Structure-activity relationship investigation of Phe-Arg mimetic region of human glutaminyl cyclase inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.04.040

Glutamyl cyclase (QC) is a promising therapeutic target because of its involvement in the pathogenesis of Alzheimer's disease. In this study, we developed novel QC inhibitors that contain 3-aminoalkyloxy-4-methoxyphenyl and 4-aminoalkyloxyphenyl groups to replace the previously developed pharmacophore. Several potent inhibitors were identified, showing IC₅₀ values in a low nanomolar range, and were further studied for in vitro toxicity and in vivo activity. Among these, inhibitors 51 and 53 displayed the most potent Aβ_N3pE?40-lowering effects in in vivo acute model with reasonable BBB penetration, without showing cytotoxicity and hERG inhibition. The molecular modeling analysis of 53 indicated that the salt bridge interaction and the hydrogen bonding in the active site provided a high potency. Given the potent activity and favorable BBB penetration with low cytotoxicity, we believe that compound 53 may serve as a potential candidate for anti-Alzheimer's agents.

Full text of DOI:10.1016/j.bmc.2018.04.040

Accepted Manuscript
Structure-Activity Relationship Investigation of Phe-Arg Mimetic Region of
Human Glutaminyl Cyclase Inhibitors
Van T.H. Ngo, Van-Hai Hoang, Phuong-Thao Tran, Nguyen Van Manh, Jihyae
Ann, Eunhye Kim, Minghua Cui, Sun Choi, Jiyoun Lee, Hee Kim, Hee-Jin Ha,
Kwanghyun Choi, Young-Ho Kim, Jeewoo Lee
PII:
S0968-0896(18)30622-9
https://doi.org/10.1016/j.bmc.2018.04.040
BMC 14323
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 March 2018
16 April 2018
17 April 2018
Please cite this article as: Ngo, V.T.H., Hoang, V-H., Tran, P-T., Van Manh, N., Ann, J., Kim, E., Cui, M., Choi,
S., Lee, J., Kim, H., Ha, H-J., Choi, K., Kim, Y-H., Lee, J., Structure-Activity Relationship Investigation of Phe-
Arg Mimetic Region of Human Glutaminyl Cyclase Inhibitors, Bioorganic & Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.bmc.2018.04.040
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Structure-Activity Relationship Investigation of Phe-Arg
Mimetic Region of Human Glutaminyl Cyclase Inhibitors
Van T.H. Ngo a, Van-Hai Hoang a, Phuong-Thao Tran b, Nguyen Van Manh a, Jihyae Ann a, Eunhye
c
c
c
d
e
e
e
Kim , Minghua Cui , Sun Choi , Jiyoun Lee , Hee Kim , Hee-Jin Ha , Kwanghyun Choi ,
Young-Ho Kim e, and 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, Vietnam
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
* To whom correspondence should be addressed.
Phone: 82-2-880-7846
Fax: 82-2-888-0649
E-mail: jeewoo@snu.ac.kr
1

ABSTRACT
Glutamyl cyclase (QC) is a promising therapeutic target because of its involvement in the
pathogenesis of Alzheimer’s disease. In this study, we developed novel QC inhibitors that contain
3-aminoalkyloxy-4-methoxyphenyl and 4-aminoalkyloxyphenyl groups to replace the previously
developed pharmacophore. Several potent inhibitors were identified, showing IC50 values in a low
nanomolar range, and were further studied for in vitro toxicity and in vivo activity. Among these,
inhibitors 51 and 53 displayed the most potent AβN3pE-40-lowering effects in in vivo acute model
with reasonable BBB penetration, without showing cytotoxicity and hERG inhibition. The
molecular modeling analysis of 53 indicated that the salt bridge interaction and the hydrogen
bonding in the active site provided a high potency. Given the potent activity and favorable BBB
penetration with low cytotoxicity, we believe that compound 53 may serve as a potential candidate
for anti-Alzheimer’s agents.
2

1. Introduction
β-Amyloid (Aβ) is a main component of the amyloid plaques found in Alzheimer’s disease
(AD), and the elevated levels of Aβ peptides and amyloid plaques are thought to be the hallmarks of
AD pathology. The brain Aβ peptides are present as a mixture of various isoforms, among which
Aβ1-40 and Aβ1-42, are most abundant in AD patients. Several recent studies found that
pyroglutamyl-β-amyloid (pE-Aβ) peptides, the less abundant but more hydrophobic form of Aβ,
appeared to be more neurotoxic than Aβ1-40 and Aβ1-42 and to promote the formation of amyloid and
tau plaques.1-3 In particular, pE-Aβ levels are highly upregulated in the brains of patients and
animal models with AD,4,5 and are shown to be the major constituents of Aβ deposit in sporadic and
familial Alzheimer’s disease (AD).6-8
Pyroglutamyl-β-amyloid (pE-Aβ) peptides are a type of the N-truncated Aβ forms
containing an N-terminal pyroglutamate at positions 3 or 11 in Aβ. Due to their increased
hydrophobicity, pE-Aβ peptides are prone to rapid aggregation and are much more resistant to
proteolytic degradation, which explains their exacerbated neurotoxicity. The formation of the
pyroglutamate from the N-terminal glutamate of Aβ is catalyzed by glutamyl cyclase (QC), which
has been implicated in the pathogenesis of AD. Moreover, the administration of QC inhibitors
reduced the brain pGlu-Aβ levels and Aβ plaques9,10 and decreased gliosis and recovered memory
deficits in AD mice.4 Given that most of the Aβ-lowering drugs that were advanced to clinical trials
have not yet proven to be effective,11,12 blocking the formation of neurotoxic Aβ species such as pE-
Aβ can provide a promising alternative to current therapeutic approaches.
Previously reported QC inhibitors were developed by a pharmacophore design based on the
N-terminal structure of its substrate Aβ3-42. The representative inhibitors developed by Probiodrug
(1, 2),13-15 Wu and colleagues (3)16 and our research group (4)17-19 are shown in Figure 1. Naturally
occurring inhibitors20,21 and other small molecule inhibitors developed by the fragment-based
approach22 were also reported; however, these inhibitors demonstrated only modest activity.
3

Currently, PQ912, developed by Probiodrug, is undergoing a clinical trial, and it has exhibited
favorable safety and tolerability. More importantly, PQ912 demonstrated a slight improvement in
synaptic and neurological functions in patients with AD in a recently completed phase IIa clinical
trial,23,24 supporting that QC is a potential therapeutic target for the treatment of AD.
H
N
O
O
O
O
S
S
N
S
N
N
N
N
H
H
N N
2
1
F
O
NH2
S
N
N
N
N
N
O
N
N
H
H
H
N
4
3
Figure 1. Representative QC inhibitors.
The Probiodrug compound 1 contains three key pharmacophores derived from the N-
14,15
terminal Glu-Phe moiety of Aβ3E-42
.
The 5-methylimidazole ring (red) mimics the N-terminal
carboxylic acid as a zinc-binding motif. The distal NH of thiourea (blue) serves as a hydrogen bond
donor, mimicking the first peptide bond from the N-terminus. The phenyl ring (green) mimics the
Phe side chain at the penultimate position to the N-terminus. Inspired by this approach, we had
previously developed a series of QC inhibitors (template A), with an extended scaffold as described
in Figure 2.18,19 The scaffold contains an additional pharmacophore that mimics the binding
interaction of the guanidine side chain of Arg at the antepenultimate position to the N-terminus. The
newly developed QC inhibitors displayed much improved potency with a range of 5 to 40-fold more
potent inhibition than 1. Specifically, compound 4 not only exhibited potent inhibition without
cytotoxicity but also significantly reduced the brain concentrations of pE-Aβ and total Aβ while
restoring cognitive functions in an AD animal model. The molecular modeling analysis of 4
4

