Discovery of 1-(3-(benzyloxy)pyridin-2-yl)-3-(2-(piperazin-1-yl)ethyl)urea: A new modulator for amyloid beta-induced mitochondrial dysfunction

Article abstract of DOI:10.1016/j.ejmech.2016.12.057

Herein, we report a new series of aliphatic substituted pyridyl-urea small molecules synthesized as potential modulators for amyloid beta (Aβ) induced mitochondrial dysfunction. Their blocking activities 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 sixteen compounds against Aβ-induced mPTP opening was superior or almost similar to that of the standard Cyclosporin A (CsA). Among them, 1-(3-(benzyloxy)pyridin-2-yl)-3-(2-(piperazin-1-yl)ethyl)urea (5x) effectively maintained mitochondrial function and cell viabilities on ATP assay, MTT assay, and ROS assay. Using CDocker algorithm, a molecular docking model presented a plausible binding mode for 5x with cyclophilin D (CypD) receptor as a major component of mPTP. Moreover, hERG and BBB-PAMPA assays presented safe cardiotoxicity and high CNS bioavailability profiles for 5x. Taken as a whole, this report presents compound 5x as a new nonpeptidyl mPTP blocker may hold a promise for further development of Alzheimer's disease (AD) therapeutics.

Full text of DOI:10.1016/j.ejmech.2016.12.057

Accepted Manuscript
Discovery of 1-(3-(benzyloxy)pyridin-2-yl)-3-(2-(piperazin-1-yl)ethyl)urea: A new
modulator for amyloid beta-induced mitochondrial dysfunction
Ahmed Elkamhawy, Jung-eun Park, Ahmed H.E. Hassan, Hyunhwa Ra, Ae Nim Pae,
Jiyoun Lee, Beoung-Geon Park, Bongjin Moon, Hyun-Mee Park, Eun Joo Roh
PII:
S0223-5234(16)31063-7
10.1016/j.ejmech.2016.12.057
EJMECH 9148
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 August 2016
Revised Date: 15 December 2016
Accepted Date: 29 December 2016
Please cite this article as: A. Elkamhawy, J.-e. Park, A.H.E. Hassan, H. Ra, A.N. Pae, J. Lee, B.-G.
Park, B. Moon, H.-M. Park, E.J. Roh, Discovery of 1-(3-(benzyloxy)pyridin-2-yl)-3-(2-(piperazin-1-
yl)ethyl)urea: A new modulator for amyloid beta-induced mitochondrial dysfunction, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.057.
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Graphical Abstract:

ACCEPTED MANUSCRIPT
Discovery of 1-(3-(benzyloxy)pyridin-2-yl)-3-(2-(piperazin-1-yl)ethyl)urea: a new
modulator for amyloid beta-induced mitochondrial dysfunction
a,b,1
a,c,1
d,e
a,c
f,g
Ahmed Elkamhawy , Jung-eun Park , Ahmed H. E. Hassan , Hyunhwa Ra , Ae Nim Pae ,
h
f,i
c
j
a,g,*
Jiyoun Lee , Beoung-Geon Park , Bongjin Moon , Hyun-Mee 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
Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea
f
Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology (KIST), Seoul
0
2792, Republic of Korea
g
Department of Biological chemistry, Korea University of Science and Technology (UST), Daejeon 305-350, 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
Advanced Analysis Center, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea
j
*
Corresponding authors: Eun Joo Roh (e-mail: r8636@kist.re.kr).
1
Both authors contributed equally to this work

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Abstract
Herein, we report a new series of aliphatic substituted pyridyl-urea small molecules synthesized
as potential modulators for amyloid beta (Aβ) induced mitochondrial dysfunction. Their blocking
activities 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 sixteen compounds against Aβ-induced mPTP opening was
superior or almost similar to that of the standard Cyclosporin A (CsA). Among them, 1-(3-
benzyloxy)pyridin-2-yl)-3-(2-(piperazin-1-yl)ethyl)urea (5x) effectively maintained
(
mitochondrial function and cell viabilities on ATP assay, MTT assay, and ROS assay. Using
CDocker algorithm, a molecular docking model presented a plausible binding mode for 5x with
cyclophilin D (CypD) receptor as a major component of mPTP. Moreover, hERG and BBB-
PAMPA assays presented safe cardiotoxicity and high CNS bioavailability profiles for 5x. Taken
as a whole, this report presents compound 5x as a new nonpeptidyl mPTP blocker may hold a
promise 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
Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic
lateral sclerosis and Huntington’s disease, are a group of neurological disorders characterized by
chronic, progressive and irreversible neuronal loss [1]. As world's population grows older
because of global increase in the life expectancy, the incidence of neurodegenerative diseases is
anticipated to increase consequently [2, 3]. This fact has been reflected in Alzheimer’s
association report estimating the number of Alzheimer's patients aged 65 or more in the US only,
to almost triple, from 5.1 million in 2015, to a 13.8 million by 2050, with a rate of one new case
every 33 seconds [4].
While the number of population afflicted with neurodegenerative disorders increases, effective
treatments are still lacking. However, over the past two decades, better understanding of the
molecular mechanisms underlying neurodegeneration offered opportunities to address new
approaches for developing novel therapeutic agents combating these diseases [1, 5-8].
Mitochondrial dysfunction has been found to be a major contributor in the pathogenesis and
prognosis of neurodegeneration [8-16]. Prolonged opening of mitochondrial permeability
transition pore (mPTP) results in depolarization of mitochondrial membrane potential, loss of
+
homeostasis and NAD , mitochondrial swelling and rupture of the outer mitochondrial
membrane and finally release of apoptotic factors and cell death [17-20].
Cyclophilin D (CypD) is a mitochondrial matrix peptidyl prolyl cis-trans isomerase (PPI).
CypD-sensitive mPTP has been found in viable human brain mitochondria [21]. The role of
CypD in induction of mPTP opening leading to cell death has been validated using CypD

