Design, synthesis, and biological evaluation of compounds with a new scaffold as anti-neuroinflammatory agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2018.02.063

Twenty-eight compounds with a new scaffold were designed and synthesized by assembling fragments derived from known agents such as stilbenes and piperazinyl pyrimidines. Many strategies have been explored to improve the druggability of these series of compounds, such as increasing the distance between two benzene rings in the scaffold and introducing functional groups at designated positions. These compounds were validated for their anti-neuroinflammatory activity in BV2 cells. Experimental results reveal that the most active compound 8b can inhibit nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) production with IC₅₀ values of 1.0, 2.6, and 0.5 μM, respectively. The compound can also significantly modulate the MAPK pathways through inhibiting the phosphorylation of JNK, ERK1/2, and p38 MAPK without disturbing NF-κB pathway. Parallel artificial membrane permeation assay demonstrated that the most active compound can overcome the blood-brain barrier (BBB). Therefore, this compound can be a promising lead for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2018.02.063

European Journal of Medicinal Chemistry 149 (2018) 129e138
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis, and biological evaluation of compounds with a new
scaffold as anti-neuroinflammatory agents for the treatment of
Alzheimer's disease
Yuying Fang 1, Wenjuan Xia 1, Bao Cheng, Pei Hua, Huihao Zhou, Qiong Gu**, Jun Xu*
Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-eight compounds with a new scaffold were designed and synthesized by assembling fragments
derived from known agents such as stilbenes and piperazinyl pyrimidines. Many strategies have been
explored to improve the druggability of these series of compounds, such as increasing the distance
between two benzene rings in the scaffold and introducing functional groups at designated positions.
These compounds were validated for their anti-neuroinflammatory activity in BV2 cells. Experimental
Received 14 December 2017
Received in revised form
18 February 2018
Accepted 20 February 2018
results reveal that the most active compound 8b can inhibit nitric oxide (NO), tumor necrosis factor-
(TNF- ), and interleukin-1 (IL-1 ) production with IC50 values of 1.0, 2.6, and 0.5 M, respectively. The
compound can also significantly modulate the MAPK pathways through inhibiting the phosphorylation
of JNK, ERK1/2, and p38 MAPK without disturbing NF- B pathway. Parallel artificial membrane perme-
ation assay demonstrated that the most active compound can overcome the blood-brain barrier (BBB).
Therefore, this compound can be a promising lead for the treatment of Alzheimer's disease.
© 2018 Elsevier Masson SAS. All rights reserved.
a
a
b
b
m
Keywords:
Fragment-based drug design
Anti-neuroinflammation
Alzheimer's disease
k
1. Introduction
detrimental cycle of neuroinflammation [9e11]. Therapeutic agents
modifying AD through the intervention of neuroinflammation is a
Alzheimer's disease (AD), a chronic neurodegenerative disorder,
is characterized by memory loss and progressive cognitive
impairment [1]. Studies on AD pathogenesis have been conducted
feasible strategy [12e15].
Recently, the piperazinyl pyrimidine derivatives were reported
as anti-neuroinflammatory agents (Fig. 1) [16e19]. Compound
for decades. It is hypothesized that acetylcholine, Amyloid
b, tau
GIBH-130 (Fig.1) can selectively suppress the IL-1b production with
protein and calcium homeostasis play major roles in the disease
progress [2]. Increasing evidence indicates that inflammation may
the IC50 value of 3.4 nM in LPS-activated microglia. GIBH-130 has
been approved by China Food and Drug Administration for clinical
trials against AD [17].
have
a causal role in AD [3]. Microglia-dominated neuro-
inflammation is a characteristic feature of AD [4]. Chronic and
sustained microglia activation can result in the increased inflam-
matory mediators including nitric oxide (NO), tumor necrosis factor
Meanwhile, resveratrol has been reported with a wide range of
biological effects related to AD including anti-inflammation [20],
anti-oxidation [21], and anti-amyloid
b (Ab) aggregation [22].
a
(TNF-
a
), and interleukin 1
b
(IL-1
b
) [5,6]. These mediators may
However, resveratrol's bioavailability is low. Therefore, we
designed and synthesized twenty-eight stilbene mimics combining
fragments derived from known inhibitors and resveratrol (Fig. 2).
The preliminary structure and activity relationship (SAR) against
neuroinflammatory has been established, and the mechanism of
action was further investigated.
directly induce neuronal apoptosis or amplify the local inflamma-
tory response [7]. The inflammatory environment might promote
the formation of senile plaques and neurofibrillary tangles, leading
to neuronal damage [8]. While neuronal damage can induce
microglial activation, which facilitates the propagation of
a
2. Chemistry
* Corresponding author.
** Corresponding author.
As shown in Scheme 1, compounds of series 4, 5, 6, and 8 were
synthesized. The intermediate acids were synthesized through a
E-mail address: guqiong@mail.sysu.edu.cn (J. Xu).
1
These authors contribute equally.
https://doi.org/10.1016/j.ejmech.2018.02.063
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

130
Y. Fang et al. / European Journal of Medicinal Chemistry 149 (2018) 129e138
ring and B-ring were not substituted (Scheme 1), such as com-
pound 4a, the inhibition of NO production was decreased
M). Based on this
(IC50 ¼ 17.8
m
M) comparing with Res (IC50 ¼ 11.1
m
SAR observation, we synthesized more A-ring and B-ring
substituted compounds. Compounds 4b and 4c, B-ring substituted
with OCH3 at C-4 or C-2, exhibited improved NO inhibitory activ-
ities (IC50 ¼ 9.5
substituted with Cl at C-2 or C-3, also exhibited improved NO
inhibitory activities (IC50 ¼ 12.5 M and 10.1 M). However, when
B-ring was substituted with Cl at C-4 (4d), the inhibitory activity
significantly reduced (IC50 > 50 M). If B-ring at C-4 was substituted
with CF3 (4g), the activity remained unchanged (IC50 ¼ 14.9 M)
comparing with 4a. If both C-2 and C-5 were substituted at B-ring
M).
mM and 7.9 mM). Compounds 4e and 4f, B-ring
m
m
m
m
Fig. 1. Structures of reported anti-inflammatory agents.
(4h), the activity would be significantly reduced (IC50 ¼ 48.1
m
Compounds 5a~5i are featured a 3-CF3 group at A-ring, and the
substituents at B-ring vary. Compounds 5g and 5h, B-ring
substituted with F or CF3 at C-4, showed improved NO inhibitory
activities (IC50 ¼ 15.6
mM and 6.5 mM) comparing with 4a. Com-
pound 5h significantly increased the inhibition of NO release. This
can be due to the stronger electron-withdrawing inductive effects
of the 4-CF3 group at B-ring. Compounds 5a~5f and 5i demon-
strated reduced NO inhibitory activities comparing with 4a.
Compound 6h, with 4-F at A-ring and 4-CF3 at B-ring, also
Fig. 2. Design strategy for the stilbene mimics.
demonstrated
(IC50 ¼ 3.0 M) comparing with 4a. While compounds 6b and 6d, B-
ring substituted with 4-OCH3 or 4-Cl, showed reduced activities
M and 25.1 M) comparing with 4a. This indicates that
a stronger electron-withdrawing group can improve the inhibitory
activity. Compounds 6c and 6f, OCH3 or Cl switched from C-4 to C-2
significantly
increased
inhibitory
activity
m
Perkin reaction [23] with commercially available phenylacetic acids
and benzaldehydes or cinnamaldehydes. The target compounds
were prepared via the condensation of the secondary aliphatic
amine 2-(piperazin-1-yl) pyrimidine and the intermediate acids
using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDC$HCl) and triethylamine mixture as solvent. Twenty-
six diphenylethene derivatives and two diphenylbutene derivatives
were synthesized. The yields ranged from 45% to 81%.
(IC50 ¼ 23.1
m
m
in B-ring, both showed increased activities (IC50 ¼ 9.8
7.3 M). Compound 6a, exhibits significantly higher activity than
compound 6g, which has 4-F at B-ring (IC50 > 50 M).
mM and
m
m
In summary, the compounds with 2,5-substitutes at B-ring (2-
OH, 5-OCH3) were unable to demonstrate inhibitory activity
(IC50 > 40 mM).
