2-Arylidene-1-indandiones as Pleiotropic Agents with Antioxidant and Inhibitory Enzymes Activities ?

Article abstract of DOI:10.3390/molecules24234411

Indandiones are a relatively new group of compounds presenting a wide range of biological activities. The synthesis of these compounds was performed via a Knoevenagel reaction between an aldehyde and 1,3-indandione and were obtained with a yield up to 54%

Full text of DOI:10.3390/molecules24234411

molecules
Article
2-Arylidene-1-indandiones as Pleiotropic Agents with
Antioxidant and Inhibitory Enzymes Activities †
Olympia Kouzi, Eleni Pontiki and Dimitra Hadjipavlou-Litina *
Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences, Aristotle University
of Thessaloniki, 54124 Thessaloniki, Greece; kpaulina2000@yahoo.com (O.K.); epontiki@pharm.auth.gr (E.P.)
* Correspondence: hadjipav@pharm.auth.gr; Tel.: +30-231-099-7627
† A part of these results has been presented at the 14th Hellenic Symposium of Medicinal Chemistry
(23–25 April 2010, Thessaloniki, Greece) and awarded.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Orazio Nicolotti
Received: 29 October 2019; Accepted: 29 November 2019; Published: 3 December 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Indandiones are a relatively new group of compounds presenting a wide range of biological
activities. The synthesis of these compounds was performed via a Knoevenagel reaction between an
aldehyde and 1,3-indandione and were obtained with a yield up to 54%. IR, 1H-Nucleic Magnetic
Resonance (NMR), 13C-NMR, LC/MS ESI+ and elemental analysis were used for the confirmation of the
structures of the novel derivatives. Lipophilicity values of compounds were calculated theoretically
and experimentally by reversed chromatography method as values RM. The novel derivatives
were studied through in vitro and in vivo experiments for their activity as anti-inflammatory and
antioxidant agents and as inhibitors of lipoxygenase, trypsin, and thrombin. The inhibition of the
carrageenin-induced paw edema (CPE) was also determined for representative structures. In the above
series of experiments, we find that all the compounds showed moderate to satisfying interaction with
the stable DPPH free radical in relation to the concentration and the time 2-arylidene-1-indandione
(
10) was the strongest. We observed moderate or very low antioxidant activities for selected
compounds in the decolorization assay with ABTS+•. Most of the compounds showed high anti-lipid
peroxidation of linoleic acid induced by AAPH.2-arylidene-1-indandione ( ) showed a strongly
inhibited soybean LOX. Only 2-arylidene-1-indandione ( ) showed moderate scavenging activity of
superoxide anion, whereas 2-arylidene-1-indandione ( ) and 2-arylidene-1-indandione ( ) showed
very strong inhibition on proteolysis. 2-arylidene-1-indandione ( ) highly inhibited serine protease
thrombin. 2-arylidene-1-indandiones ( and ) can be used as lead multifunctional molecules.
The compounds were active for the inhibition of the CPE (30–57%) with 2-arylidene-1-indandione (
7
3
8
9
8
7
,
8
9
1)
being the most potent (57%). According to the predicted results a great number of the derivatives
can cross the Blood–Brain Barrier (BBB), act in CNS and easily transported, diffused, and absorbed.
Efforts are conducted a) to correlate quantitatively the in vitro/in vivo results with the most important
physicochemical properties of the structural components of the molecules and b) to clarify the
correlation of actions among them to propose a possible mechanism of action. Hydration energy
as EHYDR and highest occupied molecular orbital (HOMO) better describe their antioxidant profile
whereas the lipophilicity as RM values governs the in vivo anti-inflammatory activity. Docking studies
are performed and showed that soybean LOX oxidation was prevented by blocking into the
hydrophobic domain the substrates to the active site.
Keywords: 2-arylidene-1,3-indandiones; lipoxygenase inhibitors; antioxidants; Lipinski “Rule of five”
1. Introduction
Inflammation is a complex protective biological response to injury or infection, representing one
of the most important defense mechanisms of an organism to pathogens, damaged cells, or irritants.
Molecules 2019, 24, 4411; doi:10.3390/molecules24234411
www.mdpi.com/journal/molecules

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Inflammatory responses are mainly associated with arachidonic acid (AA-cis 5, 8,11,14-eicosatetraenoic
acid)cascade [ ]. AA is a twenty-carbon polyunsaturated fatty acid released in response to various
stimuli [ ], by the activation of phospholipase A2 (PLA2, generally cytoplasmicPLA2) and other
lipases [ ]. AA once released from the phospholipid bilayer can be further metabolized via three major
enzymatic routes: cyclooxygenase (COX), lipoxygenase (LOX) and monooxygenase cytochrome P450
(CYP) to eicosanoids [ ]. The COX pathway leads to prostaglandins (PGs), thromboxane and other
1
2
3
4
prostanoids; the 5-, 12- and 15-LOX pathways leads to 5-, 12-, or 15-hydro(-pero)xy-eicosa-tetra-enoic
acids [H(P)ETEs] and additionally leukotrienes as well as lipoxins (LXs); and finally the cytochrome
P450 monooxygenase pathway leads to diverse epoxy-eicosatetraenoic acids (EETs) and diHETEs [5].
Inflammation is associated with many diseases such atherosclerosis, cardiovascular disorders,
neurodegeneration, osteoarthritis, rheumatoid arthritis, diabetes, allergy, infection and cancer [6].
Recent studies have established the implication of inflammatory processes in cardiovascular
diseases (initiation, progression and thrombotic complications), formerly considered a bland lipid
storage disease [7]. Coronary heart disease (CHD) is characterized as a chronic low-grade inflammatory
process. In physiological conditions vascular endothelium regulates homeostasis through the
regulation of vascular dilatation, prevention of platelet adhesion, and inhibition of thrombin generation.
Endotheliun dysfunction caused by chronic inflammation (atherothrombosis, atherosclerosis, diabetes,
or chronic arterial hypertension) or acute inflammation (physical injury of the vascular wall e.g., balloon
angioplasty) generates a pro-inflammatory environment supporting leukocyte transmigration toward
inflammatory sites [
and signaling molecules, lipid mediators, cytokines, and chemokines controlling leukocytes playing
pivotal role in inflammatory response [ ]. At the same time, thrombin is realized from the damaged
endothelium as a pro-inflammatory mediator provoking fibrin deposition and coagulation [10].
Thrombin, a serine protease plays crucial role in clotting and inflammatory process [ ]. During this
8]. By this state there is enhanced expression of growth factors, adhesion,
9
8
inflammatory process free radicals are produced from diet-derived lipids in the arterial wall and serum
producing peroxides and other reactive oxygen species [11].
The implication of reactive oxygen species (ROS) in disorders associated with oxidative
stress (e.g., coronary artery disease, inflammatory injury, cancer and cardiovascular diseases) was
acknowledged by various research groups [12–16]. It is well known that ROS e.g., hydroxyl
radical, superoxide anion and hydrogen peroxide act by attacking the macromolecules of the
organism (proteins, lipids and DNA) [17] provoking chemoresistance, proliferation, genetic instability,
radio-resistance, angiogenesis, and invasion [18].
Over the last decade the concept of “promiscuous” medicine has been primarily associated with the
capability to selectively attack multiple factors and to address multifactorial diseases. The multi-target
concept has been extensively reviewed due to the complex and deleterious consequences of ROS in
diverse diseases (atherosclerosis, inflammatory injury, cancer and cardiovascular diseases) implicating
inflammatory processes [19]. Combination of more than one activity in one molecule offers an
advantage for the therapy of the above-mentioned pathophysiological diseases.
One of the main research interests in our group has been the design of small multi-target
molecules with biological activity based on the
cinnamic acid derivatives [20 21]. Chalcones are small molecules, known as
presenting exceptional biological interest [22]. The medicinal significance of the enone moiety and of
chalcones is highlighted by numerous publications [23 27]. The bioactivity of chalcones depends on
the conjugated double bond with the carbonyl moiety, as removal of the enone group renders them
inactive. Indandiones are classified as bicyclic aromatic -diketones. They have been predominantly
α
,
β-unsaturated keto scaffold e.g., chalcones and
,
α, β-unsaturated ketones,
–
β
applied as substrates in organic and medicinal chemistry as their derivatives have interesting biological
activities such as: antitumor, antiplatelet, anti-inflammatory, antimicrobial, neuroprotective [28].
Indandiones and their derivatives process important activity against Alzheimer’s disease and cancer.
Donezepil for example acts as acetylcholineserase inhibitor for the treatment of mild (early stage) to
moderate (middle stage) Alzheimer [29] whereas indanocine combat drug resistance malignancies [30].

