 128.91, 135.06,
136.68, 141.07, 144.42, 178.00. HRMS (ES+): m/z calculated for C16H19N3OS: 302.1327
[M+H]+. Found 302.1331.
1-(3-(Benzyloxy)pyridin-2-yl)-3-(tert-butyl)thiourea (9c)
1
White solid, yield: 70%, mp: 136.5-136.9 °C, HPLC purity: 7.31 min, 96.59%, H NMR (400
MHz, CDCl3) δ = 1.63 (9H, s, 3CH3), 5.15 (2H, s, OCH2Ph), 6.82 (1H, dd, J = 5.1 Hz, 8.0 Hz,
ArH), 7.08 (1H, dd, J = 1.1 Hz, 8.0 Hz, ArH), 7.34-7.41 (5H, m, ArH), 7.72 (1H, dd, J = 1.1 Hz,
13
5.1 Hz, ArH), 8.43 (1H, s, NH), 11.82 (1H, s, NH); C NMR (100.6 MHz, CDCl3) δ = 28.41,

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54.03, 70.73, 117.12, 118.15, 127.35, 128.57, 128.90, 135.14, 136.48, 141.00, 144.50, 177.67.
HRMS (ES+): m/z calculated for C17H21N3OS: 316.1483 [M+H]+. Found 316.1488
.
1-Benzyl-3-(3-(benzyloxy)pyridin-2-yl)thiourea (9d)
1
Yellow solid, yield: 64%, mp: 122.7-124.3 °C, HPLC purity: 7.24 min, 100%, H NMR (400
MHz, CDCl3) δ = 5.02 (2H, d, J = 5.6 Hz, OCH2Ph), 5.16 (2H, s, OCH2Ph), 6.84 (1H, dd, J = 5.2
Hz, 8.0 Hz, ArH), 7.11 (1H, dd, J = 1.2 Hz, 8.0 Hz, ArH), 7.29-7.30 (1H, m, ArH), 7.34-7.42
(9H, m, ArH), 7.67 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 8.69 (1H, s, NH), 12.04 (1H, s, NH). 13C
NMR (100.6 MHz, CDCl3) δ = 49.63, 70.89, 117.69, 118.46, 127.47, 127.58, 128.69, 128.95,
135.04, 136.77, 137.58, 141.11, 144.24, 179.98. HRMS (ES+): m/z calculated for C20H19N3OS:
350.1327 [M+H]+. Found 350.1329.
1-(3-(Benzyloxy)pyridin-2-yl)-3-(3-chlorophenyl)thiourea (9e)
1
Yellow solid, yield: 79%, mp: 134.8-135.3 °C, HPLC purity: 7.52 min, 100%, H NMR (400
MHz, CDCl3) δ = 5.19 (2H, s, OCH2Ph), 6.94 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.18-7.22 (2H,
m, ArH), 7.32 (1H, t, J = 8.0 Hz, ArH), 7.37-7.42 (5H, m, ArH), 7.66 (1H, d, J = 8.0 Hz, ArH),
7.79 (1H, t, J = 2.0 Hz, ArH), 7.81 (1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 8.76 (1H, s, NH), 13.73
13
(1H, s, NH). C NMR (100.6 MHz, CDCl3) δ = 71.05, 118.16, 118.89, 122.62, 124.39, 126.04,
127.51, 128.78, 129.00, 129.61, 134.16, 134.86, 136.61, 139.99, 141.32, 143.95, 178.44. HRMS
(ES+): m/z calculated for C19H16ClN3OS: 370.0781 [M+H]+. Found 370.0786.
1-(3-(Benzyloxy)pyridin-2-yl)-3-(4-methylphenyl)thiourea (9f)

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1
Yellow solid, yield: 67%, mp: 106.4-107.2 °C, HPLC purity: 7.42 min, 98.83%, H NMR (400
MHz, CDCl3) δ = 2.36 (3H, s, CH3), 5.18 (2H, s, OCH2Ph), 6.91 (1H, dd, J = 5.2 Hz, 8.0 Hz,
ArH), 7.16 (1H, dd, J = 1.2 Hz, 8.0 Hz, ArH), 7.20 (2H, d, J = 8.0 Hz, ArH), 7.36-7.43 (5H, m,
ArH), 7.54 (2H, d, J = 8.0 Hz, ArH), 7.80 (1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 8.73 (1H, s, NH),
13
13.47 (1H, s, NH). C NMR (100.6 MHz, CDCl3) δ = 21.20, 70.92, 118.10, 118.82, 124.55,
127.56, 128.68, 128.94, 129.35, 135.08, 135.90, 136.31, 136.61, 141.18, 143.92, 178.31. HRMS
(ES+): m/z calculated for C20H19N3OS: 350.1327 [M+H]+. Found 350.1337.
