Discovery of anxiolytic 2-ferrocenyl-1,3-thiazolidin-4-ones exerting GABAA receptor interaction via the benzodiazepine-binding site

European Journal of Medicinal Chemistry 83 (2014) 57e73

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Discovery of anxiolytic 2-ferrocenyl-1,3-thiazolidin-4-ones exerting

GABA_Areceptor interaction via the benzodiazepine-binding site

ꢀ ^a

ꢀ

ꢀ ^b

ꢀ ^a

Anka Pejovic , Marija S. Denic , Dragana Stevanovic , Ivan Damljanovic ,

d

ꢀ ^c

ꢀ ^e

Mirjana Vukicevic , Kalina Kostova , Maya Tavlinova-Kirilova , Pavle Randjelovic ,

ꢀ ^f

*

ꢀ ^g

ꢀ ^b

h

Nikola M. Stojanovic , Goran A. Bogdanovic , Polina Blagojevic , Matthias D'hooghe ,

Niko S. Radulovic , Rastko D. Vukicevic

Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovica 12, 34000 Kragujevac, Serbia

Department of Chemistry, Faculty of Science and Mathematics, University of Nis, Visegradska 33, 18000 Nis, Serbia

,

, **

ꢀ ^b

ꢀ

ꢀ ^a

a

ꢀ

b

c

ꢁ

ꢀ

Department of Pharmacy, Faculty of Medicinal Sciences, University of Kragujevac, S. Markovica 69, 34000 Kragujevac, Serbia

^dInstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Bl. 9, Acad. G. Bonchev Str., Soﬁa 1113, Bulgaria

e

ꢁ

ꢀ

ꢁ

Department of Physiology, Faculty of Medicine, University of Nis, Bulev

Faculty of Medicine, University of Nis, Bulev

а

r Zorana

ꢁ

Đ

inCica 81, 18000 Nis, Serbia

f

ꢁ

ꢀ

а

r Zorana

Đ

inCica 81, 18000 Nis, Serbia

g

ꢁ

Vinca Institute of Nuclear Sciences, Laboratory of Theoretical Physics and Condensed Matter Physics, PO Box 522, 11001 Belgrade, Serbia

^hSynBioC Research Group, Department of Sustainable Organic Chemistry and Technology, Ghent University, Coupure Links 653, B-9000 Gent, Belgium

a r t i c l e i n f o

a b s t r a c t

Article history:

Herein, we report on the synthesis, spectral, crystallographic and electrochemical properties of a small

library of N-substituted 2-ferrocenyl-1,3-thiazolidin-4-ones, designed as novel GABA_Abenzodiazepine-

binding site ligands. The anxiolytic properties of the title compounds were evaluated in several

different in vivo models, whereas the involvement of the GABA_Areceptor complex in the activity of the

most potent compound, 2-ferrocenyl-3-(4-methoxyphenylethyl)-1,3-thiazolidin-4-one, was inferred

from experiments with known GABA_A-targeting agents. Ligand docking experiments revealed that the

high, dose-dependent, anxiolytic activity of the new compounds might be due to their favorable in-

teractions with the benzodiazepine-binding site of the GABA_Areceptor complex. The incorporation of the

ferrocene core and ﬁne tuning of the distance between the thiazolidinone core and an additional aro-

matic ring were judged to be crucial structural requirements for the observed anxiolytic effect.

Received 22 April 2014

Received in revised form

5 May 2014

Accepted 25 May 2014

Available online 28 May 2014

Keywords:

2-Ferrocenyl-1,3-thiazolidin-4-ones

Anxiolytic agents

Ligand docking

GABA_Areceptor complex

GABA_Abenzodiazepine-binding site

1. Introduction

GABA_Areceptor complex and exert a positive cooperative effect

that results in increased frequency of chloride channel opening

Since their discovery in the mid-1950s, drugs targeting the

g

-

(there are other modulatory sites on the GABA_Areceptor, i.e.

barbiturate, neuroactive steroid etc.) [4,5]. The ﬂow of chloride ions

causes excitation/depolarization, shunting or inhibition/hyperpo-

larization of neurons [6]. The development of novel compounds

that target the GABA_Asystem is driven by the need to improve the

effectiveness and lessen the side effects of existing drugs [2]. In that

sense, the aim of this study was the design and synthesis of a novel

class of GABA_A-targeting anxiolytics. In that respect, a small library

of 1,3-thiazolidin-4-oneeferrocene hybrids (N-substituted 2-

ferrocenyl-1,3-thiazolidin-4-ones; 13 compounds in total) was

designed on the following three premises: (i) Therapeutically

speaking, besides as anxiolytics, GABA_Aagonists are frequently

used as anticonvulsant, sedative-hypnotic, and muscle-relaxant

drugs [4]. In recent years, there is a growing tendency towards

the use of anticonvulsants as an alternative treatment for some

aminobutyric acid A receptor (GABA_Aagonists; e.g., the family of

benzodiazepine, BZD, compounds) have been and still are the ﬁrst

choice in the treatment of anxiety [1e4]. The fully functional GABA_A

receptor is a membrane-bound heteropentameric protein consist-

ing of an assembly of different subunits (usually a, b and g-sub-

types; different subunit isoforms are possible), together forming a

pore for chloride ion transport [4,5]. A number of GABA_Aagonists

bind to an allosteric site (BZD-binding pocket) located on the

* Corresponding author.

** Corresponding author.

E-mail

addresses:

nikoradulovic@yahoo.com,

vangelis0703@yahoo.com

ꢀ

(N.S. Radulovic), vuk@kg.ac.rs (R.D. Vukicevic).

http://dx.doi.org/10.1016/j.ejmech.2014.05.062

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

58

anxiety disorders in individuals who are partially responsive or

nonresponsive to conventional therapy [1,7]. It was previously

shown that certain 1,3-thiazolidin-4-one derivatives act as anti-

convulsants [8e11]; however, their possible use as anxiolytic

agents has not been pursued thus far. (ii) A number of studies have

shown that the introduction of the ferrocene core (Fc), or a formal

exchange of an existing aromatic ring with Fc, may signiﬁcantly

enhance a molecule’s (desirable) bioactive properties [12e18]. For

example, ferrocene analogues of the antimalarial drug chloroquine

are active against chloroquine-resistant strains of Plasmodium fal-

ciparum; one of these, ferroquine, made it to clinical trials [19,20].

The Fc unit might act as a hydrophobic spacer and/or lipophilicity/

bioavailability enhancer (enabling easier passage through cell

membranes) [20]. It is also known that the ferrocene Fe^2þ/Fe^3þ

redox chemistry might contribute to the bioactivity of ferrocene

derivatives [21]. (iii) Despite possessing unique features, the library

compounds isosterically resemble the known BZD-type anxiolytics

(Fig. 1), i.e., we hypothesized that they could fulﬁll known BZD-

binding site requirements, and thus could potentially have a high

afﬁnity towards the GABA_Areceptor [22].

Anxiolytic properties of all library compounds were evaluated in

several different in vivo models (light/dark, open ﬁeld, horizontal

wire and diazepam-induced sleep tests). The involvement of the

GABA_Areceptor complex was assessed using the known GABA_A-

targeting agents ﬂumazenil (competitive antagonist), picrotoxin

(GABA_Achannel blocker), pentylenetetrazol and isoniazid (con-

vulsants). In order to rationalize the obtained experimental results

and disclose structureeactivity relationships, i.e., to gain insight

into the possible interactions of the title compounds with the

GABA_Areceptor (possible involvement of BZD-binding site), ligand

docking experiments were performed based on the recently

published uniﬁed a₁b₂g₂GABA_Areceptor model (this is believed to

be the most abundant receptor subtype) [23]. Alongside with these

results, we present detailed data on the spectral and electro-

chemical properties, as well as the crystal structure of the studied

heterocycle-organometallic hybrids.

2. Results and discussion

2.1. Library design

The starting point for the library design was the notion that 1,3-

thiazolidin-4-ones, as is the case for GABA_Aagonists, are known to

act as anticonvulsants [8e11]. However, their possible use as

anxiolytic agents has not been explored previously. The idea was to

prepare a series of 1,3-thiazolidin-4-one derivatives with key

structural attributes of well-known GABA_Aagonists (BZD-type

compounds).

We started from Cook's et al. pharmacophore/receptor model

for agonists and inverse agonists at the GABA_A(BZD-binding site)

[22]. For example, the central structural features of the GABA_A

agonist diazepam (Fig. 1a), ﬁtted into the mentioned model, is the

1H-1,4-diazepin-2(3H)-one core, which is fused to a chlorobenzene

ring and bears a phenyl substituent. The hypothetical pharmaco-

phore triangle is depicted in red (edges, angles and vertices of the

triangle are deﬁned in Fig. 1b). According to Cook's et al. model,

potential GABA_Aagonists should be able to interact with the

following (sub)sites of the receptor: (i) an H-bond acceptor (A₂), (ii)

an H-bond donor (H₁), (iii) a ‘bifunctional’ hydrogen-bond donor/

acceptor site (H₂/A₃), (iv) four lipophilic pockets (L₁, L₂, L₃, and LD_i),

and (v) three sterically forbidden sites (S₁, S₂, and S₃) (Fig. 1a).

Hydrogen-bond donor sites H₁and H₂, hydrogen bond acceptor A₂

Fig. 1. a) Pharmacophore model [7] for the BZD-binding site of the GABA_Areceptor (A₁-A₃-H-bond acceptor, H₁, H₂eH-bond acceptor, S₁eS₃-sterically forbidden sites, L₁-L₃-

lipophilic pockets), with diazepam as template; the hypothetical pharmacophore triangle is given in red. b) Edges (l_A, l_B, l_C), angles ( ) and vertices (A, B, C) of the hypothetical

a, b, g

pharmacophore triangle. c) and d) Fitting of N-substituted 2-ferrocenyl-1,3-thiazolidin-4-ones into the proposed model for ligands targeting the GABA_Acomplex. (For interpretation

of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

59

and lipophilic region L₁were assigned as four basic anchor points

on the receptor protein-complex [22]. We believe that certain 1,3-

thiazolidin-4-one derivatives should also be able to ﬁt this model.

We initially placed the thiazolidinone core at the position (vertex A)

of the analogous heterocyclic moiety from the diazepam molecule

(1H-1,4-diazepin-2(3H)-one ring; Fig. 1c and d): the 1,3-

thiazolidin-4-one carbonyl unit is a strong H-bond acceptor

(possible interactions with H₁receptor (sub)site), while the diva-

lent sulfur atom could either act as a weak H-bond acceptor or

to be the most convenient one. The reaction employs readily

available substrates and is easy to perform. There are three

experimental variants of the synthesis: (i) Reaction of a-mercapto

carboxylic acids with imines obtained in a separate experiment

(as it had originally been conducted) [26]; this approach was

previously employed in the synthesis of several 2-ferrocenyl-1,3-

thiazolidin-4-ones, but the achieved yields were very poor

(1.6e10.8%) [28]; (ii) A continuous process using a water separator

and the same solvent for both steps (the synthesis of imines and

eventually may form a favorable Se

p

interaction with the appro-

their condensation with a-mercapto carboxylic acids) [27]; (iii) a

priate arene system at the receptor-binding site [24]. Now, we had

to “expand” the molecule as to ﬁt the pharmacophoric triangle.

Thus, the 1,3-thiazolidin-4-one substitution pattern was chosen in

one-pot three-component technique with the use of a suitable

dehydrating agent [29e31]. We have chosen the latter, one-pot

approach for the synthesis of the target N-substituted 2-

ferrocenyl-1,3-thiazolidin-4-ones (3aem). The method utilizes

ferrocenecarboxaldehyde (1, FcCHO), an appropriate primary

amine (2aem), thioglycolic acid and DCC as a dehydrating agent

in THF (Scheme 1). This approach was previously employed for the

synthesis of analogous compounds [29,31], with the ratio of re-

actants being amine/aldehyde/mercapto acid ¼ 1/2/3. However, in

this case the yields based on the used aldehydes are necessarily

less than 50%. This was not a reasonable approach in our case,

since FcCHO (1) was the most valuable (expensive) reactant.

Hence, we tried to improve the original protocol in order to ach-

ieve higher reaction yields with regard to FcCHO. We found that

optimal results were obtained by a 25 min ultrasonic irradiation of

the reaction mixture consisting of an amine, FcCHO and thio-

glycolic acid in the ratio 1/1/2. The corresponding N-substituted

2-ferrocenyl-1,3-thiazolidin-4-ones (3aem, Scheme 1) were ob-

tained in moderate-to-high yields, calculated by taking 1 as the

limiting reagent (See Experimental section).

