Synthesis and evaluation of 2-(3-arylureido)pyridines and 2-(3-arylureido)pyrazines as potential modulators of Aβ-induced mitochondrial dysfunction in Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2017.12.045

A series of 2-(3-arylureido)pyridines and 2-(3-benzylureido)pyridines were synthesized and evaluated as potential modulators for amyloid beta (Aβ)-induced mitochondrial dysfunction in Alzheimer's disease (AD). The blocking activities of forty one small molecules against Aβ-induced mitochondrial permeability transition pore (mPTP) opening were evaluated by JC-1 assay which measures the change of mitochondrial membrane potential (ΔΨm). The inhibitory activity of twenty five compounds against Aβ-induced mPTP opening was superior to that of the standard cyclosporin A (CsA). Six hit compounds have been identified as likely safe in regards to mitochondrial and cellular safety and subjected to assessment for their protective effect against Aβ-induced deterioration of ATP production and cytotoxicity. Among them, compound 7fb has been identified as a lead compound protecting neuronal cells against 67% of neurocytotoxicity and 43% of suppression of mitochondrial ATP production induced by 5 μM concentrations of Aβ. Using CDocker algorithm, a molecular docking model presented a plausible binding mode for these compounds with cyclophilin D (CypD) receptor as a major component of mPTP. Hence, this report presents compound 7fb as a new nonpeptidyl mPTP blocker which would be promising for further development of Alzheimer's disease (AD) therapeutics.

Full text of DOI:10.1016/j.ejmech.2017.12.045

Accepted Manuscript
Synthesis and evaluation of 2-(3-arylureido)pyridines and 2-(3-arylureido)pyrazines
as potential modulators of Aβ-induced mitochondrial dysfunction in Alzheimer's
disease
Ahmed Elkamhawy, Jung-eun Park, Ahmed H.E. Hassan, Ae Nim Pae, Jiyoun Lee,
Beoung-Geon Park, Eun Joo Roh
PII:
S0223-5234(17)31079-6
10.1016/j.ejmech.2017.12.045
EJMECH 10023
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 June 2017
Revised Date: 9 November 2017
Accepted Date: 13 December 2017
Please cite this article as: A. Elkamhawy, J.-e. Park, A.H.E. Hassan, A.N. Pae, J. Lee, B.-G. Park, E.J.
Roh, Synthesis and evaluation of 2-(3-arylureido)pyridines and 2-(3-arylureido)pyrazines as potential
modulators of Aβ-induced mitochondrial dysfunction in Alzheimer's disease, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.045.
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Graphical Abstract:
JC-1 assay = 11% increased g/r ratio
MTT viability = 115%
ATP production =104%
Protection against Aβ-induced neurocytotoxicity = 67%
Protection against Aβ-induced ATP suppression = 43%

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Synthesis and evaluation of 2-(3-arylureido)pyridines and 2-(3-
arylureido)pyrazines as potential modulators of Aβ-induced mitochondrial
dysfunction in Alzheimer’s disease
a,b,1
a,c,1
d,e
f,g
h
Ahmed Elkamhawy , Jung-eun Park , Ahmed H. E. Hassan , Ae Nim Pae , Jiyoun Lee ,
f,i
a,g,*
Beoung-Geon Park , Eun Joo Roh
a
b
c
d
e
f
Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Department of Chemistry, Sogang University, Seoul 04107, Republic of Korea
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea
Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology (KIST), Seoul
0
2792, Republic of Korea
g
Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul, Republic of Korea
Department of Global Medical Science, Sungshin Women's University, Seoul 142-732, Republic of Korea
h
i
School of Life Sciences and Biotechnology, Korea University, Seoul 02792, Republic of Korea
*
Corresponding author:
Eun Joo Roh (e-mail: r8636@kist.re.kr).
1
Both authors contributed equally to this work

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Abstract
A series of 2-(3-arylureido)pyridines and 2-(3-benzylureido)pyridines were synthesized and
evaluated as potential modulators for amyloid beta (Aβ)-induced mitochondrial dysfunction in
Alzheimer’s disease (AD). The blocking activities of forty one small molecules against Aβ-
induced mitochondrial permeability transition pore (mPTP) opening were evaluated by JC-1
assay which measures the change of mitochondrial membrane potential (∆Ψm). The inhibitory
activity of twenty five compounds against Aβ-induced mPTP opening was superior to that of the
standard cyclosporin A (CsA). Six hit compounds have been identified as likely safe in regards
to mitochondrial and cellular safety and subjected to assessment for their protective effect
against Aβ-induced deterioration of ATP production and cytotoxicity. Among them, compound
7
fb has been identified as a lead compound protecting neuronal cells against 67% of
neurocytotoxicity and 43% of suppression of mitochondrial ATP production induced by 5 µM
concentrations of Aβ. Using CDocker algorithm, a molecular docking model presented a
plausible binding mode for these compounds with cyclophilin D (CypD) receptor as a major
component of mPTP. Hence, this report presents compound 7fb as a new nonpeptidyl mPTP
blocker which would be promising for further development of Alzheimer's disease (AD)
therapeutics.
Keywords: Mitochondrial permeability transition pore (mPTP), Alzheimer’s disease (AD), β-
amyloid peptide (Aβ), pyridyl-urea, molecular docking
.

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1
. Introduction
Over the past decades, the presence of a mitochondrial channel involved in permeability
transition, understanding its formation, regulation, and roles, has been a subject of debate and
intense research [1-9]. Culminated data suggest that acute mitochondrial permeability transition
pore (mPTP) opening is a physiologic process that contributes to the regulation and cycling of
calcium in the mitochondria [9-11]. However, prolonged opening of mPTP results in loss of
mitochondrial membrane potential, termination of ATP production, deterioration of homeostasis,
swelling of mitochondria and eventually rupture of the outer mitochondrial membrane and
release of cell death factors [12-15]. The detrimental sequences of prolonged mPTP opening
have been correlated with several pathologies found in a wide range of diseases including
cardiovascular, neurological, and hepatic diseases [16-26]. Inhibition of mPTP opening has been
proved as a valid approach for cytoprotection against cell death in diseases characterized by
excessive opening of mPTP [27-30].
The molecular structure of mPTP has been always a subject of controversy. Several elements
have been suggested to be involved in mPTP formation; however, the exact structure is not
certainly confirmed. The current model of mPTP involves dimers of F /F ATPase as the core
0
1
unit of the pore; in addition to adenine nucleotide translocator (ANT) and inorganic phosphate
carrier (PiC). According to this model, there are direct interactions of F /F ATPase dimer with
0
1
CypD in the mitochondrial matrix, mitochondrial creatine kinase oligomers in intermembrane
space, voltage dependent anion channel, Bcl-2-associated X protein and Bcl-2 homologous
antagonist killer in the outer membrane. In addition, other regulatory elements exist including