indicated that the 2-aminopyridine moiety showed strong interactions with the carboxylate group of
Glu327 in the QC binding site.
O
S
N
N
R
O
N
H
N
H
B
R
O
O
S
N
N
N
H
N
O R
H
S
N
N
N
H
N
H
A
R = Arg mimetic
C
Figure 2. Design rationale for QC inhibitors with novel templates
In this study, we investigated a series of 3-aminoalkyloxy-4-methoxyphenyl (template B)
and 4-aminoalkyloxyphenyl (template C) surrogates as variants of template A. We anticipate that
these templates would provide useful information to optimize the position of Arg-mimetic region
(from template B) and to identify the significance of the 3-methoxy group (from template C) for QC
inhibition. We evaluated the human QC inhibitory activity of the synthesized compounds in vitro
and selected several potent inhibitors (IC50 < 10 nM). We further tested these compounds for in
vitro toxicity/permeability and in vivo activity in acute AD model mice. Finally, a molecular
modeling study was performed to analyze the specific binding interactions in the QC active site.
2. Results and discussion
2.1. Chemistry
In general, the final thiourea compounds were synthesized by the coupling reaction between
3-(5-methyl-1H-imidazol-1-yl)propan-1-amine and aniline intermediates obtained by the reduction
of nitro fragments prepared in Schemes 1 and 2.
The synthesis of the 3-aminoalkyloxy-4-methoxy-1-nitrobenzene fragments is described in
Scheme 1. Starting from 5-nitroguaiacol (5), the Mitsunobu reaction or Williamson reaction
5

incorporated N-protected aminoalkyl moieties into the phenolic position to provide 6-17. Among
them, the N-Boc protected amino (6-12) and phthalimide protected amino (13, 14) intermediates
were directly employed for the thiourea coupling. The bromides 15-17 were reacted with N-Boc
piperazine to give 22-24. After deprotection, the free amines (18, 21) were converted to the N-
methylpiperidine (19) and dimethylamino (29) analogues by reductive amination, respectively.
Meanwhile, the amines (20, 21) were protected with a Boc group and then N-methylated to afford
the corresponding N-Boc methylamino (27, 28) analogues.
Scheme 1. Reagents and conditions: (a) N-Boc ethanolamine, N-Boc piperidine derivatives or tert-
butyl (4-(2-hydroxyethyl)pyridin-2-yl)carbamate, DEAD, PPh3, DCM, r.t., overnight; (b) alkyl
halides, K2CO3, DMF, 100 °C, 1 h; (c) TFA, DCM; (d) ZnCl2, HCHO, NaBH3CN, MeOH, r.t.,
overnight; (e) N2H4.H2O, EtOH, r.t., overnight; (f) Boc2O, TEA, DMC; (g) CH3I, NaH, THF, 0 °C
for 27, 28; (h) N-Boc piperazine, DMF, TEA, 60 °C, 2 h.
The synthesis of 4-aminoalkyloxy-1-nitrobenzene fragment is shown in Scheme 2. The N-
Boc aminoalkyloxy analogues (32, 33) were prepared from 4-nitrophenol (30) by alkylation
reactions.
The N-Boc amido analogues (37, 38, 42, 43, 50) were synthesized from 4-nitrobenzoic acid (34), 2-
(4-nitrophenoxy)acetic acid, (35) or 4-nitro cinnamic acid (36), by coupling with the corresponding
amines. The N-aminoethyl piperazinyl (40) and piperidinyl (47-49) analogues were prepared from
6

the corresponding piperazine (39) and piperazine (44-46) precursors by alkylation with tert-butyl
(2-iodoethyl)carbamate.
Scheme 2. Reagents and conditions: (a) alkyl halide, Cs2CO3, DMF, heat; (b) N-Boc piperazine,
Cs2CO3, DMF, heat; (c) RNH2, EDC, HOBt, DMC; (d) TFA, DMC; (e) tert-Butyl (2-
iodoethyl)carbamate, NaH, DMF, 0 °C to r.t., 2 h.
The synthesis of the final thiourea compounds is illustrated in Scheme 3. The synthesized
anilines were reduced either by hydrogenation or by zinc in acetic acid to provide the corresponding
amines, which were then coupled with 3-(5-methyl-1H-imidazol-1-yl)propan-1-amine by previous
method18 to afford the corresponding thioureas. Finally, the deprotection of N-Boc and N-
phthalimide provided the final 3-aminoalkyloxy-4-methoxyphenyl (51-66) and 4-
aminoalkyloxyphenyl (68-78) derivatives, respectively. The pyrimidine 67 was synthesized from
amine 53 by reacting with 2-chloropyrimidine.
7

Scheme 3. Reagents and conditions: (a) Pd, H2, MeOH; (b) Zn, AcOH, MeOH; (c) 3-(5-methyl-1H-
imidazol-1-yl)propan-1-amine, TCDI, Et3N, DMC, r.t., overnight; (d) TFA, DMC, 0 °C to r.t.,
overnight; (e) N2H4.H2O, EtOH, r.t., overnight; (f) 2-chloropyrimidine, TEA, EtOH, reflux, 2 days.
2.2. In vitro QC Inhibition
As previously reported, to evaluate the human QC inhibition of the synthesized compounds
we performed the QC activity assays employing a fluorogenic substrate, Gln-AMC (L-glutamine 7-
amido-4-methylcoumarin), and pyroglutamyl peptidase (pGAP) as an auxiliary enzyme. 25
First, we investigated a series of 3-aminoalkyloxy-4-methoxyphenyl analogues represented
as template B (Table 1). The primary amine derivatives (51-53) exhibited similarly potent
inhibition regardless of the length of linkers with a range of IC50 = 7.9-9.0 nM, which were 3.5-fold
more potent than the parent 1. The secondary amine derivatives (54, 55) were found approximately
3-fold less active than the corresponding primary amines (52, 53). The tertiary amine derivative (56)
showed poor inhibition compared to the corresponding secondary derivative (55). Next, we
examined the cyclic amine derivatives. The piperazine derivatives (57-59) also displayed reasonable
inhibitory effect regardless of the linker length. The piperidine derivatives (60-62) exhibited better
activity than those of piperazine derivatives, and they showed similar IC50 values compared to the
primary amine derivatives (51-53). However, the N-methylation of the piperidine (63) resulted in
the loss of activity. Because the previously developed 2-aminopyridine derivatives (template A,
Figure 2) showed promising activities both in in vitro (IC50 = 4.5 nM for 4) and in vivo, we also
examined the 2-aminopyridine analogues (64-66). Unfortunately, these compounds were found to
8

be less potent than their 3-methoxyphenyl-4-aminoalkyloxy counter parts (template A). When the
aminopyridine group was replaced with a 2-aminopyrimidine (67), the compound showed similar
inhibition as the aminopyridine analogue 66.
Table 1. IC50 values for the inhibition of hQC by template B compounds
R
n
IC50 (nM)a
29.2b
Cpd#
1
2
3
4
7.9 ( 0.7)
9.0 ( 2.3)
8.8 ( 0.6)
51
52
53
3
4
4
2
3
4
2
3
4
2
26.8 ( 4.7)
22.0 ( 6.8)
46.8 ( 10.8)
18.4 ( 1.9)
15.8 ( 2.8)
15.0 ( 0.5)
7.3 ( 6.3)
8.8 ( 2.2)
7.9 ( 2.1)
20.6 ( 7.4)
54
55
56
57
58
59
60
61
62
63
2
3
4
26.4 ( 8.8)
15.0 ( 2.9)
15.9 ( 7.4)
64
65
66
9

