Design, synthesis and evaluation of novel bivalent β-carboline derivatives as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2018.06.018

To develop potent multi-target ligands against Alzheimer's disease (AD), a series of novel bivalent β-carboline derivatives were designed, synthesized, and evaluated. In vitro studies revealed these compounds exhibited good multifunctional activities. In particular, compounds 8f and 8g showed the good selectivity potency on BuChE inhibition (IC₅₀ = 1.7 and 2.7 μM, respectively), Aβ_1-42 disaggregation and neuroprotection. Compared with the positive control resveratrol, 8f and 8g showed better activity in inhibiting Aβ_1-42 aggregation, with inhibitory rate 82.7% and 85.7% at 25 μM, respectively. Moreover, compounds 8e, 8f and 8g displayed excellent neuroprotective activity by ameliorating the impairment induced by H₂O₂, okadaic acid (OA) and Aβ_1-42 without cytotoxicity in SH-SY5Y cells. Thus, the present study evidently showed that compounds 8f and 8g are potent multi-functional agents against AD and might serve as promising lead candidates for further development.

Full text of DOI:10.1016/j.bmc.2018.06.018

Accepted Manuscript
Design, synthesis and evaluation of novel bivalent β-carboline derivatives as
multifunctional agents for the treatment of Alzheimer's disease
Yifan Zhao, Feng Ye, Jian Xu, Qinghong Liao, Lei Chen, Weijia Zhang,
Haopeng Sun, Wenyuan Liu, Feng Feng, Wei Qu
PII:
S0968-0896(18)30910-6
https://doi.org/10.1016/j.bmc.2018.06.018
BMC 14410
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 May 2018
8 June 2018
13 June 2018
Please cite this article as: Zhao, Y., Ye, F., Xu, J., Liao, Q., Chen, L., Zhang, W., Sun, H., Liu, W., Feng, F., Qu,
W., Design, synthesis and evaluation of novel bivalent β-carboline derivatives as multifunctional agents for the
treatment of Alzheimer's disease, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.06.018
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Design, synthesis and evaluation of novel bivalent β-carboline
derivatives as multifunctional agents for the treatment of Alzheimer's
disease
Yifan Zhaoa, Feng Yea, Jian Xua, Qinghong Liaoa, Lei Chenb, Weijia Zhanga, Haopeng Sunc,
Wenyuan Liub, Feng Fenga,d,e *, Wei Qua,d *
a School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009,
China
b Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009,
China
c Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009,
China
d
Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University,
Nanjing 210009, China
e Jiangsu Food and Pharmaceutical Science College, Huaian 223003, China
* Corresponding author
Tel./Fax: +86 025 86185216, E-mail address: fengfeng@cpu.edu.cn (F. Feng)
Tel./Fax: +86 025 86185216, E-mail address: popoqzh@126.com (W. Qu)

Abstract
To develop potent multi-target ligands against Alzheimer's disease (AD), a series of novel bivalent
β-carboline derivatives were designed, synthesized, and evaluated. In vitro studies revealed these
compounds exhibited good multifunctional activities. In particular, compounds 8f and 8g showed
the good selectivity potency on BuChE inhibition (IC50 = 1.7 and 2.7 μM, respectively), Aβ1-42
disaggregation and neuroprotection. Compared with the positive control resveratrol, 8f and 8g
showed better activity in inhibiting Aβ1-42 aggregation, with inhibitory rate 82.7% and 85.7% at 25
μM, respectively. Moreover, compounds 8e, 8f and 8g displayed excellent neuroprotective activity
by ameliorating the impairment induced by H2O2, okadaic acid (OA) and Aβ1-42 without
cytotoxicity in SH-SY5Y cells. Thus, the present study evidently showed that compounds 8f and
8g are potent multi-functional agents against AD and might serve as promising lead candidates for
further development.
Keywords:
bivalent β-carbolines
Aβ1-42 aggregation inhibitory
neuroprotective activity
Alzheimer's disease

1. Introduction
Alzheimer's disease (AD) is a neurodegenerative disease and the most common cause of
dementia affecting millions of elderly people. It is characterized by a decline in memory,
irreversible memory loss, or other cognitive skills that affects a person's daily activities.1 The
World Alzheimer Report 2015 showed that 46.8 million people have been suffering from AD and
this number is expected to exceed 131.5 million in 2050.2 The aetiology of AD remains elusive,
but several factors, such as β-amyloid (Aβ) deposits,3 τ-protein hyperphosphorylation,4 metal
dyshomeostasis,5 oxidative stress6 and low levels of acetylcholine,7 have been greatly implicated in
the AD pathogenesis. During the past decades, a considerable number of therapies, including anti-
cholinesterases, antioxidants, anti-inflammatories, anti-amyloid aggregation agents, metal chelates,
and anti-apoptotic approaches have been explored for strategies to treat AD.8-9 However, there is
still no effective treatment to prevent or reverse AD. Three FDA-approved anti-AD agents
(donepezil, galantamine and rivastigmine)10 could only provide a symptomatic, palliative
pharmacological effect, but do not cure AD patients since their effect is moderate and wears off
after a certain treatment time.11 Consequently, development of effective drugs is urgently needed.
Among the multiple factors of AD, most of the current treatments are based on the
cholinergic dysfunction hypothesis, which states that degeneration of cholinergic neurons and a
deficiency of the neurotransmitter acetylcholine (ACh) are the ultimate reasons for the loss of
memory and decline of cognitive function.12 It is well-known that two different cholinesterase
(ChE) enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), are responsible
for the hydrolysis of ACh within the brain.13 Therefore, administration of ChE inhibitors is a
useful approach to elevate ACh levels within the brain.14 Histochemically, AChE is mostly of
neuronal origin, while BuChE is mostly of glial origin.15 Under normal conditions, ACh is
dominantly decomposed by AChE instead of BuChE. Although BuChE is considered as a minor
role in regulating brain ACh level, it has been reported to correlate with drug metabolism and
detoxification. In AD, the AChE level in the brain decreases progressively, but BuChE activity
remains the same or increases up to 165% of the normal level.16 Therefore, BuChE is a proposed
drug target for treatment of the advanced stages of AD. Selective BuChE inhibition is potentially
advantageous because it may circumvent the classical cholinergic toxicity like nausea and
vomiting that is a common side effect of AChE inhibition.17 Therefore, development of selective

BuChE inhibitors may be a new trend in the treatment of late-stage AD patients.
Aβ peptides are probably the other most extensively studied because they are central to the
amyloid hypothesis.18 The main isoforms of the Aβ peptide are Aβ42 and Aβ40. Aβ40 is present in
larger amounts in the brain, yet Aβ42 is more neurotoxic and has a higher tendency to aggregate. A
common concept of the amyloid cascade hypothesis is that the aggregation of Aβ42 is the main
initiating event that sets off the pathogenic cascade, ultimately leading to neuronal loss and
dementia.19 So design of inhibitors aimed at halting aggregation or disrupting preformed
aggregates about Aβ is the study direction of many scientists.
Because of the complexity, AD has been described as a multifactorial disease, one single
drug that acts on a specific target to produce the desired clinical effects might not be suitable for
the complex nature of AD, and the development of multi-target-directed ligands (MTDLs) agents
to treat complex disease is the mainstream of the present research of AD treatment drugs.20-23
Designing of MTDLs widely employs synthetic hybrid compounds to integrate into a single
molecular framework consisting of at least two different pharmacophoric moieties.24 MTDLs may
be generated by combining fragments endowed with complementary properties, each fragment
synergistically contributing to the overall activity profile of the resulting molecule.
Natural products are valuable sources of novel leading compounds with bioactive
properties.25-27 β-Carbolines (pyrido [3,4-b] indoles) were first discovered in plants and were
referred to as harman alkaloids because they were first isolated from Pergamum harmala.28 β-
Carboline alkaloids possessed a wide range of pharmacological properties related to a variety of
neurological disorders. Several studies have shown that β-carbolines were effective inhibitors of
both AChE and butyrylcholinesterase (BuChE).29-31 The naturally occurring harmine (A) (Fig. 1)
was the first monoamine oxidase (MAO) inhibitor used for the treatment of Parkinson's disease
(PD).32 More recently, 9-methyl β-carboline (B) (Fig. 1) gained certain interests as
neuroprotective, neurorestorative, anti-inflammatory and cognitive enhancing drug.33-34 Besides,
β-carbolines derivatives, the specific inhibitors of dual-specificity tyrosine phosphorylation-
regulated kinase 1A (DYRK1A), were proved to inhibit the phosphorylation of tau protein on
multiple sites which associated with tau pathology in Alzheimer’s disease (AD).35-36 Furthermore,
the studies indicated that naturally occurring as well as the chemically synthesized β-carboline
analogs exhibited potent AChE inhibitory activity, especially bivalent β-carbolines with IC50

values in the nanomolar range for ChEs inhibition (C) (Fig. 1).31,37 These reports supported the
notion that β-carbolines could be considered as the another useful scaffold for AD drug design.
Figure 1. Chemical structures of β-carboline analogs (A) (B) and bivalent β-carbolines (C).
Considering the catalytic active site and the peripheral anionic site of the peculiar
architecture of the enzyme, Pang et al first reported that bis-tacrine (1) possessed both optimal
AChE inhibitory potency and AChE/BuChE selectivity than tacrine itself.38-39 The bis-
galanthamine linked by alkylene (2) was more potent inhibition than galanthamine.40 The
derivatives of bis-β-carboline (3) have also been reported.37 It was evident that the increased
affinities of inhibitors 1, 2 and 3 (Fig. 2) to AChE were presented by binding to the dual-site of
the enzyme.
Figure 2 Several bivalent molecules AChE inhibitors (1, 2 and 3).
In this paper, we synthesized bivalent β-carboline derivatives modified by several series of
hydrophobic moieties (Fig. 3) as potential neuroprotective agents for AD. β-Carboline containing
molecules with hydrophobic and aromatic moieties commonly have solubility problems. As
regards the spacer tether, the introduction of hydrophilic structure piperazine or ediamine might
reduce the hydrophobicity of the entire molecule to increase the solubility. In addition, a linking
tether bearing N atoms might have the chance to establish cation-π interaction with aromatic
residues lining the wall of the gorge in the enzyme.41 Therefore, we designed that piperazine or
ediamine sidearmed-alkane spacer connecting two β-carboline fragments might expect to enhance