1-(3-(Benzyloxy)pyridin-2-yl)-3-(naphthalen-1-yl)thiourea (9g)
1
White solid, yield: 49%, mp: 152.0-154.1 °C, HPLC purity: 7.45 min, 90.33%, H NMR (400
MHz, CDCl3) δ = 5.19 (2H, s, OCH2Ph), 6.96 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.22 (1H, dd, J
= 5.2 Hz, 8.0 Hz, ArH), 7.36-7.44 (5H, m, ArH), 7.50-7.54 (2H, m, ArH), 7.56 (1H, d, J = 7.6
Hz, ArH), 7.81-7.83 (2H, m, ArH), 7.89-7.91 (1H, m, ArH), 8.00 (1H, d, J = 7.6 Hz, ArH), 8.07-
8.09 (1H, m, ArH), 8.92 (1H, s, NH), 13.79 (1H, s, NH). HRMS (ES+): m/z calculated for
C23H19N3OS: 386.1327 [M+H]+. Found 386.1336.
1-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-ethylthiourea (9h)
1
White solid, yield: 49%, mp: 116.2-116.8 °C, HPLC purity: 6.77 min, 100%, H NMR (400
MHz, CDCl3) δ = 1.34 (3H, t, J = 7.2 Hz, CH3), 3.75-3.82 (2H, m, CH2), 5.14 (2H, s, OCH2Ph),
6.86 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.03-7.09 (3H, m, ArH), 7.17 (1H, d, J = 7.2 Hz, ArH),
7.35-7.40 (1H, m, ArH), 7.77 (1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 8.56 (1H, s, NH), 11.56 (1H, s,
NH). 13C NMR (100.6 MHz, CDCl3) δ = 14.07, 40.55, 70.03, 114.29 (JC-F = 22.1 Hz), 115.61 (JC-
F = 21.1 Hz), 117.47, 118.27, 122.83 (JC-F = 2.0 Hz), 130.64 (JC-F = 8.0 Hz), 137.01, 137.57 (JC-F

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= 7.0 Hz), 140.79, 144.30, 163.00 (JC-F = 247.5 Hz), 179.22. HRMS (ES+): m/z calculated for
C15H16FN3OS: 306.1076 [M+H]+. Found 306.1078.
1-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-isopropylthiourea (9i)
1
White solid, yield: 56%, mp: 131.4-133.2 °C, HPLC purity: 7.00 min, 91.71%, H NMR (400
MHz, CDCl3) δ = 1.35 (6H, d, J = 6.8 Hz, 2CH3), 4.57-4.61 (1H, m, CH), 5.14 (2H, s, OCH2Ph),
6.85 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.05-7.08 (3H, m, ArH), 7.16 (1H, d, J = 7.6 Hz, ArH),
7.35-7.39 (1H, m, ArH), 7.77 (1H, J = 1.2 Hz, 5.2 Hz, ArH), 8.50 (1H, s, NH), 11.53 (1H, s,
13
NH). C NMR (100.6 MHz, CDCl3) δ = 22.23, 47.67, 70.02, 114.27 (JC-F = 22.1 Hz), 115.61
(JC-F = 21.1 Hz), 117.35, 118.21, 122.80 (JC-F = 3.0 Hz), 130.65 (JC-F = 9.1 Hz), 137.00, 137.58
(JC-F = 7.0 Hz), 140.80, 144.41, 163.02 (JC-F = 246.5 Hz), 178.05. HRMS (ES+): m/z calculated
for C16H18FN3OS: 350.1233 [M+H]+. Found 320.1236.
