Development of Multifunctional Pyrimidinylthiourea Derivatives as Potential Anti-Alzheimer Agents

Article abstract of DOI:10.1021/acs.jmedchem.6b00636

Starting from a screening-hit compound, via structure modifications and optimizations, a series of nonfused and nonassembly pyrimidinylthiourea derivatives (2-5) was designed, synthesized, and evaluated as novel multifunctional agents against Alzheimer's disease. Biological activity results demonstrated that compounds 5r and 5t exhibited potent inhibition and excellent selectivity toward acetylcholinesterase (AChE, 5r, IC₅₀ = 0.204 μM, SI > 196; 5t, IC₅₀ = 0.067 μM, SI > 597), specific metal-chelating ability, significant antioxidant effects, modulation of metal-induced Aβ aggregation, inhibition of ROS production by copper redox cycle, low cytotoxicity, and moderate neuroprotection to human neuroblastoma SH-SY5Y cells. Moreover, compound 5r displayed appropriate blood-brain barrier (BBB) permeability both in vitro and in vivo and could improve memory and cognitive function of scopolamine-induced amnesia mice. The multifunctional profiles of 5r and its effectivity in AD mice highlight these structurally distinct pyrimidinylthiourea derivatives as prospective prototypes in the research of innovative multifunctional drugs for Alzheimer's disease.

Full text of DOI:10.1021/acs.jmedchem.6b00636

Subscriber access provided by Northern Illinois University
Article
Development of Multifunctional Pyrimidinylthiourea
Derivatives as Potential Anti-Alzheimer Agents
Xiaokang Li, Huan Wang, Zhengyu Lu, Xinyu Zheng, Wei Ni, Jin Zhu,
Yan Fu, Fulin Lian, Naixia Zhang, Jian Li, Haiyan Zhang, and Fei Mao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00636 • Publication Date (Web): 23 Aug 2016
Downloaded from http://pubs.acs.org on August 24, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Development of Multifunctional Pyrimidinylthiourea
Derivatives as Potential AntiꢀAlzheimer Agents
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a,†
b,†
a
a
a
a
Xiaokang Li , Huan Wang , Zhengyu Lu , Xinyu Zheng , Wei Ni , Jin Zhu , Yan
b
c
c
a,
b,
a,
Fu , Fulin Lian , Naixia Zhang , Jian Li *, Haiyan Zhang *, Fei Mao *
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
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong
Zhi Road, Shanghai 201203, China
†
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.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
Starting from a screeningꢀhit compound, via structure modifications and optimizations,
a series of nonꢀfused and nonꢀassembly pyrimidinylthiourea derivatives (2ꢀ5) was
designed, synthesized and evaluated as novel multifunctional agents against
Alzheimer’s disease. Biological activity results demonstrated that compounds 5r and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
t exhibited potent inhibition and excellent selectivity toward acetylcholinesterase
(AChE; 5r: IC50 = 0.204 ꢁM, SI > 196; 5t: IC50 = 0.067 ꢁM, SI > 597), specific
metalꢀchelating ability, significant antioxidant effects, modulation of metalꢀinduced
Aβ aggregation, inhibition of ROS production by copper redox cycle, low cytotoxicity
and moderate neuroprotection to human neuroblastoma SHꢀSY5Y cells. Moreover,
compound 5r displayed appropriate bloodꢀbrain barrier (BBB) permeability both in
vitro and in vivo and could improve memory and cognitive function of
scopolamineꢀinduced amnesia mice. The multifunctional profiles of 5r and its
effectivity in AD mice highlight these structurally distinct pyrimidinylthiourea
derivatives as prospective prototypes in the research of innovative multifunctional
drugs for Alzheimer’s disease.
ACS Paragon Plus Environment

Page 3 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
Alzheimer’s disease (AD) is a chronic, fatal and neurodegenerative disease in the
brain; its mechanism has not been fully elucidated due to complex etiology. As the
most common form of dementia and the leading cause of death among the elderly,
approximately 44 million patients suffer from AD, and the number is increasing
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
ꢀ2
annually . Commercial drugs for AD treatment are unable to meet the market
demand. The failure of several candidates based on new targets in clinical trials
3
actuated new strategies for drug development . Thus, multitargetꢀdirected ligands
(MTDLs), aiming to simultaneously target multiple pathological processes involved
4ꢀ13
in the neurodegenerative cascade, were developed as a therapeutic approach
.
Acetylcholine (ACh), a major neurotransmitter widely existing in the brain, plays
1
4ꢀ15
an important role in learning, memory, cognition and maintaining consciousness
.
Studies indicated that 90% of cholinergic neurons were damaged in the brain of AD
patients, and the associated cholinergic neurotransmitter in the cerebral cortex and
1
6ꢀ18
hippocampus decreased dramatically
levels has been considered a feasible therapy for AD
activity termination is hydrolysis by AChE. Therefore, inhibitors of AChE appear to
. Thus, remedying the physiological ACh
1
4, 19
. The major pathway of ACh
20
be a beneficial approach to repair the cholinergic transmission pathway . Currently,
the therapeutic options for AD treatment are limited to four AChE inhibitors
2
1
(
AChEIs) , namely, donepezil, rivastigmine, galantamine, and huperzine A (Hup A,
approved by CFDA) and one NꢀmethylꢀDꢀaspartic acid (NMDA) receptor antagonist,
22
memantine .
An increasing body of evidence suggests that the pathogenesis of AD is an
2
3ꢀ25
extremely complex interconnected network
. In addition to the low level of the
neurotransmitter ACh, this complex multisystem disease features multiple
pathological hallmarks, including abnormal accumulation and deposition of beta
amyloid protein (Aβ), dysregulation of biometals, oxidative stress and
2
3, 25ꢀ26
neuroinflammation of the CNS
(
central nervous system)
. The high levels and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2+
2+
2+
dysregulation of biometal ions, such as Cu , Zn , and Fe , are closely implicated in
2+
2+
the pathogenesis of AD. These metal ions (especially Cu and Zn ) bind to Aβ
peptides and influence the Aβ aggregation pathway, leading to the generation of toxic
2
5ꢀ27
Aβ oligomers
. Moreover, redoxꢀactive metal ions, Cu (I/II) and Fe (II/III), which
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
are associated with Aβ, were demonstrated to produce reactive oxygen species (ROS)
under physiological conditions through Fentonꢀlike reactions. The overproduction of
2
3, 27ꢀ30
ROS leads to oxidative stress and eventually neuronal death in AD patients
.
Therefore, biometal chelators endowed with the ability to modulate metalꢀAβ
interactions, as well as the metal distribution in the brain, have been considered as a
potential therapeutic approach for AD. In addition, neurotoxic ROS and oxidative
damage of neuronal cells are generally associated with AD, so compounds with
3
1ꢀ32
antioxidant activities may be beneficial for the treatment of AD
.
Many researchers have devoted their efforts to the development of MTDLs as a
hopeful approach to combat the multifactorial nature of AD. Typically, two or three
bioactive scaffolds with beneficial antiꢀAD properties were assembled or fused to
obtain new chemical entities, which possessed multiple abilities due to the functional
superposition of each constituent fragment and additional synergetic activities (Table
33
S1, supporting information). For example, tacrine–8ꢀhydroxyquinoline hybrid ,
34
6
ꢀchlorotacrine–metalꢀAβ
modulator
hybrid ,
3
5
36
donepezil–8ꢀhydroxyquinoline–propargylamine hybrid , tacrine–flavonoid hybrid ,
37
HLA20A and M30D were all obtained by assembling AChEIs and metal chelator
fragments (Figure 1). The hybrid strategies have had success in the field of MTLDs
but possess an apparent designing flaw—excessive dependence on the structure of
limited known bioactive compounds, such as tacrine, donepezil, clioquinol, rhein,
melatonin, resveratrol, and coumarin (Table S1). Therefore, to broaden the research
extent of multifunctional antiꢀAD drugs, the discovery of nonꢀfused and nonꢀassembly
lead compounds with multiple antiꢀAD properties is extremely urgent.
Insert Figure 1 Here
ACS Paragon Plus Environment