Compounds 3aem were spectroscopically characterized by IR,

UVeVis, MS, ¹H and ¹³C NMR spectroscopy (see Experimental

section and Supporting Information). The obtained spectral data

agreed favorably with the expected structures for all compounds

[15,17,32e37]. Several well-resolved IR bands associated with

characteristic vibrations of the 2-ferrocenyl substituted 1,3-

thiazolidin-4-one coreeC]C stretching, Csp²-H stretching, C]O

stretching and the symmetrical deformation of the thiazolidinone

CH₂group (scissoring)ewere observed for nearly all compounds

[32]. An assortment of other bands, characteristic for the speciﬁc

functional groups of individual compounds, were also observable in

the corresponding spectra. For example, in the spectra of 3g, 3h and

3kem, the arrangements of the CeH out-of-plane bending bands in

the 680e900 cm^ꢀ1region reﬂected the substitution patterns of the

benzene ring, whereas the asymmetrical stretching and symmet-

rical deformation of the CH₃group in N-alkyl and methoxy

substituted compounds were observed as medium intensity bands

around 2960 cm^ꢀ1and 1380 cm^ꢀ1, respectively.

a way to match, as closely as possible, the value of angle

identity of the substituents had to comply with l_A, l_B, l_C(triangle

edges), and (triangle angles) values (Fig. 1b). We decided one of

a, while the

b

g

the substituents to be the ferrocene core, as we expected that it

would nicely ﬁt L₁/L₂(Fig. 1c) or L₃(Fig. 1d) lipophilic pockets and

could possibly act as an “activity enhancer” [12e21]. Finally, we

have chosen to vary the identity of the substituent attached to the

nitrogen atom (R, Scheme 1), in order to gain an insight into

possible SAR relationships. Two main types of R substituents were

included in the study: aliphatic (n-alkyl chains of differing lengths,

3aef; “aliphatic analogues”) and aromatic (with benzene/furan/

thiophene cores, 3gem; “aromatic analogues”). Within the “aro-

matic” series (which might be more active, e.g. due to possible

favorable

pep interaction), we have varied the length of the

e(CH₂)_ne (n ¼ 0e2) spacer between the N-atom and the aromatic

core. This was done to ﬁnely tune l_A, l_Band l_Cvalues, as well as to

probe if higher conformational freedom of the molecule would

inﬂuence the net activity. Another reason for the synthesis of 3i, 3j

(heteroaromatics) and 3g (methoxyphenyl group) was the fact that

these compounds contain additional H-acceptors (S/O-atoms) that

could increase the ligand's afﬁnity towards the receptor.

2.2. Chemistry

Among many reported protocols towards the synthesis of 1,3-

thiazolidin-4-ones [11,25], the reaction of a-mercapto carboxylic

acids with imines, known for more than sixty years [26,27], seems

In general, UVeVis spectra of 3aem were marked by the pres-

ence of the ferrocene chromophore [18]. The band around 200 nm

was the most intense one, assignable to

p /

p^*transitions, and

was characteristic for the ferrocenyl moiety. The bands at c.a.

320 nm (although not noted in for all compounds) and 430 nm,

which were about 100 times weaker than that at 200 nm, most

probably corresponded to d / p^*and ded transitions of the

ferrocene core, respectively.

The ¹H NMR spectra contained typical signals for a mono-

substituted ferrocene (dt (or m) at ~4.4 ppm, a multiplet at

~4.3 ppm and a singlet at ~4.2 ppm; last two signals were found to

overlap occasionally) [18] and were also characterized by the

presence of an ABX spin system formed by the methine (at

~5.34e5.90 ppm) and methylene protons (at ~3.60e3.81 ppm) of

the thiazolidinone scaffold [33]. Generally speaking, when

compared to the corresponding protons in 2,3-diaryl-1,3-

thiazolidin-4-ones and 3-alkyl-2-aryl-1,3-thiazolidin-4-ones, the

Scheme 1. Synthesis of N-substituted 2-ferrocenyl-1,3-thiazolidin-4-ones (3aem).

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

60

ring methine and methylene protons were slightly shifted upﬁeld

by the presence of the ferrocene unit [33,34]. Similarly, signals at

c.a. 85.0, 70.3, 69.7, 69.0, 68.5 and 67.7 ppm, observable in the ¹³C

NMR spectra of the library compounds, could be attributed to the

ferrocene moiety [15] while the other characteristic signals, at

about 169, 61 and 33 ppm were those corresponding to the

carbonyl-, methine and methylene carbons of the thiazolidinone

ring, respectively [35]. Depending on the nature of the substituent

attached to the N-atom, other expected signals in both ¹H and ¹³C

NMR spectra were noted. Thus, in the spectra of 3g, 3h and 3kem,

there were typical signals in the aromatic region, while the spectra

of 3i and 3j contained characteristic signals of the thiophen-2-yl

and furan-2-yl AMX systems.

The fragmentation pattern in the mass spectra of 3aem was a

similar one to the previously reported for 2,3-diaryl-1,3-

thiazolidin-4-ones, with the most striking differences relating to

the base peak [36]. In the cases of 3aem, the base peak was the

molecular ion as well; this suggests that the introduction of a

ferrocene unit onto the thiazolidinone core increased the stability

of corresponding [M]^þ. Other characteristic ions observable in the

MS spectra of 3aem, m/z 186 ([(C₅H₅)₂Fe]^þ), 121 ([C₅H₅Fe]^þ) and 56

([Fe]^þ), were those characteristic for a monosubstituted ferrocene

derivative [37]. Moreover, in spectra of almost all compounds, the

second most intense peak corresponded to a [FcCHNHR]^þion.

Additional abundant ions in the mass spectra of N-aryl or N-ary-

lalkyl derivatives were formed by scission of the CeN bond (eg.

[C₆H₅]^þat m/z 77 for 3k, or [C₇H₇]^þat m/z 91 for 3h).

centroids of the corresponding Cp rings; designations of the atoms

are given on Fig. 2). However, the Cp rings slightly deviated from

the parallel orientation (the dihedral angle between two the Cp

rings was 4.0(2)^ꢁ). The conformation of the thiazolidinone ring

could be described as being close to an envelope, and the present

orientation of the C14eC19 phenyl ring was inﬂuenced by an

intramolecular CeH…

the phenyl ring as a -acceptor (Fig. 1). There were no classical H-

intermolecular interactions in the crystal packing

p interaction between the C6eH6 group and

p

bonds and p…p

of 3k.

The S1eC13 heterocyclic ﬁve-membered ring (thiazolidone

core) is directly bound to the C1eC5 cyclopentadiene ring via a

single bond (C1eC11) with a bond distance of 1.504 (3) Å. However,

the longest CeC bond in the crystal structure of 3k was the

C12eC13 bond, situated within the heterocyclic ring, while the

remaining CeC bonds had p-character (Table 1). The S1 atom forms

two SeC bonds which are signiﬁcantly different in bond length,

1.849 (2) and 1.796 (3) Å for the S1eC11 and S1eC13, respectively.

Around the sulfur atom, the C11eS1eC13 angle was considerably

smaller than the rest of bond angles within the S1eC13 ﬁve-

membered ring (Table 1). The sum of bond angles around N1

(359.8^ꢁ) indicates trigonal geometry, i.e. sp²hybridization of N1.

The conformation of the thiazolidinone ring could be described

as being close to an envelope since the C11, N1, C12 and C13 atoms

were nearly coplanar (root-mean-square deviation from a mean

plane of the ﬁtted atoms was only 0.032 Å). The S1 atom was dis-

placed from the C11eN1eC12eC13 mean plane by 0.622 (4) Å. The

present ring conformation was also in agreement with the bond

character of N1eC12 since this bond is the only one (within the

2.3. Crystallographic analysis

ring) which had

p-character and consequently forced a coplanar

position of the C11, N1, C12 and C13 atoms. The dihedral angle

between the C11eN1eC12eC13 plane and the C14eC19 phenyl

ring was 58.57 (8)^ꢁ. However, it seems that the present orientation

of the C14eC19 phenyl ring was inﬂuenced by an intramolecular

The structural features of thiazolidin-4-one 3k as a represen-

tative example were also studied by a single-crystal X-ray structure

analysis (Fig. 2 and Supporting Information). Two ferrocenyl Cp

rings adopted an almost ideally eclipsed geometry with the

C1ꢀCg1ꢀCg2ꢀC6 torsion angle of only 0.2^ꢁ(Cg1 and Cg2 are the

CeH …

p

interaction between the C6eH6 group and the phenyl

ring as a

p-acceptor (Fig. 1). A perpendicular distance of the H6 on

the phenyl ring was only 2.74 Å (H6 … Cg ¼ 2.80 Å, C6eH6 …

Cg ¼ 131^ꢁ). The dihedral angle between the phenyl and the C6eC10

cyclopentadiene rings of 69.4 (1)^ꢁwas also in agreement to the

existence of this intramolecular interaction. Thus this CeH …

p was

the only apparent intramolecular interaction in the crystal struc-

ture of 3k and it probably additionally stabilized the present mo-

lecular conformation.

There were no classical H-bonds in the crystal packing of 3k. The

reason for this could be found in the fact that molecules of 3k do

not possess any signiﬁcant H-bond donors, such as OeH or NeH

(Fig. 3 and S1). The C12eO1 carbonyl group may be recognized as

the best H-bond acceptor in 3k but it formed only a single weak

hydrogen bond (C15eH15 … O1, Figure S1, Supporting Information)

with H … O distance shorter than 2.60 Å. Although 3k is comprised

of three aromatic rings, it is interesting to note that this molecule

did not form any

p … p intermolecular interactions. The C6eC10

Table 1

Selected bond lengths (Å) and bond angles (^ꢁ) in the crystal structure of 3k.

Bonds (Å)

Angles (^ꢁ)

S1eC13

S1eC11

O1eC12

N1eC12

N1eC14

N1eC11

C1eC11

C12eC13

1.796 (3)

1.840 (2)

1.215 (3)

1.360 (3)

1.437 (3)

1.464 (3)

1.504 (3)

1.513 (4)

C13eS1eC11

C12eN1eC14

C12eN1eC11

C14eN1eC11

N1eC11eS1

N1eC12eC13

C12eC13eS1

90.69 (11)

122.4 (2)

117.0 (2)

120.42 (18)

103.52 (14)

111.4 (2)

Fig. 2. Perspective view of 3k with the atom-numbering scheme. Displacement el-

lipsoids are drawn at 40% probability level. The C6eH6…

p intramolecular interaction

107.04 (18)

is represented by a dotted red line. (For interpretation of the references to colour in

this ﬁgure legend, the reader is referred to the web version of this article.)

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

61

Fig. 3. The centrosymmetric dimer of 3k formed via two C13eH13b … S1 (dashed green lines) and two C13eH13a …

interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

p

(dotted red lines) intermolecular interactions. (For

unsubstituted Cp ring participated in an intermolecular CeH …

p

¹He¹H COSY and NOESY) of 3a-m, suggesting that the geometry

with the pseudo-axial H_X(conformer B, Figs. 4 and 6) was the

preferred one in solution as opposed to the solid-state one (see also

Supporting Information). The results obtained for compound 3g are

summarized in Table 2, and Figs. 5 and 6. The use of higher mag-

netic ﬁeld (400 MHz) and DMSO-d₆as deuterated solvent enabled a

clear resolution of the ABX splitting pattern of the thiazolidinone

methine (C2H_X) and methylene protons (C5H_AH_B) (Table 2). The

larger in value (in comparison to H_B) coupling of the methine

proton H_Xwith the methylene proton H_A, appearing at lower ﬁeld,

indicated that these protons should be mutually anti-oriented. At

the same time, the methine proton showed a nOe crosspeak with

the upperﬁeld methylene proton (and vice versa), suggesting that

H_Xand H_Bare spatially near one to another. Based on the calculated

A and B geometries of 3g (MM þ force ﬁeld), the distances of H_X‒

C5H_Bin A (more than 4 Å) and B (around 3 Å) differed signiﬁcantly

in value (Fig. 6). This suggested that the geometry with the pseudo-

axial H_X(conformer B, Figs. 4 and 6) was the preferred one in so-

lution as opposed to the solid-state one; otherwise, one would

expect the H_XeH_BnOe crosspeak not to appear. Furthermore, the

ferrocene proton H5⁰should be considerably closer to the NeCH₂

(ca. 2 Å) than to the AreCH₂(ca. 4 Å) protons in conformer B, while

the opposite should be true for conformer A. Thus, the fact that H5’

interaction as a -acceptor (Figure S2, Supporting Information)

p

while the C14eC19 phenyl ring contributed to the same interaction

as a CeH donor.

Probably the most interesting association of the molecules of 3k

in solid state was the formation of centrosymmetric dimers.

