Multifunctional novel Diallyl disulfide (DADS) derivatives with β-amyloid-reducing, cholinergic, antioxidant and metal chelating properties for the treatment of Alzheimer's disease

Bioorganic & Medicinal Chemistry 23 (2015) 6389–6403

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Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Multifunctional novel Diallyl disulﬁde (DADS) derivatives with

b-amyloid-reducing, cholinergic, antioxidant and metal chelating

properties for the treatment of Alzheimer’s disease

§

⇑

Apra Manral , Vikas Saini , Poonam Meena, Manisha Tiwari

Bio-Organic Chemistry Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi 110007, India

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of novel Diallyl disulﬁde (DADS) derivatives were designed, synthesized and evaluated as chem-

ical agents, which target and modulate multiple facets of Alzheimer’s disease (AD). The results showed

that the target compounds 5a–l and 7e–m exhibited signiﬁcant anti-Ab aggregation activity, considerable

acetylcholinesterase (AChE) inhibition, high selectivity towards AChE over butyrylcholinesterase

(BuChE), potential antioxidant and metal chelating activities. Speciﬁcally, compounds 7k and 7l exhibited

Received 16 July 2015

Revised 20 August 2015

Accepted 22 August 2015

Available online 22 August 2015

highest potency towards self-induced Ab aggregation (74% and 71.4%, 25 lM) and metal chelating ability.

Keywords:

Furthermore, compounds 7k and 7l disaggregated Ab ﬁbrils generated by Cu²⁺-induced Ab aggregation by

80.9% and 78.5%, later conﬁrmed by transmission electron microscope (TEM) analysis. Besides, 7k and 7l

Diallyl disulﬁde derivatives

Alzheimer’s disease

Ab aggregation

had the strongest AChE inhibitory activity with IC₅₀values of 0.056 lM and 0.121 lM, respectively.

Furthermore, molecular modelling studies showed that these compounds were capable of binding simul-

taneously to catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. All the target

compounds displayed moderate to excellent antioxidant activity with ORAC-FL values in the range

0.546–5.86 Trolox equivalents. In addition, absorption, distribution, metabolism and excretion (ADME)

proﬁle and toxicity prediction (TOPKAT) of best compounds 7k and 7l revealed that they have drug like

properties and possess very low toxic effects. Collectively, the results strongly support our assertion that

these compounds could provide good templates for developing new multifunctional agents for AD

treatment.

AChE inhibitors

Antioxidant

Metal chelator

Molecular docking

1. Introduction

to form ﬁbrils than Ab_1–40. The peptide Ab_1–42aggregates into

oligomers and ﬁbrils in the brain and causes strong neuronal tox-

Alzheimer’s disease (AD) is a neurodegenerative disease which

has an associated impact on individuals, families, and society.

Although the exact etiology of AD is not yet fully known, it is

now widely accepted that AD is a multifactorial neurodegenerative

disorder.¹It is characterized by memory loss, language impair-

ment, personality changes and decline in intellectual ability.

Though the disease has been known for more than a 100 years,

it is still incurable due to our incomplete understanding of its

pathogenesis. Neurochemically, AD is characterized by deposition

of amyloid b (Ab)-containing neuritic plaques in the brain parench-

yma followed by neuronal loss.²Ab, a 40–42 amino acid residue, is

a product of transmembrane amyloid precursor protein (APP) that

icity.⁴Finding molecules that ubiquitously reduce the propensity

of these Ab peptides for aggregation is therefore a matter of active

research. Furthermore, the deposited Ab in the brain activates

microglia, and this interaction between microglia and Ab generates

reactive oxygen species (ROS) and cytochemokines leading to sev-

ere neuronal damage.^5,6Besides, ROS such as hydrogen peroxide

(H₂O₂) can also be generated by interaction of Ab and metal ions.⁷

Moreover, recent studies have also indicated that excessive

biometals, such as iron, zinc, and copper, exist in the brains of

AD patients and can promote the aggregation of Ab.⁸Therefore,

modulation of these biometals in the brain may exert potential

therapeutic effects on AD.

The cholinergic pathway plays an important role in the patho-

genesis of AD. Acetylcholine (ACh) can be degraded by two types

of cholinesterases, namely acetylcholinesterase (AChE) and

butyrylcholinesterase (BuChE).⁹AChE acts primarily as a regula-

tory enzyme at cholinergic synapses, while BuChE functions as

an enzyme closely related to AChE and serves as a co-regulator

is cleaved by enzymatic actions of b-secretase and

c

-secretase.³Of

the two most abundant forms of Ab, Ab_1–42has a higher propensity

⇑

Corresponding author. Tel.: +91 11 27666272; fax: +91 11 27666248.

E-mail address: mtiwari07@gmail.com (M. Tiwari).

These authors have contributed equally.

§

http://dx.doi.org/10.1016/j.bmc.2015.08.024

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A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

of cholinergic neurotransmission.^10,11AChE and BuChE inhibition

have been recognized as critical targets for the effective manage-

ment of AD as it increases the availability of synaptic ACh in the

brain regions.¹¹However, BuChE is mainly localized in the periph-

eral tissues including plasma and very small amount is present in

the brain region. Thus, higher selectivity of the compounds

towards AChE over BuChE is considered an advantage as it may

lead to lesser side effects associated with peripheral inhibition

which might be beneﬁcial for the treatment of AD.¹²Additionally,

AChE contains two binding sites: the catalytic active site (CAS) at

the bottom and the peripheral anionic site (PAS) near the entrance

of the gorge.¹³It has been indicated that PAS site of the enzyme

promotes amyloid ﬁbril formation, giving stable AChE–Ab com-

plexes, which are more toxic than single Ab peptides.¹⁴Hence, dual

binding AChE inhibitors targeting Ab aggregation appears to be

promising therapeutic lead compounds.^15–18

Interestingly, our DADS analogs show a structural resemblance

with Curcumin (Fig. 1b and c), a natural compound with a variety

of neuroprotective functions, including anti-inﬂammatory, antiox-

idant, metal chelating and promising anti-Alzheimer’s disease

activity.^32–35Literature review on structure–activity relationships

between Ab and Curcumin have suggested that the presence of

two relatively polar aromatic groups connected by a rigid linker

along with two carbonyl groups were found to be crucial for

interaction with the amyloidogenic core thus inhibiting Ab ﬁbril

formation.^36,37Based on these SAR studies, we modiﬁed our lead

compound by incorporating two keto-moieties into the core struc-

ture of DADS analogs to design a novel series of DADS derivatives

(7e–m) (Fig. 1d). Thus, in the present study, we have reported

the synthesis and evaluation of novel Diallyl disulﬁde derivatives,

5a–l and 7e–m against different processes associated with AD

including Ab anti-aggregation, biometal chelation, cholinergic

and antioxidant properties.

Due to the multi-pathogenesis of AD, the development of agents

that affect two or more relevant targets have drawn considerable

attention for their potential advancements in the treatment of

AD. In recent years many multifunctional agents have been

discovered^19–22with multiple potencies, including cholinesterase

inhibition, Ab deposit inhibition, antioxidative and metal chelating

properties. Natural products are rich and unexplored sources of

novel leading compounds with bioactive properties. One such

compound present in garlic is Diallyl disulphide (DADS) which pos-

sess diverse physiological effects and is both cardioprotective^23,24

2. Results and discussion

2.1. Preliminary screening using in silico tools

The computational tools are considered to be reliable methods

to bring down cost and time required in the drug discovery and

optimization process.³⁸The drugs used for neurological disorders

treatment, such as AD, are generally CNS acting drugs,³⁹so struc-

ture based assessment of CNS penetration proﬁle and other drug

like properties, was deliberated as the ﬁrst step towards analyzing

the applicability of novel chemical entities.

and neuroprotective.^25,26It was found that

a garlic extract

containing DADS among other constituents, prevented APP pro-

cessing and tau phosphorylation in Alzheimer’s transgenic model

Tg2576.^27–29But the potential of DADS is limited by its volatile nat-

ure therefore, to circumvent this problem; our lab took interest in

the synthesis of substituted DADS derivatives which showed

greater stability along with potent antioxidant and anti-inﬂamma-

tory activities.^30,31Taking the aforementioned reasoning into

account, we have synthesized new substituted DADS derivatives

(5a–l) via a novel scheme and evaluated them against various

targets of AD in this current report.

Consequently, we have employed in silico tool such as ADME

predictor to afford preliminary screening of hypothetical mole-

cules using Qikprop module of the Schrödinger program. A set of

over 80 hypothetical DADS derivatives, carrying diverse range of

substituents employed on phenyl ring for instance, electron with-

drawing groups such as –Cl, –F, –Br, –NO₂and electron donating

groups like –OCH₃, –OH and –NH₂varying at position (-ortho,

-meta and -para) were sketched and screened for most relevant

CNS penetration predictors namely Lipinski’s rule of ﬁve

(MW 6 500, QPlogPo/w 6 5, donorHB 6 5, accptHB 6 10), QPlogBB

(brain/blood partition coefﬁcient), Polar surface area (PSA) and

QPlogKhsa (binding to human serum albumin). Meanwhile, other

key pharmacokinetic parameters like QPPCaco (cell model for

gut-blood barrier), QPPMDCK (cell model for blood–brain barrier)

and % human-oral absorption, which mainly account for the ability

of the compound’s distribution inside the body, were also evalu-

ated. By this means, 40 molecules were ﬁltered on the basis of their

compliance with standard range of CNS active drug likeness.

These forty molecules were further screened by docking against

Ab_1–42peptide using Discovery Studio (DS) 4.0 package. Inhibition

of Ab aggregation has been considered as the primary therapeutic

strategy therefore, the ligands exhibiting afﬁnity for the b-amyloid

peptide are more effective at preventing amyloid formation and

inhibiting amyloid neurotoxicity.⁴⁰The X-ray crystal structure of

Ab_1–42peptide (PDB code: 1IYT)⁴¹was obtained from the Protein

Data Bank. Among the successfully docked ligands, the best 21 mole-

cules with highest CDOCKER interaction energies and minimization

energies were chosen for synthesis and further investigation. The

respective minimization energies and CDOCKER interaction energies

of twenty-one derivatives are provided in Table 1 and their ADME

proﬁle are summarised in Table S3 (Supplementary ﬁle).

S

(a) Diallyl Disulfide

R

S

R

(b) Substituted Diallyl Disulfide

O

H₃CO

HO

OCH₃

OH

(c) Curcumin

O

R

S

R

O

2.2. Chemistry

(d) New Diallyl Disulfide derivatives

The intermediates 2a–m, 3a–l, 4a–l and 6e–m were known

compounds and were synthesized by methods described in litera-

ture with slight modiﬁcations.^42–45The target compounds 5a–l

Figure 1. (a) Structure of Diallyl disulﬁde (b) and (c) shows structural resemblance

between DADS analogs and Curcumin. (d) Structure of new DADS analogs.

