Synthesis and biological evaluation of new cholinesterase inhibitors for Alzheimer's disease

molecules

Article

Synthesis and Biological Evaluation of New

Cholinesterase Inhibitors for Alzheimer’s Disease

Weiam Hussein ^1,2, Begüm Nurpelin Sag˘lık ^2,3, Serkan Levent ^2,3, Büs¸ra Korkut ⁴, Sinem Ilgın ⁴,

Yusuf Özkay ^2,3,

*

and Zafer Asım Kaplancıklı ²

ID

1

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Aden University, 6075 Aden, Yemen;

white.wf.rose@gmail.com

2

3

4

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey;

bnsaglik@anadolu.edu.tr (B.N.S.); serkanlevent@anadolu.edu.tr (S.L.); zakaplan@anadolu.edu.tr (Z.A.K.)

Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University, 26470

Eskis¸ehir, Turkey

Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir,

Turkey; busrakorkut@anadolu.edu.tr (B.K.); silgin@anadolu.edu.tr (S.I.)

Correspondence: yozkay@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 3779)

*

Academic Editor: Praveen P. Nekkar Rao

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Received: 29 July 2018; Accepted: 12 August 2018; Published: 14 August 2018

Abstract: Alzheimer’s disease (AD) is a neurodegenerative disorder mostly inﬂuencing the elderly,

and causes death due to dementia. The main pathogenic feature connected with the progression of

this multifactorial disease is the weakening of the cholinergic system in the brain. Cholinesterase

(ChE) inhibitors are recognized as one of the choices in the treatment of AD. The inhibition of

acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were approved as a therapeutic

strategy to reduce the symptoms of AD and prevent its progression. The capacity of BChE is not

completely known yet; rather, it is accepted to assume a part in a few disorders such as AD. Thus,

BChE inhibitors may have a greater role for the treatment of AD in the future. In the present study,

2-(9-acridinylamino)-2-oxoethyl piperazine/piperidine/morpholinecarbodithioate derivatives were

synthesized in order to investigate anticholinesterase activity. Eight derivatives demonstrated a

speciﬁc and promising action against BChE. Furthermore, compound 4n showed inhibitory activity

against both enzymes. It was found that the active compounds were well tolerated in the cytotoxicity

test. Possible interactions between the lead compound, 4n, and the BChE enzyme were determined

through a docking study. The ﬁndings obtained within this paper will contribute to the development

of new and effective synthetic anti-Alzheimer compounds, and will ideally encourage future screening

against AD.

Keywords: Alzheimer’s disease; acetylcholinesterase; butyrylcholinesterase; 9-aminoacridine;

dithiocarbamate salts; docking study

1. Introduction

Alzheimer’s disease (AD) is a neurological disorder and is one of the most common causes of

dementia in the age group above 60 years, for which there is no radical cure. It is estimated from the

world population that more than 35.6 million people are currently living with AD, and this may increase

to 65.7 million by 2030, and 115.4 million by 2050 [1]. According to a World Health Organization

(WHO) report, more than 50% of people with AD live in the developing world, and it is estimated

that this population will be 70% by 2025 [2]. To date, some cases of low levels of acetylcholine (ACh),

oxidative anxiety, dyshomeostasis of biometals, and amyloid-

β (Aβ) stores were exhibited to be related

to AD pathogenesis [ ]. In light of these ﬁndings, a few speculations were proposed to clarify the

3

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Molecules 2018, 23, 2033

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progress of AD. The most recognized theory includes the loss of cholinergic neurons in the cerebrum of

patients with AD, and a reduced action of choline acetyltransferase, which catalyzes the generation of

ACh. Thus, cognitive dysfunction occurs and neurotransmission levels decrease [

4]. In AD, although

AChE is generally the primary medication target, recent investigations were additionally centered on a

scan for BChE inhibitors [

toward the formation of A

to “damaging” plaques [

5

β

–

8

]. One of the most interesting studies showed that, when BChE progresses

plaques, its advanced role involves the conversion of “harmless” plaques

9

]. Scientists additionally discovered a relationship between a less active

variant of BChE (Ala539Thr) and a lower inclination toward growing AD [10]. Therefore, BChE

represents a proper imaging target for early diagnosis and treatment of AD. In this way, new remedial

approaches are presently in progress, and exploratory medicine surrounding this approach sped up

during the recent decade.

Carbamates are the most generally contemplated class of AChE inhibitors, on which impressive

research was performed in connection to AD. Rivastigmine, a dual AChE and BChE inhibitor,

is one of the most broadly utilized anticholinesterase agents bearing a carbamate moiety [11,12].

Dithiocarbamates have a great deal of interest in the design of new compounds, which can be obtained

via the bioisosteric substitution of a carbamate moiety with a dithiocarbamate moiety. They are critical

pharmacophores due to their lipophilicity, which is essential for the transport of drugs to the central

nervous system (CNS) through the blood–brain barrier (BBB) [13,14]. Tacrine and donepezil are other

anticholinesterase agents that selectively inhibit BChE and AChE, respectively. The use of tacrine

is limited as it may cause an increase in the level of serum aminotransferase, which is connected to

intense liver harm. On the other hand, donepezil is a more favorable AChE inhibitor since it has

some advantages such as non-hepatotoxicity, and the lowest number of side effects when consumed

once daily [15]. Thus, several studies on compounds structurally related to tacrine were performed to

discover new BChE inhibitors with reduced side effects [16–23]

It was reported that anticholinesterase agents ought to feature a central ring, a basic region,

and a suitable connection between the central ring and basic region, such as O, CH₂, CONH,

and CONH(CH₂)n, to satisfy the main structural necessity for active and strong enzyme inhibitors [15].

In addition, the bulky moieties were reported to possess good anti-BChE activity, which may be related

to the ability of BChE to ideally bind with bigger substituents, as its structure is composed of a more

open core [24]. Furthermore, there are no reported data on 9-aminoacridine and its acetylated analogs as

anticholinesterase inhibitors until this study. Based on this empirical knowledge, we aimed to improve

BChE inhibitory activity via the design of new compounds bearing the following structural properties:

9-aminoacridine as the central ring, heterocyclic rings such as piperazine, piperidine, or morpholine

as the basic region, and ﬁnally, a (thiocarbamoylthio)acetamide moiety as the connection between

the central ring and the basic region (Figure 1). Thus, the aim was to synthesize new hybrids of

9-aminoacridine and dithiocarbamates analogs as anticholinesterase agents.

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O

N

O

N

O

NH₂

Donepezil

Tacrine

N

S

R

NH

S

N

O

(A center ring/9-aminoacridine)

Tacrine like moiety

(A basic part)

Donepezil like moiety

(The connection)

(thiocarbamoylthio)acetamide part which

may enhance AChE inhibiton activity

Figure 1. Design strategy of recently synthesized derivatives.

2. Results and Discussion

2.1. Chemistry

The newly synthesized derivatives (4a

–

4u) were obtained by taking advantage of the three-step

protocol as outlined in Scheme 1 . The treatment of N-(9-acridinyl)-2-chloroacetamide derivatives (

2)

with the appropriate sodium N-substituted piperazine/piperidine/morpholine dithiocarbamates

(3) gave the target compounds with yields of 81.0–97.0%. The structures of all synthesized

compounds were elucidated using infrared (IR), high-resolution mass spectrometry (HRMS), ¹H-NMR,

and ¹³C-NMR spectroscopic methods. All compounds indicated logical, analytical, and spectroscopic

information strongly correlating with their structures.

2.2. Enzymatic Inhibition

The results of biological assays were evaluated to understand the inhibitory potency of the newly

synthesized compounds (4a

Electrophorus electricus and human cells, and BChE from equine and human cells). In order to acquire

effective knowledge about structure activity relationship (SAR), compounds 4a 4u were divided

into two classes as derivatives containing piperazine (4a 4q) and morpholine or piperidine (4r 4u).

The assay was performed in two steps. Firstly, compounds 4a

4u were tested at 10⁻³and 10⁻⁴

–4u) against cholinesterase enzymes of different species (AChE from

–

M

concentrations. The second step was performed by using 10⁻⁵–10⁻⁹M concentrations of selected

compounds that indicated more than 50% inhibitory activity at the initial concentrations. It was clearly

observed that similar results were obtained against both species of AChE and BChE. The reason for

this similarity was thought to be high degree of homology in the enzymes of the studied species

(Supplementary Materials and Table 1).

Our in-depth analysis showed that the incorporation of a 9-aminoacridine moiety into the

designed compounds contributed weakly to the AChE inhibitory activity; however, it strongly

increased the inhibitory activity toward BChE (Supplementary Materials and Table 1).

