Small multitarget molecules incorporating the enone moiety

molecules

Article

Small Multitarget Molecules Incorporating the

Enone Moiety

Thalia Liargkova ¹, Nikolaos Eleftheriadis ², Frank Dekker ², Efstathia Voulgari ³,

Constantinos Avgoustakis ³, Marina Sagnou ⁴, Barbara Mavroidi ⁴, Maria Pelecanou ⁴and

Dimitra Hadjipavlou-Litina ^1,

*

1

Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences, Aristotle

University of Thessaloniki, Thessaloniki 54124, Greece; thalialiargkova@yahoo.gr

Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of

Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands;

2

nikolaoselef@hotmail.com (N.E.); f.j.dekker@rug.nl (F.D.)

3

4

Department of Pharmaceutical Technology and Pharmaceutical Analysis, School of Pharmacy, University of

Patras, Rio Patras 26504, Greece; eﬁv48@hotmail.com (E.V.); avgoust@upatras.gr (C.A.)

Institute of Biosciences and Applications, National Center for Scientiﬁc Research “Demokritos”, Agia

Paraskevi, Athens 15310, Greece; sagnou@bio.demokritos.gr (M.S.); bmavroidi@bio.demokritos.gr (B.M.);

pelmar@bio.demokritos.gr (M.P.)

*

Correspondence: hadjipav@pharm.auth.gr; Tel.: +30-231-099-7627; Fax: +30-231-099-7679

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Received: 7 December 2018; Accepted: 28 December 2018; Published: 7 January 2019

Abstract: Chalcones represent a class of small drug/druglike molecules with different and multitarget

biological activities. Small multi-target drugs have attracted considerable interest in the last

decade due their advantages in the treatment of complex and multifactorial diseases, since “one

drug-one target” therapies have failed in many cases to demonstrate clinical efﬁcacy. In this context,

we designed and synthesized potential new small multi-target agents with lipoxygenase (LOX),

acetyl cholinesterase (AChE) and lipid peroxidation inhibitory activities, as well as antioxidant

activity based on 2-/4- hydroxy-chalcones and the bis-etheriﬁed bis-chalcone skeleton. Furthermore,

the synthesized molecules were evaluated for their cytotoxicity. Simple chalcone b4 presents

signiﬁcant inhibitory activity against the 15-human LOX with an IC₅₀value 9.5 µM, interesting

anti-AChE activity, and anti-lipid peroxidation behavior. Bis-etheriﬁed chalcone c12 is the most

potent inhibitor of AChE within the bis-etheriﬁed bis-chalcones followed by c11. Bis-chalcones c11

and c12 were found to combine anti-LOX, anti-AchE, and anti-lipid peroxidation activities. It seems

that the anti-lipid peroxidation activity supports the anti-LOX activity for the signiﬁcantly active

bis-chalcones. Our circular dichroism (CD) study identiﬁed two structures capable of interfering with

the aggregation process of Aβ. Compounds c2 and c4 display additional protective actions against

Alzheimer’s disease (AD) and add to the pleiotropic proﬁle of the chalcone derivatives. Predicted

results indicate that the majority of the compounds with the exception of c11 (144 Å) can cross the

Blood Brain Barrier (BBB) and act in CNS. The results led us to propose new leads and to conclude

that the presence of a double enone group supports better biological activities.

Keywords: chalcones; bis-ethers; bis-chalcones; multitarget; lipoxygenase inhibitors; acetylcholinesterase

inhibitors; β-amyloid peptide; Alzheimer

1. Introduction

“Small-molecule drug” refers to a compound with a low molecular weight, able to enter cells

easily, affecting other biological molecules, such as proteins, enzymes, etc., and may cause the death of

cancer cells. They strongly differ from drugs that have large molecular weight, such as monoclonal

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ww w .mdpi.com/journal/molecules

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antibodies, which are not able to get inside cells very easily. Chalcones belong to the group of

small molecules, known as -unsaturated ketones, displaying a very large number of biological

activities [ ]. Numerous published reviews underline the medicinal signiﬁcance of the enone moiety

of chalcones [ ]. This conjugation of the double bond with the carbonyl moiety seems to be

α, β

1

2–6

responsible for the biological activities of chalcones, as removal of the enone group renders them

inactive. Chalcones represent a key structural scaffold for many synthetic and natural products.

While a number of synthetic processes have been reported for the synthesis of chalcones, the general

and more widely used synthetic route involves Claisen–Schmidt condensation under acidic or basic

homogeneous conditions [7–11]. Changes in their structure have offered a high degree of diversity

which has been extremely useful and highly advantageous for medicinal chemistry purposes aiming

to develop new therapeutic agents with improved pharmacokinetic properties, as well as a better

therapeutic proﬁle.

The formation of reactive oxygen species (ROS) is characteristic of aerobic organisms. However,

in many pathophysiological conditions the excessive production of ROS overwhelms the natural

antioxidant defense mechanisms leading to an imbalance widely known as oxidative stress (OS).

Antioxidants have been found to prevent and offer therapeutic beneﬁts on inﬂammation-generated

oxidative stress which is closely related to the lesions of Alzheimer’s disease (AD) [12]. Nowadays

the involvement of free radicals in the AD brain pathology includes the presence of elevated

levels of protein oxidation, lipid peroxidation products and oxidative mitochondrial damage [12].

Hydroxy-chalcones have exhibited a very strong antioxidant proﬁle which has been directly related to

the presence of the α, β-double bond and the hydroxyl moiety [2–6]. Therefore, they may be considered

as promising structural templates to design potential therapeutic agents against the ROS-related

AD pathology.

Acetyl cholinesterase (AChE) is a serine hydrolase mainly found at neuromuscular junctions and

cholinergic brain synapses with the biological role to terminate impulse transmission at cholinergic

synapses by rapid hydrolysis of the neurotransmitter. The inhibition of brain AChE presents a very

attractive and highly promising therapeutic target in AD treatment strategies.

Chronic inﬂammation is a common phenomenon present in the background of multiple

neurodegenerative diseases, including AD. Previous studies showed that the key regulatory enzymes

in the eicosanoid pathway, i.e., cyclooxygenase-2 and 5-, 12-, and 15-lipoxygenases, appear to have an

important role in mediating the pro-inﬂammatory responses, providing further evidence that, in the

future, it may be possible to manipulate pro-inﬂammatory pathways for therapeutic purposes [13].

Lipoxygenases (LOXs) are dioxygenases which catalyze the stereoselective addition of oxygen to

arachidonic acid (AA). They are characterized by the presence of a non-heme iron in their structure.

LOXs peroxidize membrane lipids causing structural changes to the cell [14]. The most abundant LOX

isoforms in the central nervous system are 12/15-LOX. The metabolites of these enzymes, 12(S)-HETE

and 15(S)-HETE, are important secondary messengers in synaptic transmission and are involved in

learning and memory processes. 12/15-LOX have been described abundantly in neurons and in some

glial cells throughout the cerebrum, hippocampus, and basal ganglia [15

inﬂammatory reactions have been related with the both up-regulation of 12/15-LOX expression levels

and activity [15 17]. The mammalian reticulocyte 15-LOX-1 is the major enzyme which is responsible for

,16]. Oxidative stress and

,

membrane lipid peroxidation [18]. Experiments on animal AD models have provided evidence on the

importance of 15-LOX and have demonstrated its role in the etiopathology of AD. Furthermore, 15-LOX

has been found to promote brain cell survival through the synthesis of neuroprotectin D1 [19

an antiapoptotic and neuroprotective docosahexaenoic acid.

–21],

Moreover, the 5-LOX inhibitor, Zileuton, reduced Aβ formation in embryonic ﬁbroblasts. [22]

Wang et al. [23] showed that 12-LOX activation plays a key role in oxidative injury due to glutathione

(GSH) depletion. Inhibitors for 5-lipoxygenase (5-LOX) and cyclo-oxygenase 2 (COX-2 and 5-, 12-,

and 15-LOX-inhibitor in astrocytes reduced signiﬁcantly IL-6 secretion, compared to exposed glial cells

without inhibitors [13]. Treatment with an inhibitor of all LOXs, namely nordihydroguaiaretic acid,

Molecules 2019, 24, 199

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or 5-LOX inhibitors, such as zileuton, BWB70C, and inhibitors of 12/15-LOX, baicalein and AA-861

(also an inhibitor of 5-LOX) signiﬁcantly reduced, in a concentration-dependent manner, the level of

lipid and protein oxidation [24].

For many years, researchers have been struggling to develop highly speciﬁc compounds against a

particular target. However, the “one drug-one target” therapies failed to demonstrate clinical efﬁcacy

in multifactorial disorders. Consequently, the treatment of these multi-factorial diseases, such as AD,

may have to depend on and be the result of the concurrent interference with more than one pathological

pathway, to exert therapeutic beneﬁts. In the last decade the idea of a drug to be “promiscuous” has

been correlated with its ability to selectively target multiple cellular processes and to treat diseases

that stem from a combination of biological parameters. Following this approach, the researchers

succeeded in having one single molecule hit multiple targets that participate in the pathogenesis of a

multifactorial disease [25]. In this context, a single multi-target drug may have distinctive advantages

over drug combination therapies [26] and as a result an increasing number of therapeutic strategies

that are based on poly-pharmacology have been proposed for AD [27,28]. Therefore, it is evident

that the treatment of AD could beneﬁt from the use of multipotent drugs that present free radical

scavenging, anti-lipid peroxidation, anti-inﬂammatory, and AChE inhibitory activity.

Herein, we continue our efforts [29] to design and synthesize small biologically active molecules

and for this scope, the chalcone structural template was exploited as a privileged scaffold for the design

of multi-target agents. The synthesis of simple chalcones a1–4, of the bis-etheriﬁed chalcones c1–4 and

of the intermediate ether dii, as well as their antioxidant activities (DPPH, AAPH, ABTS^•+), cytotoxicity,

and inhibition against lipoxygenase and acetylcholinesterase have been already published [29]. Several

substituted aromatic and heteroaromatic aldehydes have been used in order to evaluate the effect

of steric and electronic parameters on the biological activity and to optimize the activities through

systematic modiﬁcation of the substituents. Thus, we present the synthesis and biological evaluation

of a series of 2-hydroxy- and 4-hydroxy-chalcones (Figures 1 and 2), diversely substituted, as well

as of a series of bis-chalcones combined together through an −O(CH₂)_nO− linkage leading to the

structures of bis-ethers (Figures 3–5) in which the effect of the length of the chain was studied.

Figure 1. Synthesis of 2-hydroxy- and 4-hydroxy-substituted chalcones.

Figure 2. Structures of the used aromatic aldehydes.

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Figure 3. Synthesis of the bis-ethers.

Figure 4. Synthesis and general structure of bis-etheriﬁed bis-chalcones.

Figure 5. Synthesis of the dimethyl amino substituted bis- etheriﬁed bis-chalcones.

2. Results and Discussion

2.1. Chemistry

The 4-hydroxy-substituted chalcones

a(1–13) and the 2-hydroxy-substituted chalcones b(1–4, 7–9,

11, and 13) were successfully synthesized via Claisen-Schmidt condensation, using 20% KOH as a

catalyst in a US-bath. Figure 1 shows a generalized synthetic route for the preparation of chalcones of

a

and b series, whereas the structures of the various substituted aromatic aldehydes used for those

derivatizations are presented in Figure 2. During the course of these reactions, the use of the US-bath

was found to signiﬁcantly affect the reactivity of the starting material, as well as the yields of chalcone

formation. For the synthesis of bis-etheriﬁed double chalcones

was successfully dimerized to form bis-ethers vii), by means of a Williamson reaction in dry

acetone and in the presence of the desired 1, -dibromoalkane, under basic conditions and reﬂux,

c and e, 4-hydroxyacetophenone

d(i–

ω

as shown in Figure 3. Subsequent reaction of dii, for which a 3C-ether link was employed between the

two 4-hydroxyacetophenone units, with the aromatic aldehydes of Figure 2 under Claisen-Schmidt

conditions afforded chalcones

results of our previous work on bis-etheriﬁed chalcone c3

4-dimethylamino(phenyl)acrylaldehyde with dii, and exhibited a very potent anti-lipid peroxidation

activity (100%), a series of the corresponding bis-etheriﬁed double chalcones vii) were synthesized

c(

1–

13) is shown in Figure 4. Finally, prompted by the encouraging

[

29], which resulted from the reaction of the

e(i–

in a similar manner, as shown in Figure 5. These molecules were anticipated to reveal the effect

of the carbon chain length of the bis-ether between the two 4-dimethylamino(phenyl)acryl-derived

chalcone units.

Their structures are conﬁrmed spectroscopically (IR, ¹H-NMR, ¹³C-NMR, and LC-MS) and by

elemental analysis. All the simple chalcones of series

a

and

b

present the characteristic absorption

1

in the IR (KBR cm⁻¹3280–3550 (O–H), 1720 (C=O), 1625 (C=C)). H-NMR spectroscopy revealed

through integration the right analogy of aromatic and CH= protons. The results are consistent with the

proposed (E) structures and are in agreement with our previous ﬁndings [29]. All the derivatives were

Molecules 2019, 24, 199

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taken in satisfactory yields (over 70%). The physicochemical properties of the products are described

in the experimental session.

We did not succeed to synthesize bis-etheriﬁed bis-chalcones from the 2-hydroxy-substituted

chalcones. The chromatographic data and the spectrometric studies veriﬁed the failure of the reactions

due to stereochemical reasons.

Several 2-hydroxy- and 4-hydroxy-chalcones and derivatives are known as anti-tumor anti-

inﬂammatory, anti-parasites, anti-depressive, anticonvulsant, antimicrobial, antinociceptives, and nitric

oxide synthase inhibitors, associated with diseases such as Alzheimer’s and Huntington’s [30,31]

.

Biological results from 3-hydroxy chalcones and derivatives have also been reported. The results

from simple amino ethers derived from 3-hydroxy chalcones suggested that amino alkyl side

chain of chalcone dramatically inﬂuenced the inhibitory activity against AChE. Among them,

the structural combination of the 4,6-diamino-1,2-dihydro-1,3,5-triazine and chalcone scaffolds via

ﬂexible diether linkers of varying lengths were successfully synthesized and characterized [32].

The resulting compounds were evaluated as dual-target inhibitors of dihydrofolate reductase (DHFR)

and thioredoxin reductase (TrxR), revealing the inﬂuence of linker length on biological activities.

It is worth mentioning, and aiming to rationalize our choice of aldehydes used in this study

(Figure 2) that derivatives of some heterocyclic chalcones bearing thiofuran, furan, and quinoline

moieties have presented interesting biological activities [33]. Hence, aldehydes

were used, whereas 3-hydroxychalcones and their substituted benzo-ethers have been tested as

multifunctional non-purine xanthine oxidase inhibitors [34] prompting us to use aldehydes , and 13

Diarylpentanoid analogues evaluated as nitric oxide inhibitors [35] were found to inhibit superoxide

4, 5, 10, and 12

1

,

2

.

anion and elastase release in human neutrophils [36], have inspired us to employ aldehydes 3, 7–9,

and 11 for the desired chalcone formation.

2.2. Physicochemical Studies

2.2.1. Experimental Determination of Lipophilicity as R_MValues

It is well-documented and discussed in the literature that lipophilicity is a very signiﬁcant

physicochemical property which may critically affect distribution, bioavailability, metabolic activity

and elimination, all the useful characteristics for the kinetics of biologically active compounds. Thus,

we attempted to determine experimentally the lipophilicity of the synthesized compounds from the

RPTLC method [37] as R_Mvalues. The method is considered to be a reliable, fast and convenient

method for expressing lipophilicity. Moreover, many calculated methods were used in order to predict

the logP value [38–40].

2.2.2. In Silico Determination of Lipophilicity Values as cLogP

The CLOGP program of BioByte Corp. [41] (BioByte Corp., Claremont, CA, USA) was used for the

in silico calculation of lipophilicity values in n-octanol buffer. Lipophilicity was theoretically calculated

as cLogP values.

The attempt to correlate together in one equation simultaneously the theoretically calculated

lipophilicity values calculated with C-QSAR (as cLogP) and the R_Mvalues of all the compounds did

not succeed. This disagreement may have been the result of various factors, such as the speciﬁc

chromatographic behavior of the compounds (e.g., different solvation, silanophilic interaction,

H-bridges, etc.), causing such limitations. Thus, the different nature of the hydrophilic and lipophilic

phases used in the two systems (n-octanol: water for the theoretically calculated values and

methanol-water: nujol for the RPTLC experiment) must be considered. It is difﬁcult to obtain reliable

experimental logP values outside the range of about 5 < logP < 7; therefore, it is likely that a calculated

logP value using a model is more accurate than an experimental [42].

Molecules 2019, 24, 199

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However, we succeeded to correlate the calculated lipophilicity values (as cLogP) and the

experimental R_Mvalues for the simple chalcones in the following statistically signiﬁcant equation:

R_M= 0.431 (±0.198) cLogP − 2.108 (±0.753)

(1)

N = 11, r = 0.854, q²= 0.592, s = 0.257, F_{1, 9}= 24.266, α = 0.01

2.2.3. Molecular Properties Prediction—Lipinski “Rule of Five”

Computational methods have emerged as a powerful strategy for the prediction of human

pharmacokinetic properties. In this regard, a variety of useful in silico models have been developed

with different levels of complexity for the screening of data sets of compounds, creating tools that are

faster, simpler, and more cost-effective than traditional experimental procedures [43].