Namely, two molecules within the dimer (Fig. 3) were inter-

connected by four interactions, two CeH … S and two CeH …

p.

Although these interactions are considered weak, their cumulative

effect and the fact that two molecules are in a centrosymmetric

arrangement most likely resulted in such stable dimers.

2.4. Conformational analysis

In line with the results of the crystallographic analysis of 3k,

previous NMR and X-ray studies of 2,3-diaryl-1,3-thiazolidin-4-

ones have showed that the favorable geometry of the heterocyclic

ring corresponds to an envelope conformation, with the out-of-

plane sulfur atom (conformations A and B, Fig. 4). Such geometry

minimizes strain within the ring and ensures co-planarity of the

carbonyl group and the N-aryl system (the latter enabling effective

overlap of the corresponding orbitals). It was previously established

that conformer A (Fig. 4), with a pseudo-axial C2 aryl substituent

(A_R’), represented the preferred solid-state geometry, while

conformer B (pseudo-equatorial A_R’, Fig. 4) was the dominant one

in solution [34,38]. This is in agreement with the crystal structure of

3k.

In order to explore the solution conformation (most probably

that connected to the activity of the compounds) of our library

compounds and to compare it to previous conformational features

of 1,3-thiazolidin-4-one derivatives [34,38], we have performed a

detailed 1D/2D NMR spectral study (DEPT, HSQC, HMBC (Fig. 5),

Fig. 4. 2,3-Diaryl-1,3-thiazolidin-4-ones conformers.

Fig. 5. Important HMBC interactions of 3g.

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

62

Fig. 6. Preferred solid-state (A) and solution (B) conformers of 3g, minimized using MM þ force ﬁeld. Important NOESY interactions are also shown on conformer B. Notation of

some atoms, double and CeFe bonds (conformer A) are omitted for clarity.

proton showed nOe crosspeaks with NeCH₂, but did not interact

with AreCH₂protons, corroborated the assumption that B was the

preferred 3g geometry in solution (Fig. 6).

voltammetry in acetonitrile containing 0.1 mol/L lithium perchlo-

rate as the supporting electrolyte. Since preliminary measurements

conducted with 3a showed that this compound exhibits only a

single redox couple (E_1/2¼ þ499 mV) in the potential range

between ꢀ500 and þ1000 mV (belonging to the ferrocene unit), we

performed cyclic voltammetry experiments with all other com-

pounds in the potential range between 0 and þ1000 mV. It turned

out that all thiazolidinones exhibit a reversible one-electron redox

couple at a very similar potential (E_1/2¼ þ487 to þ512, Table S1).

Since the ferrocene unit of compounds 3aem is connected to an

electron withdrawing group, these potentials are considerably

more positive than that of the unsubstituted ferrocene. As repre-

sentative examples, the voltammograms of 3a and 3k are depicted

in Figure S3. Differences between anodic and cathodic peak po-

tentials (Table S1) were close to the theoretical value; both anodic

and cathodic peak currents were proportional to the square root of

the scan rate (as illustrative examples graphs for 3a and 3k are

given in Figure S4), and their ratio is independent of the scan rate,

indicating a diffusion-controlled process.

There are two more pairs of diastereotopic protons in 3g left

unassigned. The protons of the CH₂directly attached to nitrogen

were shifted slightly downﬁeld when compared to those next to

the aromatic ring. A previous NMR study of 2-aryl-3-benzyl-1,3-

thiazolidin-4-ones pointed to a large chemical shift difference (of

approximately 1.5 ppm) between diastereotopic benzylic protons

and the downﬁeld shift of one of the protons was explained by

stronger hydrogen bonding, as well as to hindered rotation around

the N- and benzyl C-bond [34]. A similar trend was visible in the

spectra of the herein studied benzyl (3h), 2-furfuryl (3i) and 2-

thenyl (3j) derivatives, with the mentioned shift differences of

about 1.1 ppm for the geminal NeCH₂protons. The shift difference

for the corresponding protons in 3g was considerable lower

(0.37 ppm), suggesting a higher degree of conformational freedom

with respect to rotation about the NeC bond in NeCH₂CH₂.

2.5. Electrochemistry

As mentioned above, the Fe^2þ/Fe^3þredox chemistry is known to

contribute to the bioactivity of ferrocene derivatives [18]. Because

of this, we decided to evaluate the electrochemical properties of the

new compounds 3aem. This was done by means of cyclic

2.6. Pharmacological studies

The synthesized thiazolidinone derivatives 3aem were evalu-

ated for their CNS-modulating properties using the light/dark (LD),

open ﬁeld (OF), horizontal wire (HW) and diazepam-induced sleep

tests. The results of the experiments are presented in Table 3 and

Figure S5 (Supporting Information).

Table 2

¹H NMR (DMSO-d₆, 400 MHz) and ¹³C NMR (DMSO-d₆, 100 MHz) data for 3g (atom

During the LD test, all library compounds increased the time

mice spent in the brightly illuminated area, and decreased time

spent in the dark one, in a dose-dependent manner (Table 3).

However, 3g was the only compound that signiﬁcantly increased

the number of crossings between the mentioned compartments at

all applied doses, and in almost the same fashion as diazepam did.

The effects of other library compounds drastically varied in the

number of crossings and were dependent on the dose

administered.

All of the tested compounds had increased (statistical signiﬁ-

cance) the latency of the ﬁrst crossing from the illuminated

compartment to the dark one (this referred to those groups that

had transitions at all). This could be the result of an anxiolytic-like

action of the thiazolidinone derivatives that is also a characteristic

of benzodiazepines [39]. The increased latency of the ﬁrst transition

between compartments could be a reﬂection of the reduction of

exploratory activity, which was observed for most of the herein

studied compounds (the open ﬁeld test). Nevertheless, this is a

controversial parameter, commonly not discussed in studies on

labels can be found in Fig. 5).

Position

¹H NMR

¹³C NMR

1⁰

/

85.5

67.2

68.3

69.4

70.2

68.7

130.4

129.4

113.8

157.7

43.7

31.9

32.4

60.3

54.9

169.5

2⁰

4.42 (m, 1H)

4.34e4.31 (m, 2H) e overlapped

3⁰

4⁰

5⁰

4.49 (dt, J ¼ 2.4, 1.4, 1.4 Hz, 1H)

4.24 (m, 5H)

/

6.99 (AA'BB', J ¼ 8.6 Hz, 2H)

6.82 (AA'BB', J ¼ 8.6 Hz, 2H)

/

1⁰⁰, 2⁰⁰, 3⁰⁰, 4⁰⁰and 5⁰⁰

1⁰⁰⁰

2⁰⁰⁰and 6⁰⁰⁰

3⁰⁰⁰and 5⁰⁰⁰

4⁰⁰⁰

N-CH_AH_B

CH_AH_B-Ar

S-CH_AH_B-C]O

NeCHeS

OeCH₃

3.34 (ddd, J ¼ 13.6, 10.1, 5.8 Hz, 1H)

2.97 (ddd, J ¼ 13.6, 9.8, 5.8 Hz, 1H)

2.60 (ddd, J ¼ 13.2, 10.1, 5.8 Hz, 1H)

2.26 (ddd, J ¼ 13.2, 9.8, 5.8 Hz, 1H)

3.61 (dd, J ¼ 15.4, 1.6 Hz, 1H)

3.56 (dd, J ¼ 15.4, 1.0 Hz, 1H)

5.57 (br. s, 1H)

3.71 (s, 3H)

C¼O

/

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

63

Table 3

Effect of thiazolidinone derivatives 3aem, diazepam and vehicle on anxiety (LD test) and locomotor activity (OF test) in mice.

Compound

Dose (mg/kg)

Light/dark (LD) test

Open ﬁeld (OF) test

No. of squares crossed

99 11

Time spent in light box

No. of transitions

Time of the ﬁrst transition

No. of rearings

3a

3b

3c

3d

3e

3f

25

50

100

25

50

100

25

50

100

25

50

100

25

50

100

25

50

100

25

50

100

25

50

100

25

50

100

25

50

100

25

50

100

25

50

100

25

204 7^a

218 4^a

285 10^a

199 12^a

224 10^a

289 10^a

203 8^a

219 16^a

292 12^a

213 10^a

223 11^a

300 0^a

220 13^a

231 12^a

294 12^a

193 8^a

213 8^a

276 9^a

144 10^a

224 9^a

257 10^a

271 11^a

300 0^a

221 10^a

277 9^a

300 0^a

207 12^a

300 0^a

132 11^a

194 10^a

279 12^a

121 11^c

175 11^a

267 15^a

117 16

181 13^a

300 0^a

184 12^a

2.5 0.5

2.5 0.2

1.0 0.5^b

3.0 0.2^a

1.5 0.2

1.0 0.2^a

3.0 0.8^c

2.0 0.5

1.5 0.2

2.0 0.5

1.5 0.2

39 15^b

42 15^b

195 9^a

47 9^a

6.4 0.9^c

4.4 0.7

3.9 0.9

6.9 0.6^b

5.8 0.9

5.5 0.9

5.2 0.4

5.0 0.7

4.2 0.6

8.0 0.6^a

6.8 0.4^b

4.5 0.3

5.7 0.9

5.2 0.8

4.8 0.9

5.5 0.2

5.1 0.3

4.9 0.2

12.4 0.5^a

14.0 0.7^a

15.5 0.3^a

2.3 0.8

3.6 0.5

5.1 0.8

7.2 0.3^a

8.1 0.6^a

8.6 0.9^a

8.3 0.7^a

9.5 0.6^a

10.1 0.9^a

7.4 0.8^a

6.7 0.4^b

5.4 0.9

8.7 0.6^a

9.5 0.7^a

5.3 0.6

8.5 0.6^a

6.6 0.8^b

2.4 0.9

7.6 3.1^a

4.1 2.1

101

100

9

123 9^a

106 9^c

106 12^a

164 11^a

37 7^b

98

9

98 10

87 11

68 12^a

94 11^a

46 9^a

64 10

113 12^b

42 11^a

98

92

9

2

0

0^a

0

0^a

3.0 0.2^a

2.5 0.2

1.5 0.5

3.5 0.5^a

2.0 0.5

1.5 0.2

3.5 0.8^b

5.0 0.5^a

8.5 0.8^a

1.5 0.2^c

51 10^a

63 12^a

89 6^a

72 9^a

93 9.^a

101 8^a

56 8^a

61 7^a

74 8^a

84 5^a

99 15

97 10

90 11

100

9

104 10

97 13

3g

3h

3i

75 16

51 13^c

35 10^a

75 16

0

0^a

0

0^a

51 13^c

35 10^a

144 3^a

123 10^a

114 9^b

157 11^a

145 7^a

107 8^c

143 12^a

124 14^a

100 10

135 15^a

162 12^a

108 13^c

144 11^a

98 10

2.0 0.2

89 12^a

1.0 0.5^b

106 11^a

0

1

0

0^a

1^c

0^a

0

0^a

3j

57 5^a

0

0^a

3k

3l

3.0 0.2^a

2.5 0.2

1.5 0.5

2.0 0.2

2.5 0.2

1.5 0.5

2.5 0.2

1.5 0.8

54 10^a

64 10^a

71 9^a

44 6^a

50 10^a

99 8^a

32 6^a

63 7^a

3m

50

100

2

0

0^a

0

0^a

69 3^a

16

86 15

DZP

VEH

10.5 0.5^a

2.0 0.25

145 10^a

80 21

10

99

9

6

All substances (in the doses of 25, 50, 100 mg/kg for the experimental substances; DZP (2 mg/kg); Vehicle (VEH, 10 mL/kg)), were administrated 1 h prior to the experiment.

Data are presented as mean SD, n ¼ 6.

a

p < 0.001; ^bp < 0.01; ^cp < 0.05 vs. vehicle.

experimental anxiety; compounds cause a decrease in locomotion

and thus give false positive results [40].

aromatic ring (3g) caused a dose-dependent increase in the num-

ber of squares crossed.