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O

a

N

Cl

NH

2

NH₂

Cl

O

1

S

N

R

Na

S

N

S

3

b

R

NH

S

N

O

4a-4u

Comp.

R

Comp.

R

4a

4b

4c

4d

4e

4f

4g

4h

4i

Piperazinyl

4-(2-(dimethylamino)ethyl

4-(3-(dimethylamino)propyl

4-ethyl

4-(4-nitrophenyl)

4-(2-hydroxyethyl)

4-phenyl

4l

4m

4n

4o

4p

4q

4r

4s

4t

4u

Piperazinyl

Morpholinyl

Piperidinyl

4-cyclohexyl

4-benzhydryl

4-[4-(triﬂuoromethyl)benzyl]

4-(4-methylbenzyl)

4-(4-ﬂuorophenyl)

4-(2-furoyl)

4-methyl

-

4-(4-methoxyphenyl)

4-(4-chlorophenyl)

4-(pyrimidin-2-yl)

4-benzyl

2-methyl

4-benzyl

4j

4k

◦

Scheme 1. Synthesis of compounds 4a–4u. (a): (C₂H₅)₃N/THF/0 C then rt; (b) acetone/reﬂux.

Compounds in the ﬁrst class (4a

–

4q) offered inhibitory activity with an IC₅₀ranging from

4b 4e 4i 4m 4n, and 4o possessing piperazine

0.014 to 2.097 M against BChE. Compounds 4a

µ

,

with 2-dimethylaminoethyl, 3-dimethylaminopropyl, 2-hydroxyethyl, 4-chlorophenyl, 4-benzhydryl,

4-(triﬂuoromethyl)benzyl, and 4-methylbenzyl respectively, showed more potent BChE inhibitory

activity than other derivatives in the series. Furthermore, the most active compound, 4m (IC₅₀:

0.092

µM), exhibited 15.4-fold better BChE inhibition in comparison with the positive control, donepezil

(IC₅₀: 1.419

µM). In the second class of compounds, the replacement of piperazine with its bioisosters,

namely morpholine (4r) and piperidine (4s, 4t, and 4u), did not signiﬁcantly enhance the activity

against either AChE or BChE.

In terms of SAR, it can be concluded that the variation of secondary amines such as piperazine,

piperidine, and morpholine does not affect the enzyme inhibitory potency of the ﬁnal compounds.

However, the substituents on these cyclic amines are mainly responsible for the biological activity.

Although the structures of the compounds show great similarities, the presence of triﬂuoromethyl

in compound 4n signiﬁcantly inﬂuenced the activity. The better inhibitory proﬁle of 4n than

aminoacridine (Table 1) also supports this ﬁnding. Thus, it can be suggested that the triﬂuoromethyl

group possesses the ability to form interactions in the enzyme’s active site.

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−4

–4u against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes at 10 M concentration and

Table 1. Inhibitory activity (%) of compounds 4a

their half maximal inhibitory concentration (IC₅₀; µM) values against these enzymes.

AChE (Electrophorus electricus)

(%) ± SD

AChE (Human)

BChE (Equine)

BChE (Human)

(%) ± SD

−4

10 M

Compound

IC₅₀(µM)

10

M

10

M

10

M

4a

17.03 ± 0.48

32.41 ± 1.01

5.11 ± 0.09

31.64 ± 0.45

7.09 ± 0.64

4.70 ± 0.27

10.44 ± 0.66

15.23 ± 0.69

35.91 ± 1.57

2.88 ± 0.18

8.62 ± 0.22

3.90 ± 0.09

25.43 ± 1.21

* 63.21 ± 1.76

25.30 ± 1.01

48.40 ± 1.18

16.35 ± 0.39

37.33 ± 1.03

32.17 ± 1.23

15.94 ± 0.42

25.88 ± 0.74

75.18 ± 1.15

98.56 ± 2.87

97.29 ± 3.24

-

18.15 ± 0.51

35.49 ± 1.00

10.28 ± 0.22

34.71 ± 0.77

10.13 ± 0.51

8.71 ± 0.62

11.30 ± 0.58

18.39 ± 0.57

33.85 ± 1.15

5.97 ± 0.22

7.21 ± 0.18

7.29 ± 0.18

27.38 ± 1.10

* 68.39 ± 1.99

30.45 ± 1.15

49.51 ± 1.50

20.28 ± 0.51

40.70 ± 1.00

35.20 ± 1.12

14.51 ± 0.44

30.71 ± 0.52

78.22 ± 0.89

96.95 ± 2.14

98.79 ± 2.63

-

* 91.13 ± 2.94

* 82.50 ± 2.07

22.70 ± 0.70

40.19 ± 0.49

* 95.98 ± 2.88

12.80 ± 0.87

33.65 ± 0.89

12.64 ± 0.46

* 69.51 ± 1.16

27.86 ± 1.07

25.14 ± 1.42

45.88 ± 1.62

* 98.96 ± 3.47

* 99.03 ± 3.26

* 74.83 ± 2.17

48.65 ± 1.14

39.90 ± 1.36

45.30 ± 1.88

38.40 ± 0.74

* 55.00 ± 1.74

29.50 ± 0.48

87.29 ± 1.07

71.65 ± 2.16

98.61 ± 3.71

0.226 ± 0.008

0.264 ± 0.012

* 93.25 ± 2.24

* 84.18 ± 2.21

20.55 ± 0.61

45.26 ± 0.60

* 96.57 ± 2.03

15.67 ± 0.79

35.97 ± 0.80

15.58 ± 0.67

* 72.90 ± 1.08

30.77 ± 1.20

20.65 ± 1.20

42.60 ± 1.53

* 97.50 ± 2.06

* 98.25 ± 2.63

* 75.25 ± 2.08

40.71 ± 1.08

42.48 ± 1.22

43.29 ± 1.17

34.99 ± 0.63

* 54.26 ± 1.27

31.21 ± 0.50

86.12 ± 1.22

76.50 ± 2.08

99.15 ± 2.07

0.243 ± 0.009

0.222 ± 0.010

4b

-

4c

-

4d

-

4e

-

1.420 ± 0.068

1.373 ± 0.057

4f

-

4g

-

4h

-

4i

-

2.038 ± 0.068

2.097 ± 0.085

4j

-

4k

-

4l

-

4m

-

0.092 ± 0.001

0.015 ± 0.0006

1.071 ± 0.051

0.093 ± 0.002

0.014 ± 0.0005

1.069 ± 0.039

4n

45.945 ± 1.581

41.129 ± 1.694

4o

-

4p

-

4q

-

4r

-

4s

4t

-

1.515 ± 0.072

-

1.607 ± 0.062

-

4u

-

Aminoacridine

Donepezil

Tacrine

1.752 ± 0.072

0.0077 ± 0.0003

0.147 ± 0.004

1.339 ± 0.047

0.0072 ± 0.0002

0.151 ± 0.002

0.233 ± 0.009

1.683 ± 0.064

0.0068 ± 0.0002

0.221 ± 0.007

1.419 ± 0.047

0.0070 ± 0.0002

* Represents the selected compounds for second step due to more than 50% inhibitory potency at 10⁻⁴M concentration.

Molecules 2018, 23, 2033

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It was reported that BChE contains residues which permit bulkier substrates to enter into the

active site [25]. Due to the presence of an acridine ring, the synthesized compounds are big molecules;

thus, they may have a higher tendency to inhibit BChE compared to AchE. This situation is also

reﬂected in Table 1. At 10⁻⁴M concentration, all compounds possessed higher inhibition against BChE

indicating that the synthesized compounds had more selectivity toward BChE than AcHE.

,

2.3. Kinetics Characterization of BChE Inhibition

Recognizing the types of enzyme inhibitors is vital; however, much of the drug discovery process

focuses instead upon deciding whether one inhibitor is more powerful than another. The two most

basic values for evaluating the inhibitor’s power and characteristics are IC₅₀and K_i[26].

To recognize the inhibition type, enzyme kinetics studies were applied using Lineweaver–Burk

plots [27]. For this purpose, we selected compound 4n, which showed the best activity against

human BChE. There are four different points on the graphs showing the presence (at concentrations

of IC₅₀/2, IC₅₀, and 2

×

IC₅₀) and absence (control) of 4n. As seen in Figure 2 , every single straight

line intersected in the second quadrant of the coordinate axis, which characterizes a typical mixed

inhibition. In addition, a graphical examination of the corresponding Lineweaver–Burk plot shows

both increased slopes (decreased V_max) and intercepts (higher K_m) at higher inhibitor concentration.

This pattern also indicates a mixed-type inhibition.