A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

6391

Table 1

Minimization energies, CDOCKER interaction energies and % inhibition of self-induced Ab_1–42aggregation by target compounds 5a–l and 7e–m and reference compound

Curcumin

O

R

S

R

O

5a-l

7e-m

Compd

Substituent ‘R’

Minimization energy^a,b

(kcal/mol)

CDOCKER interaction energy^a,c

(PDB:1IYT)

Ab_1–42aggregation

inhibition^d(%)

5a

5b

5c

5d

5e

R = 2-F

R = 3-F

R = 3-Cl

R = 3,5-diCl

R = 2,6-diF

R = 4-Cl

R = 3-Br

R = 4-Br

14.389

16.343

34.619

14.587

12.165

14.633

17.428

16.071

2.663

ꢁ3.439

ꢁ4.338

ꢁ6.766

ꢁ4.055

ꢁ3.999

ꢁ4.514

ꢁ3.897

ꢁ3.423

ꢁ4.643

ꢁ4.258

ꢁ8.608

ꢁ10.916

ꢁ5.908

ꢁ6.766

ꢁ7.577

ꢁ4.656

ꢁ12.217

ꢁ18.004

ꢁ20.227

ꢁ20.204

ꢁ17.032

24.7434

33.54 2.79

44.16 2.10

52.41 3.04

47.49 2.52

39.54 1.79

48.16 2.10

39.41 2.04

37.49 2.52

53.01 1.04

58.54 3.79

61.21 1.04

66.89 3.04

62.41 2.08

65.62 2.82

68.74 2.23

52.18 3.07

66.44 2.07

63.54 4.79

74.16 2.10

71.41 3.04

68.97 3.45

51.5 2.68

5f

5g

5h

5i

5j

5k

5l

7e

7f

7g

7h

7i

7j

7k

7l

7m

Curcumin

R = 4-OH

R = 4-OCH₃

R = 3,4-diOCH₃

R = 4-OH, 3-OCH₃

R = 4-F

R = 4-Cl

R = 3-Br

R = 4-Br

R = 4-OH

R = 4-OCH₃

R = 3,4-diOCH₃

R = 4-OH, 3-OCH₃

R = 2,4-diOMe

13.862

18.747

8.469

ꢁ14.672

ꢁ13.486

ꢁ12.819

ꢁ14.742

ꢁ14.736

ꢁ12.604

ꢁ11.220

ꢁ23.727

31.795

ꢁ13.086

Bold values are represent the most active two compounds 7k and 7l.

a

Minimization energies and CDOCKER interaction energies were calculated using CDOCKER program of Discovery Studio.

Minimization energy signify the inherent stability of the compound.

CDOCKER interaction energy denotes binding afﬁnity between receptor–ligand complex.

b

c

d

Inhibition of self-induced Ab_1–42aggregation (25

lM) by tested inhibitors at 25

l

M by thioﬂavin-T based ﬂuorescence method. Data are presented as the mean SD of

three independent experiments.

were synthesized via the route outlined in Scheme 1. The commer-

cially available cinnamaldehydes (1a–m) were used as starting

material to synthesize corresponding cinnamic acids (2a–2m) by

Knoevenagel condensation.⁴²Cinnamic acids (2a–2l) were trans-

formed into mixed acyl anhydrides using ethylchloroformate in

tetrahydrofuran (THF) and reduced thereafter using sodium boro-

hydride/methanol at room temperature to afford corresponding

cinnamyl alcohols⁴³(3a–l). Thionyl chloride was then added to cin-

namyl alcohols in the presence of pyridine to provide cinnamyl

halides.⁴⁴Finally, cinnamyl chlorides (4a–l) were reacted with

sodium hydrosulﬁde in the presence of triethylamine hydrochlo-

ride⁴⁵to obtain corresponding substituted cinnamyl mercaptans

which further undergoes oxidation to yield title compounds (5a–l).

The general procedure for the synthesis of title compounds

7e–m is depicted in Scheme 2. The method used to prepare inter-

mediates compounds (6e–m) was similar to that of corresponding

step 3 as described in Scheme 1 with some modiﬁcations. Interme-

diates 2e–m were reﬂuxed with oxalyl chloride in THF at 65–70 °C

to obtain substituted cinnamoyl halides (6e–m). The ﬁnal com-

pounds (7e–m) were also synthesized in similar conditions as

described in corresponding step 4 of Scheme 1. The structures

and purities of the target compounds were conﬁrmed by ¹H

NMR, ¹³C NMR, MS and elemental analysis. All the target com-

pounds 5a–l and 7e–m are new, except compound 5j, which was

previously reported⁴⁶using a different synthesis procedure.

R

Cl

4a- 4l

1

R

S

5a-5l

R

4a, 5a: R = 2-F

4b, 5b: R = 3-F

4c, 5c: R = 3-Cl

4d, 5d: R = 3,5- diCl

4e, 5e: R = 2,6-diF

4f, 5f: R = 4-Cl

4g,5g: R = 3-Br

4h, 5h: R = 4-Br

4i, 5i: R = OH

4j, 5j: R = 4-OCH₃

4k, 5k: R = 3,4-diOCH₃

4l, 5l: R = 4-OH, 3-OCH₃

Scheme 1. Synthesis of ﬁnal compounds Bis[3-(substituted phenyl)prop-2-ene]

disulphide (5a–5l). Reagents and conditions: (i) NaHS, Et₃NꢀHCl in MeOH, rt for 3–

4 h.

Thioﬂavin-T (ThT) to monitor and quantify the aggregation of

Ab_1–42synthetic peptide into ﬁbrils.⁴⁷Curcumin (Cur) was used

as reference compound and the results are summarised in Table 1.

From the results, it is evident that most derivatives exhibited

moderate-to-good inhibition potencies (33–74%) compared with

2.3. Inhibition of self-mediated Ab_1–42aggregation

To examine the anti-amyloidogenic activity of the new com-

pounds, our experimental approach relies on in vitro assays with

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A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

compounds 5a and 5f bearing ortho-ﬂuoro (33.5%) and para-chloro

(48.1%) groups on their phenyl ring. On comparing the two series

5a–l and 7e–m, it was found that introduction of keto-moieties

in the core structure had favourable contribution towards superior

inhibition potencies of compounds 7e–m (52–74%) as compared to

compounds lacking keto-moiety 5a–l (33–66%), which were con-

sistent with their respective CDOCKER interaction energies. The

most potent compounds were found to be 7k (3,4-dimethoxy

substituent) and 7l (4-hydroxy, 3-methoxy substituent) with

inhibition ratio of 74.16% and 71.41%, respectively. For better

understanding of molecular interactions responsible for Ab aggre-

gation inhibitory activity of these compounds, molecular docking

studies were followed.

R

Cl

6e-6m

O

1

O

R

S

R

O

7e-7m

6e,7e: R = 4-F

6f, 7f: R = 4-Cl

6g,7g: R = 3-Br

6h,7h: R = 4-Br

2.4. Docking study of 7k and 7l with Ab_1–42peptide

Previous studies have reported that the C-terminus region of

Ab_1–42, formed by residues Gln15-Ala21 and Ala30-Ala42, have

the highest local propensity to aggregate.⁴⁸Particularly, it contains

residue Lys28, which is involved in the stabilization of the

monomer hairpin structure during b-sheet conversion.⁴⁸Hence,

molecules binding to C-terminus of Ab_1–42could decrease the

formation of b-sheet which in turn inhibits Ab aggregation.⁴⁹

Besides, this domain is important owing to the nearby presence

of Met35, a critical residue involved in oxidative stress and neuro-

toxicity.⁵⁰As shown in Figure 2, compound 7k could bind to the

C-terminus of Ab_1–42peptide by forming Van der Waals interac-

tions with important residues such as Met35, Ala31, Ile32, Gly25

and Gly29. A hydrogen bond interaction was observed between

oxygen atom of keto-moiety and hydrogen atom of amine group

of Lys28 at a close distance of 5.8 Å. The positively charged nitro-

6i, 7i:

6j, 7j:

R = 4-OH

R = 4-OCH₃

6k,7k: R = 3, 4-diOCH₃

6l, 7l: R = 4-OH, 3-OCH₃

6m,7m: R = 2, 4-diOCH₃

Scheme 2. Synthesis of ﬁnal compounds Bis[2-(substituted phenyl)-ethenyl] dithio

peroxy anhydride (7e–7m). Reagents and conditions: (i) NaHS, Et₃NꢀHCl in MeOH,

rt for 3–4 h.

Curcumin (51.5%) in a concentration ratio of 1:1 [Ab (25

hibitor (25 M)]. From the inhibition values of compounds 5a–l

lM):in-

l

and 7e–m, it was found that the type and location of the sub-

stituent on the phenyl ring had great effects on the Ab_1–42aggrega-

tion inhibitory activity. It appeared that the compounds with an

electron-donating group (such as –OCH₃, –OH etc.) on the phenyl

ring displayed higher Ab self-induced aggregation inhibitory activ-

ity, while the compounds with an electron withdrawing group

(such as –F, –Cl, and –Br) on the phenyl ring gave weaker activity.

Moreover, the electron withdrawing groups at meta position for

instance, compounds 5b and 5c featuring meta-ﬂuoro and meta-

chloro substituent, respectively, on phenyl ring displayed slightly

better inhibition value (44.1% and 52.4%) than corresponding

gen atom present on residue Lys28 was also engaged in cation–

interaction with phenyl ring of 7k, further strengthening the stabil-

ity of the compound 7k/Ab_1–42complex.

Similarly, compound 7l was also seen to be interacting with

C-terminus of Ab_1–42peptide as shown in Figure 3. Interestingly,

Lys28 residue was again found to be involved in forming major

p

interactions such as a cation–p interaction with aromatic ring of

Figure 2. 2D schematic diagram of docking model of compound 7k with Ab_1–42. Residues involved in hydrogen-bonding, charge or polar interactions are represented by

magenta-coloured circles. Van der Waals interactions are represented by green circles. The solvent accessible surface of an atom is represented by a blue halo around the

atom. Hydrogen-bond interactions as blue dashed line, p–p and cationic–p interactions are represented by an orange line with symbols indicating the interaction.