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knockout mice [22]. Also, Amyloid β (Aβ) forms a complex with CypD which enhances mPTP
formation [23]. In addition, increased CypD levels has been found in Alzheimer's affected
neurons [24]. Furthermore, recent studies proved the protective effect of CypD inhibitors against
neurodegeneration [25]. To the best of our knowledge, up-to-date, the immunosuppressant cyclic
peptide cyclosporin A (CsA) is the most significant mPTP blocker [24]. Its action is mediated via
inhibition of CypD. Unfortunately, because of its immunosuppressant activity, as well as, its
poor bioavailability and low penetration through blood brain barrier (BBB), it is used only as a
probe in research [26]. Research resulted in cyclosporin A analogs devoid of immunosuppressant
activity such as NIM811 and UNIL025 [27, 28]. Despite this improvement, these analogs are
still far from clinical significance because of side effects and inefficiency in crossing BBB. On
the other hand, few non-peptidic small molecules have been reported as mPTP inhibitors.
Among seven quinoxaline derivatives, compound 1 (Figure 1) was the best selective inhibitor for
CypD over CypA with micromolar activity [29]. However, to the best of our knowledge, there
are no reported further efforts to develop more potent or selective derivatives belonging to this
class of quinoxaline derivatives for modulation of mPTP. A series of compounds 2 (Figure 1),
having 5-(halophenyl)furan-2-yl and substituted hydantoin scaffolds linked through amide spacer
have been bond were found to inhibit calcium induced mitochondrial swelling, albeit with at
high micromolar concentration and low potency [30]. The lack of therapeutically effective small
molecule mPTP blockers stimulated our institute to launch a discovery project towards
identification of promising compounds. In this project, oxime ethers 3 (Figure 1) were developed
based on an oxime ether hit compound retrieved from screening of our institute chemical library
[
31]. This class of compounds elicited reasonable inhibition of Aβ-induced mPTP opening using
JC-1 assay. Assessment of their impact on cytochrome P450 (CYP) enzymatic activities revealed

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severe deterioration in CYP activity, which would leads to risks and complication of drug-drug
interactions and side effects. Also in this project, a series of compounds 4 containing 1,2,3-
triazole spacer as a surrogate to oxime ether were identified [32]. In comparison to oxime ether
spacer in compounds 3, the triazole spacer seems to have a function in enhancing mPTP blocking
activity. In fact, 1,2,3-triazoles linkers are known as active linkers that might accept hydrogen
bonds, as well as, participate in dipole-dipole and pi-stacking interactions [33].
Figure 1. Proposed design of N-alkyl-N-2-(3-benzyloxypyridyl)urea derivatives via replacing the
linker, modifying the aliphatic moiety and incorporating heterocyclic pyridine ring in the
aromatic moiety.

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As outlined in Figure 1, both of oxime ether in compound 3 and 1,2,3-triazole in compound 4
link 2-benzyloxyphenyl aromatic moieties, with aliphatic 3-(N-substituted-piperidinyl) or 3-(N-
substituted-pyrrolidinyl) moieties, respectively. Quinoxaline compound (1) and furyl derivatives
(2) have also aromatic moieties linked with aliphatic moieties through urea and hydrazide linkers
respectively. As previously mentioned, the linker might have effect in enhancing the mPTP
activity. Also, it can be realized that both linkers in compound 1 and furyl derivatives 2 contain
hydrogen donor and acceptor groups while the 1,2,3-triazole linker in compound 4 is lacking
hydrogen bond donation. In addition, the reported crystal structure of CsA-CypD complex (pdb
ID = 2Z6W) reveals hydrogen bonding interactions between amide groups of CsA and Arg55,
Asn102, Trp121 and His126. Accordingly, we envisioned that installing a three atoms urea linker
might maintain the three atoms linker's length and may participate in binding via hydrogen
bonding interactions. Also in our design, we switched from substituted aliphatic cyclic amines
moieties to different simple alkyl or cycloalkyl groups to investigate the impact of simple
aliphatic moiety on activity. In addition, the phenyl ring was modified to assess the isosteric
heterocyclic pyridine ring for mPTP inhibition activity.
2
. Results and discussion
.1. Chemistry
2
The target pyridyl-urea derivatives were prepared according to the sequence of reactions
depicted in Scheme 1. Commercially available starting material 2-amino-3-hydroxypyridine (6)
was subjected to the S 2 displacement with various benzyl bromides under basic conditions to
N
give 3-(benzyloxy)pyridin-2-amine derivatives (7a−f) which upon addition to various aliphatic
isocyanates afforded compounds 5a‒r and 5s‒w. 2-Chloroethyl ureas (5s and 5w) have been
used as intermediates to prepare the piperazinylethyl ureas (5x and 5y).

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Scheme 1. Reagents and conditions: (i) 1) NaOH, H O, Bu NBr, DCM, rt, 15 min, 2)
2
4
appropriate benzylbromide, DCM, rt, 18 h; (ii) appropriate isocyanate derivative, NaH (60% in
mineral oil), THF, reflux, 5−18 h; (iii) 2-chloroethyl isocyanate, DIPEA, THF, reflux, 18 h; (iv)
piperazine, K CO , acetonitrile, reflux, 20 h.
2
3
2
2
.2. Biological evaluation
.2.1. JC-1 assay (Mitochondrial membrane potential assay)
The ability of the synthesized compounds to inhibit the mitochondrial membrane potential loss
has been evaluated using JC-1 assay [34, 35]. In this assay, JC-1 which is a lipophilic fluorescent
cationic dye is used as indicator for mitochondrial membrane potential. If mitochondrial
membrane potential is maintained or recovered, it forms red fluorescent J-aggregates. This red
fluorescence is converted into green fluorescence when the mitochondrial membrane potential is
lost. The ratio of green to red fluorescence allows the measurement of the percent of
mitochondria whose membrane is affected by Aβ-induced mPTP opening. Accordingly, the
lower increased green to red ratio, the more effectiveness of the tested compound to block the
Aβ-induced mPTP opening. The ratio of green to red fluorescence is dependant only on
mitochondria membrane potential and is not affected by other factors such as mitochondrial size
or shape rendering the test highly specific. The results of the prepared compounds is summarized

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in Table 1 and visualized in Figure 2. In the course of this test, the activity to block the Aβ-
induced mPTP opening was evaluated at a single dose concentration of 5 µM. CsA was used as a
positive standard where it exerted 46% of increased green to red ratio. As shown in Figure 2,
sixteen compounds exhibited inhibitory activity superior or almost similar to that of the standard
CsA. Three of these compounds showed high efficiency exhibiting increased ratio of green to red
less than 20%. Among the tested compounds, four compounds exhibited weak or non significant
inhibitory activity ranging from 64~94%. Finally, five compounds did not show any reduction in
increased green to red ratio. In contrast, they exhibited elevation of the increased green to red
ratio. It is noted that the most potent compounds in lead series 3 and 4 elicited 41% and 13%
respectively.
Further investigation of the JC-1 assay outcome revealed mixed results. For example, while
unsubstituted benzyloxy derivative 5b bearing tert-butyl as the aliphatic moiety elicited potent
activity with increased green to red ratio of 23%, the unsubstituted benzyloxy derivative 5a
bearing cycloheptyl moiety did not show any inhibitory activity. In contrast, the 3-
chlorobenzyloxy derivative 5l bearing tert-butyl as the aliphatic moiety did not show any
reduction in increased green to red ratio, while the cycloheptyl derivative 5j bearing the same 3-
chlorobenzyloxy moiety elicited activity comparable to that of CsA. In the same event, all of the
cycloheptyl derivatives (5m and 5j) and tert-butyl derivatives (5o and 5r) bearing 4-
chlorobenzyloxy and 4-flourobenzyloxy moieties respectively, showed nearly similar or superior
to that of CsA. These results indicate that subtle stereoelectronic variations to have significant
impact of the elicited biological activity.

