Pyrazinyl ureas revisited: 1-(3-(Benzyloxy)pyrazin-2-yl)-3-(3,4-dichlorophenyl)urea, a new blocker of Aβ-induced mPTP opening for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2018.07.068

Herein, we report synthesis and evaluation of new twenty-eight pyrazinyl ureas 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). Among them, 1-(3-(benzyloxy)pyrazin-2-yl)-3-(3,4-dichlorophenyl)urea (5) effectively maintained mitochondrial function and cell viabilities on ATP assay and MTT assay. Also, hERG channel assay presented safe cardiotoxicity profile for compound 5. In addition, using CDocker algorithm, a molecular docking model presented a plausible explanation for the elicited differences in efficiencies of the synthesized compounds to reduce the green to red fluorescence as indication of mPTP closure. Hence, this report presents compound 5 as the most promising pyrazinyl urea-based mPTP blocker up to date.

Full text of DOI:10.1016/j.ejmech.2018.07.068

Accepted Manuscript
Pyrazinyl ureas revisited: 1-(3-(Benzyloxy)pyrazin-2-yl)-3-(3,4-dichlorophenyl)urea, a
new blocker of Aβ-induced mPTP opening for Alzheimer's disease
Ahmed Elkamhawy, Jung-eun Park, Ahmed H.E. Hassan, Ae Nim Pae, Jiyoun Lee,
Sora Paik, Beoung-Geon Park, Eun Joo Roh
PII:
S0223-5234(18)30633-0
10.1016/j.ejmech.2018.07.068
EJMECH 10603
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 April 2018
Revised Date: 27 July 2018
Accepted Date: 29 July 2018
Please cite this article as: A. Elkamhawy, J.-e. Park, A.H.E. Hassan, A.N. Pae, J. Lee, S. Paik, B.-G.
Park, E.J. Roh, Pyrazinyl ureas revisited: 1-(3-(Benzyloxy)pyrazin-2-yl)-3-(3,4-dichlorophenyl)urea,
a new blocker of Aβ-induced mPTP opening for Alzheimer's disease, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.068.
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Graphical Abstract:

ACCEPTED MANUSCRIPT
Pyrazinyl ureas revisited: 1-(3-(Benzyloxy)pyrazin-2-yl)-3-(3,4-dichlorophenyl)urea, a new
blocker of Aβ-induced mPTP opening for Alzheimer’s disease
a,b,1,*
a,c,1
d
e,f
g
Ahmed Elkamhawy
, Jung-eun Park , Ahmed H. E. Hassan , Ae Nim Pae , Jiyoun Lee , Sora
a,h
e
a,f,*
Paik , Beoung-Geon Park , Eun Joo Roh
a
b
c
d
e
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
Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology (KIST), Seoul
0
2792, Republic of Korea
f
Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Republic of Korea
Department of Global Medical Science, Sungshin Women's University, Seoul 142-732, Republic of Korea
g
h
Department of Fundamental Pharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul, 02447, Republic of Korea
*
Corresponding authors:
Ahmed Elkamhawy: a_elkamhawy@mans.edu.eg
Eun Joo Roh: r8636@kist.re.kr
; a.elkamhawy@gmail.com
1
Both authors contributed equally to this work

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Abstract
Herein, we report synthesis and evaluation of new twenty-eight pyrazinyl ureas 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). Among them, 1-(3-(benzyloxy)pyrazin-2-yl)-3-(3,4-
dichlorophenyl)urea (5) effectively maintained mitochondrial function and cell viabilities on
ATP assay and MTT assay. Also, hERG channel assay presented safe cardiotoxicity profile for
compound 5. In addition, using CDocker algorithm, a molecular docking model presented a
plausible explanation for the elicited differences in efficiencies of the synthesized compounds to
reduce the green to red fluorescence as indication of mPTP closure. Hence, this report presents
compound 5 as the most promising pyrazinyl urea-based mPTP blocker up to date.
Keywords: β-amyloid peptide (Aβ); Mitochondrial permeability transition pore (mPTP); Aβ-
induced neurotoxicity; Alzheimer’s disease (AD); urea; Cyclophilin D

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1
. Introduction
As the most common cause of dementia, Alzheimer’s disease (AD) comes to the fore with 60−70%
of cases [1, 2]. The excessive production of β-amyloid peptide (Aβ) and its extracellular
deposition is a significant key factor associated with Alzheimer’s disease etiology [3]. Recent
reports indicate that mitochondrial permeability transition pore (mPTP) is a confirmed target for
the soluble Aβ oligomers since it plays a key role in mitochondrial dysfunction induced by Aβ
toxicity [4-6]. The multiprotein complex (mPTP) could be formed in mitochondria under certain
pathological conditions including traumatic membrane injury, oxidative stress, ischemia, and
0 1
stroke. The recent acceptable model of mPTP shows that its core consists of F /F ATPase
dimers in direct interaction with the regulatory CypD in the mitochondrial matrix [7].
Accumulation of the soluble Aβ oligomers results in excessive calcium entry into cytosol with
consequent uncontrolled mPTP opening followed by inhibition of mitochondrial ATP production,
mitochondrial swelling and outer membrane rupture. In fact, CypD was reported to be the mPTP
component that responds to various stimuli and initiates opening of mPTP [8]. Thus,
development of novel mPTP blockers targeting CypD as a main component of the pore could
serve as a promising approach for treatment of Alzheimer’s disease [5, 9, 10]. However, some
peptidic compounds as cyclosporin A (CsA) and its analog N-methyl-4-isoleucine cyclosporin
(NIM811) which have been identified as mPTP inhibitors showed no promising therapy due to
hurdles of peptide delivery to the brain, reports of efforts to develop mPTP opening modulators
are limited in literature (Fig. 1) [11-15]. To the best of our knowledge, the therapeutic small
molecule blocker of mPTP has not been realized yet.

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Fig. 1. Previously reported chemical entities with potential mPTP inhibition activity
This encouraged our institute to launch a discovery project towards identification of promising
mPTP modulators. As a result, different chemical scaffolds-based small molecules have been
reported as potential mPTP inhibitors [16-21]. Recently, as illustrated in Fig. 2, we have found
the aromatic pyrazinyl urea derivatives to be inactive [18]. However, high activity has been
exhibited by pyrazinyl thiourea analogs during our recent investigation for pyridyl/pyrazinyl
thioureas [22]. Thus, we revisited the pyrazinyl urea chemical scaffold in order to identify a
potential promising mPTP modulating analog. Herein, various aliphatic derivatives have been
included in our investigation containing chloroethyl and piperazinethyl analogs as we noticed
previously high activity of the pyridyl derivatives of these moieties. Successfully, in this report,
the blocking activity of new seventeen pyrazinyl ureas against Aβ-induced mPTP opening has
been found superior to that of the peptidic standard CsA. The most active hits were further
assessed for their effect on ATP production as well as their protective effect against Aβ-induced
ATP production impairment. The promising candidates were subjected to MTT viability to

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evaluate their toxicity on neuronal cells. After assessment of cardiotoxicity, the promising
compound 5 has been found to be a safe mPTP modulator that could serve as a novel runner
towards AD drug development marathon.
Fig. 2. KIST reported pyrazinyl ureas with low mPTP inhibition activity and revisiting design approach of the newly
synthesized pyrazinyl ureas via modifying the eastern moiety as well as various substitutions on the benzyloxy
group.
2
2
. Results and discussion
.1. Chemistry
A concise preparation of the target compounds (1−28) was achieved as illustrated in Scheme 1.
Nucleophilic aromatic substitution of the commercially available starting material 2-amino-3-
chloropyrazine (VII) with the appropriate phenylmethoxide derivative generated by treatment of
the corresponding benzyl alcohol with sodium hydride afforded 3-aryloxypyrazin-2-amine
derivatives (VIIIa−f). Nucleophilic addition of intermediates VIII to the appropriate
electrophilic isocyanate derivative afforded the corresponding pyrazinyl ureas (1−21) while 2-
chloroethyl urea analogs (22−24) were accessed via reaction of intermediates VIIIa, VIIIc, and
VIIId with 2-chloroethyl isocyanate reagent in presence of DIPEA. Finally, a small series of