A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

6393

Figure 3. 2D schematic diagram of docking model of compound 7l with Ab_1–42peptide. Residues involved in hydrogen-bonding, charge or polar interactions are represented

by magenta-coloured circles. Van der Waals interactions are represented by green circles. The solvent accessible surface of an atom is represented by a blue halo around the

atom. Hydrogen-bond interactions as blue dashed line,

p–p

and cationic–

p

interactions are represented by an orange line with symbols indicating the interaction.

Table 2

7l and a hydrogen bond formation between sulfur atom of 7l and

hydrogen of amino group of Lys28 at a distance of 6.1 Å. Other

important residues including Met35 were also seen in receptive

zone of compound 7l by means of electrostatic and Van der Waals

interactions. Overall, we can conclude that compounds 7k and 7l

might prevent Ab aggregation by directly interacting with residue

Lys28 and thus blocking the random coil ? b-sheet conversion and

in turn Ab ﬁbril formation. Moreover, their interactions with other

important residues of C-terminus that participate in Ab_1–42

aggregation may also provide an explanation of their b-amyloid

aggregation inhibition activity.

In vitro inhibition of AChE and BuChE and oxygen radical absorbance capacity (ORAC,

Trolox equivalents) of 5a–l, 7e–7m, and donepezil

Compd

IC₅₀S.D^a

(

l

M)

Selectivity

ORAC (lM of

index^b(BuChE/ Trolox

BuChE (

l

M) AChE (lM)

AChE)

equivalents)

5a

5b

5c

5d

5e

5f

5g

5h

5i

5j

5k

5l

7e

7f

7g

7h

7i

7j

7k

7l

7m

Donepezil

>100

48.67 1.70

53.24 2.61

44.16 2.91

64.62 1.08

42.33 2.92

>2

0.54 0.03

0.87 0.04

1.43 0.08

1.72 0.05

1.30 0.29

1.39 0.11

2.35 0.14

2.41 0.16

2.41 0.17

2.31 0.05

2.69 0.09

2.35 0.14

3.09 0.01

3.38 0.05

2.04 0.03

1.98 0.04

4.47 0.27

4.29 0.65

5.24 0.37

5.86 0.98

5.09 0.72

nd^c

>1.8

>2.2

>1.5

>2.3

2.5

3.1

3.4

5.1

7.1

50.1

39.9

72.0

69.8

71.7

94.16 2.10 36.96 2.11

75.41 0.04 23.71 1.69

72.49 0.52 20.85 0.97

63.01 1.04 12.27 0.61

59.54 0.79

55.21 1.04

57.89 0.74

32.41 1.08

35.62 0.52

38.74 0.23

37.18 0.17

24.54 0.79

15.98 0.34

2.5. Antioxidant activity

8.29 0.44

1.10 0.14

1.45 0.02

0.45 0.07

0.51 0.07

0.54 0.06

0.34 0.09 109.3

0.21 1.70 116.8

0.22 0.06

The antioxidant activities of the target compounds were deter-

mined through the oxygen radical absorbance capacity assay using

ﬂuorescein (ORAC-FL).⁵¹Peroxyl radicals were thermally generated

from 2,2⁰-azobis-(amidinopropane) dihydrochloride and reacted

with ﬂuorescein to form non-ﬂuorescent products at 535 nm. The

antioxidant capacity of synthesized derivatives 5a–l and 7e–l were

determined by their competition with ﬂuorescein in the radical

capture, using a ﬂuorescence microplate reader. The vitamin E ana-

logue Trolox was used as standard, and antioxidant activity is

72.6

13.64 0.27 0.056 0.05 243.5

12.87 0.81 0.121 0.06 106.3

14.89 0.98 0.142 0.03 104.8

8.71 1.36 0.049 0.05 177.7

expressed as Trolox equivalents (lmol of Trolox/lmol of tested

compound). As shown in Table 2, all the derivatives demonstrated

modest to excellent antioxidant activity with ORAC-FL values in

range 0.546–5.86 Trolox equivalents. In particular, compounds 7l

and 7k showed the most potent antioxidant activity with ORAC-

FL values of 5.86 and 5.24 Trolox equivalents, respectively, owing

to the presence of free –OH and –OCH₃group on phenyl ring,

which might be crucial to the radical scavenging ability.

Bold values are represent the most active two compounds 7k and 7l.

a

IC₅₀values are expressed as mean standard deviation (SD) of at least three

experiments.

b

SI selectivity index; IC₅₀EqBuChE/IC₅₀EeAChE.

Not determined.

c

Most of the target compounds showed signiﬁcant AChE inhibi-

tory activities with IC₅₀values ranging from submicromolar to

micromolar, and demonstrated comparatively weaker BuChE

inhibitory activities, indicating that these derivatives were pre-

dominantly inhibiting AChE. Among the DADS derivatives series

5a–l, the compounds containing electron-drawing groups on their

phenyl ring (5a–h) showed modest AChE inhibition while their

inhibitory potency was much less for BuChE. For instance, the

2.6. Inhibition studies on AChE and BuChE

To explore the inhibition potencies of the synthesized

compounds 5a–l and 7e–m towards AChE and BuChE, modiﬁed

Ellman’s method⁵²was used. The compounds were evaluated

using AChE and BuChE obtained from electric eel and equine

serum, respectively. Donepezil was used as reference compound.

The IC₅₀values and selectivity index for the inhibition of AChE

and BuChE are summarised in Table 2.

approximate IC₅₀values of 5a–d were 48

lM, 53.2

lM, 44 lM

and 64.6 M for AChE and it exceeds >100

l

lM against BuChE.

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A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

Within this series, electron rich substituent (5i–l), displayed higher

inhibitory potency than their electron-drawing counterparts (5a–

h). For instance, the best inhibitory potency in this series was

the key residues in the binding site is shown in Figure 4. The phe-

nyl ring of compound 7k was involved in interaction with

Trp279 of PAS site, with a distance of 4.8 Å. A hydrogen bond

between the oxygen atom of carbonyl moiety and hydrogen of

OH group of Tyr334 was observed, with a close distance of 2.5 Å.

Besides, the other phenyl ring of 7k was bound to the CAS of AChE,

p–p

achieved by 3,4-dimethoxy bearing compound 5k (IC₅₀= ꢂ1.1

which exhibited 18.9 times stronger AChE inhibition than 4-bromo

containing compound 5h (IC₅₀= ꢂ20.85 M).

lM)

l

Moreover, compound 5k displayed IC₅₀= ꢂ55.21

l

M for BuChE

it being stacked against indole group of Trp84 through p–p interac-

and thus possess higher selectivity towards AChE inhibition (50

tion, with the ring to ring distance of 4.5 Å. All these interactions

clearly indicated that compound 7k can simultaneously binds to

CAS and PAS of AChE. In addition, the CDOCKER interaction energy

of compound 7k [(ꢁ56.931 kcal/mol), Supplementary material] was

a slightly higher than standard Donepezil (ꢁ55.321 kcal/mol) which

further conﬁrms its strong binding afﬁnity towards AChE enzyme.

Similarly, interaction of compound 7l with key residues of

TcAChE was also studied (Fig. 3, Supplementary material). The

folds).

In extension of the above study, new DADS derivatives (7e–m)

were synthesized to achieve more potent compounds. With intro-

duction of two carbonyl moieties into the main pharmacophore,

inhibitory activity and selectivity of all the synthesized derivatives

have increased substantially as compared to the aforementioned

series (5a–l). Particularly, compounds 7k and 7l displayed the

maximum potency and selectivity for AChE, with their IC₅₀values

binding afﬁnity can be attributed to

p–p stacking interaction

of 0.056

106.3 fold, respectively.

l

M and 0.121

l

M, and selectivity indexes of 243.5 and

between the phenyl moiety and Trp84 residue of CAS site with a

distance of 4.8 Å. Furthermore, the hydroxyl group on the same

phenyl ring was engaged in hydrogen bond formation with

Glu199 residue at a close distance of 2.1 Å. In addition, another

hydrogen bond formation occurred at PAS site between oxygen

of carbonyl moiety and Tyr121 residue (2.4 Å).

A similar docking study was performed on reported crystal

structure of Human BuChE (HuBuChE). The active site of HuBuChE

consists of residues Trp82, Ala328 and His438 forming catalytic

anionic site (CAS) and peripheral anionic site (PAS)⁵⁴having

residues Asn68, Asp70, Glu197, Ala277 and Tyr332. As shown in

Figure 5, compound 7k could occupy the large catalytic cavity of

HuBuChE, where oxygen atom of methoxy group on aromatic ring

forms hydrogen bond with Tyr332. No other notable interaction

was observed between 7k and BuChE. In addition, the calculated

CDOCKER interaction energies of compound 7k for AChE was evi-

dently higher than that for BuChE (ꢁ56.931 and ꢁ41.470 kcal/mol,

respectively), as shown in Table S4 (Supplementary material),

which explain relatively weaker inhibition of BuChE and higher

selectivity for AChE.

The best compound 7k from new series (7e–m), was compara-

tively better than the most active compound 5k from the corre-

sponding series (5a–l) in terms of inhibitory potency and about 5

times higher selectivity for AChE enzyme over BuChE. Simultane-

ously, the potency of compound 7k was found to be comparable

to standard Donepezil (IC₅₀= 0.049 lM). In addition, the results

also suggested that the inhibitory activity of compounds was inﬂu-

enced by the type of substituent on aromatic ring in both the

series. The order of activity for substituted groups on ring is as

follows: 3,4-dimethoxy > 4-hydroxy, 3-methoxy > 4-methoxy > 4-

hydroxy > bromo > chloro > ﬂuoro. These results proposed that

the electron density of aromatic ring might be associated with

the inhibitory activity of compounds towards the cholinesterase

enzymes.

2.7. Molecular docking study of 7k and 7l with AChE and BuChE

To explore the possible mode of interaction of the best deriva-

tives 7k and 7l towards TcAChE (PDB code: 1EVE) and HuBuChE

(PDB code: 1POI), molecular modelling studies were carried out

utilizing the docking program DS Client v 4.0 package. The active

site of TcAChE enzyme consists of catalytic anionic site (CAS) con-

taining Trp84 and Phe330 residues and the peripheral anionic site

(PAS) comprising mainly of Tyr70, Asp72, Tyr121, Tyr334, and

Trp279 residues.⁵³The position of compound 7k with respect to

Similarly, compound 7l was also seen to be involved through

hydrogen bond formation with Asn83, Tyr440 and Trp82. However,

taking into account the fact that only residue Trp82 forms a part of

catalytic triad of BuChE (Fig. 4, Supplementary material), the sig-

niﬁcance of these interactions in terms of inhibitory potency were

weaker than AChE which was also corroborated by their CDOCKER

interaction energies and in vitro enzyme inhibition studies. Hence,

Figure 4. 2D schematic diagram of docking model of compound 7k with TcAChE. Residues involved in hydrogen-bonding, charge or polar interactions are represented by

magenta-coloured circles. Van der Waals interactions are represented by green circles. The solvent accessible surface of an atom is represented by a blue halo around the

atom. Hydrogen-bond interactions as blue dashed line, p–p and cationic–p interactions are represented by an orange line with symbols indicating the interaction.