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mitochondrial translocator protein, protein kinase C epsilon and glycogen synthase kinase 3-beta
[31].
Despite the debate over the molecular structure of mPTP, the role of cyclophilin D (CypD) in
modulating mPTP opening is indubious [32, 33]. In fact, the suggestion of the presence of mPTP
by Haworth and Hunter [34] was later confirmed by the discovery that inhibition of CypD by
cyclosporin A (CsA) causes inhibition of transient potential [35, 36]. Although CsA is an
effective mPTP inhibitor, it has also immunosuppressant activity which limits its therapeutic use
as mPTP inhibitor [37]. In addition, being a polypeptide, its poor kinetics across blood brain
barrier (BBB) results in low bioavailability to central nervous system neurons [38]. Efforts
towards separation of immunosuppressant activity from cyclophilin inhibition activity afforded
CsA analogs free from immunosuppressant activity. In addition to other side effects of such
analogs, like NIM811 and UNIL025, they still suffer from poor penetration of BBB caused by
their polypeptidic structure [39, 40]. Few reports are found in the literature documenting efforts
to develop non-peptidic small molecule inhibitors of mPTP. Quinoxaline derivative 1 in Fig. 1,
was described by Guo et al. as a selective agent for inhibition of CypD over cyclophilin A
(CypA), albeit in micromolar activity [41]. To the best of our knowledge, there are no further
reports of developing more potent or selective quinoxaline compounds as mPTP modulators.
Furamide derivatives 2, have been reported to possess inhibitory activity on calcium induced
mitochondrial swelling via modulation of mPTP, however, at high micromolar concentration and
low potency [42]. N-phenylbenzamide 3 has been found to maintain mitochondrial potential in
models of calcium-induced mitochondrial permeability transition. However, they act on
unknown biological target(s) [43]. The selective, safe, non-peptidic, bioavailable, small molecule

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inhibitor sought for is still lacking. This situation stimulated our institute to initiate a discovery
project aiming to find promising hit molecules as mPTP modulators. In the early stage of the
project, screening of our institute chemical library using JC-1 assay resulted in identification of
quinuclidinyl oxime ether 4 as a hit compound recovering the amyloid beta (Aβ)-induced
impairment of mitochondrial membrane potential [44]. Structural modifications of the hit
compound afforded substituted pyrrolidinyl triazoles 5 as potential mPTP blockers [45]. It is
known that 1,2,3-triazoles might act as a hydrogen bond acceptor, elicit π-π stacking and/or
contribute to dipole-dipole interactions. Considering this fact, we have reported the identification
of a urea linked compound (6) as a promising lead compound recovering mitochondria from
amyloid beta induced mPTP opening and membrane potential loss [46]. Moreover, it exerted a
significant protection against amyloid beta induced mitochondrial and cellular toxicities.
<
Please Insert Fig. 1. Here>
Investigation of the crystal structure of CsA-CypD complex (Fig. 2. pdb ID = 2Z6W) indicates a
crucial role for hydrophobic interactions in addition to the known hydrogen bonding interactions
[47]. These hydrophobic interactions include CsA's methylated valine residue (Mva11) within
the critical main binding pocket (P1), CsA's methylated leucine residue side chain (Mle9) with a
flat hydrophobic surface (S2) adjacent to pocket P1 and a neighboring Ala103 residue with side
chain of 4-(2-butenyl)-4-methylthreonine residue (Bmt1) residue of CsA. The hydrogen bonding
of CsA includes with Gln63 and Asn102 in the saddle region (S1) separating the main binding
pocket P1 from a partially filled pocket (P2); hydrogen bonds with Arg55 and His126 at the edge

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of the main binding pocket P1; and Trp121 in a flattened hydrophobic surface (S2) adjacent to
the other side of the hydrophobic pocket P1.
<
Please Insert Fig. 2. Here>
Considering the pivotal role of hydrophobic interactions with the flattened hydrophobic surface
next to the main binding pocket, as well as, hydrogen bond bonding interactions, we anticipated
that incorporation of planar aromatic moieties in place of the aliphatic cyclic amines moieties of
compounds 4, 5 and 6 while conserving the urea linker would result in enhanced hydrophobic
interaction with the hydrophobic surface and would produce ligands with enhanced biological
activities. The introduced aromatic moieties could be attached to the urea moiety through a
methylene group or alternatively directly attached. Different mono or fused aromatic rings with
different substitution patterns were considered in the target molecules (7). Moreover, assessment
of the effect on the elicited biological activity by both of using different halogenation patterns of
the benzyloxy ring, and isosteric replacement of pyridine with pyrazine on the elicited biological
activity was planned.
<
Please Insert Fig. 3. Here>
2
. Results and discussion
.1. Chemistry
2
Conciseness of chemical synthesis is a desirable sensible practice. The less number of synthetic
steps, the more economic and efficient the synthetic process. Considering this point, the designed
synthetic Scheme 1 for preparation of the target compounds (7aa−7hc) involved two synthetic

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steps employing commercially available starting materials, 2-amino-3-hydroxypyridine (8a) or
2
-amino-3-chloropyrazine (8b). While 3-(benzyloxy)pyridine-2-amine (9a) is commercially
available, other 3-benzyloxy-2-pyridinamine derivatives (9b−f) were prepared via O-alkylation
of 2-amino-3-hydroxypyridine (8a) with the appropriate benzyl bromide derivative in biphasic
medium using tetra-n-butylammonium bromide as a phase transfer catalyst and sodium
hydroxide as a base [46]. Meanwhile, nucleophilic aromatic substitution of 2-amino-3-
chloropyrazine (8b) with appropriate phenylmethoxide anion generated from corresponding
benzyl alcohol and sodium hydride yielded 3-benzyloxy-2-pyrazinamine derivatives (9g and 9h).
The target urea linked derivatives (7aa‒hc) were obtained via addition reaction of the
nucleophilic amino derivatives (9a−h) to the appropriate electrophilic aromatic isocyanate
derivatives.
<
Please Insert Scheme 1. Here>
2
.2. Biological evaluation
2
.2.1. JC-1 assay (Mitochondrial membrane potential assay)
Opening of mPTP leads to depolarization of the potential across the mitochondrial membrane.
This loss of potential can be measured and quantified using a suitable indicator such as JC-1; a
lipophilic fluorescent cationic dye. In mitochondria with conserved membrane potential, it forms
aggregates eliciting characteristic red fluorescence. Upon loss of mitochondrial membrane, these
aggregates dismantle into green fluorescent monomers. This behavior is dependent only on the
mitochondrial membrane potential and is not affected by other factors such as mitochondrial size
or shape, which renders the test highly specific. Accordingly, JC-1 assay can be used for
evaluation of the effect of different molecules on the mitochondrial membrane potential via

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measuring and quantification of the ratio of red to green fluorescence. In the course of the
conducted test, mPTP opening was induced by amyloid beta (Aβ) resulting in potential loss and
shifting from red to green fluorescence. Doses of five micromolar concentrations were used to
evaluate the capability of the tested compounds to block mPTP and recover the mitochondrial
potential by measuring the decrement of green to red fluorescence. The results of the tested
compounds are shown in Table 1. CsA was used as a standard, whereby, it lowered the green to
red ratio to 46% of that of Aβ alone.
<
Please Insert Table 1. Here>
Initially, four series of unsubstituted benzyloxy and 3-fluorobenzyloxy derivatives of pyridine
and pyrazine containing compounds (7aa−7dd) were synthesized and evaluated using JC-1 assay.
As shown in Fig. 4, the assay results revealed high efficiency for pyridine over pyrazine
containing derivatives in reduction of the green to red fluorescence ratio. Most of pyridine
derivatives exerted excellent lowering of green to red fluorescence more than that of standard
CsA while the tested pyrazine compounds were ineffective in lowering the green to red
fluorescence. With only 9% green to red fluorescence, pyridine derivative 7ca was the most
potent pyridine derivative in this four series. In comparison, the analogous pyrazine derivative
7
da was the only derivative showing significant activity, despite being weak.
<
Please Insert Fig. 4. Here>