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piperazinethyl urea derivatives (25−28) were obtained via reaction of 2-chloroethyl urea analogs
(22−24) with the appropriate piperazine derivative.
Scheme 1. Reagents and conditions: a) benzyl alcohol derivative, NaH (60% in mineral oil), DMF, 100 °C, 15 h; b)
for aromatic urea derivatives: appropriate aromatic isocyanate derivative, THF, reflux, 3−6 h; for aliphatic urea
derivatives: appropriate aliphatic isocyanate derivative, NaH (60% in mineral oil), THF, reflux 5h; c) 2-chloroethyl
isocyanate, DIPEA, THF, reflux, 18 h; d) appropriate piperazine derivative, K CO , MeCN, reflux, 20 h.
2
3
2
2
.2. Biological evaluation
.2.1. Protection against loss of mitochondrial membrane potential assay (JC-1 assay)
) is essential to maintain the mitochondrial
As the mitochondrial membrane potential (∆Ψ
M
integrity, the prolonged opening of the mega-channel mPTP leads to mitochondrial membrane
potential loss. While mitochondria with conserved membrane potential are featured with an
inside negative matrix potential, the decline in mitochondrial membrane potential could be
measured and quantified using fluorescent cationic dyes. JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-
tetraethylbenzimidazolocarbocyanine iodide); a lipophilic cationic cyanine dye; offers many
advantages over other cationic dyes as its performance is specific for the mitochondrial

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membrane potential; it is not affected by other components including plasma membrane potential
(
∆Ψ ), mitochondrial size or shape [23, 24]. In mitochondria with conserved membrane potential,
p
JC-1 forms red aggregates which break up into green fluorescing monomers upon membrane
potential loss. Using the neurotoxin amyloid beta (Aβ), the protective effect of the newly
synthesized compounds to inhibit the Aβ-induced mitochondrial potential loss was successfully
quantified via measurement of the green/red fluorescence ratio in hippocampal neuronal cells. In
this assay, the standard Cyslosporin (CsA) lowered the green/red (g/r) ratio to 46% (recovery of
the mitochondrial membrane potential = 54%). As illustrated in Table 1, both of aromatic and
aliphatic derivatives possessing unsubstituted benzyloxy moiety (compounds 1−5) elicited
significant reduction of g/r ratio. However, the t-butyl derivative 3 was the less effective among
them eliciting g/r % higher than the standard CsA. While, the phenyl derivative 4 was almost as
active as the standard CsA. A slightly different pattern of activity was elicited by compounds
having 3-flourobenzyloxy moiety (compounds 6−9). Among the later derivatives, the t-butyl
derivative 7 was also the less effective among them eliciting g/r % higher than the standard CsA.
However, the phenyl derivative 8 showed increased activity. In contrast, the t-butyl derivative 11
was the most active while the 1,1,3,3-tetramethylbutyl derivative 10 became the less active
among the derivatives having 2-chlorobenzyloxy moiety (compounds 10−14). For the 3-
chlorobenzyloxy derivatives (compounds 15−18), ethylurea derivative 15 was the less active
than aromatic congeners. Converting the ethyl substituent of compound 15 into 2-chloroethyl
derivative 24 produced less active compound. However, attachment of N-methylpiperazinyl
moiety instead of the chlorine atom of compound 24 resulted in the more active compound 27.
Similarly, replacement of chlorine atom of compound 23 with piperazinyl or N-
methylpiperazinyl resulted in the more active compounds 28 and 26. This pattern of activity

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enhancement by replacing the chlorine atom with N-methylpiperazinyl moiety was less
pronounced for compounds 22 and 25 having unsubstituted benzyloxy moiety. Among all
evaluated compounds, derivative 21 possessing 4-fluorobenzyloxy moiety and 1,1,3,3-
tetramethylbutylurea moiety was the most effective eliciting 17% g/r ratio. Also, another five
compounds (2, 5, 6, 11, and 18) showed high efficiency with increased g/r ratio less than 30%. In
addition, only four compounds (15, 20, 23, and 24) exerted weak or non-significant inhibitory
activity ranging from 60~73%. Overall investigation shows that pyrazine-based scaffold could
results in potentially active chemical entities. However, the mixed activity might indicate that
subtle stereoelectronic variations significantly impact the elicited biological activity [25, 26].
Accordingly, to characterize the promising hit compounds among the synthesized series, a set of
active compounds were further assessed for ATP production (Luciferase-based assay) as well as
neurocytotoxicity assay.
Table 1. In vitro blocking activity of the newly synthesized compounds (1−28) against Aβ-induced mPTP opening
in hippocampal neuronal cell line HT-22 (JC-1 assay) at dose of 5 µM
Increased
g/r ratio
Increased
g/r ratio
(%)
Cpd
R1
R2
Cpd
R1
R2
a
a
(
%)
33
25
51
44
29
29
55
30
39
59
28
33
39
55
60
1
2
3
4
5
6
7
8
9
H
H
H
H
H
3-F
3-F
3-F
3-F
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
3-Cl
Cycloheptyl
1,1,3,3-Tetramethylbutyl
tert-Butyl
16
17
18
19
20
21
22
23
24
25
26
27
28
CsA
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-F
H
2-Cl
3-Cl
H
Phenyl
3-Fluorophenyl
3-Chlorophenyl
59
50
29
35
60
17
53
72
73
43
34
34
37
46
Phenyl
1,1,3,3-Tetramethylbutyl
1-Naphthyl
1,1,3,3-Tetramethylbutyl
3,4-Dichlorophenyl
1,1,3,3-Tetramethylbutyl
tert-Butyl
−
−
−
−
−
−
−
−
Phenyl
3-Chlorophenyl
1,1,3,3-Tetramethylbutyl
tert-Butyl
1
1
1
1
1
1
0
1
2
3
4
5
2-Cl
3-Cl
2-Cl
−
Benzyl
Phenyl
3-Chlorophenyl
Ethyl

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a
%
Increase of fluorescence-ratio (green/red) after treatment of each compound and Aβ with regard to that of Aβ
alone (100%). See the text for more detailed information. All data are reported as the average of duplicates.
2
.2.2. Assessment of ATP production and protection against Aβ-induced impairment of
ATP production (Luciferase-based assay)
Mitochondrial toxicity is a major concern for safety in drug discovery programs; in addition, the
compounds developed in this work are potential mPTP blockers whose core unit is formed of
ATP synthase dimers. Therefore, it is important to assess their impact on mitochondrial ATP
production to avoid undesirable blocking of mitochondrial energy production. Thus, a set of
highly active compounds was evaluated for potential toxicity on ATP production using
hippocampal neuronal cell line after incubation for 7 h with 5 µM concentrations of each tested
compound. As shown in Table 2, compounds 11, 13 and 28 were found to seriously impair the
mitochondrial ATP production and, therefore, were excluded from further evaluation.
Fortunately, in presence of other compounds of the evaluated set, mitochondria elicited ATP
production levels close or higher than the level of ATP production in presence of the standard
piracetam. In lieu of this, it may be concluded that these compounds does not produce unwanted
impairment of mitochondrial energy production. Next, the compounds passed mitochondrial
ATP production filter were assessed for their capacity to protect neuronal cells against Aβ-
induced deterioration of ATP production which is a major concern in of Aβ-induced
mitochondrial dysfunction. As illustrated in Fig. 3 and presented in Table 2, three compounds (1,
5
, and 12) were able to show significant promising protection against Aβ-suppression of ATP
production with 45.7, 71.2, and 71.3% of ATP recovery respectively.
Table 2. The result of ATP assay of the tested compounds
a
b
a
b
Cpd
1
ATP Production
103.4
% ATP Recovery
45.7
Cpd
18
ATP Production
85.4
% ATP Recovery
-3.5

