Discovery of highly potent human glutaminyl cyclase (QC) inhibitors as anti-Alzheimer's agents by the combination of pharmacophore-based and structure-based design

Article abstract of DOI:10.1016/j.ejmech.2021.113819

The inhibition of glutaminyl cyclase (QC) may provide a promising strategy for the treatment of early Alzheimer's disease (AD) by reducing the amount of the toxic pyroform of β-amyloid (Aβ_Ν3pE) in the brains of AD patients. In this work, we ide

Full text of DOI:10.1016/j.ejmech.2021.113819

European Journal of Medicinal Chemistry 226 (2021) 113819
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of highly potent human glutaminyl cyclase (QC) inhibitors
as anti-Alzheimer's agents by the combination of pharmacophore-
based and structure-based design
,
Nguyen Van Manh a, Van-Hai Hoang a, Van T.H. Ngo a e, Jihyae Ann a, Tae-ho Jang b,
Jung-Hye Ha b, Jae Young Song b, Hee-Jin Ha c, Hee Kim c, Young-Ho Kim c, Jiyoun Lee d,
,
*
Jeewoo Lee a
a Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 08826, Republic
of Korea
b New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, 41061, Republic of Korea
c Medifron DBT, Seoul, 08502, Republic of Korea
d Department of Global Medical Science, Sungshin University, Seoul, 01133, Republic of Korea
e Graduate Department of Healthcare Science, Dainam University, Hanoi, Viet Nam
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of glutaminyl cyclase (QC) may provide a promising strategy for the treatment of early
Received 19 July 2021
Received in revised form
29 August 2021
Accepted 31 August 2021
Available online 8 September 2021
Alzheimer's disease (AD) by reducing the amount of the toxic pyroform of b-amyloid (AbN3pE) in the
brains of AD patients. In this work, we identified potent QC inhibitors with subnanomolar IC50 values that
were up to 290-fold higher than that of PQ912, which is currently being tested in Phase II clinical trials.
Among the tested compounds, the cyclopentylmethyl derivative (214) exhibited the most potent
in vitro activity (IC50 ¼ 0.1 nM), while benzimidazole (227) showed the most promising in vivo efficacy,
selectivity and druggable profile. 227 significantly reduced the concentration of pyroform Ab and total Ab
Keywords:
in the brain of an AD animal model and improved the alternation behavior of mice during Y-maze tests.
The crystal structure of human QC (hQC) in complex with 214 indicated tight binding at the active site,
supporting that the specific inhibition of QC results in potent in vitro and in vivo activity. Considering the
recent clinical success of donanemab, which targets AbN3pE, small molecule-based QC inhibitors may also
provide potential therapeutic options for early-stage AD treatment.
Alzheimer's disease
Beta-amyloid pyroform
Glutaminyl cyclase inhibitor
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
amyloid-b (Ab) oligomers in the brain is the key molecular event in
AD pathogenesis [2]; this accumulation has been recognized as the
most promising target for directed therapies. Thus, efforts have
Alzheimer's disease (AD), the most common cause of dementia,
is a chronic neurodegenerative disorder in which the death of brain
cells causes progressive memory loss and cognitive dysfunction [1].
Currently, only four drugs, including acetylcholinesterase (AchE)
been made over the past 20 years to decrease the levels of A
monomers, oligomers, aggregates and plaques in patients by
inhibiting -producing enzymes, preventing aggregation or
increasing the clearance of A from the brain [3,4]. Unfortunately,
these approaches have failed to show unambiguous efficacy in
clinical trials [5,6]; Recently, aducanumab, an A antibody, has
received accelerated approval for AD by U.S. Food and Drug
Administration as an A -clearing treatment. It is the first therapy to
b
A
b
inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists,
b
are clinically used for AD treatment. However, they only relieve
some symptoms for a limited period of time and have no effect on
the prevention or mitigation of disease progression.
Among the proposed molecular mechanisms involved in AD, the
“amyloid hypothesis” states that the accumulation of neurotoxic
b
b
demonstrate that removing amyloid from the brain is reasonably to
reduce cognitive and functional decline in people with early AD.
Thus, antiamyloid therapies is recognized as a viable approach for
early-stage AD patients considering that the accumulation of A
peptides begins decades before any symptoms of the disease
b
* Corresponding author.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
https://doi.org/10.1016/j.ejmech.2021.113819
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
appear [7]. Moreover, numerous types of A
are neurotoxic [8,9]; thus, targeting specific forms of toxic A
species may provide a disease-modifying treatment for AD.
b
fibrils and oligomers
also been actively developed [28e30]. The first prototype of the QC
inhibitor PBD150 (1) with imidazole as a ZBG was discovered by
Probiodrug AG through fragment-based screening [31]; this pro-
totype demonstrated a reduction in brain AbN3pE through QC in-
hibition, offering a new therapeutic option for AD [22]. However, a
study using radiolabeled PBD150, [11C]PBD150, indicated that 1 did
not appear to cross the blood-brain barrier (BBB) in mice, sug-
gesting that the therapeutic effect of QC inhibition may be associ-
ated with peripheral mechanisms [32]. In the same study, the ZBG
was further optimized; 5-methylimidazole (2) was selected by
homology modeling [33], and benzimidazole (3, 4) was selected by
virtual screening [34,35]. Both exhibited stronger enzyme inhibi-
tion and better drug properties than imidazole. PQ912 (4) is the first
small molecule to have entered a clinical trial [36]. Phase I and IIa
studies indicated that PQ912 has a beneficial effect on cognitive
function and reverses AD-induced changes in theta and alpha
rhythms [37e39].
A series of diphenyl-conjugated imidazole derivatives were
investigated by the Wu group. Among them, 5 exhibited potent QC
inhibition with good in vitro BBB permeability, dramatically
reduced AbN3pE-42 in cultured cells and improved behavior in AD
model mice [40]. The N-methyltriazole-based inhibitor 6 was
discovered through three-dimensional (3D) pharmacophore and
model-based in silico methods and in vitro screening performed by
Caricasol and Rubinsztein [41]. The crystal structure of human QC
(hQC) in complex with SEN177 exhibits a binding mode that is
different from those of PBD150 and PQ912 [42]. In addition, natu-
rally occurring inhibitors [43,44] and small molecule inhibitors
generated by the fragment-based approach [45] or virtual
screening [46] have been reported and exert moderate in vitro
activity. Recently, hydrazide-bearing peptide inhibitors were re-
ported as potent transition state analogs of hQC, which was eluci-
dated by X-ray structure analysis of the enzyme-ligand complex
[47].
b
The pyroglutamate form of A
product of N-truncated A peptides, comprises a substantial pro-
portion of A peptides, accounting for more than 50% of the total A
plaques in the AD brain [10e12]. AbN3pE is a major constituent of A
b (AbN3pE), a posttranslational
b
b
b
b
species deposited in early extracellular plaques and intraneuronal
aggregates and likely plays a critical role in the early stages of AD
pathogenesis. Many studies report that AbN3pE is found upstream of
the amyloid cascade, contributing to the early stages of AD patho-
genesis and ultimately leading to clinical dementia [13]. Compared
with unmodified A
aggregation and participate in plaque seeding rather than in the
formation of oligomeric and fibrillar A assemblies [14e16].
b, AbN3pE species exhibit a higher propensity for
b
Furthermore, AbN3pE aggregates not only exhibit higher neuronal
and glial toxicities but also demonstrate greater resistance to pro-
teolytic degradation by peptidases. In addition, AbN3pE contributes
to the formation of hyperphosphorylated tau, suggesting a possible
connection between the two neurotoxic species in AD. In a recent
clinical trial, patients exhibiting early symptoms of AD were treated
with donanemab, a monoclonal antibody to AbN3pE; the treatment
resulted in a significant slowing of decline in cognition and daily
function [17], supporting that AbN3pE is an alternative therapeutic
target for AD [18].
Glutaminyl cyclase (QC), belonging to the family of metal-
dependent aminoacyltransferases, is responsible for the conver-
sion of glutaminyl or glutamyl residues at the terminus of peptides
into corresponding lactam rings and is mainly found in the hip-
pocampus and cortex of the mammalian brain [19e21]. In A
peptides, QC catalyzes the conversion of the N-truncated A pep-
b
b
tide (Ab3-40/42) generated from Ab40-42 by dipeptidyl peptidase IV
into the pyroglutamate peptide AbN3pE-40/42. QC protein and mRNA
levels are higher in the brains and cerebrospinal fluid (CSF) of AD
patients than in control brains [13,22,23] and can be detected in the
early stages of AD. Furthermore, QC knockout has been shown to
rescue cognitive function in AD model mice [24], and QC inhibitors
Recently, we investigated new classes of potent QC inhibitors
that were designed by employing bioisostere-based [48],
pharmacophore-based [49e51] and structure-based approaches
[52]. Among them, 8 and 9 (Fig. 2) were reported as representative
inhibitors and demonstrated potent QC inhibition, significantly
efficiently reduce the levels of AbN3pE and Ab plaques in the brain
[25], improving memory deficits in AD mice [22]; these findings
support QC inhibition as a viable therapeutic strategy for AD
[26,27].
reduced the concentrations of AbN3pE and total Ab in the brain and
restored cognitive functions in an AD animal model.
Small molecule-based QC inhibitors (Fig.1), mostly derived from
the N-terminal (Glu-Phe) structure of the substrate Ab3-42, have
The main pharmacophores of these inhibitors can be classified
as zinc-binding groups (ZBGs; pharmacophore A), hydrogen
Fig. 1. Reported QC inhibitors and their principal pharmacophores.
2

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
Fig. 2. Design of new QC inhibitors by the combination of pharmacophore-based and bioactive conformation-based approaches.
bonding donors (pharmacophore B) or phenyl groups (pharmaco-
phore C), as represented in Fig. 2. On the basis of pharmacophoric
analysis of the N-terminal tripeptide (Glu-Phe-Arg) of the substrate
aminopyridin-4-yl)ethanol or previously prepared 4-(2-
aminopyridin-4-yl)butanol [49] via a Mitsunobu reaction to give
12 and 13, whose nitro groups were subsequently reduced to
amines 14 and 15, respectively. The reductive amination of 14 with
aldehydes or ketones led to the formation of secondary amines
16e18. To synthesize C-region N-substituted anilines, aniline (19)
was prepared from 11 by methoxymethyl (MOM) protection fol-
lowed by reduction. The Buchwald-Hartwig reaction of 19 with
iodobenzene produced N-phenyl aniline (20), which was joined
with the 2-aminopyridine D-region via a Mitsunobu reaction to
afford 23. N-substituted anilines with (2-aminopyridin-4-yl)butyl
groups were synthesized by means of 2-nitrobenzenesulfonamide
[54,55]. Amine 19 was treated with 2-nitrobenzenesulfonyl chlo-
ride to produce a sulfonamide (24), which underwent N-mono-
alkylation by either direct alkylation with corresponding alkyl
halides or a Mitsunobu reaction with corresponding alcohols to
provide 25e37 after MOM deprotection. The Williamson reaction
of phenols 25e37 with an iodobutyl intermediate led to the for-
mation of 38e50, whose 2-nitrobenzylsulfonyl groups were
removed through a nucleophilic aromatic substitution by thio-
phenol in basic media to provide secondary anilines 51e63.
A
b3-42, 8 was discovered by inserting an Arg-mimicking group (e.g.,
2-aminopyridine in 8) as an additional pharmacophore D into
prototype inhibitor 2. In addition, based on the X-ray structure of
the hQC-PBD150 (1) complex [53], wherein the ligand resides at the
hQC active site in a bent conformation (Z-E conformation), 9 was
designed by incorporating a conformational blocker (e.g., N-
methylpiperidine in 9) into the urea surrogate 7 to maintain an
active Z-E conformation.
The two effective design approaches prompted us to combine
compounds 8 and 9 to design a new scaffold (10) with an Arg-
mimicking group and a bioactive conformation. In this work, we
synthesized
a series of N-substituted N-(4-aminoalkylphenyl)
thiourea/urea analogs as new scaffolds and investigated their
structure-activity relationships (SARs) as hQC inhibitors. The
selected potent inhibitors were further evaluated in vivo by
measuring their ability to reduce the formation of AbN3pE-42 in
acute and transgenic AD mouse models and examining their ability
to improve cognition and behavior. In addition, we determined the
crystal structure of hQC complexed with a potent inhibitor to
identify the specific binding interactions of the inhibitor-enzyme
complex and rationalize our hypothesis.
The synthesis of secondary anilines with alkyl D-regions is
shown in Scheme 2. To synthesize dimethylamino D-region de-
rivatives, 4-nitroguaialcol was condensed with dibromoalkanes to
form bromoalkyl derivatives (64e66), which were reacted with
either dimethylamine or cyclic amines to provide 67e69 or 85e87,
respectively. Nitro compounds 67e69 and 85e87 were reduced
before undergoing reductive amination with corresponding alde-
hydes or ketones to form secondary anilines 73e84 and 91e102,
respectively. The Williamson reaction of phenols 21, 25e27 and 32
with dibromoethane followed by N-alkylation with cyclic amines
provided 104 and 109e117, respectively, whose N-protecting
groups were removed to give secondary amines 118e127.
2. Results and discussion
2.1. Chemistry
In general, the final thiourea and urea compounds were syn-
thesized by a coupling reaction between A-region imidazole/
benzimidazole alkyl azide or amine and corresponding C-region
anilines, while the final
obtained by the condensation of
with corresponding C-region amines.
a
,a
0-gem-dialkyl amide compounds were
0-gem-dialkyl carboxylic acids
The synthesis of a,a
0-gem-dialkyl carboxylic acids is depicted in
a,a
Scheme 3. The protection and reduction of vanillin (128) produced
the primary alcohol 129. By using diphenyl phosphorocyanidate
[56], the primary alcohol was converted to the corresponding cy-
anide derivative 130, which was alkylated with iodomethane or
The synthesis of primary and secondary anilines with 2-
aminopyridine D-regions is illustrated in Scheme 1. Commercially
available 4-nitroguaialcol (11) was reacted with 2-(2-
3

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
Scheme 1. Synthesis of the N-substituted anilines with 2-aminopyridine pharmacophores (D)a.
a Reagents and conditions: (a) ROH, diethyl azodicarboxylate, PPh3, CH2Cl2, RT, 3 h; (b) Pd/C, H2, MeOH, RT, 2 h; (c) RCHO or R2C]O, NaBH3CN, AcOH, MeOH, 40 ꢀC, overnight; (d)
MOMBr, NaH, DMF, RT, 1 h; (e) iodobenzene, Pd(OAc)2, xantphos, Cs2CO3, dioxane, 100 ꢀC, overnight; (f) CbzCl, K2CO3, MeCN, RT, overnight; (g) HCl, MeOH, H2O, RT, 1 h; (h) Et3SiH,
Pd/C, MeOH, RT, 30 min; (i) 2-nitrobenzenesulfonyl chloride, NEt3, CH2Cl2, 0 ꢀC-RT, 4 h; (j) RX, K2CO3, DMF, 100 ꢀC, 1 h; (k) Ar(CH2)4I, K2CO3, DMF, 40 ꢀC, 5 h; (l) PhSH, K2CO3, MeCN,
RT, overnight.
dibromoalkanes to achieve
a
,
a
0-disubstituted cyanides (131e134).
[49,59]. In addition, we prepared known inhibitors 2e9 in house as
references, and their IC50 values were measured for comparison
(IC50; 2: 29.2 nM, 3: 47.9 nM, 4: 29.0 nM, 5: 118 nM, 6: 16.7 nM, 7:
162 nM, 8: 4.5 nM, 9: 6.1 nM).
The cyanides were hydrolyzed into their corresponding acids under
basic conditions and then converted to methyl esters (135e138);
this process was concomitant with MOM removal. The esters un-
derwent alkylation with the D-region iodide followed by hydrolysis
To validate our hypothesis that the combination of
pharmacophore-based and structure-based design induces a syn-
ergistic effect, we first investigated the SARs of the N-substituted
thiourea derivatives of 8 in which conformationally blocking
groups were incorporated with the proximal nitrogen of thiourea
(Table 1). The incorporation of small alkyl groups, such as methyl
(151) and ethyl (152) groups, did not have an effect on the activity.
However, QC inhibition began to increase after the substituent
exceeded a threshold size. The potency of isopropyl (153), cyclo-
butyl (154), cyclopentyl (155), and cyclohexyl (156) derivatives was
up to 9-fold higher than that of 8, and the derivatives exhibited IC50
values of 0.5e0.8 nM; the increased potency is associated with the
size of the substituent increasing, supporting that the combination
approach is effective for QC inhibition.
Because our combination approach demonstrated the proposed
synergistic effect in the analogs of N-alkyl thiourea (8), we decided
to investigate the SARs of their urea surrogates by incorporating
conformationally blocking groups of varying sizes with the prox-
imal nitrogens (structure-based design). We also introduced a va-
riety of amino groups, such as dialkylamino groups, cyclic amine
groups and 2-aminopyridine groups selected from a previous study,
as D-region pharmacophores [51]. First, we explored the SARs of
the N-substituted urea derivatives of 8 (Table 2). Similar to thiourea,
urea derivatives with small alkyl groups (158, 159) did not show
improved activity compared to unsubstituted urea (157), and the
activity started to increase when the size of the substituent
exceeded that of the isopropyl group. Isopropyl (160), cyclopropyl
(161), cyclobutyl (162), cyclopentyl (163), cyclohexyl (164), cyclo-
pentylmethyl (165), 1-methylpiperidinyl (166), and phenyl (167)
derivatives exhibited activities that were 2- to 5-fold higher than
that of 157. Compound 166 with N-(1-methylpiperidin-4-yl)urea in
to afford the a,a
0-gem-dialkyl carboxylic acids (143e146).
The synthesis of the final compounds is described in Scheme 4
and Scheme 5. Imidazole and benzimidazole A-region fragments
(147, 148 and 150) were synthesized in accordance with previous
reports [33,52], while 149 was prepared from its alcohol surrogate
[57] by using the azide-transfer reagent diphenylphosphoryl azide
(DPPA). To synthesize N-substituted thiourea derivatives, the N-
substituted anilines were coupled with 1-(3-azidopropyl)-5-
methyl-1H-imidazole (147) via Aza-Wittig coupling reactions [58]
employing carbon disulfide and then deprotected under acidic
conditions to produce 151e156. N-substituted urea derivatives
were obtained by two synthetic methods. In Method A, an Aza-
Wittig coupling reaction of A-region azides 147 and 149 with the
N-substituted anilines using carbon dioxide provided either the
final urea compounds or the penultimate intermediates that were
subsequently deprotected to form the final products. In Method B,
the N-substituted anilines were reacted with phenyl chloroformate
to afford the corresponding phenyl carbamates, which were
coupled with A-region amines 148 and 150 to provide either the
final product or the penultimate intermediates before deprotection.
Finally, the synthesis of
a,a
0-gem-dialkyl-substituted amide
derivatives was achieved by a coupling reaction between dialkyl
carboxylic acids 143e146 and amine 152 or 154 using the reagent
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) followed
by Boc deprotection.
2.2. Structure-activity relationships
The QC inhibitory activity of the synthesized compounds was
tested, and the corresponding IC50 values are shown in Tables 1e10
4

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
Scheme 2. Synthesis of the N-substituted anilines with dialkylamino pharmacophores (D)a.
a Reagents and conditions: (a) Br(CH2)nBr, K2CO3, DMF, 100 ꢀC; (b) R2NH, NEt3, DMF, heat; (c) Pd/C, H2, MeOH, RT, 2 h; (d) RCHO or R2C]O, NaBH3CN, AcOH, MeOH, 40 ꢀC; (e) Et3SiH,
Pd/C, MeOH, RT, 30 min; (f) PhSH, K2CO3, MeCN, RT, overnight.
Scheme 3. Synthesis of gem-dialkyl carboxylic acids with 2-aminopyridine pharmacophores (D)a.
a Reagents and conditions: (a) MOMBr, NEt3, CH2Cl2, RT, 1 h; (b) NaBH4, THF, RT, overnight; (c) diphenyl phosphorocyanidate, DBU, DMF, RT, 7 h; (d) MeI or Br(CH2)nBr, NaH, THF, RT
to 50 ꢀC; (e) NaOH, dioxane, H2O, reflux, 48 h; (f) conc. H2SO4, MeOH, reflux, 3 h then H2O, RT, 30 min; (g) Ar(CH2)4I, K2CO3, DMF, 70 ꢀC, 30 min; (h) NaOH, MeOH, H2O, 70 ꢀC.
5