1-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-(tert-butyl)thiourea (9j)
1
White solid, yield: 63%, mp: 157.5-159.7 °C, HPLC purity: 7.28 min, 96.35%, H NMR (400
MHz, CDCl3) δ = 1.63 (9H, s, 3CH3), 5.14 (2H, s, OCH2Ph), 6.82 (1H, dd, J = 5.2 Hz, 8.0 Hz,
ArH), 7.03-7.08 (3H, m, ArH), 7.16 (1H, J = 8.0 Hz, ArH), 7.34-7.39 (1H, m, ArH), 7.74 (1H,
13
dd, J = 1.6 Hz, 5.2 Hz, ArH), 8.40 (1H, s, NH), 11.80 (1H, s, NH). C NMR (100.6 MHz,
CDCl3) δ = 28.39, 54.07, 69.96, 114.21 (JC-F = 22.1 Hz), 115.55 (JC-F = 21.1 Hz), 117.08, 118.14,
122.75 (JC-F = 3.0 Hz), 130.62 (JC-F = 8.0 Hz), 136.78, 137.66 (JC-F = 8.0 Hz), 140.72, 144.44,
163.01 (JC-F = 247.5 Hz), 177.66. HRMS (ES+): m/z calculated for C17H20FN3OS: 334.1389
[M+H]+. Found 334.1396.

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1-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-(naphthalen-1-yl)thiourea (9k)
1
White solid, yield: 77%, mp: 163.5-166.8 °C, H NMR (400 MHz, CDCl3) δ = 5.21 (2H, s,
OCH2Ph), 6.96 (1H, dd, J = 4.8 Hz, 8.0 Hz, ArH), 7.08-7.09 (1H, m, ArH), 7.10-7.14 (1H, m,
ArH), 7.18 (1H, dd, J = 1.2 Hz, 8.0 Hz, ArH), 7.22 (1H, d, J = 7.6 Hz, ArH), 7.38-7.44 (1H, m,
ArH), 7.50-7.54 (2H, m, ArH), 7.57 (1H, d, J = 8.0 Hz, ArH), 7.81-7.85 (2H, m, ArH), 7.89-7.91
(1H, m, ArH), 8.00 (1H, d, J = 7.6 Hz, ArH), 8.07-8.09 (1H, m, ArH), 8.90 (1H, s, NH), 13.76
13
(1H, s, NH). C NMR (100.6 MHz, CDCl3) δ = 70.22, 114.44 (JC-F = 22.1 Hz), 115.76 (JC-F
=
21.1 Hz), 118.08, 118.84, 122.32, 122.97, 124.43, 125.43, 126.21, 126.64, 127.31, 128.57,
129.26, 130.75 (JC-F = 8.6 Hz), 134.27, 134.87, 137.00, 137.46 (JC-F = 7.0 Hz), 141.09, 144.19,
163.07 (JC-F = 248.5 Hz), 180.00. HRMS (ES+): m/z calculated for C23H18FN3OS: 404.1233
[M+H]+. Found 404.1238.
1-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-ethylthiourea (9l)
1
Yellow solid, yield: 19%, mp: 98.6-100.2 °C, HPLC purity: 7.02 min, 100%, H NMR (400
MHz, CDCl3) δ = 1.34 (3H, t, J = 7.6 Hz, CH3), 3.75-3.82 (2H, m, CH2), 5.25 (2H, s, OCH2Ph),
6.88 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.12 (1H, J = 1.2 Hz, 8.0 Hz, ArH), 7.28-7.33 (2H, m,
ArH), 7.41-7.44 (2H, m, ArH), 7.78 (1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 8.59 (1H, s, NH), 11.58
13
(1H, s, NH). C NMR (100.6 MHz, CDCl3) δ = 14.09, 40.57, 67.99, 117.54, 118.36, 127.36,
129.01, 129.79, 129.87, 132.67, 132.95, 137.05, 140.82, 144.36, 179.24. HRMS (ES+): m/z
calculated for C15H16ClN3OS: 322.0781 [M+H]+. Found 322.0790.