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Based on these initial results, further four series of pyridine derivatives having different
halogenation pattern on the benzyloxy moiety were prepared. Their JC-1 assay results are also
presented in Table 1 and visualized in Fig. 5. Investigating collectively the activity data of the
prepared pyridine derivatives, it can be concluded that, for the other aromatic ring linked to urea,
the two fused rings (naphthyl derivatives) generally elicited weak or no efficacy as shown for
compounds 7eg, 7fh and 7gg (Fig. 5). Other compounds having single ring moieties linked to
urea were generally more efficient in lowering the green to red fluorescence. The 2-flourophenyl
and 3-fluorophenyl urea derivatives were more efficient than the 3-chlorophenyl and 3,4-
dichlorophenyl urea derivatives. However, the 3,4-dichlorophenyl urea derivative (7fe) was
highly efficient in blocking Aβ-induced mPTP opening, eliciting a very low increase in green to
red fluorescence ratio. Considering the halogenation pattern on the benzyloxy ring, it can be
generally concluded that fluorination or chlorination at position 3 afford more efficient
compounds than chlorination at position 2 or 4. 3-Chlorobenzyloxy derivatives were generally
more active as mPTP blockers than 3-flourobenzyloxy analogs. Four highly efficient derivatives
(7fe, 7fb, 7fc and 7fa) eliciting lowering of the produced green to red fluorescence ratio to 15%
or less were 3-chlorobenzyloxy derivatives, while one derivative was 3-flourobenzyloxy (7ca),
one was 4-chlorobenzyloxy derivative (7gb), and one was unsubstituted benzyloxy derivative
(7ab).
<
Please Insert Fig. 5. Here>
2
.2.2. Assessment of effect on mitochondrial ATP production and neurocytotoxicity

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Toxicity is a major cause for termination of drug discovery programs in preclinical, Phase I and
Phase II. Therefore, it becomes imperative to assess toxicity early in drug discovery programs.
Employing front toxicity assays would allow filtering out the likely toxic molecules; thus saving
spent efforts and costs and minimizing the probability of molecules failure in later stages of
development. Mitochondrial toxicity is a major concern for safety in drugs development. In
addition to the fact that mitochondria are cell’s energy plant, the recently proposed model of
0 1
mitochondrial permeability transition pore suggested dimers of F /F ATPase as the core unit of
the pore. Accordingly, it is crucial to assess effect of the promising prepared compounds on
mitochondrial ATP production. The impact of a set of highly active compounds on ATP
production was evaluated using hippocampal neuronal cell line after incubation for 7 hours with
five micromolar concentrations of tested compound. As Table 2 shows, no decrement of ATP
production was measured for all tested compounds while the standard piracetam elicited ATP
production percent equal to 88%. This indicates absence of detrimental effects of the tested
compounds on the vital mitochondrial energy production process. Next employed toxicity filter
was set to exclude cytotoxic molecules. In addition to the fact that cytotoxicity is generally
unwanted effect for development of safe drugs, it would also worsen degenerative diseases as in
these disease patients already suffers from loss of function due to cell death. For assessment of
cytotoxicity, MTT cell viability assay using hippocampal neuronal cell line HT-22 was
employed to evaluate neurocytotoxicity of tested compounds. In the performed assay cells were
incubated with 5 µM concentrations of each tested compound for 24 hours. The results presented
in Table 2 shows that almost all tested compounds have acceptable viability values comparable
to the standard CsA (Table 2). Only one compound 7ee showed significant lowering of cells
viability and thus was excluded from further biological evaluation and testing.

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<
Please Insert Table 2. Here>
2
.2.3. Protection against Aβ-induced impairment of ATP production
In addition to Aβ-induced loss of mitochondrial potential, deterioration of ATP synthesis is
another major outcome of Aβ-induced mitochondrial dysfunction. It is demanded that candidate
molecules be also capable of lessening impairment of ATP production induced by Aβ. The set of
active compounds which elicited mPTP blocking activity better than CsA in JC-1 assay and
passed through mitochondrial and cellular toxicity filters, were subjected for evaluation of their
capacity to protect the cells against Aβ-induced deterioration of ATP production employing a
luciferase based cellular ATP assay in hippocampal neuronal cell line using piracetam as a
reference standard. As presented in Table 3 and illustrated in Fig. 6, the level of ATP production
in Aβ-injured cells was considered as the base line having an arbitrary value of zero. The heights
of bars represent the percent recovered ATP production. Thus a value of 100% equals to the
value of ATP production in the vehicle treated cells. Using a dose of 5 µM concentration, all
tested compounds showed significant protection against Aβ-suppression of ATP production,
however, with variable capacities. Two compounds (7ed and 7fa) were able to protect cells
against almost all of the Aβ-induced deterioration of ATP production (91% and 90%
respectively). Another two compounds (7ec and 7fc) elicited high capacity for maintaining
mitochondrial ATP production (78% and 69% respectively). Among tested compounds,
compounds 7ff and 7fb exhibited the lowest, however, significant capacity for protection against
Aβ-induced deterioration of ATP production (54% and 43% respectively). The culminated
results of JC-1 assay, mitochondrial and cellular toxicity assays, in addition to protection against

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Aβ-suppressed production of ATP, indicated that tested compounds are likely safe molecules
with potential capabilities to ameliorate Aβ-induced mitochondrial dysfunction.
<
Please Insert Table 3. Here>
Please Insert Fig. 6. Here>
<
2
.2.4. Protection against Aβ-induced neurocytotoxicity
Progressive neuronal cells death is the major problem manifested in neurodegenerative diseases.
This neurocytotoxicity is a consequence of complex multiple mechanisms. An effective
treatment should eventually be capable of protecting neuronal cells against this progressive
cytotoxicity. Therefore, next to affirming that tested compounds elicited significant ability to
reduce Aβ-induced mitochondrial dysfunction, assessment of compounds ability to protect
neuronal cells from Aβ-induced neurocytotoxicity was performed employing hippocampal
neuronal cell line and the results are presented in Table 3 and visualized in Fig. 6. In the figure,
the viability level of Aβ-treated cells was considered as the base line having an arbitrary value of
zero. The heights of bars represent the percent protection from Aβ-induced cytotoxicity of
hippocampal neuronal cells recovery of ATP production. A value of 100% protection of Aβ-
induced cytotoxicity equals to the measured viability level of the vehicle treated cells. At a
concentration of 5 µM, compound 7fb exhibited significantly high capacity to protect neuronal
cells from Aβ-induced cytotoxicity (inhibition of 67% of the cytotoxicity of 5 µM concentrations
of Aβ). In addition, compound 7fc showed low, however, significant capacity to protect neuronal
cells from Aβ-induced cytotoxicity (inhibition of 38% of the neuronal cytotoxicity). The
remaining evaluated compounds exhibited protective capacities lower or comparable to the

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reference Piracetam. The culminated results of successively performed biological evaluation
indicate that compound 7fb could be a promising a lead compound possessing significant
abilities to protect neuronal cells from Aβ-induced mitochondrial dysfunctions and cells death.
2
.2.5. Molecular docking
CypD, a cyclophilin family member in mitochondrial matrix, is a peptidyl prolyl cis-trans
isomerase PPI that modulates mPTP opening and loss of membrane potential. CsA is a cyclic
polypeptide that has been found to strongly bind to CypD blocking mPTP opening. Inspection of
crystal structure of CypD-CsA complex (pdb ID = 2Z6W) discloses hydrophobic interactions as
the major contributor in binding. A crucial hydrophobic binding pocket 1 formed by residues
Met61, Ala101, Phe113, Leu122 and His126 is the binding site for the CsA's methylated valine
residue (Mva11). Another less hydrophobic pocket (2), in which the CsA's α-amino-butyric acid
residue (Aba2) is interacting with Gln111 and Gly72, is located next to pocket 1. Pockets 1 and 2
are separated by a saddle formed of Gln63, Ala101, and Asn102. On the other side next to pocket
1
, a flat hydrophobic surface formed of Phe60, Ile57 and Trp121, is located. This hydrophobic
surface interacts with which the side chain of CsA's methylated leucine residue (Mle9). The side
chain of 4-(2-butenyl)-4-methylthreonine residue (Bmt1) interacts hydrophobically with a nearby
Ala103 residue. Hydrogen bonding interactions are found with amino acids Gln63 and Asn102 in
the saddle region, Arg55 and His126 at the edge of pocket 1, and Trp121 in the flattened
hydrophobic surface next to pocket 1.
A molecular modeling study was conducted to understand the difference in the tested compounds
capabilities in blocking mPTP. Sets of the pyridine derivatives highly effective for mPTP

