Discovery of novel propargylamine-modified 4-aminoalkyl imidazole substituted pyrimidinylthiourea derivatives as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2017.08.025

A series of novel propargylamine-modified pyrimidinylthiourea derivatives (1–3) were designed and synthesized as multifunctional agents for Alzheimer's disease (AD) therapy, and their potential was evaluated through various biological experiments. Among these derivatives, compound 1b displayed good selective inhibitory activity against AChE (vs BuChE, IC₅₀ = 0.324 μM, SI > 123) and MAO-B (vs MAO-A, IC₅₀ = 1.427 μM, SI > 35). Molecular docking study showed that the pyrimidinylthiourea moiety of 1b could bind to the catalytic active site (CAS) of AChE, and the propargylamine moiety interacted directly with the flavin adenine dinucleotide (FAD) of MAO-B. Moreover, 1b demonstrated mild antioxidant ability, good copper chelating property, effective inhibitory activity against Cu²⁺-induced Aβ_1?42 aggregation, moderate neuroprotection, low cytotoxicity, and appropriate blood?brain barrier (BBB) permeability in vitro and was capable of ameliorating scopolamine-induced cognitive impairment in mice. These results indicated that 1b has the potential to be a multifunctional candidate for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2017.08.025

Accepted Manuscript
Discovery of novel propargylamine-modified 4-aminoalkyl imidazole substituted
pyrimidinylthiourea derivatives as multifunctional agents for the treatment of
Alzheimer's disease
Yi-xiang Xu, Huan Wang, Xiao-kang Li, Sheng-nan Dong, Wen-wen Liu, Qi Gong,
Tian-duan-yi Wang, Yun Tang, Jin Zhu, Jian Li, Hai-yan Zhang, Fei Mao
PII:
S0223-5234(17)30628-1
10.1016/j.ejmech.2017.08.025
EJMECH 9669
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 February 2017
Revised Date: 7 August 2017
Accepted Date: 8 August 2017
Please cite this article as: Y.-x. Xu, H. Wang, X.-k. Li, S.-n. Dong, W.-w. Liu, Q. Gong, T.-d.-y.
Wang, Y. Tang, J. Zhu, J. Li, H.-y. Zhang, F. Mao, Discovery of novel propargylamine-modified 4-
aminoalkyl imidazole substituted pyrimidinylthiourea derivatives as multifunctional agents for the
treatment of Alzheimer's disease, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.08.025.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of novel propargylamine-modified 4-aminoalkyl imidazole
substituted pyrimidinylthiourea derivatives as multifunctional agents
for the treatment of Alzheimer’s Disease
Yi-xiang Xua,1, Huan Wangb,c,1, Xiao-kang Lia, Sheng-nan Dongb, Wen-wen Liua, Qi
Gongb, Tian-duan-yi Wanga, Yun Tanga, Jin Zhua, Jian Lia,*, Hai-yan Zhangb,*, Fei
Maoa,*
a Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China
University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
b CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
c University of Chinese Academy of Science, No.19A Yuquan Road, Beijing 100049,
China
1 These authors contributed equally to this work.
*
To whom correspondence should be addressed maofei@ecust.edu.cn,
hzhang@simm.ac.cn, jianli@ecust.edu.cn.

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ABSTRACT:
A series of novel propargylamine-modified pyrimidinylthiourea derivatives (1-3) were
designed and synthesized as multifunctional agents for Alzheimer’s disease (AD)
therapy, and their potential was evaluated through various biological experiments.
Among these derivatives, compound 1b displayed good selective inhibitory activity
against AChE (vs BuChE, IC50 = 0.324 µM, SI > 123) and MAO-B (vs MAO-A, IC50
= 1.427 µM, SI > 35). Molecular docking study showed that the pyrimidinylthiourea
moiety of 1b could bind to the catalytic active site (CAS) of AChE, and the
propargylamine moiety interacted directly with the flavin adenine dinucleotide (FAD)
of MAO-B. Moreover, 1b demonstrated mild antioxidant ability, good copper
chelating property, effective inhibitory activity against Cu2+-induced Aβ1−42
aggregation, moderate neuroprotection, low cytotoxicity, and appropriate blood−brain
barrier (BBB) permeability in vitro and was capable of ameliorating
scopolamine-induced cognitive impairment in mice. These results indicated that 1b
has the potential to be a multifunctional candidate for the treatment of Alzheimer’s
disease.
Keywords
: Multifunctional agents, AChE inhibitors, MAO-B inhibitors, Metal
chelating agents, Alzheimer’s disease.

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1. Introduction
Alzheimer’s disease (AD) is a progressive and chronic neurodegenerative
disorder characterized by memory loss and cognitive impairments [1,2]. The number
of dementia patients has reached over 46 million by 2015 and is expected to triple by
2050 [3]. Due to its complex etiology, the pathogenesis has not been fully elucidated,
and there have been various pathogenesis hypotheses for AD, such as cholinergic
hypothesis [4], amyloid cascade hypothesis [5,6], oxidative stress hypothesis [7], and
metal dishomeostasis hypothesis [8,9].
Among these hypotheses, the cholinergic deficit hypothesis has been generally
accepted by most researchers. This theory suggests that AD patients’ learning-memory
and cognitive ability dysfunction is closely related to the low acetylcholine (ACh)
levels in brain [10,11]. Thus, strengthening the central cholinergic activity and
increasing the level of ACh in the brain, for example, by inhibiting the activity of
acetylcholinesterase (AChE) have been considered to be effective approaches for the
treatment of AD [12,13]. Currently, the clinical first-line drugs for the treatment of
AD are mainly AChE inhibitors such as donepezil, rivastigmine, galantamine, and
huperzine A (Hup A, approved by CFDA) [14,15]. However, these drugs only
improve the memory and cognitive abilities of AD patients but fail to achieve a
radical cure [16,17].
Many studies have found that monoamine oxidase (MAO) also plays a very
important role in the pathogenesis of AD because the increase of MAO in the brain
may result in a cascade of biochemical events leading to neuronal dysfunction [18,19].
Monoamine oxidases (MAOs) are flavin adenine dinucleotide (FAD)-containing
enzymes that are responsible for the oxidative deamination of endogenous and
exogenous monoamine substances. MAOs have two functional isozymic forms,
namely, MAO-A and MAO-B [20]. MAO-A inhibitors are used as clinical
antidepressants and anti-anxiety agents, whereas MAO-B inhibitors are applied to
treat neurodegenerative disorders such as AD and Parkinson’s diseases (PD) [21,22].
According to previous studies [23,24], the activities of MAO-B in the brain and blood
platelets of AD patients were increased, while high expression levels of MAO-B

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could cause the high level of free radicals that played a key role in the pathogenesis of
AD. MAO-B inhibitors can reduce the oxidative stress response and protect the nerve
cells from oxidative damage and neurotoxicity, and therefore, MAO-B has been
another important target for the treatment of AD [25,26]. Among the MAO inhibitors,
selegiline, an irreversible and selective MAO-B inhibitor, has been reported as a
potential anti-AD agent due to its neuroprotective property in cellular and animal
models of AD [27].
In addition, recent studies have revealed that the high levels and dysregulation of
biometal ions were closely implicated in the pathogenesis of AD [8,9,28]. Metal ions
such as Cu2+, Zn2+ and Fe2+ have been shown to facilitate Aβ aggregation, leading to
the generation of toxic Aβ oligomers [29]. In particular, redox-active Cu (I/II) and Fe
(II/III) are implicated in the generation of reactive oxygen species (ROS) leading to
an increased oxidative stress [30-33]. Biometal chelators, especially Cu2+ chelators,
not only reduce the metal-induced Aβ aggregation but also reduce the level of ROS
produced by the redox metal and metal-Aβ complex [33,34]. Therefore, biometal
chelators have been considered as a potential therapeutic approach for AD.
Furthermore, neurotoxic ROS and oxidative damage of neuronal cells are also
associated with AD, so that compounds with antioxidant activities are beneficial for
the treatment of AD [35,36].
Taking the complex pathogenesis of AD and the impotency of single-target drugs
into consideration, the development of multi-target-directed ligands (MTDLs) has
been proposed to be an effective approach for the treatment of AD [37,38].
Considering that ChEs and MAOs are important targets for the treatment of AD, some
researchers have devoted their efforts to finding multifunctional agents that target
both ChEs and MAOs. For example, Ladostigil (Fig. 1), generated by joining the
carbamate moiety of rivastigmine and indolamine moiety of rasagiline, shows
bifunctional inhibitory activities of MAOs and ChEs in a single molecule, and
presently, it is in a Phase III clinical trial for the treatment of mild cognitive
impairment (MCI). This indicates that the combination of ChEs inhibitor and MAOs
inhibitor is a promising therapeutic method for AD [39-41].

