The structure-based optimization of δ-sultone-fused pyrazoles as selective BuChE inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112273

Structure-based optimization was conducted to improve the potency and selectivity of BuChE inhibitors with δ-sulfonolactone-fused pyrazole scaffold. By mimicking the hydrophobic interactions of donepezil at PAS, the introduction of a tertiary benzylamine at 5-position can significantly increase BuChE inhibitory activity. Compounds C4 and C6 were identified as high selective nanomolar BuChE inhibitors (IC₅₀ = 8.3 and 7.7 nM, respectively), which exhibited mild antioxidant capacity, nontoxicity, lipophilicity and neuroprotective activity. Kinetic studies showed that BuChE inhibition of compound C6 was mixed-type against BuChE (K_i = 24 nM) and >2000-fold selectivity for BuChE over AChE. The proposed binding mode of new inhibitors was consistent with the results of structure–activity relationship analysis.

Full text of DOI:10.1016/j.ejmech.2020.112273

Journal Pre-proof
The structure-based optimization of δ-sultone-fused pyrazoles as selective BuChE
inhibitors
Ziwen Zhang, Jingli Min, Mengdie Chen, Xia Jiang, Yingying Xu, Huali Qin, Wenjian
Tang
PII:
S0223-5234(20)30242-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112273
EJMECH 112273
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 February 2020
Revised Date: 20 March 2020
Accepted Date: 23 March 2020
Please cite this article as: Z. Zhang, J. Min, M. Chen, X. Jiang, Y. Xu, H. Qin, W. Tang, The structure-
based optimization of δ-sultone-fused pyrazoles as selective BuChE inhibitors, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112273.
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By the introduction of a tertiary benzylamine, structure-based optimization was
conducted to significantly improve the potency and selectivity of δ-sulfon-fused
pyrazole as BuChE inhibitors. Compounds C4 and C6 showed high selective
nanomolar BuChE inhibitors, mild antioxidant capacity, nontoxicity, lipophilicity and
remarkably neuroprotective activity.

The structure-based optimization of δ-sultone-fused pyrazoles
as selective BuChE inhibitors
Ziwen Zhang a,1, Jingli Min a,1, Mengdie Chen a,1, Xia Jiang a, Yingying Xu a, Huali Qin
b,*, Wenjian Tang a,*
a School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases,
Anhui Medical University, Hefei 230032, China
b School of Chemistry, Chemical Engineering and Life Science, Wuhan University of
Technology, Wuhan 430070, China
Abstract:
Structure-based optimization was conducted to improve the potency and selectivity of
BuChE inhibitors with δ-sulfonolactone-fused pyrazole scaffold. By mimicking the
hydrophobic interactions of donepezil at PAS, the introduction of a tertiary benzylamine
at 5-position can significantly increase BuChE inhibitory activity. Compounds C4 and
C6 were identified as high selective nanomolar BuChE inhibitors (IC50 = 8.3 and 7.7 nM,
respectively), which exhibited mild antioxidant capacity, nontoxicity, lipophilicity and
neuroprotective activity. Kinetic studies showed that BuChE inhibition of compound C6
was mixed-type against BuChE (Ki = 24 nM) and > 2000-fold selectivity for BuChE over
AChE. The proposed binding mode of new inhibitors was consistent with the results of
structure–activity relationship analysis.
Keywords:
Cholinesterase; SuFEx; sultone; pyrazole; structural optimization; Alzheimer’s disease
_________________
*Corresponding author.
E-mail: qinhuali@whut.edu.cn (H.L. Qin); ahmupharm@126.com (W.J. Tang).
1 Z. Zhang, J. Min and M. Chen contributed equally to this work.

1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder associated with
progressive memory loss and cognitive impairments. Nearly 47 million individuals are
affected by Alzheimer's disease in 2018, and 75 million people are affected by 2030
globally [1]. In the past decades, the biological mechanisms on AD widely studied
included amyloid plaques of amyloid-β, tau-protein aggregations, cholinergic dysfunction,
oxidative stress and biometal dyshomeostasis, et al [2-4]. Up till now, the clinical drugs
approved by FDA for AD patients are focused on acetylcholinesterase (AChE) inhibitors
such as donepezil, galantamine and rivastigmine. The nebulous and complicated
pathogenesis of AD leads to failure of many promising drug candidates, which makes
research on AD be still a hot topic [5-8].
Cholinergic system has been extensively studied for the design of anti-AD drugs [9,10].
Physiologically, the activity of ACh in the synapses can be terminated by AChE and
butyrylcholinesterase (BuChE). AChE hydrolyzes the majority of ACh in a healthy brain,
while, with BuChE in the progressive AD takes over the main role of AChE, reaching
almost 80% of the overall ChE activity [11,12]. In the AChE deficient mice, levels of
excessive ACh were alleviated by BuChE activity [13,14], moreover in the
BuChE-knockout mice, BuChE deficience augmented learning capacities and reduced
fibrillar β-amyloid in subcortical structures to lower its toxicity [15-18]. Selective BuChE
inhibition might circumvent the classical cholinergic toxicity of AChE inhibitors,
therefore, BuChE inhibitors have great potentiality for the treatment of AD, especially the
progressive AD [19,20]. Although two ChEs contain a catalytic active site (CAS), a
peripheral anionic site (PAS), and a mid-gorge recognition site between CAS and PAS,
the wider space of BuChE in acyl-binding site allows larger substrate to be recognized
and hydrolyzed [21,22]. The structural features of BuChE help to perform the rational
drug design of selective BuChE inhibitors with novel scaffolds [23-26].
The δ-sultone-fused heterocycles can be efficiently synthesized through sulfur (VI)
fluoride exchange (SuFEx), which is a rapid and powerful click chemistry reaction owing
to the balanced properties of fluoride bonding to hydrogen and sulfur (VI) [27,28].
SuFEx building blocks provided structurally versatile scaffolds in medicinal chemistry.

[29-32]. Recent, a class of novel δ-sulfonolactone-fused pyrazole scaffold was discovered
as highly selective submicromolar BuChE inhibitors [33]. SAR on
7-aryl-4,5-disubstituted δ-sultone-fused pyrazoles showed that (i) C4-, C5-,
N7-substituents affected BuChE inhibition, however compounds with CH2 at C4 position
showed better BuChE inhibition; (ii) the volume of C5-substituent affected BuChE
inhibition; (iii) BuChE inhibition of 2-substituent at N7-phenyl ring was better than that
of 3-substituent; (iv) compound 4a was identified as a most potent BuChE inhibitor (IC50
= 0.20 μM). Moreover, this scaffold showed reversible, mixed-type BuChE inhibitory
activity, which may be used to improve disease symptoms in progressive AD.
The molecular modeling results showed that the active compound is accommodated
into hBuChE via π−S interaction and hydrophobic interactions, and its δ-sultone ring falls
into the hydrolysis sub-pocket of hBuChE active site, however, it is not observed for the
interaction between the δ-sultone-fused pyrazole scaffold and PAS of BuChE. Generally,
a ChE inhibitor (reversible or pseudo-irreversible) contains a basic nitrogen atom crucial
for the interaction with the ChE binding site. Therefore, the inhibitors were designed for
optimal binding to BuChE, and targeting was confirmed through docking analysis [34,35].
Herein, we report the structure-based optimization, synthesis, and evaluation for their
ChE inhibition to further improve the potency and selectivity of this series of BuChE
inhibitors (Fig. 1).

