Synthesis, crystal structure and biological evaluation of novel 2-phenylthiazole derivatives as butyrylcholinesterase inhibitors

Article abstract of DOI:10.3184/174751918X15314837408346

To find novel butyrylcholinesterase inhibitors, three novel 2-phenylthiazole derivatives were synthesised. The synthesised compounds were characterised by NMR and single-crystal X-ray diffraction analysis. Hirshfeld surface analysis and two-dimensional fingerprint plots of the compounds were used as a theoretical approach to assess the driving force for crystal structure formation via the intermolecular interactions in the crystal lattices of the synthesised compounds. Among the three compounds, N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro- 1H-pyrazol-4-yl)-2-(4-methoxyphenyl)thiazole-4-carboxamide showed the best butyrylcholinesterase-inhibition activity with an IC₅₀ value of 75.12 μM. A docking study demonstrated that this compound interacts with the peripheral anionic site of butyrylcholinesterase.

Full text of DOI:10.3184/174751918X15314837408346

366 RESEARCH PAPER
VOL. 42 JULY, 366–370
JOURNAL OF CHEMICAL RESEARCH 2018
Synthesis, crystal structure and biological evaluation of novel
2-phenylthiazole derivatives as butyrylcholinesterase inhibitors
Da-Hua Shia,b,c*, Xiao-Dong Mab, Yu-Wei Liub, Wei Minb, Fu-Jun Yind, Zong-Ming Tangb,
Meng-Qiu Songb, Chen Lub, Xiao-Kai Songb, Wei-Wei Liub and Tong Dongb
aKey Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang 222005, P.R. China
bPharmacy School, Huaihai Institute of Technology, Lianyungang 222005, P.R. China
cCo-Innovation Center of Jiangsu Marine Bio-industry Technology, Lianyungang 222005, P.R. China
dJiangsu Institute of Marine Resources, Huaihai Institute of Technology, Lianyungang 222005, P.R. China
To find novel butyrylcholinesterase inhibitors, three novel 2-phenylthiazole derivatives were synthesised. The synthesised compounds
were characterised by NMR and single-crystal X-ray diffraction analysis. Hirshfeld surface analysis and two-dimensional fingerprint plots
of the compounds were used as a theoretical approach to assess the driving force for crystal structure formation via the intermolecular
interactions in the crystal lattices of the synthesised compounds. Among the three compounds, N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro- 1H-pyrazol-4-yl)-2-(4-methoxyphenyl)thiazole-4-carboxamide showed the best butyrylcholinesterase-inhibition activity with an
IC50 value of 75.12 µM. A docking study demonstrated that this compound interacts with the peripheral anionic site of butyrylcholinesterase.
Keywords: 2-phenylthiazole derivatives, crystal structure formation, cholinesterase, Alzheimer’s disease
Alzheimer’s disease (AD) is an age-related progressive
neurodegenerative disorder that causes gradual physical and
mental decline, eventually resulting in death. According to World
Alzheimer Report 2015, the number of people with AD and other
forms of dementia worldwide is estimated to be 46.85 million;
this will probably double by 2030 and could possibly increase
to three times the current level by 2050.1 As acetylcholine (ACh)
is a neurotransmitter associated with learning and memory,
treatment approaches have focused on the compensation of deficit
cholinergic neurotransmission in the CNS. Acetylcholinesterase
inhibitors (AChEIs) could increase the level of ACh in AD
patients through the inhibition AChE and, therefore, relieve some
symptoms experienced by patients. AChEIs such as rivastigmine,
donepezil and galantamine have been used clinically for AD
management.2 However, under normal conditions, ACh is mostly
hydrolysed by butyrylcholinesterase (BuChE) in progressed
AD. To account for an increased level of BuChE and decreased
level of AChE in the late stage of AD, development of a selective
BuChE inhibitor is of vital importance. To date, compounds with
various scaffolds have been found to selectively inhibit BuChE.3
Some thiazole derivatives have been reported to possess
BuChE-inhibition activity.4–6 We have previously synthesised
a series of 2-phenylthiazole derivatives and found that these
derivatives possess AChE- and BuChE-inhibiton activities.7,8
In this study, three novel 2-phenylthiazole derivatives were
synthesised and characterised by NMR and single-crystal X-ray
diffraction analysis. The role of intermolecular interactions in
these compounds was analysed through single-crystal structure
studies. Also, we report detailed analysis of intermolecular
interactions by Hirshfeld surface analysis. The BuChE-inhibiton
activity of the 2-phenylthiazole derivatives was studied to find
BuChE inhibitors for the treatment of AD.
and acylamination with compound 4 as starting material.
