Synthesis and pharmacochemistry of new pleiotropic pyrrolyl derivatives

Article abstract of DOI:10.3390/molecules200916354

Within the framework of our attempts to synthesize pleiotropic anti-inflammatory agents, we have synthesized some chalcones and their corresponding 3,4-pyrrolyl derivatives. Chalcones constitute a class of compounds with high biological impact. They are k

Full text of DOI:10.3390/molecules200916354

Molecules 2015, 20, 16354-16374; doi:10.3390/molecules200916354
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Pharmacochemistry of New Pleiotropic
Pyrrolyl Derivatives
Markella Konstantinidou, Alice Gkermani and Dimitra Hadjipavlou-Litina *
Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences,
Aristotle University of Thessaloniki, Thessaloniki 54124, Greece;
E-Mails: konsmark@hotmail.com (M.K.); germanialisa@gmail.com (A.G.)
* Author to whom correspondence should be addressed; E-Mail: hadjipav@pharm.auth.gr;
Tel.: +30-231-099-7627; Fax: +30-231-099-7679.
Academic Editor: Panayiotis A. Koutentis
Received: 14 July 2015 / Accepted: 3 September 2015 / Published: 10 September 2015
Abstract: Within the framework of our attempts to synthesize pleiotropic anti-inflammatory
agents, we have synthesized some chalcones and their corresponding 3,4-pyrrolyl derivatives.
Chalcones constitute a class of compounds with high biological impact. They are known for
a number of biological activities, including anti-inflammatory and free radical scavenging
activities. They inhibit several enzymes implicated in the inflammatory process, such as
lipoxygenase, cyclooxygenase (COX) and lysozymes. The synthesized pyrroles have been
studied for: (1) their in vitro inhibition of lipoxygenase; (2) their in vitro inhibition of COX; (3)
their in vitro inhibition of lipid peroxidation; (4) their interaction with the stable, N-centered,
free radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH); (5) their inhibition on interleukin-6
(IL-6); (6) their anti-proteolytic activity; and (7) their in vivo anti-inflammatory activity using
carrageenan-induced rat paw edema. Their physicochemical properties were determined
to explain the biological results. Lipophilicity was experimentally determined. 2i and 2v
were found to be promising multifunctional molecules with high antiproteolytic and
anti-inflammatory activities in combination with anti-interleukin-6 activity.
Keywords: chalcones; pyrroles; pleiotropic; antioxidant; anti-lipoxygenase;
anti-cyclooxygenase; anti-inflammatories

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1. Introduction
Inflammation is the natural response of the biological system to various stimuli. It is well established
that excessive chronic inflammation is linked to reactive oxygen species and oxidative stress [1].
Moreover, chronic inflammation leads to pathological disorders, such as atherosclerosis, rheumatoid
arthritis, neurogenerative diseases [2] and various types of cancer [3,4].
Upon appropriate stimulation of neutrophils, arachidonic acid is cleaved from membrane
phospholipids through the enzymatic activity of phospholipase A2, and it is metabolized by two
enzymes: cyclooxygenase (COX) and lipoxygenase leading to pro-inflammatory mediators, prostanoids
and leukotrienes, respectively [5]. Regarding cyclooxygenases, COX-3 is the third and most recently
discovered cyclooxygenase isozyme, the others being COX-1 and COX-2. COX-3 has been confirmed as
an alternate splice variant of COX-1. Its activity appears to be selectively inhibited by acetaminophen and
some other analgesic and antipyretic NSAIDs [6]. COX-1 is traditionally considered as a house-keeping
enzyme, and it is responsible for the production of prostanoids and proinflammatory prostaglandins,
implicated in homeostatic functions. COX-2 is the induced isoform, which catalyzes the production
of prostaglandins during the inflammatory response [7]. The differential expression of the two
isoforms, together with the fact that COX-1 is the main isoform expressed in the gastrointestinal track,
led to the design and synthesis of selective COX-2 inhibitors as potent anti-inflammatory agents that
would lack the gastro-intestinal side effects of traditional NSAIDs. Despite the initial success of selective
COX-2 inhibitors, recent studies showed that they were associated with cardiovascular side effects, and
some of them have been withdrawn from the world-wide market [8,9]. Furthermore, COX inhibition led
to an increase of LOXs metabolites [10]. Hence, another strategy, which was followed towards more
potent NSAIDs, was the design of dual inhibitors COX and LOX, which would have simultaneously
the advantage of blocking both enzymes in the arachidonic acid cascade [11].
COX-1 and COX-2 possess distinct roles in neuroinflammation. Taking into account the differential
expression of the two isoforms in central nervous system (CNS), COX-1 selective inhibition has lately
emerged as a new therapeutic target for CNS inflammatory diseases [12–14].
Nowadays, there is great interest in the use of pleiotropic drugs for the treatment of complex diseases,
in which more than one target is implicated. In the last decade, we have designed and synthesized several
potent lipoxygenase inhibitors, antioxidants and anti-inflammatories guided by our Quantitative–Structure
Activity Relationships (QSAR) results. Since inflammation is a complex phenomenon in which several
different factors are implicated, pleiotropic agents will offer additional benefits. In light of this, we
designed a number of 3,4-substituted pyrroles. The design principle was aimed at combining the synergistic
property of biological potent chalcones to get new heterocyclic entities: pyrrolyl derivatives that might
act as effective pleiotropic bioactive agents. Yet another objective of the study was to evaluate the effect
of steric and electronic parameters on anti-inflammatory activities and to optimize the activity through
systematic modification of the pyrrole 3,4-substituents.
Chalcones or 1,3-diaryl-2-propen-1-ones are α,β-unsaturated ketones and have a large number
of different biological activities. These compounds occur naturally or they are readily synthesized.
In the literature, derivatives bearing the chalcone scaffold present antibacterial [15], antifungal [16],
analgesic [17], anti-inflammatory [18], anticancer [19], antioxidant [20], immunosuppressive [21],
gastroprotective [22], antiviral [23] and antileishmanial [24] activities.