4
15.2 ( 2.0)
67
a The values indicate the mean of at least three experiments. b Refs. 14 and 18
Next, we examined a series of 4-aminoalkyloxyphenyl analogues, in which the 3-methoxy
group was removed and only contained the substituent at the 4-position and was represented as
template C (Table 2). We first tested compounds 68 and 69, which were most potent when they
contained the 3-methoxy group.18 However, it was found that compounds 68 and 69 were much less
active, by 27- and 5.5-fold respectively, than their previously reported 3-methoxy counterparts,
suggesting that the 3-methoxy group represents an important binding interaction for inhibitory
effect. We also examined inhibition of compounds 70-72 with an amido linker to compare the
structure-activity relationship of the previously developed derivatives containing a 3-methoxy
group.19 These compounds appeared to maintain similar activity compared to the previously
reported compounds,19 while the N-aminoethyl piperidine derivative (72) was found to be more
potent than that of 70, suggesting that the presence of an additional amide group may aid extra
binding interactions and that the terminal amino group may serve as an Arg-mimetic. However,
shortening or rigidifying the linker group mostly resulted in the loss of activity, as demonstrated in
73 and 74 (a 2-fold reduction than compounds 69 and 72, respectively). As a bioisostere of
methyleneoxy group in the linker, we also tested the cinnamic linker surrogates (75-78). Although
compounds containing the cinnamic linker appeared to be slightly less active than the
corresponding alkyloxy derivatives, the addition of an N-aminoethyl group to compound 75
increased the activity 3-fold to give potent inhibitor 76 with an IC50 = 6.4 nM, again suggesting that
the terminal amino group likely served as an Arg-mimetic group to interact with QC.
10

Table 2. IC50 values for inhibition of hQC by template C compounds
R
IC50 (nM)a
Cpd#
68
18.7 ( 5.8)
24.7 ( 6.5)
11.4 ( 2.0)
69
70
12.9 ( 5.9)
7.9 ( 7.3)
71
72
56.5 ( 12)
16.2 ( 4.0)
19.4 ( 9.9)
73
74
75
6.4 ( 0.6)
76
26.0 ( 9.6)
11.2 ( 3.2)
77
78
a The values indicate the mean of at least three experiments.
2.3. In vivo activity
Based on the in vitro QC inhibition of the synthesized compounds, we selected eight of the
most potent inhibitors with IC50 values less than 10 nM for further evaluation. We first examined
cytotoxicity by incubating HT-22 cells, an immortalized hippocampal neuronal cell line, with 10
11

µM of each compound, and performed MTT assays. All tested compounds were not cytotoxic,
demonstrating normal cell viability. We also evaluated the ability of hERG channel blocking for all
compounds to assess drug toxicity. All compounds showed moderate (35.1%, 62) to low inhibition
(2.8%, 53) at 10 µM, indicating that they pose a marginal to low risk for cardiotoxicity. To evaluate
the ability of the compounds to penetrate the blood-brain barrier (BBB), we carried out a parallel
artificial membrane permeability assay (PAMPA). Six compounds, 51-53 and 60-62, showed
reasonable permeability, with a range of 5.0-5.9 for –logPe. Interestingly, all of the primary amine
(51-53) and piperidine (60-62) derivatives showed reasonable values for BBB penetration
regardless of the length of the carbon linker, whereas the N-aminoethyl derivatives, 72 and 76,
exhibited very low permeability (–logPe = 10 and 9.0).
Finally, we tested each compound in acute AD model mice to evaluate QC inhibition in vivo.
We first injected human Aβ3-40 (5 µg) and each compound (25 mg/kg) successively into deep
cortical/hippocampal tissues of ICR mice (male, six weeks old) by intracerebroventricular (icv)
administration. On the next day, we measured the levels of human AβN3pE-40 in the brain extracts of
each mouse to determine the QC inhibitory activity. As described in Table 3, compounds 51-53, 62
and 76 suppressed the formation of AβN3pE-40 by 12.8% to 36.6% compared to the vehicle control.
In particular, compounds 51 and 53, with in vitro IC50 values of 7.9 and 8.8 nM, exhibited the most
potent AβN3pE-40-lowering effects by 35.3 and 36.6% reduction, respectively, indicating that the
specific inhibition of QC resulted in the reduced brain levels of AβN3pE-40. Overall, these two
compounds exhibited potent in vitro and in vivo QC inhibitory activities and good brain penetration
without potential toxicity.
Table 3. Studies of in vitro toxicity, permeability, and in vivo QC inhibition in acute model
cytotoxicity
at 10 µM
(% of control)
hERG assay
(10 µM, %
inhibition)
15.3
% inhibition of
human AβN3pE-40
formation (icv)a
35.3
in vitro
IC50 (nM)
PAMPA
(-logPe)
51
7.9
~100
5.7
12

9.0
8.8
7.3
8.8
7.9
7.9
6.4
~100
~100
~100
~100
~100
~100
~100
8.8
2.8
5.6
5.9
5.3
5.0
5.7
10.0
9.0
12.8
36.6
1.8
52
53
60
61
62
72
76
14.9
25.6
35.1
19.9
7.2
5.5
14.4
NE
22.9
a 5 µL of human Aβ3−40 in PBS (1 µg/µL) was injected into the deep cortical/hippocampus to 5 weeks old ICR mice (25
g, n = 4, male) using a stereotaxic frame to induce acute Aβ toxicity. Test compounds were administrated via icv
injection. Sandwich ELISA was performed for the quantification of the brain AβN3pE-40
2.4. Molecular modeling
To evaluate the binding interactions between the hQC and the potent inhibitor 53, we carried
out the sequential docking studies using X-ray crystal structure of hQC (PDB id: 3PBB).26 For the
initial docking study, the protonated amine form of 53 at pH 7.4 was used and placed into a Glide
SP (Standard Precision). The result exhibited that the N-3 nitrogen of the 5-methyl imidazole
chelated with zinc and formed a hydrogen bonding with Trp329. Additionally, the 5-methyl group
occupied a hydrophobic pocket composed of Trp207, Leu249, Ile303, Ile321, and Phe325. The
thiourea group caused the appropriate positioning of the phenyl ring for the hydrophobic interaction
with Ile303, while the methoxy oxygen on the phenyl ring formed a hydrogen bond with Tyr299
(Figure 3A).
Figure 3. Sequential docking and refinement of 53 in hQC.
13

(A) Glide SP docking, (B) QPLD, (C) Local optimization, and (D) Monte Carlo minimization.
Binding modes of 53 in protonated form were shown in each step. The distances between Glu327
and the terminal N from D-region of the ligands were revealed in black dashed lines.
Afterwards, Glide QM-Polarized Ligand Docking (QPLD) in Maestro was implemented,
and the result showed the amino group in the side chain shifted toward Glu327 of the hQC active
site (Figure 3B). The local optimization refinement was conducted, and the result displayed the
bended Glu327 side chain, and constituted a salt bridge with the amino group of 53 (Figure 3C). To
search for the global minimum, we performed a Monte Carlo minimization (Figure 3D). This type
of sequential optimization for the protein-ligand complexes formed hydrogen bonding as well as
salt bridge interactions, along with the H-bonding with the phenyl ring of 53 (Figure 4).
Accordingly, the imidazole ring formed π-π interactions with Trp329, and the thiourea group
showed a hydrogen bond with Gln304 while the dimethoxyphenyl group formed a H-bonding
interaction and π-π interaction with Phe325. Moreover, the protonated amine group located in the
side chain displayed a salt bridge interaction with Glu327 and H-bonding with Pro326 and Glu327.
Overall, we believe that switching the substituents in the 3-and 4-positions did not alter the binding
interactions significantly, partly due to the flexibility of the 4-aminoalkoxy chain, which is also
supported by the SAR found in the analogues with rigid and short linkers.
Figure 4. Docked and refined structure of 53 in hQC.
14

(A) Binding interactions of 53 at the active site of the hQC. Ligand is displayed as sticks with cyan
carbon atoms, and Zn2+ is in purple ball. The interacted residues are shown in light blue sticks.
Hydrogen bonds are described as black dashed lines. (B) 2D illustration of the binding interactions
between 53 with hQC. Hydrophobic interactions are indicated in light brown. Hydrogen bonds are
exposed as red- and green-dotted point with the directionality. The π-π stacking interaction is
signified in purple disc and arrow, and the salt bridge interaction is displayed as purple wedge line.
3. Conclusion
In this study, we investigated a series of QC inhibitors containing 4-aminoalkyloxy-3-
methoxyphenyl and 3-aminoalkyloxyphenyl groups as Phe-Arg mimetics of Aβ3-42. The primary
amines (51-53) and 4-piperidinyl (57-59) derivatives exhibited potent QC inhibition, demonstrating
3-4 fold more potent activity than the parent inhibitor 1 by Probiodrug. Further in vivo studies
revealed that inhibitors 51 and 53 displayed the most potent AβN3pE-40-lowering effects with 35.3
and 36.6% in vivo, respectively, with reasonable BBB penetration, which also corresponded to their
in vitro potency. The molecular modeling analysis of 53 indicated that the salt bridge interaction
and the hydrogen bonding of the protonated amine group with Glu327 and Pro326 provided a high
potency compared to the parent inhibitor 1. Given the potent QC inhibitory effect, favorable BBB
penetration, and the toxicity profile, we believe that compound 53 may serve as a potential
candidate for anti-Alzheimer’s agents.
15