220.00

Control

0.014 μΜ 0.028 μΜ

0.007 μΜ

180.00

140.00

100.00

60.00

20.00

-60

-10

40

90

140

190

240

-20.00

1/[S] μM^-1

Figure 2. Lineweaver–Burk plot for the inhibition of human butyrylcholinesterase (BChE) by

compound 4n at different concentrations of substrate butyrylthiocholine iodide (BTCI).

The calculation of the inhibition constant (K_i) is an effective tool for measuring the afﬁnity of an

enzyme for its substrate, and it is easily determined using the secondary plots of the Lineweaver–Burk

equation. Hence, a graphical investigation for the lead compound, 4n, was performed and is presented

in Figure 3. It was determined that 4n has a very close K_ivalue (0.017 µM) to that of tacrine [28],

meaning that it is able to ﬁrmly bind to the BChE active site.

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When the small molecule binds to the BChE catalytic active site (CAS), the type of inhibition of the

enzyme is classiﬁed as competitive; by contrast, when the small-molecule interacts with the peripheral

anionic site (PAS), the enzymatic inhibition is called non-competitive. Furthermore, once the active

molecule acts on both CAS and PAS, the enzymatic inhibition is designated as mixed inhibition [29].

In this manner, the enzyme kinetics study suggests that compound 4n interacts with both BChE

functional sites, CAS and PAS [30].

10

y = 130.06x + 2.2776

R² = 0.9918

8

6

4

2

0

-0.05

-0.025

0

0.025

0.05

-2

-4

-6

-8

-10

Compound 4n (μM)

Figure 3. Secondary plot for calculation of the steady-state inhibition constant (K_i= 0.0175

BChE) of compound 4n.

µM against

2.4. Molecular Docking

So as to explore a possible interacting mode of the lead compound, 4n, and to evaluate the effects

of structural modiﬁcations on the BChE enzyme activity, molecular docking was performed and

carried out using the X-ray crystal structure of human BChE in complex with tacrine (Protein Data

Bank identiﬁer (PDB ID): 4BDS) [31]. Firstly, tacrine was docked in the enzyme’s active site to validate

the docking procedure. Thus, the π–π interaction between the aromatic ring of tacrine and the indole

of Trp82, and the hydrogen bond between the 9-amino group of tacrine and the carbonyl of His438

were established, as reported in the literature [31].

The docking poses of compound 4n, showing two- and three-dimensional interactions,

are presented in Figures 4 and 5. According to the poses, there are three main patterns of interaction.

The ﬁrst one is a π–π interaction between the phenyl moiety and the imidazole of His438 in the catalytic

site of the BChE enzyme, almost identical to that of tacrine. The second pattern involves a hydrogen

bond between the amide carbonyl and the amino group of Thr120. The last and probably the most

important interaction is another hydrogen bond between the three-ﬂuoro methyl substituent of 4n and

the amino group of Gly115. These results demonstrate that 4n has a signiﬁcant potency to strongly

bind to BChE. In particular, the last interaction clariﬁes why compound 4n is more active than other

derivatives in the series. Findings of the docking analyses conﬁrmed the data reporting that the bulky

moieties have good anti-BChE activity due to their binding ability in the active site of BChE [24,25].

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Figure 4. Two-dimensional interaction of compound 4n with BChE.

Figure 5. Three-dimensional interaction of compound 4n with BChE.

2.5. MTT Cell Viability Assay and Selectivity Indexes

The active compounds (4a 4b 4e 4i 4m 4n

,

4o, and 4t) were chosen to determine their cytotoxic

activity toward a murine ﬁbroblast healthy cell line (NIH3T3). After incubating the cells with active

compounds at concentrations of 0.000316 M to1000 M for 24 h, the cell viability was measured using

µ

Molecules 2018, 23, 2033

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the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay [15]. The inhibition percentage

was calculated for each concentration of the test compounds. The IC₅₀values of the compounds were

determined using a non-linear regression analysis over the calculated percentage inhibition values,

and they are presented in Table 2. Accordingly, the cytotoxicity test proposed that the active derivatives

were nontoxic against NIH3T3 cells at their active concentrations against BChE.

Table 2. IC₅₀of selected derivatives against a murine ﬁbroblast healthy cell line (NIH3T3).

Compound

IC₅₀(µM)

26.81

4a

4b

4e

4i

25.64

324.90

>1000

214.18

>1000

4m

4n

4o

2.6. BBB Permeability and Drug-Likeness Score (DLS)

BBB permeability is exceptionally fundamental for medications that particularly target and focus

on the CNS. The failure of medication particles to penetrate the BBB constitutes a major obstacle for

CNS drug candidates and ought to be considered in new drug discovery efforts. For this reason,

the BBB permeability of the most active derivatives was computed by a CBLigand-BBB forecast

server. This predictor uses two different algorithms, AdaBoost and a support vector machine (SVM),

combining with four different ﬁngerprints, employed to predict if a compound can pass (+) or cannot

pass ( ) the BBB. As presented in Table 3, all calculations for the selected compounds resulted in

−

BBB permeability (+), which is necessary for compounds planning to act as ChE inhibitors [32].

The drug-likeness scores (DLS) were additionally calculated for all active compounds using Molsoft’s

chemical ﬁngerprint mode consisting of 5000 marketed drugs from the World Drug Index (positives)

and 10,000 carefully selected non-drug compounds (negatives) [33] According to this program,

the DLS scores were observed between 1.26 and 2.21, which establishes and fortiﬁes their therapeutic

signiﬁcance in future studies.

Table 3.

Drug-likeness score (DLS) and blood–brain barrier (BBB) permeability of the

active compounds.

Comp.

DLS

2.21

2.16

1.73

1.80

1.88

1.40

1.54

0.97

0.91

BBB Permeability

4a

+

4b

4e

4i

4m

4n

4o

Tacrine

Donepezil

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3. Materials and Methods

3.1. Chemistry

All chemicals were purchased from Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis,

MO, USA) or Merck Chemicals (Merck KGaA, Darmstadt, Germany). Melting points of the synthesized

compounds were assessed with an MP90 digital melting point apparatus (Mettler Toledo, Columbus,

OH, USA), and were uncorrected. The ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker

Fourier 300 (Bruker Bioscience, Billerica, MA, USA) in DMSO-d_6.The IR spectra were obtained on a

Shimadzu, IR Afﬁnity 1S (Shimadzu, Kyoto, Japan). The mass spectra of the compounds were taken in

negative and positive mode using electron spray ionization (ESI) in LCMS/IT/TOF (Shimadzu, Kyoto,

Japan) from the solutions of the samples in methanol. The purities of compounds were checked using

thin-layer chromatography (TLC) on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany).

3.1.1. General Procedure for the Synthesis of N-(9-Acridinyl)-2-Chloroacetamide Derivatives (2)

Chloroacetyl chloride (30 mmol, 2.4 mL) in THF (10 mL) was added dropwise while stirring to a

mixture of 9-aminoacridine (1; 20 mmol, 3.9 g) and triethylamine (40 mmol, 5.6 mL) in THF (50 mL)

at 0–5 ^◦C and stirred for 4 h. The solvent was evaporated under reduced pressure. The residue was

washed with water to remove triethylamine hydrochloride, before being dried and crystallized from

ethanol [34].

3.1.2. General Procedure for the Synthesis of Sodium N-Substituted

Piperazine/Morpholine/Piperidine Dithiocarbamates (3a–3u)

An ethanolic solution (10 mL) of sodium hydroxide (10 mmol, 0.4 g) was added to an ethanolic

solution (10 mL) of the secondary amine (10 mmol). The mixture was cooled in an ice bath; additionally,

carbon disulﬁde (100 mmol, 6.0 mL) was added dropwise with continuous stirring for 1 h at room

temperature. The precipitates were ﬁltrated and washed with diethyl ether to obtain a white to

pale-yellow-colored product in 70–90% yield [14].

3.1.3. General Procedure for the Synthesis of 2-(9-Acridinylamino)-2-Oxoethyl

4-Piperazinyl/Morpholinyl/Piperidinylcarbodithioate Derivatives (4a–4u)

An equimolar quantity (5 mmol) of appropriate sodium N,N-disubstituted dithiocarbamate (

3)

and of N-(9-acridinyl)-2-chloroacetamide ( ) in acetone (10 mL) were reﬂuxed for 8 h. The mixture was

2

cooled, and the precipitate was ﬁltrated. The residue was washed with water, before being dried and

crystallized from ethanol to obtain the ﬁnal products [14].