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Various research groups evaluated 2-arylidene-1,3-indandiones for cytotoxic properties [31
,32]. It was
later found that cytotoxicity and the fractional positive charge on the olefinic carbon atom could
be increased by placing an additional electron-attracting oxygen atom into the indane scaffold [33].
2-aryl-1,3-indandiones were found to present anti-inflammatory activities in a carrageenin edema
test [34–36] 2-Phenyl-1,3-indandione appeared to be a weak inhibitor of prostaglandin biosynthesis [37].
However, the inhibition of prostaglandin biosynthesis by 2-aryl-1,3-indandiones showed no clear
relationship with anti-inflammatory activity.
Thus, we decided to carry on our attempts to design and synthesize small biologically pleotropic
effective compounds using the 1,3-indandionemoiety as an advantaged template for the design of
multi-target agents. In our synthetic procedure several aromatic and heteroaromatic aldehydes properly
substituted have been used in our effort to define the importance and correlation of steric and electronic
parameters on the biological activities and to improve them through methodical alteration of the
substituents. Thus, we present the synthesis and in vitro and in vivo biological evaluation of a series
of 2-arylidene-1,3-indandiones (Scheme 1) diversely substituted.
Scheme 1. General synthesis of the new derivatives: 0.0068 mol of 1,3-indandione, 0.0075 mol of the
corresponding arylaldehyde in 30 mL of absolute ethanol and piperidine 0.06 mL. Yields of the products
are given in parentheses.
Some of these derivatives, compounds
kinase inhibitors, in the treatment of schistosomiasis whereas
1
,
3
,
4
,
5
,
6
,
10 are referred in the literature as tyrosinase
as a urease inhibitor. However their
3
synthesis follow a different route from the described herein procedure [38–51].
2. Results and Discussion
2.1. Chemistry
The synthesis of the title compounds was accomplished via a Knoevenagel condensation reaction
as presented in Scheme 1 as well as the structures of the various substituted aromatic aldehydes used for
those derivatives. 2-aryl-1,3-indandiones were synthesized from aryl-aldehyde and 1,3-indandionein
the presence of ethanol and piperidine as catalyst [33].

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Compounds
2,
3,
5,
9,
10
,
11,
12 were obtained in satisfactory yields (44–55%), compounds
1, 4, 6,
8
in 25–40% yield while compound
7
was obtained in lower yield under our experimental synthetic
procedure. The structure of the used aldehyde influences the yield of the reaction. It is known
that Knoevenagel condensation reactions yields are influenced by the stereochemistry of the used
aldehyde. Compounds 1, 3, 4, 5, 6, 10 have been synthesized earlier under different experimental
conditions [38–51]. We used a different synthetic technique and the structures of the known compounds
were verified according to the given in the literature spectral data, elemental analysis, or mps. In all
cases our synthetic procedure was simpler (heating in alcohol with piperidine drops for eight hours)
compared to the previous methods in which acidic conditions and heating for 21 h, microwave
irradiation or specific catalysts were referred.
Herein we synthesized them using our synthetic technique and we investigated their
biological activities.
The final products were obtained by recrystallization from ethanol or ethanol/water or with
1
preparative TLC with preparative TLC using CH3COOC2H5: petroleum ether (1:2), IR, H-NMR,
13C-NMR and elemental analysis were applied for the verification of the structures of the novel
derivatives. LC/MS. The infrared spectra of the derivatives were determined. All the products
displayed strong absorption for the carbonyl group indicating their stereo chemical homogeneity.
The carbonyl absorptions were noted in the regions of 1680, 1700, 1720–1740 cm−1 in Nujol and
1625cm−1 for the C=C. The 1H-NMR spectra disclosed that they possessed the Z configuration of the
1
olefinic protons. H-NMR spectroscopy indicated through integration the right analogy of aromatic
and CH=protons. 13C-NMR spectra indicated the keto carbons (C=O) as well as the –C=C. The findings
are in agreement with earlier publication [33]. The physicochemical properties of the novel derivatives
are reported in the experimental session.
2.2. Physicochemical Studies
2.2.1. Experimental Determination of Lipophilicity as RM Values
Lipophilicity is a physicochemical parameter indicating the relationship of a drug to its biological
activity. It affects ligand-target binding interactions, solubility, ADME (absorption, distribution,
bioavailability, metabolism, and elimination) and toxicological effects. RPTLC (reverse phase thin layer
chromatography) method was used for the experimental determination, as RM values [52]. The RPTLC
method is considered a secure, rapid and appropriate method for expressing the lipophilic character of
a compound (Table 2).
2.2.2. In Silico Determination of Lipophilicity Values as MilogP
We used the Molinspiration program to calculate in silico the lipophilicity as milogP values.
We tried to correlate the milogP values, the theoretically calculated lipophilicity in one equation with
the RM values of all the compounds (Table 1). However, this attempt, was found to be unsuccessful.
Several factors, for example different solvation, silanophilic interaction, H-bridges, might cause this
disagreement. It seems that a theoretically in silico calculated logP value is more accurate than an
experimental [53].