A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

6395

Figure 5. 2D schematic diagram of docking model of compound 7k with HuBuChE. Residues involved in hydrogen-bonding, charge or polar interactions are represented by

magenta-colored circles. Van der Waals interactions are represented by green circles. The solvent accessible surface of an atom is represented by a blue halo around the atom.

Hydrogen-bond interactions as blue dashed line.

it was reasonably concluded that both 7k and 7l exhibited admir-

able selectivity for the inhibition of AChE over BuChE.

7k and 7l by a ThT-binding assay.⁵⁶Curcumin was used as reference

compound. It could be seen from Figure 7, the ﬂuorescence of Ab

treated with Cu²⁺is 158.6% that of Ab alone (100%), which indicates

that Cu²⁺accelerates Ab aggregation. By contrast, the ﬂuorescence of

Ab treated with Cu²⁺and the tested compounds decreased dramati-

cally (7k, 80.9% inhibition of Cu²⁺-induced Ab aggregation; 7l, 78.5%

inhibition and Curcumin, 65.9% inhibition) at a concentration ratio of

2.8. Metal-chelating properties of compound 7k

The ability of compound 7k to chelate biometals such as Cu²⁺and

Fe²⁺was studied by UV–Visible spectrometry.⁵⁵In the absence of

metal ions, the UV–Vis spectrum of compound 7k showed the

absorption maximum at 288 nm and at 314 nm. When CuCl₂was

added, a red shift in the maximum absorption from 288 nm to

294 nm and 314 nm to 324 nm occurred, indicating the formation

of a 7k–Cu²⁺complex (Fig. 6a). This chelating ability can be reason-

ably attributed to the presence of dimethoxy group on phenyl ring

and keto-moieties in the core structure of the compound. Similar

results were obtained when FeSO₄was added; the peak shift in the

electronic spectra of 7k was found similar to that of Cu²⁺, indicating

that compound 7k can simultaneously bind to both bivalent cations.

The stoichiometry of the 7k–Cu²⁺complex was determined

using the molar ratio method by preparing solutions of compound

7k with increasing amounts of CuCl₂. The UV spectra were used to

obtain the absorbance of the complex of 7k and CuCl₂at different

concentrations at 324 nm. The UV spectra were recorded and trea-

ted by numerical subtraction of CuCl₂and 7k at corresponding

concentrations, plotted versus the mole fraction of 7k. As shown

in Figure 6b, initially, the absorbance increased linearly and then

became stable. The two straight lines intersected at a mole fraction

of 1.1, revealing a 1:1 stoichiometry for the complex.

1:1:2 [Ab (25 l lM):Inhibitor (50 lM)]. These results

M):Cu²⁺(25

suggested that our compounds could inhibit Cu²⁺-induced Ab_1–42

aggregation effectively by chelating Cu²⁺ions.

2.10. Inhibition of compounds 7k and 7l on Cu²⁺-induced Ab_1–42

aggregation monitored by TEM

To further conﬁrm the ability of compounds 7k and 7l to inhibit

Cu²⁺-mediated Ab_1–42aggregation, TEM analysis was performed.⁵⁷

As shown in Figure 8c, Cu²⁺(25

l

M) could accelerate the aggrega-

tion of Ab_1–42(25

lM) which was evident by presence of denser

amyloid aggregates as compared to Ab_1–42alone (Fig. 8b) under

identical conditions. However, disaggregation effects were clearly

visible upon addition of compounds 7k and 7l (50 lM each), as

few and slender Ab ﬁbrils were observed (Fig. 8e and f). The results

of TEM are consistent with ThT binding assay results which sug-

gested that compounds 7k and 7l could inhibit Cu²⁺-induced Ab

aggregation noticeably through chelating Cu²⁺ions.

2.11. Control of Cu²⁺-Ab H₂O₂production by compounds 7k

and 7l

The similar study was performed on compound 7l with Cu²⁺

and Fe²⁺ions (Fig. 5a, Supplementary ﬁle). Upon addition of CuCl₂

and FeSO₄, the curve displayed a red shift (the maximum absorp-

tion peak at 288 nm shifts to 318 nm) suggesting formation of

complex between 7l-metal ions. However, there were no signiﬁ-

cant difference between shifts in 7l–Cu²⁺and 7l–Fe²⁺peaks which

suggests that 7l binds non-selectively to both these cations. The

stoichiometric ratio of the 7l–Cu²⁺complex was determined at

318 nm (Fig. 5b, Supplementary ﬁle). The mole fraction was found

to be 1.2, suggesting a 1:1 stoichiometry for the complex.

Binding of redox active metal ions such as Cu²⁺to Ab species is

known to be involved in generation of ROS such as H₂O₂and sub-

sequent facilitation of Ab aggregation and neurotoxicity. The effect

of compounds 7k and 7l on H₂O₂production by Cu²⁺-Ab_1–42spe-

cies was examined using the HRP/Amplex Red assay.⁵⁸It was

observed from Figure 9 that addition of compounds 7k and 7l have

reduced the production of H₂O₂by about 72% and 62% as compared

to Cu²⁺-Ab_1–42species. Comparatively, the strong chelator

ethylenediamine tetraacetic acid (EDTA) showed an even more

pronounced effect (>80%). Overall, these results support not only

the strong chelating ability of compounds 7k and 7l for Cu²⁺, but

also their ability to control ROS formation and the redox properties

of both free Cu²⁺and Cu²⁺-Ab_1–42species, important multifunc-

tional features needed for the future use of these compounds.

2.9. Effect of compounds 7k and 7l on Cu²⁺-induced Ab_1–42

aggregation

To investigate the ability of the DADS derivatives to inhibit Cu²

⁺-induced Ab_1–42aggregation, we studied most potent compounds

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A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

Figure 6. (a) The UV spectrum of compound 7k (50 lM) alone (Green) or in the presence of 50 lM CuCl₂(Red) and 50 lM FeSO₄(blue). (b) Determination of the

stoichiometry of complex 7k–Cu(II) by using molar ratio method through titrating the methanol solution of compound 7k with ascending amounts of CuCl₂. The ﬁnal

concentration of the tested compound 7k was 50 l lM to 80 lM.

M, and the ﬁnal concentration of Cu²⁺ranged from 5

2.12. In vitro cytotoxicity assay

compounds did not exhibit cytotoxic effect on the cells in the range

of concentrations, which includes the highest concentration

To determine the potential cytotoxic effects of the most potent

compounds 7k and 7l, the human embryonic kidney cells (HEK

293) were treated with different concentrations of compounds

(50 lM) used in the experiment.

2.13. Toxicity prediction

(0.1–100 lM). After exposing the cells to these compounds for

48 h, the cell viability was evaluated by 3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium (MTT) assay.⁵⁹Figure 10 shows a

dose-dependent effect of tested compounds 7k and 7l on the

viability of HEK cells at different concentration in a range from

Toxicity predictor (TOPKAT module of Accelrys Discovery Stu-

dio 4.0) computes a probable value of toxicity for a submitted

chemical structure using quantitative structure–activity relation-

ship (QSTR) equation. Parameters for toxicity risk such as carcino-

genicity, mutagenicity, skin irritancy, ocular irritancy, aerobic

biodegradability and developmental toxicity potential etc., were

observed for best derivatives 7k and 7l. The results (Table S5, Sup-

plementary material) indicated that for all the default parameters,

computed probabilities of toxicity were low to intermediate in

0.1 to 100

95.4 3.6%; 25

88.6 3.1%) and (0.1

94.4 4.6%; 25 M: 92.3 5.4%; 50

l

M (0.1

M: 95.7 5.4%; 50

M: 97.3 3.6%; 1

M: 90.1 4.5%; 100

l

M: 98.9 4.7%; 1

M: 92.8 2.5%; 100

M: 96.5 4.5%; 10

l

M: 96.1 3.5%; 10

lM:

l

80.9 2.9%), respectively. The results demonstrated that new

A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

6397

the predicted ADME properties and TOPKAT analysis revealed that

the compounds possess drug-likeness.

3. Conclusions

In summary, 21 new DADS analogs (5a–l and 7e–m) have been

synthesized and evaluated as novel multifunctional agents against

AD. Most of these compounds exhibited multifunctional activity

which included a signiﬁcant ability to inhibit self-induced Ab

aggregation, potent AChE inhibition and displayed high selectivity

for AChE over BuChE. They also acted as antioxidants and biometal

chelators. Particularly, compound 7k exhibited the highest potency

towards self-induced Ab aggregation (74.16%, 25 lM) as conﬁrmed

by molecular docking studies. Compounds 7k and 7l also exhibited

signiﬁcant antioxidant (ORAC-FL value of 5.24 and 5.86, respec-

tively) and potent AChE inhibitory activity (IC₅₀= 0.056

lM and

0.121 M) along with notable selectivity ratio 243.5 and 106.3 fold,

l

respectively. Molecular modelling studies suggested that com-

pounds 7k and 7l showed simultaneous binding to both CAS and

PAS of AChE, and thus inducing a strong inhibitory effect. More-

over, compounds 7k and 7l were also found to be good biometal

chelators and inhibit metal induced Ab aggregation (80.9% and

Figure 7. ThT binding assay for Cu²⁺-induced Ab aggregation, and test compound

induced Ab disaggregation was carried out with incubation for 2 min, followed with

test compound induced Ab disaggregation upon incubation at 37 °C for 24 h. The

78.5%, at 50 lM). Compared with other recently developed struc-

tural families that feature similar multitarget proﬁles for instance,

tacrine–caffeic acid hybrids,¹⁹tacrine–ﬂavonoid hybrids²⁰and

tacrine–coumarin²¹hybrids etc., the two lead compounds 7k and

7l represent a simpler chemical structure with comparable anti-

Ab aggregation, antioxidant and metal chelating activities but

weaker cholinesterase inhibition as compared to tacrine hybrids.