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2
5
6
86.0
148.6
98.9
55.3
96.9
66.9
-59.0
71.2
32.9
19
25
27
85.7
97.2
96.1
70.0
88
-1.7
0.7
17.7
c
c
1
1
1
1
2
3
NT
28
NT
71.3
Piracetam
127
c
NT
a
%
ATP production in hippocampal neuronal cell line HT-22 after 7 h incubation with 5 µM of each compound. All
data are reported as the average of duplicates.
b
%
Recovery of ATP production by Aβ-suppressed mitochondrial ATP production in hippocampal neuronal cell
line HT-22 after 7 h incubation with 5 µM of each compound. All data are reported as the average of duplicates.
c
NT: not tested
8
6
4
2
0
0
0
0
0
1
2
5
6
12
18
19
25
27
-20
-40
-60
Fig. 3. The result of % recovery of ATP production assay at 5 µM of each compound against Aβ-suppressed
mitochondrial ATP reduction
2
.2.3. Assessment of neurocytotoxicity (MTT assay)
As a subsequent filter to exclude compound(s) possessing potential cytotoxic effects [27, 28],
analogs showed promising results in luciferase-based assay (1, 5, and 12) were subjected to MTT
cell viability assay to evaluate the level of hippocampal neuronal cells survival after incubation
for 24 h with 5 µM of each tested compound. As presented in Table 3, compound 1 elicited
potential cytotoxic effects decreasing the cells viability to 59%. Also, compound 12 elicited a

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slight decrement in cell viability, to a lesser extent that is comparable to that of the standard CsA.
Fortunately, compound 5 showed cell viability result superior to that of CsA and piracetam.
Based on previous applied consecutive filter assays, it can be assumed that this derivative has no
direct toxic effect on mitochondrial ATP production and also no cytotoxicity on hippocampal
neuronal cells viability. In addition, it has a significant protection feature against A
impairment of ATP production.
β-induced
Table 3. Results of neurocytotoxicity at 5 µM concentrations
a
Cpd
MTT viability
1
5
59
139.2
84.8
132
90
1
2
Piracetam
CsA
a
%
Viability of hippocampal neuronal cell line HT-22 after 24 h incubation with 5 µM of each compound. All data
are reported as the average of duplicates.
2
.2.4. Assessment of cardiotoxicity (hERG channel assay)
Blockade of the human ether-a-go-go related gene (hERG) potassium channel may cause sudden
drug withdrawal from markets as a result of induction the prolongation of the QT interval of the
surface electrocardiogram (ECG) which makes hERG blockade an important hurdle in lead
optimization activity as well as drug discovery process due to its potential cardiotoxicity [29, 30].
Thus, we measured the hERG inhibitory activity of compound 5. The test demonstrated a high
micromolar IC50 value equal to 11.3 µM. Hence, compound 5 doesn’t exert potential blockage
activity towards the hERG channel. Accordingly, it could be assumed as safe compound with
regard to the potential cardiotoxicity.

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2
.2.5. Molecular modelling
In the reported crystal of human CypD-CsA complex (pdb ID = 2Z6W), CsA effectively binds to
CypD eliciting hydrophobic interactions as a major contributor to the binding forces [31]. The
hydrophobic pocket 1 formed by residues Met61, Ala101, Phe113, Leu122 and His126 interacts
with the methylated valine (Mva11) of CsA. Amino acid residues Gln63 and Asn102 form a
saddle between pocket 1 and the less hydrophobic pocket 2. Also, the structure of CypD-CsA
complex shows that Phe60, Ile57 and Trp121 form a flat hydrophobic surface which interacts
with the side chain methylated leucine (Mle9) of CsA. In the crystal structure, hydrogen bonds
exist with amino acids Gln63 and Asn102 in the saddle region, Arg55 and His126 at the edge of
pocket 1, and Trp121 in the flat hydrophobic surface.
To get insights into the molecular behavior underlying the differences in the elicited biological
activities, a molecular modeling study was conducted. A set of active compounds (2, 5, 6, 11, 18
and 21), as well as, another set of the less active compounds (15, 20, 23 and 24) were docked
into the reported crystal structure of human CypD (pdb ID = 2Z6W). The retrieved poses were
refined using in situ minimization after which, the energetic terms of CypD-ligand complex were
calculated. Poses were ranked based on these calculated energetic terms. As reported earlier, the
obtained poses of this class of compounds could accommodate two general binding modes [18,
1
9, 22]. The first binding mode, favored by active compounds, features the ligand partially
buried into the hydrophobic pocket 1 eliciting favorable hydrophobic interactions. The second
binding mode, favored by inactive compounds, shows a vacant hydrophobic pocket 1 while the
ligand is forming an umbrella above it.

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Analysis of compound 5 poses which represent the set of the most active compounds shows a
strong tendency of this compound to bind in mode 1. According to the performed in silico
calculations, the pyrazine ring in binding mode 1 shown in Fig. 4A fits into the hydrophobic
pocket 1 eliciting favorable hydrophobic interactions with the aromatic rings of Phe113 and
His126, as well as, with the side chain of Ala101. Also, the pyrazine ring is involved in
electrostatic
π-cation interaction with His126. In addition, it establishes hydrogen bonding
interactions with side chain of Arg55 and backbone peptide linkage of Asn102. Meanwhile, the
urea moiety accepts hydrogen bond from His126 and donates two hydrogen bonds to Asn102. In
addition, there is an amide-
π stacking interaction between the aromatic ring of the 3,4-
dichlorophenyl moiety and the peptide linkage of Ala103-Gly104. Moreover, the two chlorine
atoms are involved in two hydrophobic interactions with side chains of Pro105 and Lys125. The
calculated energetic terms for this binding mode were -58.68 Kcal/mol for binding energy and
1
.80 Kcal/mol for the value of the ligand conformational energy above the energy of the lowest
conformer energy. On the other hand, as illustrated in Fig. 4B, if compound 5 accommodates
binding mode 2 in which it is hovering above the hydrophobic pocket 1 without docking inside,
it would interact through several favorable interactions including interactions of the 3,4-
dichlorophenyl moiety with hydrophobic and
π-cation interactions with His126, as well as,
hydrophobic interactions of the two chlorine atoms with Phe60 and Leu122. The urea moiety
donates two hydrogen bonds to Asn102 while the pyrazine ring is interacting via two hydrogen
bonds with Gln63and Gln111, in addition to π-cation and hydrophobic interactions with arg55
and Ala101 respectively. In silico calculations of the energetic terms of binding mode 2 for
compound 5 estimated binding energy value of -54.75 Kcal/mol and ligand conformational
energy above the energy of the lowest conformer energy of 2.74 Kcal/mol. The higher

