Novel Deoxyvasicinone-Donepezil Hybrids as Potential Multitarget Drug Candidates for Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.8b00699

In this study, we designed and synthesized a series of deoxyvasicinone-donepezil hybrids and determined whether they could be used as novel multitarget inhibitors for Alzheimer's disease. In vitro studies showed that most of the hybrids demonstrated moderate to potent inhibition of hAChE, BACE1, and Aβ_1-42 aggregation. In particular, the hybrids 10a, 10d, 11a, and 11j exhibited excellent inhibitory activities against hAChE (IC₅₀ = 56.14, 5.91, 3.29, and 8.65 nM, respectively), BACE1 (IC₅₀ = 0.834, 0.167, 0.129, and 0.085 μM, respectively), and Aβ_1-42 aggregation (IC₅₀ = 13.26, 19.43, 9.26, and 5.41 μM, respectively). In addition, 10a and 11a exhibited very low cytotoxicity and showed remarkable neuroprotective activity against Aβ_1-42-induced damage in SH-SY5Y cells.

Full text of DOI:10.1021/acschemneuro.8b00699

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Article
Novel deoxyvasicinone–donepezil hybrids as potential
multitarget drug candidates for Alzheimer's disease
Hong-tao Du, Xinlian Liu, Jusen Xie, and Fang Ma
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00699 • Publication Date (Web): 05 Feb 2019
Downloaded from http://pubs.acs.org on February 7, 2019
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Novel deoxyvasicinone–donepezil hybrids as potential multitarget drug candidates for
Alzheimer's disease
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Hongtao Du a, d, *, Xinlian Liu a, b, Jusen Xie a, b, Fang Ma c, *
a College of Life Science, Xinyang Normal University, Xinyang, 464000, China
b Tea Plant Biology Key Laboratory of Henan Province, Xinyang, 464000, China
c School of Geographic Sciences, Xinyang Normal University, Xinyang, 464000, China
d College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi Province, China
E-mails: duhongtao8410@163.com (H. T. Du); 13782955771@163.com (X. L. Liu); 13782956291@163.com (J. S. Xie);
mafang_1984@126.com (F. Ma).
*Corresponding authors:
Dr. Hongtao Du,
College of Life Science, Xinyang Normal University, Xinyang, 464000, China.
Email: duhongtao8410@163.com
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Abstract:
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In this study, we designed and synthesized a series of deoxyvasicinone–donepezil hybrids and
determined whether they could be used as novel multi-target inhibitors for Alzheimer's disease (AD).
In vitro studies showed that the most of the hybrids demonstrated moderate to potent inhibition of
hAChE, BACE1, and Aβ1-42 aggregation. In particular, the hybrids 10a, 10d, 11a, and 11j exhibited
excellent inhibitory activities against hAChE (IC50 = 56.14 nM, 5.91 nM, 3.29 nM, and 8.65 nM,
respectively), BACE1 (IC50 = 0.834 μM, 0.167 μM, 0.129 μM, and 0.085 μM, respectively), and
Aβ1-42 aggregation (IC50 = 13.26 μM, 19.43 μM, 9.26 μM, and 5.41 μM, respectively). In addition,
10a and 11a exhibited very low cytotoxicity and showed remarkable neuroprotective activity against
Aβ1-42–induced damage in SH-SY5Y cells.
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Keywords: Alzheimer's disease; Acetylcholinesterase; β-secretase; β-amyloid peptide;
Deoxyvasicinone
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 INTRODUCTION
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Alzheimer's disease (AD), the most common form of dementia, is a chronic and
neurodegenerative illness with a number of symptoms, including loss of memory, deterioration in the
use of language, mood swings, and loss of bodily functions leading finally to death.1-3
Epidemiological data indicate that more than 45 million people suffered from AD in 2015, a number
that is projected to triple by 2050.4 Currently, the most popular drugs for AD include one
N-methyl-D-aspartate (NMDA) receptor antagonist (memantine) and three cholinesterase (ChE)
inhibitors (donepezil, rivastigmine, and galantamine).5 Unfortunately, these drugs only partially
alleviate the symptoms of AD and cannot cure the disease. The exact causes of AD are not yet fully
known, but some factors, such as deficits of acetylcholine (ACh), β-amyloid peptide (Aβ) deposits,
tauprotein (τ) aggregation, neuroinflammation, and oxidative stress, are suggested to play significant
roles in the pathogenesis of AD.6-9 Due to the complexity of AD and the multitude of factors
potentially involved in its progression, a strategy that uses multi-target-directed ligands (MTDLs)
has drawn much attention as a mainstream therapeutic approach for treatment of AD.10-14
The deposition of Aβ plays a crucial role in the pathogenesis of AD.15 In particular, Aβ1-42 leads
to the formation of senile plaques that causes neuronal death and eventually dementia. The initial and
rate-determining step in the production of Aβ is the cleavage of amyloid precursor protein (APP) by
β-secretase (BACE1).16 Therefore, drugs with BACE1 inhibition activity reduce Aβ production and
aggregation.17 Additionally, acetylcholinesterase inhibitors (AChEIs) increase the amount of ACh by
blocking acetylcholine hydrolysis in the synaptic cleft.18 Furthermore, recent studies have shown that
AChEIs are able to influence the expression and/or metabolic processing of APP, which may affect
Aβ generation.19 Moreover, some evidence has shown that the peripheral anionic site (PAS) of AChE
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contributes to Aβ aggregation.20 Therefore, we speculate that a single molecule with inhibitory
activity against AChE, BACE1, and Aβ1-42 aggregation could be a promising potential agent for AD
therapy.
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[Fig. 1]
To design novel MTDLs against AD, a pharmacophore-combination strategy was used with
deoxyvasicinone and donepezi as the lead compounds. Deoxyvasicinone (Fig. 1), a naturally
occurring alkaloid, is composed of a quinazolinone moiety conjugated with pyrrolidine. Its
derivatives have been identified as novel cholinesterase inhibitors (Fig. 1).21-25 We have previously
reported a series of deoxyvasicinone derivatives with significant cholinesterase inhibitory activity.25
These derivatives could bind simultaneously to the catalytic active site (CAS) and the peripheral
anionic site (PAS) of hAChE. Donepezil is an AChE inhibitor used to treat AD.5 The
benzylpiperidine fragment of donepezil is considered to be a pharmacophore that can bind to the
CAS site of AChE.26-28 In addition, some research has suggested that compounds with an aryl
piperazine moiety exhibit significant BACE1 inhibition.29-31 In the current study, we describe the
design, synthesis, and biological evaluation of a series of novel deoxyvasicinone–donepezil hybrids,
in which the piperidine fragment of donepezil is replaced by the piperazine moiety (Fig. 2).
According to the aforementioned reports, these new deoxyvasicinone–donepezil hybrids may
simultaneously possess a dual AChE binding sites for inhibition and inhibitory activity against
BACE1 and Aβ1-42 aggregation. Additionally, some target compounds were also evaluated for
blood-brain barrier (BBB) permeability and protective capacity for Aβ1-42–induced H-SY5Y cell
death. Furthermore, computational studies were performed to predict binding modes in the active
pocket.
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[Fig. 2]
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 CHEMISTRY
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The compounds 8a−11m were synthesized as reported in Scheme 1. To obtain deoxyvasicinone
(2), 2-aminobenzoic acid was reacted with pyrrolidin-2-one analogues using POCl3 as a catalyst in
dry toluene. Nitration reaction of deoxyvasicinone (2) provided the product, which was reduced to
6-aminodeoxyvasicinone (3). Subsequently, an amidation reaction of the compound 3 with different
bromo-acids gave the key intermediates 4−7.25 Finally, the nucleophilic substitution reaction of
compound 4−7 with different benzylpiperazine derivatives yielded the target compounds 8a−11m.