All crude products 6a–c were purified by silica gel column
chromatography. Purity of the obtained compounds was
1
checked by TLC. The compounds were characterised by H
NMR, 13C NMR and X-ray crystallography. All the results
substantiate the proposed structures.
Crystal structure description of compounds 6a–c
The structures of compounds 6a–c were determined by X-ray
diffraction analysis. The crystal data are presented in Table 1 and
Fig. 1 and have been deposited at the Cambridge Crystallographic
Data Centre as supplementary publication numbers CCDC
1821145, CCDC 1821147 and CCDC 1821148. Compound 6a is
monoclinic: space group P21/c, a = 11.961(5) Å, b = 11.961(5) Å,
c = 11.961(5) Å, β = 99.118(6)°, V = 1528.4(11) Å3, Z = 4. Compound
6b is monoclinic: space group P2(1)/n, a = 10.7575(17) Å,
b = 10.7575(17) Å, c= 10.7575(17) Å, β= 10.7575(17)°, V = 1662.5(5)
Å3, Z = 4. Compound 6c is triclinic: space group P-1, a = 8.707(3)
Å, b = 9.302(3) Å, c = 77.576(4) Å, α = 77.576(4)°, β = 89.269(4)°,
γ = 72.078(5)°, V = 1025.0(5) Å3, Z = 2. The packing diagrams of
the compounds 6a–c viewed along the b axis are shown in Fig. 2.
There are no hydrogen bonds between the molecules in all three
compounds.
Hirshfeld surface analysis
Hirshfeld surfaces and fingerprint plots were generated for all
three compounds to understand the different intermolecular
interactions and packing modes. For compounds 6a–c, the
red regions mainly indicate a C–H···O interaction (Fig. 1
in the Electronic Supplementary Information (ESI)). H···H
intermolecular contacts with a contribution of 41.2%, 54.4%
and 46.1% of the overall contacts for compounds 6a, 6b and 6c,
respectively, are the major contributor to the Hirshfeld surface.
The O···H intermolecular contacts, as one of the strong contacts
due to the presence of C–H···O intermolecular interactions,
contribute 13.4%, 13.9% and 16.9% of the overall contacts to
the Hirshfeld surface for compounds 6a, 6b and 6c, respectively
(Fig. 2 and Table 1 in the ESI).
Results and discussion
Synthesis of compounds 6a–c
The synthetic route for compounds 6a–c is presented in
Scheme 1. Condensation reaction of 4-hydroxythiobenzamide
(1) and ethyl bromopyruvate (2) in refluxing EtOH for 4 h
gave ethyl 2-(4-hydroxyphenyl)thiazole-4-carboxylate (3).
Compound 3 was O-alkylated by treating it with methyl
iodide in DMF at 40 °C to afford compound 4. The desired
products were synthesised via saponification, acyl chlorination
Biological activity
The target compounds 6a–c were tested for BuChE (from
equine serum)-inhibiton activity using the method published
by Ellman et al.9 In this test, tacrine was used as a reference
* Correspondent. E-mail: shidahua@hhit.edu.cn

JOURNAL OF CHEMICAL RESEARCH 2018 367
H
O
O
H
O
Et H 4h
O
NH2
+
Et
O
Br
S
80
°C
O
S
N
2
O
1
3
Et
O
CH3I DMF, K2CO3, 40 oC
O
O
O
S
KOH
S
S
amine
l
SOC 2
N
N
N
CH3OH, reflux
DCM reflux DCM, reflux
O
O
O
R
S
H
O
Et
O
5
4
6
6a
6b
N
N
NH
6c
O
N
N
Scheme 1 Synthesis of 2-phenylthiazole derivatives.
Table 1 Crystallographic data for the compounds 6a, 6b and 6c.