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conformational energy of the conformer required for binding mode 2 relative to that required for
binding mode 1 translates into less accessibility of the conformer of binding mode 2. At the same
time, the lower binding energy of binding mode 2 relative to that of binding mode 1 translates
into lower kinetics for forming binding mode 2. Considering the above mentioned energetic
terms, the high efficiency of compound 5 in reducing g/r ratio would be understandable.
Fig. 4. Binding modes of compound 5 with CypD: A) Binding mode 1; B) Binding mode 2
Analysis of compound 23 poses representing the set of the less active compounds shows that if it
binds according to binding mode 1 (Fig. 5A), it would elicit favorable hydrogen bonding
interactions between pyrazine ring and all of Arg55, Gln63 and His126. It shows also
hydrophobic and π-sulfur interactions with Ala101 and Met61, respectively. The urea moiety
accepts hydrogen bonds from Arg55 while the oxygen atom of the benzyloxy moiety accepts
hydrogen bonding from His126. In comparison, the predicted binding mode 2 of compound 23
(
Fig. 5B) shows favorable hydrogen bonding between Arg55 and both of the pyrazine ring and
oxygen atom of the urea moiety, as well as, hydrophobic and -cation interactions between
π
His126 and the aromatic ring of the benzyloxy moiety. In addition, the chloro atom of the
benzyloxy moiety interacts hydrophobically with Lys125. The energetic terms for binding mode
1
of compound 23 were estimated to be -57.60 Kcal/mol for binding energy, -3050.51 Kcal/mol

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for ligand-complex energy and 3.75 Kcal/mol for conformational energy above the energy of the
lowest conformer energy. Regarding binding mode 2 of compound 23, the energetic terms of
binding mode 2 were estimated to be -46.31 for Kcal/mol binding energy, -3057.89 Kcal/mol for
ligand-complex energy and 1.82 Kcal/mol for ligand conformational energy above the energy of
the lowest conformer energy. While the lower binding energy of mode 2 relative to mode 1
predicts slower kinetics for formation of binding mode 2, the lower conformational energy of the
conformer of binding mode 2 indicates more probability of accessing that conformer. In addition,
the lower ligand-CypD complex energy predicts more stability of binding mode 2 and thus more
elapsed time accommodating this binding mode. In the light of these calculations, the lower
efficiency of compound 23 would be justifiable.
Fig. 5. Binding modes of compound 23 with CypD: A) Binding mode 1; B) Binding mode 2
3
. Conclusion
As a conclusion for revisiting of the pyrazinyl-urea chemistry as potential modulators of A
induced mitochondrial dysfunction towards novel therapies for Alzheimer’s diseases, compound
, 1-(3-(benzyloxy)pyrazin-2-yl)-3-(3,4-dichlorophenyl)urea, has been identified as a new
promising candidate featured with: (1) significant blockage of mPTP A -induced opening (2) no
toxicity on ATP production (3) no detrimental effect on the viability of hippocampal neuronal
cells (4) significant protection against A -induced impairment of ATP production (5) no
β-
5
β
β

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potential cardiotoxicity. Thus, this study presents analog 5 as a new non-peptidyl pyrazinyl-urea-
based promising mPTP blocker.
4
4
. Experimental
.1. Chemistry
General: All reactions and manipulations were performed in nitrogen atmosphere using standard
Schlenk techniques. The reaction solvents and reagents were purchased from commercial
suppliers and used without further purification. The NMR spectra were obtained on Bruker
1
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 determined by analytical HPLC using a Water ACQUITY
TM
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. General procedure of 3-(benzyloxy)pyrazin-2-amine derivatives (VIIIa−f)

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NaH (60% in mineral oil, 0.04 g, 1 mmol) was added to a stirred solution of the appropriate
benzyl alcohol derivative (1 mmol) in anhydrous DMF (3 mL) at room temperature and stirring
was continued for 1 h. Commercially available 2-amino-3-chloropyrazine (0.13 g, 1 mmol) was
added to the reaction mixture which was further 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 anhydrous Na SO , filtered, and concentrated. The residue was
2
4
purified by flash column chromatography (SiO
2
, EA/n-Hex = 1/5). Synthesis and
characterization of compounds VIIIa−c have been reported previously by our group [18, 19].
3
-(3-Chlorobenzyloxy)pyrazin-2-amine (VIIId)
1
Light yellow solid, yield: 66.0%, H NMR (400 MHz, CDCl )
δ
= 4.80 (2H, brs, NH ), 5.37 (2H,
3
2
s, OCH Ph), 7.31 (3H, d,
J
= 1.3 Hz, ArH), 7.42 (1H, d,
J = 3.1 Hz, ArH), 7.44 (1H, s, ArH),
2
7
.58 (1H, d, J = 3.1 Hz, ArH).
3
-(4-Chlorobenzyloxy)pyrazin-2-amine (VIIIe)
1
White solid, yield: 38.1%, H NMR (400 MHz, CDCl
3
)
δ
= 5.36 (2H, s, OCH
2
Ph), 6.31 (2H, brs,
= 3.0 Hz,
NH ), 7.25 (1H, d,
J
= 3.0 Hz, ArH), 7.44 (2H, d,
J
= 8.4 Hz, ArH), 7.48 (1H, d, J
2
ArH), 7.52 (2H, d,
J = 8.4 Hz, ArH).
3
-(4-Fluorobenzyloxy)pyrazin-2-amine (VIIIf)
1
Yellow solid, yield: 65.5%, H NMR (400 MHz, CDCl )
δ
= 4.78 (2H, brs, NH ), 5.36 (2H, s,
3
2
OCH
2
Ph), 7.05−7.09 (2H, m, ArH), 7.41−7.44 (3H, m, ArH), 7.56 (1H, d,
J
= 3.1 Hz, ArH).

ACCEPTED MANUSCRIPT
4
.1.2. General procedure of pyrazinyl urea compounds 1−21
For aromatic urea derivatives; the appropriate aromatic isocyanate (3.0 mmol) was added to a
solution of the appropriate 2-amino-3-benzyloxy pyrazine derivative (2.5 mmol) in THF (10
mL). The reaction was refluxed for 3−6 h. After cooling, the reaction mixture was evaporated
and the residue was purified by precipitation in cold methanol and filtered to give the target
compound(s). For aliphatic urea derivatives; the appropriate aliphatic isocyanate derivative (1.02
mmol) was added to a solution of the appropriate 2-amino-3-benzyloxy pyrazine derivative (0.85
mmol) in dry THF (5 mL) in the presence of NaH (60% in mineral oil, 68 mg, 1.71 mmol). The
reaction was refluxed for 5 h. After cooling, the reaction mixture was evaporated and the residue
was purified by flash column chromatography (SiO , EA/n-Hex = 1/4).
2
1
-(3-(Benzyloxy)pyrazin-2-yl)-3-cycloheptylurea (1)
White solid, yield: 46.2%, mp: 91.8-93.7 °C, HPLC purity: 7.33 min, 100%, H NMR (400 MHz,
CDCl3)
= 1.58−1.65 (10H, m, 5CH ), 1.96−2.00 (2H, m, CH ), 3.97−4.03 (1H, m, CH), 5.41
1
δ
2
2
(
2H, s, OCH = 3.1 Hz, ArH), 7.66
2
Ph), 7.29 (1H, s, NH), 7.36−7.44 (5H, m, ArH), 7.63 (1H, d,
J
1
3
(
1H, d, = 3.1 Hz, ArH), 9.08 (1H, s, NH). C NMR (100 MHz, CDCl ) δ = 24.00, 27.96, 35.12,
J
3
5
0.96, 68.72, 128.61, 128.67, 131.51, 131.64, 135.48, 140.10, 147.90, 153.22. HRMS
+
+
(
ES ): m/z calculated for C H N O : 341.1977 [M+H] . Found 341.1976.
19 24 4 2
1
-(3-(Benzyloxy)pyrazin-2-yl)-3-(2,4,4-trimethylpentan-2-yl)urea (2)
1
White solid, yield: 20.1%, mp: 86.7-87.1 °C, H NMR (400 MHz, CDCl )
δ
= 1.01 (9H, s,
3
3
CH
3
), 1.47 (6H, s, 2CH
3
), 1.80 (2H, s, CH
2
), 5.40 (2H, s, OCH
2
Ph), 7.15 (1H, s, NH),

