Potent human glutaminyl cyclase inhibitors as potential anti-Alzheimer's agents: Structure-activity relationship study of Arg-mimetic region

Article abstract of DOI:10.1016/j.bmc.2018.01.015

Pyroglutamate-modified amyloid β peptides (pGlu-Aβ) are highly neurotoxic and promote the formation of amyloid plaques. The pGlu-Aβ peptides are generated by glutaminyl cyclase (QC), and recent clinical studies indicate that QC represents an alternative t

Full text of DOI:10.1016/j.bmc.2018.01.015

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Potent human glutaminyl cyclase inhibitors as potential
anti-Alzheimer’s agents: Structure-activity relationship study
of Arg-mimetic region
Van T.H. Ngo a, Van-Hai Hoang a, Phuong-Thao Tran b, Jihyae Ann a, Minghua Cui c, Gyungseo Park c,
Sun Choi c, Jiyoun Lee d, Hee Kim e, Hee-Jin Ha e, Kwanghyun Choi e, Young-Ho Kim e, 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 Department of Medicinal Chemistry, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoan Kiem, Hanoi, Viet Nam
c National Leading Research Laboratory of Molecular Modeling & Drug Design, College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University,
Seoul 03760, Republic of Korea
d Department of Global Medical Science, Sungshin University, Seoul 01133, Republic of Korea
e Medifron DBT, Sandanro 349, Danwon-Gu, Ansan-City, Gyeonggi-Do 15426, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyroglutamate-modified amyloid b peptides (pGlu-Ab) are highly neurotoxic and promote the formation
of amyloid plaques. The pGlu-Ab peptides are generated by glutaminyl cyclase (QC), and recent clinical
studies indicate that QC represents an alternative therapeutic target to treat Alzheimer’s disease (AD).
We have previously developed a series of QC inhibitors with an extended pharmacophoric scaffold, ter-
med the Arg-mimetic D-region. In the present study, we focused on the structure activity relationship
(SAR) of analogues with modifications in the D-region and evaluated their biological activity. Most com-
pounds in this series exhibited potent activity in vitro, and our SAR analysis and the molecular docking
studies identified compound 202 as a potential candidate because it forms an additional hydrophobic
interaction in the hQC active site. Overall, our study provides valuable insights into the Arg-mimetic
pharmacophore that will guide the design of novel QC inhibitors as potential treatments for AD.
Ó 2018 Elsevier Ltd. All rights reserved.
Received 2 January 2018
Revised 19 January 2018
Accepted 22 January 2018
Available online xxxx
Keywords:
Glutaminyl cyclase inhibitor
Alzheimer’s disease
Beta-amyloid
1. Introduction
truncated species, such as Abn-40/42, where n ranges from 2 to 11,
is also present in the brains of patients with AD.12–14 These N-ter-
b-Amyloid (Ab) peptides are produced by the sequential cleav-
age of the amyloid precursor protein (APP), generating a heteroge-
neous mixture of peptides of various sizes. These Ab peptides not
only exhibit distinct structural and functional features1,2 but also
participate in a wide range of physiological processes, including
neurogenesis,3 neuronal survival,4–6 oxidative stress,7,8 and innate
immunity.9 Although elevated levels of Ab peptides and amyloid
plaques are thought to be the hallmarks of Alzheimer’s disease
(AD) pathology, given the diverse roles of Ab peptides, general
blockade of Ab production may not result in effective therapeutic
outcomes.10,11
minally truncated species undergo enzyme-catalyzed cyclization
to form pyroglutamate (pGlu)-Ab products that are prone to rapid
aggregation and are much more resistant to proteolytic degrada-
tion due to their increased hydrophobicity. Moreover, pGlu-Ab
peptides are more neurotoxic than Ab1–40 and Ab1–42 and promote
the formation of amyloid and tau plaques.15–17 Considering the
prion-like behavior and high neurotoxicity of these cyclized pep-
tides, strategies that specifically target pGlu-Ab peptides may offer
an efficient alternative to conventional anti-Ab strategies.
The pGlu-Ab peptides are produced by glutaminyl cyclase (QC),
which is mainly located in the pituitary, hypothalamus, and brain.18
Human QC is implicated in various pathological conditions, includ-
ing inflammation,19 septic arthritis,20 Huntington’s disease,21 and
Alzheimer’s disease.22,23 In particular, QC is upregulated in the
brains of patients and animal models with AD,22,23 and the enzy-
matic activity of QC correlates with high brain levels of Ab1–8 and
Ab1–40 in patients with AD.24 Treatment with small molecule QC
inhibitors reduces brain pGlu-Ab levels and Ab plaques25,26 and
The brain Ab pool consists of a mixture of peptides containing
30–51 amino acid residues. Ab1–40 and Ab1–42 are the most abun-
dant isoforms and are considered the major initiators of AD patho-
genesis. In addition, a significant amount of the N-terminally
⇑
Corresponding author.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
https://doi.org/10.1016/j.bmc.2018.01.015
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
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V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
decreases gliosis and recovered memory deficits in AD mice.22 In a
recently completed phase IIa clinical trial,27 the QC inhibitor PQ912
exhibited favorable safety and tolerability but also showed a slight
improvement in synaptic and neurological functions in patients
with AD,28 supporting the hypothesis that QC is a promising thera-
peutic target that represents a novel treatment strategy for AD.
Previously reported QC inhibitors have three pharmacophores
designated the A-, B- and C-regions, as shown in Fig. 1.29,30 The
A-region contains a zinc-binding motif (ZBM), the B-region contains
a hydrogen bond donor, and the C-region contains an aromatic ring
that mimics the Phe side chain at the penultimate position to the
N-terminus of the substrate Ab3E-42. Inspired by these findings,
our group previously investigated a series of QC inhibitors with
an extended scaffold based on the N-terminal tripeptide (Glu-
Phe-Arg) of Ab3E-42, and identified an additional pharmacophore,
the D-region, which mimics the binding interaction of the guanidine
moiety of Arg.31 The newly developed QC inhibitors display improved
potency, 5–40-fold increases, compared to the previously reported
inhibitor 1. According to our molecular modeling studies, the Arg
mimetic D-region forms strong interactions with the carboxylate
group of Glu327, supporting our hypothesis that the additional
pharmacophore provides an extra binding interaction. Although
compound 2 was the most potent inhibitor in our previous study
(IC50 = 0.7 nM for hQC), it displayed moderate efficacy in an in vivo
model, likely due to the low blood–brain barrier (BBB) penetration,
whereas compound 3 showed a better in vivo efficacy, reducing the
brain concentrations of Ab and restoring cognitive functions in AD
mice. Based on these findings, we aim to develop a library of
D-region-modified analogues with improved potency and BBB
penetration. All compounds in this series have the same scaffolds
in the A-, B-, and C-regions, but they contain various moieties, includ-
ing substituted piperazines, 2-aminopyridines, anilines, and phenyl
group derivatives, in the D-region. We also synthesized a group of
compounds that contain a phenyl and a benzyl linker group between
the C- and D-regions to study the conformational effect of modifica-
tions at this specific position. We evaluated the QC inhibitory activity
in vitro and the in vivo activity of several selected compounds; we
also analyzed the specific binding interactions between the selected
inhibitor and the QC active site by performing molecular docking
studies.
2. Results and discussion
2.1. Chemistry
A library of 46 compounds with modifications in the D-region
was synthesized. We first synthesized the C/D-region, 4-alkyl(or
aryl)oxy-3-methoxyaniline fragment, and then coupled them to
3-(5-methyl-1H-imidazol-1-yl)propan-1-amine, which represents
the A-region, to obtain the final compounds.
For the synthesis of the 4-phenylpiperidine D-region fragments
(Scheme 1), the protected piperdin-4-one (5) was converted to the
corresponding triflate 6,32 which underwent the Suzuki coupling
reaction with phenylboronic acid derivatives to afford compounds
7 and 8, respectively.33 The phenol 7 was reacted with 2-bromo-5-
nitroanisole through theUllmann reaction to generatethe 4-phenyl-
tetrahydropyridine 9.34 The double bond of compound 8 was
carefully reduced to piperidine 10 and then coupled with
4-nitroguaiacol in the Mitsunobu reaction to afford 4-phenylpiperi-
dine 11.
For the synthesis of 4-phenylpiperazine fragments (Scheme 2),
4-(piperazin-1-yl)phenol 12 was protected by a Boc group or reduc-
tively methylated to yield compound 13 or 14, respectively, which
underwent the Ullmann coupling reaction with 2-bromo-5-nitroa-
nisole to afford compounds 15 and 16. Methyl 3-bromobenzoate 17
was reacted with N-Boc piperazine under Buchwald-Hartwig con-
Fig. 1. Representative structures of QC inhibitors.
Scheme 1. Reagents and conditions: (a) Tf2NPh, LDA, THF, ꢀ78 °C, overnight; (b) phenylboronic acids, Pd(PPh3)4, MeCN, Na2CO3, reflux, overnight; (c) 2-bromo-5-
nitroanisole, Cs2CO3, TMEDA, CuI, DMF, 90 °C, 24 h; (d) H2, Pd/C, MeOH, r.t., 2 h; (e) 4-nitroguaiacol, DEAD, PPh3, DCM, r.t., 3 h.
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V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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Scheme 2. Reagents and conditions: (a) Boc2O, MC, r.t., overnight; (b) HCHO, HCOOH, reflux, overnight; (c) 2-bromo-5-nitroanisole, Cs2CO3, TMEDA, CuI, DMF, 90 °C, 24 h; (d)
t-butyl piperazine-1-carboxylate, BINAP, toluene, Pd(OAc)2, NaOtBu, 100 °C, 15 min; (e) LiAlH4, THF, 0 °C, 1 h; (f) 4-nitroguaiacol, DEAD, PPh3, DCM, r.t., 3 h.
Scheme 3. Reagents and conditions: (a) Br(CH2)nBr or Br(CH2)3OH or Br(CH2)nCOOCH3, Cs2CO3, DMF, 100 °C, 30 min; (b) piperazine derivatives, Cs2CO3, DMF, 70 °C, 30 min;
(c) 1-Boc-4-(2-hydroxyethyl)piperazine, DEAD, PPh3, MC, r.t., overnight; (d) NaOH, MeOH, reflux, 1 h; (e) K2Cr2O7, H2SO4, acetone, H2O, r.t., overnight; (f) EDCꢁHCl, HOBt,
DCM, NH3 (or NH(CH3)2ꢁHCl, 1-Boc-4-piperidinamine, 1-Boc piperazine), DCM, r.t., overnight; (g) TFA, DCM, r.t., overnight; (h) RBr, Cs2CO3, DMF, 100 °C, 30 min; (i) acetyl
chloride, TEA, DCM, r.t., 1 h (for compound 62).
ditions35 to yield compound 18, whose ester underwent reduction
followed by the Mitsunobu reaction with 4-nitroguaiacol to pro-
duce compound 20.
21. Meanwhile, acid 40 was synthesized from the corresponding
alcohol 27 through the unwanted b-elimination of 3-bromo-
propanoate during the O-alkylation with compound 21. The acids
(39–41) were converted to acyclic and cyclic amides (42–51) by
coupling with the corresponding amines, respectively. The N-Boc
piperidine and piperazine derivatives (28–31, 46, and 49) were
deprotected to provide the corresponding amines (52–57), which
underwent N-alkylation or N-acetylation to afford compounds
58–72, respectively.
The 4-carboxamidopiperidine fragment 75 was synthesized
from compound 21 in 3 steps (Scheme 4). For the syntheses of 4-
alkyl-2-aminopyridine fragments (Scheme 4), 2-amino-4-picoline
76 was protected and then alkylated with O-TBS 3-bromopropanol
For the synthesis of 4-alkylpiperidine, 4-alkylpiperazine and
4-amidoalkyl D-region fragments (Schemes 3), 4-nitroguaiacol 21
was condensed with 1-Boc-4-(2-hydroxyethylpiperidine) using
the Mitsunobu reaction to yield 4-ethylpiperazine derivative 28.
The Williamson reaction of compound 21 with dibromoalkanes fol-
lowed by N-alkylation with the corresponding piperazine deriva-
tives produced 4-alkylpiperazine derivatives 29–38, respectively.