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
Scheme 4. Synthesis of the final N-substituted thiourea and urea compounds.a
a Reagents and conditions: (a) CS2, Ph3P, toluene, reflux, overnight; (b) [Method A] CO2, Ph3P, toluene, reflux, overnight; [Method B] i) PhOCOCl, NEt3, CH2Cl2, RT, 5 h, ii) amine, NEt3,
RT, overnight; (c) CF3COOH, CH2Cl2, RT, overnight.
Scheme 5. Synthesis of the final gem-dialkyl amide compounds.a
a
Reagents and conditions: (a) i) 148, EDC, HCl, HOBt, NEt3, CH2Cl2, RT, ii) CF3COOH, CH2Cl2, RT, overnight; (b) i) 150, EDC, HCl, HOBt, NEt3, CH2Cl2, RT, ii) CF3COOH, CH2Cl2, RT,
overnight.
the B-region exhibited excellent inhibition with an IC50 value of
2.8 nM, which is approximately 2-fold more potent than that of 9
[52], which contains the same B-region pharmacophore. Benzyl
(168) and 4-fluorobenzyl (169) derivatives also displayed potent
inhibition with IC50 values of 1.0 and 4.3 nM, respectively. It should
be noted that in this series, the urea derivatives appeared to be less
potent than their corresponding thiourea surrogates; however, the
urea derivatives are less toxic [60,61].
6

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
Table 1
Table 3
IC50 values for the inhibition of hQC by N-alkyl/aryl-substituted urea compounds.
IC50 values for the inhibition of hQC by thiourea QC inhibitors based on 7.
Compound
X
IC50 (nM)a
14.6
Compound
151
R
IC50 (nM)a
8.9
170
(
(
(
2.2)
2.3)
5.1)
(
(
2.5)
3.5)
152
11.0
171
172
7.6
153
1.4
(
0.3)
10.9
154
155
0.8
0.5
(
(
0.4)
0.3)
173
23.8
(
7.4)
156
0.6
(
0.4)
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
Table 4
IC50 values for the inhibition of hQC by N-substituted urea compounds.
Table 2
IC50 values for the inhibition of hQC by N-alkyl/aryl-substituted urea compounds.
Compound
R
N
IC50 (nM)a
174
175
176
177
H
H
H
2
3
4
2
109
51.9
39.7
8.6
(
(
(
(
8.7)
7.2)
5.6)
0.6)
Compound
R
IC50 (nM)a
157
158
H
15.0
9.2
(
(
2.5)
4.4)
178
179
180
181
182
183
184
185
186
3
4
2
3
4
2
3
4
2
2.7
(
(
(
(
(
(
(
(
(
0.2)
0.2)
5.3)
2.3)
0.2)
0.7)
2.1)
3.4)
3.1)
159
160
14.2
7.3
(
(
3.4)
2.1)
4.2
13.3
15.5
0.71
18.3
11.9
7.9
161
162
4.7
5.0
(
(
1.3)
2.9)
163
164
6.9
3.6
(
(
2.9)
2.8)
165
166
167
168
3.4
2.8
9.3
1.0
(
(
(
(
1.8)
0.3)
4.5)
0.4)
19.3
187
188
3
4
34.1
12.8
(
(
6.0)
0.9)
169
4.3
(
1.2)
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
7

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
Table 5
Table 7
IC50 values for the inhibition of hQC by N-alkyl/aryl-substituted urea compounds.
IC50 values for the inhibition of hQC by N-alkyl/aryl-substituted urea compounds.
IC50 (nM)a
Compound
R
IC50 (nM)a
Compound
R
203
204
H
10.9
2.6
(
(
2.5)
0.8)
189
190
H
16.9
1.7
(
(
7.8)
0.8)
205
1.3
(
0.2)
191
192
3.8
1.6
(
(
1.3)
0.7)
206
207
208
209
1.0
1.8
3.5
3.2
(
(
(
(
0.2)
0.4)
1.8)
0.4)
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
Table 6
IC50 values for the inhibition of hQC by N-alkyl/aryl-substituted urea compounds.
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
Table 8
Compound
R
IC50 (nM)a
IC50 values for the inhibition of hQC by N-alkyl/aryl-substituted urea compounds.
193
194
H
13.2
13.4
(
(
1.7)
4.3)
195
196
4.8
5.5
(
(
0.8)
0.6)
197
198
199
200
201
202
1.5
0.46
1.6
3.9
1.4
3.8
(
(
(
(
(
(
0.2)
0.2)
0.1)
0.2)
0.1)
3.1)
Compound
R
IC50 (nM)a
210
211
H
18.7
15.1
(
(
2.5)
0.4)
212
20.8
(
9.6)
213
8.9
(
2.5)
214
215
216
217
0.1
(
(
(
(
0.02)
10.9)
0.2)
33.0
2.6
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
4.4
0.6)
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
Next, we examined the gem-dialkyl amide derivatives as sur-
rogates of N-substituted urea (Table 3). Gem-dimethyl (170),
cyclopropyl (171) and cyclobutyl (172) derivatives showed similar
activity to that of the corresponding N-substituted ureas (158 and
160). Unlike the N-substituted urea series, compounds in this series
were adversely affected by substituents of larger size.
Accordingly, we further explored the SAR of 10 by using a different
D-type pharmacophore, which was selected based on previous
studies [49e52]. We initially examined the SARs of dimethylami-
noalkyl analogs (Table 4) containing a dimethylamino group in the
Based on the N-substituted thiourea/urea and gem-dimethyl
amide analogs of 8 (Tables 1e3), we established a newly designed
scaffold (10), which had both a conformationally restricted B-re-
gion and a pharmacophore D-region; this scaffold provided a
synergistic effect, which was not the case for prototypes 8 and 9.
D
region. As anticipated, the unsubstituted urea derivatives
(174e176) showed moderate inhibition. Selected blocking groups,
including cyclohexyl (177e179), 1-methylpiperidine (180e182), 4-
fluorobenzyl (183e185) and 3-pyridylmethyl (186e188) groups,
8

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
Table 9
series indicated that the activity improved as the length of the
linker increased. The 4-carbon linker between pharmacophore C
(phenyl) and pharmacophore D (dimethylamino) appeared to be
optimal. Compound 182 with N-(1-methylpiperidin-4-yl)urea and
dimethylaminobutyl groups demonstrated excellent inhibition
with an IC50 value of 0.71 nM, which is ca. 40-fold higher than that
of PQ912.
IC50 values for the inhibition of hQC by N-alkyl/aryl-substituted urea compounds.
Next, we explored the SARs of cyclic amine-containing analogs
with 2-carbon linkers. A previous study indicated that cyclic
amines, such as pharmacophore D, with 2-carbon linkers exhibit
the most potent inhibition [49]. In a series of 2-aminopyridine
derivatives (Table 5), N substitution in urea improved the activity.
Interestingly, both unsubstituted and substituted ureas with 2-
carbon linkers were found to have similar activities to those of
the corresponding 4-carbon linker derivatives (157 vs 189, 164 vs
190, 166 vs 191, and 169 vs 192), suggesting that the 2-carbon linker
was sufficient for the amino pharmacophore to interact with the
anionic binding site of the enzyme.
On the other hand, for the series of 4-piperazinyl derivatives
(Table 6), the activity began to increase beyond N-ethyl urea, and
most of the derivatives displayed potent inhibition, exhibiting an
IC50 range between 0.46 and 5.5 nM. Among the 4-piperazinyl
derivatives, N-cyclopentylmethyl urea (198) exhibited the best
potency with an IC50 ¼ 0.46 nM, which is ca. 63-fold higher than
that of PQ912.
Compound
R
IC50 (nM)a
218
219
H
27.1
35.7
(
(
0.8)
19.9)
220
221
26.0
12.6
(
(
5.6)
3.6)
222
223
224
9.0
(
(
(
1.4)
7.8)
2.1)
12.6
4.0
225
226
17.1
20.0
(
(
3.7)
9.3)
For the series of 1-methyl-4-piperazinyl derivatives (Table 7), all
N-substituted urea derivatives displayed potent inhibition with an
IC50 range of 1.0e3.5 nM. As for the 4-piperazinyl derivatives, in this
series, N-cyclopentylmethyl urea (206) showed the highest inhi-
bition potency with an IC50 ¼ 1.0 nM.
227
9.9
(
2.5)
228
229
230
21.5
3.4
(
(
(
7.7)
1.4)
2.1)
For the series of 1-morpholinyl derivatives (Table 8), the activity
increased when the B-region was large (i.e., N-cyclohexyl urea), and
most derivatives, except for 1-methylpiperidinyl (215), exhibited
high potency. The cyclopentylmethyl derivative (214) also showed
exceptionally high potency with an IC50 ¼ 0.1 nM, which is ca. 190-
and 290-fold higher than that of the corresponding unsubstituted
urea (210) and PQ912, respectively. To our knowledge, 214 is the
most potent QC inhibitor reported to date.
9.5
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
Although the imidazole ring found in most QC inhibitors effec-
tively serves as a ZBG, it is known to cause toxicity problems in, for
example, cytochrome P450 (CYP) and human ether-a-go-go-related
gene (hERG) channel inhibition. This toxicity is due to the coordi-
nation of the nonbonding electron in the imidazole ring with the
heme group in CYP or with pi-stacking interactions in the central
cavity of the hERG channel. Indeed, a number of our potent
imidazole-containing inhibitors were found to be CYP and hERG
inhibitors. PQ912, the only QC inhibitor currently undergoing
clinical trials, contains a benzimidazole ring as a ZBG. Therefore, we
decided to investigate the benzimidazole surrogates of the most
potent inhibitors we identified in this work (Table 9).
The activity of the unsubstituted urea (218) was 2-fold lower
than that of the corresponding imidazole surrogate (157); never-
theless, the unsubstituted urea displayed similar activity to that of
PQ912. As shown in Table 2, the activity gradually increased as the
size of the blocking group increased above that of the isopropyl
group, providing potent inhibition with an IC50 range between 1.0
and 9.3 nM. Generally, the activities of benzimidazole derivatives
were found to be slightly lower than those of the corresponding
imidazole derivatives. In addition, gem-dialkyl surrogates of benz-
imidazole N-substituted urea were explored (Table 10). However,
most of these derivatives demonstrated moderate to weak
inhibition.
Table 10
IC50 values for the inhibition of hQC by N-alkyl/aryl-substituted urea compounds.
Compound
231
X
IC50 (nM)a
24.0
(
(
(
11.4)
9.9)
79)
232
233
42.2
471
234
691
(
53)
a
Values are the mean of at least three experiments, and numbers in parentheses
represent the standard deviation.
In summary, we investigated a total of 84 newly designed de-
rivatives as novel QC inhibitors. Most of the derivatives that we
tested exhibited the proposed synergistic effect of their parent
were incorporated with urea; these derivatives exhibited much
better activity than the unsubstituted urea. A SAR analysis of this
9

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
inhibitors (8 and 9), validating our rationale. Among the de-
rivatives, eight compounds displayed exceptionally high potencies
with subnanomolar IC50 values. Compound 214 was the most
potent inhibitor, exhibiting an IC50 value of 0.1 nM, which is ca.
2.3.2. Binding of hQC to 214
Structural analysis of hQC in complex with 214 confirmed that
214 binds to hQC. The imidazole, urea amine, benzene, and mor-
pholine groups of 214 interacted with the key residues of the ligand
binding site of hQC (Fig. 4A). The zinc ion was coordinated tetra-
hedrally with the three electron-rich side chains of Asp159, Glu202
and His330 at 2.1 Å, 2.2 Å, and 1.8 Å, respectively, and the imidazole
amine of 214 at 2.3 Å. The carbonyl group of the main Gln304 chain
interacted with the urea amine of 214 at 3.4 Å through hydrogen
bonding. Residue Phe325 interacted with the benzene ring of 214 at
4 Å by face-to-face pi-pi stacking, and the carboxylate of the Glu327
side chain was connected to the oxygen of morpholine through a
water bridge formed by hydrogen bonding. Finally, the hydroxyl
group of the Tyr299 side chain formed hydrogen bonds with the
methoxy oxygen of 214 at 3.4 Å (Fig. 4B).
1620-, 45-, and 60-fold higher than that of prototypes
7
(IC50 ¼ 162 nM), 8 (IC50 ¼ 4.5 nM) and 9 (IC50 ¼ 6.1 nM), respec-
tively. The SAR analyses indicated that when a conformational
blocker was incorporated into the urea group, the activity generally
increased with respect to its size. Among these blockers, cyclo-
pentylmethyl and 1-methylpiperidinyl exhibited the best activity,
whereas gem-dialkyl amide derivatives displayed lower potencies
than those of the corresponding N-substituted urea analogs. As
ZBGs, benzimidazole surrogates exhibited potencies that were
slightly lower than those of the corresponding imidazole groups;
however, we expected the benzimidazole surrogates to exhibit
enhanced druggable properties.
Consequently, the crystal structure of hQC in complex with 214
indicated that the principal pharmacophores of 214 interacted with
the key residues in the binding pocket of hQC via metal coordina-
tion, hydrogen bonding and pi-pi stacking interactions, resulting in
the exceptional potency of 214.
2.3. Crystal structure of the QC inhibitor
2.3.1. Structural elucidation of hQC in complex with 214
To understand how 214 binds to the binding site of the hQC
ligand, we determined the structure of hQC (amino acids 33 to 361)
in complex with 214 at a resolution of 2.85 Å; molecular replace-
ment was performed using a searching model (Protein Data Bank
(PDB) entry 3SI0). The collected data were refined and converged to
Rwork ¼ 0.25% and Rfree ¼ 0.26% using Coot and Phenix [62,63].
Structural analysis revealed that an asymmetric unit containing a
single hQC bound to a single 214 molecule forms a monomeric
biological assembly (Fig. 3). The crystal structure comprises resi-
dues Ala35-Lys361 from hQC with chain breaks in residues
148e151, 185e189, and 332 due to the lack of electron density
resulting from the flexibility of these regions. In addition, one
molecule of zinc was found at the ligand binding site, which could
interact with the ligand to enhance the overall activity. In our
structure, the hQC-ligand complex consisted of nine helices, two-
stranded antiparallel sheets, three-stranded parallel sheets, and
two free Cys residues.
2.4. Acute AD model
To assess efficacy in vivo, we evaluated imidazole 191
(IC50 ¼ 3.8 nM), 214 (IC50 ¼ 0.1 nM) and benzimidazole 227
(IC50 ¼ 9.9 nM) in an acute AD mouse model. In this study, we
injected 5
mg of human Ab3-40 together with each compound
(10 mM) into ICR mice (male, six weeks old) by intra-
cerebroventricular (ICV) injection. We determined the activity of
each compound on the following day by measuring the levels of
human AbN3pE-40 in the brain extracts of the mice. Compared to the
vehicle control, 191 and 227 suppressed the formation of AbN3pE-40
by 33.44% and 30.34%, respectively, as shown in Table 11. Unfor-
tunately, compound 214 was not effective. While the tested com-
pounds demonstrated similar in vitro activities, their in vivo
activities appeared to vary. We believe that this apparent discrep-
ancy can be attributed to the use of different substrates and enzyme
species in the in vitro assays (glutaminyl-AMC tested against hu-
man QC) and in vivo studies (human Ab3-40 tested in mice) as well
as compound-specific characteristics such as permeability, enzyme
Monomeric biological assembly of hQC (PDB entry 7CM0).
Asymmetric unit containing a single hQC bound to a single 214
molecule.
Fig. 3. Crystal structure of hQC in complex with 214.
10

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
Fig. 4. Binding of 214 to hQC. (A) 214 clearly binds to the ligand binding pocket of hQC. (B) 2D sketch of the interaction between 214 and the key binding pocket residues of hQC.
Table 11
18.6 nM and 213 nM for isoQC, which were more than 5-fold and
22-fold greater than those for QC, respectively, indicating that 227
In vivo QC inhibition in acute model studies.a
is highly specific for QC. We also determined their IC50 values in
mouse QC to be 7.6 nM and 60.7 nM, respectively, indicating that
227 is an hQC selective inhibitor.
In vitro IC50 (nM)
% Inhibition of AbN3pE-40
formation
(ICV)
(IP)
To examine the potential toxicity of each compound, we deter-
mined the cell viability of a hippocampal neuronal cell line (HT-22)
and found that both compounds were nontoxic at a concentration
191
227
3.8
9.9
33.44
30.34
21.76
20.02
a
5ug/5ul of human Ab3ꢁ40 dissolved in PBS (1 mg/mL) was injected into the deep
of 10
of each compound and found that both exhibited less than 50%
inhibition at a concentration of 10 M.
For the drug-drug interaction study, we examined CYP inhibi-
tion using five representative isozymes. Both compounds (10 M)
mM. We also examined the hERG potassium channel inhibition
cortical layer/hippocampus of 5-week-old ICR mice (25 g, n ¼ 4, male) using a
stereotaxic frame to induce acute A toxicity. Test compounds were administered
b
via ICV (10 mM) or IP (25 mg/kg) injection. Sandwich ELISA was performed to
quantify AbN3pE-40 in the brain.Fi.
m
m
displayed less than 50% inhibition of 1A2, 2C9, 2C19, and 3A4 iso-
zymes. However, 191 and 227 exhibited high and moderate inhi-
bition of the 2D6 isozyme, respectively, which is likely due to the
ionization of basic groups.
For the in vitro metabolic stability study, we determined the
stability of the two compounds in mouse and human liver micro-
somes. The percentage values of intact compounds remaining after
30 min for 191 and 227 were found to be 60.9% and 92.1% in mouse
liver microsomes and 73.2% and 97.7% in human liver microsomes,
reaction kinetics, and metabolic rate.
To examine whether these compounds exert their activity by
penetrating the BBB, we performed the same experiment by
injecting each compound (25 mg/kg) via intraperitoneal (IP) in-
jection; human Ab3-40 was injected via ICV injection prior to IP
administration. As shown in Table 11, 191 and 227 exhibited AbN3pE-
40-lowering effects with >20% inhibition (191: 24.76%, 227: 20.02%),
which is comparable to the activities observed in the ICV admin-
istration experiments, suggesting that these compounds likely
cross the BBB. However, since these experiments are an indirect
measure and compare biological effects after administration, po-
tential peripheral effects associated with QC inhibition should be
considered in future mechanistic studies.
respectively, indicating that 227 is
a metabolically stable
compound.
The parallel artificial membrane permeability assay modeling
the BBB (PAMPA-BBB) is considered one of the most powerful and
versatile physiochemical methods for screening early-stage central
nervous system (CNS)-targeted drugs [65]. The PAMPA-BBB system
models the transcellular, passive diffusion rate of drugs across the
BBB by measuring the effective permeability of an artificial lipid
membrane. As shown in Table 12, 227 exhibited reasonable
permeability with a cutoff value of 6.0 (-log Pe), whereas 191
showed very low permeability with a value of 10; these results
indicate that 227 is likely to be BBB permeable.
2.5. In vitro studies to assess drug candidacy
To explore the drug candidate potential of 191 and 227, we
evaluated their selectivity, toxicity and pharmacokinetic properties
(Table 12). First, we determined the selectivity of each compound
against isoQC, an isozyme of QC with differential substrate speci-
ficity [64], and mouse QC. 191 and 227 exhibited IC50 values of
11

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
Table 12
In vitro studies of selected QC inhibitors.
191
227
IC50 (hQC)
IC50 (isoQC)
IC50 (mQC)
Cytotoxicity (HT-22)
3.8 nM
18.6 nM
7.6 nM
100%
9.9 nM
213 nM
60.7 nM
100%
(10 mM, % of control)
hERG FP assay (10
CYP inhibition (10
m
M, % inhibition)
9.2%
35.2%
m
M, % inhibition)
1A2 (0), 2C9 (17), 2C19 (6), 2D6 (95), 3A4 (50)
1A2 (0), 2C9 (11), 2C19 (24), 2D6 (61), 3A4 (48)
Liver microsomal stability (remaining % after 30 min)
60.9 (mouse)
73.2 (human)
10.0
92.1 (mouse)
97.7 (human)
5.6
PAMPA-BBB (-logPe)
Overall, our studies indicate that 227 has a favorable druggable profile with respect to its target selectivity, toxicity and pharmacokinetic properties. Thus, we further tested it
in an AD model to evaluate its in vivo efficacy.
2.6. In vivo AD model
respectively). It also substantially reduced Ab40 and Ab42 levels (by
37.5 and 30.5%, respectively) without significant toxicity, indicating
that QC inhibition not only reduced the amount of AbN3pE in the
To evaluate the potential therapeutic effects of 227 in AD, we
performed long-term in vivo studies using 5XFAD transgenic mice,
brain but also contributed to the overall reduction in A
previously reported [68,69].
b levels, as
which is a well-known AD mouse model suitable for studying Ab42
-
induced neurodegeneration and amyloid plaque formation. This
particular AD model exhibits severe AD-related pathologies that
Next, we performed a behavior test on the 5XFAD transgenic
mice exposed to 227. Animal behavior was confirmed using a Y-
maze test, as the 5XFAD mice exhibited impaired memory [70].
Mice were treated via an infusion pump with vehicle or 50 mM 227.
After 4 weeks of treatment, we analyzed the animal's working
memory by using the Y-maze test. We found that 227-treated mice
showed significantly improvement in spontaneous alternation
compared to the vehicle-treated mice (Fig. 6). The number of arm
entries during the test period was not different between the group.
Thus, treatment with 227 improved behavioral deficits in the
result from substantially large amounts of A
the brain [66,67].
b and pyroform Ab in
227 (10 mM) was administered to 5XFAD mice every day for 4
weeks via brain infusion. Following the 4-week administration, the
concentrations of AbN3pE-40,42 and Ab40,42 were measured in the
brain by sandwich ELISA. As shown in Fig. 5, compared to the
vehicle control, 227 significantly reduced the concentrations of
A
bN3pE-40 and AbN3pE-42 in the brain (by 32.6 and 21.3%,
Fig. 5. Inhibition of the formation of human AbN3pE-40,42 and Ab40,42 in 5XFAD mice after the administration of 227.
12