Page 5 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Toward this end, we have identified a series of nonꢀfused and nonꢀassembly
multifunctional pyrimidinylthiourea derivatives as prospective antiꢀAD agents.
Among them, the aminomethylimidazoleꢀcontaining pyrimidinylthiourea derivatives
5lꢀx showed potent inhibition and excellent selectivity toward AChE, as well as mild
antioxidant properties. Integrally considering the abilities of metalꢀchelating,
modulating metalꢀinduced Aβ aggregation, free radical scavenging, low cytotoxicity
and moderate neuroprotective effect, compounds 5r and 5t were identified as the
prototype of potential multifunctional antiꢀAlzheimer agents. Compound 5r also
showed positive results in an AD mice cognitive ability test, demonstrating that our
pyrimidinylthiourea derivatives possess development potential as prospective antiꢀAD
agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Results and Discussion
The Discovery of Hit Compound 1
Because several AChEIs have successfully been approved to be marketed as
drugs and have made prominent contributions to the symptomatic improvement of AD
2
0ꢀ21
patients
, AChE inhibitory ability should be firstly considered in the process of
searching for new multifunctional antiꢀAD scaffolds. An inꢀhouse library with 630
small molecular compounds (MW < 300) was screened using the AChE inhibition
assay. A “cocktail” of tactics was imitated to simplify this process. The detailed steps
are as follows. Firstly, 630 compounds were screened through the AChE inhibition
assay in 96ꢀwell plates with 10 compounds per well. Then, each constituent in
positive wells was screened again using the same method but with one compound per
well. Finally, the positive wells, contained compounds with potential AChE inhibitory
activity. Pyrimidine derivative 1 was one of the discovered compounds and exhibited
some AChE inhibitory activity (inhibition rate: 32.15% at 40 ꢁM). Compound 1 was
designated as a hit compound because a similar disubstituted pyrimidine scaffold has
appeared in potential antiꢀAD agents, such as AChEI, Aβꢀaggregation inhibitors and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
38ꢀ41
receptor for advanced glycation end products (RAGE) inhibitors
and also has
good synthetic accessibility.
Design and Synthesis
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Firstly, the ethylamino group of hit 1 was replaced by ethyoxyl, nꢀpropyl,
benzylamino, amino, acetylamino, and different substituted thiourea moieties to
produce target compounds 2a–o. As depicted in Scheme 1, reaction of pyrazole and
commercially available 4,6ꢀdichloropyrimidine or 4ꢀaminoꢀ6ꢀchloropyrimidine gave
the key intermediate 6 and compound 2d, respectively. Nucleophilic substitution of
the Cꢀ2 chlorine of intermediate 6 provided target compounds 2a–c. Target compound
2
3
e was obtained by acetylation of 2d with acetylchloride in the presence of Et N. The
reaction of 2d with ethyl isocyanate and different substituted isothiocyanates in the
presence of NaH provided the target compounds 2f and 2g–o.
Insert Scheme 1 Here
Ethylthiourea analogue 2g was treated as lead compound for its significantly
increased activity (Table 1). The ethylthiourea moiety was demonstrated as
indispensable pharmacophore for AChE inhibitory activity, and played important role
42
43ꢀ45
in generating metal chelation and antioxidant properties
. The pyrazolyl group of
compound 2g was regarded as a modification site and was replaced by various
azaꢀaromatics, such as pyrrolyl (3a), imidazolyl (3b), 1,2,3ꢀtriazolyl (3c),
1
,2,4ꢀtriazolyl (3d), benzo[d]imidazolyl (3e), and different alicyclic amino groups,
such as pyrrolidinyl (3f), morpholino (3g), 4ꢀmethylpiperazinyl (3h), benzylamino
3i) (Scheme 2). Commercially available 4ꢀaminoꢀ6ꢀchloropyrimidine was reacted
(
with azaꢀaromatic compounds or alicyclic amines to provide intermediates 7a–i,
followed by treatment with ethyl isothiocyanate to afford target compounds 3a–i.
Insert Scheme 2 Here
Imidazolyl analogue 3b showed improved potency (Table 2) and had sufficient
modifiable sites. The imidazolyl moiety (3b) remained in the following structure
modification. The 4,6ꢀdisubstituted pyrimidine core was treated as the next
ACS Paragon Plus Environment

Page 7 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
replacement part. Then, target compounds 4a–e were designed and obtained by the
reaction of intermediates 8a–e (synthetic details, see supporting information) with
ethyl isothiocyanate in the presence of NaH (Scheme 3).
Insert Scheme 3 Here
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Having maintained the integrality of compound 3b (Table 3), a series of target
compounds 5a–k with diverse substituents at Cꢀ2 or Cꢀ4 of the imidazole ring were
designed and synthesized to improve the biological activities and to expand the SAR
studies (Scheme 4). Nucleophilic substitution of 4ꢀaminoꢀ6ꢀchloropyrimidine with
substituted imidazole derivatives afforded intermediates 9a–j, which were further
reacted with ethyl isothiocyanate in the presence of NaH to obtain compounds 5a–j.
Reduction of 5i with NaBH gave compound 5k.
4
Insert Scheme 4 Here
Finally, for the potent inhibitory activity of compound 5i against AChE, a series
of compounds 5l–x, possessing diverse aminomethyl groups on the Cꢀ4 position of
the imidazole ring, were designed and synthesized (Scheme 4). Compounds 5l–x were
prepared by reductive amination of compound 5i with diverse aliphatic amines in the
presence of NaBH (CN).
3
In total, 53 derivatives (i.e., 2a–o, 3a–i, 4a–4e, and 5a–x) were designed and
synthesized, their chemical structures are shown in Tables 1–4, and details of the
synthetic procedures and structural characterization are described in the Experimental
Section.
Cholinesterase (ChE) Inhibitory Activity
To determine the therapeutic potential of these pyrimidine derivatives for the
treatment of AD, compounds (2a–o, 3a–i, 4a–4e, and 5a–x) were assayed against rat
46
AChE (rAChE) by the modified method of Ellman et al , using huperzine A (Hup A)
as reference. The experimental results were presented as IC50 (ꢁM) or, for poorly
active compounds, as the percentage of inhibition at 40 ꢁM. The tested compounds
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
displayed poor to excellent rAChE inhibitory activity (Tables 1–4). The compounds
(5i, 5l–m, 5o–x) with good rAChE inhibition were further tested for their inhibitory
activities against rat butyrylcholinesterase (rBuChE) using the same method, and the
activity results and selectivity indexes (SI) are summarized in Table 4.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Examples of variation in the initial ethylamino group of hit 1 gave encouraging
results (Table 1). Compound 2g, with the ethylamino group replaced by an
ethylthiourea group, exhibited good inhibitory activity, with an IC50 of 8.14 ꢁM.
However, replacement of the ethylthiourea group with a similar ethylurea group was
not tolerated (2g vs 2f). Introduction of either larger (2i–o) or smaller (2h)
substituents on the Nꢀ3 atom decreased the potency. This observation indicates that
steric hindrance of the Nꢀ3 substituent of thiourea may play an important role in the
binding modes of compounds to AChE.
Insert Table 1 Here
As shown in Table 2, changing pyrazolyl into various types of azaꢀaromatics or
alicyclic amino groups remarkably affected the AChE inhibitory activities. Generally,
alicyclic amino substituents (3f–i) were not beneficial, while the introduction of
azaꢀaromatic substituents (3b–e), except pyrrolyl (3a), was favorable. Among them,
compounds 3b and 3d were relatively potent, with IC50 values of 1.59 ꢁM and 1.05
ꢁM, respectively. In the studied set of central cores (Table 3), changing the position of
the N atom in the ring (4c–d) and introducing substituents on the ring (4a–b and 4e)
were not tolerated, leading to an obvious decline in potency.
Insert Tables 2–3 Here
The data in Table 4 show that introduction alkyl groups on the Cꢀ2 position of
the imidazole ring (5a–b) was not beneficial. Compared with Cꢀ2 methyl derivative
5
a, Cꢀ4 methyl derivative 5c showed relatively increased potency, so the Cꢀ4 position
was considered the preferable modification site. Fortunately, the following
Cꢀ4ꢀsubstituted derivatives 5d–k exhibited more increased activities than prototype
3
b. The two most effective inhibitors, 5h (IC50 = 0.170 ꢁM) and 5i (IC50 = 0.166 ꢁM),
ACS Paragon Plus Environment