To circumvent this situation, all new drugs are screened for

nonspeciﬁc increases or decreases (aspects of sedation) in general

locomotion in a novel arena (OF test). Anxiety behavior in rodents,

observed during the OF test, is a consequence of individual (out of

the group) testing of animals and agoraphobia. In these situations,

rodents show a thigmotaxic behavior, identiﬁed by spontaneous

preference for the periphery of the apparatus and reduced ambu-

lation [41]. At lower doses, almost all tested substances increased

the number of squares crossed in the OF apparatus (Table 3),

whereas at higher doses a reversed effect was observed. This

indicated the possible sedative/hypnotic or muscle relaxant effect

of the library compounds. The impact on animal movement in the

OF test depended on the structure of the group attached to the

nitrogen atom. For example 3h, with a benzyl group in the side

chain, caused a suppression of the motor activity in the OF test even

at low doses. The introduction of a heteroatom into the aromatic

ring, as in 3i and 3j, resulted in an increase of locomotion in the

lowest dose (25 mg/kg), although at higher doses the mentioned

reversal of effect was noted. Elongation of the side chain with a

methylene unit and the introduction of a methoxy group onto the

To rule out the possible muscle relaxant effect, mice were sub-

mitted to an HW test. All animals treated with thiazolidinone de-

rivatives (at all doses) were capable of grasping a wire within the

10 s period (displaying 100% of activity), whereas diazepam (2 mg/

kg; causing a failure to grasp in 50% of cases) decreased the ability

of animals to grasp the wire (data not shown).

The observed depressant activity of 3aem during the OF sug-

gested central mechanisms and not a peripheral neuromuscular

blockade, as opposed to diazepam (at higher doses ꢂ 10 mg/kg) that

affects both mechanisms [42,43], as the library compounds did not

exhibit a muscle relaxant activityeven at doses as high as 100 mg/kg.

Strictly speaking, having all of this in mind and taking into ac-

count that the most reliable parameters for the assessment of

anxiolytic drugs are the increase in time spent in the illuminated

area, the number of crossings between light and dark compart-

ments and increase of locomotion [44], under our experimental

conditions, only substance 3g could be regarded as possessing

purely anxiolytic properties.

Because of the fact that the title compounds were designed to

target the GABA_Areceptor, we decided to experimentally assess its

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

64

Fig. 7. Effect of vehicle (white column), diazepam (1 mg/kg; black column) and 3g (50 mg/kg, striped column) following ﬂumazenil (FLU; 3 mg/kg by ip) treatment on the time that

the animals spent in the light compartment (ﬁrst graph), on the number of crossings between compartments (second graph) and on the time of the ﬁrst transition (third graph).

Values are expressed as mean SD, n ¼ 6, (a) p < 0.0001 vs. FLU þ vehicle, FLU þ diazepam and 3g; (b) p < 0.0001 vs. FLU þ vehicle, FLU þ diazepam; (c) p < 0.001 vs. 3g.

Fig. 8. Effect of vehicle (white column), diazepam (1 mg/kg; black column) and 3g (50 mg/kg; striped column) following picrotoxin (PIC; 1 mg/kg by ip) treatment on the time that

the animals spent in the light compartment (ﬁrst graph), on the number of crossings between compartments (second graph) and on the time of the ﬁrst transition (third graph).

Values are expressed as mean SD, n ¼ 6, (a) p < 0.0001 vs. PIC þ vehicle and 3g; (b) p < 0.0001 vs. PIC þ diazepam.

possible involvement in the anxiolytic action of the library com-

pounds. For this purpose, we have chosen 3g, the most potent

derivative with no CNS depressant or myorelaxant activity. We

were additionally encouraged by the fact that 3g and diazepam

(GABA_Aagonist) displayed similar activity in the LD test. In order to

evaluate the involvement of the GABA receptor, 3g was further

tested in the LD paradigm in combination with ﬂumazenil (FLU, a

competitive antagonist of GABA_Areceptor that antagonize both

group FLUþ3g spent in the light compartment was decreased for

almost a third of the time of animals from group 3g (Fig. 7),

whereas the time group PICþ3g animals spent in the light

compartment was decreased for more than a half of the time of

animals from group 3g (Fig. 8). Likewise, the number of crossings

between compartments and the time of the ﬁrst transition were

only partially reduced by FLU and PIC (but to a greater extent by

PIC) when compared with the values noted for the animals from

group 3g. However, compound 3g still displayed some degree of

anxiolytic activity even in FLU and PIC pretreated animals (statis-

tically signiﬁcant differences existed between 3g and vehicle pre-

treated groups). Also, statistically signiﬁcant differences between

groups that received the combination of FLU/PIC and 3g and those

that were administered with FLU/PIC and diazepam suggested the

existence of additional effects that are probably due to other

mechanisms involved in its anxiolytic action.

agonists and inverse agonists [45]) and picrotoxin (PIC,

a

noncompetitive GABA_Areceptor chloride channel antagonist (one

can rather say channel blocker) [46]) (Figs. 7 and 8) and in

pentylenetetrazol (PTZ)/isoniazid (INH)-induced convulsion tests

(Table 4).

From Figs. 7 and 8, one can see that FLU and PIC, in both the

vehicle and diazepam groups, produced an expected result: they

decreased the time animals spent in the light compartment,

decreased the number of crossings between compartments and

decreased the time of the ﬁrst transition. For the groups that

received FLU/PIC and 3g, the observed parameters were modiﬁed

compared to the results of the animals that received 3g alone, in the

same dose (50 mg/kg). For example, the time that the animals from

The hypnotic/sedative activity of the library compounds was

estimated using the diazepam (20 mg/kg)-induced sleep model.

Almost all substances had no signiﬁcant effect on the onset of sleep,

except for three compounds that caused a prolongation of this

period (3kem, having an aryl group directly attached to the

Table 4

Anticonvulsant effect of the vehicle (olive oil), 3g and diazepam in the pentylenetetrazole (PTZ) e and isoniazid (INH) e induced seizures in mice.

Substance

Dose

PTZ

INH

Seizure onset (sec)

Onset of HLTE (sec)

Seizure onset (sec)

% of living mice

in 30 min

Vehicle

3g

Diazepam

10 mL/kg

50 mg/kg

1 mg/kg

39

1

106 10

338 14^a

1324 38^a

1244 26

2207 43^a

2197 17^a

0

100

62 2^a

601 25^a

Data are presented as mean SD, n ¼ 6.

a

p < 0.001 vs. vehicle.

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

65

nitrogen atom), but all of the tested library compounds signiﬁcantly

increased, in a dose-dependent manner, the duration of the sleep.

These results suggested a central nervous system depressant ac-

tivity and possible sedative properties of these thiazolidinone-

ferrocene hybrids (Fig. S5). The tested hybrids had manifested a

profound inﬂuence on the diazepam-induced sleep. Compounds

3aef, with an alkyl chain of variable length attached to the nitrogen

atom, were the most active ones as they increased the sleeping

period, in a dose-dependent manner, almost three-fold. The lowest

effect was noted in the case of compound 3h (which possesses a

CH₂Ph group) treatment, where only its highest dose caused a

statistically signiﬁcant different prolongation of the diazepam-

induced sleep. Once again the introduction of an oxygen or a sul-

fur atom (3i and 3j) into the aromatic rings resulted in a signiﬁcant

alteration of activity. These sedative properties can be related to the

activation of benzodiazepine and/or GABA sites of the GABA re-

ceptor complex. This is in accordance with a previous report on

several thiazolidinone derivatives, tested at a dose of 100 mg/kg,

that were found to potentiate the effect of a more general sedative,

sleep-inducing agent (pentobarbital) [11]. The effects of sleep la-

tency shortening and increase of total sleep time, as well as the

enhancement of electroencephalogram power in the delta fre-

quency range are some of the hypnotic drug characteristics (at

higher doses) and it is considered to involve more pronounced

depression of the CNS than sedation [47].

activity (the results are presented in Table 4) compared to diaz-

epam (1 mg/kg), but it signiﬁcantly increased the convulsion

threshold compared to vehicle pre-treated animals. These results

further conﬁrm the possible involvement of 3g in signal trans-

duction through GABA systems.

The used dose of 3g (50 mg/kg) applied after a speciﬁc GABA_A

antagonist could have been supramaximal and in this way could

have masked its competitive effect for this receptor. Even after the

application of a nonselective antagonist (PIC), 3g exerted some

degree of anxiolytic activity in the experimental animals suggesting

that there are also other mechanisms involved in the activity of 3g.

The accumulated in vivo data regarding the possible interaction of

the library compounds and the GABA_Areceptor complex motivated

us to perform in silico experiments that could provide further (pro/

con) evidences for the proposed mechanism of action.

2.7. Molecular docking

The ligand-based [22] design of 3aem relied on the structure of

GABA_Atargeting BZD-type anxiolytics. In order to further explore

whether the library compounds ﬁt into the BZD-binding site and to

possibly rationalize the results of in vivo experiments, we have

decided to dock 3aem, as well as the appropriate standard drugs

used in biological assays, into the extracellular domain of the

GABA_Areceptor (anxiolytic agents (usually) bind to this domain).

Although the crystal structure for the GABA_Areceptor is not yet

available, there are several homology models that are in good

agreement with experimental ﬁndings [23,49]. Among them, we

have chosen a recently reported uniﬁed model of a₁b₂g₂GABA_A

receptor complex (the most abundant subunit combination), based

on the glutamate-gated chloride ion channel (Fig. 9) [23]. The BZD-

binding site is located on the extracellular surface of the receptor

(Figs. 9 and 10) and it includes amino acid residues from 6

noncontiguous regions (these are usually designated as Loops AeF,

A matter that should be kept in mind when discussing the ac-

tivity of our thiazolidinone derivatives is the differentiation of ef-

fects produced by benzodiazepine-type compounds binding to

different GABA_Areceptor subtypes (a₁, a₂and a₃) [43]. The a₁

GABA_Areceptor is known to mediate sedative, amnesic, and part of

the anticonvulsant effects, while the a₂GABA_A(and not a₃GABA_A)

receptor mediates the anxiolytic effects of diazepam. It was

observed that a₂GABA_Areceptor (expressed on motor neurons and

in the superﬁcial layer of the dorsal horns) were primarily involved

in the myorelaxant activity of diazepam, whereas at higher doses it

was observed that a₃GABA_Areceptors are also included [43]. These

results allow at least a partial explanation for the observed activity

of the tested thiazolidinone-ferrocene hybrids. One can speculate

that most of them possess higher afﬁnity for a₁GABA_Areceptors

(due to their activity in the OF and diazepam-induced sleep tests)

Fig. 10A) of subunits

a and g [23,49]. For several classical benzo-

diazepines even speciﬁc amino acid residues that contribute to the

binding are uncovered. For example, it is known that the molecule

than for a₂/a₃GABA_Areceptors that are involved in myorelaxant

activity (negative results in HW test). For example, 3h decreased

the number of crossings in the OF test more than two folds when

compared to the results of the vehicle pretreated animals (Table 3),

but it did not modify the performance of mice in the HW test. It also

prolonged, although not to a great extent, the diazepam-induced

sleep (Figure S5, Supporting information). Furthermore, other

compounds, such as 3i, 3k and 3m, decreased the number of

crossings in the OF test only at doses of 100 mg/kg, did not modify

animal performance in the HW test, but prolonged the diazepam-

induced sleep (two folds when compared to diazepam), also sug-

gesting their higher afﬁnity for a₁GABA_Arather than for a₂and a₃

GABA_Areceptors.

Anticonvulsant effects of the library compounds were also

studied in order to determine the possible interaction of 3g with

the GABA_Areceptor complex. For that reason we utilized pentyle-

netetrazole (PTZ) and isoniazid (INH) that are known to interrelate

with the GABA neurotransmitter itself and the GABA complex. PTZ

not only produces epileptiform activity but also mimics the seizure

induced behavioral changes that are very similar to temporal lobe

epilepsy in humans and, thus, it has a predictive relevance

regarding the clinical spectrum of activity of experimental com-

pounds. It is also interesting to mention that neither PTZ nor INH-

binding sites on GABA receptors belong to the GABA or PIC sites of

the GABA receptor complex [48]. Compound 3g exerted modest

Fig. 9. Uniﬁed homology model of the GABA_Areceptor complex [24] (the show

perspective from the side of the receptor's extracellular domain). a₁, b₂and g₂subunits

are given in blue, green and gray, respectively; BZD- and GABA-binding sites are

marked with arrows; the most favorable docking poses of 3g are given in red (F1eF5

families of nodes). (For interpretation of the references to colour in this ﬁgure legend,

the reader is referred to the web version of this article.)

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

66

Fig. 10. The most preferred docking mode of 3g at the BZD-binding site (F1): A e molecular surface of BZD pocket Loops AeF (Loop A e gray, Loop B e pale blue, Loop C e dark blue,

Loop D e pale cyan, Loop E e bright green, Loop F e olive green); B e overlay of 3g (solid red line) and diazepam (solid black line) at the BZD-binding site; C e amino acid residues

found in the 4 Å-radius sphere around 3g and diazepam, D e 3g and the nearby amino acid residues (4 Å-radius sphere), a simpliﬁed representations (red dashed lines group

spatially close entities). Color coding of the receptor subunits: a₁(secondary structure, amino acid residues (C) or labels (D))-dark blue, g₂(secondary structure, amino acid residues

and labels)-light gray, b₂(secondary structure)-green. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

of diazepam interacts with a₁His101, a₁Asn102 (Loop A), a₁Gly157

(Loop B), a₁Val202, a₁Ser205, a₁Thr206 and a₁Val211 (Loop C)

[23,49].