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
5XFAD mice during the Y-maze test, suggesting that 227 has
beneficial effects in a transgenic AD mouse model.
involving a potent small molecule-based QC inhibitor and AbN3pE
antibody could effectively decrease the levels of toxic AbN3pE and
The concentrations of (a) AbN3pE-40, (b) AbN3pE-42, (c) Аb40 and
(d) Аb42 in the brains of vehicle and treated mice, as measured by
sandwich ELISA. All data are represented as the mean standard
deviation. Data were analyzed by unpaired t-test (n.s.:
nonsignificant).
Ab species, providing a potential disease-modifying and thera-
peutic strategy for early-stage AD treatment.
4. Experimental section
4.1. Chemistry
3. Conclusion
All chemical reagents were commercially available. Melting
points were determined with a melting point apparatus (Buchi B-
540) and were uncorrected. Silica gel column chromatography was
performed with 230e400 mesh silica gel 60 (Merck). Preparative
layer (PLC) plates used PLC silica gel 60 F254 (1 mm, Merck). 1H and
13C nuclear magnetic resonance (NMR) spectra were obtained with
a JEOL JNM-LA 300 operated at 300 MHz and 75 MHz, a Bruker
Avance III HD 400 operated at 400 MHz and 100 MHz, a Bruker
Avance III HD 500 operated at 500 MHz and 125 MHz, a JEOL JNM-
ECA 600 operated at 600 MHz and 150 MHz, and an ultrashielded
Bruker Avance III HD 800 operated at 800 MHz and 200 MHz.
Chemical shifts were reported in units of ppm with Me4Si as a
reference standard. Mass spectra were obtained with a VG Trio-2
gas chromatographyꢁmass spectrometry (GCꢁMS) instrument
and a 6460 Triple Quad liquid chromatographyꢁMS (LCꢁMS) in-
strument. All final compounds were of >95% purity, as determined
by high-performance liquid chromatography (HPLC). HPLC was
performed with an Agilent 1120 Compact LC (G4288A) instrument
We investigated a series of novel QC inhibitors that were
designed by combining pharmacophore-based and structure-based
approaches. Comprehensive SAR analyses indicated that, based on
prototype inhibitors 2 and 7, the substitution of a conformational
blocker on thiourea/urea and the addition of an extra pharmaco-
phore D led to a dramatic improvement in potency (up to ca. 300-
fold), supporting our rationale that combining two key pharma-
cophores into one scaffold results in synergistic potency effects.
Among the 84 synthesized derivatives, eight compounds exhibited
subnanomolar IC50 values, and 214 was found to be the most potent
inhibitor with an IC50 ¼ 0.1 nM, which is 290-fold higher than that
of PQ912.
To further evaluate the selected inhibitors, we measured AbN3pE-
levels in an acute model and examined the selectivity, cytotox-
40
icity, hERG and CYP inhibition, metabolic stability and BBB
permeability of the inhibitors. The results indicated that 227
showed the most favorable efficacy, selectivity and druggable
profile; thus, it was evaluated in an AD model to determine its
in vivo efficacy. Following 4 weeks of treatment in 5XFAD trans-
genic mice, 227 significantly reduced the concentrations of pyro-
using an Agilent TC-C18 column (4.6 mm ꢂ 250 mm, 5
mm).
4.1.1. General procedure for the Williamson reaction (Procedure A)
A corresponding alkyl halide (1.5 eq) was added to a suspension
containing phenol (1.0 eq) and potassium carbonate (2.0 eq) in
anhydrous DMF. The reaction mixture was heated at 70 ꢀC for 3 h
and then cooled to room temperature and quenched by the addi-
tion of water. The mixture was extracted with EtOAc and washed
with water several times. The organic layer was concentrated in
vacuo and purified by silica gel column chromatography to obtain
the desired product.
form Ab and total Ab in the brain and restored alternation behavior
in the mice during a Y-maze test. The crystal structure of hQC in
complex with 214 confirmed that the principal pharmacophores of
214 interacted with the key residues in the binding pocket of hQC
via metal coordination, hydrogen bonding and pi-pi stacking
interactions.
In this study, a highly potent and selective QC inhibitor with
favorable drug-like properties was discovered by rational design
based on the combination of pharmacophore-based and structure-
based approaches. Although the amyloid hypothesis has faced a
major setback due to the clinical failures of antiamyloid therapies,
the recent success of donanemab may imply an alternative
approach for targeting QC. Furthermore, combination therapy
4.1.2. General procedure for the Mitsunobu-type reactions
(Procedure B) Ethyl azodicarboxylate (1.5 eq) was slowly added
to a mixture containing sulfonamide (1.0 eq), alcohol (1.5 eq) and
triphenylphosphine (1.5 eq) in CH2Cl2. The reaction mixture was
Fig. 6. Chronic treatment with 227 restores behavioral deficits in 5XFAD mice. (a) Total arm entries and (b) spontaneous alternation (%) in 5-month-old 5XFAD mice during a Y-
maze test after 4 weeks of chronic treatment with 227. All data are represented as the mean standard deviation. Data were analyzed by unpaired t-test (*p < 0.05).
13

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
stirred overnight at room temperature. The reaction was quenched
with water and extracted with CH2Cl2. The organic layer was
concentrated in vacuo, and the residue was purified by silica gel
column chromatography (EtOAc/hexane) to obtain the desired
product.
(Procedure C) Triphenylphosphine (1.3 eq) was added to a so-
lution of phenol (2) (1.0 eq) in CH2Cl2 at 0 ꢀC; this was followed by
the addition of alkyl alcohol (1.2 eq) and a solution of diethyl
azodicarboxylate (1.3 eq) in CH2Cl2. The mixture was warmed to
room temperature and stirred overnight. The reaction was
quenched with water and extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and purified by silica gel
column chromatography to produce the desired product.
4.1.8. General procedure for C alkylation (Procedure I)
A solution of 130 (1.0 eq) in anhydrous THF kept at 0 ꢀC was
added to a cooled suspension of sodium hydride (1.1 eq) in anhy-
drous THF. After stirring for 10 min, a solution of iodomethane or
dibromoalkane (1.05 eq) was added to the mixture. The reaction
mixture was stirred at room temperature or at 50 ꢀC until the initial
material was consumed. After completion, the reaction mixture
was quenched with water and extracted twice with EtOAc. The
combined organic layer was washed with water and brine and
concentrated in vacuo. The residue was purified by silica gel column
chromatography (EtOAc/n-hexane) to produce the
tuted cyanide derivative.
a,a
0-disubsti-
4.1.9. General procedure for the hydrolysis of nitrile groups and
methyl esterification (Procedure J)
4.1.3. General procedure for N-alkylation (Procedure D)
An equivalent amount of water and excess NaOH were added to a
Triethyl amine (for amines) (5.0 eq) or potassium carbonate (for
sulfonamides) (3.0 eq) was added to an amine solution (1.5 eq) or a
sulfonamide solution with anhydrous DMF as the solvent. Then, the
corresponding alkyl halide (1.0 eq) was added, and the reaction
mixture was heated at 100 ꢀC until the initial material was
consumed. The reaction mixture was cooled to room temperature
and quenched by the addition of water. The mixture was extracted
with EtOAc and washed with water several times. The organic layer
was concentrated in vacuo, and the residue was purified by silica
gel column chromatography to obtain the desired product.
solution of
a,a
0-disubstituted cyanide in dioxane. The reaction
mixture was refluxed for 48 h and then cooled to room temperature.
The mixture was washed twice with EtOAc, acidified with a dilute
HCl solution and extracted several times with EtOAc. The combined
organic layer was washed with water and concentrated in vacuo to
yield
following step without further purification. A mixture of the ob-
tained
0-gem-dialkyl carboxylic acid (2 mmol) in MeOH (10 mL)
a,a
0-gem-dialkyl carboxylic acid, which was used in the
a,a
and concentrated H2SO4 (0.5 mL) was heated at reflux for 3 h. The
reaction was cooled to room temperature; this was followed by the
addition of water (1 mL) and stirring for an additional 30 min. The
resulting mixture was diluted with water and extracted with EtOAc
twotimes. Thecollected organic layer wasdried over MgSO4, and the
solvent was evaporated in vacuo. The residue was purified by silica
gel column chromatography to obtain the desired compound.
4.1.4. General procedure for MOM deprotection (Procedure E)
Concentrated HCl (20.0 eq) was added to a solution of MOM-
protected compound (1.0 eq) in MeOH. The reaction mixture was
stirred at room temperature until the initial material was
consumed, and then the solvent was evaporated in vacuo. The
residue was purified by silica gel column chromatography to pro-
duce the desired product.
4.1.10. General procedure for the hydrolysis of methyl esters
(Procedure K)
NaOH (5.0 eq) was added to a mixture of methyl ester (1.0 eq) in
MeOH/H2O (1:1). The reaction mixture was heated at 70 ꢀC for 4 h
and then concentrated in vacuo to remove MeOH. The residue was
diluted with water, cooled to 0 ꢀC and acidified with a 1 N HCl
solution. The mixture was diluted with water and extracted with
EtOAc two times. The collected organic layer was dried over MgSO4,
and the solvent was evaporated in vacuo. The residue was purified
by silica gel column chromatography to obtain the desired
compound.
4.1.5. General procedure for the reduction of nitro groups to amine
groups (Procedure F)
A nitro compound was dissolved in MeOH, and then 10% Pd/C
was added. The mixture was stirred at room temperature under
hydrogen gas until the initial material was consumed. The crude
mixture was filtered through Celite, washed with MeOH, and then
concentrated. The product was used for the following step without
further purification or was purified by silica gel column chroma-
tography if necessary.
4.1.11. General procedure for Aza-Wittig coupling reactions
(Procedure L) Triphenylphosphine (1.2 eq) was added to a so-
lution of N-substituted aniline (1.0 eq) and azide 147 or 149 (1.1 eq)
in toluene under stirring. The reaction mixture was degassed and
aerated with carbon dioxide. After stirring at 80 ꢀC overnight under
a carbon dioxide atmosphere, the mixture was concentrated in
vacuo and purified by silica gel column chromatography (CH2Cl2/
MeOH) to yield the desired product.
(Procedure M) A solution containing azide 147 (1.0 eq) and
triphenylphosphine (1.2 eq) in toluene was stirred for 30 min under
a nitrogen atmosphere. Then, a solution of carbon disulfide (1.1 eq)
in toluene was added. The mixture was stirred at room temperature
for 1 h, and N-substituted aniline (1.0 eq) was added. After stirring
at 40 ꢀC overnight, the mixture was concentrated in vacuo and
purified by silica gel column chromatography (MeOH/CH2Cl2) to
yield the desired product.
4.1.6. General procedure for reductive amination (Procedure G)
Acetic acid (5.0 eq) was added to a mixture of primary arylamine
(1.0 eq) and ketone or aldehyde (1.0 eq) in MeOH. This was followed
by the addition of sodium cyanoborohydride (3.0 eq). After stirring
at 40 ꢀC overnight, the reaction mixture was basified with a satu-
rated NaHCO3 solution and then extracted with CH2Cl2. The organic
layer was concentrated in vacuo and purified by silica gel column
chromatography using CH2Cl2/MeOH as the eluent to obtain the
corresponding secondary amines.
4.1.7. General procedure for desulfurization (Procedure H)
Thiophenol (2.0 eq) was added to a mixture of sulfonamide (1.0
eq) and excess potassium carbonate in CH3CN. The reaction mixture
was stirred at room temperature overnight, diluted with a NaOH
solution and extracted with CH2Cl2 several times. The combined
organic layer was washed with water, dried over MgSO4 and pu-
rified by silica gel column chromatography. The experiment was
carried out in a fume hood. The aqueous solution was collected and
detoxified with hydrogen peroxide.
4.1.12. General procedure for the coupling reaction with phenyl
chloroformate (Procedure N)
A solution of phenyl chloroformate (1.0 eq) in anhydrous CH2Cl2
was added dropwise to an ice-cooled solution of aniline (1.0 eq) and
14

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
triethyl amine (1.1 eq) in anhydrous CH2Cl2 under nitrogen gas. The
reaction mixture was stirred at room temperature until the initial
material was consumed (approximately 5 h). Then, a solution of
primary amine 148 or 150 (1.1 eq) in anhydrous CH2Cl2 was added
dropwise, followed by the addition of triethylamine (3.0 eq). The
reaction mixture was stirred at room temperature overnight. The
reaction was quenched with water and extracted with CH2Cl2 two
times. The combined organic layers were dried over MgSO4,
concentrated in vacuo, and purified by silica gel column chroma-
tography to obtain the desired product.
desired product by following the procedure P, yield 71%, white
solid, mp 161e162 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.75 (d,
J ¼ 5.49 Hz, 1H), 7.52 (s, 1H), 7.03 (d, J ¼ 8.22 Hz, 1H), 6.65-6.61 (m,
3H), 6.50 (dd, J ¼ 1.47, 5.49 Hz, 1H), 6.42 (s, 1H), 5.83-5.74 (m, 1H),
4.07 (t, J ¼ 5.49 Hz, 2H), 3.87 (t, J ¼ 7.14 Hz, 2H), 3.80 (s, 3H), 3.56 (t,
J ¼ 6.78 Hz, 2H), 2.60 (t, J ¼ 6.96 Hz, 2H), 2.16 (d, J ¼ 0.90 Hz, 3H),
1.97 (quintet, J ¼ 7.14 Hz, 2H), 1.83-1.81 (m, 4H), 1.07 (s, 3H), 1.07 (d,
J ¼ 6.75 Hz, 6H); HRMS (ESI) calc. for C27H39N6O2S [MþH]þ
511.2850, found: 511.2853.
4.1.18. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-cyclobutyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)thiourea
(154)
4.1.13. General procedure for the amide coupling reaction with EDC
(Procedure O)
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC)-HCl
Prepared from compound 55 by following the procedure M to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 85%, white
(1.5 eq) was added to a solution of carboxylic acid (1.0 eq) and
primary amine 152 or 154 (1.0 eq) in anhydrous CH2Cl2 at 0 ꢀC. This
was followed by the addition of hydroxybenzotriazole (HOBt) (1.5
eq). The mixture was warmed to room temperature and stirred
overnight. The reaction was quenched with water and extracted
with CH2Cl2 two times. The combined organic layers were washed
with brine, dried over MgSO4 and concentrated in vacuo. The res-
idue was purified by silica gel column chromatography to obtain
the desired product.
solid, decompose at 70 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.75 (d,
J ¼ 5.31 Hz, 1H), 7.52 (s, 1H), 7.04 (d, J ¼ 8.22 Hz, 1H), 6.66-6.61 (m,
3H), 6.50 (dd, J ¼ 5.49, 1.47 Hz, 1H), 6.46 (s, 1H), 5.64-5.59 (m, 1H),
4.06 (t, J ¼ 5.49 Hz, 2H), 3.86 (t, J ¼ 7.35 Hz, 2H), 3.82 (s, 3H), 3.54 (t,
J ¼ 6.96 Hz, 2H), 2.59 (t, J ¼ 6.93 Hz, 2H), 2.21-2.12 (m, 2H), 2.16 (d,
J ¼ 0.93 Hz, 3H), 1.97-1.88 (m, 2H), 1.83-1.79 (m, 4H) 1.77-1.70 (m,
2H), 1.57-1.45 (m, 2H); HRMS (ESI) calc. for C28H39N6O2S [MþH]þ
523.2850, found: 523.2865.
4.1.14. General procedure for Boc deprotection (Procedure P)
Trifluoroacetic acid (20.0 eq) was added dropwise to a solution
of Boc-protected compound in CH2Cl2 at 0 ꢀC. The mixture was
warmed to room temperature and stirred until the initial material
was consumed; then, the solvent was evaporated. The residue was
dissolved in MeOH and then neutralized by a strongly basic ion-
exchange column to obtain the desired product.
4.1.19. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-cyclopentyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)thiourea
(155)
Prepared from compound 56 by following the procedure M to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 91%, white
solid, mp 125e126 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.89 (d,
4.1.15. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-methyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)thiourea (151)
Prepared from compound 51 by following the procedure M to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 86%, white
J ¼ 5.13 Hz, 1H), 7.27 (s, 1H), 6.85 (d, J ¼ 8.43 Hz, 1H), 6.70 (s, 1H),
6.59 (dd, J ¼ 8.43, 2.37 Hz, 1H), 6.52 (d, J ¼ 2.13 Hz, 1H), 6.50 (dd-
like, J ¼ 5.49 Hz,1H), 6.37 (s,1H), 5.73-5.68 (m, 1H), 5.21 (t-like,1H),
4.63 (brs, 2H), 4.02 (t, J ¼ 5.85 Hz, 2H), 3.81 (s, 3H), 3.78 (t,
J ¼ 7.14 Hz, 2H), 3.57 (brm, 2H), 2.58 (t, J ¼ 6.96 Hz, 2H), 2.12 (d,
J ¼ 0.93 Hz, 3H), 1.96-1.75 (m, 8H), 1.55-1.44 (m, 4H), 1.25-1.15 (m,
2H); HRMS (ESI) calc. for C29H41N6O2S [MþH]þ 537.3006, found:
537.3000.
solid, mp 124e125 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.75 (d,
J ¼ 5.31 Hz, 1H), 7.54 (d, J ¼ 1.11 Hz, 1H), 6.99 (d, J ¼ 8.43 Hz, 1H),
6.81 (d, J ¼ 2.58 Hz, 1H), 6.73 (dd, J ¼ 8.40, 2.73 Hz, 1H), 6.65 (s, 1H),
6.49 (dd, J ¼ 5.31, 1.47 Hz, 1H), 6.45 (s, 1H), 4.04 (t, J ¼ 5.67 Hz, 1H),
3.89 (t, J ¼ 7.32 Hz, 2H), 3.82 (s, 3H), 3.55 (s, 3H), 3.54 (d, J ¼ 6.93 Hz,
2H), 2.58 (t, J ¼ 6.96 Hz, 2H), 2.18 (d, J ¼ 0.90 Hz, 3H), 1.96 (quintet,
J ¼ 7.14 Hz, 2H),1.82-1.80 (m, 4H); HRMS (ESI) calc. for C25H35N6O2S
[MþH]þ 483.2537, found: 483.2547.
4.1.20. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-cyclohexyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)thiourea
(156)
Prepared from compound 57 by following the procedure M to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 75%, white
solid, mp 124e125 ꢀC (white solid, 75%, mp ¼ 88e89 ꢀC): 1H NMR
4.1.16. 3-(3-(1H-Imidazole-1-yl)propyl)-1-(4-(4-(2-aminopyridin-
4-yl)butoxy)-3-methoxyphenyl)-1-ethylthiourea (152)
Prepared from compound 52 by following the procedure M to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 85%, white
(500 MHz, CD3OD)
d
7.75 (d, J ¼ 5.40 Hz, 1H), 7.50 (s, 1H), 7.02 (d,
J ¼ 8.30 Hz, 1H), 6.63-6.60 (m, 3H), 6.50 (d, J ¼ 4.50 Hz, 1H), 6.45 (s,
1H), 5.38 (t, J ¼ 11.70 Hz, NH), 4.06 (t, J ¼ 5.45 Hz, 2H), 3.86 (t,
J ¼ 7.20 Hz, 2H), 3.80 (s, 3H), 3.56 (t, J ¼ 6.65 Hz, 2H), 2.60 (t,
J ¼ 7.15 Hz, 2H), 2.16 (s, 1H), 1.94-1.91 (m, 4H), 1.82-1.79 (m, 4H),
1.76-1.73 (m, 2H), 1.59-1.56 (m, 1H), 1.43-1.35 (m, 2H), 1.05-0.97 (m,
solid, mp 125e126 ꢀC: 1H NMR (500 MHz, CD3OD)
d 7.74 (d,
J ¼ 5.40 Hz, 1H), 7.53 (s, 1H), 7.01 (d, J ¼ 8.45 Hz, 1H), 6.76 (d,
J ¼ 2.20 Hz, 1H), 6.70 (dd, J ¼ 8.32, 2.30 Hz, 1H), 6.64 (s, 1H), 6.50 (d,
J ¼ 5.45 Hz, 1H), 6.45 (s, 1H), 4.20 (q, J ¼ 7.05 Hz, 2H), 4.05 (t,
J ¼ 5.80 Hz, 2H), 3.87 (t, J ¼ 7.35 Hz, 2H), 3.82 (s, 3H), 3.53 (t,
J ¼ 7.30 Hz, 2H), 2.58 (t, J ¼ 7.15 Hz, 2H), 2.18 (s, 3H), 1.95 (quintet,
J ¼ 7.05 Hz, 2H), 1.82-1.79 (m, 4H), 1.15 (t, J ¼ 7.05 Hz, 3H); HRMS
(ESI) calc. for C26H37N6O2S [MþH]þ 497.2693, found: 497.2673.
2H), 0.93-0.89 (m, 1H); 13C NMR (400 MHz, CD3OD)
d 181.65,
159.46, 153.97, 150.35, 149.19, 145.84, 136.48, 133.53, 130.14, 124.96,
122.19, 113.55, 113.50, 113.14, 108.57, 68.55, 59.63, 55.32, 42.11,
34.51, 31.56, 30.01, 28.45, 26.44, 25.66, 25.22, 7.74; HRMS (ESI) calc.
for C30H43N6O2S [MþH]þ 551.3163, found: 551.3171.
4.1.17. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-isopropyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)thiourea
(153)
Prepared from compound 53 by following the procedure M to
afford the penultimate intermediate which was deprotected to the
4.1.21. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (157)
Prepared from compound 15 by following the procedure N to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 92%, white
15