4. Experimental
4.1. Chemistry
4.1.1. General
All chemical reagents were commercially available. Melting points were determined on a melting
point Buchi B-540 apparatus and are uncorrected. Silica gel column chromatography was
1
performed on silica gel 60, 230-400 mesh, Merck. H NMR spectra were recorded on a 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
−MS instrument and a 6460 Triple Quad LC−MS
spectra were recorded on a VG Trio-2 GC
instrument. All final compounds were assessed for purity by high performance liquid
chromatography (HPLC) on Agilent 1120 Compact LC (G4288A) system via the following
conditions. Column: Agilent TC-C18 column (4.6 mm × 250 mm, 5 µm). Mobile phase A: MeOH,
Mobile phase B: 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 ≥95%.
4.1.2. General procedure
4.1.2.1. Mitsunobu reaction (Procedure 1)
Triphenylphosphine (1.3 eq) was added under nitrogen to a solution of 5-nitroguaiacol (1.0 eq) in
CH2Cl2, followed by adding alcohol (1.2 eq) and a solution of diethyl azodicarboxylate (1.3 eq) in
CH2Cl2. 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 give desired product.
4.1.2.2. Williamson reaction (Procedure 2)
Alkyl halide (4.0 eq) was added to a suspension of 5-nitroguaiacol or 4-nitrophenol (1.0 eq) and
Cs2CO3 (2.0 eq) in anhydrous DMF. The reaction mixture was heated to 100 °C for 1 h and then
cooled to room temperature before quenched by H2O. The mixture was extracted with EtOAc (2 x
16

50 mL). The organic layer was washed by H2O three times, dried by MgSO4 and concentrated. This
concentration was then purified by column chromatography to get product.
4.1.2.3. Deprotection of phthalimide group (Procedure 3)
Hydrazine monohydrate was added to a solution of phthalimide compound in ethanol and stirred at
room temperature overnight. The precipitate was filtered and washed with EtOH. The filtrate was
collected and concentrated in vacuo. The residue was then purified by PLC (MeOH/CH2Cl2) to give
product.
4.1.2.4. N-Alkylation (Procedure 4)
A mixture of alkyl halide, amine, and excess base in DMF was stirred at 60 °C for 30 min. The
mixture reaction was quenched by H2O and extracted by EtOAc several times. The combined
organic layer was washed with H2O and brine and concentrated in vacuo. The residue was purified
by column chromatography.
4.1.2.5. Boc protection (Procedure 5)
To a suspension of starting material amine (1.0 eq) in CH2Cl2 was added triethylamine (1.2 eq) and
di-tert-butyl dicarbonate (2.5 eq) in CH2Cl2 under ice bath. The mixture was stirred at room
temperature until starting material was consumed, by checking with TLC. The mixture was diluted
with H2O and extracted with CH2Cl2 several times. The combined organic layers were washed with
10% aqueous NaHCO3 solution, H2O and brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by flash chromatography to get the desired product.
4.1.2.6. Boc deprotection (Procedure 6)
17

Trifluoroacetic acid (10 eq) was added to the solution of Boc-protected compound (1.0 eq) in
CH2Cl2 (DCM:TFA = 1:1 (v/v)). Then, the mixture was stirred at room temperature until the
starting material consumed and evaporated. The residue was dissolved in MeOH and purified by
ion-exchange column to get desired product or carried to the next step without further purification.
4.1.2.7. Reductive methylation of amine (Procedure 7)
To a stirred solution of amine (1 eq) in MeOH (5 mL) containing 37% aqueous formaldehyde (3 eq)
at room temperature was added a solution of sodium cyanoborohydride (1 eq) and zinc chloride (0.5
eq) in MeOH (5 mL). After the reaction mixture was stirred at room temperature for overnight, the
solution was taken up in 0.1 N NaOH (10 mL), and most of the MeOH was evaporated under
reduced pressure. After the aqueous solution was extracted with EtOAc (20 mL x 3), the combined
extracts were washed with H2O and brine, dried over MgSO4 and evaporated until dry . The residue
was distilled in vacuo to give the desired product.
4.1.2.8. EDC coupling (Procedure 8)
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide HCl (EDC-HCl, 1.1 eq) and N,N-
diisopropylethylamine (2.2 eq) were added to a solution of amine (1.0 eq), acid (1.0 eq) and 1-
hydroxybenzotriazole (HOBt, 1.1 eq) in CH2Cl2. The mixture was stirred for 24 h at room
temperature under nitrogen. The solvent was removed in vacuo and the residue purified by column
chromatography on silica gel, eluting with CH2Cl2/MeOH to provide the desired compound.
4.1.3. Intermediate compounds
4.1.3.1. tert-Butyl (2-(2-methoxy-5-nitrophenoxy)ethyl)carbamate (6). The title compound was
prepared from 5-nitroguaiacol (5) according to procedure 1 as a yellow solid in 99% yield. 1H NMR
(300 MHz, CDCl3) δ 7.95 (dd, J = 8.97, 2.55 Hz, 1H), 7.76 (d, J = 2.58 Hz, 1H), 6.93 (d, J = 8.97
18

Hz, 1H), 5.07 (br, NH), 4.15 (t, J = 5.31 Hz, 2H), 3.96 (s, 3H), 3.61 (q, J = 5.49 Hz, 2H), 1.45 (s,
9H).
4.1.3.2. tert-Butyl 4-(2-(2-methoxy-5-nitrophenoxy)ethyl)piperidine-1-carboxylate (7). The title
compound was prepared from compound 5 according to procedure 1 as a yellow oil in 63% yield.
1H NMR (300 MHz, CDCl3) δ 7.93 (dd, J = 8.97, 2.58 Hz, 1H), 7.73 (d, J = 2.55 Hz, 1H), 6.92 (d, J
= 8.97 Hz, 1H), 4.35-4.17 (m, 3H), 3.96 (s, 3H), 2.72 (t, J = 11.73 Hz, 2H), 1.85-1.80 (m, 2H),
1.75-1.72 (m, 2H), 1.46 (s, 9H), 1.46-1.45 (m, 2H), 1.36-1.30 (m, 2H).
4.1.3.3. tert-Butyl 4-(3-(2-methoxy-5-nitrophenoxy)propyl)piperidine-1-carboxylate (8). The title
compound was prepared from compound 5 according to procedure 1 as a yellow oil in 76% yield.
1H NMR (300 MHz, CDCl3) δ 7.92 (dd, J = 8.97, 2.73 Hz, 1H), 7.73 (d, J = 2.73 Hz, 1H), 6.92 (d,
J = 8.97 Hz, 1H), 4.15-4.05 (m, 4H), 3.96 (s, 3H), 3.48 (m, 1H), 2.72 (t, J = 12.06 Hz, 2H), 1.95 (p,
J = 6.60 Hz, 2H), 1.72 (d, J = 13.53 Hz, 2H), 1.45 (s, 9H), 1.45-1.39 (m, 2H), 1.17-1.11 (m, 2H).
4.1.3.4. tert-Butyl 4-(4-(2-methoxy-5-nitrophenoxy)butyl)piperidine-1-carboxylate (9). The title
compound was prepared from compound 5 according to procedure 1 as a brown oil in 63% yield.
1H NMR (300 MHz, CDCl3) δ 7.92 (dd, J = 8.79, 2.55 Hz, 1H), 7.73 (d, J = 2.76 Hz, 1H), 6.91 (d,
J = 8.79 Hz, 1H), 4.15-4.06 (m, 4H), 3.96 (s, 3H), 2.71 (t, J = 12.09 Hz, 2H), 1.91 (p, J = 6.96 Hz,
2H), 1.68-1.64 (m, 2H), 1.51-1.47 (m, 3H), 1.45 (s, 9H), 1.35-1.28 (m, 2H), 1.15-1.02 (m, 2H).
4.1.3.5. tert-Butyl (4-(2-(2-methoxy-5-nitrophenoxy)ethyl)pyridin-2-yl)carbamate (10). The title
compound was prepared from compound 5 according to procedure 1 as a yellow solid in 59% yield.
1H NMR (300 MHz, CDCl3) δ 8.17 (d, J = 5.13 Hz, 1H), 7.94-7.90 (m, 2H), 7.73 (d, J = 2.55 Hz,
1H), 7.43 (br, NH), 6.94 (dd, J = 5.13, 1.47 Hz, 1H), 6.92 (d, J = 8.97 Hz, 1H), 4.33 (t, J = 6.78 Hz,
2H), 3.96 (s, 3H), 3.19 (t, J = 6.78 Hz, 2H), 1.53 (s, 9H).
19