Compound
6a
6b
6c
Formula
FW
C15H16N2O2S2
320.42
C17H20N2O2S
316.41
C22H20N4O3S
420.48
Crystal shape/colour
Crystal size (mm)
T (K)
Block/yellow
0.24 × 0.21 × 0.19
296(2)
Block/white
0.23 × 0.21 × 0.18
296(2)
Block/yellow
0.23 × 0.22 × 0.2
296(2)
0.71073
Monoclinic
P21/c
0.71073
Monoclinic
P21/n
0.71073
Triclinic
P-1
λ (Mo Kα) (Å)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
11.961(5)
8.925(4)
14.501(6)
90.00
10.7575(17)
8.3288(13)
18.773(3)
90.00
8.707(3)
9.302(3)
13.644(4)
77.576(4)
89.269(4)
72.078(5)
1025.0(5)
2
α (°)
β (°)
99.118(6)
90.00
1528.4(11)
4
98.739(2)
90.00
1662.5(5)
4
γ (°)
V (Å3)
Z
µ(Mo Kα) (mm–1)
Tmin
0.353
0.9200
0.203
0.9548
0.190
0.9576
Tmax
Dc (g cm–3)
0.9359
1.392
0.9644
1.264
0.9630
1.359
Measured reflections
Independent reflections
Rint
8074
2981
0.0229
8984
3755
0.0864
5646
3935
0.0384
Data/restraints/parameters
Goodness of fit on F2
R1 [I ≥ 2σ(I)]
wR2 [I ≥ 2σ(I)]
R1 (all data)
2981/0/192
1.062
0.0349
3755/0/201
0.886
0.0455
3935/0/278
0.932
0.0579
0.0927
0.0432
0.1074
0.0900
0.1407
0.1409
wR2 (all data)
Large diff. peak and hole (e Å–3)
0.0992
0.21 and −0.28
0.1244
0.16 and −0.20
0.1845
0.24 and −0.23

368 JOURNAL OF CHEMICAL RESEARCH 2018
Fig. 1 Molecular structure of compound 6a, 6b and 6c at 30% probability ellipsoids.
Fig. 2 Packing diagrams of the compounds 6a, 6b and 6c viewed along the b axis.

JOURNAL OF CHEMICAL RESEARCH 2018 369
3.89 (s, 3H), 1.45 (t, J = 7.1 Hz, 3H); 13C NMR (126 MHz, CDCl3): δ
168.8, 161.63, 161.56, 147.9, 128.5, 126.2, 125.8, 114.3, 61.5, 55.4, 14.4.
HRMS (ESI) m/z calcd for C13H13NNaO3S [M + Na]+: 286.0508; found:
286.0521.
Table 2 In vitro BuChE-inhibitory activity of 2-phenylthiazole derivatives
Compound
IC50 (µM)a
6a
6b
6c
Tacrine
95.61 2.12
80.99 0.87
75.12 1.21
0.05 0.01
Synthesis of 2-(4-methoxyphenyl)thiazole-4-carboxylic acid (5)
KOH (0.83 g) was added to a solution of compound 4 (1 g) in anhydrous
methanol (15 mL) and refluxed for 9 h. After reaction, the solution was
neutralised to pH 5 and poured into cool water (100 mL). The residue
was filtered and dried to obtain compound 5 as a white solid: yield:
a50% inhibitory concentration (means SD of three experiments) of BuChE from equine
serum.
1
85%; m.p. 157–159 °C; H NMR (500 MHz, CDCl3): δ 8.20 (s, 1H),
drug and the BuChE-inhibition results are presented in Table 2.
All three compounds showed BuChE-inhibition activity, with
IC50 values of of 95.61, 80.99 and 75.12 µM, respectivley. A
docking study of the most potent anti-BuChE compound 6c was
conducted to identify the possible interactions between it and the
enzyme active site. A docking study was performed on reported
crystal structure of human BuChE (HuBuChE, PDB code
1P0I) using the Molecular Operating Environment (MOE).10,11
The molecular docking of the compound 6c interacts with the
peripheral anionic site (PAS) of BuChE (Fig. 4 in the ESI).11
7.93 (d, J = 8.7 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 3.88 (s, 3H); 13C NMR
(126 MHz, CDCl3): δ 169.0, 162.3, 162.0, 146.5, 128.5, 126.8, 125.1,
114.5, 55.5. HRMS (ESI) m/z calcd for C11H9NNaO3S [M + Na]+:
258.0195; found: 258.0202.
Synthesis of compounds 6a–c; general procedure
Sulfoxide chloride (0.45 mL) was added to a solution of compound
5 (0.4 g) in dichloromethane (10 mL) and refluxed for 10 h. When the
reaction was completed, the solvent was evaporated under reduced
pressure. The residual mass was dissolved into dichloromethane
(10 mL). Then the amine was added to the solution and refluxed for
another 6 h. When the reaction was finished, the solvent was evaporated
under reduced pressure. The residual was purified using column
chromatography (CH2Cl2:CH3OH = 15:1) to obtain 2-phenylthiazole
derivatives 6a–c. The compounds were grown at room temperature from
organic solvent (methanol and dichloromethane) for single crystals of
compounds 6a, 6b and 6c, respectively.