Fig. 1. The design strategy of structural optimization in this study.
2. Results and Discussion
2.1. Chemistry
According to our recent work, δ-sultone-fused pyrazoles as the lead scaffold was used
to synthesize the following compounds (series A–D).
Firstly, to know that C5-/N7-substituents of δ-sultone-fused pyrazoles affected BuChE
inhibition, series A were synthesized as shown in Scheme 1 through the direct annulation
of ethenesulfonyl fluoride (ESF) under equimolar amounts of pyrazolones and NaHCO3
and 5 mol% DBU in DCM at room temperature for 12 h with good yields.
Secondly, to introduce a basic tertiary amine group, ethyl chloroacetoacetate was used
to synthesize the intermediate 5 in dioxane rather than ethanol by condensation reaction.
The key intermediate 6 was efficiently synthesized through Sulfur(VI) Fluoride Exchange
(SuFEx), and was substituted by a secondary amine to give series B and C as shown in
Scheme 2.
Finally, compounds containing different substituent of the benzylamine at 5-positon
were prepared to further study the SAR between the tertiary amine group and BuChE
inhibition, for example series C and D as shown in Scheme 2.
O O
O
S
O
NHNH3Cl
O
O
R2
(i)
(ii)
R1
N
+
N
R2
R1
N
N
R1
OEt
R2
1
2
3
A1−A14
Scheme 1. Synthesis of compounds Series A1–A14
Reaction conditions and reagents: (i) EtOH, AcOH, NaHCO3, reflux, 1.5 h; (ii) ESF
(1.0 equiv.), DBU (30 mol%), CH2Cl2, 12 h, r.t.

O
NHNH3Cl
R1
O
O
N
(i)
Cl
+
N
Cl
OEt
R1
5
1
4
O O
O O
S
S
O
O
R2
N
(ii)
(iii)
N
N
N
N
Cl
R3
R1
R1
6
Series B, C, D
Scheme 2. Synthesis of compounds Series B1–B14, C1–C10 and D1–D10 (for the
intermediate 6, B1: R1 = Cl; B8: R1 = F)
Reaction conditions and reagents: (i) dioxane, 60ºC, 12 h; (ii) ESF (1.0 equiv.), DBU
(30 mol%), CH2Cl2, 12 h, r.t.; (iii) HNR2R3, CH3CN, K2CO3, r.t.
2.2. Inhibition of Equine BuChE and Electrophorus electricus AChE
The δ-sulfonolactone-fused pyrazoles were evaluated for their activity with equine
BuChE (eqBuChE) and Electrophorus electricus AChE (EeAChE), using modified
Ellman’s method.
Table 1. ChE inhibitory activity of compounds A1–A14. a
O O
S
1
3
4
O
2
7
N
N
6
R2
R1
5
IC50, µM (or inhibition% at 20 µM)
Compd.
R1
R2
AChE b
na d
BuChE c
1.19±0.47
0.18±0.01
2-F
Me
A1
A2
2-Cl
cyclopropyl
na d

3-Cl
2-F
cyclopropyl
cyclopropyl
cyclopropyl
phenyl
na d
na d
na d
na d
na d
na d
na d
na d
na d
na d
na d
na d
0.70±0.34
0.14±0.02
0.37±0.09
0.18±0.04
0.20±0.05
0.47±0.24
0.16±0.04
0.17±0.03
1.58±0.38
3.69±2.90
0.13±0.07
8.78±0.52
A3
A4
3-F
A5
2-F
A6
2-F
4-Cl-phenyl
4-Br-phenyl
4-F-phenyl
phenyl
A7
2-F
A8
2-F
A9
2-Cl
2-Cl
2-Cl
2-Cl
3-Cl
A10
A11
A12
A13
A14
4-Cl-phenyl
4-Br-phenyl
4-F-phenyl
4-MeO-phenyl
a Each IC50 value is the mean ± SEM from at least three independent experiments.
b AChE from electric eel. na, no inhibitory activity (%) against EeAChE at 20 µM.
c
BuChE from horse serum. Positive control compounds had the following results
(compound: EeAChE IC50, eqBuChE IC50): donepezil: 0.025 µM, 10.38 µM;
rivastigmine: > 20 µM, 0.56 µM.
d na = no inhibitory activity
As shown in Table 1, compounds A1–A10 and A13 (series A) exhibited the
submicromolar inhibitory effect on BuChE (IC50 values: 0.1–0.7 μM). Compounds with
2-substituent at R1 position showed better BuChE inhibition than those with the
corresponding 4-/3-substituent (A2 > A3; A4 > A5). For the 2-substituted group of
N7-benzene ring, the activity of 2-F substituent is superior to that of 2-Cl substituent (A4
> A2; A7 > A11; A8 > A12; A9 > A13; except for A10 ≈ A6). Moreover, for the

substituent on the benzene ring at R2 position, the order of BuChE inhibitory activity is
4-F > H > 4-Cl > 4-Br (for example, A9 > A6 > A7 > A8 for R1 = 2-F; A13 > A10 > A11
> A12 for R1 = 2-Cl). Based on the above SAR analysis, it was obvious that compounds
with 2-F or 2-Cl at R1 position exhibited better BuChE inhibitory activity. Due to a basic
group being important for ChE inhibitor, the introduction of a tertiary amine at R2
position may improve the BuChE inhibitory activity. Therefore, series B were
synthesized and evaluated for their ChE activity.
Table 2. ChE inhibitory activity of compounds B1–B14. a
O O
S
1
O
3
4
7
N
R2
N
N
R3
R1
5
IC50, µM (or inhibition % at 20 µM)
Compd.
R1
NR2R3
AChE b
BuChE c
Cl
CH3
2-Cl
2-Cl
na d
0.20±0.01
B1
B2
na d
na d
na d
0.07±0.02
0.05±0.02
0.04±0.02
N CH2Ph
CH2CH3
2-Cl
2-Cl
B3
B4
N CH2Ph
CH2CH2OH
N CH2Ph
na d
na d
0.41±0.10
2.22±0.63
N
O
2-Cl
2-Cl
B5
B6
B7
2-Cl
na d
0.46±0.23
N
NCH2Ph

na d
na d
0.19±0.15
0.08±0.05
Cl
CH3
2-F
2-F
B8
B9
N CH2Ph
CH2CH3
2-F
2-F
na d
na d
0.08±0.01
0.04±0.02
B10
B11
N CH2Ph
CH2CH2OH
N CH2Ph
na d
na d
na d
0.15±0.06
2.17±1.01
0.68±0.11
N
O
2-F
2-F
2-F
B12
B13
B14
N
NCH2Ph
a Each IC50 value is the mean ± SEM from at least three independent experiments.
b AChE from electric eel. na, no inhibitory activity (%) against EeAChE at 20 µM.
c
BuChE from horse serum. Positive control compounds had the following results
(compound, EeAChE IC50, eqBuChE IC50): donepezil, 0.025 µM, 10.38 µM;
rivastigmine, > 60 µM, 0.56 µM.
d na = no inhibitory activity
As shown in Table 2, when both R2 and R3 are aliphatic substituent, the activity isn’t
improved compared to those of series A. The introduction of an aromatic group increased
the activity (B7 > B6, B14 > B13). According to the structure characteristics of donepezil,
the introduction of a tertiary benzylamine at 5-position can significantly increase BuChE
inhibitory activity. Some of δ-sultone-fused pyrazoles (series B) exhibited nanomolar
inhibition against BuChE. Another substituent of a tertiary benzylamine affected BuChE
inhibition, for example, the order of IC50 values for BuChE inhibition is hydroxyethyl >
ethyl > methyl (B4 > B3 > B2 for R1 = 2-Cl; B11 > B10 > B9 for R1 = 2-F). To further
study the relationship between the substituted benzyl and BuChE inhibition, a series of
substituted phenyl-methyl derivatives were prepared and evaluated their ChE inhibitory

