Design, synthesis, and biological evaluation of rutacecarpine derivatives as multitarget-directed ligands for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.05.055

A series of 3-amino-substituted rutacecarpine derivatives were synthesized to identify novel multitarget-directed ligands (MTDLs) for the treatment of Alzheimer's disease (AD). Biological evaluation showed that most of the synthesized compounds inhibited butyrylcholinesterase (BuChE) and exerted antioxidant effects. Among the synthesized compounds, 6n was subjected to further biological evaluation. Lineweaver–Burk plotting and molecular modeling illustrated that 6n bound simultaneously to the peripheral anionic site (PAS) and catalytic sites (CAS) of BuChE. Furthermore, 6n modulated Aβ aggregation; chelated biometals; presented good absorption, distribution, metabolism, excretion, and toxicity properties; and showed remarkable neuroprotective activity. Previous research has shown that the optimized compound 6n has considerable potential for development as an MTDL for the treatment of AD.

Full text of DOI:10.1016/j.ejmech.2019.05.055

Accepted Manuscript
Design, synthesis, and biological evaluation of rutacecarpine derivatives as
multitarget-directed ligands for the treatment of Alzheimer's disease
Mingfei Wu, Jie Ma, Lijun Ji, Min Wang, Jianfei Han, Zeng Li
PII:
S0223-5234(19)30465-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.055
EJMECH 11365
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 March 2019
Revised Date: 22 April 2019
Accepted Date: 19 May 2019
Please cite this article as: M. Wu, J. Ma, L. Ji, M. Wang, J. Han, Z. Li, Design, synthesis, and
biological evaluation of rutacecarpine derivatives as multitarget-directed ligands for the treatment of
Alzheimer's disease, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.05.055.
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Design, synthesis, and biological
evaluation of
rutacecarpine
ligands for the
derivatives
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multitarget-directed
treatment of Alzheimer's disease
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Mingfei Wu , Jie Ma , Lijun Ji , Min Wang , Jianfei Han , Zeng Li
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School of Pharmacy, Anhui Medical University, Hefei 230032, China
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The Central Hospital of Wuhan, Tongji Medical College of HUST, Wuhan 430000, China
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Corresponding author. E-mail addresses: lizeng@ahmu.edu.cn (Z. Li).
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Abstract
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A series of 3-amino-substituted rutacecarpine derivatives were synthesized to identify
novel multitarget-directed ligands (MTDLs) for the treatment of Alzheimer's disease
(AD). Biological evaluation showed that most of the synthesized compounds inhibited
butyrylcholinesterase (BuChE) and exerted antioxidant effects. Among the
synthesized compounds, 6n was subjected to further biological evaluation.
Lineweaver–Burk plotting and molecular modeling illustrated that 6n bound
simultaneously to the peripheral anionic site (PAS) and catalytic sites (CAS) of
BuChE. Furthermore, 6n modulated Aβ aggregation; chelated biometals; presented
good absorption, distribution, metabolism, excretion, and toxicity properties; and
showed remarkable neuroprotective activity. Previous research has shown that the
optimized compound 6n has considerable potential for development as an MTDL for
the treatment of AD.
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Keywords: Alzheimer's disease, Rutacecarpine, Multitarget-directed ligands,
Cholinesterases, Docking study, Drug-likeness prediction
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1. Introduction
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Alzheimer's disease (AD) is the most common cause of dementia among the elderly,
and its main clinical manifestations are progressive memory loss and severe cognitive
decline [1]. Its exact pathogenic mechanism remains unclear. Current research
suggests that different factors are involved in the development of AD. The main
pathological features of this disease include cholinergic neurotransmitter dysfunction
in the hippocampal and cortical regions of the brain and the abnormal deposition of
β-amyloid protein (Aβ), as well as oxidative stress, biometallic dysregulation, and
neuroinflammation [2-4].
The reduction in the amount of cholinergic neurotransmitters is crucial for the
pathogenesis of AD [5]. Acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE) are two important types of cholinesterases (ChEs) in the central nervous
system that can rapidly hydrolyze acetylcholine [6]. Under normal physiological
conditions, AChE activity precedes BuChE activity. However, as AD progresses, the
hydrolytic activity of AChE gradually decreases, whereas the activity of BuChE
drastically increases [7]. ChE inhibitors, including tacrine, donepezil, galantamine,
and rivastigmine, have been developed in recent years [8]. These inhibitors, however,
induce numerous side effects, such as hepatotoxicity and digestive tract reactions, that
severely affect therapeutic goals [9, 10]. Therefore, the development of effective and
selective BuChE inhibitors may be an important approach for AD treatment and for
avoiding the adverse reactions caused by AChE inhibitors during treatment [11, 12].
Neurofibrillary lesions composed of amyloid plaques formed through excessive Aβ
production and deposition are a primary cause of AD [13]. Numerous plaques that are
produced via Aβ aggregation are deposited in the hippocampal and basal ganglia
regions of the brains of patients with AD [14]. Aβ oligomers are neurotoxic and can
continuously activate inflammatory factors, such as IL-6 and NFTα [15]. Moreover,
Aβ itself can act as an oxygen free radical donor that produces reactive oxygen

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species (ROS) that directly affect the normal physiological functions of neurocytes
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[16, 17]. Furthermore, the biophysical disorders of biometallic ions, such as Cu ,
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Fe and Zn , are closely related to the pathogenesis of AD [18]. Metal ions,
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especially Cu , have a strong affinity for Aβ and catalyze ROS production by
participating in plaque formation [19]. Excessive ROS accumulation can result in
oxidative stress and neuronal damage.
A growing body of evidence shows that the etiology of AD is extremely complex
and involve different pathogenic factors that are correlated each other [20]. Therefore,
the development of multitarget-directed ligands (MTDLs) that target multiple
neuropathological processes has become a new approach for the treatment of AD [21].
Natural components have the advantages of low toxicity, limited side effects, and
extensive pharmacological functions. Rutaecarpine (Ru, Figure 1A) is an important
ingredient in the Chinese traditional medicine rutaceous evodia, which belongs to the
pentacyclic indolopyridoquinazolinone alkaloid family [22]. Ru has multiple
pharmacological activities, including antitumor, antioxidant, neuroprotection, and
anti-inflammatory activities [23, 24]. The pharmacophore feature of Ru is similar to
that of tacrine (9-amino-1, 2, 3, 4-tetrahydroacridine, THA, Figure 1A), which was
first approved for use as an AChE inhibitor in 1993 [25]. Moreover, a series of novel
Ru
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3-aminoalkanamide-substituted Ru, have been synthesized and shown to exert strong
inhibitory activity when applied as selective AChE inhibitors [26-28].
A series of novel sulfonamide-based compounds with reversible and selective
BuChE inhibitory effects [29, 30] at low nanomolar concentrations (IC = 4.91 nM)
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(Figure 1B) have been reported. Although Ru and sulfonamide groups exhibit ChE
inhibitory activities, their combinations have not been applied as anti-AD agents.
Thus, we designed and synthesized sulfonylamide-substituted Ru derivatives, a series
of selective BuChE inhibitors, by hybridizing the pharmacophores of Ru with the
sulfonamide group into one molecule (Figure 1C).
We aimed to establish Ru-based compounds on the basis of our preliminary work
and literature review [26-28]. We focused on the synthesis of Ru derivatives with