In order to identify more active compounds, based on the
scaffold of GIBH-130 (Fig. 1), we designed and synthesized new
compounds (8a and 8b) by increasing the distance between A-ring
and B-ring. Compounds 8a and 8b, A-ring substituted with 4-F or 3-
3. Results and discussion
3.1. Inhibition of NO, IL-1
b, and TNF-a production in LPS-induced
BV2 cell lines and SAR study
CF3, did show significantly increased activities (IC50 ¼ 7.9
1.0 M), which are better than resveratrol.
These compounds were further validated for their anti-
neuroinflammatory efficacy with TNF- and IL-1 assay experi-
ments [19,20]. The results are listed in Table 1. Compound 8b was
the most potent compound against NO, TNF- , and IL-1 produc-
tion with the IC50 values of 1.0 M, 2.6 M, and 0.5 M, respectively,
mM and
m
The twenty-eight target compounds were evaluated for their
anti-neuroinflammatory activity. Their effects on NO production
was tested in LPS-induced BV2 microglia cell lines. Resveratrol
(Res) was used as the positive control. The IC50 values for each
tested compound were calculated and listed in Table 1. The pre-
liminary structure and activity relationship (SAR) was analyzed.
Compounds 4b, 4c, 4f, 5h, 6a, 6c, 6e, 6f, 6h, 8a, and 8b showed
higher inhibition of NO generation in BV2 cells (Table 1). When A-
a
b
a
b
m
m
m
and was further investigated for anti-neuroinflammatory mecha-
nism of action.
Scheme 1. Synthesis of stilbene mimics 4a-4h, 5a-5i, 6a-6i, and 8a-8b. Regents and conditions: (i) Et3N, Ac2O, 90 ꢀC, overnight; (ii) 2-(piperazin-1-yl)pyrimidine, Et3N, EDC$HCl,
CH2Cl2, r.t, overnight.

Y. Fang et al. / European Journal of Medicinal Chemistry 149 (2018) 129e138
131
Table 1
Assay results of the compounds validated for NO, IL-1
b
and TNF-
a
production, and permeability (Pe ꢁ 10ꢂ6 cm sꢂ1) of the compounds.
d
ID
R1/R
R2
NO
IC50
IL-1
IC50
b
TNF-
IC50
a
Pe ( ꢁ 10ꢂ6 cm sꢂ1
)
(m
M)a
(
m
M)b
(m
M)c
4a
4b
4c
4d
4e
4f
R1 ¼ H
H
17.8 2.5
9.5 0.3
7.9 1.4
>50
12.6 4.1
10.1 2.6
14.9 0.9
48.1 0.5
>50
35.4 1.2
16.9 0.1
21.3 0.5
>50
16.2 2.4
17.6 2.2
21.6 0.4
36.4 1.2
14.3 0.2
10.3 0.3
12.3 0.1
7.4 0.2
11.2 0.2
9.4 0.2
8.2 0.4
14.1 0.3
4-OCH3
2-OCH3
4-Cl
3-Cl
2-Cl
3.4 0.6
31.6 0.8
>50
7.8 2.2
9.3 1.7
11.3 1.6
11.4 1.3
4g
4h
4-CF3
2-OH, 5-OCH3
5a
5b
5c
5d
5e
5f
5g
5h
5i
R1 ¼ 3-CF3
H
39.8 1.5
22.4 2.7
31.7 3.1
26.9 1.7
37.2 0.8
19.9 1.8
15.6 2.3
6.5 1.4
25.3 2.3
15.2 1.8
41.2 0.5
21.9 0.2
15.2 1.3
18.4 0.5
31.2 0.4
4.2 1.0
>50
>50
7.2 0.3
14.7 0.6
9.7 0.3
5.1 0.4
10.7 0.4
15.1 0.2
12.3 0.7
9.3 0.2
12.7 0.3
4-OCH3
2-OCH3
4-Cl
3-Cl
2-Cl
26.5 2.6
34.7 1.3
43.1 0.5
26.7 0.4
25.1 2.2
19.8 0.7
6.9 1.2
>50
4-F
4-CF3
2-OH, 5-OCH3
42.6 2.7
6a
6b
6c
6d
6e
6f
6g
6h
6i
R1 ¼ 4-F
H
8.1 2.0
23.1 0.5
9.9 2.1
25.1 1.6
11.0 1.5
7.3 2.4
>50
10.2 1.7
42.9 0.8
18.4 2.3
36.4 2.1
11.9 3.4
5.2 0.6
25.7 1.2
8.9 1.1
>50
15.2 0.4
41.3 0.2
23.7 1.0
17.8 0.3
9.4 0.6
10.9 1.7
37.5 0.7
6.6 2.5
>50
11.2 0.5
10.5 0.2
13.8 0.1
7.8 0.3
9.4 0.3
11.3 0.7
14.3 0.7
7.6 0.5
6.4 0.2
4-OCH3
2-OCH3
4-Cl
3-Cl
2-Cl
4-F
4-CF3
2-OH, 5-OCH3
3.0 0.7
46.8 0.4
8a
8b
Res
R ¼ 4-F
R ¼ 3-CF3
e
e
e
e
7.9 2.1
1.0 0.8
11.1 1.3
2.4 0.9
0.5 1.2
8.7 2.9
5.9 1.2
2.6 0.3
9.5 1.1
14.9 0.2
12.6 0.3
1.1 0.05
a
Concentration (
Concentration (
Concentration (
m
m
m
M) for 50% inhibition of NO release in BV2 cells. The IC50 values are the mean SEM for at least three experimental determinations.
M) for 50% inhibition of IL-1 release in BV2 cells. The IC50 values are the mean SEM for at least three experimental determinations.
M) for 50% inhibition of TNF- release in BV2 cells. The IC50 values are the mean SEM for at least three experimental determinations.
b
c
b
a
d
Compounds were dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). Values are expressed as the means SD at least three independent experiments,
compounds with permeabilities Pe > 4.7 ꢁ 10ꢂ6 cm sꢂ1 could cross the BBB by passive diffusion.
3.2. Compound 8b inhibited the LPS-induced iNOS production
LPS-induced IkBa phosphorylation. Fig. 4C, 4D, and 4E indicate the
compound 8b does not inhibit p65 translocation from cytoplasma
to the nucleus. This concludes that compound 8b didn't disturb the
NF-kB pathway in LPS-induced BV2 cells.
Inducible nitric oxide synthase (iNOS) is over expressed in
activated BV2 microglia cells. The BV2 cells were harvested after
16 h incubation with or without LPS (1 mg/mL) and various con-
centrations of 8b. As shown in Fig. 3, the iNOS could not be detected
in BV2 cells without LPS. With adding LPS, the iNOS was signifi-
cantly expressed. However, when BV2 cells were incubated with
8b, LPS-induced iNOS expression was suppressed in a dose-
dependent manner.
3.4. Compound 8b suppressed LPS-induced MAPK activation
Mitogen-activated protein kinases (MAPKs) modulate inflam-
matory activation in microglia cells. Proteins in MAPK family, such
as JNK, ERK1/2, and p38, are regarded as the targets of anti-
inflammatory agents [18,24,25]. To investigate the effects of com-
pound 8b against MAPKs activation in LPS-stimulated cells, the BV2
cells were pretreated with compound 8b for 30 min, then incu-
3.3. Compound 8b didn't disturb NF-kB pathway
Nuclear factor
k
B (NF-
k
B) is a key transcription factor that reg-
bated with LPS (1 mg/mL) for 16 h. Western blot analysis exhibited
ulates the expression of inflammatory cytokines in the activated
compound 8b significantly suppressed LPS-induced phosphoryla-
tion of JNK, ERK1/2, and p38 in a concentration dependent manner
(Fig. 5). These results suggest that 8b inhibits neuroinflammatory
activity through modulating MAPK signal transduction pathway in
LPS-stimulated BV2 cells.
microglial cells. When BV2 cells are exposed to LPS, the NF-
pathway is activated. When the NF- B pathway is activated, I
k
B
B
a
k
k
protein is phosphorylated and p65 translocates from cytoplasm to
the nucleus. Fig. 4A and B indicate the compound 8b does not cause

132
Y. Fang et al. / European Journal of Medicinal Chemistry 149 (2018) 129e138
Fig. 3. Compound 8b suppressed LPS-induced iNOS expression in BV2 cells. BV2 cells were incubated with 8b for 30 min, then incubated with LPS (1 mg/mL) for 16 h. GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) was used as reference. The data are presented as mean SEM of three independent experiments. ***p < 0.001 relative to the negative
control.