For 4-oxopiperidine and piperazine derivatives, the acids (39 and
41) were obtained by the hydrolysis of corresponding esters (25
and 26), which were prepared by the O-alkylation of compound
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V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Scheme 4. Reagents and conditions: (a) 2-(2-bromoethyl)isoindoline-1,3-dione, K2CO3, DMF, 100 °C, 30 min; (b) N2H4ꢁH2O, EtOH, r.t., overnight; (c) 1-Boc piperidine-4-
carboxylic acid, EDCꢁHCl, HOBt, DCM, r.t., overnight; (d) Boc2O, t-BuOH, r.t., overnight; (e) Br(CH2)3OTBS, n-BuLi, THF, ꢀ78 °C; (f) TBAF, THF, r.t., 2 h, then 4-nitroguaiacol,
DEAD, PPh3, MC; (g) RI, Cs2CO3 (or NaH), DMF, heat.
to generate compound 78. After deprotection of compound 78, the
Mitsunobu reaction was performed with 4-nitroguaiacol followed
by N-alkylation with the corresponding alkyl iodides to generate
compounds 80–82, respectively.
The synthesis of the final compounds is described in Scheme 5.
The 4-nitroguaiacol fragments prepared above were reduced by
either reacting them with zinc powder in acidic medium or hydro-
genation to yield the corresponding anilines 83–125, respectively.
All synthesized anilines were coupled in situ with 3-(5-methyl-1H-
imidazol-1-yl)propan-1-amine36 via isothiocyanate to provide the
final thioureas 162, 171–175, 179–182, and 196, as well as the pre-
cursors that were converted to the final compounds 160–161, 166–
168, 170, 177, 184–186, 188–191, 193–195, 199–203, and 205 by
Boc deprotection, 164–165, 176, 187, 192, and 204 by TBS depro-
tection and 163 and 169 by benzoyl deprotection, respectively.
The amide final compounds 178 and 181 were synthesized from
ester 157 by condensation with the corresponding amines. The
1-alkyl-4-phenylpiperazines containing the final compounds
197–198 were prepared from compound 195 by N-alkylation
followed by deprotection.
Scheme 5. Reagents and conditions: (a) Zn, MeOH, AcOH, r.t., 2 h; (b) Pd/C, H2, MeOH, r.t., 3 h; (c) 3-(5-methyl-1H-imidazol-1-yl)propan-1-amine, TCDI, TEA, DCM, r.t.,
overnight; (d) TFA, DCM, r.t., overnight; (e) RBr, NaH, DMF, 0 °C to 100 °C, 30 min; (f) HCl, MeOH, r.t., overnight or TFA:H2O (9:1), DCM, r.t., overnight; (g) NaOH, MeOH, H2O,
reflux, 30 min; (h) NH3 or NH(CH3)2, MeOH, r.t., overnight.
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V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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2.2. In vitro QC inhibition
of the Group I compounds, with IC50 values of 3.8 and 3.6 nM,
respectively, suggesting that their terminal amino groups may be
involved in an additional ionic interaction. When we varied the
length of the spacer between the piperazine substituent and the
C-ring oxygen (compounds 163–168, n = 1–3), we did not observe
any particular trend in the inhibitory effect, suggesting that speci-
fic interactions, such as the ionic interaction inside the binding
pocket, may be more important than steric effects. The 4-phenyl
(171), benzyl (172) and pyrimidine (173–175) derivatives showed
moderate inhibition (IC50 = 12–21.6 nM), and the introduction of a
halogen at the 5-position of the pyrimidine further reduced the
activity. The two piperidine surrogates (176 and 177) were slightly
less active than the corresponding piperazine derivatives (163 and
166).
We performed QC activity assays using a fluorogenic substrate,
Gln-AMC (L-glutamine 7-amido-4-methylcoumarin), and pyroglu-
tamyl peptidase (pGAP) as an auxiliary enzyme37 to evaluate the
ability of the D-region-modified library to inhibit QC. We first
investigated a group of compounds containing the modified piper-
azine ring in the D-region and summarized their structures and
in vitro inhibition as Group I in Table 1. The incorporation of a
methyl group at the 2- or 3-position of the piperazine (160 and
161) led to a slight reduction in activity, probably due to steric
hindrance. Among the 4-N substituted piperazine analogues
(162–175), compounds with a relatively small sized substituent
displayed slightly better activity than compounds with aromatic
and heteroaromatic rings. Specifically, compounds with an alky-
lamine substituent (167 and 170) appeared to be the most potent
Next, we examined the amidoalkyl derivatives in the D-region
since the aminoalkyl derivatives showed potent inhibitory activity
Table 1
IC50 values for the inhibition of hQC by Group I compounds.
Compound
R
n
IC50 (nM)a
1
2
0
2
29.2b
0.7c
160
161
2
2
5.8
9.9
( 1.0)
( 0.7)
162
2
30.8
( 4.2)
163
164
165
166
167
168
169
2
3
4
2
3
4
2
7.4
23.5
4.8
7.5
3.8
( 1.2)
( 9.8)
( 1.0)
( 6.8)
( 1.9)
( 0.6)
( 3.1)
8.6
17.3
170
2
3.6
( 1.6)
171
172
2
2
21.6
12.7
( 5.0)
( 2.9)
173
174
175
176
177
2
2
2
2
2
12
( 3.2)
( 24)
40.3
66.8
9.0
( 45.4)
( 3.7)
( 2.2)
10.6
a
b
c
Values indicate the means of at least three experiments.
Ref. [30].
Ref. [31].
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V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
in our previous study.31 As described in Table 2, compounds with a
primary amide (178–180), tertiary amide (181–183) and piper-
azinyl amide (184–186) appeared to be less potent than the previ-
ously reported aminoalkyl derivatives, probably due to the
decreased basicity of the amide nitrogen that is thought to be
involved in the salt bridge interaction with the enzyme. The amide
surrogate of 4-aminoethylpiperazinyl derivative (188) exhibited a
comparable activity to the compound without the amide group
(166) because both compounds contain the terminal amine.
Among the N-(piperidin-4-yl)amido derivatives (189–194), the N-
(aminoethyl)piperidinyl derivative (193) was the most potent in
the series, with an IC50 of 4.5 nM. In addition, (piperidin-4-yl)car-
bamoyl derivative 190 exhibited comparable activity, even without
the 4-aminoethyl group (IC50 = 5.5 nM), whereas its reverse amide
(194) showed reduced inhibition (IC50 = 15.7 nM). Interestingly,
the length of the spacer between the C-region oxygen and the
D-region (n = 1–3) appears to affect the inhibitory activity of
compounds within this series; compounds with the ethylene
spacer (n = 2) generally displayed better potency, likely due to
the location of the amide nitrogen.
in this series displayed slightly decreased activity compared to
the compounds with a flexible linker. The piperidine analogue
195 and the piperazine analogues 199, 200, and 202 displayed sim-
ilar inhibitory activities. Compounds with a benzyl linker (201 and
202) showed a comparable activity to the compounds with a phe-
nyl linker. Overall, the conformational rigidity between the two
regions did not appear to have a significant impact on QC
inhibition.
Finally, we evaluated the inhibitory activity of analogues con-
taining the 2-aminopyridine group containing and summarized
these results in Table 4. The 2-aminoethylamino substituent in
compound 205 improved the inhibitory activity (IC50 = 1.8 nM)
compared to its parent compound 3 (IC50 = 4.2 nM), which was also
observed with the piperidine derivatives. In contrast, the 2-methy-
lamino pyridine derivative (203) and the 2-hydroxyethylamino
pyridine derivative (204) exhibited slightly decreased activity
compared to compound 3.
2.3. In vivo activity
Next, we modified the linker that connects the C- and the
D-regions to confer a rigid conformation and evaluated the inhibi-
tory activity of these derivatives (Table 3). In general, compounds
Based on the in vitro QC inhibition data, we selected 20 com-
pounds with IC50 values less than 10 nM for further in vivo studies.
We first screened these compounds at one fixed concentration
Table 2
IC50 values for the inhibition of hQC by Group II compounds.
Compound
n
IC50 (nM)a
178
179
180
181
182
183
1
2
3
1
2
3
89.7
19.0
53.8
117
35.4
51.0
( 17)
( 11.0)
( 5.7)
( 19.4)
( 4.0)
( 4.0)
184
185
186
1
2
3
15.0
7.7
7.0
( 2.5)
( 1.8)
( 1.1)
187
1
48.7
7.8
( 3.5)
188
1
( 4.1)
189
190
191
1
2
3
10.3
5.5
16.7
( 1.5)
( 0.2)
( 1.1)
192
1
17.7
( 6.0)
193
194
1
2
4.5
( 4.8)
( 2.0)
15.7
a
Values indicate the means of at least three experiments.
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V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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Table 3
IC50 values for the inhibition of hQC by Group III compounds.
Table 4
IC50 values for the inhibition of hQC by Group IV compounds.
Compound
R
IC50 (nM)a
8.5
Compound
R
IC50 (nM)a
4.2b
195
( 0.5)
( 11.4)
( 8.3)
3
196
197
17.0
16.1
203
204
13.4
8.7
( 5.5)
( 3.4)
198
12.0
( 6.4)
199
200
201
9.4
( 3.4)
( 1.7)
( 4.1)
205
1.8
( 0.7)
9.9
11.5
a
202
6.2
( 2.9)
Values indicate the means of at least three experiments.
Ref. [31].
b
a
Values indicate the means of at least three experiments.
appeared to suppress the formation of AbN3pE-40 by 13.5–30% com-
pared to the vehicle control. In particular, compound 202, which
showed the potent inhibition in vitro with an IC50 value of 6.2
nM, exhibited the most potent AbN3pE-40-lowering effects (30%).
Because compound 202 contains a benzylic linker with a piperi-
dine moiety, this potent in vivo activity may be attributed to its
high BBB penetration.
We performed a parallel artificial membrane permeability assay
(PAMPA)38 that can be translated to the ability of the compounds
to penetrate the blood–brain barrier (BBB). The four most active
compounds in vivo, 185, 190, 199, and 202, showed reasonable
permeability, with a range of 4.9–5.8 for ꢀlogPe, supporting the
(10 lM) in an immortalized hippocampal neuronal cell line (HT-
22) to evaluate cytotoxicity, and found that none of the com-
pounds, with the exception of compound 170, were cytotoxic.
We successively injected human Ab3–40 (5 lg) and each compound
(25 mg/kg) into deep cortical/hippocampal tissues of ICR mice
(male, six weeks old) by intracerebroventricular (icv) administra-
tion to assess the in vivo activity of the selected compounds. We
measured the levels of human AbN3pE-40 in the brain extracts of
these mice on the next day to determine the QC inhibitory activity.