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anti-AD activities, including antioxidant, Aβ aggregation inhibition, and
neuroprotective effects, as well as metal-chelating properties [31, 32]. We performed
enzyme studies, molecular modeling , and drug-likeness evaluation to characterize the
derivatives. Moreover, we summarized the structure–activity relationship of the
derivatives to provide basic information for the development of anti-AD MTDLs
based on the parental structure of Ru.
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2. Results and discussion
2.1. Chemistry
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The intermediate 3-amino-substituted Ru was prepared in accordance with the
modified Bergman synthetic procedure [33]. A series of benzenesulfonyl chloride and
thiazolesulfonyl chloride fragments replaced H in the 3-amino-substituted Ru to form
an
acylamino
group.
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reaction
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the
target
benzenesulfonylamino-substituted or thiazolesulfonamide-substituted Ru derivatives.
As described in Scheme 1, isatoic anhydride was nitrated in concentrated H SO in
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the presence of KNO following the method reported by Iqbal [34]. Then, as shown in
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Schemes 2, a solution of nitroisoleic anhydride 1 in anhydrous pyridine was reacted
through the one-pot method to obtain compound 2 in accordance with the Bergman
method. The cyclization of compound 2 under acidic conditions afforded compound 3.
Compound 3 was dissolved in isopropanol and reduced with 80% hydrazine hydrate
and 10% Pd/C to provide compound 4. The CF H group of compound 4 was
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eliminated to obtain intermediate 5 with alcoholic KOH. Finally, intermediate 5 with a
series of benzenesulfonyl chloride or thiazolesulfonyl chloride fragments was
refluxed in anhydrous pyridine to induce an amide substitution reaction and acquire
the desired derivatives 6a–p, 7a–c, and 8a.
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2.2. In vitro ChE inhibition and structure–activity relationships

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The anti-ChE activity of the synthetic derivatives 6a–6p, 7a–7c, 8a and the parental
structure Ru were evaluated by using the method described by Ellman et al. [35] with
tacrine and donepezil as reference drugs. The inhibitory activities of the compounds
against AChE and BuChE are summarized in Table 1. Structure–activity relationships
were explored, and active compounds with the highest activity were identified.
The experimental results revealed that all of the synthetic derivatives presented
better inhibitory activity and selective effect on BuChE than the original parental
structure (Ru). The compounds presented a weak or nonexistent inhibitory effect on
AChE. Compounds 6m, 6n, and 6o showed the best inhibitory activity against BuChE
with the IC values of 3.82 ± 0.68, 3.60 ± 0.34, and 3.95 ± 0.72 µM, respectively.
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group could drastically strengthen the inhibitory activity of the derivatives. The
inhibitory activities of the reference drugs tacrine and donepezil against AChE and
BuChE were quantified under the same assay conditions. Tacrine and donepezil
provided IC values of 0.28 ± 0.026 and 0.04 ± 0.007 µM for AChE, respectively,
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and 0.02 ± 0.01 and 7.87 ± 1.24 µM for BuChE, respectively.
Structure–activity relationship analysis showed that the derivatives with
sulfonamide groups connected to different aromatic nuclei expressed different degrees
of ChE inhibitory activity. The inhibitory activity of derivatives with benzene rings
was strong and was 20-fold higher than that of derivatives with thiophene rings. For
example, the inhibitory activity of compounds 6a–6p (IC : 3.60 ± 0.34–25.78 ± 1.31
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µM) against BuChE was significantly stronger than that of compounds 7a–7c and 8a
(IC ≥ 60.34 ± 4.03 µM). Moreover, the electronegativity of the substituent base on
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the aromatic ring had a significant effect on the inhibitory activity of the compounds,
and the compounds with strong electron withdrawing group on the benzene ring had
significant inhibitory activity for BuChE, such as compounds 6i, 6k, 6l, 6m, 6n and
6o. In addition, with the volume increase of the substituent of aromatic ring, the
inhibitory activity for BuChE enhanced observably. The inhibitory activities of
derivatives against BuChE gradually increased (IC value: 6k = 7.57 ± 0.77 µM, 6l =
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8.28 ± 0.91 µM, 6o = 3.95 ± 0.72 µM) when −F, −CF , or −OCF was introduced into
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the C4 positions of their benzene rings. Moreover, the introduction of substituents at
the C2, C3, and C4 positions of benzene rings resulted in inhibitory activities with
different intensities. The effectiveness of the derivative with a −CF in the C3 position
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was 2-fold that of the derivative with a −CF in the C4 position (IC value: 6m =
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3.82 ± 0.68 µM, 6l = 8.28 ± 0.91 µM). This trend was also demonstrated by other
compounds. The effectiveness of the derivative with a −OCF substituent in the C2
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position was better than that of the derivative with a −OCF substituent in the C4
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position (IC value: 6n = 3.60 ± 0.34 µM, 6o = 3.95 ± 0.72 µM).
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2.3. Kinetic study of BuChE inhibition
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Compound 6n, the derivative with the highest inhibitory activity, was subjected to
enzyme kinetics analysis to determine the kinetics of BuChE inhibition. The slopes
and intercepts of the reciprocal Lineweaver–Burk plot presented in Figure 2 increased
with the increase in inhibitor concentration. The intersection of each trend line in the
fourth quadrant indicated a mixed-type inhibitory mode. This result suggests that
compound 6n bound to BuChE at the catalytic active site (CAS) and peripheral
anionic (PAS) site.
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2.4. Docking analysis of compounds through enzyme and molecular dynamics
simulation
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Molecular modeling can be used to determine interactions between ligands and
receptors [36, 37]. The most stable conformation in the active site was illustrated and
analyzed through 2D and 3D diagrams and on the basis of the
-CDOCKER-INTERACTION-ENERGY scores generated by the CDOCKER program,
which was verified as docking protocol by re-docking of the co-crystallized ligand
92H into 5NN0 active site. The docking pose of 92H was compared with the initial
pose using root mean square deviation (RMSD) and 92H docked almost at the same
position (RMSD = 0.3193 Å, see supporting information for details, Table S1). The
highest score provided by -CDOCKER_INTERACTION_ENERGY for compound 6n

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within BuChE was 45.9637 kcal/mol. The H bond surface of the BuChE receptor with
compound 6n indicates that compound 6n has strong affinity for BuChE (Figure 3A).
The 2D diagram shows that one conventional H bond interaction was generated by
connecting the F atom of trifluoromethoxy group at the C2 position of the aromatic
ring with the OH group of THR120 (distance = 2.12 Å). Three C–H bond interactions
were also produced between the F atoms of the trifluoromethoxy group with the CH2
group of GLY116 (distance = 2.82 Å), GLY121 (distance = 2.82 Å) and THR120
(distance = 3.00 Å). Moreover, three halogen interactions occurred between F atoms
of the trifluoromethoxy group with the C=O group of THR82 (distance = 3.00 and
2.98 Å) and GLY115 (distance = 2.91 Å). One π–sulfur interaction occurred in
sulfonamide with TRP82 (distance = 4.24 Å). One π–π t–shaped interaction generated
in benzene ring with TRP82 (distance = 4.15 Å). The above results confirm that the
introduction of the side-chain substituent of the sulfonamide aromatic group
drastically improved the binding between compound 6n and BuChE. Furthermore, the
quinazolinocarboline core structure of compound 6n created a C–H bond interaction
with SER287 (distance = 2.82 Å) and three hydrophobic interactions, which included
one π–sigma interactions with PRO285 (distance = 2.61 Å) and two π–π stacked
interactions with TYP332 (distance = 5.91 and 5.64 Å) ( (Figure 3B).
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Molecular dynamics (MD) simulation was performed to verify the structural
constancy of the system 6n-BuChE [38]. The convergence and stability of the system
were monitored by evaluating the RMSD values of the backbone atoms with respect
to the initial conformation [39]. The system 6n-BuChE were subjected to the 10000
ps MD simulations. Figure 4 shows that after 7000 ps, the wave range of RMSD was
within 1 Å, which corresponds to a near-identical degree of postural similarity. The
MD simulation results suggest that compound 6n and BuChE formed a stable
combination and were consistent with the results of molecular docking.
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2.5. DPPH radical scavenging activity
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scavenger that can be used to monitor a chemical reaction that involves free radicals
[40, 41]. The DPPH assay was performed with ascorbic acid and donepezil as
reference antioxidants to evaluate the ability of the synthesized compounds to
scavenge activated oxygen species. As shown in Table 1, all of the synthesized
compounds exhibited mild free radical scavenging activity with the DPPH RP of
35.18%–59.32% at a concentration of 100 µM.
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2.6. Determination of intracellular ROS production
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The extensive accumulation of endogenous ROS can accelerate the progression of
AD by causing oxidative stress and triggering nerve damage [42]. The antioxidant
potency of compound 6n against intracellular ROS generation was investigated in
H O -treated SH-SY5Y and PC12 cells through the 2',7'-dichlorodihydrofluorescein
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diacetate (DCFH-DA) assay [43]. Cells were treated with compound 6n at
concentrations of 1, 5, 10, 20, and 50 µM and then exposed to H O (250 µM) for 18
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h. Intracellular ROS content was measured by using the DCFH-DA probe (10 µM).
As shown in Figure 5A and 5B, the amount of intracellular ROS generation in
control group increased significantly relative to that in blank group with the
percentage of 119.61% in SH-SY5Y cells model and 136.41% in PC12 cells model,
respectively. ROS levels in 6n treated group almost reduced to the blank group level
in a concentration-independent manner (see supporting information for details, Table
S2). Therefore, compound 6n can effectively reduce H O -induced intracellular ROS
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accumulation. This result provides further evidence that compound 6n is a promising
neuroantioxidant.
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2.7. Effect on Aβ peptide aggregation
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The self-assembly of Aβ peptide into fibers promotes the formation of senile
plaques and ROS by inducing aggregation effects and participating in redox reactions
in the body [44]. Therefore, the ability of the representative compound 6n to inhibit
self-mediated Aβ aggregation was assessed through the thioflavin T (ThT)