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Table 1. In vitro blocking activity of the new ureas against Aβ-induced mPTP opening (JC-1
assay) at single dose concentration of 5 µM.
Increased
Increased
g/r ratio
Cpd
R1
R2
g/r ratio
Cpd
R1
R2
a
a
(
%)
(%)
5
5
5
a
b
c
H
H
3-F
3-F
3-F
3-F
2-Cl
2-Cl
2-Cl
3-Cl
3-Cl
3-Cl
4-Cl
cycloheptyl
tert-butyl
cycloheptyl
130
23
91
31
14
44
41
21
49
46
50
176
19
5n
5o
5p
5q
5r
4-Cl
4-Cl
4-F
4-F
4-F
H
3-Cl
2-Cl
4-Cl
4-F
H
ethyl
tert-butyl
cycloheptyl
ethyl
tert-butyl
94
35
28
127
49
169
73
41
131
64
16
48
5
d
1,1,3,3-tetramethylbutyl
ethyl
tert-butyl
cycloheptyl
1,1,3,3-tetramethylbutyl
ethyl
cycloheptyl
1,1,3,3-tetramethylbutyl
tert-butyl
5
5
e
f
g
5s
5t
−
−
−
−
−
−
−
−
5
5
h
5u
5v
5w
5x
5y
CsA
5
5
i
j
5
k
5
l
3-Cl
−
5
m
cycloheptyl
46
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.
2
1
1
1
1
1
00
80
60
40
20
00
8
6
4
2
0
0
0
0
0
Figure 2. Percentage increase in the fluorescence ratio (green/red) after treatment of each
compound and Aβ with respect to that of Aβ alone (100%). Y-axis represents the percent of
increased green to red fluorescence ratio of each compound. Dashed line represents the percent
of increased green to red fluorescence ratio of Cyclosporin A (CsA) used as a control.

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2
.2.2. ROS production and MTT cell viability assay
In drug discovery processes, it is beneficial to perform a front in vitro testing of toxicities. This
would enable early detection and exclusion of potential toxic molecules; thus reducing the
number of unfavorable compounds and focusing efforts on the more likely safe candidates.
Accordingly, the intrinsic ability of a set of the most active compounds in JC-1 assay to induce
production of reactive oxygen species (ROS) and cytotoxicity was assessed in neuro-2a cells
using 10-dose 3-fold serial dilutions. As Table 2 shows, all tested compounds did not show
significant increase of hydrogen peroxide levels up to a dose of 10 µM concentration which is
double the dose used in JC-1 assay. In comparison, at 10 µM concentration, the standard
menadione elicited approximately seven times-elevation of hydrogen peroxide level with IC =
5
0
2
.688 µM. This indicates that these compounds can’t induce ROS formation which is an
important consideration in assessment of toxicity. In addition, almost all tested compounds
exhibited excellent cell viability in cytotoxicity tests using MTT assay with doses up to 10 µM
concentration. At this dose, the standard staurosporine elicited only 1.3% viability with IC =
5
0
0
.0113 µM.
Table 2. Results of induction of ROS production and cytotoxicity up to 10 µM concentrations
c
ROS
MTT viability
%
Viability
ROS
MTT viability
%
b
Viability
Cpd
Fold of H O
Cpd
Fold of H O
2
2
a
b
b
2
2
a
b
IC50
IC50
IC50
IC50
c
c
Production
Production
5
5
5
b
d
e
0.92
1.02
1.14
0.94
> 10
> 10
> 10
> 10
106.1
101.4
96.6
> 10
> 10
> 10
> 10
5o
6p
5x
0.91
0.91
0.97
6.94
> 10
> 10
> 10
2.688
93.1
93.2
101.8
> 10
> 10
> 10
5
f
95.8
Menadione
5
m
1.15
> 10
85.25
> 10 Staurosporine
1.3
0.0116
a
Folds of produced H O by neuro-2a cells after 6 hours incubation with 10 µM concentration of
2
2
each compound relative to non-treated.
b
IC expressed in µM concentration.
5
0
c
%
Viability of neuro-2a cells after 72 hours incubation with10 µM concentration of each
compound.

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2
.2.3. Assay for cellular ATP levels (Luciferase-based assay)
To identify likely-safe and promising compounds for further testing and development, the next
filter in the adopted discovery workflow was rationalized on that promising compounds should
not impair ATP production, and preferably, protect neurons against Aβ-induced impairment of
ATP production. This is because a main role for the mitochondria is the production of ATP.
Candidates able to block detrimental Aβ-induced mPTP opening should eventually repair
mitochondrial dysfunction by promoting ATP generation. A luciferase-based assay for cellular
ATP levels was carried out to validate the effect of selected set of the most active compounds on
mitochondrial function [36]. As shown in Table 3, among the tested compounds, compounds 5b
and 5x, bearing unsubstituted benzyloxy group, possessed highest inhibitory activity against Aβ-
induced ATP reduction as well as reasonable cell viability, which are comparable to those of
piracetam. Thus, compounds 5b and 5x were advanced to further investigations.
Table 3. The result of ATP assay of the tested compounds
a
b
a
b
Cpd
ATP Viability
% Recovery
Cpd
ATP Viability
% Recovery
5
5
5
b
d
e
103
30
102
129
105
72
-140
41
45
47
5o
6p
5x
104
99
78
13
40
87
5
f
Piracetam
88
127
5
m
a
%
Viability of hippocampal neuronal cell line HT-22 was calculated based on ATP production
after 7 hours incubation with 5 µM of each compound.
b
%
Recovery of ATP production by Aβ-suppressed mitochondrial ATP production in
hippocampal neuronal cell line HT-22 after 7 hours incubation with 5 µM of each compound.
2
.2.4. Protection against Aβ-induced ROS production and cytotoxicity
Aβ induces cytotoxicity via multiple mechanisms, and furthermore, the exact Aβ toxic
polymorph and species are still controversial [37-42]. Despite these controversies about the
mechanisms of Aβ-induced cytotoxicity, protection against Aβ-induced cytotoxicity is a