Importantly, ADME and TOPKAT analysis suggested that the lead

compounds 7k and 7l exhibit appropriate drug like properties

and were largely non-toxic. Moreover, cytotoxicity assays per-

incubations were carried out with following reagents: (a) 25

M Cu²⁺; (c) 25 M Cu²⁺, and 50

25 M Ab_1–42and 25 M Ab_1–42, 25

M Cu²⁺, and 50

(d) 25 M Ab_1–42, 25 M compound 7k; (e) 25 M Ab_1–42, 25 lM

l

M Ab_1–42alone; (b)

l

M Curcumin;

l

Cu²⁺, and 50

lM compound 7l. Values are reported as the mean SD of three

independent experiments. ^*p < 0.05, ^**p < 0.01.

compound 7k. The compound 7l was also found to be non-toxic for

most part, with a few exceptions, for instance, it was found to be a

skin irritant which could be due to the presence of phenolic group

in its structure and therefore, needs further optimization. Overall,

formed at concentration (0.1–100 lM) did not indicate toxicity.

Overall, the multifunctional properties highlight compounds 7k

and 7l as interesting candidates for further development as lead

Figure 8. TEM images for Cu²⁺-induced Ab aggregation and test compound induced Ab disaggregation. The incubations were carried out with following reagents: (a) 25

Ab_1–42alone at 0 h; (b) 25 M Ab_1–42alone at 24 h, 37 °C; (c) 25 M Ab_1–42and 25 M Ab_1–42, 25 M Curcumin; (e) 25 M Ab_1–42, 25

M Cu²⁺; (d) 25 M Cu²⁺and 50 M Cu²⁺

and 50 M compound 7k; (f) 25 M Ab_1–42, 25 M compound 7l.

M Cu²⁺and 25

lM

l

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A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

4.2. General procedure for the synthesis of intermediates 2a–

2m (Scheme 1)

3 g (28.6 mmol) of malonic acid was added to a stirred solution

of (13 mmol) substituted aromatic aldehydes (1a–1m) in pyridine

(30 mL) and piperidine (3 mL). The mixture was heated to temper-

ature of 90–105 °C for 5–6 h under reﬂux. After cooling, the mix-

ture was poured into cold water and acidiﬁed with concd HCl.

The precipitated white crystals were ﬁltered, washed with 4ꢃ

200 mL water and dried at 45 °C and were further identiﬁed

through ESI-MS and NMR (overall yield 70–80%).

4.3. General procedure for the synthesis of intermediates 3a–3l

To a 250 mL round-bottom ﬂask containing 10 mmol of substi-

tuted cinnamic acid (2a–2l), 30 mL of dry THF and 19.9 mmol tri-

ethylamine at ꢁ7 °C, 10 mmol of ethyl chloroformate was added

dropwise. After being stirred for an additional 30 min at the same

temperature, the mixture was ﬁltered with suction and cake was

washed with THF. The reaction mixture was allowed to warm to

10 °C and 2.7 g (30 mmol) of anhydrous sodium borohydride was

added in one portion. To this suspension, 15 mL of absolute metha-

nol was added dropwise over 1 h at the same temperature. After

being stirred at room temperature for additional 1 h, the reaction

mixture was poured into water and extracted with dichloro-

methane. The combined organic layer were dried over anhydrous

Na₂SO₄and concentrated. The residue obtained was puriﬁed by

column chromatography using hexane/ethyl acetate (60/40) as

eluent to afford the pure compounds 3a–3l (overall yield 60–

75%) and were further identiﬁed through ESI-MS and NMR.

Figure 9. Shows production of H₂O₂from reactions of Ab, Cu²⁺, and compound

upon addition of ascorbate, as determined by an HRP/Amplex-Red assay. [Ab]

= 200 nM, [Cu²⁺] = 400 nM, [chelator] = 800 nM, [ascorbate] = 10 mM, [Amplex

Red] = 50 nM, [HRP] = 0.1 U/mL, and k_ex/k_em= 530/590 nm). Values are reported as

the mean SEM of three independent experiments.

molecules directed towards the development of novel drugs for the

treatment of AD.

4. Experimental section

4.1. Chemistry

4.4. General procedure for the synthesis of 4a–4l

All organic solvents and common reagents were procured from

Merck India Ltd. All the aromatic aldehydes and other reagents

were procured from Aldrich Chemical Company, Inc. USA. The

chemical reactions were monitored by TLC using commercially

available alumina plates coated with silica gel 60 F254 (Merck).

The purity of all organic compounds was conﬁrmed by TLC, ¹H

NMR, ¹³C NMR, Mass spectroscopy and elemental analysis. ¹H

NMR and ¹³C NMR spectras were recorded in Jeol instrument oper-

ating at 400 MHz (¹H) in CDCl₃and at 100 MHz (¹³C) using TMS as

an internal standard. The chemical shifts are reported in parts per

million (d) downﬁeld from TMS and coupling constants are

reported in hertz (Hz). Proton coupling patterns are abbreviated

as single (s), broad singlet (br s), double (d), double doublet (dd)

triplet (t) and multiple (m). Mass spectra were recorded on a Qstar

(Applied biosystem) ESI-MS mass spectrometer.

To a solution of thionyl chloride (6 mL) in dichloromethane,

13 mmol of substituted cinnamyl alcohols (3a–3m) was slowly

added in portions. The mixture was stirred for 30 min at 8 °C and

allowed to warm to room temperature followed by stirring for

3–4 h till evolution of SO₂is ceased. The solution was dried care-

fully using rota vapour under reduced pressure and the product

obtained 4a–4l was kept as it is without puriﬁcation by air tight

cork and used immediately to prepare ﬁnal product.

4.5. General procedure for the synthesis of ﬁnal compounds

5a–5l

1.12 g of sodium hydrosulﬁde (10 mmol) was dissolved in

methanol in the presence of triethylamine hydrochloride solution

(10 mmol) followed by addition of dried compound 4a–4l, respec-

tively. It was kept on continuous stirring for 4 h at room tempera-

ture under N₂atmosphere. The reaction mixture was poured into

water and extracted with dichloromethane. The combined organic

layer was separated, dried over anhydrous sodium sulphate and

evaporated under reduced pressure. The residue obtained was

puriﬁed by column chromatography using mixture of hexane/ethyl

acetate (60/40) as eluent to afford the title compounds (5a–5l).

110

Comp.7k

Comp.7l

100

90

80

70

4.5.1. Bis[3-(2-ﬂuorophenyl)prop-2-ene]disulphide (5a)

Intermediate 4a was treated according to general procedure to

give the desired product 5a as dull white solid (68% yield). Mp

156–158 °C. ¹H NMR (400 MHz, CDCl₃), d (ppm): 7.38 (d, 2H,

J = 8.2 Hz), 7.09–7.12 (m, 2H), 6.93–6.98 (m, 4H), 6.63 (d, 2H,

J = 9.1 Hz), 6.23–6.29 (m, 2H), 3.51 (d, 4H, J = 6.2 Hz). ¹³C NMR

(100 MHz, CDCl₃): 161.3, 129.8, 128.5, 127.2, 124.9, 124.0, 121.1,

115.5, 33.3. MS m/z: 334.0 [M⁺]; Anal. Calcd for C₁₈H₁₆F₂S₂: C,

64.64; H, 4.82; F, 11.36; S, 19.17; Found: C, 64.43; H, 4.75; S, 19.34.

0

50

100

150

concentration (µM)

Figure 10. Effect of compounds 7k and 7l on the viability of HEK 293 cells. The cells

were incubated with the indicated concentrations of the tested compounds for 48 h.

The cell viability was assessed by MTT assay. Percentages of the cell viability are

presented as mean SEM from 3 independent experiments.

A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

6399

4.5.2. Bis[3-(3-ﬂuorophenyl)prop-2-ene]disulphide (5b)

453.9 [M⁺, 49.3], 455.9 [(M+2)⁺, 100], 457.9 [(M+4)⁺, 54.9], Anal.

Calcd for C₁₈H₁₆Br₂S₂: C, 47.38; H, 3.53; Br, 35.03; S, 14.06; Found:

C, 47.26; H, 3.44; S, 14.43

Intermediate 4b was treated according to general procedure to

give the desired product 5b as dull white solid (52% yield). Mp

154–156 °C. ¹H NMR (400 MHz, CDCl₃), d (ppm): 7.09–7.11 (m,

6H), 6.93 (dd, 2H, J = 6.8 and 1.4 Hz), 6.61 (d, 2H, J = 8.6 Hz),

6.29–6.36 (m, 2H), 3.52 (d, 4H, J = 5.8 Hz). ¹³C NMR (100 MHz,

CDCl₃): 161.3, 136.5, 129.8, 127.2, 124.9, 121.0, 115.7, 115.5,

33.09. MS m/z: 334.0 [M⁺]; Anal. Calcd for C₁₈H₁₆F₂S₂: C, 64.64;

H, 4.82; F, 11.36; S, 19.17; Found: C, 64.23; H, 4.79; S, 19.66.

4.5.9. Bis[3-(4-hydroxyphenyl)prop-2-ene]disulphide (5i)

Intermediate 4i was treated according to general procedure to

give the desired product 5i as white solid (32% yield). Mp 146–

148 °C. ¹H NMR (400 MHz, DMSO-d₆), d (ppm): 12.4 (br s, 2H),

7.12 (d, 4H, J = 6.7 Hz), 6.64 (t, 4H, J = 5.6 Hz), 6.44 (d, 2H,

J = 9.6 Hz), 6.15–6.09 (m, 2H), 3.46 (d, 4H, J = 6.2 Hz). ¹³C NMR

(100 MHz, CDCl₃): 154.2, 128.9, 127.6, 127.4, 121.2, 115.9, 33.2.

MS m/z: 330.0 [M⁺]; Anal. Calcd for C₁₈H₁₈O₂S₂: C, 65.42; H,

5.49; O, 9.68; S, 19.41 Found: C, 65.54; H, 5.28; O, 9.62; S, 19.33.

4.5.3. Bis[3-(3-chlorophenyl)prop-2-ene]disulphide (5c)

Intermediate 4c was treated according to general procedure to

give the desired product 5c as white solid (62% yield). Mp 165–

168 °C. ¹H NMR (400 MHz, CDCl₃), d (ppm): 7.34 (s, 2H), 7.14–

7.19 (m, 6H), 6.53 (d, 2H, J = 8.7 Hz), 6.23–6.28 (m, 2H), 3.56 (d,

4H, J = 6.6 Hz). ¹³C NMR (100 MHz, CDCl₃): 134.9, 132.8, 129.6,

128.7, 127.3, 126.8, 126.7, 32.7. MS m/z: 366.1 [M⁺]; Anal. Calcd

for C₁₈H₁₆Cl₂S₂: C, 58.85; H, 4.39; Cl, 19.30; S, 17.46 Found: C,

58.35; H, 4.19; S, 17.43.