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fluorometry method [45]. As shown in Table 2, the inhibitory potency of Ru (11.25%)
against Aβ (1-42) peptide self-aggregation was lower than that of the reference drug
donepezil (33.78%). The Ru derivative 6n prevented self-mediated Aβ aggregation
with an inhibition rate of 58.95%. The inhibition rate of 6n was superior to that of Ru
and the reference drug donepezil. Therefore, the structural modification of the
parental structure Ru significantly improved biological activity.
Transmission electron microscopy (TEM) was employed to explore whether the
morphology of Aβ aggregates had changed [46, 47]. Aβ (1-42) samples incubated for
0 h alone did not aggregate and appeared dispersed (Figure 6A). However, after 48 h
of incubation, the majority of Aβ (1-42) self-aggregated into amyloid fibers, and
numerous mature and bulky fibers were observed (Figure 6B). Aggregation in Aβ
(1-42) samples incubated with compound 6n and donepezil decreased. Compound 6n
inhibited Aβ aggregation more effectively than donepezil (Figures 6C and 6D) as
indicated by the thin and short fibers observed in Aβ (1-42) samples incubated with
compound 6n. TEM results further demonstrate that compound 6n could inhibit Aβ
aggregation and are consistent with the ThT assay results.
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2.8. Metal-chelating property
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The chelation of antioxidants with biometal ions has become a strategy for the
treatment of AD because it can prevent amyloid plaque formation and reduce
oxidative stress levels [48, 49]. The chelation activity of compound 6n against
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biometal ions, such as Cu , Fe , Zn , Al , Mg and Ca , in amyloid plaques was
investigated through UV–vis spectrometry.
Figure 7A shows that the UV spectra of the methanolic solution of compound 6n
changed after the addition of CuCl solution relative to those of the methanolic
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solutions of compound 6n after the addition of FeSO , ZnCl , AlCl , MgCl , and
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CaCl . The reduction in maximum absorption intensity at 352 and the appearance of a
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shoulder at 370 nm upon the addition of CuCl solution to the compound 6n solution
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be attributed to the presence of the carbonyl and sulfonamide groups in the
compound.
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ratio method by using CuCl solutions with increasing concentrations to titrate a
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methanolic solution of compound 6n (20 µM). Figure 7B shows that absorbance
gradually increased at 382 nm and then stabilized. The intersection of two straight
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2.9. Oil/water partition coefficient assay and ADMET prediction of active compounds
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The physicochemical parameters of a drug are correlated with the membrane
permeability of the body. Lipinski's rule of five is used as the rule of thumb when
assessing whether a compound can be used as an oral drug [50, 51]. For example, the
oil/water partition coefficient, which is expressed as a log P value, can reflect the
absorption of a drug in an organism [52]. The oil/water partition coefficients of
compounds that showed strong potency in bioactivity assessment were measured.
Table 3 shows that the log P values of compounds 6k, 6l, 6h, 6m, 6n, 6i, 6o, and 7b
ranged from 2.25 to 3.62. These results imply that the active compounds were
lipophilic (log P < 5).
The absorption, distribution, metabolism, excretion, and toxicity (ADMET)
properties of the selected eight compounds were also predicted by using DS 2017 R2
[53]. The different descriptors of ADMET characteristics have different prediction
levels. Compounds with low prediction levels have good ADMET properties. Table 4
and Figure 8 show that these compounds likely demonstrate good absorption in the
human intestine (HIA levels of 0), good solubility in water at 25 °C (solubility levels
of 1 and 2) and moderate blood–brain barrier permeability. Moreover, the results show
that these compounds are noninhibitors of CYP2D6 (CYP2D6 level of 0) and are
highly likely to bind to plasma proteins. In addition, the data revealed that the selected
compounds met the PSA2D and ALogP98 requirements within the 95% and 99%

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confidence limit ellipses of the BBB permeation and HIA models, respectively.
2.10. Neuroprotection study
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Neurotoxicity caused by H O -induced oxidative damage considerably accelerates
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the progression of neurodegenerative diseases [54]. Therefore, the potential protective
effects of the synthetic derivatives 6a–6p, 7a–7c, and 8a against H O -induced
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oxidative stress were evaluated by using PC12 cells. PC12 cells were pretreated for 4
h with different concentrations (1, 5, 10, 30, and 50 µM) of the compounds prior to
treatment with H O (250 µM). Cell viability was measured after 24 h of treatment by
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using the MTT assay. As seen in Table 5, the survival rate of PC12 cells incubated
without the derivatives fell in the range of 35.69%–50.26%. The viability of PC12
cells incubated with various concentrations of compounds, especially compounds 6h,
6i, 6l, 6o, 6n, 6p, and 7b, increased in a concentration-dependent manner. The
neuroprotective effect of compounds 6h, 6o and 6n were higher than that of reference
drug quercetin. The protective effects of compounds 6a, 6b, 6c, 6f, 6g, 6j, 6k, 6m, 7a,
and 7c were weaker than that of the control treatment. Compounds 6d, 6e, and 8a did
not show a significant protective effect against H O -induced cytotoxicity at all
2
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concentrations.
The human neuroblastoma SH-SY5Y cell line was also used to assess
H O -induced cytotoxicity and Aβ, which contribute to neuronal cell death, to validate
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the neuroprotective potency of compound 6n. Figure 9 shows that the viability of
SH-SY5Y cells treated with compound 6n (10, 30, and 50 µM) significantly increased
relative to that of cells treated with the control. Survival rate increased in a
dose-dependent manner. Moreover, comparing the H O (Figure 9A) and Aβ (Figure
2
2
9B) groups revealed that the cell viability of the H O group (60.58%) was higher
2
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than that of the Aβ group (57.84%) with compound 6n at 30 µM (see supporting
information for details, Table S3).
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3. Conclusions

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A series of novel 3-sulfonamide-substituted Ru derivatives were designed,
synthesized, and biologically evaluated for use as novel MTDLs for the development
of potential anti-AD agents. The results of biological evaluation showed that most of
the synthesized compounds presented selective inhibitory activity against BuChE.
Compound 6n was identified as the most potent BuChE inhibitor with IC = 3.60 ±
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0
0.34 µM. The results of enzyme kinetics analysis suggest that compound 6n inhibited
BuChE through a mixed-type mode by binding to the CAS and PAS. Molecular
modeling and MD simulation results showed that compound 6n could bind strongly to
BuChE. Moreover, compound 6n could effectively reduce H O -induced intracellular
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ROS accumulation. This result demonstrates that compound 6n could act as a novel
antioxidant. Furthermore, compound 6n may potentially prevent the production of
amyloid plaques by strongly inhibiting self-induced Aβ aggregation with a percentage
of inhibition of 58.95%. This result was also confirmed through TEM. Compound 6n
2+
could selectively chelate with biometal ion Cu and showed good drug-likeness and
ADMET properties as indicated by its physicochemical parameters. It also
demonstrated remarkable neuroprotective effects against H O - and Aβ-induced
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neurotoxicity toward PC12 and SH-SY5Y cell lines. The above results strongly
suggest that compound 6n is a promising MTDL. The further structural optimization
of 3-sulfonamide-substituted Ru derivatives as potential lead candidates is
encouraged for the development of new anti-AD drugs.
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4. Experimental section
4.1. Chemistry
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All common chemicals were obtained from commercial sources and used without
further purification. Reaction progress was monitored using analytical thinlayer
chromatography (TLC) on precoated silica gel HSGF254 plates(Qingdao Haiyang
Chemical Plant, China). Column chromatography was performed on silica gel
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(200-300 mm; Qingdao Dingkang Chemical Inc.). H NMR and C NMR spectra
were measured using TMS (δ 0 ppm) as the internal standard in dimethyl sulfoxide