2-(9-Acridinylamino)-2-oxoethyl 4-(2-(dimethylamino)ethyl)piperazine-1-carbodithioate (4a). Yield: 88.5%;

◦

melting poing (m.p.): 200.6 C; FTIR (ATR, cm⁻¹): 3269 (amide N–H), 2769–2972 (aliphatic C–H), 1662

(C=O), 1463–1506 (C=N and C=C), 1232 (C=S), 756 (out-of-plane C–H bending); ¹H-NMR (300 MHz,

DMSO-d₆; δ, ppm): 2.17–2.18 (6H, d, J = 2.4 Hz, N(CH₃)₂), 2.43–2.44 (4H, t, J = 2.5 Hz, CH₂–CH₂),

2.48–2.55 (4H, m, piperazine C_3,5–H), 3.97 and 4.25 (4H, two bs, piperazine C_2,6–H), 4.62 (2H, s,

COCH₂), 7.57–7.62 (2H, t, J = 7.5 Hz, Ar–H), 7.82–7.87 (2H, t, J = 7.7 Hz, Ar–H), 8.14–8.17 (2H, d, J = 8.7

Hz, Ar–H), 8.25–8.28 (2H, d, J = 8.6 Hz, Ar–H), 11.06 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ

, ppm): 45.7 (CH₃), 50.4 (CH₂), 51.6 (CH₂), 52.9 (CH₂), 55.2 (CH₂), 56.8 (CH₂), 123.2 (C), 125.4 (CH),

126.2 (CH), 129.6 (CH), 130.9 (CH), 140.6 (C), 149.4 (C-9 in 9-aminoacridine), 167.4 (C=O), 194.9 (C=S);

HRMS (m/z): [M + H]⁺calculated for C₂₄H₂₉N₅OS₂: 468.1886, found 468.1874.

2-(9-Acridinylamino)-2-oxoethyl 4-(3-(dimethylamino)propyl)piperazine-1-carbodithioate (4b). Yield: 85.7%;

◦

m.p.: 153.8 C; FTIR (ATR, cm⁻¹): 3340 (amide N–H), 2777–2947 (aliphatic C–H), 1653 (C=O), 1411–1458

(C=N and C=C), 1209–1263 (C=S), 758 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ

, ppm): 1.47–1.56 (2H, m, CH₂–CH₂–CH₂), 2.08 (6H, s, N(CH₃)₂), 2.15–2.20 (2H, t, J = 7.1 Hz,

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CH₂–CH₂CH₂N(CH₃)₂), 2.24–2.29 (2H, d, J = 7.4 Hz, CH₂CH₂CH₂), 2.41–2.42 (4H, d, J = 0.9, piperazine

C_{3 5}–H), 3.98 and 4.26 (4H, two s, piperazine C_{2 6}–H), 4.65 (2H, s, COCH₂), 7.57–7.62 (2H, q, J = 7.6 Hz,

,

Ar–H), 7.81–7.87 (2H, m, Ar–H), 8.14–8.17 (2H, d, J = 8.7 Hz, Ar–H), 8.26–8.29 (2H, d, J = 8.7 Hz, Ar–H),

11.19 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm): 24.9 (CH₂), 45.7 (CH₂), 50.4 (CH₃), 51.6

(CH₂), 52.9 (CH₂), 55.2 (CH₂), 56.7 (CH₂), 123.2 (C), 125.4 (CH), 126.2 (CH), 129.6 (CH), 130.9 (CH),

140.6 (C), 149.4 (C-9 in 9-aminoacridine), 167.4 (C=O), 194.9 (C=S); HRMS (m/z): [M + H]⁺calculated

for C₂₅H₃₁N₅OS₂: 482.2043, found 482.2047.

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2-(9-Acridinylamino)-2-oxoethyl 4-ethylpiperazine-1-carbodithioate (4c). Yield: 81.0%; m.p.: 221.7 C;

FTIR (ATR, cm⁻¹): 3232 (amide N–H), 2358–2974 (aliphatic C–H), 1654 (C=O), 1435–1508 (C=N and

C=C), 1219–1271 (C=S), 732–758 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm):

0.98–1.03 (3H, t, J = 7.2 Hz, CH₂CH₃), 2.33–2.40 (2H, q, J = 7.2 Hz, CH₂CH₃), 2.47–2.50 (4H, m,

piperazine C_3,5–H), 3.98 and 4.26 (4H, two s, piperazine C_2,6–H), 4.65 (2H, s, COCH₂), 7.57–7.62 (2H, t,

J = 7.5 Hz, Ar–H), 7.81–7.87 (2H, t, J = 7.6 Hz, Ar–H), 8.14–8.17 (2H, d, J = 8.7 Hz, Ar–H), 8.26–8.29

(2H, d, J = 8.6 Hz, Ar–H), 11.19 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm): 12.3 (CH₃), 50.9

(CH₂), 51.1 (CH₂), 51.5 (CH₂), 52.2 (CH₂), 123.2 (C), 125.4 (CH), 126.2 (CH), 129.6 (CH), 130.9 (C), 140.7

(C), 149.4 (C-9 in 9-aminoacridine), 167.4 (C=O), 195.0 (C=S); HRMS (m/z): [M + H] ⁺calculated for

C₂₂H₂₄N₄OS₂: 425.1464, found 425.1459.

2-(9-Acridinylamino)-2-oxoethyl 4-(4-nitrophenyl)piperazine-1-carbodithioate (4d). Yield: 90.5%; m.p.:

248.2 ^◦C; FTIR (ATR, cm⁻¹): 3238 (amide N–H), 2976 (aliphatic C–H), 1670 (C=O), 1417–1598 (C=N

and C=C), 1213–1280 (C=S),752 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆; δ, ppm):

3.74 (4H, s, piperazine C_3,5–H), 4.19 and 4.42 (4H, two bs, piperazine C_2,6–H), 4.68 (2H, s, COCH₂),

6.93–6.96 (2H, d, J = 9.5 Hz, Ar–H), 7.59–7.63 (2H, t, J = 6.6 Hz, Ar–H), 7.82–7.87 (2H, t, J = 7.7 Hz, Ar–H),

8.07–8.10 (2H, d, J = 9.4 Hz, Ar–H), 8.14–8.17 (2H, d, J = 8.7 Hz, Ar–H), 8.27–8.30 (2H, d, J = 8.5 Hz,

Ar–H), 11.10 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm): 45.2 (CH₂), 48.9 (CH₂), 50.6 (CH₂),

112.3 (C), 123.2 (CH), 125.4 (CH), 126.2 (CH), 126.3 (CH), 129.6 (CH), 130.9 (CH), 137.3 (C), 140.6 (C),

149.4 (C), 154.2 (C-9 in 9-aminoacridine), 167.3 (C=O), 195.4 (C=S); HRMS (m/z): [M + H]⁺calculated

for C₂₆H₂₃N₅O₃S₂: 518.1315, found 518.1299.

2-(9-Acridinylamino)-2-oxoethyl 4-(2-hydroxyethyl)piperazine-1-carbodithioate (4e). Yield: 84.4%; m.p.:

◦

219.2 C; FTIR (ATR, cm⁻¹): 3226 (amide N–H), 2978 (aliphatic C–H), 1660 (C=O), 1433–1558 (C=N and

C=C), 1228 (C=S), 758 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm): 2.49–2.50

(2H, t, J = 1.7 Hz, OH–CH₂–CH₂–), 2.61 (4H, s, piperazine C_3,5–H), 3.54 (2H, s, OH–CH₂–CH₂–), 4.00

and 4.28 (4H, s, piperazine C_2,6–H), 4.64 (2H, s, COCH₂), 7.58–7.62 (2H, t, J = 7.4 Hz, Ar–H), 7.82–7.87

(2H, t, J = 7.4 Hz, Ar–H), 8.14–8.17 (2H, d, J = 8.6 Hz, Ar–H), 8.26–8.28 (2H, d, J = 8.6 Hz, Ar–H),

11.10 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm): 50.2 (CH₂), 51.4 (CH₂), 52.9 (CH₂), 58.8

(CH₂), 59.8 (CH₂), 123.2 (C), 125.4 (CH), 126.2 (CH), 129.6 (CH), 130.9 (CH), 140.6 (C), 149.4 (C-9 in

9-aminoacridine), 167.4 (C=O), 195.1 (C=S); HRMS (m/z): [M + H] ⁺calculated for C₂₂H₂₄N₄O₂S₂:

441.1413, found 441.1406.