Page 9 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
were nearly 10ꢀfold more potent than 3b. Compounds 5l–x possessed aminomethyl
moieties on the Cꢀ4 position of the imidazole ring and exhibited good potency.
Among them, compound 5t was the most potent, with an IC50 value of 0.067 ꢁM,
more potent than the approved drug Hup A (0.082 ꢁM).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Insert Table 4 Here
As presented in Table 4, compounds 5i, 5l–m, 5o–x displayed poor to
micromolar potency against rBuChE and excellent selectivity toward rAChE.
Compound 5v was the strongest rBuChE inhibitor and displayed the lowest SI value
of 59. The most potent compound, 5t, exhibited weak BuChE inhibitory activity,
indicating 5t was an outstanding selective AChE inhibitor over BuChE.
SAR Studies
Based on hit 1, the key transitionary compounds of structural optimization are
illustrated in Figure 2. The SAR studies of the 53 derivatives provided important
insight into the essential structural requirements for effective AChE inhibition. An
analysis of the data shown in Tables 1ꢀ4 reveals noteworthy observations of the SARs
for compounds 2a–o, 3a–i, 4a–4e and 5a–x: (1) the thiourea moiety is indispensable
pharmacophore for AChE inhibitory activity; replacement of thiourea moiety with
urea or other groups decreases the activity dramatically (2g vs 1, 2f, 2a–e). There is an
extremely rigid requirement for the steric hindrance of the Nꢀ3 substituents of
thiourea; an ethyl group (2g) is optimal, smaller (2h) or bigger (2i–o) substituents
result in obvious loss of potency; (2) considering the improved potency and sufficient
modifiable sites, the imidazole ring is the most suitable substituent at the Cꢀ6 position
of the pyrimidine ring (3b vs 2g, 3a, 3c, 3e–i; 3b vs 3d); (3) the 4,6ꢀdisubstituted
pyrimidine core is critical for AChE inhibition; changing the N atom position or
introduction other substituents lead to remarkable decline of inhibitory activity (3b vs
4
a–e); and (4) the Cꢀ4 position on imidazole ring was a promising substituted site (5c
vs 5a; 5d–k vs 3b); compounds 5l–x with protonatable aminomethyl moieties are
potent AChE inhibitors. The Nꢀmonosubstituted aminomethyl derivatives 5l–o
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
display relatively weak inhibitory potency. For Nꢀdisubstituted aminomethyl
derivatives, the potencies increase in the order of diethylaminomethyl >
dimethylaminomethyl > benzyl(methyl)aminomethyl > dipropylaminomethyl (5q >
5p > 5s > 5r); for the cyclic aminomethyl derivatives, the potencies decrease
following the augment of the cycle size, pyrrolidinylmethyl > piperidinylmethyl >
azepanylmethyl (5t > 5u > 5v). In brief, the SARs are highly explicit. Analogue 5t
represented the most robust combination, leading to 121ꢀfold potency improvement
compared to the lead compound 2g.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Insert Figure 2 Here
Free Oxygen Radical Scavenging Ability
To evaluate the antioxidant activities of compounds 5i, 5l–m, 5o–x, the oxygen
radical absorbance capacity assay (ORACꢀFL) was performed using fluorescein as the
fluorescent probe and AAPH (2,2'ꢀazobisꢀ(amidinopropane)dihydrochloride) as the
4
7ꢀ48
peroxyl radical inducer
. Trolox, a vitamin E analogue, was used as a standard. CQ
was tested as reference, and compound 5y (without thiourea fragment, supporting
information) was measured as a negative control. The results (Table 4) indicated that
all the tested compounds exhibited mild antioxidant ability with ORAC values of 1.17
to 1.48, except compound 5i, which featured an aldehyde group on the imidazole ring
(0.31). Compound 5w displayed the highest activity, with an ORACꢀFL value of 1.48.
Moreover, the thiourea fragment played a crucial role in scavenging ROS (5t vs 5y).
Metal Binding Properties of 5r and 5t
A UVꢀvis spectroscopy assay was performed to evaluate the ability of
2
+
2+
2+
compounds 5r and 5t to chelate biometals, such as Cu , Fe , and Zn . The results
showed that when CuCl was added to the solutions of 5r and 5t, the maximum
absorption both decreased dramatically at 290 nm, demonstrating the formation of
2
2+
2+
5
rꢀCu and 5tꢀCu complexes (Figures 3A, 3B). There were only vague optical shift
and maximum absorption fluctuation when FeSO
4
, FeCl
3
2
and ZnCl were added,
2
+
3+
2+
indicating 5r and 5t had poor chelation abilities to Fe , Fe and Zn . Compound 5t
ACS Paragon Plus Environment

Page 11 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was selected to test its binding property to other biological relevant metals, such as
2
+
2+
2+
2+
2+
Mg , Ca , Mn , Ni , Co , and exhibited very weak chelation ability (Figure S2,
supporting information). A competition experiment was performed to verify the metal
2+
ion selectivity of 5t, the results demonstrated that Cu could competitively bind to 5t
(Figure S3, supporting information). Compound 5y, without thiourea fragment, was
tested as negative control to determine the role of thiourea moiety played in the
chelating experiment. As expected, the absorption curve exhibited a slight shift when
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CuCl
2
, FeSO
4
, FeCl
3
and ZnCl
2
were added (Figure 3C). The above results proved
2+
that target compound 5t could selectively chelate Cu , and this property was
determined by the thiourea fragment. Clioquinol (CQ) was selected as control (Figure
3
D).
Insert Figure 3 Here
Dot Blot Assay and TEM Assay
The modulation of metalꢀinduced Aβ aggregation by compounds 5r and 5t were
49ꢀ50
4,
studied through a dot blot assay
, and a transmission electron microscopy (TEM)
13
assay was then performed to examine the morphological changes of Aβ species. In
the dot blot assay, samples were probed by A11 antibody to determine oligomeric Aβ,
and total Aβ was detected by 6E10 as the loading control. A representative scan graph
and a statistical histogram are presented in Figure 4A; the grey density of the spots is
proportional to the Aβ oligomerization level. After 24 h incubation, Aβ aggregated
extremely, compounds 5r and 5t showed no effect on Aβ selfꢀaggregation.
Epigallocatechin gallate (EGCG) was tested as positive control. To verify this result,
we performed a thioflavine T (ThT) fluorescence assay and achieved similar estimates
(Figure S4, supporting information). Most importantly, the dot blot assay clearly
2+
showed that Cu could accelerate Aβ aggregation, and this process was significantly
regulated by compounds 5r and 5t.
Further visual morphological changes of the Aβ samples were observed by TEM
2+
(
Figure 4B). The TEM images demonstrated that the Cu ꢀtreated sample of fresh Aβ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
produced more fibrils than the untreated sample (Figure 4B, sequences 2 and 3). Aβ
aggregates obviously decreased and fewer Aβ fibrils were detected when compounds
5
r, 5t and CQ were incubated with the sample (Figure 4B, sequences 4, 5 and 6).
These TEM results were consistent with the results of the dot blot assay and jointly
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2+
demonstrated that compounds 5r and 5t can indeed modulate Cu ꢀinduced Aβ
aggregation.
Insert Figure 4 Here
Inhibition of ROS Produced by Copper Redox Cycling
Increased generation of ROS is a significant pathological feature of AD patients,
and clinical evidence indicated that redoxꢀactive metal ions were closely involved in
3
1ꢀ32
the high oxidative stress caused by ROS
produce ROS in the presence of trace amounts of metals, especially copper
Considering their biological activities as metal chelators, the abilities of compounds
r and 5t to halt Cu(II)ꢀrelated redox under aerobic conditions were evaluated,
. Moreover, Aβ could spontaneously
5
1ꢀ54
.
5
utilizing the model of the Cuꢀascorbate redox system described by Faller and
55
coworkers (Figure 5A). Hydroxyl radicals (OH·), produced by copper redox cycling
in the presence of ascorbate, were measured by coincubation with
coumarinꢀ3ꢀcarboxylic acid (CCA), which generated fluorescent 7ꢀhydroxyꢀCCA
with the oxidation of OH·. As shown in Figure 5B, the OH· in the copperꢀascorbate
system increased steadily with time and reached a plateau at approximately 800
seconds. However, this process was entirely inhibited when compounds 5r and 5t
were coincubated with the Cuꢀascorbate system, indicating that the ligands were
56
capable of halting copper redox cycling by generating a redoxꢀinactive complex .
The documented metal chelator ethylenediaminetetraacetic acid (EDTA) was
measured as a control.
Insert Figure 5 Here
MTT assay
To investigate the safety of the pyrimidinylthiourea derivatives, the cytotoxicity
ACS Paragon Plus Environment

Page 13 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of compounds 5r and 5t on the human neuroblastoma cell line SHꢀSY5Y was studied
using a 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) assay to
determine cell death. Marketed antiꢀAD drugs Hup A and donepezil (Don) were tested
as references. As depicted in Figure 6A, compounds 5r and 5t, like Hup A and Don,
exhibited no cytotoxicity at concentrations of 10 ꢁM and 30 ꢁM after incubation for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
4 h. After increasing the concentration to a relatively high level of 100 ꢁM, 5r, 5t
and Don decreased the cell viability (p < 0.01, p < 0.01 and p < 0.001, respectively),
while Hup A had no obvious influence on cell viability. Therefore, compounds 5r and
5
t were proved to be low cytotoxic.
Next, to determine whether compounds 5r and 5t can protect neuronal cells from
toxic Aβ species, cell viability experiments were conducted using the same cell line.
2
+
As shown in Figure 6B, incubation of SHꢀSY5Y cells with Aβ in the presence of Cu
caused significant toxicity, its cell viability was approximately 39% lower than the
vehicle group (P < 0.001) and 11% lower than the Aβ alone group (P < 0.001).
Compounds 5r and 5t could protect the human neuronal SHꢀSY5Y cells from
2
+
Cu ꢀinduced Aβ aggregate toxicity, leading to significant increase in cell viability
2+
compared with the Aβ + Cu group (P < 0.001 and P < 0.001). CQ was tested as
positive control. Moreover, compounds 5r and 5t could not interact with Aβ directly
to restrain the cytotoxicity of Aβ aggregates. These results are highly consistent with
2+
the results from the dot blot assay. Compounds 5r and 5t in the presence of Cu or
2
+
Cu alone did not influence the SHꢀSY5Y cell viability in the tested concentrations.
Insert Figure 6 Here
In Vitro BloodꢀBrain Barrier Permeation Assay and Drugꢀlikeness Evaluation
Bloodꢀbrain barrier (BBB) permeability is the first requirement for developing
1
3, 57
antiꢀAD drugs. The parallel artificial membrane permeation assay (PAMPA)
was
used to predict passive BBB permeation of optimized compounds 5p–x. From the
results presented in Table 5, it can be observed that the most potent AChE inhibitor,
compound 5t, exhibited poor BBB permeation (CNS−). Compounds 5rꢀs and 5uꢀv,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
which possess compensatory hydrophobic substituents on the nitrogen atom of the
Cꢀ4 aminomethyl of the imidazole ring, have P values above the threshold for good
e
BBB permeation (CNS+), indicating they could cross the BBB. Among them,
compounds 5r–s exhibited comparatively better BBB permeability than 5u–v.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Insert Table 5 Here
To determine the potential druggability of compounds 5r–s, their drugꢀlike
58
physicochemical properties were evaluated . As shown in Table 6, compounds 5r–s
59ꢀ60
both obey the Lipinski’s rule of five
and fulfil the restrictions of topological polar
6
1
62
surface area (tPSA) and melting point (MP) for CNS drugs. Compound 5r shows a
relatively high value of clogP (calculated logarithm of the octanolꢀwater partition
63
coefficient) , and the corresponding hydrochloride 5r·HCl displays better water
solubility than 5s·HCl. Therefore, compound 5r·HCl was selected to conduct further
animal cognitive ability experiments.
Insert Table 6 Here
Cognitive Improvement in Mice Model of AD
The effects of compounds 5r·HCl and 5t·HCl on cognitive improvement were
assessed in a scopolamine (Scop)ꢀinduced amnesia animal model using a passageway
water maze; Hup A at 0.1 mg/kg was used as a positive control. Scop can block the
64
cholinergic pathway by antagonizing the muscarinic receptor , which provides a
typical AD model to explore the role of the cholinergic system in cognition and to
6
5
evaluate anticholinergic drug candidates preclinically . In the experiment, all mice
received 2 training sessions daily for 4 days before a cognitive ability test; then, the
trained mice were randomly allocated into 5 groups: control (Con), Scop, Scop +
5
t·HCl, Scop + 5r·HCl, and Scop + Hup A. As shown in Figure 7, the Scop group
exhibited longer escape latency and more frequent error than the Con group
salineꢀinjected group). Compound 5r·HCl, administered to mice by oral gavage at a
(
dose of 200 mg/kg, rescued the escape latency and reduced the number of errors
induced by scopolamine. The effectivity of 5r·HCl for symptom improvement in AD
ACS Paragon Plus Environment