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Pyrroles exists both in natural and synthetic products, and their ability to form chelate complexes
with metals explains their significance in biological systems. Characteristic examples of tetra-pyrrole
structures are heme, chlorophyll and vitamin B12. Pyrrole derivatives with various actions are reported
in the literature: antimalarial [25], antimicrobial [26], anti-inflammatory [27], antioxidant [28],
HIV fusion inhibitors [29], anticancer [30–32], inhibitors of dipeptidyl peptidase IV [33], selective
potassium-competitive acid blockers [34] and inhibitors of HMG-CoA reductase [35]. Diarylpyrrolizines
have been investigated as COX and LOX inhibitors [36]. Pyrroles with known anti-inflammatory
activity, e.g., ketorolac and tolmetin, are characterized by an aroyl pyrrole moiety. A carbonyl group
in the vicinal diaryl substitution is the structural feature in the design of COX-2 inhibitors. Earlier
publications refer to the influence of vicinal aryl, aroyl and carbonyl unit combinations on enzyme
inhibitory activity within the synthesized pyrroles [36].
From a synthetic point of view, chalcones are useful synthons for heterocyclic compounds, since the
α,β-unsaturated moiety can be easily exploited towards the synthesis of heterocycles with numerous
reagents. With this in mind, we synthesized and used a number of chalcones, which presented antioxidant
and anti-lipoxygenase activities as precursors for the synthesis of our pyrrolyl derivatives.
2. Results and Discussion
2.1. Chemistry
The synthesis of chalcones was accomplished by a Claisen–Schmidt condensation between a
substituted aromatic ketone and a suitable substituted aromatic aldehyde [37], as depicted in Scheme 1.
Chalcones were used as precursors for the synthesis of aryl-aroyl-substituted pyrrolyl derivatives via a
van Leusen reaction [38] with tosylmethyl isocyanide (TosMIC) to give 3-aroyl-4-arylpyrroles in an
intermolecular cyclization. The ring carbons C2 and C5 originate from TosMIC, whereas C3 and C4
are derived from the chalcones that act as Michael acceptors. The used chalcones present a variation of
interesting preliminary anti-inflammatory biological activities and physicochemical properties, and
thus, we have chosen them as promising synthons (Table 1).
(a) MeOH, KOH 15% w/v, ultrasound bath, yields 57%–78%; (b) TosMIC, NaH, DMSO/Et2O, ice-bath,
yields 17%–98%.
Scheme 1. Derivatives via a van Leusen reaction.

Molecules 2015, 20
Table 1. Chemical structures, physicochemical and reaction data of chalcones 1i–1vi.
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#
No.
1i
Ar1
Ar2
Rf
m.p. (°C) Yield (%)
0.7
48–50
57
75
88
78
1ii [39]
1iii
0.6 113–115
0.8 141–143
1iv [40]
0.8
48–50
1v [41]
1vi [42]
0.7
0.5
48–50
48–50
86
66
Cl
# Dichloromethane.
The structural characteristics of the known synthesized chalcones, Compounds 1ii [39], 1iv [40], 1v [41],
1vi [42], were in agreement with the literature. The structure of the new 1i and 1iii compounds (with
yields from 57%–88%) have been identified by their physicochemical and spectroscopic data. The
final products were obtained in good yields (30%–96%), with the only exception being Compound 2iv,
which was obtained in a lower yield (17%). The final products were recrystallized from cold ethyl
1
acetate. IR, H-NMR, 13C-NMR and elemental analysis supported the proposed structures of the
synthesized pyrrolyl derivatives. The physical data of the synthesized compounds are given in detail in
the Experimental Section.
2.2. Physicochemical Studies
2.2.1. Determination of Lipophilicity
Since lipophilicity is a significant physicochemical property determining the absorption, distribution,
metabolism and excretion properties (ADME) of drugs, we attempted to determine experimentally the
lipophilicity as RM values with reverse-phase thin layer chromatography (RPTLC) [43]. Lipophilicity
was also theoretically calculated as Clog P values [44,45]. According to the calculated Clog P values,
the most lipophilic compounds were 2iii (6.37) and 2v (6.04). However, this observation was not supported
by the RM values, since Compound 2iii presented very low lipophilicity (−0.061). Furthermore, the
lipophilicity of Compound 2v could not be determined experimentally under the reported conditions.
Attempts to correlate Clog P and RM values failed. From our results (Table 2), it can be concluded that
RM values could not be used as a successful relative measure of the overall lipophilic/hydrophilic

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properties of these molecules. Tentatively, this can be attributed to the different nature of the hydrophilic
and lipophilic phases in the two systems. Clog P refers to n-octanol/water, whereas RM in this case
refers to methanol/water. Moreover, it is likely that pyrrole derivatives form hydrogen bonds within
the hydrophilic phase.
Table 2. Chemical structures, physicochemical and reaction data of pyrrole derivatives 2i–2vi.
#
No.
2i
Ar1
Ar2
Clog P
RM
Rf
m.p. (°C)
Yields (%)
5.90
0.091
0.7
165–166
49
2ii
2iii
2iv
5.05
6.37
5.26
0.073
0.6
219–220
233–235
270–272
96
43
17
−0.061 0.9
−0.149 0.8
2v
6.04
4.07
Nd
0.9
285–286
247–248
30
32
2vi
−0.685 0.7
Nd: not determined under these conditions; # chloroform/ethyl acetate 2:1.
2.2.2. Theoretical Calculation of Physicochemical Properties
The physicochemical properties were determined with the program Spartan v.5.1.3. (Wavefunction
Inc., Irvine, CA, USA) (Table 3) in the conformation of minimum energy. The values of overall molecular
refractivity (CMR) and molar refractivity of substituents (MR) (Table 4) were predicted with the
C-QSAR program of Biobyte [45].
Table 3. Theoretical calculation of the properties associated with energy and charge
distribution for pyrrole derivatives 2i–2vi.
No. Ε(ΗΟΜΟ) (eV) Ε(LUΜΟ) (eV) ΔΕ(ΗΟΜΟ-LUMO) (eV) SM2 (eV) Elpot_MIN (eV) Elpot_MAX (eV) Dipole (D)
2i
−7.95
−7.12
−7.78
−7.21
−7.29
−7.50
2.42
2.77
2.45
2.41
2.31
-1.91
10.37
9.89
10.23
9.62
9.6
2.12
2.02
0.75
2.53
2.43
9.85
−2.16
−2.30
−2.70
−2.26
−2.13
−2.12
3.12
2.86
3.16
2.95
3.01
1.63
6.06
4.33
6.37
4.13
4.99
4.14
2ii
2iii
2iv
2v
2vi
5.59

