Tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one scaffold derivatives: Synthesis and biological evaluation as selective BuChE inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.02.002

BuChE inhibitors play important roles in treatment of patients with advanced Alzheimer's disease (AD). A series of tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one derivatives were synthesized and evaluated as acetylcholinesterase (AChE) and butyrylch

Full text of DOI:10.1016/j.ejmech.2018.02.002

Accepted Manuscript
Tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one scaffold derivatives: Synthesis
and biological evaluation as selective BuChE inhibitors
Shi-Chao Chen, Guo-Liang Qiu, Bo Li, Jing-Bo Shi, Xin-Hua Liu, Wen-Jian Tang
PII:
S0223-5234(18)30124-7
10.1016/j.ejmech.2018.02.002
EJMECH 10179
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 October 2017
Revised Date: 30 January 2018
Accepted Date: 2 February 2018
Please cite this article as: S.-C. Chen, G.-L. Qiu, B. Li, J.-B. Shi, X.-H. Liu, W.-J. Tang, Tricyclic
pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one scaffold derivatives: Synthesis and biological evaluation
as selective BuChE inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.02.002.
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ACCEPTED MANUSCRIPT
Novel tricyclic scaffolds were discovered as selective BuChE inhibitors.
Compounds 3f and 3o with dihalogen and a 6-ethyl substituent exhibited the most
potent activity. Compound 3o showed remarkable neuroprotective activity.

ACCEPTED MANUSCRIPT
Tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one scaffold
derivatives: Synthesis and biological evaluation as selective
BuChE inhibitors
Shi-Chao Chen1, Guo-Liang Qiu1, Bo Li, Jing-Bo Shi, Xin-Hua Liu*, Wen-Jian Tang*
School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Institute
of Innovative Drugs, Anhui Medical University, Hefei 230032, China.
Abstract: BuChE inhibitors play important roles in treatment of patients with advanced
Alzheimer’s
disease
(AD).
A
series
of
tricyclic
pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one derivatives were synthesized and evaluated
as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. Some
derivatives showed selective BuChE inhibitory activity, which was influenced by the
volumes of the substituted groups at the C6 position and halogen substituents at the
benzene ring of tricyclic scaffold. Among them, compounds 3f and 3o with dihalogen and
6-ethyl substituent showed the most potent activity (IC50 = 2.95, 2.04 µM, and
mixed-type, non-competitive inhibition against BuChE, respectively). Eutomer (6R)-3o
exhibited better BuChE inhibitory activity than (6S)-3o. Compound 3o exhibited
nontoxic, good ADMET properties, and remarkable neuroprotective activity. Docking
studies revealed the same binding orientation within the active site of target enzyme.
Compound 3o was nicely bound to BuChE via three hydrogen bonds, one Alkyl
interaction and three Pi-Alkyl interactions. The selective BuChE inhibitors had a
potential use in progressive neurodegenerative disorder.
Keywords: tricyclic; pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one; acetylcholinesterase;
butyrylcholinesterase; Alzheimer’s disease; kinetic study
_________________
*Corresponding author.
Tel. & fax: +86 551 65161115. E-mail: xhliuhx@163.com; ahmupharm@126.com.
1 S.C. Chen and G.L. Qiu contributed equally to this work.

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1. Introduction
Alzheimer's disease (AD) is a chronic neurodegenerative disease that is characterized
by progressive deterioration of memory and other cognitive functions [1]. As summarized
in World Alzheimer Report 2016, 47 million people are living with dementia worldwide,
and this number is estimated to increase to more than 131 million by 2050 [2].
β-Amyloid oligomerization [3], tau-protein hyperphosphorylation [4], cholinergic
dysfunction [5], and oxidative stress [6], etc. are all responsible for pathogenesis of AD
[7]. As the most effective therapeutic strategies, cholinergic dysfunction hypothesis
directly contributes to cognitive decline [8]. It has been found that amyloid protein
plaques can be caused by both cholinesterases (ChEs), named as acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE), their inhibitors can decrease those plaques
[9]. Moreover, abnormalities in cholinergic system are also closely related to other
neurodegenerative disorders, such as PD, dementia with Lewy bodies and vascular
dementia [10,11].
Therefore, administration of ChE inhibitors is a useful approach to elevate ACh levels
within the brain [12,13]. ACh could be hydrolyzed by both AChE and BuChE, but
BuChE is less substrate specific than AChE [14]. ACh levels increased 16-fold in
wild-type mice treated with a selective AChE inhibitor, while no increase with a selective
BuChE inhibitor [15]. AChE plays the major role in the hydrolysis of ACh in the healthy
brain, while BuChE takes over the hydrolysis of ACh in the AChE deficient brain [16,17].
In AChE deficient mice, levels of excessive hippocampal ACh were alleviated by BuChE
activity [18]. To avoid the adverse effects caused by suppression of AChE, development
of effective and selective BuChE inhibitors to elevate ACh level in progressed AD is
potentially advantageous [19,20]. Although the absence of BuChE has no significant
adverse effects on health individuals, the recent finding that BuChE-catalyzed ghrelin
hydrolysis influences mouse aggression and social stress may have potential implications
for humans [21]. Marketed AD drugs except for memantine are all ChEs inhibitors, but
donepezil and galantamine are selective AChE inhibitors, only rivastigmine is a dual
AChE-BuChE inhibitor.
BuChE is a potential therapeutic target for restoring ACh levels in the brain, improving

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cognitive impairment, reducing adverse effects in progressed AD patients. Although there
are different types of scaffolds with BuChE inhibition, scaffolds of selective BuChE
inhibitors are far from abundance [22,23]. As the multifactorial neurodegenerative
disease, another trend of AD therapy is focused on multiple targeted ligands, where
mostly ChE inhibition is combined with additional biological properties, along affecting
monoamine oxidases (MAOs) metabolism as well as mitochondrial functions, and
antioxidant properties [24−29]. Moreover, inhibition of enzymatic activities of MAOs
and ChEs has been pursued to identifying novel therapeutic agents with a potential
disease-modifying effect [30−32]. Thus, it is expected to discover more and more
potential ChE inhibitors with diverse structures.
In
our
recent
work,
a
novel
tricyclic
scaffold
of
pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one was found to be a selective hMAO-B
inhibitor [33,34], which are used alone or in combination to treat Alzheimer’s and
Parkinson’s diseases [35,36]. Based on multi-target property of AD therapy, ChEs
inhibitory activities of this tricyclic scaffold were screened to discover novel ChE
inhibitors, but they showed weak inhibitory activities for AChE and BuChE. When 2-aryl
group of the tricyclic scaffold was substituted by methyl group, new tricyclic compounds
exhibited moderate inhibitory activities against ChE and weak inhibitory activities
against MAOs. It implied that 2-substituent of tricyclic scaffold may lead to the change
of enzymatic inhibitory activities between MAOs and ChEs (Fig. 1). Herein, based on
above rational analysis, a series of pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one
derivatives were designed and synthesized as selective cholinesterase inhibitors. Further,
the most potent compounds will be used to study the preliminary mechanism.
(Fig. 1)
2. Results and discussion
2.1. Chemistry
According to our recent works [33,34], pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one
derivatives (3a–3s, as shown in Table 1) were synthesized according to the protocol