Thus, chemical structures and SMILES notations of the title compounds were obtained and

entered in the online Molinspiration software version 2016.10 (ww w .molinspiration.com, Bratislava,

Slovak Republic, Bratislava University) [44] to calculate various molecular properties e.g., partition

coefﬁcient (log P), topological polar surface area (TPSA), hydrogen bond donors and acceptors,

rotatable bonds, number of atoms, molecular weight, and violations of Lipinski’s rule of ﬁve, in order

to evaluate the drug likeness of chalcones and intermediates [45]. In the discovery, “the rule of ﬁve”

predicts that poor absorption or permeation is more likely when there are more than ﬁve H-bond

donors, 10 H-bond acceptors, the molecular weight (MW) is greater than 500, and the calculated Log P

is greater than 5. It is a rule of thumb to evaluate drug-likeness or determine if a chemical compound

with a certain pharmacological or biological activity has properties that would make it a likely orally

active drug in humans.

LogP values of all the title compounds except a3–13, b3–13 and di–div, were found to be more

than 5 and are in clear violation of Lipinski’s rule of ﬁve, suggesting poor permeability across cell

membrane. As shown in Table 1, logP values range from 2.41 to 9.44. LogBB (BB, Blood Brain Barrier)

is another important in silico parameter to identify CNS active agents. For the calculation of the

logBB values we use the cLogP values. For in silico prediction [46], compound with logBB value

more than 0.3 is considered to have high absorption through BBB whereas between 0.3–0.1 and less

than

−

0.1 is considered to be moderate and less absorbed through BBB. LogP values of tacrine and

nordihydroguaiaretic acid (NDGA) the standard drugs were found to be well under 5 justifying their

oral use. Twenty-nine (29) derivatives were found to present molecular weight less than 500. Thus,

these molecules could be easily transported, diffused, and absorbed in comparison to large molecules.

Counting the number of hydrogen bond acceptors (O and N atoms) and the number of hydrogen bond

donors (NH and OH) in the synthesized compounds it seems that both follow the Lipinski’s rule of

ﬁve (less than 10 and 5, respectively). Within the series of the examined derivatives, only compounds

a3–13 and b3–div seem to be orally active in accordance to Lipinski’s rule of ﬁve.

Topological polar surface area is highly correlated with the hydrogen bonding of a compound.

It is used as a signiﬁcant indicator of the bioavailability of a bioactive molecule. TPSA of the derivatives

was observed in the range of 37.3–144.26 Å and is well below the limit of 160 Å indicating good oral

bioavailability. The upper limit for TPSA for a molecule to penetrate the brain is around 90 Å. Results

indicate that all structures with the exception of c11 (144 Å) can cross the BBB.

Molecules 2019, 24, 199

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Table 1. Molecular properties prediction-Lipinski “Rule of Five”.

Compounds mi Log P ^a

n Atoms

n ^cO, N

n Violations

Nrotb ^e

Volume ^g

h

TPSA ^b

n ^dOH, NH

mol. Wt ^f

LogBB

a1 *

a2 *

a3 *

a4 *

a5

a6

a7

a8

a9

a10

a11

a12

a13

b1

b2

b3

b4

b7

b8

b9

b11

b13

di

Dii *

diii

div

dv

dvi

dvii

c1 *

c2 *

c3 *

c4 *

c5

5.79

5.06

3.95

4.31

3.05

2.43

3.85

4.4

46.53

40.54

37.3

49.33

37.3

26

24

22

21

16

20

19

20

16

22

15

17

26

24

22

21

19

20

22

17

22

23

24

25

26

27

28

55

51

47

45

35

43

41

43

35

47

37

46

48

49

50

51

52

15

22

3

2

3

2

3

5

2

3

2

5

2

4

6

4

6

4

6

10

6

4

6

2

4

1

2

1

0

2

0

2

4

1

0

1

0

1

2

1

2

1

2

0

6

5

3

4

3

5

2

3

6

5

3

4

5

3

7

8

9

10

11

12

13

18

16

12

14

12

16

12

15

17

18

19

20

21

0

409.28

316.36

293.37

274.32

230.04

265.31

250.3

264.32

329.19

214.22

295.29

200.24

224.26

409.28

316.36

293.37

274.32

250.3

264.32

239.19

295.29

224.26

298.34

312.37

326.39

340.42

354.45

368.74

382.5

324.95

290.26

283.19

253.86

200.58

245.06

237.29

253.85

255.17

191.44

260.62

189.02

209.87

324.95

290.26

283.19

253.86

237.29

253.85

255.17

260.62

209.87

278.11

294.92

311.72

328.52

345.32

362.12

378.93

689.61

620.23

606.09

547.43

440.87

529.83

514.28

547.4

0.55275

0.483

0.3135

0.43115

0.1940

−0.03325

0.31955

0.38155

0.3955

−0.01035

−0.17895

0.23795

0.2498

0.62405

0.5543

37.3

4.32

2.41

3.81

3.32

3.33

6.21

5.48

4.37

4.73

4.27

4.82

4.74

4.23

3.75

3.47

3.74

4.01

4.52

5.03

5.53

6.04

9.72

9.42

8.66

8.98

7.13

5.82

8.55

9.04

8.98

5.85

8.5

50.44

83.12

33.37

37.3

46.53

40.54

37.3

83.12

37.3

52.61

71.08

59.09

52.61

76.66

52.61

78.89

144.26

78.89

52.61

59.09

38.91

80.91

0.3848

0.50245

0.39085

0.45285

0.4668

−0.10765

0.3211

0.15095

0.2083

0.25325

0.33385

0.416

0.50435

0.5803

1.2791

1.1396

0.8007

1.03755

0.5648

0.2561

0.81435

0.93835

0.9678

0.1563

-0.1812

0.3113

0.67485

0.74335

0.93565

1.0178

1.00995

1.0921

1.17425

1.053785

1.6613

858.62

672.78

626.8

588.7

500.64

570.69

540.66

568.71

698.45

468.5

630.65

496.56

488.58

612.77

640.82

654.85

668.88

682.9

c6

c7

c8

c9

550.05

422.58

560.95

455.7

459.44

589.29

622.89

639.69

656.5

673.3

690.1

191.53

287.90

c10

c11

c12

c13

ei

eiii

eiv

ev

evi

evii

tacrine

NDGA

6.29

7.69

8.51

8.79

9

9.17

9.31

9.44

3.05

3.48

696.93

198.27

302.37

5

a

Logarithm of partition coefﬁcient between n-octanol and water (milog P); ^bTopological polar surface area (TPSA);

c

d

e

Number of hydrogen bond acceptors (n-ON); Number of hydrogen bond donors (n-OHNH); Number of

f

g

h

rotatable bonds (n-rotb); Molecular weight; Molecular volume; blood brain barrier; * these compounds

referred in [29].

2.3. Biological Assays

A number of chalcone derivatives have been obtained and evaluated herein as pleitropic potential

antioxidant agents, inhibitors of AChE and Lipoxygenase in vitro. Among them we included also

a1–4, c1–4 and dii for which earlier we performed a number of in vitro assays [29]. For the sake of

comparison, we include them in the tables and in the discussion, and simultaneously we investigate

their behavior in some additional assays. Numerous studies in patients with Alzheimer’s disease (AD)

showed a signiﬁcant enhancement of lipid peroxidation in their brain. Thus, radical scavengers

and antioxidants could offer in the treatment of AD patients targeting brain lipid peroxidation.

As we already mentioned free radicals are highly implicated in lipoxygenase induction, inﬂammation,

and neurodegenerative diseases such as AD and cancer. Compounds possessing these activities in

combination with antioxidant activity might be protective against these diseases and lead to active and

useful agents. Thus, it would be interesting to determine their antioxidant activities in comparison

Molecules 2019, 24, 199

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to well-known antioxidants, i.e., nordihydroguaiaretic acid (NDGA) and Trolox (Tables 2 and 3,

Figures 6 and 7).

Table 2. Antioxidant activity of the tested compounds % reducing activity (RA %); ABTS^•+

—

decolorization assay%; % anti-lipid peroxidation (AAPH%); and % inhibition of lipid peroxidation of

liposomes (% Inhb. of liposomes LP).

•+

RA% 100 µM

ABTS Inhb. %

AAPH%

@ 100 µM

% Inhb. of Liposomes

Compound

20 min

60 min

@ 100 µM

LP @ 100 µM

a1 *

a2 *

a3 *

a4 *

a5

a6

a7

a8

a9

a10

a11

a12

a13

b1

b2

b3

b4

b7

b8

b9

b11

b13

di

Dii *

diii

div

dv

dvi

dvii

c1 *

c2 *

c3 *

c4 *

c5

1

3

6

4

17

3

2

6

2

1

10

3

9

3

0

15

20

0

3

8

No

12

No

12

No

2

14

6

No

40

No

96

*

nt

2

18

7

17

15

19

6

10

23

7

24

7

46

45

72

30

13

17

20

4

*

23

45

31

32

33

24

55

45

38

89

44

100

82

29

25

17

31

15

22

20

29

30

18

12

27

42

22

70

95

46

100

82

75

32

77

68

71

47

72

14

64

6

No

5

12

No

no

99

no

No

11

No

17

No

98

No

73

25

79

88

96

88

2

0

10

No

no

36

No

17

55

8

14

50

no

No

no

nt

81

3

19

No

Nt

nt

74

nt

4

nt

77

nt

64

nt

69

nt

c6

c7

c8

c9

c10

c11

c12

c13

ei

eiii

eiv

ev

evi

evii

Trolox

Tacrine

NDGA

93

nt

Nt

81

nt

83

nt

No, no activity under the reported experimental conditions. Means within each column differ signiﬁcantly (p < 0.05);

nt, not tested; * [29].

Molecules 2019, 24, 199

9 of 31

Table 3. In vitro inhibition of soybean lipoxygenase (IC₅₀

of acetyl-cholinesterase (IC₅₀M or AChE Inh. %); experimentally-determined lipophilicity R_M

theoretically calculated lipophilicity values cLogP.

µM or LOX Inh. %) and in vitro inhibition

µ

;

R_M± (SD) ^¥

Compound % LOX Inhb. @ 100 µM/IC₅₀µM

% AChE Inhb. @ 100 µM/IC₅₀µM

cLogP

a1 *

a2 *

a3 *

a4 *

a5

14

10

7

26

0.34

0.36

5.51

5.06

3.58

4.13

2.60

1.91

3.41

3.81

3.90

2.13

3.15

2.63

2.96

5.97

5.52

4.04

4.59

3.87

4.27

4.36

3.61

3.42

3.31

3.68

3.97

4.49

5.02

5.59

6.08

10.88

7.92

9.03

5.98

5.54

7.59

8.39

8.58

5.04

7.08

6.04

6.69

7.55

8.21

8.74

9.27

9.79

10.4

56/100 µM

50/100 µM

−0.69

−0.26

−0.62

−0.65

−0.64

−0.68

−0.82

−0.06

−1.09

−0.81

−0.73

−0.91

0.04

−0.66

−0.03

−0.85

−0.79

−0.37

−0.91

0.43

44

9

60

25

38

a6

No

63/87 µM

57/100 µM

a7

a8

4

14

a9

16

23

36

2

48

27

a10

a11

a12

a13

b1

56/100 µM

42

23

25

89/100 µM

44

89/56 µM

b2

44

100 ^#

b3

100/57 µM

82 ^#

b4

82/65 µM

b7

b8

29

25

29

25

16

31

14

22

20

29

18

12

27

b9

16

31

14

12

b11

b13

di

0.67

Dii *

diii

div

dv

dvi

dvii

c1 *

c2 *

c3 *

c4 *

c5

no

41

17

7

17

−0.23

−0.37

0.23

−0.53

0.32

5

41

−0.36

−0.68

−0.89

0.77

0.43

−0.97

−0.77

−0.85

−0.86

0.7

−0.88

−0.96

0.34

23

100/55 µM

93/56 µM

100/55 µM

32

71/49 µM

95/52 µM

58/100 µM

51/100 µM

No

94/56 µM

100/58 µM

85/74 µM

69/76 µM

100/52 µM

100/48 µM

50.5/100 µM

71/57.5 µM

47

c6

c7

98/54 µM

41

c8

c9

26.5

24.5

c10

c11

c12

c13

ei

eiii

eiv

ev

57/85 µM

95/50 µM

52/96 µM

76/65 µM

77/62.5 µM

27

0.56

−0.07

23

7

72/76 µM

14

64/62 µM

6/85 µM

98/0.03 µM

0.13

Nd

evi

evii

Tacrine

NDGA

65/76 µM

12

93/0.5 µM

#

¥

* [29

]

% activity at 0.001

µ

M; SD < 10%; Means within each column differ signiﬁcantly (p < 0.05); nd: not

determined under the experimental conditions.

Molecules 2019, 24, 199

10 of 31

150

100

50

0

Figure 6. The residual enzyme activities % (human h-15-LOX-1) resulting from the tested compounds

at 50 µM.

Figure 7. IC₅₀value for human h-15-LOX-1 from compound b4.

In vitro antioxidant activity has been determined with the aid of a large number of different

assays in order to take into consideration signiﬁcant factors such as solubility or steric hindrance

which may be important in different milieu, and reveal their signiﬁcance. All the recorded methods

are associated with the generation of various radicals. Two approaches are referred: (i) the scavenging

by hydrogen- or electron donation of a preformed free radical, and (ii) the presence of an antioxidant

system during the generation of the radical.

The chalcones were studied for the antioxidant activity (reducing activity RA%) by the use of the

stable free radical 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) at concentrations 0.05 mM after 20

and 60 min (Table 2). In this procedure, the dominant chemical reaction involved is the reduction of

the DPPH radical by single electron transfer from the antioxidant. Thus, phenolic compounds, e.g.,

nordihydroguaiaretic acid (NDGA), giving phenoxide anions are effective antioxidants. Considering

the simple chalcones of

activity. From the derivatives of

a

series, the 4-OH substituted derivatives, exhibited very low/limited reducing

series, the 2-OH substituted chalcones, only compound b3 (72%) the

b

4-dimethyl-amino-phenyl acryl derivative presented very high ability, followed by b4, whereas all the

other simple tested chalcones exhibited low or no activity. For the sake of comparison, we measured

the RA% values for the intermediate double ethers

present any activity by themselves.

d which were included in the Table 2. They did not

The decolorization assay is used to evaluate the antioxidant activity. The ABTS^•+cationic radical is

derived from the direct oxidation of ABTS by potassium persulfate. No involvement of an intermediary

radical is observed. The addition of electron-donating antioxidants reduce ABTS^•+. The radical is

formed prior to the addition of the antioxidant and does not take place continually in the presence

of the antioxidant. Again, we observed very low or no antioxidant activities for both series

Chalcone b3 showed the highest activity among the and simple chalcones. The intermediate ethers

did not show any activity. All the bis-etheriﬁed bis-chalcones presented limited activity with the

exception of compounds c3 ei eiv evi, and evii (Table 2). The structural characteristics highlight the

a and b.

a

b

d

,

importance of the 4-dimethyl-amino-phenyl) acryl group (Figure 2, structure 3). The presence of this

moiety is correlated with higher antioxidant activity. The role of the length of the ether chain within

Molecules 2019, 24, 199

11 of 31

the derivatives of subgroup

e

is not well deﬁned, e.g., c3 (99%, n = 3), ei (98%, n = 2), eiv (73%, n = 5),

ev (25%, n = 6), evi (79%, n = 7), and evii (88%, n = 8).

The water soluble 2,2⁰-azobis(2-amidinopropane) hydrochloride (AAPH) generates in vitro free

radicals through spontaneous thermal decomposition. The experimental conditions employed in or

study signiﬁcantly resemble to cellular lipid peroxidation due to the activity of the peroxyl radicals.

Overall, chalcones

activity at 100 M concentration. The b1

the chalcones. The inhibiting activities for the rest

ethers are moderate inhibitors. It seems that the bis-chalcones

agents compared to the corresponding simple chalcones. This might be correlated with the presence of

the double C=C–COCH₃group. Among the bis-etheriﬁed bis-chalcones, evii exhibited the lowest

a,

b, and

c

present anti-lipid peroxidation activity. Chalcones a5

b3, and b4 (82–100%) are the most active inhibitors among

chalcones ranged from 15–44%. The intermediate

are more potent anti-lipid peroxidation

–13 exhibit moderate

µ

,

b

c

e

activity followed by ev. Both have a long ether chain in their structure.

We used liposomes to investigate the antioxidant activity of the bis-chalcones. The undertaken

results showed high potency for c3, c12, eiv, and evi. The observed % inhibition activities were found

to be in agreement with the results taken from the AAPH assays. Considering chalcones c2, c4, and ei,

the results obtained were not able to provide any additional information.