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Table 1. Lipophilicity values: experimental RM%. EHYDR Hydration Energy. E(HOMO). Interaction with the stable radical 1,1-diphenyl-picrylhydrazyl (DPPH),
In vitro lipoxygenase (LOX) inhibitory activity at 100 µM.
RA% 100
µM
20 min
RA% 100
µM
60 min
RA% 200
µM
20 min
RA% 200
µM
60 min
LOX
(% Inhibition
100 µM)
RA% 50 µM RA% 50 µM
a
RM (±SD) b
Compd.
EHYDR
E(HOMO)
20 min
60 min
1
2
3
4
5
6
7
8
9
0.031 ± 0
0.031 ± 0
0.014 ± 0
−0.615 ± 0.02
0.158 ± 0
−0.527 ± 0
−0.196 ± 0.02
*
−1.038 ± 0
−0.103 ± 0
−0.196 ± 0.02
−0.134 ± 0.02
-
−20.48
−17.37
−7.67
−6.53
−6.71
−9.86
−7.30
−9.31
−5.68
−5.71
−12.71
−12.72
−
−
0.310777
0.309938
0.291110
2
2
20
5
21
6
27
4
4
13
2
3
81
0
2
23
10
25
6
35
5
3
16
16
37
26
44
22
40
7
16
16
40
32
48
23
50
7
2
4
6
7
32.6
45
35.7
35
26.4
17.7
67.3
3.6
no
no
38.6
58.9
93
61
23
57
15
75
0
75
32
72
18
85
0
−
−
−
−
−
−
−
−
−
0.306356
0.302600
0.271983
0.303773
0.314300
0.306356
0.314300
0.279993
0.279598
3
1
6
6
10
11
12
17
2
3
100
19
17
87
85
19
17
93
71
10
4
71
10
6
NDGA
83
94
96
a
RM values are the average of at least 5 measurements, b SD standard deviation < 10%; * could not be measured under the reported experimental conditions.

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2.2.3. Molecular Properties Prediction-Lipinski “Rule of Five”
Traditional experimental procedures for the determination of human pharmacokinetic properties
have been progressively replaced by the predicted since the computational tools that are in use are
faster, simpler and more cost-effective [54]. Presently, a variety of use fulin silico methods have been
developed for screening hundreds of data sets of compounds. In this regard, we obtained and entered in
the online Molinspiration software version 2016.10 (www.molinspiration.com) [55] chemical structures
and simplified molecular-input line-entry system (SMILES) notations of the synthesized indandiones
to calculated various molecular properties., partition coefficient (logP), topological polar surface
area (TPSA), hydrogen bond donors and acceptors, rotatable bonds, number of atoms, molecular
weight, and to define the violations of Lipinski’s rule of five, in order to evaluate the drug-likeness of
indandiones [56]. According to Lipinski’s rule poor absorption or permeation is more likely observed
when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight (MW) is greater
than 500 and the calculated LogP value is greater than 5. It is a rule of thumb to delineate if a chemical
entity with a certain biological activity has drug-likeness, properties that would support its behavior
as an orally active drug in humans.
LogP values of all derivatives, as shown in Table 2, range from 2.52 to 5.94, except for 8, 9, and 10.
Since their lipophilicity was found to be less than 5, they did not violate “the rule of five” suggesting
satisfactory permeability across cell membrane. LogBB is another important in silico descriptor to
identify CNS active agents. LogP values were used for the theoretical calculation of the logBB values.
For in silico prediction [57], compound with logBB value higher than 0.3 is considered to have high
absorption through BBB whereas logBB values between 0.3 to
to be moderate and less absorbed through BBB.
−
0.1 and lower than 0.1 are considered
−
LogP values of 2-arylidene-1,3-indandionesand of the standard drug nordihydroguaretic acid
(NDGA) were found to be under 5 defining their use per os. Furthermore, all derivatives were found
to present MW less than 500. Thus, these compounds compared to large molecules, could be easily
transported, diffused, and absorbed. The number of hydrogen bond acceptors (O and N atoms) and
the number of hydrogen bond donors (NH and OH) in the synthesized compounds obey the Lipinski’s
rule of five (less than 10 and 5 respectively). Within the data set compounds
orally active following Lipinski’s rule of five (Table 2).
1–7 and 11, 12 seem to be
TPSA is used as a significant indicator of the bioavailability of a bioactive molecule.
This parameteris highly correlated with the hydrogen bonding properties of a molecule. TPSA of the
derivatives was observed in the range of 34.14–54.37 A0. These values are well below the limit of
160A0 supporting the presence of good oral bioavailability. The upper limit for TPSA for a molecule to
penetrate the brain is around 90 Å. The calculated results indicate that all structures are able cross BBB.

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Table 2. Molecular properties prediction-Lipinski “Rule of five”. Drug-likeness of the synthesized compounds.
No O,N
No OH, NH
No
Violations
No Rotational
Bonds e
Volume
TPSA b
logBBB h
Compd.
milogP a
No Atoms
MW g
c
d
f
1
2
3
4
5
6
7
8
9
2.79
3.16
2.52
2.89
3.64
3.75
4.62
5.35
5.94
5.94
3.09
3.04
34.14
34.14
47.28
34.14
34.14
37.38
43.38
43.38
54.37
54.37
49.93
49.93
17
18
18
18
20
23
25
27
27
27
21
21
2
2
3
2
2
3
3
3
3
3
3
3
0
0
0
0
0
0
0
0
3
1
1
1
0
0
0
0
0
0
0
1
1
1
0
0
1
1
1
1
2
3
3
4
3
3
1
1
200.71
217.27
208.12
209.99
237.41
283.32
290.39
325.07
350.38
350.38
238.97
238.97
240.28
254.31
238.24
234.25
260.29
303.36
326.35
419.27
362.47
362.47
273.29
273.29
0.255
0.312
0.082
0.270
0.387
0.371
0.446
0.560
0.541
0.541
0.144
0.144
10
11
12
No Atoms denotes the number of atoms in the compound; a Logarithm of partition coefficient between-octanol and water (milogP); b Topological polar surface area (TPSA); c Number of
hydrogen bond acceptors (n-ON); d Number of hydrogen bond donors (n-OHNH); e Number of rotatable bonds (n-rotb); f Molecular Volume; g Molecular Weight; h blood–brain barrier
(logBBB = 0.155 logP − 0.01TPSA + 0.164) [57].