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
solid, mp 55e56 ꢀC: 1H NMR (300 MHz, CD3OD)
d
8.42 (s, 1H), 7.72
4.1.26. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-cyclobutyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (162)
Prepared from compound 55 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 93%, white
(d, J ¼ 6.78 Hz, 1H), 7.12 (d, J ¼ 2.37 Hz, 1H), 7.07 (s, 1H), 6.83 (s,
J ¼ 8.31 Hz, 1H), 6.67 (d, J ¼ 2.40 Hz, 1H), 6.75-6.72 (m, 2H), 4.15 (t,
J ¼ 7.14 Hz, 2H), 3.98 (t, J ¼ 5.67 Hz, 2H), 3.81 (s, 3H), 3.24 (t,
J ¼ 6.75 Hz, 2H), 2.70 (t, J ¼ 7.14 Hz, 2H), 2.32 (d, J ¼ 1.11 Hz, 3H),
2.02 (quintet, J ¼ 6.96 Hz, 2H), 1.90-1.80 (m, 4H); 13C NMR
solid, mp 71e72 ꢀC: 1H NMR (300 MHz, CDCl3)
d
7.88 (d, J ¼ 5.13 Hz,
(400 MHz, CD3OD)
d
159.47, 157.33, 154.00, 149.90, 145.90, 144.19,
1H), 7.24 (s, 1H), 6.79 (d, J ¼ 8.43 Hz, 1H), 6.65 (s, 1H), 6.58 (dd,
J ¼ 8.43, 2.37 Hz, 1H), 6.52 (d, J ¼ 2.19 Hz, 1H), 6.45 (dd-like,
J ¼ 4.05 Hz, 1H), 6.30 (s, 1H), 4.93-4.85 (m,1 H), 4.29 (s, 2H), 4.06 (t,
J ¼ 5.85 Hz, 1H), 3.98 (t, J ¼ 6.06 Hz, 2H), 3.78 (s, 3H), 3.73 (t,
J ¼ 7.32 Hz, 2H), 3.05 (q, J ¼ 6.75 Hz, 2H), 2.53 (t, J ¼ 6.96 Hz, 2H),
2.06 (d, J ¼ 0.72 Hz, 3H), 2.06-2.01 (m, 2H), 1.81-1.65 (m, 8H), 1.53-
1.35 (m, 2H); HRMS (ESI) calc. for C28H39N6O3 [MþH]þ 507.3078,
found: 507.3084.
136.76, 133.52, 127.63, 125.15, 114.57, 113.52, 111.70, 108.53, 105.36,
69.34, 55.06, 41.90, 36.62, 34.49, 31.02, 28.56, 26.40, 7.69; HRMS
(ESI) calc. for C24H33N6O3 [MþH]þ 453.2609, found: 453.2614.
4.1.22. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-methyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (158)
Prepared from compound 51 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 94%, white
4.1.27. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-cyclopentyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (163)
Prepared from compound 56 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 93%, white
solid, mp 54e55 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.65 (d,
J ¼ 5.49 Hz, 1H), 7.44 (d, J ¼ 1.11 Hz, 1H), 6.86 (d, J ¼ 8.43 Hz, 1H),
6.76 (d, J ¼ 2.37 Hz, 1H), 6.67 (dd, J ¼ 8.40, 2.37 Hz, 1H), 6.55 (s, 1H),
6.39 (dd, J ¼ 5.49, 1.47 Hz, 1H), 6.35 (s, 1H), 3.92 (t, J ¼ 5.49 Hz, 2H),
3.80 (t, J ¼ 7.14 Hz, 2H), 3.72 (s, 3H), 3.08 (s, 3H), 3.03 (t, J ¼ 6.57 Hz,
2H), 2.47 (t, J ¼ 6.96 Hz, 1H), 2.08 (d, J ¼ 0.9 Hz, 3H), 1.82-1.69 (m,
6H); HRMS (ESI) calc. for C25H35N6O3 [MþH]þ 467.2765, found:
467.2761.
solid, mp 69e70 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.66 (d,
J ¼ 5.13 Hz, 1H), 7.35 (s, 1H), 6.88 (d, J ¼ 8.25 Hz, 1H), 6.63-6.59 (m,
2H), 6.55 (s, 1H), 6.40 (d, J ¼ 5.13 Hz, H0, 6.36 (s, 1H), 4.58 (quintet,
J ¼ 8.04 Hz, 1H), 3.95 (t-like, 2H), 3.77 (t, J ¼ 7.14 Hz, 2H), 3.72 (s,
3H), 3.01 (t, J ¼ 6.42 Hz, 2H), 2.49 (t-like, 2H), 2.07 (s, 3H), 1.80-1.65
(m, 8H), 1.45-1.38 (m, 4H), 1.18e1.23 (m, 2H); HRMS (ESI) calc. for
4.1.23. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-ethyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (159)
Prepared from compound 52 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 93%, white
C
29H41N6O3 [MþH]þ 521.3235, found: 521.3234.
4.1.28. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-cyclohexyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (164)
Prepared from compound 57 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 89%, white
solid, mp 51e52 ꢀC: 1H NMR (500 MHz, CD3OD)
d 7.75 (d,
J ¼ 5.45 Hz, 1H), 7.52 (s, 1H), 6.97 (d, J ¼ 8.50 Hz, 1H), 6.80 (d,
J ¼ 2.25 Hz, 1H), 6.75 (dd, J ¼ 8.40, 2.30 Hz, 1H), 6.64 (s, 1H), 6.49
(dd, J ¼ 5.30, 0.75 Hz, 1H), 6.45 (s, 1H), 4.04 (t, J ¼ 5.35 Hz, 2H), 3.88
(t, J ¼ 7.25 Hz, 2H), 3.82 (s, 3H), 3.63 (q, J ¼ 7.05 Hz, 2H), 3.12 (t,
J ¼ 6.70 Hz, 2H), 2.58 (t, J ¼ 7.00 Hz, 2H), 2.18 (s, 3H), 1.87-1.79 (m,
6H), 1.08 (t, J ¼ 7.10 Hz, 3H); HRMS (ESI) calc. for C26H37N6O3
[MþH]þ 481.2922, found: 481.2927.
solid, mp 68e69 ꢀC: 1H NMR (500 MHz, CD3OD)
d 7.75 (d,
J ¼ 5.35 Hz, 1H), 7.51 (s, 1H), 6.98 (d, J ¼ 8.35 Hz, 1H), 6.72 (d,
J ¼ 2.05 Hz, 1H), 6.70 (dd, J ¼ 8.30, 2.15 Hz, 1H), 6.64 (s, 1H), 6.49 (d,
J ¼ 5.15 Hz, 1H), 6.45 (s, 1H), 4.67 (quintet, J ¼ 7.50 Hz, 1H), 4.04 (t,
J ¼ 5.70 Hz, 2H), 3.86 (t, J ¼ 7.20 Hz, 2H), 3.81 (s, 3H), 3.11 (t,
J ¼ 6.70 Hz, 2H), 2.58 (t, J ¼ 7.10 Hz, 2H), 2.16 (s, 3H), 1.85-1.82 (m,
9H), 1.55-1.47 (m, 4H), 1.34-1.28 (m, 3H); HRMS (ESI) calc. for
4.1.24. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-isopropyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (160)
Prepared from compound 53 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 82%, white
C
30H43N6O3 [MþH]þ 535.3391, found: 535.3382.
4.1.29. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-(cyclopentylmethyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)
urea (165)
solid, decompose at 69 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.75 (d,
J ¼ 6.06 Hz, 1H), 7.52 (s, 1H), 6.98 (d, J ¼ 8.22 Hz, 1H), 6.72-6.68 (m,
2H), 6.64 (s, 1H), 6.50 (d, J ¼ 5.49 Hz, 1H), 6.46 (s, 1H), 4.71 (hept,
J ¼ 6.78 Hz, 1H), 4.05 (t-like, 2H), 3.86 (t, J ¼ 6.96 Hz, 2H), 3.82 (s,
3H), 3.10 (t, J ¼ 6.78 Hz, 2H), 2.59 (t-like, 2H), 2.17 (d, J ¼ 0.90 Hz,
3H), 1.83-1.78 (m, 6H), 1.04 (d, J ¼ 6.78 Hz, 6H); HRMS (ESI) calc. for
Prepared from compound 58 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 78%, white
solid, mp 98e99 ꢀC: 1H NMR (300 Mhz, CD3OD)
d 7.75 (d,
J ¼ 5.31 Hz, 1H), 7.51 (s, 1H), 6.99 (d, J ¼ 8.43 Hz, 1H), 6.81 (d,
J ¼ 2.40 Hz, 1H), 6.78 (dd, J ¼ 2.37, 8.25 Hz, 1H), 6.64 (s, 1H), 6.50
(dd, J ¼ 1.44, 5.49 Hz, 1H), 6.44 (s, 1H), 4.05 (t, J ¼ 5.70 Hz, 2H), 3.89
(t, J ¼ 7.14 Hz, 2H), 3.81 (s, 3H), 3.57 (d, J ¼ 7.71 Hz, 2H), 3.13 (t,
J ¼ 6.78 Hz, 2H), 2.60 (t, J ¼ 6.96 Hz, 2H), 2.17 (d, J ¼ 0.93 Hz, 3H),
2.02-1.97 (m, 1H), 1.87-1.78 (m, 6H), 1.67-1.60 (m, 4H), 1.56-1.48 (m,
2H), 1.27-1.20 (m, 2H); HRMS (ESI) calc. for C30H43N6O3 [MþH]þ
535.3391, found: 535.3403.
C
27H39N6O3 [MþH]þ 495.3078, found: 495.3062.
4.1.25. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-cyclopropyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (161)
Prepared from compound 54 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 79%, white
solid, mp 65e66 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.75 (d,
J ¼ 5.31 Hz, 1H), 7.58 (s, 1H), 6.91 (d, J ¼ 8.40 Hz, 1H), 6.76 (d,
J ¼ 2.40 Hz, 1H), 6.68-6.65 (m, 2H), 6.49 (d, J ¼ 5.49 Hz, 1H), 6.44 (s,
1H), 4.01-3.93 (m, 4H), 3.81 (s, 3H), 3.23 (t, J ¼ 6.78 Hz, 2H), 2.93-
2.89 (m, 1H), 2.58 (t-like, 2H), 2.21 (d, J ¼ 1.08 Hz, 3H), 1.93 (quintet,
J ¼ 6.78 Hz, 2H), 1.85-1.75 (m, 4H), 0.83-0.78 (m, 2H), 0.54-0.50 (m,
2H); HRMS (ESI) calc. for C27H37N6O3 [MþH]þ 493.2922, found:
493.2915.
4.1.30. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
3-(3-(5-methyl-1H-imidazole-1-yl)propyl)-1-(1-methylpiperidin-4-
yl)urea (166)
Prepared from compound 60 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 89%, white
solid, mp 67e68 ꢀC: 1H NMR (300 MHz, CDCl3)
d
8.87 (d, J ¼ 5.13 Hz,
16

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
1H), 7.24 (s, 1H), 6.74 (d, J ¼ 8.43 Hz, 1H), 6.65 (s, 1H), 6.56 (dd,
J ¼ 8.73, 2.40 Hz, 1H), 6.51 (d, J ¼ 2.19 Hz, 1H), 6.45 (dd, J ¼ 5.31,
1.29 Hz, 1H), 6.29 (s, 1H), 4.40-4.32 (m, 3H), 4.06 (t, J ¼ 5.85 Hz, 1H),
3.95 (t, J ¼ 6.03 Hz, 2H), 3.76 (s, 3H), 3.73 (t, J ¼ 8.76 Hz, 2H), 3.08 (q,
J ¼ 6.60 Hz, 2H), 2.75 (d, J ¼ 11.52 Hz, 2H), 2.51 (t, J ¼ 7.32 Hz, 2H),
2.15 (s, 3H), 2.06 (d, J ¼ 0.93 Hz, 3H), 2.06-1.96 (m, 2H),1.80-1.70 (m,
8H), 1.34 (qd, J ¼ 11.73, 3.66 Hz, 2H); 13C NMR (400 MHz, CD3OD)
J ¼ 7.22 Hz, 2H), 3.18 (t, J ¼ 6.56 Hz, 2H), 2.56 (brt, 2H), 2.11 (s, 3H),
1.84 (quintet, J ¼ 7.16 Hz, 2H), 1.78 (br, 4H), 1.51 (s, 6H); HRMS (FAB)
calc. for C27H38N5O3 [MþH]þ 480.2975, found: 480.2971.
4.1.35. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
N-(3-(5-methyl-1H-imidazole-1-yl)propyl)
cyclopropanecarboxamide (171)
d
159.61, 158.35, 153.74, 150.10, 149.04, 146.16, 136.59, 129.88,
Prepared from compound 144 by following the procedure O to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 85%, white
127.45, 125.15, 123.32, 114.65, 113.48, 113.14, 108.50, 68.61, 55.27,
54.84, 52.38, 44.62, 42.01, 37.52, 34.47, 31.03, 30.43, 28.43, 26.45,
7.69; HRMS (FAB) calc. for C30H44N7O3 [MþH]þ 550.3506, found:
550.3512.
solid, mp 54e55 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.65 (d,
J ¼ 5.49 Hz, 1H), 7.41 (s, 1H), 6.87-6.82 (m, 3H), 6.55 (s, 1H), 6.39 (d,
J ¼ 5.31 Hz, J ¼ 5.31 Hz, 1H), 6.35 (s, 1H), 3.92 (brt, 2H), 3.75 (t,
J ¼ 7.50 Hz, 2H), 3.73 (s, 3H), 3.05 (t, J ¼ 6.78 Hz. 2H), 2.48 (brt, 2H),
2.06 (s, 3H), 1.75-1.70 (m, 6H), 1.35 (dd, 2H), 0.94 (dd, 2H); HRMS
(ESI) calc. for C27H36N5O3 [MþH]þ 478.2813, found: 478.2825.
4.1.31. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
3-(3-(5-methyl-1H-imidazole-1-yl)propyl)-1-phenylurea (167)
Prepared from compound 23 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 94%, white
4.1.36. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
N-(3-(5-methyl-1H-imidazole-1-yl)propyl)
solid, mp 89e90 ꢀC: 1H NMR (400 MHz, CD3OD)
d 7.74 (d,
J ¼ 5.48 Hz, 1H), 7.54 (s, 1H), 7.35-7.18 (m, 5H), 6.92 (d, J ¼ 8.56 Hz,
1H), 6.87 (d, J ¼ 2.28 Hz, 1H), 6.77 (dd, J ¼ 8.44, 2.36 Hz, 1H), 6.67 (s,
1H), 6.49 (d, J ¼ 5.15 Hz, 1H), 6.44 (s, 1H), 4.01 (t, J ¼ 7.52 Hz, 2H),
3.92 (t, J ¼ 6.48 Hz, 2H), 3.78 (s, 3H), 3.18 (t, J ¼ 6.56 Hz, 2H), 2.56 (t,
J ¼ 7.00 Hz, 2H), 2.20 (s, 3H), 2.02-1.90 (m, 2H), 1.89-1.79 (m, 4H);
cyclobutanecarboxamide (172)
Prepared from compound 145 by following the procedure O to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 92%, white
solid, mp 51e52 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.74 (d,
HRMS (ESI) calc. for
529.2955.
C
30H37N6O3 [MþH]þ 529.2922, found:
J ¼ 5.31 Hz, 1H), 7.75 (d, J ¼ 0.90 Hz, 1H), 6.94-6.88 (m, 3H), 6.60 (s,
1H), 6.47 (dd, J ¼ 5.28, 1.26 Hz, 1H), 6.43 (s, 1H), 3.98 (t-like, 2H),
3.81 (s, 3H), 3.66 (7.14 Hz, 2H), 3.17 (t, J ¼ 6.42 Hz, 2H), 2.76-2.68 (m,
2H), 2.54-2.42 (m, 4H), 2.01 (d, J ¼ 0.90 Hz, 3H), 1.90-1.77 (m, 8H);
4.1.32. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-benzyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (168)
Prepared from compound 61 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 88%, white
HRMS (FAB) calc. for
492.2970.
C
28H38N5O3 [MþH]þ 492.2975, found:
4.1.37. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
N-(3-(5-methyl-1H-imidazole-1-yl)propyl)
solid, mp 81e82 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.73 (d,
J ¼ 5.13 Hz, 1H), 7.53 (s, 1H), 7.29-7.18 (m, 5H), 6.85 (d, J ¼ 8.22 Hz,
1H), 6.65 (s, 1H), 6.61-6.56 (m, 2H), 6.47 (dd, J ¼ 5.49, 1.47 Hz, 1H),
6.43 (s, 1H), 4.78 (s, 2H), 3.95 (t, J ¼ 5.67 Hz, 2H), 3.89 (t, J ¼ 7.32 Hz,
2H), 3.16 (t, J ¼ 6.57 Hz, 2H), 2.55 (t, J ¼ 6.78 Hz, 2H), 2.17 (d,
J ¼ 0.90 Hz, 3H), 1.86 (quintet, J ¼ 7.14 Hz, 2H), 1.81-1.76 (m, 4H);
cyclopentanecarboxamide (173)
Prepared from compound 146 by following the procedure O to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 95%, white
solid, mp 61e62 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.74 (d,
HRMS (ESI) calc. for
543.3077.
C
31H39N6O3 [MþH]þ 543.3078, found:
J ¼ 5.28 Hz, 1H), 7.37 (s, 1H), 6.95-6.86 (m, 3H), 6.61 (s, 1H, 6.47 (d,
J ¼ 5.31 Hz, 1H), 6.43 (s, 1H), 3.97 (t-like, 2H), 3.80 (s, 3H), 3.67 (t,
J ¼ 7.32 Hz, 2H), 3.15 (t, J ¼ 6.57 Hz, 2H), 2.55-2.45 (m, 4H), 2.03 (d,
J ¼ 0.72 Hz, 3H), 2.00-1.94 (m, 2H), 1.83-1.70 (m, 10H); HRMS (FAB)
calc. for C29H40N5O3 [MþH]þ 506.3131, found: 506.3132.
4.1.33. 1-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
1-(4-fluorobenzyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea
(169)
Prepared from compound 62 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 73%, white
4.1.38. 1-(4-(2-(Dimethylamino)ethoxy)-3-methoxyphenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)urea (174)
From compound 70, procedure N, yield 60%, white solid, mp
solid, mp 85e86 ꢀC: 1H NMR (300 MHz, CDCl3)
d
7.75 (d, J ¼ 5.13 Hz,
85e86 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.66 (s, 1H), 7.36 (s, 1H), 7.15
1H), 7.54 (s, 1H), 7.23 (dd, J ¼ 5.31 Hz, 2H), 7.01 (t, J ¼ 8.79 Hz, 2H),
6.88 (d, J ¼ 8.43 Hz, 1H), 6.65 (s, 1H), 6.60-6.54 (m, 2H), 6.49 (dd,
J ¼ 1.47, 5.49 Hz, 1H), 6.44 (s, 1H), 5.61 (t, J ¼ 6.96 Hz, NH), 4.76 (s,
2H), 3.98 (t, J ¼ 6.96 Hz, 2H), 3.91 (t, J ¼ 7.32 Hz, 2H), 3.69 (s, 3H),
3.17 (q, J ¼ 6.60 Hz, 2H), 2.56 (t, J ¼ 7.14 Hz, 2H), 3.17 (s, 3H), 1.90
(quintet, J ¼ 6.60 Hz, 2H), 1.79-1.69 (m, 4H); HRMS (ESI) calc. for
(d, J ¼ 2.4 Hz, 1H), 6.75 (d, J ¼ 8.4 Hz, 1H), 6.72 (s, 1H), 6.60 (dd,
J ¼ 2.4, 8.4 Hz, 1H), 5.80 (br, 1H), 4.02 (t, J ¼ 6.0 Hz, 2H), 3.88 (t,
J ¼ 7.0 Hz, 2H), 3.78 (s, 3H), 3.19-3.13 (m, 2H), 2.70 (t, J ¼ 6.0 Hz, 2H),
2.29 (s, 6H), 2.15 (s, 3H), 1.86 (m, 2H); 13C NMR (400 MHz, CD3OD)
d
157.35, 150.20, 143.90, 137.94, 134.05, 128.15, 125.90, 115.33,
111.53, 105.08, 67.68, 57.84, 54.86, 44.61, 44.86, 36.71, 31.04, 7.79;
C
31H38FN6O3 [MþH]þ 561.2984, found: 561.2983.
HRMS (ESI) calc. for
376.2341.
C
19H30N5O3 [MþH]þ 376.2343, found:
4.1.34. 2-(4-(4-(2-Aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-
2-methyl-N-(3-(5-methyl-1H-imidazole-1-yl)propyl)propanamide
(170)
4.1.39. 1-(4-(3-(Dimethylamino)propoxy)-3-methoxyphenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)urea (175)
Prepared from compound 143 by following the procedure O to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 91%, white
From compound 71, procedure N, yield 67%, white solid, mp
96e97 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.36 (s, 1H), 7.00 (d,
J ¼ 2.4 Hz, 1H), 6.96 (s, 1H), 6.74 (d, J ¼ 8.4 Hz, 1H), 6.70 (s, 1H), 6.58
(dd, J ¼ 2.4, 8.4 Hz, 1H), 5.28 (br, 1H), 3.96 (t, J ¼ 6.6 Hz, 2H), 3.85 (t,
J ¼ 7.2 Hz, 2H), 3.77 (s, 3H), 3.16-3.14 (m, 2H), 2.39 (t, J ¼ 7.4 Hz, 2H),
2.18 (s, 6H), 2.12 (s, 3H), 1.96-1.85 (m, 4H); 13C NMR (400 MHz,
solid, mp 58e59 ꢀC: 1H NMR (400 MHz, CD3OD)
d 7.74 (d,
J ¼ 5.32 Hz, 1H), 7.47 (s, 1H), 6.91-6.89 (m, 3H), 6.64 (s, 1H), 6.48 (d,
J ¼ 5.28 Hz, 1H), 6.44 (s, 1H), 3.99 (brt, 2H), 3.79 (s, 3H), 3.78 (t,
17