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outlined in Scheme 1. The key intermediate 2-pyrazoline (1) was obtained by the
cyclization reaction of excess hydrazine hydrate with the styrene ketones, which were
prepared through Claisen-Schmidt condensation of substituted salicylaldehyde with
acetone. The acylaton of compound
1
with α-bromoacyl chloride gave
2-pyrazoline-1-ethanone derivatives (2). The title compounds 3a−3s were obtained
through the cyclization reaction of compound 2 in the presence of NaHCO3 catalysis in
ethanol. The total yield was 14% − 23%. Compounds 3t−3x were synthesized according
to the literature procedure [34].
To further study a possible influence of chirality at C-6 on ChE inhibitory activity, two
enantiomers of compound 3o were synthesized by the acylation of
2-bromo-4-chloro-6-(3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenol with chiral pure
2-chloropropionic acid and subsequent cyclization reaction in 32% and 34% yield
respectively (Scheme 2).
(Scheme 1)
(Scheme 2)
2.2. Inhibitory activity against AChE and BuChE
The inhibitory potency of compounds 3a–3x was assessed by Ellman’s assay on
Electrophorus electricus AChE (EeAChE) and equine BuChE (eqBuChE) [37]. The IC50
values were obtained and compared to the reference donepezil, a selective AChE
inhibitor, which was the only one of four FDA-approved AChEIs that simultaneously
binds to catalytic active and peripheral anionic sites, providing moderate inhibition of Aβ
aggregation [38,39]. The IC50 values of all tested compounds against EeAChE and
eqBuChE were summarized in Table 1.
(Table 1)
Enzymatic assays revealed that 26 tricyclic compounds exhibited inhibitory activities
against cholinesterases, among them, some compounds showed potent BuChE inhibition.
It was obvious from the data that compounds 3f and 3o exhibited the best activities

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against BuChE with IC50 values of 2.95 and 2.04 µM, respectively, surpassing that of the
positive control donepezil (IC50 = 11.81 µM). Inspection of the chemical structures, it can
be concluded that the BuChE inhibitory activity was related to the substituent groups at
C2, C6, C8 and C10 positions of benzoxazepinone moiety (Table 1). From Table 1, it is
intuitive that the volume of the substituted group at the C2 position of the pyrazole ring
plays an important role in the activity. The methyl group at the C2 position can increase
the inhibitory activity against BuChE. For example, compound 3b has an IC50 value of
5.95 µM against BuChE; when the C2 position is furan (compound 3u), the inhibition
rate is 23% at 20 µM.
Compounds with halogen substituents at the benzene ring in the benzoxazepinone
moiety have great influence on the BuChE inhibition, for example, compounds 3a–3o
with 8- or/and 10-halogen substitutions at the benzene ring were found better in terms of
potency than the corresponding compounds 3p–3s with methyl or methoxyl substituents.
Compound 3o with 8-bromo and 10-chloro substituents showed more potent BuChE
inhibitory activity (IC50 = 2.04 µM) than the non-halogen substituted compounds 3p–3s
(inhibition rate at 20 µM < 5%).
From Table 1, it is observed that the volumes of the substituted groups at the C6
position of the seven-membered ring have significant influences on the BuChE activity.
Along with the big substituent, the inhibitory activity was increased significantly. When
the substituents at the R2 position are H, methyl and ethyl respectively, the BuChE
inhibitory activity were increased gradually. For example, compound 3a has an inhibition
31% at 20 µM against BuChE; when the R2 position is methyl (compound 3b), the IC50
value was changed to 5.95 µM; when the R2 position is ethyl (compound 3c), the IC50
value was changed to 4.67 µM. This trend can also be observed among others compounds
(3g, 3h, 3i; 3j, 3k, 3l; 3m, 3n, 3o except for 3e < 3d < 3f).
To observe the influence of chiral C-6 on ChE inhibitory activity, enantiomers (6R)-3o
and (6S)-3o were synthesized. Regarding BuChE affinity, an eudismic ratio equal to 18
was observed for both racemates. Eutomer (6R)-3o exhibited much better BuChE
inhibitory activity than isomer (6S)-3o (IC50 = 1.14 and 20.42 µM, respectively).

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2.3 Kinetic Study of eqBuChE inhibition
The kinetic studies for the most active compounds 3f and 3o were carried out at three
fixed inhibitor concentrations (5 µM, 10 µM and 20 µM). In each case, the kinetic type of
enzyme inhibition was obtained through the modified Ellman’s method and
Lineweaver−Burk secondary plots [40]. As shown in Fig. 2A, overlaid reciprocal
Lineweaver−Burk plots showed both higher slopes (decreased Vmax) and intercepts
(higher Km) at increasing inhibitor concentrations, a trend that is generally ascribed to a
mixed-type inhibition. In the same way, Fig. 2B showed all lines intersect the x-axis, it
confirmed the typical characteristics of non-competitive inhibitors. The dissociation
constants Ki for compounds 3f (Fig. 2C) and 3o (Fig. 2D) were estimated to be 7.60 µM
and 1.50 µM, respectively.
(Fig. 2.)
2.4 Cytotoxicity assays and Neuroprotective effect against H2O2-induced cell death in
PC12 neurons
The differentiated PC12 cells and H2O2 were selected as in vitro model and oxidative
agent to assess neuronal differentiation and other neurobiochemical and neurobiological
studies [41,42]. The neuroprotective activity of compound 3o and donepezil against
oxidative stress-induced cell death in differentiated PC12 neurons was evaluated. Thus,
differentiated PC12 cells were pretreated with different concentrations (10, 25 and 50 µM)
for 3 h, before treatment with H2O2 (300 µM), and cell viability was measured after 24 h
by using the MTT assay. As shown in Fig. 3, compound 3o (3A) and donepezil (3B) at
the test concentrations (1–50 µM) had no obviously cytotoxicity in PC-12 cells, and the
relative cell viabilities of treated cells were all more than 90%. Neuroprotective activity
of the most potent compound 3o against eqBuChE was assessed by subjecting PC12 cells
to H2O2-induced damage. As shown in Fig. 4, the percent of cell viability was calculated
in comparison to control group. Compound 3o exhibited remarkable neuroprotective
activity at 25 µM (cell viability = 70%, and p < 0.05 vs H2O2 treatment alone).

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(Fig. 3.)
(Fig. 4.)
2.5. Oil/water partition coefficient assessment and ADMET prediction
To evaluate the drug-like properties of active compounds, Lipinski's rule of five were
used to predict the ADME profiles for active compounds, i.e., log P (o/w) (octanol-water
partition coefficient), number of hydrogen bond donor atom, number of hydrogen bond
acceptor atom, et al. As an important parameter to predict the ability to cross blood-brain
barrier (BBB), the log P with the optimum central nervous system penetration was around
2.0 ± 0.7 [43,44]. The log P values of compounds 3b, 3c, 3f, 3i, 3l, 3n and 3o were 2.49,
1.12, 1.08, 1.05, 1.11, 2.18 and 1.57, respectively (Table 2), which suggested that the
active compounds are lipophilicity (log P < 5).
The ADMET properties were considered critical parameters for drug candidates [45].
The active compounds were analyzed with the ADMET prediction tools of DS 2017R2.
As shown in Table 3 and Fig. 5, they showed good absorption, good solubility, and
medium to high penetrant. Predicted ADMET properties of active compounds are in an
agreement with the log P values. These results indicated that compounds 3n and 3o were
possible sufficiently lipophilic to pass the BBB in vivo.
(Table 2)
(Table 3)
2.6. Molecular docking
In order to gain more understanding of the structure–activity relationships for the
BuChE, based on the X-ray crystal structure of human BuChE (hBuChE PDB ID: 1P0I),
molecular docking was performed on the binding model using the Discovery Studio
2017R2 software [46,47]. The docking calculation of compounds 3a–3o, (6R)-3o and
(6S)-3o was depicted in Table 4, the results showed that compounds 3a–3o, (6R)-3o and
(6S)-3o
had
nice
binding
affinity
to
BuChE
and
their