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2.3. Biological Evaluation
In this study, the novel derivatives were evaluated: (i) in vitro for their antioxidant profile and
(ii) in vivo on the inhibition of the carrageenin-induced edema. They were also studied in vitro as
possible inhibitors of soybean lipoxygenase, trypsin, and thrombin.
The ability of the novel derivatives to present antioxidative ability in vitro was studied applying
various biological protocols in which different radicals were generated. Factors such as solubility or
steric hindrance are important and influence the experimental conditions. Two different methods were
adopted: (i) the first measure the scavenging ability by donating a hydrogen- or an electron on a free
radical used as anindicator of antioxidant activity and (ii) in the second a free radical is generated from
an antioxidant system. The in vitro antioxidant activity was measured in terms of: (a) the interaction
with the stable free radical DPPH; (b) the ABTS+• radical cation reduction-decolorization ability,
(c) scavenging of superoxide anion and (d) the anti-lipid peroxidation activity.
The novel derivatives were studied for their interaction with the sTable 2,2-diphenyl-1-picrylhydrazyl
free radical (DPPH)at concentration 50 µM, 100 µM and 200 µM after 20 and 60 min (Table 1) [58–62]. In this
assay the DPPH radical is reduced transferring one electron from the antioxidant. Phenolic compounds such
as catechol and derivatives generate phenoxide anions with strong antioxidant capacity. Nordihydroguaretic
acid (NDGA), the reference compound acts by this mechanism. Compound 10 presents the highest
scavenging activity at 100
presented medium activities depending from time and concentration. The presence of the heteroatom
O or S seems to influence the activity e.g., derivatives , and . Replacement of phenyl ring ( ) by a
thienyl group ( ) decreases antioxidant behavior. Both thienyl derivatives and exhibit very low activities.
On the contrary the presence of the furyl ring ( ) enhances reduction. Additionally, the presence of a
-dimethyl-amino group reduces the scavenging ability whereas its absence in ( ), increases the activity.
The presence of two conjugated double bonds ( ) leads to lower activity compared to the simpler compound
4. Compounds and 10 present the highest milogP values (5.94). Both include a phenolic hydrogen
in their structure. However, compound 10 is the more active within the series since it generates more
easily phenoxide anions. On the contrary in compound steric reasons are possible to lead to a decrease.
µM after 20 min correlated with the presence of the phenolic OH. Compounds 3,
4, 5, 7
1,
2
3
4
1
1
2
3
π
6
5
5
9
9
Lipophilicity seems to influence the interaction of compound 10.
The radical cation ABTS+• is directly generated through potassium persulfate by oxidation with no
participation of an intermediary radical and then the reduction is followed by adding electron-donating
antioxidants. In this assay the radical is generated before adding the antioxidant (decolorization assay).
The compounds presented low to moderate antioxidant activity with most potent 10 and 7. It seems
that lipophilicity influences the activity, since both are presenting high lipophilicity values. To correlate
the activity with the physicochemical parameters it was found that hydration energy as EHYDR plays
important role (Equation (1)).
log % ABTS+• = 0.104 (± 0.036) EHYDR + 2.175 (±0.366)
(1)
n = 8, r = 0.945, r2 = 0.893, q2 = 0.762, s = 0.149, F1,8 = 5.356, α = 0.1
In the water soluble 2,20-azobis(2-amidinopropane) hydrochloride (AAPH) the free radicals are
produced in vitro through spontaneous thermal decomposition resembling to lipid peroxidation in cells
due to the action of the peroxyl radicals. Among the synthesized derivatives compounds
1, 2, 9, and
10 showed remarkable activity at final concentration 100 M. Compounds 11 and presented no
µ
,
5,
4
3
activity under the experimental conditions. From the experimental results it can be concluded that the
3,5-di-tert-butyl-group offers protection against lipid peroxidation. Electronic parameters expressed by
the highest occupied molecular orbital (HOMO) describes better the biological activity (Equation (2)).
log AAPH % = −22.042(± 8.731) HOMO − 4.853 (2.622)
(2)
n = 8, r = 0.930, r2 = 0.864, q2 = 0.567, s = 0.146, F1,8 = 5.783, α = 0.1

Molecules 2019, 24, 4411
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The compounds have been tested to scavenge superoxide anion radicals. However, no results
were recorded under the experimental conditions. Only showed 20% scavenging activity at 100 M.
Manyresearchersrevealedthehomologyofsoybeanlipoxygenasetomammalianlipoxygenase[63,64]
Thus, we used soybean LOX in our in vitro experiments. Compound is was found to be most potent
followed by compounds 11 12. Insertion of a methyl group ( ) favors the inhibitory activity
while replacement of a phenyl group from a thienyl does not seem to affect the activity ( and ).
The heteroatom in the 5membered ring (furyl/thienyl) influences LOX inhibition ( ). The presence of
two conjugated double bonds ( ) leads to lower activity compared to the simpler compound . Indolyl
3
µ
.
7
2,
3,
4,
,
2 > 1
1
4
2
> 3
5
4
derivatives in position 3- (12) present stronger inhibition than derivatives in 5-position (11). From the
literature it has been found that lipophilicity is strongly associated with lipoxygenase inhibition [65].
However, in this series it seems that there is no correlation between lipophilicity and LOX inhibition.
The combination of 1,3-indandione moiety with the double bond seems to be more significant feature and
nodefinition is found for the importance of any other specific structural characteristics. Perusal of the
antioxidant results as well as LOX inhibition did not support any correlation among these activities.
Serine proteases are implicated in the pathophysiology of inflammatory diseases and trypsin
is well known years ago [66]. Trypsin has been associated with host defense reactions and tissue
damage [67]. In this study, the novel derivatives have been evaluated towards trypsin and thrombin.
Compounds 8 and 9 present high activity. On the contrary compounds 1, 2, 5, 10, and 12 do not present
any activity under the experimental conditions. Thienylderivatives did not show any activity while
the furyl-derivatives seem to be medium inhibitors.
It is well known that indandione derivatives exert their anticoagulant activity indirectly by
inhibiting prothrombin synthesis in the liver [68]. It has been found that antioxidant compounds
presenting antithrombotic effects play pivotal role in the therapy treatment of thrombosis based on
vascular oxidative stress or inflammation [69]. Derivatives
as possible thrombin inhibitors. Among the tested derivatives compound
1
,
2
,
5
,
8
,
9
,
10, and 12 were tested in vitro
presented 100% inhibition
8
followed by compounds 3 and 10.
Carrageenin-induced rat paw edema (CPE) model was used to study the in vivo anti-inflammatory
activity of the tested derivatives and the results are presented in Table 3. Compound presented the
highest anti-inflammatory activity from all the compounds, exhibiting even higher activity compared to the
indomethacin used as reference compound. Compounds 10, and 12 presented small differences in their
1
2, 7, 8,
biological activity. The presence of the thienyl group as well as of the furyl leads to higher anti-inflammatory
activity (1 and 3) while substitution with a phenyl group leads to two-fold decrease of the activity (28%).
Table 3. % Anti-lipid peroxidation (AAPH), Decolorization activity ABTS+·% assays. In vitro inhibition of
trypsin induced proteolysis (% Trypsin Inh -Iptr%), thrombin inhibition TH%, In vivoanti-inflammatory
activity (CPE %).
Compd.
AAPH% 100 µM
ABTS +. % 100 µM
Iptr% 100 µM
TH% 100 µM
CPE a
%
1
2
3
4
5
6
7
8
90
92
no
no
no
9
82
79
96
85
no
31
93
9
2
no
no
17
4
no
29
33
85
98
no
6
no
-
39
no
-
19
no
100
0
27
-
57 **
45 *
50 *
28 *
-
20
19
no
22
37
16
no
39
8
-
41 **
46 *
-
42 *
-
9
10
11
12
Trolox
no
91
no
-
42 **
Indomethacin
Salycilic Acid
Inogatran
47
54
98
a
* p < 0.01, ** p < 0.05. % of reduction of the rat paw edema (CPE %) induced by carrageenin at the dose of
0.01 mmol/Kg/body weight.no: no action under the experimental conditions.