[Scheme 1]
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 RESULTS AND DISCUSSION
In Vitro hAChE and hBuChE Inhibition Assays
Inhibition of hAChE and hBChE by synthesized derivatives 8a−11m was achieved using
Ellman’s method, wherein tacrine and donepezil were used as reference compounds.32, 33 First, we
screened all of the compounds at a concentration of 1 μM, and IC50 values were measured for those
with over 50% inhibitory activity against ChEs in the primary screen. The IC50 values and their
selectivity index are depicted in Table 1.
The tested target compounds showed fascinating inhibitory activity against hAChE. Almost all of
the compounds display high selectivity for hAChE over hBChE (except 11f). Most compounds are
potent inhibitors of hAChE, with IC50 values in the low nanomolar range. Among of them, 13
compounds exhibit significantly higher inhibitory activity than tacrine (IC50 = 76.53 nM).
Importantly, compounds 9a, 9b, 10d, 11a, 11b, 11c, 11f, and 11i have similar or higher inhibitory
activity against hAChE than donepezil (IC50 = 18.54 nM). In particular, compound 11a showed
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excellent inhibitory potential and selectivity towards hAChE (IC50 = 3.29 nM; BuChE/AChE > 303).
Furthermore, some structure-activity relationships were surveyed. The length of the alkyl spacer
significantly influenced hAChE inhibitory activity. Compounds 11a−11d (n= 2) exhibited more
potent AChE inhibitory than compounds 8a−8d (n = 1), compounds 9a−9d (n = 3), and compounds
10a−10d (n = 4). Thus, lengthening or shortening the alkyl spacer reduced AChE inhibition. In
addition, the methyl substituted compounds (except 11f and 11g) showed better inhibitory activity
than the corresponding unsubstituted hybrids, when the hybrids had same chain linkers. On the
contrary, hAChE inhibitory activity decreased in the presence of electron-withdrawing substituents,
such as –F, –CF3, –Cl. These results imply that increasing the strength of the electron-donating group
could improve hAChE inhibitory activity.
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BACE1 enzymatic assay
BACE1 inhibitory activity of the synthetic hybrids was tested using a FRET-based assay kit.29
The inhibitory activity of the test compounds was evaluated at a concentration of 10 μM. The IC50
values were then calculated for those with a BACE1 inhibition rate over 50% (Table 1). As shown in
Table 1, 17 compounds exhibited good BACE1 inhibitory activity with IC50 values ranging from
0.028 μM to 9.03 μM. Notably, 11 hybrids displayed interesting BACE1 inhibitory activity at
concentrations below 1 µM. Among them, compounds 9a and 10c are the most potent hybrids
against BACE1 (IC50 = 0.028 μM and 0.085 μM, respectively), similar to OM99-2 (IC50 = 0.016
μM).
Inhibition of Aβ1-42 self-aggregation assay
The Thioflavin-T (ThT) fluorescence assay was used to evaluate the inhibitory potency of the
derivatives on Aβ1-42 self-aggregation.34 Resveratrol and curcumin were used as controls (Table 1).
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Most of compounds exhibited moderate or strong inhibitory activity. The compounds 10a, 10d, 11a,
and 11j exhibited significant inhibition of Aβ1-42 aggregation (IC50 =13.26 nM, 19.43 nM, 9.26 nM,
and 5.41 nM, respectively), similar to or higher than the inhibitory activity of resveratrol (IC50
=10.92 nM) and curcumin (IC50 =14.25 nM). Compound 11j displayed the strongest inhibitory
activity, 2-fold better than that of resveratrol.
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Inhibition of AChE induced Aβ1-42 peptide aggregation assay
Accumulating evidences have indicated that AChE exerts its non-cholinergic function by
promoting the aggregation of Aβ peptide, and compounds binding to the PAS of AChE can inhibit
AChE accelerated Aβ aggregation. Thus, we evaluated the AChE induced Aβ1-42 aggregation
inhibition by compounds 10a, 11a and 11j using the ThT assay. As displayed in Fig. 3, Aβ1-42
aggregation was significant decrease after Co-incubation of synthesized compounds and Aβ1-42 with
AChE for 24 h. The result clearly demonstrates that these compounds can interfere with the AChE
induced Aβ1-42 aggregation.
[Fig. 3]
Kinetic characterization of AChE inhibition
To investigate the mechanistic aspects of inhibition of AChE activity by synthesized compounds,
the potent compound 11a was chosen for an enzyme kinetic study. From the Lineweaver-Burk plot
(Fig. 4), we observed that both increased slopes (decreased Vmax) and intercepts (higher Km) at
increasing concentrations of compound 11a, indicating a mixed-type inhibition. The result revealed
that compound 11a could bind to the CAS as well as the PAS of AChE.
[Fig .4]
In vitro cytotoxicity assay
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The compounds 10a, 10d, 11a, and 11j with potential inhibitory activity against AChE, BACE1,
and Aβ1-42 aggregation were selected to evaluate the potential for toxicity in the human neuronal cell
line SH-SY5Y. SH-SY5Y cells were exposed to 10a, 10d, 11a, and 11j at different concentrations
(0.1, 1, 10, and 100 μM) for 48 h, and cell viability was determined using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay.35 As depicted in Fig. 5A,
compounds 10a, 11a, and 11j show no cytotoxicity at concentrations of 0.1 μM to 1 μM after
incubation for 48 h. Compound 11a had no significant effect on cell viability at a concentration of 10
μM. When the concentrations were increased to 100 μM, compounds 10a, 11a, and 11j displayed
significant effects on SH-SY5Y cell viability; however, the cell viability was still above 78%. These
results suggest that compounds 10a, 11a, and 11j exhibit very low toxicity to SH-SY5Y cells in the
range of tested concentrations.
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To assess hepatotoxicity of synthesized compounds, MTT assay of compounds 10a, 11a and 11j
on liver hepatocellular cells (HepG2) was performed. As shown in Fig. 5B, compounds 10a and 11a
were no cytotoxicity at concentrations of 1– 10 μM after incubation for 48 h. When the concentration
of compounds 10a and 11a at 100 μM, the cell viability was still above 88%. Unfortunately,
compounds 11j resulted hepatotoxicity at 10 μM concentration, meanwhile a significant reduction of
52 % was observed at 100 μM. These means that compounds 10a and 11a were weakly
hepatotoxicity.
[Fig. 5]
The neuroprotective effect of 10a, 11a, and 11j on Aβ1-42–induced damage in SH-SY5Y cells
was assessed using a cell viability assay at three different concentrations: 1 μM, 5 μM, and 10 μM.
As shown in Fig. 6, a significant decrease in cell viability (54.5%, ### p < 0.001) was observed when
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SH-SY5Y cells were treated with Aβ1-42 for 48 h. However, it is obvious that the tested compounds
imparted significant neuroprotection at concentrations between 5 μM and 10 μM. The most potent
compound 11j induced marked cellular recovery at the lowest concentration of 1μM (**p < 0.01),
when compared with the Aβ1-42 treated group. Thus, it seems reasonable to propose that the tested
hybrids exhibit neuroprotection against Aβ1-42-induced neurotoxicity through their ability to block
Aβ1-42 aggregation.
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[Fig. 6]
Physicochemical Properties.