activity (Table 3).
Table 3. ChE inhibitory activity of compounds C1–C10. a
IC50, µM (or inhibition% at 20 µM)
RP of DPPH
assay d
Compd.
R1
AChE b
na e
BuChE c
0.09±0.08
3-F
3-Cl
5.0%
4.7%
0.8%
5.5%
10.2%
22.2%
1.8%
5.2%
3.2%
5.5%
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
na e
0.19±0.10
3-Br
na e
0.33±0.17
4-F
na e
0.0083±0.0015
0.09±0.05
4-Cl
na e
4-Br
43.3±4.7%
na e
0.0077±0.0006
0.09±0.05
2,4-Cl
4-CH3
4-OCH3
3,4-OCH3
na e
0.17±0.09
na e
0.26±0.13
na e
0.56±0.36
a Each IC50 value is the mean ± SEM from at least three independent experiments.
b AChE from electric eel. na, no inhibitory activity (%) against EeAChE at 20 µM.
c
BuChE from horse serum. Positive control compounds had the following results
(compound, EeAChE IC50, eqBuChE IC50): donepezil, 0.025 µM, 10.38 µM;
rivastigmine, > 60 µM, 0.56 µM.
d RP of 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) (%)ꢀ=ꢀreduction percentage of DPPH,

compounds at a concentration of 100ꢀμM.
e na = no inhibitory activity
As shown in Table 3, the substituent of benzene ring at 5-positon of scaffold affected
the BuChE inhibitory activity: (i) the activity of compounds with 4-halogen substituent is
superior to that of compounds with 3-halogen substitution (C4 > C1; C5 > C2; C6 > C3);
(ii) compounds with 4-halogen substituent exhibited better BuChE inhibition (C4 > C7,
C5 > C7, C6 > C7; C4 > C8, C5 > C8, C6 > C8); (iii) compounds C4 (4-F) and C6
(4-Br) had the best BuChE activity (IC50 = 8.3 and 7.7 nM, respectively).
Table 4. ChE inhibitory activity of compounds D1–D10. a
O O
S
1
3
4
O
R1
N
7
N
R2
N
5
F
IC50, µM (or inhibition% at 20 µM)
RP of DPPH
assay d
Compd.
R1
R2
AChE b
na e
BuChE c
-CH2CH2OCH3
-CH2CH2OCH3
-CH2CH2OCH3
-CH2CH2OCH3
-CH2CH3
-H
-F
D1
D2
D3
D4
B11
D5
D6
D7
0.24±0.12
0.11±.0.09
0.09±0.04
0.15±0.02
0.08±0.01
0.07±0.01
0.04±0.01
0.07±0.01
2.3%
6.3%
3.6%
4.8%
5.4%
0.6%
4.2%
12.9%
na e
-Cl
-Br
-H
na e
na e
na e
-CH2CH3
-F
na e
-CH2CH3
-Cl
-Br
na e
-CH2CH3
na e

-CH3
-CH3
-CH3
-CH3
-H
-F
na e
na e
B10
D8
0.08±0.05
0.04±0.02
0.02±0.01
0.07±0.02
1.8%
5.7%
6.1%
0.2%
-Cl
-Br
na e
D9
33.6±14%
D10
a Each IC50 value is the mean ± SEM from at least three independent experiments.
b AChE from electric eel. na, no inhibitory activity (%) against EeAChE at 20 µM.
c
BuChE from horse serum. Positive control compounds had the following results
(compound, EeAChE IC50, eqBuChE): donepezil, 0.025 µM, 10.38 µM; rivastigmine, >
60 µM, 0.56 µM.
d
RP of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) (%)ꢀ=ꢀreduction percentage of DPPH,
compounds at a concentration of 100ꢀμM.
e na = no inhibitory activity
The data in Table 4 showed that BuChE inhibition was affected by another substituent
(R1) of the benzylamine at 5-positon: (i) the activity significantly decreased when the
hydroxyethyl group was methylated (B11 > D1; C4 > D2; C5 > D3; C6 > D4); (ii)
compounds B9, B10, D5–D10 (R1 = -Me or -Et) with smaller substituent exhibited
similar BuChE inhibitory activity (IC50 values for 24 – 78 nM). In all in, by simulating
the structural characteristics of Donepezil, introduction of a tertiary benzylamine group
significantly increased BuChE inhibitory activity of δ-sultone-fused pyrazoles to
nanomole level.
2.3. Molecular docking model of C6 with hBuChE
It has been confirmed that there are two flexible amino acids Leu286 and Val288 in
the acyl pocket of hBuChE compared to hAChE. Simultaneously, the two flexible amino
acids allows binding of bulkier ligands which may, therefore, be selective for huBuChE
[36]. A docking model of hBuChE with compound C6 (Fig. 2A and 2B) showed that the
tertiary benzylamine is extended by a hydroxyethyl chain, and compound C6 is nicely

bound to hBuChE via a strong hydrogen bond interaction between the the hydroxyl unit
with Asn68, p–π interaction between the benzyl ring with Leu286, Val288 and Phe329 in
the acyl subpocket, and a π–anion interaction between the S atom of sultone ring with
His438 in the hydrolysis sub-pocket. In addition, a π–anion interaction is observed
between the pyrazole ring at N7 position with Asp70
.
Fig. 2. (A) 3D mode of the pocket surface of compound C6 with receptor hBuChE (PDB
ID: 5LKR) performed using Discovery Studio Client v18.1.0. (B) 2D mode of the
interaction of compound C6 with receptor hBuChE (conventional H bond and C–H bond,
halogen, π–anion, alkyl, and π–alkyl are represented by green, light green, brown, pink
and light pink lines, respectively) performed using Discovery Studio Client v18.1.0.
2.4. DPPH radical scavenging activity
1,1-Diphenyl-2-picrylhydrazyl (DPPH) is an extremely effective free radical
scavenger that can be used to monitor a chemical reaction that involves free radicals
[37,38]. The DPPH assay was performed with ascorbic acid and donepezil as reference
antioxidants to evaluate the ability of the synthesized compounds to scavenge activated
oxygen species. As shown in Tables 3 and 4, some of the synthesized compounds
exhibited mild free radical scavenging activity with the RP of DPPH for 0.2%-22.2% at a
concentration of 100ꢀμM, amongst them, compound C6 showed the best antioxidant

activity, while methylation of ethoxyl group (D4) led its activity to decrease.
2.5. Kinetic study of eqBuChE inhibition
Compound C6, the derivative with the highest inhibitory activity, was subjected to
enzyme kinetics analysis to determine the kinetics of BuChE inhibition. As shown in Fig.
3A, the plots of the remaining enzyme activity versus the concentration of enzyme (0,
0.045, 0.090, 0.18, 0.36 and 0.72 U/mL) in the presence of different concentrations of
compound C6 for the catalysis of butyrylcholine gave a series of straight lines. In case of
compound C6 all the lines intersected at the same point. Increasing the inhibitor
concentration resulted in a decrease in the slope of the lines, which indicated that
compound C6 were reversible BuChE inhibitors. The slopes and intercepts of the
reciprocal Lineweaver–Burk plot presented in Fig. 3B increased with the increase in
inhibitor concentration. The intersection of each trend line in the fourth quadrant
indicated a mixed-type inhibitory mode. As shown in Fig. 3C, the dissociation constants
Ki for compound C6 from the Lineweavere-Burk secondary plots were estimated to be 24
nM.
Kinetic studies of eqBuChE inhibition Kinetic studies were performed with the same
test conditions, using six concentrations of substrate (0.1-1 mM) and four concentrations
of inhibitor (0-0.08μM). Apparent inhibition constants and kinetic parameters were
calculated within the “Enzyme kinetics” module of Prism 5. The effect of concentrations
of compound C6 on the activity for the catalysis of BuChE at 37ºC was also studied.
Assay conditions were same as described in assay protocol except that the final
concentration of enzyme was varied (0-0.72 U/mL). Concentrations of compound C6 (0,
0.02, 0.04, 0.08 μM) were used respectively, for the determination of reversible as well as
irreversible binding of inhibitors at enzyme.