4.1.3.6. tert-Butyl (4-(3-(2-methoxy-5-nitrophenoxy)propyl)pyridin-2-yl)carbamate (11).
The title compound was prepared from compound 5 according to procedure 2 as a yellow solid in
1
65% yield. H NMR (300 MHz, CDCl3) δ 8.15 (d, J = 5.10 Hz, 1H), 7.93 (dd, J = 8.79, 2.55 Hz,
1H), 7.84 (s, 1H), 7.70 (d, J = 2.58 Hz, 1H), 7.64 (s, 1H), 6.92 (d, J = 8.79 Hz, 1H), 6.84 (dd, J =
5.13, 2.28 Hz, 1H), 4.11 (t, J = 6.24 Hz, 2H), 3.97 (s, 3H), 2.86 (t, J = 7.14 Hz, 2H), 2.27 (p, J =
6.24 Hz, 2H), 1.52 (s, 9H).
4.1.3.7. tert-Butyl (4-(4-(2-methoxy-5-nitrophenoxy)butyl)pyridin-2-yl)carbamate (12). The title
compound was prepared from compound 5 according to procedure 2 as a yellow solid in 65% yield.
1H NMR (300 MHz, CDCl3) δ 8.11 (d, J = 5.31 Hz, 1H), 7.92 (dd, J = 8.97, 2.76 Hz, 1H), 7.80 (br,
NH), 7.72 (d, J = 2.55 Hz, 1H), 7.21 (br, NH), 6.91 (d, J = 8.79 Hz, 1H), 6.83 (dd, J = 4.95, 1.47 Hz,
1H), 4.11 (t, J = 6.06 Hz, 2H), 3.96 (s, 3H), 2.72 (t, J = 7.68 Hz, 2H), 1.89-1.81 (m, 4H), 1.52 (s,
9H).
4.1.3.8. 2-(3-(2-Methoxy-5-nitrophenoxy)propyl)isoindoline-1,3-dione (13).
The title compound was prepared from compound 5 according to procedure 2 as a yellow solid in
1
61% yield. H NMR (300 MHz, CDCl3) δ 7.90-7.81 (m, 3H), 7.75-7.61 (m, 3H), 6.84 (d, J = 8.97
Hz, 1H), 4.18 (t, J = 6.03 Hz, 2H), 3.96 (d, J = 6.60 Hz, 2H), 3.73 (s, 3H), 2.31 (p, J = 6.24 Hz,
2H).
4.1.3.9. 2-(4-(2-Methoxy-5-nitrophenoxy)butyl)isoindoline-1,3-dione (14). The title compound was
1
prepared from compound 5 according to procedure 2 as a yellow solid in 94% yield. H NMR (300
MHz, CDCl3) δ 7.91 (dd, J = 8.97, 2.55 Hz, 1H), 7.86-7.81 (m, 2H), 7.75-7.69 (m, 3H), 6.90 (d, J =
8.97 Hz, 1H), 4.14-4.10 (m, 2H), 3.94 (s, 3H), 3.81-3.77 (m, 2H), 1.94-1.91 (m, 4H).
20

4.1.3.10. 2-(2-Bromoethoxy)-1-methoxy-4-nitrobenzene (15). The title compound was prepared
1
from compound 5 according to procedure 2 as a yellow solid in 73% yield. H NMR (300 MHz,
CDCl3) δ 7.97 (dd, J = 8.76, 2.55 Hz, 1H), 7.77 (d, J = 2.58 Hz, 1H), 6.95 (d, J = 8.97 Hz, 1H), 4.42
(t, J = 6.42 Hz, 2H), 3.98 (s, 3H), 3.72 (t, J = 6.21 Hz, 2H).
4.1.3.11. 2-(3-Bromopropoxy)-1-methoxy-4-nitrobenzene (16). The title compound was prepared
1
from compound 5 according to procedure 2 as a yellow solid in 71% yield. H NMR (300 MHz,
CDCl3) δ 7.94 (dd, J = 8.97, 2.58 Hz, 1H), 7.78 (d, J = 2.58 Hz, 1H), 6.93 (d, J = 8.76 Hz, 1H), 4.28
(t, J = 7.14 Hz, 2H), 3.96 (s, 3H), 3.80 (t, J = 6.21 Hz, 2H), 2.37 (p, J = 6.06 Hz, 2H).
4.1.3.12. 2-(4-Bromobutoxy)-1-methoxy-4-nitrobenzene (17). The title compound was prepared
1
from compound 5 according to procedure 2 as a yellow solid in 63% yield. H NMR (300 MHz,
CDCl3) δ 7.93 (dd, J = 8.97, 2.55 Hz, 1H), 7.74 (d, J = 2.58 Hz, 1H), 6.92 (d, J = 8.97 Hz, 1H), 4.14
(t, J = 5.85 Hz, 2H), 3.96 (s, 3H) 3.53 (t, J = 6.24 Hz, 2H), 2.10-2.05 (m, 4H).
4.1.3.13. 4-(4-(2-Methoxy-5-nitrophenoxy)butyl)piperidine (18). The title compound was prepared
1
from compound 7 according to procedure 6 as a red solid in 75% yield. H NMR (300 MHz,
CD3OD) δ 7.91 (dd, J = 8.97, 2.76 Hz, 1H), 7.76 (d, J = 2.58 Hz, 1H), 7.09 (d, J = 8.97 Hz, 1H),
4.14 (t, J = 5.85 Hz, 2H), 3.93 (s, 3H), 3.06-3.02 (m, 2H), 2.65-2.56 (m, 2H), 1.80-1.74 (m, 5H),
1.29-1.18 (m, 2H).
4.1.3.14. 4-(2-(2-Methoxy-5-nitrophenoxy)ethyl)-1-methylpiperidine (19). The title compound was
1
prepared from compound 18 according to procedure 7 as a white solid in 45% yield. H NMR (300
MHz, CDCl3) δ 7.93 (dd, J = 8.40, 2.58 Hz, 1H), 7.73 (d, J = 2.73 Hz, 1H), 6.92 (d, J = 8.97 Hz,
1H), 4.14 (t, J = 6.60 Hz, 2H), 3.96 (s, 3H), 3.12 (d, J = 11.88 Hz, 2H), 2.45 (s, 3H), 2.26 (t, J =
9.90 Hz, 2H), 1.89-1.83 (m, 4H), 1.56-1.47 (m, 3H)
21