Lipoxygenases (LOXs) catalyze the metabolism of arachidonic acid to leukotrienes, important

inﬂammatory mediators [47]. Recently published research showed that AD brains had higher 5-LOX

protein levels than did healthy controls [48]. It has been found the activation of brain lipoxygenases

is an early event in the pathogenesis of Alzheimer’s disease [49]. Furthermore 12/15-LOX activity

was increased in pathologically affected areas of AD brains [50] and, consequently, 5-lipoxygenase

(5-LOX) acts as a modulator of Aβ peptides formation in vivo [48]. Some new studies points to a new

role for 5-LOX in regulating endogenous tau metabolism in the central nervous system supporting

the hypothesis that the inhibition of 5-LOX could be beneﬁcial for Alzheimer’s disease-related

tau neuropathology [51].

In our experiments we used soybean lipoxygenase. Designing agents to modulate activities of the

variety of so closely homologous enzymes, such as different LOXs, require an intimate knowledge

of their 3D structures, as well as information about metabolism of the potential xenobiotics. Thus far

only the structures of soybean isozymes LOX-1 and LOX-3 have been determined for native enzymes,

and several structures of their and rabbit 15-LOX (from reticulocytes) molecular complexes with

inhibitors are known. Due to lack of sufﬁciently-puriﬁed human enzymes most of the research has

been done on soybean LOX [52,53]. Although this enzyme is not identical to the mammalian one,

it seems that there is a sufﬁcient qualitative correlation between values for enzyme inhibition with the

two enzymes [54–56].

Thus, we used the isozyme LOX-1 in our assays which only uses free fatty acids as substrates [57

exhibits maximal activity at pH 9.0 and converts linoleic acid into to 13-hydroperoxylinoleic acid [57

producing a conjugated diene that absorbs at 234 nm.

]

LOXs contain a “non-heme” iron per molecule in the enzyme active site as high-spin Fe²⁺in

the native state and the high spin Fe³⁺in the activated state. Nordihydroguaiaretic acid (NDGA),

referred as known inhibitor of soybean LOX, has been used as a reference compound (IC₅₀0.5 µM/93%

at 100 µM).

Perusal of the %/IC₅₀₀_sinhibition values (Table 3 ) shows that the simple 2-OH substituted

chalcones are less potent than the corresponding 4-OH-substituted. Chalcone c11 was found to be the

most active inhibitor within all the series followed by: c11

the inhibitory activities in terms of IC₅₀values were in the range 50–100 µM. Compounds b1, b3 and

c13 b4 were found to be almost equipotent inhibitors The chain length of the ether linker does not

> c6 > b1, b3 > ei > c13, b4 > evi > c10 and

,

affect the degree of inhibition of the soybean LOX within the e compounds and that the three-carbon

chain provides a structural advantage over the longer (seven-carbon) or shorter (two-carbon) linkers.

For the sake of comparison, the intermediate bis-ethers d were tested and found to be inactive.

Molecules 2019, 24, 199

12 of 31

Lipophilicity is referred to as an important physichochemical property for lipoxygenase

inhibition [58]. However, in this dataset lipophilicity does not seem to correlate with LOX inhibition,

since the most active inhibitors present high lipophilicity value, but the activity does not exhibit

any apparent trend related to lipophilicity values. Thus, the presence of the two enone groups in

combination to the ether linkage is correlated with higher inhibitory activity. The length of the ether

chain seems to be of minor importance. The results derived from the chalcones, for which aldehydes

1–4 [29] and 11 and 13 were used as synthetic blocks, were more signiﬁcant. The role of any other

speciﬁc structural characteristics are not well deﬁned.

A large number of bioactive compounds inhibit lipoxygenase activity and, as a consequence,

several mechanisms of action have been proposed [59]. However, most of the LOX inhibitors are

antioxidants or free radical scavengers. Thus, LOX inhibitors: a) can reduce the iron species in the

active site to the catalytically inactive ferrous form or b) can act as ligands for Fe³⁺

.

Herein, the results from the anti-lipid peroxidation activity studies support the anti-LOX activity

for the signiﬁcantly active bis-chalcones c11 c6 b1 b3 ei c13 b4 evi, and c10. From the literature

,

it is clear that there are no strict structural requirements for lipoxygenase inhibition, thus. There is

no universally accepted approach to evaluate the relative potency of different substances to cause

lipoxygenase inhibition.

Due to the key role of 15-LOX-1 in AD, we decided to test b3, ei, eiv, c3, c2, c13, a3, b1, and b4

as inhibitors of recombinant h-15-LOX-1, since these chalcones exhibited signiﬁcant results against

soybean LOX (Figure 6). PD-146176 has been reported by Parke-Davis (now Pﬁzer) [60] as an inhibitor

of h-15-LOX-1. The residual enzyme activity was measured after 10 min pre-incubation with the tested

compounds at room temperature. As reference standard compounds the known inhibitors, PD-146176

(15-LOX) and Zileuton (5-LOX), were also tested. The results of the screening are shown in Figure 6.

It seems that the simple chalcone b4 presents the highest activity within the group and in comparison

to the reference PD-146176. Compound c11 follows

IC₅₀value, which was found to be 9.5 µM, whereas for PD-146176 it was 16 µM.

We investigated, in vitro, the inhibitory activity of , and derivatives on acetylcholinesterase

. Thus, we found it interesting to determine its

a

,

b,

c

e

activity using acetylthiocholine as a substrate [29]. The potential of these compounds to act as

acetylcholinesterase inhibitors can be considered beneﬁcial for their prospective nootropic action and

may contribute to the mechanisms of action of reported structurally related hydroxyl chalcones [61].

All the

with the exception of c6

7–48% (Table 3). It should to be noticed that simple chalcones b3 and b4 presented high inhibitory

activities. They exhibited 100% or 82% inhibition at 0.001 M, whereas b1 gave IC₅₀value 100 M.

Unfortunatelly, we did not succeed to determine the IC₅₀values of b3 and b4, which constantly

presented high inhibition independently of the decrease of the concentration. All the rest of the

chalcones were much less efﬁcient in their inhibitory action. The bis-chalcone c6 did not present any

c

and

e

bis-chalcone derivatives present signiﬁcant IC₅₀values as it is shown in Table 3

,

eiii, and ev. The simple chalcones are less active with a % inhibition range

a

µ

b

activity. On the contrary, bis-chalcone c12 was found to be the most active, followed by c12

c8 > evi > eiv > c10 > c5.

> c7 > ei >

Again it is observed, as it has been found from our published results [29], that the transformation

of a simple to a bis-chalcone with an ether linkage leads to more potent analogues (a3 c3) [29].

The chain length of the ether linker does not affect the inhibition of AChE induced by compounds

/

e

and that the three-carbon chain provides structural advantage over the longer (seven-carbon) or shorter

(two-carbon) linkers.

Perusal of logP and IC₅₀or % AChE values in Table 3 reveal that the role of lipophilicity on the

inhibition of AChE is also not well deﬁned in this series of compounds.

The cytotoxicity of the synthesized derivatives against L929 mouse ﬁbroblasts cells was

determined using the propidium iodide (PI) ﬂuorescence method [29] in the presence of different

concentrations (1–100

µM) of these compounds. The results are presented in Table 4 in the form of the

% cell survival values as PI% for the examined compounds. All tested chalcones

a(5–12) showed low

Molecules 2019, 24, 199

13 of 31

cytotoxicities in the whole area of concentrations examined (from 1

µ

M to 100

c6

, c11, and c12 follow. For these compounds cell toxicity started increasing

µ

M), with the noticeable

exception of a7

,

a11, and a12. Considering the bis-chalcone ethers c5

c8

,

, c9, and c10, they showed low

cytotoxicity. Chalcones c7

,

signiﬁcantly at the concentration of 20 µM, reaching the highest values at 100 µM.

Table 4. Cytotoxicity of chalcones and bis-etheriﬁed chalcones on L929 cells (24-h incubation) expressed

as PI % values.

1 µM Average

(% pi) ± σ

10 µM Average 20 µM Average 50 µM Average

100 µM Average

(% pi) ± σ

Compound

(% pi) ± σ

5.5 ± 0.71

27.5 ± 8.9

19 ± 1.4

(% pi) ± σ

a1 *

a2 *

a3 *

a4 *

a5

a6

a7

1 ± 0.6

5 ± 2.8

32 ± 4.9

1 ± 0.5

3.5 ± 0.7

63 ± 11.2

14 ± 3.5

15 ± 0.8

14 ± 0.7

8.5 ± 2.1

12 ± 4.2

29.5 ± 0.6

28.32 ± 2.91

3.25 ± 2.6

9.24 ± 6.49

1.84 ± 2.05

2.7 ± 0.68

2.39 ± 1.08

18.81 ± 2.57

8.12 ± 0.32

16.78 ± 5.61

13 ± 4.5

51 ± 9.9

11.36 ± 0.62

1.71 ± 2.08

6.85 ± 3.12

1.39 ± 0.1

0.55 ± 0.09

3.03 ± 0.88

3.13 ± σ 2.43

0.88 ± 0.31

4.94 ± 1.02

9 ± 4.5

No ± No

36.15 ± 2.33

6.67 ± 1.35

60.95 ± 0.01

8.15 ± 0.34

7.59 ± 0.13

17.15 ± 0.32

67.84 ± 6.45

63.54 ± 6.16

33.22 ± 9.65

42 ± 3.5

36.15 ± 2.33

6.67 ± 1.35

60.95 ± 0.01

8.15 ± 0.34

7.59 ± 0.13

17.15 ± 0.32

67.84 ± 6.45

63.54 ± 6.16

33.22 ± 9.65

42 ± 3.5

1.66 ± 0.59

9.27 ± 1.9

2.95 ± 1.23

0.59 ± 0.83

2.25 ± 0.38

6.26 ± 3

a8

a9

a10

a11

a12

di

dii *

diii

div

dv

dvi

c1 *

c2 *

c3 *

c4 *

c5

c6

c7

c8

c9

c10

c11

c12

3.25± 2.11

9.05 ± 0.58

8.5 ± 4.24

20 ± 3.12

14.06 ± 1.36

4.89 ± 0.51

4.47 ± 0.59

0.37 ± 0.52

38.5 ± 6.9

17 ± 1.4

20.76 ± 3.51

11.86 ± 2.98

8.05 ± 3.58

1.79 ± 1.53

53.5 ± 0.71

43 ± 2.8

32.14 ± 6.99

17.05 ± 1.46

9.69 ± 0.57

2.55 ± 0.5

74 ± 5.6

32.14 ± 6.99

17.05 ±1.46

9.69 ± 0.57

2.55 ± 0.5

74 ± 5.6

8.56 ± 3.97

6.83 ± 2.46

1.92 ± 1.02

49.5 ± 7.8

29 ± 4.2

55 ± 4.2

81 ± 0.8

90 ± 1

100 ± 0

29.5 ± 3.5

0.32 ± 0.45

1.12 ± 1.15

22.5 ± 11.64

35.72 ± 10.23

12.5 ± 10.31

18.10 ± 11.82

6.4 ± 0.4

42 ± 4.6

46 ± 5.6

67 ± 9.2

0.97 ± 0.2

2.2 ± 1.66

28.73 ± 11.78

29.05 ± 9.62

15.88 ± 6.31

13.48 ± 10.36

19.07 ± 7.23

31.31 ± 4.84

3.02 ± 1.36

1.16 ± 0.81

25.4 ± 8.42

35.69 ± 5.85

28.76 ± 2.74

25.93 ± 9.73

34.63 ± 0.31

53.79 ± 16.95

7.26 ± 0.3

3.03 ± 0.02

28.84 ± 5.62

38.7 ± 4.96

29.53 ± 6.87

32.11 ± 9.38

64.53 ± 3.07

84.03 ± 2.25

7.26 ± 0.3

3.03 ± 0.02

28.84 ± 5.62

38.7 ± 4.96

29.53 ± 6.87

32.11 ± 9.38

64.53 ± 3.07

84.03 ± 2.25

13.22 ± 0.64

* ref [29]; Means within each column differ signiﬁcantly (p < 0.05).

The pathogenesis of Alzheimer’s disease (AD) is widely associated with the aggregation of

-amyloid peptide (A ) either to soluble oligomers or to higher-order polymeric insoluble ﬁbrils [62 63].

As a result, one of the main therapeutic strategies against AD targets the aggregation process of A

and a great number of small molecules are being investigated for their potential to intervene in

the assembly of A and inhibit its neurotoxicity in vitro and in vivo 64]. Circular dichroism (CD)

β

,

β,

β

[

spectroscopy is commonly used to probe the secondary structure of polypeptides and to monitor

conformational changes and interactions of polypeptides with small molecules. The typical aggregation

process of A

β

produces characteristic CD spectra over time that reﬂect the conformational changes that

-sheet oligomers, and then gradually to larger

-sheet formations that ﬁnally produce amyloid ﬁbrils [65]. CD is, therefore, a valuable tool to monitor

occur as the random coil monomers are converted into

β

the aggregation process of Aβ and to study the effect of potential aggregation inhibitors. To further

investigate the pleiotropic effects of chalcones against AD, selected compounds from our library,

were subjected to CD studies in order to evaluate their potential to interfere with the aggregation

process of Aβ40. Figure 8 shows the results of the CD experiments performed over a period of 40 days.

Molecules 2019, 24, 199

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The aggregation spectra of plain A

β40 show at day 0 the typical random coil peak (negative maximum

at 198 nm) the intensity of which gradually decreases as the peptide forms β–sheet structures (negative

maximum at 222 nm, day 15) that further aggregate to higher order polymeric ﬁbrils. The precipitation

of the insoluble ﬁbrils from solution eventually results in the loss of CD signal (days 30 and 40) [66].

As can be seen in Figure 8, the presence in the A

apart from a noted delay in the overall process, did not signiﬁcantly change the ﬁbrillization course

of A 40, and the CD signal almost reached baseline at day 40. On the contrary, addition of bis-ether

dii modiﬁed the aggregation course of A 40 by stabilizing a -sheet structure that did not seem to

evolve further. This suggests that the chain length of the ether linker affects the degree of interaction

of the compounds with A and that the three-carbon chain provides a structural advantage over

β40 solution of intermediate bis-ethers di and dvi,

β

the longer (seven-carbon) or shorter (two-carbon) linkers. In view of the above results, chalcone

derivatives bearing a three-carbon chain linker, speciﬁcally c2, c3, and c4, were subsequently studied

in order to assess the effect of further derivatization and the presence of the chalcone moiety in the

structure. As shown in Figure 8, in the case of c3, no signiﬁcant effect on the aggregation process of

A

β

40 was observed, while on the contrary the presence of compounds c2 and c4, which include the

intact chalcone moiety in their structure, stabilized -sheet assemblies that did not evolve further into

insoluble ﬁbrillar aggregates, as was also observed in the case of compound dii. Overall, our CD study

identiﬁed three new structures capable of interfering with the aggregation process of A , out of which

β

compounds c2 and c4 display additional protective actions against AD and add to the pleiotropic

proﬁle of the chalcone derivatives.

Molecules 2019, 24, 199

15 of 31

5

0

Aβ40 (50 μΜ)

-5

0 day

3 day

9 day

15 day

30 day

40 day

-10

-15

-20

190

200

210

220

230

240

250

260

Wavelength (nm)

5

0

5

0

5

0

Aβ40 + di

Aβ40 + dii

Aβ40 + dvi

-5

0 day

3 day

9 day

15 day

30 day

40 day

0 day

3 day

9 day

15 day

30 day

40 day

0 day

3 day

9 day

15 day

30 day

40 day

-10

-15

-20

-10

-15

-20

-10

-15

-20

190

200

210

220

230

240

250

260

190

200

210

220

230

240

250

260

190

200

210

220

230

240

250

260

nm

5

0

5

0

5

0

Aβ40 + C2

Aβ40 + C4

Aβ40 + C3

-5

-10

-15

-20

-10

-15

-20

0 day

3 day

9 day

15 day

30 day

40 day

0 day

3 day

9 day

15 day

30 day

40 day

0 day

3 day

9 day

15 day

30 day

40 day

-10

-15

-20

190

200

210

220

230

240

250

260

200

210

220

230

nm

240

250

260

190

200

210

220

230

240

250

260

nm

Figure 8. CD spectra of A

and bis-etheriﬁed bis-halcones c2

β

40 (50

µ

M) in phosphate buffer (PB 10 mM, pH 7.33) in the absence or presence of 50

µ

M of intermediate bis-ethers di

,

dii

,

dvi

,

◦

,

c3, and c4 (1:1 ratio). Spectra were recorded for a period of 40 days at 33 C. Representative spectra from n= 3 independent

experiments are presented.

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3. Experimental Section

3.1. Materials and Instruments

All chemicals, solvents, and chemical and biochemical reagents were of analytical grade and

purchased from commercial sources (Merck, Merck KGaA, Darmstadt, Germany, Fluka Sigma-Aldrich

Laborchemikalien GmbH, Hannover, Germany, Alfa Aesar, Karlsruhe, Germany and Sigma,

St. Louis, MO, USA). Soybean lipoxygenase, sodium linoleate, and 2,2-azobis-(2-amidinopropane)

dihydrochloride (AAPH) were obtained from Sigma Chemical, Co. (St. Louis, MO, USA). All starting

materials were obtained from commercial sources (Merck, Merck KGaA, Darmstadt, Germany,

Fluka Sigma-Aldrich Laborchemikalien GmbH, Hannover, Germany, Alfa Aesar, Karlsruhe, Germany,

and Sigma, St. Louis, MO, USA) and used without further puriﬁcation. Melting points (uncorrected)

were determined on a MEL-Temp II (Lab. Devices, Holliston, MA, USA). For the in vitro tests,

UV–VIS spectra were obtained on a 554 double-beam spectrophotometer Perkin-Elmer (Perkin-Elmer

Corporation Ltd., Lane Beaconsﬁeld, Bucks, UK). Infrared spectra (KBr pellets) were recorded with

Perkin-Elmer 597 spectrophotometer (Perkin-Elmer Corporation Ltd., Lane Beaconsﬁeld, Bucks, UK).