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Table 4. Theoretical calculation of molar refractivity, surface area and volume for pyrrole
derivatives 2i–2vi.
No CMR MR (Ar1) MR (Ar2) Surface Area (Å2)
Volume (Å3)
376.32
2i
9.723
5.250
4.860
6.491
4.274
4.274
3.614
2.395
2.586
2.395
2.586
3.077
2.586
381.77
351.86
430.45
345.60
363.97
317.63
2ii 9.902
2iii 11.266
2iv 9.328
2v 10.404
2vi 8.611
343.56
421.74
341.68
359.95
314.83
2.3. Biological Evaluation of Antioxidant and Anti-Inflammatory Activity
In the present study, six new pyrrole derivatives were synthesized as pleiotropic agents against several
targets implicated in inflammation. As starting materials, we used a number of promising chalcones,
which have been tested as antioxidants/anti-inflammatories.
Taking into account the multifactorial character of oxidative stress and inflammation, we decided to
evaluate in vitro the chalcones, as well as the pyrrolyl derivatives with regard to their antioxidant ability,
as well as to their ability to inhibit enzymes implicated in the inflammation process, such as: (i) LOX;
(ii) COX-2; (iii) interleukin-6 and (iv) trypsin.
Many non-steroidal anti-inflammatory drugs have been reported to act either as inhibitors of free
radical production or as radical scavengers [46]. Consequently, lipid peroxidation inhibitors [47] could
be expected to offer protection in inflammation and to lead to potentially effective agents.
We used several different assays to measure the antioxidant activity in vitro: (1) the interaction with the
stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH); (2) the non-enzymatic method of superoxide
radical scavenging activity; and (3) the inhibition of heme-dependent lipid peroxidation of linoleic acid.
The antioxidant ability of a compound should be tested in a variety of milieus to clarify the impact of
factors, such as solubility or steric hindrance, which may be of overriding importance in some cases.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a lipophilic, free, stable radical, which absorbs at 517 nm
(violet color). Upon reduction, decolorization is observed that is stoichiometric to the number of received
electrons and is representative of the antioxidant ability of compounds. The interaction/reducing activity
(RA) of the examined compounds with the stable free radical DPPH is shown in Table 5. For the sake
of comparison, RA % values for nordihydroguaiaretic acid (NDGA) were included in the table. This
interaction, which indicates their radical scavenging ability in an iron-free system, was measured at
100 and 1000 μM after 20 and 60 min, whereas the final concentration of DPPH was stable at 50 μΜ. With
the exception of 1i and 1ii, which interact with DPPH, all of the other chalcones did not have any effect.
Considering the pyrrolyl derivatives, the results, given in Table 6, indicate no action or very low
activity under these experimental conditions. It is possible that the tested compounds failed to interact
with the DPPH radical owing to steric factors. Compound 2ii presented remarkable reducing activity at
both concentrations, which was higher than that of the reference compound NDGA. Compound 2ii was
additionally tested at a 10 μΜ concentration and presented 35% and 40% interaction at 20 and 60 min,
respectively. By examining the structures of the compounds, it is likely that the antioxidant ability of
Compound 2ii correlated with the presence of the double bond, a feature that is missing from any other
pyrrolyl derivative.

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Table 5. Reducing ability measured as the interaction with DPPH (reducing activity %
(RA %)); inhibition of rat paw edema induced by carrageenan (CPE %).
RA % (100 μΜ) RA % (1000 μΜ)
CPE %
(0.01 mmol/kg Body Weight)
Comp.
20 min 60 min 20 min 60 min
1i
1ii
1iii
1iv
1v
43
31
na
na
na
na
81
2
5
na
42
na
na
na
na
96
41 c
69 d
86 d
40 d
44 d
13
na
na
na
na
83
38
na
na
na
na
96
1vi
NDGA
Indomethacin
42 c
−
53 d
na: no activity under the reported experimental conditions; c p < 0.05; d p < 0.01 performed with Student’s t-test.
Table 6. Reducing ability measured as interaction with DPPH (RA %); superoxide radical
•−
scavenging activity (O2 %); inhibition of heme-dependent lipid peroxidation of linoleic
acid (LP, IC50 values).
•−
RA % (100 μΜ) RA % (1000 μΜ)
O2
%
LP
Comp.
(100 μΜ) IC50 (μΜ)
20 min 60 min 20 min 60 min
2i
2ii *
2iii
2iv
2v
na
86
na
na
na
na
81
na
88
na
na
na
na
83
na
99
4
na
96
5
10
na
57
na
57
76
na
40
na
100
72
8
na
na
95.6
150
2vi
na
86
na
−
−
−
NDGA
Tolmetin
Caffeic acid
−
−
3
3
4
9
−
−
−
−
86.1
6
* Thirty five percent (20 min) and 40% (60 min) at 10 μΜ, na: no activity under the reported experimental conditions.
Superoxide anion radical is an active form of oxygen, and it is the product of the one-electron
reduction of molar oxygen. Herein, superoxide radicals were formed by mixing phenazine methosulfate
(PMS) with NADH and air-oxygen. The production of radicals was estimated with the nitroblue-
tetrazolium method (NBT). From the tested pyrroles, Compounds 2i and 2iii were equipotent (57%)
scavengers. The most active compounds were 2iv and 2vi with 76 and 87%, respectively. Worth noting
was that derivative 2vi presented comparable activity to caffeic acid, a known reference compound.
Ar2 substitution for the two most active derivatives was a phenyl group, whereas for Compound 2iv,
Ar1 was a naphthyl group, and for Compound 2vi, Ar1 was an indolyl group. It should to be mentioned
that Compound 2v (Ar1 = naphthyl, Ar2 = 4-chlorophenyl) was inactive under the reported experimental
conditions. It seems that lipophilicity does not significantly influence the superoxide anion scavenging
activity. In contrast, the dipole moment appeared to be important. The values of the dipole, as calculated
by the program Spartan v.5.1.3. (Wavefunction Inc.), were similar for Compounds 2iv and 2vi
(4.13 and 4.14 D, respectively), whereas for Compound 2v, the value of the dipole was 4.99 D. Hence,
the increase of dipole values correlated with a reduction in the ability of compounds to scavenge
superoxide anion radicals.