All in silico experiments were performed as blind dockings,

covering the entire extracellular domain of the receptor. We

assumed that by limiting the search space to the BZD-binding

pocket only we could lose valuable information on other possible

favorable interactions between 3aem and the GABA_Areceptor, or

could even get false “positive results”; it is known that several

have a unique GABA_Areceptor binding site (Fig. 9) [50]. Addition-

ally, blind docking also allowed validation of the docking experi-

ments. Although the entire extracellular domain of the receptor

was explored, the most favorable calculated pose for diazepam

(binding energy ꢀ8.6 kcal/mol) was in agreement with the previ-

ous experimental ﬁndings (Fig. 10AeC). This mainly referred to the

mutual spatial arrangement and the distance between diazepam

and several a₁/g₂subunit amino acid residues, experimentally

conﬁrmed to be important for the binding. For example, a₁His101,

a₁Asn102, a₁Val202, a₁Ser205, a₁Val211 “fell” within the 4 Å radius

compounds (e.g. GABA (g-aminobutyric acid), picrotoxin) do not

Fig. 11. The most preferred F4-docking mode of 3g: Ae3g (solid red line) and the nearby amino acid residues (4 Å-radius sphere), B e a simpliﬁed representations of A (red dashed

lines group spatially close entities). Color coding of the receptor subunits: a₁(secondary structure, amino acid residues (A) or labels (B)) e dark blue, b₂(secondary structure, amino

acid residues and labels) e green, g₂(secondary structure) e light gray. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version

of this article.)

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

67

sphere around diazepam; the criteria that functional groups sepa-

rated by less than 7 Å have the potential to interact, and that those

within 4 Å may form salt bridges or hydrogen bonds, are generally

accepted [23,49].

The main results of more than 150 individual docking experi-

ments are summarized in Figs. 9e11, S6. With a few exceptions

(which were not, energetically speaking, among the most favorable

ones), all found binding modes for 3aem clustered them into ﬁve

different families of poses (F1eF5; these designations will also be

used for the appropriate regions of the receptor further on, Fig. 9).

afﬁnity (binding energies) for the BZD-binding site were 3g and 3h

(Fig. S6), and in vivo results pointed to 3g as the most promising

new anxiolytic (for this reason further discussion will be mainly

focused on this compound). The opposite was true for 3d, 3e and

3kem. One should note that the length of the N-alkyl chain, or the

presence/absence of a spacer between the N-atom and the aromatic

core, seems to be critical for interaction with the BZD-binding

pocket. One of the explanations (within the “aromatic” series)

could be found in the fact that spacers confer a certain degree of

conformational freedom to the molecule. Thus, critical structural

features of the active molecules may adopt favorable, diazepam-

like spatial arrangements. Without the spacer, the molecule is

much more constrained and possibly certain structural attributes

(aromatic core) cannot avoid unfavorable interactions with

“forbidden” regions of the receptor (Fig. 1). A similar reasoning

could be true for 3d and 3e (N-octyl and N-decyl derivatives).

Hit compound 3g was docked within the BZD-binding site in a

similar way as diazepam or ﬂumazenil molecules were. The resi-

dues of the following 15 amino acids were within the 4 Å sphere

(strong interactions expected) [49] around 3g: a₁HIS101, a₁ASN102

(Loop A); a₁LYS155, a₁VAL202, a₁SER204, a₁GLY207, a₁GLU208,

a₁TYR209, a₁VAL211 (Loop C); g₂PHE77 (Loop D); g₂ARG144 (Loop

E); g₂THR193, g₂ARG194, g₂SER195 and g₂ARG197 (Loop F), Fig.10C

and D. Several residues, namely a₁HIS101, a₁VAL202, a₁SER204,

a₁TYR209, a₁VAL211, g₂PHE77, are known to be important for BZD

binding [23,49]. Additional 20 residues were within a 7 Å-sphere

(possible interactions) around 3g: a₁PHE99, a₁GLY103, a₁GLU137,

a₁PRO153, a₁SER158, a₁TYR159, a₁GLY200, a₁GLN203, a₁SER205,

a₁THR206, a₁VAL210, a₁MET212, a₁THR213, g₂TYR58, g₂ASN60,

g₂THR142, g₂GLU189, g₂ASP192, g₂LEU198 and g₂TRP196. Among

this, a₁TYR159 (Loop B), a₁GLY200 (Loop C), a₁SER205 (Loop C) and

a₁THR206 (Loop C) are important for the binding of diazepam-type

anxiolytics to the GABA_Areceptor [23,49].

During the design of compounds 3a-m, we assumed two

possible “overlay-modes” of diazepam and the title compounds

(Fig. 1C and D). According to the docking results, the orientation

shown in Fig. 1D was energetically (more) favorable: the ferrocene

core pointed towards the same direction as did the diazepam

phenyl group. This was true for both input conformers of 3g (A and

B, Fig. 4 and S6). Interestingly, despite the different overall geom-

etries of 3g-A and 3g-B, the ferrocene moieties of both of them

perfectly “overlapped” within the BZD-binding site. This could

indicate that the ferrocene core ﬁtted perfectly within this part of

the binding pocket. In fact, the ferrocene core introduced quite

unique structural features (“length”, volume, hydrophobicity) to

the molecule. For example, the distance between the two, (almost)

parallel, aromatic Cp rings was around 3.5 Å. While one of the Cp

rings was positioned in a similar manner as the diazepam phenyl

group (Fig. 10 and S6), the second one enabled additional binding

interactions, not possible in the case of diazepam (Fig. 10C and D).

Interestingly, F4-docking poses (interface of a₁and b₂subunits,

Figs. 9 and 11) were positioned in the pocket that was rather

analogous to the BZD-binding site: it included almost the same a₁

amino-acid residues (vicinity of a₁Loops A, B and C).

Modes belonging to F1 family (at the interface of a₁/g₂subunits)

placed the molecules into the BZD-binding site (Fig. 9 and

Fig. 10AeC). F2 modes were situated at the “top” of Loops B and E,

while those from F3, and F4 and F5 families occupied pockets at the

interface of a₁/b₂and g₂/b₂subunits, respectively (Fig. 9). All

docked compounds within all families had binding energies (for the

search criteria, see Experimental section) from ꢀ6.0 to ꢀ9.2 kcal/

mol. Nevertheless, in most cases, for a single compound, the best

F1eF5 modes were mutually comparable and usually differed in

less than 0.5 kcal/mol.

As discussed in previous subsections, the 2,3-disubstituted 1,3-

thiazolidin-4-one ring is known to predominantly exist in one of

the two different geometries (Fig. 4). Although AutoDock Vina [51]

includes the possibility of a ﬂexible dock (single bonds could be

regarded as rotatable), it does not allow ring geometry to be

changed. For this reason, the docking experiments (ﬂexible ligands)

were performed with two different input geometries of 3aem

(these corresponded to the preferred solution and solid-state

conformers). Generally speaking, the input geometry had (some)

inﬂuence on the net docking results, but not as pronounced as one

might expect. The same families of binding sites (F1eF5) were

found for both input geometries, but the binding energies of the

conformer pairs differed to some level. For example, mutual overlay

of the best F1eF5 modes for compound 3g, calculated starting from

the two different input conformers (A and B, Fig. 4), are shown in

Figure S6. It is interesting that, according to the calculated values of

the corresponding binding energies, conformer B ﬁtted better into

F1eF3 regions of the receptor (Fig. 9), while geometry A showed

more afﬁnity towards F4/F5.

Except for 3d, 3e (aliphatic N-substituents) and 3kem (without

a “spacer” between the N-atom and the aromatic core), the library

compounds were successfully docked into the BZD-binding site

(Fig. 10). According to the obtained results, compounds with a

(CH₂)_xAr substituent (x ¼ 1, 2; “aromatic” analogues) attached to

the N-atom should better interact with the GABA_A-BZD-binding

pocket. The binding energy calculated for derivatives from the

“aliphatic series” (3aec and 3f) ranged from ꢀ6.4 (3f) to ꢀ7.4 kcal/

mol (3a), whereas for “aromatic” analogues (3gej), it was found to

be from ꢀ7.1 to ꢀ8.8 kcal/mol. In general, all of the docked com-

pounds/geometries displayed a unique, energetically favorable F1-

pose. Contrary to that, multiple different orientations of a single

ligand were allowed within F2eF5 regions. This suggested that

F2eF5 pockets were “too big” for the studied compounds, and that

different types of other ligands might also ﬁt (this was, for example,

the case with GABA_A-targeting drugs we used in vivo and in silico

experiments; docking results are not shown). In other words, one

could expect these regions to be much less speciﬁc (in respect to

the geometry, volume and spatial distribution of structural char-

acters) and even unimportant for the activity (otherwise a large

number of different compounds could induce channel opening).

Thus, one could assume that the anxiolytic activity of the title

compounds might be (at least partially) due to their strong afﬁnity

towards the GABA_A-BZD-binding site. Hence, it seems that the re-

sults of the in silico simulations corroborate those of the in vivo

experiments. The two compounds with the highest calculated

The calculated binding energies of the most favorable F1 poses

for 3g (ꢀ8.8 kcal/mol), diazepam (ꢀ8.6 kcal/mol) and ﬂumazenil

(ꢀ8.5 kcal/mol) suggested that 3g should have a comparable or

even stronger afﬁnity towards the GABA_Areceptor when compared

to the mentioned two. Nevertheless, the standard anxiolytics were

(slightly) more active in in vivo assays. One of the possible expla-

nations for such discrepancy could be sought in the fact that (the

most) favorable 3g F1eF5 poses were all of comparable binding

energy. This means that one could expect, under in vivo conditions,

several different “F1eF5 type” ligand-receptor complexes to be

formed. As it is reasonable to assume that only those with 3g bound

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

68

to the BZD site (F1-type ligandereceptor complex) would actually

enable channel opening, the active concentration of 3g would then

necessarily be much lower when compared to the nominally

applied one. We could even roughly estimate the hypothetical

fractional distribution of the different docking poses (and different

types of ligand-receptor complexes), based on the differences in the

corresponding binding energies, under the approximation that

they follow the Boltzmann distribution. Under these assumptions

(we approximated body temperature to be 310 K; all generated

F1eF5 poses were taken into account), less than 10% of 3g receptor

associated molecules (c.a. 75% of which in the geometry that was

preferred in solution) would be bound to the BZD pocket and

induce channel opening. The remaining 3g-receptor complexes

would have differently oriented 3g within F2eF5 regions and might

not result in a “positive outcome” (channel opening). In other

words, the majority of 3g molecules might be bound to the receptor

stronger than diazepam, but in a way that does not induce the

appropriate receptor response.