(2-(4-Methoxyphenyl)thiazol-4-yl)(thiomorpholino)methanone
(6a): White crystals; yield 79%; m.p. 99–101 °C; 1H NMR (500 MHz,
CDCl3): δ 7.87 (d, J = 8.8 Hz, 2H), 7.83 (s, 1H), 6.97 (d, J = 8.8 Hz,
2H), 4.17 (s, 1H), 4.06 (s, 1H), 3.87 (s, 3H), 2.78 (s, 4H); 13C NMR (126
MHz, CDCl3): δ 167.2, 163.0, 161.5, 151.0, 128.1, 126.0, 123.5, 114.4,
55.5, 50.2, 45.5, 28.3, 27.5. HRMS (ESI) m/z calcd for C15H17N2O2S2
[M + H]+: 321.0726; found: 321.0723.
(2- (4-Methoxyphenyl)thiazol-4-yl)(2-methylpiperidin-1-yl)
methanone (6b): Yellow crystals; yield 43%; m.p. 113–115 °C;
1H NMR (500 MHz, CDCl3): δ 7.89 (d, J = 8.8 Hz, 2H), 7.70 (s, 1H),
6.96 (d, J = 8.8 Hz, 2H), 5.20–4.05 (m, 1H), 3.87 (s, 3H), 3.01 (s, 1H),
1.87–1.49 (m, 6H), 1.34 (d, J = 5.8 Hz, 3H); 13C NMR (126 MHz,
CDCl3): δ 166.9, 163.3, 161.4, 151.9, 128.1, 126.3, 121.7, 114.3, 55.4,
53.4, 42.2, 30.5, 26.0, 19.1. HRMS (ESI) m/z calcd for C17H20N2NaO2S
[M + Na]+: 339.1138; found: 339.1149.
Conclusion
Three 2-phenylthiazole derivatives 6a–c were synthesised
and tested for their BuChE-inhibition ability in vitro. The
data showed that compound 6c had the best BuChE-inhibiton
acitivity, and it interacted with the PAS of BuChE. These results
suggest that compound 6c could be a promising lead candidate
against AD.
Experimental
All synthetic reagents were purchased from Aladdin Industrial
Corporation. All reagents were AR grade and were used without
any purification. BuChE from equine serum, 5,5′-dithio-bis(2-
nitrobenzoic acid) (DTNB) and butyrylcholine iodide (BTCI) were
purchased from Sigma-Aldrich. TLC was performed on glass-backed
silica gel sheets (silica gel 60 GF254) and visualised under UV light
(254 nm). All the melting points were determined by Kohler melting
point apparatus. 1H NMR and 13C NMR spectra were recorded
at 500 MHz using Bruker Avance III. The X-ray single crystal
diffraction data were recorded on a Bruker SMART APEX-II CCD
diffractometer. High-resolution mass spectra (HRMS) were recorded
using HPLC 1260-6230 TOF MASS.
N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-(4-
methoxyphenyl)thiazole-4-carboxamide (6c): Yellow crystals; yield 72%;
m.p. 191–193 °C; 1H NMR (500 MHz, CDCl3): δ 8.81 (s, 1H), 8.07 (s, 1H),
7.93 (d, J = 8.9 Hz, 2H), 7.53–7.39 (m, 4H), 7.32 (d, J = 7.2 Hz, 1H), 6.96
(d, J = 8.9 Hz, 2H), 3.87 (s, 3H), 3.12 (s, 3H), 2.39 (s, 3H); 13C NMR (126
MHz, CDCl3): δ 168.2, 161.7, 159.7, 149.9, 149.3, 134.9, 129.2, 128.3, 126.8,
125.6, 124.1, 122.7, 114.4, 108.5, 55.5, 36.4, 12.7. HRMS (ESI) m/z calcd
for C22H20N4NaO3S[M + Na]+: 443.1148; found: 443.1163.
Synthesis of ethyl 2-(4-hydroxyphenyl)thiazole-4-carboxylate (3)
A mixture of 4-hydroxythiobenzamide (3.93 g, 25.67 mmol) and
ethyl bromopyruvate (5.00 g, 25.64 mmol) were combined in ethanol
(50 mL). The reaction mixture was refluxed for 4 h, after which the
reaction mixture was cooled and distilled water (100 mL) was added.