ACCEPTED MANUSCRIPT
7
.35−7.43 (5H, m, ArH), 7.61 (1H, d,
J
= 3.1 Hz, ArH), 7.63 (1H, d,
J
= 3.1 Hz, ArH), 9.05 (1H,
+
+
s, NH). HRMS (ES ): m/z calculated for C H N O : 357.2290 [M+H] . Found 357.2291.
2
0
28
4
2
1
-(3-(Benzyloxy)pyrazin-2-yl)-3-(tert-butyl)urea (3)
1
Yellow solid, yield: 16.3%, mp: 96.9-97.7 °C, HPLC purity: 6.77 min, 100%, H NMR (400
MHz, CDCl3)
m, ArH), 7.61 (1H, d,
NMR (100 MHz, CDCl
35.55, 140.21, 147.84, 152.77. HRMS (ES ): m/z calculated for C16
δ
= 1.43 (9H, s, 3CH ), 5.41 (2H, s, OCH Ph), 7.18 (1H, s, NH), 7.36−7.43 (5H,
3
2
13
J
= 3.1 Hz, ArH), 7.65 (1H, d, J = 3.1 Hz, ArH), 9.01 (1H, s, NH). C
3
) δ = 29.06, 50.74, 68.65, 128.48, 128.52, 128.65, 131.31, 131.57,
+
1
H
20
N
4
O
2
: 301.1664
+
[
M+H] . Found 301.1667.
1
-(3-(Benzyloxy)pyrazin-2-yl)-3-phenylurea (4)
White solid, yield: 91.8%, mp: 139.4-140.0 °C, HPLC purity: 6.80 min, 100%, H NMR (400
MHz, CDCl3) = 5.43 (2H, s, OCH Ph), 7.09 (1H, t, = 7.4 Hz, ArH), 7.33 (2H, t, = 7.5 Hz,
ArH), 7.36−7.45 (5H, m, ArH), 7.49 (1H, s, NH), 7.57 (2H, dd,
= 1.1 Hz, 8.6 Hz, ArH), 7.72
= 3.1 Hz, 14.8 Hz, ArH), 11.28 (1H, s, NH). C NMR (100 MHz, CDCl = 68.99,
20.37, 123.88, 128.70, 128.75, 129.01, 131.45, 132.35, 135.34, 138.01, 139.50, 148.08, 151.63.
1
δ
J
J
2
J
1
3
(2H, dd,
J
3
) δ
1
+
+
HRMS (ES ): m/z calculated for C H N O : 321.1351 [M+H] . Found 321.1349.
1
8
16
4
2
1
-(3-(Benzyloxy)pyrazin-2-yl)-3-(3,4-dichlorophenyl)urea (5)
White solid, yield: 87.1%, mp: 151.1-151.8 °C, HPLC purity: 7.65 min, 100%, H NMR (400
MHz, CDCl = 5.44 (2H, s, OCH
Ph), 7.32−7.45 (7H, m, ArH), 7.53 (1H, s, NH), 7.73-7.74
3
2H, m, ArH), 7.76 (1H, d, = 2.4 Hz, ArH), 11.42 (1H, s, NH). C NMR (100 MHz, CDCl ) δ
1
3
)
δ
2
1
3
(
J

ACCEPTED MANUSCRIPT
=
69.08, 119.37, 121.63, 126.80, 128.70, 128.75, 130.41, 131.30, 132.65, 132.80, 135.23,
+
1
37.64, 139.09, 148.10, 151.36. HRMS (ES ): m/z calculated for C H Cl N O : 389.0572
1
8
14
2
4
2
+
[
M+H] . Found 389.0570.
1
-(3-(3-Fluorobenzyloxy)pyrazin-2-yl)-3-(2,4,4-trimethylpentan-2-yl)urea (6)
1
White solid, yield: 56.2%, mp: 112.5-113.8 °C, HPLC purity: 7.83 min, 100%, H NMR (400
MHz, CDCl = 1.02 (9H, s, 3CH ), 1.48 (6H, s, 2CH ), 1.81 (2H, s, CH ), 5.40 (2H, s,
OCH Ph), 7.04 (1H, td,
= 2.2 Hz, 8.4 Hz, ArH), 7.11−7.14 (2H, m, ArH+NH), 7.20 (1H, d,
.7 Hz, ArH), 7.32−7.38 (1H, m, ArH), 7.60 (1H, d, = 3.1 Hz, ArH), 7.65 (1H, d, = 3.1 Hz,
ArH), 9.04 (1H, s, NH); C NMR (100 MHz, CDCl3) = 29.57, 31.47, 31.65, 51.68, 54.52,
3
)
δ
3
3
2
2
J
J =
7
J
J
1
3
δ
6
1
7.75, 115.31 (JC-F = 21.7 Hz), 115.44 (JC-F = 20.8 Hz), 123.88, 130.26 (JC-F = 8.0 Hz), 131.25,
31.78, 138.00 (JC-F = 7.3 Hz), 140.11, 147.48, 152.51, 162.83 (JC-F = 245.2 Hz). HRMS
+
+
(
ES ): m/z calculated for C H FN O : 375.2196 [M+H] . Found 375.2198.
20 27 4 2
1
-(3-(3-Fluorobenzyloxy)pyrazin-2-yl)-3-(tert-butyl)urea (7)
1
White solid, yield: 75.9%, mp: 102.8-103.5 °C, HPLC purity: 6.78 min, 100%, H NMR (400
MHz, CDCl3)
1H, m, ArH), 7.15 (1H, s, NH), 7.19 (1H,
d, = 3.1 Hz, ArH), 7.66 (1H, d,
CDCl3) = 29.60, 50.77, 67.71, 115.42 (JC-F = 21.5 Hz), 115.42 (JC-F = 20.9 Hz), 123.87, 130.25
JC-F = 8.0 Hz), 131.27, 131.85, 137.99, 140.13, 147.51, 152.71, 162.83 (JC-F = 245.6 Hz). HRMS
δ
= 1.43 (9H, s, 3CH ), 5.40 (2H, s, OCH Ph), 7.03−7.05 (1H, m, ArH), 7.10−7.13
3
2
(
J
= 7.4 Hz, ArH), 7.32−7.36 (1H, m, ArH), 7.60 (1H,
1
3
J
J = 3.1 Hz, ArH), 8.99 (1H, s, NH); C NMR (100 MHz,
δ
(
(
+
+
4 2
ES ): m/z calculated for C16H19FN O : 319.1510 [M+H] . Found 319.1577.