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(DMSO-d6) solutions with a Bruker 400 MHz instrument (Bruker, Germany). The
MS spectra was recorded on Q Exactive™ Plus mass spectrometry (Thermofisher,
USA).
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4.1.1. Intermediate 3-amino-substituted rutaecarpine (5)
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To a solution of isatoic anhydride (20 g, 122.7 mmol) in con. H SO (30 mL) at
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0 °C was added Potassium nitrate (12.40 g, 122.7 mmol). The reaction was stirred for
1 h. The mixture was poured into cold water, and the precipitate formed was collected
and dried to afford nitro compound 1. Compound 1 dissolving (5.2 g) in the
anhydrous pyridine (100 mL) was dropwise add with the mixture solution of
trifluoroacetic anhydride (3.7 mL) and anhydrous pyridine (4 mL) at room
temperature. The stirring was refluxed for 30 min. Then, tryptamine (4.0 g) was added
and refluxed for additional 1 h. The reaction solution was cooled and poured into ice
water. The precipitate was filtrated and dried to give compound 2. A solution of
compound 2 (8.0 g) in acetic acid (40 mL) and of hydrochloric acid (6 mL) was
refluxed for 1 h. After cooling, ice water was add to precipitate compound 3.
compound 3 (4.0 g) was hydrogenated by 10% Pd/C (1.2 g) and 80% hydrazine
hydrate (10mL) in isopropanol at 80℃ for 1h. After the reaction was completed, Pd/C
was filtered off. The hydrazine hydrate was evaporated to remove to obtain compound
4. Compound 4 (3.7 g) with KOH (4.0 g) were added to a solution of water (15 mL)
and ethanol (50 mL), and the mixture was refluxed for 1 h. After cooling the reaction
mixture, a large amount of yellow-brown precipitate was formed, and the precipitate
was filtered and recrystallizated by hot methanol to give intermediate
1
3-amino-substituted rutaecarpine 5 as pale yellow powder, in a yield of 35%. H
NMR (400 MHz, DMSO) δ 11.73 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.45 (dd, J = 8.3,
3.7 Hz, 2H), 7.28 (s, 1H), 7.22 (t, J = 7.3 Hz, 1H), 7.08 (dd, J = 16.5, 8.7 Hz, 2H),
13
5.69 (s, 2H), 4.42 (t, J = 6.7 Hz, 2H), 3.13 (t, J = 6.7 Hz, 2H). C NMR (101 MHz,
DMSO) δ 160.94, 148.11, 141.35, 138.75, 138.57, 128.20, 128.01, 125.60, 124.45,
122.86, 122.32, 119.99, 119.99, 116.18, 112.80, 107.67, 41.24, 19.52. HRMS (ESI)

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m/z [M + H] : 303.1238 calcd for C H BrN O S: 303.1235;
24 17 4 3
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4.1.2. General procedure for the preparation of desired derivatives 6a-6p, 7a-7c, and
8a.
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A solution of the appropriate acid halide fragments (4.8 mmol) in dry pyridine (5
mL) was dropwise added to a well-stirred mixture of the intermediate
3-amino-substituted rutaecarpine 5 (4 mmol) in dry pyridine (50 mL) at room
temperature. The reaction was stirred for 30 min and monitored by TLC. After the
reaction completed, the pH was adjusted to neutral with dilute hydrochloric acid. The
pyridine and residual sulfonyl chloride were distilled under reduced pressure. The
mixture was extracted with an ethyl acetate-water system. The aqueous layer was
discarded and ethyl acetate was evaporated to obtain crude product. The crude product
was washed with water and dried under vacuum. The solid residue was purified by
flash chromatography on silica gel with ethyl acetate/petroleum ether (1:1) elution to
provide the desired derivatives 6a-6p, 7a-7c, and 8a.
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4.1.2.1. 3-(4-methoxybenzenesulfonamide)-rutaecarpine (6a)
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The intermediate 5 was treated with 4-methoxybenzenesulfonyl chloride according
to the general procedure mentioned above to get compound 6a as white powder, in a
1
yield of 65%; H NMR (400 MHz, DMSO-D6) δ 11.80 (s, 1H), 10.50 (s, 1H), 7.85 –
7.77 (m, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.8 Hz,
1H), 7.53 – 7.49 (m, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.21 (t, J = 7.7 Hz, 1H), 7.04 (t, J
13
= 7.6 Hz, 3H), 4.36 (t, J = 6.8 Hz, 2H), 3.73 (s, 3H), 3.10 (t, J = 6.8 Hz, 2H). C
NMR (101 MHz, DMSO) δ 163.02, 160.67, 144.85, 144.30, 139.06, 136.25, 131.25,
129.37, 128.19, 127.54, 127.46, 125.36, 125.11, 121.60, 120.33, 120.17, 117.99,
+
116.39, 114.97, 112.96, 56.09, 56.09, 41.37, 19.35, 19.11. HRMS (ESI) m/z [M + H] :
473.1273 calcd for C H BrN O S: 473.1275;
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4.1.2.2. 3-(2,4-dimethoxybenzenesulfonamide)-rutaecarpine (6b)

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The intermediate 5 was treated with 2,4-dimethoxybenzenesulfonyl chloride
according to the general procedure mentioned above to get compound 6b as brown
1
powder, in a yield of 63%; H NMR (400 MHz, DMSO) δ 11.82 (s, 1H), 10.31 (s, 1H),
7.85 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.56 (s, 2H), 7.45 (d, J
= 8.2 Hz, 1H), 7.25 (d, J = 7.5 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 6.71 – 6.50 (m, 2H),
4.39 (t, J = 6.8 Hz, 2H), 4.03 (q, J = 7.0 Hz, 1H), 3.88 (s, 3H), 3.77 (s, 3H), 3.14 (t, J
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= 6.8 Hz, 2H), 1.99 (s, 1H), 1.19 (dd, J = 18.3, 11.2 Hz, 2H). C NMR (101 MHz,
DMSO) δ 165.13, 160.67, 158.44, 144.61, 143.88, 139.02, 136.57, 132.58, 127.90,
127.58, 126.83, 125.21, 121.51, 120.23, 118.67, 117.85, 115.55, 112.94, 105.54, 99.66,
+
60.24, 56.72, 56.19, 41.34, 19.35, 14.54. HRMS (ESI) m/z [M + H] : 503.1374 calcd
for C H BrN O S: 503.1276;
2
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4.1.2.3. 3-(3,4-dimethoxybenzenesulfonamide)-rutaecarpine (6c)
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The intermediate 5 was treated with 3,4-dimethoxybenzenesulfonyl chloride
according to the general procedure mentioned above to get compound 6c as white
1
powder, in a yield of 46%; H NMR (400 MHz, DMSO-D6) δ 11.80 (s, 1H), 10.45 (s,
1H), 7.84 (s, 1H), 7.55 (ddd, J = 14.5, 11.1, 5.0 Hz, 3H), 7.41 (d, J = 8.3 Hz, 1H),
7.30 (d, J = 10.5 Hz, 2H), 7.21 (t, J = 7.6 Hz, 1H), 7.04 (t, J = 8.7 Hz, 2H), 4.36 (t, J
13
= 6.0 Hz, 2H), 3.31 (d, J = 2.3 Hz, 9H), 3.10 (t, J = 6.2 Hz, 2H). C NMR (101 MHz,
DMSO) δ 161.44, 160.62, 158.91, 145.16, 144.86, 139.09, 137.08, 135.27, 129.68,
128.69, 128.60, 128.40, 128.08, 127.47, 125.33, 125.16, 121.65, 121.51, 121.32,
120.34, 120.17, 118.84, 118.61, 118.16, 117.45, 112.98, 41.39, 19.33. HRMS (ESI)
+
m/z [M + H] : 503.1378 calcd for C H BrN O S: 503.1377;
2
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4.1.2.4. 3-(4-nitrobenzenesulfonamide)-rutaecarpine (6d)
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The intermediate 5 was treated with 4-nitrobenzenesulfonyl chloride according to
the general procedure mentioned above to get compound 6d as yellow powder, in a
1
yield of 52%; H NMR (400 MHz, DMSO-D6) δ 11.81 (s, 1H), 10.94 (s, 1H), 8.34 (d,
J = 8.8 Hz, 2H), 7.98 (d, J = 8.6 Hz, 2H), 7.83 (d, J = 2.2 Hz, 1H), 7.59 (dd, J = 8.4,