The precipitated solid was filtered, washed with distilled water
(50 mL) and dried under reduced pressure to obtain compound 3
as: White solid; yield 89%; m.p. 92–93 °C; 1H NMR (500 MHz,
DMSO-d6): δ 10.11 (s, 1H), 8.44 (s, 1H), 7.80 (m, 2H), 6.90 (m, 2H),
4.33 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H); 13C NMR (126 MHz,
DMSO-d6): δ 168.0, 160.7, 159.9, 146.5, 128.1, 127.7, 123.6, 115.9, 60.6,
14.1. HRMS (ESI) m/z calcd for C12H11NNaO3S [M + Na]+: 272.0352;
found: 272.0362.
X-ray crystallography
Crystals of compound 6a–c were grown by slow evaporation of
methanol and dichloromethane at room temperature, respectively.
Diffraction intensities for the compounds were collected at 296(2)
K using a Bruker SMART APEX-II CCD area-detector with Mo
Kα radiation (λ = 0.71073 Å). The collected data were reduced with
the SAINT program,12 and multi-scan absorption corrections were
performed using the SADABS program.13 Structures were solved
by direct methods. The compounds were refined against F2 by full-
matrix least-squares methods using the SHELXTL package.14 All
of the non-hydrogen atoms were refined anisotropically. Hydrogen
atoms were placed in calculated positions and constrained to ride on
their parent atoms. The Mercury programs15 were used to describe the
molecular structures. The crystallographic data for the compounds
are summarised in Table 1. Crystallographic data for the compounds
have been deposited with the Cambridge Crystallographic Data Center
(numbers CCDC 1821145, CCDC 1821147 and CCDC 1821148).
Synthesis of ethyl 2-(4-methoxyphenyl)thiazole-4-carboxylate (4)
Compound 3 (1.00 g, 4.02 mmol) was combined with methyl iodide
(8.04 mmol), K2CO3 (1.11 g, 8.04 mmol) and DMF (8 mL). The
reaction mixture was heat to 40 °C for 8 h. The reaction mixture was
cooled and DMF was evaporated under reduced pressure. The residue
was extracted with ethyl acetate (50 mL), filtered and evaporated
under reduced pressure to yield the crude product, which was purified
by column chromatography using petroleum ether and ethyl acetate
(4:1) as an eluent to obtain compound 4 as a white solid: yield 89%;
1
m.p. 93–95 °C ; H NMR (500 MHz, CDCl3): δ 8.11 (s, 1H), 7.97 (d,
J = 8.6 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 4.47 (q, J = 7.1 Hz, 2H),

370 JOURNAL OF CHEMICAL RESEARCH 2018
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Acknowledgements
The financial support of the Natural Science Foundation of
Jiangsu Province (BK20141246), a project funded by Jiangsu
Key Laboratory of marine pharmaceutical compound screening
(2015HYB07, HY201603), research and innovation program
for graduate students of Huaihai Institute of Technology
(XKYCXX2017-13) and a project funded by the priority
academic program development of Jiangsu higher education
institutions are gratefully acknowledged.
5
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Electronic Supplementary Information
The methods and results for the biological test, molecular
modelling study and Hirshfeld surface analysis are available
through:
10 Y. Nicolet, O. Lockridge, P. Masson, J.C. Fontecilla-Camps and F. Nachon,
J. Biol. Chem., 2003, 278, 41141.
http://ingentaconnect.com/content/stl/jcr/2018/00000042/00000007/art00006
11 P. Meena, V. Nemaysh, M. Khatri, A. Manral, P.M. Luthra and M. Tiwari,
Bioorg. Med. Chem., 2015, 23, 1135.
12 Bruker, SMART (version 5.628) and SAINT (version 6.02). Bruker AXS
Inc, Madison, WI, 1998.
13 G.M. Sheldrick, SADABS: program for empirical absorption correction of
area detector. University of Göttingen: Göttingen, 1996.
14 G.M. Sheldrick, SHELXTL V5.1. Software Reference Manual. Bruker
AXS, Madison, WI, 1997.
15 C.F. Macrae, I.J. Bruno, J.A. Chisholm, P.R. Edgington, P. McCabe, E.
Pidcock, L. Rodriguez-Monge, R. Taylor, J. van de Streek and P.A. Wood,
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Received 5 February 2018; accepted 22 June 2018
Paper 1805240
https://doi.org/10.3184/174751918X15314837408346
Published online: 20 July 2018
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