To determine the potential drug-likeness of compounds 10a, 11a, and 11j, their physicochemical
properties, such as the calculated octanol/water partition coefficient (cLogP), topological polar
surface area (tPSA), the logarithm of brain to blood concentration ratio (LogBB), and the verification
of Lipinski's rule of five,36, 37 were predicted (Table 2). It can be seen that compounds 10a, 11a, and
11j obey Lipinski’s rule of five, fulfill the restrictions of topological polar surface area (tPSA), and
present good BBB permeability (LogBB). Those results indicate that the selected hybrids (10a, 11a,
and 11 j) are eligible to be orally-available drug candidates for the central nervous system (CNS).
Molecular docking
To further investigate the binding mode of the compounds with their target enzymes, molecular
docking simulations of the highest potent hybrid 11a with hAChE (PDB code: 4EY7),38 BACE1
(PDB code: 4D8C),39 and Aβ1-42 (PDB code: 1IYT)40 were performed using the Surflex-Dock
program in Sybyl-X 2.0 Software (Fig. 7).
As depicted in Fig. 7A. 11a was able to simultaneously bind to both the enzymatic catalytic
active site (CAS) and the peripheral anionic site (PAS). This is similar to the way that the amide
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group of deoxyvasicinone binds to the PAS through a cation−π interaction with Trp286 (3.2 Å), and
the carbonyl group creates two hydrogen bonds with residues Phe295 (2.2 Å) and Arg296 (2.1 Å). In
mid-gorge sites, a hydrogen bond with the residue Tyr124 (2.0 Å) and a cation−π interaction with
Phe338 (3.8 Å) were observed, and could enhance the interaction with hAChE. In addition, the
benzene ring of piperazine was able to bind to the CAS sites via a π−π stacking interaction with
Trp86 (3.0 Å). This means that enhancing the electron-donating group of the benzene ring could
intensify hAChE inhibitory activity, consistent with our previous results.
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As shown in Fig. 7B, a cation−π interaction was found between residue Try71 and the amino
group of piperazine (4.3 Å), which may improve the potency of BACE-1 inhibitory activity.
Additionally, the hybrid 11a formed four hydrogen bonds with residues Thr72 (2.99 Å and 3.4 Å),
Try18 (3.4 Å) and Gly34 (3.5 Å), which may contribute to improving the interaction between small
ligand with BACE1, eventually leading to good inhibitory potency against BACE-1.
Examination of Fig. 7C shows that the benzene ring of piperazine interacts with the residue
Phe20 via a π−π stacking interaction at a distance of 3.1 Å. In addition, the carbonyl oxygen and the
amino group of piperazine forms hydrogen bonds with Lys16 at distances of 2.1 Å and 2.0 Å
respectively, and the amino group of deoxyvasicinone moiety interacts with Gln15 via another
hydrogen bond (1.9 Å). These results indicate that the π−π stacking interaction and three hydrogen
bond interactions play important roles in the effective inhibition of Aβ1-42 aggregation.
[Fig. 7]
 CONCLUSIONS
In summary, we designed and synthesized 25 deoxyvasicinone–donepezil hybrids as
multi-functional agents for treatment AD. Among them, 13 compounds exhibited similar to or higher
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inhibitory activity against hAChE than the standard drugs tacrine (IC50 = 76.53 nM) and donepezil
(IC50 = 18.54 nM). Moreover, 17 compounds exhibited good to potential BACE1 inhibitory activities
with IC50 values from 0.028 μM to 9.03 μM. In addition, the compounds 10a, 10d, 11a, and 11j
displayed significant inhibition of Aβ1-42 aggregation (IC50 =13.26 nM, 19.43 nM, 9.26 nM, and 5.41
nM, respectively), and possessed good hAChE and BACE1 inhibitory activities. Importantly,
compounds 10a, 11a, and 11j showed remarkable neuroprotective activity against Aβ1-42–induced
damage in SH-SY5Y cells. In addition, compounds 10a and 11a were low toxicity. Moreover, these
compounds should be able to penetrate the blood-brain barrier (BBB). Thus, compounds 10a and 11a
should be considered to be effective and promising multi-functional agents against AD.
 METHODS
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General Remarks
All reagents and solvents were obtained from commercial suppliers and used without further
purification. Silica GF254 for thin-layer chromatography (TLC) were produced by Qingdao Marine
Chemical Company (China). Column chromatography was performed on silica gel 200-300 mesh
Qingdao Marine Chemical Company (China).
1H NMR and 13C NMR spectra were recorded on a 600 MHz NMR spectrometer （ JEOL
ECZ600R/S3）equipped with a 14.09 T superconducting magnet and a 5.0 mm 600MHz broadband
Z-gradient high resolution ROYAL probe (JEOL RESONANCE Inc., Japan). Chemical shifts are
reported in parts per million (ppm, δ) using CDCl3 as solvent and tetramethylsilane (TMS) as a
reference. Multiplicities are reported as follows: singlet (s), doublet (d), triplet (t), multiplet (m).
High resolution mass spectra (HRMS) were recorded on Agilent 6520 Q-TOF LC/MS and Varian
7.0T FTMS (MALDI).
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Preparation of intermediates 2−7
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The compound 2 and 3 was synthesized according to a previously described method [25]. Then, a
suspension of compound 3 (0.509 g, 2.5 mmol) in dichloromethane (30 mL) was added slowly to a
solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.566g, 3 mmol) and
bromo-acid (2.7 mmol) in dichloromethane (40 mL). The mixture was refluxed for 24 h and
concentrated in vacuum. The reaction solution was washed in order with water, brine, dried over
anhyd Na2SO4, and concentrated in vacuum to give compound 4−7.
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Compound 5. 1H NMR (600 MHz, Chloroform-d) δ 8.68 (s, 1H), 8.29 (dd, J = 8.9, 2.5 Hz, 1H),
8.08 (d, J = 2.6 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 4.26 – 4.18 (m, 2H), 3.76 (t, J = 5.6 Hz, 2H), 3.18
(t, J = 7.9 Hz, 2H), 2.69 (t, J = 5.6 Hz, 2H), 2.35 – 2.26 (m, 2H). 13C NMR (150 MHz, Chloroform-d)
δ 170.0, 160.8, 158.4, 145.6, 136.6, 127.7, 127.0, 120.7, 115.6, 59.1, 46.5, 37.9, 32.4, 19.6.
Compound 6. 1H NMR (600 MHz, Chloroform-d) δ 8.59 (dd, J = 9.0, 2.7 Hz, 1H), 7.97 (d, J =
2.6 Hz, 1H), 7.67 (d, J = 9.0 Hz, 1H), 4.25 – 4.19 (m, 2H), 3.98 (t, J = 7.0 Hz, 2H), 3.19 (t, J = 7.9
Hz, 2H), 2.67 (dd, J = 8.5, 7.7 Hz, 2H), 2.35 – 2.27 (m, 2H), 2.22 (m, 2H). 13C NMR (150 MHz,
Chloroform-d) δ 174.5, 160.8, 158.7, 145.7, 137.9, 127.5, 127.2, 120.5, 114.5, 48.9, 46.6, 32.8, 32.4,
19.6, 17.9.
Compound 7. 1H NMR (600 MHz, Chloroform-d) δ 8.45 (s, 1H), 8.39 (dd, J = 8.9, 2.5 Hz, 1H),
8.11 (d, J = 2.5 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 4.19 (t, J = 7.3 Hz, 2H), 3.56 (t, J = 6.2 Hz, 2H),
3.24 – 3.16 (m, 2H), 2.47 (t, J = 7.0 Hz, 2H), 2.36 – 2.28 (m, 2H), 1.94 – 1.84 (m, 4H). 13C NMR
(151 MHz, Chloroform-d) δ 171.1, 160.8, 158.3, 145.5, 136.9, 127.7, 126.9, 120.5, 115.3, 46.6, 44.6,
36.5, 32.3, 31.9, 22.72, 19.6.