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Fig. 1. Chemical structures of ladostigil, rivastigmine and rasagiline
Inspired by the MTDLs strategy, we designed and synthesized a series of novel
pyrimidinylthiourea derivatives as multifunctional agents for the potential treatment
of AD in our previous work. These derivatives exhibited potent inhibition and good
selectivity toward AChE, specific metal-chelating ability, mild antioxidant effects,
low cytotoxicity, and good blood–brain barrier (BBB) permeability and could
improve memory and cognitive function of scopolamine-induced cognitive
impairment mice [42]. In this work, we combined the imidazole-substituted
pyrimidinylthiourea moiety (AChE inhibitor’s pharmacophore) and the
propargylamine moiety (MAO-B inhibitor’s pharmacophore) of selegiline [27] or
pargyline [43] into a single molecule (Fig. 2). These derivatives showed good
inhibition for both AChE and MAO-B, as well as the abilities of metal-chelating,
modulating metal induced Aβ aggregation, free radical scavenging, mild antioxidant
properties, neuroprotection and low cytotoxicity.
Fig. 2. Design strategy for the new series of propargylamine-modified
pyrimidinylthiourea derivatives.
2. Results and discussion
2.1. Design and synthesis
To study the SAR of new compounds and find the optimal compound, structural

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modifications were performed. First, “R” at the N-atom of propargylamine was
modified with hydrogen, methyl, ethyl, propyl, butyl, acetyl, benzyl, cyanomethyl and
propargyl to study the effect of different sizes of substitution on multiple functions
(compounds 1a-i). Next, to obtain the optimal linker between imidazole ring and
propargylamine, the length of carbon spacer (“n”) was changed from one to three or a
ring (compounds 2a-d, 3a-b, and 1j). The synthesis of propargylamine-modified
pyrimidinylthiourea derivatives (1-3) was carried out following the sequence of
reactions depicted in Scheme 1. The nucleophilic substitution reaction of imidazole
derivatives (4a-c) and 4-amino-6-chloropyrimidine in the presence of inorganic base
(K2CO3 or Cs2CO3) provided intermediates 5a-c, followed by the condensation
reaction with ethylisothiocyanate in the presence of NaH affording the key
intermediates 6a-c. The target compounds 1a-b and 1j were obtained by reductive
amination of intermediate 6a with diverse propargylamines in the presence of
NaBH3(CN). Next, the nucleophilic substitution reaction of 1a with haloalkane or
acetylchloride in the presence of Et3N provided compounds 1c-i. To obtain
compounds 2a-d and 3a-b, the intermediates 7b-c were synthesized by the
bromination reaction of 6b-c with PBr3. Then, the nucleophilic substitution of 7b-c
with propargylamine or N-methyipropargaylamine afforded the target compounds
2a-b and 3a-b. Similarly, the nucleophilic substitution reaction of 2a with haloalkane
in the presence of Et3N furnished the target compounds 2c-d.
The details of the synthetic procedures and structural characterizations of target
compounds (1H NMR, 13C NMR, and HRMS spectroscopy) and intermediates (1H
NMR) are described in the experimental section. The purities of all target compounds
were determined by HPLC.

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R1
N
R1
N
R1
N
H
H
b
H2N
N
N
N
N
a
HN
N
N
S
N
N
4a-c
4a: R1 = CHO
5a-c
6a-c
5a: R1 = CHO
5b: R1 = CH2CH2OH
5c: R1 = CH2CH2CH2OH
6a: R1 = CHO
6b: R1 = CH2CH2OH
6c: R1 = CH2CH2CH2OH
4b: R1 = CH2CH2OH
4c: R1 = CH2CH2CH2OH
N
CHO
R2
c
H
N
H
N
H
H
N
N
N
N
N
N
S
N
N
S
N
N
6a
1a-i
1a: R2 = H
1b: R2 = CH3
1c: R2 = CH2CH3
1d: R2 = CH2CH2CH3
1e: R2 = CH2CH2CH2CH3
1f: R2 = C(O)CH3
1g: R2 = CH2Ph
1h: R2 = CH2CN
1i: R2 = CH2CCH
d
N
CHO
N
N
H
N
H
N
H
H
c
N
N
N
N
N
S
N
N
S
N
N
6a
1j
OH
Br
N
n
n
n
R3
f
e
H
N
H
N
H
H
H
H
N
N
N
N
N
N
N
N
N
N
S
N
N
S
N
N
S
N
N
6b-c
7b-c
2a-d, 3a-b
2a: n = 2, R3 = H
2b: n = 2, R3 = CH3
6b: n = 2
6c: n = 3
7b: n = 2
7c: n = 3
d
2c: n = 2, R3 = CH2CH3
2d: n = 2, R3 = CH2CN
3a: n = 3, R3 = H
3b: n = 3, R3 = CH3
Scheme 1. Synthetic route to compounds 1a-j, 2a-d and 3a-b. Reagents and
conditions: (a) (i) for 5a: 4-amino-6-chloropyrimidine, K2CO3, DMF, 100 °C, 12 h; (ii)
for 5b-c: 4-amino-6-chloropyrimidine, Cs2CO3, DMF, 140 °C, 12 h; (b) CH3CH2NCS,
NaH, DMF, rt, 0.5 h; (c) 2-Propynylamine, N-methylpropargylamine or
1-(prop-2-yn-1-yl)piperazine, NaBH3(CN), MeOH:DCE = 1:1 (v:v), AcOH, rt, 12 h;
(d) (i) for 1c-e and 1g-i: RX, acetonitrile, Et3N, reflux, 12 h; (ii) for 1f: acetyl chloride,
DCM, Et3N, rt; (e) PBr3, DCM, rt, overnight; (f) 2-Propynylamine or
N-methylpropargylamine, K2CO3, rt, overnight.
2.2. Biological activity
2.2.1. In vitro inhibition studies of AChE and BuChE
To evaluate the therapeutic potential of these propargylamine-modified
pyrimidinylthiourea derivatives for AD, compounds 1a−j, 2a−d, and 3a−b were

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assayed against rat ChE (rChE) by the modified method of Ellman et al. [44], using
huperzine A (Hup A) as a reference compound. As shown in Table 1, all of our target
compounds exhibited desirable inhibitory activity to rAChE and poor inhibitory
activity against rBuChE (SI> 30), indicating that these derivatives were selective
AChE inhibitors. Examination of the IC50 values of compounds 1a-i shows that
unsubstituted compound 1a (IC50 = 0.354 µM for AChE) and methyl substituted
compound 1b (IC50 = 0.324 µM for AChE) exhibited higher activity than compounds
1c-i (IC50 > 0.4 µM for AChE) with larger substitutions. This suggested that the size
of the substitution at the N-atom of propargylamine has a moderate influence on the
AChE inhibitory activity, and the appropriate substituents at the N-atom of
propargylamine appeared to be methyl and hydrogen. Then, the influence of the linker
length to AChE inhibitory activity was also studied; however, compounds 1a-b, 2a-b
and 3a-b exhibited similar inhibitory activities for AChE. The inhibitory activity
result of compound 1j shows that the use of the piperazine ring as the linker led to a
decrease in AChE inhibitory activity.
Table 1.
Structures, rAChE and rBuChE inhibitory activities, and selectivity index for rAChE
of the target compounds.
IC50 (µM)a for
IC50 (µM)a for rBuChE
(Inhibition at 40 µM)
Compd
SIb
n
R
rAChE
1
1
1
1
1
1
H
0.354 ± 0.057
0.324 ± 0.022
0.482 ± 0.019
0.485 ± 0.025
0.675 ± 0.072
0.515 ± 0.070
>40 (15.24%)
>40 (12.59%)
>40 (10.49%)
>40 (7.75%)
>40 (13.42%)
>40 (5.15%)
>113
>123
>83
1a
1b
1c
1d
1e
1f
CH3
CH2CH3
(CH2)2CH3
(CH2)3CH3
C(O)CH3
>83
>59
>78