1-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-isopropylthiourea (9m)

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1
Yellow solid, yield: 72.3%, mp: 72.5-73.8 °C, HPLC purity: 7.21 min, 93.24%, H NMR (400
MHz, CDCl3) δ = 1.36 (6H, d, J = 6.8 Hz, 2CH3), 4.57-4.63 (1H, m, CH), 5.25 (2H, s, OCH2Ph),
6.87 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.11 (1H, d, J = 8.0 Hz, ArH), 7.29-7.33 (2H, m, ArH),
7.41-7.44 (2H, m, ArH), 7.78 (1H, d, J = 5.2 Hz, ArH), 8.53 (1H, s, NH), 11.54 (1H, s, NH). 13C
NMR (100.6 MHz, CDCl3) δ = 22.23, 47.63, 67.98, 117.53, 118.37, 127.30, 129.03, 129.74,
129.85, 132.64, 132.96, 137.02, 140.74, 144.32, 178.00. HRMS (ES+): m/z calculated for
C16H18ClN3OS: 336.0937 [M+H]+. Found 336.0945.
1-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-(3-chlorophenyl)thiourea (9n)
1
Yellow solid, yield: 83%, mp: 127.8-128.5 °C, HPLC purity: 7.70 min, 100%, H NMR (400
MHz, CDCl3) δ = 5.29 (2H, s, OCH2Ph), 6.94 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.10-7.12 (1H,
m, ArH), 7.19-7.35 (4H, m, ArH), 7.43-7.46 (2H, m, ArH), 7.66 (1H, d, J = 8.0 Hz, ArH), 7.78
(1H, t, J = 2.4 Hz, ArH), 7.85 (1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 8.76 (1H, s, NH), 13.72 (1H, s,
NH). 13C NMR (100.6 MHz, CDCl3) δ = 68.27, 118.23, 119.03, 122.60, 124.38, 126.05, 127.40,
129.17, 129.62, 129.88, 130.04, 132.48, 133.12, 134.16, 136.95, 139.97, 141.06, 143.92, 178.44.
HRMS (ES+): m/z calculated for C19H15Cl2N3OS: 404.0391 [M+H]+. Found 404.0397.
1-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-(4-methylphenyl)thiourea (9o)
1
Yellow solid, yield: 15%, mp: 138.9-142.7 °C, HPLC purity: 7.58 min, 91.48%, H NMR (400
MHz, CDCl3) δ = 2.36 (3H, s, CH3), 5.29 (2H, s, OCH2Ph), 6.92 (1H, dd, J = 5.2 Hz, 8.0 Hz,
ArH), 7.07-7.14 (3H, m, ArH), 7.18-7.19 (1H, m, ArH), 7.21 (2H, d, J = 8.0 Hz, ArH), 7.38-7.40
(1H, m, ArH), 7.54 (2H, d, J = 8.0 Hz, ArH), 7.82 (1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 8.74 (1H, s,
13
NH), 13.47 (1H, s, NH). ). C NMR (100.6 MHz, CDCl3) δ = 21.11, 68.12, 117.88, 118.71,

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124.90, 127.41, 129.09, 129.35, 129.84, 129.96, 132.57, 133.02, 136.13, 136.21, 137.00, 141.02,
144.14, 178.73. HRMS (ES+): m/z calculated for C20H18ClN3OS: 384.0937 [M+H]+. Found
384.0947.
1-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-(naphthalen-1-yl)thiourea (9p)
1
White solid, yield: 87%, mp: 116.5-117.3 °C, HPLC purity: 7.63 min, 94.06%, H NMR (400
MHz, CDCl3) δ = 5.33 (2H, s, OCH2Ph), 6.96 (1H, dd, J = 4.8 Hz, 8.0 Hz, ArH), 7.22-7.25 (2H,
m, ArH), 7.34-7.35 (2H, m, ArH), 7.43-7.49 (2H, m, ArH), 7.50-7.58 (3H, m, ArH), 7.82 (1H, d,
J = 8.0 Hz, ArH), 7.86 (1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 7.89-7.91 (1H, m, ArH), 8.0 (1H, d, J
= 6.0 Hz, ArH), 8.07-8.10 (1H, m, ArH), 8.92 (1H, s, NH), 13.78 (1H, s, NH). 13C NMR (100.6
MHz, CDCl3) δ = 68.24, 118.17, 118.96, 122.32, 122.69, 123.49, 124.37, 125.44, 126.21, 126.63,
127.16, 127.26, 127.43, 127.76, 128.52, 129.24, 129.97, 132.86, 134.59, 137.03, 141.10, 144.20,