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The anti-lipid peroxidation activity was determined through the inhibition of heme-dependent lipid
peroxidation. The peroxidation of sodium linoleate is induced from a mixture of heme and hydrogen
peroxide, which produces free radicals from the side chains of amino acids. The assay is performed at
pH 7.4 with a phosphate buffer, and the products of peroxidation are identified with the thiobarbituric
acid method (TBA) [48]. In particular, malonic dialdehyde, a final product of lipid peroxidation, reacts
with TBA and forms a colored product, whose absorbance is measured at 535 nm. Pyrrolyl derivatives
presented moderate inhibitory activity, with the exception of 2i (IC50 = 40 μΜ). Compounds 2ii and
2vi were inactive under these experimental conditions. In general, pyrrolyl derivatives were less active
than the reference compound: caffeic acid. Compounds 2ii and 2vi with low Clog P values are weak
inhibitors of lipid peroxidation. It seems that the increase of volume of Ar1, measured as molar
refractivity MR (Ar1), enhances IC50 values and reduces the inhibitory activity (2i and 2iii; and 2ii, 2iv
and 2vi). For derivatives 2iv and 2v, the presence of an electron-acceptor, e.g., a chloro-substituent on
the phenyl ring (Ar2), diminishes the anti-lipid peroxidation activity.
Taking into account the multifactorial character of oxidative stress and inflammation, the pyrrolyl
derivatives were evaluated for their anti-inflammatory activity. The following biological assays were
performed in vitro: (1) inhibition of soybean lipoxygenase; (2) inhibition of COX-1; (3) inhibition
of COX-2; (4) inhibition of IL-6 and (5) inhibition of the proteolytic activity of trypsin. For the
anti-inflammatory in vivo assay, we used rat paw edema induced by carrageenan.
Lipoxygenase catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes.
LTB4 generation is important in the pathogenesis of neutrophil-mediated inflammatory diseases [49]
with a marked relation to the severity of cardiovascular diseases, asthma and cancer. We evaluated the
synthesized pyrroles for their ability to inhibit soybean LOX by the UV absorbance-based enzyme
assay [50]. All pyrroles presented very low or no action under these experimental conditions (Table 7).
The main reason for the lack of inhibitory activity was attributed to be the bulk of molecules. We
succeeded at determining the IC50 value only for Compound 2i. The inhibition of COX-2, as a peroxidase,
was determined by a colorimetric assay with the oxidation of N,N,N′,N′-tetramethyl-p-phenylenediamine
(TMPD). Compounds 2iii, 2iv and 2vi were inactive under the experimental conditions (Table 7).
The most potent COX-2 inhibitor at a 100 μΜ concentration was Compound 2v (Ar1 = naphthyl,
Ar2 = p-chlorophenyl), followed by 2i. The structurally-related 2iv (Ar1 = naphthyl, Ar2 = phenyl) was
inactive. It is possible that electron-acceptor substituents influence the activity. Indomethacin, which
was included in the study for the sake of comparison, presented 95% inhibition at 100 μΜ.
The assay used for the inhibition of IL-6 is based on an immunometric double antibody method
(sandwich technique). In this assay, Compounds 2i and 2v were studied, which were previously proven
to be the most potent COX-2 inhibitors. Both compounds gave satisfactory biological responses, both
in high and in low concentrations of IL-6 (Table 8). It should to be mentioned that both compounds
have been tested at 10 μΜ, a concentration significantly lower than the concentration used in the
COX-2 assay. Both Compounds 2i and 2v exhibited better results compared to indomethacin.
Preliminary results (data not given) support that Compound 2v is a double COX-1 (75% at
concentration of 100 μΜ) and COX-2 inhibitor, whereas Compound 2iii is a selective COX-1 inhibitor
(75% at concentration of 100 μΜ), without any activity against COX-2. More experiments are in
progress to delineate accurately the inhibition profile.

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Table 7. Inhibition of soybean lipoxygenase (LOX IC50 μΜ or % at 100 μM); percent
inhibition of cycloxygenase-2 (COX-2 % at 100 μΜ); inhibition of trypsin (TP IC50 μΜ);
inhibition of rat paw edema induced by carrageenan (CPE %).
LOX IC50 (μΜ)/%
COX-2 %
TP IC50 CPE % (0.01 mmol/kg Body Weight)
Comp.
(100 μM)
1000 μΜ
na
(100 μM)
(μΜ)
60
na
9
or ED50 mmol/kg Body Weight
2i
2ii
67.2
47
na
na
81
nt
0.061 c
89% d
0.0525(89%) c
0.064 (75%) c
0.063 (78%) c
nt
2iii
na
2iv
10%
65
8
2v
32%
NDGA
Tolmetin
Indomethacin
Salicylic acid
43 μΜ
nt
190 μΜ
nt
nt
76%
53% d
−
95
-
nt
−
100
nt
c
d
na: no activity under these experimental conditions, nt: not tested; p < 0.05; p < 0.01 performed with
Student’s t-test.
Table 8. Percent inhibition of interleukin-6 (IL-6).
IL-6
pg/mL
1500
750.0
375.0
187.5
93.7
Comp. 2i (10 μΜ)
Comp. 2v (10 μΜ)
IMA (10 μΜ)
% Inhibition
% Inhibition
% Inhibition
99
98
99
99
na
na
na
100
99
99
na
100
na
na
na
na
na
na
na
46.8
na
23.4
na
na: no activity under these experimental conditions; IMA: indomethacin.
Trypsin is an enzyme with an active role in inflammation. Thus, we studied the anti-proteolytic
ability of the pyrrole derivatives, using salicylic acid as a reference compound. Compounds 2i and 2iv
presented IC50 values of 60 and 65 μΜ, respectively, whereas Compounds 2iii and 2v were much more
potent, with IC50 values of 9 and 8 μΜ, respectively. Comparing Compounds 2iv and 2v, the insertion
of an electron-acceptor substituent (Cl) on the phenyl ring (substituent Ar2) and the simultaneous
increase of the volume of substituent Ar2 (expressed as molar refractivity) led to an enhancement of
the anti-proteolytic activity.
The pyrrolyl derivatives, as well as their precursor chalcones were tested in vivo for the inhibition of
rat paw edema induced by carrageenan. Chalcones 1iii and 1ii highly reduced the rat paw edema at
0.01 mmol/kg body weight, whereas 1i, 1iv, 1v and 1vi presented lower and almost equal response. It
should to be noted that the most potent chalcones 1ii and 1iii are the precursors of the two most active
pyrrolyl derivatives. All pyrrolyl derivatives at 0.01 mmol/kg body weight showed anti-inflammatory
activity comparable or higher than indomethacin, which was the reference compound, and tolmetin,
which was included in the study as a pyrrolyl NSAID compound. Compound 2ii showed an inhibition
of 89%. The two naphthyl-substituted derivatives 2iv and 2v presented almost equivalent inhibition