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1
1
1
2
2
2
2
3
3
CH2Ph
CH2CN
CH2CCH
H
0.585 ± 0.012
0.707 ± 0.044
0.718 ± 0.034
0.228 ± 0.027
0.392 ± 0.037
1.337 ± 0.184
0.597 ± 0.011
0.381 ± 0.052
0.459 ± 0.052
>40 (9.28%)
>68
>57
1g
1h
1i
>40 (5.48%)
>40 (4.97%)
>40 (8.73%)
>40 (8.91%)
>40 (2.44%)
>40 (3.02%)
>40 (12.74%)
>40 (6.70%)
>56
>175
>102
>30
2a
2b
2c
2d
3a
3b
CH3
CH2CH3
CH2CN
H
>67
>105
>87
CH3
0.482 ± 0.029
>40 (7.30%)
>83
1j
0.098 ± 0.016
36.4 ±1.838
>371
Hup A
aResults are expressed as the mean ± SD of at least three independent experiments.
bSelectivity index for AChE is defined as IC50(rBuChE)/IC50(rAChE).
2.2.2. In vitro inhibition studies of MAOs
To confirm the multipotent biological functions of these new compounds,
inhibitory activities against the recombinant human MAOs (hMAO-A and hMAO-B)
were determined using clorgyline and pargyline as the reference compounds,
respectively. The corresponding IC50 values or the percentage of inhibition at 50 µM
are shown in Table 2. The data showed that only a fraction of the tested compounds
could effectively inhibit MAO-A or MAO-B. Among the synthesized compounds, 1b
was the most potent selective inhibitor against MAO-B (IC50 = 1.472 µM, SI > 35),
and 1a exhibited the highest potency for both MAO-A and MAO-B (IC50 = 4.327 µM
and 5.236 µM, respectively). The other compounds, except 1a-b, 2b and 3b, showed
weak activities with IC50 values over 50 µM. This result suggested that the
enlargement of the substitution at the N-atom of propargylamine dramatically
decrease the inhibitory activity for MAOs. For unsubstituted compounds (1a, 2a and
3a), once the linker length changed from 1 to 3 carbon atoms, the inhibitory activities
for MAO-A and MAO-B decreased sharply (1a: IC50 = 4.327 µM for MAO-A, IC50 =

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5.236 µM for MAO-B; 2a and 3a: IC50 > 50 mM for both MAO-A and MAO-B); for
N-methyl substituted compounds (1b, 2b and 3b), the linker length affected the
selectivity toward MAO-A and MAO-B: compound 2b with a 2 carbon atoms-long
linker exhibited inhibitory activities for both MAO-A and MAO-B, whereas 1b and
3b, with 1 and 3 carbon atoms-long linkers, respectively, were good selective
inhibitors against MAO-B. This meant that the selectivity of the compounds towards
MAO-A and MAO-B is closely related to the length of the linker.
Table 2.
Structures, hMAO-A and hMAO-B inhibitory activities, and selectivity index for
hMAO-B of the target compounds.
IC50 (µM)a for
IC50 (µM)a for
Compd
hMAO-A
hMAO-B
SIb
ORACc
n
R
(Inhibition at 50 µM) (Inhibition at 50 µM)
1
1
1
1
1
1
1
1
1
2
2
2
2
3
H
4.327 ± 0.299
>50 (20.22%)
>50 (6.71%)
>50 (1.76%)
>50 (2.43%)
>50 (4.83%)
>50 (17.82%)
>50 (2.01%)
>50 (15.47%)
>50 (54.24%)
9.003 ± 0.982
>50 (13.03%)
>50 (13.92%)
>50 (31.39%)
5.236 ± 0.527
1.427 ± 0.272
>50 (35.12%)
>50 (24.31%)
>50 (20.11%)
>50 (1.17%)
>50 (18.44%)
>50 (15.35%)
>50 (42.11%)
>50 (19.23%)
12.435 ± 1.082
>50 (49.4%)
>50 (24.94%)
>50 (36.59%)
0.826
>35
1.175 ± 0.061
1.126 ± 0.015
1.383 ± 0.061
0.448 ± 0.222
0.238 ± 0.013
1.167 ± 0.035
n.t.
1a
1b
1c
1d
1e
1f
CH3
CH2CH3
(CH2)2CH3
(CH2)3CH3
C(O)CH3
CH2Ph
CH2CN
CH2CCH
H
1g
1h
1i
0.494 ± 0.031
0.585 ± 0.070
1.272 ± 0.053
1.243 ± 0.016
n.t.
2a
2b
2c
2d
3a
CH3
0.724
CH2CH3
CH2CN
H
0.556 ± 0.029
0.186 ± 0.004

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3
CH3
>50 (63.77%)
3.229 ± 0.557
>15
0.447 ± 0.169
1.028 ± 0.061
3b
1j
>50 (9.22%)
>50 (13.29%)
n.t.d
n.t.
n.t.
Clorgyline
Pargyline
12.850 ± 0.495（nM）
n.t.
0.188 ± 0.061
aResults are expressed as the mean ± SD of at least two experiments. bSelectivity
c
index for MAO-B is defined as IC50(MAO-A)/IC50(MAO-B). The mean ± SD of
three independent experiments. Data are expressed as µmol of Trolox equivalent/µmol
of tested compounds. dNo test.
2.2.3. Free oxygen radical scavenging ability
Given that reactive radical species have been identified to be closely related to
AD [45], compounds that can decrease reactive radical species may exert potential
therapeutic effects. The oxygen radical absorbance capacity assay (ORAC-FL) was
performed to evaluate the antioxidant activity of these target compounds. This assay
used
fluorescein
as
the
fluorescent
probe,
AAPH
(2,2’-azobis-(amidinopropane)dihydrochloride) as the peroxyl radical inducer, and the
water-soluble analogue of vitamin E, Trolox, as the standard [46,47]. ORAC values
were expressed as Trolox equivalents calculated from the antioxidant of tested
compounds vs Trolox (the ORAC value of Trolox = 1.00). The results (Table 2)
indicated that half of the tested compounds exhibited mild antioxidant ability with
ORAC values of 1.028−1.383. The modification of the “R” substituent at the
N-position and changes in the length of the linker obviously affected the antioxidant
activity (1c vs 1d-e; 1a and 2a vs 3a): unsubstituted and small-size substituent
compounds with 1−2 carbon atoms linker length exhibited relatively good antioxidant
activity (ORAC values > 1), whereas the rest of the tested compounds displayed weak
antioxidant activity (ORAC values < 1). Compound 1c displayed the best antioxidant
activity, with an ORAC value of 1.383.
2.2.4. Metal chelating effect

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Based on above biological evaluation results, compound 1b was the most
promising compound with excellent multiple functions (IC50 = 0.324 µM for AChE;
IC50 = 1.472 µM for MAO-B; ORAC value of 1.126 for antioxidant activity).
Therefore, compound 1b was chosen for further biological evaluation.
The metal chelating activity of compound 1b with copper, zinc, and iron was
measured by UV−vis spectroscopy assay. In our previous work [42], we found that
thiourea fragment played the key role in the generation of the metal chelating action,
and the pyrimidinylthiourea derivatives could selectively chelate Cu2+. As expected,
when CuCl2 was added to the solution of 1b, the maximum absorption decreased
dramatically at 290 nm (Fig. 3), demonstrating the formation of 1b−Cu2+ complexes,
while only vague optical shift and maximum absorption fluctuation were observed
when FeSO4, FeCl3, and ZnCl2 were added, indicating that compound 1b has better
selectivity towards Cu2+.
Fig. 3. UV spectra of compound 1b (40 µM) alone and in the presence of CuCl2 (20
µM), FeSO4 (20 µM), FeCl3 (20 µM), or ZnCl2 (20 µM) in buffer (20 mM HEPES,
150 mM NaCl, pH 7.4).
2.2.5. Inhibition of ROS produced by copper redox cycling
Many researchers have found that redox-active metal ions were closely involved
in the high oxidative stress caused by ROS [31], which is a proposed pathogenesis of
AD. To evaluate the ability of compound 1b to inhibit the ROS produced by
Cu(II)-related redox, the model of the Cu-ascorbate redox system described by Faller
and co-workers was used (Fig. 4A) [48], using the metal chelator