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blocking (7ab, 7ac, 7ca, 7fa, 7fb, 7fc, 7fe, 7gb and 7hb), pyridine derivatives ineffective for
mPTP blocking (7ea, 7fh, 7gf and 7hc) and pyrazine derivatives (7ba, 7bb, 7da, 7db, 7dc and
7
dd) were docked into the reported crystal structure of human CypD (pdb ID = 2Z6W) after
appropriate preparation of receptor and ligands. CDocker algorithm, which is a CHARMM force
field based docking method implemented in Accerlys Discovery Studio 4.0, was used as a
flexible ligand docking method to predict the binding mode of different ligands to CypD. This
algorithm uses explicit all-atoms CHARMM force field calculations which give more accurate
prediction of correct pose than grid-based algorithms. Refinements of the poses were done using
in situ minimizations of the resulting poses. The binding energies and complexes energies were
calculated. Selection of the most probable binding modes was based on these calculated energy
terms.
Analysis of calculated binding modes revealed two general binding modes for this class of
compounds with CypD. The first general binding mode is characterized by part of the ligand
being buried into the hydrophobic pocket 1, which is binding site for methylated valine residue
(Mva11) of CsA. The buried part of the molecule is in almost hydrophobic interactions with
residues forming the pocket. This binding mode is generally favored by effective mPTP blockers.
In the second general binding mode, the hydrophobic pocket is vacant while the ligand is docked
above it. The second binding mode is more frequently encountered in ineffective mPTP blockers
as the preferable binding mode.
The different binding modes for the effective mPTP blocker 7ab are illustrated in Fig. 7. Out of
twenty retrieved different poses, compound 7ab showed nineteen poses belonging to general

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binding mode 1. Only one pose was belonging to binding mode 2. The calculated energy terms
for binding mode 2 were -55.99 kcal/mol for binding energy, -53.22 kcal/mol for total binding
energy and -3075.61 kcal/mol for complex energy. Four binding poses belonging to binding
mode 1 were superior to binding mode 2 in terms of binding energy, total binding energy and
complex stability. This indicates the tendency for compound 7ab to bind in mode 1 rather than
mode 2.
<
Please Insert Fig. 7. Here>
As shown in Fig. 7, three distinctive subtypes of binding modes can be identified under the
general binding mode 1. In binding mode 1 subtype 1 (Fig. 7A), the eastern aromatic moiety is
docked into the hydrophobic pocket 1. The overall best pose in terms of energy for compound
7
ab docked in this pose (-58.06 kcal/mol for binding energy, -56.36 kcal/mol for total binding
energy and -3076.99 kcal/mol for complex energy) belongs to this subtype of binding mode.
Binding mode 1 subtype 2 (Fig. 7B) is characterized by pyridine ring docked into the
hydrophobic pocket 1. This subtype of mode 1 was detected in ten poses out of the generated
twenty poses for compound 7ab. Despite the binding energy of the best pose of this subtype of
binding mode 1 is slightly higher than best pose for subtype 1 (-58.23 kcal/mol for binding
energy), the total binding energy and complex energy favors subtype 1 over this subtype (-53.61
kcal/mol for total binding energy and -3068.71 kcal/mol for complex energy). It is noted that
poses belonging to this subtype showed rotation of the pyridine ring within the pocket resulting
in different orientations of benzyloxy and eastern aromatic moiety groups.

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In subtype 3 of binding mode 1 (Fig. 7C), the benzyloxy moiety of compound 7ab is docked into
the hydrophobic pocket 1. The calculated energy terms (-55.57 kcal/mol for binding energy, -
5
0.26 kcal/mol for total binding energy and -3069.01 kcal/mol for complex energy) indicate that
mode 1 subtype 3 is energetically much less favored than mode 2 and other mode 1 subtypes.
Binding mode 2 is illustrated in Fig. 7D. This mode, which has been detected only in one pose
for compound 7ab, is characterized by hydrophobic pocket 1 being unfilled. The ligand in this
mode docked above the pocket. Considering all of previously mentioned binding modes in
conjunction with their calculated energy terms, the potent activity of compound 7ab can be
attributed to dominating binding mode 1 subtype 1.
Compound 7ba is shown in Fig. 8 as a representative example for ineffective compounds. The
highest scoring binding modes were for binding mode 1 subtype 3 (Fig. 8A) and binding modes
2
(Fig. 8B). As mentioned previously, binding mode 2 contribution leads to ineffective mPTP
blockers. In addition, inspection of binding mode 1 revealed high ligand energy for the docked
conformations of compound 7ba which would disfavor this binding mode. The calculated energy
terms for the pose of compound 7ba shown in Fig. 8A revealed high ligand energy for the
docked pose (20.58 kcal/mol).
<
Please Insert Fig. 8. Here>
3
. Conclusion

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In this study, a new series of N-benzyl and N-aryl-N′-heteroaryl urea derivatives was prepared
and evaluated as protective agents against Aβ-induced mitochondrial dysfunctions and
neurocytotoxicity. Out of forty one prepared compounds, twenty five derivatives protected
mitochondria more effectively than CsA from Aβ-induced loss of mitochondrial membrane
potential (∆Ψm). It is noteworthy that these newly synthesized compounds, in contrast to
quinoxaline derivatives 1, furamide derivatives 2 and N-phenylbenzamide 3, were evaluated in a
model of Aβ-induced loss of mitochondrial membrane potential which is more relevant to
Alzheimer’s disease while other compounds were evaluated in models of calcium-induced
mitochondrial swelling which is non-relevant to Alzheimer’s disease. In addition, these new
compounds have elicited more efficient protective activity than quinuclidinyl oxime ether 4,
pyrrolidinyl triazoles 5, and urea linked compound (6). Moreover, evaluation of their protection
of neuronal cells from Aβ-induced deterioration of ATP production and cytotoxicity identified
compound 7fb as a lead compound protecting neuronal cells against 67% of neurocytotoxicity
and 43% of impairment of mitochondrial ATP production induced by 5 µM concentrations of Aβ.
Furthermore, in silico docking simulations provided rational explanation of the observed mPTP
blocking activity of this series. In summary, this study presents 2-(3-arylureido)pyridines and 2-
(3-arylureido)pyrazines as new modulators of Aβ-induced mitochondrial dysfunctions and
identified compound 7fb as a promising lead compound for development of new AD
therapeutics.
4
. Experimental
.1. Chemistry
4

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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 9a has been purchased from Alfa
1
Aesar Co. The NMR spectra were obtained on Bruker Avance 300 or 400. H NMR spectra were
referenced to tetramethylsilane (δ = 0.00 ppm) as an internal standard and are reported as 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
TM
determined by analytical HPLC using a Water ACQUITY UPLC (CORTECS ) 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 (9b−f).
Compounds 9b−9f have been synthesized as reported by our group in a previous work [46].
4
.1.2. General procedure of 3-(benzyloxy)pyrazin-2-amine derivatives (9g and 9h).
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