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CDOCKER_INTERACTION_ENERGY had the same trend as the BuChE inhibitory
activities, which further proved the correlation between the BuChE inhibitory activity and
binding energy.
(Table 4)
Among
them,
compound
3o
showed
the
maximum
-CDOCKER_INTERACTION_ENERGY, which suggested that it was mostly easy to
bind to BuChE. The 2D and 3D binding models of compound 3o with BuChE were
depicted in Fig. 6. The amino acid residues which had interactions with BuChE as well as
bond lengths were labeled. In the binding models, compound 3o was nicely bound to
BuChE via three hydrogen bonds with His438 (distance
= 2.36 Å), Gly116 (distance =
3.04 Å) and Ser198 (distance 2.85 Å). Specifically, two conventional hydrogen bond
=
interactions arose between the C=O group at the C5 position of ligand 3o and the NH
groups of His438 and Gly116. Moreover, carbon hydrogen bond interaction also arose
between the C=O group of ligand 3o and the CH2 group of Ser198. The above
observations can explain the potent BuChE inhibition of compound 3o. In addition,
compound 3o was also nicely bound to BuChE via one Alkyl interaction and three
Pi-Alkyl interactions. The Alkyl interaction was formed between the end group of
Leu125 and bromine, which increased inhibitory activities by strengthening the binding
affinity. Three Pi-Alkyl interactions were formed between the end group of Trp82 and the
methyl of the C6 position and 8-bromo substituent. Furthermore, the H-bonds surface of
compound 3o and receptor BuChE was also depicted in Fig. 7. These docking results,
along with the biological assay data, suggested that compound 3o possessed higher
inhibitory activity than other compounds, which will help us carry out the next structure
optimization.

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(Fig. 6.)
(Fig. 7.)
3. Conclusions
In this work, we reported design and synthesis of
a
series of
pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one derivatives and evaluated in vitro AChE
and BuChE activities. Some derivatives showed selective BuChE inhibitory activity. The
SARs analysis showed that the volumes of the substituted groups at the C6 position and
halogen substituent at the benzene ring have significant influences for the BuChE activity.
Among them, compounds bearing dihalogen and 6-ethyl substituent showed the most
potent activity (IC50 = 2.95 and 2.04 µM, respectively). Eutomer (6R)-3o exhibited much
better BuChE inhibitory activity than isomer (6S)-3o (IC50 = 1.14 and 20.42 µM,
respectively). Kinetic studies on substrate-enzyme relationship revealed that compounds
3f and 3o showed mixed-type and non-competitive inhibition against BuChE (Ki = 7.60
and 1.50 µM, respectively). The active compounds were found to be nontoxic at their
effective concentrations against BuChE and to have good ADMET property predictions.
Compound 3o demonstrated remarkable neuroprotective activity. Docking studies
showed that these potent compounds had same binding orientation within the active site
of target enzyme. Compound 3o was nicely bound to BuChE via three hydrogen bonds
with His438, Gly116 and Ser198, one Alkyl interaction and three Pi-Alkyl interactions.
The active compounds are selective BuChE inhibitors with a potential use against
progressive neurodegenerative disorder.
4. Experimental section
4.1. Chemistry
All reagents were purchased from commercial sources and were used without further
purification. Melting points (uncorrected) were determined on a XT4MP apparatus (Taike
Corp., Beijing, China). 1H NMR and 13C NMR spectra were recorded on Bruker AV-600 or

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AV-300 MHz instruments in CDCl3. 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). Optical rotations: WZZ-2S digital automatic polarimeter. High
resolution mass spectra (HRMS) were obtained on an Agilent 1260-6221 TOF mass
spectrometry. Column and thin-layer chromatography (CC and TLC, resp.) were
performed on silica gel (200-300 mesh) and silica gel GF254 (Qingdao Marine Chemical
Factory) respectively.
4.1.1. General procedure for the preparation of compounds 3a–3s
To a solution of salicylaldehyde (10 mmol) in acetone (20 mL) was added 40% NaOH
aqueous solution (2 mL) dropwise and the reaction was stirred at 60 ºC for 2 h. The
mixture was poured into cold water and neutralized with 2 M HCl to a pH in the range of
5~6. The resulting precipitate was collected, washed with water and dried to give
chalcones. The chalcone was treated with 5 times excess of hydrazine hydrate in ethanol
and refluxed for 5 h. The reaction mixture was then poured into ice-cold water. The solid
was filtered, washed and recrystallized from ethanol to afford respective pyrazoline (1). A
solution of compound 1 in CH2Cl2 (20 mL) was added α-bromoacyl chloride (5.0 mmol)
and 4-dimethylaminopyridine (6.0 mmol) and the reaction was stirred overnight. The
reaction mixture was washed with water and brine, dried with anhydrous Na2SO4,
filtrated and concentrated in vacuo. The resulting residue was then purified by column
chromatography to give product 2. NaHCO3 (3.0 mmol) was added to an ethanol (20 mL)
solution of compound 2 (2.0 mmol), then the reaction mixture was stirred at 70 ºC until
the disappearance of starting material (monitored by TLC). EtOAc (100 mL) was added
to the reaction mixture, then washed with water and brine, dried with anhydrous Na2SO4,
filtrated and concentrated in vacuo. The residue was purified by chromatography on a
silica gel column (petroleum/EtOAc, 1:1 → 1:2) to give title compounds 3a–3s.
4.1.1.1.
8,10-Dichloro-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one (3a).
White powder, yield, 20%; m.p. 217–219°C; 1H NMR (300 MHz, CDCl3) δ 7.46 (d, J =