ACCEPTED MANUSCRIPT
1
-(3-(3-Fluorobenzyloxy)pyrazin-2-yl)-3-phenylurea (8)
1
White solid, yield: 51.9%, mp: 136.0-136.5 °C, HPLC purity: 6.80 min, 100%, H NMR (400
MHz, CDCl3) = 5.43 (2H, s, OCH Ph), 7.03−7.16 (3H, m, ArH), 7.22 (1H, d, = 7.7 Hz, ArH),
= 1.1 Hz, 8.7 Hz, ArH), 7.70 (1H, d,
δ
J
2
7
=
δ
.31−7.37 (3H, m, ArH), 7.50 (1H, s, NH), 7.58 (2H, dd,
J
J
1
3
3.1 Hz, ArH), 7.76 (1H, d,
J = 3.1 Hz, ArH), 11.26 (1H, s, NH). C NMR (100 MHz, CDCl )
3
= 68.04, 115.44 (JC-F = 13.1 Hz), 115.65 (JC-F = 12.1 Hz), 120.38, 123.93, 124.08 (JC-F = 3.0
Hz), 129.00, 130.36 (JC-F = 9.1 Hz), 131.73, 132.30, 137.81 (
JC-F = 7.0 Hz), 137.95, 139.46,
+
1
47.78, 151.58, 162.87 (
J
C-F = 246.5). HRMS (ES ): m/z calculated for C18
H
15FN
4
O
2
: 339.1257
+
[
M+H] . Found 339.1271.
1
-(3-(3-Fluorobenzyloxy)pyrazin-2-yl)-3-(3-chlorophenyl)urea (9)
1
White solid, yield: 91.9%, mp: 146.5-147.4 °C, H NMR (400 MHz, DMSO-d6
)
δ
= 5.49 (2H, s,
= 1.9 Hz, 8.4 Hz, ArH), 7.29−7.49 (5H, m,
= 2.1 Hz, 11.68 Hz, ArH), 7.83 (1H, d, = 3.0 Hz, ArH), 7.95 (1H, d,
.0 Hz, ArH), 9.16 (1H, s, NH), 11.09 (1H, s, NH). C NMR (100 MHz, CDCl = 68.13,
15.53 ( C-F = 21.6 Hz), 115.68 ( C-F = 21.1 Hz), 118.26, 120.25, 123.89, 124.11, 129.98, 130.38
OCH Ph), 6.86−6.90 (1H, m ArH), 7.17 (1H, td,
J
2
ArH), 7.61 (1H, dt,
J
J
J =
1
3
3
1
3
) δ
J
J
(JC-F = 9.4 Hz), 131.66, 132.59, 134.61, 139.25, 147.82, 151.38, 162.85 (JC-F = 245.4 Hz).
1
-(3-(2-Chlorobenzyloxy)pyrazin-2-yl)-3-(2,4,4-trimethylpentan-2-yl)urea (10)
1
White solid, yield: 86.1%, mp: 100.0-101.2 °C, H NMR (400 MHz, CDCl )
δ = 1.02 (9H, s,
3
3
7
3
CH ), 1.48 (6H, s, 2CH ), 1.80 (2H, s, CH ), 5.51 (2H, s, OCH Ph), 7.14 (1H, s, NH),
3 3 2 2
.28−7.31 (2H, m, ArH), 7.41-7.46 (2H, m, ArH), 7.61 (1H, d,
J
= 3.1 Hz, ArH), 7.66 (1H, d,
J =
1
3
.1 Hz, ArH), 9.05 (1H, s, NH). C NMR (100 MHz, CDCl ) δ = 29.58, 31.48, 31.64, 51.68,
3

ACCEPTED MANUSCRIPT
5
1
4.51, 66.03, 126.98, 129.73, 129.85, 130.17, 131.34, 131.73, 133.26, 133.97, 140.10, 147.56,
52.56.
1
-(3-(2-Chlorobenzyloxy)pyrazin-2-yl)-3-(tert-butyl)urea (11)
1
White solid, yield: 85.2%, mp: 149.8-153.3 °C, H NMR (400 MHz, CDCl )
δ = 1.43 (9H, s,
3
3
CH ), 5.51 (2H, s, OCH Ph), 7.16 (1H, brs, NH), 7.28−7.33 (2H, m, ArH), 7.41−7.45 (2H, m,
3
2
1
3
ArH), 7.61 (1H, d,
J
= 3.1 Hz, ArH), 7.66 (1H, d, J = 3.1 Hz, ArH), 9.01 (1H, s, NH); C NMR
(100 MHz, CDCl
3
)
δ
= 29.06, 50.77, 66.05, 126.98, 129.76, 129.88, 130.18, 131.36, 131.80,
1
33.26, 134.01, 140.12, 147.60, 152.76.
1
-Benzyl-3-(3-(2-chlorobenzyloxy)pyrazin-2-yl)urea (12)
1
White solid, yield: 80.0%, mp: 107.8-108.8 °C, H NMR (400 MHz, CDCl )
δ
= 4.53 (2H, d,
J
=
3
5
7
1
1
.8 Hz, CH Ph), 5.44 (2H, s, OCH Ph), 7.19−7.28 (7H, m, ArH+NH), 7.34−7.39 (3H, m, ArH),
2 2
1
3
.57 (2H, s, ArH), 9.38 (1H, s, NH). C NMR (100 MHz, CDCl3)
δ = 43.88, 66.25, 127.03
27.26, 127.38, 128.63, 129.82, 130.02, 130.42, 131.87, 131.96, 133.16, 134.16, 138.81, 139.75,
47.74, 154.40.
1
-(3-(2-Chlorobenzyloxy)pyrazin-2-yl)-3-phenylurea (13)
1
White solid, yield: 43.0%, mp: 198.8-196.7 °C, H NMR (400 MHz, DMSO-d6
)
δ
= 5.54 (2H, s,
= 7.6 Hz, 8.3 Hz, ArH), 7.38−7.40 (2H,
= 7.6 Hz, ArH), 7.71−7.73 (1H, m, ArH), 7.83
OCH Ph), 7.06 (1H, t,
J
= 7.4 Hz, ArH), 7.33 (2H, dd,
m, ArH), 7.50−7.53 (1H, m, ArH), 7.56 (2H, d,
1H, d, = 3.0 Hz, ArH), 7.96 (1H, d, = 3.0 Hz, ArH), 8.94 (1H, s, NH), 10.91 (1H, s, NH); C
J
2
J
1
3
(
J
J

ACCEPTED MANUSCRIPT
NMR (100 MHz, CDCl
3
)
δ
= 66.43, 120.40, 123.92, 127.06, 129.01, 129.87, 130.11, 130.52,
1
31.67, 132.67, 133.05, 134.25, 137.96, 139.47, 147.88, 151.60.
1
-(3-(2-Chlorobenzyloxy)pyrazin-2-yl)-3-(3-chlorophenyl)urea (14)
1
White solid, yield: 83.0%, mp: 169.5-170.6 °C, H NMR (400 MHz, DMSO-d6
)
δ
= 5.54 (2H, s,
= 8.7 Hz,
= 2.0 Hz, ArH), 7.85 (1H,
OCH Ph), 7.11 (1H, d,
J
= 7.3 Hz, ArH), 7.33−7.40 (3H, m, ArH), 7.45 (1H, d,
ArH), 4.50−7.53 (1H, m, ArH), 7.71-7.73 (1H, m, ArH), 7.79 (1H, t,
d, = 2.9 Hz, ArH), 7.97 (1H, d, = 2.9 Hz, ArH), 9.09 (1H, s, NH), 11.0 (1H, s, NH); C NMR
100 MHz, CDCl = 66.51, 118.25, 120.23, 123.86, 127.06, 129.89, 129.96, 130.16, 130.57,
31.59, 132.65, 132.98, 134.59, 139.25, 147.91, 151.41.
J
2
J
1
3
J
J
(
3
) δ
1
1
-(3-(3-Chlorobenzyloxy)pyrazin-2-yl)-3-ethylurea (15)
1
White solid, yield: 65.2%, mp: 126.5-127.5 °C, H NMR (300 MHz, DMSO-d6
)
δ
= 1.11 (3H, t,
J
= 7.2 Hz, CH ), 2.26−7.27 (2H, m, CH ), 5.42 (2H, s, OCH Ph), 7.37−7.44 (2H, m, ArH),
3 2 2
7
.46−7.48 (1H, m, ArH), 7.67 (1H, brs, ArH), 7.71 (1H, d,
J
= 3.0 Hz, ArH), 7.80 (1H, d,
= 15.25, 34.88,
7.79, 126.59, 128.62, 128.75, 129.99, 131.65, 131.91, 134.55, 137.48, 139.89, 147.57, 154.12.
J = 3.0
1
3
3
Hz, ArH), 8.52 (1H, s, NH), 8.81 (1H, s, NH); C NMR (100 MHz, CDCl ) δ
6
1
-(3-(3-Chlorobenzyloxy)pyrazin-2-yl)-3-phenylurea (16)
1
White solid, yield: 82.5%, mp: 148.0-148.5 °C, H NMR (400 MHz, DMSO-d6
)
δ
= 5.46 (2H, s,
= 7.8 Hz, ArH), 7.40−7.44 (2H, m, ArH),
= 8.2 Hz, ArH), 7.69 (1H, s, ArH), 7.81 (1H, d,
OCH Ph), 7.06 (1H, t,
J
= 7.4 Hz, ArH), 7.33 (2H, t,
= 6.6 Hz, ArH), 7.57 (2H, d,
.0 Hz, ArH), 7.93 (1H, d, = 3.0 Hz, ArH), 9.05 (1H, s, NH), 10.97 (1H, s, NH); C NMR
J
2
7
3
.51 (1H, d,
J
J
J =
1
3
J