Fig. 4. Assay result for 8b on LPS-induced NF-
kB activation. BV2 cells were incubated with 8b for 30 min, then incubated with LPS (1 mg/mL) for 16 h. The data are presented as
mean SEM of three independent experiments. no significance, **p < 0.01, ***p < 0.001 relative to the negative control.
3.5. The compounds could penetrate BBB
decrease compounds' ability to penetrate BBB. Since the sub-
stituents of most compounds are lipophilic, such as CF3, Cl and
OCH3, the aqueous solubility of these compounds is low. Among the
compounds, compounds 5d-5f and 5h with substituents at both A
and B ring (3-CF3 at A-ring and Cl or CF3 at B-ring) have even lower
solubility. In addition, the calculations indicate that all compounds
have good intestinal absorption after oral administration and can
easily be bound to the plasma proteins. Compounds of series 4, 5
and 8 don't show liver toxicity, but nearly all compounds of series 6
(4-F at A-ring) have liver toxicity. While compounds of series 4 and
8 show no inhibition of CYP2D6, some compounds of series 5 and 6
inhibit CYP2D6. Compounds 5d, 5e and 5f with 3-CF3 at A-ring
inhibit CYP2D6 no matter what position the Cl at B-ring is. And
compound 5g with 4-F at B-ring can also inhibit CYP2D6. Com-
pounds 6f and 6h, B-ring substituted with 2-Cl or 4-CF3, inhibit
CYP2D6 as well. The most active compound 8b has improved BBB
penetration, but its solubility is low. The compound can be further
optimized for druggability.
Parallel artificial membrane permeation assay (PAMPA) experi-
ments [26] were conducted to measure the capacity of penetrating
blood-brain barrier (BBB) for our compounds. This method was
used to predict compounds' passive diffusion through BBB with
high throughput and reproducibility. As shown in Table 1, our
compounds can penetrate BBB. Comparing with resveratrol, the
positive control [27], 8b (the most potent compound) can better
penetrate CNS.
3.6. Preliminary ADMET study
ADMET properties were predicted for all the compounds by
means of Discovery Studio 2.5 software package. As shown in
Table 2, most of the synthesized compounds can penetrate BBB
with high possibility, but compounds 4h, 5i and 6i with 2-OH at B-
ring can penetrate BBB with medium possibility, which could be
due to the substituent. The hydrophilic hydroxyl group (OH) may

Y. Fang et al. / European Journal of Medicinal Chemistry 149 (2018) 129e138
133
Fig. 5. Effects of 8b on LPS-induced phosphorylation of MAPKs. BV2 cells were pretreated with 8b for 30 min and then with LPS (1
m
g/mL). The whole-cell lysate was analyzed by
Western blot for both phosphorylated and nonphosphorylated JNK, ERK1/2, and p38 MAPK. The data are presented as mean SEM of three independent experiments. *p < 0.05,
**p < 0.01, ***p < 0.001, no significance relative to the negative control.
Table 2
ADMET properties for all compounds and resveratrol.
ID
R1/R
R2
BBBa
Absorptionb
Solubilityc
Hepatotoxicityd
CYP2D6e
PPBf
4a
4b
4c
4d
4e
4f
R1 ¼ H
H
1
1
1
1
1
1
1
2
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
4-OCH3
2-OCH3
4-Cl
3-Cl
2-Cl
4g
4h
4-CF3
2-OH, 5-OCH3
5a
5b
5c
5d
5e
5f
5g
5h
5i
R1 ¼ 3-CF3
H
1
1
1
0
0
0
1
0
2
0
0
0
0
0
0
0
0
0
2
2
2
1
1
1
2
1
2
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
0
0
1
1
1
1
1
1
1
1
1
4-OCH3
2-OCH3
4-Cl
3-Cl
2-Cl
4-F
4-CF3
2-OH, 5-OCH3
6a
6b
6c
6d
6e
6f
6g
6h
6i
R1 ¼ 4-F
H
1
1
1
1
1
1
1
1
2
0
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
2
0
1
1
1
1
1
1
0
1
0
0
0
0
0
1
0
1
0
1
1
1
1
1
1
1
1
1
4-OCH3
2-OCH3
4-Cl
3-Cl
2-Cl
4-F
4-CF3
2-OH, 5-OCH3
8a
8b
Res
R ¼ 4-F
R ¼ 3-CF3
e
e
e
e
1
1
2
0
0
0
2
2
3
0
0
0
0
0
0
1
1
0
a
BBB is blood brain barrier penetration and predicts blood-brain penetration after oral administration. 0 (very high), 1 (high), 2 (medium).
Absorption predicts human intestinal absorption (HIA) after oral administration. 0 (good).
b
c
d
e
f
Solubility predicts the solubility of each compound in water at 25 ꢀC. 1 (very low), 2 (low), 3 (good).
Hepatotoxicity predicts potential liver toxicity of compounds. 0 (nontoxic), 1 (toxic).
CYP2D6 predicts CYP2D6 enzyme inhibition using 2D chemical structure. 0 (non-inhibitor), 1 (inhibitor).
PPB predicts whether a compound is likely to be highly bound to carrier proteins in the blood. 0 (binding ability < 90%), 1 (binding ability > 90%).
4. Conclusion
(piperazinyl pyrimidine). In order to improve the activity, the dis-
tance between two benzene rings needs to be increased. The
druggability of this new series can be improved by introducing
functional groups at designated positions. These compounds are
capable of penetrating BBB, therefore, they are promising leads for
Our work has demonstrated that novel anti-neuroinflammatory
agents can be discovered by assembling fragments derived from
known agents such as resveratrol (stilbene) and GIBH-130

134
Y. Fang et al. / European Journal of Medicinal Chemistry 149 (2018) 129e138
further development as a new anti-AD therapeutic solution.
5. Experimental section
NMR (100 MHz, CDCl3):
d
169.7, 160.5, 158.3, 156.7 (2 ꢁ C), 134.9,
134.2, 129.8 (2 ꢁ C), 129.5, 127.8 (4 ꢁ C), 126.9, 126.6, 112.6 (2 ꢁ C),
109.3, 54.1, 42.5 (4 ꢁ C). ESI-HRMS [MþH]þ m/z ¼ 401.1965, calcd
for C24H24N4O2, 401.1972.
5.1. Synthesis
5.3.3. Synthesis of (E)-3-(2-methoxyphenyl)-2-phenyl-1-(4-
(pyrimidin-2-yl)piperaz-in-1-yl)prop-2-en-1-one (4c)
Materials and equipment. Reagents were used without further
purification unless specified. Solvents were dried and redistilled
prior to use with usual methods. The 1H NMR and 13C NMR spectra
were recorded using tetramethylsilane (TMS) as the internal stan-
Following the general method above, 4c was obtained as a white
solid (63%). 1H NMR (400 MHz, CDCl3):
7.62 (1H, s), 7.56e7.47 (2H, m), 7.38 (1H, t, J ¼ 7.8 Hz), 7.26e7.20
(1H, m), 7.04 (1H, s), 6.89 (2H, m), 6.70 (1H, t, J ¼ 7.5 Hz), 6.57e6.50
(1H, m), 3.82 (3H, s), 3.82e3.52 (8H, m). 13C NMR (100 MHz, CDCl3):
d
8.33 (2H, d, J ¼ 4.7 Hz),
dard on
a
BrukerBioSpin GmbH spectrometer (AvanceIII,
Switzerland) at 400 MHz and 100 MHz. Coupling constants are
given in Hz. High-resolution mass spectra were obtained using a
Shimadzu LCMS-IT-TOF mass spectrometer. Flash column chro-
matography was performed using silica gel (200e300 mesh) pur-
chased from Qingdao Haiyang Chemical Co. Ltd. All reaction
progress was monitored by thin layer chromatography (TLC) on
precoated silica gel GF254 (Qingdao Haiyang Chemical Co. Ltd) and
the spots were detected under UV light (254 nm).
d
170.0, 161.5, 157.7 (2 ꢁ C), 157.6, 136.5, 134.9, 132.1, 130.0, 129.7,
128.9, 128.2, 125.5, 124.5, 124.4, 123.5, 120.2, 110.6, 110.4, 55.4, 43.6
(4 ꢁ C). ESI-HRMS [MþH]þ m/z ¼ 401.1965, calcd for C24H24N4O2,
401.1972.