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desirable feature for development of an effective Alzheimer’s disease therapy. Accordingly,
assays of the overall protective activity of prepared compounds against Aβ-induced cytotoxicity
were incorporated in the adopted workflow. Next to JC-1 assay, compound’s triggered ROS
production and cytotoxicity assay, and the luciferase-based ATP assay; subsequent filters were
applied to test the capability of compounds for protection of hippocampal neuronal cells against
Aβ induced ROS production and cytotoxicity. In this regard, MTT assay was carried out to
evaluate the mitochondrial dehydrogenase activity as well as a CM-H DCFDA-fluorescent assay
2
to measure the cellular reactive oxygen species (ROS) resulting from Aβ toxicity [43, 44]. As
listed in Table 4, compound 5x reduced Aβ-induced toxicity in the MTT assay with 81%
compared to 29% of the standard piracetam. It also reduced Aβ-induced ROS production with
9
7% inhibition value. These data suggested that compound 5x as a potential mPTP blocker
maintaining mitochondrial function and cell viability.
Table 4. Results of MTT assay, ROS assay and BBB diffusion assay of 5b and 5x
d
%
Inhibition of Aβ-
induced ROS
b
% Inhibition of Aβ-
induced cytotoxicity
Effective Permeability
Compound
a
% Viability
c
-6
P (10 cm/sec)
e
5
5
b
x
95
97
129
92
133
132
11
81
29
45.14
20.63
Piracetam
Progesterone
Lidocaine
Theophylline
a
48.81
26.56
0.29
Percent inhibition of Aβ-induced ROS production by Aβ-treated hippocampal cell line HT-22
after 6 hours incubation with 5 µM of each compound.
b
%
Viability of hippocampal cell line HT-22 after 24 hours incubation with at 5 µM of each
compound.
c
%
Viability of Aβ-suppressed hippocampal cell line HT-22 after 24 hours incubation with at 5
µM of each compound.
d
Effective permeability (Pe) was measured at 50 µM concentrations after 4 hours of passive
diffusion using Pion PAMPA-BBB.
2
.2.5. Parallel artificial membrane permeability assay (artificial BBB diffusion assay)

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Molecules to be developed for treatment of CNS disorders must be able to overcome the natural
obstacle of blood brain barrier (BBB) to reach their actions targets within CNS. Without
appropriate capacity to penetrate BBB, in vitro active candidates would be useless in vivo.
Therefore, compounds 5b and 5x were submitted for evaluation of their tendency to cross blood-
brain barrier using parallel artificial membrane permeability assay (PAMPA) [45, 46]. Three
standard drugs were used for calibration and comparison. The following effective permeability
-6
(
P ) ranges were established in this assay: no penetration of BBB (P < 0.4×10 cm/sec),
e
e
-6
-6
penetration of BBB (P > 0.4×10 cm/sec), low CNS bioavailability (P < 10×10 cm/sec), and
e
e
-6
high CNS bioavailability (P > 10×10 cm/sec). As shown in Table 4, theophylline which poorly
e
-6
diffuse across BBB, had measured effective permeability P = 0.29×10 cm/sec, while lidocaine
e
-6
and progesterone which highly cross BBB had effective permeability of P = 26.56×10 and
e
-6
4
8.81×10 cm/sec respectively. In comparison, compounds 5X and 5b had measured effective
-6
-6
permeability P = 20.63×10 and 45.14×10 cm/sec respectively indicating their high CNS
e
bioavailability.
2
.2.6. Molecular Modeling Study
As mentioned before, CypD is a mitochondrial peptidyl prolyl cis-trans isomerase PPI that plays
an important role in mPTP opening and loss of membrane potential [22]. The cyclic polypeptide
CsA effectively binds and inhibits CypD. Investigation of the reported crystal of human CypD-
CsA complex (pdb ID = 2Z6W) reveals hydrophobic interactions as the major contributor in
binding [47]. CypD residues interacting with bound CsA can be differentiated into distinctive
regions. First, there is a hydrophobic pocket 1 formed by residues Met61, Ala101, Phe113,
Leu122 and His126. In this pocket, the CsA's methylated valine residue (Mva11) elegantly fits.

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A saddle formed of Gln63 and Asn102, separates pocket 1 from another less hydrophobic pocket
2
in which the CsA's α-amino-butyric acid residue (Aba2) is interacting with Gln111 and Gly72.
On the other side next to pocket 1, there is a flattened hydrophobic surface formed of Phe60,
Ile57 and Trp121, with which the side chain of CsA's methylated leucine residue (Mle9) is
interacting. In addition, side chain of the 4-(2-butenyl)-4-methylthreonine residue (Bmt1) shows
hydrophobic interaction with a nearby Ala103 residue. Hydrogen bonding interactions are
observed mainly 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.
In order to understand the outcome of mitochondrial membrane potential assay, molecular
modeling study was conducted to get insights into the molecular factors that might underlie the
observed biological activity. A set of most effective mPTP blockers (5b, 5d, 5e, 5h, 5m, 5o, 5p,
and 5x) and another set of ineffective mPTP blockers (5a, 5c, 5l, 5n, 5q, 5s and 5v) were docked
into the reported crystal structure of human CypD (pdb ID = 2Z6W) after appropriate preparation
of receptor and ligands. The CHARMm force field based docking CDocker algorithm
implemented in Accerlys Discovery Studio 4.0 was used as a flexible docking method to predict
the binding mode of different ligands to CypD [48]. This algorithm is characterized by
employing explicit all-atoms CHARMm force field calculations which outperforms algorithms
based on grid-based approximations in accurate prediction of correct pose. The resulting poses
were further refined by in situ minimizations and the binding energies and complexes energies
were calculated. Selection of the most probable binding modes was based on these calculated
energy terms.

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The predicted binding modes for this series of compounds can be generally grouped under two
broad binding modes. The first one, which is more preferable by active ligands, is characterized
by part of ligand being bound within the hydrophobic pocket 1 eliciting interactions with the
residues forming this pocket. On the other hand, the second mode, which is more prevalent in
inactive ligands, shows no binding within this hydrophobic pocket 1. Instead, the docked ligand
tends to flip out of binding pocket 1 exhibiting bind above the pocket.
The different binding modes for the most effective mPTP blocker 5e are illustrated in Figure 3.
Out of twenty retrieved different poses, compound 5e elicited nineteen poses belonging to
general mode 1 and only one pose belonging to general mode 2. The energy terms for binding
mode 1 in the best calculated pose were estimated to be -52.55 kcal/mol for binding energy, -
4
9.77 kcal/mol for total binding energy and -2933.91 kcal/mol for complex energy. In
comparison, the energy terms for only one detected pose belonging to general binding mode 2
were -44.99 kcal/mol for binding energy, -38.22 kcal/mol for total binding energy and -2929.09
kcal/mol for complex energy. Total binding poses belonging to mode 1, which were superior to
binding mode 2, are eight poses in terms of binding energy, nine poses in terms of total binding
energy and eight poses with in terms of complex stability. This indicates a high tendency of
compound 5e to bind in mode 1 rather than mode 2.