ACCEPTED MANUSCRIPT
(100 MHz, CDCl
3
)
δ
= 68.01, 120.40, 123.94, 126.67, 128.71, 128.86, 129.01, 130.06, 131.75,
1
32.29, 134.61, 137.34, 137.93, 139.46, 147.75, 151.57.
1
-(3-(3-Chlorobenzyloxy)pyrazin-2-yl)-3-(3-fluorophenyl)urea (17)
1
White solid, yield: 91.5%, mp: 158.4-159.8 °C, H NMR (400 MHz, DMSO-d6
)
δ
= 5.46 (2H, s,
= 6.5 Hz, ArH),
= 3.0 Hz, ArH), 7.95 (1H, d, = 3.0
Hz, ArH), 9.19 (1H, s, NH), 11.06 (1H, s, NH); C NMR (100 MHz, CDCl = 68.06, 107.62
C-F = 26.1 Hz), 110.51 ( C-F = 21.3 Hz), 115.52, 126.68, 128.80 ( C-F = 16.8 Hz), 129.94,
30.06, 131.67, 132.55, 134.62, 137.27, 139.27, 139.57 (JC-F = 10.6 Hz), 147.78, 151.40, 163.11
JC-F = 242.6 Hz).
OCH2Ph), 6.86−6.88 (1H, m, ArH), 7.28-7.41 (4H, m, ArH), 7.50 (1H, d,
.58−7.61 (1H, m, ArH), 7.68 (1H, brs, ArH), 7.83 (1H, d,
J
7
J
J
1
3
3
) δ
(J
J
J
1
(
1
-(3-(3-Chlorobenzyloxy)pyrazin-2-yl)-3-(3-chlorophenyl)urea (18)
1
White solid, yield: 93.8%, mp: 151.5-152.0 °C, H NMR (400 MHz, DMSO-d6
)
δ
= 5.47 (2H, s,
OCH
.82−7.83 (2H, m ArH), 7.95 (1H, d,
NMR (100 MHz, DMSO- 6 = 67.35, 118.21, 119.17, 123.11, 126.98, 128.24, 128.30, 130.59,
30.90, 132.81, 133.36, 133.53, 133.73, 139.26, 139.58, 140.57, 148.97, 151.75.
2
Ph), 7.11 (1H, dd, J
= 1.1 Hz, 7.9 Hz, ArH), 7.33−7.52 (5H, m, ArH), 7.69 (1H, s, ArH),
1
3
7
J = 3.0 Hz, ArH), 9.19 (1H, s, NH), 11.11 (1H, s, NH); C
d ) δ
1
1
-(3-(4-Chlorobenzyloxy)pyrazin-2-yl)-3-(2,4,4-trimethylpentan-2-yl)urea (19)
1
White solid, yield: 70.1%, mp: 56.1-59.2 °C, HPLC purity: 8.11 min, 93.94%, H NMR (300
MHz, DMSO- = 0.96 (9H, s, 3CH ), 1.39 (6H, s, 2CH ), 1.73 (2H, s, CH ), 5.41 (2H, s,
OCH Ph), 7.45 (2H, d, = 8.4 Hz, ArH), 7.57 (2H, d, = 8.4 Hz, ArH), 7.69 (1H, d, = 3.0 Hz,
d
6
)
δ
3
3
2
2
J
J
J

ACCEPTED MANUSCRIPT
ArH), 7.78 (1H, d,
J
= 3.0 Hz, ArH), 8.12 (1H, s, NH), 8.91 (1H, s, NH). HRMS
+
+
(
ES ): m/z calculated for C H ClN O : 391.1901 [M+H] . Found 391.1901.
20
27
4
2
1
-(3-(4-Chlorobenzyloxy)pyrazin-2-yl)-3-(naphthalen-1-yl)urea (20)
1
White solid, yield: 60.8%, mp: 132.2-136.6 °C, H NMR (400 MHz, DMSO-d6 + Acetone- 6
d
)
δ
=
t,
5.54 (2H, s, OCH Ph), 7.47 (2H, d,
J
= 8.4 Hz, ArH), 7.54 (1H, t,
= 7.2 Hz, ArH), 7.65−7.74 (4H, m, ArH), 7.87 (1H, d,
= 3.0 Hz, ArH), 7.99 (1H, d,
= 2.6 Hz, ArH), 8.18−8.25 (2H, m, ArH), 9.06 (1H, brs, NH), 11.85
J
= 7.9 Hz, ArH), 7.59 (1H,
2
J
J
J
= 8.2
Hz, ArH), 8.08 (1H, d,
4 2
1H, brs, NH). HRMS (ES ): m/z calculated for C22H17ClN O : 405.1118 [M+H] . Found
J
+
+
(
4
05.1117.
1
-(3-(4-Fluorobenzyloxy)pyrazin-2-yl)-3-(2,4,4-trimethylpentan-2-yl)urea (21)
1
White solid, yield: 68.4%, mp: 110.6-111.6 °C, HPLC purity: 7.83 min, 100%, H NMR (400
MHz, CDCl3) = 1.02 (9H, s, 3CH ), 1.48 (6H, s, 2CH ), 1.80 (2H, s, CH ), 5.37 (2H, s,
OCH Ph), 7.07 (2H, t,
3
dd, = 3.1 Hz, 16.4 Hz, ArH), 9.04 (1H, s, NH); C NMR (100 MHz, CDCl ) δ = 29.57, 31.46,
δ
3
3
2
J
= 8.7 Hz, ArH), 7.14 (1H, s, NH), 7.39−7.43 (2H, m, ArH), 7.62 (2H,
2
1
3
J
3
3
1.65, 51.64, 54.50, 67.91, 115.60 (JC-F = 22.1 Hz), 130.58 (JC-F = 9.1 Hz), 131.25, 131.38 (JC-F =
+
.0 Hz), 131.63, 140.12, 147.63, 152.56, 162.83 (JC-F = 247.5 Hz). HRMS (ES ): m/z calculated
+
for C H FN O : 375.2196 [M+H] . Found 375.2202.
2
0
27
4
2
4
.1.3. General procedure of chloroethyl urea derivatives 22−24
2
-Chloroethyl isocyanate (0.1 mL, 1.1 mmol) was added to a solution of the appropriate 2-
amino-3-benzyloxy pyrazine derivative (0.92 mmol) in dry THF (5 mL) in the presence of