5.3.4. Synthesis of (E)-3-(4-chlorophenyl)-2-phenyl-1-(4-
(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one (4d)
5.2. General procedure for the synthesis of (E)-2,3-diphenylacrylic
acids and its derivatives (1a~1h, 2a~2i, 3a~3i, 7a, and 7b)
Following the general method above, 4d was obtained as a light
yellow solid (77%). 1H NMR (400 MHz, CDCl3):
d 8.30 (2H, d,
J ¼ 4.7 Hz), 7.39e7.28 (5H, m), 7.19e7.13 (2H, m), 7.06 (2H, d,
According to previously reported methods [28,29], benzalde-
hydes or cinnamaldehyde (20 mmol, 1 eq.) and 2-phenylacetic acid
(20 mmol, 1 eq.) were dissolved in acetic anhydride (20 mL), and
then triethylamine (20 mmol, 1 eq.) was added slowly. The mixture
solution was stirred at 90 ꢀC overnight. Water (2 mL) was added
and the reaction was allowed to continue for 15 min. After, K2CO3
(26 g) in water (150 mL) was added slowly and the mixture was
stirred at 60 ꢀC for 1 h. The reaction mixture was cooled to 4 ꢀC and
acidified with 12N HCl. The aqueous solution was extracted with
CH2Cl2 (3 ꢁ 100 mL), and the obtained organic phases were com-
bined and washed with brine, dried, and then evaporated. Pure
compounds were obtained by further crystallization from EtOAc.
J ¼ 8.5 Hz), 6.74 (1H, s), 6.52 (1H, t, J ¼ 4.7 Hz), 3.83e3.46 (8H, m).
13C NMR (100 MHz, CDCl3):
d
170.1, 161.5, 157.7 (2 ꢁ C), 138.1, 135.1,
133.6, 130.7 (2 ꢁ C), 129.3, 128.9 (2 ꢁ C), 128.7 (2 ꢁ C), 128.4 (4 ꢁ C),
110.4, 43.6 (4 ꢁ C). ESI-HRMS [MþH]þ m/z ¼ 405.1475, calcd for
C23H21N4OCl, 405.1477.
5.3.5. Synthesis of (E)-3-(3-chlorophenyl)-2-phenyl-1-(4-
(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one (4e)
Following the general method above, 4e was obtained as a white
solid (68%). 1H NMR (400 MHz, CDCl3):
d
8.30 (2H, d, J ¼ 4.7 Hz),
7.40e7.30 (5H, m), 7.21e7.16 (1H, m), 7.16e7.13 (1H, m), 7.10 (1H, d,
J ¼ 7.9 Hz), 7.00 (1H, d, J ¼ 7.7 Hz), 6.73 (1H, s), 6.53 (1H, t,
J ¼ 4.7 Hz), 3.91e3.54 (8H, m). 13C NMR (100 MHz, CDCl3):
d 169.9,
5.3. General method for the synthesis of (E)-2,3-diphenyl-1-(4-
(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one and its derivatives
(4a~4h, 5a~5i, 6a~6i) and (2E,4E)-2,5-diphenyl-1-(4-(pyrimidin-2-
yl)piperazin-1-yl)penta-2,4-dien-1-one and derivatives (8a, 8b)
161.5, 157.7 (2 ꢁ C), 138.8, 137.0, 134.9, 134.0, 129.3 (2 ꢁ C), 129.0,
128.9 (2 ꢁ C), 128.6 (2 ꢁ C), 128.5, 127.9, 127.4, 110.4, 43.7 (4 ꢁ C).
ESI-HRMS [MþH]þ m/z ¼ 405.1475, calcd for C23H21N4OCl,
405.1477.
The intermediates (1 eq.) were reacted with 2-(piperazin-1-yl)
pyrimidine (1.5 eq.) in the presence of EDC$HCl (1 eq.) and Et3N (1
eq.) using CH2Cl2 (20 mL) as a solvent. The reaction mixture was
stirred under room temperature overnight. The solvent was
removed under reduced pressure, and the residue was purified by
flash chromatography (EtOAc/dichloromethane ¼ 1/10) to give
pure product.
5.3.6. Synthesis of (E)-3-(2-chlorophenyl)-2-phenyl-1-(4-
(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one (4f)
Following the general method above, 4f was obtained as a light
yellow solid (64%). 1H NMR (400 MHz, CDCl3):
d 8.31 (2H, d,
J ¼ 4.7 Hz), 7.41 (1H, d, J ¼ 7.9 Hz), 7.31e7.28 (2H, m), 7.27e7.21 (3H,
m), 7.20e7.14 (1H, m), 7.00e6.97 (2H, m), 6.91 (1H, s), 6.55e6.49
(1H, t, J ¼ 4.7 Hz), 3.89e3.57 (8H, m). 13C NMR (100 MHz, CDCl3):
5.3.1. Synthesis of (E)-2,3-diphenyl-1-(4-(pyrimidin-2-yl)
piperazin-1-yl)prop-2-en-1-one (4a)
d
169.7, 161.5, 157.7 (2 ꢁ C), 138.8, 134.4, 134.2, 134.1, 130.9, 129.2,
128.9, 128.7 (2 ꢁ C), 128.6 (2 ꢁ C), 128.2, 127.3, 126.4, 110.4, 46.7,
Following the general method above, 4a was obtained as a white
43.9, 43.6, 41.8. ESI-HRMS [MþH]þ m/z ¼ 405.1489, calcd for
solid (81%). 1H NMR (400 MHz, CDCl3):
d
8.29 (2H, t, J ¼ 4.7 Hz),
C
23H21N4OCl, 405.1477.
7.36e7.26 (5H, m), 7.20e7.12 (5H, m), 6.78 (1H, s), 6.51 (1H, t,
J ¼ 4.7 Hz), 3.69 (8H, m). 13C NMR (100 MHz, CDCl3):
d 170.5, 161.7,
157.8 (2 ꢁ C), 137.4, 135.6, 135.3, 130.7, 129.5 (2 ꢁ C), 128.9 (4 ꢁ C),
128.3 (2 ꢁ C), 128.3, 128.0, 110.5, 43.7 (4 ꢁ C). ESI-HRMS [MþH]þ m/
z ¼ 371.1859, calcd for C23H22N4O, 371.1866.
5.3.7. Synthesis of (E)-2-phenyl-1-(4-(pyrimidin-2-yl)piperazin-1-
yl)-3-(4-(trifluoro-methyl)phenyl)prop-2-en-1-one (4g)
Following the general method above, 4g was obtained as a white
solid (74%). 1H NMR (400 MHz, CDCl3):
d
8.31 (2H, d, J ¼ 4.7 Hz), 7.46
5.3.2. Synthesis of (E)-3-(4-methoxyphenyl)-2-phenyl-1-(4-
(pyrimidin-2-yl)piperaz-in-1-yl)prop-2-en-1-one (4b)
(2H, d, J ¼ 8.3 Hz), 7.33 (5H, s), 7.25 (2H, d, J ¼ 8.2 Hz), 6.81 (1H, s),
6.54 (1H, t, J ¼ 4.7 Hz), 3.89e3.49 (8H, m). 13C NMR (100 MHz,
Following the general method above, 4b was obtained as a white
CDCl3):
d
169.7, 161.4, 157.7 (2 ꢁ C), 139.7, 138.8, 134.8, 129.6 (2 ꢁ C),
solid (71%). 1H NMR (400 MHz, CDCl3):
d
8.19 (2H, d, J ¼ 4.7 Hz),
129.0 (2 ꢁ C), 128.8, 128.6 (3 ꢁ C), 125.1 (3 ꢁ C), 125.0, 110.5, 43.6,
7.32e7.26 (2H, m), 7.25e7.17 (3H, m), 6.99 (2H, d, J ¼ 8.7 Hz), 6.63
43.5, 43.5, 43.4. ESI-HRMS [MþH]þ m/z ¼ 439.1744, calcd for
(3H, m), 6.41 (1H, t, J ¼ 4.7 Hz), 3.68 (3H, s), 3.66e3.17 (8H, m). 13
C
C24H21N4OF3, 439.1740.