As described in Table 5, compounds 185, 190, 199 and 202
Table 5
QC inhibition in acute model-based studies in vivo.a
In vitro
IC50 (nM)
Cytotoxicity at 10
(% of control)
lM
% inhibition of human AbN3pE-40
formation (icv)
PAMPA
(ꢀlogPe)
hERG FP at 10
(% inhibition)
lM
160
161
163
165
166
167
168
170
176
185
186
188
190
193
195
199
200
202
204
205
5.8
9.9
7.4
4.8
7.5
3.8
9.7
3.6
9.0
7.7
7.0
7.8
5.5
4.5
8.5
9.4
9.9
6.2
8.7
1.8
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
51.1
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
ꢂ100
3.01
NEb
NE
5.8
5.3
6.0
8.7
5.7
10.0
10.0
NT
6.7
5.7
5.7
10.0
5.6
6.4
5.6
4.9
5.6
5.8
10.0
10.0
33.4
2.5
9.7
NT
2.5
28.9
21.1
NT
8.2
1.7
1.9
14.0
5.0
17.8
39.7
52.8
72.8
40.1
44.4
31.5
NTc
5.43
NE
NE
NT
0.19
18.7
8.07
NE
13.5
NE
NE
16.1
NE
30.0
NE
NE
a
Five microliters of human Ab3ꢀ40 in PBS (1 lg/lL) were injected into the deep cortical/hippocampal tissues of 5-week-old ICR mice (25 g, n = 4, males) using a stereotaxic
frame to induce acute Ab toxicity. Test compounds were administered via an icv. A sandwich ELISA was performed to quantify the brain AbN3pE-40 level.
b
NE = not effective
NT = not tested.
c
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8
V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
hypothesis that the in vivo activity of these compounds resulted
from good BBB penetration and QC inhibition. In contrast, the com-
pounds that showed potent in vitro activity but were ineffective
in vivo, such as 167, 168, 188, 204 and 205, exhibited very low per-
meability (ꢀlogPe = 10).
over, the phenyl ring located between the C- and D-regions showed
additional interactions with Phe325 (Fig. 3).
p–p
3. Conclusion
We performed an hERG channel assay for all compounds to
assess potential drug toxicity. Although compounds 185 and 190
slightly inhibited the hERG channel by less than 5%, compounds
199 and 202 moderately inhibited the hERG channel by 52.8%
In the present study, we synthesized and evaluated the biolog-
ical activity of the D-region-modified analogues based on the pre-
viously developed lead compounds 2 and 3. In general, the
modification of the piperazinyl group maintained or slightly
reduced QC inhibition in vitro compared to its parent compound
2, and the rigidification of the linker between the C- and D-regions
did not appear to affect biological activity. Compared to the lead
compound 3, the addition of an aminoethyl group to the 2-
aminopyridine ring of the D-region slightly improved the in vitro
activity up to 2.5-fold. When we tested compounds with low IC50
values (<10 nM) in mice, four compounds with high membrane
penetration (ꢀlogPe = 4.9–5.8) displayed good in vivo activity. In
particular, compound 202 reduced AbN3pE-40 formation in the brain
by 30% compared to the vehicle-treated control. According to the
molecular docking study of compound 202, the benzyl linker
between the C- and D-regions participated in an additional
hydrophobic interaction with Phe325 in the active site. We believe
that our SAR studies added valuable information regarding the
D-region pharmacophore, and we will continue our efforts in lead
optimization to identify QC inhibitors with better penetration and
in vivo activity without any potential toxicity.
and 40.1%, respectively, at 10 lM. Although compound 202 moder-
ately inhibited the hERG channel, overall, this compound exhibited
potent in vitro and in vivo activities and good brain penetration;
therefore, we decided to perform a molecular docking study with
compound 202.
2.4. Molecular modeling
We performed sequential molecular modeling studies using the
X-ray crystal structure of hQC (PDB id: 3PBB)39 to investigate the
interactions between hQC and compound 202. The initial docking
study was conducted using the piperidine protonated form of com-
pound 202 at pH 7.4, utilizing Glide SP (Standard Precision). The
presence of the 5-methyl imidazole in the A-region chelated zinc
and formed an H-bond with the indole NH of Trp329, as well as
several hydrophobic interactions with Leu249, Trp207, and
Ile321. The thiourea group in the B-region contributed to the
appropriate positioning of the C-region phenyl ring for the
hydrophobic interaction with Tyr299. Interestingly, the phenyl
ring located between the C- and D-regions participated in a
hydrophobic interaction with Pro324. The piperidine ring of the
D-region participated in a hydrophobic interaction with Pro326
(Fig. 2A).
4. Experimental
4.1. Chemistry
4.1.1. General
Subsequently, we performed Glide QM-Polarized Ligand Dock-
ing (QPLD) in Maestro. The piperidine ring in the D-region moved
toward Glu327 of the hQC active site (Fig. 2B). The local optimiza-
tion refinement further shifted the Glu327 side chain toward the
piperidine ring of compound 202 (Fig. 2C). We conducted Monte
Carlo minimization to identify the global minimum (Fig. 2D). This
type of sequential optimization of the protein-ligand complexes
induced a remarkable change in the orientation of the Glu327 side
chain, leading to the formation of a salt bridge interaction, along
with the H-bond with the D-region of compound 202. Overall,
the A-region maintained its binding position and interactions
throughout the optimization procedure, whereas the phenyl ring
in the C-region formed an additional H-bond with Tyr299. More-
All chemical reagents were commercially available. Silica gel
column chromatography was performed on silica gel 60, 230–
400 mesh, Merck. 1H NMR spectra were recorded on an a JEOL
JNM-LA 300 at 300 MHz, Bruker Analytik, DE/AVANCE Digital 400
at 400 MHz, a Bruker Analytik, DE/AVANCE Digital 500 at 500
MHz, and a JEOL JNM-ECA-600 at 600 MHz. Mass spectra were
recorded on a VG Trio-2 GC–MS instrument and a 6460 Triple Quad
LC–MS instrument. Melting points were determined on a melting
point Buchi B-540 apparatus and are uncorrected. All final com-
pounds were assessed for purity by high performance liquid chro-
matography (HPLC) on Agilent 1120 Compact LC (G4288A) system
via the following conditions. Column: Agilent TC-C18 column (4.6
mm ꢃ 250 mm, 5
lm). Mobile phase A: MeOH, Mobile phase B:
Fig. 2. Binding modes of compound 202 in hQC after (A) Glide SP docking, (B) QPLD, (C) local optimization, and (D) Monte Carlo minimization. Binding modes of the
protonated form of compound 202 are shown in each step. Interactions with Glu327 are highlighted in red-dotted boxes, and the distances between Glu327 and the terminal
N from the D-region of the ligands are marked with black dashed lines.
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V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
Fig. 3. Refined structure of compound 202 docked with hQC. (A) Binding interactions of compound 202 at the active site of the hQC. Compound 202 is displayed as sticks with
magenta carbon atoms, and Zn2+ is depicted as a purple ball. The interacting residues are depicted as light blue sticks. Hydrogen bonds are depicted as black dashed lines. (B)
A 2D representation of the interactions of compound 202 with the active site residues of hQC. Hydrophobic interactions are marked in light brown. Hydrogen bonds are
shown as red-dotted arrows with the indicated directionality. The
blue wedged line.
p-p stacking interaction is marked as a blue disc and arrow, and the salt bridge interaction is displayed as
0.1% TFA in water (v/v) in 30 min. Wavelength: 254 nM. Flow: 0.7
mL/min. According to the HPLC analyses, all final compounds
showed a purity of ꢄ95%.
stirred at room temperature for 30 min and then filtered through
a Celite filter. The filtrate was portioned between H2O (10 mL)
and DCM (30 mL). The organic layer was separated, dried over
MgSO4, concentrated, and purified by column chromatography to
provide the product.
4.1.2. General procedure
Procedure 4.2: The nitro compound or alkene derivative was dis-
solved in MeOH (or mixture of MeOH and THF) and then 10% Pd/C
was added. The mixture was stirred at room temperature under
hydrogen gas until all starting material was consumed (confirmed
by TCL). The crude mixture was filtered through Celite filter,
washed with MeOH (3 ꢃ 50 mL) and then concentrated. The pro-
duct was subjected to the next step without further purification.
4.1.2.1. Suzuki coupling (Procedure 1). A solution containing the tri-
flate compound (1.0 equiv) and boronic compound (1.0 equiv) in
acetonitrile and sodium carbonate (1.5 equiv) was added to a dried
two-neck flask. Then, the mixture was degassed and back-filled
with dry nitrogen before a suspension of tetrakis(triphenylphos-
phine)palladium(0) (5% mol) in acetonitrile was added. The reac-
tion was refluxed overnight, then cooled to room temperature,
quenched with water, extracted with EtOAc (2 ꢃ 50 mL), dried
over MgSO4, and concentrated. The concentrate was purified by sil-
ica gel chromatography with EA:n-hexane to obtain the product.
4.1.2.5. Williamson reaction (Procedure 5). Alkyl halide (4.0 equiv)
was added to a suspension of 4-nitroguanicol (1.0 equiv) and
cesium carbonate (2.0 equiv) in anhydrous DMF. The reaction mix-
ture was heated to 100 °C for 1 h and then cooled to room temper-
ature before being quenched with water. The mixture was
extracted with EtOAc (2 ꢃ 50 mL). The organic layer was washed
with water 3 times, dried with MgSO4 and concentrated. The con-
centrate was purified by column chromatography to obtain the
product.
4.1.2.2. Ullmann reaction (Procedure 2). A dried two-neck flask was
charged with aryl halide (1 equiv), phenol compound (1 equiv),
cesium carbonate (2 equiv) and N,N0-dimethylethylenediamine
(0.2 equiv) in anhydrous DMF. The reaction was degassed and
back-filled with dry nitrogen before CuI (0.1 equiv) in DMF was
added. The reaction was stirred at 90–100 °C for 24 h, cooled to
room temperature, quenched with NaHCO3 and extracted with
EtOAc (2 ꢃ 50 mL). The organic layer was washed with water 3
times, dried over MgSO4, and concentrated. The concentrate was
purified by silica gel chromatography with EtOAc:n-hexane to
obtain the desired product.
4.1.2.6. Boc protection and deprotection. Procedure 6.1: Triethy-
lamine (1.2 equiv) and di-tert-butyl dicarbonate (2.5 equiv) in
DCM were added to a suspension of the starting amine material
(1.0 equiv) in DCM in an ice bath. The mixture was stirred at room
temperature until starting material was consumed (confirmed by
TLC). Water was added to the mixture and subsequently extracted
with DCM. The organic layer was washed with a 10% aqueous
NaHCO3 solution, water and brine, dried over MgSO4, filtered,
and concentrated in vacuo. The residue was purified by flash chro-
matography to obtain the desired product.
Procedure 6.2: Trifluoroacetic acid (10 equiv) was added to the
solution of the boc-protected compound (1.0 equiv) in DCM
(DCM:TFA = 1:1 (v/v)). Then, the mixture was stirred at room tem-
perature until the starting material was consumed and evaporated.
The residue was dissolved in MeOH and purified on an ion-
4.1.2.3. Mitsunobu reaction (Procedure 3). Triphenylphosphine (1.3
equiv) was added to a solution of 4-nitroguanicol (1.0 equiv) in
DCM under a nitrogen atmosphere, followed by the addition of a
primary alcohol (1.2 equiv) and a solution of diethyl azodicarboxy-
late (1.3 equiv) in DCM. After the solution was stirred for 30 min at
room temperature, the reaction was poured onto a column of silica
and was eluted with EtOAc:n-hexane to yield the desired product.
4.1.2.4. Reduction (Procedure 4). Procedure 4.1: AcOH (5 equiv) and
Zn dust (5 equiv) were added to a solution of a nitro compound
in MeOH (10 mL) at room temperature. The reaction mixture was
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10
V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
exchange column to obtain the desired product or subjected to the
next step without further purification.
4.1.3.3. 1-(4-(2-(4-Acetylpiperazin-1-yl)ethoxy)-3-methoxyphenyl)-
3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (162). Starting
with compound 111 as following the general procedure 8, com-
pound 162 was obtained as white solid, 87% yield, mp = 55–56 °C.
1H NMR (300 MHz, CDCl3) d 7.63 (s, 1H), 7.39 (s, 1H), 6.89 (d, J =
9.15 Hz, 1H), 6.75–6.72 (m, 3H), 5.97 (s, 1H), 4.15 (t, J = 5.89 Hz,
2H), 3.90 (t, J = 7.14 Hz, 2H), 3.81 (s, 3H), 3.69–3.62 (m, 4H), 3.49
(t, J = 4.95 Hz, 2H), 2.87 (t, J = 5.67 Hz, 2H), 2.60 (t, J = 5.1 Hz, 2H),
2.56 (t, J = 5.10 Hz, 2H), 2.18 (s, 3H), 2.09 (s, 3H), 2.05 (p, J = 7.14
4.1.2.7. TBDMS deprotection (Procedure 7). A solution of conc. HCl
(0.1 mL) was added to the solution of t-butyl dimethyl silyl ether
dissolved in MeOH (10 mL). The mixture was stirred at room tem-
perature overnight and then concentrated. The concentrate was
dissolved in DCM and washed with water. The organic layer was
concentrated to obtain the desired product or purified by flash
chromatography.
Hz, 2H). MS (ESI) m/z 475 [M+H]+. HRMS (FAB) m/z calc. for C23H34
-
N6O3S [M+H]+ 475.2492, found: 475.2495. Anal. HPLC 96.7% (Rt =
3.76 min).