4.1.3.15. 3-(2-Methoxy-5-nitrophenoxy)propan-1-amine (20). The title compound was prepared
from compound 13 according to procedure 3 as a light yellow solid in 94% yield. 1H NMR (300
MHz, CDCl3) δ 7.87 (dd, J = 8.43, 2.43 Hz, 1H), 7.72 (d, J = 2.43 Hz, 1H), 6.88 (d, J = 8.46 Hz,
1H), 4.13 (t, J = 5.82 Hz, 2H), 3.93 (s, 3H), 2.62 (t, J = 6.12 Hz, 2H), 1.98-1.88 (m, 2H).
4.1.3.16. 4-(2-Methoxy-5-nitrophenoxy)butan-1-amine (21). The title compound was prepared from
1
compound 14 according to procedure 3 as a white solid in 85% yield. H NMR (400 MHz, CDCl3)
δ 7.88 (dd, J = 8.40, 2.40 Hz, 1H), 7.71 (d, J = 2.40 Hz, 1H), 6.88 (d, J = 8.88 Hz, 1H), 4.13 (t, J =
5.80 Hz, 2H), 3.93 (s, 3H), 2.79 (t, J = 6.68 Hz, 2H), 2.00-1.83 (m, 4H)
4.1.3.17. tert-Butyl 4-(2-(2-methoxy-5-nitrophenoxy)ethyl)piperazine-1-carboxylate (22). The title
compound was prepared from compound 15 according to procedure 4 as a white solid in 75% yield.
1H NMR (300 MHz, CDCl3) δ 7.94 (dd, J = 8.40, 2.58 Hz, 1H), 7.80 (d, J = 2.58 Hz, 1H), 6.92 (d, J
= 8.97 Hz, 1H), 4.24 (t, J = 5.85 Hz, 2H), 3.95 (s, 3H), 3.47 (t, J = 4.95 Hz, 4H), 2.90 (t, J = 5.85
Hz, 2H), 2.56 (t, J = 4.74 Hz, 4H), 1.46 (s, 9H).
4.1.3.18. tert-Butyl 4-(3-(2-methoxy-5-nitrophenoxy)propyl)piperazine-1-carboxylate (23). The title
compound was prepared from compound 16 according to procedure 4 as a white solid in 89% yield.
1H NMR (300 MHz, CDCl3) δ 7.92 (dd, J = 9.15, 2.55 Hz, 1H), 7.77 (d, J = 2.19 Hz, 1H), 6.91 (d, J
= 9.15 Hz, 1H), 4.18 (t, J = 6.57 Hz, 2H), 3.95 (s, 3H), 3.44 (t, J = 4.95 Hz, 2H), 2.57 (t, J = 6.60
Hz, 4H), 2.41 (t, J = 4.95 Hz, 4H), 2.08 (p, J = 6.78 Hz, 2H), 1.46 (s, 9H).
4.1.3.19. tert-Butyl 4-(4-(2-methoxy-5-nitrophenoxy)butyl)piperazine-1-carboxylate (24). The title
compound was prepared from compound 17 according to procedure 4 as a white solid in 75% yield.
1H NMR (300 MHz, CDCl3) δ 7.93 (dd, J = 8.97, 2.55 Hz, 1H), 7.73 (d, J = 2.55 Hz, 1H), 6.92 (d, J
22

= 8.79 Hz, 1H), 4.15 (d, J = 6.96 Hz, 2H), 3.95 (s, 3H), 3.44 (t, J = 5.13 Hz, 4H), 2.45-2.37 (m, 6H),
1.96 (p, J = 6.75 Hz, 2H), 1.74 (p, J = 7.89 Hz, 2H), 1.46 (s, 9H).
4.1.3.20. tert-Butyl (2-(2-methoxy-5-nitrophenoxy)ethyl)carbamate (25). The title compound was
prepared from compound 20 according to procedure 5 as an opaque semi-solid in 72% yield, which
was used for the next step without further purification.
4.1.3.21. tert-Butyl (3-(2-methoxy-5-nitrophenoxy)propyl)carbamate (26). The title compound was
prepared from compound 21 according to procedure 5 as a white solid in 62% yield, which was
used for the next step without further purification.
4.1.3.22. tert-Butyl (2-(2-methoxy-5-nitrophenoxy)ethyl)(methyl)carbamate (27). The title
compound was prepared from compound 25 according to procedure 4 as a light yellow solid in 51%
yield. 1H NMR (300 MHz, CDCl3) δ 7.87 (dd, J = 8.46, 2.40 Hz, 1H), 7.72 (d, J = 2.43 Hz, 1H),
6.87 (d, J = 8.61 Hz, 1H), 4.14 (t, J = 6.03 Hz, 2H), 3.93 (s, 3H), 2.92 (t, J = 6.12 Hz, 2H), 2.78 (s,
3H), 2.01-1.90 (m, 2H), 1.44 (s, 9H).
4.1.3.23. tert-Butyl (3-(2-methoxy-5-nitrophenoxy)propyl)(methyl)carbamate (28). The title
compound was prepared from compound 26 according to procedure 4 as a light yellow solid in 55%
1
yield. H NMR (300 MHz, CDCl3) δ 7.89 (dd, J = 8.79, 2.73 Hz, 1H), 7.72 (d, J = 2.73 Hz, 1H),
6.89 (d, J = 8.97 Hz, 1H), 4.14 (t, J = 6.21 Hz, 2H), 3.94 (s, 3H), 2.65 (t, J = 6.12 Hz, 2H), 2.80 (s,
3H), 2.01-1.87 (m, 4H), 1.44 (s, 9H).
4.1.3.24. 4-(2-Methoxy-5-nitrophenoxy)-N,N-dimethylbutan-1-amine (29). The title compound was
1
prepared from compound 21 according to procedure 7 as a red semi-solid in 21% yield. H NMR
(300 MHz, CDCl3) δ 7.93 (dd, J = 8.97, 2.73 Hz, 1H), 7.74 (d, J = 2.58 Hz, 1H), 6.92 (d, J = 8.97
23

Hz, 1H), 4.14 (t, J = 6.24 Hz, 2H), 3.96 (s, 3H), 2.62 (t, J = 8.61 Hz, 2H), 2.40 (s, 6H), 1.95 (p, J =
6.60 Hz, 2H), 1.82 (p, J = 7.32 Hz, 2H).
4.1.3.25. 1-(2-Bromoethoxy)-4-nitrobenzene (31). The title compound was prepared from
compound 30 according to procedure 2 as a yellow solid in 64% yield. 1H NMR (300 MHz, CDCl3)
δ 8.20 (m, 2H), 6.97 (m, 2H), 4.36 (d, J = 6.03 Hz, 2H), 3.68 (d, J = 6.24 Hz, 2H).
4.1.3.26. tert-Butyl 4-(2-(4-nitrophenoxy)ethyl)piperazine-1-carboxylate (32). The title compound
1
was prepared from compound 31 according to procedure 4 as a white solid in 61% yield. H NMR
(300 MHz, CDCl3) δ 8.19 (2H, m), 6.97 (2H, m), 4.19 (t, J = 5.67 Hz, 2H), 3.45 (m, 4H), 2.85 (t, J
= 5.70 Hz, 2H), 2.52 (m, 4H), 1.45 (s, 9H).
4.1.3.27. tert-Butyl (4-(4-(4-nitrophenoxy)butyl)pyridin-2-yl)carbamate (33). The title compound
1
was prepared from compound 30 according to procedure 2 as a white solid in 91% yield. H NMR
(300 MHz, CDCl3) δ 8.19 (d, J = 9.15 Hz, 2H), 8.11-8.09 (m, 2H), 7.86 (s, 1H), 6.94 (d, J = 9.15
Hz, 2H), 6.82 (dd, J = 5.73, 1.47 Hz, 1H), 4.06 (t, J = 5.67 Hz, 2H), 2.68 (t, J = 6.06 Hz, 2H), 1.86-
1.81 (m, 4H), 1.51 (s, 9H).
4.1.3.28. tert-Butyl 4-(2-(4-nitrophenoxy)acetyl)piperazine-1-carboxylate (37). The title compound
was prepared from compound 35 according to procedure 8 as a yellow solid in 81% yield. 1H NMR
(300 MHz, CDCl3) δ 8.17 (d, J = 9.33 Hz, 2H), 6.99 (d, J = 9.33 Hz, 2H), 4.75 (s, 2H), 3.53-3.49
(m, 2H), 3.46-3.43 (m, 2H), 3.40-3.34 (m, 4H), 1.39 (s, 9H).
4.1.3.29. tert-Butyl (E)-4-(3-(4-nitrophenyl)acryloyl)piperazine-1-carboxylate (38). The title
compound was prepared from commercial available 36 according to procedure 8 as a white solid in
1
80% yield. H NMR (300 MHz, CDCl3) δ 8.25 (d, J = 8.79 Hz, 2H), 7.70 (d, J = 15.36 Hz, 1H),
24