the inhibitory activity on the enzyme. The potential anti-AD activity of these novel compounds
were evaluated by multiple biological analysis, including AChE and BuChE inhibition, Aβ1-42
disaggregation, protective effect against H2O2, okadaic acid (OA), Aβ1-42 induced SH-SY5Y cell
injury and cytotoxic effects on SH-SY5Y cells. Additionally, molecular modeling studies were
also performed to afford insight into the binding mode and the structure-activity relationships of
the novel bivalent β-carboline derivatives.
AChE and BuChE inhibitions
N2-bivalent β-carboline
N9-bivalent β-carboline
multifunctional agents for AD (a, b)
Figure 3. Design strategy for novel bivalent β-carboline derivatives
2. Results and discussion
2.1. Chemistry
The synthetic routes for the bivalent β-carboline derivatives are outlined in Schemes 1–3.
Two different series of bivalent β-carboline were synthesized: one series connected via the indole
nitrogen (N9-series) in Schemes 2 and the other with hydrophobic moieties between piperidine
nitrogen (N2-series) in Schemes 3. Some important intermediates monovalent β-carboline
derivatives listed in Schemes 1.
Using commercially available material L-tryptophan were esterified with methanol in the
presence of SOCl2 under room temperature condition to give 2.42 Intermediates 3a-c were

prepared in the presence of 5% trifluoroacetic acid by Pictet–Spengler cyclization, as previously
reported,43 according to described methods, intermediates 3b–c were oxidized with KMnO4 in
THF to obtain 4b–c (Scheme 1).44
To prepare bivalent derivatives 6b-i of the N9-series, the monovalent β-carboline 4b-c
reacted with 1,3-dibromopropane, 1,4-dibromobutaneto in DMF to give the intermediates 5b-e.
Finally, the target compounds 6b-i were obtained by the general reaction of compounds 5b-e with
piperazine or N,N'-dimethylethanediamine in the presence of anhydrous K2CO3 in CH3CN at
85 °C (Scheme 2).
To prepare bivalent derivatives 8a-j of the N2-series, the monovalent β-carbolines derivatives
7a-h provided a key intermediate for preparation of these derivatives of bivalent β-carbolines
derivatives in our research as shown in Scheme 3. Reaction of 2 equiv of 7a-h and 1 equiv of
piperazine or N,N'-dimethylethanediamine were carried out, in presence of potassium carbonate
(K2CO3) and potassium iodide (KI), to afford derivatives 8a-j.
Scheme 1. Reagents and conditions: (a) CH3OH and SOCl2, 0 °C to room temperature, 15 h; (b) CH2Cl2 and 5%
TFA, room temperature, 24 h; (c) KMnO4, THF, room temperature, 24 h;

Scheme 2. Reagents and conditions: (d) NaH, DMF, 1,3-dibromopropane or 1,4-dibromobutane, 0 °C to room
temperature, 5 h; (e) CH3CN, K2CO3, piperazine or N,N'-dimethylethanediamine reflux, 15-20 h;
Scheme 3. Reagents and conditions: (f) CHCl3, NaHCO3, chloroacetyl chloride, 3-chloropropionyl chloride or 4-
chlorobutyryl chloride, 0 °C to room temperature, 3 h; (g) piperazine or N,N'-dimethylethanediamine, K2CO3, KI,
CHCl3, reflux, 24 h.
2.2. In vitro inhibition of AChE and BuChE
AChE (E.C.3.1.1.7, Type VI-S, from Electric Eel) and BuChE (E.C.3.1.1.8, Type VI-S, from
equine serum) were employed to determine the ChEs inhibitory activity using the method of
Ellman assay,45 and donepezil, a well-known AChE inhibitor, was used as the positive control.
The AChE and BuChE inhibitory activities of the synthesized bivalent β-carboline derivatives
were listed in Table 1, Table 2, expressed as IC50 values.
These results revealed many compounds exhibited moderate inhibitory effect against BuChE
and displayed good selectivity for eqBuChE over eeAChE. The N9-bivalent β-carbolines (Table 1)
exhibited moderate to strong inhibitory effect against eqBuChE, and the target compounds were
more potential than the monovalent β-carbolines 4b, 4c, which was consistent with our design
strategy. The evaluation of the pharmacological results from the N9-bivalent β-carbolines revealed
that there was no substantial difference of the inhibitory activities between methyl and phenyl of
substituents at the pyridine residue, which linked bivalent β-carbolines in same number of atoms.
Among the N9-series, compounds (6b, 6g) with the chain of three carbon atoms showed
better inhibitory activity than those with the chain of four carbon atoms (6d, 6i). 6b containing
methyl group at 1-position performed the moderate potent inhibitory activity for BuChE (IC50 =

0.7 μM), which was 4 times stronger than those of the reference compounds donepezil (IC50 = 3.2
μM), 6g containing phenyl group at 1-position showed the moderate potent inhibitory activity for
BuChE and displayed high selectivity for BuChE (IC50 = 0.8 μM, SI: 183.8). Noticeably,
compound 6b contained a piperazine ring while 6g contained an ethylenediamine groups, which
implied that the special steric hindrance of 6g need some simple linkers.
The N2-bivalent β-carbolines showed moderate inhibitory properties for BuChE, and the
bivalent β-carbolines were more potential than the monovalent β-carbolines 3a, 3b, 3c.
Interestingly, compounds with the methyl groups of substituents at the pyridine residue was
slightly stronger for BuChE inhibition than those the phenyl and non-groups. Among the N2-series,
8f (IC50 = 1.7 μM) and 8g (IC50 = 2.8 μM) showed the moderate potent inhibitory activity for
BuChE.
The spacer tether in bivalent β-carbolines derivatives was different from other bis-molecules
inhibitors. The optimal tether length among our bivalent β-carbolines was a chain of 8-12 atoms,
which was longer than other bis-molecule AChE inhibitors.46-51 An intuitive reason inferred that
the β-carbolines molecule possessed the special steric hindrance, therefore the longer tether chain
was needed for optimal binding to BuChE.
Table 1
Inhibitory activities of AChE and BuChE by 4b, 4c, donepezil and bivalent β-carbolines.
Comp.
IC50 (μM)
AChEa
SI* (AChE/BuChE b)
Aβ1-42 aggregation
inhibition (%)c
BuChEa
0.7 ± 0.4
15.4 ± 6.0
9.7 ± 3.3
1.5 ± 0.7
n.a
6b
11.2 ± 5.1
99.1 ± 34.5
11.6 ± 3.9
9.6 ± 5.1
n.a
16.0
6.4
n.ad
6c
n.a
6d
1.2
n.a
6e
6.4
65.5 ± 1.6
n.de
6f
n.a
6g
147.1 ± 37.6
n.a
0.8 ± 0.3
n.a
183.8
n.a
25.4 ± 5.3
n.d
6h
6i
15.7 ± 4.3
n.a
1.8 ± 0.7
n.a
8.7
71.5 ± 0.7
n.d
4b
n.a
4c
n.a
n.a
n.a
n.d
donepezil
resveratrol
0.05 ± 0.01
n.d
3.2 ± 0.7
n.d
0.02
n.d
n.d
57.8 ± 5.2
a
Inhibitor concentration (mean ± SEM of three experiments) required for 50% inactivation of AChE、BuChE.
b
Selectivity index means IC50 (AChE)/IC50 (BuChE).

c
The thioflavin-T fluorescence method was used to evaluate the inhibiting ability of Aβ1-42 self-aggregation. The
values are expressed as the mean ± SD of at least three independent experiments. All experiments were performed
in the presence of 25 μM compounds.
d
n.a. Not active.
e
n.d. Not determined.
Table 2
Inhibitory activities of AChE and BuChE by 3a, 3b, 3c, donepezil and bivalent β-carbolines
Comp.
IC50 (μM)
AChE
SI*(AChE/BuChE)
Aβ1-42 aggregation
Inhibition (%)
BuChE
n.a
8a
8b
8c
8d
8e
8f
n.a
25.4 ± 13.0
n.a
n.a
0.9
n.a
n.d
27.0 ± 23.4
20.9 ± 5.8
9.0 ± 3.7
6.0 ± 3.7
1.7 ± 0.8
2.8 ± 0.7
14.6 ± 5.6
5.7 ± 1.9
n.a
59.2 ± 2.7
50.8 ± 6.6
72.3 ± 2.4
65.6 ± 1.3
82.7 ± 1.6
85.7 ± 1.7
65.1 ± 2.8
41.9 ± 5.9
n.d
39.8 ± 19.0
19.9 ± 5.1
18.0 ± 7.12
44.2 ± 13.2
64.0 ± 13.2
487.7 ± 36.5
n.a
4.3
3.3
10.6
15.8
4.4
85.6
n.a
8g
8h
8i
8j
3a
3b
3c
n.a
n.a
n.a
n.d
n.a
n.a
n.a
n.d
n.a
n.a
n.a
n.d
donepezil
resveratrol
0.05 ± 0.01
n.d
3.2 ± 0.7
n.d
0.02
n.d
n.d
57.8 ± 5.2
a
Inhibitor concentration (mean ± SEM of three experiments) required for 50% inactivation of AChE、BuChE.
b
c
Selectivity index means IC50 (AChE)/IC50 (BuChE).
The thioflavin-T fluorescence method was used to evaluate the inhibiting ability of Aβ1-42 self-aggregation. The
values are expressed as the mean ± SD of at least three independent experiments. All experiments were performed
in the presence of 25 μM compounds.
d
n.a. Not active.
e
n.d. Not determined.
2.3. Inhibition of Aβ1-42 self-aggregation by compounds
Thioflavin T (ThT) fluorescence assay was used to assess the ability of the selected
compounds to inhibit the self-induced Aβ1-42 aggregation. Resveratrol (Res) was used as positive
control (Table 1, Table 2 and Figure 4). From the results, we found that most of compounds