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3.5 Hz, 2H), 7.53 (dd, J = 8.8, 2.2 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.21 (t, J = 7.6
1
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Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 4.36 (t, J = 6.8 Hz, 2H), 3.10 (t, J = 6.8 Hz, 2H). C
NMR (101 MHz, DMSO) δ 160.60, 150.39, 145.18, 145.03, 144.89, 139.09, 135.14,
128.74, 128.74, 128.44, 128.09, 127.45, 125.33, 125.25, 125.25, 125.20, 121.66,
+
120.38, 120.21, 118.20, 117.45, 112.98, 41.39, 19.33. HRMS (ESI) m/z [M + H] :
488.1013 calcd for C H BrN O S: 488.1012;
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4.1.2.5. 3-(3-nitrobenzenesulfonamide)-rutaecarpine (6e)
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The intermediate 5 was treated with 3-nitrobenzenesulfonyl chloride according to
the general procedure mentioned above to get compound 6e as pale brown powder, in
1
a yield of 58%; H NMR (400 MHz, DMSO-D6) δ 11.80 (s, 1H), 10.89 (s, 1H), 8.50
(s, 1H), 8.41 (dd, J = 8.2, 1.1 Hz, 1H), 8.11 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 8.3 Hz,
1H), 7.20 (t, J = 7.6 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 4.35 (t, J = 6.8 Hz, 2H), 3.09 (t,
1
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J = 6.8 Hz, 2H). C NMR (101 MHz, DMSO) δ 160.59, 148.37, 145.21, 144.97,
141.11, 139.09, 135.03, 133.05, 131.99, 128.45, 128.27, 128.21, 127.44, 125.31,
125.18, 121.89, 121.65, 120.35, 120.18, 118.19, 117.66, 112.97, 41.38, 19.32. HRMS
+
(ESI) m/z [M + H] : 488.1013 calcd for C H BrN O S: 488.1011;
2
4
17
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3
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4.1.2.6. 3-(4-bromobenzenesulfonamide)-rutaecarpine (6f)
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The intermediate 5 was treated with 4-bromobenzenesulfonyl chloride according to
the general procedure mentioned above to get compound 6f as white powder, in a
1
yield of 59%; H NMR (400 MHz, DMSO-D6) δ 11.80 (s, 1H), 10.50 (s, 1H), 7.85 –
7.77 (m, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.8 Hz,
1H), 7.53 – 7.49 (m, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.21 (t, J = 7.7 Hz, 1H), 7.04 (t, J
13
= 7.6 Hz, 3H), 4.36 (t, J = 6.8 Hz, 2H), 3.73 (s, 3H), 3.10 (t, J = 6.8 Hz, 2H). C
NMR (101 MHz, DMSO) δ 160.64, 145.06, 144.67, 139.08, 138.88, 135.59, 133.00,
133.00, 129.13, 129.13, 128.34, 127.86, 127.54, 127.49, 125.34, 125.16, 121.63,
+
120.36, 120.19, 118.12, 117.03, 112.98, 41.39, 19.34. HRMS (ESI) m/z [M + H] :
523.0252 calcd for C H BrN O S: 523.0253;
2
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17
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4.1.2.7. 3-(2,6-dichlorobenzenesulfonamide)-rutaecarpine (6g)
4
4
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The intermediate 5 was treated with 2,6-dichlorobenzenesulfonyl chloride
according to the general procedure mentioned above to get compound 6g as white
1
powder, in a yield of 65%; H NMR (400 MHz, DMSO) δ 11.86 (s, 1H), 10.92 (s, 1H),
8.00 (s, 4H), 7.89 (s, 1H), 7.60 (dt, J = 8.9, 5.1 Hz, 3H), 7.47 (d, J = 8.2 Hz, 1H), 7.26
1
3
(t, J = 7.6 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 4.41 (s, 2H), 3.15 (t, J = 6.7 Hz, 2H). C
NMR (101 MHz, DMSO) δ 160.62, 145.14, 144.80, 143.55, 139.09, 135.31, 133.35,
133.03, 128.40, 128.14, 127.91, 127.47, 127.18, 127.18, 125.33, 125.15, 122.42,
121.66, 120.34, 120.17, 118.14, 117.17, 112.97, 41.38, 19.32. HRMS (ESI) m/z [M +
+
H] : 511.1036 calcd for C H BrN O S: 511.1038;
24
17
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4.1.2.8. 3-(3,5-dichlorobenzenesulfonamide)-rutaecarpine (6h)
4
4
4
4
4
4
4
4
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The intermediate 5 was treated with 3,5-dichlorobenzenesulfonyl chloride
according to the general procedure mentioned above to get compound 6h as brown
1
powder, in a yield of 52%; H NMR (400 MHz, DMSO-D6) δ 11.81 (s, 1H), 10.81 (s,
1H), 7.93 (s, 1H), 7.81 (s, 1H), 7.71 (s, 2H), 7.63 – 7.50 (m, 3H), 7.42 (d, J = 8.2 Hz,
1H), 7.21 (t, J = 7.5 Hz, 1H), 7.03 (t, J = 7.3 Hz, 1H), 4.36 (t, J = 6.7 Hz, 2H), 3.10 (t,
1
3
J = 6.7 Hz, 2H). C NMR (101 MHz, DMSO) δ 160.61, 145.27, 145.01, 142.68,
139.11, 135.69, 135.69, 134.96, 133.36, 128.48, 128.19, 127.47, 125.67, 125.67,
125.33, 125.18, 121.67, 120.37, 120.18, 118.22, 117.67, 112.99, 41.40, 19.33. HRMS
+
(ESI) m/z [M + H] : 511.0384 calcd for C H BrN O S: 511.0389;
2
4
17
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3
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4.1.2.9. 3-(3-chloro-4-fluorobenzenesulfonamide)-rutaecarpine (6i)
4
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The intermediate 5 was treated with 3-chloro-4-fluorobenzenesulfonyl chloride
according to the general procedure mentioned above to get compound 6i as white
1
powder, in a yield of 48%; H NMR (400 MHz, DMSO-D6) δ 11.81 (s, 1H), 10.73 (s,
1H), 7.94 (d, J = 6.6 Hz, 1H), 7.81 (s, 1H), 7.75 – 7.68 (m, 1H), 7.64 – 7.50 (m, 4H),
7.42 (d, J = 8.2 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 4.36 (t, J =