Molecules 2019, 24, 4411
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Correlation of the biological activity with the physicochemical parameters showed that lipophilicity
as RM values governs the activity (Equation (3)):
logCPE% = 0.349 (± 0.117)RM + 1661(± 0.031)
(3)
n = 6, r = 0.972, r2 = 0.945, q2 = 0.898, s = 0.022, F1,6 = 3.892, α = 0.1
2.4. Computational Studies—Docking Simulation Soybean Lipoxygenase
Molecular Modeling of the Synthesized Derivatives in Soybean LOX
In silico docking has been performed for all the synthesized derivatives in accordance with the
biological results. The favored docking position for the most promising indandione is shown in
Figure 1. Compound has an AutoDockVina score of 10.2 binding to soybean LOX (PDB code:
7
7
−
3PZW). It is well known that the results from the in vitro inhibition of soybean lipoxygenase represent
experimental values while docking scores are based on algorithms and scoring function calculations,
so a 1 to 1 correlation is difficult to be reached. Docking describes the ligand binds to the protein.
It seems that the novel compounds interact with the soybean lipoxygenase (SLOX) through allosteric
interactions. Compound
7 present weak hydrogen bonds with ARG533 and THR529 with the two
carbonyl groups of the 1H-indene-1,3(2H)-dione template and hydrophobic ones with the rest of
the molecule. Since most LOX inhibitors act as antioxidants or by scavenging free radicals [70] and
the oxidation of the enzyme occurs via a carbon-centered radical on a lipid chain, it is possible that
compound 7 extendsinto the hydrophobic domain by blocking the substrates to the binding site and
thus preventing oxidation.
ARG533
THR529
Figure 1. Preferred docking pose of 7(depicted in blue) bound to soybean lipoxygenase (LOX-1).

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3. Experimental Section
3.1. Materials and Instruments
All chemicals, solvents, chemical and biochemical reagents were of analytical grade and purchased
from commercial sources (Merck, Fluka, and Sigma-Aldrich Laborchemikalien GmbH, Hannover,
Germany and Alfa Aesar, Karlsruhe, Germany). Soybean lipoxygenase, albumin, trypsin, thrombin,
sodium linoleate, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), sodium linoleate,
2,2-azinobis-2-methyl-propanimidamine HCl (AAPH) were obtained from Sigma Chemical, Co.
(St. Louis, MI, USA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), nordihydroguairetic acid (NDGA),
salycilic acid, warfarin, inogatran are purchased from the Aldrich Chemical Co. (Milwaukee, WI,
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, Ultaviolet-Vis spectra were obtained on a Perkin–Elmer 554 double
beam spectrophotometer (Perkin-Elmer Corporation Ltd., Lane Beaconsfield, Bucks, UK). Infrared
spectra (film as Nujol mulls or KBr pellets) were recorded with Perkin–Elmer 597 spectrophotometer
(Perkin–Elmer Corporation Ltd., Lane Beaconsfield, Bucks, UK).
The 1H-NMR spectra were recorded at 300 MHz on a Bruker AM-300 spectrometer
(Bruker AnalytischeMesstechnik 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 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 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 FlukaChemie 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 RPTLC TLC-Silica gel 60 F254 DC
Kieselgel, Merck (20 × 20 cm) plates were used.
3.2. Chemistry General Procedure
In a round bottom flask 0.0068 mol 1,3-indandione and 0.0075 mol of the corresponding
arylaldehyde were added and diluted in 30 mL of absolute ethanol using piperidine (0.06 mL)
as catalyst. The mixture was heated reflux for approximately 8 h monitoring with TLC (petroleum
ether/ethyl acetate, 7/3) for the completion of the reaction. The excess of the solvent was removed with
rotary evaporator. The residue was filtered and purified either with recrystallization with EtOH 95◦or
ethanol/water or with preparative TLC using CH3COOC2H5: petroleum ether (1:2).
3.2.1. (Z)-2-(thiophen-3-ylmethylene)-1H-indene-1,3(2H)-dione (1)
◦
The precipitate was recrystallized from EtOH 95◦. Yield: 36.8%; m.p. 176–177 C; IR (Nujol)
1
(cm−1): 1720, 1680, 1600, 730; H-NMR (300 MHz, CDCl3):
δ (ppm) 7.78–7.81 (m, 2H, aromatics),
7.88 (d, 1H, aromatic, J = 5.5 Hz), 7.97–8.03 (m, 5H, aromatic and C=CH); 13C-NMR (75 MHz, CDCl3):
123.0, 123.1, 128.6, 134.9, 135.2, 136.3, 138.2, 141.7, 150.6, 173.6, 186.2, 187.3, 200.5, 203.5; Anal. C, H.
Calcd %: (C14H8O2S) C: 69.98, H: 3.36 Found %: C: 70.4, H: 3.31 [38–41].
3.2.2. (Z)-2-((3-methylthiophen-2-yl)methylene)-1H-indene-1,3(2H)-dione (2)
The residue was recrystallized from EtOH 95◦. Yield: 48.5%; m.p. 171–172 ◦C; IR (Nujol) (cm−1):
1720, 1680, 730; 1H-NMR (300 MHz, CDCl3):
δ (ppm) 2.60 (s, 3H, CH3), 7.07 (d, 1H, aromatic, J= 4.5 Hz),