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
CD3OD)
d
157.33, 149.96, 143.95, 136.56, 133.79, 127.66, 125.14,
2H), 2.87-2.77 (m, 4H), 2.35 (s, 6H), 2.18 (s, 3H), 2.17 (s, 3H), 2.13-
2.07 (m, 2H), 1.88-1.79 (m, 4H), 1.43-1.41 (m, 2H); HRMS (ESI) calc.
for C25H41N6O3 [MþH]þ 473.3235, found: 473.3245.
114.74, 111.56, 105.19, 67.81, 56.15, 54.45, 43.95, 41.91, 36.62, 31.03,
26.70, 7.59; HRMS (ESI) calc. for C20H32N5O3 [MþH]þ 390.2500,
found: 390.2502.
4.1.45. 1-(4-(3-(Dimethylamino)propoxy)-3-methoxyphenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)-1-(1-methylpiperidin-4-yl)
urea (181)
4.1.40. 1-(4-(4-(Dimethylamino)butoxy)-3-methoxyphenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)urea (176)
From compound 72, procedure N, yield 57%, white solid, mp
101e102 ꢀC: 1H NMR (300 MHz, CDCl3),
d 7.41 (br, NH), 7.33 (s, 1H),
From compound 78, procedure L, yield 72%, white solid, mp
100e101 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.51 (s, 1H), 7.01 (d,
7.06 (d, J ¼ 2.22 Hz, 1H), 6.71-6.68 (m, 2H), 6.59 (dd, J ¼ 2.19,
8.25 Hz, 1H), 5.60 (t, J ¼ 4.59 Hz, NH), 3.92 (t, J ¼ 6.42 Hz, 2H), 3.85
(t, J ¼ 6.78 Hz, 2H), 3.74 (s, 3H), 3.14 (q, J ¼ 5.68 Hz, 2H), 2.27 (t,
J ¼ 7.32 Hz, 2H), 2.15 (s, 6H), 2.11 (s, 3H), 1.87 (quintet, J ¼ 6.78 Hz,
2H), 1.76-1.69 (quintet, J ¼ 6.78 Hz, 2H), 1.60 (quintet, J ¼ 6.96 Hz,
J ¼ 8.22 Hz, 1H), 6.72-6.69 (m, 2H), 6.64 (s, 1H), 4.31-4.29 (m, 1H),
4.07 (t, J ¼ 6.06 Hz, 2H), 3.88 (t, J ¼ 7.32 Hz, 2H), 3.77 (s, 3H), 3.13 (t,
J ¼ 6.57 Hz, 2H), 2.87 (d, J ¼ 11.52 Hz, 2H), 2.55 (t, J ¼ 7.14 Hz, 2H),
2.27 (s, 6H), 2.18 (s, 3H), 2.17 (d, J ¼ 0.93 Hz, 3H), 2.12-2.07 (m, 2H),
1.98 (quintet, J ¼ 6.03 Hz, 2H), 1.84-1.79 (m, 4H), 1.44-1.31 (m, 2H);
2H); 13C NMR (400 MHz, CD3OD)
d 157.29, 149.91, 144.20, 136.67,
HRMS (ESI) calc. for
487.3397.
C
26H43N6O3 [MþH]þ 487.3391, found:
133.61, 127.55, 125.25, 114.46, 111.67, 105.34, 69.32, 58.88, 55.05,
43.77, 41.82, 36.59, 31.02, 26.92, 23.43, 7.70; HRMS (ESI) calc. for
C
21H34N5O3 [MþH]þ 404.2656, found: 404.2652.
4.1.46. 1-(4-(4-(Dimethylamino)butoxy)-3-methoxyphenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)-1-(1-methylpiperidin-4-yl)
urea (182)
4.1.41. 1-Cyclohexyl-1-(4-(2-(dimethylamino)ethoxy)-3-
methoxyphenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea
(177)
From compound 82, procedure L, yield 63%, white solid, mp
From compound 73, procedure L, yield 73%, white solid, mp
97e98 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.30 (s, 1H), 6.82 (d,
111e112 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.25 (s, 1H), 6.83 (d,
J ¼ 8.4 Hz, 1H), 6.70 (s, 1H), 6.62 (dd, J ¼ 2.4, 8.4 Hz, 1H), 6.56 (d,
J ¼ 2.4 Hz,1H), 4.40 (m,1H), 4.06 (t, J ¼ 6.0Hz,1H), 4.02 (t, J ¼ 6.8 Hz,
2H), 3.80 (s, 3H), 3.77 (t, J ¼ 7.2 Hz, 2H), 3.14 (q, J ¼ 6.8 Hz, 2H), 2.81-
2.78 (m, 2H), 2.33-2.31 (m, 2H), 2.21 (s, 6H), 2.19 (s, 3H), 2.05 (m,
2H),1.88-1.80 (m, 4H),1.78-1.74 (m, 2H),1.67-1.58 (m, 2H),1.45-1.35
(m, 2H); HRMS (ESI) calc. for C27H45N6O3 [MþH]þ 501.3548, found:
501.3550.
J ¼ 8.25 Hz, 1H), 6.64 (s, 1H), 6.60 (dd, J ¼ 2.37, 8.43 Hz, 1H), 6.52 (d,
J ¼ 2.19 Hz, 1H), 4.35-4.27 (m, 1H), 4.05 (t, J ¼ 6.60 Hz, 2H), 3.99 (t,
J ¼ 6.06 Hz, NH), 3.77 (s, 3H), 3.75 (t, J ¼ 7.32 Hz, 2H), 3.11 (q,
J ¼ 6.42 Hz, 2H), 2.43 (t, J ¼ 6.96 Hz, 2H), 2.19 (s, 6H), 2.12 (d,
J ¼ 0.72 Hz, 3H), 2.01 (quintet, J ¼ 6.78 Hz, 2H), 1.81 (quintet,
J ¼ 6.93 Hz, 2H), 1.67-1.63 (m, 2H), 1.51-1.47 (m, 1H), 1.37e1.24 (m,
2H), 1.01-0.80 (m, 3H); HRMS (ESI) calc. for C25H40N5O3 [MþH]þ
458.3126, found: 458.3129.
4.1.47. 1-(4-(2-(Dimethylamino)ethoxy)-3-methoxyphenyl)-1-(4-
fluorobenzyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (183)
From compound 75, procedure L, yield 68%, white solid, mp
4.1.42. 1-Cyclohexyl-1-(4-(3-(dimethylamino)propoxy)-3-
methoxyphenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea
(178)
95e96 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.27 (s, 1H), 7.13-7.09 (m,
From compound 77, procedure L, yield 71%, white solid, mp
2H), 6.90 (t, J ¼ 8.79 Hz, 2H), 6.75 (d, J ¼ 8.40 Hz, 1H), 6.64 (s, 1H),
6.48 (dd, J ¼ 2.19, 8.43 Hz, 1H), 6.34 (d, J ¼ 2.40 Hz, 1H), 4.68 (s, 2H),
4.27 (t, J ¼ 5.67 Hz, NH), 4.03 (t, J ¼ 6.06 Hz, 2H), 3.81 (t, J ¼ 8.61 Hz,
2H), 3.65 (s, 3H), 3.17 (q, J ¼ 6.60 Hz, 2H), 2.73 (t, J ¼ 5.85 Hz, 2H),
2.27 (s, 6H), 2.07 (s, 3H), 1.85 (quintet, J ¼ 7.14 Hz, 2H); HRMS (ESI)
calc. for C26H35FN5O3 [MþH]þ 484.2718, found: 484.2722.
123e124 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.25 (s, 1H), 6.83 (d,
J ¼ 8.25 Hz, 1H), 6.64 (s, 1H), 6.60 (dd, J ¼ 2.37, 8.43 Hz, 1H), 6.52 (d,
J ¼ 2.18 Hz, 1H), 4.35-4.27 (m, 1H), 4.05 (t, J ¼ 6.60 Hz, 2H), 3.98 (t,
J ¼ 5.85 Hz, NH), 3.77 (s, 3H), 3.73 (t, J ¼ 7.14 Hz, 2H), 3.11 (q,
J ¼ 6.39 Hz, 2H), 2.42 (t, J ¼ 6.96 Hz, 2H), 2.18 (s, 6H), 2.07 (s, 3H),
2.00 (quintet, J ¼ 6.60 Hz, 2H), 1.81-1.74 (m, 4H), 1.67-1.63 (m, 2H),
1.51-1.47 (m, 1H), 1.33-1.25 (m, 2H), 1.02-0.97 (m, 2H), 0.90-0.80 (m,
1H); HRMS (ESI) calc. for C26H42N5O3 [MþH]þ 472.3282, found:
472.3294.
4.1.48. 1-(4-(3-(Dimethylamino)propoxy)-3-methoxyphenyl)-1-(4-
fluorobenzyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (184)
From compound 79, procedure L, yield 75%, white solid, mp
78e79 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.34 (s, 1H), 7.20-7.15 (m,
4.1.43. 1-Cyclohexyl-1-(4-(4-(dimethylamino)butoxy)-3-
methoxyphenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea
(179)
2H), 6.96-6.90 (m, 2H), 6.81 (d, J ¼ 8.4 Hz, 1H), 6.71 (s, 1H), 6.52 (dd,
J ¼ 2.4, 8.4 Hz, 1H), 6.41 (d, J ¼ 2.4 Hz, 1H), 4.74 (s, 2H), 4.31 (t,
J ¼ 5.7 Hz,1H), 4.04 (t, J ¼ 6.8 Hz, 2H), 3.84 (t, J ¼ 8.8 Hz, 2H), 3.70 (s,
3H), 3.23-3.16 (m, 2H), 2.46 (t, J ¼ 7.2 Hz, 2H), 2.25 (s, 6H), 2.13 (s,
3H), 2.02-1.95 (m, 2H), 1.91-1.82 (m, 2H); HRMS (ESI) calc. for
From compound 81, procedure L, yield 69%, white solid,
154e155 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.51 (s, 1H), 6.99 (d,
J ¼ 8.4 Hz, 1H), 6.70-6.63 (m, 3H), 4.31-4.23 (m, 1H), 4.05 (t,
J ¼ 6.2 Hz, 2H), 3.88-3.83 (m, 2H), 3.81 (s, 3H), 3.10 (t, J ¼ 6.6 Hz,
2H), 2.42-2.37 (m, 2H), 2.24 (s, 6H), 2.17 (s, 3H), 1.83-1.69 (m, 10H),
1.59-1.52 (m, 1H), 1.39-1.35 (m, 2H), 1.12-0.90 (m, 3H); HRMS (FAB)
calc. for C27H44N5O3 [MþH]þ 486.3444, found: 486.3441.
C
27H37FN5O3 [MþH]þ 498.2875, found: 498.2885.
4.1.49. 1-(4-(4-(Dimethylamino)butoxy)-3-methoxyphenyl)-1-(4-
fluorobenzyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (185)
From compound 83, procedure L, yield 63%, white solid, mp
4.1.44. 1-(4-(2-(Dimethylamino)ethoxy)-3-methoxyphenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)-1-(1-methylpiperidin-4-yl)
urea (180)
86e87 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.53 (s, 1H), 7.23-7.18 (m,
2H), 7.01-6.95 (m, 2H), 6.89 (d, J ¼ 8.3Hz, 1H), 6.65 (s, 1H), 6.60-6.55
(m, 2H), 4.76 (s, 2H), 3.98 (t, J ¼ 5.9 Hz, 2H), 3.89 (t, J ¼ 7.2 Hz, 2H),
3.69 (s, 3H), 3.16 (t, J ¼ 6.8 Hz, 2H), 2.41-2.35 (m, 2H), 2.23 (s, 6H),
2.17 (s, 3H), 1.91-1.61 (m, 6H); HRMS (ESI) calc. for C28H39FN5O3
[MþH]þ 512.3031, found: 512.3042.
From compound 74, procedure L, yield 58%, white solid, mp
110e111 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.51 (s, 1H), 7.03 (d,
J ¼ 8.25 Hz, 1H), 6.74-6.60 (m, 2H), 6.64 (s, 1H), 4.31 (m, 1H), 4.13 (t,
J ¼ 5.5 Hz, 2H), 3.89-3.84 (m, 2H), 3.81 (s, 3H), 3.11 (t, J ¼ 6.8 Hz,
18