4.5.10. Bis[3-(4-methoxyphenyl)prop-2-ene]disulphide (5j)

Intermediate 4j was treated according to general procedure to

give the desired product 5j as white solid (48% yield). Mp 151–

153 °C. ¹H NMR (400 MHz, CDCl₃),

d (ppm): 7.27(d, 4H,

J = 6.9 Hz), 6.82 (t, 4H, J = 5.9 Hz), 6.48 (d, 2H, J = 8.8 Hz), 6.19–

6.23 (m, 2H), 3.81 (s, 6H), 3.62 (d, 4H, J = 6.7 Hz). ¹³C NMR

(100 MHz, CDCl₃): 162.4, 128.6, 127.5, 127.2, 121.9, 114.6, 56.0,

33.0. MS m/z: 358.1 [M⁺]; Anal. Calcd for C₂₀H₂₂O₂S₂: C, 67.00; H,

6.19; O, 8.93; S, 17.89 Found: C, 67.34; H, 6.23; O, 8.66; S, 17.99.

4.5.4. Bis[3-(3,5-dichlorophenyl)prop-2-ene]disulphide (5d)

Intermediate 4d was treated according to general procedure to

give the desired product 5d as yellow solid (42% yield). Mp 155–

158 °C. ¹H NMR (400 MHz, CDCl₃), d (ppm): 7.22–7.26 (m, 6H),

6.65 (d, 2H, J = 8.9 Hz), 6.23–6.27 (m, 2H), 3.61 (d, 4H, J = 5.9 Hz).

¹³C NMR (100 MHz, CDCl₃): 137.9, 134.6, 128.6, 128.2, 125.43,

121.33, 33.2. MS m/z: 435.9 [M⁺]; Anal. Calcd for C₁₈H₁₄Cl₄S₂: C,

49.56; H, 3.23; Cl, 32.51; S, 14.70 Found: C, 49.86; H, 3.45; S,

14.44. Found: C, 49.34; H, 3.23; S, 14.69.

4.5.11. Bis[3-(3,4-dimethoxyphenyl)prop-2-ene]disulphide (5k)

Intermediate 4k was treated according to general procedure to

give the desired product 5k as white solid (36% yield). Mp 134–

136 °C. ¹H NMR (400 MHz, CDCl₃), d (ppm): 6.85–6.79 (m, 4H),

6.65 (d, 2H, J = 7.9 Hz), 6.52 (d, 2H, J = 15.7 Hz), 6.33–6.39 (m, 2H),

3.73 (s, 12H), 3.62 (d, 4H, J = 5.7 Hz). ¹³C NMR (100 MHz, CDCl₃):

149.4, 148.1, 128.2, 127.5, 121.9, 114.6, 119.7, 115.9, 56.04, 33.09.

MS m/z: 418.1 [M]⁺; Anal. Calcd for C₂₂H₂₆O₄S₂: C, 63.16; H, 6.26;

O, 15.29; S, 15.32 Found: C, 63.34; H, 6.23; O, 15.78; S, 15.99.

4.5.5. Bis[3-(2,6-diﬂuorophenyl)prop-2-ene]disulphide (5e)

Intermediate 4e was treated according to general procedure to

give the desired product 5e as dull white solid (52% yield). Mp

169–172 °C. ¹H NMR (400 MHz, CDCl₃), d (ppm): 6.88–7.09 (m,

6H), 6.78 (d, 2H, J = 6.7 Hz), 6.20–6.23 (m, 2H), 3.54 (d, 4H,

J = 6.7 Hz). ¹³C NMR (100 MHz, CDCl₃): 163.4, 131.0, 128.6, 121.5,

115.5, 33.1. MS m/z: 370.0 [M⁺]; Anal. Calcd for C₁₈H₁₄F₄S₂: C,

58.36; H, 3.81; F, 20.52; S, 17.31; Found: C, 58.56; H, 3.75; S, 17.46.

4.5.12. Bis[3-(4-hydroxy, 3-methoxyphenyl)prop-2-ene]

disulphide (5l)

Intermediate 4l was treated according to general procedure to

give the desired product 5l as white solid (32% yield). Mp 144–

147 °C. ¹H NMR (400 MHz, CDCl₃), d (ppm): 9.6 (br s, 2H), 6.69–

6.77 (m, 4H), 6.57 (d, 2H, J = 7.2 Hz), 6.49 (d, 2H, J = 15.7 Hz),

6.31–6.36 (m, 2H), 3.73 (s, 6H), 3.55 (d, 4H, J = 5.7 Hz). ¹³C NMR

(100 MHz, CDCl₃): 149.4, 142.1, 128.5, 127.5, 121.6, 119.6, 119.7,

115.8, 56.4, 33.09. MS m/z: 390.1 [M⁺]; Anal. Calcd for

4.5.6. Bis[3-(4-chlorophenyl)prop-2-ene]disulphide (5f)

Intermediate 4f was treated according to general procedure to

give the desired product 5f as white solid (58% yield). Mp 162–

164. °C ¹H NMR (400 MHz, CDCl₃), d (ppm): 7.24–7.26 (m, 8H),

6.51 (d, 2H, J = 8.9 Hz), 6.18–6.22 (m, 2H), 3.68 (d, 4H, J = 5.9 Hz).

¹³C NMR (100 MHz, CDCl₃): 132.9, 129.3, 128.0, 127.6, 124.2,

121.3, 34.3. MS m/z: 366.0 [M⁺]; Anal. Calcd for C₁₈H₁₆Cl₂S₂: C,

58.85; H, 4.39; Cl, 19.30; S, 17.46 Found: C, 58.44; H, 4.23; S, 17.19.

C₂₂H₂₆O₄S₂, C, 61.51; H, 5.68; O, 16.39; S, 16.42 Found: C, 61.67;

H, 5.83; O, 16.66; S, 16.59.

4.6. General procedure for the synthesis of intermediates 6e–6m

(Scheme 2)

4.5.7. Bis[3-(3-bromophenyl)prop-2-ene]disulphide (5g)

Intermediate 4g was treated according to general procedure to

give the desired product 5g as light brown solid (48% yield). Mp

187–189 °C. ¹H NMR (400 MHz, CDCl₃), d (ppm): 7.48 (d, 2H,

J = 8.6 Hz), 7.31 (dd, 2H, J = 8.1 and 1.3 Hz), 7.13–7.19 (m, 4H), 6.59

(d, 2H, J = 8.6 Hz), 6.24–6.35 (m, 2H), 3.43 (d, 4H, J = 5.6Hz). ¹³C

NMR (100 MHz, CDCl₃): 137.3, 133.5, 129.8, 128.9, 127.2, 126.6,

123.9, 121.0, 33.3. MS m/z: 453.9 [M⁺, 49.8], 455.9 [(M+2)⁺, 100],

457.9 [(M+4)⁺, 55.2]; Anal. Calcd for C₁₈H₁₆Br₂S₂: C, 47.38; H, 3.53;

Br, 35.03; S, 14.06; Found: C, 47.32; H, 3.66; S, 14.24.

In a double necked round bottom ﬂask (RB ﬂask), 1.94 g

(10 mmol) of the compounds 2e–2m obtained in step 1 was taken

and dissolved in 30 mL of tetrahydrofuran (THF). 2 mL (20 mmol)

of oxalyl chloride was subsequently added to the RB ﬂask using

dropper. The reﬂuxing was performed for 4 h at 60–70 °C. The solu-

tion was dried carefully under reduced pressure using rota vapour

and the product obtained was used directly in the next step.

4.7. General procedure for the synthesis of Bis[2-(substituted

phenyl)-ethenyl] dithioperoxyanhydride (7e–7m)

4.5.8. Bis[3-(4-bromophenyl)prop-2-ene]disulphide (5h)

Intermediate 4h was treated according to general procedure to

give the desired product 5h as light brown solid (44% yield). Mp

176–178 °C. ¹H NMR (400 MHz, CDCl₃), d (ppm): 7.44 (d, 4H,

J = 8.9 Hz), 7.11 (t, 4H, J = 7.5 Hz), 6.49 (d, 2H, J = 8.1 Hz), 6.29–

6.33 (m, 2H), 3.32 (d, 4H, J = 6.9 Hz). ¹³C NMR (100 MHz, CDCl₃):

133.4, 131.07, 128.6, 127.60, 127.9, 125.5, 121.7, 33.15. MS m/z:

1.12 g of sodium hydrosulﬁde (10 mmol) was dissolved in

methanol in the presence of triethylamine hydrochloride solution

(10 mmol) followed by addition of dried compounds 6e–6m,

respectively. After continuous stirring for 4 h at room temperature

under N₂atmosphere, the reaction mixture was poured into water

and extracted with dichloromethane. The combined organic layer

6400

A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

was separated, dried over anhydrous sodium sulphate and evapo-

rated under reduced pressure. The residue obtained was puriﬁed

by column chromatography using mixture of chloroform/methanol

(99/1) as eluent to yield the desired product (7e–7m).

146–148 °C. ¹H NMR (400 MHz, DMSO-d₆) d (ppm): 7.65 (d, 2H,

J = 15.9 Hz), 7.45 (dd, 4H, J = 8.6 and 1.8 Hz), 6.87 (d, 4H, J = 7.6

and 1.7 Hz), 6.31 (d, 2H, J = 15.2 Hz), 3.81 (s, 6H).¹³

C NMR

(100 MHz, DMSO-d₆): 182.0, 160.6, 151.0, 127.3, 122.4, 119.9,

111.0, 55.8. MS m/z: 386.4 [M⁺]; Anal. Calcd for C₂₀H₁₈O₄S₂: C,

62.15; H, 4.69; O, 16.56; S, 16.59 Found: C, 62.24; H, 4.62; O,

16.34; S, 16.44.

4.7.1. Bis[2-(4-ﬂuorophenyl)-ethenyl] dithioperoxyanhydride

(7e)

Intermediate 6e was treated according to general procedure to

give the desired product 7e as white solid (49% yield). 164–167 °C.

¹H NMR (400 MHz, CDCl₃), d (ppm): 7.34 (d, 2H, J = 15.6 Hz), 7.28

(dd, 4H, J = 7.9 and 1.4 Hz), 7.05 (dd, 4H, J = 8.6 and 1.8 Hz), 6.44

(d, 2H, J = 15.2 Hz). ¹³C NMR (100 MHz CDCl₃): 180.0, 161.3,

158.8, 127.2, 126.9, 124.9, 124.0, 115.7, 115.5. MS m/z: 362.0

[M⁺]. Anal. Calcd for C₁₈H₁₂F₂O₂S₂: C, 59.65; H, 3.34; F, 10.48; O,

8.83; S, 17.70 Found: C, 59.61; H, 3.80; O, 8.90; S, 17.82.