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2.0 Hz, 1H), 7.14 (s, 1H), 5.83 (m, 1H, 11b-H), 5.01 (d, J = 16.8 Hz, 1H, 6-Hb), 4.43 (d,
J = 16.8 Hz, 1H, 6-Ha), 3.42 (dd, J = 17.9, 9.1 Hz, 1H, 1-Hb), 3.27 (dd, J = 17.9, 11.4 Hz,
1H, 1H-a), 2.23 (s, 3H, 2-CH3). TOF-HRMS: m/z [M + Na]+ calcd for C12H10Cl2N2NaO2:
307.0012; found: 307.0014.
4.1.1.2.
8,10-Dichloro-2-methyl-6-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H
1
)-one (3b). White powder, yield, 18%; m.p. 228–230°C; H NMR (600 MHz, CDCl3) δ
7.43 (d, J = 2.2 Hz, 1H), 7.08 (d, J = 1.8 Hz, 1H), 5.69 (t, J = 10.6 Hz, 1H, 11b-H), 5.10
(q, J = 6.9 Hz, 1H, 6-Hb), 3.34 (dd, J = 10.5, 4.6 Hz, 2H, 1-H), 2.20 (s, 3H, 2-CH3), 1.57
13
(s, 3H, 6-CH3); C NMR (150 MHz, CDCl3) δ 166.6, 157.8, 150.3, 132.6, 130.3, 129.4,
129.1, 124.8, 78.3 (C-6), 57.3 (11b-C), 42.0 (C-1), 17.3 (6-CH3), 16.2 (2-CH3).
TOF-HRMS: m/z [M + Na]+ calcd for C13H12Cl2N2NaO2: 321.0168; found: 321.0166.
4.1.1.3.
8,10-Dichloro-6-ethyl-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-
1
one (3c). White powder, yield, 17%; m.p. 219–221°C; H NMR (300 MHz, CDCl3) δ
7.37 (d, J = 2.1 Hz, 1H), 6.98 (d, J = 1.6 Hz, 1H), 5.71 (t, J = 10.6 Hz, 1H, 11b-H), 4.58
(t, J = 6.5 Hz, 1H, 6-Ha), 3.50 (dd, J = 17.4, 11.1 Hz, 1H, 1-Hb), 3.24 (dd, J = 17.6, 10.0
Hz, 1H, 1-Ha), 2.16 (s, 3H, 2-CH3), 2.14 – 2.01 (m, 2H, 6-CH2CH3), 1.14 (t, J = 7.4 Hz,
3H, 6-CH2CH3). TOF-HRMS: m/z [M + Na]+ calcd for C14H14Cl2N2NaO2: 335.0325;
found: 335.0323.
4.1.1.4.
8,10-Dibromo-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one
(3d). White powder, yield, 21%; m.p. 223–225°C; 1H NMR (300 MHz, CDCl3) δ 7.76 (d,
J = 1.9 Hz, 1H), 7.31 (s, 1H), 5.83 (m, 1H, 11b-H), 5.00 (d, J = 16.8 Hz, 1H, 6-Hb), 4.44
(d, J = 16.8 Hz, 1H, 6-Ha), 3.42 (dd, J = 18.0, 8.9 Hz, 1H, 1-Hb), 3.27 (dd, J = 18.0, 11.4
13
Hz, 1H, 1-Ha), 2.23 (s, 3H, 2-CH3); C NMR (75 MHz, CDCl3) δ 163.5, 157.6, 153.9,
136.2, 134.8, 127.2, 118.5, 117.8, 71.5 (C-6), 55.8 (11b-C), 39.5 (C-1), 16.2 (2-CH3).
TOF-HRMS: m/z [M + Na]+ calcd for C12H10Br2N2NaO2: 394.9001; found: 394.9002.

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4.1.1.5.
8,10-Dibromo-2-methyl-6-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H
1
)-one (3e). White powder, yield, 18%; m.p. 243–245°C; H NMR (300 MHz, CDCl3) δ
7.72 (d, J = 1.9 Hz, 1H), 7.25 (d, J = 1.1 Hz, 1H), 5.68 (t, J = 10.7 Hz, 1H, 11b-H), 5.07
(q, J = 6.9 Hz, 1H, 6-Hb), 3.34 (d, J = 10.6 Hz, 2H, 1-H), 2.19 (s, 3H, 2-CH3), 1.62 (s,
3H, 6-CH3); 13C NMR (75 MHz, CDCl3) δ 166.5, 158.0, 152.0, 135.9, 132.2, 128.6,
118.4, 116.6, 78.0 (C-6), 57.4 (11b-C), 42.4 (C-1), 17.3 (6-CH3), 16.2 (2-CH3).
TOF-HRMS: m/z [M + Na]+ calcd for C13H12Br2N2NaO2: 408.9158; found: 408.9160.
4.1.1.6.
8,10-Dibromo-6-ethyl-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-
one (3f). White powder, yield, 20%; m.p. 224–226°C; 1H NMR (300 MHz, CDCl3) δ 7.66
(d, J = 1.7 Hz, 1H), 7.14 (d, J = 1.3 Hz, 1H), 5.72 (t, J = 10.6 Hz, 1H, 11b-H), 4.56 (dd, J
= 7.6, 5.5 Hz, 1H, 6-Ha), 3.52 (dd, J = 17.6, 11.2 Hz, 1H, 1-Hb), 3.23 (dd, J = 17.7, 9.8
Hz, 1H, 1-Ha), 2.15 (s, 3H, 2-CH3), 2.09 (m, 2H, 6-CH2CH3), 1.15 (t, J = 7.4 Hz, 3H,
6-CH2CH3). TOF-HRMS: m/z [M + Na]+ calcd for C14H14Br2N2NaO2: 422.9314; found:
422.9311.
4.1.1.7. 10-Chloro-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one
1
(3g). White powder, yield, 23%; m.p. 186–188°C; H NMR (300 MHz, CDCl3) δ 7.35
(dd, J = 8.5, 2.3 Hz, 1H), 7.21 (d, J = 2.0 Hz, 1H), 7.14 (d, J = 8.5 Hz, 1H), 5.85–5.73 (m,
1H, 11b-H), 4.96 (d, J = 16.6 Hz, 1H, 6-Hb), 4.39 (d, J = 16.6 Hz, 1H, 6-Ha), 3.44 (dd, J
= 17.9, 9.3 Hz, 1H, 1-Hb), 3.26 (dd, J = 17.9, 11.3 Hz, 1H, 1-Ha), 2.23 (s, 3H, 2-CH3);
13C NMR (75 MHz, CDCl3) δ 164.1, 157.9, 156.7, 132.7, 130.7, 130.6, 125.3, 123.1, 72.8
(C-6), 55.8 (11b-C), 39.7 (C-1), 16.2 (2-CH3). TOF-HRMS: m/z [M + Na]+ calcd for
C12H11ClN2NaO2: 273.0401; found: 273.0394.
4.1.1.8.
10-Chloro-2-methyl-6-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-on
1
e (3h). White powder, yield, 21%; m.p. 199–202°C; H NMR (300 MHz, CDCl3) δ 7.24