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ethylenediaminetetraacetic acid (EDTA) as the positive control. Hydroxyl radicals
(OH·), produced by copper redox cycling in the presence of ascorbate, could oxidize
coumarin-3-carboxylic acid (CCA) to fluorescent 7-hydroxy CCA so that the
fluorescence values could indicate the amount of OH·. As shown in Fig. 4B, the
fluorescence value of the copper-ascorbate system increased steadily with time and
reached a plateau at approximately 800 s. However, the value changed little when
compound 1b was coincubated with the Cu-ascorbate system and was the same as the
value of the sample without Cu(II). The result showed that compound 1b could halt
copper redox cycling.
Fig. 4. (A) Oxygen reduction by copper redox cycling in the presence of ascorbate. (B)
Fluorescence intensity of the copper-ascorbate-1b, copper-ascorbate-EDTA,
copper-ascorbate, and ascorbate alone systems. CCA (50 µM) and ascorbate (150 µM)
were incubated in each system. [Cu2+] = 5 µM, [1b] = [EDTA] = 15 µM. PBS buffer
(20 mM KH2PO4, 150 mM NaCl); N = 3; pH = 7.4; 37 °C.
2.2.6. Dot blot assay and TEM assay
A dot blot assay was performed to evaluate the modulation of metal-induced Aβ
aggregation by compound 1b, and a transmission electron microscopy (TEM) assay
was then performed to examine the morphological changes of the Aβ species. In the
dot blot assay, the samples were probed by the A11 antibody to determine the
oligomeric Aβ, and total Aβ was detected by 6E10 as the loading control. A
representative scan graph and a statistical histogram are shown in Fig. 5A; the gray

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density of the spots is proportional to the level of the Aβ oligomerization. The results
showed that Aβ aggregated spontaneously, and compound 1b showed no effect on the
Aβ self-aggregation after 24 h incubation. Epigallocatechin gallate (EGCG) was
tested as the positive control. A thioflavine T (ThT) fluorescence assay was performed
to verify this result (Fig. S1, Supporting Information). The dot blot assay showed that
Cu2+ could accelerate Aβ aggregation and that this process was significantly inhibited
by compound 1b.
The TEM experiment showed that the aggregation of Aβ in the presence of Cu2+
produced more fibrils than self-induced Aβ aggregation (Fig. 5B, images 2 and 3). As
expected, fewer Aβ fibrils were detected when compound 1b and clioquinol (CQ)
were incubated with the sample after incubation for 24 h at 37 °C (Fig. 5B, images 4
and 5), indicating that 1b can efficiently inhibit Cu2+-induced Aβ aggregation.
Fig. 5. Inhibition of compound on Cu2+-induced Aβ1−42 aggregation. (A) Scan and bar
graph of the dot blot assay. Aggregated Aβ was detected by A11 antibody, and total
Aβ was probed by 6E10 antibody. Data are shown as the mean ± SEM, ###p < 0.001 vs
Aβ fresh, *p < 0.05, **p < 0.01 vs Aβ alone, $p < 0.05, $$p < 0.01 vs Aβ + Cu2+, N = 4.
(B) TEM image analysis of copper induced Aβ1−42 aggregation studies ([Aβ1−42] =
[Cu2+] = 25 µM, [compd] = 50 µM; HEPES (20 µM) and NaCl (150 µM); pH 6.6;
37 °C). (1) fresh Aβ1−42, (2) Aβ1−42 + Cu2+, (3) Aβ1−42 alone, (4) Aβ1−42 + Cu2+ + 1b, (5)
Aβ1−42 + Cu2+ + CQ.

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2.2.7. MTT Assay
To investigate the safety of the propargylamine-modified 4-aminoalkyl imidazole
substituted pyrimidinylthiourea derivatives, the cytotoxicity of compound 1b on rat
primary
cortical
neuron
(PCN)
was
studied
using
a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and
donepezil (Don) tested as the reference. The result is depicted in Fig. 6A. Similar to
Don, compound 1b exhibited no cytotoxicity at concentrations of 10 µM and 30 µM
after incubation for 24 h. When the concentration increased to a relatively high level
of 100 µM, 1b showed a significant but mild neurotoxicity (*p < 0.05 vs Ctrl)
compared to Don (***p < 0.001 vs Ctrl). Therefore, the MTT assay showed that
compound 1b was weakly cytotoxic.
Neurotoxicity, neuronal damage and neuronal cell apoptosis caused by the toxic
Aβ oligomer are important causes of AD. To determine whether compound 1b can
protect neuronal cells from toxic Aβ species, cell viability experiments were
conducted using PCNs. EGCG (inhibitor of Aβ self-aggregation) and CQ (inhibitor of
Cu2+-induced Aβ aggregation) were tested as positive controls. The results are shown
in Fig. 6B. Incubation of PCNs with Aβ in the presence of Cu2+ caused higher
cytotoxicity than the Aβ alone group (*p < 0.05). Compared to the Aβ + Cu2+ group,
compound 1b, like CQ, could protect the PCNs from Cu2+-induced Aβ neurotoxicity,
leading to a significant increase in cell viability ($$p < 0.01). Moreover, compound 1b
could not interact with Aβ directly to restrain the cytotoxicity produced by the Aβ-self
aggregates. These results were highly consistent with the results of the dot blot assay.

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Fig. 6. (A) Cell viability of primary cortical neurons exposed to compound 1b and
ff
Don at di erent concentrations (range 10−100 µM) for 24 h. Vehicle-treated cells
were used as controls. The results are expressed as the percentage of viable cells
observed after treatment with compound vs vehicle-treated cells (100%) and are
*
shown as the mean ± SEM; p < 0.05, ***p < 0.001 vs Ctrl, N = 3; (B) Protection of
compound 1b from Cu2+-induced Aβ1-42 neurotoxicity. Data are shown as the mean ±
SEM; N = 3. Experimental conditions: [Aβ1−42] = [Cu2+] =1 µM, [compd] = 2 µM.
###p < 0.001 vs Ctrl, *p < 0.05 vs Aβ alone, $$p < 0.01 vs Aβ + Cu2+.
2.2.8. In vitro blood−brain barrier permeation assay
To evaluate the target compounds’ BBB penetrability, we carried out the parallel
artificial membrane permeation assay (PAMPA) [49]. First, to validate the assay, the
values of permeability (Pe) of 13 commercial drugs were determined and compared to
the reported values. A plot of the experimental data versus reference values gave a
strong linear correlation, Pe (exp.) = 0.947Pe (bibl.) – 0.8152 (R2 = 0.9651, Fig. S2,
Supporting Information). Based on this equation and the limit established by Di et al.
for blood−brain barrier permeation, we concluded that compounds with Pe values
greater than 3.08 × 10−6 cm s−1 could cross the blood−brain barrier by passive
permeation (CNS+). Pe values from 1.13×10−6 cm s−1 to 3.08 × 10−6 cm s−1 were
classified as “CNS±”, uncertain BBB permeation. The results of PAMPA-BBB assay
are presented in Table 3. It can be observed that compounds 1b, 2a-b and 3a-b could
cross the BBB, whereas compound 1a exhibited uncertain BBB permeation (CNS±).
It can also be seen that the compounds’ BBB permeation ability is related to the
length of the linker (3b > 2b > 1b).
Table 3.
Permeability results (Pe×10−6 cm s−1) from the PAMPA-BBB assay for compounds
1a-b, 2a-b and 3a-b and their prediction of BBB penetration.
Compd
1a
Pe (10−6 cm s−1)a
2.78 ± 0.12
predictionb
CNS±
1b
3.16 ± 0.10
CNS+

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2a
2b
3a
3b
3.24 ± 0.08
3.33 ± 0.12
3.26 ± 0.21
3.98 ± 0.09
CNS+
CNS+
CNS+
CNS+
aValues are expressed as the mean ± SD of at least three independent experiments.
bCompounds with Pe > 3.08 × 10−6 cm s−1 could cross the BBB by passive di usion
(CNS+). Compounds with Pe < 1.13 × 10−6 cm s−1 could not cross the BBB (CNS−),
and compounds with 1.13 × 10−6 cm s−1 < Pe < 3.08 × 10−6 cm s−1 show uncertain
BBB permeation (CNS±).
2.2.9. Improvement of cognition in scopolamine-induced dementia mice by 1b.
To analyze the in vivo effect of compound 1b on cognitive impairment,
scopolamine-induced cognitive performance deficits in the channel water maze were
employed, with huperzine A (Hup A) as a positive control. As shown in Fig. 7, mice
treated with scopolamine (4.5 mg/kg, i.p.) required longer time to find the platform
(escape latency) and committed more errors (entering no-exit paths) than those of the
control group (###p < 0.001) in the water maze task. Compound 1b·HCl, administered
to mice orally at a dose of 30 mg/kg, could ameliorate scopolamine-induced learning
and memory deficits, with the mice in this group showing shorter escape latency and
less frequent errors compare to the scopolamine group (*p < 0.05). Therefore,
compound 1b·HCl has the potential to be a lead anti-AD agent due to its symptomatic
improvement effect in the AD mice model.
Fig. 7. Effect of compound 1b·HCl (30 mg/kg) on scopolamine-induced cognitive
deficit mice, with Hup A (0.1 mg/kg) as positive control: (A) Escape latency of mice