ACCEPTED MANUSCRIPT
Figure 3. Different Binding modes of compound 5e; A) Binding mode 1 subtype 1; in which the
pyridine ring docks into the hydrophobic pocket 1. B) Binding mode 1 subtype 2; in which the
aliphatic moiety docks into the hydrophobic pocket 1. C) Binding mode 1 subtype 3; in which
the aromatic ring of the benzyloxy moiety docks into the hydrophobic pocket 1. D) Binding
mode 2; in which the hydrophobic pocket 1 is vacant while compound 5e is docked above it.
Within the general binding mode 1, three different sub-modes were identified. As shown in
Figure 3A, in binding mode 1 subtype 1, the aromatic pyridine ring docks into the hydrophobic
pocket 1. This subtype of mode 1 was detected in seven out of the eight poses having energy
terms superior to binding mode 2. The best pose in terms of energy belongs to this subtype of
binding mode. In this pose, the pyridine ring is involved in interactions with residues of pocket 1
forming pi-pi T-shaped interaction with Phe113, pi-alkyl interaction with Ala101 and Met61 and
pi-cation interaction with Arg55 and His126. The urea linker is involved in hydrogen bonding

ACCEPTED MANUSCRIPT
interactions with Arg55. The aliphatic ethyl moiety is directed towards the hydrophobic surface
adjacent to pocket 1. The benzyloxy moiety is involved in two non-classical hydrogen bonding
interactions between Asn102 and Gln63 in the saddle region with benzylic hydrogen and the
aromatic ring respectively. Fluorine substitution on the benzyloxy ring is involved in halogen
interaction with side chain of Arg55. It should be noted that poses belonging to this subtype
shared the criteria of pyridine ring docked into the pocket, albeit, it was rotated within the pocket
resulting in different orientations of benzyloxy and aliphatic urea groups.
In subtype 2 of binding mode 1(Figure 3B), which has been detected in five poses, the aliphatic
moiety is docked into the hydrophobic pocket 1. Despite the calculated energy terms of the best
scoring pose in binding mode 1 subtype 2 (-45.52 kcal/mol for binding energy, -43.30 kcal/mol
for total binding energy and -2931.64 kcal/mol for complex energy) are much less favorable than
that for the best performing pose in binding mode 1 subtype 1, it is still energetically more
favorable than mode 2, especially in terms of total binding energy and complex energy. Although
the docked aliphatic ethyl moiety into the hydrophobic pocket, the more pronounced interactions
were benzyloxy moiety pi-cation interactions with Arg55, non classical pi-hydrogen bond with
Gln63, pi-alkyl interaction with Ala101, halogen-hydrogen bond with NH of Asn102, halogen-
acceptor interaction with CO of Asn102, and Hydrogen bond with His126. The urea linker was
involved in hydrogen bonding interaction with Arg55.
In subtype 3 of binding mode 1 (Figure 3C), which has been detected only in one pose, the
benzyloxy moiety is docked into the hydrophobic pocket 1. The most pronounced interactions in
this mode are two pi-cation interactions between benzyloxy ring with Arg55 and His126, pi-alkyl

ACCEPTED MANUSCRIPT
interaction of benzyloxy ring with Ala101, halogen acceptor interaction of fluorine atom on
benzyloxy ring with Gln63, pi-alkyl interaction of pyridine ring with Ala103, and hydrogen bond
of urea with Gly72. The calculated energy terms (-31.08 kcal/mol for binding energy, -27.97
kcal/mol for total binding energy and -2917.78 kcal/mol for complex energy) indicate that mode
1
subtype 3 is energetically much less favored than all of mode 2 and other mode 1 subtypes.
Binding mode 2 is illustrated in Figure 3D. This mode, which has been detected only in one
pose, is characterized by unoccupied hydrophobic pocket 1. The ligand in this mode docks above
the pocket. The most pronounced interactions in this mode are hydrogen bonds between urea
with Arg55, benzylic hydrogens with Asn102, and halogen-hydrogen bond between the fluorine
with His126. Considering all of previously mentioned binding modes in conjunction with their
calculated energy terms, the potent activity of compound 5e can be attributed to dominating
binding mode 1 subtype 1.
Similar analysis of calculated binding modes of the active and inactive sets revealed the
anticipated binding modes behind the biological performance of tested compounds for blocking
mPTP. For example, the high efficiency of compound 5x to block mPTP was attributed to a
variant of binding mode 1 subtype 3 (Figure 4A), where the benzyloxy group is docked into the
hydrophobic pocket 1. In this variant, the pyridyl ring is facing the hydrophobic surface adjacent
to the hydrophobic pocket 1, and the urea linker along with the ethyl chain are extended to direct
the piperazine ring towards the less hydrophobic pocket which in turn shows interactions non-
classical and classical hydrogen bonding interactions between Gly72 in the pocket and Asn102
in the saddle with piperazine ring. Other pronounced interactions are non-classical hydrogen

ACCEPTED MANUSCRIPT
bond interactions of hydrogens in ethyl chain with Gln63 in the saddle, classical hydrogen
bonding interactions between Arg55 and urea linker and pyridyl's nitrogen, pi-alkyl interactions
of benzyloxy ring with both of Leu122 and Ala101, in addition to pi-cation interaction with
His126. It is noted that there is pi-lone pair interaction between the benzyloxy ring and the
oxygen atom in the urea linker which contributes to the stabilization of this bent conformation.
The calculated energy terms (-66.38 kcal/mol for binding energy, -52.28 kcal/mol for total
binding energy, and -2930.12 kcal/mol for complex energy), along with elicited binding mode,
provide a good explanation for the high efficiency of this ligand in blocking mPTP.