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(75% and 78%, respectively), indicating that the bulky substituents significantly influence the biological
activity. For Compounds 2i, 2iii, 2iv and 2v, we determined their ED50 values. Considering these
results, 2iii with ED50 = 0.052 mmol/kg body weight was the most potent anti-inflammatory agent.
The finding supports the primary idea of this design. Two mono-substituted pyrroles (Scheme 2),
2-benzoylpyrrole and 3-benzoylpyrrole, which, at a dose of 0.44 mmol/kg, showed 34% and 43%
inhibition of carrageenan-induced rat paw edema, respectively, were previously studied for their ability
to inhibit carrageenan-induced edema [51].
N
H
O
2-benzoylpyrrole
3-benzoylpyrrole
Scheme 2. Structures of mono-substituted pyrrole derivatives.
The insertion of the substituent in position 3 rather than 2 favors the inhibitory activity. Although
the role of the physicochemical properties is not entirely clear for the biological results, a comparison
of in vitro and in vivo results underlines a correlation between them and, in particular, among the
results from the inhibition of COX-2, trypsin, IL-6 and antioxidant activity. Comparing the data from
mono- and bis-substituted pyrrolyl derivatives, a second bulky substituent in position 4 significantly
improved the anti-inflammatory activity in vivo. The absence of a free carboxylic group in the
synthesized derivatives is considered to be important owing to its implication in adverse side effects.
Such adverse side effects may occur with “classic” non-steroid anti-inflammatory drugs, in which
tolmetin belongs.
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, Merck KGaA, Darmstadt, Germany, Fluka Sigma-Aldrich
Laborchemikalien GmbH, Hannover, Germany, Alfa Aesar, Karlsruhe, Germany and Sigma, St. Louis,
MO, USA).
For the biological assays, soybean lipoxygenase, pancreatic bovine trypsin, sodium linoleate, free
radical DPPH, PMS, NADH, nitroblue tetrazolium, caffeic acid, heme, H2O2, trichloroacetic acid, acetic
acid, salicylic acid (SA), nordihydroguaiaretic acid (NDGA), N,N,N′,N′-tetramethyl-p-phenylenediamine
(TMPD) and arachidonic acid were obtained from Sigma Chemical, Co. (St. Louis, MO, USA).
Carrageenan, type K, was commercially available. Cycloxygenase-2 (COX-2) and interleukin-6 (IL-6)
EIA Kit, Item No. 583371 were purchased from Cayman. Trolox was obtained from Aldrich Chemical Co.
(Milwaukee, WI, USA). All starting materials were obtained from commercial sources, and they were
used without further purification.

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Melting points (uncorrected) were determined on an MEL-Temp II (Lab Devices, Holliston,
MA, USA). For the in vitro tests, UV-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 a Perkin-Elmer 597 spectrophotometer
(Perkin-Elmer Corporation Ltd.). The 1H-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 in CDCl3 or DMSO solutions with tetramethylsilane as
the internal reference, unless otherwise stated. Chemical shifts are expressed in δ (ppm) and coupling
constants J in Hz. Mass spectra were determined on a LC-MS 2010 EV Shimadzu (Shimadzu Scientific
Instruments, Inc., Columbia, MA, USA), using MeOH as a 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.). 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 (PLC), silica gel 60 F254, plates 2 mm, Merck KGaA ICH078057, were
used. For the experimental determination of the lipophilicity reverse phase thin layer chromatography
(RPTLC), TLC-silica gel 60 F254 DC Kieselgel, Merck (20 × 20 cm) plates were used.
3.2. Chemistry General Procedure
3.2.1. Chemistry General Procedure for Chalcones
A modified Claisen–Schmidt condensation was followed. Equimolar amounts of a suitable
substituted acetophenone (10 mmol) and of a suitable substituted aromatic aldehyde (10 mmol) were
used. Each reagent was dissolved in methanol, and the total volume of the used solvent was 20 mL.
The methanolic solution of the substituted acetophenone was slowly added under stirring in the
methanolic solution of the aldehyde, followed by the addition of 5 mL of potassium hydroxide
(15% w/v). The reaction was carried out in an ultrasound bath and has been monitored by thin-layer
chromatography. The reaction mixture was cooled by an ice bath and was acidified with an aqueous
solution of hydrochloric acid (10%). The precipitate was filtered off and washed repeatedly with water.
In case no precipitate was formed, the mixture was extracted with 3 × 40 mL of dichloromethane or
chloroform; the organic layer was collected, dried over sodium sulfate, evaporated to dryness and was
recrystallized with methanol. Chalcones 1ii, 1iv–vi are referred to in the literature.
3-(3-Phenoxyphenyl)-1-(thien-2-yl)prop-2-en-1-one (1i): the modified general method was followed
using 3-phenoxybenzaldehyde and 2-acetylthiophene. IR (Nujol) (ν cm−1): 3100m (Ar-H), 1650s
1
(C=O), 1580m (C=C). H-NMR (300 MHz, CDCl3): δ (ppm) 7.03–7.44 (m, 10H, 9 × Ph H,
13
1 × COCH=, 1 × thienyl Η), 7.86–7.68 (m, 4H, 3 × thienyl H and 1 × CH=). C-NMR (75.5 MHz,
CDCl3): δ (ppm) 172.9, 154.6, 153.2, 145.4, 143.3, 136.6, 134.0, 131.9, 130.3, 129.9, 128.2, 123.7,
123.5, 122.3, 120.8, 119.1, 118.1, 77.4, 77.0, 76.6. Anal. C, H. (C19H14O2S); Calc. % C: 74.18, H:
4.60; found % C: 74.17, H: 4.61.