ACCEPTED MANUSCRIPT
temperature and stirring was continued for 1 h. 2-Amino-3-chloropyrazine (8b, 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 (SiO , EA/n-Hex = 1/5).
2
3
-(Benzyloxy)pyrazin-2-amine (9g)
1
Yellow solid, yield: 53.8%, H NMR (400 MHz, CDCl
3
) δ = 5.45 (2H, s, OCH
2
Ph), 6.20 (2H, br,
2
NH ), 7.38-7.48 (7H, m, ArH). Reported [48, 49].
3
-(3-Fluorobenzyloxy)pyrazin-2-amine (9h)
1
Light orange solid, yield: 75.6%, H NMR (400 MHz, DMSO-d ) δ = 5.39 (2H, s, OCH Ph),
6
2
6
.36 (2H, br, NH ), 7.14 (1H, td, J = 2.6 Hz, 9.0 Hz, ArH), 7.25 (1H, d, J = 3.1 Hz, ArH), 7.32
2
(1H, d, J = 7.6 Hz, ArH), 7.37-7.44 (2H, m, ArH), 7.49 (1H, d, J = 3.1 Hz, ArH).
4
.1.3. General procedure of final urea compounds (7aa−7hc)
2
-Amino-3-benzyloxy pyridine or pyrazine derivative (2.5 mmol) was dissolved in dry THF (10
mL), isocyanate derivative (3.0 mmol) was added to the reaction mixture. The reaction 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 the target compounds.
1
-(3-(Benzyloxy)pyridin-2-yl)-3-(2-fluorophenyl)urea (7aa)

ACCEPTED MANUSCRIPT
1
White solid, yield: 91.1%, mp: 133.4-134.1 °C, HPLC purity: 7.21 min, 100%, H NMR (400
MHz, CDCl ) δ = 5.13 (2H, s, OCH Ph), 6.88 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH), 6.99-7.01 (1H,
3
2
m, ArH), 7.07-7.16 (3H, m, ArH), 7.38-7.43 (5H, m, ArH), 7.56 (1H, s, NH), 7.88 (1H, dd, J =
1
3
1
.2 Hz, 5.1 Hz, ArH), 8.30-8.35 (1H, m, ArH), 12.23 (1H, s, NH). C NMR (100.6 MHz,
CDCl ) δ = 70.92, 114.74 (J = 18.5 Hz), 117.03, 118.04, 121.72, 123.20 (JC-F = 7.4 Hz),
3
C-F
1
2
24.41, 127.32, 127.83, 128.78, 128.94, 135.07, 137.12, 141.44, 143.55, 152.19, 152.97 (JC-F =
+
+
3 2
06.5 Hz). HRMS (ES ): m/z calculated for C19H16FN O : 338.1305 [M+H] . Found 338.1335.
1
-(3-(Benzyloxy)pyridin-2-yl)-3-(3-fluorophenyl)urea (7ab)
1
White solid, yield: 92.9%, mp: 122.8-123.5 °C, HPLC purity: 8.12 min, 100%, H NMR (400
MHz, CDCl ) δ = 5.13 (2H, s, OCH Ph), 6.74-6.77 (1H, m, ArH), 6.89 (1H, dd, J = 5.1 Hz, 8.0
3
2
Hz, ArH), 7.15 (1H, dd, J = 1.1 Hz, 8.0 Hz, ArH), 7.25-7.27 (m, 1H, ArH), 7.38-7.43 (5H, m,
ArH), 7.27 (1H, s, NH), 7.50-7.55 (2H, m, ArH), 7.86 (1H, dd, J = 1.1 Hz, 5.1 Hz, ArH), 12.0
1
3
(
1H, s, NH). C NMR (100.6 MHz, CDCl ) δ = 70.92, 99.13, 107.43 (J = 26.0 Hz), 109.93
3 C-F
(JC-F = 21.3 Hz), 115.40, 117.03, 118.09, 127.83, 128.82, 128.96, 129.83, 134.99, 136.92, 141.55,
+
1
3
43.67, 152.14, 152.92 (JC-F = 206.6 Hz). HRMS (ES ): m/z calculated for C19
H16FN
3
O
2
:
+
38.1305 [M+H] . Found 338.1382.
1
-(3-(Benzyloxy)pyridin-2-yl)-3-(3,4-dichlorophenyl)urea (7ac)
1
White solid, yield: 83.7%, mp: 134.4-135.0 °C, HPLC purity: 7.61 min, 98.50%, H NMR (400
MHz, CDCl ) δ = 5.13 (2H, s, OCH Ph), 6.91 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH), 7.17 (1H, dd, J
3
2
=
1.2 Hz, 8.0 Hz, ArH), 7.36 (1H, d, J = 8.8 Hz, ArH), 7.38-7.42 (5H, m, ArH), 7.47 (1H, dd, J =
2
.5 Hz, 8.8 Hz, ArH), 7.54 (1H, s, NH), 7.80 (1H, d, J = 2.5 Hz, ArH), 7.86 (1H, dd, J = 1.2 Hz,

ACCEPTED MANUSCRIPT
1
3
5
1
1
3
.1 Hz, ArH), 12.04 (1H, s, NH). C NMR (100.6 MHz, CDCl
3
) δ = 70.95, 117.21, 118.21,
19.34, 121.57, 126.26, 127.83, 128.85, 128.97, 130.37, 132.55, 134.93, 136.88, 138.25, 141.58,
+
+
43.49, 152.04. HRMS (ES ): m/z calculated for C H Cl N O : 388.0619 [M+H] . Found
1
9
15
2
3
2
88.0638.
1
-(3-(Benzyloxy)pyrazin-2-yl)-3-(2-fluorophenyl)urea (7ba)
1
White solid, yield: 69.4%, mp: 159.9-163.4 °C, HPLC purity: 7.38 min, 100%, H NMR (400
MHz, CDCl ) δ = 5.38 (2H, s, OCH Ph), 6.96-7.09 (3H, m, Ph), 7.32-7.40 (5H, m, ArH), 7.43
1H, s, NH), 7.67 (1H, d, J = 3.1 Hz, ArH), 7.72 (1H, d, J = 3.1 Hz, ArH), 8.21-8.23 (1H, m,
3
2
(
1
3
ArH), 11.56 (1H, s, NH). C NMR (100.6 MHz, CDCl ) δ = 69.00, 114.82 (J = 18.9 Hz),
3
C-F
1
21.84, 123.77 (JC-F = 7.3 Hz), 124.48, 128.72, 131.63, 132.60, 135.32, 139.24, 148.02, 151.47,
+
+
1
63.02 (JC-F = 287.5 Hz). HRMS (ES ): m/z calculated for C H FN O : 339.1857 [M+H] .
18 15 4 2
Found 339.1277.
1
-(3-(Benzyloxy)pyrazin-2-yl)-3-(3-fluorophenyl)urea (7bb)
1
White solid, yield: 76.8%, mp: 134.4-134.8 °C, HPLC purity: 7.30 min, 97.91%, H NMR (400
MHz, DMSO-d ) δ = 5.47 (2H, s, OCH Ph), 6.86-6.88 (1H, m, ArH), 7.26-7.41 (5H, m, ArH),
6
2
7
.53-7.60 (3H, m, ArH), 7.83 (1H, d, J = 3.0 Hz, ArH), 7.93 (1H, d, J = 3.0 Hz, ArH), 9.00 (1H,
1
3
s, NH), 11.01 (1H, s, NH). C NMR (100.6 MHz, CDCl ) δ = 70.04, 107.61 (J = 25.9 Hz),
3
C-F
1
1
10.46 (JC-F = 21.2 Hz), 115.51, 128.75, 129.97 (JC-F = 9.2 Hz), 131.97, 132.60, 135.26, 139.32,
+
39.58, 148.12, 151.42, 163.39 (JC-F = 287.8 Hz). HRMS (ES ): m/z calculated for
+
C
18
H
15FN
4
O
2
: 339.1857 [M+H] . Found 339.1269.