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in water maze; (B) Number of errors of mice in water maze. Data are shown as the
mean ± SEM: ###p < 0.001 vs Ctrl, *p < 0.05, **p < 0.01, ***p < 0.001 vs Scop, N = 16.
Ctrl: control; Scop: scopolamine; HupA: huperzine A.
2.3. Molecular docking studies of AChE and MAO-B
To confirm the rationality of compound design and explore the interaction mode
of target compounds with enzymes, molecular docking simulations of 1b with mice
AChE (mAChE) and human MAO-B (hMAO-B) were performed using Glide in
Schrödinger software. The X-ray crystallographic structure of mAChE complexed
with tacrine (PDB code 5EIE), and human MAO-B complexed with
7-(3-chlorobenzyloxy)-4-formylcoumarin (PDB code 2V60) were obtained from the
Protein Data Bank. As shown in Fig. 8A, compound 1b interacts with the CAS of
AChE via π-π interactions and hydrogen bond interactions. The two strong hydrogen
bonds between the thiourea-NH (1.7 Å) and carbonyl of His 447 (2.2 Å) may play an
important role in the positioning and the stabilization of the ligand inside the CAS.
Moreover, its pyrimidine ring could interact with Trp86 via face-to-face π-π stacking
interaction with the distance of 4.1 Å. The results also validated the hypothesis that
the thiourea and pyrimidine fragments were key groups for maintaining the AChE
activity [42].
Examination of Fig. 8B shows that the propargylamine group of 1b has a
favorable fit into the substrate cavity of the enzyme, oriented to the flavin adenine
dinucleotide (FAD) cofactor in close proximity (4.64 Å), and is properly adopted
between Tyr398 (3.32 Å) and Tyr435 (3.29 Å), forming face-to-face cation-π stacking
interactions in a “sandwich” form. Additionally, the face-to-edge π-π stacking
interaction (4.9 Å) between the pyrimidine ring and Trp326 and the hydrogen bond
(2.0 Å) between thiourea-NH and carbonyl of Ile199 could stabilize the ligand in the
catalysis gorge of the enzyme. Moreover, the ethyl group of thiourea may be
embedded into the hydrophobic pocket in the entrance cavity formed by Pro104,
Phe103, Pro102, Ile199, Ile316, Leu167 and Phe168.

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Fig. 8. (A) 3D docking model of compound 1b with mAChE. (B) 3D docking model
of compound 1b with hMAO-B. Compound 1b is illustrated using blue sticks, and the
FAD cofactor is depicted using yellow sticks. π-π stacking interactions are highlighted
by green lines, and hydrogen bonds are shown with purple dash lines.
3. Conclusions
Multi-target-directed ligands (MTDLs), aiming to simultaneously target multiple
pathological processes involved in the neurodegenerative cascade, have been
developed as effective agents for the treatment of Alzheimer’s disease. Our previous
studies obtained the pyrimidinylthiourea derivatives as MTDLs with ChE inhibitory
activity, specific metal-chelating ability and mild antioxidant effect. In the present
study, by introducing the MAO-B inhibitor’s pharmacophore propargylamine into the
pyrimidinylthiourea scaffold, a series of novel propargylamine-modified 4-aminoalkyl
imidazole substituted pyrimidinylthiourea derivatives have been designed,
synthesized, and evaluated as potential multifunctional anti-AD agents. The activities
of all synthesized compounds were evaluated against rAChE, rBuChE, hMAO-A(B),
and antioxidant ability. The SAR studies revealed that the size of the substitution at
the N-atom of propargylamine strongly influences the AChE and MAOs inhibition
and the antioxidant activity. Among all derivatives, compound 1b displayed good
selective inhibitory activities against AChE (IC50 = 0.324 µM, SI > 123) and MAO-B
(IC50 = 1.427 µM, SI > 35) and demonstrated mild antioxidant ability (ORAC =
1.126), good copper chelating property, effective inhibitory activity against

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Cu2+-induced Aβ1−42 aggregation, low cytotoxicity and moderate neuroprotection
against Cu2+-induced Aβ1−42 neurotoxicity in primary cultured cortical neurons and
appropriate blood−brain barrier (BBB) permeability in vitro. The molecular docking
simulations of 1b with mAChE and hMAO-B also confirmed the rationality of our
design strategy. Furthermore, compound 1b·HCl could improve the memory and
cognitive function of Scop-induced dementia mice. Taken together, these results
showed that the new compound 1b can be considered a potential multifunctional
agent for AD therapy.
4. Experimental section
4.1. Chemistry
4.1.1. General methods
All common reagents and solvents were obtained from commercial suppliers and
used without purification. Reaction progress was monitored using analytical thin layer
chromatography (TLC), HSGF 254 (150−200 µm thickness; Yantai Huiyou Co.,
China), and the spots were detected under UV light (254 nm). Melting points were
measured in capillary tubes on a SGWX−4 melting point apparatus without correction.
13
1H NMR and C NMR spectra were recorded on a Bruker AMX 400 and AMX 500
spectrometer in DMSO-d6, CD3COCD3 or CD3OD with TMS as internal standard.
Chemical shifts were reported in parts per million (ppm, δ) downfield from
tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), and broad (br). High-resolution mass spectra
(HRMS) were obtained by electric ionization (EI) and electrospray ionization (ESI)
using a Waters GCT Premie and Waters LCT. All target compounds were purified to ≥
95% purity as determined by an Agilent 1100 with a quaternary pump and diode array
detector (DAD). The eluent was CH3OH:H2O = 80:20 (v:v), flow rate was 0.5
mL/min, and the relative purity of each compound calculated at 254 nm.
4.1.2. General synthetic procedure for 5a-c
A
mixture
of
imidazole
derivatives
(4a-c,
10
mmol),
4-amino-6-chloropyrimidine (1.30 g, 10 mmol), and K2CO3 (2.07 g, 15 mmol, for 5a)
or Cs2CO3 (3.91 g, 12 mmol, for 5b-c) was stirred in DMF (20 mL) at 100 °C (for 5a)

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or at 140 °C (for 5b-c) for 12 h. Water (100 mL) was then added. The mixture was
extracted with EtOAc (3 × 50 mL). The organic layer was separated and dried over
Na2SO4. Removal of the solvents produced a residue which was purified using
column chromatography and eluted with a mixture of MeOH:CH2Cl2 (1:30, v:v) to
afford 5a-c.
4.1.2.1. 1-(6-Aminopyrimidin-4-yl)-1H-imidazole-4-carbaldehyde (5a) [42].
1
White solid, 46% yield. H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.70 (s, 1H),
8.64 (s, 1H), 8.37 (s, 1H), 7.35 (s, 2H), 6.72 (s, 1H).
4.1.2.2. 2-(1-(6-Aminopyrimidin-4-yl)-1H-imidazol-4-yl)ethan-1-ol (5b). White
solid, 58% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.33 (s, 1H), 8.29 (s, 1H), 7.54 (s,
1H), 7.16 (s, 2H), 6.50 (s, 1H), 4.64 (br, 1H), 3.70–3.60 (m, 2H), 2.66 (t, J = 6.9 Hz,
2H).
4.1.2.3. 3-(1-(6-Aminopyrimidin-4-yl)-1H-imidazol-4-yl)propan-1-ol (5c). White
1
solid, 41% yield. H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J = 1.1 Hz, 1H), 8.29 (s,
1H), 7.50 (s, 1H), 7.13 (s, 2H), 6.50 (d, J = 0.7 Hz, 1H), 4.47 (t, J = 5.2 Hz, 1H), 3.50
– 3.38 (m, 2H), 2.59–2.51 (m, 2H), 1.81–1.69 (m, 2H).
4.1.3. General synthetic procedure for 6a-c
NaH (60% dispersion in mineral oil, 96 mg, 2.4 mmol) was added to a solution
of corresponding intermediates 5a-c (2 mmol) in DMF (10 mL). The solution was
stirred for 5 min, and then, ethyl isothiocyanate (174 mg, 2 mmol) was added. The
reaction mixture was stirred at room temperature for 0.5 h. Water was added, and the
mixture was extracted with EtOAc (3 × 60 mL). The organic layer was separated and
dried over Na2SO4. Removal of the solvents produced a residue, which was purified
using column chromatograph and eluted with a mixture of MeOH:CH2Cl2 (1:40, v:v)
to afford corresponding intermediates 6a-c.
4.1.3.1. 1-Ethyl-3-(6-(4-formyl-1H-imidazol-1-yl)pyrimidin-4-yl)thiourea (6a)
1
[42]. White solid, 60% yield. H NMR (400 MHz, DMSO-d6) δ 11.13 (t, J = 5.1 Hz,
1H), 11.10 (s, 1H), 9.88 (s, 1H), 8.83 (s, 1H), 8.61 (s, 2H), 7.42 (s, 1H), 3.72–3.57 (m,
2H), 1.23 (t, J = 7.2 Hz, 3H).
4.1.3.2.
1-Ethyl-3-(6-(4-(2-hydroxyethyl)-1H-imidazol-1-yl)pyrimidin-4-yl)