4.1.2.8. Thiourea coupling (Procedure 8). A solution of the amine
(1.0 equiv) in anhydrous DCM was added to a solution of 1,10-thio-
carbonyldiimidazole (1.02 equiv) in anhydrous DCM in a dropwise
manner under nitrogen gas at room temperature. The reaction
mixture was stirred at room temperature until the starting mate-
rial was consumed. Then, the solution of 3-(5-methyl-1H-imida-
zol-1-yl)propan-1-amine (1.1 equiv) in anhydrous DCM was
added dropwise, followed by the addition of triethylamine (3.0
equiv), and stirred at room temperature until the reaction was
complete (monitored with TLC). The mixture was washed with
water 2 times, the combined organic layer was dried over MgSO4,
concentrated, and purified by column chromatography.
4.1.3.4. 1-(4-(2-(4-(2-Hydroxyethyl)piperazin-1-yl)ethoxy)-3-meth-
oxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(163). An excess solution of 10% NaOH was added in solution of
155 in MeOH (10 mL). The mixture was heated to reflux for 30
min, diluted with DCM, washed with water. The organic layer
was concentrated, purified by column chromatography to afford
compound 163 as a white solid, 35% yield, mp = 64–65 °C. 1H
NMR (300 MHz, CDCl3) d 7.70 (s, 1H), 7.36 (s, 1H), 6.89 (d, J =
8.79 Hz, 1H), 6.75–6.72 (m, 3H), 5.99 (s, 1H), 4.15 (t, J = 6.06 Hz,
2H), 3.89 (t, J = 7.14 Hz, 2H), 3.83 (s, 3H), 3.69–3.60 (m, 4H), 2.87
(t, J = 5.85 Hz, 2H), 2.63–2.54 (m, 10H), 2.18 (s, 3H), 2.05 (p, J =
7.32 Hz, 2H). MS (ESI) m/z 477 [M+H]+. HRMS (FAB) m/z calc. for
4.1.2.9. EDC coupling (Procedure 9). EDC.HCl (1.1 equiv) and N,N-
diisopropylethylamine (2.2 equiv) were added to a solution of the
amine compound (1.0 equiv), acid compound (1.0 equiv) and HOBt
(1.1 equiv) in DCM. The mixture was stirred for 24 h at room tem-
perature under nitrogen. The solvent was removed in vacuo and
the residue purified by column chromatography on silica gel eluted
with DCM:MeOH to produce the desired compound.
C
23H36N6O3S [M+H]+ 477.2647, found: 477.2633. Anal. HPLC
98.5% (Rt = 3.63 min).
4.1.3.5. 1-(4-(3-(4-(2-Hydroxyethyl)piperazin-1-yl)propoxy)-3-meth-
oxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (1 6
4). Starting with compound 149 as following the general proce-
dure 7, compound 164 was obtained as off white solid, 47% yield,
mp = 78–79 °C. 1H NMR (500 MHz, CD3OD) d 7.58 (s, 1H), 6.95 (d,
J = 8.50 Hz, 1H), 6.90 (s, 1H), 6.75 (dd, J = 2.15, 8.50 Hz, 1H), 6.66
(s, 1H), 4.05 (t, J = 6.05 Hz, 2H), 3.98 (t, J = 7.20 Hz, 2H), 3.80 (s,
3H), 3.68 (t, J = 6.00 Hz, 2H), 3.60 (t, J = 6.35 Hz, 2H), 2.58–2.55
(m, 8H), 2.54 (t, J = 6.00 Hz, 4H), 2.21 (s, 3H), 2.06 (p, J = 7.10 Hz,
2H), 1.97 (p, J = 6.95 Hz, 2H). MS (ESI) m/z 491 [M+H]+. HRMS
(ESI) calc. for C24H38N6O3S [M+H]+ 491.2799, found 491.2795. Anal.
HPLC 100% (Rt = 2.95 min).
4.1.2.10. N-Alkylation (Procedure 10). A mixture of the alkyl halide,
nitrogen-containing compound and excess base (NaH for com-
pounds 80 and 81, Cs2CO3 for other compounds) in DMF was stir-
red at 60 °C for 30 min. The reaction was quenched with water and
extracted with EA. The organic layer was washed with water and
brine, concentrated and purified by column chromatography.
4.1.3. Final compounds
4.1.3.1. 1-(3-Methoxy-4-(2-(2-methylpiperazin-1-yl)ethoxy)phenyl)-
4.1.3.6. 1-(4-(4-(4-(2-Hydroxyethyl)piperazin-1-yl)butoxy)-3-meth-
oxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(165). Starting with compound 150 as following the general proce-
dure 7, compound 165 was obtained desired product as a white
solid, 84% yield, mp = 58–59 °C. 1H NMR (300 MHz, CD3OD) d
7.59 (s, 1H), 6.95 (d, J = 8.43 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H), 6.76
(dd, J = 2.22, 8.43 Hz, 1H), 6.66 (s, 1H), 4.03 (t, J = 6.45 Hz, 2H),
3.97 (t, J = 7.14 Hz, 2H), 3.80 (s, 3H), 3.68 (t, J = 6.03 Hz, 2H), 3.61
(t, J = 6.78 Hz, 2H), 2.54–2.41 (m, 12H), 2.22 (d, J = 0.90 Hz, 3H),
2.05 (p, J = 4.95 Hz, 2H), 1.79–1.73 (m, 4H). MS (ESI) m/z 505 [M
+H]+. HRMS (ESI) calc. for C25H40N6O3S [M+H]+ 505.2955, found
505.2957. Anal. HPLC 98.4% (Rt = 2.99 min).
3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(160). Starting
with compound 126 as following the general procedure 6.2, com-
pound 160 was obtained as a white solid, 42% yield, mp = 77–78 °C.
1H NMR (300 MHz, CDCl3) d 7.60 (s, 1H), 7.37 (s, 1H), 6.89 (d, J =
8.43 Hz, 1H), 6.75–6.73 (m, 3H), 5.92 (s, 1H), 4.13 (t, J = 6.42 Hz,
2H), 3.89 (t, J = 6.96 Hz, 2H), 3.83 (s, 3H), 3.66 (q, J = 7.53 Hz, 2H),
3.22–3.13 (m, 1H), 2.93–2.78 (m, 5H), 2.56–2.41 (m, 3H), 2.18 (d,
J = 0.93 Hz, 3H), 2.05 (p, J = 7.32 Hz, 2H), 1.08 (d, J = 5.88 Hz, 3H).
MS (ESI) m/z 447 [M+H]+. HRMS (FAB) m/z calc. for C22H34N6O2S
[M+H]+ 447.2542, found: 447.2535. Anal. HPLC 97.7% (Rt = 3.45
min).
4.1.3.2. 1-(4-(2-(3,5-Dimethylpiperazin-1-yl)ethoxy)-3-methoxyphe-
nyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (161). Start-
ing with compound 127 as following the general procedure 6.2,
compound 161 was obtained as white solid, 78% yield, mp = 58–
59 °C. 1H NMR (300 MHz, CD3OD) d 7.60 (s, 1H), 6.95 (d, J = 8.61
Hz, 1H), 6.94 (d, J = 2.37 Hz, 1H), 6.75 (dd, J = 8.43, 2.40 Hz, 1H),
6.67 (s, 1H), 4.14 (t, J = 5.13 Hz, 2H), 3.98 (t, J = 6.96 Hz, 2H), 3.81
(s, 3H), 3.59 (t, J = 6.96 Hz, 2H), 3.11 (br, 4H), 2.84 (t, J = 5.31 Hz,
2H), 2.22 (d, J = 0.90 Hz, 3H), 2.08–1.94 (m, 4H), 1.16 (d, J = 6.21
4.1.3.7. 1-(4-(2-(4-(2-Aminoethyl)piperazin-1-yl)ethoxy)-3-methoxy-
phenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(166). Starting with compound 128 as the general procedure 6.2,
compound 166 was obtained as white solid, 85% yield, mp = 81–
82 °C. 1H NMR (300 MHz, CD3OD) d 7.59 (d, J = 0.93 Hz, 1H), 6.96
(d, J = 8.43 Hz, 1H), 6.92 (d, J = 2.40 Hz, 1H), 6.75 (dd, J = 8.40, 2.37
Hz, 1H), 6.66 (s, 1H), 4.14 (t, J = 5.49 Hz, 2H), 3.97 (t, J = 6.96 Hz,
2H), 3.81 (s, 3H), 3.60 (t, J = 6.75 Hz, 2H), 2.82 (t, J = 5.49 Hz, 2H),
2.79 (t, J = 6.60 Hz, 2H), 2.68 (br, 4H), 2.55 (br, 4H), 2.47 (t, J = 6.42
Hz, 2H), 2.22 (d, J = 0.93 Hz, 3H), 2.03 (p, J = 6.96 Hz, 2H). MS (ESI)
m/z 476 [M+H]+. HRMS (FAB) m/z calc. for C23H37N7O2S [M+H]+
476.2808, found: 476.2823. Anal. HPLC 96.5% (Rt = 3.52 min).
Hz, 6H). MS (ESI) m/z 461 [M+H]+. HRMS (FAB) m/z calc. for C23H36
-
N6O2S [M+H]+ 461.2699, found: 461.2705. Anal. HPLC 95.7% (Rt =
3.48 min).
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V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
11
4.1.3.8. 1-(4-(3-(4-(2-Aminoethyl)piperazin-1-yl)propoxy)-3-methox-
yphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
4.1.3.13. 1-(4-(2-(4-Benzylpiperazin-1-yl)ethoxy)-3-methoxyphenyl)-
3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (172). Starting
(167). Starting with compound 129 as following the general proce-
dure 6.2, compound 167 was obtained as a red solid, 65% yield, mp
= 107–108 °C. 1H NMR (500 MHz, CD3OD) d 7.58 (s, 1H), 6.95 (d, J =
8.55 Hz, 1H), 6.91 (d, J = 2.05 Hz, 1H), 6.75 (dd, J = 2.25, 8.35 Hz,
1H), 6.66 (s, 1H), 4.03 (t, 2H, J = 6.20 Hz), 3.98 (t, J = 7.20 Hz, 2H),
3.80 (s, 3H), 3.60 (t, J = 6.60 Hz, 2H), 2.74 (t, J = 6.70 Hz, 2H),
2.59–2.55 (m, 8H), 2.48–2.44 (m, 4H), 2.23 (s, 3H), 2.09 (p, J =
7.10 Hz, 2H), 1.99 (p, J = 6.95 Hz, 2H). MS (ESI) m/z 490 [M+H]+.
with compound 93 as following the general procedure 8, com-
pound 172 was obtained as a white solid, 47% yield, mp = 78–80
°C. 1H NMR (300 MHz, CD3OD) d 7.60 (s, 1H), 7.32–7.26 (m, 5H),
6.96 (d, J = 8.58 Hz, 1H), 6.90 (d, J = 0.75 Hz, 1H), 6.76 (dd, J =
2.37, 8.40 Hz, 1H), 6.66 (s, 1H), 4.14 (t, J = 5.49 Hz, 2H), 3.99 (t, J
= 7.14 Hz, 2H), 3.79 (s, 3H), 3.59 (t, J = 5.49 Hz, 2H), 3.53 (s, 2H),
2.83 (t, J = 5.49 Hz, 2H), 2.66–2.53 (m, 8H), 2.21 (s, 3H), 2.05 (p, J
= 7.14 Hz, 2H). MS (FAB) m/z 523 [M+H]+. HRMS (FAB) m/z calcd
for C28H38N6O2S [M+H]+ 523.2855, found: 523.2861. Anal. HPLC
95.7% (Rt = 3.32 min).
HRMS (ESI) calc. for
490.2968. Anal. HPLC 100% (Rt = 2.87 min).
C
24H39N7O2S [M+H]+ 490.2959, found
4.1.3.9. 1-(4-(4-(4-(2-Aminoethyl)piperazin-1-yl)butoxy)-3-methoxy-
phenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
4.1.3.14.