Y. Fang et al. / European Journal of Medicinal Chemistry 149 (2018) 129e138
135
5.3.8. Synthesis of (E)-3-(2-hydroxy-5-methoxyphenyl)-2-phenyl-
1-(4-(pyrimidin-2-yl)piperazin-1-yl) prop-2-en-1-one (4h)
Following the general method above, 4h was obtained as a light
J ¼ 8.0 Hz), 7.16e7.10 (2H, m), 6.95 (1H, d, J ¼ 7.7 Hz), 6.80 (1H, s),
6.55 (1H, t, J ¼ 4.7 Hz), 3.73 (8H, m). 13C NMR (100 MHz, CDCl3):
d
168.2, 160.4, 156.7 (2 ꢁ C), 136.0, 135.3, 134.7, 133.3, 131.1, 129.3,
yellow solid (64%). 1H NMR (400 MHz, CDCl3):
d
8.30 (2H, d,
128.6, 128.4, 128.2,127.4, 126.3,124.6, 124.5, 124.2,124.1, 109.6, 42.7,
J ¼ 4.7 Hz), 7.41e7.37 (2H, m), 7.31e7.26 (3H, m), 6.96 (1H, d,
J ¼ 8.9 Hz), 6.78 (1H, dd, J ¼ 8.9, 3.0 Hz), 6.66 (1H, s), 6.52 (1H, t,
J ¼ 4.7 Hz), 6.49 (1H, d, J ¼ 3.0 Hz), 3.88e3.51 (8H, m), 3.43 (3H, s).
42.6, 42.5, 42.4. ESI-HRMS [MþH]þ m/z ¼ 473.1355, calcd for
C24H20N4OF3Cl, 473.1351.
13C NMR (100 MHz, CDCl3):
d
169.8, 161.4, 157.7 (2 ꢁ C), 156.6, 142.7,
5.3.14. Synthesis of (E)-3-(2-chlorophenyl)-1-(4-(pyrimidin-2-yl)
piperazin-1-yl)-2-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (5f)
Following the general method above, 5f was obtained as a light
139.6, 134.7, 129.2, 128.8 (2 ꢁ C), 128.7 (2 ꢁ C), 128.3, 124.5, 122.8,
115.7, 114.4, 110.4, 55.2, 46.4, 43.4, 41.7, 38.9. ESI-MS [M þ H]þ m/
z ¼ 417.1, C24H24N4O3.
yellow solid (61%). 1H NMR (400 MHz, CDCl3):
d 8.33 (2H, t,
J ¼ 4.8 Hz), 7.58 (1H, s), 7.51e7.46 (2H, m), 7.42 (1H, dd, J ¼ 8.0,
0.9 Hz), 7.36 (1H, t, J ¼ 7.8 Hz), 7.19 (1H, td, J ¼ 7.7, 1.4 Hz), 7.04e6.95
(2H, m), 6.91 (1H, dd, J ¼ 7.9, 1.3 Hz), 6.54 (1H, t, J ¼ 4.7 Hz),
5.3.9. Synthesis of (E)-3-phenyl-1-(4-(pyrimidin-2-yl)piperazin-1-
yl)-2-(3-(trifluor-o-methyl)phenyl)prop-2-en-1-one (5a)
Following the general method above, 5a was obtained as a light
3.89e3.57 (8H, m). 13C NMR (100 MHz, CDCl3):
d 169.0, 161.5, 157.7
yellow solid (71%). 1H NMR (400 MHz, CDCl3):
d
8.30 (2H, d,
(2 ꢁ C), 137.2, 135.3, 134.1, 133.5, 132.0, 130.6, 129.5, 129.4, 129.1,
128.9, 126.6, 125.5, 125.5, 124.9, 124.9, 110.5, 43.5 (4 ꢁ C). ESI-HRMS
[MþH]þ m/z ¼ 473.1354, calcd for C24H20N4OF3Cl, 473.1351.
J ¼ 4.7 Hz), 7.61 (1H, s), 7.56e7.50 (2H, m), 7.41 (1H, t, J ¼ 7.8 Hz),
7.23e7.16 (3H, m), 7.09 (2H, dd, J ¼ 7.3, 2.1 Hz), 6.88 (1H, s), 6.54
(1H, t, J ¼ 4.7 Hz), 3.72 (8H, m). 13C NMR (100 MHz, CDCl3):
d 169.7,
161.4, 157.7 (2 ꢁ C), 136.3, 135.5, 134.4, 132.3, 132.0,131.4, 129.3
(2 ꢁ C), 128.4 (3 ꢁ C), 125.7 (2 ꢁ C), 124.8 (2 ꢁ C), 110.5, 43.6 (4 ꢁ C).
ESI-HRMS [MþH]þ m/z ¼ 439.1735, calcd for C24H21N4OF3,
439.1740.
5.3.15. Synthesis of (E)-3-(4-fluorophenyl)-1-(4-(pyrimidin-2-yl)
piperazin-1-yl)-2-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (5g)
Following the general method above, 5g was obtained as a light
yellow solid (61%). 1H NMR (400 MHz, CDCl3):
d 8.32 (2H, d,
J ¼ 4.7 Hz), 7.62 (1H, s), 7.59e7.52 (2H, m), 7.46 (1H, t, J ¼ 7.7 Hz),
5.3.10. Synthesis of (E)-3-(4-methoxyphenyl)-1-(4-(pyrimidin-2-yl)
piperazin-1-yl)-2-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (5b)
Following the general method above, 5b was obtained as a light
7.12e7.05 (2H, m), 6.95e6.87 (2H, m), 6.83 (1H, s), 6.54 (1H, t,
J ¼ 4.7 Hz), 3.60 (8H, m). 13C NMR (100 MHz, CDCl3):
d 169.5, 163.7,
161.4, 157.7 (2 ꢁ C), 136.1, 135.5, 132.2, 131.5, 131.1, 131.0, 130.8,
130.5, 130.4, 129.4, 125.6, 125.0, 115.6, 115.4, 110.5, 43.6 (4 ꢁ C). ESI-
HRMS [MþH]þ m/z ¼ 457.1649, calcd for C24H20N4OF4, 457.1646.
yellow solid (62%). 1H NMR (400 MHz, CDCl3):
d 8.33 (2H, d,
J ¼ 4.7 Hz), 7.66 (1H, s), 7.60e7.55 (2H, m), 7.46 (1H, t, J ¼ 7.8 Hz),
7.04 (2H, t, J ¼ 5.7 Hz), 6.82 (1H, s), 6.77e6.73 (2H, m), 6.55 (1H, t,
J ¼ 4.7 Hz), 3.80 (3H, s), 3.66 (8H, m). 13C NMR (100 MHz, CDCl3):
5.3.16. Synthesis of (E)-1-(4-(pyrimidin-2-yl)piperazin-1-yl)-2-(3-
(trifluoromethyl)phenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-
1-one (5h)
d
170.1, 161.4, 159.7, 157.7 (2 ꢁ C), 136.8, 133.4, 132.4, 131.9, 130.8
(2 ꢁ C), 129.3, 126.8, 125.7, 125.7, 124.7, 124.6, 113.8 (2 ꢁ C), 110.5,
55.2, 43.6 (4 ꢁ C). ESI-HRMS [MþH]þ m/z ¼ 469.1847, calcd for
Following the general method above, 5h was obtained as a light
C
25H23N4O2F3, 469.1846.
yellow solid (76%). 1H NMR (400 MHz, CDCl3):
d 8.32 (2H, d,
J ¼ 4.7 Hz), 7.60 (2H, d, J ¼ 8.9 Hz), 7.55e7.43 (4H, m), 7.22 (2H, d,
5.3.11. Synthesis of (E)-3-(2-methoxyphenyl)-1-(4-(pyrimidin-2-yl)
piperazin-1-yl)-2-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (5c)
Following the general method above, 5c was obtained as a light
J ¼ 8.3 Hz), 6.89 (1H, s), 6.55 (1H, t, J ¼ 4.7 Hz), 3.73 (8H, m). 13C
NMR (100 MHz, CDCl3):
d
169.0, 161.4, 157.7 (2 ꢁ C), 138.1, 137.9,
135.7, 132.1, 131.7, 131.4, 130.2, 129.5 (4 ꢁ C) 125.5, 125.5, 125.3,
125.3, 125.2, 110.6, 43.6 (4 ꢁ C). ESI-HRMS [MþH]þ m/z ¼ 507.1618,
calcd for C25H20N4OF6, 507.1614.
yellow solid (70%). 1H NMR (400 MHz, CDCl3):
d 8.30 (2H, d,
J ¼ 4.7 Hz), 7.60 (1H, s), 7.53e7.46 (2H, m), 7.35 (1H, t, J ¼ 7.8 Hz),
7.24e7.18 (1H, m), 7.01 (1H, s), 6.89e6.83 (2H, m), 6.68 (1H, t,
J ¼ 7.5 Hz), 6.52 (1H, t, J ¼ 4.7 Hz), 3.87e3.52 (11H, overlay). 13C
5.3.17. Synthesis of (E)-3-(2-hydroxy-5-methoxyphenyl)-1-(4-
(pyrimidin-2-yl)piper-azin-1-yl)-2-(3-(trifluoromethyl)phenyl)
prop-2-en-1-one (5i)
NMR (100 MHz, CDCl3):
d
170.0, 161.5, 157.7 (2 ꢁ C), 157.6, 136.5,
134.9, 132.1, 130.0, 129.7, 128.9, 128.2, 125.5, 125.5, 124.5, 124.4,
123.5, 120.2, 110.6, 110.4, 55.4, 43.6 (4 ꢁ C). ESI-HRMS [MþH]þ m/
z ¼ 469.1845, calcd for C25H23N4O2F3, 469.1846.