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thiourea (6b). White solid, 41% yield. 1H NMR (400 MHz, CD3OD) δ 8.69 (d, J = 0.9
Hz, 1H), 8.52 (d, J = 1.2 Hz, 1H), 7.64 (s, 1H), 6.98 (d, J = 0.9 Hz, 1H), 3.84 (t, J =
6.7 Hz, 2H), 3.74 (q, J = 7.3 Hz, 2H), 2.82 (t, J = 6.7 Hz, 2H), 1.31 (t, J = 7.3 Hz,
3H).
4.1.3.3.
1-ethyl-3-(6-(4-(3-hydroxypropyl)-1H-imidazol-1-yl)pyrimidin-4-yl)
thiourea (6c). White solid, 35% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.19 (t, J =
5.0 Hz, 1H), 10.92 (s, 1H), 8.74 (s, 1H), 8.35 (s, 1H), 7.42 (s, 1H), 7.25 (s, 1H), 4.48
(t, J = 5.2 Hz, 1H), 3.70–3.59 (m, 2H), 3.49–3.40 (m, 2H), 2.56 (t, J = 7.5 Hz, 2H),
1.81–1.71 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H).
4.1.4. General synthetic procedure for 1a-b and 1j
Propargylamine (0.75 mmol) and AcOH (0.75 mmol) corresponding to a solution
of compound 6a (138 mg, 0.5 mmol) in MeOH:DCE = 1:1 were added. The solution
was stirred for 10 min, and then, NaBH3(CN) (62.8 mg, 1 mmol) was added. The
mixture was stirred at room temperature for 12 h, and the solvent was evaporated to
dryness under reduced pressure. The residue was purified on a silica gel column using
mixtures of MeOH:CH2Cl2 as the eluent, obtaining the target compounds 1a-b and 1j.
4.1.4.1.
1-Ethyl-3-(6-(4-((prop-2-yn-1-ylamino)methyl)-1H-imidazol-1-yl)
pyrimidin-4-yl)thiourea
(1a).
The
corresponding
propargylamine
was
1
2-Propynylamine. White solid, 53% yield. Mp 166–167 °C; H NMR (400 MHz,
CD3OD) δ 8.71 (d, J = 1.0 Hz, 1H), 8.55 (d, J = 1.2 Hz, 1H), 7.76 (s, 1H), 7.02 (d, J =
1.0 Hz, 1H), 3.86 (s, 2H), 3.74 (q, J = 7.3 Hz, 2H), 3.46 (d, J = 2.5 Hz, 2H), 2.67 (t, J
13
= 2.5 Hz, 1H), 1.31 (t, J = 7.3 Hz, 3H). C NMR (125 MHz, DMSO-d6) δ 179.18,
159.95, 157.33, 154.44, 143.17, 134.64, 112.55, 94.29, 82.74, 73.79, 45.03, 39.69,
36.83, 13.65. HRMS (EI): m/z calcd C14H17N7S (M+) 315.1266, found 315.1269.
HPLC purity: 98.0%.
4.1.4.2. 1-Ethyl-3-(6-(4-((methyl(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl)
pyrimidin-4-yl)thiourea
(1b).
The
corresponding
propargylamine
was
1
N-methylpropargylamine. White solid, 30% yield. Mp 183–184 °C; H NMR (400
MHz, CD3OD) δ 8.71 (d, J = 1.0 Hz, 1H), 8.54 (d, J = 1.3 Hz, 1H), 7.78 (s, 1H), 7.02
(d, J = 1.0 Hz, 1H), 3.74 (q, J = 7.3 Hz, 2H), 3.65 (s, 2H), 3.39 (d, J = 2.4 Hz, 2H),

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13
2.71 (t, J = 2.4 Hz, 1H), 2.38 (s, 3H), 1.31 (t, J = 7.3 Hz, 3H). C NMR (125 MHz,
DMSO-d6) δ 179.17, 159.94, 157.33, 154.39, 141.15, 134.70, 113.88, 94.46, 79.13,
75.84, 52.34, 44.53, 41.15, 39.69, 13.65. HRMS (EI): m/z calcd C15H19N7S (M+)
329.1423, found 329.1425. HPLC purity: 99.1%.
4.1.4.3. 1-Ethyl-3-(6-(4-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)-1H-imidazol
-1-yl)pyrimidin-4-yl)thiourea (1j). The corresponding propargylamine was
1
1-(prop-2-yn-1-yl)piperazine. Yellow solid, 28% yield. Mp 160−162 °C; H NMR
(400 MHz, CD3COCD3) δ 11.35 (br, 1H), 9.79 (s, 1H), 8.71 (s, 1H), 8.36 (d, J = 1.2
Hz, 1H), 7.61 (s, 1H), 7.32 (s, 1H), 3.82–3.61 (m, 2H), 3.51 (s, 2H), 3.27 (d, J = 2.4
13
Hz, 2H), 2.69 (t, J = 2.4 Hz, 1H), 2.55 (br, 8H), 1.29 (t, J = 7.2 Hz, 3H). C NMR
(100 MHz, DMSO-d6) δ 179.18, 159.95, 157.34, 154.40, 140.94, 134.62, 113.86,
94.44, 79.36, 75.67, 54.95, 52.33, 51.05, 45.96, 39.73, 13.68. HRMS (EI): m/z calcd
C18H24N8S (M+) 384.1845, found 384.1847. HPLC purity: 96.1%.
4.1.5. General synthetic procedure for 1c-i
To a solution of compound 1a (158 mg, 0.5 mmol) in CH3CN (8 mL),
corresponding RX (0.55 mmol), Et3N (101 mg, 1 mmol), and a catalytic amount of KI
were added. The reaction mixture was refluxed for 12 h, and the solvent was
evaporated to dryness under reduced pressure. The residue was purified on a silica gel
column using mixtures of MeOH:CH2Cl2 as the eluent, affording the target
compounds 1c-i.
4.1.5.1. 1-Ethyl-3-(6-(4-((ethyl(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl)
pyrimidin-4-yl)thiourea (1c). Iodoethane was used as the corresponding RX. White
1
solid, 27% yield. Mp 163−164 °C; H NMR (400 MHz, DMSO-d6) δ 11.18 (br, 1H),
10.91 (s, 1H), 8.75 (s, 1H), 8.39 (s, 1H), 7.54 (s, 1H), 7.28 (s, 1H), 3.68–3.60 (m, 2H),
3.58 (s, 2H), 3.38 (s, 2H), 3.14 (s, 1H), 2.59–2.52 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H),
1.03 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 179.18, 159.95, 157.35,
154.37, 141.10, 134.75, 113.90, 94.43, 78.86, 75.77, 50.07, 46.45, 40.77, 39.73, 13.68,
12.47. HRMS (EI): m/z calcd C16H21N7S (M+) 343.1579, found 343.1583. HPLC
purity: 98.0%.
4.1.5.2. 1-Ethyl-3-(6-(4-((prop-2-yn-1-yl(propyl)amino)methyl)-1H-imidazol-1-yl)