General procedure for the preparation of compounds 8a−11m.
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To a stirred solution of compound 4 (161 mg, 0.5 mmol), K2CO3 (140 mg, 1 mmol), and
1-benzylpiperazine (176 mg, 1 mmol) in acetonitrile (50 mL) was refluxed. When the compound 4
disappeared (as detected by TLC), the solvents were removed under reduced pressure. Distilled
water (25 mL) was then added, and the mixture was extracted with chloroform (20 mL × 3). The
organic solvent phase was washed by water (20 mL × 2), brine (20 mL × 2), dried over anhyd
Na2SO4, and evaporated under vacuum. The compounds 8a were purified by a flash chromatography
on silica gel. The compound 8b−11m were prepared according to the general procedure.
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N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-2-(4-benzylpiperazin-1-yl)acetamide
(8a). White solid, yield: 63%, m.p. 168−170 °C. 1H NMR (600 MHz, Chloroform-d) δ 11.39 (s, 1H),
8.16 (d, J = 2.2 Hz, 1H), 8.15 (dd, J = 8.7, 2.5 Hz, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.35 – 7.30 (m, 4H),
7.26 (dt, J = 7.6, 2.0 Hz, 1H), 3.58 (s, 2H), 3.32– 3.29 (m, 2H), 3.16 (t, J = 7.9 Hz, 2H), 2.75 – 2.61
(m, 10H), 2.30 (dt, J = 15.2, 8.0 Hz, 2H). 13C NMR (150 MHz, Chloroform-d) δ 170.4, 160.4, 157.8,
144.9, 137.5, 136.9, 128.8, 127.9, 127.3, 126.8, 126.4, 120.5, 114.8, 62.5, 60.5, 52.9, 52.1, 46.2, 32.1,
19.5. HRMS (ESI): Calcd. for C24H28N5O2 [M+H]+ 418.2243, found 418.2239.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-2-(4-(4-methylbenzyl)piperazin-1-yl)a
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cetamide (8b). White solid, yield: 70 %, m.p. 193−194 °C. H NMR (600 MHz, Chloroform-d) δ
11.34 (s, 1H), 8.11 (d, J = 2.1 Hz, 1H), 8.09 (dd, J = 8.6, 2.5 Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H), 7.18
(d, J = 8.0 Hz, 2H), 7.07 (d, J = 7.7 Hz, 2H), 3.49 (s, 2H), 3.29 – 3.25 (m, 2H), 3.10 (t, J = 7.9 Hz,
2H), 2.68 – 2.54 (m, 10H), 2.27 (s, 3H), 2.25 (dt, J = 15.2, 8.0 Hz, 2H). 13C NMR (150 MHz,
Chloroform-d) δ169.4, 159.9, 157.6, 144.7, 135.8, 135.4, 133.9, 128.3, 128.1, 126.9, 126.1, 119.9,
114.6, 63.4, 61.7, 52.6, 51.4, 45.6, 31.5, 20.2, 18.7. HRMS (ESI): Calcd. for C25H30N5O2 [M+H]+
432.2401, found 432.2395.
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cetamide (8c). White solid, yield: 48 %, m.p. 186−188 °C. H NMR (600 MHz, Chloroform-d) δ
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11.35 (s, 1H), 8.20 (dd, J = 8.8, 2.5 Hz, 1H), 8.11 (d, J = 2.5 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.32
– 7.28 (m, 2H), 7.03 – 6.97 (m, 2H). 3.52 (s, 2H), 3.30– 3.26 (m, 2H), 3.14 (t, J = 7.9 Hz, 2H), 2.74
– 2.60 (m, 10H), 2.28 (dt, J = 15.2, 8.0 Hz, 2H). 13C NMR (150 MHz, Chloroform-d) δ 170.8, 161.8
(d, J = 245.0 Hz), 158.3, 144.9, 136.8, 133.4, 130.0, 127.4, 126.5, 120.3, 115.1, 114.9, 62.5, 60.4,
53.0, 51.9, 45.4, 33.1, 19.8. HRMS (ESI): Calcd. for C24H27FN5O2 [M+H]+ 436.2149, found
436.2136.
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azin-1-yl)acetamide (8d). Faint yellow, yield: 65 %, m.p. 153−154 °C. H NMR (600 MHz,
Chloroform-d) δ 11.35 (s, 1H), 8.22 (dd, J = 8.8, 2.5 Hz, 1H), 8.13 (d, J = 2.5 Hz, 1H), 7.82 (d, J =
7.6 Hz, 2H), 7.60 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 7.6 Hz, 2H), 3.63 (s, 2H), 3.34– 3.30 (m, 2H), 3.16
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(t, J = 7.9 Hz, 2H), 2.74 – 2.60 (m, 10H), 2.30 (dt, J = 15.2, 8.0 Hz, 2H). C NMR (150 MHz,
Chloroform-d) δ 170.3, 160.5, 158.5, 145.2, 139.8, 137.5, 130.3, 129.3, 126.9, 126.7, 124.7, 124.2 (q,
J = 230 Hz), 120.5, 114.9, 62.6, 60.6, 53.3, 51.9, 45.6, 31.3, 21.0. HRMS (ESI): Calcd. for
C26H29F3N5O2 [M+H]+ 500.2273, found 500.2261.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-4-(4-benzylpiperazin-1-yl)butanamid
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e (9a). White solid, yield: 68 %, m.p. 185−187 °C. H NMR (600 MHz, Chloroform-d) δ 11.39 (s,
1H), 8.57 (dd, J = 9.0, 2.7 Hz, 1H), 7.97 (d, J = 2.6 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.26 – 7.20 (m,
4H), 7.15 – 7.1 (m, 1H), 3.70 (s, 2H), 3.18 (t, J = 7.9 Hz, 2H), 2.96 – 2.92 (m, 2H), 2.76 – 2.55 (m,
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12H), 2.32 (dt, J = 15.2, 8.0 Hz, 2H), 2.04– 1.98 (m, 2H). C NMR (150 MHz, Chloroform-d) δ
174.5, 160.8, 158.6, 145.7, 137.9, 137.2, 129.1, 128.4, 127.5, 127.1, 126.6, 120.5, 114.5, 62.8, 55.5,
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54.2, 52.3, 46.5, 32.7, 32.4, 19.58, 18.9. HRMS (ESI): Calcd. for C26H32N5O2 [M+H]+ 446.2556,
found 446.2560.