1-(3-Methoxy-4-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)
ethoxy)phenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(173). Starting with compound 94 as following the general proce-
dure 8, compound 173 was obtained as white solid, 94% yield, mp
= 71–72 °C. 1H NMR (300 MHz, CDCl3) d 8.31 (d, J = 4.74 Hz, 2H),
7.77 (s, 1H), 7.42 (s, 1H), 6.91 (d, J = 8.97 Hz, 1H), 6.76–6.74 (m,
3H), 6.50 (t, J = 4.74 Hz, 1H), 6.08 (br, 1H), 4.25–4.15 (m, 2H), 3.95–
3.80 (m, 9H), 3.66 (dd, J = 13.74, 6.60 Hz, 2H), 2.91 (t, J = 5.85 Hz,
2H), 2.75–2.60 (m, 4H), 2.18 (s, 3H), 2.13–1.97 (m, 2H). MS (ESI) m/
z 511 [M+H]+. HRMS (FAB) m/z calc. for C25H34N8O2S [M+H]+
511.2604, found: 511.2609. Anal. HPLC 97.5% (Rt = 3.58 min).
(168). Starting from compound 130 as following the general pro-
cedure 6.2, compound 168 was obtained as a white solid, 71% yield,
mp = 131–132 °C. 1H NMR (300 MHz, CD3OD) d 7.59 (d, J = 0.90 Hz,
1H), 6.95 (d, J = 8.31 Hz, 1H), 6.89 (d, J = 2.40 Hz, 1H), 6.76 (dd, J =
2.58, 6.03 Hz, 1H), 6.66 (s, 1H), 4.03 (t, J = 5.88 Hz, 2H), 3.97 (t, J =
7.14 Hz, 2H), 3.80 (s, 3H), 3.59 (q, J = 6.42 Hz, 2H), 2.75 (t, J = 6.60
Hz, 2H), 2.53–2.47 (br, 12H), 2.22 (d, J = 1.11 Hz, 3H), 2.03 (p, J =
7.50 Hz, 2H), 1.77–1.67 (m, 4H). MS (ESI) m/z 504 [M+H]+. HRMS
(ESI) calc. for C25H41N7O2S [M+H]+ 504.3115, found 504.3127. Anal.
HPLC 99.5% (Rt = 2.99 min).
4.1.3.10.
1-(4-(2-(4-(2-(2-Hydroxyethoxy)ethyl)piperazin-1-yl)
4.1.3.15. 1-(4-(2-(4-(5-Fluoropyrimidin-2-yl)piperazin-1-yl)ethoxy)-
3-methoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(174). Starting with compound 95 as following the general proce-
dure 8, compound 174 was obtained as white solid, 78% yield, mp
= 71–72 °C. 1H NMR (300 MHz, CDCl3) d 8.20 (s, 2H), 7.79 (s, 1H),
7.42 (s, 1H), 6.95–6.87 (m, 1H), 6.80–6.68 (m, 3H), 6.10 (br, 1H),
4.25–4.13 (m, 2H), 3.95–3.86 (m, 2H), 3.83 (s, 3H), 3.82–3.75 (m,
4H), 3.72–3.60 (m, 2H), 2.95–2.85 (m, 2H), 2.70–2.60 (m, 4H),
2.18 (s, 3H), 2.13–1.97 (m, 2H). MS (ESI) m/z 529 [M+H]+. HRMS
ethoxy)-3-methoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
thiourea (169). Starting with compound 156 as following the
experiment procedure used for compound 163 to obtained com-
pound 169 as a white solid, 50% yield, mp = 64–65 °C. 1H NMR
(300 MHz, CD3OD) d 7.66 (s, 1H), 6.96 (d, J = 8.43 Hz, 1H), 6.93
(d, J = 2.19 Hz, 1H), 6.75 (dd, J = 8.43, 2.4 Hz, 1H), 6.70 (s, 1H),
4.18 (t, J = 5.31 Hz, 2H), 3.99 (t, J = 7.14 Hz, 2H), 3.81 (s, 3H), 3.66
(t, J = 4.92 Hz, 4H), 3.62–3.52 (m, 4H), 2.87 (t, J = 5.31 Hz, 2H),
2.75 (br, 10H), 2.23 (s, 3H), 2.04 (p, J = 7.14 Hz, 2H). MS (ESI) m/z
521 [M+H]+. HRMS (FAB) m/z calc. for C25H40N6O4S [M+H]+
521.2905, found: 521.2896. Anal. HPLC 99.3% (Rt = 3.09 min).
(FAB) m/z calc. for
529.2508. Anal. HPLC 97.3% (Rt = 3.56 min).
C
25H33FN8O2S [M+H]+ 529.2509, found:
4.1.3.16. 1-(4-(2-(4-(5-Chloropyrimidin-2-yl)piperazin-1-yl)ethoxy)-
3-methoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(175). Starting with compound 96 as following the general proce-
dure 8, compound 175 was obtained as white solid, 35% yield, mp
= 87–88 °C. 1H NMR (300 MHz, CDCl3) d 8.22 (s, 2H), 7.72 (s, 1H),
7.40 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.76–6.72 (m, 3H), 6.04 (br,
1H), 4.18 (t, J = 5.9 Hz, 2H), 3.89 (m, 2H), 3.83–3.80 (m, 7H), 3.65
(m, 2H), 2.89 (t, J = 5.9 Hz, 2H), 2.65 (t, J = 5.1 Hz, 2H), 2.18 (s,
3H), 2.05 (m, 2H). MS (ESI) m/z 545 [M+H]+. HRMS (FAB) m/z calc.
for C25H33ClN8O2S [M+H]+ 545.2214, found: 545.2220. Anal. HPLC
99.0% (Rt = 3.73 min).
4.1.3.11. 1-(4-(2-(4-(2-(2-Aminoethoxy)ethyl)piperazin-1-yl)ethoxy)-
3-methoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(170). Starting with compound 131 as following the general proce-
dure 6.2, compound 170 was obtained as white solid, 73% yield,
mp = 57–58 °C. 1H NMR (300 MHz, CD3OD) d 7.60 (s, 1H), 6.96 (d,
J = 8.43 Hz, 1H), 6.92 (d, J = 2.73 Hz, 1H), 6.75 (dd, J = 8.43, 2.37
Hz, 1H), 6.66 (s, 1H), 4.14 (t, J = 5.49 Hz, 2H), 3.97 (t, J = 7.14 Hz,
2H), 3.81 (s, 3H), 3.63–3.57 (m, 4H), 3.50 (t, J = 5.13 Hz, 2H),
2.84–2.80 (m, 4H), 2.61 (br, 10H), 2.22 (d, J = 0.93 Hz, 3H), 2.03
(p, J = 6.96 Hz, 2H). MS (ESI) m/z 520 [M+H]+. HRMS (FAB) m/z calc.
for C25H41N7O3S [M+H]+ 520.3070, found: 520.3076. Anal. HPLC
96.1% (Rt = 3.35 min).
4.1.3.17. 1-(4-(2-(1-(2-Hydroxyethyl)piperidin-4-yl)ethoxy)-3-meth-
oxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
4.1.3.12. 1-(3-Methoxy-4-(2-(4-phenylpiperazin-1-yl)ethoxy)phenyl)-
3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(171). Starting
(176). Starting with compound 151 as following the general proce-
dure 7, compound 176 was obtained as a pale red solid, 62% yield,
mp = 62–63 °C. 1H NMR (300 MHz, CD3OD) d 7.58 (d, J = 1.11 Hz,
1H), 6.95 (d, J = 8.61 Hz, 1H), 6.90 (d, J = 2.19 Hz, 1H), 6.75 (dd, J
= 2.40, 8.43 Hz, 1H), 6.66 (s, 1H), 4.05 (t, J = 6.21 Hz, 2H), 3.99 (t,
J = 7.32 Hz, 2H), 3.81 (s, 3H), 3.67 (t, J = 6.24 Hz, 2H), 3.59 (t, J =
6.78 Hz, 2H), 2.98–2.95 (m, 2H), 2.53 (t, J = 6.21 Hz, 2H), 2.21 (d,
J = 1.08 Hz, 3H), 2.14–1.98 (m, 5H), 1.79–1.68 (m, 4H), 1.37–1.28
with compound 92 as following the general procedure 8, com-
pound 171 was obtained as white solid, 50% yield, mp = 64–65
°C. 1H NMR (300 MHz, CD3OD) d 7.60 (s, 1H), 7.22 (t, J = 7.97 Hz,
2H), 7.00–6.92 (m, 4H), 6.83 (t, J = 6.15 Hz, 1H), 6.76 (dd, J = 8.40,
2.73 Hz, 1H), 6.67 (s, 1H), 4.19 (t, J = 5.31 Hz, 2H), 3.97 (t, J = 7.14
Hz, 2H), 3.82 (s, 3H), 3.59 (t, J = 6.57 Hz, 2H), 3.20 (t, J = 4.95 Hz,
4H), 2.88 (t, J = 5.52 Hz, 2H), 2.80 (t, J = 4.95 Hz, 4H), 2.22 (d, J =
0.93 Hz, 3H), 2.03 (p, J = 6.96 Hz, 2H). MS (ESI) m/z 509 [M+H]+.
HRMS (FAB) m/z calc. for C27H37N6O2S [M+H]+ 509.2699, found:
509.2693. Anal. HPLC 96.9% (Rt = 3.81 min).
(m, 2H). MS (ESI) m/z 476 [M+H]+. HRMS (ESI) m/z calcd for C24H37
-
N5O3S [M+H]+ 476.2690, found: 476.2677. Anal. HPLC 100.0% (Rt =
2.96 min).
Please cite this article in press as: Ngo V.T.H., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.015

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V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
4.1.3.18. 1-(4-(2-(1-(2-Aminoethyl)piperidin-4-yl)ethoxy)-3-methox-
yphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
5.97 (t, J = 7.5 Hz, 1H), 4.33 (t, J = 7.2 Hz, 2H), 3.87 (t, J = 7.5 Hz,
2H), 3.80 (s, 3H), 3.64 (q, J = 7.5 Hz, 2H), 3.04 (s, 3H), 2.95 (s, 3H),
2.88 (t, J = 6.9 Hz, 2H), 2.15 (d, J = 0.6 Hz, 3H), 2.02 (p, J = 6.9 Hz,
2H). MS (ESI) m/z 420 [M+H]+. HRMS (ESI) calc. for C20H30N5O3S
[M+H]+ 420.2064, found 420.2065. Anal. HPLC 97.4% (Rt = 4.56
min).
(177). Starting with compound 132 as following the general proce-
dure 6.2, compound 177 was obtained as an off white solid, 76%
yield, mp = 76–77 °C. 1H NMR (300 MHz, CD3OD) d 7.58 (d, J =
1.08 Hz, 1H), 6.95 (d, J = 8.43 Hz, 1H), 6.90 (d, J = 2.19 Hz, 1H),
6.75 (dd, J = 2.40, 8.43 Hz, 1H), 6.66 (t, J = 2.55 Hz, 1H), 4.05 (t, J
= 6.42 Hz, 2H), 3.99 (t, J = 7.14 Hz, 2H), 3.81 (s, 3H), 3.59 (t, J =
7.14 Hz, 2H), 2.94 (br, 2H), 2.78 (t, J = 6.42 Hz, 2H), 2.45 (t, J =
7.32 Hz, 2H), 2.21 (d, J = 1.11 Hz, 3H), 2.04–1.98 (m, 5H), 1.79–
1.68 (m, 4H), 1.37–1.28 (m, 2H). MS (ESI) m/z 475 [M+H]+. HRMS
(ESI) calc. for C24H38N6O2S [M+H]+ 475.2850, found 475.2837. Anal.
HPLC 99.5% (Rt = 2.89 min).
4.1.3.24. 4-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
thioureido)phenoxy)-N,N-dimethylbutanamide (183). Starting with
compound 100 as following the general procedure 8, compound
183 was obtained as white solid, 60% yield, mp = 70–72 °C. 1H
NMR (300 MHz, CDCl3) d 7.59 (s, 1H), 7.38 (s, 1H), 6.96 (d, J =
8.25 Hz, 1H), 6.74–6.69 (m, 3H), 5.98 (s, 1H), 4.14 (t, J = 6.24 Hz,
2H), 3.91 (t, J = 7.14 Hz, 2H), 3.82 (s, 3H), 3.69 (q, J = 6.21 Hz, 2H),
3.02 (s, 3H), 2.95 (s, 3H), 2.55 (t, J = 6.96 Hz, 2H), 2.17 (d, J = 0.90
Hz, 3H), 2.14 (p, J = 6.78 Hz, 2H), 2.09 (p, J = 7.32 Hz, 2H). MS
(FAB) m/z 434 [M+H]+. HRMS (FAB) m/z calcd for C21H31N5O3S [M
+H]+ 434.2226, found: 434.2228. Anal. HPLC 98.6% (Rt = 3.40 min).