Following the general method above, 5i was obtained as a light
yellow solid (52%). 1H NMR (400 MHz, CDCl3):
d 8.32 (2H, d,
J ¼ 4.7 Hz), 7.72 (1H, s), 7.59e7.51 (2H, m), 7.40 (1H, t, J ¼ 7.8 Hz),
6.98 (1H, d, J ¼ 8.9 Hz), 6.81 (1H, dd, J ¼ 8.9, 3.0 Hz), 6.73 (1H, s),
6.55 (1H, t, J ¼ 4.7 Hz), 6.41 (1H, d, J ¼ 3.0 Hz), 3.86e3.53 (8H, m),
5.3.12. Synthesis of (E)-3-(4-chlorophenyl)-1-(4-(pyrimidin-2-yl)
piperazin-1-yl)-2-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (5d)
Following the general method above, 5d was obtained as a light
3.46 (3H, s). 13C NMR (100 MHz, CDCl3):
d 169.6, 169.1, 161.4, 157.7
yellow solid (79%). 1H NMR (400 MHz, CDCl3):
d
8.32 (2H, d,
(2 ꢁ C), 156.9, 142.7, 138.0, 132.3, 129.1, 128.8, 126.2, 125.6, 125.5,
124.9, 124.9, 123.1, 115.9, 114.4, 110.6, 55.3, 43.6 (4 ꢁ C). ESIꢂMS
[M þ H]þ m/z ¼ 485.1, calcd for C25H23F3N4O3.
J ¼ 4.7 Hz), 7.63 (1H, s), 7.59 (1H, d, J ¼ 7.7 Hz), 7.53 (1H, d,
J ¼ 7.8 Hz), 7.46 (1H, t, J ¼ 7.7 Hz), 7.23e7.17 (2H, m), 7.04 (2H, d,
J ¼ 8.4 Hz), 6.82 (1H, s), 6.55 (1H, t, J ¼ 4.7 Hz), 3.72 (8H, m). 13C
NMR (100 MHz, CDCl3):
d
169.4, 161.4, 157.7 (2 ꢁ C), 136.3, 136.0,
5.3.18. Synthesis of (E)-2-(4-fluorophenyl)-3-phenyl-1-(4-
(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one (6a)
134.2, 132.9, 132.2, 130.6, 130.5 (2 ꢁ C), 129.4, 128.6 (2 ꢁ C), 125.6,
125.5, 125.1, 125.1, 110.6, 43.6, 43.6, 43.5, 43.5. ESI-HRMS [MþH]þ
m/z ¼ 473.1358, calcd for C24H20N4OF3Cl, 473.1351.
Following the general method above, 6a was obtained as a light
yellow solid (73%). 1H NMR (400 MHz, CDCl3):
d 8.29 (2H, d,
J ¼ 4.7 Hz), 7.35e7.28 (2H, m), 7.20e7.10 (5H, m), 7.02e6.93 (2H, m),
5.3.13. Synthesis of (E)-3-(3-chlorophenyl)-1-(4-(pyrimidin-2-yl)
piperazin-1-yl)-2-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (5e)
Following the general method above, 5e was obtained as a light
6.76 (1H, s), 6.51 (1H, t, J ¼ 4.7 Hz), 3.90e3.43 (8H, m). 13C NMR
(100 MHz, CDCl3):
d
170.2, 163.6, 161.5, 157.7 (2 ꢁ C), 136.1, 134.9,
131.4, 130.7, 130.7, 130.6, 129.3 (2 ꢁ C), 128.2 (2 ꢁ C), 128.0, 116.0,
115.8, 110.5, 43.6 (4 ꢁ C). HR-ESI-MS [MþH]þ m/z ¼ 389.1754, calcd
for C23H21N4OF, 389.1772.
yellow solid (76%). 1H NMR (400 MHz, CDCl3):
d
8.32 (2H, d,
J ¼ 4.7 Hz), 7.63e7.52 (3H, m), 7.46 (1H, t, J ¼ 7.7 Hz), 7.22 (1H, d,

136
Y. Fang et al. / European Journal of Medicinal Chemistry 149 (2018) 129e138
5.3.19. Synthesis of (E)-2-(4-fluorophenyl)-3-(4-methoxyphenyl)-
1-(4-(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one (6b)
Following the general method above, 6b was obtained as a light
5.3.25. Synthesis of (E)-2-(4-fluorophenyl)-1-(4-(pyrimidin-2-yl)
piperazin-1-yl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (6h)
Following the general method above, 6h was obtained as a light
yellow solid (67%). 1H NMR (400 MHz, CDCl3):
d
8.29 (2H, d,
yellow solid (71%). 1H NMR (400 MHz, CDCl3):
d 8.32 (2H, d,
J ¼ 4.7 Hz), 7.35e7.31 (2H, m), 7.01 (4H, m), 6.74e6.69 (3H, m), 6.51
J ¼ 4.7 Hz), 7.48 (2H, d, J ¼ 8.0 Hz), 7.36e7.29 (2H, m), 7.25 (2H, d,
(1H, t, J ¼ 4.7 Hz), 3.76 (3H, s), 3.74e3.44 (8H, m). 13C NMR
J ¼ 8.0 Hz), 7.07e6.99 (2H, m), 6.80 (1H, s), 6.55 (1H, t, J ¼ 4.7 Hz),
(100 MHz, CDCl3):
d
170.5, 161.5, 159.4, 157.7 (2 ꢁ C), 134.0, 132.4,
3.86e3.49 (8H, m). 13C NMR (100 MHz, CDCl3):
d 169.5, 163.9, 161.5,
130.8, 130.8, 130.7, 130.6, 130.6, 128.7, 127.3, 116.0, 115.8, 113.74,
113.7, 110.5, 55.1, 43.6 (4 ꢁ C). HR-ESI-MS [MþH]þ m/z ¼ 419.1861,
calcd for C24H23N4O2F, 419.1878.
157.7 (2 ꢁ C), 138.6, 138.5, 130.7, 130.6, 130.5, 129.5 (2 ꢁ C), 128.9,
125.3, 125.2, 125.2, 125.2, 116.3, 116.1, 110.6, 43.6 (4 ꢁ C). HR-ESI-MS
[MþH]þ m/z ¼ 457.1639, calcd for C24H20N4OF4, 457.1646.