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6.5 Hz, 2H), 3.10 (t, J = 6.5 Hz, 2H). C NMR (101 MHz, DMSO) δ 160.69, 152.73,
149.02, 144.86, 144.33, 139.05, 136.26, 131.00, 128.16, 127.59, 127.53, 125.35,
125.12, 121.54, 121.10, 120.33, 120.18, 118.01, 116.53, 112.96, 111.60, 109.81, 56.22,
+
56.17, 41.37, 19.33. HRMS (ESI) m/z [M + H] : 495.0682 calcd for C H BrN O S:
24
17
4
3
495.0680;
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4.1.2.10. 3-(4-tert-butylbenzenesulfonamide)-rutaecarpine (6j)
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4
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4
4
4
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The intermediate 5 was treated with 4-tert-butylbenzenesulfonyl chloride according
to the general procedure mentioned above to get compound 6j as white powder, in a
1
yield of 53%; H NMR (400 MHz, DMSO-D6) δ 11.80 (s, 1H), 10.64 (s, 1H), 7.86 (s,
1H), 7.69 (d, J = 8.4 Hz, 2H), 7.62 – 7.53 (m, 4H), 7.41 (d, J = 8.3 Hz, 1H), 7.21 (t, J
= 7.5 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 4.36 (t, J = 6.8 Hz, 2H), 3.50 (s, 1H), 3.10 (t,
1
3
J = 6.8 Hz, 2H), 1.20 (s, 10H). C NMR (101 MHz, DMSO) δ 160.67, 156.58,
144.86, 144.24, 139.06, 137.08, 136.19, 128.27, 127.52, 127.17, 126.99, 126.99,
126.77, 126.77,125.36, 125.14, 121.64, 120.36, 120.19, 118.01, 116.00, 112.97, 41.39,
+
35.35, 31.15, 31.15, 31.15, 19.36. HRMS (ESI) m/z [M + H] : 499.1787 calcd for
C H BrN O S: 499.1786;
24
17
4
3
4
84
4.1.2.11. 3-(4-fluorobenzenesulfonamide)-rutaecarpine (6k)
4
4
4
4
4
4
4
4
4
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93
The intermediate 5 was treated with 4-fluorobenzenesulfonyl chloride according to
the general procedure mentioned above to get compound 6k as white powder, in a
1
yield of 61%; H NMR (400 MHz, DMSO-D6) δ 11.80 (s, 1H), 10.65 (s, 1H), 7.80
(dd, J = 8.4, 3.9 Hz, 3H), 7.61 – 7.48 (m, 3H), 7.44 – 7.34 (m, 3H), 7.21 (t, J = 7.6 Hz,
13
1H), 7.04 (t, J = 7.4 Hz, 1H), 4.36 (t, J = 6.8 Hz, 2H), 3.10 (t, J = 6.8 Hz, 2H). C
NMR (101 MHz, DMSO) δ 166.13, 163.62, 160.64, 145.03, 144.61, 139.08, 136.00,
135.73, 130.27, 130.18, 128.29, 127.87, 127.49, 125.34, 125.15, 121.61, 120.34,
+
120.18, 118.09, 117.22, 117.00, 112.97, 41.38, 19.33. HRMS (ESI) m/z [M + H] :
461.1069 calcd for C H BrN O S: 461.1068;
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4.1.2.12. 3-[4-(trifluoromethyl)benzenesulfonamide]-rutaecarpine (6l)
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02
03
The intermediate 5 was treated with 4-(trifluoromethyl)benzenesulfonyl chloride
according to the general procedure mentioned above to get compound 6l as pale
1
brown powder, in a yield of 47%; H NMR (400 MHz, DMSO-D6) δ 11.81 (s, 1H),
10.87 (s, 1H), 7.94 (s, 1H), 7.82 (d, J = 2.2 Hz, 1H), 7.56 (ddd, J = 10.8, 8.4, 3.6 Hz,
1H), 7.41 (d, J = 8.3 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 4.36 (s,
1
3
1H), 3.11 (t, J = 6.7 Hz, 1H). C NMR (101 MHz, DMSO) δ 160.66, 151.61, 145.00,
144.57, 139.09, 138.75, 135.88, 129.77, 129.77, 128.33, 127.92, 127.51, 125.35,
125.14, 121.98, 121.98, 121.65, 120.35, 120.18, 118.09, 116.99, 112.98, 41.38, 19.34.
+
HRMS (ESI) m/z [M + H] : 511.1042 calcd for C H BrN O S: 511.1041;
24
17
4
3
5
04
4.1.2.13. 3-[3-(trifluoromethyl)benzenesulfonamide]-rutaecarpine (6m)
5
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The intermediate 5 was treated with 3-(trifluoromethyl)benzenesulfonyl chloride
according to the general procedure mentioned above to get compound 6m as pale
1
brown powder, in a yield of 52%; H NMR (400 MHz, DMSO-D6) δ 11.81 (s, 1H),
10.77 (s, 1H), 8.00 (dd, J = 12.5, 6.7 Hz, 3H), 7.78 (dd, J = 10.8, 5.0 Hz, 2H), 7.59
(dd, J = 8.5, 3.2 Hz, 2H), 7.52 (dd, J = 8.8, 2.5 Hz, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.21
(ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.04 (ddd, J = 8.0, 7.1, 0.9 Hz, 1H), 4.36 (t, J = 6.9 Hz,
2H), 3.98 (q, J = 7.1 Hz, 1H), 3.49 (s, 1H), 3.10 (t, J = 6.9 Hz, 2H), 1.95 (s, 1H), 1.17
1
3
(dt, J = 14.2, 6.8 Hz, 3H). C NMR (101 MHz, DMSO) δ 160.60, 145.12, 144.80,
140.88, 139.09, 135.50, 131.56, 131.16, 130.56, 130.31, 128.36, 128.24, 127.47,
125.33, 125.16, 123.66, 122.39, 121.62, 120.36, 120.18, 118.15, 117.50, 112.97,
+
60.23, 41.38, 21.21, 19.33, 14.52. HRMS (ESI) m/z [M + H] : 511.1037 calcd for
C H BrN O S: 511.1039;
24
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4
3
5
17
4.1.2.14. 3-[2-(trifluoromethoxy)benzenesulfonamide]-rutaecarpine (6n)
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The intermediate 5 was treated with 2-(trifluoromethoxy)benzenesulfonyl chloride
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powder, in a yield of 61%; H NMR (400 MHz, DMSO) δ 11.86 (s, 1H), 10.98 (s, 1H),
8.02 (d, J = 7.2 Hz, 1H), 7.88 – 7.73 (m, 2H), 7.68 – 7.52 (m, 5H), 7.46 (d, J = 8.3 Hz,
1H), 7.25 (t, J = 7.5 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 4.40 (t, J = 6.7 Hz, 2H), 3.37 (s,
1
3
1H), 3.15 (t, J = 6.7 Hz, 2H). C NMR (101 MHz, DMSO) δ 160.61, 145.58, 144.97,
144.41, 139.07, 136.18, 135.42, 131.67, 131.49, 128.24, 128.09, 127.51, 127.23,
125.35, 125.14, 121.57, 121.57, 120.35, 120.18, 119.03, 118.07, 116.17, 112.96, 41.37,
+
19.33. HRMS (ESI) m/z [M + H] : 527.0987 calcd for C H BrN O S: 527.0988;
2
4
17
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3
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4.1.2.15. 3-[4-(trifluoromethoxy)benzenesulfonamide]-rutaecarpine (6o)
5
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36
The intermediate 5 was treated with 4-(trifluoromethoxy)benzenesulfonyl chloride
according to the general procedure mentioned above to get compound 6o as white
1
powder, in a yield of 54%; H NMR (400 MHz, DMSO-D6) δ 11.81 (s, 1H), 10.75 (s,
1H), 7.85 (dd, J = 17.2, 5.6 Hz, 3H), 7.66 – 7.48 (m, 5H), 7.42 (d, J = 8.0 Hz, 1H),
7.21 (dd, J = 8.1, 7.2 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 4.36 (t, J = 6.8 Hz, 2H), 3.10 (t,
1
3
J = 6.8 Hz, 2H). C NMR (101 MHz, DMSO) δ 160.63, 145.15, 144.80, 143.60,
139.11, 135.36, 133.35, 133.03, 128.41, 128.15, 127.93, 127.49, 127.19, 127.19,
125.35, 125.17, 122.44, 121.68, 120.36, 120.19, 118.16, 117.19, 112.99, 41.39, 19.34.
+
HRMS (ESI) m/z [M + H] : 527.0984 calcd for C H BrN O S: 527.0988;
24
17
4
3
5
37
4.1.2.16. 3-(4-acetylbenzenesulfonamide)-rutaecarpine (6p)
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46
The intermediate 5 was treated with 4-acetylbenzenesulfonyl chloride according to
the general procedure mentioned above to get compound 6p as brown powder, in a
1
yield of 48%; H NMR (400 MHz, DMSO-D6) δ 11.80 (s, 1H), 10.81 (s, 1H), 8.06 (d,
J = 8.2 Hz, 2H), 7.85 (dd, J = 17.5, 4.9 Hz, 3H), 7.63 – 7.46 (m, 3H), 7.41 (d, J = 8.3
Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 4.35 (t, J = 6.8 Hz, 2H),
1
3
3.10 (t, J = 6.8 Hz, 2H), 2.46 (s, 3H). C NMR (101 MHz, DMSO) δ 197.68, 160.62,
145.05, 144.65, 143.26, 140.32, 139.07, 135.56, 129.62, 129.62, 128.35, 127.78,
127.52, 127.52, 125.33, 125.16, 121.62, 120.35, 120.18, 118.11, 116.94, 112.97, 41.37,
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27.44, 27.44, 19.33. HRMS (ESI) m/z [M + H] : 485.1272 calcd for C H BrN O S:
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485.1274;
4.1.2.17. 3-(2-thiophenesulfonamide)-rutaecarpine (7a)
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The intermediate 5 was treated with 2-thiophenesulfonyl chloride according to the
general procedure mentioned above to get compound 7a as white powder, in a yield of
1
43%; H NMR (400 MHz, DMSO-D6) δ 11.82 (s, 1H), 10.75 (s, 1H), 7.88 (s, 2H),
7.56 (dt, J = 14.8, 5.8 Hz, 4H), 7.42 (d, J = 8.3 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.12
13
– 7.00 (m, 2H), 4.38 (t, J = 6.7 Hz, 2H), 3.11 (t, J = 6.8 Hz, 2H), -0.10 (s, 4H). C
NMR (101 MHz, DMSO) δ 160.69, 145.07, 144.70, 140.05, 139.09, 135.75, 134.15,
133.06, 128.24, 128.24,127.96, 127.53, 125.36, 125.17, 121.59, 120.38, 120.20,
+
118.11, 117.09, 112.99, 41.40, 19.36. HRMS (ESI) m/z [M + H] : 449.0728 calcd for
C H BrN O S: 449.0736;
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4.1.2.18. 3-(5-bromothiophene-2-sulfonamide)-rutaecarpine (7b)
5
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The intermediate 5 was treated with 5-bromothiophene-2-sulfonyl chloride
according to the general procedure mentioned above to get compound 7b as white
1
powder, in a yield of 40%; H NMR (400 MHz, DMSO-D6) δ 11.83 (s, 1H), 10.90 (s,
1H), 7.87 (d, J = 2.3 Hz, 1H), 7.66 – 7.51 (m, 3H), 7.42 (d, J = 8.3 Hz, 1H), 7.39 –
7.34 (m, 1H), 7.31 – 7.25 (m, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H),
1
3
4.38 (t, J = 6.8 Hz, 2H), 3.12 (t, J = 6.7 Hz, 2H). C NMR (101 MHz, DMSO) δ
160.68, 145.22, 144.96, 140.88, 139.11, 135.21, 133.77, 131.96, 128.40, 128.08,
127.50, 125.35, 125.19, 121.66, 120.38, 120.20, 119.86, 118.20, 117.37, 113.00, 41.42,
+
19.35. HRMS (ESI) m/z [M + H] : 528.9815 calcd for C H BrN O S: 528.9812;
2
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17
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4.1.2.19. 3-(5-chlorothiophene-2-sulfonamide)-rutaecarpine (7c)
5
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The intermediate 5 was treated with 5-chlorothiophene-2-sulfonyl chloride
according to the general procedure mentioned above to get compound 7c as pale
1
brown powder, in a yield of 37%; H NMR (400 MHz, DMSO-D6) δ 11.83 (s, 1H),
10.91 (s, 1H), 7.88 (d, J = 2.3 Hz, 1H), 7.67 – 7.53 (m, 3H), 7.47 – 7.38 (m, 2H), 7.25