ACCEPTED MANUSCRIPT
Figure 4. The most favorable binding modes of selected examples of active ligands. A)
Compound 5x binding mode 1 subtype 3, in which the benzyloxy moiety is docked into the
hydrophobic pocket 1. B) Compound 5b binding mode 1 subtype 1, in which the pyridine is
docked into the hydrophobic pocket 1 with 90° rotation within the pocket. C) Compound 5h
binding mode 1 subtype 1, in which the pyridine is docked into the hydrophobic pocket 1 D)
Compound 5m binding mode 1 subtype 2, in which the cycloheptyl ring is docked into the
hydrophobic pocket 1.
The most probable binding modes for selected examples of active compounds from the docked
active set are displayed in Figure 4. As illustrated, Compounds 5b and 5h elicit their activities
via different variants of binding mode 1 subtype 1. The pyridine rings of the two docked ligands
show 90° rotation within the hydrophobic pocket in relation to each other. Accordingly, the
aliphatic moiety and benzyloxy moiety occupy different orientations. Also, as illustrated,
compound 5m elicit activity through a variant of binding mode 1 subtype 2. In this case, the
cycloheptyl ring of the ligand is docked into the hydrophobic pocket 1.
Application of this protocol of docking and analysis to the set of inefficient mPTP blockers
revealed general dominance of binding mode 2. Accordingly, it can be anticipated that this mode
might be responsible for their observed inefficiency as mPTP blockers. As Figure 5A illustrates,
in some compounds exemplified by compound 5a, binding mode 2 dominates exclusively. While
in some compounds, exemplified by compound 5c with very weak blocking activity (91%
increased green to red in JC-1 assay), both binding mode 1 subtype 1 (Figure 5B) and mode 2
(Figure 5C) were detected in close calculated energy terms for both modes (for mode 1 subtype
1
, -54.34 kcal/mol for binding energy, -50.54 kcal/mol for total binding energy and -2926.6
kcal/mol for complex energy; for mode 2, -52.23 kcal/mol for binding energy, -48.04 kcal/mol
for total binding energy and -2925.59 kcal/mol for complex energy).

ACCEPTED MANUSCRIPT
Figure 5. The most favorable binding modes of selected inefficient mPTP blocker; A)
Compound 5a binding mode 2, in which no binding within the hydrophobic pocket is observed.
B) Compound 5c binding mode 1 subtype 1, in which pyridine ring is docked into the
hydrophobic pocket. C) Compound 5c binding mode 2, in which the hydrophobic pocket is
vacant.
The current modeling study revealed clearly distinguishable binding modes that can provide an
explanation for the observed activity of the tested series of 3-((benzyloxy) pyridin-2-yl) aliphatic
urea derivatives.
+
2
.2.7. hERG K channel binding affinity (Cardiotoxicity measurement)

ACCEPTED MANUSCRIPT
Recently some small molecules were withdrawn from the market because they induced the
prolongation of the QT interval of the surface electrocardiogram (ECG) by blocking the human
ether-a-go-go related gene (hERG) potassium channel. Blockade of the hERG channel is a
significant hurdle in lead optimization activity due to its potential cardiotoxicity [49, 50]. Thus,
we measured the hERG affinity of compound 5x. The test demonstrated an excellent result (IC50
value > 28.7 µM), which means that the compound under test doesn’t have blockage activity
towards the hERG channel and then it is considered as safe compound with regard to the
potential cardiotoxicity.
3
. Conclusion
In this study, compound 5x has been discovered as a new mPTP modulator efficiently protecting
mitochondria against Aβ induced mitochondrial membrane depolarization. It elicited good in
vitro profile with regard to the assays associated with mitochondrial functions and cell viabilities.
Moreover, the in silico study provided rational explanation of the observed activity. In vitro
cardiotoxicity test of 5x showed promising results. Therefore, compound 5x, a potential mPTP
blocker, may serve as a good lead for further development of new AD therapeutics.
4
. Experimental
.1.Chemistry
4
General: All reactions and manipulations were performed in nitrogen atmosphere using standard
Schelenk techniques. The reaction solvents purchased from Aldrich Co., TCI and Alfa and used
without any other purification. The NMR spectra were obtained on Bruker Avance 300 or 400.
1
H NMR spectra were referenced to tetramethylsilane (δ = 0.00 ppm) as an internal standard and

ACCEPTED MANUSCRIPT
are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t =
1
3
triplet, dd = doublet of doublet, m = multiplet). C NMR spectra were referenced to the residual
CDCl (δ = 77.0 ppm). Column chromatography was performed on Merck Silica Gel 60
3
(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 Waters ACQUITY UPLC (CORTECS ) with C18
column (2.1 mm × 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, 30/70
gradient in 10 min, and flow rate of 0.3 mL/min. 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)pyridin-2-amine derivatives (7a−f)
Sodium hydroxide (6 g, 0.15 mol) was dissolved in water (10 mL) and mixed with
dichloromethane (15 mL). 2-Amino-3-hydroxy pyridine (6, 3 g, 27.24 mmol) was added to the
reaction mixture in portions, under stirring, followed by the catalyst, tetrabutylammonium
bromide (156 mg, 0.48 mmol). The reaction mixture was stirred for 15 min at 25−30 °C, and
treated with a solution of the appropriate benzyl bromide derivative (5.4 g, 28.61 mmol) in
dichloromethane (10 mL). The reaction mixture was stirred 18 h at 25 °C and then diluted with
water (15 mL). The organic phase was separated, and the aqueous phase was extracted with
dichloromethane. The organic extracts were combined, washed with water and brine, dried over
sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by

ACCEPTED MANUSCRIPT
column chromatography (SiO , EA/n-Hex = 1/2). For 3-(benzyloxy)pyridin-2-amine 7a, it has been
2
purchased from Alfa Aesar Co.
3
-(3-fluorobenzyloxy)pyridin-2-amine (7b)
1
Light pink solid, yield: 70%, H NMR (400 MHz, CDCl ) δ = 4.68 (2H, brs, NH ), 5.06 (2H, s,
3
2
OCH Ph), 6.58 (1H, dd, J = 5.1 Hz, 7.8 Hz, ArH), 6.93 (1H, dd, J = 1.1 Hz, 7.8 Hz, ArH), 7.04
2
(1H, td, J = 2.4 Hz, 8.4 Hz ArH), 7.14 (1H, dd, J = 2.0 Hz, 9.4 Hz, ArH), 7.18 (1H, d, J = 7.6
Hz, ArH), 7.36 (1H, td, J = 5.8 Hz, 8.0 Hz, ArH), 7.69 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH).
3
-(3-chlorobenzyloxy)pyridin-2-amine (7c)
1
Light yellow solid, yield: 66%, H NMR (400 MHz, DMSO-d ) δ = 5.13 (2H, s, OCH Ph), 5.72
6
2
(
2H, brs, NH ), 6.47 (1H, dd, J = 5.0 Hz, 7.7 Hz, ArH), 7.07 (1H, d, J = 7.8 Hz, ArH), 7.37−7.47
2
(3H, m, ArH), 7.51 (1H, dd, J = 1.2 Hz, 5.0 Hz, ArH), 7.58 (1H, s, ArH). Reported [51].
3
-(2-chlorobenzyloxy)pyridin-2-amine (7d)
1
Yellow solid, yield: 88%, H NMR (400 MHz, CDCl ) δ = 4.73 (2H, brs, NH ), 5.18 (2H, s,
3
2
OCH Ph), 6.59 (1H, dd, J = 5.1 Hz, 7.8 Hz, ArH), 6.96 (1H, d, J = 7.8 Hz, ArH), 7.27−7.31 (2H,
2
m, ArH), 7.40−7.43 (1H, m, ArH), 7.46−7.48 (1H, m, ArH), 7.69 (1H, d, J = 5.0 Hz, ArH).
Reported [51].
3
-(4-chlorobenzyloxy)pyridin-2-amine (7e)