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
4.1.50. 1-(4-(2-(Dimethylamino)ethoxy)-3-methoxyphenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)-1-(pyridin-3-ylmethyl)urea
(186)
2.06 (s, 3H), 1.81-1.74 (m, 4H), 1.65 (d, J ¼ 13.20 Hz, 2H), 1.49 (d,
J ¼ 12.45 Hz, 1H), 1.37-1.24 (m, 2H), 1.01-0.88 (m, 2H), 0.84-0.75 (m,
1H); HRMS (FAB) calc. for C28H39N6O3 [MþH]þ 507.3084, found:
507.3084.
From compound 76, procedure L, yield 70%, white solid, mp
91e92 ꢀC: 1H NMR (300 MHz, CD3OD)
d
8.48 (dd, J ¼ 2.0, 5.0 Hz,1H),
8.40 (d, J ¼ 2.0 Hz, 1H), 7.63 (d, J ¼ 7.8 Hz, 1H), 7.34 (s, 1H),
7.23e7.7.19 (m, 1H), 6.75 (d, J ¼ 8.4 Hz, 1H), 6.71 (s, 1H), 6.53 (dd,
J ¼ 2.4, 8.4 Hz, 1H), 6.44 (d, J ¼ 2.4 Hz, 1H), 4.80 (s, 2H), 4.34 (t,
J ¼ 5.9 Hz, 2H), 3.82 (t, J ¼ 7.0 Hz, 2H), 3.71 (s, 3H), 3.23-3.16 (m, 2H),
2.95 (t, J ¼ 5.7 Hz, 2H), 2.33 (s, 6H), 2.13 (s, 3H), 1.91-1.82 (m, 2H);
4.1.55. 1-(4-(2-(2-Aminopyridin-4-yl)ethoxy)-3-methoxyphenyl)-
3-(3-(5-methyl-1H-imidazole-1-yl)propyl)-1-(1-methylpiperidin-4-
yl)urea (191)
Prepared from compound 17 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 89%, white
HRMS (ESI) calc. for
467.2753.
C
25H35N6O3 [MþH]þ 467.2765, found:
solid, mp 85e86 ꢀC: 1H NMR (300 MHz, CDCl3)
d
7.98 (d, J ¼ 5.1 Hz,
1H), 7.29 (s, 1H), 6.82 (d, J ¼ 8.4 Hz, 1H), 6.70 (s, 1H), 6.64-6.57 (m,
3H), 6.44 (s, 1H), 4.44-4.37 (m, 3H), 4.20 (t, J ¼ 7.0 Hz, 2H), 4.06 (t,
J ¼ 6.0 Hz,1H), 3.81 (s, 3H), 3.79 (t, J ¼ 7.3 Hz, 2H), 3.13 (q, J ¼ 6.6 Hz,
2H), 3.02 (t, J ¼ 6.8 Hz, 2H), 2.85-2.79 (m, 2H), 2.20 (s, 3H), 2.12 (s,
3H), 2.11-2.02 (m, 2H), 1.85-1.75 (m, 4H), 1.46-1.35 (m, 2H); 13C
4.1.51. 1-(4-(3-(Dimethylamino)propoxy)-3-methoxyphenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)-1-(pyridin-3-ylmethyl)urea
(187)
From compound 80, procedure L, yield 56%, white solid, mp
87e88 ꢀC: 1H NMR (300 MHz, CD3OD)
d
8.41 (dd, J ¼ 1.30, 4.75 Hz,
NMR (400 MHz, CD3OD) d 159.58, 158.32, 150.26, 150.13, 148.75,
1H), 8.35 (d, J ¼ 1.45 Hz, 1H), 7.74 (d, J ¼ 7.90 Hz, 1H), 7.53 (s, 1H),
7.38 (dd, J ¼ 4.90 Hz, 1H), 6.92 (d, J ¼ 8.45 Hz, 1H), 6.66 (dd, J ¼ 2.35,
8.15 Hz,1H), 6.64 (s,1H), 6.60 (dd, J ¼ 2.35, 8.50 Hz,1H), 4.81 (s, 2H),
4.02 (t, J ¼ 6.10 Hz, 2H), 3.91 (t, J ¼ 7.20 Hz, 2H), 3.72 (s, 3H), 3.16 (t,
J ¼ 6.55 Hz, 2H), 2.52 (t, J ¼ 7.45 Hz, 2H), 2.25 (s, 6H), 2.17 (d,
J ¼ 0.60 Hz, 3H), 1.98 (quintet, J ¼ 6.25 Hz, 2H), 1.89 (quintet,
J ¼ 6.75 Hz, 2H); HRMS (ESI) calc. for C26H37N6O3 [MþH]þ 481.2922,
found: 481.2928.
146.25, 136.58, 130.23, 127.46, 125.17, 123.36, 114.96, 113.76, 113.65,
109.18, 68.57, 55.44, 54.81, 52.49, 44.63, 41.95, 37.52, 34.72, 31.04,
30.40, 7.74; HRMS (FAB) calc. for C28H40N7O3 [MþH]þ 522.3193,
found: 522.3198.
4.1.56. 1-(4-(2-(2-Aminopyridin-4-yl)ethoxy)-3-methoxyphenyl)-
1-(4-fluorobenzyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea
(192)
Prepared from compound 18 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 88%, white
4.1.52. 1-(4-(4-(Dimethylamino)butoxy)-3-methoxyphenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)-1-(pyridin-3-ylmethyl)urea
(188)
solid, mp 70e71 ꢀC: 1H NMR (300 MHz, CDCl3)
d
7.97 (d, J ¼ 5.3 Hz,
From compound 84, procedure L, yield 76%, white solid, mp
1H), 7.30 (s, 1H), 7.19-7.15 (m, 2H), 6.96-6.90 (m, 2H), 6.75 (d,
J ¼ 8.4 Hz, 1H), 6.70 (s, 1H), 6.57 (dd, J ¼ 1.1, 5.4 Hz, 1H), 6.51 (dd,
J ¼ 2.4, 8.4 Hz,1H), 6.43-6.41 (m, 2H), 4.82 (s, 2H), 4.42 (br, 2H), 4.28
(t, J ¼ 5.9 Hz, 1H), 4.16 (t, J ¼ 7.0 Hz, 2H), 3.81 (t, J ¼ 7.3 Hz, 2H), 3.69
(s, 3H), 3.19 (q, J ¼ 6.6 Hz, 2H), 3.00 (t, J ¼ 7.0 Hz, 2H), 2.15 (s, 3H),
1.93-1.81 (m, 2H); HRMS (FAB) calc. for C29H34FN6O3 [MþH]þ
533.2676, found: 533.2679.
102e103 ꢀC: 1H NMR (300 MHz, CD3OD)
d
8.40 (d, J ¼ 3.30 Hz, 1H),
8.35 (s, 1H), 7.75 (d, J ¼ 8.25 Hz,1H), 7.53 (s, 1H), 6.92 (d, J ¼ 8.43 Hz,
1H), 6.66-6.65 (m, 2H), 6.60 (dd, J ¼ 2.37, 8.43 Hz, 1H), 4.01 (t,
J ¼ 5.88 Hz, 2H), 3.92 (t, J ¼ 7.14 Hz, 2H), 3.72 (s, 3H), 3.17 (t,
J ¼ 6.60 Hz, 2H), 2.40 (t, J ¼ 7.68 Hz, 2H) 2.23 (s, 6H), 2.17 (d,
J ¼ 0.90 Hz, 2H), 1.84-1.66 (m, 6H); HRMS (ESI) calc. for C27H39N6O3
[MþH]þ 495.3078, found: 495.3083.
4.1.57. 1-(3-Methoxy-4-(2-(piperazin-1-yl)ethoxy)phenyl)-3-(3-(5-
methyl-1H-imidazole-1-yl)propyl)urea (193)
4.1.53. 1-(4-(2-(2-Aminopyridin-4-yl)ethoxy)-3-methoxyphenyl)-
3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (189)
Prepared from compound 88 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 82%, white
Prepared from compound 14 by following the procedure N to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 80%, white
solid, mp 68e69 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.60 (d,
solid, mp 99e100 ꢀC: 1H NMR (300 MHz, CDCl3)
d
7.95 (s, 1H), 7.91
J ¼ 0.93 Hz, 1H), 7.15 (d, J ¼ 2.37 Hz, 1H), 6.87 (d, J ¼ 8.61 Hz, 1H),
6.74 (dd, J ¼ 8.61, 2.58 Hz, 1H), 6.67 (s, 1H), 4.09 (t, J ¼ 5.52 Hz, 2H),
4.00 (t, J ¼ 7.14 Hz, 2H), 3.80 (s, 3H), 3.21 (t, J ¼ 6.78 Hz, 2H), 3.03 (t,
J ¼ 4.95 Hz, 4H), 2.81 (t, J ¼ 5.31 Hz, 2H), 2.72 (br, 4H), 2.23 (d,
J ¼ 0.90 Hz, 3H), 1.94 (quintet, J ¼ 7.14 Hz, 2H); 13C NMR (400 MHz,
(d, J ¼ 5.13 Hz, 1H), 7.37 (s, 1H), 7.17 (d, J ¼ 2.19 Hz, 1H), 6.71 (s, 1H),
6.67 (d, J ¼ 8.58 Hz, 1H), 6.58-6.51 (m, 2H), 6.38 (s, 1H), 5.91 (t,
J ¼ 5.49 Hz, 1H), 4.09 (t, J ¼ 7.14 Hz, 2H), 3.85 (t, J ¼ 6.96 Hz, 2H),
3.75 (s, 3H), 3.15 (q, J ¼ 6.06 Hz, 2H), 2.91 (t, J ¼ 6.93 Hz, 2H), 2.13 (s,
3H), 1.87 (quintet, J ¼ 6.42 Hz, 2H); 13C NMR (400 MHz, CD3OD)
CD3OD) d 157.26,150.11,143.75,136.73,134.15,127.61,125.21,115.21,
d
159.54, 157.30, 150.31, 150.08, 146.15, 143.87, 136.70, 133.96,
111.40, 105.02, 67.42, 57.31, 54.99, 52.68, 41.89, 36.61, 31.04, 7.72;
127.56, 115.22, 113.81, 111.66, 109.15, 105.45, 69.32, 55.11, 41.91,
36.66, 34.87, 31.04, 7.67; HRMS (FAB) calc. for C22H29N6O3 [MþH]þ
425.2301, found: 425.2305.
HRMS (ESI) calc. for C21H33N6O3 [MþH]þ 417.2609, found: 417.2620.
4.1.58. 1-(3-Methoxy-4-(2-(piperazin-1-yl)ethoxy)phenyl)-1-
methyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (194)
Prepared from compound 123 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 85%, white
4.1.54. 1-(4-(2-(2-Aminopyridin-4-yl)ethoxy)-3-methoxyphenyl)-
1-cyclohexyl-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (190)
Prepared from compound 16 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 91%, white
solid, mp 67e68 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.45 (d,
J ¼ 1.11 Hz, 1H), 6.95 (d, J ¼ 8.43 Hz, 1H), 6.73 (d, J ¼ 2.40 Hz, 1H),
6.65 (dd, J ¼ 8.43, 2.40 Hz, 1H), 6.55 (s, 1H), 4.08 (t, J ¼ 5.67 Hz, 2H),
3.80 (t, J ¼ 7.14 Hz, 2H), 3.45 (s, 3H), 3.45 (t, J ¼ 6.96 Hz, 2H), 2.77 (t,
J ¼ 4.95 Hz, 4H), 2.73 (t, J ¼ 5.49 Hz, 2H), 2.53 (br, 4H), 2.09 (d,
J ¼ 1.08 Hz, 3H), 1.87 (quintet, J ¼ 6.96 Hz, 2H); HRMS (ESI) calc. for
solid, mp 169e170 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.92 (d,
J ¼ 5.13 Hz, 1H), 7.23 (s, 1H), 6.77 (d, J ¼ 8.25 Hz, 1H), 6.64 (s, 1H),
6.59-6.50 (m, 3H), 6.40 (s, 1H), 4.39 (s, 2H), 4.35-4.27 (m, 1H), 4.15
(t, J ¼ 6.96 hz, 2H), 3.94 (t, J ¼ 5.85 Hz, 1H), 3.77 (s, 3H), 3.73 (t,
J ¼ 7.14 Hz, 2H), 3.06 (q, J ¼ 6.03 Hz, 2H), 2.97 (t, J ¼ 6.96 Hz, 2H),
C
22H35N6O3 [MþH]þ 431.2765, found: 431.2777
19

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
4.1.59. 1-Ethyl-1-(3-methoxy-4-(2-(piperazin-1-yl)ethoxy)phenyl)-
3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (195)
solid, mp 92e93 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.52 (s, 1H), 7.02
(d, J ¼ 8.22. 1H), 6.73 (m, 2H), 6.64 (m, 1H), 4.35 (m, 1H), 4.18 (t,
J ¼ 5.49, 2H), 3.93 (m, 3h), 3.81 (s, 3H), 3.13 (t, J ¼ 6.78, 2H), 2.87 (m,
8H), 2.61 (m, 4H), 2.18 (s, 3H), 2.17 (s, 3H), 2.13 (m, 2H),1.84 (m, 4H),
1.45 (m, 2H); HRMS (ESI) calc. for C27H43N7O3 [MþH]þ 514.3500,
found: 514.3503.
Prepared from compound 124 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 90%, white
solid, mp 56e57 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.44 (d,
J ¼ 0.72 Hz, 1H), 6.92 (d, J ¼ 8.43 Hz, 1H), 6.72 (d, J ¼ 2.40 Hz, 1H),
6.67 (dd, J ¼ 8.43 Hz, 2.40 Hz, 1H), 6.55 (s, 1H), 4.07 (t, J ¼ 5.49 Hz,
2H), 3.79 (t, J ¼ 7.14 Hz, 2H), 3.72 (s, 3H), 3.54 (q, J ¼ 7.14 Hz, 2H),
3.03 (t, J ¼ 6.78 Hz, 2H), 2.76 (t, J ¼ 4.92 Hz, 4H), 2.72 (t, J ¼ 5.49 Hz,
2H), 2.51 (br, 4H), 2.08 (d, J ¼ 0.93 Hz, 3H), 1.74 (t, J ¼ 7.14 Hz, 2H),
0.99 (t, J ¼ 7.14 Hz, 3H); HRMS (ESI) calc. for C23H37N6O3 [MþH]þ
445.2922, found: 445.2922.
4.1.64. 1-(3-Methoxy-4-(2-(piperazin-1-yl)ethoxy)phenyl)-3-(3-
(5-methyl-1H-imidazole-1-yl)propyl)-1-phenylurea (200)
Prepared from compound 127 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 94%, white
solid, mp 122e123 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.56 (s, 1H),
4.1.60. 1-Isopropyl-1-(3-methoxy-4-(2-(piperazin-1-yl)ethoxy)
phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (196)
Prepared from compound 125 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 83%, white
7.34 (t, J ¼ 7.86 Hz, 2H), 7.24 (dd, J ¼ 8.79, 1.47 Hz, 2H), 7.19 (td,
J ¼ 8.61, 1.29 Hz, 1H), 6.97 (d, J ¼ 8.73 Hz, 1H), 6.89 (d, J ¼ 2.37 Hz,
1H), 6.78 (dd, J ¼ 8.40, 2.37 Hz, 1H), 6.66 (s, 1H), 4.14 (t, J ¼ 5.49 Hz,
2H), 3.94 (t, J ¼ 7.14 Hz, 2H), 3.77 (s, 3H), 3.20 (t, J ¼ 6.78 Hz, 2H),
2.84 (t, J ¼ 4.92 Hz, 4H), 2.79 (t, J ¼ 5.49 Hz, 2H), 2.59 (br, 4H), 2.20
(d, J ¼ 0.72 Hz, 3H), 1.91 (quintet, J ¼ 7.32 Hz, 2H); HRMS (ESI) calc.
for C27H37N6O3 [MþH]þ 493.2922, found: 493.2941.
solid, mp 61e63 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.52 (d,
J ¼ 1.11 Hz, 1H), 7.03 (d, J ¼ 8.61 Hz, 1H), 6.73 (s, 1H), 6.71 (dd,
J ¼ 8.67, 2.37 Hz,1H), 6.64 (s,1H), 4.71 (hept, J ¼ 6.57 Hz,1H), 4.18 (t,
J ¼ 5.49 Hz, 2H), 3.87 (t, J ¼ 7.14 Hz, 2H), 3.81 (s, 3H), 3.10 (t,
J ¼ 6.78 Hz, 2H), 2.80 (t, J ¼ 4.95 Hz, 4H), 2.83 (t, J ¼ 5.49 Hz, 2H),
2.86 (br, 4H), 2.17 (d, J ¼ 1.11 Hz, 3H), 1.82 (quintet, J ¼ 7.14 Hz, 2H),
1.04 (d, J ¼ 6.78 Hz, 3H). HRMS (ESI) calc. for C24H39N6O3 [MþH]þ
459.3078, found: 459.3081.
4.1.65. 1-(4-Fluorobenzyl)-1-(3-methoxy-4-(2-(piperazin-1-yl)
ethoxy)phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea
(201)
Prepared from compound 101 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 70%, white
4.1.61. 1-Cyclohexyl-1-(3-methoxy-4-(2-(piperazin-1-yl)ethoxy)
phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (197)
Prepared from compound 99 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 73%, white
solid, mp 111e112 ꢀC: 1H (300 MHz, CD3OD)
d 7.53 (s, 1H), 7.23 (dd,
J ¼ 5.31, 8.58 Hz, 2H), 7.01 (t, J ¼ 8.79 Hz, 2H), 6.92 (d, J ¼ 8.25 Hz,
1H), 6.65 (s, 1H), 6.61-6.56 (m, 2H), 4.76 (s, 2H), 4.13 (t, J ¼ 5.49 Hz,
2H), 3.92 (t, J ¼ 7.32 Hz, 2H), 3.69 (s, 3H), 3.18 (t, J ¼ 6.75 Hz, 2H),
2.86 (t, J ¼ 4.74 Hz, 4H), 2.80 (t, J ¼ 5.31 Hz, 2H), 2.58 (br, 4H), 2.17
(d, J ¼ 0.90 Hz, 3H), 1.88 (quintet, J ¼ 7.14 Hz, 2H); HRMS (ESI) calc.
for C28H38FN6O3 [MþH]þ 525.2984, found: 525.3000.
solid, mp 97e98 ꢀC: 1H (300 MHz, CD3OD)
d 7.50 (s, 1H), 7.03 (d,
J ¼ 8.61 Hz, 1H), 6.70-6.68 (m, 2H), 6.64 (s, 1H), 4.37-4.27 (m, 1H),
4.19 (t, J ¼ 5.49 Hz, 2H), 3.88 (t, J ¼ 7.89 Hz, 2H), 3.81 (s, 3H), 3.12 (t,
J ¼ 6.60 Hz, 2H), 2.88-2.81 (m, 6H), 2.61 (br, 4H), 2.17 (d, J ¼ 1.08 Hz,
3H), 1.83-1.73 (m, 6H),1.57-1.50 (m, 1H), 1.39-1.28 (m, 2H),1.18-0.83
(m, 3H); HRMS (ESI) calc. for C27H43N6O3 [MþH]þ 499.3391, found:
499.3384.
4.1.66. 1-(3-Methoxy-4-(2-(piperazin-1-yl)ethoxy)phenyl)-3-(3-(5-
methyl-1H-imidazole-1-yl)propyl)-1-(pyridin-3-ylmethyl)urea
(202)
4.1.62. 1-(Cyclopentylmethyl)-1-(3-methoxy-4-(2-(piperazin-1-yl)
ethoxy)phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea
(198)
Prepared from compound 102 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 55%, white
Prepared from compound 126 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 68%, white
solid, mp 152e153 ꢀC: 1H (300 MHz, CD3OD)
d
8.41 (dd, J ¼ 1.47,
4.77 Hz, 1H), 8.35 (d, J ¼ 1.47 Hz, 1H), 7.74 (dt, J ¼ 6.03, 2.01 Hz, 1H),
7.53 (s, 1H), 7.38-7.30 (m, 2H), 6.94 (d, J ¼ 8.43 Hz, 1H), 6.67 (d,
J ¼ 2.37 Hz, 1H), 6.64 (s, 1H), 6.61 (dd, J ¼ 2.37, 8.40 Hz, 1H), 4.13 (t,
J ¼ 5.49 Hz, 2H), 3.92 (t, J ¼ 7.14 Hz, 2H), 3.72 (s, 3H), 3.18 (t,
J ¼ 6.60 Hz, 2H), 2.85 (t, J ¼ 4.95 Hz, 4H), 2.78 (t, J ¼ 5.49 Hz, 2H),
2.57 (br, 4H), 2.17 (d, J ¼ 1.08 Hz, 3H), 1.91 (quintet, J ¼ 6.78 Hz, 2H);
solid, mp 130e131 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.52 (s, 1H),
7.03 (d, J ¼ 8.40 Hz, 1H), 6.82 (d, J ¼ 2.22 Hz, 1H), 6.79 (dd, J ¼ 2.37,
8.40 Hz, 6.64 (s, 1H), 4.18 (t, J ¼ 5.49 Hz, 2H), 3.90 (t, J ¼ 7.32 Hz, 2H),
3.81 (s, 3H), 3.58 (d, J ¼ 7.71 Hz, 2H), 3.13 (t, J ¼ 6.78 Hz, 2H), 2.87-
2.80 (m, 6H), 2.61 (br, 4H), 2.18 (d, J ¼ 0.90 Hz, 3H), 1.99e1.96 (m,
1H), 1.85-1.80 (p, J ¼ 7.32 Hz, 2H), 1.64-1.51 (m, 6H), 1.28-1.25 (m,
HRMS (ESI) calc. for
508.3036.
C
27H37N7O3 [MþH]þ 508.3031, found:
2H); 13C NMR (400 MHz, CD3OD)
d 158.68, 150.68, 147.91, 136.59,
134.63, 127.45, 125.11, 120.97, 114.06, 112.38, 66.47, 57.30, 55.20,
53.62, 53.25, 44.50, 42.01, 38.60, 37.53, 31.11, 29.85, 24.92, 7.71;
4.1.67. 1-(3-Methoxy-4-(2-(4-methylpiperazin-1-yl)ethoxy)
phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (203)
From compound 89, procedure N, yield 77%, white solid, mp
HRMS (ESI) calc. for
499.3395.
C
27H43N6O3 [MþH]þ 499.3391, found:
98e99 ꢀC: 1H NMR (300 Mhz, CDCl3)
d 7.40 (s, 1H), 7.06 (s, 2H), 6.78
4.1.63. 1-(3-Methoxy-4-(2-(piperazin-1-yl)ethoxy)phenyl)-3-(3-(5-
methyl-1H-imidazole-1-yl)propyl)-1-(1-methylpiperidin-4-yl)urea
(199)
Prepared from compound 100 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 78%, white
(d, J ¼ 8.55 Hz, 1H), 6.74 (s, 1H), 6.63 (dd, J ¼ 8.50, 2.25 Hz, 1H), 5.35
(br, NH), 4.09 (t, J ¼ 6.15 Hz, 2H), 3.91 (t, J ¼ 6.85 Hz, 2H), 3.79 (s,
3H), 3.20 (q, J ¼ 5.95 Hz, 2H), 2.8 (t, J ¼ 6.10 Hz, 2H), 2.59 (br, 4H),
2.44 (br, 4H), 2.26 (s, 3H), 2.15 (s, 3H), 2.02-0.99 (m, 1H), 1.93 (p,
J ¼ 6.70 Hz, 2H); HRMS (ESI) calc. for C22H35N6O3 [MþH]þ 431.2765,
found: 431.2773.
20