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5-[4-(Dimethylamino)phenyl]-1-phenylpenta-2,4-dien-1-one (1ii): The modified synthetic method was
used. The analytical data, as well as the spectral analysis were in agreement with those given in
the literature [39].
3-{4-[(4-Bromobenzyl)oxy]phenyl}-1-(thien-2-yl)prop-2-en-1-one (1iii): the modified general method
was followed using 4-[(4-bromobenzyl)oxy]benzaldehyde and 2-acetylthiophene. IR (Nujol) (ν cm−1):
3100m (Ar-H), 1680s (C=O), 1580m (C=C). 1H-NMR (300 MHz, CDCl3): δ (ppm) 5.07-5.11 (br, 2H,
OCH2), 6.97–7.32 (m, 6H, 4 × Ph H, 1 × γ-thienyl-Η, 1 × H COCH=), 7.51–7.85 (m, 7H, 4 × Ph H,
1 × β-thienyl-H, 1 × δ-thienyl-H, 1 × H CH=CH). 13C-NMR: (75.5 MHz, CDCl3): δ (ppm) 180.7,
160.6, 157.7, 143.7, 142.2, 135.5, 133.5, 131.8, 131.5, 130.3, 129.0, 128.5, 128.2, 128.0, 127.3, 123.6,
119.7, 115.3, 109.3. [Μ+] 399 (11), 111 (46), 83 (7); Anal. C, H. (C20H15O2BrS); Calc. % C: 60.15, H:
3.75; found % C: 59.81, H: 3.85.
3-(Naphth-1-yl)-1-phenylprop-2-en-1-one (1iv): The modified synthetic method was used. The
analytical data, as well as the spectral analysis were in agreement with those given in literature [40].
3-(Naphth-1-yl)-1-(4-chloro)phenylprop-2-en-1-one (1v): The modified synthetic method was used. The
analytical data, as well as the spectral analysis were in agreement with those given in the literature [41].
3-(1H-Indol-5-yl)-1-phenylprop-2-en-1-one (1vi): The modified synthetic method was used. The
analytical data, as well as the spectral analysis were in agreement with those given in the literature [42].
3.2.2. General Method for the Synthesis of Pyrrolyl Derivatives
The reaction was carried out according to a modified literature method [38]. All glassware and all
used solvents were dry. Equimolar quantities of the appropriate chalcones and TosMIC were dissolved
in a mixture of dry DMSO and dry diethyl ether (1:2.5). The reaction mixture was added dropwise
under stirring into a suspension of sodium hydride (30 equiv. of the chalcone) in dry diethyl ether,
to keep the hydrogen formation under control. The reaction was carried out in an ice bath for 24 h.
In continuation, the mixture was poured in 500 mL ice water. The pH of the solution was strongly basic.
The organic phase was separated. The aquatic phase was extracted three times with ethyl acetate, dried
over sodium sulfate and concentrated under vacuum. The residue was treated with cold methanol and
recrystallized (cold ethyl acetate) to give the pure product.
[4-(3-Phenoxyphenyl)-1H-pyrrol-3-yl](thien-2-yl)methanone (2i): The above mentioned general method
was followed using chalcone 1i. IR (Nujol) (ν cm−1): 3350m (N-H), 1640s (C=O), 1580m (C=C).
1H-NMR (300 MHz, CDCl3): δ (ppm) 8.00–8.07 (m, 1H, thienyl H), 7.58–7.62 (m, 1H, 1 × thienyl H),
7.37–7.39 (m, 3H, 3 × Ph H), 7.29–7.32 (m, 1H, 1 × Ph H), 7.03–7.27 (m, 6H, 3 × Ph H, 2 × pyrrolyl H,
13
1 × thienyl H), 6.84–6.99 (m, 3H, 3 × Ph H). C-NMR (75.5 MHz, CDCl3): δ (ppm) 165.0, 133.1,
132.6, 129.6, 129.0, 127.5, 125.0, 123.9, 122.9, 119.3, 118.6, 118.5, 117.0. Anal. C, H, N. (C21H15NO2S);
Calc. % C: 72.39, H: 4.34, N: 4.04; found % C: 72.22, H: 4.57, N: 3.73.
(E)-{4-[4-(Dimethylamino)styryl]-1H-pyrrol-3-yl}(phenyl)methanone (2ii): The general method was
followed using chalcone 1ii. IR (Nujol) (ν cm−1): 3295m (N-H), 1681s (C=O), 1614m (C=C). 1H-NMR

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(300 MHz, CDCl3): δ (ppm) 7.80–7.82 (m, 2H, 2 × Ph H), 7.37–7.58 (m, 5H, 5 × Ph H), 7.10–7.12
(m, 2H, 2 × vinyl H, 6.75–6.89 (m, 4H, 2 × Ph H, 2 × pyrrolyl H), 2.96 (s, 6H, 2 × methyl H). 13C-NMR
(75.5 MHz, CDCl3): δ (ppm) 164.0, 131.1, 129.0, 128.0, 127.8, 127.3, 127.0, 116.0, 113.0, 22.0. Anal.
C, H, N. (C21H20N2O); Calc. % C: 79.72, H: 6.37, N: 8.85; found % C: 80.05, H: 6.37, N: 8.73.
(4-{4-[(4-Bromobenzyl)oxy]phenyl}-1H-pyrrol-3-yl)(thien-2-yl)methanone (2iii): The general method
was followed using chalcone 1iii. IR (Nujol) (ν cm−1): 3500–3450br s (N-H), 1710s (C=O), 1580m
1
(C=C). H-NMR (300 MHz, CDCl3): δ (ppm) 8.51–8.53 (br, 1H, 1 × thienyl Η), 7.58–7.68 (m, 3H,
2 × Ph H, 1 × thienyl H), 7.49–7.52 (m, 2H, 2 × Ph H), 7.29–7.40 (m, 3H, 2 × Ph H, 1 × thienyl H),
7.07–7.11 (br, 1H, 1 × pyrrolyl H), 6.88–7.05 (m, 4H, 3 × Ph H, 1 × pyrrolyl H), 5.00 (s, 2H,
13
2 × CH2O). C-NMR (75.5 MHz, CDCl3): δ (ppm) 183.0, 133.0, 132.0, 131.0, 130.0, 129.0, 127.0,
125.0, 118.0, 111.0, 77.0. Anal. C, H, N. (C22H16BrNO2S); Calc. % C: 60.28, H: 3.68, N: 3.19; found
% C: 60.15, H: 3.93, N: 3.10.
[4-(Naphth-1-yl)-1H-pyrrol-3-yl](phenyl)methanone (2iv): The general method was followed using
1
chalcone 1iv. IR (Nujol) (ν cm−1): 3450s (N-H), 1680s (C=O), 1620m (C=C). H-NMR (300 MHz,
CDCl3/DMSO-d6): δ (ppm) 11.4 (s, 1H, 1 × pyrrolyl H), 8.93–9.10 (m, 2H, 2 × naphthyl H), 8.06–8.26
(m, 3H, 3 × naphthyl H), 7.90–7.93 (m, 2H, 2 × Ph H), 7.79–7.81 (m, 1H, 1 × Ph H), 7.69–7.75
(m, 2H, 2 × Ph H), 7.49–7.59 (m, 2H, 2 × naphthyl H), 7.13–7.21 (br, 1H, 1 × pyrrolyl H), 6.90–7.02
(m, 1H, 1 × pyrrolyl H). 13C-NMR (75.5 MHz, CDCl3/DMSO-d6): δ (ppm) 173.0, 130.5, 128.0, 127.3,
127.2, 126.8, 126.4, 126.0, 125.7, 125.0, 124.7, 124.6, 119.7. Anal. C, H, N. (C21H15NO); Calc. % C:
75.22, H: 4.32, N: 4.62; found % C: 75.49, H: 4.63, N: 4.58.
(4-Chlorophenyl)[4-(naphth-1-yl)-1H-pyrrol-3-yl]methanone (2v): The general method was followed
1
using chalcone 1v. IR (Nujol) (ν cm−1): 3200w (N-H), 3090m (Ar-H), 1700s (C=O). H-NMR
(300 MHz, CDCl3): δ (ppm) 8.76–8.96 (m, 2H, 2 × naphthyl H), 8.22–8.28 (m, 1H, 1 × naphthyl H),
8.06–8.10 (br, 1H, 1 × naphthyl H), 7.86–7.96 (m, 3H, 2 × Ph H, 1 × naphthyl H), 7.6–7.53 (m, 4H,
2 × Ph H, 2 × naphthyl H), 7.08–7.11 (m, 1H, 1 × pyrrolyl H), 6.95-6.99 (m, 1H, 1 × pyrrolyl H).
13C-NMR (75.5 MHz, CDCl3): δ (ppm) 174.0, 145.0, 141.6, 128.0, 127.0, 124.8, 122.8, 112.0, 107.4,
104.7, 100.4. Anal. C, H, N. (C21H14ClNO); Calc. % C: 74.64, H: 3.96, N: 4.58; found % C: 74.82, H:
4.08, N: 4.19.
[4-(1H-Indol-5-yl)-1H-pyrrol-3-yl](phenyl)methanone (2vi): The general method was followed using
1
chalcone 1vi. IR (Nujol) (ν cm−1): 3200m (N-H), 1710s (C=O). H-NMR (300 MHz, DMSO-d6):
δ (ppm) 10.97 (s, 1H, 1 × indolyl H), 10.22 (s, 1H, 1 × pyrrolyl H), 7.99–8.03 (br s, 1H, 1 × indolyl H),
7.56–7.73 (m, 7H, 7 × indolyl/Ph H), 7.06–7.24 (m, 3H, 2 × pyrrolyl H, 1 × indolyl H), 6.80–6.81
(br s, 1H, 1 × indolyl H). 13C-NMR (75.5 MHz, DMSO-d6): δ (ppm) 192.0, 142.0, 135.0, 131.0, 129.0,
128.0, 127.0, 126.0, 125.0, 124.0, 122.0, 120.0, 117.0, 114.0, 110.0. Anal. C, H, N. (C19H14N2O);
Calc. % C: 79.70, H: 4.93, N: 4.89; found %: C: 79.73, H: 5.19, N: 4.85.