ACCEPTED MANUSCRIPT
1
-Benzyl-3-(3-(3-fluorobenzyloxy)pyridin-2-yl)urea (7ca)
1
White solid, yield: 74.4%, mp: 90.6-91.8 °C, HPLC purity: 6.80 min, 99.47%, H NMR (400
MHz, CDCl ) δ = 4.62 (2H, d, J = 5.8 Hz, OCH Ph), 5.09 (2H, s, OCH Ph), 6.79 (1H, dd, J = 5.1
3
2
2
Hz, 8.0 Hz, ArH), 7.05 (1H, dd, J = 1.3 Hz, 8.0 Hz, ArH), 7.07-7.10 (2H, m, ArH), 7.17 (1H, d, J
7.5 Hz, ArH), 7.26-7.27 (1H, m, ArH), 7.31-7.39 (5H, m, ArH), 7.45 (1H, s, NH), 7.74 (1H,
=
1
3
dd, J = 1.3 Hz, 5.1 Hz, ArH), 9.90 (1H, s, NH). C NMR (100.6 MHz, CDCl ) δ = 43.75, 69.95,
3
1
1
14.61 (JC-F = 22.0 Hz), 115.66 (JC-F = 20.8 Hz), 116.38, 117.62, 123.14, 127.05, 127.33, 128.55,
30.58 (JC-F = 8.1 Hz), 137.43, 137.67, 141.07, 144.16, 155.08, 163.00 (JC-F = 245.9 Hz). HRMS
+
+
(
3 2
ES ): m/z calculated for C20H18FN O : 352.1461 [M+H] . Found 352.1489.
1
-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-phenylurea (7cb)
1
White solid, yield: 78.2%, mp: 97.4-100.7 °C, HPLC purity: 6.98 min, 96.84%, H NMR (400
MHz, CDCl ) δ = 5.12 (2H, s, OCH Ph), 6.87 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH), 7.06-7.12 (4H,
3
2
m, ArH), 7.19 (1H, d, J = 7.7 Hz, ArH), 7.32-7.42 (3H, m, ArH), 7.49 (1H, brs, NH), 7.61 (2H, d,
1
3
J = 7.5 Hz, ArH), 7.86 (1H, dd, J = 1.2 Hz, 5.1 Hz, ArH), 11.82 (1H, s, NH). C NMR (100.6
MHz, DMSO-d ) δ = 69.08, 114.66 (JC-F = 22.2 Hz), 114.88 (JC-F = 21.2 Hz), 117.58, 118.16,
19.33, 119.67, 122.86, 123.78, 128.73, 128.86, 130.43 (JC-F = 8.1 Hz), 137.08, 138.68, 138.95
6
1
+
(
JC-F = 7.5 Hz), 141.49, 143.28, 152.52, 162.22 (JC-F = 242.3 Hz). HRMS (ES ): m/z calculated
+
for C H FN O : 338.1305 [M+H] . Found 338.1328.
1
9
16
3
2
1
-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-(2-fluorophenyl)urea (7cc)
White solid, yield: 71.7%, mp: 118.8-119.3 °C, HPLC purity: 7.19 min, 99.35%, H NMR (400
MHz, CDCl ) δ = 5.13 (2H, s, OCH Ph), 6.74-6.77 (1H, m, ArH), 6.89 (1H, dd, J = 5.1 Hz, 8.0
1
3
2

ACCEPTED MANUSCRIPT
Hz, ArH), 7.06-7.13 (3H, m, ArH), 7.18 (1H, d, J = 7.7 Hz, ArH), 7.27-7.28 (2H, m, ArH), 7.37-
7
.41 (1H, m, ArH), 7.51 (1H, m, NH), 7.52-7.56 (1H, m, ArH), 7.87 (1H, dd, J = 1.2 Hz, 5.1 Hz,
1
3
ArH), 11.98 (1H, s, NH). C NMR (100.6 MHz, CDCl ) δ = 70.06, 114.65 (J = 22.0 Hz),
3
C-F
1
1
14.75 (JC-F = 18.8 Hz), 115.74 (JC-F = 20.9 Hz), 117.01, 118.08, 121.74, 123.21, 123.29, 124.45,
27.27, 137.41, 141.17, 143.52, 152.12, 152.97 (JC-F = 242.4 Hz), 163.02 (JC-F = 245.9 Hz).
+
+
HRMS (ES ): m/z calculated for C H F N O : 356.1210 [M+H] . Found 356.1231.
1
9
15
2
3
2
1
-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-(3-fluorophenyl)urea (7cd)
1
White solid, yield: 73.7%, mp: 134.2-135.2 °C, HPLC purity: 7.08 min, 99.02%, H NMR (400
MHz, CDCl ) δ = 5.13 (2H, s, OCH Ph), 6.88 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH), 7.00-7.02 (1H,
3
2
m, ArH), 7.06-7.11 (5H, m, ArH), 7.14 (1H, d, J = 7.92 Hz, ArH), 7.37-7.41 (1H, m, ArH), 7.54
(1H, s, NH), 7.90 (1H, dd, J = 1.2 Hz, 5.1 Hz, ArH), 8.30-8.35 (1H, m, ArH), 12.20 (1H, s, NH).
1
3
C NMR (100.6 MHz, CDCl ) δ = 70.10, 99.14, 107.42 (JC-F = 25.9 Hz), 109.96 (JC-F = 21.2 Hz),
3
1
9
1
14.65 (JC-F = 22.0 Hz), 115.41, 115.77 (JC-F = 20.9 Hz), 117.03, 118.12, 123.20, 129.88 (JC-F
=
.4 Hz), 130.65 (JC-F = 8.1 Hz), 137.21, 137.50, 140.20 (JC-F = 10.6 Hz), 141.27, 143.61, 152.07,
+
+
63.14 (JC-F = 242.9 Hz). HRMS (ES ): m/z calculated for C19
H
15
F
2
N
3
O
2
: 356.1210 [M+H] .
Found 356.1230.
1
-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-(3,4-dichlorophenyl)urea (7ce)
1
White solid, yield: 74.6%, mp: 141.8-142.5 °C, HPLC purity: 7.60 min, 92.91%, H NMR (400
MHz, CDCl ) δ = 5.13 (2H, s, OCH Ph), 6.91 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH), 7.08-7.11 (2H,
3
2
m, ArH), 7.13 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH), 7.18 (1H, d, J = 7.8 Hz, ArH), 7.36-7.40 (2H,
m, ArH), 7.47 (1H, dd, J = 2.5 Hz, 8.8 Hz, ArH), 7.52 (1H, s, NH), 7.80 (1H, d, J = 2.5 Hz, ArH),