2-(9-Acridinylamino)-2-oxoethyl 4-phenylpiperazine-1-carbodithioate (4f). Yield: 94.5%; m.p.: oil; FTIR

(ATR, cm⁻¹): 3352 (amide N–H), 2881 (aliphatic C–H), 1660 (C=O), 1417–1506 (C=N and C=C), 1228

(C=S), 756–883 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm): 3.31–3.35 (4H,

d, J =9.4 Hz, piperazine C_3,5–H), 4.15 and 4.42 (4H, two bs, piperazine C_2,6–H), 4.66 (2H, s, COCH₂),

6.78–6.83 (1H, t, J = 7.2 Hz, Ar–H), 6.94–6.97 (2H, d, J = 8.0 Hz, Ar–H), 7.21–7.26 (2H, t, J = 7.9 Hz,

Ar–H), 7.58–7.63 (2H, t, J = 7.6 Hz, Ar–H), 7.82–7.86 (2H, t, J = 6.7 Hz, Ar–H), 8.14–8.17 (2H, d, J = 8.7 Hz,

Ar–H), 8.27–8.30 (2H, d, J = 8.5 Hz, Ar–H), 11.08 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm):

48.1 (CH₂), 50.1 (CH₂), 51.3 (CH₂), 115.9 (C), 119.7 (CH), 123.2 (CH), 125.4 (CH), 126.2 (CH), 129.5 (CH),

129.6 (CH), 130.9 (CH), 140.6 (C), 149.4 (C), 150.5 (C-9 in 9-aminoacridine), 167.4 (C=O), 195.3 (C=S);

HRMS (m/z): [M + H] ⁺calculated for C₂₆H₂₄N₄OS₂: 473.1464, found 473.1471.

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◦

2-(9-Acridinylamino)-2-oxoethyl 4-methylpiperazine-1-carbodithioate (4g). Yield: 95.3%; m.p.: 243.2 C;

FTIR (ATR, cm⁻¹): 3226 (amide N–H), 2783–2976 (aliphatic C–H), 1651 (C=O), 1435–1571(C=N and

C=C), 1224–1288 (C=S), 758 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm): 2.21

(3H, s, CH₃), 2.42–2.45 (4H, t, J = 4.5 Hz, piperazine C_3,5–H), 3.99 and 4.27 (4H, two s, piperazine

C_2,6–H), 4.61 (2H, s, COCH₂), 7.58–7.63 (2H, t, J = 7.5 Hz, Ar–H), 7.82–7.87 (2H, t, J = 7.3 Hz, Ar–H),

8.15–8.18 (2H, d, J = 8.7 Hz, Ar–H), 8.25–8.28 (2H, d, J = 8.6 Hz, Ar–H), 10.94 (1H, s, –NH–); ¹³C-NMR

(75 MHz, DMSO-d₆; δ, ppm): 45.6 (CH₂), 50.3 (CH₃), 51.6 (CH₂), 54.5 (CH₂), 123.2 (CH), 125.3 (CH),

126.3 (CH), 129.3 (CH), 130.9 (CH), 140.5 (CH), 149.4 (C-9 in 9-aminoacridine), 167.4 (C=O), 195.1 (C=S);

HRMS (m/z): [M + H]⁺calculated for C₂₁H₂₂N₄OS₂: 411.1308, found 411.1307.

2-(9-Acridinylamino)-2-oxoethyl 4-(4-methoxyphenyl)piperazine-1-carbodithioate (4h). Yield: 89.4%; m.p.:

243.8 ^◦C; FTIR (ATR, cm⁻¹): 3284 (amide N–H), 3057–2945 (aliphatic C–H), 1664 (C=O), 1431–1508

(C=N and C=C), 1215 (C=S), 707–756 (out-of-plane C–H bending), ¹H-NMR (300 MHz, DMSO-d₆;

δ,

ppm): 3.16–3.35 (4H, s, piperazine C_3,5–H), 3.68 (3H, s, OCH₃), 4.14 and 4.42 (4H, two s, piperazine

C_2,6–H), 4.66 (2H, s, COCH₂), 6.81–6.84 (2H, d, J = 8.5 Hz, Ar–H), 6.90–6.93 (2H, d, J = 8.4 Hz, Ar–H),

7.59–7.64 (2H, t, J = 7.3 Hz, Ar–H), 7.82–7.87 (2H, t, J = 7.4 Hz, Ar–H), 8.16–8.18 (2H, d, J = 8.6 Hz,

Ar–H), 8.27–8.30 (2H, d, J = 8.6 Hz, Ar–H), 10.97 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm):

49.8 (CH₂), 51.4 (CH₂), 55. 7 (CH₃), 60.21 (CH₂), 114.8 (C), 118.7 (CH), 123.2 (CH), 125.3 (CH), 126.3

(CH), 129.6 (CH), 130.9 (CH), 140.5 (C), 144.9 (C), 149.4 (C), 153.9 (C-9 in 9-aminoacridine), 167.4 (C=O),

195.3 (C=S); HRMS (m/z): [M + H] ⁺calculated for C₂₇H₂₆N₄O₂S₂: 503.1570, found 503.1547.

2-(9-Acridinylamino)-2-oxoethyl 4-(4-chlorophenyl)piperazine-1-carbodithioate (4i). Yield: 97.0%; m.p.:

213.6 ^◦C; FTIR (ATR, cm⁻¹): 3223 (amide N–H), 2706–2912 (aliphatic C–H), 1672 (C=O), 1417–1573

(C=N and C=C), 1226 (C=S), 815 (para aromatic substitution), 756 (out-of-plane C–H bending); ¹H-NMR

(300 MHz, DMSO-d₆; δ, ppm): 2.44–2.50 (4H, q, J = 5.8 Hz, piperazine C_3,5–H), 4.02 and 4.45 (4H,

two bs, piperazine C_2,6–H), 4.60 (2H, s, COCH₂), 6.97–7.00 (2H, d, J = 9.1 Hz, Ar–H), 7.25–7.28 (2H,

d, J = 9.0 Hz, Ar–H), 7.57–7.62 (2H, t, J = 7.5 Hz, Ar–H), 7.81–7.85 (2H, t, J = 5.9 Hz, Ar–H), 8.14–8.17

(2H, d, J = 8.7 Hz, Ar–H), 8.27–8.30 (2H, d, J = 8.6 Hz, Ar–H), 11.22 (1H, s, –NH–); ¹³C-NMR (75 MHz,

DMSO-d₆; δ, ppm): 45.6 (CH₂), 49.8 (CH₂), 51.0 (CH₂), 117.3 (CH), 118.0 (CH), 123.2 (CH), 125.2 (CH),

126.2 (CH), 129.2 (CH), 129.3 (CH), 129.6 (CH), 130.9 (CH), 140.7 (C), 149.4 (C-9 in 9-aminoacridine),

167.4 (C=O), 195.3 (C=S); HRMS (m/z): [M + H] ⁺calculated for C₂₆H₂₃ClN₄OS₂: 507.1075, found

507.1069.

2-(9-Acridinylamino)-2-oxoethyl 4-(pyrimidin-2-yl)piperazine-1-carbodithioate (4j). Yield: 84.3%; m.p.:

248.4 ^◦C; FTIR (ATR, cm⁻¹): 3263 (amide N–H), 2852–2897 (aliphatic C–H), 1662 (C=O), 1425–1583

(C=N and C=C), 1222–1257 (C=S), 759–792 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ

, ppm): 3.91 (4H, s, piperazine C_3,5–H), 4.13 and 4.38 (4H, two s, piperazine C_2,6–H), 4.68 (2H, s,

COCH₂), 6.67–6.70 (1H, t, J = 4.7 Hz, Ar–H), 7.58–7.63 (2H, t, J = 7.6 Hz, Ar–H), 7.82–7.87 (2H, t,

J = 7.4 Hz, Ar–H), 8.14–8.18 (2H, d, J = 8.7 Hz, Ar–H), 8.27–8.30 (2H, d, J = 8.6 Hz, Ar–H), 8.39–8.41

(2H, d, J = 4.7 Hz, Ar–H), 11.16 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm): 43.0 (CH₂), 49.8

(CH₂), 51.2 (CH₂), 111.1 (C), 123.2 (CH), 125.4 (CH), 126.2 (CH), 129.6 (CH), 130.9 (CH), 140.6 (C), 149.4

(CH), 158.48 (C), 161.3 (C-9 in 9-aminoacridine), 167.4 (C=O), 195.4 (C=S). HRMS (m/z): [M + H] ⁺

calculated for C₂₄H₂₂N₆OS₂: 475.1369, found 475.1366.