ACCEPTED MANUSCRIPT
(d, J = 2.3 Hz, 1H), 7.13 (d, J = 1.7 Hz, 1H), 6.98 (d, J = 8.6 Hz, 1H), 5.74 (t, J = 10.5 Hz,
1H, 11b-H), 4.95 (q, J = 6.8 Hz, 1H, 6-Hb), 3.36 (d, J = 11.4 Hz, 2H, 1-H), 2.18 (s, 3H,
2-CH3), 1.52 (d, J = 6.8 Hz, 3H, 6-CH3); 13C NMR (75 MHz, CDCl3) δ 166.8, 157.9,
154.1, 130.3, 129.5, 129.0, 126.4, 123.90, 76.3 (C-6), 57.5 (11b-C), 42.6 (C-1), 17.4
(6-CH3), 16.2 (2-CH3). TOF-HRMS: m/z [M + H]+ calcd for C13H14ClN2O2: 265.0738;
found: 265.0739.
4.1.1.9.
10-Chloro-6-ethyl-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one
(3i). White powder, yield, 19%; m.p. 188–190°C; 1H NMR (300 MHz, CDCl3) δ 7.23 (dd,
J = 8.7, 2.2 Hz, 1H), 7.10 (s, 1H), 7.00 (d, J = 8.6 Hz, 1H), 5.74 (t, J = 10.5 Hz, 1H,
11b-H), 4.59 (dd, J = 9.4, 4.0 Hz, 1H, 6-Ha), 3.42 (dd, J = 17.7, 11.1 Hz, 1H, 1-Hb), 3.30
(dd, J = 17.7, 9.9 Hz, 1H, 1-Ha), 2.17 (s, 3H, 2-CH3), 2.10 (ddd, J = 14.7, 7.5, 4.1 Hz, 1H,
6-CH2CH3), 1.92–1.72 (m, 1H, 6-CH2CH3), 1.11 (t, J = 7.4 Hz, 3H, 6-CH2CH3); 13C
NMR (75 MHz, CDCl3) δ 166.3, 157.9, 154.8, 129.4, 128.4, 126.8, 123.0, 101.5, 81.3
(C-6), 57.9 (11b-C), 43.5 (C-1), 24.0 (6-CH2CH3), 16.1 (2-CH3), 10.4 (6-CH2CH3).
TOF-HRMS: m/z [M + H]+ calcd for C14H16ClN2O2: 279.0895; found: 279.0896.
4.1.1.10.
10-Bromo-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one
(3j).
White powder, yield, 17%; m.p. 214–216°C; 1H NMR (300 MHz, CDCl3) δ 7.50 (dd, J =
8.5, 1.8 Hz, 1H), 7.36 (s, 1H), 7.08 (d, J = 8.5 Hz, 1H), 5.86 – 5.75 (m, 1H, 11b-H), 4.97
(d, J = 16.6 Hz, 1H, 6-Hb), 4.40 (d, J = 16.6 Hz, 1H, 6-Ha), 3.45 (dd, J = 17.9, 9.4 Hz,
13
1H, 1-Hb), 3.26 (dd, J = 18.0, 11.3 Hz, 1H, 1-Ha), 2.23 (s, 3H, 2-CH3); C NMR (75
MHz, CDCl3) δ 163.9, 157.7, 157.0, 133.4, 132.9, 128.0, 123.3, 118.0, 72.5 (C-6), 55.6
(11b-C), 39.5 (C-1), 16.1 (2-CH3). TOF-HRMS: m/z [M + Na]+ calcd for
C12H11BrN2NaO2: 316.9896; found: 316.9899.
4.1.1.11.
10-Bromo-2-methyl-6-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-on
1
e (3k). White powder, yield, 18%; m.p. 218–220°C; H NMR (300 MHz, CDCl3) δ 7.39

ACCEPTED MANUSCRIPT
(dd, J = 8.5, 2.1 Hz, 1H), 7.27 (s, 1H), 6.92 (d, J = 8.6 Hz, 1H), 5.74 (t, J = 10.5 Hz, 1H,
11b-H), 4.94 (q, J = 6.8 Hz, 1H, 6-Hb), 3.46–3.24 (m, 2H, 1-H), 2.18 (s, 3H, 2-CH3),
1.52 (d, J = 6.8 Hz, 3H, 6-CH3); 13C NMR (75 MHz, CDCl3) δ 166.5, 157.8, 154.5, 132.3,
130.4, 129.2, 124.0, 116.1, 75.9 (C-6), 57.3 (11b-C), 42.6 (C-1), 17.2 (6-CH3), 16.0
(2-CH3). TOF-HRMS: m/z [M + H]+ calcd for C13H14BrN2O2: 309.0233; found:
309.0241.
4.1.1.12.
10-Bromo-6-ethyl-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one
(3l). White powder, yield, 16%; m.p. 195–198°C; 1H NMR (300 MHz, CDCl3) δ 7.37 (dd,
J = 8.6, 2.2 Hz, 1H), 7.24 (s, 1H), 6.94 (d, J = 8.6 Hz, 1H), 5.74 (t, J = 10.5 Hz, 1H,
11b-H), 4.58 (dd, J = 9.4, 4.0 Hz, 1H, 6-Ha), 3.43 (dd, J = 17.6, 11.1 Hz, 1H, 1-Hb), 3.30
(dd, J = 17.6, 10.0 Hz, 1H, 1-Ha), 2.17 (s, 3H, 2-CH3), 2.14–2.01 (m, 1H, 6-CH2CH3),
1.91–1.76 (m, 1H, 6-CH2CH3), 1.11 (t, J = 7.4 Hz, 3H, 6-CH2CH3). TOF-HRMS: m/z [M
+ H]+ calcd for C14H16BrN2O2: 323.0390; found: 323.0388.
4.1.1.13.
8-Bromo-10-chloro-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-on
e (3m). White powder, yield, 15%; m.p. 218–221°C; 1H NMR (300 MHz, CDCl3) δ 7.61
(d, J = 2.3 Hz, 1H), 7.18 (d, J = 2.1 Hz, 1H), 5.83 (dd, J = 11.2, 9.0 Hz, 1H, 11b-H), 4.99
(d, J = 16.8 Hz, 1H, 6-Hb), 4.44 (d, J = 16.8 Hz, 1H, 6-Ha), 3.42 (dd, J = 18.1, 8.9 Hz,
13
1H, 1-Hb), 3.27 (dd, J = 18.0, 11.3 Hz, 1H, 1-Ha), 2.22 (s, 3H, 2-CH3); C NMR (75
MHz, CDCl3) δ 163.5, 157.7, 153.4, 134.4, 133.4, 131.3, 124.3, 117.4, 71.6 (C-6), 55.9
(11b-C), 39.5 (C-1), 16.2 (2-CH3). TOF-HRMS: m/z [M + H]+ calcd for C12H11BrClN2O2:
328.9687; found: 328.9692.
4.1.1.14.
8-Bromo-10-chloro-2-methyl-6-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-
5(6H)-one (3n). White powder, yield, 16%; m.p. 205–207°C; 1H NMR (300 MHz, CDCl3)
δ 7.58 (d, J = 2.3 Hz, 1H), 7.12 (d, J = 2.2 Hz, 1H), 5.68 (t, J = 10.6 Hz, 1H), 5.08 (q, J =
6.9 Hz, 1H, 6-Hb), 3.34 (d, J = 10.6 Hz, 2H, 1-H), 2.20 (s, 3H, 2-CH3), 1.61 (d, J = 6.9