exhibited moderate or strong inhibitory activity of self-mediated Aβ1-42 aggregation ranging from
25.4% to 85.7% at 25 μM compared to resveratrol (57.8% at 25 μM). The result was shown in Fig.
4, in N9-series only compounds 6e and 6i exhibited significant inhibition of Aβ1-42 aggregation
(65.5% and 71.5%, respectively), while compound 8d-8h exhibited significant inhibition activity
(65.1% and 85.7%, respectively) in N2-series. The N2-bivalent β-carboline derivatives presented a
better inhibitory potency on self-induced Aβ1-42 aggregation with percentages of inhibition than
the N9-series. The result indicated the partial reduction in the piperidine ring and the two pyridine
nitrogens connections increased inhibition activities of Aβ1-42 aggregation.
Further, compound 6i with a phenyl substituent in the piperidine ring displayed the strongest
inhibitory activity (71.5%) in the N9-series, which was 1.23-fold better than resveratrol. In the N2-
series, compounds 8f and 8g showed excellent inhibition of Aβ1-42 aggregation with percentage
inhibition 82.7% and 85.7%, which was 1.43 and 1.48-fold better than resveratrol, respectively.
Noticeably, the most potent BuChE inhibitors (8f and 8g) were also proved to have the most
inhibition activities of Aβ1-42 aggregation in the N2-series. Therefore, structure-activity
relationship profiles were similar in both test systems.
Figure 4. Inhibition of self-induced Aβ1–42 aggregation by compounds comparing with that of resveratrol. The
thioflavin-T fluorescence method was used and the measurements were carried out in the presence of 25 μM test
compound. The mean ± SD values from three independent experiments were shown.
2.4. Cytotoxicity of synthetic compounds in SH-SY5Y cells
To determine the potential cytotoxic effects of synthesized novel compounds on neuronal cell
line SH-SY5Y, IC50 of compounds 6e, 6i, 8d, 8e, 8f and 8g for BuChE was less than 10 μM and
the inhibitory activity of Aβ1-42 aggregation was higher than 65% based on the results of the above

two experiments. Compounds 6b and 6g had the most potent BuChE inhibitory activity. Therefore,
these eight most potent compounds were selected, with different concentrations ranging from 1,
2.5, 5, 10 μM. After exposing the cells to these compounds for 24 h, the cell viability was
evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Fig. 5 showed
different effects of tested compounds on the viability of SH-SY5Y cells, and compounds 6b, 6e, 6i
and 8d proved to be neurotoxic in the concentration of 1-10 μM, while compounds 6g, 8e, 8f and
8g showed weak neurotoxicity, for instance, cell viability (%), 6g (1μM: 98.2 ± 1.0 %; 2.5 μM:
93.2 ± 1.4 %; 5 μM: 93.7 ± 0.7 %), 8e (1 μM: 103.1 ± 0.9 %; 2.5 μM: 99.9 ± 3.6 %; 5 μM: 102.0 ±
8.1 %; 10 μM: 90.9 ± 2.2 %), 8f (1 μM: 106.2 ± 92.8 %; 2.5 μM: 92.8 ± 1.3 %; 5 μM: 92.4 ±
0.9 %) and 8g (1 μM: 98.6 ± 0.9 %; 2.5 μM: 99.1 ± 0.7 %; 5 μM: 100.9 ± 1.7 %; 10 μM: 90.8 ±
4.2 %) . These results demonstrated that compounds 6g, 8e, 8f and 8g did not almost display the
cytotoxic effect on the cell at concentration of 1, 2.5, 5 μM.
Figure 5. Effect of test compounds on the viability of SH-SY5Y cells. The cells were incubated with the indicated
concentrations of the tested compounds for 24 h. The cell viability was assessed by MTT assay. Percentages of the
cell viability are presented as mean ± SD from 3 independent experiments
2.5. Neuroprotection against Aβ1-42-induced toxicity in SH-SY5Y cells
Aβ-induced apoptotic neuronal cell death is considered as a decisive episode in the pathology
of AD.52 To consolidate the results obtained from Aβ1-42 inhibition studies, further experiments
were conducted to assess the potential of 6g, 8e, 8f and 8g against Aβ1-42 induced
neurodegeneration in SH-SY5Y neuroblastoma cells, using a cell viability assay (Fig. 6A). A
marked decline in cell viability (54.2%, p < 0.0001) was observed after treatment with Aβ1-42 (30
μM) for 48 h, as compared to untreated control (Fig. S8A). Compounds 6g did not display the

neuroprotection effect (Fig. S9), but it was evident from the results that the other three compounds
imparted marked neuroprotection at the given concentrations (1 and 5 μM). Compound 8e
exhibited significantly neuroprotective effects at concentrations of 1 and 5 μM with cell viability
of 95.4% (p < 0.0001) and 85.4% (p < 0.001), respectively. While compounds 8f and 8g only
revealed a high effect at concentration of 1 μM (p < 0.001), when compared with the only Aβ1-42
(30 μM) treated control. Thus, it seemed reasonable to propose that neuroprotection against Aβ1-42
induced neurotoxicity shown by these compounds was due to their anti-Aβ aggregation action.
2.6. Neuroprotective effect against H2O2-induced cell damage in SH-SY5Y cells
The neuroprotective potential of the compounds on H2O2-induced oxidative stress in SH-
SY5Y cells were assessed by cell viability assay at two different concentrations 1 and 5 μM.
Treatment of cells with H2O2 (300 μM) (Fig. S8B) for 24 h, resulted in a marked decrease in cell
viability (57.2%, p < 0.001) as compared to untreated control. The co-treatment of cells with
compounds diminished H2O2-induced neurotoxicity. As showed in Fig. 6B, the selected
compounds exhibited neuroprotective effects at concentrations of 1 and 5μM compared with the
H2O2 only treated control, with compounds 8f showing the highest protective capability at 1 μM (p
< 0.001). Compound 8e and 8g showed a significant effective at 1 and 5 μM, with cell viability of
82.2% and 76.0%, 90.4% and 85.5%, respectively. Furthermore, compound 8f exhibited
significantly neuroprotective effects and the cell viability was 100.2%. When the concentration
was 5 μM, the cell viabilities decreased to 42.1%.
2.7. Neuroprotective effect against OA-induced cell damage in SH-SY5Y cells
Given that one of the most relevant molecular changes in AD is tau-phosphorylation, and
okadaic acid (OKA) is played a pivotal role in activating tau-phosphorylation.53 Thus, we assessed
the ability of these molecules to prevent cell death mediated by OKA on the human neuroblastoma
SH-SY5Y cells.As shown in Fig. S8C, the cell viability was decreased to 53.1% after treatment
with 15 nM OKA. The neuroprotective effect of 1 and 5μM concentrations of the selected
compounds, on cell death-induced by 15 nM OKA, was analyzed. Results in Fig. 6C demonstrate
that, compounds 8e, 8f and 8g were able to revert the cell death induced by OKA at 20 nM, which
causes 50-60% cell death in the absence of compound treatment. The most neuroprotective
compound were 8e and 8g showed a significant effect at the concentrations of 1 μM, with cell
viability of 97.1% and 100.8%, respectively. While 8f showed similar protective ability at the

concentration of 1 μM (p < 0.001) and 5 μM (p < 0.001).
Noticeably, in the three neuroprotective experiment, the three compounds at the
concentration of 1 μM generally showed better activity than that of 5 μM. We inferred that the
compounds may have interfered with some functions of the cells at 5 μM and therefore could not
successfully resist Aβ1-42, H2O2, OA-induced cell damage. At 1 μM concentration, the compounds
did not show any cytotoxicity and could fully exert their protective effect. Because in the
cytotoxicity experiment (Fig. 5), the compounds exhibited weak cytotoxicity at 5 μM, merely the
effect was not intuitive in the MTT experiment. Actually most compounds at 10 μM tended to
inhibit cell proliferation. However, this conjecture is further desired to be verified.
Figure 6. Determination of the viability of SH-SY5Y cells by the MTT assay after treatment with Aβ1-42 (A), H2O2
(B), OA (C) in the absence or presence of the indicated concentrations of (1 and 5 μM) compounds 8e, 8g and 8f.
All data are expressed as mean ± SEM of three experiments and each included triplicate sets. #p < 0.05, ##p < 0.01,
###p < 0.001, ####p < 0.0001 vs. control; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Aβ1-42, H2O2,
OA.
2.8. Molecular modeling study
To explore a possible interacting mode of compound 8f and 8g with the enzyme BuChE, the
crystal structure of the human BuChE inhibitor (PDB id: 4TPK) was taken for further molecular
modeling. A molecular modeling study was performed using CDOCKER within Discovery Studio
(DS, BiOVIA). As showed in Fig. 7, compound 8f contacted with the key residues Trp82 through
π-π stacking interaction and interacted with Trp82 through π-alkyl interactions. Besides, it formed
H-bond with Thr284 and His438 and interacted with Met437 and Ala328 through alkyl
interactions and also engaged in electrostatic interactions with residues Asp70. Compound 8g
formed H-bond stacking with Lys131 and contacted with Tyr61 and His126 through π-alkyl

stacking. Besides, it interacted with Leu29 through alkyl interactions and also engaged in π-cation
interaction with residues His126. But molecular docking of 8f or 8g with human AChE (PDB id:
4ey7) was failed. The reason may be that compounds 8f and 8g with a flexible linker are easily
folded as shown in Fig. 7. The folded molecules are suitable for relatively spherical and large
cavity of BuChE, but not stretched and slender enough to fit the narrow gorge of AChE.
Furthermore, the active sites of BuChE are a ∼20 Å deep gorge, which composed of the peripheral
anionic site (Asp70), the choline-binding pocket (Trp82), and the acyl-binding pocket (Trp231,
Leu286 and Val288), as well as the catalytic serine and histidine (Ser198 and His438). It should
be noted that the important interactions with 8f may be responsible for the better inhibitory for
BuChE.
Figure 7. Binding mode prediction of 8f (A) and 8g (B) with BuChE (PDB id: 4TPK). Compounds are shown in
paleturquoise stick mode; key residues are shown in spring green line modes. π-π stacking is depicted in yellow
dot line; π-alkyl and electrostatic interactions are in blue dot line; alkyl is in pink dot line; π-cation is in orange dot
line and H-bond is in green dot line.
3. Conclusion
In conclusion, a series of novel bivalent β-carboline derivatives were designed, synthesized,
and evaluated as multi-functional agent for therapy AD. In this study, we have shown that the
combination of two identical pharmacophores into a single molecule can unexpectedly result in
designed multiple ligands having new inhibitory activities against clinically relevant targets that
are not accessible by their monomeric analogues.
With the progress of AD, the AChE levels in the brain decreases progressively, but BuChE