Fig. 3. Relationship between eqBuChE inhibition and various concentrations of
compound C6 (A). Lineweaver-Burk plots of eqBuChE inhibition kinetics of compound
C6 (B). The Lineweaver-Burk secondary plots of compound C6 (C). Reciprocals of
enzyme activity (eqBuChE) vs reciprocals of substrate (butyrylthiocholine iodide) with
different concentrations (0-0.08 μM) of inhibitor.
2.6. Comparison of oil/water partition coefficient assay and ADMET prediction with hit
The physicochemical parameters of a drug are correlated with the membrane
permeability of the body. Lipinski's rule of five is used as the rule of thumb when
assessing whether a compound can be used as an oral drug [37,38]. For example, the
oil/water partition coefficient, which is expressed as a log P value, can reflect the
absorption of a drug in an organism [39]. The oil/water partition coefficients of

compounds that showed strong potency in bioactivity assessment were measured. As an
important parameter, the log P (o/w) (octanol-water partition coefficient) with the
optimum central nervous system penetration was around 2.0 ± 0.7 [40,41]. Meanwhile,
the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of
the selected five compounds were also predicted by using Discovery Studio Client
v18.1.0 [42]. The different descriptors of ADMET characteristics have different
prediction levels. Table 5 shows that the log P values of compounds B9, B10, B11, C4,
and C6 ranged from 1.78 to 2.42. These results indicate that, according to the hit
compound, the modified compounds still retains good lipophilicity (log P < 5). These
compounds and the hit compound likely demonstrate good absorption in the human
intestine (HIA levels of 0), good solubility in water at 25ºC (solubility levels of 1 and 2)
and moderate blood-brain barrier permeability. Moreover, the results show that these
compounds are noninhibitors of CYP2D6 (CYP2D6 level of 0) and are highly likely to
bind to plasma proteins.
Table 5. Log P values and ADMET properties of active compounds.
Compound
Hit
Log P a
2.09
1.78
2.00
2.05
2.42
1.93
Ab
0
Dc
2
Md
0
Ee
1
ALogP98f
3.06
PSA2Dg
60.14
2
1
1
2
2
2
0
2
0
1
3.891
4.24
63.493
63.493
84.308
84.308
84.308
B9
0
1
0
1
B10
B11
0
1
0
1
3.352
4.016
3.557
0
1
0
1
C4
0
1
0
1
C6
a Octanol–water partition coefficients of some compounds were measured by the shake
flask method with slight modification.
b Absorption: Intestinal absorption.
c Distribution: Aqueous solubility and blood-brain barrier penetration.
d Metabolism: CYPA2D6.

e Excretion: Plasma protein binding.
f ALogP98: Predicted octanol/water.
g PSA: polar surface area, 2D: two-dimensional.
2.7. Cytotoxicity assays and neuroprotective study
To examine the cytotoxicity of the representative compound C6, the inherent toxicity
towards human hepatoblastoma HepG2 cells and human normal liver cells LO2 were
studied using the MTT assay at four different concentrations of compound C6 ranging
from 5~50 μM [43]. As shown in Fig. 4A and Fig. 4B, the viability of the modified cells
of C6 was almost absent at the concentrations of 10 μM and 25 μM, respectively. With
increasing concentrations to 50 µM, C6 decreased cell viability (82.8% and 95.3%,
respectively). The results show that the target compound C6 has a wide range of
therapeutic safety for HepG2 cells and LO2 cells. In addition, these preliminary results
should encourage further research to explain the neuroprotective mechanisms of
compound C6.
Fig. 4. Cytotoxicity of donepezil, compound C6 tested at concentrations in the range
1–50 µM in HepG2 (A) cells and LO2 (B) cells for 24 h. Untreated cells were used as
control. Results are expressed as percentage of cell survival vs untreated cell (control)
and shown as mean ± SD (n = 3)
The protective effects of compound C6 against free radicals damage were assessed by
measuring the ability of the compound to protect against H2O2 injury according to the
reported protocol [44]. After 100 µM H2O2 exposure, cell viability as determined by

MTT reduction was obviously decreased to 39.8 % (P < 0.01 vs control), manifesting
high sensitivity to H2O2-induced injury. However, compound C6 showed protective
effects in a dose-dependent manner against H2O2-induced PC12 cell injury (Fig. 5). At a
concentration of 25 µM, compound C6 showed neuroprotective effects and was slightly
stronger than the positive control donepezil, with a cell viability of 49.6%. When the
concentration was reduced to 6.25 µM, the cell viabilities decreased to 40.9%. It clarified
that the compound C6 could capture the hydroxyl radical, generated by H2O2.
Fig. 5. Neuroprotective effect on PC12 neurons of compound C6. Results represent
mean ± SEM (n = 3). Statistical significance was calculated using one-way ANOVA and
Bonferroni post hoc tests. ###p < 0.001 compared with the control group; *p < 0.05
compared with H2O2 group; **p < 0.01 compared with H2O2 group; ***p < 0.001
compared with H2O2 group.
3. Conclusion
In summary, the structure-based optimization of δ-sulfonolactone-fused pyrazole
scaffold was carried out to improve the potency and selectivity of BuChE inhibitors, and
48 compounds were designed and synthesized through the stepwise guidance of
structure-activity relationship. By mimicking the hydrophobic interactions of donepezil at
PAS, the introduction of a tertiary benzylamine at 5-position can significantly increase
BuChE inhibitory activity. Compounds C4 and C6 with 4-F/4-Br-substituted

benzylamine at C-5 were identified as high selective nanomolar BuChE inhibitors (IC50 =
8.3 and 7.7 nM, respectively). The scavenging ability of compound C6 has mild values
(22.2% for DPPH) comparing to ascorbic acid at a concentration of 100ꢀμM. Kinetic
studies indicate that the inhibition of BuChE by compound C6 is reversible and mixed
competitive (Ki = 24 nM). Compound C6 has been found to be non-toxic to HepG2 cells
and LO2 cells, up to 50 μM, with good predictions of ADMET properties, and showing
significant neuroprotective activity. Molecular docking showed that compound C6 was
nicely bound to hBuChE via a strong hydrogen bond interaction with Asn68,
p–π interaction with Leu286, Val288 and Phe329, and a π–anion interaction with His438,
Asp70. Compound C6 has significant potential and can be further developed as a
promising therapy for AD treatment.
4. Experimental section
4.1. Chemistry
4.1.1. General Information
All chemicals, reagents and solvents were purchased from commercial sources and used
without further purification. Reactions were checked by thin-layer chromatography (TLC)
on precoated silica gel plates (Qingdao Marine Chemical Factory, GF254); spots were
visualized by UV at 254 nm. Melting points are determined on a XT4MP apparatus (Taike
Corp., Beijing, China) and are not corrected. The purity (relative content) of active
compounds was determined by HPLC through area normalization method. 1H NMR and
13C NMR spectra were recorded on Bruker AV-300, AV-400 or AV-600 MHz instruments
using DMSO-d6 and CDCl3 as solvent. Chemical shifts are reported in parts per million (δ)
downfield from the signal of tetramethylsilane (TMS) as internal standards. Coupling
constants are reported in Hz. The multiplicity is defined by s (singlet), d (doublet), t
(triplet), or m (multiplet). High resolution mass spectra (HRMS) were obtained on an
Agilent 1260-6221 TOF mass spectrometry.
4.1.2. General procedure for the preparation of compounds 6 (B8) and A1–A14
To a solution of phenylhydrazine hydrochloride (10.0 mmol) in ethanol (20 mL) was