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7,77–7,98 (m, 5H, aromatics), 8,14 (s, 1H, C=CH); 13C-NMR (75 MHz, CDCl3): 15.4, 103.9, 122.9, 123,
131, 132, 134, 134.7, 135, 136.8, 140.3, 151.9, 161.1, 189.7, 216.1; Anal. C, H. Calcd %: (C15H10O2S) C:
70.84, H: 3.96 Found %: C: 70.98, H: 3.91.
3.2.3. (Z)-2-((5-methylfuran-2-yl)methylene)-1H-indene-1,3(2H)-dione (3)
◦
The precipitate was recrystallized from EtOH 95◦. Yield: 54.4%; m.p. 130–136 C; IR (Nujol)
(cm−1): 1750, 1710, 1675; 1H-NMR (300 MHz, CDCl3):
δ (ppm) 2.37–2.47 (m, 3H, CH3), 6.38-6.39 (br,
1H, aromatic), 7,69–7,95 (m, 6H, aromatics and C=CH), 8,56 (s, 1H, C=CH); 13C-NMR (75 MHz, CDCl3):
14.5, 112.2, 122.8, 129.1, 134.6, 142.3, 148.0, 155.7, 167.7, 180.2, 182.9, 186.0, 195.0, 199.8, 201.1; LC/MS
(ESI): 237 [M]; Anal. C, H. Calcd %: (C15H10O3) C: 75.62, H: 4.23 Found%: C: 75.68, H: 4.50 [51].
3.2.4. 2-benzylidene-1H-indene-1,3(2H)-dione (4)
The residue was recrystallized from EtOH 95◦. Yield: 27.9%; m.p. 150–152 ◦C; IR (Nujol) (cm−1):
1730, 1680, 1580; 1H-NMR (300 MHz, CDCl3):
δ (ppm) 7.53–7.55 (m, 4H, aromatics), 7.82–7.85 (m, 2H,
aromatics), 7.92-8.02 (m, 3H, aromatics), 8.47 (d, 1H, C=CH, J = 7.3 Hz); 13C-NMR (75 MHz, CDCl3):
125.5, 126.0, 127.1, 127.3, 128.3, 128.5, 129.0, 130.2, 130.4, 134.0, 137.9, 138.2, 142.9, 193.3, 193.4 [42–44].
3.2.5. (Z)-2-(3-phenylallylidene)-1H-indene-1,3(2H)-dione (5)
The residue was purified with preparative TLC using CH3COOC2H5: petroleum ether (1:2).
Yield: 47%; m.p.165-167 ◦C; IR (Nujol) (cm−1): 1740, 1700, 1580; 1H-NMR (300 MHz, CDCl3):
δ (ppm)
6.48–6.99 (m, 2H, CH=CH), 7.33–8.10 (br, 9H, aromatics), 9.31 (d, 1H, J = 8.4 Hz); 13C-NMR (75 MHz,
CDCl3): 125.5, 125.8, 126.4, 126.5, 127.2, 127.3, 127.6, 127.8, 128.4, 128.5, 129.8, 137.0, 138.1, 138.2, 143.2,
144.0, 192.7, 192.8; Anal. C, H. Calcd %: (C18H12O2) C: 83.06, H: 4.65, Found %: C: 83, H: 4.60 [45–47].
3.2.6. (Z)-2-(3-(4-(dimethylamino)phenyl)allylidene)-1H-indene-1,3(2H)-dione (6)
The residue was recrystallized from EtOH 95◦/water. Yield: 26.5%; m.p. 85–87 ◦C; IR (Nujol)
(cm−1): 1710, 1670, 1620; 1H-NMR (300 MHz, CDCl3):
(ppm) 2.89–3.15 (m, 6H, N(CH3)2), 7.02 (d,1H,
δ
J = 8.24 Hz), 7.20 (d, 1H, J = 8.19 Hz), 7.35–7.90 (m, 8H, aromatics), 8.30 (d, 1H, J = 9.1 Hz); 13C-NMR
(75MHz, CDCl3): 28.0, 39.9, 112.1, 112.3, 123.4, 125.6, 125.9, 127.2, 127.3, 128.3, 128.4, 131.0, 133.0, 136.0,
138.1, 138.2, 145.0, 151.2, 192.6, 192.7; Anal. C, H, N. Calcd %: (C20H17NO2) C: 79.19, H: 5.65, N: 4.62
Found %: C: 79.31, H: 5.28, N: 4.22 [48].
3.2.7. (Z)-2-(3-phenoxybenzylidene)-1H-indene-1,3(2H)-dione (7)
The residue was recrystallized from EtOH 95◦. Yield: 13%; m.p. 143–145 ◦C; IR (Nujol) (cm−1):
1730, 1680, 1620, 1150; 1H-NMR (300MHz, CDCl3):
δ (ppm) 7.36–7.59 (m, 3H, aromatics), 7.81–8.22 (m,
5H, aromatics), 7.07–7.24 (m, 5H, aromatics), 9.96 (s, 1H, C=CH);13C-NMR (75 MHz, CDCl3): 114.0,
118.9, 119.0, 120.1, 124.5, 125.6, 125.8, 126.0, 127.2, 127.3, 129.5, 129.6, 130.5, 132.0, 135.3, 138.1, 138.2,
142.6, 154.4, 156.9, 192.9, 193.1; LC/MS (ESI): 327 [M + 1]; Anal. C, H. Calcd %: (C22H14O3) C: 80.97, H:
4.32, Found %: C: 81.29, H: 4.28.
3.2.8. (Z)-2-(4-((4-bromobenzyl)oxy)benzylidene)-1H-indene-1,3(2H)-dione (8)
The residue was recrystallized from EtOH 95◦. Yield: 32.45%; m.p. 222–224 ◦C; IR (Nujol) (cm−1):
1
1720, 1670, 1570, 1250; H-NMR (300 MHz, CDCl3):
δ (ppm) 5.13 (s, 2H, OCH2), 7.06–7.08 (m, 2H,
aromatics), 7.31–7.34 (m, 2H, aromatics), 7.53–7.56 (m, 2H, aromatics), 7.78–7.98 (m, 4H, aromatics),
7.99–8.00 (m, 2H, aromatics), 8.53–8.56 (m, 1H, C=CH); 13C-NMR (75 MHz, CDCl3): 70.5, 115.5, 115.6,
121.0, 125.7, 125.9, 127.3, 127.9, 127.9, 129.1, 129.3, 130.5, 131.5, 131.7, 131.9, 132.0, 134.3, 138.1, 138.2,
142.8, 159.4, 193.0, 193.2; LC/MS (ESI): 445 [M + K]+; Anal. C, H. Calcd %: (C23H15BrO3) C: 65.89, H:
3.61, Found %: C: 66.01, H: 3.58.