ACCEPTED MANUSCRIPT
Hz, 3H, 6-CH3); 13C NMR (75 MHz, CDCl3) δ 166.60, 157.9, 151.5, 133.2, 131.9, 129.5,
125.7, 118.1, 78.2 (C-6), 57.5 (11b-C), 42.3 (C-1), 17.4 (6-CH3), 16.2 (2-CH3).
TOF-HRMS: m/z [M + H]+ calcd for C13H13BrClN2O2: 342.9843; found: 342.9847.
4.1.1.15.
8-Bromo-10-chloro-6-ethyl-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(
1
6H)-one (3o). White powder, yield, 14%; m.p. 227–229°C; H NMR (300 MHz, CDCl3)
δ 7.52 (dd, J = 2.4, 0.5 Hz, 1H), 7.01 (dd, J = 2.4, 0.8 Hz, 1H), 5.72 (dd, J = 10.9, 10.3
Hz, 1H, 11b-H), 4.56 (dd, J = 7.5, 5.5 Hz, 1H, 6-Ha), 3.52 (dd, J = 17.5, 11.2 Hz, 1H,
1-Hb), 3.23 (dd, J = 16.8, 10.0 Hz, 1H, 1-Ha), 2.15 (s, 3H, 2-CH3), 2.13–2.03 (m, 2H,
13
6-CH2CH3), 1.15 (t, J = 7.4 Hz, 3H, 6-CH2CH3); C NMR (75 MHz, CDCl3) δ 165.8,
157.8, 152.6, 132.8, 128.8, 127.8, 126.8, 115.9, 81.40 (C-6), 58.76 (11b-C), 45.3 (C-1),
23.9 (6-CH2CH3), 16.1 (2-CH3), 10.3 (6-CH2CH3). TOF-HRMS: m/z [M + H]+ calcd for
C14H15BrClN2O2: 342.9843; found: 342.9847.
4.1.1.16.
8-Methoxy-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one (3p).
1
White powder, yield, 19%; m.p. 180–182°C; H NMR (300 MHz, CDCl3) δ 7.17 (t, J =
8.0 Hz, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 5.95– .82 (m, 1H, 11b-H),
4.99 (d, J = 16.8 Hz, 1H, 6-Hb), 4.40 (d, J = 16.8 Hz, 1H, 6-Ha), 3.90 (s, 3H, OCH3),
3.49 (dd, J = 18.0, 9.0 Hz, 1H, 1-Hb), 3.22 (dd, J = 18.0, 11.4 Hz, 1H, 1-Ha), 2.21 (s, 3H,
2-CH3). TOF-HRMS: m/z [M + H]+ calcd for C13H15N2O3: 247.1077; found: 247.1085.
4.1.1.17.
8-Methoxy-2-methyl-6-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-o
ne (3q). White powder, yield, 19%; m.p. 172–174°C; 1H NMR (300 MHz, CDCl3) δ 7.14
(t, J = 8.0 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.82 (d, J = 7.8 Hz, 1H), 5.76 (t, J = 10.7 Hz,
1H, 11b-H), 5.13 (q, J = 7.1 Hz, 1H, 6-Hb), 3.88 (s, 3H, OCH3), 3.45 (dd, J = 17.7, 10.4
Hz, 1H, 1-Hb), 3.22 (dd, J = 17.6, 11.2 Hz, 1H, 1-Ha), 2.20 (s, 3H, 2-CH3), 1.50 (d, J =
7.0 Hz, 3H, 6-CH3). TOF-HRMS: m/z [M + H]+ calcd for C14H17N2O3: 261.1234; found:
261.1244.

ACCEPTED MANUSCRIPT
4.1.1.18.
2-Methyl-10-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one
(3r).
1
White powder, yield, 21%; m.p. 176–178°C; H NMR (300 MHz, CDCl3) δ 7.17 (d, J =
8.1 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 7.02 (s, 1H), 5.89 – 5.74 (m, 1H, 11b-H), 4.95 (d, J
= 16.7 Hz, 1H, 6-Hb), 4.37 (d, J = 16.7 Hz, 1H, 6-Ha), 3.51 (dd, J = 17.9, 9.5 Hz, 1H,
1-Hb), 3.21 (dd, J = 17.9, 11.3 Hz, 1H, 1-Ha), 2.36 (s, 3H, 10-CH3), 2.22 (s, 3H, 2-CH3).
TOF-HRMS: m/z [M + H]+ calcd for C13H15N2O2: 231.1128; found: 231.1123.
4.1.1.19.
2-Methyl-6-methyl-10-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-on
1
e (3s). White powder, yield, 20%; m.p. 146–148°C; H NMR (300 MHz, CDCl3) δ 7.09
(d, J = 8.1 Hz, 1H), 6.94 (d, J = 8.2 Hz, 2H), 5.75 (t, J = 10.6 Hz, 1H, 11b-H), 4.96 (q, J
= 6.9 Hz, 1H, 6-Hb), 3.42 (dd, J = 17.8, 10.1 Hz, 1H, 1-Hb), 3.30 (dd, J = 17.4, 11.0 Hz,
1H, 1-Ha), 2.34 (s, 3H, 10-CH3), 2.18 (s, 3H, 2-CH3), 1.49 (d, J = 6.9 Hz, 3H, 6-CH3);
13C NMR (75 MHz, CDCl3) δ 167.5, 158.0, 152.9, 133.7, 130.1, 129.0, 126.4, 122.7, 76.4
(C-6), 57.7 (11b-C), 42.0 (C-1), 21.2 (10-CH3), 17.7 (6-CH3), 16.3 (2-CH3). TOF-HRMS:
m/z [M + H]+ calcd for C14H17N2O2: 245.1285; found: 245.1291.
4.1.2. General procedure for the preparation of compounds 3t–3x
To a solution of aromatic methyl ketones (10 mmol) and salicylaldehyde (11 mmol) in
ethanol (20 mL) was added 40% NaOH solution (2 mL) dropwise and the reaction was
carried out at 60 ºC for 5 h. The mixture was poured into cold water and neutralized with
2 M HCl to a pH in the range of 5~6. The resulting precipitate was collected, washed
with water and dried to give chalcones. The chalcone was treated with 5 times excess of
hydrazine hydrate in ethanol and refluxed for 5 h. The reaction mixture was then poured
into ice-cold water. The solid was filtered, washed and recrystallized from ethanol to
afford respective pyrazoline (1). A solution of compound 1 in CH2Cl2 (20 mL) was added
α-bromoacyl chloride (4.0 mmol) and 4-dimethylaminopyridine (5.0 mmol) and the
reaction was stirred overnight. The reaction mixture was washed with water and brine,
dried with anhydrous Na2SO4, filtrated and concentrated in vacuo. The resulting residue