Page 15 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mice model identifies it as a potential lead antiꢀAD agent, especially for the treatment
of cholinergic deficiency in the early stages of the disease.
Insert Scheme 7 Here
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Measuring 5r in Plasma and Brain Samples
Liquid chromatography tandem mass spectroscopy (LCꢀMS/MS) was used to
identify and quantify the plasma and brain (cortex, hippocampus) levels of compound
68ꢀ69
5
r after oral administration
(5r·HCl at 200 mg/kg). As shown in Table 7,
compound 5r was detected in the brain tissues (cortex, 296.3 ng/g; hippocampus,
79.1 ng/g) and plasma (1738.9 ng/g). LogBB values were used to evaluate BBB
5
permeability; two vital brain areas showed the logBB values of −0.85 (cortex) and
−0.49 (hippocampus), which were within the established threshold that defines a
70
compound as crossing the BBB (log BB ≥ −1) . Therefore, compound 5r crosses
the bloodꢀbrain barrier.
Insert Table 7 Here
CONCLUSIONS
By understanding the heterogeneity and the multifactorial nature of the disease, a
single chemical entity against multiple diseaseꢀrelevant targets might offer a potential
approach to the treatment of AD. Given that most of the drugs marketed for AD are
cholinergic agents, AChE inhibitory activity was recognized as a prospective focus
point to guide MTDL design strategy. The deficiency of novel bioactive scaffolds
largely hindered the development of multifunctional antiꢀAD agents, especially
because many researchers continue to concentrate on the strategy of fusing or
assembling pharmacophores of limited known bioactive entities. Therefore, it is
urgent to search for a new scaffold as the hit compound for multifunctional antiꢀAD
agent design.
Through an in vitro HTS campaign, hit 1, with some AChE inhibitory activity,
was screened out. Then, a series of pyrimidinylthiourea derivatives was rationally
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
designed, synthesized and assayed. The SAR studies revealed that the thiourea
fragment acted as an indispensable pharmacophore for AChE inhibitory activity and
played curial role in generating metalꢀchelating and antioxidant properties.
Compounds 5r and 5t exhibited multiple antiꢀAD properties, and showed low
cytotoxicity and moderate neuroprotection to human neuroblastoma SHꢀSY5Y cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
By changing the R , R substituents (Scheme 4) on the nitrogen atom of the Cꢀ4
aminomethyl on the imidazole ring, the water solubility, lipophilicity and BBB
permeability could be modified. Compound 5r exerted appropriate BBB permeation
and fulfilled drugꢀlike rules. Its hydrochloride was used to conduct the animal
cognitive ability experiment and showed a remarkable improvement of memory and
cognitive function in a Scopꢀinduced amnesia animal model.
In conclusion, this work clearly validates the nonꢀfused and nonꢀassembly
multifunctional ligands design strategy and discovers a series of novel
pyrimidinylthiourea derivatives with multiple antiꢀAD profiles. These compounds can
be considered as potential prototypes to guide the future research of innovative
multifunctional drugs for Alzheimer’s disease.
Experimental Section
Chemistry. General Method. Reagents and solvents were purchased from
Adamasꢀbeta, J&K, Energy Chemical, Target Molecule, TCI, Alfa Aesar, and Acros
and were used without further purification. Analytical thinꢀlayer chromatography
(TLC) was performed using HSGF 254 (150ꢀ200 ꢁm thickness; Yantai Huiyou Co.,
China), and spots were visualized with UV light. Yields were not optimized. Melting
points were measured in capillary tubes on a SGW Xꢀ4 melting point apparatus
without correction. Nuclear magnetic resonance (NMR) spectroscopy was performed
on a Bruker AMXꢀ400 NMR (IS as TMS). 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). Highꢀresolution mass spectra (HRMS) were obtained by electric ionization (EI)
ACS Paragon Plus Environment

Page 17 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
and electrospray ionization (ESI) using a Waters GCT Premie and Waters LCT. HPLC
data analysis of compounds 2–5 was performed on an Agilent 1100 with a quaternary
pump and diodeꢀarray detector (DAD). The peak purity was verified by UV spectra.
All analogs were confirmed to be ≥95% pure (Table S2, supporting information).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NꢀEthylꢀ6ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀamine (1). A mixture of 6 (390 mg, 2
mmol), ethylamine (4 mmol), and Cs
mL) at 40–50 °C overnight. Water (60 mL) was added. The mixture was extracted
with EtOAc (3 × 60 mL). The organic layer was separated and dried over Na SO
2 3
CO (1.30 g, 4 mmol) were stirred in DMF (10
2
4
.
Removal of the solvents produced a residue which was purified using column
chromatography, eluted with a mixture of EtOAc/petroleum ether (1:3, v/v), to afford
1
compound 1 as yellow solid. 58% yield. mp 119−121 °C. H NMR (400 MHz, CDCl
3
)
δ 8.53 (d, J = 2.6 Hz, 1H), 8.40 (s, 1H), 7.75 (d, J = 0.9 Hz, 1H), 7.26 (s, 1H), 6.91 (s,
1H), 6.46 (dd, J = 2.6, 1.7 Hz, 1H), 5.38 (s, 1H), 3.52–3.31 (m, 2H), 1.34–1.22 (m,
+
3
H). HRMS (EI) m/z calcd C
9
H
11
N
5
(M ) 189.1014, found 189.1015.
4
ꢀEthoxyꢀ6ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidine (2a). To a solution of NaH (60%
dispersion in mineral oil, 240 mg, 6 mmol) in stirred anhydrous DMF (10 mL) was
added anhydrous EtOH (263 ꢁL, 4.5 mmol) and intermediate 6 (542 mg, 3 mmol).
The reaction mixture was stirred at room temperature for 4 h. Water (50 mL) was
added and the mixture was extracted with EtOAc (3 × 60 mL). The organic layer was
2 4
separated and dried over Na SO . Removal of the solvents produced a residue which
was purified using column chromatography, eluted with
a
mixture of
1
EtOAc/petroleum ether (1:50, v/v), to afford 2a (280 mg, 49%) as a brown oil. H
3
NMR (400 MHz, CDCl ) δ 8.60 (d, J = 0.5 Hz, 1H), 8.57–8.50 (m, 1H), 7.76 (d, J =
1
.0 Hz, 1H), 7.25 (d, J = 0.9 Hz, 1H), 6.48 (dd, J = 2.6, 1.7 Hz, 1H), 4.47 (q, J = 7.1
+
Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H). HRMS (EI) m/z calcd C H N O (M ) 190.0855,
9
10
4
found 190.0854.
4ꢀPropylꢀ6ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidine (2b). A mixture of 6 (90 mg, 0.5 mmol)
and ferrous acetylacetonate (25.4 mg, 0.1 mmol) in anhydrous THF were stirred at
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢀ
2
30 °C for 10 min, with protection of N . Then nꢀpropylmagnesium bromide (2M in
THF, 0.5 mL, 1 mmol) was added by syringe. Saturated NH Cl aqueous solution was
4
added 1h later. The mixture was extracted with EtOAc. The organic layer was
separated and dried over Na
2 4
SO . Removal of the solvents produced a residue which
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was purified using column chromatography, eluted with
a
mixture of
1
EtOAc/petroleum ether (1:15, v/v), to afford 2b as a brown oil. 15% yield. H NMR
(400 MHz, Acetoneꢀd
6
) δ 8.90 (s, 1H), 8.65 (d, J = 2.6 Hz, 1H), 7.85 (s, 1H), 7.82 (s,
1
3
H), 6.60 (s, 1H), 2.82 (t, J = 14.1, 5.9 Hz, 2H), 1.90–1.73 (m, 2H), 0.99 (t, J = 7.4 Hz,
+
12 4
H). HRMS (EI) m/z calcd C10H N (M ) 188.1062, found 188.1063.
NꢀBenzylꢀ6ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀamine (2c). A mixture of 6 (390 mg, 2
mmol), benzylamine (4 mmol), and Cs CO (1.30 g, 4 mmol) were stirred in DMF (10
mL) at 40–50 °C overnight. Water (60 mL) was added. The mixture was extracted
with EtOAc (3 × 60 mL). The organic layer was separated and dried over Na SO
2
3
2
4
.
Removal of the solvents produced a residue which was purified using column
chromatography, eluted with a mixture of EtOAc/petroleum ether (1:3, v/v). To afford
1
2
c as yellow solid. 60% yield. mp 167–170 °C H NMR (400 MHz, DMSOꢀd
6
) δ 8.54
(d, J = 2.5 Hz, 1H), 8.39 (s, 1H), 8.25 (s, 1H), 7.84 (s, 1H), 7.38–7.31 (m, 4H),
7
.30–7.19 (m, 1H), 7.02 (s, 1H), 6.56 (s, 1H), 4.62 (s, 2H). HRMS (EI) m/z calcd
+
C
14
H
13
N
5
(M ) 251.1171, found 251.1170.
6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀamine (2d). A mixture of pyrazole (0.75 g, 10
mmol), 4ꢀaminoꢀ6ꢀchloropyrimidine (1.30 g, 10 mmol) and Cs CO (3.91 g, 12 mmol)
2
3
were stirred in DMF (20 mL) for 12 h at 120 °C. Water (100 mL) was added. The
mixture was extracted with EtOAc (3 × 60 mL). The organic layer was separated and
2 4
dried over Na SO . Removal of the solvents produced a residue which was purified
using column chromatography, eluted with a mixture of EtOAc/petroleum ether (1:1,
1
v/v), to afford 2d (835 mg, 52%) as a yellow solid. mp 235–237 °C. H NMR (400
6
MHz, DMSOꢀd ) δ 8.48–8.43 (m, 1H), 8.33 (d, J = 0.8 Hz, 1H), 7.82 (t, J = 1.4 Hz,
1H), 7.20 (s, 2H), 7.15–7.08 (m, 1H), 6.57 (d, J = 1.0 Hz, 1H). HRMS (EI) m/z calcd
+
C
7
H
7
N
5
(M ) 161.0701, found 161.0702.
ACS Paragon Plus Environment