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pyrimidin-4-yl)thiourea (1d). 1-Iodopropane was used as the corresponding RX.
White solid, 30% yield. Mp 158−159 °C; 1H NMR (400 MHz, CD3OD) δ 8.71 (d, J =
1.0 Hz, 1H), 8.53 (d, J = 1.3 Hz, 1H), 7.75 (s, 1H), 7.02 (d, J = 1.0 Hz, 1H), 3.74 (q, J
= 7.3 Hz, 2H), 3.69 (s, 2H), 3.43 (d, J = 2.4 Hz, 2H), 2.65 (t, J = 2.4 Hz, 1H),
2.61–2.51 (m, 2H), 1.62–1.51 (m, 2H), 1.31 (t, J = 7.3 Hz, 3H), 0.93 (t, J = 7.4 Hz,
3H). 13C NMR (125 MHz, DMSO-d6) δ 179.17, 159.92, 157.31, 154.36, 141.51,
134.65, 113.60, 94.38, 79.14, 75.51, 54.39, 50.37, 41.28, 39.69, 20.05, 13.63, 11.70.
HRMS (EI): m/z calcd C17H23N7S (M+) 357.1736, found 357.1734. HPLC purity:
98.9%.
4.1.5.3.
1-(6-(4-((Butyl(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl)
pyrimidin-4-yl)-3-ethylthiourea (1e). 1-Iodobutane was used as the corresponding RX.
1
Faint yellow solid, 28% yield. Mp 112−114 °C; H NMR (400 MHz, CD3OD) δ 8.71
(d, J = 0.9 Hz, 1H), 8.53 (d, J = 1.2 Hz, 1H), 7.76 (s, 1H), 7.03 (d, J = 0.9 Hz, 1H),
3.74 (q, J = 7.3 Hz, 2H), 3.70 (s, 2H), 3.44 (d, J = 2.4 Hz, 2H), 2.66 (t, J = 2.4 Hz,
1H), 2.63–2.58 (m, 2H), 1.58–1.47 (m, 2H), 1.41–1.34 (m, 2H), 1.31 (t, J = 7.3 Hz,
3H), 0.94 (t, J = 7.3 Hz, 3H).
13C NMR (100 MHz, DMSO-d6) δ 179.18, 159.95,
157.34, 154.37, 141.44, 134.69, 113.68, 94.38, 79.08, 75.63, 52.07, 50.37, 41.29,
39.73, 28.97, 19.94, 13.88, 13.67. HRMS (EI): m/z calcd C18H25N7S (M+) 371.1892,
found 371.1895. HPLC purity: 96.6%.
4.1.5.4.
N-((1-(6-(3-Ethylthioureido)pyrimidin-4-yl)-1H-imidazol-4-yl)
methyl)-N- (prop-2-yn-1-yl)acetamide (1f). Reagents used were compound 1a (158
mg, 0.5 mmol), acetyl chloride (47 mg, 0.75 mmol), Et3N (101 mg, 1 mmol), DCM (5
mL), rt, 12 h. Purification involved the use of MeOH/CH2Cl2 as eluent. Compound 1f:
1
white solid, 45% yield. Mp 197−199 °C; H NMR (400 MHz, CD3OD) δ 8.73 (8.72)
(d, J = 0.8 Hz, 1H), 8.58 (8.55) (d, J = 1.1 Hz, 1H), 7.88 (7.79) (s, 1H), 7.04 (7.02) (d,
J = 0.8 Hz, 1H), 4.70 (4.65) (s, 2H), 4.25 (d, J = 2.4 Hz, 2H), 3.75 (q, J = 7.3 Hz, 2H),
2.81 (2.66) (t, J = 2.3 Hz, 1H), 2.30 (2.25) (s, 3H), 1.33 (t, J = 7.3 Hz, 3H). 13C NMR
(125 MHz, DMSO-d6) δ 179.16, 169.65 (169.57), 159.94, 157.34, 154.31, 140.33
(139.86), 135.42 (134.84), 113.78 (113.63), 94.61 (94.46), 79.97 (79.61), 74.83
(73.86), 44.46 (41.92), 39.69 (s), 37.48 (33.55), 21.49 (21.38), 13.63. HRMS (ESI):

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m/z calcd C16H19N7OS [M+H]+ 358.1405, found 358.1448. HPLC purity: 98.5%.
4.1.5.5.
1-(6-(4-((Benzyl(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl)
pyrimidin-4-yl)-3-ethylthiourea (1g). (Bromomethyl)benzene was used as the
1
corresponding RX. White solid, 48% yield. Mp 146−147 °C; H NMR (400 MHz,
CD3OD) δ 8.71 (d, J = 0.9 Hz, 1H), 8.55 (d, J = 1.3 Hz, 1H), 7.78 (s, 1H), 7.40 (d, J =
7.0 Hz, 2H), 7.31 (t, J = 7.3 Hz, 2H), 7.25 (t, J = 7.2 Hz, 1H), 7.03 (d, J = 0.9 Hz, 1H),
3.84–3.62 (m, 6H), 3.33 (s, 2H), 2.71 (t, J = 2.3 Hz, 1H), 1.31 (t, J = 7.3 Hz, 3H).
13C
NMR (125 MHz, DMSO-d6) δ 179.17, 159.92, 157.31, 154.34, 141.23, 138.51,
134.79, 128.62, 128.23, 127.03, 113.80, 94.46, 78.77, 76.10, 56.60, 49.90, 41.05,
39.69, 13.64. HRMS (EI): m/z calcd C21H23N7S (M+) 405.1736, found 405.1737.
HPLC purity: 98.0%.
4.1.5.6. 1-(6-(4-(((Cyanomethyl)(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl)
pyrimidin-4-yl)-3-ethylthiourea (1h). 2-Bromoacetonitrile was used as the
1
corresponding RX. Yellow solid, 36% yield. Mp 151−152 °C; H NMR (400 MHz,
CD3OD) δ 8.72 (d, J = 0.9 Hz, 1H), 8.57 (d, J = 1.2 Hz, 1H), 7.84 (s, 1H), 7.02 (d, J =
0.9 Hz, 1H), 3.82–3.77 (m, 4H), 3.74 (q, J = 7.3 Hz, 2H), 3.51 (d, J = 2.4 Hz, 2H),
2.79 (t, J = 2.4 Hz, 1H), 1.31 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ
179.16, 159.95, 157.35, 154.32, 139.49, 135.12, 116.01, 114.71, 94.56, 78.45, 76.67,
49.67, 42.22, 40.77, 39.69, 13.64. HRMS (ESI): m/z calcd C16H18N8S [M+H]+
355.1409, found 355.1454. HPLC purity: 97.1%.
4.1.5.7. 1-(6-(4-((Di(prop-2-yn-1-yl)amino)methyl)-1H-imidazol-1-yl) pyrimidin
-4-yl)-3-ethylthiourea (1i). 3-Bromoprop-1-yne was used as the corresponding RX.
1
White solid, 36% yield. Mp 151−153 °C; H NMR (400 MHz, DMSO-d6) δ 11.17 (t,
J = 5.2 Hz, 1H), 10.91 (s, 1H), 8.73 (s, 1H), 8.40 (s, 1H), 7.56 (s, 1H), 7.27 (s, 1H),
3.67 – 3.60 (m, 2H), 3.59 (s, 2H), 3.41 (d, J = 1.8 Hz, 4H), 3.20 (s, 2H), 1.22 (t, J =
7.2 Hz, 3H).13C NMR (100 MHz, DMSO-d6) δ 179.17, 159.96, 157.36, 154.35,
140.50, 134.93, 114.23, 94.52, 79.09, 75.90, 49.57, 41.28, 39.73, 13.69. HRMS (EI):
m/z calcd C17H19N7S (M+) 353.1423, found 353.1402. HPLC purity: 98.9%.
4.1.6. General synthetic procedure for 7b-c
PBr3 (541 mg, 2 mmol) was dropped to a solution of compounds 6b-c (2 mmol)

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in DCM (30 mL). The mixture was stirred at room temperature overnight, and the
solvent was evaporated to dryness under reduced pressure. The residue was purified
on a silica gel column using mixtures of MeOH:CH2Cl2 as the eluent to afford the
target compounds 7b-c.
4.1.6.1.
1-(6-(4-(2-Bromoethyl)-1H-imidazol-1-yl)pyrimidin-4-yl)-3-ethyl
thiourea (7b). White solid, 28% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.19 (t, J =
5.6 Hz, 1H), 10.98 (s, 1H), 8.75 (s, 1H), 8.40 (s, 1H), 7.61 (s, 1H), 7.27 (s, 1H), 3.76
(t, J = 6.9 Hz, 2H), 3.69–3.58 (m, 2H), 3.11 (t, J = 6.9 Hz, 2H), 1.22 (t, J = 7.2 Hz,
3H).
4.1.6.2.
1-(6-(4-(3-Bromopropyl)-1H-imidazol-1-yl)pyrimidin-4-yl)-3-ethyl
thiourea (7c). White solid, 30% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.27 (s, 1H),
11.03 (t, J = 5.4 Hz, 1H), 9.88 (s, 1H), 8.88 (s, 1H), 8.00 (d, J = 1.3 Hz, 1H), 7.46 (s,
1H), 4.36 (t, J = 7.3 Hz, 2H), 3.69–3.58 (m, 2H), 3.02 (t, J = 7.0 Hz, 2H), 2.63–2.54
(m, 2H), 1.21 (t, J = 7.2 Hz, 3H).
4.1.7. General synthetic procedure for 2a-b and 3a-b
A mixture of 7b-c (0.5 mmol) and K2CO3 (172 mg, 1.25 mmol) was stirred
overnight in 2-propynylamine or N-methylpropargylamine (1 mL) at room
temperature. The mixture was then evaporated to dryness under reduced pressure. The
residue was purified on a silica gel column using mixtures of MeOH:CH2Cl2 as the
eluent to afford the corresponding compounds 2a-b and 3a-b.
4.1.7.1.
1-Ethyl-3-(6-(4-(2-(prop-2-yn-1-yl)amino)ethyl)-1H-imidazol-1-yl)
1
pyrimidin-4-yl)thiourea (2a). Yellow solid, 70% yield. Mp 159−160 °C; H NMR
(400 MHz, CD3OD) δ 8.70 (d, J = 0.9 Hz, 1H), 8.52 (d, J = 1.3 Hz, 1H), 7.63 (s, 1H),
6.98 (d, J = 0.9 Hz, 1H), 3.74 (q, J = 7.3 Hz, 2H), 3.44 (d, J = 2.5 Hz, 2H), 3.00 (t, J =
7.2 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 2.5 Hz, 1H), 1.31 (t, J = 7.3 Hz, 3H).
13C NMR (100 MHz, DMSO-d6) δ 179.20, 159.95, 157.31, 154.44, 142.94, 134.47,
112.06, 94.17, 83.09, 73.53, 47.37, 39.73, 37.32, 28.04, 13.69. HRMS (EI): m/z calcd
C15H19N7S (M+) 329.1423, found 329.1424. HPLC purity: 99.0%.
4.1.7.2.
1-Ethyl-3-(6-(4-(2-(methyl(prop-2-yn-1-yl)amino)ethyl)-1H-imidazol
1
-1-yl)pyrimidin-4-yl) thiourea (2b). Yellow solid, 68% yield. Mp 162−164 °C; H