ACCEPTED MANUSCRIPT
DIPEA (0.5 mL, 2.75 mmol). The reaction mixture was refluxed for 18 h and evaporated after
cooling. The residue was purified by flash column chromatography (SiO , EA/
n
-Hex = 1/4).
2
1
-(3-(Benzyloxy)pyrazin-2-yl)-3-chloroethylurea (22)
1
White solid, yield: 71.3%, mp: 112.2-112.6 °C, H NMR (400 MHz, CDCl )
δ
= 3.67−3.74 (4H,
3
m, 2CH ), 5.41 (2H, s, OCH Ph), 7.36−7.43 (6H, m, ArH+NH), 7.67 (1H, d,
J = 3.1 Hz, ArH),
2
2
1
3
7
6
.70 (1H, d,
J
= 3.1 Hz, ArH), 9.45 (1H, s, NH); C NMR (100 MHz, CDCl
3
)
δ
= 41.93, 43.86,
8.86, 128.65, 128.71, 131.72, 132.17, 133.03, 139.60, 140.93, 154.34.
1
-(3-(2-Chlorobenzyloxy)pyrazin-2-yl)-3-chloroethylurea (23)
1
White solid, yield: 83.4%, mp: 134.1-134.6 °C, H NMR (400 MHz, CDCl )
δ
= 3.67−3.75 (4H,
3
m, 2CH ), 5.52 (2H, s, OCH Ph), 7.27−7.35 (2H, m, ArH), 7.36 (1H, brs, NH), 7.43−7.46 (2H,
2
2
1
3
m, ArH), 7.69 (1H, d,
J
= 3.1 Hz, ArH), 7.72 (1H, d,
J
= 3.1 Hz, ArH), 9.46 (1H, s, NH);
C
NMR (100 MHz, CDCl3)
δ
= 41.94, 43.87, 66.29, 127.02, 129.83, 130.04, 130.46, 131.93,
1
32.19, 133.11, 134.21, 139.55, 147.74, 154.29.
1
-(3-(3-Chlorobenzyloxy)pyrazin-2-yl)-3-chloroethylurea (24)
1
White solid, yield: 84.4%, mp: 114.0-114.5 °C, H NMR (400 MHz, CDCl )
δ = 3.68−3.74 (4H,
3
m, 2CH ), 5.39 (2H, s, OCH Ph), 7.31−7.35 (4H, m, ArH+NH), 7.41 (1H, brs, ArH), 7.67 (1H,
2
2
1
3
d,
CDCl3)
39.55, 147.61, 154.26
J
= 3.1 Hz, ArH), 7.72 (1H, d,
J = 3.1 Hz, ArH), 9.44 (1H, s, NH); C NMR (100 MHz,
δ
= 41.95, 43.85, 67.88, 126.62, 128.65, 128.80, 130.02, 132.03, 132.12, 134.58, 137.40,
1

ACCEPTED MANUSCRIPT
4
.1.4. General procedure of piperazinethyl urea derivatives 25−28
K CO (406.8 mg, 2.94 mmol) and the appropriate piperazine derivative (1.96 mmol) were
2
3
dissolved in the solution of the appropriate chloroethyl urea derivative (1.96 mmol) in MeCN (5
mL). The reaction mixture was refluxed for 20 h and quenched by addition of water after cooling,
then extracted with ethyl acetate. The organic layer was dried over anhydrous Na SO , filtered
2
4
and concentrated under reduced pressure. The residue was purified by flash column
chromatography (SiO , DCM/MeOH = 5/1).
2
1
-(3-(Benzyloxy)pyrazin-2-yl)-3-(2-(4-methylpiperazin-1-yl)ethyl)urea (25)
1
White solid, yield: 45.4%, mp: 110.8-115.5 °C, H NMR (400 MHz, CDCl )
δ = 2.32 (3H, s,
3
CH ), 2.48−2.60 (10H, m, 5CH ), 3.47−3.51 (2H, m, CH ), 5.41 (2H, s, OCH Ph), 7.34−7.44
3
2
2
2
(6H, m, ArH+NH), 7.65 (1H, d,
J
= 3.1 Hz, ArH), 7.69 (1H, d,
J
= 3.1 Hz, ArH), 9.31 (1H, s,
1
3
NH); C NMR (100 MHz, CDCl )
δ
= 37.18, 45.93, 52.61, 55.22, 56.68, 68.75, 128.60, 128.68,
3
1
31.71, 131.80, 135.46, 139.89, 147.89, 154.25.
1
-(3-(2-Chlorobenzyloxy)pyrazin-2-yl)-3-(2-(4-methylpiperazin-1-yl)ethyl)urea (26)
1
White solid, yield: 25.1%, mp: 130.7-131.9 °C, H NMR (300 MHz, DMSO-d6
)
δ
= 2.15 (3H, s,
CH ), 2.31−3.43 (10H, m, 5CH ), 3.31−3.34 (2H, m, CH ), 5.50 (2H, s, OCH Ph), 7.37−7.40
3
2
2
2
(
2H, m, ArH), 7.49−7.54 (1H, m, ArH), 7.67−7.70 (1H, m, ArH), 7.75 (1H, d,
J
= 3.0 Hz, ArH),
1
3
7
δ
1
3
.80 (1H, d, J = 3.0 Hz, ArH), 8.38 (1H, s, NH), 9.08 (1H, s, NH); C NMR (100 MHz, CDCl )
= 37.21, 46.13, 52.80, 55.40, 56.67, 66.17, 126.99, 129.79, 129.96, 130.36, 131.79, 131.95,
33.19, 134.13, 139.86, 147.68, 154.18.
1
-(3-(3-Chlorobenzyloxy)pyrazin-2-yl)-3-(2-(4-methylpiperazin-1-yl)ethyl)urea (27)

ACCEPTED MANUSCRIPT
1
White solid, yield: 27.8%, mp: 93.9-97.6 °C, HPLC purity: 4.06 min, 100%, H NMR (400 MHz,
CDCl3)
δ
= 2.30 (3H, s, CH ), 2.32−2.61 (8H, m, 4CH ), 3.33−3.51 (4H, m, 2CH ), 5.39 (2H, s,
3
2
2
OCH Ph), 7.30−7.34 (4H, m, ArH+NH), 7.41 (1H, brs, ArH), 7.64 (1H, d,
J
= 1.0 Hz, ArH),
2
1
3
7
5
1
.70 (1H, d,
J = 3.1 Hz, ArH), 9.31 (1H, s, NH); C NMR (100 MHz, CDCl ) δ = 37.21, 46.14,
3
2.81, 54.65, 55.40, 56.66, 126.52, 128.60, 128.71, 128.76, 139.92, 131.72, 132.03, 139.89,
+
+
47.46, 154.58. HRMS (ES ): m/z calculated for C H25ClN O : 405.1806 [M+H] . Found
1
9
6
2
4
05.1815.
1
-(3-(2-Chlorobenzyloxy)pyrazin-2-yl)-3-(2-(piperazin-1-yl)ethyl)urea (28)
1
White solid, yield: 29.5%, mp: 104.0-106.5 °C, HPLC purity: 3.85 min, 100%, H NMR (300
MHz, DMSO- 6
d
)
δ
= 1.25 (s, 1H), 2.51−2.69 (12H, m, 6CH ), 5.50 (2H, s, OCH Ph), 7.37−7.40
2
2
(2H, m, ArH), 7.44 (1H, m, ArH), 7.57 (1H, m, ArH), 7.69 (1H, d,
J
= 3.0 Hz, ArH), 7.78 (1H, d,
+
J
= 3.0 Hz, ArH), 8.11 (1H, s, NH), 8.91 (1H, s, NH). HRMS (ES ): m/z calculated for
+
C H23ClN O : 391.1649 [M+H] . Found 391.1640
.
18
6
2
4
4
.2. Biological evaluation
.2.1. Cell culture
HT-22 (mouse hippocampal cells) cells were grown in Dulbecco’s Modified Eagle’s Medium
DMEM, GIBCO) supplemented with 10% (v/v) FBS and antibiotics (100 µg/mL
penicillin/streptomycin mix) in a humidified atmosphere at 37 °C with 5% CO2.
(
4
.2.2. Protection against loss of mitochondrial membrane potential assay