Page 19 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Nꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)acetamide (2e). To a solution of 2d (161 mg,
1
mmol) and Et N (278 ꢁL, 2 mmol) in CH Cl (20 mL) stirred in ice bath was added
3 2 2
AcCl (142 ꢁL, 2 mmol) dropwise. The reaction mixture was stirred at room
temperature overnight. Remove the precipitate by filtration, and evaporate the solvent
to dryness under reduced pressure. The residue was purified on a silica gel column
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
using mixtures of EtOAc/petroleum ether (1:3, v/v) as eluent, obtaining target
1
compound 2e. White solid. 58% yield. mp 232–235 °C; H NMR (400 MHz, CDCl
3
)
δ 8.73 (s, 1H), 8.65 (d, J = 0.9 Hz, 1H), 8.62 (br, 1H), 8.55 (d, J = 2.2 Hz, 1H), 7.82 (d,
J = 0.9 Hz, 1H), 6.51 (dd, J = 2.7, 1.6 Hz, 1H), 2.29 (s, 2H). HRMS (EI) m/z calcd
+
C
9
H
9
N
5
O (M ) 203.0807, found 203.0808.
General
procedure
for
the
synthesis
of
1ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀsubstituted thioureas (2f–m). To
a
solution of 2d (1 mmol) in DMF (10 mL) was added NaH (60% dispersion in mineral
oil, 48 mg, 1.2 mmol), stirred for 5 min, and then corresponding isocyanate and
isothiocyanates were added. The reaction mixture was stirred at room temperature for
0
.5 h. Water was added and the mixture was extracted with EtOAc (3 × 50 mL). The
organic layer was separated and dried over Na SO . Removal of the solvents produced
2
4
a residue which was purified using column chromatography, eluted with a mixture of
MeOH/CH Cl , to afford corresponding target compounds in good yield.
2
2
1ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylurea (2f). White solid. 45% yield. mp
1
195–197 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 9.75 (s, 1H), 8.64 (s, 1H), 8.62 (d, J =
2
.7 Hz, 1H), 8.09 (s, 1H), 7.93 (d, J = 19.3 Hz, 1H), 7.49 (s, 1H), 6.62 (dd, J = 2.6,
1
.6 Hz, 1H), 3.20 (m, J = 14.1, 7.1 Hz, 2H), 1.10 (t, J = 7.2 Hz, 2H). HRMS (EI) m/z
+
calcd C10
H
12
N
6
O (M ) 232.1073, found 232.1074.
1
ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (2g). White solid. 40% yield.
1
6
mp 201–203 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.30 (t, J = 5.1 Hz, 1H), 11.09 (s,
1H), 8.73 (t, J = 7.5 Hz, 1H), 8.63 (dd, J = 2.7, 0.5 Hz, 1H), 7.94 (d, J = 1.0 Hz, 1H),
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
.64 (d, J = 0.9 Hz, 1H), 6.64 (dd, J = 2.7, 1.6 Hz, 1H), 3.79–3.53 (m, 2H), 1.28–1.06
+
(m, 3H). HRMS (EI) m/z calcd C H N S (M ) 248.0844, found 248.0845.
10
12
6
1ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀmethylthiourea (2h). White solid. 63%
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
yield. mp 255–257 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.22 (d, J = 4.4 Hz, 1H),
11.16 (s, 1H), 8.72 (d, J = 0.9 Hz, 1H), 8.63 (d, J = 2.6 Hz, 1H), 7.94 (d, J = 1.1 Hz,
1
H), 7.62 (d, J = 0.9 Hz, 1H), 6.64 (dd, J = 2.7, 1.6 Hz, 1H), 3.12 (d, J = 4.6 Hz, 3H).
+
HRMS (EI) m/z calcd C H N S (M ) 234.0688, found 234.0687.
9
10
6
1
ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀpropylthiourea (2i). White solid. 74%
1
yield. mp 166–168 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.38 (s, 1H), 11.14 (s, 1H),
8
.75 (s, 1H), 8.63 (s, 1H), 7.95 (s, 1H), 7.64 (s, 1H), 6.64 (s, 1H), 3.59 (dd, J = 12.7,
6.5 Hz, 2H), 1.77–1.55 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). HRMS (EI) m/z calcd
+
C H N S (M ) 262.1001, found 262.1002.
11
14
6
1
ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀisopropylthiourea (2j). White solid. 76%
1
yield. mp 180–182 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.30 (d, J = 7.5 Hz, 1H),
1
7
1
2
1.07 (s, 1H), 8.75 (d, J = 0.8 Hz, 1H), 8.66–8.50 (m, 1H), 7.94 (d, J = 1.0 Hz, 1H),
.64 (d, J = 0.9 Hz, 1H), 6.64 (dd, J = 2.7, 1.6 Hz, 1H), 4.42 (m, J = 13.2, 6.6 Hz, 1H),
+
.28 (d, J = 6.6 Hz, 6H). HRMS (EI) m/z calcd C H N S (M ) 262.1001, found
11
14
6
62.1002.
1ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀbutylthiourea (2k). White solid. 68%
1
yield. mp 142–144 °C; H NMR (400 MHz, DMSO) δ 11.36 (t, J = 4.8 Hz, 1H), 11.13
(
s, 1H), 8.75 (s, 1H), 8.63 (s, 1H), 7.95 (s, 1H), 7.63 (s, 1H), 6.64 (s, 1H), 3.62 (dd, J
12.8, 6.5 Hz, 2H), 1.70–1.56 (m, 2H), 1.44–1.30 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H).
HRMS (EI) m/z calcd C12 S (M+) 276.1157, found 276.1158.
=
16 6
H N
1
ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀ(tertꢀbutyl)thiourea (2l). White solid.
1
6
69% yield. mp 148–151 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.60 (s, 1H), 10.86 (s,
1H), 8.74 (s, 1H), 8.66–8.48 (m, 1H), 7.93 (t, J = 7.6 Hz, 1H), 7.62 (d, J = 0.9 Hz,
ACS Paragon Plus Environment