◦

2-(9-Acridinylamino)-2-oxoethyl 4-benzylpiperazine-1-carbodithioate (4k). Yield: 91.4%; m.p.: 222.3 C; FTIR

(ATR, cm⁻¹): 3248 (amide N–H), 2818–2908 (aliphatic C–H), 1668 (C=O), 1417–1510 (C=N and C=C),

1230 (C=S), 731–756 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm): 3.53 and

3.99 (4H, two bs, piperazine C₂,₆–H), 4.28 (2H, s, C₆H₅–CH₂–), 4.62 (2H, s, COCH₂), 7.26–7.32 (5H, m,

Ar–H), 7.57–7.62 (2H, t, J = 7.5 Hz, Ar–H), 7.82–7.87 (2H, t, J = 7.5 Hz, Ar–H), 8.14–8.17 (2H, d, J = 8.7 Hz,

Ar–H), 8.25–8.28 (2H, d, J = 8.6 Hz, Ar–H), 11.05 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm):

50.4 (CH₂), 51.7 (CH₂), 52.4 (CH₂), 61.8 (CH₂), 123.2 (C), 125.3 (CH), 126.2 (CH), 127.6 (CH), 128.7 (CH),

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129.4 (CH), 129.6 (CH), 130.9 (CH), 138.0 (C), 140.6 (C), 149.4 (C-9 in 9-aminoacridine), 167.4 (C=O),

195.0 (C=S); HRMS (m/z): [M + H]⁺calculated for C₂₇H₂₆N₄OS₂: 487.1621, found 487.1622.

2-(9-Acridin-ylamino)-2-oxoethyl 4-cyclohexylpiperazine-1-carbodithioate (4l). Yield: 88.4%; m.p.: 225.4^◦C;

FTIR (ATR, cm⁻¹): 3309 (amide N–H), 2357–2931 (aliphatic C–H), 1676 (C=O), 1469–1566 (C=N and

C=C), 1230–1274 (C=S), 754–866 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm):

1.03–1.18 (6H, m, cyclohexyl –CH₂–), 1.54–1.57 (1H, d, J = 11.2 Hz, cyclohexyl –CH–), 1.72 (4H, s,

cyclohexyl –CH₂–), 2.60 (4H, s, piperazine C_3,5–H), 3.94 and 4.24 (4H, two bs, piperazine C_2,6–H), 4.62

(2H, s, COCH₂), 7.57–7.62 (2H, t, J = 7.5 Hz, Ar–H), 7.82–7.87 (2H, t, J = 7.6 Hz, Ar–H), 8.14–8.17 (2H,

d, J = 8.7 Hz, Ar–H), 8.25–8.28 (2H, d, J = 8.6 Hz, Ar–H), 11.04 (1H, s, –NH–); ¹³C-NMR (75 MHz,

DMSO-d₆; δ, ppm): 25.7 (CH₂), 26.3 (CH₂), 28.8 (CH₂), 48.6 (CH₂), 50.9 (CH₂), 52.2 (CH₂), 62.7 (CH),

123.2 (C), 125.4 (CH), 126.2 (CH), 129.6 (CH), 130.9 (CH), 140.6 (C), 149.4 (C-9 in 9-aminoacridine), 167.4

(C=O), 194.7 (C=S); HRMS (m/z): [M + H]⁺calculated for C₂₆H₃₀N₄OS₂: 479.1934, found 479.1929.

◦

2-(9-Acridinylamino)-2-oxoethyl 4-benzhydrylpiperazine-1-carbodithioate (4m). Yield: 84.3%; m.p.: 137.0 C;

FTIR (ATR, cm⁻¹): 3246 (amide N–H), 2355–2920 (aliphatic C–H), 1656 (C=O), 1427–1571 (C=N and

C=C), 1226–1284 (C=S), 705–758 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm):

2.44–2.50 (4H, d, J = 5.8 Hz, piperazine C_3,5–H), 4.02 and 4.28 (4H, two s, piperazine C_2,6–H), 4.40 (1H,

s, C₁₂H₁₀–CH–), 4.60 (2H, s, COCH₂), 7.18–7.23 (3H, t, J = 7.0 Hz, Ar–H), 7.29–7.32 (4H, t, J = 7.4 Hz,

Ar–H), 7.44 (4H, s, Ar–H), 7.57–7.62 (2H, t, J = 7.6 Hz, Ar–H), 7.82–7.88 (2H, m, Ar–H), 7.15–7.18 (2H, d,

J = 8.7 Hz, Ar–H), 7.24–7.27 (2H, d, J = 8.6 Hz, Ar–H), 11.02 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ

, ppm): 51.4 (CH₂), 60.7 (CH₂), 72.7 (CH₂), 74.6 (CH), 117.9 (C), 123.1 (CH), 125.4 (CH), 126.3 (CH),

127.6 (CH), 128.1 (CH), 129.1 (CH), 131.2 (CH), 140.9 (C), 142.6 (C), 149.1 (C-9 in 9-aminoacridine), 167.4

(C=O), 195.1 (C=S); HRMS (m/z): [M + H]⁺calculated for C₃₃H₃₀N₄OS₂: 563.1934, found 563.1924.

2-(9-Acridinylamino)-2-oxoethyl 4-(4-(triﬂuoromethyl)benzyl)piperazine-1-carbodithioate (4n). Yield: 87.7%;

◦

m.p.: 161.5 C; FTIR (ATR, cm⁻¹): 3232 (amide N–H), 2814–2920 (aliphatic C–H), 1653 (C=O), 1415–1510

(C=N and C=C), 1219 (C=S), 756 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm):

2.48–2.52 (4H, t, J = 5.10 Hz, piperazine C_3,5–H), 3.62 and 4.00 (4H, two s, piperazine C_2,6–H), 4.29 (2H,

s, C₆H₅–CH₂–), 4.62 (2H, s, COCH₂), 7.55–7.57 (2H, d, J = 7.9 Hz, Ar–H), 7.60–7.62 (2H, d, J = 7.7 Hz,

Ar–H), 7.68–7.71 (2H, d, J = 8.2 Hz Ar–H), 7.82–7.87 (2H, t, J = 7.8 Hz, Ar–H), 7.14–7.17 (2H, d, J = 8.8 Hz,

Ar–H), 7.25–7.27 (2H, d, J = 8.6 Hz, Ar–H), 11.01 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm):

52.4 (CH₂), 60.8 (CH₂), 72.7 (CH₂), 125.0 (q, J = 214.9 Hz, CF₃), 123.2 (CH), 125.3 (CH), 125.6 (q, J =3.6 Hz,

triﬂuoromethylphenyl C_3,3₀), 126.2 (CH), 129.6 (CH), 129.9 (q, J = 39.8 Hz, triﬂuoromethylphenyl C₄),

130.0 (CH), 130.1 (C), 140.6 (C), 143.2 (C), 149.4 (C-9 in 9-aminoacridine), 167.4 (C=O), 195.1 (C=S);

HRMS (m/z): [M + H]⁺calculated for C₂₈H₂₅F₃N₄OS₂: 555.1495, found 555.1493.

2-(9-Acridinylamino)-2-oxoethyl 4-(4-methylbenzyl)piperazine-1-carbodithioate (4o). Yield: 91.0%; m.p.:

205.8 ^◦C; FTIR (ATR, cm⁻¹): 3255 (amide N–H), 2792–2902 (aliphatic C–H), 1654 (C=O), 1435–1508

(C=N and C=C), 1220 (C=S), 759 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm):

2.27 (3H, s, –CH₃), 2.47–2.50 (4H, m, piperazine C_3,5–H), 3.47 (2H, s,C₆H₅–CH₂), 3.98 and 4.26 (4H,

two s, piperazine C_2,6–H), 4.63 (2H, s, COCH₂), 7.11–7.14 (2H, d, J = 7.9 Hz, Ar–H), 7.18–7.20 (2H,

d, J =7.9 Hz, Ar–H), 7.56–7.61 (2H, t, J = 7.5 Hz, Ar–H), 7.86–7.81 (2H, t, J = 7.4 Hz, Ar–H), 8.14–8.17

(2H, d, J = 8.7 Hz, Ar–H), 8.25–8.28 (2H, d, J = 8.6 Hz, Ar–H), 11.15 (1H, s, –NH–); ¹³C-NMR (75 MHz,

DMSO-d₆; δ, ppm): 21.2 (CH₃), 50.4 (CH₂), 51.6 (CH₂), 52.4 (CH₂), 61.5 (CH₂), 123.2 (C), 125.4 (CH),

126.2 (CH), 129.3 (CH), 129.4 (CH), 129.6 (CH), 130.9 (CH), 134.8 (C), 36.7 (C), 140.6 (C), 149.4 (C-9

in 9-aminoacridine), 167.4 (C=O), 195.0 (C=S); HRMS (m/z): [M + H]⁺calculated for C₂₈H₂₈N₄OS₂:

501.1777, found 501.1774.