7.65 (d, J = 8.61 Hz, 2H), 7.02 (d, J = 15.57 Hz, 1H), 3.72-3.64 (m, 4H), 3.42-3.36 (m, 4H), 1.48 (s,
9H).
4.1.3.30. (E)-3-(4-Nitrophenyl)-1-(piperazin-1-yl)prop-2-en-1-one (39). The title compound was
1
prepared from compound 38 according to procedure 6 as a yellow oil in 94% yield. H NMR (300
MHz, CD3OD) δ 8.27 (d, J = 8.79 Hz, 2H), 7.89 (d, J = 8.58 Hz, 2H), 7.71 (d, J = 15.57 Hz, 1H),
7.39 (d, J = 15.57 Hz, 1H), 4.00-3.95 (m, 4H), 3.39-3.22 (m, 4H).
4.1.3.31. tert-Butyl (E)-(2-(4-(3-(4-nitrophenyl)acryloyl)piperazin-1-yl)ethyl)carbamate (40). The
title compound was prepared from compound 39 according to procedure 4 as a pale yellow solid in
54% yield. 1H NMR (300 MHz, CDCl3) δ 8.27 (d, J = 8.79 Hz, 2H), 7.72-7.65 (m, 3H), 7.03 (d, J =
15.57 Hz, 1H), 3.76-3.66 (m, 4H), 3.27 (q, J = 5.31 Hz, 2H), 2.53-2.47 (m, 6H), 1.46 (s, 9H).
4.1.3.32. tert-Butyl 4-(4-nitrobenzamido)piperidine-1-carboxylate (41). The title compound was
1
prepared from compound 34 according to procedure 8 as a white solid in 80% yield. H NMR (300
MHz, CDCl3) δ 8.30 (d, J = 8.79 Hz, 2H), 7.95 (d, J = 8.79 Hz, 2H), 6.18 (d, J = 7.50 Hz, NH),
4.15-4.10 (m, 3H), 2.94 (t, J = 12.24 Hz, 2H), 2.06 (t, J = 4.59 Hz, 2H), 1.46 (s, 9H), 1.42-1.39 (m,
2H).
4.1.3.33. tert-Butyl 4-(2-(4-nitrophenoxy)acetamido)piperidine-1-carboxylate (42). The title
compound was prepared from compound 35 according to procedure 8 as a white solid in 82% yield.
1H NMR (300 MHz, CDCl3) δ 8.25 (d, J = 9.15 Hz, 2H), 7.02 (d, J = 9.33 Hz, 2H), 6.32 (br, NH),
4.54 (s, 2H), 4.13-4.09 (m, 3H), 2.88 (t, J = 12.09 Hz, 2H), 1.93-1.90 (m, 2H), 1.43 (s, 9H), 1.38-
1.33 (m, 2H).
25

4.1.3.34. tert-Butyl (E)-4-(3-(4-Nitrophenyl)acrylamido)piperidine-1-carboxylate (43). The title
compound was prepared from compound 36 according to procedure 8 as a white solid in 83% yield.
1H NMR (300 MHz, CDCl3) 8.25 (d, J = 8.79 Hz, 2H), 7.70 (d, J = 15.36 Hz, 1H), 6.65 (d, J = 8.61
Hz, 2H), 6.54 (d, J = 15.57 Hz, 1H), 5.76 (d, J = 7.89 Hz, NH), 4.13 (m, 3H), 2.93 (t, J = 11.73 Hz,
2H). 2.05-1.98 (m, 2H), 1.46 (s, 9H), 1.36-1.32 (m, 2H).
4.1.3.35. 4-Nitro-N-(piperidin-4-yl)benzamide (44). The title compound was prepared from
1
compound 41 according to procedure 6 as a pale yellow oil in 89% yield. H NMR (300 MHz,
CDCl3) δ 8.31 (d, J = 8.79 Hz, 2H), 7.94 (d, J = 8.79 Hz, 2H), 6.05 (d, J = 7.89 Hz, NH), 4.14-4.04
(m, 1H), 3.16 (td, J = 12.45, 2.55 Hz, 2H), 2.81 (dt, J = 12.27, 2.55 Hz, 2H), 2.08-2.08 (m, 2H),
1.51-1.38 (m, 2H).
4.1.3.36. 2-(4-Nitrophenoxy)-N-(piperidin-4-yl)acetamide (45). The title compound was prepared
1
from compound 42 according to procedure 6 as a a brown semi-solid in 92% yield. H NMR (300
MHz, CDCl3) δ 8.25 (d, J = 8.97 Hz, 2H), 7.02 (d, J = 9.03 Hz, 2H), 6.32 (br, NH), 4.54 (s, 2H),
4.13-4.09 (m, 1H), 3.16-3.10 (m, 2H), 2.78 (t, J = 12.09 Hz, 2H), 2.00-1.95 (m, 2H), 1.48-1.39 (m,
2H).
4.1.3.37. (E)-3-(4-Nitrophenyl)-N-(piperidin-4-yl)acrylamide (46). The title compound was
prepared from compound 43 according to procedure 6 as a yellow solid in 94% yield. 1H NMR (300
MHz, CD3OD) δ 8.27 (d, J = 8.79 Hz, 2H), 7.80 (d, J = 8.79 Hz, 2H), 7.65 (d, J =15.93 Hz, 1H),
6.77 (d, J = 15.90 Hz, 1H), 4.08-4.01 (m, 1H), 3.53-3.43 (m, 2H), 3.19 (dt, J= 12.99, 3.12 Hz , 2H),
2.18-2.14 (m, 2H), 1.79-1.65 (m, 2H).
4.1.3.38. tert-Butyl (2-(4-(4-nitrobenzamido)piperidin-1-yl)ethyl)carbamate (47). The title
compound was prepared from compound 44 according to procedure 4 as an opaque semi-solid in 51%
26

yield. 1H NMR (300 MHz, CDCl3) δ 8.31 (d, J = 8.61 Hz, 2H), 7.93 (d, J = 8.79 Hz, 2H), 6.07 (d, J
= 8.07 Hz, NH), 4.98 (br, NH), 4.02-3.95 (m, 1H), 3.24 (q, J = 6.69 Hz, 2H), 2.93-2.90 (m, 2H),
2.51 (t, J = 6.06 Hz, 2H), 2.26 (t, J = 11.55 Hz, 2H), 2.08-2.04 (m, 2H), 1.63-1.59 (m, 2H), 1.45 (s,
9H).
4.1.3.39. tert-Butyl (2-(4-(2-(4-nitrophenoxy)acetamido)piperidin-1-yl)ethyl)carbamate (48). The
title compound was prepared from compound 45 according to procedure 4 as a red semi-solid in 55%
yield. 1H NMR (300 MHz, CDCl3) δ 8.24 (d, J = 9.15 Hz, 2H), 7.01 (d, J = 9.15 Hz, 2H), 6.38 (br,
NH), 5.12 (br, NH), 4.53 (s, 2H), 3.94-3.90 (m, 1H), 3.25-3.23 (m, 2H), 2.93-2.91 (m, 2H), 2.54-
2.51 (m, 2H), 2.24-2.20 (m, 2H), 1.96-1.94 (m, 2H), 1.62-1.58 (m, 2H), 1.42 (s, 9H).
4.1.3.40. tert-Butyl (E)-(2-(4-(3-(4-nitrophenyl)acrylamido)piperidin-1-yl)ethyl)carbamate (49).
The title compound was prepared from compound 46 according to procedure 4 as a pale yellow
solid in 21% yield. 1H NMR (300 MHz, CD3OD) δ 8.27 (d, J = 8.97 Hz, 2H), 7.80 (d, J = 8.79 Hz,
2H), 7.60 (d, J =15.90 Hz, 1H), 6.79 (d, J = 15.72 Hz, 1H), 4.08-4.01 (m, 1H), 3.53-3.43 (m, 2H),
2.93-2.87 (m, 2H), 2.55-2.51 (m, 2H), 2.42-2.39 (m, 2H), 1.96-1.89 (m, 2H), 1.65-1.55 (m, 2H),
1.42 (s, 9H).
4.1.3.41. tert-Butyl (4-((2-(4-nitrophenoxy)acetamido)methyl)pyridin-2-yl)carbamate (50). The title
compound was prepared from compound 35 according to procedure 8 as a white solid in 65% yield.
1H NMR (300 MHz, CDCl3) δ 8.24 (d, J = 9.15 Hz, 2H), 8.19 (d, J = 5.31 Hz, 1H), 7.88 (s, 1H),
7.06 (d, J = 9.15 Hz, 2H), 6.87 (dd, J = 5.31, 1.47 Hz, 1H), 4.64 (s, 2H), 4.53 (d, J = 6.03 Hz, 2H),
1.50 (s, 9H).
4.1.4. Final compounds
4.1.4.1. General procedure for final compound
27