Page 21 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
16 6
H), 6.64 (dd, J = 2.7, 1.6 Hz, 1H), 1.56 (s, 8H). HRMS (EI) m/z calcd C12H N S
+
(M ) 276.1157, found 276.1158.
1ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀcyclohexylthiourea (2m). White solid.
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
80% yield. mp 195–197 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.44 (d, J = 7.7 Hz,
1
H), 11.08 (s, 1H), 8.76 (s, 1H), 8.63 (d, J = 2.7 Hz, 1H), 7.94 (d, J = 1.0 Hz, 1H),
.64 (d, J = 0.8 Hz, 1H), 6.64 (dd, J = 2.6, 1.6 Hz, 1H), 4.21 (s, 1H), 1.98 (m, J = 8.6
7
Hz, 2H), 1.69 (m, 2H), 1.56 (m, J = 6.0 Hz, 1H), 1.49–1.22 (m, 5H). HRMS (EI) m/z
+
calcd C14
H
18
N
6
S (M ) 302.1314, found 302.1313.
1
ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀbenzylthiourea (2n). White solid. 72%
1
6
yield. mp 217–219 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.74 (t, J = 5.5 Hz, 1H),
11.28 (s, 1H), 8.71 (d, J = 0.9 Hz, 1H), 8.65–8.48 (m, 1H), 7.94 (t, J = 8.4 Hz, 1H),
7.67 (d, J = 0.9 Hz, 1H), 7.51–7.18 (m, 5H), 6.65–6.53 (m, 1H), 4.93 (d, J = 5.6 Hz,
+
2
H). HRMS (EI) m/z calcd C15
H
14
N
6
S (M ) 310.1001, found 310.1002.
1
ꢀ(6ꢀ(1HꢀPyrazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀ(3ꢀchlorophenyl)thiourea (2o). White
1
solid. 75% yield. mp 191–194 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 13.50 (s, 1H),
11.52 (s, 1H), 8.83 (d, J = 24.3 Hz, 1H), 8.64 (t, J = 15.7 Hz, 1H), 8.04–7.89 (m, 2H),
7
.74 (s, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.46 (t, J = 8.1 Hz, 1H), 7.29 (dd, J = 35.4, 19.6
+
Hz, 1H), 6.67 (dd, J = 2.6, 1.6 Hz, 1H). HRMS (EI) m/z calcd C14
H11ClN
6
S (M )
330.0454, found 330.0452.
General procedure for the synthesis of different 1ꢀPyrimidinꢀ3ꢀethylthioureas
aꢀl. To a solution of corresponding intermediates 7aꢀl (1 mmol) in DMF (8 mL) was
3
added NaH (60% dispersion in mineral oil, 48 mg, 1.2 mmol). The solution was
stirred for 5 min and then ethyl isothiocyanate (87 mg, 1 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 × 50 mL). Organic layer was separated and
2 4
dried over Na SO . Removal of the solvents produced a residue which was purified
using column chromatography, eluted with a mixture of MeOH/CH
2 2
Cl , to afford
corresponding target compounds in good yield.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
ꢀ(6ꢀ(1HꢀPyrrolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (3a). White solid. 60% yield.
1
mp 226–229 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.25 (t, J = 5.0 Hz, 1H), 10.81 (s,
6
1
H), 8.69 (s, 1H), 7.58–7.44 (m, 2H), 7.24 (d, J = 0.7 Hz, 1H), 6.54–6.26 (m, 2H),
+
3.77–3.54 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H). HRMS (EI) m/z calcd C11
H
13
N
5
S (M )
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
247.0892, found 247.0892.
1
ꢀ(6ꢀ(1HꢀImidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (3b). White solid. 54%
1
yield. mp 221–223 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.12 (d, J = 54.2 Hz, 1H),
6
1
1
1
0.98 (s, 1H), 8.77 (d, J = 0.8 Hz, 1H), 8.47 (d, J = 15.4 Hz, 1H), 7.75 (t, J = 1.4 Hz,
H), 7.29 (t, J = 16.7 Hz, 1H), 7.23–6.97 (m, 1H), 3.76–3.56 (m, 1H), 1.31–1.08 (m,
+
H). HRMS (EI) m/z calcd C H N S (M ) 248.0844, found 248.0845.
1
0
12
6
1ꢀ(6ꢀ(1Hꢀ1,2,3ꢀTriazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (3c). White solid. 48%
1
yield. mp 252–254 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.26 (s, 1H), 11.22 (s, 1H),
6
9
.17–8.77 (m, 2H), 8.06 (d, J = 1.0 Hz, 1H), 7.88 (s, 1H), 3.90–3.48 (m, 2H), 1.24 (t,
+
J = 7.2 Hz, 3H). HRMS (EI) m/z calcd C
9
H
11
N
7
S (M ) 249.0797, found 249.0796.
1
ꢀ(6ꢀ(1Hꢀ1,2,4ꢀTriazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (3d). White solid. 50%
1
yield. mp 246–247 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.22 (d, J = 10.9 Hz, 1H),
9
3
.45 (s, 1H), 8.81 (d, J = 0.9 Hz, 1H), 8.40 (s, 1H), 7.61 (d, J = 0.9 Hz, 1H),
+
9 11 7
.76–3.56 (m, 1H), 1.23 (t, J = 7.2 Hz, 1H). HRMS (EI) m/z calcd C H N S (M )
249.0797, found 249.0796.
1
4
1
ꢀ(6ꢀ(1HꢀBenzo[d]imidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (3e). White solid.
1
6% yield. mp 227–230 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.21 (s, 1H), 11.02 (s,
H), 8.92 (s, 1H), 8.86 (s, 1H), 8.17 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.64
(s, 1H), 7.56–7.09 (m, 3H), 3.81–3.55 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H). HRMS (EI)
+
m/z calcd C14
H
14
N
6
S (M ) 298.1001, found 298.1002.
1ꢀEthylꢀ3ꢀ(6ꢀ(pyrrolidinꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (3f). Yellow solid. 44% yield.
1
mp 194–196 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.57 (t, J = 5.0 Hz, 1H), 10.27 (s,
1
H), 8.29 (d, J = 38.6 Hz, 1H), 6.06 (s, 1H), 3.73–3.53 (m, 2H), 3.53–2.99 (m, 4H),
ACS Paragon Plus Environment

Page 23 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
1
17 5
.96 (d, J = 24.4 Hz, 3H), 1.24–1.06 (m, 3H). HRMS (EI) m/z calcd C11H N S (M )
2
51.1205, found 251.1204.
1ꢀEthylꢀ3ꢀ(6ꢀmorpholinopyrimidinꢀ4ꢀyl)thiourea (3g). Yellow solid. 54% yield. mp
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
168–170 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.46 (s, 1H), 10.32 (s, 1H), 8.31 (s,
1
H), 6.35 (s, 1H), 3.71–3.64 (m, 3H), 3.64–3.53 (m, 2H), 3.52–3.42 (m, 3H), 1.19 (t,
+
J = 7.2 Hz, 3H). HRMS (EI) m/z calcd C11
H
17
N
5
OS (M ) 267.1154, found 267.1155.
1
4
8
1
ꢀEthylꢀ3ꢀ(6ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (3h). Yellow solid.
1
1% yield. mp 169–172 °C; H NMR (400 MHz, CDCl ) δ 11.52 (s, 1H), 8.33 (s, 1H),
3
.27 (s, 1H), 5.79 (s, 1H), 3.82–3.71 (m, 2H), 3.71 (s, 4H), 2.55 (s, 3H), 2.40 (s, 3H),
+
.32 (t, J = 7.3 Hz, 3H). HRMS (EI) m/z calcd C12
H
20
N
6
S (M ) 280.1470, found
280.1473.
1
ꢀ(6ꢀ(Benzylamino)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (3i). Yellow solid. 51% yield.
1
mp 207–211 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.51 (t, J = 5.0 Hz, 1H), 10.33 (s,
1
3
H), 8.19 (s, 1H), 8.00 (s, 1H), 7.59–6.98 (m, 5H), 6.11 (s, 1H), 4.46 (s, 2H),
17 5
.70–3.40 (m, 2H), 1.16 (t, J = 7.1, 2.8 Hz, 3H). HRMS (EI) m/z calcd C14H N S
+
(M ) 287.1205, found 287.1206.
General procedure for the synthesis of target compounds 4aꢀe. To a solution of
corresponding intermediates 8aꢀe (1 mmol) in DMF (8 mL) was added NaH (60%
dispersion in mineral oil, 48 mg, 1.2 mmol). The solution was stirred for 5 min and
then ethyl isothiocyanate (87 mg, 1 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 × 50 mL). Organic layer was separated and dried over Na SO .
2
4
Removal of the solvents produced a residue which was purified using column
2 2
chromatography, eluted with a mixture of MeOH/CH Cl , to afford corresponding
target compounds 4aꢀe in good yield.
1ꢀ(6ꢀ(1HꢀImidazolꢀ1ꢀyl)ꢀ2ꢀmethylpyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (4a). White solid.
1
6
0% yield. mp 237–239 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.33 (s, 1H), 10.90 (s,
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
2
H), 8.41 (s, 1H), 7.73 (s, 1H), 7.18 (s, 1H), 7.13 (s, 1H), 3.75–3.51 (m, 2H), 2.59 (s,
+
H), 1.25 (t, J = 7.2 Hz, 3H). HRMS (EI) m/z calcd C H N S (M ) 262.1001, found
11
14
6
62.1002.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1ꢀ(6ꢀ(1HꢀImidazolꢀ1ꢀyl)ꢀ5ꢀmethylpyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (4b). White solid.
1
5
1
3
2
8% yield. mp 198–200 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.37 (s, 1H), 9.50 (s,
H), 8.71 (s, 1H), 8.14 (s, 1H), 7.63 (s, 1H), 7.14 (s, 1H), 3.79–3.48 (m, 2H), 2.26 (s,
+
H), 1.23 (t, J = 7.2 Hz, 3H). HRMS (EI) m/z calcd C H N S (M ) 262.1001, found
11
14
6
62.1002.
1
ꢀ(2ꢀ(1HꢀImidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (4c). White solid. 65%
1
yield. mp 242–245 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.10 (s, 1H), 10.45 (s, 1H),
8.55 (d, J = 5.8 Hz, 1H), 8.49 (s, 1H), 7.84 (s, 1H), 7.17 (s, 1H), 7.09 (d, J = 5.8 Hz,
+
1H), 3.77–3.50 (m, 2H), 1.28 (t, 3H). HRMS (EI) m/z calcd C H N S (M )
10
12
6
2
48.0844, found 248.0845.
1
ꢀ(4ꢀ(1HꢀImidazolꢀ1ꢀyl)pyrimidinꢀ2ꢀyl)ꢀ3ꢀethylthiourea (4d). White solid. 68%
1
yield. mp 244–246 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 10.90 (d, J = 71.8 Hz, 1H),
1
0.72 (s, 1H), 8.72 (d, J = 5.6 Hz, 1H), 8.66 (s, 1H), 8.01 (s, 1H), 7.55 (d, J = 5.7 Hz,
1
H), 7.20 (s, 1H), 3.81–3.54 (m, 2H), 1.22 (t, J = 19.2, 12.1 Hz, 3H). HRMS (EI) m/z
+
calcd C10
H
12
N
6
S (M ) 248.0844, found 248.0845.
1ꢀ(4ꢀ(1HꢀImidazolꢀ1ꢀyl)ꢀ6,7ꢀdihydroꢀ5Hꢀcyclopenta[d]pyrimidinꢀ2ꢀyl)ꢀ3ꢀethylthio
1
urea (4e). White solid. 63% yield. mp 285–286 °C. H NMR (400 MHz, DMSOꢀd
6
) δ
1
2
3
0.91 (s, 1H), 10.60 (s, 1H), 8.45 (s, 1H), 7.89 (s, 1H), 7.21 (s, 1H), 3.73–3.54 (m,
H), 3.22–3.09 (m, 2H), 3.05–2.88 (m, 2H), 2.22–2.06 (m, 2H), 1.24 (t, J = 7.2 Hz,
+
H). HRMS (EI) m/z calcd C13
procedure
H
16
N
6
S (M ) 288.1157, found 288.1158.
General
for
the
synthesis
of
1ꢀ(6ꢀ(1HꢀImidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthioureas 5aꢀj. To a solution of
corresponding intermediates 9aꢀj (1 mmol) in DMF (8 mL) was added NaH (60%
dispersion in mineral oil, 48 mg, 1.2 mmol). The solution was stirred for 5 min, then
ACS Paragon Plus Environment