4.7.7. Bis[2-(3, 4-dimethoxy phenyl)-ethenyl] dithioperoxyanhydride

(7k)

Intermediate 6k was treated according to general procedure to

give the desired product 7k as dull white solid (42.0% yield). Mp

162–164 °C. ¹H NMR (400 MHz, DMSO-d₆) d (ppm): 7.63 (d, 2H,

J = 15.6 Hz), 7.06 (dd, 2H, J = 8.8 and 1.8 Hz), 6.78–6.84 (m, 4H),

6.26 (d, 2H, J = 15.8 Hz), 3.82 (s, 12H). ¹³C NMR (100 MHz,

DMSO-d₆) d: 182.0, 157.8, 146.3, 144.1, 128.3, 127.0, 115.9,

115.4, 111.0, 55.8. MS m/z: 446.0 [M⁺]; Anal. Calcd for

4.7.2. Bis[2-(4-chlorophenyl)-ethenyl] dithioperoxyanhydride

(7f)

C₂₂H₂₂O₆S₂: C, 59.17; H, 4.97; O, 21.50; S, 14.36 Found: C, 59.10;

Intermediate 6f was treated according to general procedure to

give the desired product 7f as white solid (56% yield). Mp 162–

H, 4.44; O, 21.64; S, 14.39.

164 °C. ¹H NMR (400 MHz, CDCl₃)

d

(ppm): 7.55 (d, 2H,

4.7.8. Bis[2-(4-hydroxy, 3-methoxy phenyl)-ethenyl] dithioperoxy-

anhydride (7l)

J = 15.9 Hz), 7.33 (dd, 4H, J = 7.6 and 1.6 Hz), 7.24 (d, 4H, J = 8.2

and 1.7 Hz), 6.32 (d, 2H, J = 15.6 Hz). ¹³C NMR (100 MHz, CDCl₃):

180.2, 152.3, 134.9, 132.8, 129.6, 128.6, 127.3, 126.7. MS m/z:

393.6 [M⁺]; Anal. Calcd for C₁₈H₁₂Cl₂O₂S₂: C, 54.69; H, 3.06; Cl,

17.94; O, 8.09; S, 16.22 Found: C, 54.12; H, 3.30; O, 8.67; S, 16.72.

Intermediate 6l was treated according to general procedure to

give the desired product 7l as yellow solid (38.0% yield). Mp

145–149 °C. ¹H NMR (400 MHz, DMSO-d₆), d (ppm): 9.80 (br s,

2H), 7.61 (d, 2H, J = 15.7 Hz), 7.09 (dd, 2H, J = 8.7 and 1.8 Hz),

7.011 (d, 2H, J = 2.4), 6.84 (d, 2H, J = 8.1 Hz), 6.26 (d, 2H,

J = 15.6 Hz), 3.88 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) d: 180.0,

151.0, 149.1, 144.6, 127.3, 127.0, 122.4, 115.9, 115.4, 111.0,

109.6, 55.89. MS m/z: 418.0 [M⁺]; Anal. Calcd for C₂₀H₁₈O₆S₂: C,

57.40; H, 4.34; O, 22.94; S, 15.32 Found: C, 57.24; H, 4.56; O,

22.76; S, 15.16.

4.7.3. Bis[2-(3-bromophenyl)-ethenyl] dithioperoxyanhydride

(7g)

Intermediate 6g was treated according to general procedure to

give the desired product 7g as white solid (43% yield). Mp 175–

178 °C. ¹H NMR (400 MHz, CDCl₃),

d (ppm): 7.59 (d, 2H,

J = 15.1 Hz), 7.45 (s, 2H), 7.24 (dd, 2H, J = 7.8 and 1.3 Hz), 7.09–

7.11 (m, 2H), 6.65 (d, 2H, J = 15.2 Hz). ¹³C NMR (100 MHz, CDCl₃),

d (ppm): 182.0, 151.5, 136.9, 133.8, 131.7, 129.6, 128.4, 121.56.

MS m/z: 481.3 [M⁺, 49.8], 483.3 [(M+2)⁺, 100], 485.3 [(M+4)⁺,

55.2]; Anal. Calcd for C₁₉H₁₆Br₂O₂S₂: C, 45.62; H, 3.22; Br, 31.94;

O, 6.40; S, 12.82 Found: C, 45.72; H, 3.44; O, 6.49; S, 12.65.

4.7.9. Bis[2-(2,4-dimethoxyphenyl)-ethenyl] dithioperoxyanhydride

(7m)

Intermediate 6m was treated according to general procedure to

give the desired product 7m as dull white solid (46.2% yield). Mp

166–169 °C. ¹H NMR (400 MHz, DMSO-d₆) d (ppm): 7.93 (d, 2H,

J = 15.6 Hz), 7.15 (dd, 2H, J = 8.7 and 2.2 Hz), 6.51 (d, 2H,

J = 15.7 Hz), 6.23–6.32 (m, 4H), 3.84 (s, 12H). ¹³C NMR (400 MHz,

DMSO-d₆): 180.0, 168.5, 167.5, 151.0, 127.3, 122.4, 111.0, 109.6,

55.8. MS m/z: 446.0 [M⁺]; Anal. Calcd for C₂₂H₂₂O₆S₂: C, 59.17; H,

4.97; O, 21.50; S, 14.36 Found: C, 59.26; H, 4.86; O, 21.38; S, 14.24.

4.7.4. Bis[2-(4-bromophenyl)-ethenyl] dithioperoxyanhydride

(7h)

Intermediate 6h was treated according to general procedure to

give the desired product 7h as white solid (45% yield). Mp 171–

175 °C. ¹H NMR (400 MHz, CDCl₃),

d (ppm): 7.51(d, 2H,

J = 15.7 Hz), 7.39 (dd, 4H, J = 6.9 and 1.4 Hz), 7.26 (dd, 4H, J = 7.2

and 1.6 Hz), 6.27 (d, 2H, J = 15.2 Hz). ¹³C NMR (100 MHz, CDCl₃),

d (ppm): 182.0, 156.5, 133.9, 131.7, 129.6, 128.4, 121.5. MS m/z:

481.3 [M⁺, 49.4], 483.3 [(M+2)⁺, 100], 485.3 [(M+4)⁺, 55.9]; Anal.

Calcd for C₁₉H₁₆Br₂O₂S₂: C, 45.62; H, 3.22; Br, 31.94; O, 6.40; S,

12.82 Found: C, 45.71; H, 3.18; O, 6.55; S, 12.77.

4.8. Biological assays

4.8.1. Thioﬂavin T (ThT) assay

Commercially available peptides were ﬁrst treated with hex-

aﬂuoroisopropanol (HFIP) at 5 mg/mL to avoid self-aggregation.

The clear solution containing the dissolved peptide was then ali-

quoted in micro centrifuge tube. The HFIP was allowed to evapo-

rate under a stream of nitrogen until a clear ﬁlm remained in the

test tube. The pre-treated Ab_1–42samples were then dissolved in

DMSO in order to have a stable stock solution (Ab = 5 mM). For

the inhibition of self-mediated Ab_1–42aggregation experiment,

the Ab stock solution was diluted with 50 mM phosphate buffer

4.7.5. Bis[2-(4-hydroxyphenyl)-ethenyl] dithioperoxyanhydride

(7i)

Intermediate 6i was treated according to general procedure to

give the desired product 7i as dull white solid (38% yield). Mp

157–159 °C. ¹H NMR (DMSO-d₆), d (ppm): 13.4 (s, 2H), 7.57 (d,

2H, J = 15.8 Hz), 7.09 (dd, 4H, J = 8.8 and 1.6 Hz), 6.93 (dd, 4H,

J = 7.6 and 1.8 Hz), 6.88 (d, 2H, J = 15.6 Hz). ¹³C NMR (100 MHz,

CDCl₃): 180.0, 156.5, 152.2, 129.6, 127.6, 127.5, 115.6. MS m/z:

358.2 [M⁺]; Anal. Calcd for C₁₈H₁₄O₄S₂: C, 60.32; H, 3.94; O,

17.85; S, 17.89 Found C, 60.54; H, 3.87; O, 17.85; S, 17.77.

(pH 7.4) to 25

25 M, ﬁnal concentration) with or without the tested compound

(25 M) was incubated at 37 °C for 48 h. To quantify amyloid ﬁbril

lM before use. A mixture of the peptide (10 lL,

l

formation, the thioﬂavin-T ﬂuorescence method was used. Blanks

containing 50 mM phosphate buffer (pH 7.4) instead of Ab with

or without inhibitors were also carried out. After incubation, sam-

4.7.6. Bis[2-(4-methoxy phenyl)-ethenyl]

dithioperoxyanhydride (7j)

ples were diluted to a ﬁnal volume of 200

NaOH buffer (pH 8.0) containing thioﬂavin-T (5

l

L with 50 mM glycine–

M). Then the

l

Intermediate 6j was treated according to general procedure to

give the desired product 7j as dull white solid (44% yield). Mp

ﬂuorescence intensities were recorded 5 min later (excitation,

450 nm; emission, 485 nm). The percent inhibition of aggregation

A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

6401

was calculated by the expression (1 ꢁ IFi/IFc) ꢃ 100% in which IFi

and IFc are the ﬂuorescence intensities obtained for Ab in the pres-

ence and absence of inhibitors after subtracting the background,

respectively. Each measurement was run in triplicate.

The effects of compounds on metal-induced Ab_1–42aggregation

were also determined by using thioﬂavin T. The Ab_1–42stock solu-

inhibitor-free assay systems were utilized to measure the full

activity. The percent inhibition was calculated by the following

expression: (1 ꢁ Ai/Ac) ꢃ 100, where Ai and Ac are the absorbances

obtained for AChE in the presence and absence of the inhibitors,

respectively, after subtracting the respective background. Each

experiment was performed in triplicate, and the mean standard

deviation was calculated. Data from concentration-inhibition

experiments of the inhibitors were calculated by nonlinear regres-

sion analysis, using the Graph Pad Prism 5 program.

tion was diluted with 25

NaCl. 25 M Ab_1–42was incubated with or without 25

and 50 M tested compounds at 37 °C for 24 h. The sample was

diluted to a ﬁnal volume of 40 L with 50 mM glycine–NaOH

lM HEPES (pH 6.6) containing 150

lM

l

M copper

l

buffer (pH 8.0) containing thioﬂavin T (5

l

M) and assayed as

4.8.4. Metal binding studies

described above.