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3.3. Physicochemical Studies
Reverse-phase thin layer chromatography (RPTLC) was used for the determination of RM values.
Silica gel plates were saturated with 5% v/v liquid paraffin in petroleum ether, and the mobile phase
was methanol/water (75/25 v/v) with the addition of ammonia drops. Spots were detected under UV
light. RM values were determined by the corresponding Rf values (from five measurements for each
compound) using the equation [43]:
RM = log[(1/Rf) − 1]
(1)
Lipophilicity was theoretically calculated as Clog P values using the program of Biobyte
Corporation [45]. For the calculation of physicochemical properties of the pyrrolyl derivatives, the
Spartan program v.5.1.3 (Wavefunction Inc.) was used. Briefly, for each compound: (1) the structure
was optimized with the quantum mechanics semi-empirical method AM1 (Austin Model 1); and
(2) conformers were randomly reproduced with the Monte Carlo method by selecting all possible
bonds that could rotate and by applying the dynamic field of molecular mechanics, Merck (MMFF94).
Calculations were performed on the conformer with the minimum energy using quantum mechanics
methods in level 6-31G* (and for compounds bearing bromium: 3-21G*) and semi-empirical in
level AM1-SM2.
The C-QSAR program [45] was also used for the calculation of the values of overall molar
refractivity (CMR) and of the molar refractivity of substituents (MR).
3.4. Biological Assays
For the in vitro biological assays, the tested compounds, as stock solutions (10 mM), were dissolved
in DMSO. Final solutions of several concentrations were prepared for the assays given below.
The results were the mean of at least two different experiments, and the standard deviation did not
exceed 10%.
3.4.1. Biological Assays in Vitro
Interaction with the Stable Free Radical 2,2-Diphenyl-1-picrylhydrazyl
To a solution of DPPH in absolute ethanol (50 μΜ final concentration), the appropriate volume
of the compounds (final concentrations of 100 and 1000 μΜ) dissolved in DMSO was added. The
absorbance was measured at 517 nm after 20 and 60 min at room temperature [52].
Superoxide Radical Scavenging Activity
The superoxide anion radicals were produced non-enzymatically by mixing phenazine methosulfate
(PMS) with NADH and air-oxygen. The production of superoxide radicals was estimated with the
nitroblue-tetrazolium (NBT) method with 3 μΜ PMS, 78 μΜ NADH and 25 μΜ NBT in 19 μΜ
phosphate buffer pH 7.4. The absorbance was measured at 560 nm at room temperature. The tested
compounds were added in the mixture 2 min before the addition of NADH [52].