4.1.3.19. 2-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
thioureido)-phenoxy)acetamide (178). Starting with compound
157 as following the reaction with NH3 to obtained compound
178, 62% yield, mp = 147–149 °C. 1H NMR (300 MHz, CD3OD) d
7.59 (s, 1H), 7.02 (d, J = 2.37 Hz, 1H), 7.00 (d, J = 8.43 Hz, 1H),
6.79 (dd, J = 2.40, 8.61 Hz, 1H), 6.66 (s, 1H), 4.48 (s, 2H), 4.00 (t, J
= 7.32 Hz, 2H), 3.85 (s, 3H), 3.61 (t, J = 6.78 Hz, 2H), 2.22 (d, J =
0.90 Hz, 3H), 2.08 (p, J = 7.14 Hz, 2H). MS (FAB) m/z 378 [M+H]+.
HRMS (FAB) m/z calcd for C17H23N5O3S [M+H]+ 378.1600 found:
378.1604. Anal. HPLC 97.0% (Rt = 3.99 min).
4.1.3.25. 1-(3-Methoxy-4-(2-oxo-2-(piperazin-1-yl)ethoxy)phenyl)-3-
(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (184). Starting with
compound 133 as following the general procedure 6.2, compound
184 was obtained as white solid, 34% yield, mp = 124–125 °C. 1H
NMR (300 MHz, CDCl3) d 7.76 (s, 1H), 7.40 (s, 1H), 6.91 (d, J =
8.43 Hz, 1H), 6.77 (d, J = 2.22 Hz, 1H), 6.70 (s, 1H), 6.69 (dd, J =
8.07, 2.37 Hz, 1H), 6.21 (t, J = 7.14 Hz, 1H), 4.77 (s, 2H), 3.90 (t, J
= 7.35 Hz, 2H), 3.82 (s, 3H), 3.65 (t, J = 7.14 Hz, 2H), 3.39–3.55 (m,
4H), 2.88–2.84 (m, 4H), 2.18 (d, J = 0.75 Hz, 3H), 2.03 (p, J = 7.14
Hz, 2H). MS (ESI) m/z 447 [M+H]+. HRMS (FAB) calc. for C21H31N6-
O3S [M+H]+ 447.2173, found 447.2165. Anal. HPLC 95.7% (Rt = 4.34
min).
4.1.3.20. 3-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
thioureido)phenoxy)propanamide (179). Starting with compound
97 as following the general procedure 8, compound 179 was
obtained as white solid, 78% yield, mp = 115–117 °C. 1H NMR
(300 MHz, CD3OD) d 7.64 (s, 1H), 6.98 (d, J = 8.58 Hz, 1H), 6.92
(d, J = 2.37 Hz, 1H), 6.75 (dd, J = 8.43, 2.40 Hz, 1H), 6.69 (s, 1H),
4.24 (t, J = 6.24 Hz, 2H), 3.98 (t, J = 7.14 Hz, 2H), 3.80 (s, 3H), 3.59
(t, J = 7.14 Hz, 2H), 2.67 (t, J = 6.24 Hz, 2H), 2.22 (d, J = 0.93 Hz,
3H), 2.03 (p, J = 7.14 Hz, 2H). MS (ESI) m/z 392 [M+H]+. MS (HRMS)
calc. for C18H26N5O2S [M+H]+ 392.1751, found 392.1750. Anal.
HPLC 99.2% (Rt = 3.22 min).
4.1.3.26. 1-(3-Methoxy-4-(3-oxo-3-(piperazin-1-yl)propoxy)phenyl)-
3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(185). Starting
with compound 134 as following the general procedure 6.2, com-
pound 185 was obtained as white solid, 31% yield, mp = 155–
157 °C. 1H NMR (300 MHz, CDCl3) d 7.65 (s, 1H), 6.97 (d, J = 8.61
Hz, 1H), 6.93 (d, J = 3.00 Hz, 1H), 6.74 (dd, J = 8.40, 2.37 Hz, 1H),
6.68 (s, 1H), 4.27 (t, J = 6.24 Hz, 2H), 3.98 (t, J = 7.14 Hz, 2H), 3.80
(s, 3H), 3.71–3.57 (m, 6H), 2.97 (t, J = 4.95 Hz, 2H), 2.88 (t, J =
5.85 Hz, 4H), 2.22 (d, 1.11 Hz, 3H), 2.04 (p, J = 7.14 Hz, 2H). MS
(ESI) m/z 461 [M+H]+. HRMS (FAB) calc. for C22H33N6O3S [M+H]+
461.2329, found 461.2323. Anal. HPLC 95.0% (Rt = 3.15 min).
4.1.3.21. 4-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
thioureido)phenoxy)butanamide (180). Starting with compound 98
as following the general procedure 8, compound 180 was obtained
as white solid, 20% yield, mp = 158–160 °C. 1H NMR (300 MHz,
CD3OD) d 7.56 (s, 1H), 6.92 (d, J = 8.61 Hz, 1H), 6.86 (s, 1H), 6.71
(d, J = 8.61 Hz, 1H), 6.63 (s, 1H), 4.00 (t, J = 6.24 Hz, 2H), 3.96 (t, J
= 7.14 Hz, 2H), 3.77 (s, 3H), 3.57 (t, J = 6.78 Hz, 2H), 2.39 (t, J =
7.32 Hz, 2H), 2.17 (d, J = 0.9 Hz, 3H), 2.04 (p, J = 7.32 Hz, 4H). MS
(FAB) m/z 406 [M+H]+. HRMS (FAB) m/z calcd for C19H27N5O3S [M
+H]+ 406.1913, found: 406.1907. Anal. HPLC 99.6% (Rt = 3.32 min).
4.1.3.27. 1-(3-Methoxy-4-(4-oxo-4-(piperazin-1-yl)butoxy)phenyl)-3-
(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (186). Starting with
compound 135 as following the general procedure 6.2, compound
186 was obtained as a white solid, 39% yield, mp = 91–93 °C. 1H
NMR (300 MHz, CD3OD) d 7.59 (s, 1H), 6.96 (d, J = 8.61 Hz, 1H),
6.92 (d, J = 2.37 Hz, 1H), 6.76 (dd, J = 2.37, 8.61 Hz, 1H), 6.66 (s,
1H), 4.06 (t, J = 6.21 Hz, 2H), 3.99 (t, J = 7.50 Hz, 2H), 3.81 (s, 3H),
3.61–3.55 (m, 6H), 2.80–2.77 (m, 4H), 2.61 (t, J = 7.32 Hz, 2H),
2.22 (d, J = 0.93 Hz, 3H), 2.08 (p, J = 6.78 Hz, 4H). MS (FAB) m/z
475 [M+H]+. HRMS (FAB) m/z calcd for C23H34N6O3S [M+H]+
475.2491, found: 475.2483. Anal. HPLC 95.5% (Rt = 3.43 min).
4.1.3.22. 2-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
thioureido)phenoxy)-N,N-dimethylacetamide (181). Starting with
compound 157 as following the reaction with dimethylamine,
compound 181 was obtained as a white solid, 78% yield, mp =
120–122 °C. 1H NMR (400 MHz, CD3OD) d 7.91 (s, 1H), 6.95 (d, J
= 2.16 Hz, 1H), 6.91 (dd, J = 8.56 Hz, 1H), 6.81 (s, 1H), 6.73 (dd, J
= 8.52, 2.40 Hz, 1H), 4.79 (s, 2H), 4.03 (t, J = 7.20 Hz, 2H), 3.84 (s,
3H), 3.61 (t, J = 6.88 Hz, 2H), 3.09 (s, 3H), 2.96 (s, 3H), 2.25 (d, J =
0.76 Hz, 3H), 2.07 (p, J = 7.00 Hz, 2H). MS (ESI) m/z 406 [M+H]+.
HRMS (FAB) for C19H28N5O3S [M+H]+ 406.1907, found 406.1908.
Anal. HPLC 95.0% (Rt = 4.06 min).
4.1.3.28. 1-(4-(2-(4-(2-Hydroxyethyl)piperazin-1-yl)-2-oxoethoxy)-3-
methoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(187). Starting with compound 152 as following the general proce-
dure 7, compound 187 was obtained as a white solid, 44% yield,
mp = 90–91 °C. 1H NMR (300 MHz, CD3OD) d 7.59 (s, 1H), 6.87–
6.84 (m, 2H), 6.71 (dd, J = 2.52, 9.03 Hz, 1H), 6.68 (s, 1H), 4.41 (s,
2H), 3.98 (t, J = 7.23 Hz, 2H), 3.82 (s, 3H), 3.68 (t, J = 6.03 Hz, 2H),
3.59–3.55 (m, 2H), 2.87 (t, J = 5.13 Hz, 4H), 2.52–2.48 (m, 6H),
2.21 (d, J = 1.2 Hz, 3H), 2.02 (p, J = 6.96 Hz, 2H), MS (ESI) m/z 491
4.1.3.23. 3-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
thioureido)phenoxy)-N,N-dimethylpropanamide (182). Starting with
compound 99 as following the general procedure 8, compound 182
was obtained as white solid, 63% yield, mp = 181–183 °C. 1H NMR
(300 MHz, CDCl3) d 7.57 (s, 1H), 7.36 (s, 1H), 6.94 (d, J = 8.4 Hz, 1H),
6.71 (dd, J = 7.8, 3.0 Hz, 1H), 6.70 (s, 1H), 6.68 (d, J = 2.1 Hz, 1H),
Please cite this article in press as: Ngo V.T.H., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.015

V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
13
[M+H]+. HRMS (ESI) calc. for C23H34N6O4S [M+H]+ 491.2435, found
491.2456. Anal. HPLC 97.5% (Rt = 2.99 min).
1.88–1.82 (m, 2H), 1.66–1.54 (m, 2H). MS (ESI) m/z 505 [M+H]+.
HRMS (ESI) calc. for
24H36N6O4S [M+H]+ 505.2592, found
C
505.2583. Anal. HPLC 100.0% (Rt = 2.97 min).
4.1.3.29. 1-(4-(2-(4-(2-Aminoethyl)piperazin-1-yl)-2-oxoethoxy)-3-
methoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(188). Starting with compound 136 as following the general proce-
dure 6.2, compound 188 was obtained as a white solid, 24% yield,
mp = 79–81 °C .1H NMR (400 MHz, CD3OD) d 7.58 (s, 1H), 6.97 (d, J
= 2.28 Hz, 1H), 6.94 (d, J = 8.52 Hz, 1H), 6.75 (dd, J = 2.28, 8.48 Hz,
1H), 6.66 (s, 1H), 4.78 (s, 2H), 3.99 (t, J = 7.16 Hz, 2H), 3.82 (s,
3H), 3.61–3.59 (m, 6H), 2.76 (t, J = 6.44 Hz, 2H), 2.51 (t, J = 3.16
Hz, 2H), 2.47 (t, J = 4.68 Hz, 4H), 2.21 (d, J = 0.96 Hz, 3H), 2.07 (p,
4.1.3.34. N-(1-(2-Aminoethyl)piperidin-4-yl)-2-(2-methoxy-4-(3-(3-
(5-methyl-1H-imidazol-1-yl)propyl)thioureido)phenoxy)acetamide
(193). Starting with compound 140 as following the general proce-
dure 6.2, compound 193 was obtained as a white solid, 77% yield,
mp = 125–126 °C. 1H NMR (500 MHz, CD3OD) d 7.59 (s, 1H), 7.06
(d, J = 1.75 Hz, 1H), 7.00 (d, J = 8.55 Hz, 1H), 6.79 (dd, J = 2.15,
8.50 Hz, 1H), 6.66 (s, 1H), 4.49 (s, 2H), 3.99 (t, J = 7.20 Hz, 2H),
3.87 (s, 3H), 3.79–3.76 (m, 1H), 3.59 (t, J = 6.60 Hz, 2H), 2.90 (d, J
= 11.35 Hz, 2H), 2.82 (t, J = 6.55 Hz, 2H), 2.49 (t, J = 6.60 Hz, 2H),
2.22 (s, 3H), 2.19–2.14 (m, 2H), 2.07 (p, J = 7.10 Hz, 2H), 1.89–
1.87 (m, 2H), 1.62 (q, J = 8.95 Hz, 2H). MS (ESI) m/z 504 [M+H]+.