All nitro compounds were reduced by either hydrogenation using 10% Pd/C or zinc powder in
acidic medium to obtain the corresponding amines, respectively. The amines were converted into
the isothiocyanates by 1,1’-thiocarbonyldiimidazole (1.02 eq) in anhydrous CH2Cl2, and then
coupled with 3-(5-methyl-1H-imidazol-1-yl)propan-1-amine (1.1 eq) to afford the corresponding
thiourea, respectively. The Boc deprotection by following the general procedure 6 provided the
final compounds.
4.1.4.2.
N-(3-(2-Aminoethoxy)-4-methoxyphenyl)-N’-(3-(5-methyl-1H-imidazol-1-
o
1
yl)propyl)thiourea (51). mp = 58-59 C. H NMR (300 MHz, CDCl3) δ 7.60 (s, 1H), 7.37 (s, 1H),
6.91 (d, J = 8.43 Hz, 1H), 6.78-6.72 (m, 3H), 5.93 (br, 1H), 4.02 (t, J = 5.13 Hz, 2H), 3.91 (t, J =
7.14 Hz, 2H), 3.88 (s, 3H), 3.69 (q, J = 6.24 Hz, 2H), 3.14 (t, J = 5.13 Hz, 2H), 2.17 (d, J = 0.93 Hz,
3H), 2.09 (p, J = 7.14 Hz, 2H). MS (FAB) m/z 364 [M+H]+. HRMS (FAB) m/z calcd for
C17H25N5O2S [M + H]+ 364.1807, found: 364.1818. Anal. HPLC 99.33% (Rt = 3.723 min).
4.1.4.3.
N-(3-(3-Aminopropoxy)-4-methoxyphenyl)-N’-(3-(5-methyl-1H-imidazol-1-
o
1
yl)propyl)thiourea (52). mp = 154-155 C. H NMR (300 MHz, CDCl3) δ 7.79 (br, 1H), 7.38 (s,
1H), 6.88-6.85 (m, 1H), 6.76-6.72 (m, 3H), 6.10 (br, 1H), 4.09 (t, J = 6.03 Hz, 2H), 3.91 (t, J =
7.14 Hz, 2H), 3.86 (s, 3H), 3.68 (q, J = 6.24 Hz, 2H), 2.93 (t, J = 6.60 Hz, 2H), 2.17 (d, J = 0.90 Hz,
3H), 2.09-1.92 (m, 4H). MS (FAB) m/z 378 [M+H]+. HRMS (FAB) m/z calcd for C18H27N5O2S [M
+ H]+ 378.1964, found: 378.1972. Anal. HPLC 99.53% (Rt = 3.641 min)
4.1.4.4.
N-(3-(4-Aminobutoxy)-4-methoxyphenyl)-N’-(3-(5-methyl-1H-imidazol-1-
o
yl)propyl)thiourea (53). mp = 55-56 C. 1H NMR (300 MHz, CDCl3) δ 7.60 (br, 1H), 7.43 (s, 1H),
6.80 (d, J = 8.43 Hz, 1H), 6.76-6.70 (m, 3H), 6.00 (br, 1H), 4.00 (t, J = 6.60 Hz, 2H), 3.91 (t, J =
7.14 Hz, 2H), 3.87 (s, 3H), 3.68 (q, J = 6.42 Hz, 2H), 2.79 (t, J = 6.78 Hz, 2H), 2.17 (d, J = 0.90 Hz,
3H), 2.09 (p, J = 7.14 Hz, 2H), 1.92 (p, J = 6.78 Hz, 2H), 1.66-1.59 (m, 2H). MS (FAB) m/z 392
28

[M+H]+. HRMS (FAB) m/z calcd for C19H29N5O2S [M + H]+ 392.2120, found: 392.2127. Anal.
HPLC 100.00% (Rt = 3.199 min).
4.1.4.5.
N-(4-Methoxy-3-(3-(methylamino)propoxy)phenyl)-N’-(3-(5-methyl-1H-imidazol-1-
o
1
yl)propyl)thiourea (54). mp = 88-89 C. H NMR (300 MHz, CDCl3) δ 7.42 (s, 1H), 6.86 (d, J =
8.43 Hz, 1H), 6.78 (dd, J = 8.43, 2.37 Hz, 1H), 6.73 (d, J = 2.19 Hz, 1H), 6.68 (s, 1H), 6.09 (br, 1H),
4.07 (t, J = 6.24 Hz, 2H), 3.89 (t, J = 6.96 Hz, 2H), 3.87 (s, 3H), 3.66 (q, J = 6.60 Hz, 2H), 2.83 (t, J
= 6.57 Hz, 2H), 2.47 (s, 3H), 2.17 (s, 3H), 2.09-2.02 (m, 4H). MS (ESI) m/z 392 [M+H]+. HRMS
(ESI) calcd for C19H29N5O2S [M + H]+ 392.2115, found 392.2097. Anal. HPLC 98.5% (Rt = 3.222
min).
4.1.4.6.
N-(4-Methoxy-3-(4-(methylamino)butoxy)phenyl)-N’-(3-(5-methyl-1H-imidazol-1-
o
yl)propyl)thiourea (55). mp = 76-77 C. 1H NMR (300 MHz, CD3OD) δ 7.43 (d, J = 0.93 Hz, 1H),
6.85 (d, J = 8.43 Hz, 1 H), 6.84 (d, J = 2.40 Hz, 1H), 6.69 (dd, J = 8.61, 2.37 Hz, 1H), 6.57 (s, 1H),
3.92-3.85 (m, 4H), 3.73 (s, 3H), 3.49 (t, J = 6.96 Hz, 2H), 2.67 (t, J = 7.32 Hz, 2H), 2.37 (s, 3H),
2.12 (d, J = 0.90 Hz, 3H), 1.93 (p, J = 7.14 Hz, 2H), 1.75-1.58 (m, 4H). MS (ESI) m/z 406 [M+H]+.
HRMS (ESI) calcd for C20H31N5O2S [M + H]+ 406.2271, found 406.2262. Anal. HPLC 97.6% (Rt =
3.012 min).
4.1.4.7.
N-(3-(4-(Dimethylamino)butoxy)-4-methoxyphenyl)-N’-(3-(5-methyl-1H-imidazol-1-
o
1
yl)propyl)thiourea (56). mp = 51-52 C. H NMR (300 MHz, CD3OD) δ 7.58 (s, 1H), 6.96 (d, J =
8.61 Hz, 1H), 6.91 (d, J = 2.40 Hz, 1H), 6.77 (dd, J = 8.58, 2.55 Hz, 1H). 6.66 (s, 1H), 4.01 (t, J =
6.06 Hz, 2H), 3.97 (t, J = 7.32 Hz, 2H), 3.82 (s, 3H), 3.61 (t, J = 6.96 Hz, 2H), 2.42 (t, J = 7.71 Hz,
2H), 2.25 (s, 6H), 2.21 (d, J = 0.90 Hz, 3H), 2.05 (p, J = 7.14 Hz, 2H), 1.81-1.75 (m, 2H), 1.72-1.67
29