activity increases up to 165% of the normal level, the inhibition of BuChE may be more effective
in AD.16 According to the data, all the target compounds showed fascinating inhibitory activity
against BuChE and high selectivity for eqBuChE over eeAChE, and part of compounds showed
promising Aβ1-42 disaggregation potential and neuroprotective activity in numerous in vitro studies.
Among them, compounds 8f and 8g appeared as most active and selective BuChE inhibitors,
which endowed with good Aβ1-42 disaggregation (82.7% and 85.7%). Furthermore, these
derivatives 8e, 8f and 8g exerted potential neuroprotective effect against H2O2, OA and Aβ-
induced cytotoxicity in SH-SY5Y cell line. This is the first report that bivalent β-carboline has
good activity in Aβ1-42 aggregation inhibition and neuroprotection for AD. Additionally,
derivatives 8e, 8f and 8g came out as nontoxic chemical entities in MTT assay which was
performed by using SH-SY5Y cells. Therefore, it can be concluded that 8f and 8g might be useful
as lead molecules for further development for AD.
Although the structure-activity relationships for BuChE/Aβ1-42 aggregation inhibition
activities and the molecular targets of these analogues remain to be fully elucidated, the findings
in this study have revealed a novel bivalent β-carboline analogues that exhibits strong beneficial
effects for AD treatment, which may provide a new avenue for drug design in treating AD. And
further investigations about evaluating compound 8f and 8g in vivo and developing structura
refinements are in progress and will be reported in due course.
4. Experimental section
4.1. Chemistry
All chemicals (reagent grade) used were purchased from Aladdin Shanghai Biochemical
Technology Co., Ltd. or Energy Chemical Reagent Co., Ltd. (China). Reaction progress was
monitored using analytical thin layer chromatography (TLC) on precoated silica gel GF254
(Qingdao Haiyang Chemical Plant, Qingdao, China) plates and the spots were detected under UV
light (λ=254 nm). Melting point was measured on an XT-4 micro melting point apparatus and
uncorrected.1H and 13C NMR spectra were measured on a Bruker 300 and 500 MHz spectrometer
at 298T and referenced to TMS. Chemical shifts were reported in ppm using the residual solvent
line as internal standard. Splitting patterns were designed as s (singlet), d (doublet), t (triplet), m
(multiplet). MS spectra data were obtained on an Agilent-6210 LC-MS spectrometer. Column
chromatography was performed on silica gel (200-300 mesh; Qingdao Marine Chemical Inc.)

4.1.1. General procedure for the preparation of methyl L-tryptophan 2
To a stirred solution of L-tryptophan (5.0 g, 24.5 mmol) in MeOH (250 mL) was added
SOCl2 (4mL, 55.1 mmol) dropwise at 0 °C over 1 h. The mixture was further stirred 12 h at room
temperature. MeOH was concentrated and poured enough diethyl ether to precipitate, collected by
filtration and washed with diethyl ether to get methyl L-tryptophan 2.
4.1.2. General procedure for the preparation of 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid
methyl esters (3a–c)
L-tryptophan methyl ester (1.0 g, 4.58 mmol), and aldehydes (formaldehyde for 3a,
acetaldehyde for 3b, benzaldehyde for 3c) were dissolved in CH2Cl2 (70 mL) and cooled to 0 °C
in an ice bath, To this solution, 5% triflouroacetic acid (TFA, 2 mL, 26.8 mmol) was added
dropwise and the mixture was stirred at room temperature for about 24 h under N2 atmosphere and
the complete reaction was monitored by TLC layer. The reaction mixture was then basified with
dilute NH4OH solution and extracted with CH2Cl2 (3 × 50 mL). The organic layer was washed
with water, brine, dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue
was purified using column chromatography on silica gel eluted with PE/EtOAc to obtain the
corresponding procedure.
4.1.2.1. 1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid methyl esters (3a).
L-tryptophan methyl ester was treated with formaldehyde (4 mL, 109 mmol) according to the
general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with PE/EtOAc (1:2) to obtain the product 3a as nigger-
brown solid in 63.5 % yield, m.p.: 215-216 °C. ESI-MS m/z: 231.1 [M+H]+; 1H NMR (300 MHz,
CDCl3) δ 8.00 (s, 1H), 7.48 (d, J = 7.8Hz, 1H), 7.32 (d, J = 7.8Hz, 1H), 7.15 (m, J = 8.4Hz, J =
6.9Hz, 2H), 4.17 (d, J = 9Hz, 1H), 3.80 (s, 3H), 3.13 (m, J = 4.8Hz, J = 4.2Hz, 1H), 2.97-2.89 (m,
1H), 2.18 (brs, 2H).
4.1.2.2. 1-Methyl-1,2,3,4-tetrahydro-β-carboline-3- carboxylic acid methyl esters (3b).
L-tryptophan methyl ester was treated with acetaldehyde (2 mL, 35.6 mmol) according to the
general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with PE/EtOAc (1:2) to obtain the product 3b as nigger-
brown solid in 62.5 % yield, m.p.: 218-219 °C. ESI-MS m/z: 245.0 [M+H]+; 1H NMR (300 MHz,
CDCl3) δ 8.12 (s, 1H), 7.50 (d, J = 7.2Hz, 1H), 7.30 (t, J = 7.2Hz, 1H), 7.15 (m, J = 6.9Hz, J =

7.8Hz, 2H), 4.27 (d, J = 6.6Hz, 1H), 3.84 (s, 6H), 3.17 (d, J = 6.3Hz, 1H), 2.90-2.80 (m, 1H), 2.06
(s, 2H), 1.52 (d, J = 6.6Hz, 3H).
4.1.2.3. 1- Phenyl-1,2,3,4-tetrahydro-β-carboline-3- carboxylic acid methyl esters (3c).
L-tryptophan methyl ester was treated with benzaldehyde (2 mL, 19.6 mmol) according to
the general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with PE/EtOAc (3:1) to obtain the product 3c as nigger-brown
solid in 48.4 % yield, m.p.: 215-216 °C. ESI-MS m/z: 307.01[M+H]+; 1H NMR (300 MHz, CDCl3)
δ 7.56 (d, J = 7.8Hz, 1H), 7.50 (s, 1H), 7.39 (s, 5H), 7.21 (s, 1H), 7.15 (t, J = 3.9Hz, 2H), 5.25 (s,
1H), 3.9 (m, J =3.9Hz, 1H), 3.84 (s, 3H), 3.49 (s, 1H),3.29-3.24 (m, 1H), 3.08-2.99 (m, 1H).
4.1.3. General procedure for the preparation of β-carboline-3-carboxylic acid methyl esters (4b
and 4c)
A suspension of the corresponding 1,2,3,4-tetrahydro-β-carboline-3- carboxylic acid methyl
esters (3b and 3c) (1 mmol) and 2.5 times KMnO4 (2.5 mmol) in anhydrous THF (100 mL) , the
mixture was stirred for 12 h at room temperature , remove the KMnO4 by filtration and evaporated
the THF under reduced pressure to give the corresponding procedure, the material was used
without further purification for the following steps.
4.1.3.1. 1-Methyl-β-carboline-3-carboxylic acid methyl ester (4b).
Intermediate 3b (1 g, 4.09 mmol) was oxidized according to the general procedure to obtain
the desired product 4b as white solid in 96.5% yield, m.p.: 250-252 °C. ESI/MS m/z: 241.1
[M+H]+; 1H NMR (300 MHz, CDCl3 ) δ 8.81 (s, 1H),8.58 (brs, 1H), 8.21 (d, J = 7.8Hz, 2H), 7.60
(m, 2H), 7.38 (m, 1H), 4.07 (s, 3H), 2.92 (s, 3H).
4.1.3.2. 1-Phenyl-β-carboline-3-carboxylic acid methyl ester (4c).
Intermediate 3c (1 g, 4.09 mmol) was oxidized according to the general procedure to obtain
the desired product 4c as white solid in 99.3% yield, m.p.: 254-255 °C. ESI/MS m/z: 303.1
[M+H]+; 1H NMR (300 MHz, CDCl3) δ 8.89 (s, 1H), 8.23 (d, J = 7.8Hz, 1H), 7.95 (d, J = 7.2Hz,
2H), 7.64-7.47 (m, 5H), 7.39 (t, J = 6.9Hz, 1H), 4.07 (s, 3H).
4.1.4. General procedure for the preparation of 9-bromoalkyl substituted β-carbolines (5b-e)
A mixture of β-carboline 4b and 4c (4 mmol) and anhydrous DMF (75 mL) was stirred at
room temperature for 20 min and 3 times NaH were added when cooled to 0 °C in an ice bath, The
mixture was stirred for 20 min, 2 times the appropriate alkylating reagent was added into the

mixture. The mixture was stirred at room temperature for 6h. After completion of the reaction as
indicated by TLC, the solution was poured into H2O (100 mL) and extracted with CH2Cl2. The
organic phase was washed with water and brine, then dried over anhydrous sodium sulfate, filtered
and evaporated. The resulting solid was purified by silica column chromatography with PE/EtOAc
as the eluent to successfully afford the desirable products.
4.1.4.1. 9-Bromopropane -1-methyl-β-carboline-3-carboxylic acid methyl ester (5b)
Intermediate 4b (200 mg, 0.83 mmol) was mixture with 1,3-dibromopropane(170 μL, 1.66
mmol) according to the general procedure to give the desired the residue, the residue was purified
using column chromatography on silica gel eluted with PE/EtOAc (2:1) to obtain the product 5b
as yellow oil in 54.8% yield. ESI/MS m/z: 385.1 [M+Na]+; 1H NMR (300 MHz, CDCl3) δ 8.75 (s,
1H), 8.16 (d, J = 7.8Hz, 1H), 7.59 (t, J = 8.4Hz, J = 7.2Hz, 1H), 7.42 (d, J = 8.4Hz, 1H), 7.34 (t, J
= 7.5Hz, 1H), 5.16 (m, 3H), 4.74 (t, J = 10.5Hz, J = 6.9Hz, 2H), 4.04 (s, 3H), 3.47 (t, J = 6Hz,
1H), 3.06 (s, 3H), 2.39 (t, J = 8.4Hz, J = 6.3Hz, 1H).
4.1.4.2. 9-Bromobutane -1-methyl-β-carboline-3-carboxylic acid methyl ester (5c)
Intermediate 4b was mixture with 1,4-dibromobutane (198 μL, 1.66 mmol) according to the
general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with PE/EtOAc (2:1) to obtain the product 5c as yellow oil in
54.4 % yield. ESI/MS m/z: 375.1 [M+H]+; 1H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H), 8.16 (d, J =
8.1Hz, 1H), 7.62 (t, J = 7.2Hz, 1H), 7.48(d, J = 8.4Hz, 1H), 7.34(t, J = 7.5Hz, 1H), 4.59 (t, J =
7.5Hz, 2H), 4.05 (s, 3H), 3.41 (t, J = 6.3Hz, 1H), 3.11(s, 3H), 2.05-1.94(m, 4H).
4.1.4.3. 9-Bromopropane -1-Phenyl -β-carboline-3-carboxylic acid methyl ester (5d)
Intermediate 4c (200 mg, 0.66 mmol) was mixture with 1,3-dibromopropane (134 μL, 1.32
mmol) according to the general procedure to give the desired the residue, the residue was purified
using column chromatography on silica gel eluted with PE/EtOAc (5:1) to obtain the product 5d
1
as light yellow solid in 69.6% yield, m.p.: 132-134 °C, ESI/MS m/z: 447.1 [M+Na]+; H NMR
(300 MHz, CDCl3) δ 8.94 (s, 1H), 8.16 (d, J = 7.8Hz, 1H), 7.66-7.58 (m, 3H), 7.55-7.48 (m, 3H),
7.45-7.37 (m, 2H), 4.66-4.54 (m, 2H), 4.05 (s, 3H), 3.61 (t, J = 6.6Hz, 1H), 2.89 (t, J = 6Hz, 1H),
2.42 (t, J = 6Hz, 1H), 1.89 (t, J = 6.6Hz, 1H).
4.1.4.4. 9-Bromobutane -1-Phenyl -β-carboline-3-carboxylic acid methyl ester (5e)
Intermediate 4e was mixture with 1,4-dibromobutane (158 μL, 1.32 mmol) according to the