ACCEPTED MANUSCRIPT
1
Yellow solid, yield: 53%, H NMR (400 MHz, CDCl ) δ = 4.66 (2H, brs, NH ), 5.03 (2H, s,
3
2
OCH Ph), 6.59 (1H, dd, J = 5.1 Hz, 7.8 Hz, ArH), 6.93 (1H, dd, J = 1.3 Hz, 7.8 Hz, ArH),
2
7
.29−7.46 (4H, m, ArH), 7.69 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH). Reported [52].
3
-(4-fluorobenzyloxy)pyridin-2-amine (7f)
1
White solid, yield: 26%, H NMR (400 MHz, CDCl ) δ = 4.64 (2H, brs, NH ), 5.02 (2H, s,
3
2
OCH Ph), 6.59 (1H, dd, J = 5.1 Hz, 7.8 Hz, ArH), 6.93 (1H, dd, J = 1.1 Hz, 7.8 Hz, ArH), 7.08
2
(2H, t, J = 8.7 Hz, ArH), 7.37−7.40 (2H, m, ArH), 7.68 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH).
Reported [51].
4
2
6
.1.2. General procedure of urea compounds (5a−r)
-amino-3-benzyloxy pyridine derivative (0.85 mmol) and sodium hydride (60% in mineral oil,
8 mg, 1.71 mmol) was dissolved in dry THF (5 mL), aliphatic isocyanate derivative (1.02
mmol) was added to the reaction mixture. The reaction was refluxed for 5−18 h. After cooling,
the reaction mixture was evaporated and the residue was purified by flash column
chromatography (SiO , EA/n-Hex = 1/4) to afford the title final compounds 5a−r.
2
1
-(3-(benzyloxy)pyridin-2-yl)-3-cycloheptylurea (5a)
1
White solid, yield: 80%, mp: 111−112 °C, HPLC purity: 3.97 min, 92.14%, H NMR (400 MHz,
CDCl ) δ = 1.56−1.66 (10H, m, 5CH ), 1.95−2.00 (2H, m, CH ), 4.01−4.03 (1H, m, CH), 5.09
3
2
2
(
2H, s, OCH Ph), 6.76−6.79 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH), 7.06 (1H, d, J = 8.0 Hz, ArH),
2
1
3
7
.33 (1H, s, NH), 7.35−7.39 (5H, m, ArH), 7.76 (1H, d, J = 5.1 Hz, ArH), 9.53 (1H, s, NH); C
NMR (100.6 MHz, CDCl ) δ = 24.05, 28.01, 35.18, 50.70, 70.66, 116.04, 117.38, 127.74,
3

ACCEPTED MANUSCRIPT
+
1
28.63, 128.86, 135.26, 137.09, 141.29, 144.52, 153.99. HRMS (ES ): m/z calculated for
+
C H N O : 362.1845 [M+Na] . Found 362.1846.
20
25
3
2
1
-(3-(benzyloxy)pyridin-2-yl)-3-(tert-butyl)urea (5b)
1
White solid, yield: 21%, mp: 78−79 °C, HPLC purity: 1.90 min, 99.54%, H NMR (400 MHz,
CDCl ) δ = 1.44 (9H, s, 3CH ), 5.09 (2H, s, OCH Ph), 6.77 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH),
3
3
2
7
.05 (1H, dd, J = 1.3 Hz, 8.0 Hz, ArH), 7.23 (1H, s, NH), 7.35−7.39 (5H, m, ArH), 7.74 (1H, dd,
1
3
J = 1.3 Hz, 5.1 Hz, ArH), 9.46 (1H, s, NH); C NMR (100.6 MHz, CDCl ) δ = 29.18, 50.42,
3
7
0.60, 115.90, 117.31, 127.64, 128.58, 128.83, 135.32, 137.02, 141.20, 144.59, 153.60. HRMS
+
+
(
ES ): m/z calculated for C H N O : 322.1532 [M+Na] . Found 322.1529.
17 21 3 2
1
-(3-(3-fluorobenzyloxy)pyridin-2-yl)-3-cycloheptylurea (5c)
1
White solid, yield: 71%, mp: 186−189 °C, HPLC purity: 8.30 min, 94.48%, H NMR (400 MHz,
CDCl ) δ = 1.57−1.68 (10H, m, 5CH ), 1.95−2.01 (2H, m, CH ), 4.02−4.04 (1H, m, CH), 5.08
3
2
2
(
2H, s, OCH Ph), 6.78 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH), 7.03 (1H, dd, J = 1.3 Hz, 8.0 Hz,
2
ArH), 7.06−7.09 (2H, m, ArH), 7.16 (1H, d, J = 7.7 Hz, ArH), 7.31 (1H, s, NH), 7.34−7.39 (1H,
1
3
m, ArH), 7.78 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH), 9.51 (1H, s, NH); C NMR (100.6 MHz,
CDCl ) δ = 29.59, 36.54, 42.02, 42.58, 51.09, 69.81, 114.52 (J = 22.0 Hz), 115.56 (JC-F = 21.0
3
C-F
Hz), 115.79, 117.34, 122.96, 130.52 (JC-F = 8.1 Hz), 137.29, 137.75, 140.96, 144.63, 153.12,
+
+
1
62.96 (JC-F = 245.9 Hz). HRMS (ES ): m/z calculated for C H N O : 396.2836 [M+K] .
17 21 3 2
Found 396.2121.
1
-(3-(3-fluorobenzyloxy)pyridin-2-yl)-3-(2,4,4-trimethylpentan-2-yl)urea (5d)