added saturated NaHCO3 solution (2 mL), acetic acid (5 mL) and ethyl acetoacetate (10.0
mmol), the reaction was heated for 1.5 h at reflux. The mixture was poured into EtOAc
(100 mL) and was washed with water and saline solution sequentially, dried over
anhydrous MgSO4 for 2 h, filtrated, concentrated to 10 mL, then allowed to stand
overnight in cold storage. The mixture was collected by filtration to give the title product
3.
Ethyl chloroacetoacetate (10.0 mmol) were added to the dioxane (20 mL) solution of
phenylhydrazine hydrochloride (10.0 mmol) and the reaction was heated to reflux for 12
h. The mixture was poured into EtOAc (100 mL) and was washed with water and saline
solution sequentially, dried over anhydrous MgSO4 for 2 h, filtrated, concentrated to 10
mL, then allowed to stand overnight in refrigeration. The mixture was filtered and
collected to get the title product 5.
To a solution of an oven-dried reaction tube (20 mL) was charged with ethenesulfonyl
fluoride (ESF, 0.5 mmol) and the pyrazolone (3 or 5, 0.5 mmol, 1.0 equiv), DBU (180
mg dissolved in 10 mL CH2Cl2, 2 mL, 30 mol%), and NaHCO3 (42 mg, 0.5 mmol, 1.0
equiv). The resulting mixture was stirred at room temperature for 12 h with monitoring
by TLC. The crude products were purified by column chromatography on silica gel
(petroleum ether / EtOAc, 10: 1 to 8: 1) to give the title products 6 and A1–A14.
4.1.2.1
7-(2-fluorophenyl)-5-methyl-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
1
2,2-dioxide (A1). Yellow powder, yield 71%; m.p. 132-133ºC; H NMR (400 MHz,
CDCl3) δ 7.54 – 7.33 (m, 2H), 7.30 – 7.13 (m, 2H), 3.50 (t, J = 6.6 Hz, 2H), 3.12 (t, J =
6.5 Hz, 2H), 2.26 (s, 3H).
4.1.2.2
7-(2-chlorophenyl)-5-cyclopropyl-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (A2). Light yellow powder, yield 73%; m.p. 112-113ºC; 1H NMR (400 MHz,
CDCl3) δ 7.66 (t, J = 2.0 Hz, 1H), 7.52 (dd, J = 8.2, 1.1 Hz, 1H), 7.35 (t, J = 8.1 Hz, 1H),
7.26 (s, 1H), 3.52 (t, J = 6.6 Hz, 2H), 3.21 (t, J = 6.6 Hz, 2H), 1.90 – 1.65 (m, 1H), 1.09 –
0.79 (m, 4H). TOF-HRMS: m/z [M + H]+ calcd for C14H13ClN2O3S: 325.0408; found:
325.0402.

4.1.2.3 7-(3-chlorophenyl)-5-cyclopropyl-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (A3). Light yellow powder, yield 61%; m.p. 182-183ºC; 1H NMR (400 MHz,
CDCl3) δ 7.50 (t, J = 4.8 Hz, 1H), 7.44 – 7.32 (m, 3H), 3.50 (t, J = 6.5 Hz, 2H), 3.22 (t, J
= 6.5 Hz, 2H), 1.85 – 1.72 (m, 1H), 0.98 – 0.89 (m, J = 3.8, 2.2 Hz, 4H). TOF-HRMS:
m/z [M + H]+ calcd for C14H13ClN3O2S: 325.0408; found: 325.0402.
4.1.2.4 5-cyclopropyl-7-(2-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (A4). Light yellow powder, yield 81%; m.p. 152-153ºC; 1H NMR (400 MHz,
CDCl3) δ 7.48 – 7.32 (m, J = 8.4, 7.8, 4.2 Hz, 2H), 7.26 – 7.15 (m, J = 10.4, 4.3 Hz, 2H),
3.50 (t, J = 6.5 Hz, 2H), 3.21 (t, J = 6.5 Hz, 2H), 1.85 – 1.70 (m, J = 6.7 Hz, 1H), 1.06 –
0.82 (m, J = 4.0, 2.2 Hz, 4H). TOF-HRMS: m/z [M + H]+ calcd for C14H13FN2O3S:
309.0704; found: 309.0704.
4.1.2.5 5-cyclopropyl-7-(3-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (A5). Light yellow powder, yield 75%; m.p. 122-123ºC; 1H NMR (400 MHz,
CDCl3) δ 7.52 – 7.48 (m, J = 7.8, 1.8 Hz, 1H), 7.44 – 7.29 (m, 3H), 3.50 (t, J = 6.5 Hz,
2H), 3.22 (t, J = 6.5 Hz, 2H), 1.93 – 1.70 (m, 1H), 1.05 – 0.82 (m, 4H). TOF-HRMS: m/z
[M + H]+ calcd for C14H13FN2O3S: 309.0704; found: 309.0698.
4.1.2.6
7-(2-fluorophenyl)-5-phenyl-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (A6). Light yellow powder, yield 71%; m.p. 141-142ºC; 1H NMR (400 MHz,
CDCl3) δ 7.73 – 7.66 (m, 2H), 7.60 – 7.51 (m, J = 7.7, 1.7 Hz, 1H), 7.48 – 7.34 (m, 4H),
7.31 – 7.20 (m, 2H), 3.51 (t, J = 6.4 Hz, 2H), 3.38 (t, J = 6.5 Hz, 2H). TOF-HRMS: m/z
[M + H]+ calcd for C17H13FN2O3S: 345.0704; found: 345.0701.
4.1.2.7
5-(4-chlorophenyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (A7). Light yellow powder, yield 74%; m.p. 105-106ºC; 1H NMR (300 MHz,
CDCl3) δ 7.73 – 7.61 (m, 2H), 7.62 – 7.38 (m, 4H), 7.36 – 7.14 (m, 2H), 3.56 (t, J = 6.4