general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with PE/EtOAc (5:1) to obtain the product 5e as light yellow
solid in 64.64 % yield, m.p.: 178-180 °C. ESI/MS m/z: 459.1 [M+H]+; 1H NMR (300 MHz,
CDCl3) δ 8.94 (s, 1H), 8.27 (d, J = 7.8Hz, 1H), 7.68-7.63 (m ,3H), 7.55-7.48(m, 4H), 7.42(d, J =
7.5Hz ,1H), 4.05 (s, 3H), 3.98 (t, J = 7.8Hz, 2H), 3.14 (t, J = 6.9Hz, 2H), 1.59 (t, J = 7.5Hz, 2H),
1.42-1.35 (m, 2H).
4.1.5. General procedure for the preparation of chloro-3,4-dihydro-1H -pyrido[3,4-b]indol-2(9H )-
yl)alkanone 3-carboxylic acid methyl ester (7a-h)
A mixture of Intermediate 3a, 3b and 3c and anhydrous CHCl3 (75 mL) was stirred at 0 °C in
an ice bath for 20 min, chloroacetyl/chloropropionyl/chlorobutyryl chloride was added dropwise
into the mixture. The mixture was stirred at room temperature under N2 for 6 h. After completion
of the reaction as indicated by TLC, the mixture was then diluted with CH2Cl2 washed with a
solution of NaHCO3, dried over anhydrous Na2SO4, filtered and evaporated. The resulting solid
was purified by silica column chromatography with PE/EtOAc as the eluent to successfully afford
the desirable products.
4.1.5.1 3-Chloro-3,4-dihydro-1H–pyrido[3,4-b]indol-2(9H)-yl) propan-1-one 3-carboxylic acid
methyl ester (7a)
Intermediate 3a (200 mg, 0.86 mmol) was mixture with 3-chloropropionyl chloride (124 μL,
1.30 mmol) according to the general procedure to give the desired the residue, the residue was
purified using column chromatography on silica gel eluted with CHCl2/ MeOH (50:1) to obtain
1
the product 7a as light yellow oil in 56.3% yield. ESI/MS m/z: 320.1 [M+Na]+. H NMR (300
MHz, CDCl3) δ 8.30 (s, 1H), 7.45 (d, J = 7.2Hz, 1H), 7.30 (d, J = 7.5Hz, 1H), 7.25 (m, J = 7.2Hz,
J = 7.5Hz, 2H), 5.45 (d, J = 6.9Hz, 1H), 4.87 (d, J = 6.5Hz, 1H), 3.95 (m, J = 6.6Hz, 2H), 3.78-
3.60 (m, 4H), 3.04 (m, J = 6.6Hz, J = 7.5Hz, 2H), 2.40 (s, 1H).
4.1.5.2 4-Chloro-3,4-dihydro-1H -pyrido[3,4-b]indol-2(9H)-yl) butan-1-one 3-carboxylic acid
methyl ester (7b)
Intermediate 3a was mixture with 4-chlorobutyryl chloride (149 μL, 1.30 mmol) according to
the general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with CHCl2/ MeOH (50:1) to obtain the product 7b as light
1
yellow oil in 60.3% yield. ESI/MS m/z: 357.1 [M+Na]+; H NMR (300 MHz, CDCl3) δ 8.34 (s,

1H), 7.54 (d, J = 5.4Hz ,1H), 7.30 (d, J = 7.2Hz, 1H), 7.18 (m, J = 7.2Hz, J = 8.1Hz ,2H), 5.69 (d,
J = 6.6Hz, 1H), 5.04 (d, J = 5.7Hz, 1H), 3.75-3.59 (m, 6H), 3.04-2.99 (m, 1H), 2.75 (t, J = 7.5Hz,
J = 6.9Hz, 1H), 2.57 (t, J = 7.2Hz, 1H), 2.28 (t, J = 6.6Hz, 2H), 2.09 (s, 1H).
4.1.5.3 2-Chloro-1-methyl-3,4-dihydro-1H -pyrido[3,4-b]indol-2(9H)-yl) ethanone 3-carboxylic
acid methyl ester (7c)
Intermediate 3b (200 mg, 0.82 mmol) was mixture with chloroacetyl chloride (97 μL, 1.23
mmol) according to the general procedure to give the desired the residue, the residue was purified
using column chromatography on silica gel eluted with CH2Cl2/ MeOH (50:1) to obtain the
product 7c as light yellow solid in 60.9% yield, m.p.: 175-176 °C, ESI-MS m/z:343.7 [M+Na]+;
1H NMR (300 MHz, CDCl3) δ 8.46 (s, 1H), 7.54 (d, J = 7.2Hz, 1H), 7.34 (d, J = 7.2Hz, 1H), 7.18
(t, J = 7.5Hz, 2H),5.58 (d, J = 5.4Hz, 1H), 4.96 (d, J = 5.4Hz, 1H),4.35 (d, J = 12.3Hz, 1H), 4.24
(d, J = 12.3Hz, 1H), 3.68 (s, 3H), 3.57 (s, 1H), 3.68 (d, J = 15.6Hz, 1H), 1.54 (m, J = 6.0Hz, 3H).
4.1.5.4 3-Chloro-1-methyl-3,4-dihydro-1H–pyrido[3,4-b]indol-2(9H)-yl) propan-1-one 3-
carboxylic acid methyl ester (7d)
Intermediate 3b was mixture with 3-chloropropionyl chloride (117 μL, 1.23 mmol) according
to the general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with CH2Cl2/ MeOH (50:1) to obtain the product 7d as light
yellow solid in 62.2% yield, m.p.: 145-147 °C, ESI-MS m/z: 357.8 [M+Na]+; 1H NMR (300 MHz,
CDCl3) δ 8.32 (s, 1H), 7.54 (d, J = 7.2Hz, 1H), 7.35 (d, J = 7.5Hz, 1H), 7.20 (m, J = 7.2Hz, J =
7.5Hz, 2H), 5.67 (d, J = 6.9Hz, 1H), 4.96 (d, J = 6.3Hz, 1H),3.96 (m, J = 6.6Hz, 2H), 3.73-3.56
(m, 4H), 3.04 (t, J = 6.6Hz, J = 7.5Hz, 2H), 1.80 (s, 1H), 1.52 (m, J = 6.6Hz, 3H).
4.1.5.5 4-Chloro-1-methyl-3,4-dihydro-1H -pyrido[3,4-b]indol-2(9H)-yl) butan-1-one 3-
carboxylic acid methyl ester (7e)
Intermediate 3b was mixture with 4-chlorobutyryl chloride (140 μL, 1.23 mmol) according to
the general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with CH2Cl2/ MeOH (50:1) to obtain the product 7e as light
1
yellow solid in 60.3% yield, m.p.: 158-160 °C, ESI-MS m/z: 347.2[M-H]-; H NMR (300 MHz,
CDCl3) δ 8.67 (s, 1H), 7.55 (d, J = 5.7Hz, 1H), 7.32 (d, J = 7.2Hz, 1H), 7.18 (m, J = 7.2Hz, J =
8.1Hz, 2H), 5.69 (d, J = 6.6Hz, 1H), 5.04 (d, J = 5.7Hz, 1H), 3.73-3.55 (m, 6H), 3.04-2.99 (m,
1H), 1.80 (s, 1H), 2.75 (t, J = 7.5Hz, J = 6.9Hz, 1H), 2.57 (t, J = 7.2Hz, 1H), 2.28 (t, J = 6.6Hz,