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utanamide (9b). White solid, yield: 61 %, m.p. 161−163 °C. H NMR (600 MHz, Chloroform-d) δ
11.39 (s, 1H), 8.38 (dd, J = 9.0, 2.7 Hz, 1H), 7.77 (d, J = 2.6 Hz, 1H), 7.58 (d, J = 9.0 Hz, 1H), 7.25
(d, J = 7.8 Hz, 2H), 7.17 (d, J = 7.8 Hz, 2H), 3.72 (s, 2H), 3.19 (t, J = 7.9 Hz, 2H), 2.98 – 2.94 (m,
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2H), 2.78 – 2.56 (m, 12H), 2.75 (s, 3H), 2.32 (dt, J = 15.2, 8.0 Hz, 2H), 2.08 – 2.02 (m, 2H). C
NMR (150 MHz, Chloroform-d) δ 172.8, 160.8, 158.4, 145.4, 137.5, 136.4, 129.2, 128.5, 127.7,
127.1, 126.6, 120.9, 115.3, 62.6, 53.5, 53.3, 52.3, 46.4, 32.4, 32.3, 21.13, 19.5, 18.6. HRMS (ESI):
Calcd. for C27H34N5O2 [M+H]+ 460.2713, found 460.2709.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-4-(4-(4-fluorobenzyl)piperazin-1-yl)b
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utanamide (9c). White solid, yield: 49 %, m.p. 170−172°C. H NMR (600 MHz, Chloroform-d) δ
11.41 (s, 1H), 8.18 (dd, J = 8.8, 2.5 Hz, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.34
(dd, J = 8.6, 5.5 Hz, 2H), 7.08 (t, J = 8.7 Hz, 2H), 3.58 (s, 2H), 3.15 (t, J = 7.9 Hz, 2H), 2.98 – 2.94
(m, 2H), 2.78 – 2.64 (m, 12H), 2.39 (dt, J = 15.2, 8.0 Hz, 2H), 2.12 – 2.06 (m, 2H). 13C NMR (150
MHz, Chloroform-d) δ 170.9, 162.2 (d, J = 265.0 Hz), 160.9, 158.3, 145.4, 137.4, 133.8, 130.7,
127.8, 126.9, 121.1, 115.3, 115.2. 63.2, 55.8, 54.6, 52.8, 46.5, 33.7, 31.4, 19.7, 18.2. HRMS (ESI):
Calcd. for C26H31FN5O2 [M+H]+ 464.2462, found 464.2457.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-4-(4-(4-(trifluoromethyl)benzyl)piper
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azin-1-yl)butanamide (9d). Faint yellow, yield: 71 %, m.p. 207−209 °C. H NMR (600 MHz,
Chloroform-d) δ 11.38 (s, 1H), 8.22 (dd, J = 8.8, 2.5 Hz, 1H), 8.13 (d, J = 2.5 Hz, 1H), 7.81 (d, J =
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7.6 Hz, 2H), 7.63 (d, J = 8.8 Hz, 1H), 7.22 (d, J = 7.6 Hz, 2H), 3.66 (s, 2H), 3.18 (t, J = 7.9 Hz, 2H),
3.02 – 2.98 (m, 2H), 2.78 – 2.58 (m, 12H), 2.35 (dt, J = 15.2, 8.0 Hz, 2H), 2.06 – 1.98 (m, 2H). 13C
NMR (150 MHz, Chloroform-d) δ 170.7, 160.5, 158.3, 145.4, 139.8, 137.4, 130.4, 129.5, 127.1,
126.8, 124.6, 124.1 (q, J = 241 Hz), 120.8, 115.2, 62.8, 56.2, 55.3, 52.4, 46.2, 33.1, 30.7, 19.9, 18.8.
HRMS (ESI): Calcd. for C27H31F3N5O2 [M+H]+ 514.2430, found 514.2422.
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N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-5-(4-benzylpiperazin-1-yl)pentanami
de (10a). White solid, yield: 54 %, m.p. 176−178 °C. 1H NMR (600 MHz, Chloroform-d) δ 11.36 (s,
1H), 8.06 (dd, J = 8.8, 2.5 Hz, 1H),, 7.69 (d, J = 2.5 Hz, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.36 – 7.30 (m,
4H), 7.24 – 7.21 (m, 1H), 3.60 (s, 2H), 3.15 (t, J = 8.0 Hz, 2H), 2.80 – 2.54 (m, 12H), 2.35-2.31 (m,
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2H). 2.27 (dt, J = 15.2, 8.0 Hz, 2H), 2.02 – 1.94 (m, 4H). C NMR (150 MHz, Chloroform-d) δ
170.2, 160.5, 159.5, 147.4, 137.8, 141.5, 133.2, 128.5, 127.5, 127.1, 124.0, 120.9, 115.2, 60.8, 55.3,
54.3, 51.3, 44.5, 31.2, 30.8, 19.8, 18.1, 17.2. HRMS (ESI): Calcd. for C27H34N5O2 [M+H]+ 460.2713,
found 460.2719.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-5-(4-(4-methylbenzyl)piperazin-1-yl)p
entanamide (10b). White solid, yield: 66 %, m.p. 169−171 °C. 1H NMR (600 MHz, Chloroform-d) δ
11.40 (s, 1H), 8.18 (dd, J = 8.8, 2.5 Hz, 1H), 7.88 (d, J = 2.5 Hz, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.18
(d, J = 8.0 Hz, 2H), 7.09 (d, J = 7.7 Hz, 2H), 3.58 (s, 2H), 3.17 (t, J = 8.0 Hz, 2H), 2.80 – 2.54 (m,
12H), 2.38-2.34 (m, 2H). 2.31 (s, 3H), 2.27 (dt, J = 15.2, 8.0 Hz, 2H), 2.00 – 1.96 (m, 4H). 13C NMR
(150 MHz, Chloroform-d) δ 170.5, 160.5, 158.4, 145.6, 137.4, 136.8, 134.6, 129.2, 128.7, 127.5,
126.3, 120.5, 115.0, 61.2, 55.6, 54.3, 51.5, 44.8, 31.4, 30.7, 19.7, 18.3, 17.8, 16.9. HRMS (ESI):
Calcd. for C28H36N5O2 [M+H]+ 474.2869, found 474.2858.
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N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-5-(4-(4-fluorobenzyl)piperazin-1-yl)p
entanamide (10c). White solid, yield: 57 %, m.p. 194−196 °C. 1H NMR (600 MHz, Chloroform-d) δ
11.38 (s, 1H), 8.16 (dd, J = 8.8, 2.5 Hz, 1H), 7.91 (d, J = 2.5 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.36
(dd, J = 8.6, 5.5 Hz, 2H), 7.15 (t, J = 8.7 Hz, 2H), 3.62 (s, 2H), 3.17 (t, J = 8.0 Hz, 2H), 2.72 – 2.46
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(m, 12H), 2.36-2.32 (m, 2H). 2.28 (dt, J = 15.2, 8.0 Hz, 2H), 2.00 – 1.96 (m, 4H). C NMR (150
MHz, Chloroform-d) δ 170.4, 161.06 (d, J = 245.0 Hz), 159.74, 158.39, 146.2, 138.1, 134.2, 131.7,
128.2, 127.36, 120.82, 115.12, 114.81, 61.95, 55.87, 54.90, 50.2, 43.7, 31.8, 29.9, 19.94, 18.6, 18.3,
17.5. HRMS (ESI): Calcd. for C27H33FN5O2 [M+H]+ 478.2618, found 478.2615.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-5-(4-(4-(trifluoromethyl)benzyl)piper
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azin-1-yl)pentanamide (10d). Faint yellow, yield: 65 %, m.p. 211−213 °C. H NMR (600 MHz,
Chloroform-d) δ 11.38 (s, 1H), 8.22 (dd, J = 8.8, 2.5 Hz, 1H), 8.13 (d, J = 2.5 Hz, 1H), 7.74 (d, J =
7.6 Hz, 2H), 7.56 (d, J = 8.8 Hz, 1H), 7.16 (d, J = 7.6 Hz, 2H), 3.66 (s, 2H), 3.30– 3.28 (m, 2H), 3.18
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(t, J = 7.9 Hz, 2H), 2.77 – 2.59 (m, 10H), 2.27 (dt, J = 15.2, 8.0 Hz, 2H), 2.00 – 1.96 (m, 4H). C
NMR (150 MHz, Chloroform-d) δ 170.5, 160.2, 158.1, 145.1, 139.9, 137.3, 130.5, 129.4, 126.9,
126.8, 124.6, 124.2 (q, J = 232 Hz), 120.5, 114.9, 62.1, 55.8, 54.2, 50.7, 43.2, 32.1, 30.7, 19.8, 18.6,
17.7. HRMS (ESI): Calcd. for C28H33F3N5O2 [M+H]+ 528.2586, found 528.2579.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-benzylpiperazin-1-yl)propanami
de (11a). White solid, yield: 68 %, m.p. 184−185 °C. 1H NMR (600 MHz, Chloroform-d) δ 11.41 (s,
1H), 8.23 – 8.14 (m, 2H), 7.62 (d, J = 8.7 Hz, 1H), 7.38 – 7.32 (m, 4H), 7.30 – 7.27 (m, 1H), 4.27 –
4.19 (m, 2H), 3.60 (s, 2H), 3.18 (t, J = 7.9 Hz, 2H), 2.78 – 2.56 (m, 12H), 2.30 (dt, J = 15.2, 8.0 Hz,
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2H). C NMR (150 MHz, Chloroform-d) δ 170.8, 160.7, 158.2, 145.3, 137.9, 137.2, 129.2, 128.3,
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127.7, 127.2, 126.7, 120.8, 115.2, 62.9, 53.6, 53.3, 52.4, 46.5, 32.4, 32.4, 19.6. HRMS (ESI): Calcd.