Germany). The spots on TLC were visualized by UV light (254 nm)

and by spraying with 50% (v/v) aqueous H₂SO₄or phosphomolybdic

acid (12 g) in EtOH (250 mL) followed by heating. Chromatographic

separations were carried out using silica gel 60 (particle size dis-

tribution 40e63 mm) purchased from Merck (Darmstadt, Germany),

whereas silica gel 60 on Al plates, layer thickness 0.2 mm (Merck)

was used for TLC. Proton (¹H) and Carbon (¹³C) NMR spectra were

recorded on a Bruker Avance III 400 spectrometer (400 MHz for ¹H,

100 MHz for ¹³C) and a Varian Gemini 200 spectrometer (200 MHz

for ¹H, 50 MHz for ¹³C). Solutions were prepared in either deuter-

ochloroform (CDCl₃) or deuterated dimethylsulfoxide (DMSO-d₆)

with chemical shifts (in ppm) referenced to TMS and/or deuterated

solvent as an internal standard. 2D experiments (¹He¹H COSY,

NOESY, HSQC and HMBC) were run on the Bruker Avance III 400

spectrometer with the usual pulse sequences. The IR measure-

ments were carried out on a PerkineElmer Spectrum One FT-IR

spectrometer using KBr disks. UV spectra (in CH₃CN) were

measured using a UV-1650 PC Shimadzu spectrophotometer. High-

resolution mass spectrometry (HRMS) analysis was performed us-

ing a JEOL Mstation JMS 700 instrument (JEOL, Germany). The GC/

MS analyses were performed on a HewlettePackard 6890N gas

chromatograph equipped with fused silica capillary column DB-

5MS (5% phenylmethylsiloxane, 30 m ꢃ 0.25 mm, ﬁlm thickness

3. Conclusions

Herein we reported the design, synthesis, spectral, crystallo-

graphic and electrochemical characterization of a small library of N-

substituted 2-ferrocenyl-1,3-thiazolidin-4-ones (3aem, 13 com-

pounds in total). These compounds were designed starting from the

structure of benzodiazepine-type anxiolytics, known to act via the

GABA_A-BZD-binding site. Substituents introduced onto the 1,3-

thiazolidin-4-one core were chosen in a way as to enable favor-

able interactions with the BZD-binding pocket. This turned out to

be especially true for the ferrocenyl substituent, which allowed

acquiring BZD-analogs with two aromatic (Cp) rings in close

proximity (c.a. 3.5 Å), “positioned” in parallel. We assumed that

such an arrangement would enable additional favorable in-

teractions with the benzodiazepine-binding site. In vivo experi-

ments (light/dark, open ﬁeld, horizontal wire and diazepam-

induced sleep tests; the involvement of the GABA_A-receptor com-

plex in the activity of the most potent compound (3g) was evalu-

ated using known GABA_A-targeting agents) conﬁrmed that the

0.25

mm, Agilent Technologies, USA) and coupled with a 5975B

mass selective detector from the same company. If necessary,

alongside the GC analyses, the purity was determined by high

performance liquid chromatography (HPLC). HPLC was performed

using an Agilent Technologies HPLC system 1200 series (Wald-

bronn, Germany) equipped with a quaternary pump, vacuum

degasser, thermostated autosampler, thermostated column

compartment and a diode array detector. Chromatographic sepa-

ration was carried out using Eclipse Plus C18 column

(50 mm ꢃ 4.6 mm, particle size 1.8

mm; Agilent Technologies,

Waldbronn, Germany). Purity of all final compounds was 95% or

higher. The microanalyses were carried out by the microanalyses

service of the Institute of Organic Chemistry, Bulgarian Academy of

Sciences. Cyclic voltammetry experiments were performed at room

temperature in a standard three-electrode cell using an Autolab

potentiostat (PGSTAT 302 N). The working electrode was a plat-

inum disk (2 mm diameter; Metrohm). The counter electrode was a

platinum wire, whereas an Ag/AgCl electrode was used as the

reference. Prior to experiments, the working electrode was pol-

ished using Metrohm polishing kit 6.2802.000 (extremely ﬁne

aluminum oxide on a cloth), followed by washing with distilled

water. Melting points were measured on a Mel-Temp cap. melting-

points apparatus, model 1001, and the given values are uncorrec-

ted. Ultrasonic cleaner Elmasonic S 10, 30 W was used for the ul-

trasonically supported synthesis.

designed

compounds,

especially

2-ferrocenyl-3-(4-

methoxyphenylethyl)-1,3-thiazolidin-4-one (3g), possess (strong)

anxiolytic properties. The docking experiments (favorable geome-

tries of 3a-m were inferred from crystallographic and NMR ana-

lyses) corroborated the assumptions made during the design of 3a-

m and justiﬁed the introduction of the ferrocene core into the

molecules; this metallocene seems to perfectly ﬁt into the BZD-

binding site. Alongside the ferrocene core, both in vivo and in sil-

ico experiments conﬁrmed that the introduction of CH₂-spacers

between the 1,3-thiazolidin-4-one N-atom and an additional

(hetero)aromatic ring was important for their activity. To the best of

our knowledge, hybrids of ferrocene and 1,3-thiazolidin-4-one

were not previously studied for anxiolytic properties. Thus, the

herein presented data might be regarded as a start of a new chapter

in the design of new thiazolidinone-ferrocene based anxiolytics. It

is also reasonable to expect that further work on related ferrocene

containing BZD-analogues might also result in even better GABA_A-

targeting compounds.

4.1.2. General procedure for the synthesis of 2-

Ferrocenylthiazolidin-4-ones (3aem)

An ice-cooled solution of the corresponding primary amine

(1 mmol) and ferrocenecaboxaldehyde (214 mg, 1 mmol) in THF

(2 mL) was irradiated in an ultrasonic bath for 5 min, followed by

the addition of thioglycolic acid (184 mg, 2 mmol). After further

irradiation for 5 min, DCC (206 mg, 1 mmol) was added to the

resulting mixture and irradiation continued for another 15 min

under the same conditions. DCU was removed by ﬁltration, the

solvent evaporated and the residue taken up in EtOAc (30 mL). The

organic layer was washed with 5% aq. solution of citric acid, H₂O, 5%

aq. solution of NaHCO₃and brine, successively, and dried overnight

(anh. Na₂SO₄). After the evaporation of the solvent, the crude

mixture was puriﬁed by column chromatography (SiO₂/hexane -

EtOAc 9:1, v/v).

4. Experimental section

4.1. Chemistry

4.1.1. General

All commercially available chemicals and solvents were used

without further puriﬁcation. TLC experiments were performed on

alumina-backed silica gel 40 F254 plates (Merck, Darmstadt,

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

69

4.1.2.1. 3-Butyl-2-ferrocenyl-1,3-thiazolidin-4-one (3a). Yield 71%,

orange oil; IR (neat): n_max3094.9 (arC-H), 2957.6 ((CH₃)_as), 2930.9

4.1.2.4. 2-Ferrocenyl-3-octyl-1,3-thiazolidin-4-one (3d). Yield 71%,

yellow solid, mp 62 ^ꢁC; IR (KBr): n_max3092.9 (arC-H), 2954.1

((CH₃)_as), 2923.6 ((CH₂)_as), 2852.4 ((CH₂)_s), 1661.6 (C]O), 1440.6

((CH₂)_as), 2871.3 ((CH₃)_s), 1669.4 (C]O), 1442.0 (

1410.0, 1377.0 ( (CH₃)_s), 1297.0, 1105.8, 819.6; UVeVis (CH₃CN):

l_max(log ε) 422 (2.56), 202 (4.51) nm; ¹H NMR (200 MHz, CDCl₃):

5.51 (br. s, 1H, NeCHeS), 4.41 (m, 1H, HeC (5⁰)), 4.14e4.31

d(CH₂)_scissoring),

d

(

d

(CH₂)_scissoring), 1402.0, 1379.5 (

821.5; UVeVis (CH₃CN): l_max(log ε) 422 (2.61), 329 (2.82), 203

(4.80) nm; ¹H NMR (200 MHz, CDCl₃):

5.52 (br. s, 1H, NeCHeS),

d(CH₃)_s), 1307.2, 1105.1, 1002.0,

d

(overlapping peaks, 8H, HeC (1⁰⁰), HeC (2⁰⁰), HeC (3⁰⁰), HeC (4⁰⁰),

HeC (5⁰⁰), HeC (2⁰), HeC (3⁰), HeC (4⁰)), 3.61 (AA', 2H, SCH₂C]O),

3.36 (ddd, J ¼ 13.8, 8.4, 5.4 Hz, 1H, CH_AH_BN), 2.82 (ddd, J ¼ 13.8, 8.4,

5.4 Hz, 1H, CH_AH_BN), 1.11e1.40 (m, 4H,CH₂CH₂CH₃), 0.83 (t,

4.43 (m, 1H, HeC (5⁰)), 4.19e4.33 (overlapping peaks, 8H, HeC (1⁰⁰),

HeC (2⁰⁰), HeC (3⁰⁰), HeC (4⁰'), HeC (5⁰'), HeC (2⁰), HeC (3⁰), HeC

(4⁰)), 3.62 (AA', 2H, SCH₂C]O), 3.33 (ddd, J ¼ 13.9, 8.6, 5.3 Hz, 1H,

CH_AH_BN), 2.84 (ddd, J ¼ 13.9, 8.6, 5.3 Hz, 1H, CH_AH_BN), 1.1e1.31 (m,

12H, (CH₂)₆CH₃), 0.87 (t, J ¼ 6.5 Hz, 3H, CH₃); ¹³C NMR (50 MHz,

J ¼ 6.8 Hz, 3H, CH₃); ¹³C NMR (50 MHz, CDCl₃):

d 170.2 (C]O), 85.1

(C (1⁰)), 70.0, 69.8 (C (2⁰), C (5⁰)), 69.0 (C (1⁰⁰), C (2⁰⁰), C (3⁰⁰), C (4⁰⁰), C

(5⁰⁰)), 68.0, 67.7 (C (3⁰), C (4⁰)), 61.3 (NeCHeS), 42.0 (CH₂N), 33.4

(SCH₂C]O), 29.0 (CH₂CH₂N), 19.9 (CH₂CH₃), 13.7 (CH₃); MS (EI,

70 eV) m/z (%): 343 [M]^þ.(100), 310 (2.4), 270 (61.7), 230 (6), 213

(7.8), 199 (5.1), 186 (17.3), 166 (8.2), 148 (7.9), 121 (28.4), 97 (2.7), 77

(2.2), 56 (10.1), 41 (2.6); HRMS (ESI): m/z calculated for

CDCl₃): d

170.1 (C]O), 85.1 (C (1⁰)), 70.0, 69.8 (C (2⁰), C (5⁰)), 69.0 (C

(1⁰'), C (2⁰'), C (3⁰'), C (4⁰'), C (5⁰')), 68.4, 67.7 (C (3⁰), C (4⁰)), 61.4

(NeCHeS), 42.4 (CH₂N), 33.4 (SCH₂C]O), 31.7, 29.1, 29.0, 26.9, 26.7

(CH₂CH₂CH₂CH₂CH₂CH₂CH₃), 22.6 (CH₂CH₃), 14.0 (CH₃); MS (EI,

70 eV) m/z (%): 399 [M]^þ.(100), 366 (2.3), 326 (56.2), 291 (1.9), 260

(2.4), 230 (10), 213 (10.2), 199 (6.6), 186 (20.4),166 (8.4), 148 (6), 121

(27.7), 97 (2.5), 79 (2), 56 (7.4), 41 (6.5); HRMS (ESI): m/z calculated

for C₂₁H₂₉FeNOS þ H^þ[M þ H^þ]: 400.13975. Found: 400.13968;

Anal. Calcd for C₂₁H₂₉FeNOS: C, 63.16%; H, 7.32%; Fe, 13.98%; N,

3.51%; S, 8.03%. Found: C, 63.09%; H, 7.15%; N, 3.49%; S, 8.00%.

C

₁₇H₂₁FeNOSþH^þ[MþH^þ]: 344.07715. Found: 344.07709; Anal.

Calcd for C₁₇H₂₁FeNOS: C, 59.48%; H, 6.17%; Fe, 16.27%; N, 4.08%; S,

9.34%. Found: C, 59.14%; H, 6.28%; N, 3.73%; S, 9.54%.

4.1.2.2. 2-Ferrocenyl-3-pentyl-1,3-thiazolidin-4-one (3b). Yield 78%,

orange solid, mp 90 ^ꢁC; IR (KBr): n_max3091.6 (arC-H), 2952.9

((CH₃)_as), 2930.2 ((CH₂)_as), 2871.6 ((CH₃)_s), 2856.4 ((CH₂)_a), 1662.6

4.1.2.5. 3-Dodecyl-2-ferrocenyl-1,3-thiazolidin-4-one (3e). Yield 90%,

yellow solid, mp 70 ^ꢁC; IR (KBr): n_max2959.8 ((CH₃)_as), 2920.9

(C]O), 1458.7 (

1104.3, 1001.8, 820.2; UVeVis (CH₃CN): l_max(log ε) 422 (2.40), 315

(2.68), 203 (4.71) nm; ¹H NMR (200 MHz, CDCl₃):

5.52 (br. s, 1H,

d

(CH₂)_scissoring), 1401.9, 1380.6 (

d

(CH₃)_s), 1307.9,

((CH₂)_as), 2851.9 ((CH₂)_a), 1664.1 (C]O), 1466.0 (

1402.0, 1384.6 ( (CH₃)_s), 1287.5, 1122.7; UVeVis (CH₃CN): l_max(log

ε) 430 (2.61), 324 (2.74), 203 (4.71) nm; ¹H NMR (200 MHz, CDCl₃):

5.52 (br. s, 1H, NeCHeS), 4.44 (m, 1H, HeC (5⁰)), 4.17e4.34

d(CH₂)_scissoring),

d

NeCHeS), 4.43 (m, 1H, HeC (5⁰)), 4.21e4.32 (overlapping peaks,

8H, HeC (1⁰⁰), HeC (2⁰⁰), HeC (3⁰⁰), HeC (4⁰⁰), HeC (5⁰'), HeC (2⁰),

HeC (3⁰), HeC (4⁰)), 3.62 (AA', 2H, SCH₂C]O), 3.35 (ddd, J ¼ 13.9,

8.7, 5.2 Hz, 1H, CH_AH_BN), 2.84 (ddd, J ¼ 13.9, 8.7, 5.2 Hz, 1H,

d

(overlapping peaks, 8H, HeC (1⁰'), HeC (2⁰'), HeC (3⁰'), HeC (4⁰'),

HeC (5⁰'), HeC (2⁰), HeC (3⁰), HeC (4⁰)), 3.62 (AA', 2H, SCH₂C]O),

3.33 (ddd, J ¼ 14.0, 8.6, 5.4 Hz, 1H, CH_AH_BN), 2.83 (ddd, J ¼ 14.0, 8.6,

5.4 Hz, 1H, CH_AH_BN), 1.04e1.41 (m, 20H, (CH₂)₁₀), 0.87 (t, J ¼ 6.5 Hz,

CH_AH_BN), 1.1e1.48 (m, 6H, (CH₂)₃), 0.84 (t, J ¼ 6.8 Hz, 3H, CH₃); ¹³

C

NMR (50 MHz, CDCl₃):

d

170.1 (C]O), 85.1 (C (1⁰)), 70.0, 69.8 (C (2⁰),

3H, CH₃); ¹³C NMR (50 MHz, CDCl₃): 170.1 (C]O), 85.2 (C (1⁰)),

d

C (5⁰)), 69.0 (C (1⁰⁰), C (2⁰⁰), C (3⁰⁰), C (4⁰⁰), C (5⁰⁰)), 68.4, 67.7 (C (3⁰), C

(4⁰)), 61.4 (NeCHeS), 42.3 (CH₂N), 33.4 (SCH₂C]O), 28.8, 26.6, 22.2

(CH₂CH₂CH₂CH₃), 13.9 (CH₃); MS (EI, 70 eV) m/z (%): 357 [M]^þ.