2H), 2.09 (s, 1H), 1.66 (dd, J = 6.6Hz, 3H).
4.1.5.6 2-Chloro-1-phenyl-3,4-dihydro-1H -pyrido[3,4-b]indol-2(9H)-yl) ethanone 3-carboxylic
acid methyl ester (7f)
Intermediate 3c (200 mg, 0.65 mmol) was mixture with chloroacetyl chloride(77 μL, 1.23
mmol) according to the general procedure to give the desired the residue, the residue was purified
using column chromatography on silica gel eluted with CH2Cl2/ MeOH (100:1) to obtain the
product 7f as light yellow solid in 71.22% yield, m.p.: 185-186 °C, ESI-MS m/z: 405.8[M+Na]+;
1H NMR (300 MHz, CDCl3) δ 8.16 (s, 1H), 7.62 (d, J = 6.9Hz, 1H), 7.30-7.17 (m, 7H), 6.99 (s,
1H),4.90 (d, J = 5.2Hz, 1H), 4.33 (dd, J = 12.3Hz, 2H), 3.70 (d, J = 15.9Hz, 1H), 3.24-2.19 (m,
1H), 2.98 (s, 4H).
4.1.5.7 3-Chloro-1-phenyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)propan-1-one3-carboxylic
acid methyl ester (7g)
Intermediate 3c was mixture with 3-chloropropionyl chloride (93 μL, 0.97 mmol) according
to the general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with CH2Cl2/ MeOH (100:1) to obtain the product 7g as light
1
yellow oil in 63.6 % yield. ESI-MS m/z: 419.8 [M+Na]+; H NMR (300 MHz, CDCl3) δ 8.18 (s,
1H), 7.62 (d, J = 6.6Hz, 1H), 7.27-7.21 (m, 7H), 7.06 (s, 1H), 4.83 (d, J = 6.3Hz, 1H), 3.9-3.66 (m,
3H), 3.13-2.94 (m, 6H).
4.1.5.8 4-Chloro-1-phenyl-3,4-dihydro-1H -pyrido[3,4-b]indol-2(9H)-yl) butan-1-one 3-
carboxylic acid methyl ester (7h)
Intermediate 3c was mixture with 4-chlorobutyryl chloride (112 μL, 0.97 mmol) according to
the general procedure to give the desired the residue, the residue was purified using column
chromatography on silica gel eluted with CH2Cl2/ MeOH (100:1) to obtain the product 7h as light
yellow oil in 61.5% yield, ESI-MS m/z: 409.2 [M-H]-; 1H NMR (300 MHz, CDCl3) δ 8.50 (s, 1H),
7.63 (d, J = 5.1Hz, 1H), 7.23-7.18 (m, 7H), 7.09 (s, 1H), 4.93 (d, J = 6Hz, 1H), 3.67 (s, 3H), 3.17-
3.05 (m, 2H), 2.90 (s, 2H), 2.70 (m, 2H), 2.23 (m, 2H).
4.1.6 General procedure for the preparation of N-9 substituted bivalent β-carbolines (6b-i)
4.1.6.1 Bis( 9-propane-1-methyl-β-carboline 3-carboxylic acid methyl ester)-piperazine (6b)
To a solution of intermediate 5b (200 mg, 0.55 mmol) and piperazine (23.85 mg, 0.27 mmol)
in CH3CN (20 mL), K2CO3 (191 mg, 1.38 mmol) were added. The mixture was refluxed for 20 h,

then cooled to rt, filtered, and evaporated. The residue was purified by silica gel flash column
chromatography with CHCl3/CH3OH (10:1) to afford 6b, as white solid in 25.13 % yield, m.p.:
219-220 °C. IR (KBr) ν 2925, 2811, 1735, 1623, 1555, 1433, 1350, 1261, 1188, 1137, 1056, 1006,
790, 753, 733, 637, 433 cm-1. 1H NMR (300 MHz, CDCl3) δ 8.86 (s, 2H), 8.21 (d, J = 7.2Hz, 2H),
7.62 (m, 2H), 7.38-7.28 (m, 2H), 4.07 (s, 6H), 3.17(s, 6H), 2.53-2.18 (m, 10H), 2.05-1.84 (m,
10H). 13C-NMR (125 MHz, CDCl3) δ 166.39 (2C), 142.31 (2C), 141.62 (2C), 137.01 (2C), 136.55
(2C), 129.13 (2C), 128.39 (2C), 121.87 (2C), 120.47 (2C), 116.56 (2C), 110.59 (2C), 55.57 (2C),
53.55 (2C), 52.74 (2C), 46.03 (2C), 42.99 (2C), 28.06 (2C), 23.87 (2C). HRMS (ESI) (m/z) calcd
for C38H42N6O4 [M+Na]+ 669.3268, found 669.3175.
4.1.6.2 N,N'-Dimethyl-bis(9-propane-1-methyl-β-carboline 3-carboxylic acid methyl ester)-
ethylenediamine (6c)
The derivative 6c was prepared from 5b and N,N'-dimethylethylenediamine(30 μL, 0.27
mmol) according to procedure 6b as yellow solid in 27.84 % yield, m.p.: 204-206 °C. IR (KBr) ν
2925, 2854, 1706, 1623, 1577, 1490, 1457, 1347, 1159, 1058, 816, 784, 749, 732, 634, 434 cm-1.
1H NMR (500 MHz, CDCl3) δ 8.81 (s, 2H), 8.20 (d, J = 7.5Hz , 2H), 7.63-7.57(m, 4H), 7.35 (t, J
= 7Hz, 1H), 7.31 (s, 1H), 4.68(t, J = 6.5Hz ,4H), 4.08(s, 6H), 3.17 (s, 6H), 2.47-2.45(m, 8H), 2.26
(s, 6H), 2.03 (t, J = 6.5Hz, 4H), 13C-NMR (125 MHz, CDCl3) δ 166.39 (2C), 141.94 (2C), 141.55
(2C), 136.92 (2C), 136.60 (2C), 133.53 (2C), 129.27 (2C), 128.68 (2C), 121.70 (2C), 120.69 (2C),
116.51 (2C), 110.28 (2C), 55.82 (2C), 54.93 (2C), 52.81 (2C), 43.15 (2C), 42.53 (2C), 28.69 (2C),
24.4 (2C). HRMS (ESI) (m/z) calcd for C38H44N6O4 [M+Na]+ 671.3424, found 671.3334.
4.1.6.3 Bis( 9-butane-1-methyl-β-carboline 3-carboxylic acid methyl ester)-piperazine (6d)
The derivative 6d was prepared from 5c (200 mg, 0.53 mmol) and piperazine (23 mg, 0.26
mmol) according to procedure 6b as yellow solid in 25.0% yield, m.p.: 178-180 °C. IR (KBr) ν
2951, 2811, 1720, 1623, 1488, 1475, 1340, 1305, 1253, 1157, 1123, 1061, 897, 787, 748, 730, 637,
436 cm-1. 1H NMR (300 MHz, CDCl3) δ 8.79 (s, 2H), 8.20 (d, J = 7.8Hz ,2H), 7.63 (t, J = 7.8Hz,
2H), 7.55 (d, J = 8.4Hz, 2H), 7.35(t, J = 7.5Hz ,2H), 4.06 (s, 6H), 3.15(s, 6H), 2.43-2.37 (m, 10H),
1.98-1.90(m, 4H), 1.71-1.62 (m, 10H). 13C-NMR (125MHz, DMSO) δ 166.35 (2C), 141.94 (2C),
141.80 (2C), 136.47 (2C), 136.22 (2C), 129.03 (2C), 128.52 (2C), 122.33 (2C), 121.24 (2C),
120.93 (2C), 116.43 (2C), 111.35 (2C), 79.75 (2C), 57.11 (2C), 53.44 (2C), 52.37 (2C), 44.59 (2C),
28.43 (2C), 23.71 (2C), 23.51 (2C). HRMS (ESI) (m/z) calcd for C40H46N6O4 [M+Na]+ 697.3581,

found 697.3479.
4.1.6.4 N,N'-Dimethyl-bis( 9-butane-1-methyl-β-carboline 3-carboxylic acid methyl ester)-
ethylenediamine (6e)
The derivative 6e was prepared from 5c and N,N'-dimethylethylenediamine(29 μL, 0.26
mmol) according to procedure 6b as yellow oil in 23.84% yield. IR (KBr) ν 2951, 2854, 1709,
1618, 1560, 1433, 1306, 1256, 1142, 1056, 892, 792, 756, 732, 631, 434 cm-1. 1H NMR (300 MHz,
CDCl3) δ 8.75 (s, 2H), 8.16 (d, J = 7.8Hz, 2H), 7.60 (t, J = 7.8Hz, 2H), 7.49 (d, J = 5.1Hz, 2H),
7.32(t, J = 7.8Hz, 2H), 4.57(t, J = 7.8Hz, 4H), 4.05 (s, 6H), 3.11(s, 6H), 2.37 (m, J = 6.9Hz, J
=9.9Hz, 8H), 2.18(s, 6H), 1.92-1.82 (m, 4H), 1.63-1.53 (m, 4H); 13C-NMR (75 MHz, CDCl3) δ
166.87 (2C), 142.19 (2C), 141.21 (2C), 137.62 (2C), 136.72 (2C), 128.87 (2C), 128.56 (2C),
121.67 (2C), 121.62 (2C), 120.69 (2C), 115.87 (2C), 110.41 (2C), 58.14 (2C), 55.34 (2C), 52.62
(2C), 45.45 (2C), 43.10 (2C), 29.03 (2C), 24.65 (2C), 23.57 (2C). HRMS (ESI) (m/z) calcd for
C40H48N6O4 [M+Na]+ 699.3737, found 699.3633.
4.1.6.5 Bis( 9-propane-1-phenyl -β-carboline 3-carboxylic acid methyl ester)-piperazine (6f)
The derivative 6f was prepared from 5d (200 mg, 0.47 mmol) and piperazine (20 mg, 0.23
mmol) according to procedure 6b as yellow solid in 13.7 % yield, m.p.: 174-176 °C. IR (KBr) ν
2948, 2880, 1706, 1675, 1556, 457, 1353, 1256, 1209, 1186, 1051, 1021, 756, 706, 621, 454 cm-1.
1H NMR (300 MHz, CDCl3) δ 8.94 (s, 2H), 8.25 (d, J = 7.5Hz, 2H), 7.64 (m, 6H), 7.51(m, 8H),
7.38 (t, J = 9.3Hz ,2H), 4.05 (s, 6H), 2.09 (m, 8H), 1.81 (m, 4H), 1.71(m, 4H), 1.50 (m, 4H). 13C-
NMR (125 MHz, CDCl3) δ 166.82 (2C), 161.01 (2C), 160.86 (2C), 160.69 (2C), 143.95 (2C),
142.32 (2C), 139.53 (2C), 136.98 (2C), 135.67 (2C), 139.65 (2C),130.41 (2C), 129.89 (2C),
128.87 (2C), 121.92 (2C), 121.71 (2C)，120.93 (2C), 116.90 (2C), 110.50 (2C), 57.42 (2C),
53.06 (2C), 45.68 (2C), 39.97 (2C), 26.80 (2C), 23.54 (2C). HRMS (ESI) (m/z) calcd for
C48H46N6O4 [M+Na]+ 793.3581, found 793.3492.
4.1.6.6 N,N'-Dimethyl-bis( 9-propane -1- phenyl -β-carboline 3-carboxylic acid methyl ester)-
ethylenediamine (6g)
The derivative 6g was prepared from 5d and N,N'-dimethylethylenediamine (25 μL,0.23
mmol) according to procedure 6b as yellow oil in 25.57% yield. IR (KBr) ν 2928, 2856, 1710,
1
1656, 1565, 1450, 1347, 1259, 1159, 1052, 1044, 775, 784, 745, 630, 443 cm-1. H NMR (500
MHz, CD3OD) δ 8.96 (s, 2H), 8.35 (d, J = 7.5Hz, 2H), 7.68-7.59 (m, 14H), 7.54(d, J = 3.5Hz, 2H),