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
4.1.68. 1-Isopropyl-1-(3-methoxy-4-(2-(4-methylpiperazin-1-yl)
ethoxy)phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea
(204)
4.1.72. 1-(4-Fluorobenzyl)-1-(3-methoxy-4-(2-(4-methylpiperazin-
1-yl)ethoxy)phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)
urea (208)
Prepared from compound 121 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 49%, white
Prepared from compound 97 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 55%, white
solid, mp 151e152 ꢀC: 1H NMR (300 MHz, CDCl3)
d
7.30 (s, 1H), 6.88
solid, mp 121e122 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.54 (s, 1H),
(d, J ¼ 8.43 Hz, 1H), 6.70 (s, 1H), 6.66 (dd, J ¼ 2.37, 8.43 Hz, 1H), 6.58
(d, J ¼ 2.40 Hz, 1H), 4.83-4.79 (m, 1H), 4.16 (t, J ¼ 6.21 Hz, 2H), 3.99
(t, J ¼ 6.06 Hz, NH), 3.81 (s, 3H), 3.78 (t, J ¼ 7.32 Hz, 2H), 3.16 (q,
J ¼ 6.60 Hz, 2H), 2.89 (t, J ¼ 6.03 Hz. 2H), 2.63 (br, 4H), 2.45 (br, 4H),
2.27 (s, 3H), 2.12 (d, J ¼ 0.90 Hz, 3H), 1.86 (quintet, J ¼ 6.96 Hz, 2H),
1.03 (d, J ¼ 6.78 Hz, 6H); HRMS (ESI) calc. for C25H41N6O3 [MþH]þ
473.3235, found: 473.3236.
7.23 (dd, J ¼ 5.79, 8.61 Hz, 2H), 7.01 (t, J ¼ 8.79 Hz, 2H), 6.92 (d,
J ¼ 8.43 Hz, 1H), 6.65 (s, 1H), 6.61-6.55 (m, 2H), 4.77 (s, 2H), 4.13 (t,
J ¼ 5.70 Hz, 2H), 3.92 (t, J ¼ 7.14 Hz, 2H), 3.69 (s, 3H), 3.18 (t,
j ¼ 6.78 Hz, 2H), 2.82 (t, J ¼ 5.49 Hz, 2H), 2.65 (br, 4H), 2.51 (br, 4H),
2.27 (s, 3H), 2.18 (d, J ¼ 0.90 Hz, 3H), 1.90 (quintet, J ¼ 6.57 Hz, 2H);
HRMS (ESI) calc. for
539.3157.
C
29H40FN6O3 [MþH]þ 539.3140, found:
4.1.73. 1-(3-Methoxy-4-(2-(4-methylpiperazin-1-yl)ethoxy)
phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)-1-(pyridin-3-
ylmethyl)urea (209)
4.1.69. 1-Cyclohexyl-1-(3-methoxy-4-(2-(4-methylpiperazin-1-yl)
ethoxy)phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea
(205)
Prepared from compound 98 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 45%, white
Prepared from compound 95 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 54%, white
solid, mp 129e130 ꢀC: 1H NMR (300 MHz, CDCl3)
d 4.43 (dd,
solid, mp 115e116 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.52 (s, 1H), 7.03
J ¼ 4.77, 1.65 Hz, 1H), 8.34 (d, J ¼ 1.65 Hz, 1H), 7.57 (dt-like,
J ¼ 7.86 Hz, 1H), 7.27 (s, 1H), 7.16 (dd, J ¼ 7.89, 4.95 Hz, 1H), 6.74 (d,
J ¼ 8.43 Hz, 1h), 6.66 (s, 1H), 6.47 (dd, J ¼ 8.25, 2.40 Hz. 1H), 6.38 (d,
J ¼ 2.40 Hz, 1H), 4.74 (s, 2H), 4.28 (t-like, 1H), 4.05 (t, J ¼ 6.21 Hz,
2H), 3.76 (t, J ¼ 7.14 Hz, 2H), 3.65 (s, 3H), 3.14 (q, J ¼ 6.39 Hz, 2H),
2.79 (t, J ¼ 6.03 Hz, 2H), 2.56-2.40 (m, 8H), 2.22 (s, 3H), 2.07 (d,
J ¼ 0.72 Hz, 3H), 1.81 (quintet, J ¼ 6.93 Hz, 2H); HRMS (ESI) calc. for
(d, J ¼ 8.61 Hz, 1H), 6.70-6.67 (m, 2H), 6.64 (s, 1H), 4.31-4.27 (m,
1H), 4.19 (t, J ¼ 5.52 Hz, 2H), 3.88 (t, J ¼ 7.14 Hz, 2H), 3.83 (s, 3H),
3.11 (t, J ¼ 6.78 Hz, 2H), 2.87 (t, J ¼ 5.49 Hz, 2H), 2.69 (br, 4H), 2.53
(br, 4H), 2.29 (s, 3H), 2.17 (d, J ¼ 0.93 Hz, 3H),1.89-1.72 (m, 6H),1.59-
1.55 (m, 1H), 1.43-1.28 (m, 2H), 1.11-0.90 (m, 3H); HRMS (ESI) calc.
for C28H45N6O3 [MþH]þ 513.3548, found: 513.3540.
C
28H40N7O3 [MþH]þ 522.3187, found: 522.3203.
4.1.74. 1-(3-Methoxy-4-(2-morpholinoethoxy)phenyl)-3-(3-(5-
methyl-1H-imidazole-1-yl)propyl)urea (210)
4.1.70. 1-(Cyclopentylmethyl)-1-(3-methoxy-4-(2-(4-
methylpiperazin-1-yl)ethoxy)phenyl)-3-(3-(5-methyl-1H-
imidazole-1-yl)propyl)urea (206)
From compound 90, procedure N, yield 61%, white solid, mp
64e65 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.40 (s, 1H), 7.36 (s, 1H), 7.09
Prepared from compound 96 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 77%, white
(d, J ¼ 2.37 Hz, 1H), 6.73 (d, J ¼ 8.61 Hz, 1H), 7.70 (s, 1H), 6.56 (dd,
J ¼ 8.61, 2.40 Hz, 1H), 4.04 (t, J ¼ 6.03 Hz, 2H), 3.87 (t, J ¼ 6.78 Hz,
2H), 3.75 (s, 3H), 3.66 (t, J ¼ 4.38 Hz, 4H), 3.14 (q, J ¼ 6.06 Hz, 2H),
2.73 (t, J ¼ 5.85 Hz, 2H), 2.51 (t, J ¼ 4.59 Hz, 4H), 2.13 (s, 3H), 1.89
solid, mp 137e138 ꢀC: 1H NMR (300 MHz, CD3OD)
d 7.52 (s, 1H),
(quintet, J ¼ 6.60 Hz, 2H); 13C NMR (400 MHz, CD3OD)
d 157.22,
7.03 (d, J ¼ 8.43 Hz, 1H), 6.82 (d, J ¼ 2.40 Hz, 1H), 6.79 (dd, J ¼ 2.37,
8.22 Hz, 1H), 6.64 (s, 1H), 4.18 (t, J ¼ 5.31 Hz, 2H), 3.90 (t, J ¼ 7.32 Hz,
2H), 3.81 (s, 3H), 3.58 (d, J ¼ 7.50 Hz, 2H), 3.12 (t, J ¼ 6.78 Hz, 2H),
2.85 (t, J ¼ 5.49 Hz, 2H), 2.68 (br, 4H), 2.49 (br, 4H), 2.27 (s, 3H), 2.17
(d, J ¼ 0.93 Hz, 3H), 2.01-1.91 (m, 1H), 1.88 (quintet, J ¼ 7.14 Hz, 2H),
1.67-1.48 (m, 6H), 1.27-1.20 (m, 2H); HRMS (ESI) calc. for
150.16, 143.85, 136.70, 134.09, 127.58, 125.14, 115.32, 111.47, 105.09,
67.39, 66.28, 57.49, 54.98, 53.81, 41.93, 36.64, 31.04, 7.71; HRMS
(ESI) calc. for C21H32N5O4 [MþH]þ 418.2449, found: 418.2463.
4.1.75. 1-(3-Methoxy-4-(2-morpholinoethoxy)phenyl)-1-methyl-3-
(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (211)
C
28H45N6O3 [MþH]þ 513.3548, found: 513.3557.
From compound 118, procedure L, yield 56%, white solid, mp
67e69 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.30 (s, 1H), 6.82 (d,
4.1.71. (1-(3-Methoxy-4-(2-(4-methylpiperazin-1-yl)ethoxy)
phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)-1-(1-
methylpiperidin-4-yl)urea (207)
J ¼ 8.43 Hz, 1H), 6.71-6.65 (m, 3H), 4.28 (t, J ¼ 5.88 Hz, 1H), 4.10 (t,
J ¼ 6.03 Hz, 2H), 3.78 (s, 3H), 3.77 (t, J ¼ 7.32 Hz, 2H), 3.67 (t,
J ¼ 4.95 Hz, 4H), 3.16 (s, 3H), 3.11 (q, J ¼ 6.24 Hz, 2H), 2.79 (t,
J ¼ 6.03 Hz, 2H), 2.54 (t, J ¼ 4.56 Hz, 4H), 2.08 (d, J ¼ 0.93 Hz, 3H),
1.80 (quintet, J ¼ 6.96 Hz, 2H); HRMS (ESI) calc. for C22H34N5O4
[MþH]þ 432.2605, found: 432.2608.
Prepared from compound 122 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 44%, white
solid, mp 120e121 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.31 (s, 1H), 6.85
(d, J ¼ 8.4 Hz, 1H), 6.71 (s, 1H), 6.64 (dd, J ¼ 2.2, 8.3 Hz, 1H), 6.57 (d,
J ¼ 2.2 Hz, 1H), 4.41 (m, 1H), 4.13 (t, J ¼ 6.0 Hz, 2H), 4.06 (t,
J ¼ 5.7 Hz,1H), 3.81 (s, 3H), 3.79 (t, J ¼ 7.3 Hz, 2H), 3.15 (q, J ¼ 6.2 Hz,
2H), 2.88-2.74 (m, 4H), 2.60-2.47 (m, 8H), 2.29 (s, 3H), 2.21 (s, 3H),
2.13 (s, 3H), 2.12-2.03 (m, 2H), 1.87-1.75 (m, 4H), 1.46-1.35 (m, 2H);
4.1.76. 1-Isopropyl-1-(3-methoxy-4-(2-morpholinoethoxy)phenyl)-
3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (212)
From compound 119, procedure L, yield 73%, white solid, mp
92e93 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.30 (s, 1H), 6.91 (d,
J ¼ 8.40 Hz, 1H), 6.81 (s, 1H), 6.66-6.58 (m, 2H), 4.26 (t, J ¼ 5.67 Hz,
2H), 4.81-4.75 (m, 1H), 4.10 (t, J ¼ 4.59 Hz, NH), 3.91 (t, J ¼ 7.14 Hz,
2H), 3.81 (s, 3H), 3.79 (t, J ¼ 4.41 Hz, 4H), 3.15 (q, J ¼ 6.24 Hz, 2H),
2.98 (t, J ¼ 5.67 Hz, 2H), 2.75 (t, J ¼ 4.41 Hz, 4H), 2.17 (s, 3H), 1.87 (p,
J ¼ 6.60 Hz, 2H), 1.03 (d, J ¼ 6.78 Hz, 6H); HRMS (ESI) calc. for
13C NMR (400 MHz, CD3OD)
d 158.35, 150.32, 148.71, 136.58, 130.33,
127.44, 125.12, 123.26, 114.69, 113.74, 66.95, 56.65, 55.21, 54.78,
54.23, 52.74, 52.24, 44.61, 44.41, 42.04, 37.53, 31.01, 30.31, 7.69
HRMS (FAB) calc. for
528.3660.
C
28H46N7O3 [MþH]þ 528.3657, found:
C
24H38N5O4 [MþH]þ 460.2918; found: 460.2924.
21

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
4.1.77. 1-Cyclohexyl-1-(3-methoxy-4-(2-morpholinoethoxy)
phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (213)
From compound 91, procedure L, yield 72%, white solid, mp
[MþH]þ 509.2876, found: 509.2878.
4.1.82. 1-((1H-Benzo[d]imidazole-5-yl)methyl)-3-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)urea (218)
78e79 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.25 (s, 1H), 6.82 (d,
J ¼ 8.22 Hz, 1H), 6.65 (s, 1H), 6.60 (dd, J ¼ 2.37, 8.40 Hz, 1H), 6.53 (d,
J ¼ 2.37 Hz, 1H), 4.36-4.28 (m, 1H), 4.12 (t, J ¼ 6.03 Hz, 2H), 3.95 (t,
J ¼ 5.85 Hz, NH), 3.77 (s, 3H), 3.76 (t, J ¼ 7.14 Hz, 2H), 3.68 (t,
J ¼ 4.41 Hz, 4H), 3.11 (q, J ¼ 6.60 Hz, 2H), 2.82 (t, J ¼ 5.85 Hz, 2H),
2.56 (t, J ¼ 4.56 Hz, 4H), 2.07 (d, J ¼ 0.93 Hz, 3H), 1.81 (quintet,
J ¼ 6.78 Hz, 2H), 1.70-1.63 (m, 4H), 1.52-1.47 (m, 1H), 1.34-1.25 (m,
2H), 1.01-0.94 (m, 2H), 0.89-0.80 (m, 1H); HRMS (ESI) calc. for
Prepared from compound 15 by following the procedure N to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 75%, white
solid, mp 115e116 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.13 (s, 1H), 7.74
(d, J ¼ 4.95 Hz, 1H), 7.75-7.74 (m, 2H), 7.25 (d, J ¼ 8.40 Hz, 1H), 7.15
(d, J ¼ 2.37 Hz, 1H), 6.82 (d, J ¼ 8.61 Hz, 1H), 6.74 (dd, J ¼ 8.61,
2.40 Hz, 1H), 6.48 (d, J ¼ 5.31 Hz, 1H), 6.43 (s, 1H), 4.49 (s, 2H), 3.97
(br, 2H), 3.80 (s, 3H), 2.56 (t-like, 2H), 1.78-1.77 (m, 4H); HRMS (ESI)
calc. for C25H29N6O3 [MþH]þ 461.2296, found: 461.2312.
C
27H42N5O4 [MþH]þ 500.3231; found: 500.3238.
4.1.78. 1-(Cyclopentylmethyl)-1-(3-methoxy-4-(2-
morpholinoethoxy)phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)
propyl)urea (214)
4.1.83. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-methylurea (219)
Prepared from compound 51 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 77%, white
From compound 120, procedure L, yield 88%, white solid, mp
67e68 ꢀC: 1H NMR (400 MHz, CD3OD)
d 7.53 (s, 1H), 7.01 (d,
J ¼ 8.4 Hz, 1H), 6.81 (d, J ¼ 2.8 Hz, 1H), 6.76 (dd, J ¼ 2.0, 8.0 Hz, 1H),
6.64 (s, 1H), 4.16 (t, J ¼ 5.6 Hz, 2H), 3.87 (t, J ¼ 7.2 Hz, 2H), 3.80 (s,
3H), 3.69-3.67 (m, 4H), 3.55 (d, J ¼ 7.6 Hz, 2H), 3.10 (t, J ¼ 6.4 Hz,
2H), 2.81 (t, J ¼ 5.6 Hz, 2H), 2.62-2.59 (m, 4H), 2.16 (s, 3H), 1.98 (m,
1H), 1.82 (m, 2H), 1.68-1.58 (m, 4H), 1.51-1.47 (m, 2H), 1.26-1.20 (m,
solid, mp 100e102 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.01 (s, 1H),
7.63 (d, J ¼ 5.31 Hz, 1H), 7.43 (s, 1H), 7.42 (d, J ¼ 8.43 Hz, 1H), 7.08
(dd, J ¼ 8.25, 1.47 Hz, 1H), 6.84 (d, J ¼ 8.40 Hz, 1H), 6.75-6.69 (m,
2H), 6.38-6.36 (dd, J ¼ 5.31, 1.29 Hz, 1H), 6.33 (s, 1H), 4.31 (s, 2H),
3.89 (t, J ¼ 5.85 Hz, 2H), 3.64 (s, 3H), 3.15 (s, 3H), 2.45 (t, J ¼ 6.96 Hz,
2H); 13C NMR (600 MHz, CDCl3)
d 157.60, 150.41, 147.89, 136.56,
134.75, 126.96, 126.83, 120.99, 113.69, 112.23, 66.98, 66.90, 57.44,
56.10, 54.08, 53.81, 42.25, 38.62, 37.96, 31.65, 30.18, 25.26, 9.16;
2H),1.69-1.65 (m, 4H); 13C NMR (400 MHz, CD3OD)
d 159.56,158.73,
153.75, 150.35, 147.94, 146.12, 141.38, 136.20, 135.21, 131.78, 131.69,
128.69, 128.58, 122.14, 119.47, 113.74, 113.51, 111.34, 108.48, 68.62,
55.16, 44.10, 36.72, 34.47, 28.41, 26.37; HRMS (ESI) calc. for
HRMS (FAB) calc. for
500.3242.
C
27H42N5O4 [MþH]þ 500.3237, found:
C
26H31N6O3 [MþH]þ 475.2452, found: 475.2467.
4.1.79. 1-(3-Methoxy-4-(2-morpholinoethoxy)phenyl)-3-(3-(5-
methyl-1H-imidazole-1-yl)propyl)-1-(1-methylpiperidin-4-yl)urea
(215)
4.1.84. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-ethylurea (220)
Prepared from compound 52 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 77%, white
From compound 92, procedure L, yield 67%, white solid, mp
102e103 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.35 (s, 1H), 6.83 (d,
J ¼ 8.43 Hz, 2H, 6.68 (s, 1H), 6.60 (dd, J ¼ 2.37, 8.25 Hz, 1H), 6.52 (d,
J ¼ 2.19 Hz, 1H), 4.51-4.46 (m, 1H), 4.12 (t, J ¼ 5.88 Hz, 2H), 3.77 (s,
3H), 3.75-3.72 (m, 2H), 3.69 (t, J ¼ 4.74 Hz, 4H), 3.18-3.08 (m, 6H),
2.82 (t, J ¼ 5.85 Hz, 2H), 2.57 (t, J ¼ 4.59 Hz, 4H), 2.45 (s, 3H), 2.08 (s,
3H), 1.82-1.75 (m, 6H); HRMS (ESI) calc. for C27H43N6O4 [MþH]þ
515.3340; found: 515.3347.
solid, mp 153e154 ꢀC: 1H NMR (500 MHz, CD3OD)
d 8.11 (s,1H), 7.73
(d, J ¼ 5.35 Hz, 1H), 7.51 (d, J ¼ 8.30 Hz, 1H), 7.47 (s, 1H), 7.16 (d,
J ¼ 8.15 Hz, 1H), 6.95 (d, J ¼ 8.15 Hz, 1H), 6.80 (s, 1H), 6.79 (dd,
J ¼ 8.60, 2.30 Hz, 1H), 6.48 (d, J ¼ 5.35 Hz, 1H), 6.44 (s, 1H), 4.39 (s,
2H0, 4.00 (t, J ¼ 5.55 Hz, 2H), 3.76 (s, 3H), 3.66 (q, J ¼ 7.15 Hz, 2H),
2.56 (t, J ¼ 6.90 Hz, 2H), 1.79e77 (m, 4H), 1.10 (t, J ¼ 7.05 Hz, 3H);
4.1.80. 1-(4-Fluorobenzyl)-1-(3-methoxy-4-(2-morpholinoethoxy)
phenyl)-3-(3-(5-methyl-1H-imidazole-1-yl)propyl)urea (216)
From compound 93, procedure L, yield 72%, white solid, mp
HRMS (ESI) calc. for
489.2614.
C
27H33N6O3 [MþH]þ 489.2609, found:
111e112 ꢀC: 1H NMR (300 MHz, CD3OD)
d
7.47 (s, 1H), 7.13 (dd,
4.1.85. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-isopropylurea
(221)
J ¼ 5.49, 8.79 Hz, 2H), 6.92 (t, J ¼ 8.79 Hz, 2H), 6.83 (d, J ¼ 8.43 Hz,
1H), 6.55 (s, 1H), 6.52 (d, J ¼ 2.37 Hz, 1H), 6.50 (dd, J ¼ 2.55, 8.40 Hz,
1H), 4.69 (s, 2H), 4.04 (t, J ¼ 5.49 Hz, 2H), 3.82 (t, J ¼ 7.14 Hz, 2H),
3.60 (s, 3H), 3.59 (t, J ¼ 4.68 Hz, 4H), 3.06 (t, J ¼ 6.84 Hz, 2H), 2.71 (t,
J ¼ 5.49 Hz, 2H), 2.51 (t, J ¼ 4.59 Hz, 4H), 2.08 (d, J ¼ 0.90 Hz, 3H),
1.81 (quintet, J ¼ 6.78 Hz, 2H); HRMS (ESI) calc. for C28H37FN5O4
[MþH]þ 526.2824, found: 526.2823.
Prepared from compound 53 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 72%, white
solid, mp 105e106 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.01 (s, 1H),
7.64 (d, J ¼ 5.49 Hz, 1H), 7.42 (d, J ¼ 8.25 Hz, 1H), 7.35 (s, 1H), 7.05
(dd, J ¼ 1.47, 8.25 Hz, 1H), 6.89 (d, J ¼ 8.43 Hz, 1H), 6.67-6.63 (m,
3H), 6.38 (d, J ¼ 5.49 Hz, 1H), 6.33 (s, 1H), 4.67-4.62 (m, 1H), 4.28 (s,
2H), 3.92 (t, J ¼ 6.69 Hz, 2H), 3.67 (s, 3H), 2.46 (t, J ¼ 7.14 Hz, 2H),
1.70-1.68 (m, 4H), 0.97 (d, J ¼ 6.78 Hz, 6H); HRMS (ESI) calc. for
4.1.81. 1-(3-Methoxy-4-(2-morpholinoethoxy)phenyl)-3-(3-(5-
methyl-1H-imidazole-1-yl)propyl)-1-(pyridin-3-ylmethyl)urea
(217)
From compound 94, procedure L, yield 71%, white solid, mp
C
28H35N6O3 [MþH]þ 503.2765, found: 503.2771.
100e102 ꢀC: 1H NMR (300 MHz, CDCl3)
d
8.47 (dd, J ¼ 4.77, 1.65 Hz,
1H), 8.37 (d, J ¼ 1.65 Hz, 1H), 7.63 (dt, J ¼ 7.86, 2.01 Hz, 1H), 7.34 (s,
1H), 7.20 (dd, J ¼ 7.68, 4.77 Hz, 1H), 6.79 (d, J ¼ 8.43 Hz, 1H), 6.71 (s,
1H), 6.51 (dd, J ¼ 8.49, 2.37 Hz, 1H), 6.43 (d, J ¼ 2.40 Hz, 1H), 4.78 (s,
2H), 4.31 (t, J ¼ 5.85 Hz, 1H), 4.10 (t, J ¼ 6.06 Hz, 2H), 3.81 (t,
J ¼ 7.14 Hz, 2H), 3.72-3.69 (m. 7H), 3.18 (q, J ¼ 6.42 Hz, 2H), 2.82 (t,
J ¼ 6.06 Hz, 2H), 2.57 (t, J ¼ 4.74 Hz, 4H), 2.12 (d, J ¼ 0.93 Hz, 3H),
1.86 (quintet, J ¼ 6.96 Hz, 2H); HRMS (FAB) calc. for C27H37N6O4
4.1.86. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-cyclobutylurea
(222)
Prepared from compound 55 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 83%, white
solid, 85e86 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.78 (s, 1H), 7.79 (d,
22