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– 7.14 (m, 2H), 7.04 (t, J = 7.5 Hz, 1H), 4.38 (t, J = 6.8 Hz, 2H), 3.11 (t, J = 6.8 Hz,
1
3
2H). C NMR (101 MHz, DMSO) δ 160.67, 145.23, 145.00, 139.11, 138.27, 135.94,
135.17, 133.04, 128.61, 128.40, 128.16, 127.50, 125.35, 125.19, 121.65, 120.38,
+
120.20, 118.21, 117.48, 112.99, 41.41, 19.35. HRMS (ESI) m/z [M + H] : 483.0343
calcd for C H BrN O S: 483.0346;
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4.1.2.20. 3-(2-carbomethoxy-3-thiophenesulfonamide)-rutaecarpine (8a)
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The intermediate 5 was treated with 2-carbomethoxy-3-thiophenesulfonyl chloride
according to the general procedure mentioned above to get compound 8a as white
1
powder, in a yield of 36%; H NMR (400 MHz, DMSO-D6) δ 11.80 (s, 1H), 10.51 (s,
1H), 7.94 (d, J = 5.3 Hz, 1H), 7.83 (d, J = 2.1 Hz, 1H), 7.55 (dt, J = 8.9, 5.6 Hz, 3H),
7.48 – 7.38 (m, 2H), 7.20 (t, J = 7.6 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 4.36 (t, J = 6.8
1
3
Hz, 2H), 3.31 (s, 1H), 3.10 (t, J = 6.8 Hz, 2H). C NMR (101 MHz, DMSO) δ 160.65,
160.34, 144.93, 144.34, 142.56, 139.07, 135.66, 133.22, 132.68, 131.05, 128.22,
127.53, 127.25, 125.36, 125.12, 121.60, 120.34, 120.18, 118.03, 116.19, 112.97,
+
53.73, 41.38, 19.35. HRMS (ESI) m/z [M + H] : 507.0784 calcd for C H BrN O S:
24
17
4
3
507.0785;
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89
4.2. AChE and BuChE inhibition assay
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The ChE inhibition activity of the test compounds was performed adopting the
method of Ellman et al. with modification. Acetylcholinesterase (AChE,
C3389-2KU, from electric eel)
was purchased from SigmaAldrich.
from equine serum),
Butylcholinesterase (BuChE, B128570-2KU,
5,5-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent, D105559-1g),
acetylthiocholine iodide (ATCI, A100869-1g), S-Butyrylthiocholine iodide (BTCI,
B100871-1g) and donepezil hydrochloride (D129948-1g) was obtained from Aladdin.
Tacrine and donepezil were used as reference drugs. The compounds were dissolved
in DMSO and then diluted in 0.1 M pH 8 phosphate buffer to provide different
concentration (DMSO 0.1%). Briefly, 50µL DTNB, 10 µL of AChE or BuChE

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(2U/ml), 10 µL different concentration of the test or standard compounds and 430 µL
phosphate buffer were preincubated in 48-well plates at 37℃ for 30 min. Further, 50
µL the substrate ATCI or BTCI was added. The mixture was incubated for additional
30 min. Changes in absorbance were measured at 412 nm in a Biotek Synergy HTX
Multi-Mode reader. To determine the IC (concentration of the compound resulting
5
0
in 50% inhibition of the enzyme activity), log inhibitor concentration versus percent
of inhibition curve was analyzed.
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4.3. Kinetic characterization of BuChE inhibition
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Enzyme kinetic studies was done in a similar manner as enzyme inhibition assay.
Substrate (BTCI) in the presence of different concentrations of inhibitor (0−8µM)
were measured at different times to determine the inhibition model and reciprocal
plots of 1/V versus 1/[S].
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4.4. Molecular modeling and dynamic simulation
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The Discovery Studio 2017 R2 (DS, BIOVIA Software, Inc., San Diego,CA, United
States) was applied for molecular docking to gain the interaction between ligands and
receptors. The X-ray crystal structure of the BuChE (PDB ID: 5NN0, co-crystallized
with 92H) was obtained from Protein Data Bank. The structure of BuChE derived
from the complex 5NN0 was prepared by removal of the original ligand and water
molecules, and all polar hydrogen and CHARMm force fields were then added. And
the treated protein was prepared using Prepare Protein protocol. The ligand was
processed by Full Minimization of the Small Molecular protocol. Then the ligand was
docked into the active site of protein using the CDOCKER program with default
parameters. The docked conformation with the highest score of
-CDOCKER_INTERACTION_ENERGY values was selected and analyzed.
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MD simulations in explicit solvent water were performed. The system was
minimized with fixed positions for all heavy atoms of BuChE and compound 6n for