4.01 (d, J = 6.5Hz, 6H), 3.26(t, J =6Hz, 2H), 3.11 (t, J = 6Hz, 2H), 2.94-2.77 (m, 4H), 2.3-2.2 (m,
4H), 2.06 (m, J=10Hz, J=3.5Hz, 4H), 1.96(s, 3H), 1.92(s, 3H). 13C-NMR (125 MHz, DMSO) δ
166.30 (2C), 143.99 (2C), 142.42 (2C), 139.69 (2C), 136.56 (2C), 135.44 (2C), 130.18 (2C),
129.78 (4C), 129.40 (4C), 129.10 (2C), 128.70 (2C), 122.60 (2C), 125.38 (2C), 121.22 (2C),
117.04 (2C), 111.59 (2C), 58.78 (2C), 55.26 (2C), 54.16 (2C), 52.05 (2C), 42.08 (2C), 26.21 (2C).
HRMS (ESI) (m/z) calcd for C48H48N6O4 [M+Na]+ 795.3737, found 795.3634.
4.1.6.7 Bis( 9-butane-1- phenyl -β-carboline 3-carboxylic acid methyl ester)-piperazine (6h)
The derivative 6h was prepared from 5e (200 mg, 0.47 mmol) and piperazine (19 mg, 0.22
mmol) according to procedure 6b as yellow solid in 13.6% yield, m.p.: 238-241 °C. IR (KBr) ν
2951, 2805, 1712, 1548, 1483, 1454, 1355, 1253, 1193, 1155, 1111, 769, 751, 704, 646, 449 cm-1.
1H NMR (300 MHz, CDCl3) δ 8.93 (s, 2H), 8.26 (d, J = 7.8Hz, 2H), 7.66-7.61 (m ,6H), 7.55-
7.49(m, 8H), 7.38(t, J = 7.8Hz ,2H), 4.01 (s, 6H), 3.95(t, J =7.8 Hz , 4H), 2.27 (m, 8H), 2.02(t, J =
7.5Hz, 4H), 1.46-1.36 (m, 4H), 1.08-0.98(m, 4H). 13C-NMR (125 MHz, DMSO) δ 167.12 (2C),
166.05 (2C) ,157.88 (2C), 144.07 (2C), 142.77 (2C), 139.64 (2C), 137.21 (2C), 132.37 (2C),
130.64 (2C), 129.51 (2C), 128.96 (2C), 128.82 (2C), 128.41 (2C), 128.20 (2C), 121.65 (2C),
120.76 (2C), 116.75 (2C), 111.39 (2C), 57.66 (2C), 54.55 (2C), 52.99 (2C), 57.36 (2C), 49.92
(2C), 42.49 (2C), 27.30 (2C). HRMS (ESI) (m/z) calcd for C50H50N6O4 [M+Na]+ 821.3894, found
821.3793.
4.1.6.8 N,N'-Dimethyl-bis( 9-butane-1- phenyl -β-carboline 3-carboxylic acid methyl ester)-
ethylenediamine (6i)
The derivative 6i was prepared from 5e and N,N'-dimethylethylenediamine (25 μL, 0.23
mmol) according to procedure 6b as yellow oil in 14.1% yield. IR (KBr) ν 2928, 2854, 1715, 1620,
1553, 1430, 1357, 1262, 1111, 1051, 788, 750, 701, 620, 443 cm-1. 1H NMR (300 MHz, CDCl3) δ
8.94 (s, 2H), 8.26 (d, J = 9.0Hz, 2H), 7.66-7.60 (m ,6H), 7.56-7.46 (m, 8H), 7.40 (m, J =8.0Hz, J
= 7.5Hz, 2H), 4.50 (t, J = 6.5Hz, 2H),4.07 (t, J = 4.5Hz, 6H), 4.38 (t, J = 6.5Hz, 2H) ,2.68-2.35 (m,
8H), 2.18-2.10 (m, 6H), 1.44-1.29 (m, 8H). 13C-NMR (125 MHz, CDCl3) δ 166.84 (2C), 166.16
(2C) ,144.22 (2C), 142.65 (2C), 139.42 (2C), 136.95 (2C), 135.60 (2C), 132.09 (2C), 129.65 (2C),
128.97 (2C), 128.93 (2C), 128.43 (2C), 128.37 (2C), 121.77 (2C), 120.95 (2C), 116.91 (2C),
111.09 (2C), 110.52 (2C), 57.24 (2C), 54.58 (2C), 52.55 (2C), 57.36 (2C), 44.42 (2C), 29.82 (2C),
26.11 (2C) . HRMS (ESI) (m/z) calcd for C50H52N6O4 [M+Na]+ 823.4050, found 823.3802.

4.1.7 General procedure for the preparation of N-2 substituted bivalent β-carbolines (8a-j)
4.1.7.1 Bis(1-(1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl ester) propan-1-one)-
piperazine (8a)
To a solution of intermediate 7a (200.00 mg, 0.62 mmol) and piperazine (26 mg, 0.311 mmol)
in CHCl3 (40 mL), K2CO3 (172 mg, 1.25 mmol) and KI (17.08 mg, 0.102 mmol) were added. The
mixture was refluxed for 24 h, then cooled to rt, filtered, and evaporated. The residue was purified
by silica gel flash column chromatography with CH2Cl2/CH3OH (10:1) to afford 8a, as white solid
in 20.5% yield, m.p.: 227-228 °C. IR (KBr) ν 2951, 2818, 1749, 1647, 1621, 1447, 1334, 1220,
1199, 1027, 1006, 891, 861, 748, 657 cm-1. 1H NMR (300 MHz, CDCl3) δ 8.44-8.34 (m, 2H), 7.52
(d, J = 7.5Hz, 2H), 7.33 (d, J = 7.5Hz, 2H), 7.15 (m, J = 7.5Hz, J = 7.2Hz, 4H), 5.91 (d, J =
5.4Hz ,1H), 5.61 (d, J = 5.4Hz ,1H), 5.24-5.09 (m, 1H) 4.95-4.87 (m, 1H), 3.65 (m, 6H), 3.54-3.45
(m, 2H), 3.39-3.30 (m, 4H), 3.19-3.03 (m, 2H), 2.66-2.43 (m,8H), 1.92 (brs, 2H). 13C-NMR (75
MHz, CDCl3) δ 171.73 (2C), 170.08 (2C), 136.47 (2C), 129.55 (2C), 128.44 (2C), 122.06 (2C),
119.63 (2C), 118.01 (2C), 111.48 (2C), 104.97 (2C), 62.22 (2C), 55.34 (2C), 52.86 (2C), 52.65
(2C), 52.49 (2C), 39.51 (2C), 24.18 (2C). HRMS (ESI) (m/z) calcd for C34H38N6O6 [M+H]+
627.2853, found 627.2927.
4.1.7.2 Bis(1-(1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl ester) butan-1-one)-
piperazine (8b)
The derivative 8b was prepared from 7b and piperazine according to procedure 8a as yellow
solid in 19.6% yield, m.p.: 207-210 °C. IR (KBr) ν 2950, 2817, 1745, 1647, 1614, 1451, 1327,
1
1219, 1202, 1027, 1004, 860, 748, 659 cm-1. H NMR (300 MHz, CDCl3) δ 8.56-8.37 (m, 2H),
7.51 (d, J = 7.2Hz, 2H), 7.32 (d, J = 7.8Hz, 2H), 7.15 (m, J = 8.4Hz, J = 7.2Hz, 4H), 5.27-5.19 (m,
1H), 4.43-4.38 (m, 1H), 3.60 (d, J = 7.5Hz, 6H), 3.50-3.43 (m, 2H), 3.14-3.00 (m, 2H), 2.83-2.61
(m,16H), 2.24 (brs, 4H).13C-NMR (75MHz, CDCl3) δ 172.01 (2C), 170.67 (2C), 135.73 (2C),
127.61 (2C), 125.82 (2C), 121.32 (2C), 119.53 (2C), 117.40 (2C), 110.83 (2C), 106.06 (2C),
54.76 (2C), 52.50 (2C), 50.30 (2C), 41.53 (2C), 38.67 (2C), 30.78 (2C), 29.55 (2C), 22.68 (2C).
HRMS (ESI) (m/z) calcd for C36H42N6O6 [M+H]+ 655.3166, found 655.3253.
4.1.7.3 Bis(1-(1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl ester) ethyl
ketone)-piperazine (8c)
The derivative 8c was prepared from 7c and piperazine according to procedure 8a as yellow