ACCEPTED MANUSCRIPT
was then purified by column chromatography to give product 2. NaHCO3 (3.0 mmol) was
added to an ethanol (20 mL) solution of compound 2 (2.0 mmol), then the reaction
mixture was stirred at 70 ºC until the disappearance of starting material (monitored by
TLC). EtOAc (100 mL) was added to the reaction mixture, then washed with water and
brine, dried with anhydrous Na2SO4, filtrated and concentrated in vacuo. The residue was
purified by column chromatography (petroleum/EtOAc, 1:1 → 1:2) to give title
compounds 3t–3x.
4.1.2.1. 10-Bromo-2-furan-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one
(3t). White powder, yield, 13%; m.p. 213–215°C; 1H NMR (300 MHz, CDCl3) δ 7.60 (d,
J = 1.5 Hz, 1H, Fu-H), 7.52 (dd, J = 8.5, 2.2 Hz, 1H, Ar-H), 7.43 (d, J = 1.8 Hz, 1H,
Fu-H), 7.16–7.05 (m, 2H, Ar-H), 6.58 (dd, J = 3.5, 1.7 Hz, 1H, Fu-H), 5.99– .85 (m, 1H,
11b-H), 5.02 (d, J = 16.7 Hz, 1H, 6-Hb), 4.45 (d, J = 16.6 Hz, 1H, 6-Ha), 3.83 (dd, J =
17.7, 9.3 Hz, 1H, 1-Hb), 3.67 (dd, J = 17.7, 11.5 Hz, 1H, 1-Ha); 13C NMR (75 MHz,
CDCl3) δ 164.2, 157.0, 147.8, 146.3, 145.2, 133.6, 132.7, 128.1, 123.3, 118.2, 113.4,
112.5, 72.8 (C-6), 55.5 (11b-H), 35.4 (C-1). TOF-HRMS: m/z [M + H]+ calcd for
C15H12BrN2O3: 347.0026; found: 347.0029.
4.1.2.2.
8,10-Dichloro-2-furan-6-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-
1
one (3u). White powder, yield, 15%; m.p. 249–253°C; H NMR (300 MHz, CDCl3) δ
7.58 (s, 1H, Fu-H), 7.45 (d, J = 2.1 Hz, 1H, Ar-H), 7.17 (s, 1H, Fu-H), 7.09 (d, J = 3.4 Hz,
1H, Ar-H), 6.62 – 6.53 (m, 1H, Fu-H), 5.81 (t, J = 10.7 Hz, 1H, 11b-H), 5.17 (q, J = 7.0
Hz, 1H, 6-Hb), 3.74 (dd, J = 10.7, 3.4 Hz, 2H, 1-H), 1.62 (d, J = 7.0 Hz, 3H, 6-CH3).
TOF-HRMS: m/z [M + H]+ calcd for C16H13Cl2N2O3: 351.0298; found: 351.0302.
4.1.2.3.
8,10-Dibromo-2-(4-fluoro-phenyl)-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6
H)-one (3v). White powder, yield, 12%; m.p. 276–279°C; 1H NMR (300 MHz, CDCl3) δ
7.91 (dd, J = 7.7, 5.4 Hz, 2H), 7.79 (s, 1H), 7.39 (s, 1H), 7.16 (t, J = 8.2 Hz, 2H),
6.09–5.93 (m, 1H, 11b-H), 5.06 (d, J = 16.8 Hz, 1H, 6-Hb), 4.50 (d, J = 16.8 Hz, 1H,

ACCEPTED MANUSCRIPT
13
6-Ha), 3.80 (dd, J = 17.5, 8.9 Hz, 1H, 1-Hb), 3.69 (dd, J = 17.6, 11.5 Hz, 1H, 1-Ha); C
NMR (75 MHz, CDCl3) δ 163.8, 154.9, 153.9, 136.4, 134.6, 129.7, 127.2, 126.7, 118.7,
117.8, 116.4, 116.1, 71.8 (C-6), 56.4 (11b-H), 35.6 (C-1). TOF-HRMS: m/z [M + Na]+
calcd for C17H11Br2FN2NaO2: 474.9064; found: 474.9066.
4.1.2.4.
10-Bromo-2-(4-fluoro-phenyl)-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-o
1
ne (3w). White powder, yield, 14%; m.p. 181–184°C; H NMR (300 MHz, CDCl3) δ
7.97–7.83 (m, 2H), 7.52 (dd, J = 8.5, 2.2 Hz, 1H), 7.44 (s, 1H), 7.21–6.92 (m, 3H),
6.05–5.89 (m, 1H, 11b-H), 5.03 (d, J = 16.7 Hz, 1H, 6-Hb), 4.47 (d, J = 16.7 Hz, 1H,
6-Ha), 3.82 (dd, J = 17.4, 9.3 Hz, 1H, 1-Hb), 3.68 (dd, J = 17.4, 11.5 Hz, 1H, 1-Ha).
TOF-HRMS: m/z [M + H]+ calcd for C17H13BrFN2O2: 375.0139; found: 375.0130.
4.1.2.5.
10-Chloro-2-(4-fluoro-phenyl)-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-o
1
ne (3x). White powder, yield, 12%; m.p. 209–211°C; H NMR (300 MHz, CDCl3) δ
7.99–7.86 (m, 2H), 7.42–7.27 (m, 2H), 7.15 (t, J = 8.7 Hz, 3H), 6.03–5.89 (m, 1H,
11b-H), 5.02 (d, J = 16.7 Hz, 1H, 6-Hb), 4.45 (d, J = 16.7 Hz, 1H, 6-Ha), 3.82 (dd, J =
17.4, 9.3 Hz, 1H, 1-Hb), 3.68 (dd, J = 17.4, 11.5 Hz, 1H, 1-Ha). TOF-HRMS: m/z [M +
H]+ calcd for C17H13ClFN2O2: 331.0644; found: 331.0650.
4.1.3. General procedure for the preparation of compounds (6R)-3o and (6S)-3o
To
a
CH2Cl2
(5
mL)
solution
of
2-bromo-4-chloro-6-(3-methyl-4,5-dihydro-1H-pyrazol-5-yl)phenol (150 mg, 0.52 mmol)
was added 2-chloropropionic acid (67 mg, 0.55 mmol), EDC·HCl (195 mg, 1.0 mmol)
and HOBt (135 mg, 1.0 mmol), the mixture was stirred 30 min at room temperature.
After NEt3 (140 µL) was added, the reaction solution was stirred overnight. The mixture
was washed twice with water, dried with Na2SO4, filtered, and concentrated in vacuo.
The residue was dissolved in ethanol (10 mL), NaHCO3 (84 mg, 1.0 mmol) was added to
the solution. The reaction mixture was stirred at 60–70 ºC until the disappearance of
starting material (TLC monitoring). EtOAc (100 mL) was added to the reaction mixture,

ACCEPTED MANUSCRIPT
then washed with water and brine, dried with anhydrous Na2SO4, filtrated and
concentrated in vacuo. The residue was purified by column chromatography
(petroleum/EtOAc, 1:1 → 1:3) to give enantiomers (6R)-3o and (6S)-3o.
4.1.3.1.
8-Bromo-10-chloro-6R-ethyl-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-
20
D
[α
]
5(6H)-one ((6R)-3o). White powder, yield, 32%;
= ‒202º (c = 0.164, CHCl3); m.p.
219–221°C; 1H NMR (600 MHz, CDCl3) δ 7.51 (d, J = 1.8 Hz, 1H), 7.00 (s, 1H), 5.71 (t,
J = 10.6 Hz, 1H, 11b-H), 4.55 (dd, J = 8.1, 4.9 Hz, 1H, 6-Ha), 3.52 (dd, J = 17.5, 11.2 Hz,
1H, 1-Hb), 3.22 (dd, J = 17.4, 10.0 Hz, 1H, 1-Ha), 2.15 (s, 3H, 2-CH3), 2.13–2.03 (m, 2H,
6-CH2CH3), 1.13 (t, J = 7.4 Hz, 3H, 6-CH2CH3).
4.1.3.2.
8-Bromo-10-chloro-6S-ethyl-2-methyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxazepin-
20
D
[α
]
5(6H)-one ((6S)-3o). White powder, yield, 34%;
= +167º (c = 0.154, CHCl3); m.p.
215–218°C; 1H NMR (600 MHz, CDCl3) δ 7.5c2 (d, J = 2.0 Hz, 1H), 7.00 (s, 1H), 5.71 (t,
J = 10.6 Hz, 1H, 11b-H), 4.55 (dd, J = 8.2, 4.9 Hz, 1H, 6-Ha), 3.56 – 3.47 (m, 1H, 1-Hb),
3.22 (dd, J = 17.5, 10.0 Hz, 1H, 1-Ha), 2.15 (s, 3H, 2-CH3), 2.13–2.03 (m, 2H,
6-CH2CH3), 1.13 (t, J = 7.4 Hz, 3H, 6-CH2CH3).
4.2. EeAChE and eqBuChE inhibition assays
Enzymatic inhibition assays were performed on AChE from electric eel
(C3389-500UN; Sigma) and BuChE from equine serum (C4290-1KU; Sigma), according
to the spectrophotometric Ellman’s method as previously described. The experiment was
performed in 48-well plates in a final volume of 500 µL. Each well contained 0.036
U/mL of EeAChE or eqBuChE, and 0.1 M pH 8 phosphate buffer. They were
preincubated for 20 min at different compound concentrations at 37 ºC. Then 0.35 mM
acetylthiocholine iodide (ATCh; A5751-1G; Sigma) or 0.5 mM butyrylthiocholine iodide
(20820-1G; Sigma) and 0.35 mM 5,5’-ditiobis-2-nitrobenzoico (DTNB; D8130-1G;