The 1H Nucleic Magnetic Resonance (NMR) spectra were recorded at 300 MHz on a Bruker AM-300

spectrometer (Bruker Analytische Messtechnik GmbH, Rheinstetten, Germany) in CDCl3 or DMSO

using tetramethylsilane as an internal standard unless otherwise stated. 13C-NMR spectra were

obtained at 75.5 MHz on a Bruker AM-300 spectrometer (Bruker, Hamburg, Germany) in CDCl3 or

DMSO solutions with tetramethylsilane as internal reference unless otherwise stated. Chemical shifts

are expressed in ppm and coupling constants J in Hz. Mass spectra were determined on a LC-MS

2010 EV Shimadzu (Shimadzu, Kiyoto, Japan) using MeOH as the solvent. Elemental analyses for C

and H gave values acceptably close to the theoretical values ( 0.4%) in a Perkin-Elmer 240B CHN

±

analyzer (Perkin-Elmer Corporation Ltd., Lane Beaconsﬁeld, Bucks, UK). Reactions were monitored

by thin layer chromatography on 5554 F254 silica gel/TLC cards (Merck and Fluka Chemie GmbH

Buchs, Steinheim, Switzerland). For preparative thin layer chromatography (prep TLC) silica gel

60 F254, plates 2 mm, Merck KGaA ICH078057 were used. For the experimental determination of

the lipophilicity using reverse phase thin layer chromatography (RP-TLC) TLC-Silica gel 60 F254 DC

Kieselgel, Merck (Merck, Merck KGaA, Darmstadt, Germany) (20 × 20 cm) plates were used.

3.2. Chemistry General Procedure

3.2.1. Synthesis of 4-Hydroxy-Chalcones (a1–13)

A modiﬁed Claisen–Schmidt condensation was performed between 4-hydroxy acetophenone

and the suitable substituted aryl aldehyde at a molar ratio 1:1 in absolute ethanol (10 mL) [29].

Three milliliters (3 mL) aqueous KOH (20%) was added. The mixture was stirred at room temperature

in a US-bath. The end of the reaction was monitored by TLC. The mixture was treated with aqueous

HCl 10% and adjusted to acidic pH. The precipitate was either ﬁltered and washed with cold water

or extracted with CHCl₃(30 mL

brine and dried under anhydrous MgSO₄. The product was evaporated to dryness and puriﬁed by

recrystallization from a proper solvent.

×

3). The combined organic layers were washed with water and

(E)-3-(4-((4-bromobenzyl) oxy) phenyl)-1-(4-hydroxyphenyl) prop-2-en-1-one (a1) [29 ]. The crude product was

recrystallized from ethanol: yield: 76%; yellow solid; R_f(hexane:acetone 2:1): 0.47; m.p.: 117–119 ^◦C.

(E)-1-(4-hydroxyphenyl)-3-(3-phenoxyphenyl) prop-2-en-1-one (a2) [29]. The mixture was heated in a

◦

microwave oven for 15 min (50 watt, 60 C). The crude product was recrystallized from ethanol: yield:

69%; yellow solid; R_f(hexane:acetone 2:1): 0.5; m.p.: 193–195 ^◦C.

(2E,4E)-5-(4-(dimethylamino) phenyl)-1-(4-hydroxyphenyl) penta-2,4-dien-1-one (a3) [29]. The crude product

was crystallized from ethanol: yield: 79%; dark brown solid; R_f(hexane:acetone 2:1): 0.55, 0, 55; m.p.:

220–222 ^◦C.

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(E)-1-(4-hydroxyphenyl)-3-(naphthalen-1-yl) prop-2-en-1-one (a4) [29 ]. The crude product was recrystallized

from PE/EA: yield: 84%; yellow solid; R_f(hexane:acetone 2:1): 0.52; m.p.: 148–150 ^◦C.

(E)-1-(4-hydroxyphenyl)-3-(thiophen-2-yl) prop-2-en-1-one (a5). The crude product was recrystallized from

methanol: yield: 96%; yellow solid; R_f(hexane:acetone 2:1): 0.41; m.p.: 162–164 ^◦C [67].

(E)-3-(2,7a-dihydro-1H-indol-3-yl)-1-(4-hydroxyphenyl) prop-2-en-1-one (a6). The crude product was

crystallized from ethanol: yield: 77%; R_f(hexane:acetone 2:1): 0.36; m.p.: 212–213 ^◦C; IR (KBr, cm⁻¹):

1610, 1640; LC-MS (m/z): (C₁₇H₁₅NO₂₎289 [M + Na]⁺, 298 [M + CH₃OH]⁺; ¹H-NMR (300 MHz, CDCl₃)

δ

: 10.05 (s, 1H, OH

4H, aromatic protons, =CH), 7.29–7.37 (m, 3H, aromatic protons), 6.86–7.14 (m, 3H, aromatic protons,

CO–CH=); ¹³C-NMR (75 MHz, CDCl₃)

: 184.30 (C=O), 161.87 (C–O ), 143.92 (CH–C=O), 135.82,

−

), 8.32 (m, 2H, aromatic protons), 7.85 (m, 1H, aromatic proton), 7.41–7.53 (m,

−

δ

−

124.51, 123.06, 122.02, 111.46, 106.92, 77.19, 76.91, 76.59. Elemental analysis: expected % (C₁₇H₁₅NO₂):

C 76.96, H 5.7, N 5.28; found % (C₁₇H₁₅NO₂): C 76.73, H 5.9, N 5.43.

(2E,4E)-1-(4-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-one (a7). Methanol was used as a solvent. The mixture

◦

was heated at 35 C in US-bath. The crude product was recrystallized from ethanol: yield: 78%;

R_f(hexane:acetone 2:1): 0.46; m.p.: 164–166 ^◦C [68].

(2E,4E)-1-(4-hydroxyphenyl)-4-methyl-5-phenylpenta-2,4-dien-1-one (a8). Methanol was used as a solvent.

The crude product was recrystallized from ethanol and treated with diethylether. Yield: 74%;

R_f(hexane:acetone 2:1): 0.42; m.p.: 100–102 ^◦C; IR (KBr, cm⁻¹): 1610,1640; LC-MS (m/z): (C₁₈H₁₆O₂)

1

265 [M + 1]⁺, 306 [M + CH3CN + H]⁺; H-NMR (300 MHz, CDCl₃)

δ: 7.90–8.04 (m, 2H, aromatic

protons), 7.81 (d, 1H, CH=, J = 15 Hz), 7.41(d, 1H, CH=, J = 15 Hz), 7.17–7.31 (m, 6H, aromatic protons),

6.83–6.86 (m, 2H, aromatic protons), 2.47 (s, 3H, CH₃); ¹³C-NMR (75 MHz, CDCl₃)

: 193.1 (C=O),

163.1 (C–O ), 146.1, 145.2, 142.1, 134.2, 131.9, 129.8, 127.4, 125.2, 124,9, 116.4, 15.7 (CH₃). elemental

analysis: expected % (C₁₈H₁₆O₂): C 81.79, H 6.10; found % (C₁₈H₁₆O₂): C 81.67, H 6.04.

δ

−

(2E,4E)-4-bromo-1-(4-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-one (a9). Methanol was used as asolvent.

The mixture was heated at 35 ^◦C in an ultrasound bath. The crude product was recrystallized from

ethanol: yield: 73%; yellow semi-solid; R_f(hexane:acetone 2:1): 0.42; m.p.: 88–89 ^◦C; IR (KBr, cm⁻¹):

1

1610,1650; LC-MS (m/z): (C₁₇H₁₃BrO₂) 363 [M + CH₃OH + H]⁺; H-NMR (300 MHz, CDCl₃)

δ:

7.89–8.04 (m, 3H, aromatic protons), 7.77–7.80 (m, 1H, =CH–CO), 7.60–7.66 (m, 1H, aromatic proton),

7.36–7.93 (m, 3H, aromatic protons and =CH

−

), 7.02–7.09 (m, 1H, CH=C (Br)), 6.88–7.01 (m, 3H,

δ: 196.73 (C=O), 162.59 (C–OH), 130.83, 130.59, 130.43,

aromatic protons); ¹³C-NMR (75 MHz, CDCl₃)

129.50 (C–C=O), 128.90 (Ph–C=), 121.83 (C–Br), 114.32, 114.22, 114.12. Elemental analysis: expected %

(C₁₇H₁₃BrO₂): C 62.03, H 3.98; found % (C₁₇H₁₃BrO₂): C 59.97, H 4.18.

(E)-3-(furan-2-yl)-1-(4-hydroxyphenyl) prop-2-en-1-one (a10). The crude product was recrystallized from

ethanol: yield: 81%; light yellow solid; R_f(hexane:acetone 2:1): 0.4; m.p.: 153–155 ^◦C [69].

(2E,4E)-1-(4-hydroxyphenyl)-5-(4-nitrophenyl) penta-2,4-dien-1-one (a11). The crude product was

recrystallized from ethanol: yield: 82%; dark brown solid; R_f(hexane:acetone 2:1): 0.2; m.p.: 182–183

^◦C; IR (KBr, cm⁻¹): 1620,1710; LC-MS (m/z): (C₁₇H₁₃NO₄) 297 [M + 1]⁺, 342 [M + CH₃CH₂OH]⁺,

337 [M + CH₃CN]⁺; ¹H-NMR (300 MHz, CDCl₃)

δ: 8.12–8.13 (m, 2H, aromatic protons), 7.99 (m, 2H,

aromatic protons), 7.51–7.59 (m, 4H aromatic and CH= protons), 7.36–7.38 (m, 2H aromatic protons),

7.02–7.09 (m, 2H aromatic and CH=, protons), 6.92–6.93(m, 2H, aromatic protons); ¹³C-NMR (75 MHz,

CDCl₃) δ: 192.80 (C=O), 162.74 (C–OH), 146.50, 146.12, 141.97, 136.42, 129.40, 127.93, 127.54 (CH–CO),

125.83, 124.70, 116.63. Elemental analysis: expected % (C₁₇H₁₃NO₄): C 69.15, H 4.44, N 4.74; found %

(C₁₇H₁₃NO₄): C 69.25, H 4.21, N 5.01.

(E)-1-(4-hydroxyphenyl)-3-(5-methylfuran-2-yl) prop-2-en-1-one (a12). The crude product was recrystallized

◦

from ethanol: yield: 89%; orange-yellow solid; R_f(hexane:acetone 2:1): 0.44; m.p.: 157–159 C; IR (KBr,

cm⁻¹): 1610, 1640; ¹H-NMR (300 MHz, CDCl₃)

δ

: 8.01–8.05 (m, 2H, aromatic protons), 7.52 (d, 1H,

CH=, J = 15 Hz), 7.36 (d, 1H, CH=, J = 15 Hz), 6.92–6.66 (m, 4H, aromatic protons), 5.77 (s, 1H, OH),

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2.39 (s, 3H, CH₃); ¹³C-NMR (75 MHz, CDCl₃)

δ

: 188.37 (C=O), 160.04, 150.42, 130.99, 130.38, 117.94,

117.39, 115.35, 114.93, 109.70, 109.21, 23.04 (

(C₁₄H₁₂O₃): C 77.89, H 5.03; found % (C₁₃H₁₂O₂): C 78.1, H 5.68.

−

CH₂), 14.02 (CH₃); elemental analysis: expected %

−

(E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one (a13). The crude product was crystallized from ethanol:

yield: 74%; white solid; R_f(hexane:acetone 2:1): 0.48; m.p.: 122–123 ^◦C [70].

3.2.2. Synthesis of 2-Hydroxy-Chalcones (b1–4, b7–9, b11, b13)

A modiﬁed Claisen–Schmidt condensation was performed between 2-hydroxy acetophenone and

the suitable substituted aryl aldehyde at a molar ratio 1:1 in absolute ethanol (10 mL). Three milliliters

(3 mL) of aqueous KOH (20%) was added. The mixture was stirred at room temperature in a US-bath.

The end of the reaction was monitored by TLC. The mixture was treated with aqueous HCl 10% and

adjusted to acidic pH. The precipitate was either ﬁltered and washed with cold water or extracted with

CHCl₃(30 mL

×

3). The combined organic layers were washed with water and brine and dried under

anhydrous MgSO₄. The product was evaporated to dryness and puriﬁed by recrystallization from a

proper solvent.

(E)-3-(4-((4-bromobenzyl) oxy) phenyl)-1-(2-hydroxyphenyl) prop-2-en-1-one (b1). The crude product was

◦

crystallized from acetone: yield: 70%; bright yellow solid; R_f(dichloromethane): 0.8; m.p.: 76 C;

1

IR (KBr, cm⁻¹): 3060, 3100,1680, 1580, 1100; H-NMR (300 MHz, CDCl₃)

δ

: 8.06–8.18 (s, 1H, OH),

OCH₂); ¹³C-NMR (75 MHz, CDCl₃)

193.62 (C=O), 163.57, 163.37 (C–OH), 160.82, 145.09, 136.00, 135.40, 132.00, 131.86, 130.54, 130.31, 129.05,

127.82, 122.30 (C–Br), 120.10, 118.76, 118.60, 118.00, 115.36, 115.10, 77.42, 77.00, 76.57, 69.46 ( CH₂–O );

6.91–7.92 (m, 14H, aromatic and =CH–CO protons), 5.17 (s, 2H,

−

δ:

−

elemental analysis: expected % (C₂₂H₁₇BrO₃): C 64.56, H 4.19; found % (C₂₂H₁₇BrO₃): C 54.44, H 4.02.

(E)-1-(2-hydroxyphenyl)-3-(3-phenoxyphenyl) prop-2-en-1-one (b2). The reaction was assisted with

microwave at 65 ^◦C, 50 watt for 15 min. The crude product was recrystallized from ethanol: yield:

68%; yellow solid; R_f(dichloromethane): 0.8; m.p.: 76 ^◦C; IR (KBr, cm⁻¹): 3100,1700, 3050; ¹H-NMR

(300 MHz, CDCl₃)

(br, 2H aromatic protons), 7.41–7.54 (m, 3H aromatic protons), 6.91–7.18 (m, 3H, aromatic and CH=,

protons); ¹³C-NMR (75 MHz, CDCl₃)

: 187.21 (C=O), 163.60, 163.37, 160.82, 144.84, 141.89, 132.64,

δ: 12.90 (s, 1H, OH), 7.82–7.90 (m, 7H aromatic and CH= protons), 7.67–7.7.72

δ

129.21, 128.82, 128.56, 127.27, 126.95, 125.43, 77.42; elemental analysis: expected % (C₂₁H₁₆O₃): C 79.73,

H 4.78; found % (C₂₁H₁₆O₃): C 79.76, H 4.39.

(2E,4E)-5-(4-(dimethylamino)phenyl)-1-(2-hydroxyphenyl)penta-2,4-dien-1-one (b3) The crude product was

crystallized from methanol/chloroform Yield: 71%; Dark purple semi-solid; R_f(dichloromethane): 0.9;

m.p.: semi-solid; IR (KBr, cm⁻¹): 3100, 3060, 1720, 1580, 1300; ¹H-NMR (300 MHz, CDCl₃)

δ

: 12.28 (s,

1H, OH), 7.67–7.7.72 (br, 2H aromatic protons), 7.41–7.54 (m, 3H aromatic protons), 6.91–7.18 (m, 3H,

aromatic and CH=, protons) 6.50–6.85 (m, 4H aromatic and CH=, protons), 2.63–3.27 (m, 6H, 2 CH₃);

¹³C-NMR (75 MHz, CDCl₃)

: 190.28 (C=O), 153.89, 152.37, 136.40, 132.00, 130.67, 130.44, 123.75, 121.74,

×

δ

118.86, 118.4, 117.97, 111.71, 110.94, 110.61, 77.40, 77.00, 76.54, 40.00 (CH₃); elemental analysis: expected

% (C₁₉H₁₉NO₂): C 77.79, H 6.53, N 4.77; found % (C₁₉H₁₉NO₂): C 77.56, H 6.14, N 4.84.

(E)-1-(2-hydroxyphenyl)-3-(naphthalen-1-yl) prop-2-en-1-one (b4). The crude product was recrystallized

with ﬂash chromatography hexane/acetone (2:1): yield: 68%; yellow solid; R_f(dichloromethane): 0.9;

m.p.: 84–85 ^◦C [71].

(2E,4E)-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-one (b7). Methanol was used as a solvent. The mixture

was heated at 35 ^◦C in an ultrasound bath. The crude product was recrystallized from acetone: yield:

72%; bright yellow solid; R_f(hexane:acetone 2:1): 0.48; m.p.: 82–83 ^◦C [72].

(2E,4E)-1-(2-hydroxyphenyl)-4-methyl-5-phenylpenta-2,4-dien-1-one (b8). Methanol was used as solvent.