Hz, 2H), 3.41 (t, J = 6.3 Hz, 2H). TOF-HRMS: m/z [M + H]+ calcd for C17H12ClFN2O3S:
379.0314; found: 379.0314.
4.1.2.8
5-(4-bromophenyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (A8). Light yellow powder, yield 82%; m.p. 136-137ºC; 1H NMR (300 MHz,
CDCl3) δ 7.65 – 7.41 (m, 6H), 7.39 – 7.23 (m, 2H), 3.56 (t, J = 6.4 Hz, 2H), 3.41 (t, J =
6.5 Hz, 2H). TOF-HRMS: m/z [M + H]+ calcd for C17H12BrFN2O3S: 422.9809; found:
422.9808.
4.1.2.9
7-(2-fluorophenyl)-5-(4-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
1
2,2-dioxide (A9). Yellow powder, yield 91%; m.p. 142-145ºC; H NMR (400 MHz,
CDCl3) δ 7.73 – 7.66 (m, 2H), 7.58 – 7.52 (m, 1H), 7.49 – 7.42 (m, 1H), 7.29 (t, J = 7.7
13
Hz, 2H), 7.14 (t, J = 8.7 Hz, 2H), 3.55 (t, J = 6.4 Hz, 2H), 3.40 (t, J = 6.4 Hz, 2H). C
NMR (101 MHz, CDCl3) δ 163.18, 156.31, 148.56, 146.05, 131.03, 128.78, 128.69(2C),
128.16, 125.03, 124.29, 117.12, 116.08(2C), 93.85, 44.87, 20.08. TOF-HRMS: m/z [M +
H]+ calcd for C17H12F2N2O3S: 363.0609; found: 363.0604.
4.1.2.10
7-(2-chlorophenyl)-5-phenyl-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
1
2,2-dioxide (A10). Yellow powder, yield 78%; m.p. 104-106ºC; H NMR (400 MHz,
CDCl3) δ 7.73 (d, J = 7.1 Hz, 2H), 7.54 (ddd, J = 14.6, 7.4, 2.0 Hz, 2H), 7.48 – 7.37 (m,
13
5H), 3.54 (t, J = 6.3 Hz, 2H), 3.44 (t, J = 6.2 Hz, 2H). C NMR (101 MHz, CDCl3) δ
149.02, 146.12, 134.04, 132.52, 132.00, 131.14, 130.71, 129.64 (2C), 129.01, 128.85,
127.86, 126.92 (2C), 93.64, 45.00, 20.18. TOF-HRMS: m/z [M + H]+ calcd for
C17H13ClN2O3S: 361.0408; found: 361.0402.
4.1.2.11
7-(2-chlorophenyl)-5-(4-chlorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
1
2,2-dioxide (A11). Yellow powder, yield 83%; m.p. 172-173ºC; H NMR (400 MHz,
CDCl3) δ 7.70 – 7.63 (m, 2H), 7.56 (dd, J = 7.8, 1.7 Hz, 1H), 7.53 – 7.37 (m, 5H), 3.55 (t,

J = 6.4 Hz, 2H), 3.43 (t, J = 6.4 Hz, 2H). TOF-HRMS: m/z [M + H]+ calcd for
C17H12Cl2N2O3S: 395.0018; found: 395.0019.
4.1.2.12
5-(4-bromophenyl)-7-(2-chlorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
1
2,2-dioxide (A12). Yellow Powder, yield 68%; m.p. 110-112ºC; H NMR (400 MHz,
CDCl3) δ 7.66 – 7.53 (m, 5H), 7.53 – 7.38 (m, 3H), 3.55 (t, J = 6.5 Hz, 2H), 3.42 (t, J =
6.4 Hz, 2H). TOF-HRMS: m/z [M + H]+ calcd for C17H12BrClN2O3S: 438.9513; found:
438.9508.
4.1.2.13
7-(2-chlorophenyl)-5-(4-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (A13). Light yellow powder, yield 81%; m.p. 141-143ºC; 1H NMR (400 MHz,
CDCl3) δ 7.75 – 7.64 (m, 2H), 7.56 (dd, J = 7.8, 1.6 Hz, 1H), 7.51 (dd, J = 7.4, 2.1 Hz,
1H), 7.44 (td, J = 7.4, 1.9 Hz, 2H), 7.14 (t, J = 8.7 Hz, 2H), 3.55 (t, J = 6.5 Hz, 2H), 3.42
(t, J = 6.4 Hz, 2H). TOF-HRMS: m/z [M + H]+ calcd for C17H12ClFN2O3S: 379.0314;
found: 379.0313.
4.1.2.14
7-(4-chlorophenyl)-5-(4-methoxyphenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
1
2,2-dioxide (A14). Light yellow powder, yield 61%; m.p. 92-95ºC; H NMR (600 MHz,
CDCl3) δ 7.78 – 7.57 (m, 4H), 7.48 – 7.41 (m, 2H), 7.03 – 6.95 (m, 2H), 3.86 (s, 3H),
3.55 (t, J = 6.5 Hz, 2H), 3.40 (t, J = 6.5 Hz, 2H). TOF-HRMS: m/z [M + H]+ calcd for
C18H15ClN2O4S: 391.0514; found: 391.0507.
4.1.3. General procedure for the preparation of compounds B1–B14
To a solution of compound 6 (1 mmol) in MeCN (20 mL) was added the secondary
amine (HNR2R3, 1.2 equiv) and K2CO3 (1.2 equiv). The suspension solution was stirred
at room temperature for 12 h, and subjected to column chromatography on silica gel
(CH2Cl2 / methanol, 50: 1) to obtain the title products B1–B14.

4.1.3.1
5-(chloromethyl)-7-(2-chlorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
1
2,2-dioxide (B1). Yellow powder, yield 51%; m.p. 192-195ºC; H NMR (400 MHz,
CDCl3) δ 7.56 – 7.52 (m, 1H), 7.47 – 7.37 (m, 3H), 4.60 (s, 2H), 3.53 (t, J = 6.6 Hz, 2H),
3.30 (t, J = 6.5 Hz, 2H). TOF-HRMS: m/z [M + H]+ calcd for C12H10Cl2N2O3S: 332.9862;
found: 332.9863.
4.1.3.2
5-((benzyl(methyl)amino)methyl)-7-(2-chlorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-
1
c]pyrazole 2,2-dioxide (B2). Colorless oil, yield 51%; H NMR (600 MHz, CDCl3) δ
7.52 (d, J = 8.1 Hz, 1H), 7.43 – 7.26 (m, 8H), 3.57 (s, 2H), 3.55 (s, 2H), 3.46 (t, J = 6.6
13
Hz, 2H), 3.21 (t, J = 6.5 Hz, 2H), 2.26 (s, 3H). C NMR (101 MHz, CDCl3) δ 148.41,
145.84, 138.76, 133.96, 132.11, 131.01, 130.64, 129.51 (2C), 129.38 (2C), 128.53,
127.77, 127.49, 95.12, 62.40, 54.59, 45.17, 42.65, 18.69. TOF-HRMS: m/z [M + H]+
calcd for C20H20ClN3O3S: 418.0987; found: 418.0992.
4.1.3.3
5-((benzyl(ethyl)amino)methyl)-7-(2-chlorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]
1
pyrazole 2,2-dioxide (B3). Colorless oil, yield 53%; H NMR (400 MHz, CDCl3) δ 7.52
(dd, J = 5.1, 4.5 Hz, 1H), 7.45 – 7.36 (m, 3H), 7.36 – 7.26 (m, 5H), 3.73 – 3.65 (m, J =
10.7, 6.3 Hz, 6H), 3.43 (t, J = 6.5 Hz, 2H), 3.06 (t, J = 6.5 Hz, 2H), 2.78 (t, J = 5.3 Hz,
2H). 13C NMR (101 MHz, CDCl3) δ 147.82, 145.67, 138.22, 133.65, 131.83, 130.90,
130.51, 129.29 (2C), 129.27 (2C), 128.48, 127.63, 127.48, 94.76, 58.99, 58.70, 55.84,
50.89, 44.80, 18.32. TOF-HRMS: m/z [M + H]+ calcd for C21H22ClN3O4S: 448.1092;
found: 448.1089.
4.1.3.4
5-((benzyl(2-hydroxyethyl)amino)methyl)-7-(2-chlorophenyl)-4,7-dihydro-3H-[1,2]oxathi
1
ino[6,5-c]pyrazole 2,2-dioxide (B4). Colorless oil, yield 68%; H NMR (400 MHz,
CDCl3) δ 7.51 (d, J = 7.9 Hz, 1H), 7.45 – 7.32 (m, 6H), 7.26 (s, 2H), 3.60 (s, 4H), 3.43 (t,