2-(9-Acridinylamino)-2-oxoethyl 4-(4-ﬂuorophenyl)piperazine-1-carbodithioate (4p). Yield: 96.4%; m.p.:

227.7 ^◦C; FTIR (ATR, cm⁻¹): 3257 (amide N–H), 2800–2906 (aliphatic C–H), 1653 (C=O), 1433 (C=N

and C=C), 1213 (C=S), 1029 (C–F), 825 (1,4-disubstituted benzene), 756 (out-of-plane C–H bending);

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¹H-NMR (300 MHz, DMSO-d₆;

δ, ppm): 3.24–3.27 (4H, t, J = 4.8 Hz, piperazine C_3,5–H), 4.16 and

4.39 (4H, two s, piperazine C_2,6–H), 4.67 (2H, s,COCH₂), 6.94–6.99 (2H, m, Ar–H), 7.04–7.10 (2H, t,

J = 8.9 Hz, Ar–H), 7.58–7.63 (2H, t, J = 7.6 Hz, Ar–H), 7.82–7.87 (2H, t, J = 7.7 Hz, Ar–H), 8.15–8.18

(2H, d, J = 8.7 Hz, Ar–H), 8.27–8.30 (2H, d, J = 8.6 Hz, Ar–H), 11.07 (1H, s, –NH–); ¹³C-NMR (75 MHz,

DMSO-d₆; δ, ppm): 49.0 (CH₂), 50.1 (CH₂), 51.36 (CH₂), 115.9 (d, J =21.8 Hz, ﬂuorophenyl C_3,3₀), 117.9

(d, J = 7.5 Hz, ﬂuorophenyl C_2,2₀), 123.2 (CH), 125.4 (CH), 126.2 (CH), 129.6 (CH), 130.9 (CH), 140.6

(CH), 147.4 (d, J = 2.3 Hz, ﬂuorophenyl C₁), 149.4 (C-9 in 9-aminoacridine), 156.8 (d, J = 234.8 Hz,

ﬂuorophenyl C₄), 167.4 (C=O), 195.3 (C=S); HRMS (m/z): [M + H]⁺calculated for C₂₆H₂₃FN₄OS₂:

490.62, found 491.13; HRMS (m/z): [M + H]⁺calculated for C₂₆H₂₃FN₄OS₂: 491.1370, found 491.1354.

2-(9-Acridinylamino)-2-oxoethyl 4-(furan-2-carbonyl)piperazine-1-carbodithioate (4q). Yield: 95.5%; m.p.:

218.9 ^◦C; FTIR (ATR, cm⁻¹): 3255 (amide N–H), 2895–2995 (aliphatic C–H), 1653 (furan-2-carbonyl),

1

1616 (amide C=O), 1423 (C=N and C=C), 1215 (C=S), 758 (out-of-plane C–H bending); H-NMR

(300 MHz, DMSO-d₆; δ, ppm): 3.88 (4H, s, piperazine C_3,5–H), 4.12 and 4.36 (4H, two s, piperazine

–CH₂), 4.68 (2H, s, COCH₂), 6.64–6.65 (1H, m, Ar–H), 7.07–7.08 (2H, d, J = 3.4 Hz, Ar–H), 7.57–7.63 (2H,

t, J = 7.6 Hz, Ar–H), 7.81–7.87 (3H, t, J = 8.1 Hz, Ar–H), 8.14–8.17 (2H, d, J = 8.7 Hz, Ar–H), 8.27–8.30

(2H, d, J = 8.6 Hz, Ar–H), 11.21 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm): 49.6 (CH₂), 51.1

(CH₂), 111.9 (C), 116.6 (CH), 123.2 (CH), 125.4 (CH), 126.2 (CH), 129.6 (CH), 130.9 (CH), 140.6 (CH),

145.6 (C), 147.1 (C), 149.4 (C), 159.0 (C-9 in 9-aminoacridine), 167.4 (C=O), 195.7 (C=S); HRMS (m/z):

[M + H]⁺calculated for C₂₅H₂₂N₄O₃S₂: 491.1206, found 491.1186.

◦

2-(9-Acridinylamino)-2-oxoethyl morpholine-4-carbodithioate (4r). Yield: 89.2%; m.p.: 237.8 C; FTIR

(ATR, cm⁻¹): 3232 (amide N–H), 2864–2949 (aliphatic C–H), 1653 (amide C=O), 1417–1423 (C=N

and C=C), 1273 (C=S), 1001 (C–O–C of morpholine), 864–758 (out-of-plane C–H bending); ¹H-NMR

(300 MHz, DMSO-d₆; δ, ppm): 3.71–3.72 (4H, d, J = 4.3 Hz, morpholine C_3.5–H), 4.01 and 4.25 (4H,

two bs, morpholine C_2,6–H), 4.65 (2H, s, COCH₂), 7.58–7.63 (2H, t, J = 7.5 Hz, Ar–H), 7.82–7.87 (2H, t,

J = 7.6 Hz, Ar–H), 8.15–8.18 (2H, d, J = 8.7 Hz, Ar–H), 8.26–8.29 (2H, d, J = 8.6 Hz, Ar–H), 11.06 (1H,

s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm): 50.9 (CH₂), 51.8 (CH₂), 66.1 (CH₂), 123.2 (C), 125.3

(CH), 126.2 (CH), 129.6 (CH), 130.9 (CH), 140.6 (C), 149.4 (C-9 in 9-aminoacridine), 167.4 (C=O), 195.7

(C=S); HRMS (m/z): [M + H]⁺calculated for C₂₀H₁₉N₃O₂S₂: 398.0991, found 398.0977.

2-(9-Acridinylamino)-2-oxoethyl piperidine-1-carbodithioate (4s). Yield: 84.4%; m.p.: 265.4 ^◦C; FTIR (ATR,

cm⁻¹): 3255 (amide N–H), 2904–2983 (aliphatic C–H), 1653 (amide C=O), 1417 (C=N and C=C), 1217

(C=S), 1001 (C–N of piperidine), 758 (out-of-plane C–H bending); ¹H-NMR (300 MHz, DMSO-d₆;

δ,

ppm): 1.64 (6H, s, piperidine –CH₂), 3.96 and 4.25 (4H, two s, piperidine CH₂–N–CH₂), 4.62 (2H,

s, COCH₂), 7.57–7.61 (2H, t, J = 6.6 Hz, Ar–H), 7.82–7.86 (2H, t, J = 6.9 Hz, Ar–H), 8.14–8.16 (2H,

d, J = 8.3 Hz, Ar–H), 8.26–8.29 (2H, d, J = 8.5 Hz, Ar–H), 11.14 (1H, s, –NH–); ¹³C-NMR (75 MHz,

DMSO-d₆; δ, ppm): 23.9 (CH₂), 26.0 (CH₂), 51.7 (CH₂), 53.1 (CH₂), 123.2 (C), 125.4 (CH), 126.2 (CH),

129.6 (CH), 130.9(CH), 140.7 (C), 149.4 (C-9 in 9-aminoacridine), 167.5 (C=O), 193.7 (C=S); HRMS (m/z):

[M + H]⁺calculated for C₂₁H₂₁N₃OS₂: 396.1199, found 396.1184.

◦

2-(9-Acridinylamino)-2-oxoethyl 2-methylpiperidine-1-carbodithioate (4t). Yield: 93.3%; m.p.: 265.4 C; FTIR

(ATR, cm⁻¹): 3255 (amide N–H), 2924 (aliphatic C–H), 1653 (amide C=O), 1417–1463 (C=N and C=C),

1

1215–1263 (C=S), 1001 (C–N of piperidine), 758 (out-of-plane C–H bending); H-NMR (300 MHz,

DMSO-d₆; δ, ppm): 1.19–1.25 (9H, m, piperidine –CH₂, –CH₃), 4.26–4.30 (3H, m, piperidine –CH₂,

–CH–), 4.61 (2H, s, COCH₂), 7.60 (2H, t, J = 7.5 Hz, Ar–H), 7.85 (2H, t, J = 7.5 Hz, Ar–H), 8.16 (2H, d,

J = 8.6 Hz, Ar–H), 8.28 (2H, d, J = 8.7 Hz, Ar–H), 10.94 (1H, s, –NH–); ¹³C-NMR (75 MHz, DMSO-d₆;

δ

, ppm): 18.5 (CH₃), 18.7 (CH₂), 25.7 (CH₂), 26.2 (CH₂), 52.2 (CH₂), 54.6 (CH), 123.2 (C), 125.4 (CH),

126.2 (CH), 129.6 (CH), 130.9 (CH), 140.3 (C), 149.4 (C-9 in 9-aminoacridine), 167.6 (C=O), 194.3 (C=S);

HRMS (m/z): [M + H]⁺calculated for C₂₂H₂₃N₃OS₂: 410.1355, found 410.1338.