J = 5.44 Hz, 2H). MS (ESI) m/z 490 [M+H]+. HRMS (ESI) calc. for C23
-
H35N7O3S [M+H]+ 490.2595, found 490.2595. Anal. HPLC 98.8% (Rt
= 2.87 min).
HRMS (ESI) calc. for
C
24H37N7O3S [M+H]+ 504.2751, found
4.1.3.30. 2-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
504.2750. Anal. HPLC 100.0% (Rt = 2.80 min).
thioureido)phenoxy)-N-(piperidin-4-yl)acetamide
(189). Starting
with compound 177 as following the general procedure 6.2, com-
pound 189 was obtained as a white solid, 41% yield, mp = 57–58 °C.
1H NMR (300 MHz, CD3OD) d 7.59 (d, J = 1.11 Hz, 1H), 7.07 (d, J =
2.40 Hz, 1H), 7.01 (d, J = 8.61 Hz, 1H), 6.80 (dd, J = 2.40, 8.43 Hz,
1H), 6.66 (s, 1H), 4.49 (s, 2H), 4.00 (t, J = 7.32 Hz, 2H), 3.87 (s,
3H), 3.84–3.81 (m, 1H), 3.62 (t, J = 6.78 Hz, 2H), 3.05 (d, J = 12.81
Hz, 2H), 2.68 (t, J = 11.91 Hz, 2H), 2.22 (d, J = 1.08 Hz), 2.06 (p, J =
6.96 Hz, 2H), 1.88–1.84 (m, 2H), 1.50–1.42 (m, 2H). MS (ESI) m/z
461 [M+H]+. HRMS (ESI) calc. for C22H32N6O3S [M+H]+ 461.2329,
found 461.2318. Anal. HPLC 98.5% (Rt = 2.96 min).
4.1.3.35. N-(2-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)pro-
pyl)thioureido)phenoxy)-ethyl)piperidine-4-carboxamide
(194). Starting with compound 141 as following the general proce-
dure 6.2, compound 194 was obtained as a white solid, 54% yield.
1H NMR (300 MHz, CD3OD) d 7.51 (d, J = 1.29 Hz, 1H), 6.89 (d, J =
2.40 Hz, 1H), 6.87 (d, J = 8.61 Hz, 1H), 6.69 (dd, J = 2.40, 8.61 Hz,
1H), 6.57 (s, 1H), 3.98 (t, J = 5.67 Hz, 2H), 3.91 (t, J = 7.32 Hz, 2H),
3.71 (s, 3H), 3.52–3.41 (m, 4H), 3.28–3.24 (m, 2H), 2.89 (td, J =
12.27 Hz, 3.66, Hz, 2H), 2.41–2.38 (m, 1H), 2.13 (d, J = 0.90 Hz,
3H), 1.99 (quint, J = 6.75 Hz, 2H), 1.83–1.78 (m, 2H), 1.76–1.71
(m, 2H). MS (FAB) m/z 475 [M+H]+. HRMS (FAB) m/z calcd for C22
34N6O3S [M+H]+ 475.2491, found: 475.2491. Anal. HPLC 99.6% (Rt
= 4.29 min).
-
4.1.3.31. 3-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
thioureido)phenoxy)-N-(piperidin-4-yl)propanamide (190). Starting
with compound 138 as following the general procedure 6.2, com-
pound 190 was obtained as a white solid, 24% yield, mp = 89–90 °C.
1H NMR (300 MHz, CD3OD) d 7.58 (s, 1H), 6.97 (d, J = 8.43 Hz,
1H), 6.92 (d, J = 2.19 Hz, 1H), 6.77 (dd, J = 2.37, 8.61 Hz, 1H), 6.66
(s, 1H), 4.25 (t, J = 6.03 Hz, 2H), 4.06–4.04 (m, 1H), 3.99 (t, J =
7.32 Hz, 2H), 3.81 (s, 3H), 3.61 (t, J = 7.14 Hz, 2H), 3.08–3.03 (m,
2H), 2.71–2.60 (m, 4H), 2.21 (d, J = 0.93 Hz, 3H), 2.05 (p, J = 6.96
Hz, 2H), 1.88–1.85 (m, 2H), 1.46–1.41 (m, 2H). MS (FAB) m/z 475
[M+H]+. HRMS (FAB) m/z calcd for C23H34N6O3S [M+H]+ 475.2491,
found: 475.2477. Anal. HPLC 97.8% (Rt = 3.91 min).
H
4.1.3.36. 1-(3-Methoxy-4-(4-(piperazin-1-yl)phenoxy)phenyl)-3-(3-
(5-methyl-1H-imidazol-1-yl)propyl)thiourea (195). Starting with
compound 142 as following the general producer 6.2, compound
195 was obtained as a white solid, 69% yield, mp = 88–90 °C. 1H
NMR (300 MHz, CD3OD) d 7.60 (s, 1H), 7.09 (d, J = 2.37 Hz, 1H),
6.92–6.89 (m, 2H), 6.86–6.81 (m, 3H), 6.76 (dd, J = 2.55, 8.58 Hz,
1H) 6.66 (s, 1H), 4.01 (t, J = 7.14 Hz, 2H), 3.78 (s, 3H), 3.63 (t, J =
6.54 Hz, 2H), 3.06–3.02 (m, 4H), 2.97–2.85 (m, 4H), 2.22 (s, 3H),
2.07 (p, J = 6.96 Hz, 2H). MS (ESI) m/z 481 [M+H]+. HRMS (ESI) calc.
for C25H32N6O2S [M+H]+ 481.2380, found 481.2394. Anal. HPLC
96.6% (Rt = 2.91 min).
4.1.3.32. 4-(2-Methoxy-4-(3-(3-(5-methyl-1H-imidazol-1-yl)propyl)
thioureido)phenoxy)-N-(piperidin-4-yl)butanamide (191). Starting
with compound 139 as following the general procedure 6.2, com-
pound 191 was obtained as white solid, 87% yield, mp = 97–98 °C.
1H NMR (300 MHz, CD3OD) d 7.58 (d, J = 0.90 Hz, 1H), 6.93 (d, J =
8.61 Hz, 1H), 6.92 (s, 1H), 6.74 (dd, J = 8.43, 2.40 Hz, 1H), 6.66 (s,
1H), 4.01 (t, J = 6.06 Hz, 2H), 3.97 (t, J = 7.32 Hz, 2H), 3.81 (s, 3H),
3.79–3.74 (m, 1H), 3.59 (t, J = 6.96 Hz, 2H), 3.08 (dt, J = 9.90, 2.94
Hz, 2H), 2.72 (td, J = 12.09, 2.04 Hz, 2H), 2.38 (t, J = 7.14 Hz, 2H),
2.21 (d, J = 0.9 Hz, 3H), 2.10–1.99 (m, 4H), 1.88 (d, J = 10.08 Hz,
2H), 1.41 (qt, J = 11.70, 2.91 Hz, 2H). MS (ESI) m/z 489 [M+H]+.
4.1.3.37. 1-(3-Methoxy-4-(4-(4-methylpiperazin-1-yl)phenoxy)phe-
nyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea (196). Start-
ing with compound 88 as following the general producer 8,
compound 196 was obtained as a white solid, 49% yield, mp =
104–106 °C. 1H NMR (300 MHz, CD3OD) d 7.63 (s, 1H), 7.10 (d, J
= 2.37 Hz, 1H), 6.95–6.92 (m, 2H), 6.89 (s, 1H), 6.86 (s, 1H), 6.84–
6.81 (m, 2H), 6.79 (dd, J = 2.37, 8.43 Hz, 1H), 4.02 (t, J = 7.23 Hz,
2H), 3.78 (s, 3H), 3.63 (t, J = 6.93 Hz, 2H), 3.14 (t, J = 4.95 Hz, 4H),
2.66 (t, J = 4.95 Hz, 4H), 2.36 (s, 3H), 2.23 (d, J = 0.90 Hz, 3H), 2.10
(p, J = 6.78 Hz, 2H). MS (ESI) m/z 495 [M+H]+. HRMS (ESI) calc. for
HRMS (FAB) calc. for
C
24H37N6O3S [M+H]+ 489.2642, found
489.2659. Anal. HPLC 98.5% (Rt = 3.52 min).
C
26H34N6O2 [M+H]+ 495.2537, found 495.2532. Anal. HPLC 99.8%
(Rt = 2.99 min).
4.1.3.33. N-(1-(2-Hydroxyethyl)piperidin-4-yl)-2-(2-methoxy-4-(3-
(3-(5-methyl-1H-imidazol-1-yl)propyl)thioureido)phenoxy)ac-
etamide (192). Starting with compound 153 as following the gen-
eral procedure 7, compound 192 was obtained as a white solid,
41% yield, mp = 104–106 °C. 1H NMR (300 MHz, CDCl3) d 7.59 (s,
1H), 7.05 (d, J = 2.19 Hz, 1H), 7.01 (d, J = 8.40 Hz, 1H), 6.80 (dd, J
= 2.37, 8.43 Hz, 1H), 6.66 (s, 1H), 4.49 (s, 2H), 4.00 (t, J = 7.32 Hz,
2H), 3.87 (s, 1H), 3.85–3.82 (m, 1H), 3.69 (t, J = 6.06 Hz, 2H), 3.61
(t, J = 6.96 Hz, 2H), 2.95 (d, J = 12.09 Hz), 2.54 (t, J = 6.06 Hz, 2H),
2.22 (d, J = 0.72 Hz, 3H), 2.16–2.13 (m, 2H), 2.08 (p, J = 7.32 Hz),
4.1.3.38.
1-(4-(4-(4-(2-Hydroxyethyl)piperazin-1-yl)phenoxy)-3-
methoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(197). Starting with compound 159 as following the general pro-
ducer 7, compound 197 was obtained as a white solid, 89% yield,
mp = 66–67 °C. 1H NMR (300 MHz, CD3OD) d 7.92 (s, 1H), 7.12 (d,
J = 2.19 Hz, 1H), 6.97–6.94 (m, 2H), 6.87–6.84 (m, 2H), 6.82–6.77
(m, 3H), 4.07 (t, J = 7.14 Hz, 2H), 3.82 (t, J = 5.52 Hz, 2H), 3.75 (s,
3H), 3.24–3.22 (m, 6H), 3.04 (t, J = 4.56 Hz, 4H), 2.91 (t, J = 5.67
Hz, 2H), 2.26 (d, J = 0.90 Hz, 3H), 2.11 (p, J = 7.32 Hz, 2H). MS
Please cite this article in press as: Ngo V.T.H., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.015

14
V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
(ESI) m/z 525 [M+H]+. HRMS (ESI) calc. for C27H36N6O3S [M+H]+
525.2642, found 525.2633. Anal. HPLC 96.3% (Rt = 2.92 min).
4.1.3.44. 1-(3-Methoxy-4-(4-(2-(methylamino)pyridin-4-yl)butoxy)
phenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(203). Starting with compound 147 as following the general proce-
dure 6.2, compound 203 was obtained as a white solid, 83% yield,
mp = 56–58 °C. 1H NMR (300 MHz, CD3OD) d 7.79 (d, J = 4.95 Hz,
1H), 7.59 (d, J = 0.90 Hz, 1H), 6.93 (d, J = 8.79 Hz, 1H), 6.89 (d, J =
2.55 Hz, 1H), 6.75 (dd, J = 2.37, 8.01 Hz, 1H), 6.66 (s, 1H), 6.45
(dd, J = 1.29, 5.31 Hz, 1H), 6.35 (s, 1H), 4.04 (t, J = 5.64 Hz, 2H),
3.94 (t, J = 7.14 Hz, 2H), 3.80 (s, 3H), 3.61 (t, J = 6.57 Hz, 2H), 2.82
(s, 3H), 2.57 (t, J = 7.29 Hz, 2H), 2.21 (d, J = 0.90 Hz, 3H), 2.07 (p,
J = 7.32 Hz, 2H), 1.80–1.78 (m, 4H). MS (ESI) m/z 483 [M+H]+.