◦

The mixture was heated at 35 C in an ultrasound bath. The crude product was crystallized from

acetone: yield: 67%; yellow semi-solid; R_f(hexane:acetone 2:1): 0.68; m.p.: semi-solid; IR (KBr, cm⁻¹):

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1

3150, 1620; LC-MS (m/z): (C₁₉H₁₆O₂) 266 [M + 1]⁺, 297 [M + CH₃OH]⁺, 288 [M + Na]⁺, H-NMR

(300 MHz, CDCl₃) : 7.97–7.99 (m, 2H, CH=C), 7.61–7.62 (m, 4H aromatic protons), 7.12–7.37 (m, 3H,

aromatic protons, =CH–CO), 6.92–7.02 (m, 2H, aromatic protons), 6.4–6.69 (m, 2H, aromatic proton,

Ph–CH=), 2.02 (s, 3H, CH₃); ¹³C-NMR (75 MHz, CDCl₃)

: 196.97 (C=O), 162.73 (C–OH), 132.02

CH–C=O), 131.82, 130.88, 130.45, 127.97, 127.70, 123.40, 119.25, 118.74, 116.28, 16.20 (CH₃); elemental

δ

(

−

analysis: expected % (C₁₉H₁₆O₂): C 81.79, H 6.10; found % (C₁₉H₁₆O₂): C 82.01, H 5.99.

(2E,4E)-4-bromo-1-(2-hydroxyphe_◦nyl)-5-phenylpenta-2,4-dien-1-one (b9). Methanol was used as a solvent.

The mixture was heated at 35 C in an ultrasound bath. The crude product was crystallized from

ethanol: yield: 65%; yellow semi-solid; R_f(hexane:acetone 2:1): 0.48; m.p.: semi-solid; IR (KBr,

cm⁻¹): 3150, 1690, 1610; LC-MS (m/z): (C₁₇H₁₃BrO₂) 331 [M + 1]⁺, 376 [M + CH₃CH₂OH]⁺; ¹H-NMR

(300 MHz, CDCl₃)

7.60–7.66 (m, 1H), 7.54 (d, 1H, CH=, J = 15 Hz), 7.36–7.44 (m, 2H), 7.02–7.09 (m, 1H), 6.89–7.01 (m,

3H, aromatic protons); ¹³C-NMR (75 MHz, CDCl₃)

: 196.73 (C=O), 162.59 (C–OH), 130.83, 130.71,

δ: 7.89–8.04 (m, 4H, aromatic and OH, protons), 7.80 (d, 1H, CH=, J = 15 Hz),

δ

130.59, 130.43, 129.50, 128.90, 128.30, 121.83, 114.32, 114.22, 114.12 (C–Br); elemental analysis: expected

% (C₁₇H₁₃BrO₂): C 62.03, H 3.98; found % (C₁₇H₁₃BrO₂): C62.33, H 4.15.

(2E,4E)-1-(2-hydroxyphenyl)-5-(4-nitrophenyl) penta-2,4-dien-1-one (b11). The crude product was crystallized

◦

from methanol: yield: 76%; R_f(hexane:acetone 2:1): 0.65; m.p.: 146–148 C; IR (KBr, cm⁻¹): 3200,

1710,1620; LC-MS (m/z): (C₁₇H₁₃NO₄) 297 [M + 1]⁺, 342 [M + CH₃CH₂OH]⁺, 307 [M + CH₃CN]⁺;

¹H-NMR (300 MHz, CDCl3)

δ

: 8.12–8.13 (m, 1H, OH proton), 7.99–8.01 (m, 2H, aromatic proton),

7.51–7.59 (m, 4H, aromatic and CH=, protons), 7.36–7.38 (m, 2H aromatic protons), 7.02–7.09 (m,

2H aromatic protons), 6.92–6.93 (m, 2H aromatic and CH=, protons); ¹³C-NMR (75 MHz, CDCl3)

191.97 (C=O), 163.40 (C–OH), 146.45 (C–NO₂), 146.12 ( C=C ), 141.97, 135.90, 129.67, 127.93, 127.54,

δ:

−

125.83, 124.70, 117.90; elemental analysis: expected % (C₁₇H₁₃NO₄): C 69.15, H 4.44, N 4.74; found %

(C₁₇H₁₃NO₄): C 69.36, H 4.72, N 4.86.

(E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one (b13). The crude product was recrystallized from

ethanol: yield: 78%; R_f(hexane:acetone 2:1): 0.56; m.p.: 75–78 ^◦C [73].

3.2.3. Synthesis of bis-Ethers (di–dvii)

4-hydroxy acetophenone and 1, 3-dibromoalkane in molar ratio 2:1 was diluted in 50 mL acetone.

Anhydrous K₂CO₃was added and the mixture was reﬂuxed for approximately 18 h. the reaction was

completed judged by the negative alcoholic ferric chloride (3%) test. The mixture was evaporated to

dryness. The solid was treated with water, ﬁltered of and recrystallized from absolute ethanol [28].

0

1,1 -((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))diethanone (di) [74]. The crude product was recrystallized

from ethanol: yield: 92%; white crystals; R_f(hexane:acetone 2:1): 0.4; m.p.: 161 ^◦C.

1,1⁰-((propane-1, 3-diyl bis-(oxy))bis(4,1-phenylene))diethanone (dii). The crude product was crystallized

from ethanol: yield: 90%; white crystals; R_f(hexane:acetone 2:1): 0.53; m.p.: 115–117 ^◦C [29].

0

1,1 -((butane-1,4-diylbis(oxy))bis(4,1-phenylene))diethanone (diii) [43]. The crude product was crystallized

from ethanol: yield: 87%; white crystals; R_f(hexane:acetone 2:1): 0.42; m.p.: 142 ^◦C [74].

1,1⁰-((pentane-1,5-diylbis(oxy))bis(4,1-phenylene))diethanone (div). The crude product was crystallized

from ethanol: yield: 82%; white crystals; R_f(hexane:acetone 2:1): 0.41; m.p.: 87 ^◦C; IR (KBr, cm⁻¹):

1

1620, 1700, 3040; H-NMR (300 MHz, CDCl₃)

δ

: 7.90–7.91 (d, 4H, aromatic protons), 6.89–6.91 (m,

CH₂–O), 3.68–3.72 (m, 2H, CH₂), 2.53 (s, 6H, 2 CH₃),

); ¹³C-NMR (75 MHz, CDCl₃)

: 196.78 (C=O), 162.93 (C–O ), 87.90, 77.30,

C–CH₂–O), 28.87, 26.36, 22.56 ( CH₂); elemental analysis: expected % (C₂₁H₂₄O₄):

C 74.09, H 7.11; found % (C₂₁H₂₄O₄): C 74.05, H 6.94.

4H, aromatic protons), 4.02–4.05 (m, 4H,

1.85–1.92 (m, 4H, CH₂

77.04, 76.79 (

−

×

−

δ

−

1,1⁰-((hexane-1,6-diylbis(oxy))bis(4,1-phenylene))diethanone (dv) [74]. The crude product was crystallized

from ethanol: yield: 89%; white crystals; Rf (hexane:acetone 2:1): 0.42; m.p.: 121 ^◦C.

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1,1⁰-((heptane-1,7-diylbis(oxy)) bis-(4,1-phenylene)) diethanone (dvi). The crude product was crystallized

from ethanol: yield: 94%; white crystals; R_f(hexane:acetone 2:1): 0.45; m.p.: 111 ^◦C; IR (KBr, cm⁻¹):

1610, 1700, 3030; ¹H-NMR (300 MHz, CDCl₃)

δ

: 7.92–8.00 (m, 4H, aromaticand CH=, protons), 6.88–6.90

(m, 4H, aromatic and CH=, protons), 4.29–4.31 (m, 4H, CH₂–O), 2.56 (s, 6H, 2 CH₃), 1.86–1.91 (m, 4H,

aliphatic protons), 1.44–1.48 (m, 4H, aliphatic protons), 1.31–1.32 (m, 2H, CH₂

); ¹³C-NMR (75 MHz,

CDCl₃) : 197.15 (C=O), 196.70, 165.40, 163.25 (C–O), 130.04, 128.35, 127.80, 67.43, 30.24, 23.01 (CH₃),

22.65 ( CH₂); elemental analysis: expected % (C₂₃H₂₈O₄): C 74.97, H 7.66; found % (C₂₃H₂₈O₄):

C 74.94, H 7.61.

×

−

δ

−

1,1⁰-((octane-1,8-diylbis(oxy)) bis-(4,1-phenylene))diethanone (dvii). The crude product was crystallized

from ethanol: yield: 93%; white crystals; R_f(hexane:acetone 2:1): 0.46; m.p.: 123 ^◦C; IR (KBr, cm⁻¹):

1

1610, 1690, 3040; H-NMR (300 MHz, CDCl₃)

δ

: 7.98–8.07 (m, 4H, aromatic and CH=, protons),

6.95–7.02 (m, 4H, aromatic and CH=, protons), 4.38–4.40 (m, 4H, aliphatic protons), 2.53 (s, 6H,

CH₃), 1.98–2.01 (m, 4H, aliphatic protons), 1.45–1.49 (m, 4H, aliphatic protons), 1.29–1.30 (m, 4H,

aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃)

: 196.80 (C=O), 162.95 ( C–O ), 129.34, 128.70, 127.35,

67.83 ( CH₂–O ), 29.74, 28.80, 23.62; elemental analysis: expected % (C₂₄H₃₀O₄): C 75.36, H 7.91;

2

×

δ

−

found % (C₂₄H₃₀O₄): C 75.31, H 7.88.

3.2.4. Synthesis of bis-Etheriﬁed Double Chalcones (c1–13)

A Claisen–Schmidt condensation was performed between (1,1⁰-((propane-1,3-diyl-bis(oxy))

bis-(4,1-phenylene))diethanone) and the appropriate substituted aromatic aldehyde at a molar ratio 1:2

in absolute ethanol (10 mL). Three milliliter (3 mL) aqueous KOH (20%) was added. The mixture was

stirred at room temperature in a US-bath. The end of the reaction was monitored by TLC. After the

completion of the reaction the mixture was treated with aqueous HCl 10% and adjusted to acidic pH.

The precipitate was either ﬁltered and washed with cold water or extracted with CHCl₃(30 mL

The combined organic layers were washed with water and brine and dried under anhydrous MgSO₄.

The product was evaporated to dryness and puriﬁed by recrystallization from a proper solvent.

×

3).

(2E,2⁰E)-1,1⁰-((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(3-(4-((4-bromobenzyl)oxy)phenyl)prop-2-en-1-one)

(

c1). The crude product was crystallized from acetone: yield: 89%; yellow solid; R_f(hexane:acetone

2:1): 0.5; m.p.: 197–199 ^◦C [29].

(2E,2⁰E)-1,1⁰-((propane-1,3-diylbis (oxy))bis (4,1-phenylene))bis(3-(3-phenoxyphenyl)prop-2-en-1-one) (c2).

The crude product was crystallized from ethanol: yield: 72%; yellow solid; R_f(hexane:acetone 2:1):

0.68; m.p.: 172–174 ^◦C [29].

(2E,2⁰E, 4E,4⁰E)-1,1⁰-((propane-1,3-diylbis-(oxy)) bis(4,1- phenylene)) bis-(5-(4-(dimethylamino) phenyl)

penta-2,4- dien-1-one) (c3). The crude product was recrystallized from ethanol: yield: 79%; Dark brown

solid; R_f(hexane:acetone 2:1): 0.55, 0.62; m.p.: 156–158 ^◦C [29].

(2E,2⁰E)-1, 1⁰-((propane-1,3-diylbis-(oxy)) bis-(4,1-phenylene)) bis-(3-(naphthalen-1-yl) prop-2-en-1-one) (c4).

The crude product was recrystallized from PE/EA: yield: 74%; yellow solid; R_f(hexane:acetone 2:1):

0.64; m.p.: 167–168 ^◦C [29].

(2E,2⁰E)-1,1⁰-((propane-1,3-diyl bis-(oxy)) bis-(4,1-phenylene)) bis-(3-(thiophen-2-yl) prop-2-en-1-one) (c5).

The crude product was recrystallized with ethanol: yield: 83%; light yellow solid; R_f(hexane:acetone

2:1): 0.4; m.p.: 132 ^◦C; IR (KBr, cm⁻¹): 1610, 1650; ¹H-NMR (300 MHz, CDCl₃)

δ: 8.27–8.25 (m, 4H,

phenyl aromatic and heteroaromatic protons

7.28–7.34 (m, 6H, aromatic protons,), 7.05–7.14 (m, 2H, aromatic protons), 6.91–7.03 (m, 6H, aromatic

and CH= protons), 4.19–4.29 (m, 4H, aliphatic protons), 2.30–2.36 (m, 2H, aliphatic protons); ¹³C-NMR

−

CH–S), 7.93–7.98 (m, 4H, aromatic and CH= protons),

(75 MHz, CDCl₃)

128.92, 127.54, 120.87, 115.01, 67.42 (

(C₂₉H₂₄O₄S₂): C 67.8, H 4.38; found % (C₂₉H₂₄O₄S₂): C 67.6, H 4.18.

δ

: 187.02 (C=O), 162.64 (

−

C–O–), 139.45 (

−

C–S), 135.24, 132.01, 130.89, 130.53,

−

CH₂–O

−

), 24.90 ( CH₂

−

); elemental analysis: expected %

Molecules 2019, 24, 199

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0

(2E,2 E)-1,1 -((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(3-(1H-indol-3-yl)prop-2-en-1-one) (c6). The crude

product was recrystallized with ethanol: yield: 81%; white solid; R_f(hexane:acetone 2:1): 0.42; m.p.:

197 C; IR (KBr, cm⁻¹): 1610, 1640; LC-MS (m/z): (C₃₇H₃₀N₂O₄) 606 [M + K]⁺, 640 [M + CH₃CN + Na]⁺;

◦

¹H-NMR (300 MHz, CDCl₃)

δ: 10.05 (br, 2H,

−

NH), 7.87–7.93 (m, 4H, aromatic protons), 7.67–7.79

(m, 4H, aromatic and CH= protons), 7.39–7.44 (m, 2H aromatic protons), 7.16–7.28 (m, 6H aromatic

protons), 6.99–7.11 (m, 6H aromatic and CH= protons), 4.21–4.27 (m, 4H, aliphatic protons), 2.28–2.35

(m, 2H, aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃)

δ: 191.63 (C=O), 163.24 (

−

C–O), 145.53, 129.92,

126.37, 118.16, 117.82, 114.40, 113.20, 112.73, 64.92 (C–N), 28.70 ( CH₂); elemental analysis: expected

% (C₃₇H₃₀N₂O₄): C 82.20, H 5.97; found % (C₃₇H₃₀N₂O₄): C 82.0, H 5.71.

−

(2E,2⁰E,4E,4⁰E)-1,1⁰-((propane-1,3-diylbis(oxy)) bis(4,1-phenylene))bis (5-phenylpenta-2,4-dien-1-one) (c7).

The crude product was recrystallized with ﬂash chromatography hexane/acetone (2:1): yield: 74%;

orange-yellow solid; R_f(hexane:acetone 2:1): 0.9; m.p.: 98–100 ^◦C; IR (KBr, cm⁻¹): 1600, 1650, 1710;

LC-MS (m/z): (C₃₇H₃₂O₄) 542 [M + 1]⁺=, 587 [M + CH₃CH₂OH]⁺, 540[M

−

1]⁺; ¹H-NMR (300 MHz,

: 7.88–8.06 (m, 8H, aromatic protons), 7.79 (d, 2H, CH=, J = 15 Hz), 7.53–7.66 (m, 2H, aromatic

CDCl₃)

δ

protons), 7.47–7.53 (m, 2H, aromatic protons), 7.30–7.43 (br, 4H, aromatic protons), 7.10 (d, 2H, =CH, J

= 15 Hz), 6.90–7.03 (m, 8H aromatic protons), 4.22–4.27 (m, 4H, aliphatic protons), 2.29–2.36 (m, 2H,

aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃)

131.04, 128.82, 127.60, 124.72, 116.25, 64.60, 29.42 (

δ

: 190.12 (C=O), 163.77 (

CH₂); elemental analysis: expected % (C₃₇H₃₂O₄):

C 82.20, H 5.97; found % (C₃₇H₃₂O₄): C 82.0, H 5.71.

−

C–O), 143.97 (

−

CH=CH

−

),

−

0

(2E,2 E,4E,4 E)-1,1 -((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(4-methyl-5-phenylpenta-2,4-dien-1-one)

(

c8). The crude product was recrystallized with ethanol: yield: 72%; yellow solid; R_f(hexane:acetone

2:1): 0.46; m.p.: 90–92 ^◦C; IR (KBr, cm⁻¹): 1610, 1720; LC-MS (m/z): (C₃₉H₃₆O₄) 570 [M + 1]⁺, 615 [M

+ CH₃CH₂OH]⁺; H-NMR (300 MHz, CDCl₃)

δ: 7.98–8.05 (m, 4H, aromatic protons), 7.91–7.95 (m,

1

4H, aromatic protons), 7.80 (d, 2H, CH=, J = 15 Hz), 7.51–7.66 (br, 4H aromatic protons), 7.36–7.42 (m,

4H, aromatic protons), 7.05 (d, 2H, =CH, J = 15 Hz), 6.92–7.00 (m, 8H, aromatic protons), 4.21–4.27

(m, 4H, aliphatic protons), 2.55 (s, 6H, 2

(75 MHz, CDCl₃) : 191.02 (C=O), 164.78 (C–O), 143.97, 129.84, 128.82, 127.60, 124.72, 115.20, 64.60,

29.42 ( CH₂); elemental analysis: expected % (C₃₉H₃₆O₄): C 82.20, H 5.97; found % (C₃₉H₃₆O₄):

C 82.0, H 5.71.