5.3.20. Synthesis of (E)-2-(4-fluorophenyl)-3-(2-methoxyphenyl)-
1-(4-(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one (6c)
Following the general method above, 6c was obtained as a light
5.3.26. Synthesis of (E)-2-(4-fluorophenyl)-3-(2-hydroxy-5-
methoxyphenyl)-1-(4-(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-
one (6i)
yellow solid (61%). 1H NMR (400 MHz, CDCl3):
d
8.30 (2H, d,
Following the general method above, 6i was obtained as a yel-
J ¼ 4.7 Hz), 7.32e7.28 (2H, m), 7.23e7.17 (1H, m), 6.97e6.89 (4H, m),
low solid (54%). 1H NMR (400 MHz, CDCl3):
d
8.32 (2H, d, J ¼ 4.7 Hz),
6.86 (1H, d, J ¼ 8.3 Hz), 6.68 (1H, t, J ¼ 7.4 Hz), 6.52 (1H, t, J ¼ 4.7 Hz),
7.77e7.69 (1H, m), 7.57e7.51 (1H, m), 7.43e7.35 (2H, m), 7.02e6.95
(3H, m), 6.80 (1H, dd, J ¼ 8.9, 3.0 Hz), 6.64 (1H, s), 6.54 (1H, t,
J ¼ 4.7 Hz), 3.72 (8H, m), 3.50 (3H, s). 13C NMR (100 MHz, CDCl3):
3.90e3.56 (overlap, 11H). 13C NMR (100 MHz, CDCl3):
d 169.5, 162.4,
160.4, 160.0, 156.7, 156.6, 134.3, 130.5, 129.5, 129.4, 129.1, 128.3,
125.6, 122.9, 119.1, 114.7, 114.5, 109.4, 109.4, 54.4, 42.7 (4 ꢁ C). HR-
ESI-MS [MþH]þ m/z ¼ 419.1868, calcd for C24H23N4O2F, 419.1878.
d
169.6, 167.6, 161.4, 157.7, 157.7, 142.7, 138.5, 130.8, 130.7, 130.6,
129.2, 128.7, 124.6, 122.9, 115.8, 115.6, 115.5, 114.6, 110.5, 55.3, 43.8,
43.8, 43.6, 43.4. ESI-MS [MþH]þ m/z ¼ 435.1, C24H23FN4O3.
5.3.21. Synthesis of (E)-3-(4-chlorophenyl)-2-(4-fluorophenyl)-1-
(4-(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one (6d)
5.3.27. Synthesis of (2E,4E)-2-(4-fluorophenyl)-5-phenyl-1-(4-
(pyrimidin-2-yl)pipe-razin-1-yl)penta-2,4-dien-1-one (8a)
Following the general method above, 8a was obtained as a
Following the general method above, 6d was obtained as a light
yellow solid (76%). 1H NMR (400 MHz, CDCl3):
d 8.31 (2H, d,
J ¼ 4.7 Hz), 7.32e7.27 (2H, m), 7.21e7.16 (2H, m), 7.09e6.98 (4H, m),
yellow solid (58%). 1H NMR (400 MHz, CDCl3):
d 8.28 (2H, t,
6.73 (1H, s), 6.54 (1H, t, J ¼ 4.7 Hz), 3.86e3.46 (8H, m). 13C NMR
J ¼ 4.7 Hz), 7.46e7.39 (2H, m), 7.37e7.33 (2H, m), 7.30 (2H, t,
J ¼ 7.3 Hz), 7.26e7.21 (1H, m), 7.15e7.08 (2H, m), 7.00 (1H, dd,
J ¼ 15.5, 11.1 Hz), 6.81 (1H, d, J ¼ 15.6 Hz), 6.68 (1H, d, J ¼ 10.8 Hz),
6.52 (1H, t, J ¼ 4.7 Hz), 3.91e3.43 (8H, m). 13C NMR (100 MHz,
(100 MHz, CDCl3):
d
169.9, 163.7, 161.5, 157.7 (2 ꢁ C), 136.8, 133.8,
133.4, 130.8, 130.6, 130.6 (2 ꢁ C), 130.5, 129.4, 128.5 (2 ꢁ C), 116.2,
116.0, 110.5, 43.6 (4 ꢁ C). HR-ESI-MS [MþH]þ m/z ¼ 423.1358, calcd
for C23H20N4OFCl, 423.1382.
CDCl3):
d
170.1, 161.5, 161.1, 157.7 (2 ꢁ C), 137.8, 136.6, 135.6, 131.7,
131.7, 130.8, 130.7, 128.7 (2 ꢁ C), 128.4, 126.9 (2 ꢁ C), 123.9, 115.9,
115.7, 110.5, 43.6 (4 ꢁ C). HR-ESI-MS [MþH]þ m/z ¼ 415.1912, calcd
for C25H23N4OF, 415.1929.
5.3.22. Synthesis of (E)-3-(3-chlorophenyl)-2-(4-fluorophenyl)-1-
(4-(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one (6e)
Following the general method above, 6e was obtained as a light
yellow solid (65%). 1H NMR (400 MHz, CDCl3):
d
8.30 (2H, t,
5.3.28. Synthesis of (2E,4E)-5-phenyl-1-(4-(pyrimidin-2-yl)
piperazin-1-yl)-2-(3-(trifluoromethyl)phenyl)penta-2,4-dien-1-one
(8b)
J ¼ 4.7 Hz), 7.32e7.27 (2H, m), 7.19e7.14 (1H, m), 7.16e7.08 (2H, m),
7.06e6.95 (3H, m), 6.71 (1H, s), 6.53 (1H, t, J ¼ 4.7 Hz), 3.86e3.43
(8H, m). 13C NMR (100 MHz, CDCl3):
d
169.7, 163.8, 161.5, 157.7
Following the general method above, 8b was obtained as a
(2 ꢁ C), 137.6, 136.8, 134.2, 130.8, 130.6, 130.5, 129.5, 129.2, 129.0,
128.0, 127.3, 116.1, 115.9, 110.5, 43.6 (4 ꢁ C). HR-ESI-MS [MþH]þ m/
z ¼ 423.1374, calcd for C23H20N4OFCl, 423.1382.
yellow solid (45%). 1H NMR (400 MHz, CDCl3):
d 8.32 (2H, d,
J ¼ 4.7 Hz), 7.75 (1H, s), 7.66 (2H, t, J ¼ 8.2 Hz), 7.62e7.55 (1H, m),
7.42e7.23 (5H, m), 7.00 (1H, dd, J ¼ 13.9, 12.3 Hz), 6.88 (1H, d,
J ¼ 15.5 Hz), 6.76 (1H, d, J ¼ 10.9 Hz), 6.54 (1H, m), 3.54 (8H, m). 13
C
5.3.23. Synthesis of (E)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-1-
(4-(pyrimidin-2-yl)piperazin-1-yl)prop-2-en-1-one (6f)
NMR (100 MHz, CDCl3):
d
169.8, 161.6, 157.8 (2 ꢁ C), 139.0, 136.6,
136.4, 135.0, 133.1, 132.4, 129.3, 128.9 (2 ꢁ C), 128.7, 127.0 (2 ꢁ C),
125.8, 125.6, 124.8, 124.7, 123.4, 110.5, 43.6 (4 ꢁ C). HR-ESI-MS
[MþH]þ m/z ¼ 465.1888, calcd for C26H23N4OF3, 465.1897.
Following the general method above, 6f was obtained as a light
yellow solid (61%). 1H NMR (400 MHz, CDCl3):
d 8.31 (2H, d,
J ¼ 4.7 Hz), 7.43e7.38 (1H, m), 7.30e7.25 (2H, m), 7.19e7.15 (1H, m),
7.03e6.90 (4H, m), 6.88 (1H, s), 6.53 (1H, t, J ¼ 4.7 Hz), 3.76 (8H, m).
5.4. Cell culture
13C NMR (100 MHz, CDCl3):
137.6,134.1,134.0,130.8,130.6,130.5,129.3,129.1,127.4,126.5,115.8,
115.6, 110.5, 43.5 (4 ꢁ C). HR-ESI-MS [MþH]þ m/z ¼ 423.1368, calcd
for C23H20N4OFCl, 423.1382.
d
169.5, 163.7, 161.5, 161.2, 157.7 (2 ꢁ C),
The murine microglial cell line BV-2 (Cell Resource Center of
Chinese Academy of Medical Science, Beijing, China) was cultured
in Dulbecco's modified Eagle's medium (Gibco) supplemented with
10% (v/v) fetal bovine serum (Hyclone), 100 U/mL of penicillin, and
5.3.24. Synthesis of (E)-2,3-bis(4-fluorophenyl)-1-(4-(pyrimidin-2-
yl)piperazin-1-yl)prop-2-en-1-one (6g)
100 mg/mL of streptomycin (Hyclone). These cells were grown in a
humidified incubator containing 5% CO2 at 37 ꢀC and subcultured
Following the general method above, 6g was obtained as a light
every two days.
yellow solid (78%). 1H NMR (400 MHz, CDCl3):
d 8.30 (2H, d,
J ¼ 4.7 Hz), 7.35e7.29 (2H, m), 7.12e7.06 (2H, m), 7.01e6.96 (2H, m),
5.5. Inhibiting NO production in LPS-stimulated BV2 microglial cells
6.93e6.84 (2H, m), 6.74 (1H, s), 6.52 (1H, t, J ¼ 4.7 Hz), 3.66 (8H, m).