ACCEPTED MANUSCRIPT
1
3
7
7
1
3
.87 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH), 12.01 (1H, s, NH). C NMR (100.6 MHz, CDCl ) δ =
0.14, 114.66 (JC-F = 22.3 Hz), 115.81 (JC-F = 20.9 Hz), 117.18, 118.23, 119.35, 121.58, 123.19,
26.32, 130.38, 130.67 (JC-F = 8.3 Hz), 137.18, 137.37, 138.20, 141.30, 143.47, 151.98, 162.97
+
+
(
JC-F = 245.9 Hz). HRMS (ES ): m/z calculated for C H Cl FN O : 406.0525 [M+H] . Found
19 14 2 3 2
4
06.0552.
1
-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-(4-methylphenyl)urea (7cf)
1
White solid, yield: 72.2%, mp: 136.5-137.1 °C, HPLC purity: 7.19 min, 98.50%, H NMR (400
MHz, CDCl ) δ = 2.32 (3H, s, CH ), 5.12 (2H, s, OCH Ph), 6.86 (1H, dd, J = 5.1 Hz, 8.0 Hz,
ArH), 7.08-7.09 (3H, m, ArH), 7.12 (2H, m, ArH), 7.18 (1H, m, ArH), 7.37-7.39 (1H, m, ArH),
3
3
2
1
3
7
.48 (3H, m, ArH+NH), 7.86 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH), 11.71 (1H, s, NH). C NMR
(
100.6 MHz, CDCl ) δ = 20.85, 70.04, 114.63 (J = 22.0 Hz), 115.71 (JC-F = 20.8 Hz), 116.68,
3
C-F
1
1
17.90, 123.18, 129.44, 130.61 (JC-F = 8.1 Hz), 132.98, 135.88, 137.27, 137.62, 141.20, 144.89,
+
52.32, 163.02 (JC-F = 246.0 Hz). HRMS (ES ): m/z calculated for C H FN O : 352.1461
20
18
3
2
+
[
M+H] . Found 352.1483.
1
-(3-(3-Fluorobenzyloxy)pyridin-2-yl)-3-(4-trifluoromethylphenyl)urea (7cg)
1
White solid, yield: 85.4%, mp: 156.5-157.3 °C, HPLC purity: 7.39 min, 93.73%, H NMR (400
MHz, CDCl ) δ = 5.29 (2H, s, OCH Ph), 6.92 (1H, dd, J = 5.5 Hz, 8.0 Hz, ArH), 7.06-7.15 (3H,
3
2
m, ArH), 7.19 (1H, d, J = 7.6 Hz, ArH), 7.37-7.42 (1H, m, ArH), 7.55 (1H, s, NH), 7.58 (2H, d, J
8.4 Hz, ArH), 7.73 (2H, d, J = 8.4 Hz, ArH), 7.89 (1H, dd, J = 1.2 Hz, J = 5.2 Hz, ArH), 12.13
=
+
+
(
1H, s, NH). HRMS (ES ): m/z calculated for C20
15 4 3 2
H F N O : 428.0998 [M+Na] . Found
4
28.0990.

ACCEPTED MANUSCRIPT
1
-Benzyl-3-(3-(3-fluorobenzyloxy)pyrazin-2-yl)urea (7da)
1
White solid, yield: 51.7%, mp: 121.7-121.4 °C, HPLC purity: 7.08 min, 96.93%, H NMR (400
MHz, DMSO-d ) δ = 4.47 (2H, d, J = 6.0 Hz, OCH Ph), 5.45 (2H, s, OCH Ph), 7.15-7.17 (1H, m,
6
2
2
ArH), 7.24-7.26 (1H, m, ArH), 7.32-7.37 (5H, m, ArH), 7.40-7.48 (2H, m, ArH), 7.73 (1H, d, J
1
3
=
3.0 Hz, ArH), 7.80 (1H, d, J = 3.0 Hz, ArH), 8.69 (1H, s, NH), 9.27 (1H, s, NH). C NMR
(
100.6 MHz, CDCl ) δ = 43.88, 67.89, 115.43 (JC-F = 21.7 Hz), 115.54 (JC-F = 20.9 Hz), 124.02,
3
1
1
3
27.26, 127.37, 128.63, 130.32 (JC-F = 8.1 Hz), 131.90 (JC-F = 4.8 Hz), 137.93, 138.80, 139.77,
+
47.64, 154.34, 162.88 (JC-F = 242.2 Hz). HRMS (ES ): m/z calculated for C19
H17FN
4
O
2
:
+
53.1414 [M+H] . Found 353.1491.
1
-(3-(3-Fluorobenzyloxy)pyrazin-2-yl)-3-(3-fluorophenyl)urea (7db)
1
White solid, yield: 82.5%, mp: 152.3-153.4 °C, HPLC purity: 7.24 min, 100%, H NMR (400
MHz, DMSO-d ) δ = 5.48 (2H, s, OCH Ph), 6.87-6.90 (1H, m, ArH), 7.14-7.16 (1H, m, ArH),
6
2
7
.28-7.49 (5H, m, ArH), 7.59 (1H, d, J = 11.9 Hz, ArH), 7.84 (1H, d, J = 3.0 Hz, ArH), 7.95
1
3
(
1H, d, J = 3.0 Hz, ArH), 9.18 (1H, s, NH), 11.08 (1H, s, NH). C NMR (100.6 MHz, CDCl
3
) δ
=
67.40, 106.50, 109.83, 115.15, 115.49, 124.24, 130.73, 130.92, 133.17, 139.60, 139.70,
+
1
3
40.92, 149.06, 151.73, 161.52, 163.93, 167.06. HRMS (ES ): m/z calculated for C H F N O :
18 14 2 4 2
+
57.1163 [M+H] . Found 357.1176.
1
-(3-(3-Fluorobenzyloxy)pyrazin-2-yl)-3-(3,4-dichlorophenyl)urea (7dc)
1
White solid, yield: 63.6%, mp: 162.8-163.7 °C, HPLC purity: 7.67 min, 94.31%, H NMR (400
MHz, CDCl ) δ = 5.45 (2H, s, OCH Ph), 7.06-7.08 (1H, m, ArH), 7.13-7.16 (1H, m, ArH), 7.22
3
2

ACCEPTED MANUSCRIPT
(1H, d, J = 7.60 Hz, ArH), 7.35-7.39 (2H, m, ArH), 7.43-7.46 (1H, m, ArH), 7.47 (1H, s, NH),
13
7
.75 (1H, m, ArH), 7.77-7.78 (2H, m, ArH), 11.40 (1H, s, NH). C NMR (100.6 MHz, CDCl ) δ
3
=
68.11, 107.61, 110.51, 115.51, 115.64 (JC-F = 23.2 Hz), 124.08, 129.99, 130.37 (JC-F = 8.2 Hz),
1
2
4
31.65, 132.56, 137.75, 139.27, 139.57 (JC-F = 11.0 Hz), 147.80, 151.41, 162.98, 163.10 (JC-F
=
+
+
42.9 Hz). HRMS (ES ): m/z calculated for C H Cl FN O : 407.0478 [M+H] . Found
18
13
2
4
2
07.0496.
1
-(3-(3-Fluorobenzyloxy)pyrazin-2-yl)-3-(4-methylphenyl)urea (7dd)
1
White solid, yield: 77.8%, mp: 173.0-173.5 °C, HPLC purity: 7.38 min, 100%, H NMR (400
MHz, CDCl ) δ = 2.32 (3H, s, CH ), 5.44 (2H, s, OCH Ph), 7.06-7.09 (1H, m, ArH), 7.15 (3H, d,
3
3
2
J = 8.5 Hz, ArH), 7.22 (1H, d, J = 7.7 Hz, ArH), 7.34-7.38 (1H, m, ArH), 7.43 (1H, s, NH), 7.45
(
(
(
2H, d, J = 8.4 Hz, ArH), 7.70 (1H, d, J = 3.1 Hz, ArH), 7.77 (1H, d, J = 3.1 Hz, ArH), 11.15
1
3
1H, s, NH). C NMR (100.6 MHz, CDCl3) δ = 19.83, 66.98, 114.45 (J = 21.8 Hz), 114.57
C-F
JC-F = 21.0 Hz), 119.47, 123.02, 128.47, 129.27, 130.71, 131.13, 132.51, 134.24, 136.79,
+
1
38.48, 146.71, 150.59, 163.10 (JC-F = 243.0 Hz). HRMS (ES ): m/z calculated for
+
C
19
H
17FN
4
O
2
: 353.1414 [M+H] . Found 353.1447.
1
-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-phenylurea (7ea)
1
White solid, yield: 93.3%, mp: 153.0-153.7 °C, HPLC purity: 7.26 min, 97.68%, H NMR (300
MHz, DMSO-d ) δ = 5.31 (2H, s, OCH Ph), 7.02-7.09 (2H, m, ArH), 7.32 (2H, t, J = 7.5 Hz,
6
2
ArH), 7.38-7.43 (2H, m, ArH), 7.53-7.90 (4H, m, ArH), 7.68-7.71 (1H, m, ArH), 7.96 (1H, d, J
1
3
=
5.1 Hz, ArH), 8.09 (1H, s, NH), 11.69 (1H, s, NH). C NMR (100.6 MHz, CDCl
3
) δ = 68.09,
1
16.90, 118.12, 120.28, 123.43, 127.33, 128.94, 129.49, 129.86, 130.04, 132.77, 133.36, 137.27,