4.1.3.39. 1-(4-(4-(4-(2-Aminoethyl)piperazin-1-yl)phenoxy)-3-meth-
oxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(198). Starting with compound 158 as following the general pro-
ducer 6.2, compound 198 was obtained as a white solid, 75% yield,
m.p = 75–77 °C. 1H NMR (300 MHz, CD3OD) d 7.59 (s, 1H), 7.09 (d, J
= 2.37 Hz, 1H), 6.96–6.93 (m, 2H), 6.84–6.81 (m, 3H), 6.79 (dd, J =
2.22, 8.43 Hz, 1H), 6.66 (s, 1H), 4.01 (t, J = 7.50 Hz, 2H), 3.78 (s, 3H),
3.61 (t, J = 7.14 Hz, 2H), 3.13 (t, J = 5.10 Hz, 4H), 2.82 (t, J = 6.39 Hz,
2H), 2.66 (t, J = 5.13 Hz, 4H), 2.53 (t, J = 6.60 Hz, 2H), 2.22 (d, J =
0.90 Hz, 3H), 2.07 (p, J = 7.14 Hz, 2H). MS (ESI) m/z 524 [M+H]+.
HRMS (ESI) calc. for C27H37N7O2S [M+H]+ 524.2802, found
524.2808. Anal. HPLC 99.5% (Rt = 2.75 min).
HRMS (ESI) calc. for
483.2534. Anal. HPLC 99.2% (Rt = 2.96 min).
C
25H34N6O2S [M+H]+ 483.2537, found
4.1.3.45. 1-(4-(4-(2-((2-Hydroxyethyl)amino)pyridin-4-yl)butoxy)-3-
methoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(204). Starting with compound 154 as following the general proce-
dure 7, compound 204 was obtained as a white solid, 68% yield,
mp = 103–104 °C. 1H NMR (300 MHz, CD3OD) d 7.79 (d, J = 5.28
Hz, 1H), 7.59 (s, 1H), 6.93–6.90 (m, 2H), 6.75 (dd, J = 2.55, 9.12
Hz, 1H), 6.66 (s, 1H), 6.46 (d, J = 5.31 Hz, 1H), 6.41 (s, 1H), 4.00 (t,
J = 5.97 Hz, 2H), 3.97 (t, J = 7.14 Hz, 2H), 3.80 (s, 3H), 3.70 (t, J =
5.49 Hz, 2H), 3.59 (q, J = 6.87 Hz, 2H), 3.37 (t, J = 5.49 Hz, 2H),
2.57 (m, 2H), 2.21 (d, J = 0.90 Hz, 3H), 2.02 (p, J = 6.96 Hz, 2H),
4.1.3.40.
1-(3-Methoxy-4-(4-(1,2,3,6-tetrahydropyridin-4-yl)phe-
noxy)phenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(199). Starting with compound 143 as following the general proce-
dure 6.2, compound 199 was obtained as a white solid, 91% yield,
mp = 95–97 °C. 1H NMR (300 MHz, CD3OD) d 7.60 (s, 1H), 7.34 (d, J
= 8.79 Hz, 2H), 7.15 (d, J = 2.40 Hz, 1H), 6.99 (d, J = 8.61 Hz, 1H),
6.84–6.80 (m, 3H), 6.67 (s, 1H), 6.08 (s, 1H), 4.02 (t, J = 7.14 Hz,
2H), 3.76 (s, 3H), 3.64 (t, J = 6.78 Hz, 2H), 3.44 (q, J = 3.12 Hz, 2H),
3.04 (t, J = 5.88 Hz, 2H), 2.47–2.43 (m, 2H), 2.23 (d, J = 0.93 Hz,
3H), 2.08 (p, J = 7.14 Hz, 2H). MS (ESI) m/z 478 [M+H]+. HRMS
(ESI) calc. for C26H31N5O2S [M+H]+ 478.2271, found 478.2288. Anal.
HPLC 98.6% (Rt = 2.91 min).
1.79 (m, 4H). MS (ESI) m/z 513 [M+H]+. HRMS (ESI) calc. for C26H36
-
N6O3S [M+H]+ 513.2642, found 513.2633. Anal. HPLC 98.1% (Rt =
2.95 min).
4.1.3.46. 1-(4-(4-(2-((2-Aminoethyl)amino)pyridin-4-yl)butoxy)-3-
methoxyphenyl)-3-(3-(5-methyl-1H-imidazol-1-yl)propyl)thiourea
(205). Starting with compound as 148 following the general proce-
dure 6.2, compound 205 was obtained as a white solid, 46% yield,
mp = 100–102 °C. 1H NMR (500 MHz, DMSO) d 9.51 (br, 1H), 7.82
(d, 1H, J = 5.05 Hz), 7.76 (br, 1H), 7.53 (s, 1H), 6.97 (s, 1H), 6.89
(d, 1H, J = 9.05 Hz), 6.76 (d, 1H, J = 8.15 Hz), 6.60 (s, 1H), 6.33–
6.27 (m, 3H), 3.93 (t, 2H, J = 5.55 Hz), 3.89 (t, 2H, J = 7.05 Hz),
3.71 (s, 3H), 3.43 (q, J = 5.34 Hz, 2H), 3.19 (q, 2H, J = 6.20 Hz),
2.66 (t, 2H, J = 6.25 Hz), 2.47–2.45 (m, 2H), 2.14 (s, 3H), 1.92 (p,
2H, J = 7.00 Hz), 1.68–1.61 (m, 4H). MS (ESI) m/z 512 [M+H]+. HRMS
(ESI) calc. for C26H37N7O2S [M+H]+ 512.2802, found 512.2800. Anal.
HPLC 97.1% (Rt = 2.80 min).
4.1.3.41. 1-(3-Methoxy-4-(4-(piperidin-4-yl)phenoxy)phenyl)-3-(3-
(5-methyl-1H-imidazol-1-yl)propyl)thiourea (200). Starting with
compound 144 as following the general procedure 6.2, compound
200 was obtained as a white solid, 61% yield, mp = 87–89 °C. 1H
NMR (400 MHz, CD3OD) d 7.59 (s, 1H), 7.15–7.12 (m, 3H), 6.94
(d, J = 8.40 Hz, 1H), 6.82–6.80 (m, 3H), 6.66 (s, 1H), 4.01 (t, J =
7.28 Hz, 2H), 3.78 (s, 3H) 3.63 (t, J = 6.96 Hz, 2H), 3.14–3.11 (m,
2H), 2.74–2.71 (m, 2H), 2.64–2.62 (m, 1H), 2.23 (d, J = 0.64 Hz,
3H), 2.14–2.00 (m, 4H), 1.63–1.57 (m, 2H). MS (ESI) m/z 480 [M
+H]+. HRMS (ESI) calc. for C26H33N5O2S [M+H]+ 480.2428, found
480.2424. Anal. HPLC 97.7% (Rt = 2.90 min).
4.1.3.42. 1-(3-Methoxy-4-(3-(piperazin-1-yl)benzyloxy)phenyl)-3-(3-
(5-methyl-1H-imidazol-1-yl)propyl)thiourea (201). Starting with
compound 145 as following the general procedure 6.2, compound
201 was obtained as a white solid, 75% yield, mp = 106–108 °C. 1H
NMR (300 MHz, CD3OD) d 7.59 (s, 1H), 7.23 (t, J = 7.86 Hz, 1H), 7.06
(s, 1H), 6.97 (d, J = 7.89 Hz, 1H), 6.93–6.90 (m, 3H), 6.71 (dd, J =
7.89, 1.83 Hz, 1H), 6.66 (s, 1H), 5.07 (s, 2H), 3.96 (t, J = 7.14 Hz,
2H), 3.84 (s, 3H), 3.59 (t, J = 6.96 Hz, 2H), 3.13 (t, J = 4.38, 4H),
2.96 (t, J = 5.31 Hz, 4H), 2.21 (s, 3H), 2.02 (p, J = 7.14 Hz, 2H). MS
(ESI) m/z 495 [M+H]+. HRMS (FAB) calc. for C26H35N6O2S [M+H]+
495.2537 found 495.2542. Anal. HPLC 95.0% (Rt = 3.15 min).
4.2. Molecular modeling
The X-ray crystal structure of human glutaminyl cyclase (PDB
ID: 3PBB)39 was prepared using the Protein Preparation Wizard
in Maestro v.10.2 (Schrödinger, LLC, New York, NY, USA). During
the preparation process, bond orders were assigned, zero-order
bonds to Zn2+ were created, and hydrogen atoms were added. All
hydrogen atoms were energy minimized with the optimized
potential for liquid simulation (OPLS) 2005 force field. The proto-
nation states of the ligand molecules were predicted using the
pKa prediction module in ACD/I-Lab web server (ACD/Labs, Tor-
onto, ON, Canada). The 3D structure of compound 202 was gener-
ated by LigPrep v.3.4 in Maestro and the resulting structure was
energy minimized in implicit solvent with OPLS 2005 force field
in Maestro. The prepared ligand molecules were docked to hQC
using Glide v.6.7 in Maestro. The grid for the active site was gener-
ated using the centroid of the co-crystallized ligand, PBD150, and
the grid box size was set as default. The metal coordination con-
straint was set to the tetrahedral geometry for Zn2+. Glide SP dock-
ing was performed with the maximum number of 30 poses per
ligand. The resulting top 5 poses of compound 202 were selected
and used for the following QM-Polarized Ligand Docking (QPLD)
process. The partial charges of the docked ligands were calculated
using Jaguar with the option of accurate QM level. Then, the
4.1.3.43. 1-(3-Methoxy-4-(3-(piperidin-4-yl)benzyloxy)phenyl)-3-(3-
(5-methyl-1H-imidazol-1-yl)propyl)thiourea (202). Starting with
compound 146 as following the general procedure 6.2, compound
202 was obtained as a white solid, 68% yield, mp = 166–167 °C. 1H
NMR (300 MHz, CD3OD) d 7.58 (d, J = 1.08 Hz, 1H), 7.33–7.26 (m,
3H), 7.19 (tt, J = 6.24 Hz, 1H), 6.97 (d, J = 8.61 Hz, 1H), 6.94 (d, J =
2.37 Hz, 1H), 6.72 (dd, J = 8.43, 2.37 Hz, 1H), 6.66 (s, 1H), 3.96 (t,
J = 7.14 Hz, 2H), 3.83 (s, 3H), 3.59 (t, J = 6.78 Hz, 2H), 3.25 (br,
1H), 3.21 (br, 1H), 2.84 (td, J = 12.45, 2.94 Hz, 2H), 2.74 (tt, J =
11.91, 3.66 Hz, 1H), 2.21 (d, J = 0.90 Hz, 3H), 2.03 (p, J = 7.14 Hz,
2H), 1.90–1.86 (br, 2H), 1.75 (p, J = 8.40, 3.84 Hz, 2H). MS (ESI)
m/z 494 [M+H]+. HRMS (FAB) calc. for C27H36N5O2S [M+H]+
494.2584, found 494.2595. Anal. HPLC 97.7% (Rt = 3.18 min).
Please cite this article in press as: Ngo V.T.H., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.015

V.T.H. Ngo et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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5 Å of the docked ligand were minimized by local optimization
refinement. The side chain conformations of the selected protein
residues were predicted and minimized along with the docked
ligand during this process. The resulting structures were further
energy minimized using Monte Carlo sampling algorithm in Mae-
stro in 2500 steps. All figures of the molecular structures were gen-
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computational studies were performed on an Intel Xeon Octa-Core
2.67 GHz workstation with Linux CentOS release 6.7.
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Acknowledgements
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government (MSIP)
(NRF-2017R1A2B4011094) and the Midcareer Researcher Program
(NRF-2017R1A2B4010084 to S.C.) funded by the Ministry of
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2018.01.015.
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Please cite this article in press as: Ngo V.T.H., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.015