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3.2.9. (Z)-2-(3,5-di-tert-butyl-2-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (9)
The product was recrystallized from EtOH 95◦. Yield: 47%; m.p. 52–54 ◦C; IR (Nujol) (cm−1):
1
3500, 1750, 1710, 1600; H-NMR (300 MHz, CDCl3):
δ (ppm) 1.12–1.64 (m,18H,2(CH3)3), 7.34–7.60
(m, 4H, aromatics), 9.86 (s, 1H, C=CH), 11.64 (s, 1H, OH); 13C-NMR (75 MHz, CDCl3): 29.2, 31.0, 31.3,
31.5, 34.0, 34.2, 35.0, 35.2, 119.9, 120.9, 127.8, 131.8, 132.0, 132.4, 136.0, 137.0, 137.6, 138.0, 140.7, 141.0,
141.6, 159.0, 197.0, 197.3; Anal. C, H. Calcd %: (C24H26O3) C: 79.53, H: 7.23, Found %: C: 79.89, H: 7.61.
3.2.10. (Z)-2-(3,5-di-tert-butyl-4-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (10)
The precipitate was recrystallized from EtOH 95◦. Yield: 51.5%; m.p. 98–100 ◦C; IR (Nujol) (cm−1):
3350, 1710, 1680, 1625; 1H-NMR (300 MHz, CDCl3):
δ
(ppm) 1.35–1.78 (m, 18H, 2
×
C(CH3)3), 7.73–8.52
(m, 5H, aromatics and HC=CH), 9.85 (s, 1H, OH); 13C-NMR (75 MHz, CDCl3): 30.4, 30.5, 34.0, 34.1,
124.8, 125.0, 125.7, 125.9, 127.1, 127.3, 127.6, 132.3, 136.0, 136.2, 138.1, 138.2, 142.6, 153.0, 193.1, 193.4.;
Anal. C, H. Calcd %: (C24H26O3) C: 79.53, H: 7.23, Found %: C: 79.88, H: 7.55 [49,50].
3.2.11. (Z)-2-((1H-indol-5-yl)methylene)-1H-indene-1,3(2H)-dione (11)
The residue was recrystallized from EtOH 95◦. Yield: 51.5%; m.p. 223–225 ◦C; IR (Nujol) (cm−1):
3300, 1720, 1670, 1620; 1H-NMR (300 MHz, CDCl3):
δ (ppm) 6.74 (s, 1H, aromatic), 7.29–7.30 (m, 1H,
aromatic), 7.49–7.61 (m, 2H, aromatic), 7,78–7,80 (m, 2H, aromatic), 8.00–8,02 (m, 2H, aromatic),
8.47–8.50 (m, 2H, aromatic), 8.98 (s, 1H, C=CH); 13C-NMR (75 MHz, CDCl3): 100.8, 104.9, 105.9, 108.9,
111.0, 123.0, 129.0, 134.0, 134.9, 137.2, 144.0, 151.4, 158.5, 175.2, 180.6, 185.5, 186.6, 187.5; LC/MS (ESI):
313 [M+K]+; Anal. C, H, N. Calcd %: (C18H11NO2) C: 79.11, H: 4.06, N: 5.13 Found %: C: 79.22, H: 4.24,
N: 5.32.
3.2.12. (Z)-2-((1H-indol-3-yl)methylene)-1H-indene-1,3(2H)-dione (12)
◦
The precipitate was recrystallized from EtOH 95◦. Yield: 44.1%; m.p. 283–285 C; IR (Nujol)
(cm−1): 3250, 1720, 1650, 1590; 1H-NMR (300 MHz, CDCl3):
δ
(ppm) 6.89–6.99 (m, 1H), 7.38–8.03 (m, 8H
aromatics), 8,35(s, 1H, C=CH), 9.8 (s, 1H, NH); 13C-NMR (75 MHz, CDCl3): 126.2, 127.0, 134.0, 137.0,
139.8, 146.0, 148.0, 150.8, 156.0, 157.7, 166.0, 167.2, 168.9, 174.0, 180.3, 187.7, 189.3, 204.4; LC/MS (ESI):
311 [M+K]+ (272); Anal. C, H, N. Calcd %: (C18H11NO2) C: 79.11, H: 4.06, N: 5.13 Found %: C: 79.16,
H: 4.19, N: 5.04.
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 [55
]
(Table 2).
3.3.2. Determination of RMValues
Reversed phase TLC (RPTLC) 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).
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 Rf values were recorded and
used for the determination of RM using the equation RM = log [(1/Rf) − 1] [59,60,62] (Table 1).
3.4. Biological Assays
3.4.1. 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

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appropriate buffer with the tested compound diluted under sonication) was prepared from which
several dilutions were made with the appropriate buffer.
Determination of the Reducing Activity of the Stable Radical 1,1-Diphenyl-picrylhydrazyl (DPPH)
To a solution of DPPH in absolute ethanol an equal volume of the compounds with final
concentrations of 50, 100 and 200 µM from a stock solution of 10 mM (in 1% DMSO and ethanol) was
added. After 20 and 60 min the absorbance was recorded at 517 nm at room temperature [59
,60,62]
(Table 1).
ABTS+•—Decolorization Assay for Antioxidant Activity
The radical cation ABTS+• was generated from an ABTS stock solution in water (7 mM) which
was mixed with potassium persulfate (2.45 mM) and left in the dark at room temperature for 12–16 h
before use. The results are recorded after 1min of the mixing solutions at 734 nm and compared to the
appropriate standard inhibitor Trolox [60] (Table 3).
Inhibition of Linoleic Acid Peroxidation
The oxidation reaction was started with 2,20-Azobis(2-amidinopropane) dihydrochloride (AAPH)
as a free radical initiator. Linoleic acid sodium salt was oxidized at 234 nm to a conjugated diene
hydroperoxide resulting an increased absorption. Trolox was used as reference compound (93%)
(Table 3).
Measurement of Superoxide Anion Radical Scavenging Activity
The superoxide anion producing system was set up by mixing PMS, NADPH, and air oxygen.
The production of superoxide anion was estimated by the nitrobluetetrazole NBT method. The reaction
mixture containing compounds, 3 µM PMS, 78 µM NADPH, and 25 µM NBT in 19 µM phosphate
buffer pH 7.4 was incubated for 2 min at room temperature and the absorption was measured at 560 nm
against a blank containing PMS. The tested compounds were pre-incubated for 2 min before adding
NADPH. [59] Caffeic acid was used as an appropriate standard (76%).
Soybean Lipoxygenase Inhibition Study in Vitro
In vitro study was performed as reported previously by our group [58
–60,62]. Sodium linoleate
(0.1 mM) and 0.2 mL of enzyme solution (1/9 104 w/v in saline) were used for the incubation of the
×
tested derivatives. Sodium linoleate was converted to 13-hydroperoxylinoleic acid at 234 nm using
NDGA as reference compound [58–60,62]. The results are given in Table 1.
In Vitro Inhibition of Trypsin Induced Proteolysis
The Kunitz method was used after modification using bovine albumin was used as substrate
for trypsin. In this assay, first 200
µL of 0.075 mg/mL trypsin and 780
µL of phosphate buffer pH 7.6
including the tested compounds (20
µL in DMSO, final concentration 100
µM) were pre-incubated
at 37 ◦C for 20 min and then added 1 mL of albumin (stock solution 6 g/100 mL in phosphate buffer
0.1 M, pH 7.6) and incubated at 37 ◦C for 30 min. Secondly 1mLof 5% trichloroacetic acid was added
to the incubated solution to stop the enzyme’s reaction and allowed to set at room temperature for
1 h. Then the solution was filtered, and the absorption of the filtrate was measured at 280 nm using
salicylic acid as a reference compound [61] (Table 3).
In Vitro Inhibition of Thrombin
As a substrate tosyl-Gly-Pro-Arg-pNA acetate salt was used at 1 mM final concentration.
Compounds were dissolved at a final concentration of 0.1m in a Tris-buffer (0.05 M Tris, 0.154 M NaCl,
ethanol 5%, pH = 8.0). Three minutes after the addition of bovine thrombin (2.5 unit/mg), the reaction