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Inhibition of Heme-Dependent Lipid Peroxidation of Linoleic Acid
Firstly, 0.4 mL of heme (50 μΜ), 0.25 mL of sodium linoleate (400 μΜ), 0.1 mL of the solutions of
the tested compounds (concentrations varying from 10–100 μΜ in DMSO) and 0.25 mL of H2O2 (500 μΜ)
were incubated for 10 min in 37 °C in a phosphate buffer (KH2PO4–KOH 50 mM, pH 7.4). The
peroxidation products were identified with the reaction of thiobarbituric acid, by adding successively
0.5 mL of 40% trichloroacetic acid (TCA) and 0.5 mL of 2% of thiobarbituric acid (TBA) [52]. The
addition was followed by stirring and heating at 90 °C for 20 min, and then, 1 mL of acetic acid and
1 mL of chloroform were added and the absorbance measured at 535 nm. IC50 values were determined.
Inhibition of Soybean Lipoxygenase
The tested compounds were dissolved in DMSO, and the final concentration was 100 μΜ. The
compounds were incubated at room temperature with sodium linoleate as a substrate (0.1 mL) in a
buffer solution of Tris:HCl (pH 9.00) and 0.2 mL of a solution of soybean lipoxygenase [52]. The
conversion of linoleic acid into 13-hydroperoxylinoleic acid was measured at 234 nm and was
compared to the reference compound nordihydroguaiaretic acid (NDGA).
Inhibition of Cycloxygenase-2 (COX-2)
COX-2 inhibitory activity was determined by using arachidonic acid (AA) as a substrate in buffer
(0.1 M Tris pH 8.5) and TMPD as a co-substrate. Ten microliters of the DMSO solution of the tested
compounds (final concentration 100 μM) were added in a buffer (Tris-HCl pH 8.5), followed by the
addition of 0.75 μΜ heme, 80 μΜ arachidonic acid, COX-2 and 128 μΜ TMPD. The mixture
was incubated for 5 min at 37 °C, and after vigorous shaking, the absorbance was measured at
590 nm [52,53].
Inhibition of Interleukin-6 (IL-6) (Interleukin-6 (Mouse))
The method was based on an immunometric double antibody sandwich technique. Each well of the
microwell plate was coated with a specific for mouse IL-6 rat monoclonal antibody. Tested compounds
at 10 μΜ and standards were incubated in the wells, and after rinsing, a second monoclonal antibody
was added, which was conjugated with biotin and detected the captured IL-6. The recognition of
double antibody sandwiches was done with HRP-conjugated streptavidin. The concentration of IL-6
was determined by measuring the enzymatic activity of HRP using the chromogenic substrate TMB
(3,3′,5,5′-tetramethylbenzidine). The absorbance of the reaction product was measured at 450 nm, and
the intensity of the color was directly proportional to the amount of bound HRP-streptavidin conjugate,
which was proportional to the concentration of IL-6.
Inhibition of the Proteolytic Activity of Trypsin
The tested compounds (concentrations varying from 10 μΜ–100 μΜ in DMSO) were dissolved in a
phosphate buffer (0.1 M K2HPO4–NaH2PO4·2H2O, pH = 7.6), and 0.2 mL of trypsin 0.075 mg/mL
were added. The mixture was incubated at room temperature for 20 min, and then, 1 mL of bovine

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albumin (6 g/100 mL) was added. The mixture was incubated at 37 °C for 30 min. The reaction was
stopped with the addition of 1 mL of trichloroacetic acid (5% w/w) Separation of the phases followed,
with measurement of the supernatants at 280 nm at room temperature [54]. IC50 values were determined.
3.4.2. Biological Assay in Vivo
Inhibition of Rat Paw Edema Induced by Carrageenan
Edema was induced in the right hind paw of Fischer 344 rats (150–200 g). In the study, both males
and females were included, with the exemption of pregnant females. 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 maintenance. One hour before the onset of the experiment, food
and water were removed. The tested compounds (dose 0.01 mmol/kg body weight) were diluted in
water with a few drops of Tween 80, and they were administered intraperitoneally simultaneously with
the intradermal injection of 0.1 mL 2% of carrageenan in water. The animals were euthanatized 3.5 h
after the carrageenan administration. The difference between the weight of injected and uninjected
paws was calculated for each animal and was compared to control animals, which were treated with
water [52]. Indomethacin was used as a reference compound in 0.01 mmol/kg. The results were
expressed as the percent inhibition of the edema (CPE %).
4. Conclusions
In the present study, six pyrrolyl derivatives were designed and synthesized using chalcones as the
starting material. Lipophilicity as RM values was determined, and physicochemical properties were
calculated. The biological activity of the pyrrolyl derivatives was evaluated in antioxidant and
anti-inflammatory assays. Compound 2iii was found to be a potent selective COX-1 inhibitor, and as
mentioned in recent literature, such compounds could be useful in neurodegenerative diseases,
including Parkinson’s and Alzheimer’s disease, in which the production of prostaglandins in neurons
and neuroglia is increased. Compound 2v presented satisfactory anti-inflammatory activity, inhibiting
COX-2, IL-6 and trypsin. The presence of an electron-acceptor substituent (Cl) on the phenyl ring on
Ar2 and the increase of the volume of Ar2 simultaneously improve the anti-proteolytic activity
significantly. A carbonyl group into the vicinal diaryl substitution in 3,4-positions of the pyrrolyl ring
was found to be the structural feature for the pharmacophore. The presented combination of biological
activities of 2v support that this pyrrolyl derivative is a promising pleiotropic bioactive molecule.
Additional studies of the physicochemical properties could lead to useful conclusions for the future
design of compounds with the ability to interact with multiple biological targets. Further investigation
is in progress.
Acknowledgments
We thank E. Evgenidou, Department of Chemistry, Aristotle University of Thessaloniki, for
providing the MS spectra. We also thank Biobyte and A. Leo for free use of C-QSAR and support.

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Author Contributions
M.K. performed her MSc thesis working on this research. D.H.-L. supervised the research
and contributed to the writing, design, synthesis, biological evaluation and analysis of the data.
A.G. has been involved in the synthesis and biological evaluation. All authors read and approved the
final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
Abbreviations
AA: arachidonic acid; Clog P: theoretically-calculated lipophilicity; CMR: calculated molar
refractivity; CNS: central nervous system; COX: cyclooxygenase; CPE: carrageenan-induced paw
edema; DPPH: 2,2-diphenyl-1-picrylhydrazyl; HIV: human immunodeficiency virus; HMG-CoA
reductase: 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; HRP-streptavidin: horseradish
peroxidase-labeled streptavidin; IL-6: interleukin-6; IMA: indomethacin; LOX: lipoxygenase; LP:
lipid peroxidation; LTB4: leukotriene B4; MR: molar refractivity of substituent; NADH: nicotinamide
adenine dinucleotide; NBT: nitroblue-tetrazolium; NDGA: nordihydroguaiaretic acid; NSAIDs:
non-steroid anti-inflammatory drugs; PMS: phenazine methosulfate; RA: reducing activity; RPTLC:
reverse-phase thin layer chromatography; TBA: thiobarbituric acid; TCA: trichloroacetic acid;
TMB: 3,3′,5,5′-tetramethylbenzidine; TMPD: N,N,N′,N′-tetramethyl-p-phenylenediamine; TosMIC:
p-toluenesulfonylmethyl isocyanide.
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Sample Availability: Samples of the compounds 1i–1vi and 2i–2vi are available from the authors.
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