(100), 324 (2.8), 284 (67), 264 (1.6), 249 (2.6), 230 (6.9), 213 (10.4),

199 (6.5), 186 (18.8), 166 (8), 148 (6.6), 121 (26.4), 97 (2.5), 77 (2), 56

(7.3), 43 (3.3); HRMS (ESI): m/z calculated for C₁₈H₂₃FeNOS þ H^þ

70.0, 69.8 (C (2⁰), C (5⁰)), 69.0 (C (1⁰⁰), C (2⁰⁰), C (3⁰⁰), C (4⁰⁰), C (5⁰⁰)),

68.4, 67.7 (C (3⁰), C (4⁰)), 61.4 (NeCHeS), 42.4 (CH₂N), 33.4 (SCH₂C]

O), 31.8, 29.1e29.6, 26.9, 26.7 ((CH₂)₉CH₂CH₃), 22.6 (CH₂CH₃), 14.1

(CH₃); MS (EI, 70 eV) m/z (%): 455 [M]^þ.(100), 422 (1.1), 382 (32.3),

347 (0.8), 310 (0.7), 288 (3.1), 230 (10.1), 213 (7.1), 199 (6.8), 186

(13.9), 166 (6.6), 148 (4.6), 121 (17.4), 97 (1.8), 69 (1.8), 55 (4.3), 43

(8.5); HRMS (ESI): m/z calculated for C₂₅H₃₇FeNOS þ H^þ[M þ H^þ]:

456.20235. Found: 456.20239; Anal. Calcd for C₂₅H₃₇FeNOS: C,

65.92%; H, 8.19%; Fe, 12.26%; N, 3.08%; S, 7.04%. Found: C, 66.50%; H,

7.85%; N, 3.54%; S, 7.52%.

[M

þ

H^þ]: 358.09280. Found: 358.09283; Anal. Calcd for

C

₁₈H₂₃FeNOS: C, 60.51%; H, 6.49%; Fe, 15.63%; N, 3.92%; S, 8.95%.

Found: C, 60.83%; H, 6.64%; N, 3.64%; S, 8.80%.

4.1.2.3. 2-Ferrocenyl-3-hexyl-1,3-thiazolidin-4-one (3c). Yield 72%,

yellow solid, mp 72 ^ꢁC; IR (KBr): n_max3095.2 (arC-H), 2954.3

((CH₃)_as), 2926.8 ((CH₂)_as), 2856.8 ((CH₂)_s), 1671.0 (C]O), 1441.0

4.1.2.6. 3-Hexadecyl-2-ferrocenyl-1,3-thiazolidin-4-one (3f). Yield 62%,

orange solid, mp 75 ^ꢁC; IR (KBr): n_max3090.8 (arC-H), 2951.1 ((CH₃)_as),

2917.6 ((CH₂)_as), 2870.7 ((CH₃)_s), 2849.1 ((CH₂)_a), 1664.1 (C]O),

(

d

(CH₂)_scissoring), 1409.3, 1377.2 (

1000.7, 818.2; UVeVis (CH₃CN): l_max(log ε) 430 (2.33), 203 (4.89)

nm; ¹H NMR (200 MHz, CDCl₃):

5.52 (br. s, 1H, NeCHeS), 4.43 (m,

d(CH₃)_s), 1298.2, 1225.7, 1105.9,

1464.6

1002.2, 823.0; UVeVis (CH₃CN): l_max(log ε) 441 (2.20), 324 (2.25),

203 (4.72) nm; ¹H NMR (200 MHz, CDCl₃):

5.52 ((br. s,1H, NeCHeS),

(d(CH₂)_scissoring), 1402.3, 1380.9 (d(CH₃)_s), 1307.9, 1104.9,

d

1H, HeC (5⁰)), 4.16e4.33 (overlapping peaks, 8H, HeC (1⁰⁰), HeC

(2⁰⁰), HeC (3⁰⁰), HeC (4⁰⁰), HeC (5⁰'), HeC (2⁰), HeC (3⁰), HeC (4⁰)),

3.62 (AA', 2H, SCH₂C]O), 3.31 (ddd, J ¼ 13.9, 8.6, 5.3 Hz, 1H,

CH_AH_BN), 2.80 (ddd, J ¼ 13.9, 8.6, 5.3 Hz,1H, CH_AH_BN),1.08e1.46 (m,

8H, (CH₂)₄CH₃), 0.85 (t, J ¼ 6.5 Hz, 3H, CH₃); ¹³C NMR (50 MHz,

d

4.43 (m, 1H, HeC (5⁰)), 4.18e4.32 (overlapping peaks, 8H, HeC (1⁰⁰),

HeC (2⁰⁰), HeC (3⁰⁰), HeC (4⁰⁰), HeC (5⁰'), HeC (2⁰), HeC (3⁰), HeC (4⁰)),

3.62 (AA', 2H, SCH₂C]O), 3.33 (ddd, J ¼ 13.8, 8.4, 5.4 Hz, 1H, CH_AH_BN),

2.82 (ddd, J ¼ 13.8, 8.4, 5.4 Hz, 1H, CH_AH_BN), 1.03e1.39 (m, 28H,

(CH₂)₁₄CH₃), 0.88 (t, J ¼ 6.6 Hz, 3H, CH₃); ¹³C NMR (50 MHz, CDCl₃):

CDCl₃): d

170.1 (C]O), 85.1 (C (1⁰)), 70.0, 69.8 (C (2⁰), C (5⁰)), 69.0 (C

(1⁰⁰), C (2⁰⁰), C (3⁰⁰), C (4⁰⁰), C (5⁰⁰)), 68.4, 67.7 (C (3⁰), C (4⁰)), 61.4

(NeCHeS), 42.4 (CH₂N), 33.4 (SCH₂C]O), 31.3, 26.8, 26.4

(CH₂CH₂CH₂CH₂CH₃), 22.4 (CH₂CH₃), 13.9 (CH₃); MS (EI, 70 eV) m/z

(%): 371 [M]^þ.(100), 338 (2.4), 298 (58), 263 (2.1), 240 (2.2), 213

(10.6), 199 (5.5), 186 (18.5), 166 (7.8), 148 (6.4), 121 (28.2), 97 (2.6),

77 (2), 56 (8.7), 43 (5.5); HRMS (ESI): m/z calculated for

d

170.1 (C]O), 85.2 (C (1⁰)), 70.0, 69.8 (C (2⁰), C (5⁰)), 69.0 (C (1⁰⁰), C

(2⁰⁰), C (3⁰'), C (4⁰⁰), C (5⁰⁰)), 68.5, 67.8 (C (3⁰), C (4⁰)), 61.4 (NeCHeS),

42.4 (CH₂N), 33.5 (SCH₂C]O), 31.9, 29.2e29.7, 26.9, 26.7

((CH₂)₁₃CH₂CH₃), 22.7 (CH₂CH₃), 14.1 (CH₃); MS (EI, 70 eV) m/z (%):

511 [M]^þ.(100), 478 (0.6), 438 (18.7), 397 (0.4), 366 (0.5), 344 (1.4),

324 (0.9), 287 (1.6), 260 (1.4), 230 (11), 213 (6.2), 199 (7.1), 186 (11.4),

166 (6.3), 148 (4.2), 121 (12.9), 97 (1.6), 69 (2.4), 57 (5.3), 43 (12.3);

HRMS (ESI): m/z calculated for C₂₉H₄₅FeNOS þ H^þ[M þ H^þ]:

512.26495. Found: 512.26501; Anal. Calcd for C₂₉H₄₅FeNOS: C,

C

₁₉H₂₅FeNOS þ H^þ[M þ H^þ]: 372.10845. Found: 372.10840; Anal.

Calcd for C₁₉H₂₅FeNOS: C, 61.46%; H, 6.79%; Fe, 15.04%; N, 3.77%; S,

8.64%. Found: C, 61.31%; H, 6.90%; N, 3.65%; S, 8.83%.

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

70

68.08%; H, 8.87%; Fe, 10.92%; N, 2.77%; S, 6.27%. Found: C, 67.83%; H,

7.85%; N, 2.88%; S, 6.26%.

1300.6, 1232.5, 1105.7, 1038.9, 822.9, 703.0; UVeVis (CH₃CN): l_max

(log ε) 430 (2.32), 322 (2.33), 202 (4.89) nm; ¹H NMR (200 MHz,

CDCl₃):

d

7.21 (dd, J ¼ 5.0, 1.2 Hz, 1H, HeC (5⁰'')), 6.93 (dd, J ¼ 5.0,

4.1.2.7. 2-Ferrocenyl-3-(4-methoxyphenethyl)-1,3-thiazolidin-4-one

(3g). Yield 82%, ocher solid, mp 130 ^ꢁC; IR (KBr): n_max3100.2 (arC-

H), 2965.2 ((OCH₃)_as), 2924.1 ((CH₂)_as), 2837.6 ((CH₂)_a), 1667.6 (C]

3.4 Hz, 1H, HeC (4⁰'')), 6.86 (br. d, J ¼ 3.4 Hz, 1H, HeC (3⁰'')), 5.45 (br.

s,1H, NeCHeS), 4.95 (br. d, J ¼ 15.3 Hz,1H, CH_AH_BN), 4.43 (dt, J ¼ 2.4,

1.2, 1.2 Hz, 1H, HeC (5⁰)), 4.20e4.32 (overlapping peaks, 8H, HeC

(1⁰⁰), HeC (2⁰⁰), HeC (3⁰⁰), HeC (4⁰⁰), HeC (5⁰⁰), HeC (2⁰), HeC (3⁰),

HeC (4⁰)), 3.87 (d, J ¼ 15.3 Hz, 1H, CH_AH_BN), 3.64 (AA', 2H, SCH₂C]

O), 1511.0 (arC ¼ arC), 1458.2 (

d

(CH₂)_scissoring), 1401.1, 1304.5

(d

(CH₃)_s), 1240.7, 1176.9, 1029.2, 817.0 (g

(arC-H)); UVeVis (CH₃CN):

l_max(log ε) 438 (2.18), 431 (2.18), 197 (5.08) nm; MS (EI, 70 eV) m/z

(%): 421 [M]^þ.(100), 388 (0.2), 348 (12.5), 314 (7.1), 287 (14.8), 255

(1.8), 226 (9), 199 (7), 186 (9.5), 166 (5.4), 148 (2.4), 135 (16.5), 121

(36.6), 105 (4.3), 91 (5.9), 77 (6.6), 65 (2), 56 (8.2), 39 (1.1); HRMS

(ESI): m/z calculated for C₂₂H₂₃FeNOSþH^þ[MþH^þ]: 406.09280.

Found: 406.09286; Anal. Calcd for C₂₂H₂₃FeNOS: C, 62.71%; H,

5.50%; Fe, 13.25%; N, 3.32%; S, 7.62%. Found: C, 62.59%; H, 5.31%; N,

3.60%; S, 7.58%.