solid in 17.15% yield, m.p.: 238-239 °C. IR (KBr) ν 2931, 2850, 1741, 1654, 1459, 1436, 1310,
1274, 1222, 1201, 1165, 1048, 860, 746, 616, 491, 436 cm-1. 1H NMR (300 MHz, CDCl3) δ 8.48
(s, 1H), 8.41 (s, 1H), 7.53 (t, J = 7.5Hz, 2H), 7.33 (d, J = 7.5Hz, 2H), 7.15(t, J = 7.5Hz, 4H), 5.75-
5.28 (m, 2H), 3.70-3.48 (m, 8H), 3.40-3.00 (m, 6H), 2.70-2.40 (brs, 8H), 2.0(s, 2H), 1.72-1.52 (m,
6H). 13C-NMR (75 MHz, CDCl3) δ 171.63 (2C), 170.87 (2C), 136.78 (2C), 133.04 (2C), 126.42
(2C), 121.42 (2C), 119.19 (2C), 117.85 (2C), 110.69 (2C), 104.43 (2C), 62.86 (2C), 56.30 (2C),
53.50 (2C), 52.52 (2C), 47.53 (2C), 46.27 (2C), 22.67 (2C), 19.26 (2C). HRMS (ESI) (m/z) calcd
for C36H42N6O6 [M+H]+ 655.3166, found 655.3253.
4.1.7.4 N,N'-Dimethyl-bis(1-(1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl
ester) ethyl ketone)-ethylenediamine (8d)
The derivative 8d was prepared from 7c and N,N'-dimethylethylenediamine according to
procedure 8a as yellow solid in 21% yield, m.p.: 210-213 °C. IR (KBr) ν 2957, 2854, 1744, 1654,
1
1459, 1310, 1274, 1222, 1165, 1025, 746, 491, 436 cm-1. H NMR (300 MHz, CDCl3) δ 7.54-
7.46(m, 4H), 7.17-7.08 (m, 4H), 6.06-5.38 (m, 2H), 3.80 (s, 2H), 3.75-3.56 (m, 6H), 3.50-3.24 (m,
6H), 3.23-2.80 (m, 4H), 2.68-2.46 (m, 4H), 2.33 (s, 6H), 1.71-1.51 (m, 6H). 13C-NMR (75 MHz,
CDCl3) δ 172.31 (2C), 170.63 (2C), 135.92 (2C), 126.15 (2C), 121.42 (2C), 118.93 (2C), 117.70
(2C), 111.02 (2C), 106.59 (2C), 104.73 (2C), 54.12 (2C), 52.15 (2C), 51.61 (2C), 50.02 (2C),
48.27 (2C), 42.45 (2C), 22.30 (2C), 19.83 (2C). HRMS (ESI) (m/z) calcd for C36H44N6O6
[M+Na]+ 679.3322, found 679.3223.
4.1.7.5 Bis(1-(1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl ester) propan-1-
one)-piperazine (8e)
The derivative 8e was prepared from 7d and piperazine according to procedure 8a as yellow
solid in 18.6% yield, m.p.: 201-204 °C. IR (KBr) ν 2929, 2821, 1738, 1633, 1454, 1310, 1272,
1222, 1163, 1088, 796, 745, 431 cm-1. 1H NMR (300 MHz, CDCl3) δ 8.92-8.22 (m, 2H), 7.52 (t, J
= 6.9Hz, 2H), 7.34 (d, J = 7.2Hz, 2H), 7.15 (t, J = 6.9Hz, 4H), 5.40-4.90 (m, 2H), 3.70-3.50 (m,
8H), 3.35-3.04 (m, 4H), 2.93-2.46 (m, 16H), 1.65 (d, J = 6Hz ,3H), 1.53 (d, J = 6Hz, 3H). 13C-
NMR (75 MHz, CDCl3) δ 172.02 (2C), 171.25 (2C), 135.93 (2C), 134.60 (2C), 126.10 (2C),
121.63 (2C), 119.38 (2C), 118.01 (2C), 110.84 (2C), 104.62 (2C), 53.50 (2C), 52.53 (2C), 51.57
(2C), 49.75 (2C), 47.32 (2C), 28.97 (2C), 22.02 (2C), 21.30 (2C), 19.04 (2C). HRMS (ESI) (m/z)
calcd for C38H46N6O6 [M+H]+ 683.3479, found 683.3567.

4.1.7.6 N,N'-Dimethyl-bis(1-(1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl
ester) propan-1-one)- ethylenediamine (8f)
The derivative 8f was prepared from 7d and N,N'-dimethylethylenediamine according to
procedure 8a as yellow solid in 22% yield, m.p.: 174-176 °C. IR (KBr) ν 2951, 2916, 2850, 1738,
1
1639, 1434, 1310, 1271, 1203, 1162, 1022, 747, 604, 431 cm-1. H NMR (300 MHz, CDCl3) δ
7.42 (t, J = 7.2Hz, 4H), 7.11 (t, J = 7.2Hz, 4H), 5.51-4.90 (m, 2H), 4.36 (s, 1H), 4.19(s, 1H), 3.70-
3.59 (m, 4H), 3.47 (s, 6H), 3.05-2.60 (m, 14H), 2.50 (d, J = 11.1Hz, 2H), 2.30 (t, J = 11.7Hz, J =
6.6Hz, 2H), 1.67-1.27 (m, 6H). 13C-NMR (75 MHz, CDCl3) δ 178.97 (2C), 172.03 (2C), 136.18
(2C), 126.44 (2C), 121.43 (2C), 118.92 (2C), 117.32 (2C), 110.71 (2C), 105.69 (2C), 103.47 (2C),
53.52 (2C), 51.88 (2C), 49.68 (2C), 48.15 (2C), 45.91 (2C), 41.54 (2C), 30.22 (2C), 22.33 (2C),
19.17 (2C). HRMS (ESI) (m/z) calcd for C38H48N6O6 [M+H]+ 685.3635, found 685.3731.
4.1.7.7 Bis(1-(1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl ester) butan-1-
one)-piperazine (8g)
The derivative 8g was prepared from 7e and piperazine according to procedure 8a as yellow
solid in 19.1% yield, m.p.: 162-165 °C. IR (KBr) ν 2947, 2817, 2245, 1739, 1631, 1453, 1308,
1270, 1202, 1161, 1008, 733, 430 cm-1. 1H NMR (300 MHz, CDCl3) δ 8.75-8.48 (m, 2H), 7.49 (d,
J = 6.9Hz, 2H), 7.31 (d, J = 7.5Hz, 2H), 7.12 (t, J = 7.5Hz, 4H), 5.37-4.99 (m, 2H), 3.71-3.47 (m,
8H), 3.32-2.88 (m, 4H), 2.60-2.13 (m, 20H), 1.62 (m, 3H), 1.49 (t, J = 7.2Hz ,3H). 13C-NMR
(125MHz, CDCl3) δ 175.53 (2C), 171.62 (2C), 136.22 (2C), 133.40 (2C), 129.56 (2C), 128.16
(2C), 126.36 (2C), 120.02 (2C), 118.21 (2C), 111.47 (2C), 57.64 (2C), 56.84 (2C), 54.16 (2C),
52.35 (2C), 51.65 (2C), 32.23 (2C), 30.12 (2C), 24.08 (2C), 22.70 (2C), 21.58 (2C). HRMS (ESI)
(m/z) calcd for C40H50N6O6 [M+H]+ 711.7192, found 711.3878.
4.1.7.8 N,N'-dimethyl-bis(1-(1-phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl
ester) ethyl ketone)-ethylenediamine (8h)
The derivative 8h was prepared from 7f and N,N'-dimethylethylenediamine according to
procedure 8a as yellow solid in 24.54% yield, m.p.: 173-175 °C. IR (KBr) ν 2951, 2854, 1735,
1
1647, 1462, 1308, 1272, 1238, 1059, 899, 741, 710, 434 cm-1. H NMR (300 MHz, CDCl3) δ
7.56-6.92 (m, 20H), 5.65-5.32 (m, 1H), 4.69-4.43 (m, 1H), 3.64-3.43 (m, 6H), 3.29-2.95 (m, 8H),
2.62-2.52 (m, 2H), 2.29 (s, 3H), 2.20 (s, 3H), 1.77 (s, 2H). 13C-NMR (125 MHz, CDCl3) δ 188.02
(2C), 170.4 (2C), 136.57 (2C), 136.50 (2C), 129.30 (2C), 128.73 (2C), 127.91 (2C), 127.06 (2C),

126.89 (2C), 122.73 (2C), 121.90 (2C), 119.46 (2C), 118.45 (2C), 118.19 (2C), 111.90 (2C),
110.99 (2C), 54.40 (2C), 53.36 (2C), 52.47 (2C), 51.94 (2C), 42.71 (2C), 25.99 (2C), 21.22 (2C).
HRMS (ESI) (m/z) calcd for C46H48N6O6 [M+N]+ 803.3635, found 803.3547.
4.1.7.9 Bis(1-(1-phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl ester) propan-1-
one)-piperazine (8i)
The derivative 8i was prepared from 7g and piperazine according to procedure 8a as yellow
solid in 18.2% yield, m.p.: 225-227 °C. IR (KBr) ν 2928, 2850, 1646, 1624, 1459, 1420, 1308,
1272, 1238, 1215, 1009, 899, 741, 710, 537, 495 cm-1.1H NMR (300 MHz, CDCl3) δ 8.16 (s, 2H),
7.60 (d, J = 7.8Hz, 2H), 7.29-7.27 (m, 4H), 7.23-7.14 (m, 12H), 7.09 (s, 2H), 4.91 (d, J = 6Hz,
2H), 3.65 (d, J = 15.9Hz, 2H), 2.95 (s, 6H), 2.82 (d, J = 6Hz, 4H), 2.73 (d, J = 6Hz, 4H), 2.53 (brs,
8H), 1.94 (brs, 4H). 13C-NMR (125 MHz, CDCl3) δ 176.05 (2C), 172.01 (2C), 170.91 (2C),
139.45 (2C), 136.47 (2C), 130.26 (2C), 129.36 (2C),128.45 (2C), 128.19 (2C), 128.00 (2C),
126.51 (2C), 122.42 (2C), 119.74 (2C), 118.61 (2C), 110.98 (2C), 107.66 (2C), 54.02 (2C), 53.24
(2C), 52.74 (2C), 51.98 (2C), 51.14 (2C), 31.88 (2C), 29.79 (2C), 21.22 (2C). HRMS (ESI) (m/z)
calcd for C48H50N6O6 [M+Na]+ 829.3792, found 829.3717.
4.1.7.10 Bis(1-(1-phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl ester) butan-1-
one)-piperazine (8j)
The derivative 8j was prepared from 7h and piperazine according to procedure 8a as yellow
solid in17.2% yield, m.p.: 243-246 °C. IR (KBr) ν 2954, 2854, 1737, 1654, 1452, 1311, 1275,
1
1240, 1165, 1025, 1012, 746, 704, 503, 434 cm-1. H NMR (300 MHz, CDCl3) δ 8.71 (s, 2H),
7.50-7.09 (m, 16H), 6.35 (s, 2H), 5.14 (s, 2H), 3.58 (s, 6H), 3.23 (d, J = 15.3Hz, 2H), 2.73-2.21
(m, 18H), 1.84-1.73(m, 4H). 13C-NMR (125 MHz, CDCl3) δ 175.53 (2C), 171.62 (2C), 141.56
(2C), 136.22 (2C), 133.40 (2C), 129.56 (2C), 128.16 (2C), 126.36 (2C), 122.33 (2C), 120.02 (2C),
118.21 (2C), 111.47 (2C), 57.64 (2C), 56.84 (2C), 56.42 (2C), 54.16 (2C), 52.35 (2C), 51.65 (2C),
32.23 (2C), 30.12 (2C), 24.08 (2C), 22.70 (2C), 21.58 (2C). HRMS (ESI) (m/z) calcd for
C50H54N6O6 [M+H]+ 835.4105, found 835.4185.
4.2. Biological evaluation
4.2.1. Cholinesterase inhibition assay in vitro
AChE (E.C.3.1.1.7, Type VI-S, from Electric Eel) and BuChE (E.C.3.1.1.8, Type VI-S, from
equine serum), 5,5’-dithiobis-2-nitrobenzoic acid (Ellman's reagent, DTNB), acetylthiocholine