ACCEPTED MANUSCRIPT
1
White solid, yield: 18%, mp: 107−111 °C, H NMR (400 MHz, CDCl ) δ = 1.05 (9H, s, 3CH ),
3
3
1
.49 (6H, s, 2CH ), 1.82 (2H, s, CH ), 5.07 (2H, s, OCH Ph), 6.77 (1H, dd, J = 5.1 Hz, 7.9 Hz,
3 2 2
ArH), 7.02 (1H, dd, J = 1.2 Hz, 7.9 Hz, ArH), 7.04−7.09 (2H, m, ArH), 7.16 (1H, d, J = 7.7 Hz,
ArH), 7.18 (1H, s, NH), 7.33−7.37 (1H, m, ArH), 7.75 (1H, dd, J = 1.2 Hz, 5.1 Hz, ArH), 9.50
1
3
(
1H, s, NH); C NMR (100.6 MHz, CDCl ) δ = 29.69, 30.23, 31.50, 31.57, 31.66, 51.83, 52.07,
3
5
4.19, 69.82, 114.49 (JC-F = 21.9 Hz), 115.55 (JC-F = 20.9 Hz), 115.82, 117.29, 123.04, 130.52
+
(
JC-F = 8.1 Hz), 137.24, 137.80, 140.87, 144.53, 153.33, 162.97 (JC-F = 245.9 Hz). HRMS (ES ):
+
m/z calculated for C H FN O : 396.2064 [M+Na] . Found 396.2060.
2
1
28
3
2
1
-(3-(3-fluorobenzyloxy)pyridin-2-yl)-3-ethylurea (5e)
1
Yellow solid, yield: 39%, mp: 55−56 °C, HPLC purity: 4.08 min, 95.66%, H NMR (400 MHz,
CDCl ) δ = 1.25 (3H, t, J = 7.2 Hz, CH ), 3.40−3.44 (2H, m, CH ), 5.08 (2H, s, OCH Ph), 6.79
3
3
2
2
(1H, dd, J = 5.1 Hz, 8.0 Hz, ArH), 7.03−7.08 (3H, m, ArH), 7.16 (1H, d, J = 7.6 Hz, ArH),
1
3
7
.34−7.38 (2H, m, ArH+NH), 7.77−7.78 (1H, m, ArH), 9.42 (1H, s, NH); C NMR (100.6
MHz, CDCl ) δ = 15.37, 34.71, 69.89, 114.60 (JC-F = 21.9 Hz), 115.62 (JC-F = 21.1 Hz),
3
1
16.20, 117.48, 123.14, 130.56 (JC-F = 8.1 Hz), 137.37, 137.66, 141.02, 144.31, 154.87, 162.99
+
+
(
JC-F = 245.9 Hz). HRMS (ES ): m/z calculated for C H FN O : 312.1125 [M+Na] . Found
15 16 3 2
3
12.1118.
1
-(3-(3-fluorobenzyloxy)pyridin-2-yl)-3-(tert-butyl)urea (5f)
1
White solid, yield: 32%, mp: 108−109 °C, HPLC purity: 2.02 min, 99.93%, H NMR (400 MHz,
CDCl ) δ = 1.46 (9H, s, 3CH ), 5.08 (2H, s, OCH Ph), 6.77 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH),
3
3
2
7
.02 (1H, dd, J = 1.3 Hz, 8.0 Hz, ArH), 7.04−7.09 (2H, m, ArH), 7.16 (1H, d, J = 7.6 Hz, ArH),

ACCEPTED MANUSCRIPT
7
.20 (1H, s, NH), 7.33−7.39 (1H, m, ArH), 7.75 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH), 9.5 (1H, s,
1
3
NH); C NMR (100.6 MHz, CDCl ) δ = 29.17, 50.45, 69.80, 114.46 (J = 21.9 Hz), 115.54
3
C-F
(JC-F = 21.0 Hz), 115.86, 117.34, 123.01, 130.51 (JC-F = 8.1 Hz), 137.31, 137.81, 140.91, 144.56,
+
1
53.52, 162.98 (JC-F = 245.9 Hz). HRMS (ES ): m/z calculated for C H FN O : 340.1438
17
20
3
2
+
[
M+Na] . Found 340.1434.
1
-(3-(2-chlorobenzyloxy)pyridin-2-yl)-3-cycloheptylurea (5g)
1
White solid, yield: 72%, mp: 120−121 °C, HPLC purity: 6.99 min, 96.82%, H NMR (400 MHz,
CDCl ) δ = 1.56−1.68 (10H, m, 5CH ), 1.97−1.98 (2H, m, CH ), 4.01−4.05 (1H, m, CH), 5.20
3
2
2
(
2H, s, OCH Ph), 6.81 (1H, dd, J = 5.1 Hz, 8.0 Hz, ArH), 7.07 (1H, dd, J = 1.3 Hz, 8.0 Hz,
2
ArH), 7.28−7.32 (2H, m, ArH), 7.33 (1H, s, NH), 7.41−7.44 (2H, m, ArH), 7.78 (1H, dd, J = 1.3
1
3
Hz, ArH), 9.52 (1H, s, NH); C NMR (100.6 MHz, CDCl ) δ = 24.05, 28.01, 35.18, 50.72,
3
6
1
7.82, 116.11, 117.51, 127.27, 129.34, 129.76, 129.87, 132.95, 133.20, 137.39, 141.04, 144.48,
+
+
53.95. HRMS (ES ): m/z calculated for C H ClN O : 396.1455 [M+Na] . Found 396.1448.
2
0
24
3
2
1
-(3-(2-chlorobenzyloxy)pyridin-2-yl)-3-(2,4,4-trimethylpentan-2-yl)urea (5h)
1
Pink solid, yield: 90%, mp: 110‒112 °C, H NMR (400 MHz, CDCl ) δ = 1.03 (9H, s, 3CH ),
3
3
1
.49 (6H, s, 2CH ), 1.82 (2H, s, CH ), 5.19 (2H, s, OCH Ph), 6.79 (1H, dd, J = 5.1 Hz, 8.0 Hz,
3 2 2
ArH), 7.04 (1H, dd, J = 1.3 Hz, 8.0 Hz, ArH), 7.21 (1H, s, NH), 7.29‒7.32 (2H, m, ArH),
1
3
7
.41‒7.45 (2H, m, ArH), 7.75 (1H, dd, J = 1.3 Hz, 5.1 Hz, ArH), 9.51 (1H, s, NH); C NMR
(
100.6 MHz, CDCl ) δ = 29.69, 31.51, 31.66, 51.87, 54.19, 67.75, 115.91, 117.44, 127.25,
3
+
1
29.19, 129.70, 129.78, 133.03, 137.26, 140.90, 144.55, 153.37. HRMS (ES ): m/z calculated
+
for C H ClN O : 412.1768 [M+Na] . Found 412.1761.
2
1
28
3
2