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NMR (400 MHz, CD3OD) δ 8.70 (d, J = 0.9 Hz, 1H), 8.51 (d, J = 1.3 Hz, 1H), 7.65 (s,
1H), 6.98 (d, J = 0.9 Hz, 1H), 3.74 (q, J = 7.3 Hz, 2H), 3.44 (d, J = 2.4 Hz, 2H),
2.87–2.77 (m, 4H), 2.69 (t, J = 2.4 Hz, 1H), 2.40 (s, 3H), 1.31 (t, J = 7.3 Hz, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 179.19, 159.95, 157.33, 154.43, 142.66, 134.39,
112.06, 94.19, 78.88, 75.88, 54.29, 44.83, 41.21, 39.73, 26.04, 13.68. HRMS (ESI):
m/z calcd C16H21N7S [M+H]+ 344.1613, found 344.1657. HPLC purity: 99.0%.
4.1.7.3.
1-Ethyl-3-(6-(4-(3-(prop-2-yn-1-yl)amino)propyl)-1H-imidazol-1-yl)
1
pyrimidin-4-yl)thiourea (3a). Yellow solid, 65% yield. Mp 118−120 °C; H NMR
(400 MHz, DMSO-d6) δ 11.19 (t, J = 5.3 Hz, 1H), 10.92 (s, 1H), 8.73 (s, 1H), 8.35 (d,
J = 1.1 Hz, 1H), 7.41 (d, J = 15.7 Hz, 1H), 7.24 (s, 1H), 3.68–3.59 (m, 2H), 3.30 (d, J
= 2.3 Hz, 2H), 3.03 (t, J = 2.3 Hz, 1H), 2.62–2.52 (m, 4H), 1.78–1.68 (m, 2H), 1.22 (t,
J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 179.20, 159.96, 157.32, 154.45,
144.41, 134.53, 111.57, 94.17, 82.72, 73.79, 47.30, 39.73, 37.28, 28.28, 25.48, 13.69.
HRMS (EI): m/z calcd C16H21N7S (M+) 343.1579, found 343.1573. HPLC purity:
98.3%.
4.1.7.4. 1-Ethyl-3-(6-(4-(3-(methyl(prop-2-yn-1-yl)amino)propyl)-1H-imidazol
1
-1-yl)pyrimidin-4-yl) thiourea (3b). Yellow solid, 71% yield. Mp 122−124 °C; H
NMR (400 MHz, DMSO-d6) δ 11.19 (t, J = 5.3 Hz, 1H), 10.92 (s, 1H), 8.74 (s, 1H),
8.35 (d, J = 1.1 Hz, 1H), 7.44 (s, 1H), 7.24 (s, 1H), 3.68–3.60 (m, 2H), 3.30 (d, J =
2.3 Hz, 2H), 3.10 (t, J = 2.3 Hz, 1H), 2.56–2.52 (m, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.19
(s, 3H), 1.80–1.67 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)
δ 179.20, 159.93, 157.29, 154.45, 144.35, 134.50, 111.57, 94.17, 79.04, 75.63, 54.43,
44.92, 41.24, 39.69, 26.33, 25.41, 13.65. HRMS (EI): m/z calcd C17H23N7S (M+)
357.1736, found 357.1739. HPLC purity: 98.5%.
4.1.8. General synthetic procedure for 2d-c
To a solution of compound 2a (164 mg, 0.5 mmol) in CH3CN (8 mL),
corresponding RX (0.55 mmol), Et3N (101 mg, 1 mmol), and a catalytic amount of KI
were added. The reaction mixture was refluxed for 12 h, and the solvent was
evaporated to dryness under reduced pressure. The residue was purified on a silica gel
column using mixtures of MeOH:CH2Cl2 as the eluent, affording the target

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compounds 2c-d.
4.1.8.1 1-Ethyl-3-(6-(4-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)-1H-imidazol-1-yl)
pyrimidin-4-yl)thiourea (2c). Iodoethane was used as the corresponding RX. White
solid, 33% yield. Mp 154−155 °C; 1H NMR (400 MHz, CD3OD) δ 8.72 (s, 1H), 8.54
(s, 1H), 7.70 (s, 1H), 7.02 (s, 1H), 3.75 (q, J = 7.3 Hz, 2H), 3.71 (d, J = 1.8 Hz, 2H),
3.10 – 3.04 (m, 2H), 2.93 – 2.84 (m, 4H), 2.83 (s, 1H), 1.32 – 1.30 (m, 3H), 1.20 (t, J
= 7.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 179.18, 159.96, 157.35, 154.43,
134.49, 112.22, 94.22, 51.87, 46.93, 40.70, 40.11, 29.02, 13.68. HRMS (EI): m/z
calcd C17H23N7S (M+) 357.1736, found 357.1738. HPLC purity: 97.5%.
4.1.8.2. 1-(6-(4-(2-((Cyanomethyl)(prop-2-yn-1-yl)amino)ethyl)-1H-imidazol-1
-yl)pyrimidin-4-yl)-3-ethylthiourea (2d). 2-Bromoacetonitrile was used as the
1
corresponding RX. White solid, 35% yield. Mp 159−160°C; H NMR (400 MHz,
DMSO-d6) δ 11.19 (t, J = 5.3 Hz, 1H), 10.95 (s, 1H), 8.74 (s, 1H), 8.36 (d, J = 1.3 Hz,
1H), 7.52 (s, 1H), 7.25 (s, 1H), 3.84 (s, 2H), 3.68–3.59 (m, 2H), 3.45 (d, J = 2.4 Hz,
2H), 3.30 (t, J = 2.4 Hz, 1H), 2.86–2.80 (m, 2H), 2.77–2.71 (m, 2H), 1.22 (t, J = 7.2
Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 179.20, 159.98, 157.37, 154.45, 141.97,
134.58, 116.14, 112.34, 94.27, 78.43, 76.69, 51.74, 42.57, 41.21, 39.73, 25.70, 13.70.
HRMS (EI): m/z calcd C17H20N8S (M+) 368.1532, found 368.1515. HPLC purity:
95.8%.
4.2. Biological activity
4.2.1. In vitro inhibition of AChE and BuChE
The enzyme inhibition activity of the tested compounds was performed using the
method of Ellman et al. with slight modification [44]. The rat cortex was
homogenized in cold 75 mM sodium phosphate buffer (pH 7.4) as the AChE source,
and the rat serum was collected as the BuChE source. 5,5′-dithiobis (2-nitrobenzoic
acid) (DTNB), acetylthiocholine iodide and butyrylthiocholine iodide were purchased
from Sigma. Huperzine A (Hup A) was used as reference compound. 1 µL of the
tested compounds with an appropriate concentration (1 nM to 10 mM) was added into
the assay solution, which consisted of 50 µL of 0.1 M phosphate buffer, 50 µL of
0.2% DTNB, 109 or 99 µL of deionized water, 10 µL of rat cortex homogenate or 20