Page 25 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ethyl isothiocyanate (87 mg, 1 mmol) was added. The reaction mixture was stirred at
room temperature for 0.5 h. Water was added and the mixture was extracted with
2 4
EtOAc (3 × 50 mL). Organic layer was separated and dried over Na SO . Removal of
the solvents produced a residue which was purified using column chromatography,
eluted with a mixture of MeOH/CH Cl , to afford corresponding target compounds
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
5
aꢀj in good yields.
1
ꢀEthylꢀ3ꢀ(6ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (5a). White
1
solid. 60% yield. mp 201–204 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.29–11.04 (m,
1
H), 10.99 (s, 1H), 8.80 (s, 1H), 7.60 (d, J = 1.5 Hz, 1H), 7.33 (s, 1H), 6.94 (t, J =
1
7.7 Hz, 1H), 3.77–3.55 (m, 2H), 2.58 (s, 3H), 1.26–1.17 (m, 4H). HRMS (EI) m/z
+
calcd C11
H
14
N
6
S (M ) 262.1001, found 262.1002.
1ꢀEthylꢀ3ꢀ(6ꢀ(2ꢀethylꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (5b). White solid.
1
6
8% yield. mp 166–168 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.18 (t, J = 5.1 Hz,
1
H), 11.01 (s, 1H), 8.80 (s, 1H), 7.56 (d, J = 1.3 Hz, 1H), 7.28 (s, 1H), 7.00 (d, J = 1.3
Hz, 1H), 3.73–3.50 (m, 2H), 2.96 (q, J = 7.4 Hz, 2H), 1.39–1.10 (m, 6H). HRMS (EI)
+
m/z calcd C12
H
16
N
6
S (M ) 276.1157, found 276.1158.
1
ꢀEthylꢀ3ꢀ(6ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (5c). White solid.
1
6
6
9% yield. mp 252–255 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.19 (s, 1H), 10.95 (s,
1H), 8.76 (d, J = 24.7 Hz, 1H), 8.34 (d, J = 0.7 Hz, 1H), 7.38 (d, J = 35.7 Hz, 1H),
7.23 (s, 1H), 3.79–3.54 (m, 2H), 2.19 (s, 3H), 1.22 (t, J = 7.2 Hz, 3H). HRMS (EI)
+
m/z calcd C11
H
14
N
6
S (M ) 262.1001, found 262.1001.
1
6
1
ꢀ(6ꢀ(4ꢀChloroꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (5d). White solid.
1
0% yield. mp 241ꢀ250 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.15 (s, 1H), 11.06 (s,
H), 8.75 (t, J = 17.8 Hz, 1H), 8.45 (d, J = 1.5 Hz, 1H), 7.89 (d, J = 1.5 Hz, 1H), 7.29
(d, J = 0.7 Hz, 1H), 3.81–3.50 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H). HRMS (EI) m/z calcd
+
C
10
H11ClN
6
S (M ) 282.0454, found 282.0459.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
6
1
ꢀ(6ꢀ(4ꢀBromoꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea (5e). White solid.
1
3% yield. mp 262–265 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.15 (s, 1H), 11.05 (s,
6
H), 8.78 (s, 1H), 8.45 (s, 1H), 7.93 (d, J = 1.0 Hz, 1H), 7.29 (s, 1H), 3.79–3.46 (m,
+
2H), 1.22 (t, J = 7.2 Hz, 3H). HRMS (EI) m/z calcd C10
found 325.9947.
H11BrN
6
S (M ) 325.9949,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6
1
2
3
ꢀEthylꢀ3ꢀ(6ꢀ(4ꢀiodoꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (5f). White solid.
1
8% yield. mp 268–270 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.15 (t, J = 5.0 Hz,
6
H), 11.02 (s, 1H), 8.77 (s, 1H), 8.39 (s, 1H), 7.94 (s, 1H), 7.27 (s, 1H), 3.72–3.56 (m,
+
H), 1.22 (t, J = 7.2 Hz, 3H). HRMS (EI) m/z calcd C10
H11IN
6
S (M ) 373.9811, found
73.9812.
1ꢀEthylꢀ3ꢀ(6ꢀ(4ꢀ(trifluoromethyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (5g).
1
White solid. 64% yield. mp 262–264 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.14 (t, J
6
=
5.1 Hz, 1H), 11.08 (s, 1H), 8.83 (s, 1H), 8.63 (s, 1H), 8.42 (s, 1H), 7.41 (s, 1H),
+
3
.75–3.51 (m, 2H), 1.37–1.13 (m, 3H). HRMS (EI) m/z calcd C11
16.0718, found 306.0721.
H F
11 3
N
6
S (M )
3
1
5
1
ꢀEthylꢀ3ꢀ(6ꢀ(4ꢀnitroꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (5h). White solid.
1
0% yield. mp 243–244 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.16 (s, 1H), 11.12 (s,
6
H), 8.86 (s, 2H), 8.62 (s, 1H), 7.47 (s, 1H), 3.78–3.50 (m, 2H), 1.23 (t, J = 7.1 Hz,
+
3H). HRMS (EI) m/z calcd C10
H
11
N
7
O
2
S (M ) 293.0695, found 293.0696.
1
ꢀEthylꢀ3ꢀ(6ꢀ(4ꢀformylꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (5i). Yellow
1
6
solid. 48% yield. mp 212–213 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.14 (t, J = 5.1
Hz, 1H), 11.10 (s, 1H), 9.88 (s, 1H), 8.83 (s, 1H), 8.62 (s, 2H), 7.43 (s, 1H), 3.72–3.57
+
(m, 2H), 1.23 (t, J = 7.2 Hz, 3H). HRMS (EI) m/z calcd C11
H
12
N
6
OS (M ) 276.0793,
found 276.0795.
Methyl 1ꢀ(6ꢀ(3ꢀethylthioureido)pyrimidinꢀ4ꢀyl)ꢀ1Hꢀimidazoleꢀ4ꢀcarboxylate (5j).
1
White solid. 53% yield. mp 240–243 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.15 (d,
J = 5.4 Hz, 1H), 11.04 (s, 1H), 8.82 (s, 1H), 8.54 (d, J = 1.2 Hz, 1H), 8.36 (d, J = 1.2
ACS Paragon Plus Environment