J = 6.4 Hz, 2H), 3.19 (t, J = 6.3 Hz, 2H), 2.58 (d, J = 6.7 Hz, 2H), 1.11 (t, J = 6.9 Hz, 3H).
TOF-HRMS: m/z [M + H]+ calcd for C21H22ClN3O3S: 432.1143; found: 432.1145.
4.1.3.5
7-(2-chlorophenyl)-5-(morpholinomethyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (B5). Colorless oil, yield 63%; 1H NMR (400 MHz, CDCl3) δ 7.55 – 7.49 (m,
1H), 7.45 – 7.34 (m, 3H), 3.76 – 3.69 (m, 4H), 3.55 (s, 2H), 3.50 (t, J = 6.5 Hz, 2H), 3.27
(t, J = 6.5 Hz, 2H), 2.51 (s, 4H). TOF-HRMS: m/z [M + H]+ calcd for C16H18FN3O3S:
384.0779; found: 384.0778.
4.1.3.6
7-(2-chlorophenyl)-5-((4-methylpiperazin-1-yl)methyl)-4,7-dihydro-3H-[1,2]oxathiino[6,
1
5-c]pyrazole 2,2-dioxide (B6). Colorless oil, yield 55%; H NMR (400 MHz, CDCl3) δ
7.52 (d, J = 7.8 Hz, 1H), 7.47 – 7.32 (m, 3H), 3.57 (s, 2H), 3.48 (t, J = 6.5 Hz, 2H), 3.26
(t, J = 6.4 Hz, 2H), 2.52 (d, J = 28.1 Hz, 8H), 2.30 (s, J = 13.3 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 147.70, 145.74, 133.92, 131.98, 131.00, 130.60, 129.49, 127.75, 95.07,
55.66, 55.17 (2C), 52.86 (2C), 45.92, 45.12, 18.78. TOF-HRMS: m/z [M + H]+ calcd for
C17H21ClN4O3S: 397.1096; found: 397.1092.
4.1.3.7
5-((4-benzylpiperazin-1-yl)methyl)-7-(2-chlorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,
1
5-c]pyrazole 2,2-dioxide (B7). Colorless oil, yield 54%; H NMR (400 MHz, CDCl3) δ
7.51 (d, J = 7.7 Hz, 1H), 7.46 – 7.34 (m, J = 16.8, 7.4 Hz, 3H), 7.34 – 7.27 (m, J = 14.5,
4.0 Hz, 5H), 3.55 (d, J = 15.1 Hz, 4H), 3.48 (t, J = 6.5 Hz, 2H), 3.26 (t, J = 6.5 Hz, 2H),
2.53 (s, 8H). 13C NMR (101 MHz, CDCl3) δ 147.51, 145.55, 137.44, 133.74, 131.82,
130.81, 130.42, 129.34 (2C), 129.31 (2C), 128.27, 127.56, 127.23, 94.95, 62.89, 55.49,
52.93(2C), 52.87(2C), 44.94, 18.61. TOF-HRMS: m/z [M + H]+ calcd for C23H25ClN4O3S:
473.1409; found: 473.1409.
4.1.3.8
5-(chloromethyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole

1
2,2-dioxide (B8). Yellow powder, yield 61%; m.p. 162-163ºC; H NMR (400 MHz,
CDCl3) δ 7.53 – 7.40 (m, 2H), 7.29 (d, J = 7.2 Hz, 1H), 7.24 (d, J = 9.0 Hz, 1H), 4.61 (s,
2H), 3.55 (t, J = 6.6 Hz, 2H), 3.31 (t, J = 6.6 Hz, 2H). TOF-HRMS: m/z [M + H]+ calcd
for C12H10ClFN2O3S: 317.0157; found: 317.0158.
4.1.3.9
5-((benzyl(methyl)amino)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-
c]pyrazole 2,2-dioxide (B9). Light yellow oil, yield 66%; 1H NMR (400 MHz, CDCl3) δ
7.49 – 7.36 (m, 2H), 7.32 (s, 5H), 7.25 – 7.18 (m, 2H), 3.57 (s, 2H), 3.55 (s, 2H), 3.46 (t,
J = 6.4 Hz, 2H), 3.20 (t, J = 6.3 Hz, 2H), 2.26 (s, 3H).
13C NMR (101 MHz, CDCl3) δ
156.06, 148.92, 145.47, 138.79, 130.52, 129.13, 128.33 (2C), 127.81 (2C), 127.25,
124.72, 124.19, 116.87, 95.25, 62.38, 54.66, 44.93, 42.64, 18.53. TOF-HRMS: m/z [M +
H]+ calcd for C20H20FN3O3S: 402.1282; found: 402.1284.
4.1.3.10
5-((benzyl(ethyl)amino)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]
1
pyrazole 2,2-dioxide (B10). Light yellow oil, yield 71%; H NMR (400 MHz, CDCl3) δ
7.48 – 7.34 (m, 3H), 7.31 (d, J = 3.5 Hz, 4H), 7.25 – 7.19 (m, 2H), 3.60 (s, 4H), 3.42 (t, J
= 6.4 Hz, 2H), 3.17 (t, J = 6.4 Hz, 2H), 2.59 (dd, J = 13.5, 6.6 Hz, 2H), 1.12 (t, J = 6.9 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 156.20, 149.52, 145.55, 130.60, 129.25 (2C), 128.72,
128.37 (2C), 127.95, 127.16, 124.86, 124.36, 117.02, 95.42, 58.43, 51.56, 48.15, 45.05,
18.72, 12.10. TOF-HRMS: m/z [M + H]+ calcd for C21H22FN3O3S: 416.1439; found:
416.1442.
4.1.3.11
5-((benzyl(2-hydroxyethyl)amino)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathi
1
ino[6,5-c]pyrazole 2,2-dioxide (B11). Colorless oil, yield 73%; H NMR (400 MHz,
CDCl3) δ 7.51 – 7.38 (m, 2H), 7.37 – 7.26 (m, 6H), 7.23 (d, J = 9.6 Hz, 1H), 3.68 (m,
6H), 3.42 (t, J = 6.5 Hz, 2H), 3.05 (t, J = 6.5 Hz, 2H), 2.78 (t, J = 5.3 Hz, 2H). 13C NMR
(101 MHz, CDCl3) δ 156.18, 148.57, 145.65, 138.54, 130.78, 129.39 (2C), 128.64 (2C),