×

CH₃), 2.29–2.36 (m, 2H, aliphatic protons); ¹³C-NMR

δ

−

(2E,2⁰E,4E,4⁰E)-1,1⁰-((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(4-bromo-5-phenylpenta-2,4-dien-1-one)

c9). The crude product was recrystallized with ethanol: yield: 70%; light yellow solid; R_f(hexane:acetone

2:1): 0.46; m.p.: 120–122 ^◦C; IR (KBr, cm⁻¹): 1610, 1700; LC-MS (m/z): (C₃₇H₃₀Br₂O₄) 700 [M + 1]⁺,

745 [M + CH₃CH₂OH]⁺; ¹H-NMR (300 MHz, CDCl₃)

: 7.83–7.94 (br, 4H, aromatic and CH= protons),

(

δ

7.46–7.68 (m, 6H, aromatic protons), 7.15–7.28 (m, 6H, aromatic protons), 6.74–6.92 (m, 8H, aromatic

and =CH protons), 4.20–4.24 (m, 4H, aliphatic protons), 2.34–2.39 (m, 2H, aliphatic protons); ¹³C-NMR

(75 MHz, CDCl₃,)

116.25 (C–Br), 115.07, 64.60, 29.13 (

H 4.33; found % (C₃₇H₃₀Br₂O₄): C63.78, H 4.55.

δ

: 189.60 (C=O), 164.70 (

−

C–O), 143.97, 133.25, 131.04, 128.82, 127.60, 124.72, 122.01,

); elemental analysis: expected % (C₃₇H₃₀Br₂O₄): C 63.63,

−

CH₂

−

0

(2E,2 E)-1,1 -((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(3-(furan-2-yl)prop-2-en-1-one) (c10). The crude

product was crystallized with PE/EA Yield: 89%; Light yellow solid; R_f(hexane:acetone 2:1): 0.42; m.p.:

◦

159–160 C; IR (KBr, cm⁻¹): 1610,1640,1730; LC-MS (m/z): (C₂₉H₂₄O₆) 470 [M + 1]⁺, 533 [M + CH₃CN +

Na]⁺; ¹H-NMR (300 MHz, CDCl₃,)

6.92–6.98 (m, 4H, aromatic and =CH protons), 6.66–6.71 (br, 2H), 6.48–6.53 (br, 2H), 4.21–4.29 (m, 4H,

aliphatic protons), 2.28–2.38 (m, 2H, aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃,)

: 188.08 (C=O),

162.81 ( C–O), 144.74 ( CH₂–C–O), 131.60, 131.32, 130.46, 129.89, 119.08, 116.24, 114.59, 112.31, 64.34,

29.41 ( CH₂); elemental analysis: expected % (C₂₉H₂₄O₆): C 74.35, H 5.16; found % (C₂₉H₂₄O₆):

C 74.43, H 4.77.

δ: 7.95–8.03 (m, 4H, aromatic and CH= protons), 7.41–7.62 (m, 6H),

δ

−

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(2E,2⁰E,4E,4⁰E)-1,1⁰-((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(5-(4-nitrophenyl)penta-2,4-dien-1-one)

(

c11). The crude product was recrystallized with ethanol: yield: 87%; dDark brown solid; R_f(hexane:

◦

acetone 2:1): 0.34; m.p.: 278–280 C; IR (KBr, cm⁻¹): 1620, 1730; LC-MS (m/z): (C₃₇H₃₀N₂O₈) 630 [M

−

1]⁺, 654 [M + Na]⁺; ¹H-NMR (300 MHz, CDCl₃,)

δ: 8.21–8.25 (m, 4H, aromatic protons), 7.96–8.01 (m,

2H, aromatic and CH= protons), 7.90–7.96 (m, 4H), 7.53–7.66 (m, 4H, aromatic protons), 7.10–7.23 (m,

2H), 6.90–7.05 (m, 8H, aromatic and =CH, protons), 4.21–4.28 (m, 4H, aliphatic protons), 2.29–2.38 (m,

2H, aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃)

142.44, 130.72, 126.94, 125.60, 123.42, 121.70, 114.69, 66.01, 26.42 ( CH₂); elemental analysis: expected

% (C₃₇H₃₀N₂O₈): C 70.47, H 4.79, N 4.44; found % (C₃₇H₃₀N₂O₈): C70.67, H 4.53, N 4.13.

δ

: 191.31 (C=O), 164.02 (

−

C–O), 146.07 ( C–NO₂),

−

(2E,2⁰E)-1,1⁰-((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(3-(5-methylfuran-2-yl)prop-2-en-1-one) (c12).

The crude product was recrystallized with PE/EA: yield: 90%; orange-yellow solid; R_f(hexane:acetone

◦

2:1): 0.44; m.p.: 107–109 C; IR (KBr, cm⁻¹): 1620, 1680; LC-MS (m/z): (C₃₁H₂₈O₆) 498 [M + 1]⁺,

456 [M

−

CH₃CN]⁺; ¹H-NMR (300 MHz, CDCl₃)

δ: 7.98–8.08 (m, 2H, aromatic protons), 7.86–7.96

(m, 2H aromatic and CH= protons), 7.31–7.57 (m, 2H aromatic protons), 6.91–7.17 (m, 6H, aromatic

protons), 6.11–6.59 (m, 4H aromatic and =CH protons), 4.14–4.32 (m, 4H, aliphatic protons), 2.56 (s,

6H, 2

162.20 (

28.47 (

(C₃₁H₂₈O₆): C 74.79, H 5.29.

×

CH₃), 2.28–2.41 (m, 2H, aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃)

δ

: 188.28 (C=O),

C–O), 131.49, 130.40, 128.28, 122.28, 118.17, 117.78, 117.08, 113.78, 110.18, 67.27 ( CH₂–O),

CH₂), 14.05 ( CH₃); elemental analysis: expected % (C₃₁H₂₈O₆): C 74.98, H 5.68; found %

−

(2E,2⁰E)-1,1⁰-((propane-1,3-diylbis(oxy))bis(4,1-phenylene))bis(3-phenylprop-2-en-1-one) (c13). The crude

product_◦was recrystallized with PE/EA: yield: 81%; white solid; R_f(hexane:acetone 2:1): 0.45; m.p.:

148–150 C; IR (KBr, cm⁻¹): 1630,1700; LC-MS (m/z): (C₃₃H₂₈O₄) 490 [M + 1]⁺, 512 [M + Na]⁺; ¹H-NMR

(300 MHz, CDCl₃) δ: 8.12–8.16 (m, 4H, aromatic protons), 7.76–7.78 (m, 2H aromatic and CH= protons),

7.51–7.59 (m, 6H, aromatic protons), 7.38–7.45 (m, 6H, aromatic protons), 7.06–7.09 (m, 4H, aromatic

and CH= protons), 4.02–4.07 (m, 4H, aliphatic protons), 2.16–2.21 (m, 2H, aliphatic protons); ¹³C-NMR

(75 MHz, CDCl₃)

115.35, 64.92 ( CH₂–O), 24.87 (

found % (C₃₃H₂₈O₄): C 74.79, H 5.29.

δ

: 190.02 (C=O), 163.54 (

−

C–O), 144.76 (

−

C–Ph), 134.18, 127.00, 126.89, 126.42, 119.62,

−

CH₂); elemental analysis: expected % (C₃₃H₂₈O₄): C 74.98, H 5.68;

−

3.2.5. Synthesis of bis-Etherified Double Chalcones of (E)-3-(4-(Dimethylamino) Phenyl-Acryl-Aldehyde

(ei, eiii–evii)

A Claisen—Schmidt condensation was performed between (E)-3-(4-(dimethylamino) phenyl)

acryl-aldehyde and the appropriate ether at a molar ratio 2:1 in absolute ethanol (10 mL).

◦

Three milliliters (3 mL) aqueous KOH (40%) was added. The mixture was stirred at 45 C in a

US-bath. The end of the reaction was monitored by TLC. After the completion of the reaction the

mixture was treated with aqueous HCl 25% and adjusted to acidic pH. The precipitate was either

ﬁltered and washed with cold water and then extracted with CHCl₃(30 mL

×

3). The combined

organic layers were washed with water and brine and dried under anhydrous MgSO₄. The product

was evaporated to dryness and puriﬁed by recrystallization from a proper solvent. Chalcone eii has

been already synthesized and referred to as c3 [29].

(2E,4E)-5-(4-(Dimethylamino)phenyl)-1-(4-(2-(4-((2E,4E)-5-(4-(dimethylamino)phenyl)penta-2,4-dien-1-yl)

phenoxy)ethoxy)phenyl)penta-2,4-dien-1-one (ei). The crude product was recrystallized with ethanol:

yield: 84%; brown solid; R_f(hexane:acetone 2:1): 0.25; m.p.: 101 ^◦C; IR (KBr, cm⁻¹): 1610, 1670, 1710;

¹H-NMR (300 MHz, CDCl₃)

δ

: 7.90–8.05 (br, 12H aromatic protons), 7.89 (d, 2H, CH=, J = 15 Hz),

7.40 (d, 2H, =CH, J = 15 Hz), 6.98–7.06 (br, 8H, aromatic protons) 4.34 (s, 4H, aliphatic protons),

2.99–3.09 (br, 12H, 4 : 196.59 (C=O), 162.98 ( C–O), 149.19,

CH₃); ¹³C-NMR (75 MHz, CDCl₃)

130.78, 130.39, 114.49, 106.58, 98.98, 59.18, 26.67 ( CH₂), 16.02 (CH₃); elemental analysis: expected %

(C₄₀H₄₀N₂O₄): C 80,24, H 7.07,N 4.68; found % (C₄₀H₄₀N₂O₄): C 80.02, H 6.98, N 4.34.

×

δ

−

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(2E,2⁰E,4E,4⁰E)-1,1⁰-((butane-1,4-diylbis (oxy))bis(4,1-phenylene)) bis(5-(4-(dimethylamino)phenyl)penta-

2,4-dien-1-one) (eiii). The crude product was recrystallized with ethanol: yield: 85%; brown solid;

R_f(hexane:acetone 2:1): 0.29; m.p.: 138 ^◦C; IR (KBr, cm⁻¹): 1610, 1680; ¹H-NMR (300 MHz, CDCl₃)

δ:

7.93–8.09 (br, 12H, aromatic and CH=, protons), 7.402–7.445 (m, 4H aromatic protons), 6.88–7.08 (br,

8H, aromatic and CH=, protons), 4.18–4.23 (m, 4H, aliphatic protons), 2.99–3.15 (br, 12H, 4

2.05–2.11 (m, 4H, aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃)

: 196.78 (C=O), 162.43 (

149.60, 139.54, 122.87, 122.25, 121.63, 112.37, 63.08, 41.33, 26.12 ( CH₂); elemental analysis: expected

×

CH₃),

C–O),

δ

−

% (C₄₂H₄₂N₂O₄): C 78.72, H 6.92, N 4.37; found % (C₄₂H₄₄N₂O₄): C 78.57, H 6.82, N 4.31.

(2E,2⁰E,4E,4⁰E)-1,1⁰-((pentane-1,5-diylbis(oxy))bis(4,1-phenylene))bis(5-(4-(dimethylamino)phenyl)penta-

2,4-dien-1-one) (eiv). The crude product _◦was recrystallized with ethanol: yield: 80%; brown solid;

1

R_f(hexane:acetone 2:1): 0.35; m.p.: 117 C; IR (KBr, cm⁻¹): 1610, 1680, 1710; H-NMR (300 MHz,

CDCl₃,

protons), 4.23–4.28 (m, 4H, aliphatic protons), 2.835 (s, 12H, 4

protons), 1.45–1.49 (m, 2H, aliphatic protons); ¹³C-NMR (CDCl₃, 75 MHz)

C–O), 149.83, 146.07, 132.15, 128.17, 125.96, 125.02, 113.70, 111.34, 68.90, 41.54, 26.73 (

elemental analysis: expected % (C₄₃H₄₆N₂O₄): C 78.87, H 7.08, N 4.28; found % (C₄₃H₄₆N₂O₄): C 78.45,

H 6.94, N 4.09.

δ

: 7.89–7.99 (m, 12H, aromatic and CH= protons), 6.79–6.90 (m, 12H, aromatic and CH=

CH₃), 1.80–1.97 (m, 4H, aliphatic

: 196.52 (C=O), 163.65

CH₂);

×

δ

(

−

(2E,2⁰E,4E,4⁰E)-1,1⁰-((hexane-1,6-diylbis(oxy)) bis(4,1-phenylene)) bis(5-(4-(dimethylamino) phenyl)penta-

2,4-dien-1-one) (ev). The crude product was recrystallized with ethanol: yield: 91%; brown solid;

◦

1

R_f(hexane:acetone 2:1): 0.43; m.p.: 108 C; IR (KBr, cm⁻¹): 1605, 1710, 1710; H-NMR (300 MHz,

CDCl₃)

protons), 4.10–4.19 (m, 4H, aliphatic protons) 2.740–2.99 (br, 12H, 4

aliphatic protons), 1.462–1.510 (m, 4H, aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃,)

(C=O), 162.94 ( C–O), 130.90, 130.22, 125.58, 118.98, 114.67, 68.21, 39.65 (CH₃), 28.90, 27.07, 25.48;

δ

: 7.88–7.98 (m, 12H, aromatic and CH=, protons), 6.88–6.96 (m, 12H, aromatic and CH=

CH₃), 1.882–1.925 (m, 4H,

: 196.72

×

δ

−

elemental analysis: expected % (C₄₄H₄₈N₂O₄): C 79.01, H 7.23, N 4.19; found % (C₄₄H₄₈N₂O₄): C 78.91,

H 7.19, N 4.14.

(2E,2⁰E,4E,4⁰E)-1,1⁰-((heptane-1,7-diylbis(oxy)) bis(4,1-phenylene))bis(5-(4-(dimethylamino) phenyl)penta-

2,4-dien-1-one) (evi). The crude product was recrystallized with ethanol: yield: 90%; brown solid;

R_f(hexane:acetone 2:1): 0.46; m.p.: 104 ^◦C; IR (KBr, cm⁻¹): 1605, 1690; ¹H-NMR (300 MHz, CDCl₃)

δ

: 7.89–7.98 (m, 12H, aromatic and CH= protons) 6.88–6.96 (m, 12H, aromatic and CH= protons),

4.00–4.05 (m, 4H, aliphatic protons), 2.56–2.89 (br, 12H, 4 CH₃), 1.78–1.85 (m, 4H, aliphatic protons)

1.47–1.52 (m, 4H), 1.22–1.26 (m, 2H, aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃)

: 196.76 (C=O),

163.04 ( C–O), 151.58, 130.55, 130.12, 114.09, 68.10, 58.39, 40.55, 29.00 ( CH₂–); elemental analysis:

×

δ

−

expected % (C₄₅H₅₀N₂O₄): C 79.15, H 7.38, N 4.10; found % (C₄₅H₅₀N₂O₄): C 78.94, H 7.29, N 4.03.

(2E,2⁰E,4E,4⁰E)-1,1⁰-((octane-1,8-diylbis(oxy)) bis(4,1-phenylene)) bis(5-(4-(dimethylamino) phenyl)penta-

2,4-dien-1-one) (evii). The crude product was recrystallized with ethanol: yield: 93%; brown solid;

◦

1

R_f(hexane:acetone 2:1): 0.5; m.p.: 112 C; IR (KBr, cm⁻¹): 1610,1710; H-NMR (300 MHz, CDCl₃)

δ

: 7.90–8.05 (m, 12H, aromatic and CH= protons), 6.75–6.88 (m, 12H, aromatic and CH= protons),

4.30–4.38 (m, 4H, aliphatic protons), 2.99–3 (br, 12H, 4 CH₃), 2.32–2.36 (m, 4H, aliphatic protons),

1.75–1.80 (m, 4H, aliphatic protons), 1.43–1.45 (m, 4H, aliphatic protons); ¹³C-NMR (75 MHz, CDCl₃)

196.80 (C=O), 162.95 ( C–O), 131.52, 129.34, 128.70, 127.35, 67.83, 42.67, 29.74, 28.80, 23.62 ( CH₂

×

δ

:

−

);

elemental analysis: expected % (C₄₆H₅₂N₂O₄): C 79.28, H 7.52, N 4.02; found % (C₄₆H₅₂N₂O₄): C 79.02,

H 7.47, N 3.98.

3.3. Physicochemical Studies

3.3.1. Molecular Properties Prediction-Lipinski “Rule of Five”

Compounds were subjected to molecular properties prediction, drug-likeness by molinspiration

[44] (Table 1).