Page 27 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Hz, 1H), 7.40 (s, 1H), 3.82 (s, 3H), 3.72–3.55 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H).
+
HRMS (EI) m/z calcd C H N O S (M ) 306.0899, found 306.0900.
12
14
6
2
1ꢀEthylꢀ3ꢀ(6ꢀ(4ꢀ(hydroxymethyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea (5k).
To a solution of compound 5i (276mg, 1 mmol) in MeOH:CH Cl = 1:1 (5:5 mL) was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
4
added NaBH (45.4 mg, 1.2 mmol), and the mixture was stirred at room temperature
for 5 h, and the solvent was evaporated to dryness under reduced pressure. The
residue was purified on a silica gel column using mixtures of MeOH/CH Cl (1:20,
2
2
v/v) as eluent, obtaining the target compound 5k. White solid, 50% yield. mp
1
2
1
4
38–240 °C; H NMR (400 MHz, DMSOꢀd
6
) δ 11.19 (t, J = 5.0 Hz, 1H), 10.93 (s,
H), 8.75 (s, 1H), 8.39 (s, 1H), 7.52 (s, 1H), 7.27 (s, 1H), 5.13 (t, J = 5.7 Hz, 1H),
.43 (d, J = 5.6 Hz, 2H), 3.75–3.50 (m, 2H), 1.31–1.14 (m, 3H). HRMS (EI) m/z
+
calcd C11
H
14
N
6
OS (M ) 278.0950, found 287.0952.
General
procedure
for
the
synthesis
of
1
ꢀ(6ꢀ(4ꢀ(Aminomethyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthioureas 5lꢀx.
To a solution of compound 5i (138 mg, 0.5 mmol) in MeOH:DCE = 1:1 was added
corresponding alicyclic amines (1 mmol), AcOH (1 mmol) and NaBH (CN) (62.8 mg,
mmol) in sequence. The mixture was stirred at room temperature for 12 h, and
solvent was evaporated to dryness under reduced pressure. The residue was purified
3
1
2 2
on a silica gel column using mixtures of MeOH/CH Cl as eluent, obtaining the target
compounds 5lꢀx.
1
ꢀEthylꢀ3ꢀ(6ꢀ(4ꢀ((methylamino)methyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea
5l). Reagents used were compound 5i (138 mg, 0.5 mmol), methylamine (1 mmol, 2
M in THF), AcOH (60 mg, 1 mmol) and NaBH (CN) (62.8 mg, 1 mmol). Purification
involved the use of MeOH/CH Cl (1:5, v/v) as eluent. Compound 5l: yellow solid.
0% yield. mp 114–117 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.19 (s, 1H),
.85–8.69 (m, 1H), 8.59–8.36 (m, 1H), 7.66 (s, 1H), 7.33–7.22 (m, 1H), 3.77 (s, 2H),
(
3
2
2
1
4
8
6
3.70–3.57 (m, 3H), 2.40 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H). HRMS (EI) m/z calcd
+
C
12
H
17
N
7
S (M ) 291.1266, found 291.1267.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
ꢀEthylꢀ3ꢀ(6ꢀ(4ꢀ((ethylamino)methyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)thiourea
(
5m). Reagents used were compound 5i (138 mg, 0.5 mmol), ethylamine (1 mmol, 2
M in THF), AcOH (60 mg, 1 mmol) and NaBH (CN) (62.8 mg, 1 mmol). Purification
(1:10, v/v) as eluent. Compound 5m: yellow solid.
3
2 2
involved the use of MeOH/CH Cl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
55% yield. mp 105–108 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.20 (t, J = 5.0 Hz,
6
1
3
1
H), 8.77 (d, J = 15.5 Hz, 1H), 8.53–8.32 (m, 1H), 7.59 (s, 1H), 7.39–7.23 (m, 1H),
.71 (s, 2H), 3.68–3.59 (m, 3H), 2.70–2.58 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H),
+
.13–1.02 (m, 3H). HRMS (EI) m/z calcd C13
H
19
N
7
S (M ) 305.1423, found 305.1424.
1
ꢀ(6ꢀ(4ꢀ((Cyclohexylamino)methyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthio
urea (5n). Reagents used were compound 5i (138 mg, 0.5 mmol), cyclohexylamine
114 ꢁL, 1 mmol), AcOH (60 mg, 1 mmol) and NaBH (CN) (62.8 mg, 1 mmol).
(1:20, v/v) as eluent. Compound 5n:
) δ 11.19 (t, J
(
3
2 2
Purification involved the use of MeOH/CH Cl
1
white solid. 50% yield. mp 171–174 °C; H NMR (400 MHz, DMSOꢀd
6
=
5.0 Hz, 1H), 8.75 (s, 1H), 8.38 (d, J = 13.9 Hz, 1H), 7.44 (d, J = 108.6 Hz, 1H),
7
.28 (s, 1H), 3.70 (d, J = 20.8 Hz, 2H), 3.69–3.58 (m, 2H), 1.87 (d, J = 11.5 Hz, 2H),
1
.75–1.63 (m, 2H), 1.56 (d, J = 10.4 Hz, 1H), 1.30–0.98 (m, 9H). HRMS (ESI) m/z
+
calcd C H N S [M+H] 360.1970, found 360.1973.
17
26
7
1
ꢀ(6ꢀ(4ꢀ((Benzylamino)methyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea
5o). Reagents used were compound 5i (138 mg, 0.5 mmol), Benzylamine (109 ꢁL, 1
mmol), AcOH (60 mg, 1 mmol) and NaBH (CN) (62.8 mg, 1 mmol). Purification
(1:20, v/v) as eluent. Compound 5o: yellow solid.
(
3
2 2
involved the use of MeOH/CH Cl
1
5
8
1
7
6% yield. mp 182–185 °C; H NMR (400 MHz, CDCl ) δ 11.28 (s, 1H), 9.72 (s, 1H),
3
.63 (s, 1H), 8.42 (d, J = 20.8 Hz, 1H), 7.48 (s, 1H), 7.42–7.30 (m, 4H), 7.25 (d, J =
0.7 Hz, 2H), 6.82 (s, 1H), 3.89 (s, 2H), 3.84 (s, 2H), 3.81–3.73 (m, 2H), 1.36 (t, J =
+
.3 Hz, 3H). HRMS (ESI) m/z calcd C H N S [M+H] 368.1657, found 368.1659.
1
8
21
7
1
ꢀ(6ꢀ(4ꢀ((Dimethylamino)methyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiour
ea (5p). Reagents used were compound 5i (138 mg, 0.5 mmol), Dimethylamine (1
mmol, 2M in THF), AcOH (60 mg, 1 mmol) and NaBH (CN) (62.8 mg, 1 mmol).
3
ACS Paragon Plus Environment

Page 29 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2 2
Purification involved the use of MeOH/CH Cl (1:10, v/v) as eluent. Compound 5p:
1
white solid. 42% yield. mp 181–183 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.17 (t, J
6
=
5.2 Hz, 1H), 11.02 (s, 1H), 8.79 (s, 1H), 8.52 (s, 1H), 7.82 (s, 1H), 7.33 (s, 1H),
3.99 (s, 1H), 3.72–3.58 (m, 1H), 2.57 (s, 6H), 1.30–1.16 (m, 3H). HRMS (EI) m/z
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
calcd C H N S (M ) 305.1423, found 305.1425.
13
19
7
1
ꢀ(6ꢀ(4ꢀ((Diethylamino)methyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiourea
5q). Reagents used were compound 5i (138 mg, 0.5 mmol), Diethylamine (103 ꢁL, 1
mmol), AcOH (60 mg, 1 mmol) and NaBH (CN) (62.8 mg, 1 mmol). Purification
(1:10, v/v) as eluent. Compound 5q: white solid.
(
3
2 2
involved the use of MeOH/CH Cl
1
4
8% yield. mp 152–155 °C; H NMR (400 MHz, DMSOꢀd ) δ 11.17 (t, J = 5.3 Hz,
6
1
H), 10.92 (s, 1H), 8.76 (s, 1H), 8.41 (s, 1H), 7.63 (s, 1H), 7.31 (s, 1H), 3.72 (s, 2H),
3.68–3.54 (m, 2H), 2.63 (s, 4H), 1.24 (t, J = 7.1 Hz, 3H), 1.07 (t, J = 6.7 Hz, 6H).
+
HRMS (EI) m/z calcd C15
H
23
N
7
S (M ) 333.1736, found 333.1738.
1
ꢀ(6ꢀ(4ꢀ((Dipropylamino)methyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethylthiour
ea (5r). Reagents used were compound 5i (138 mg, 0.5 mmol), Dipropylamine (137
L, 1 mmol), AcOH (60 mg, 1 mmol) and NaBH (CN) (62.8 mg, 1 mmol).
Purification involved the use of MeOH/CH Cl (1:15, v/v) as eluent. Compound 5r:
white solid. 58% yield. mp 158–161 °C; H NMR (400 MHz, CDCl ) δ 11.32 (s, 1H),
ꢁ
3
2
2
1
3
9
.93 (s, 1H), 8.65 (s, 1H), 8.40 (s, 1H), 7.51 (s, 1H), 6.96 (s, 1H), 3.91–3.73 (m, 2H),
3.69 (s, 2H), 2.57–2.44 (m, 4H), 1.63–1.48 (m, 4H), 1.38 (t, J = 7.3 Hz, 3H), 0.90 (t, J
+
= 7.3 Hz, 6H). HRMS (EI) m/z calcd C17
H
27
N
7
S (M ) 361.2049, found 361.2050.
1
ꢀ(6ꢀ(4ꢀ((Benzyl(methyl)amino)methyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)pyrimidinꢀ4ꢀyl)ꢀ3ꢀethyl
thiourea (5s). Reagents used were compound 5i (138 mg, 0.5 mmol),
NꢀMethylbenzylamine (129 ꢁL, 1 mmol), AcOH (60 mg, 1 mmol) and NaBH (CN)
(1:15, v/v) as
3
(
2 2
62.8 mg, 1 mmol). Purification involved the use of MeOH/CH Cl
1
eluent. Compound 5s: white solid. 50% yield. mp 153–155 °C; H NMR (400 MHz,
DMSOꢀd ) δ 11.19 (s, 1H), 10.92 (s, 1H), 8.76 (s, 1H), 8.40 (s, 1H), 7.58 (s, 1H),
6
7.45–7.29 (m, 3H), 7.26 (d, J = 6.4 Hz, 1H), 3.64 (m, J = 13.8, 7.0 Hz, 2H), 3.56 (s,
ACS Paragon Plus Environment