ACCEPTED MANUSCRIPT
+
1
38.55, 141.28, 143.83, 152.31. HRMS (ES ): m/z calculated for C19
H
16ClN
3
2
O : 354.1009
+
[
M+H] . Found 354.1072.
1
-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-(2-fluorophenyl)urea (7eb)
1
Yellow solid, yield: 78.1%, mp: 138.1-139.4 °C, HPLC purity: 7.44 min, 100%, H NMR (400
MHz, DMSO-d ) δ = 5.32 (2H, s, OCH Ph), 7.08 (2H, m, ArH), 7.18 (1H, t, J = 8.0 Hz, ArH),
6
2
7
7
.26-7.31 (1H, dd, J = 1.2 Hz, 8.0 Hz, ArH), 7.41-7.43 (2H, m, ArH), 7.53-7.55 (1H, m, ArH),
.57 (1H, d, J = 8.0 Hz, ArH), 7.69-7.71 (1H, m, ArH), 7.92 (1H, d, J = 8.0 Hz, ArH), 8.20-8.24
1
3
(
1H, m, ArH), 8.31 (1H, s, NH), 12.01 (1H, s, NH). C NMR (100.6 MHz, CDCl
3
) δ = 68.10,
14.74 (JC-F = 19.1 Hz), 117.10, 118.24, 121.72, 123.23 (JC-F = 7.3 Hz), 124.41, 127.20, 127.31,
29.49, 129.85, 130.04, 132.78, 133.37, 137.42, 141.20, 143.54, 152.16, 152.96 (JC-F = 242.4
1
1
+
+
Hz). HRMS (ES ): m/z calculated for C H ClFN O : 372.0915 [M+H] . Found 372.0987.
1
9
15
3
2
1
-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-(3-fluorophenyl)urea (7ec)
White solid, yield: 76.0%, mp: 137.1-138.5 °C, HPLC purity: 7.35 min, 95.53%, H NMR (400
MHz, CDCl ) δ = 5.24 (2H, s, OCH Ph), 6.91-6.94 (1H, m, ArH), 7.18 (1H, d, J = 8.4 Hz, ArH),
.30-7.54 (8H, m, ArH+NH), 7.80 (1H, s, ArH), 7.88 (1H, d, J = 4.8 Hz, ArH), 12.01 (1H, s,
1
3
2
7
1
3
NH). C NMR (100.6 MHz, CDCl ) δ = 68.16, 100.84, 107.43 (J = 25.7 Hz), 109.95 (JC-F
=
3
C-F
2
1
0.6 Hz), 115.41, 117.11, 118.27, 127.33, 129.52, 129.89, 130.10, 132.68, 133.40, 137.23,
+
40.16, 141.32, 143.66, 152.11, 163.24 (JC-F = 250.4 Hz). HRMS (ES ): m/z calculated for
+
C H ClFN O : 372.0915 [M+H] . Found 372.0982.
19
15
3
2
1
-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-(3-chlorophenyl)urea (7ed)

ACCEPTED MANUSCRIPT
1
Yellow solid, yield: 61.9%, mp: 121.7-122.6 °C, HPLC purity: 7.58 min, 100%, H NMR (400
MHz, DMSO-d ) δ = 5.31 (2H, OCH Ph), 7.07-7.11 (2H, m, ArH), 7.34 (1H, t, J = 8.0 Hz, ArH),
6
2
7
.40-7.43 (2H, m, ArH), 7.45-7.48 (1H, m, ArH), 7.53-7.57 (2H, m, ArH), 7.68-7.70 (1H, m,
ArH), 7.82 (1H, t, J = 2.0 Hz, ArH), 7.98 (1H, dd, J = 1.2 Hz, 4.8 Hz, ArH), 8.24 (1H, s, NH),
1
3
1
1
1
1.82 (1H, s, NH). C NMR (100.6 MHz, CDCl ) δ = 68.16, 117.15, 118.10, 118.30, 120.04,
3
23.61, 127.33, 129.51, 129.88, 130.09, 132.69, 133.39, 134.48, 137.21, 139.85, 141.31, 143.60,
+
+
2 3 2
52.09. HRMS (ES ): m/z calculated for C19H15Cl N O : 388.0619 [M+H] . Found 388.0652.
1
-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-(3,4-dichlorophenyl)urea (7ee)
1
White solid, yield: 90.0%, mp: 135.0-135.7 °C, H NMR (400 MHz, CDCl ) δ = 5.24 (2H, s,
3
OCH Ph), 6.74-6.78 (1H, m, ArH), 6.91 (1H, dd, J = 5.2 Hz, 8.0 Hz, ArH), 7.17 (1H, d, J = 7.6
2
Hz, ArH), 7.25-7.27 (1H, m, ArH), 7.30-7.35 (2H, m, ArH), 7.43-7.46 (2H, m, ArH), 7.52-7.55
1
3
(
2H, m, ArH+NH), 7.88 (1H, d, J = 8.0 Hz, ArH), 11.98 (1H, s, NH). C NMR (100.6 MHz,
CDCl ) δ = 68.21, 117.30, 118.42, 119.32, 121.55, 126.27, 127.32, 129.52, 129.89, 130.12,
3
+
1
30.35, 132.54, 132.64, 133.42, 137.18, 138.22, 141.34, 143.45, 152.03. HRMS (ES ): m/z
+
calculated for C19
H14Cl N
3 3
O
2
: 422.0230 [M+H] . Found 422.0236.
1
-(3-(2-Chlorobenzyloxy)pyridin-2-yl)-3-(4-methylphenyl)urea (7ef)
1
White solid, yield: 89.8%, mp: 151.6-152.8 °C, HPLC purity: 7.47 min, 97.54%, H NMR (400
MHz, CDCl ) δ = 2.32 (3H, s, CH ), 5.23 (2H, s, OCH Ph), 6.89 (1H, dd, J = 5.2 Hz, 8.0 Hz,
3
3
2
ArH), 7.13-7.15 (3H, m, ArH), 7.31-7.34 (2H, m, ArH), 7.43-7.47 (2H, m, ArH), 7.48 (2H, d, J
13
=
8.4 Hz, ArH), 7.50 (1H, s, NH), 7.87 (1H, dd, J = 1.2 Hz, 5.2 Hz, ArH), 11.71 (1H, s, NH). C
NMR (100.6 MHz, CDCl ) δ = 20.86, 68.05, 116.76, 118.02, 120.40, 127.32, 129.44, 129.84,
3