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2-(9-Acridinylamino)-2-Oxoethyl 4-Benzylpiperidine-1-Carbodithioate (4u). Yield: 90.7%; m.p.: 265.4 ^◦C;

FTIR (ATR, cm⁻¹): 3261 (amide N–H), 2852–2922 (aliphatic C–H), 1653 (amide C=O), 1452–1570

1

(C=N and C=C), 1261 (C=S), 1016 (C–N of piperidine), 758 (out-of-plane C–H bending); H-NMR

(300 MHz, DMSO-d₆; δ, ppm): 1.22–1.26 (5H, m, piperidine –CH₂, –CH), 3.04–3.08 (2H, m, piperidine

–CH₂–), 3.20–3.25 (2H, m, piperidine –CH₂–), 3.97 (2H, s, C₆H₅–CH₂–), 4.61 (2H, s, COCH₂), 7.58 (2H, t,

J = 7.6 Hz, Ar–H), 7.67 (2H, t, J = 7.5 Hz, Ar–H), 7.77–7.86 (5H, m, benzyl –CH), 8.15 (2H, d, J = 8.7 Hz,

Ar–H), 8.28 (2H, d, J = 8.7 Hz, Ar–H); ¹³C-NMR (75 MHz, DMSO-d₆;

δ, ppm): 37.1 (CH), 42.0 (CH₂),

48.7 (CH₂), 50.6 (CH₂), 54.6 (CH₂), 122.4 (C), 125.5 (CH), 126.0 (CH), 126.3 (CH), 128.7 (CH), 129.5 (CH),

130.3 (CH), 130.9 (CH), 132.2 (CH), 140.3 (C), 149.2 (C-9 in 9-aminoacridine), 167.6 (C=O), 193.9 (C=S);

HRMS (m/z): [M + H]⁺calculated for C₂₈H₂₇N₃OS₂: 486.1668, found 486.1654.

3.2. AChE/BChE Activity Assay

All enzymes were obtained from Sigma-Aldrich (Steinheim, Germany). Ellman’s reagent and the

substrates were obtained from Fluka (Buchs, Switzerland). The chemicals used to prepare the buffer

solution were purchased from Merck (Darmstadt, Germany).

The enzyme inhibitory activities of the synthesized compounds were investigated by applying

Ellman’s method [35]. Enzyme solutions were dissolved in gelatin solution (1%; 2.5 units/mL).

Compounds 4a

and 10⁻⁴M. The enzyme solution (20

L/well) and inhibit_◦or solution (20

with buffer (140 L/well, pH 8 0.1) and incubated at 25 C for 5 min. Then, the reaction was

– M

4u and reference agents were prepared in 2% DMSO at concentrations of 10⁻³

µ

µL/well) were mixed

µ

±

initiated by the addition of Ellman’s reagent 5,5⁰-Dithiobis(2-nitrobenzoic acid) (DTNB; 20

µL/well,

10 mM) and the substrate (10 L/well, 75 mM). The absorbance was measured for 10 min at 412 nm.

µ

The enzyme solution was processed as a control. All readings were adjusted with blank-reading.

All assays were performed in four independent wells. The same procedure was applied for further

concentrations (10⁻⁵–10⁻⁹M) of reference agents and synthesized compounds displaying

≥

50%

inhibition at initial concentrations (10⁻³and 10⁻⁴M). The IC₅₀values were calculated by applying

regression analyses using Microsoft Excel 2013 [36]. Absorbance differences between the two readings

were taken, and percentage inhibition rates were calculated according to the following formula:

[(A(C) − A(B)) − (A(I) − A(B))]

% Inhibition =

× 100.

(A(C) − A(B))

Blank (B): the well in which the inhibitor compound and substrate were not added;

Control (C): the well where the inhibitor compound was not added;

A(B): the difference in absorbance reading for the blank;

A(C): the difference in absorbance reading for the control;

A(I): the difference in absorbance reading for the inhibitor compounds.

3.3. Enzyme Kinetics

In the kinetics studies, the assay protocol speciﬁed for the inhibition assay was identical. However,

unlike the inhibition method, the concentrations of the most active inhibitor compound, 4n, were used

at the calculated IC₅₀/2, IC₅₀, and 2

at different concentrations in the range of 150–0.2929 mM were prepared and used. Measurements were

performed one of two ways, in the presence and in the absence of the inhibitor. Firstly, the solutions of

×

IC₅₀values. The substrate BTCI solution with 10 serial dilutions

compound 4n at three different concentrations were added to the wells (20

BChE (20 L/well), the mixture was incubated under the same conditions as the activity assay. Then,

the reaction was initiated by the addition of Ellman’s reagent (DTNB; 20 L/well, 10 mM) and the

substrate (10 L/well, 75 mM). The absorbance was measured for 10 min at 412 nm. The enzyme

µL/well). After addition of

µ

solution was processed as a control. All readings were adjusted with blank-reading. The results were

analyzed as Lineweaver–Burk plots using Microsoft Ofﬁce Excel 2013 [15].

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3.4. Molecular Docking

The docking study was used to discover the binding modes of the most active compound, 4n

,

to the BChE active site. The structure of Homo sapiens BChE (PDB ID: 4BDS) [32], including tacrine in

the active site, was retrieved from the Protein Data Bank server (ww w .pdb.org). The structure of 4n

was assembled with the Schrödinger Maestro (Schrödinger, 2016) interface [37], and was then directed

to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [38]. The LigPrep

3.8 [39] software was used to accurately prepare compound 4n for the assignment of protonation

regions at pH 8.0

±

1.0 and the atom types. Bond orders and hydrogen atoms in the structure were

ﬁxed as required. The Glide 7.1 program [40] was used to generate the grid, and docking analyses

were carried out using the single-precision docking mode (SP).

3.5. Cell Viability Assay and Selectivity Indexes

Healthy murine embryonic ﬁbroblast cells (NIH3T3 cell line) were used for the cytotoxicity tests.

The cell line and selected compounds were prepared for an MTT assay as reported previously [41].

The optical densities (OD) of 96 well plates were recorded at 540 nm. The percentage inhibition was

determined for each concentration according to the formula below, and the IC₅₀values were calculated

by applying regression analyses using Microsoft Excel 2013 [15].

(OD o f sample)

(OD o f solvent)

% Inhibition = 100 −

× 100.

3.6. BBB Permeability and Drug-Likeness Score (DLS)

With a speciﬁc end goal of assessing some important biological parameters for the active

compounds (4a 4b 4e 4i 4m 4n 4o, and 4t), BBB permeability and DLS were predicted using

,

an online BBB Predictor and Molsoft’s chemical ﬁngerprints mode, respectively [42,43].

4. Conclusions

Due to the complicated nature of AD, scientists are still trying to discover a medication

to cure this disease. One possible way of overcoming this disease is through the discovery

of new compounds that may be useful in its treatment. Hence, 21 new derivatives of

2-(9-acridinylamino)-2-oxoethyl-piperazinyl/piperidinyl/morpholinylcarbodithioate (4a

designed, synthesized, and screened for their inhibition potential toward ChEs. Eight derivatives (4a

4b 4e 4i 4m 4n 4o, and 4t) demonstrated a speciﬁc and promising action against BChE, from which

–4u) were

,

only one compound (4n) was able to inhibit both enzymes. BChE kinetics and molecular modeling

studies were applied to investigate the binding mode of the lead compound, 4n, to the enzyme’s

active site. Moreover, the result of cytotoxic activity against NIH3T3 cells indicated that the active

derivatives showed no cytotoxicity, suggesting a safety proﬁle of these compounds. Similarly, the BBB

permeability and DLS studies indicated the relative success of the most active derivatives.

Supplementary Materials: The following are available online, Table S1: Inhibitory Activity (%) of the compounds

4a–4u against AChE and BChE., Table S2: Inhibitory activity (%) and IC₅₀values of the selected compounds

against AChE and BChE.

Author Contributions: Y.Ö. and Z.A.K. conceived and designed the experiments; W.H. performed the synthesis;

S.L. performed the analysis; B.N.S. performed the activity tests; B.N.S. performed the docking studies; B.K. and

S.I. performed the toxicity tests; W.H., B.N.S., S.L., B.K., S.I., Y.Ö., and Z.A.K. wrote the paper.

Funding: This study was ﬁnancially supported by the Anadolu University Scientiﬁc Projects Fund, Project

No: 1805S191.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Molecules 2018, 23, 2033

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Synthesis and biological evaluation of new cholinesterase inhibitors for Alzheimer's disease

DOI: 10.3390/molecules23082033

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