13C NMR (100 MHz, CDCl3):
d
170.0, 163.6, 161.5, 161.1, 157.7 (2 ꢁ C),
Microglia cell-based assays for the concentration-dependent
activity of the compounds were performed as previously
described [16,17]. The BV2 microglial cells were plated in 96-well
136.1, 131.2, 131.1, 131.0, 131.0, 130.6, 130.5, 129.6, 116.1, 115.9, 115.4,
115.2, 110.5, 43.6 (4 ꢁ C). HR-ESI-MS [MþH]þ m/z ¼ 407.1687, calcd
for C23H20N4OF2, 407.1678.
microplates at a density of 4 ꢁ 105 cells/mL (100
mL/well). After

Y. Fang et al. / European Journal of Medicinal Chemistry 149 (2018) 129e138
137
48 h, the medium was removed, and 80
The cells were pretreated with various concentrations of com-
pounds (10 L) for 30 min, followed by stimulation with LPS (1 g/
mL, 10 L) of
L, Sigma) for 24 h at 37 ꢀC. The same volume (50
Griess assay agent (Beyotime Biotechnology) was added into the
96-well plates and mixed with the culture medium (50 L). The
m
L of DMEM was added.
values gave
a
strong linear correlation, Pe (exp.) ¼ 1.4574Pe
(lit.) ꢂ 1.0773 (R2 ¼ 0.9427). From this equation and the limit
established by Di et al. [26] (Pe (lit.) ¼ 4.0 ꢁ 10ꢂ6 cm/s) for BBB
m
m
m
m
permeation, we concluded that compounds with a perme-
ability > 4.7 ꢁ 10ꢂ6 cm/s could cross the BBB.
m
mixture solution was incubated at room temperature for 10 min,
and the absorbance at 540 nm was measured in a multifunctional
microplate reader. The percent of inhibition of NO production was
calculated with this formula: (FL ꢂ FC)/(FL ꢂ F0) ꢁ 100%, where
FC ¼ absorbance of the cells treated with the tested compound and
LPS, FL ¼ absorbance of the cells treated with LPS, and F0 ¼ absor-
bance of the normal cells. The IC50 was defined as the concentration
of the compound that reduced 50% of NO production with LPS and
was calculated by nonlinear regression of at least three indepen-
dent experiments.
5.8. Western blot assay
BV-2 cells were seeded on 6-well plate, incubated with or
without 8b for 30 min, then treated with or without LPS(1 g/ml)
m
for 16 h. The cell lysates were prepared with RIPA lysis buffer fixed
with protease inhibitor cocktail and phosphatase inhibitor. Cyto-
plasmic and nucleus proteins were extracted by Nucleus and
Cytoplasmic Extraction Reagent (Key GEN BioTEC, Nanjing, China)
according to manufacturer's protocol. The concentrations of protein
were determined by Pierce™ Rapid Gold BCA Protein Assay Kit
(ThermoFisher Scientific). A total of 45 mg protein of each sample
5.6. Inhibiting TNF-
microglial cells
a and IL-1b production in LPS-stimulated BV2
was electrophoresed in 10% SDS-PAGE, then transferred to poly-
vinylidene difluoride (PVDF) western membrane (Milipore). The
membranes were blocked using 5% (W/V) skim milk in TBST (Tris-
buffered saline with 0.1% Tween 20) for 2 h, then the membranes
were incubated with primary antibodies at 4 ꢀC overnight. Washed
by TBST for three times, the membranes were incubated with
corresponding secondary antibody at room temperature for 2 h.
Finally immunoreactive signals were detected using Chem-
iluminescence imager (Tanon 5200, Shanghai, China). Antibodies
against phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/
The procedure is almost the same as above. The cells were
plated in 96-well microplates at a density of 4 ꢁ 105 cells/mL (100
mL/well). After 48 h, the medium was removed, and 80 mL of DMEM
was added. The compound solutions (10
first, and then 10 L of 1 g/mL LPS solution was added. The cells
were incubated at 37 ꢀC for 24 h. The levels of TNF-
and IL-1 in
mL) were added to the cells
m
m
a
b
the culture medium were measured using ELISA kits (Boster Bio-
logical Technology Co. Ltd) according to the manufacturer's in-
structions. The percent of inhibition of TNF-a or IL-1b production
Tyr182), phospho-JNK (Thr183/Tyr185), and phospho-I
kBa (Ser32),
p38, ERK, JNK, I , NF- B p65 were purchased from Cell Signaling
k
Ba
k
was calculated with this formula: (FL ꢂ FC)/(FL ꢂ F0) ꢁ 100%, where
FC ¼ absorbance of the cells treated with the tested compound and
LPS, FL ¼ absorbance of the cells treated with LPS, and F0 ¼ absor-
bance of the normal cells. The IC50 was defined as the concentration
Technology. The anti-iNOS and anti-Lamin B antibodies were pur-
chased from Abcam. The anti-GAPDH antibody was purchased from
Sigma-Aldrich.
of the compound that reduced 50% of TNF-a or IL-1b production
with LPS and was calculated by nonlinear regression of at least
three independent experiments.
5.9. ADMET prediction
ADMET refers to absorption, distribution, metabolism, excre-
tion, and toxicity properties of the drug in the body. ADMET de-
scriptors module were used to predict the ADMET properties of the
target compounds using Discovery studio (version 2.5, Accelrys Inc.,
San Diego, CA.). The ADMET descriptors module estimates the
aqueous solubility, blood brain barrier penetration (BBB), cyto-
chrome P450 (CYP450) 2D6 inhibition, hepatotoxicity, human in-
testinal absorption (HIA) and plasma protein binding (PPB) of a set
of compounds.
5.7. BBB permeation assay
The BBB penetration of compounds was evaluated using the
parallel artificial membrane permeation assay (PAMPA) described
by Di et al. [22] Porcine brain lipid (PBL) was obtained from Avanti
Polar Lipids. The donor microplate (PVDF membrane, pore size
0.45 mm) and acceptor microplate were both from Millipore. The
96-well UV plate (COSTAR) was from Corning Incorporated. The
acceptor 96-well microplate was filled with 300
and the filter membrane was impregnated with 4
m
L PBS/EtOH (7:3),
Conflicts of interest
m
L PBL in
dodecane (20 mg/mL). Compounds were dissolved in DMSO at
5 mg/mL and diluted 50-fold in PBS/EtOH (7:3) to a final concen-
The authors declare no competing interest.
tration of 100 mg/mL. Then, 200 mL of the solution was added to the
Acknowledgements
donor wells. The acceptor filter plate was carefully placed on the
donor plate to form a sandwich, which was left undisturbed for
10 h at 25 ꢀC. After incubation, the donor plate was carefully
removed, and the concentration of compounds in the acceptor
wells was determined using the UV plate reader (Flexstation 3).
Every sample was analyzed at five wavelengths in four wells and in
at least three independent runs. Pe was calculated by the following
This work was supported in part by the National Natural Science
Foundation of China (81001372, 81573310, 81473138), the Medical
Scientific Research Foundation of Guangdong Province (No.
A2014212), the Science and Technology Planning Project of
Guangdong Province (2016A020217005), the National Science and
Technology Major Project of the Ministry of Science and Technology
of China (2017ZX09305010), and the Strategic New Industry
Development Foundation of Shenzhen (CXZZ20150325140421385).
expression: Pe ¼ ꢂVd ꢁ Va/[(Vd
þ
Va)A ꢁ t] ꢁ ln(1 ꢂ drugacceptor
/
drugequilibrium), where Vd is the volume of donor well; Va, volume in
acceptor well; A, filter area; t, permeation time; drugacceptor, the
absorbance obtained in the acceptor well; and drugequilibrium, the
theoretical equilibrium absorbance. The results are given as the
mean standard deviation. In the experiment, 13 quality control
standards of known BBB permeability were included to validate the
analysis set [27]. A plot of the experimental data versus literature
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.02.063.

138
Y. Fang et al. / European Journal of Medicinal Chemistry 149 (2018) 129e138
[16] Z. Wei, G. Zhong, X. Rao, et al., Identification of aminopyridazine-derived
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b