for C25H30N5O2 [M+H]+ 432.2400, found 432.2398.
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ropanamide (11b). White solid, yield: 71 %, m.p. 215−216 °C. 1H NMR (600 MHz, Chloroform-d) δ
11.39 (s, 1H), 8.20 – 8.08 (m, 2H), 7.59 (d, J = 8.7 Hz, 1H), 7.21 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 7.7
Hz, 2H), 4.25 – 4.14 (m, 2H), 3.52 (s, 2H), 3.15 (t, J = 7.9 Hz, 2H), 2.87 – 2.65 (m, 12H), 2.32 (s,
3H), 2.27 (dt, J = 15.2, 8.0 Hz, 2H). 13C NMR (150 MHz, Chloroform-d) δ 170.7, 160.7, 158.1,
145.2, 137.2, 136.7, 134.8, 129.1, 128.9, 127.6, 126.7, 120.8, 115.1, 62.6, 53.5, 53.3, 52.3, 46.4, 32.4,
32.3, 21.1, 19.5. HRMS (ESI): Calcd. for C26H32N5O2 [M+H]+ 446.2556, found 446.2552.
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ropanamide (11c). White solid, yield: 69 %, m.p. 148−149 °C. 1H NMR (600 MHz, Chloroform-d) δ
11.41 (s, 1H), 8.22 (dd, J = 8.8, 2.5 Hz, 1H), 8.13 (d, J = 2.5 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.32
(dd, J = 8.6, 5.5 Hz, 2H), 7.02 (t, J = 8.7 Hz, 2H), 4.28 – 4.16 (m, 2H), 3.55 (s, 2H), 3.18 (dd, J = 8.3,
7.6 Hz, 2H), 2.81 – 2.62 (m, 12H), 2.30 (dt, J = 15.2, 8.0 Hz, 2H). 13C NMR (150 MHz,
Chloroform-d) δ 170.7, 162.0 (d, J = 245.0 Hz), 160.7, 158.1, 145.3, 137.2, 133.7, 130.5, 127.6,
126.7, 120.8, 115.1, 115.1, 62.0, 53.5, 53.2, 52.3, 46.4, 32.4, 32.3, 19.6. HRMS (ESI): Calcd. for
C25H29FN5O2 [M+H]+ 450.2305, found 450.2303.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-(4-(trifluoromethyl)benzyl)piper
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azin-1-yl)propanamide (11d). Faint yellow, yield: 75 %, m.p. 173−174 °C. H NMR (600 MHz,
Chloroform-d) δ 11.38 (s, 1H), 8.20 (dd, J = 8.8, 2.5 Hz, 1H), 8.11 (d, J = 2.5 Hz, 1H), 7.72 (d, J =
7.6 Hz, 2H), 7.58 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 7.6 Hz, 2H), 4.26 – 4.12 (m, 2H), 3.69 (s, 2H),
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3.19 (t, J = 7.9 Hz, 2H), 2.84– 2.62 (m, 12H), 2.28 (dt, J = 15.2, 7.9 Hz, 2H). 13C NMR (150 MHz,
Chloroform-d) δ 170.6, 160.7, 158.2, 145.3, 139.8, 137.4, 130.4, 129.5, 126.9, 126.8, 124.8, 124.3 (q,
J = 227 Hz), 120.8, 115.1, 62.2, 53.5, 53.3, 52.3, 46.4, 32.4, 32.3, 19.5. HRMS (ESI): Calcd. for
C26H29F3N5O2 [M+H]+ 500.2273, found 500.2268.
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ropanamide (11e). Yellow, yield: 52 %, m.p. 195−197 °C. H NMR (600 MHz, Chloroform-d) δ
11.40 (s, 1H), 8.20 (dd, J = 8.8, 2.5 Hz, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.43
(d, J = 8.0 Hz, 2H), 7.29 (d, J = 7.7 Hz, 2H), 4.26 – 4.14 (m, 2H), 3.60 (s, 2H), 3.18 (t, J = 7.9 Hz,
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2H), 2.86 – 2.40 (m, 12H), 2.27 (dt, J = 15.2, 8.0 Hz, 2H). C NMR (150 MHz, Chloroform-d) δ
170.5, 160.3, 158.3, 145.4, 137.5, 136.8, 132.4, 130.2, 128.6, 127.1, 126.5, 120.1, 115.1, 62.2, 53.3,
53.1, 52.5, 46.8, 32.2, 31.4, 19.8. HRMS (ESI): Calcd. for C25H29ClN5O2 [M+H]+ 466.2010, found
466.2009.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-(3-methylbenzyl)piperazin-1-yl)p
ropanamide (11f). White solid, yield: 77 %, m.p. 206−208 °C. 1H NMR (600 MHz, Chloroform-d) δ
11.38 (s, 1H), 8.20 (d, J = 2.5 Hz, 1H), 8.14 (dd, J = 8.8, 2.5 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.22
(t, J = 7.5 Hz, 1H), 7.19 – 7.13 (m, 2H), 7.09 (d, J = 7.4 Hz, 1H), 4.29 – 4.16 (m, 2H), 3.56 (s, 2H),
3.18 (t, J = 7.9 Hz, 2H), 2.80 – 2.60 (m, 12H), 2.35 (s, 3H), 2.30 (dt, J = 15.2, 8.0 Hz, 2H). 13C NMR
(150 MHz, Chloroform-d) δ 170.7, 160.7, 158.2, 145.2, 137.9, 137.6, 137.2, 129.9, 128.2, 128.0,
127.6, 126.7, 126.3, 120.8, 115.2, 62.9, 53.5, 53.2, 52.3, 46.5, 32.4, 32.3, 21.4, 19.6. HRMS (ESI):
Calcd. for C26H32N5O2 [M+H]+ 446.2556, found 446.2558.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-(3-fluorobenzyl)piperazin-1-yl)p
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ropanamide (11g). White solid, yield: 48 %, m.p. 216−217 °C. 1H NMR (600 MHz, Chloroform-d) δ
11.39 (s, 1H), 8.20 (dd, J = 8.8, 2.5 Hz, 1H), 8.15 (d, J = 2.5 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.31
– 7.26 (m, 1H), 7.16 – 7.09 (m, 2H), 6.95-6.92 (m, 1H), 4.29 – 4.18 (m, 2H), 3.58 (s, 2H), 3.18 (t, J
= 7.9 Hz, 2H), 2.78 – 2.56 (m, 12H), 2.30 (dt, J = 15.2, 8.0 Hz, 2H). 13C NMR (150 MHz,
Chloroform-d) δ 170.7, 162.9 (d, J = 245.6 Hz), 160.7, 158.2, 145.2, 140.8, 137.2, 129.7, 127.6,
126.7, 124.5, 120.8, 115.6, 115.1, 114.0, 62.2, 53.5, 53.3, 52.3, 46.4, 32.4, 32.3, 19.6. HRMS (ESI):
Calcd. for C25H29FN5O2 [M+H]+ 450.2305, found 450.2313.