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1,000 steps. And the system was gradually heated to 300 K during 100 ps. The
equilibration process was started with a 100 ps simulation at 300K, while keeping all
the heavy atoms of BuChE and compound 6n fixed. This was followed by
unconstrained minimization consisting of 1,000 steps. The production simulations
were performed for 10000 ps with a 2 fs time step. Therefore, a 10000 ps dynamic
calculation was performed in the NTP ensemble, with pressure constant at 1 atm and
temperature constant at 300 K. RMSD properties of molecular dynamics trajectories
was analyzed by Analyze Trajectory procedure.
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4.5. Radical scavenging activity (DPPH assay)
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The antioxidant capacity of the test compounds were evaluated by DPPH method in
which DPPH free radical chould be scavenged by antioxidant. Brefly, 150 µL of the
compound (100 µM) with 150 uL of DPPH (140 µM) was mixed and incubated in a
96-well plate for 2 hours in the dark at 37 °C. The relevant absorbance of the reaction
mixture was measured at 520 nm using a microplate reader (BioTek Synergy HT).
The reducing percentage (RP) of DPPH was determined by the formula : RP = (1 −
A /A ) × 100%, where A /A are DPPH absorbance in the presence and absence of
c
0
c
0
inhibitors, respectively. Ascorbic acid was used as a standard for DPPH
determination.
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4.6. Determination of the intracellular ROS production
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Intracellular ROS production can cause oxidative stress and accelerate the
progression of neurodegenerative diseases. 2', 7' - dichlorofluorescein diacetate
(DCFH-DA) assay was applied to determine ROS formation. Briefly, SH-SY5Y cells
4
were seeded in 96-well plates (1 × 10 cells per well) and incubated at 37 ° C
containing 5% CO for 24 hours. After cells being incubated in the presence or
2
absence of different concentrations of the test compound for 6 hours, the cells were
washed with PBS. Then the cells were exposed to H O (250 µM) or Aβ (5 µM) for 24
2
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hours. The cells were washed with PBS again and incubated with DCFH-DA (10 µM)

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for 1h in the dark. At the end of the incubation, DCFH-DA was removed. The
fluorescence intensity of the dichlorofluorescein (DCF) from the cells was measured
at 488 nm excitation and 525 nm emission wavelengths using Synergy HTX
fluorescence microplate reader. Results were expressed as the percentage of
intracellular ROS compared to untreated control cells.
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4.7. Effect on Aβ peptide aggregation
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Aggregation of Aβ peptide in brain was considered to be an inportant factor of the
pathogenesis of AD. Inhibition of self-induced Aβ aggregation was investigated using
thioflavin T (ThT) fluorescence assay as described. Aβ(1-42) peptide (MB10425,
Meilun Biotechnology Co., LTD) was prepared in phosphate buffer (pH 7.4)
containing 1% ammonium hydroxide. The test compound was prepared in DMSO to
obtain a 10 mM solution and diluted further with phosphate buffer. Briefly, 10 µL of
Aβ(1-42) was incubated in the absence and presence of 10 µL of the test compound to
obtain final concentration of 20 µM Aβ(1–42) and 100 µM the test compound. The
mixture was incubated at room temperature. After 48 h incubation, 5 µM ThT
(prepared in 50 mM glycine-NaOH buffer; pH 8.5) was added to make the volume of
200 µL. Fluorescence intensity of the solution was measured at 450 nm excitation and
485 nm emission wavelengths using Synergy HTX fluorescence microplate reader.
The percentage inhibition of the self-induced Aβ(1–42) aggregation for the test
compound was calculated by the following formula: (1 − IF / If ) × 100%, in which
i
o
IF and IF are fluorescence intensities in the presence and absence of inhibitors,
i
o
respectively.
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4.8. Metal-chelating research
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The metal binding property of the test compound was investigated using a UV-vis
spectrophotometer with a wavelength range of 200-600 nm. The UV absorption of the
selected compound (20 µM) in the absence and presence of CuCl , FeSO , ZnCl ,
2
4
2
AlCl , MgCl and CaCl (20 µM) was recorded in a 1 cm quartz cell for 1 hour. The
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final volume of the reaction mixture was 2 mL. The molar ratio method was carried
out to determine the stoichiometry of the complex. A solution of the test compound
(20 µM) was titrated by a molar solution of increasing amount of CuCl2.
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4.9. Oil/water partition coefficient assay and ADMET prediction
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Using the classical shake flask method, the oil/water partition coefficient of the
compounds were tested. The same amount of oleic phase (noctanol) and aqueous
phase (PBS pH 7.4) were mixed. And the mixture was shaken by ultrasonic (400 W,
40 kHz) and allowed to stand for 24 h to obtain a saturated solution of noctanol. An
appropriate amount of the compounds was added. After sealing, the test compounds
was shaken at 37 °C for 48 h to make it fully equilibrated in the two phases. Then the
mixture was measured with an ultraviolet spectrophotometer.
The predictive ADMET pharmacokinetic properties, including human intestinal
absorption (HIA), aqueous solubility, blood-brain-barrier penetration (BBB),
cytochrome P450 2D6 (CYP2D6) enzyme inhibition, plasma protein binding (PPB)
were analyzed quantitatively for the selected eight ligands using ADMET descriptors
tools in Discovery Studio 2017 R2. The selected eight ligands were processed as
described above and input to computer program. Parameters of ADMET prediction
program were set in the normal mode.
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4.10. Neuroprotection assay
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4.10.1. Neuroprotective effects of all compounds against H O -induced PC12cell
2
2
death
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Viability of PC12 cell (rat adrenal chromaffin differentiated cell line) of all
compounds against H O -induced cytotoxicity was detected by
2
2
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. PC12
4
cells were seeded in 96-well plates (1 × 10 cells per well) and incubated at 37 °C
containing 5% CO . After 24 hours, cells were incubated with different concentrations
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of the test compounds for 3 hours, then the cells were exposed with H O (250 µM)
2
2
for 24 hours. Cell viability was measured by MTT assay. Briefly, 20 mL of 0.5 mg/mL
MTT reagent was added after removal of the previous medium and incubated for 4 h.
After 4 h, the medium was removed and the crystal of the formazan was then
dissolved 150 mL DMSO. The relevant absorbance was measured at 570 nm on the
Biotek Synergy HTX Multi-Mode reader. Cell viability are expressed as the
percentage of untreated control cells (PC12 cells in the absence of test compound and
H O ).
2
2
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4.10.2. Neuroprotective test of the selected compound against H O or Aβ-induced
2 2
SH-SY5Y cell death
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The Aβ(1-42) peptide was incubated in serum medium at a final concentration of 5
µM for 24 hours to induce Aβ aggregation for neuroprotective test. Neuroprotective
test of the selected compound against H O or Aβ-induced SH-SY5Y cell (human
2
2
neuroblastoma cell) death was performed in a same manner as described above.
4.11. Statistical analysis
7
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22
Data are reported as mean ± SEM of at least three independent experiments and
data analysis was performed with GraphPad Prism 6 software.
7
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24
Abbreviation
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30
Alzheimer's disease (AD)
β-amyloid protein (Aβ)
Acetylcholinesterase (AChE)
Butyrylcholinesterase (BuChE)
Cholinesterase (ChE)
Reactive oxygen species (ROS)

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Multitarget-directed ligands (MTDLs)
Rutaecarpine (Ru)
Tacrine (THA)
Studio Discovery 2017 R2 (DS)
Molecular dynamics (MD)
Root-mean-square deviation (RMSD)
1,1-Diphenyl-2-picrylhydrazyl (DPPH)
2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA)
Dichlorofluorescein (DCF)
Thioflavin T (Th T)
Transmission Electron Microscopy (TEM)
Absorption, distribution, metabolism, excretion, and toxicity (ADMET)
human intestinal absorption (HIA)
blood-brain-barrier penetration (BBB),
cytochrome P450 2D6 (CYP2D6)
plasma protein binding (PPB)
5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)
acetylthiocholine iodide (ATCI)
S-Butyrylthiocholine iodide (BTCI)
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
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Conflict of interest
We declare that we have no conflict of interest.
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Acknowledgments
We thank the General Financial Grants from the China Postdoctoral Science
Foundation (2015M571916), the National Nature Science Foundation of China
(81302701)for financial support of this study.