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
J ¼ 5.69 Hz, 1H), 7.41 (d, J ¼ 8.25 Hz, 1H), 7.36 (s, 1H), 6.98 (d,
J ¼ 8.04 Hz, 1H), 6.74 (d, J ¼ 8.40 Hz, 1H), 6.56 (s, 1H), 6.42 (d,
J ¼ 5.13 Hz, 1H), 6.30 (s, 1H), 4.92 (quintet, J ¼ 7.68 Hz, 1H), 4.59 (t,
J ¼ 5.31 Hz, 1H), 4.36 (d, J ¼ 5.67 Hz, 2H), 3.74 (s, 3H), 2.49 (t,
J ¼ 6.60 Hz, 2H), 2.03-1.98 (m, 2H), 1.80-1.65 (m, 6H), 1.51-1.35 (m,
2H); HRMS (FAB) calc. for C29H35N6O3 [MþH]þ 515.2771, found:
515.2770. .
(m, 2H), 1.85-1.81 (m, 2H), 1.69 (m, 4H), 1.40-1.28 (m, 2H); HRMS
(ESI) calc. for C30H38N7O3 [MþH]þ 544.3031, found: 544.3031.
4.1.91. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-(1-
methylpiperidin-4-yl)urea (227)
Prepared from compound 60 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 73%, white
4.1.87. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-cyclopentylurea
(223)
solid, mp 120e121 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.10 (s, 1H),
7.73 (d, J ¼ 5.49 Hz, 1H), 7.50 (d, J ¼ 8.25 Hz, 1H), 7.44 (s, 1H), 7.13
(dd, J ¼ 8.25,1.47 Hz,1H), 6.96 (d, J ¼ 9.15 Hz,1H), 6.77-6.74 (m, 2H),
6.48 (dd, J ¼ 5.49, 1.47 Hz, 1H), 6.43 (s, 1H), 4.38 (s, 2H), 4.38-4.30
(m, 1H), 4.02 (t-like, 2H), 3.78 (s, 3H), 2.19 (s, 3H), 2.19-2.00 (m, 2H),
1.75e1.85 (m, 6H), 1.50-1.43 (m, 2H); 13C NMR (400 MHz, CD3OD)
Prepared from compound 56 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 73%, white
solid, mp 88e89 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.01 (s, 1H), 7.65
(d, J ¼ 5.85 Hz, 1H), 7.42 (d, J ¼ 8.43 Hz, 1H), 7.35 (s, 1H), 7.06 (dd,
J ¼ 1.47, 8.43 Hz, 1H), 6.89 (d, J ¼ 8.97 Hz, 1H), 6.67-6.64 (m, 2H),
6.39 (dd, J ¼ 1.44, 5.49 Hz, 1H), 6.34 (s, 1H), 4.67-4.56 (m, 1H), 4.28
(s, 2H), 3.92 (t, J ¼ 5.49 Hz, 2H), 3.68 (s, 3H), 2.48 (t, J ¼ 6.78 Hz, 2H),
1.77-1.68 (m, 6H), 1.41-1.39 (m, 4H), 1.31-1.21 (m, 2H); HRMS (FAB)
calc. for C30H37N6O3 [MþH]þ 529.2927, found: 529.2935.
d
163.07, 158.19, 156.45, 155.70, 155.46, 150.37, 149.17, 141.41, 139.36,
135.05, 129.48, 123.16, 122.08, 114.46, 113.63, 113.33, 112.81, 110.79,
110.34, 68.45, 55.30, 53.84, 50.27, 43.96, 42.20, 34.75, 28.43, 28.24,
26.00; HRMS (FAB) calc. for C31H40N7O3 [MþH]þ 558.3193, found:
558.3191.
4.1.88. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-cyclohexylurea
(224)
4.1.92. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-benzylurea (228)
Prepared from compound 61 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 91%, white
Prepared from compound 57 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 81%, white
solid, mp 114e116 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.12 (s, 1H), 7.71
solid, mp 122e123 ꢀC: 1H NMR (300 MHz, CDCl3)
d
7.95 (s, 1H), 7.90
(d, J ¼ 5.49 Hz, 1H), 7.51 (d, J ¼ 8.43 Hz, 1H), 7.50 (s, 1H), 7.27-7.17
(m, 6H), 6.83 (d, J ¼ 8.61 Hz, 1H), 6.62 (dd, J ¼ 8.43, 2.40 Hz, 1H),
6.56 (d, J ¼ 2.37 Hz, 1H), 6.46 (dd, J ¼ 6.39, 1.47 Hz, 1H), 6.42 (s, 1H),
6.07 (t, J ¼ 6.03 Hz,1H), 4.81 (s, 2H), 4.43 (d, J ¼ 5.85 Hz, 2H), 3.93 (t-
like, 2H), 3.59 (s, 3H), 2.53 (t, J ¼ 6.78 Hz, 2H), 1.80-1.70 (m, 4H);
(d, J ¼ 4.95 Hz, 1H), 7.50 (d, J ¼ 8.43 Hz, 1H), 7.43 (s, 1H), 7.06 (d,
J ¼ 8.25 Hz, 1H), 6.77 (d, J ¼ 8.40 Hz, 1H), 6.67 (dd, J ¼ 8.43, 2.19 Hz,
1H), 6.62 (d, J ¼ 2.04 Hz, 2H), 6.49 (d, J ¼ 4.77 Hz, 1H), 6.35 (s, 1H),
4.52 (t, J ¼ 5.67 Hz, 1H), 4.50-4.43 (m, 4H), 3.99 (t, J ¼ 6.06 Hz, 2H),
3.80 (s, 3H), 2.56 (t, J ¼ 6.78 Hz, 2H), 1.82-1.68 (m, 6H), 1.54 (d,
J ¼ 12.63 Hz, 1H), 1.29 (q, J ¼ 12.81 Hz, 2H), 1.09-0.98 (m, 2H), 0.90-
0.86 (m, 1H); HRMS (ESI) calc. for C31H39N6O3 [MþH]þ: 543.3078,
found: 543.3083.
HRMS (ESI) calc. for
551.2762.
C
32H35N6O3 [MþH]þ 551.2765, found:
4.1.93. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-(4-fluorobenzyl)
urea (229)
4.1.89. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-
(cyclopentylmethyl)urea (225)
Prepared from compound 62 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 84%, white
Prepared from compound 58 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 81%, white
solid, mp 100e102 ꢀC: 1H NMR (300 MHz, CDCl3)
d 7.97 (s, 1H), 7.88
(d, J ¼ 5.31 Hz,1H), 7.52 (d, J ¼ 7.86 Hz,1H), 7.46 (s,1H), 7.21-7.16 (m,
2H), 7.11 (dd-like, J ¼ 8.04 Hz, 1H), 6.92 (t, J ¼ 8.61 Hz, 2H), 6.57 (d,
J ¼ 8.43 Hz, 1H), 6.55 (dd, J ¼ 8.40, 2.37 Hz, 1H), 6.47 (d, J ¼ 6.24 Hz,
1H), 6.43 (d, J ¼ 2.22 Hz, 1H), 6.31 (s, 1H), 4.78 (m, 3H), 4.48 (d,
J ¼ 5.88 Hz, 2H), 4.38 (brs, 2H), 3.93 (t, J ¼ 5.67 Hz, 2H), 3.64 (s, 3H),
2.53 (t, J ¼ 6.78 Hz, 2H), 1.85-1.70 (m, 4H); HRMS (ESI) calc. for
solid, mp 84e86 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.10 (s, H), 7.74 (d,
J ¼ 5.49 Hz, 1H), 7.52-7.46 (m, 2H), 7.17 (dd, J ¼ 2.22, 8.43 Hz, 2H),
6.97 (d, J ¼ 8.97 Hz, 1H), 6.81-6.79 (m, 2H), 6.48 (dd, J ¼ 1.56,
5.49 Hz, 1H), 6.43 (s, 1H), 4.39 (s, 2H), 4.01 (t, J ¼ 6.06 Hz, 2H), 3.76
(s, 3H), 3.61 (d, J ¼ 7.68 Hz, 2H), 2.56 (t, J ¼ 6.06 Hz, 2H), 2.01-1.98
(m, 1H), 1.82-1.78 (m, 4H), 1.70-1.61 (m, 4H), 1.55-1.49 (m, 2H), 1.30-
1.22 (m, 2H); HRMS (ESI) calc. for C31H39N6O3 [MþH]þ 543.3078,
found: 543.3091.
C
32H34FN6O3 [MþH]þ 569.2671, found: 569.2677.
4.1.94. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-(pyridin-3-
ylmethyl)urea (230)
4.1.90. 3-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-1-(piperidin-4-yl)
urea (226)
Prepared from compound 63 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 74%, white
Prepared from compound 59 by following the procedure L to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 88%, white
solid, mp 165e166 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.39-8.34 (m,
solid, mp 137e138 ꢀC: 1H NMR (300 MHz, CD3OD)
d
8.01 (s, 1H),
2H), 7.75-7.71 (m, 3H), 7.53-7.49 (m, 2H), 7.37 (dd, J ¼ 4.95 Hz,
8.07 Hz, 1H), 7.19 (d, J ¼ 8.25 Hz, 1H), 6.88 (d, J ¼ 8.22 Hz, 1H), 6.63-
6.60 (m, 2H), 6.47-6.40 (m, 2H), 4.87 (s, 2H), 4.42 (s, 2H), 3.96 (s,
2H), 3.65 (s, 3H), 2.53 (t, J ¼ 7.14 Hz, 2H), 1.81-1.71 (m, 4H); HRMS
(ESI) calc. for C31H34N7O3 [MþH]þ 552. 2718; found: 552.2712.
7.63 (d, J ¼ 5.3 Hz, 1H), 7.41 (d, J ¼ 8.3 Hz, 1H), 7.35 (s, 1H), 7.04 (dd,
J ¼ 1.5, 8.3 Hz, 1H), 6.87 (d, J ¼ 8.8 Hz, 1H), 6.68-6.64 (m, 2H), 6.37
(dd, J ¼ 1.3, 5.3 Hz, 1H), 6.34 (s, 1H), 4.44-4.36 (m, 1H), 4.29 (s, 2H),
3.92 (m, 2H), 3.72 (s, 3H), 3.10-3.01 (m, 2H), 2.77-2.69 (m, 2H), 2.46
23

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
4.1.95. N-((1H-Benzo[d]imidazole-5-yl)methyl)-2-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)-2-
compounds and 25
m
L of pGAPase. After incubation in 96-well black
plates (Greiner, Austria) for 10 min at 37 ꢀC, the reaction was started
methylpropanamide (231)
by adding 50 mL of the hQC solution.
Prepared from compound 143 by following the procedure O to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 83%, white
4.2.2. Cytotoxicity assays
solid, mp 106e107 ꢀC: 1H NMR (400 MHz, CD3OD)
d 8.11 (s,1H), 7.74
HT-22 (mouse hippocampal cells) cells were grown in Dulbec-
co's Modified Eagle's Medium (DMEM, GIBCO) supplemented with
10% (vol/vol) fetal bovine serum and antibiotics (100 mg/mL
penicillin/streptomycin mix) in a humidified atmosphere at 37 ꢀC
with 5% CO2. 5000 HT-22 cells per well were seeded into a clear 96-
well plate one day prior to assay. Medium was removed from the
(d, J ¼ 5.32 Hz, 1H), 7.49 (d, J ¼ 8.40 Hz, 1H), 7.48 (s, 1H), 7.13 (d,
J ¼ 8.24 Hz, 1H), 6.89-6.83 (m, 2H), 6.77 (s, 1H), 6.47 (d, J ¼ 5.12 Hz,
1H), 6.43 (s, 1H), 2.04 (s, 2H), 3.97 (brt, 2H), 3.59 (s, 3H), 2.54 (br,
2H), 1.76 (br, 4H), 1.53 (s, 6H); HRMS (FAB) calc. for C28H34N5O3
[MþH]þ 488.2662, found: 488.2661.
plate, and cells were treated with 25
m
L solution of each compound
at 10
m
M and incubated at 37 ꢀC for 18 h. 15
mL of 3-[4,5-
4.1.96. N-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)
cyclopropanecarboxamide (232)
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) so-
lution (5 mg/mL) was added to each well and incubated for 3 h. The
formazan formed was dissolved in dimethyl sulfoxide (DMSO) and
absorbance at 570 nm was measured using a microplate reader
(Sunrise, TECAN).OD values from each well were subtracted with
vehicle control and cell viability was calculated by using the signals
from vehicle control as 100%.
Prepared from compound 144 by following the procedure O to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 89%, white
solid, mp 86e87 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.11 (s, 1H), 7.73
(d, J ¼ 4.77 Hz, 1H), 7.51 (d, J ¼ 8.40 Hz, 1H), 7.44 (s, 1H), 7.12 (d,
J ¼ 8.25 Hz, 1H), 6.98-6.87 (m, 3H), 6.47 (dd, J ¼ 5.49, 1.44 Hz, 1H),
6.43 (s, 1H), 4.41 (s, 2H), 3.98 (brt, 2H), 3.77 (s, 3H), 2.55 (t,
J ¼ 6.78 Hz, 2H), 1.78-1.76 (m, 4H), 1,48 (dd, J ¼ 6.96, 3.84 Hz, 2H),
1.07 (dd, J ¼ 6.78, 3.66 Hz, 2H); HRMS (FAB) calc. for C28H32N5O3
[MþH]þ 486.2505, found: 486.2509.
4.2.3. hERG assay
The affinity of small molecules for the hERG channel was
determined using s Predictor hERG fluorescence polarization assay
kit (Invitrogen., USA). The assay is based on the principle of fluo-
rescence polarization where a red fluorescent tracer is displaced
from the hERG channel by compounds that bind to the channel.
Sample triplicates were then run on fluorescence polarization
following the protocol of the manufacturer. Fluorescence at 530/
25-excitation and a 590/35-emission of each well were on a plate
reader, Infinite F200 pro (Techan, Austria).
4.1.97. N-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)
cyclobutanecarboxamide (233)
Prepared from compound 145 by following the procedure O to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 89%, white
solid, mp 77e78 ꢀC: 1H NMR (300 MHz, CD3OD)
d 8.10 (s, 1H), 7.74
(d, J ¼ 5.31 Hz, 1H), 7.45 (d, J ¼ 8.43 Hz, 1H), 7.42 (s, 1H), 7.03 (d,
J ¼ 8.40 Hz, 1H), 6.95-6.87 (m, 3H), 6.48 (d, J ¼ 5.31 Hz, 1H), 6.44 (s,
1H), 4.43 (s, 2H), 3.99 (brt, 2H), 3.72 (s, 3H), 2.81-2.72 (m, 2H), 2.56-
2.42 (m, 4H), 1.94-1.78 (m, 6H); HRMS (FAB) calc. for C29H34N5O3
[MþH]þ 500.2662, found: 500.2672.
4.2.4. Animals and treatment
For acute model study, ICR mice were utilized. ICR mice were
obtained from SAMTACO (Korea). Five microgram in 5
mL of human
A
b3ꢁ40 (62-0-99, American peptide) in PBS was injected into the
deep cortical/hippocampus to 5 weeks old ICR mice (25 g, n ¼ 4,
male) using stereotaxic frame(myNeuroLab, USA). After mice were
anaesthetized with Zoletil50 and 2% Rumpun (1:2 ratio, 0.4 mL/kg)
i.m., the animal was placed into a stereotaxic frame. A small incision
(1 cm) was made over the midline of the skull and exposed the
landmarks of the cranium (bregma and lambda). A small hole was
drilled through the skull. Human Аb3-40 was injected into the deep
cortical/hippocampus by using the following coordinates: AP
-2.0 mm, ML 1.2 mm, DV 1.0 mm from the bregma. Using a Ham-
4.1.98. N-((1H-Benzo[d]imidazole-5-yl)methyl)-1-(4-(4-(2-
aminopyridin-4-yl)butoxy)-3-methoxyphenyl)
cyclopentanecarboxamide (234)
Prepared from compound 146 by following the procedure O to
afford the penultimate intermediate which was deprotected to the
desired product by following the procedure P, yield 83%, white
solid, mp 77e78 ꢀC: 1H NMR (400 MHz, CD3OD)
d 8.08 (s, 1H), 7.74
(d, J ¼ 5.28 Hz, 1H), 7.44 (d, J ¼ 7.88 Hz, 1H), 7.41 (s, 1H), 7.02 (d,
J ¼ 8.20 Hz, 1H), 6.92-6.84 (m, 3H), 6.48 (d, J ¼ 5.12 Hz, 1H), 6.44 (s,
1H), 4.41 (s, 2H), 3.98 (brt, 2H), 3.68 (s, 3H), 3.55 (brt, 2H), 2.49-2.46
(m, 2H), 2.00-1.96 (m, 2H), 1.77-1.71 (m, 8H); HRMS (FAB) calc. for
ilton 25 mL syringe with 27-gauge guide needle, a 5 mL volume
containing human Ab3ꢁ40 was delivered. Test compounds were
administrated via deep cortical/hippocampus or i.p. injection.
For transgenic mice model study, a total of 12 (n ¼ 6 for vehicle;
n ¼ 6 for compound treatment) 5XFAD mice (Jackson lab, ME) was
utilized. 5XFAD mice co-express a total of five FAD mutations,
including APP with three FAD mutations [K670 N/M671L (Swedish),
I716V (Florida) and V717I (London)] and PS1 with two mutations
(M146L and L286V) under the control of neuronal specific Thy1
promoter. In this study, we followed the national guidelines
inconducting animal experiments. All procedures for animal tests
were approved by the Medifron Animal Care and Use Committee
(approval number, Medifron 2017-1, IACUC). All surgical procedures
were performed with care to minimize pain and discomfort.To
evaluate the test compounds on AD model mice, the test com-
pounds were administrated via intraperitoneal injection to mice to
32 week old male 5xFAD mice for 4weeks. Mice were housed
individually with a 12:12 h light: dark cycle (07:00e19:00 h).
C
30H36N5O3 [MþH]þ 514.2818, found: 514.2824.
4.2. Biological study
4.2.1. In vitro QC activity assay
For testing the inhibition activities of test compounds, QC ac-
tivity was fluorometrically evaluated. The used buffer consisted of
25 mM HEPES (Sigma), with pH 7.0, adjusted with HCl. The sub-
strate H-Gln-AMC hydrobromide
(L-glutamine 7-amido-4-
methylcoumarin, BACHEM, Switzerland) was use in a concentra-
tion of 0.4 mM. The auxiliary enzyme pGAPase (50 units, Qiagen,
Germany) was prediluted 1:250 in HEPES. The human QC (10
mL, rhQPCT, R&D systems) was prediluted 1:250 in HEPES. A typical
reaction mixture consisted of 25 L of substrate, 50 L of the test
mg/
m
m
24

N. Van Manh, V.-H. Hoang, V.T.H. Ngo et al.
European Journal of Medicinal Chemistry 226 (2021) 113819
(2019) 73e88.
4.2.5. Y maze test
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Height: 10 cm, Width 5 cm) having identical angle between each
other. The mice were positioned at the center of maze and let to
move freely for 8 min with the maze. The entering order of mice in
the pathway was observed and the entering latency time was
determined when four limbs was entered within the pathway. To
determine the spatial memory, the determined spontaneous
alteration behavior was calculated by following empirical formula
and the actual alteration was assigned to one time at the time that
mice was entered three pathways continuously.
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4.2.6. Ab Sandwich ELISAs
Sandwich ELISA was performed for the quantification of N3pE-
b40 (for acute model mouse brain), Ab42 (for TG mouse brain), and
A
N3pE-Ab42 (for TG mouse brain). The mouse brains were
completely lysed with RIPAbuffer using a sonicator (SONICS, USA),
and ultracentrifuged at 100,000ꢂg, 4 ꢀC for 1 h. Protein concen-
trations were determined using a BCA kit (Thermo scientific).
Sample duplicates were then run on Ab42 and N3pE-Ab42 specific
sandwich ELISAs following the protocol of the manufacturer
(#27418 for N3pE-Ab40, #27711 for Ab42, #27716 for N3pE-Ab42, IBL
Japan). Optical densities at 450 nm of each well were read on a plate
reader, Safire (Tecan, Switzerland). The concentration of the sample
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4.2.7. Statistical analyses
Data was expressed as mean
SEM. All experiments were
repeated at least 3 times. Statistical analyses between groups were
performed using SPSS software(SPSS, Chicago, IL). For group com-
parisons of variables, we analyzed data with either One-way
ANOVA with Bonferroni post-hoc test or independent samples t-
test in SPSS. (ns: p > 0.05, *: p < 0.05, **: p < 0.01,***: p < 0.001).
Declaration of competing interest
[24] S. Jawhar, O. Wirths, S. Schilling, S. Graubner, H.U. Demuth, T.A. Bayer, T. A.
Overexpression of glutaminyl cyclase, the enzyme responsible for pyroglu-
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knock-out rescues the behavioral phenotype in 5XFAD mice, J. Biol. Chem. 286
(2011) 4454e4460.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113819.
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