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N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-(3-(trifluoromethyl)benzyl)piper
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azin-1-yl)propanamide (11h). Faint yellow, yield: 64 %, m.p. 193−195 °C. H NMR (600 MHz,
Chloroform-d) δ 11.38 (s, 1H), 8.22 (dd, J = 8.8, 2.5 Hz, 1H), 8.14 (d, J = 2.5 Hz, 1H), 7.64 (s, 1H),
7.63 (d, J = 8.8 Hz, 1H), 7.54 (dd, J = 14.0, 7.7 Hz, 2H), 7.45 (t, J = 7.7 Hz, 1H), 4.28 – 4.18 (m,
2H), 3.64 (s, 2H), 3.18 (t, J = 7.9 Hz, 2H), 2.86 – 2.63 (m, 12H), 2.30 (dt, J = 15.2, 7.9 Hz, 2H). 13C
NMR (150 MHz, Chloroform-d) δ 170.7, 160.7, 158.2, 145.3, 139.2, 137.2, 132.3, 130.6, 128.7,
127.6, 126.7, 125.5, 124.2(q, J = 231.9 Hz), 124.0, 120.8, 115.1, 62.2, 53.5, 53.3, 52.3, 46.4, 32.4,
32.3, 19.5. HRMS (ESI): Calcd. for C26H29F3N5O2 [M+H]+ 500.2273, found 500.2271.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-(3-chlorobenzyl)piperazin-1-yl)p
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ropanamide (11i). Yellow, yield: 55 %, m.p. 166−168 °C. H NMR (600 MHz, Chloroform-d) δ
11.39 (s, 1H), 8.18 (dd, J = 8.8, 2.5 Hz, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.68 (s, 1H), 7.61 (d, J = 8.8
Hz, 1H), 7.30 – 7.26 (m, 2H), 6.96 (tdd, J = 8.4, 2.6, 1.0 Hz, 1H), 4.26 – 4.14 (m, 2H), 3.62 (s, 2H),
3.18 (t, J = 7.9 Hz, 2H), 2.80 – 2.59 (m, 12H), 2.30 (dt, J = 15.2, 8.0 Hz, 2H). 13C NMR (150 MHz,
Chloroform-d) δ 170.7, 162.9 (d, J = 245.6 Hz), 160.7, 158.2, 145.2, 140.8, 137.2, 129.7, 127.6,
126.7, 124.5, 120.8, 115.6, 115.1, 114.0, 62.2, 53.5, 53.3, 52.3, 46.4, 32.4, 32.3, 19.6. HRMS (ESI):
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Calcd. for C25H29ClN5O2 [M+H]+ 466.2010, found 466.2012.
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N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-(2-methylbenzyl)piperazin-1-yl)p
ropanamide (11j). White solid, yield: 62 %, m.p. 207−209 °C. 1H NMR (600 MHz, Chloroform-d) δ
11.42 (s, 1H), 8.25 – 8.16 (m, 2H), 7.64 (d, J = 8.7 Hz, 1H), 7.28 (d, J = 7.2 Hz, 1H), 7.21 – 7.13 (m,
3H), 4.30 – 4.12 (m, 2H), 3.55 (s, 2H), 3.18 (t, J = 7.9 Hz, 2H), 2.78 – 2.56 (m, 11H), 2.39 (s, 3H),
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2.30 (dt, J = 15.2, 7.9 Hz, 2H). C NMR (150 MHz, Chloroform-d) δ 170.8, 160.7, 158.2, 145.2,
137.6, 137.2, 136.1, 130.3, 129.8, 127.6, 127.2, 126.8, 125.5, 120.8, 115.2, 60.7, 53.6, 53.2, 52.5,
46.5, 32.4, 32.3, 19.6, 19.2. HRMS (ESI): Calcd. for C26H32N5O2 [M+H]+ 446.2556, found
446.2551.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-(2-fluorobenzyl)piperazin-1-yl)-
propanamide (11k). White solid, yield: 49 %, m.p. 187−188 °C. 1H NMR (600 MHz, Chloroform-d)
δ 11.33 (s, 1H), 8.25 (d, J = 2.5 Hz, 1H), 8.03 (dd, J = 8.8, 2.5 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H),
7.40 (td, J = 7.5, 1.8 Hz, 1H), 7.29 – 7.25 (m, 1H), 7.13 (td, J = 7.5, 1.2 Hz, 1H), 7.06 (ddd, J = 9.6,
8.2, 1.2 Hz, 1H), 4.30 – 4.14 (m, 2H), 3.66 (d, J = 1.3 Hz, 2H), 3.18 (t, J = 7.9 Hz, 2H), 2.78 – 2.56
(m, 12H), 2.30 (dt, J = 15.2, 7.9 Hz, 2H). 13C NMR (150 MHz, Chloroform-d) δ 170.7, 161.4 (d, J =
246.5 Hz), 160.6, 158.2, 145.2, 137.1, 131.5, 129.0, 127.5, 126.6, 124.4, 123.9, 120.8, 115.3, 115.3,
55.2, 53.5, 53.1, 52.2, 46.5, 32.4, 32.3, 19.5. HRMS (ESI): Calcd. for C25H29FN5O2 [M+H]+
450.2305, found 450.2302.
N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-(2-(trifluoromethyl)benzyl)piper
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azin-1-yl)propanamide (11l). Faint yellow, yield: 70 %, m.p. 177−179 °C. H NMR (600 MHz,
Chloroform-d) δ 11.24 (s, 1H), 8.21 (d, J = 2.5 Hz, 1H), 8.08 (dd, J = 8.8, 2.5 Hz, 1H), 7.74 (d, J =
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7.7 Hz, 1H), 7.61 (t, J = 7.9 Hz, 2H), 7.50 (t, J = 7.5 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 4.26 – 4.13 (m,
2H), 3.73 (s, 2H), 3.15 (t, J = 7.9 Hz, 2H), 2.85 – 2.61 (m, 12H), 2.30 (dt, J = 15.2, 7.9 Hz, 2H). 13C
NMR (150 MHz, Chloroform-d) δ 170.7, 160.7, 158.2, 145.3, 137.4, 137.0, 131.7, 130.3, 128.7,
127.6, 126.9, 126.8, 125.9, 124.3 (q, J = 227.5 Hz), 120.8, 115.4, 58.1, 53.7, 53.3, 52.5, 46.5, 32.4,
32.3, 19.5. HRMS (ESI): Calcd. for C26H29F3N5O2 [M+H]+ 500.2273, found 500.2265.