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was ended by adding 1 mL acetic acid 50%. The absorption of the released p-nitro-aniline was measured
at 405 nm (Table 3) [71]. Warfarin and Inogatran were used as reference compounds.
3.4.2. Biological in Vivo Assays
Inhibition of the Carrageenin-Induced Edema
Edema was induced in the right hind paw of Fisher 344 rats (150–200 g) by the intradermal
injection of 0.1 mL 2% carrageenin in water. Both sexes were used while females pregnant were
excluded. Each group was composed of 6–15 animals. The animals, which have been bred in our
laboratory, were housed under standard conditions and received a diet of commercial food pellets
and water ad libitum during the maintenance but they were entirely fasted during the experiment
period. Our studies were in accordance with recognised guidelines on animal experimentation.
(guidelines for the care and use of laboratory animals published by the Greek Government 160/1991,
based on EU regulations 86/609). Rats were kept in the Centre of the School of Veterinary Medicine
(EL54 BIO42), Aristotle University of Thessaloniki, which is registered by the official state veterinary
authorities (presidential degree 56/2013, in harmonization with the European Directive 2010/63/EEC).
The experimental protocols were approved by the Animal Ethics Committee of the Prefecture of Central
Macedonia (no. 270079/2500). The tested compounds 0.01 mmol/kg body weight were suspended in
water, with a few drops of Tween 80 and ground in a mortar before use and were given intraperitoneally
simultaneously with the carrageenin injection. The rats were euthanized 3.5 h after carrageenin
injection. The difference between the weight of the injected and uninjected paws was calculated for
each animal. The change in paw weight was compared with that in control animals (treated with
water) and expressed as a percent inhibition of the edema CPE % values Table 1 [58,59]. Indomethacin
in 0.01 mmol/kg (47%). Values CPE % are the mean from two different experiments with a standard
error of the mean less than 10% (Table 3).
3.5. Computational Methods. Molecular Docking Studies on Soybean Lipoxygenase
UCSF Chimera was used for the visualization of the protein (PDB code: 3PZW) [72].
Water molecules were removed, missing residues were added with Modeler [73], hydrogen atoms
and AMBER99SB-ILDN charges were added, and the charge on iron was set to +2.0, with no restraint
applied to the iron atom and the ligands. Open Babel was used to generate and minimize ligand 3D
coordinates using the MMFF94 force field [74]. Ligand topologies and parameters were generated by
ACPYPE (AnteChamberPYthon Parser interfacE) [75] using Antechamber [76]. Energy minimizations
were carried out using the AMBER99SB-ILDN force field [77] with GROMACS 4.6. Docking was
performed with AutoDockVina (1.1.2) [78] applying a grid box of size 100 Å, 70 Å, 70 Åin X, Y,
Z dimensions. The generation of docking input files and the analysis of the docking results was
accomplished by UCSF Chimera. Docking was carried out with an exhaustiveness value of 10 and a
maximum output of 20 docking modes.
4. Conclusions
The designed and synthesized indandiones present multi-target activity against different targets
such as inflammation, inhibition of enzymes, scavenging of free radicals, and inhibition of lipid
peroxidation. In Table 4 the most significant indandiones in terms of activitiesare summarized.
Table 4. Comparison table with multi-target pleiotropic compounds.
Compd.
% LOX Inh. 100 µM
% IPr 100 µM
% TH 100 µM
AAPH% 100 µM
% CPE
7
8
9
67.3
82
79
96
41
46
85
98
100

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In the DPPH assay the novel derivatives showed medium antioxidant activity with small
differences dependent on time and concentration. The compounds moderately reduced at 100
the ABTS radical cation (ABTS•+) while they inhibited lipid peroxidation according to their structure.
Compound possessing the bromo-benzyloxy group seems to be the best trypsin and thrombin
inhibitor. Compound presents the better lipoxygenase inhibition while compound the best
µM
8
7
1
anti-inflammatory activity in carrageenin rat paw edema. Thus, they can be considered to be lead
compounds with multifunctional profile.
The pharmacokinetic prediction study offers useful results for the design and optimization of
the ADME profile of the new derivatives. Thus, indandionesobeying Lipinski’s rule of five are
expectedto be easily transported, diffused, absorbed being orally active with good oral bioavailability.
All structures can cross BBB and act in CNS whereas the docking supports that the extension scaffold
of compound 7 into the hydrophobic domain blocks approach of substrates to the active site and hence
prevents oxidation by soybean LOX.
Author Contributions: O.K. working in this research synthesized and biologically evaluated the new compounds
as a part of her master thesis, D.H.-L. designed and supervised the research and contributed in the writing, design,
synthesis, biological evaluation, and analysis of the data; E.P. contributed in the writing, biological evaluation,
docking studies, and analysis of data.
Funding: This research received no external funding.
Acknowledgments: E. Pontiki would like to thank A. Patsilinakos from the Department of Chemistry and
Drug Technologies, “Sapienza” University of Rome, Italy. We would like to thank Chr. Litina for creative
language proofreading.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AA: Arachidonic acid; AAPH: 2,20-azinobis(2-amidinopropane) hydrochloride; ABTS: 2,20-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid; ADME: Absorption, distribution, bioavailability, metabolism and
elimination; CHD: Coronary heart disease; Clog P:Theoretical calculated lipophilicity; COX: Cycloxygenase;
CPE: Carrageenin-induced rat paw edema; CYP: Cytochrome P450; DPPH: 1,1-diphenyl-picrylhydrazyl;
H(P)ETEs: 15-hydro(-pero)xy-eicosa-tetra-enoicacids; LOX: Lipoxygenase; LXs: Lipoxins; NADPH: reduced
nicotinamide adenine dinucleotide phosphate; NDGA: Nordihydroguaretic acid; NBT: nitroblue tetrazolium; PGs:
Prostaglandins; PMS: phenazine methosulfate; ROS: Reactive Oxygen Species; RPTLC: Reverse Phase Thin Layer
Chromatography; SMILES: simplified molecular-input line-entry system; SLOX: soybean lipoxygenase; TCA:
Trichloroacetic Acid.
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