127.92, 127.60, 124.95, 124.26, 117.10, 95.23, 59.26, 59.00, 56.13, 51.30, 44.90, 18.49.
TOF-HRMS: m/z [M + H]+ calcd for C21H22FN3O4S: 432.1388; found: 432.1392.
4.1.3.12
7-(2-fluorophenyl)-5-(morpholinomethyl)-4,7-dihydro-3H-[1,2]oxathiino[6,5-c]pyrazole
2,2-dioxide (B12). Colorless oil, yield 58%; 1H NMR (400 MHz, CDCl3) δ 7.48 (dd, J =
7.1, 1.5 Hz, 2H), 7.30 – 7.26 (m, 2H), 3.76 (t, J = 5.7, 4.3 Hz, 4H), 3.61 (s, 2H), 3.54 (t, J
= 6.5 Hz, 2H), 3.31 (t, J = 6.5 Hz, 2H), 2.58 (s, 4H). 13C NMR (101 MHz, CDCl3) δ
156.02, 147.57, 145.48, 130.64, 127.80, 124.76, 124.09, 116.87, 95.36, 66.94 (2C), 56.05,
53.60 (2C), 44.87, 18.58. TOF-HRMS: m/z [M + H]+ calcd for C16H18FN3O4S: 368.1075;
found: 368.1080.
4.1.3.13
7-(2-fluorophenyl)-5-((4-methylpiperazin-1-yl)methyl)-4,7-dihydro-3H-[1,2]oxathiino[6,
1
5-c]pyrazole 2,2-dioxide (B13). Colorless oil, yield 61%; H NMR (400 MHz, CDCl3) δ
7.46 – 7.37 (m, 2H), 7.20 (dd, J = 16.9, 8.1 Hz, 2H), 3.55 (s, 2H), 3.48 (t, J = 6.5 Hz, 2H),
3.21 (t, J = 6.5 Hz, 2H), 2.60 (s, 8H), 2.37 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 156.20,
147.87, 145.62, 130.80, 128.01, 124.92, 124.29, 117.05, 95.53, 55.42, 54.94 (2C), 52.34
(2C), 45.51, 45.06, 19.36. TOF-HRMS: m/z [M + H]+ calcd for C17H21FN4O3S: 381.1391;
found: 381.1392.
4.1.3.14
5-((4-benzylpiperazin-1-yl)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[1,2]oxathiino[6,
1
5-c]pyrazole 2,2-dioxide (B14). Colorless oil, yield 58%; H NMR (400 MHz, CDCl3) δ
7.48 – 7.37 (m, 2H), 7.35 – 7.30 (m, 5H), 7.25 – 7.19 (m, 2H), 3.58 (s, 2H), 3.56 (s, 2H),
3.49 (t, J = 6.5 Hz, 2H), 3.25 (t, J = 6.5 Hz, 2H), 2.57 (s, 8H). 13C NMR (101 MHz,
CDCl3) δ 156.18, 148.11, 145.58, 130.73, 129.55 (2C), 128.56, 128.49 (2C), 127.99,
127.46, 124.89, 124.31, 117.02, 95.54, 63.06, 55.69, 53.09 (4C), 45.07, 18.82.
TOF-HRMS: m/z [M + H]+ calcd for C23H25FN4O3S: 457.1704; found: 457.1701.

4.1.4. General procedure for the preparation of compounds C1–C10
K2CO3 (1.2 equiv) was added to a solution of compound 6 (1 mmol, 1.2 equiv)
and the hydroxyethyl benzylamine (HNR2R3, 1 mmol) in MeCN (20 mL), then the
reaction mixture was stirred at room temperature for 12 h, and subjected to column
chromatography on silica gel (CH2Cl2 / methanol, 50: 1) to obtain the title products
C1–C10 .
4.1.4.1
5-(((3-fluorobenzyl)(2-hydroxyethyl)amino)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[
1,2]oxathiino[6,5-c]pyrazole 2,2-dioxide (C1). Light yellow oil, yield 69%; purity,
99.8%. 1H NMR (400 MHz, CDCl3) δ 7.51 – 7.37 (m, J = 14.0, 6.4 Hz, 2H), 7.32 – 7.24
(m, 3H), 7.08 (t, 2H), 6.96 (t, J = 7.4 Hz, 1H), 3.71 (d, J = 13.2 Hz, 6H), 3.45 (d, J = 6.2
Hz, 2H), 3.11 (t, J = 6.1 Hz, 2H), 2.81–2.73 (m, 2H). TOF-HRMS: m/z [M + H]+ calcd
for C21H21F2N3O4S: 450.1294; found: 450.1296.
4.1.4.2
5-(((3-chlorobenzyl)(2-hydroxyethyl)amino)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[
1,2]oxathiino[6,5-c]pyrazole 2,2-dioxide (C2). Light yellow oil, yield 66%; purity,
99.9%. 1H NMR (400 MHz, CDCl3) δ 7.51 – 7.39 (m, 2H), 7.33 (s, 1H), 7.29 – 7.18 (m,
5H), 3.70 (s, 2H), 3.68 (s, 2H), 3.67 (s, 2H), 3.45 (t, J = 6.5 Hz, 2H), 3.09 (t, J = 6.5 Hz,
2H), 2.78 (t, J = 5.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 156.13, 148.34, 145.66,
140.85, 134.38, 130.81, 129.90, 129.25, 127.86, 127.69, 127.40, 124.95, 124.18, 117.08,
95.19, 59.43, 58.56, 56.32, 51.23, 44.87, 18.52. TOF-HRMS: m/z [M + H]+ calcd for
C21H21ClFN3O4S: 466.0998; found: 466.0997.
4.1.4.3
5-(((3-bromobenzyl)(2-hydroxyethyl)amino)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[
1,2]oxathiino[6,5-c]pyrazole 2,2-dioxide (C3). Light yellow oil, yield 70%; purity,
99.2%. 1H NMR (400 MHz, CDCl3) δ 7.51 – 7.37 (m, 5H), 7.28 (s, 1H), 7.24 – 7.16 (m,
3H), 3.69 (s, 2H), 3.67 (s, 2H), 3.66 (s, 2H), 3.46 (t, J = 6.5 Hz, 2H), 3.08 (t, J = 6.5 Hz,

2H), 2.79 (t, J = 5.3 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 156.18, 148.41, 145.70,
141.26, 132.21, 130.83, 130.65, 130.24, 127.91, 127.89, 124.97, 124.23, 122.69, 117.12,
95.18, 59.49, 58.57, 56.40, 51.24, 44.92, 18.59. TOF-HRMS: m/z [M + H]+ calcd for
C21H21BrFN3O4S: 510.0493; found: 510.0489.
4.1.4.4
5-(((4-fluorobenzyl)(2-hydroxyethyl)amino)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[
1,2]oxathiino[6,5-c]pyrazole 2,2-dioxide (C4). Light yellow oil, yield 53%; purity,
99.5%. 1H NMR (400 MHz, CDCl3) δ 7.51 – 7.41 (m, 2H), 7.32 – 7.25 (m, 4H), 7.03 (t, J
= 8.1 Hz, 2H), 3.70 (s, 2H), 3.68 (s, 4H), 3.47 (t, J = 6.2 Hz, 2H), 3.10 (t, J = 6.2 Hz, 2H),
2.78 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 162.28, 156.18, 148.49, 145.68, 134.21,
130.89 (2C), 130.79, 127.88, 124.96, 124.21, 117.11, 115.46 (2C), 95.15, 59.30, 58.14,
55.96, 51.03, 44.88, 18.53. TOF-HRMS: m/z [M + H]+ calcd for C21H21F2N3O4S:
450.1294; found: 450.1294.
4.1.4.5
5-(((4-chlorobenzyl)(2-hydroxyethyl)amino)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[
1,2]oxathiino[6,5-c]pyrazole 2,2-dioxide (C5). Light yellow oil, yield 58%; purity,
1
99.7%. H NMR (400 MHz, CDCl3) δ 7.55 – 7.38 (m, 3H), 7.32 – 7.27 (m, 3H), 7.25 –
7.21 (m, 2H), 3.68 (s, 2H), 3.66 (s, 4H), 3.45 (t, J = 6.5 Hz, 2H), 3.08 (t, J = 6.5 Hz, 2H),
13
2.76 (t, J = 5.3 Hz, 2H). C NMR (101 MHz, CDCl3) δ 156.00, 148.28, 145.51, 136.93,
133.13, 130.68, 130.48 (2C), 128.60 (2C), 127.72, 124.81, 124.04, 116.95, 94.99, 59.20,
58.11, 55.93, 50.94, 44.72, 18.38. TOF-HRMS: m/z [M + H]+ calcd for C21H21ClFN3O4S:
466.0998; found: 466.0999.
4.1.4.6
5-(((4-bromobenzyl)(2-hydroxyethyl)amino)methyl)-7-(2-fluorophenyl)-4,7-dihydro-3H-[
1,2]oxathiino[6,5-c]pyrazole 2,2-dioxide (C6). Light yellow oil, yield 61%; purity,