Molecules 2019, 24, 199

24 of 31

3.3.2. Determination of R_MValues

Reversed phase TLC (RP-TLC) was performed on silica gel plates impregnated with 5% (v/v)

liquid parafﬁn in light petroleum ether. The mobile phase was a methanol/water mixture (70/30,

v/v) [29]. The plates were developed in closed chromatography tanks saturated with the mobile

phase at 24 ^◦C. Spots were detected under UV light. Five individual measurements of R_fvalues were

recorded and used for the determination of R_Musing the equation R_M= log [(1/R_f) − 1]. (Table 3).

3.4. Biological In Vitro Assays

Each in vitro experiment was performed at least in triplicate and the standard deviation of

absorbance was less than 10% of the mean. For the in vitro assays, a stock solution (1% DMSO in the

appropriate buffer with the tested compound diluted under sonication) was prepared from which

several dilutions were made with the appropriate buffer.

3.4.1. Determination of the Reducing Activity Using the Stable Radical 1,1-Diphenyl-

Picrylhydrazyl (DPPH)

To a solution of DPPH (ﬁnal concentration 0.05 mM) in absolute ethanol an equal volume of the

compounds solution in DMSO (stock solution 10 mM) dissolved in ethanol was added. Absolute

ethanol was used as a control. The concentrations of the solutions of the compounds were 0.05 and

0.1 mM. The absorbance was recorded at 517 nm at room temperature after 20 and 60 min [29] (Table 2 ).

3.4.2. Inhibition of Linoleic Acid Lipid Peroxidation

In an aqueous solution spectrophotometrically at 234 nm the production of conjugated diene

hydro peroxide by oxidation of sodium linoleate was monitored and recorded according to our

previous published experimental conditions [29], using 10

added to the UV cuvette containing 930 L of 0.05 M phosphate buffer, pH 7.4 pre-thermostated at

37 ^◦C, under air by the addition of 50

L of 40 mM AAPH solution, which was used as a free radical

initiator. Ten microliters (10 L) of the appropriate solutions of the tested compounds were added in

µL of the 16 mM sodium linoleate solution

µ

the mixture, whereas same level of DMSO was used as a blank for the measurement of lipid oxidation.

◦

The rate of oxidation at 37 C was monitored by recording the increase in absorption at 234 nm caused

by conjugated diene hydro peroxides. The results were compared to the appropriate standard inhibitor

Trolox (93%) (Table 2).

3.4.3. In Vitro Lipid Peroxidation Assay

Egg phosphatidylcholine (20 mg) in chloroform (2 mL) was dried under vacuum in a rotary

evaporator to give a thin homogeneous ﬁlm and further dispersed in normal saline (5 mL) with

a vortex mixer. The mixture was sonicated to obtain a homogeneous suspension of liposomes.

Lipid peroxidation was initiated by adding 0.05 mM ascorbic acid to a mixture containing liposome

(0.1 mL), 150 mM potassium chloride, 0.2 mM ferric chloride, and the tested compounds in a total

volume of 0.4 mL ﬁnal concentration of 100 µM. The reaction mixture was incubated for 40 min at

37 ^◦C. After incubation, the reaction was terminated by adding 1 mL of ice-cold 0.25 M hydrochloric

acid containing 20% w/v trichloroacetic acid, 0.4% w/v of thiobarbituric acid, and 0.05% w/v butylated

hydroxytoluene. After heating at 80 ^◦C for 20 min, the samples were cooled and the pink chromogen

was extracted with a constant amount of butan-1-ol, and the absorbance of the upper organic layer

was measured at 532 nm [75] (Table 2).

3.4.4. ABTS^•+—Decolorization Assay for Antioxidant Activity

•+

Stock solutions of the tested compounds in DMSO (10 mM) were diluted. For the study, the ABTS

solution was diluted with ethanol to an absorbance of 0.70 at 734 nm. Ten milliliter (10 mL) aliquots

Molecules 2019, 24, 199

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+

of each sample were added into the ABTS

·

solution and the absorbance was recorded at room

temperature [76]. Trolox was used as a standard.

3.4.5. Soybean Lipoxygenase Inhibition Study In Vitro

In vitro study was evaluated as reported previously [29]. The tested compounds as stock solutions

were dissolved in DMSO. Ten microliters (10

linoleate (100 mM) and 0.2 mL of enzyme solution (1/9

room temperature (ﬁnal volume 1 mL). The conversion of sodium linoleate to 13-hydroperoxylinoleic

acid at 234 nm was recorded. The results were compared with the appropriate standard inhibitor

µ

L) were incubated at room temperature with sodium

×

10⁻⁴w/v in saline) in buffer pH 9 (tris) at

NDGA (IC₅₀= 0.5

µM). Several concentrations were used for the determination of IC₅₀values.

The results are given in Table 3 expressed as IC₅₀values or % inhibition at 100 µM.

3.4.6. Human h-15-LOX-1 Screening UV Assay [77]

The screening UV assay of h-15-LOX-1 was also studied by the formation of 13(S)-HPODE at

234 nm. The assay buffer consists of HEPES titrated to pH 7.5 using a concentrated aqueous solution

of NaOH. Linoleic acid (LA) was used as a substrate. The linear absorbance increase in absence of

the inhibitor was set as 100%, whereas the absorbance increase in absence of the enzyme was set to

0%. All experiments were performed in triplicate and the average triplicate values and their standard

deviations are plotted. The results for representative compounds are given in Figure 6 . The vertical

axis describes the residual enzyme activity percent % of the human h-15-LOX-1, whereas the horizontal

axis describes the concentration of 50 µM of the compounds. The half maximal inhibitor concentration

(IC₅₀) of the inhibitors for h-15-LOX-1 was determined using the same assay (Figures 6 and 7).

3.4.7. Inhibition of Acetyl-Cholinesterase

A modiﬁed procedure which was followed in our previous publication was performed [29].

A stock solution (10 mM) of the compounds diluted in DMSO was used (ﬁnal concentration 100 µM).

The inhibitory activity was measured by the change in absorbance at 405 nm in phosphate buffer pH

8 (0.1 M. The thiol ester acetylthioline was used as a substrate (0.01 M). It was hydrolyzed by AChE

(3.5 U/mL) to produce thiocholine and acetate. The thiocholine reduces DTNB (0.01 M in phosphate

0.1 M pH 7) liberating nitrobenzoate, which absorbs at 405 nm. The absorbance is measured with

Perkin-Elmer multilabel plate reader Victor X3. Tacrine was used as the reference compound.

3.4.8. Evaluation of the Cytotoxicity

L929 mouse ﬁbroblasts cells were cultured in EMEM supplemented with 10% horse serum, 2 mM

L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium bicarbonate,

◦

and 100

µ

g/mL penicillin–streptomycin at 37 C in a humidiﬁed atmosphere with 5% CO₂. L929 cells

were plated into 24-well plates at a density 5104 cells/well and allowed to attach and grow for 24 h.

The supernatant in each well then replaced with medium containing various concentrations (1, 10, 20,

50 and 100 µM) of compounds a5–12, c5–12, di, and dii–dvi, which presented the more interesting

results in the in vitro experiments [29]. After 24-h incubation, the supernatant was removed and the

cells were washed with PBS. The cells were detached with 0.25% trypsin, transferred to FACS tubes,

and then centrifuged (1600 rpm for 5 min) and the pellet washed with PBS. After washing, the cells

in the pellet were incubated with 5 µL propidium iodide (PI) solution (1 mg/mL) for 1 min. The PI

ﬂuorescence (cell death) was determined with ﬂow cytometry, FACS Calibur, Coulter Epics XL-MCL

(Beckman, Inc., Mount Holly, NJ, USA). The analysis of ﬂow cytometry data was performed with the

WinMDI analysis program (Table 4).

Molecules 2019, 24, 199

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3.4.9. Circular Dichroism Studies

Preparation of A

β

stocks and solutions. Amyloid peptide A

β40 was purchased from AnaSpec Co.

(Fremont, CA, USA) (>95% pure). Aβ40 was gently dissolved without vortexing in Type 1 (Milli-Q)

water to a ﬁnal concentration of 100

µ

M. Solutions of A in phosphate buffer (PB, 10 mM, pH 7.33)

β

were prepared by adding equal volumes of PB to aliquots of the aqueous solution stock to achieve

ﬁnal concentration of 50 M. The proper amount of the compounds c c3 c4 di dii, and dvi were

added to the A solutions to achieve concentration of 50 M. The secondary structural changes of Aβ

µ

2

,

β

µ

were monitored for 40 days by CD. During this period, the amyloid peptide solutions were kept at an

incubator at 33 ^◦C under quiescent conditions [66].

Circular dichroism measurements. CD spectra were recorded on a JASCO J-715 spectropolarimeter

(Jasco Co., Tokyo, Japan), at 33 ^◦C in the range of 190–260 nm with a 1 mm path length quartz cuvette.

Each spectrum was the average of three scans at a speed of 100 nm·min⁻¹and a resolution of 0.5 nm.

Three independent experiments were run for each condition and in each case solutions of plain A

β

were run as control (Figure 8). The analysis of the CD data was performed using the OriginPro

9 program [66].

4. Conclusions

In conclusion, a, b, c, and e chalcones led to a new and promising class of antioxidant and

anti-lipoxygenase enones with anti-AChE activities. In Table 5 are summarized the most signiﬁcant

chalcones in terms of activities. It should to be noticed that the simple chalcone b4 presents signiﬁcant

inhibitory activity against the 15-human LOX with an IC₅₀value of 9.5 µM, interesting anti-AChE

activity and anti-lipid peroxidation behavior. Bis-etheriﬁed chalcone c12 is the most potent inhibitor of

AChE within the bis-etheriﬁed bis-chalcones following, by c11 which also is found to present interesting

inhibition on 15-human LOX. Bis-chalcones c11 and c12 were found to combine anti-LOX, anti-AChE,

and anti-lipid peroxidation activities. Thus, they can be used as lead multifunctional molecules.

Table 5. Summary comparison table of biological activities.

% Soybean LOX Inhb.

@ 100 µM/IC₅₀µM

% AChE Inhb. @

100 µM/IC₅₀µM

h-15-LOX-1 Inhb.

AAPH%

@ 100 µM

% Inhb. of Liposomes

Compound

IC₅₀µM

LP @ 100 µM

100 ^#

b3

b4

c2 *

c3 *

c4 *

c11

c12

100/57 µM

82/65 µM

100/55 µM

93/56 µM

100/55 µM

95/50 µM

52/96 µM

82 ^#

9.5 µM

82

71/49 µM

95/52 µM

58/100 µM

100/52 µM

100/48 µM

70

95

68

81

74

(2nd potent at 50 µM)

* ref [29]; ^#% activity at 0.001 µM.

It seems that the anti-lipid peroxidation activity supports the anti-LOX activity for the signiﬁcantly

active bis-chalcones. The presence of a double enone group supports better biological results.

Our CD study identiﬁed also that bis-etheriﬁed chalcones c2 and c4 are capable of interfering with

the aggregation process of Aβ and offer to the AD treatment as anti-AChE and anti-LOX inhibitors.

Since the molecular weight of 29 derivatives was found to be less than 500, these molecules are

anticipated to be easily transported, diffused, and absorbed and they are predicted to be orally active,

as they obeyed Lipinski’s rule of ﬁve. TPSA of the derivatives was observed to be well below the limit

of 160 Å and indicated good oral bioavailability. The prediction indicates that all structures with the

exception of c11 (144 Å) can cross BBB and act in CNS. The pharmacokinetic prediction study seems

quite interesting and useful to the design and optimization of the pharmacokinetic proﬁle of the new

derivatives. As we have recently reported [29], the most probable sites of metabolism for the enzymes

CYP1A1 and CYP1A2 are the hydrogens of the −OCH₂CH₂CH₂O− linkage. Therefore, it appears

possible that the molecules of the c/e groups act as prodrugs. Further investigation is in progress to

study this possibility.

Molecules 2019, 24, 199

27 of 31

Mechanistic studies attempting to determine the actual mode of the antioxidative, anti-LOX,

and anti-AChE action are under way.

Author Contributions: T.L., working on this research, synthesized and biologically evaluated the new compounds

as a part of her PhD thesis, D.H.-L. supervised the research and contributed in the writing, design, synthesis,

biological evaluation, and analysis of the data; F.D. and N.E. designed and performed the human LOX experiments;

E.V. and C.A. performed the cytotoxicity experiments; M.S., B.M., and M.P. designed, performed and wrote the

CD experiments with Aβ.

Funding: This research received no external funding.

Acknowledgments: We would like to thank Eleni Koliarmou and Evaggelia-Eirini Vlachou, Department of

Chemistry, Aristotle University of Thessaloniki, for providing the MS spectra. We are thankful to BioByte and

Albert Leo for free use of C-QSAR and support.

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

Abbreviations

AA

Arachidonic Acid

2,2 -Azobis-(2-amidinopropane) hydrochloride

2,2-Diphenyl-1-picrylhydrazyl radical

Lipoxygenase

0

AAPH

DPPH

LOX

NDGA

RPTLC

TCA

Nordihydroguaiaretic acid

Reverse-phase thin layer chromatography

Trichloroacetic acid

References

1.

2.

3.

4.

5.

6.

7.

8.

9.

Di Carlo, G.; Mascolo, N.; Izzo, A.A.; Capasso, F. Flavonoids: Old and new aspects of a class of natural

therapeutic drugs. Life Sci. 1999, 65, 337–353. [CrossRef]

Gupta, D.; Jaina, D.K.; Trivedi, P. Recent advances in chalcones as antiinfective agents. Int. J. Chem. Sci. 2010

8, 649–654.

,

Rahman, M.A. Chalcone: A valuable insight into the recent advances and potential pharmacological activities.

Chem. Sci. J. 2011, 29, 1–16. [CrossRef]

Sinha, S.; Medhi, B.; Sehga, R. Chalcones as an emerging lead molecule for antimalarial therapy: A review.

J. Mod. Med. Chem. 2013, 1, 64–77.

Yadav, V.R.; Prasad, S.; Sung, B.; Aggarwal, B.B. The role of chalcones in suppression of NF-κB-mediated

inﬂammation and cancer. Int. Immunopharmacol. 2011, 11, 295–309. [CrossRef] [PubMed]

Nasir, S.; Bukhari, A.; Jasamai, M.; Jantan, I. Synthesis and biological evaluation of chalcone derivatives

(mini review). Med. Chem. 2012, 12, 1394–1403.

Nowakowska, Z. A review of anti-infective and anti-inﬂammatory chalcones. Eur. J. Med. Chem. 2007

42, 125–137. [CrossRef] [PubMed]

,

Katsori, A.M.; Hadjipavlou-Litina, D. Recent progress in therapeutic applications of chalcones. Expert Opin.

Ther. Pat. 2011, 21, 1575–1596. [CrossRef]

Guida, A.; Lhouty, M.H.; Tichit, D.; Figueras, F.; Geneste, P. Hydrotalcites as base catalysts. Kinetics of

Claisen-Schmidt condensation, intramolecular condensation of acetonylacetone and synthesis of chalcone.

Appl. Catal. A 1997, 164, 251–264. [CrossRef]

10. Romanelli, G.; Pasquale, G.; Sathicq, A.; Thomas, H.; Autino, J.; Vazquez, P. Synthesis of chalcones catalyzed

by aminopropylated silica sol–gel under solvent-free conditions. J. Mol. Catal. A Chem. 2011, 340, 24–32.

[CrossRef]

11. Eddarir, S.; Cotelle, N.; Bakkour, Y.; Rolando, C. An efﬁcient synthesis of chalcones based on the Suzuki

reaction. Tetrahedron Lett. 2003, 44, 5359–5363. [CrossRef]

12. Dhar, D.N. Chemistry of Chalcones and Related Compounds; John Wiley & Sons, Inc.: New York, NY, USA,

1981; p. 213.

13. Pihlaja, R.; Haaparanta-Solin, M.; Rinne, O.M. The anti-inﬂammatory effects of lipoxygenase and

cyclo-oxygenase inhibitors in inﬂammation-induced human fetal glia cells and the A

β degradation capacity

of human fetal astrocytes in an ex vivo assay. Front. Neurosci. 2017, 11, 299. [CrossRef] [PubMed]

Molecules 2019, 24, 199

28 of 31

14. Czapski, G.A.; Czubowicz, K.; Strosznajder, J.B.; Strosznajder, R.P. The lipoxygenases: Their regulation and

implication in Alzheimer’s disease. Neurochem. Res. 2016, 41, 243–257. [CrossRef] [PubMed]

15. Chinnici, C.M.; Yao, Y.; Ding, T.; Funk, C.D.; Praticò, D. Absence of 12/15 lipoxygenase reduces brain

oxidative stress in apolipoprotein e-deﬁcient mice. Am. J. Pathol. 2005, 167, 1371–1377. [CrossRef]

16. Piomelli, D. Eicosanoids in synaptic transmission. Crit. Rev. Neurobiol. 1994, 8, 65–83. [PubMed]

17. Yao, Y.; Clark, C.M.; Trojanowski, J.Q.; Lee, V.M.; Praticò, D. Elevation of 12/15 lipoxygenase products in AD

and mild cognitive impairment. Ann. Neurol. 2005, 58, 623–626. [CrossRef] [PubMed]

18. Brash, A.R. Lipoxygenases: Occurrence, functions, catalysis, and acquisition of substrate. J. Biol. Chem. 1999

274, 23679–23682. [CrossRef] [PubMed]

,

19. Ka, M.H.; Choi, E.H.; Chun, H.S.; Lee, K.G. Antioxidative activity of volatile extracts isolated from Angelica

tenuissimae roots, peppermint leaves, pine needles, and sweet ﬂag leaves. J. Agric. Food Chem. 2005

53, 4124–4129. [CrossRef] [PubMed]

,

20. Molina-Jimenez, M.F.; Sanchez-Reus, M.I.; Andres, D.; Cascales, M.; Benedi, J. Neuroprotective effect of

fraxetin and myricatin against rotenone-induced apoptosis in neuroblastoma cells. Brain Res. 2004, 1009, 9–16.