179.99. HRMS (ES+): m/z calculated for C23H18ClN3OS: 420.0937 [M+H]+. Found 420.0950.
1-(3-(3-Chlorobenzyloxy)pyridin-2-yl)-3-(tert-butyl)thiourea (9q)
1
White solid, yield: 67%, mp: 128.1-128.6 °C, HPLC purity: 7.47 min, 99.00%, H NMR (400
MHz, CDCl3) δ = 1.63 (9H, s, 3CH3), 5.12 (2H, s, OCH2Ph), 6.83 (1H, dd, J = 5.2 Hz, 8.0 Hz,
ArH), 7.03 (1H, J = 1.2 Hz, 8.0 Hz, ArH), 7.27-7.28 (1H, m, ArH), 7.32-7.35 (3H, m, ArH), 7.73
(1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 8.39 (1H, s, NH), 11.80 (1H, s, NH). 13C NMR (100.6 MHz,
CDCl3) δ = 28.39, 54.07, 69.92, 117.09, 118.13, 125.34, 127.35, 128.79, 130.31, 134.82, 136.81,
137.18, 140.69, 144.43, 177.65. HRMS (ES+): m/z calculated for C17H20ClN3OS: 350.1094
[M+H]+. Found 350.1102.

ACCEPTED MANUSCRIPT
1-(3-(3-Chlorobenzyloxy)pyridin-2-yl)-3-(3-chlorophenyl)thiourea (9r)
1
White solid, yield: 78%, mp: 115.9-117.5 °C, HPLC purity: 7.66 min, 100%, H NMR (400
MHz, CDCl3) δ = 5.16 (2H, s, OCH2Ph), 6.94 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.15 (1H, J =
1.2 Hz, 5.2 Hz, ArH), 7.20-7.23 (1H, m, ArH), 7.30-7.38 (5H, m, ArH), 7.64-7.67 (1H, m, ArH),
7.78 (1H, t, J = 2.0 Hz, ArH), 7.84 (1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 8.72 (1H, s, NH), 13.70
13
(1H, s, NH). C NMR (100.6 MHz, CDCl3) δ = 70.21, 118.16, 118.87, 122.59, 124.37, 125.50,
126.07, 127.52, 128.99, 129.63, 130.40, 134.71, 134.91, 136.89, 136.93, 139.94, 141.02, 143.88,
178.41. HRMS (ES+): m/z calculated for C19H15Cl2N3OS: 404.0391 [M+H]+. Found 404.0394.
1-(3-(3-Chlorobenzyloxy)pyridin-2-yl)-3-(4-methylphenyl)thiourea (9s)
1
Yellow solid, yield: 21%, mp: 127.1-127.5 °C, HPLC purity: 7.54 min, 100%, H NMR (400
MHz, CDCl3) δ = 2.36 (3H, s, CH3), 5.15 (2H, s, OCH2Ph), 6.91 (1H, dd, J = 4.8 Hz, 8.0 Hz,
ArH), 7.12 (1H, dd, J = 1.2 Hz, 8.0 Hz, ArH), 7.21 (2H, d, J = 8.4 Hz, ArH), 7.29-7.31 (1H, m,
ArH), 7.35-7.38 (3H, m, ArH), 7.54 (2H, d, J = 8.4 Hz, ArH), 7.82 (1H, dd, J = 1.2 Hz, 4.8 Hz,
13
ArH), 8.70 (1H, s, NH), 13.45 (1H, s, NH). C NMR (100.6 MHz, CDCl3) δ = 21.22, 70.13,
117.84, 118.61, 124.84, 125.50, 127.52, 128.95, 129.35, 130.38, 134.89, 136.13, 136.19, 136.98,
140.97, 144.07, 178.64. HRMS (ES+): m/z calculated for C20H18ClN3OS: 384.0937 [M+H]+.
Found 384.0953.
1-(3-(3-Chlorobenzyloxy)pyridin-2-yl)-3-(naphthalen-1-yl)thiourea (9t)
1
White solid, yield: 90%, mp: 153.0-154.1 °C, H NMR (400 MHz, CDCl3) δ = 5.19 (2H, s,
OCH2Ph), 6.96 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.18 (1H, dd, J = 1.2 Hz, 8.0 Hz, ArH), 7.33-
7.41 (4H, m, ArH), 7.50-7.54 (2H, m, ArH), 7.57 (1H, d, J = 7.6 Hz, ArH), 7.81-7.85 (2H, m,