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Sigma) were added. The DTNB produces the yellow anion 5-thio-2-nitrobenzoic acid
along with the enzymatic degradation of acetylthiocholine or butyrylthiocholine. Changes
in absorbance were measured at 410 nm after 20 min in a Biotek Synergy HTX
Multi-Mode reader. The IC50 values were determined graphically from inhibition curves
(log inhibitor concentration vs percent of inhibition). A control experiment was performed
under the same conditions without inhibitor and the blank contained buffer, DMSO,
DTNB, and substrate.
4.3. Oil/water partition coefficient assessment and ADMET prediction
Octanol–water partition coefficients of title compounds were measured by the shake
flask method with slight modification. The aqueous phase was replaced by PBS (pH =
7.4). Both the octanol and the aqueous phase were saturated with each other before use.
The assay mixture containing test compounds was shaken at 37 ºC. After 24 h, the
mixture was centrifuged at 4000 rpm for 30 min, followed by the measured with
ultraviolet spectrophotometer. The logP values were calculated.
The active compounds were analyzed with the ADMET prediction tools of DS 2017R2.
The pharmacokinetic properties are HIA (human intestinal absorption), PPB (plasma
protein binding), Cytochrome P450 2D6 binding (CYP2D6), Aqueous solubility, and
BBB. ADMET screening in a stepwise manner is summarized as follows. HIA: there are
four prediction levels of 0–3 represent good, moderate, low, and very low absorption
respectively. The data displayed that these compounds all had good absorption (HIA
levels of 0, score < 6.126). Aqueous solubility: solubility levels of 0–5 represent
extremely low; no, very low, but possible; yes, low; yes, good; yes, optimal; no, too
soluble respectively, and we found these compounds have good solubility (solubility
level of 2 and 3). BBB: BBB levels of 0–4 represent very high, high, medium, low, and
undefined penetration respectively. The results showed that four compounds had high
penetrant and three compounds had medium penetrant. CYP2D6 binding activity:
CYP2D6 is involved in the metabolism of a wide range of substrates in the liver and its
inhibition by a drug constitutes a major of drug-drug interaction. The level of 0 and 1
reflect as non-inhibitor and inhibitor. The results showed that active compounds were
non-inhibitors of CYP2D6 (CYP2D6 level of 0). PPB: PPB levels of 0, 1, and 2 reflect

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on binding as < 90%, binding as > 90%, and binding as > 95%. Based on PSA2D and
ALogP98 standard, the active compounds were found to fulfill the set standard at both
95% and 99% confidence limit ellipses for the BBB penetration and HIA models,
respectively, in conformity with the data presented.
4.3. Cytotoxicity Assays
PC12 cells were cultured in DMEM containing 10% fetal bovine serum, 100 units/mL
penicillin, and 100 µg/mL streptomycin at 37 ºC in a 5% CO2 humidify atmosphere. Cell
cytotoxicity was evaluated by methyl thiazolyl tetrazolium (MTT) assay. PC12 cells were
inoculated at 1 × 104 cells per well in 96-well plate. After cultured for 24 h, the cells were
treated with different compounds which were diluted in DMEM for 24 h. Then 20 µL of
0.5 mg/mL MTT reagent was added into the cells and incubated for 4 h. After 4 h, cell
culture was removed and then 150 µL DMSO was added to dissolve the formazan. The
optical density was measured at 570 nm (OD570). Cell viability was calculated from three
independent experiments. The density of formazan formed in blank group was set as
100% of viability. Cell viability (%) = compound (OD570 / blank (OD570) × 100%
Blank: cultured with fresh medium only.
Compound: treated with compounds or donepezil.
4.4. Neuroprotection assay
Neuroprotective activity was evaluated as described in the literature. Differentiated
PC12 cells were incubated with different concentrations of the compound 3o for 3 h
before treatment with H2O2 (300 µM). Cell viability was measured after 24 h by using the
MTT assay. Briefly, 20 µL of 0.5 mg/mL MTT reagent was added into the cells and
incubated for 4 h. After 4 h, cell culture was removed and then 150 µL DMSO was added
to dissolve the formazan. The optical density was measured at 570 nm (OD570) on the
Biotek Synergy HTX Multi-Mode reader. Results were adjusted considering OD
measured in the blank.

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4.5. Kinetic studies of eqBuChE inhibition
Kinetic studies were performed with the same test conditions, using six concentrations
of substrate (from 0.1 to 1 mM) and four concentrations of inhibitor (0–20 µM). Apparent
inhibition constants and kinetic parameters were calculated within the “Enzyme kinetics”
module of Prism 5.
4.6. Molecular docking study
A structure based in silico procedure was applied to discover the binding modes of the
active compounds to BuChE enzyme active site. The CDOCKER of Discovery Studio
2017R2 (DS) was conducted to explain SAR of series compounds and further guide the
design of more effective and concrete BuChE inhibitors. The ligand binding to the crystal
structure of hBuChE with PDB ID: 1P0I was selected as template. The target enzyme was
prepared with Prepare Protein of DS to ensure the integrity of target. The ligand was
processed by Full Minimization of the Small Molecular in DS. Then the title compounds
were docked into the active site of protein using CDOCKER. The view results of
molecular docking were extracted after the program running end, each docking result was
analyzed for interaction and their different pose. The binding energies of most potent
compounds were clearly observed and tabulated in Table 3. The lowest
-CDOCKER_INTERACTION_ENERGY values of those poses were regarded as the
most stable and picked to analysis binding interactions with target enzyme visualized.
Acknowledgments
Financial support was provided by the National Natural Science Funding of China
(21572003, 20802003), Anhui University Natural Science Research Project
(KJ2016A339), and University Project of Introduction and Cultivation of Leading Talents
(gxfxZD2016044).
Notes
The authors declare no competing financial interest.

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BuChE, butyrylcholinesterase; AChE, acetylcholinesterase; AD, Alzheimer’s disease;
ADMET, absorption, distribution, metabolism, excretion and toxicity in
pharmacokinetics; ChE, cholinesterase; PD, Parkinson disease; MAO, monoamine
oxidase; EeAChE, Electrophorus electricus AChE; eqBuChE, equine BuChE; AChEIs,
AChE inhibitors; BBB, blood-brain barrier; HIA, human intestinal absorption; PPB,
plasma protein binding; CYP2D6, Cytochrome P450 2D6.
Appendix A. Supplementary material
Representative 1H and 13C NMR spectra can be found at http://
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