[CrossRef]

21. Lukiw, W.J.; Cui, J.G.; Marcheselli, V.L.; Bodker, M.; Botkjaer, A.; Gotlinger, K.; Serhan, C.N.; Bazan, N.G.

A role for docosahexaenoic acid-derived neuroprotectin D1 in neural cell survival and Alzheimer disease.

J. Clin. Invest. 2005, 115, 2774–2783. [CrossRef]

22. Firuzi, O.; Zhuo, J.; Chinnici, C.M.; Wisniewski, T.; Praticò, D. 5-Lipoxygenase gene disruption reduces

amyloid-

β pathology in a mouse model of Alzheimer’s disease. FASEB J. 2008, 22, 1169–1178. [CrossRef]

[PubMed]

23. Wang, H.; Li, J.; Follett, P.L.; Zhang, Y.; Cotanche, D.A.; Jensen, F.E.; Volpe, J.J.; Rosenberg, P.A.

12-Lipoxygenase plays a key role in cell death caused by glutathione depletion and arachidonic acid

in rat oligodendrocytes. Eur. J. Neurosci. 2004, 20, 2049–2058. [CrossRef] [PubMed]

24. Czubowicz, K.; Czapski, G.A.; Cies´lik, M.; Strosznajder, R.P. Lipoxygenase inhibitors protect brain cortex

macromolecules against oxidation evoked by nitrosative stress. Folia Neuropathol. 2010, 48, 283–292.

25. Csermely, P.; Agoston, V.; Pongor, S. The efﬁciency of multi-target drugs: The network approach might help

drug design. Trends Pharmacol. Sci. 2005, 26, 178–182. [CrossRef] [PubMed]

26. Anighoro, A.; Bajorath, J.; Rastelli, G. Polypharmacology: Challenges and opportunities in drug discovery.

J. Med. Chem. 2014, 57, 7874–7887. [CrossRef]

27. Geldenhuys, W.J.; Van der Schyf, C.J. Rationally designed multitargeted agents against neurodegenerative

diseases. Curr. Med. Chem. 2013, 20, 1662–1672. [CrossRef]

28. Cavalli, A.; Bolognesi, M.L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini, M.; Melchiorre, C.

Multi-target-directed ligands to combat neurodegenerative diseases. J. Med. Chem. 2008, 51, 347–372.

[CrossRef]

29. Liargkova, T.; Hadjipavlou-Litina, D.J.; Koukoulitsa, C.; Voulgari, E.; Avgoustakis, C. Simple chalcones and

bis-chalcones ethers as possible pleiotropic agents. J. Enzyme Inhib. Med. Chem. 2015, 31, 302–313. [CrossRef]

30. Chavan, B.B.; Gadekar, A.S.; Mehta, P.P.; Vawhal, P.K.; Kolsure, A.K.; Chabukswar, A.R. Synthesis and

medicinal signiﬁcance of chalcones—A review. Asian J. Biomed. Pharm. Sci. 2015, 6, 1–7.

31. Yazdan, S.K.; Vidya, S.D.; Afzal, B.S. Chemical and biological potentials of chalcones: A review.

Org. Med. Chem. 2015, 1, 1–8. [CrossRef]

32. Ng, H.L.; Ma, X.; Chew, E.H.; Chui, W.K. Design, synthesis, and biological evaluation of coupled bioactive

scaffolds as potential anticancer agents for dual targeting of dihydrofolate reductase and thioredoxin

reductase. J. Med. Chem. 2017, 60, 1734–1745. [CrossRef] [PubMed]

33. Zheng, C.J.; Jiang, S.M.; Chen, Z.H.; Ye, B.J.; Piao, H.R. Synthesis and anti-bacterial activity of some

heterocyclic chalcone derivatives bearing thiofuran, furan, and quinoline moieties. Arch. Pharm. Chem.

Life Sci. 2011, 344, 689–695. [CrossRef] [PubMed]

34. Xie, Z.; Luo, X.; Zou, Z.; Zhang, X.; Huang, F.; Li, R.; Liao, S.; Liu, Y. Synthesis and evaluation

of hydroxychalcones as multifunctional non-purine xanthine oxidase inhibitors for the treatment of

hyperuricemia. Bioorg. Med. Chem. Lett. 2017, 27, 3602–3606. [CrossRef] [PubMed]

35. Mohd Faudzia, S.M.; Leonga, S.W.; Abas, F.; Mohd Aluwid, M.F.F.; Rullahd, K.; Lamd, K.W.; Ahmade, S.;

Thamf, C.L.; Shaaria, K.; Lajis, N.H. Synthesis, biological evaluation and QSAR studies of diarylpentanoid

analogues as potential nitric oxide inhibitors. MedChemComm 2015, 6, 1069–1080. [CrossRef]

Molecules 2019, 24, 199

29 of 31

36. Hwang, T.L.; Yeh, S.H.; Leu, Y.L.; Chern, C.Y.; Hsu, H.C. Inhibition of superoxide anion and elastase release in

human neutrophils by 30-isopropoxychalcone via a cAMP-dependent pathway. Br. J. Pharm. 2006, 148, 78–87.

[CrossRef]

37. Rekker, R. Hydrophobic Fragmental Constant, Its Derivation and Application: A Means of Characterizing Membrane

Systems; Elsevier Eds. Scientiﬁc Co.: New York, NY, USA, 1977.

38. Les´niewska, M.A.; Gdaniec, Z.; Muszalska, I. Calculation procedures and HPLC method for analysis of the

lipophilicity of acyclovir esters. Drug Dev. Ind. Pharm. 2015, 41, 663–669. [CrossRef]

39. Główka, F.; Roman´ski, M.; Siemia˛tkowska, A. Determination of partition coefﬁcients n-octanol/water

for treosulfan and its epoxy-transformers: An example of a negative correlation between lipophilicity of

unionized compounds and their retention in reversed-phase chromatography. J. Chromatogr. B Anal. Technol.

Biomed. Life Sci. 2013, 923, 92–97. [CrossRef]

40. Bakht, M.A.; Alajm, M.F.; Alam, P.; Alam, A.; Alam, P.; Ajarba, T.M. Theoretical and experimental study on

lipophilicity and wound healing activity of ginger compounds. Asian Pac. J. Trop. Biomed. 2014, 4, 329–333.

[CrossRef]

41. BioByte. Available online: ww w .biobyte.com (accessed on 7 November 2018).

42. Sakuratani, Y.; Kasai, K.; Noguchi, Y.; Yamada, J. Comparison of predictivities of log P calculation models

based on experimental data for 134 simple organic compounds. QSAR Comb. Sci. 2006, 26, 109–116.

[CrossRef]

43. Canavan, N. FDA and drug companies alike want ADME-tox testing performed earlier and earlier in a

drug’s life cycle. Drug Discov. Dev. 2007, 10, 34–36.

44. Molinspiration Cheminformatics. Available online: ww w .molinspiration.com (accessed on 20 April 2018).

45. Lipinski, C.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to

estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 1997

,

23, 3–25. [CrossRef]

46. Rishton, G.M.; LaBonte, K.; Williams, A.J.; Kassam, K.; Kolovanov, E. Computational approaches to the

prediction of bloodbrain barrier permeability: A comparative analysis of central nervous system drugs

versus secretase inhibitors for Alzheimer’s disease. Curr. Opin. Drug. Discov. Devel. 2006, 9, 303–313.

[PubMed]

47. Crooks, S.W.; Stockley, R.A. Leukotriene B4. Int. J. Biochem. Cell Biol. 1998, 30, 173–178. [CrossRef]

48. Chu, J.; Praticò, D. 5-Lipoxygenase as an endogenous modulator of amyloid beta formation in vivo

.

Ann. Neurol. 2011, 69, 34–46. [CrossRef] [PubMed]

49. Praticò, D. Oxidative stress hypothesis in Alzheimer’s disease: A reappraisal. Trends Pharmacol. Sci. 2008

29, 609–615. [CrossRef] [PubMed]

,

50. Pratico, D.; Zhukareva, V.; Yao, Y.; Uryu, K.; Funk, C.D.; Lawson, J.A.; Trojanowski, J.Q.; Lee, V.M.

12/15 lipoxygenase is increased in Alzheimer’s disease. Possible involvement in Brain oxidative stress.

Am. J. Pathol. 2004, 164, 1655–1662. [CrossRef]

51. Chu, J.; Li, J.G.; Ceballos-Diaz, C.; Golde, T.; Praticò, D. The inﬂuence of 5-lipoxygenase on Alzheimer’s

disease-related tau pathology: In vivo and in vitro evidence. Biol. Psychiatry 2013, 74, 321–328. [CrossRef]

[PubMed]

52. Skrzypczak-Jankun, E.; Zhou, K.; Jankun, J. Inhibition of lipoxygenase by ( )- epigallocatechin gallate: X-ray

−

analysis at 2.1 A reveals degradation of EGCG and shows soybean LOX-3 complex with EGC instead. Int. J.

Mol. Med. 2003, 12, 415–420. [CrossRef]

53. Skrzypczak-Jankun, E.; Chorostowska-Wynimko, J.; Selman, S.H.; Jankun, J. Lipoxygenases—A challenging

problem in enzyme inhibition and drug development. Curr. Enzyme Inhib. 2007, 3, 119–132. [CrossRef]

54. Gleason, M.M.; Rojas, C.J.; Learn, K.S.; Perrone, M.; Bilder, G.E. Characterization and inhibition of

15-lipoxygenase in human monocytes–Comparison with soybean 15-lipoxygenase. Am. J. Physiol. 1995

258, 1301–1307. [CrossRef]

,

55. Lapenna, D.; Ciofani, G.; Pierdomenico, S.D.; Giamberardino, M.A.; Cuccurullo, F. Dihydrolipoic acid

inhibits 15-lipoxygenase-dependent lipid peroxidation. Free Rad. Biol. Med. 2003, 35, 1203–1209. [CrossRef]

56. Nuhn, P.; Büge, A.; Köhler, T.; Lettau, H.; Schneider, R. Trends bei der entwicklung von lipoxygenase-

hemmern. Pharmazie 1991, 46, 81–88. [PubMed]

57. Maccarrone, M.; van Aarie, P.G.M.; Veldink, G.A.; Vliegenthart, J.F.G. In vitro oxygenation of soybean

biomembranes by lipoxygenase-2. Biochim. Biophys. Acta 1994, 1190, 164–169. [CrossRef]

Molecules 2019, 24, 199

30 of 31

58. Pontiki, E.; Hadjipavlou-Litina, D. Lipoxygenase inhibitors: A comparative QSAR study review and

evaluation of new QSARs. Med. Res. Rev. 2008, 28, 39–117. [CrossRef] [PubMed]

59. Steinhilber, D.; Hofmann, B. Recent advances in the search for novel 5-lipoxygenase inhibitors. Basic Clin.

Pharmacol. Toxicol. 2014, 114, 70–77. [CrossRef] [PubMed]

60. Sendobry, S.M.; Cornicelli, J.A.; Welch, K.; Bocan, T.; Tait, B.; Trivedi, B.K.; Colbry, N.; Dyer, R.D.;

Feinmark, S.J.; Daugherty, A. Attenuation of diet-induced atherosclerosis in rabbits with a highly selective

15-lipoxygenase inhibitor lacking signiﬁcant antioxidant properties. Br. J. Pharmacol. 1997, 120, 1199–1206.

[CrossRef] [PubMed]

61. Kang, J.E.; Cho, J.K.; Marcus, J.; Long, C.; Ryu, H.W.; Kim, J.H.; Kim, H.J.; Yuk, H.J.; Kim, D.W.; Hun, K.

Inhibitory evaluation of sulfonamide chalcones on

β-secretase and acylcholinesterase. Park Mol. 2013

,

18, 140–153. [CrossRef] [PubMed]

62. Murphy, M.P.; LeVineIII, H. Alzheimer’s disease and the

[CrossRef] [PubMed]

β-amyloid peptide. J. Alzheimers Dis. 2010, 19, 311.

63. Barten, D.M.; Albright, C.F. Therapeutic strategies for Alzheimer’s disease. Mol. Neurobiol. 2008, 37, 171–186.

[CrossRef]

64. Fezoui, Y.; Teplow, D. Kinetic studies of amyloid

36948–36954. [CrossRef]

β-protein ﬁbril assembly. J. Biol. Chem. 2002, 277,

65. Habchi, J.; Chia, S.; Limbocker, R.; Mannini, B.; Ahn, M.; Perni, M.; Hansson, O.; Arosio, P.; Kumita, J.R.;

Challa, P.K.; et al. Systematic development of small molecules to inhibit speciﬁc microscopic steps of A 42

aggregation in Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 2017, 114, 200–208. [CrossRef] [PubMed]

β

66. Matis, I.; Delivoria, D.C.; Mavroidi, B.; Papaevgeniou, N.; Panoutsou, S.; Bellou, S.; Papavasileiou, K.D.;

Linardaki, Z.I.; Stavropoulou, A.V.; Vekrellis, K.; et al. An integrated bacterial system for the discovery of

chemical rescuers of disease-associated protein misfolding. Nat. Biomed. Eng. 2017, 1, 838–852. [CrossRef]

67. Mete, E.; Comez, B.; Gul, H.I.; Gulcin, I.; Supuran, C.T. Synthesis and carbonic anhydrase inhibitory activities

of new thienyl-substituted pyrazoline benzenesulfonamides. J. Enzyme Inhib. Med. Chem. 2016, 31, 1–5.

[CrossRef] [PubMed]

68. Dimmock, J.R.; Kandepu, N.M.; Hetherington, M.; Quail, J.W.; Pugazhenthi, U.; Sudom, A.M.;

Chamankhah, M.; Rose, P.; Pass, E.; Allen, T.M.; et al. Cytotoxic activities of mannich bases of chalcones and

related compounds. J. Med. Chem. 1998, 41, 1014–1026. [CrossRef] [PubMed]

69. Cassidy, J.E.; Witcher, W.J. The polarographic characteristics of furfurylideneacetophenone and some of its

para derivatives. J. Phys. Chem. 1959, 63, 1824–1826. [CrossRef]

70. Amin, A.N.; El-Khouly, M.E.; Subbaiyan, N.K.; Zandler, M.E.; Supur, M.; Fukuzumi, S.; D’Souza, F. Syntheses,

electrochemistry, and photodynamics of ferrocene_azadipyrromethane donor_acceptor dyads and triads.

J. Phys. Chem. 2011, 115, 9810–9819. [CrossRef] [PubMed]

71. Natarajan, C.; Tharmaraj, P. Cobalt(II), nickel(II) and copper(II) complexes of 1-(2-hydroxyphenyl)-3-

(1-naphthalenyl)-2-propen-1-one and 1-(1-hydroxy-2-naphthalenyl)-3 (1-naphthalenyl)-2-propen-1-one.

Syn. React. Inorg. Metalorg. Chem. 2006, 20, 151–166. [CrossRef]

72. Silva, A.M.S.; Pinto, D.C.G.A.; Travares, H.R.; Cavaleiro, J.A.S.; Jimeno, M.L.; Elguero, J. Novel (E)-

0

and (Z)-2-styrylchromones from (E,E)-2 -hydroxycinnamylideneacetophenones—xanthones from daylight

photooxidative cyclization of (E)-2-styrylchromones. Eur. J. Org. Chem. 1998, 9, 2031–2038. [CrossRef]

73. Hofmann, E.; Webster, J.; Do, T.; Kline, R.; Snider, L.; Hauser, Q.; Higginbottom, G.; Campbell, A.; Ma, L.;

Paula, S. Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and radical scavengers.

Bioorg. Med. Chem. 2016, 24, 578–587.

74. Sodani, R.S.; Choudhary, P.C.; Sharma, H.O.; Verma, B.L. Syntheses and reactions of 4⁰-[(

ω-bromoalkyl)

0

000

oxy]-and 4 , 4 -(polymethylenedoxy)-bis substituted chalcones. J. Chem. 2010, 7, 763–769. [CrossRef]

75. Prabhakar, K.R.; Veerapur, V.P.; Bansal, P.; Vipan, P.K.P.; Reddy, K.M.; Barik, A.; Reddy, B.K.D.;

Reddanna, P.; Priyadarsinib, K.I.; Unnikrishnana, M.K. Identiﬁcation and evaluation of antioxidant,

analgesic/anti-inﬂammatory activity of the most active ninhydrin–phenol adducts synthesized.

Bioorg. Med. Chem. 2006, 14, 7113–7120. [CrossRef] [PubMed]

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Small multitarget molecules incorporating the enone moiety

DOI: 10.3390/molecules24010199

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