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N-(9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(4-(2-chlorobenzyl)piperazin-1-yl)p
ropanamide (11m). Faint yellow, yield: 68 %, m.p. 213−215 °C. 1H NMR (600 MHz, Chloroform-d)
δ 11.33 (s, 1H), 8.20 (d, J = 2.5 Hz, 1H), 8.05 (dd, J = 8.8, 2.5 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H),
7.42(d, J = 7.5, 1H), 7.26 – 7.23 (m, 1H), 7. 22-7.18 (m, 2H), 4.28 – 4.14 (m, 2H), 3.64 (d, J = 1.3
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Hz, 2H), 3.18 (t, J = 7.9 Hz, 2H), 2.80 – 2.62 (m, 12H), 2.27 (dt, J = 15.2, 7.9 Hz, 2H). C NMR
(150 MHz, Chloroform-d) δ 170.7, 160.6, 158.4, 145.2, 137.6, 136.5, 134.4, 130.5, 128.7, 128.3,
127.4, 126.3, 125.6, 120.7, 115.1, 61.3, 54.8, 53.6, 52.7, 46.8, 32.9, 32.4, 19.9. HRMS (ESI): Calcd.
for C25H29ClN5O2 [M+H]+ 466.2010, found 466.2015.
In vitro hAChE and hBuChE inhibition assay
Inhibitory activity was measured using Ellman's assay.32 hAChE (form human erythrocytes),
hBuChE (from human serum), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide
(ATCI), and butyrylthiocholine iodide (BTCI) were obtained from Sigma-Aldrich. The assay was
performed as described in the following procedure: At least 5 different concentrations (1 × 10–5–10–9
M) of each test compound were measured, each concentration in triplicate. Then, 50 μL of hAChE
(0.02 unit/mL) or hBChE (0.02 unit/mL) and 10 μL of the compound was incubated at 37°C for 6
min; next, 30 μL of 0.01 M substrate (ATCI or BTCI solution) was added, and the solution was
further incubated at 37°C for 12 min. Finally, 150 μL of 0.01 M DTNB was added, and the activity
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was measured at a wavelength of 415 nm using an Evolution 300 PC UV-Vis Spectrophotometer.
The IC50 value was calculated using Origin 8.0 software.
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BACE1 enzymatic assay
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Human BACE1 activity was assayed using a BACE1 (β-secretase) FRET (Fluorescense
Resonance Energy Transfer) assay kit.33 OM99-2 was used in the assay as a positive control. The test
compounds were dissolved in DMSO and further diluted with the assay buffer (50 mM sodium
acetate; pH 4.5). 10 μL of BACE1 enzyme (1 Units/mL) and 10 μL of each tested compound were
placed in 96-well plates. Then, 10 μL of BACE1 substrate (Rh-EVNLDAEFK-Quencher) was added
to start the reaction (the final concentration of DMSO in each sample and control wells was equal or
less than 5%). The mixture was incubated at room temperature for 1 h in the dark, and 10 μL of 2.5
M sodium acetate was then applied to stop the reaction. The fluorescence was monitored at 545 nm
(excitation wave length, Exc) and 585 nm (emission wavelength, Em).
Aβ1-42 self-aggregation inhibition assay
Inhibition of Aβ1-42 self-aggregation was measured using the thioflavin T fluorescence method.34
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) pretreated Aβ1-42 samples (Sigma-Aldrich) were dissolved
with PBS (pH 7.4) to give a 40 mM solution. Meanwhile, the ThT solution was prepared in PBS (pH
7.4) at a concentration of 10 μM. Then, 10 μL of 40 μM Aβ1-42 solution was added to each well
containing 10 μL of different concentrations of test compounds. After incubation at 30 °C for 24 h,
the samples were diluted to a final volume of 200 μL with ThT solution. Then, a 300 s time scan of
fluorescence intensity was performed (λexc = 446 nm; λem = 490 nm), and values at plateau were
averaged after subtracting the background fluorescence of the thioflavin T solution.
Kinetic characterization of AChE inhibition
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To measure the mechanism of AChE inhibition by synthesized compounds, different
concentrations of compound 11a.(1 – 10 nM) were reacted with different substrate (AChE)
oncentrations (0.1 – 0.5 mM). Then, the Vm and Km values of the inhibition were measured by
plotting the Lineweaver-Burk plot.
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Inhibition of AChE induced Aβ1-42 peptide aggregation assay
Inhibition of AChE induced Aβ1-42 peptide aggregation were conducted by co-incubation of
synthesized compounds (1 μM and 5 μM) and Aβ1-42 peptide with AChE (10 μM). For control
experiment, the test compound is absent. We have monitored the aggregation of Aβ1-42 peptide by
ThT at 37 °C for 24 h . The excitation wavelength was 446 nm and emission was ranging between
490 nm
In vitro cytotoxicity assay
The human neuroblastoma SH-SY5Y and HepG2 cell lines were obtained from the Procell Life
Science & Technology Co., Ltd. Cells were grown in Dulbecco's modified Eagle's medium (DMEM)
containing 10% (v/v) fetal bovine serum (FBS, Hyclone), 50 U/mL penicillin, and 50 mg/mL
streptomycin, under a humidified atmosphere of 5% CO2 at 37 °C. The toxic effects of the
compounds were assessed using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium
bromide) assay as described previousl.35 Briefly, SH-SY5Y or HepG2 cells were seeded in 96-well
plates at 1 × 104 per well and cultured at 37 °C for 24 h. Then, cells were exposed to the tested
compounds at various concentrations (0.1–100 μM) for 48 h. The final concentration of DMSO in
culture media did not exceed 0.05% (v/v) and no alterations of the cells were observed. The MTT
reagent was added to each well and incubated for an additional 4 h followed by solubilization of
formazan crystals in DMSO. Absorbance was measured at 570 nm using a microculture plate reader.
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Protection of SH-SY5Y cells against Aβ1-42–induced damage
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SH-SY5Y cells were seeded in 96-well plates at 1 × 104 per well and cultured at 37°C for 24 h.
Aβ1-42 (20 μM) with or without test compounds 10a, 11a, and 11j (1 μM, 5 μM, and 10 μM) were
diluted with medium and added into individual wells. Then, plates were again incubated for an
additional 48 h at 37 °C. Cell viability was determined using MTT assay protocol, and expressed as a
percentage of the control cells.
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Molecular docking
Docking was performed by using the Surflex-Dock program in Sybyl-X 2.0 Software. The 3D
structures of ligands were drawn in the Sybyl package. Atom types were checked, hydrogen atoms
were added, and then Gasteiger-Marsili charges were assigned with Sybyl-X 2.0 Software. The
protein structures of hAChE (PDB code: 4EY7), BACE1 (PDB code: 4D8C), and Aβ1-42 (PDB code:
1IYT) were derived from the RCSB Protein Data Bank (https://www.rcsb.org/). To conduct
molecular docking studies, the ligand of the crystal structure was extracted, hydrogen atoms were
added, and side-chain amides were checked. Then, the protomol was generated. PyMOL was used to
visualize the results of docking.41
 AUTHOR INFORMATION
Corresponding Authors
*Tel: +86-0376- 6391565. E-mail: wangjunru@nwsuaf.edu.cn
Author Contributions
Hongtao Du and Fang Ma take charge of the research; Fang Ma and Xinlian Liu synthesized
compounds; Hongtao Du and Jusen Xie carried out biological activity tests. Hongtao Du and Fang
Ma written the manuscript. All authors have given approval to the final version of the manuscript.
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Acknowledgments
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This work was supported by grants from the Program for Natural Science Foundation of China
(Grant No. 21602178), Nanhu Scholars Program for Young Scholars of XYNU, and the study and
utilization of active components of special plants (Grant No. 2018083). We thank Dr. qiuju Zhou
(Analysis and Testing Center, Xinyang Normal University) for her help in the compounds structure
analyses. We also thank LetPub (www.letpub.com) for its linguistic assistance during the preparation
of this manuscript.
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Conflicts of interest
The authors declare no conflict of interest about this article.

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