The metal binding studies were carried out in a Perkin-Elmer

Multimode Reader (Thermo Scientiﬁc). The UV absorption of the

tested compounds 7k and 7l, in the absence or presence of CuCl₂

and FeSO₄, was recorded with wavelength ranging from 200 to

600 nm after incubating for 30 min in methanol at room tempera-

4.8.2. Oxygen radical absorbance capacity (ORAC-FL) assay

The reaction was carried out in 75 mM phosphate buffer (pH

7.4), and the ﬁnal reaction mixture was 200

lL. Antioxidant

(20 L) and FL (120 L; 70 mM, ﬁnal concentration) solutions were

l

ture. The ﬁnal volume of reaction mixture was 200

lL, and the ﬁnal

placed in a black 96-well microplate (96F untreat, Nunc). The mix-

ture was preincubated for 15 min at 37 °C, and then AAPH solution

(60 lL, 12 mM, ﬁnal concentration) was added rapidly using a mul-

tichannel pipette. The microplate was immediately placed in the

reader and the ﬂuorescence recorded every minute for 80 min

(excitation, 485 nm; emission, 520 nm). Samples were measured

concentrations of tested compound and metals were 50 lM. The

stoichiometry of the compounds 7k–Cu²⁺and 7l–Cu²⁺complex

were determined by titrating the methanol solution of tested com-

pound with ascending concentration of CuCl₂. The ﬁnal concentra-

tion of tested compound was 50

lM, and the ﬁnal concentration of

Cu²⁺ranged from 5 to 80

lM. The UV spectra were recorded and

at ten different concentrations (1–10

using phosphate buffer instead of the tested compound and eight

calibration solutions using Trolox (1–8 M, ﬁnal concentration)

l

M). A blank (FL + AAPH)

treated by numerical subtraction of CuCl₂and tested compound

at corresponding concentrations, plotted versus the mole fraction

of tested compound.

l

as antioxidant were also carried out in each assay. A blank using

phosphate buffer instead of the tested compound was also carried

out. All of the reaction mixtures were prepared in triplicate, and at

least three independent runs were performed for each sample. The

antioxidant curves (ﬂuorescence vs time) were normalized to the

curve of the blank. The area under the ﬂuorescence decay curve

(AUC) was calculated using the following equation:

4.8.5. TEM assay

Ab_1–42peptide (Sigma) stock was diluted with 20 mM phos-

phate buffer (pH 7.4) at 4 °C to 40 mM before use. For the inhibi-

tion of metal-induced Ab_1–42aggregation experiment, it was ﬁrst

incubated with Cu²⁺for 2 min followed by incubation in the pres-

ence and absence of compounds 7k, 7l and Curcumin at a concen-

tration of 50

l

M, at 37 °C for 24 h. The ﬁnal concentration of Ab_1–42

M. Aliquots (5 mL) of the samples were placed

and Cu²⁺were 25

l

i ¼ 80

P

AUC ¼ 1 þ ðf_i=f₀Þ

on a carbon coated copper/rhodium grid for 2 min at room temper-

ature. Each grid was stained with uranyl acetate (1%, 5 mL) for

2 min. Excess staining solution was removed and the specimen

was transferred for imaging with transmission electron micro-

scopy (JEOL JEM-1400).

i ¼ 1

where f₀is the initial ﬂuorescence reading at 0 min and f_i

is the ﬂuorescence reading at time i. The net AUC was calculated

by the following equation: AUCsample – AUCblank. The regression

equations between the net AUC and the Trolox concentrations were

calculated. The ORAC-FL value for each sample was calculated using

the standard curve, and the ORAC-FL value of each tested compound

is thus expressed as Trolox equivalents.

4.8.6. Hydrogen peroxide assay

Hydrogen peroxide production was determined using an HRP/

Amplex Red assay. A general protocol from Invitrogen’s Amplex

Red Hydrogen Peroxide/Peroxidase Assay was followed. Brieﬂy,

reagents were added directly to a 96-well plate in the following

4.8.3. Inhibition of AChE and BuChE

order to give a 100 lL ﬁnal solution: CuCl₂(400 nM), phosphate

AChE and BuChE inhibitory activities of the test compounds

were determined by the modiﬁed Ellman’s method. Brieﬂy, Stock

solutions of tested compounds (10 mM) were prepared in ethanol

and diluted using 0.1 M KH₂PO₄/K₂HPO₄buffer (pH 8.0) to afford a

ﬁnal concentration range between 0.01 and 100 lM. Enzyme

solutions were prepared by dissolving lyophilized powder in dou-

buffer, Ab peptide (200 mM), compounds (800 nM, 1% v/v DMSO),

sodium ascorbate (10 mM). The reaction was allowed to incubate

for 30 min at room temperature. After this incubation, 50 mM of

freshly prepared working solution containing 100 nM Amplex

Red (Sigma) and 0.2 U/mL HRP (Sigma) in phosphate buffer was

added to each well, and the reaction was allowed to incubate for

30 min at room temperature. Fluorescence was measured using a

Tecan plate reader (k_ex/k_em= 530/590). Error bars represent stan-

dard deviations for at least three measurements.

ble-distilled water. The assay solution consisted of 1 mL of 0.1 M

phosphate buffer KH₂PO₄/K₂HPO₄, 25

3.1.1.7, from electric eel) or 25 L of BuChE (0.06 U/mL, E.C.

3.1.1.8, from equine serum) and 100 L of various concentrations

of test compounds which was allowed to stand for 5 min before

100 L of 0.01 M DTNB were added. A positive control of donepezil

was used in the same range of concentrations. The reaction was

started by addition of 20 L of the 0.075 M substrate solution

lL of AChE (0.22 U/mL, E.C.

l

4.8.7. Cell toxicity assay

l

The toxicity effect of 7k and 7l on human embryonic kidney

(HEK 293) cells was examined according to the previous method.⁵⁹

The HEK 293 cells were cultured in Dulbecco’s minimum essential

medium (DMEM) supplemented with 10% fetal bovine serum

(FBS), 1% penicillin–streptomycin solution (10,000 units of

penicillin and 10 mg of streptomycin in 0.9% NaCl) at 37 °C in a

humidiﬁed atmosphere containing 5% CO₂. HEK cells were plated

in 96-well plate at a density 5000 cells/well and cultured for

l

(acetylthiocholine/butyrylthiocholine) and exactly 2 min after sub-

strate addition the absorption was measured at 25 °C at 412 nm.

The non-enzymatic hydrolysis of acetylthiocholine/butyrylthio-

choline iodide was also measured in enzyme-free assay systems,

and the results were employed as blank. In control experiments,

6402

A. Manral et al. / Bioorg. Med. Chem. 23 (2015) 6389–6403

24 h. Next, cells were exposed to the tested compounds at different

concentrations (0.1–100 M) for 48 h. Stock solution of tested

The acceptability of the compounds based on the Lipinski’s rule

of ﬁve was also estimated from the results.

l

compound was prepared in DMSO and diluted in complete med-

ium to give ﬁnal concentrations. Cytotoxicity assay was performed

after 48 h incubation with compounds in triplicates. The MTT

reagent was added to each well for additional 4 h followed by sol-

ubilisation of formazan crystals in DMSO. The absorbance of each

well was measured using a microculture plate reader with a test

wavelength of 570 nm and a reference wavelength of 630 nm.

Results are expressed as the mean SEM of three independent

experiments.

Virtual toxicity risk assessment was performed using TOPKAT

module of Accelrys Discovery studio 4.0. TOPKAT accurately and

rapidly predicts the toxicity of chemicals from their 2D molecular

structure. It employs a range of robust, cross-validated Quantita-

tive Structure Toxicity Relationship (QSTR) models for assessing

various measures of toxicity. Compounds 7k and 7l were moni-

tored for dose dependent toxicity parameters including FDA and

NTP rodent carcinogen models, skin irritancy, ocular irritancy,

mutagenicity, aerobic biodegradability and developmental toxicity

potential.

4.9. In silico studies

Acknowledgements

4.9.1. Molecular docking studies

The coordinates for NMR structure of Ab_1–42peptide (PDB ID:

1IYT), the X-ray crystal structures of the enzyme Torpedo califor-

nica AChE (TcAChE) complexed with donepezil (PDB ID: 1EVE)

and Human BuChE (HuBuChE) complexed with echothiophate

(PDB ID: 1POI) were obtained from the RCSB protein data bank.

Docking studies were performed using Discovery Studio (DS) Client

v 4.0 package (Accelrys Inc., San Diego, CA). The ligand molecules

were sketched using the Build Fragment tool in DS followed by

the addition of hydrogen atoms. Valency of the ligand molecules

were monitored, followed by clean geometry application.

CHARMm force ﬁeld was applied to the ligand followed by energy

minimization using steepest descent and conjugate gradient till a

derivative of 0.001 was achieved. For docking studies, initial pro-

tein was prepared by removing all water molecules, heteroatoms,

any co-crystallized solvent and the ligand. Proper bonds, bond

orders, hybridization and charges were assigned using protein

model tool. CHARMm force ﬁeld was applied using the receptor

ligand interactions tool. The ligand donepezil was deleted and a

site sphere was built to deﬁne the active site of TcAChE enzyme

which spans the entire region, from the catalytic site extending

all the way to the peripheral site. The center of the grid box was

placed near the active site gorge (AChE [2.570, 64.382, 68.125,

9.533]; BuChE [132.13, 113.46, 40.88, 10.6] and Ab_1–42[8.838,

ꢁ2.0781, ꢁ10.7316, 5.0]). The CDOCKER program DS 4.0 software

was used to perform docking simulations. CDOCKER generates ran-

dom conformations of ligands within the active site through high-

temperature molecular dynamics to generate 10 docked ligand

poses. The resultant docking orientations, within 2.0 Å in root

mean square deviation (rmsd) in tolerance of each other, were

clustered together to represent the most favourable free energy

of binding. Finally, the top-posed docking conformations were

submitted to post-docking energy minimization on Discovery

Studio 4.0. Further, the enzyme–ligand complexes were ranked

based on a scoring function, CDOCKER interaction energy in

kcal/mol.

The authors wish to gratefully acknowledge scientiﬁc contribu-

tions from Prof. Vani Brahmachari. Financial assistance from the

University of Delhi is acknowledged. The author Apra Manral

wishes to acknowledge the Senior Research Fellowship awarded

by the University Grants Commission – Govt. of India. The facilities

provided by ACBR and the University of Delhi are gratefully

acknowledged.

Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.bmc.2015.08.024.

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Article Doi

Multifunctional novel Diallyl disulfide (DADS) derivatives with β-amyloid-reducing, cholinergic, antioxidant and metal chelating properties for the treatment of Alzheimer's disease

DOI: 10.1016/j.bmc.2015.08.024

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Full text of DOI:10.1016/j.bmc.2015.08.024

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