O); ¹³C NMR (50 MHz, CDCl₃): 170.2 (C]O), 138.1 (C (2⁰'')), 126.8,

d

126.6, 125.4 (C (3⁰''), C (4⁰''), C (5⁰'')), 84.3 (C (1⁰)), 70.6, 69.9 (C (2⁰), C

(5⁰)), 69.0 (C (1⁰⁰), C (2⁰⁰), C (3⁰⁰), C (4⁰⁰), C (5⁰⁰)), 68.4, 67.7 (C (3⁰), C

(4⁰)), 60.2 (NeCHeS), 39.9 (CH₂N), 33.2 (SCH₂C]O); MS (EI, 70 eV)

m/z (%): 383 [M]^þ.(100), 334 (0.3), 308 (11.4), 290 (1.2), 275 (6), 245

(1.6), 230 (9.2), 213 (26.3), 186 (8.3), 166 (4.7), 121 (27.8), 97 (32.7),

56 (10.4), 45 (3.4); HRMS (ESI): m/z calculated for

C

₁₈H₁₇FeNOS₂þ H^þ[M þ H^þ]: 383.01010. Found: 383.01002; Anal.

Calcd for C₁₈H₁₇FeNOS₂: C, 56.40%; H, 4.47%; Fe, 14.57%; N, 3.65%; S,

16.73%. Found: C, 56.12%; H, 4.28%; N, 3.47%; S, 16.94%.

4.1.2.8. 3-Benzyl-2-Ferrocenyl-1,3-thiazolidin-4-one (3h). Yield 80%,

yellow solid, mp 105 ^ꢁC; IR (KBr): n_max3086.7 (arC-H), 2923.9

((CH₂)_as), 1669.6 (C]O), 1495.0 (arC ¼ arC), 1434.7 (

1399.4, 1299.3, 1105.7, 817.1, 746.3 ( (arC-H)), 698.4; UVeVis

(CH₃CN): l_max(log ε) 430 (2.24), 322 (2.38), 202 (4.78) nm; ¹H NMR

(200 MHz, CDCl₃):

7.30 (overlapping peaks, 3H, HeC (3⁰''), HeC

d

(CH₂)_scissoring),

4.1.2.11. 2-Ferrocenyl-3-phenyl-1,3-thiazolidin-4-one (3k). Yield 61%,

light orange solid, mp 146 ^ꢁC; IR (KBr): n_max3099.4 (arC-H), 2909.6

((CH₂)_as), 1673.8 (C]O), 1592.0 (arC ¼ arC), 1494.9 (arC ¼ arC),

g

d

1454.1 (d(CH₂)_scissoring), 1401.9, 1276.5, 1215.8, 1025.7, 810.9, 692.1

(4⁰''), HeC (5⁰'')), 7.13 (m, 2H, HeC (2⁰''), HeC (6⁰'')), 5.34 (br. s, 1H,

NeCHeS), 4.96 (br. d, J ¼ 15.1 Hz, 1H, CH_AH_BN), 4.41 (m, 1H, HeC

(5⁰)), 4.27 (m, 1H, HeC (4⁰)), 4.15e4.23 (overlapping peaks, 6H, HeC

(1⁰'), HeC (2⁰'), HeC (3⁰'), HeC (4⁰'), HeC (5⁰'), HeC (3⁰)), 4.04 (dt,

J ¼ 2.2, 1.1, 1.1 Hz, 1H, HeC (2⁰)), 3.72 (AA', 2H, SCH₂C]O), 3.61 (d,

(ф(arCearC)); UVeVis (CH₃CN): l_max(log ε) 439 (2.19), 431 (2.18),

203 (4.92) nm; ¹H NMR (200 MHz, CDCl₃):

d

7.28 (m, 3H, over-

lapping peaks, HeC (3⁰''), HeC (4⁰''), HeC (5⁰'')), 6.96 (dd, J ¼ 8.0,

1.7 Hz, 2H, HeC (2⁰''), HeC (6⁰'')), 5.90 (br. s, 1H, NeCHeS), 4.47 (dt,

J ¼ 2.5, 1.3, 1.3 Hz,1H, HeC (5⁰)), 4.2 (tdd, J ¼ 2.5, 1.3, 0.9 Hz, 1H, HeC

(4⁰)), 4.15 (s, 5H, HeC (1⁰⁰), HeC (2⁰⁰), HeC (3⁰⁰), HeC (4⁰⁰), HeC (5⁰⁰)),

3.99 (td, J ¼ 2.5, 2.5,1.3, 1H, HeC (3⁰)), 3.81 (AA', 2H, SCH₂C]O), 3.70

(dt, J ¼ 2.5, 1.3, 1.3 Hz, 1H, HeC (2⁰)); ¹³C NMR (50 MHz, CDCl₃):

J ¼ 15.1 Hz, 1H, CH_AH_BN); ¹³C NMR (50 MHz, CDCl₃):

d 170.5 (C]O),

135.7 (C (1⁰'')), 128.5, 127.8 (C (2⁰''), C (3⁰''), C (5⁰''), C (6⁰'')), 127.4 (C

(4⁰'')), 84.6 (C (1⁰)), 70.5, 69.8 (C (2⁰), C (5⁰)), 68.9 (C (1⁰⁰), C (2⁰⁰), C

(3⁰⁰), C (4⁰⁰), C (5⁰⁰)), 68.2, 67.6 (C (3⁰), C (4⁰)), 60.5 (NeCHeS), 45.2

(CH₂N), 33.3 (SCH₂C]O); MS (EI, 70 eV) m/z (%): 377 [M]^þ.(100),

344 (0.8), 304 (16.4), 269 (6.8), 237 (6.5), 213 (26.4), 186 (7.9), 166

(6), 146 (6.4), 121 (25.3), 91 (21.2), 65 (4.9), 56 (10), 39 (1.3); HRMS

(ESI): m/z calculated for C₂₀H₁₉FeNOSþH^þ[MþH^þ]: 378.06150.

Found: 378.06143; Anal. Calcd for C₂₀H₁₉FeNOS: C, 63.67%; H,

5.08%; Fe, 14.80%; N, 3.71%; S, 8.50%. Found: C, 63.91%; H, 4.96%; N,

3.89%; S, 8.78%.

d

170.3 (C]O), 137.1 (C (1⁰'')), 129.0 (C (3⁰''), C (5⁰'')), 127.8 (C (4⁰'')),

127.7 (C (2⁰''), C (6⁰'')), 85.3 (C (1⁰)), 70.4, 69.4 (C (2⁰), C (5⁰)), 68.8 (C

(1⁰'), C (2⁰'), C (3⁰'), C (4⁰'), C (5⁰')), 68.3, 67.2 (C (3⁰), C (4⁰)), 63.9

(NeCHeS), 33.6 (SCH₂C]O); MS (EI, 70 eV) m/z (%): 363 [M]^þ.(100),

345 (0.1), 321 (2.3), 303 (0.1), 290 (34.6), 269 (3), 255 (6), 224 (15.4),

186 (10.4), 145 (4), 121 (20.2), 104 (3.5), 77 (7.8), 56 (8.9), 39 (1.1);

HRMS (ESI): m/z calculated for C₁₉H₁₇FeNOS þ H^þ[M þ H^þ]:

364.04585. Found: 364.04578; Anal. Calcd for C₁₉H₁₇FeNOS: C,

62.82%; H, 4.72%; Fe, 15.37%; N, 3.86%; S, 8.83%. Found: C, 53.01%; H,

4.83%; N, 3.55%; S, 8.98%.

4.1.2.9. 2-Ferrocenyl-3-furfuryl-1,3-thiazolidin-4-one

(3i). Yield

99%, light orange oil; IR (neat): n_max2924.1 ((CH₂)_as), 1680.3 (C]O),

1503.8 (arC ¼ arC), 1400.3, 1301.4, 1228.7, 1046.0, 1008.7, 821.5,

738.7; UVeVis (CH₃CN): l_max(log ε) 431 (2.28), 422 (2.39), 204

4.1.2.12. 2-Ferrocenyl-3-(m-tolyl)-1,3-thiazolidin-4-one

(3l). Yield

63%, light yellow solid, mp 138 ^ꢁC; IR (KBr): n_max3079.4 (arC-H),

2920.3 ((CH₂)_as), 1673.8 (C]O), 1586.6 (arC ¼ arC), 1490.8 (arC ¼ arC),

(4.82) nm; ¹H NMR (200 MHz, CDCl₃):

d

7.37 (dd, J ¼ 1.8, 0.7 Hz, 1H,

HeC (5⁰'')), 6.30 (dd, J ¼ 3.2, 1.8 Hz, 1H, HeC (4⁰'')), 6.18 (br. d,

J ¼ 3.2 Hz, 1H, HeC (3⁰'')), 5.48 (br. s, 1H, NeCHeS), 4.81 (br. d,

J ¼ 15.6 Hz, 1H, CH_AH_BN), 4.43 (dt, J ¼ 2.4, 1.3, 1.3 Hz, 1H, HeC (5⁰)),

4.18e4.34 (overlapping peaks, 8H, HeC (1⁰'), HeC (2⁰'), HeC (3⁰'),

HeC (4⁰'), HeC (5⁰'), HeC (2⁰), HeC (3⁰), HeC (4⁰)), 3.70 (d,

J ¼ 15.6 Hz, 1H, CH_AH_BN), 3.65 (AA', 2H, SCH₂C]O); ¹³C NMR

1455.9 (d(CH₂)_scissoring), 1365.1, 1300.1, 1215.7, 1106.3, 1000.1, 820.8

(g

(arC-H)), 692.4 (ф(arCearC)); UVeVis (CH₃CN): l_max(log ε) 431

(2.23), 204 (4.96) nm; ¹H NMR (200 MHz, CDCl₃):

d

7.18 (t, J ¼ 7.6,

7.6 Hz, 1H, HeC (5⁰'')), 7.05 (br. d, J ¼ 7.6 Hz, 1H, HeC (4⁰'')), 6.79

(overlapping peaks, 2H, HeC (2⁰''), HeC (6⁰'')), 5.88 (br. s, 1H,

NeCHeS), 4.47 (dt, J ¼ 2.5, 1.3, 1.3 Hz, 1H, HeC (5⁰)), 4.21 (tdd, J ¼ 2.5,

1.3, 0.5 Hz, 1H, HeC (4⁰)), 4.15 (s, 5H, HeC (1⁰'), HeC (2⁰'), HeC (3⁰'),

HeC (4⁰'), HeC (5⁰')), 4.01 (td, J ¼ 2.5, 2.5, 1.3, 1H, HeC (3⁰)), 3.81 (AA',

2H, SCH₂C]O), 3.71 (dt, J ¼ 2.5, 1.3, 1.3 Hz, 1H, HeC (2⁰)), 2.27 (s, 3H,

(50 MHz, CDCl₃): d

170.2 (C]O), 149.5 (C (2⁰'')), 142.3 (C (5⁰'')), 110.2,

108.6 (C (3⁰''), C (4⁰'')), 84.3 (C (1⁰)), 70.7, 69.8 (C (2⁰), C (5⁰)), 69.0 (C

(1⁰'), C (2⁰'), C (3⁰'), C (4⁰'), C (5⁰')), 68.4, 67.6 (C (3⁰), C (4⁰)), 60.7

(NeCHeS), 38.1 (CH₂N), 33.2 (SCH₂C]O); MS (EI, 70 eV) m/z (%):

367 [M]^þ.(100), 334 (0.3), 320 (0.3), 292 (9), 259 (3.2), 244 (5.1),

230 (9.6), 213 (28.6), 186 (7.8), 166 (4.5), 146 (2.1), 129 (6.5), 121

(26.4), 94 (2.6), 81 (21.3), 56 (10.1), 39 (1.4); HRMS (ESI): m/z

calculated for C₁₈H₁₇FeNO₂S þ H^þ[M þ H^þ]: 368.04077. Found:

368.04075; Anal. Calcd for C₁₈H₁₇FeNO₂S: C, 58.87%; H, 4.67%; Fe,

15.21%; N, 3.81v; S, 8.73%. Found: C, 58.59%; H, 4.73%; N, 3.68%; S,

8.53%.

CH₃); ¹³C NMR (50 MHz, CDCl₃): 170.4 (C]O), 139.0 (C (1⁰'')), 137.1 (C

d

(3⁰'')), 128.8, 128.7, 128.4, 124.8 (C (2⁰''), C (4⁰''), C (5⁰''), C (6⁰'')), 85.5 (C

(1⁰)), 70.5, 69.4 (C (2⁰), C (5⁰)), 68.9 (C (1⁰'), C (2⁰'), C (3⁰'), C (4⁰'), C (5⁰')),

68.3, 67.2 (C (3⁰), C (4⁰)), 64.0 (NeCHeS), 33.6 (SCH₂C]O), 21.2 (CH₃);

MS (EI, 70 eV) m/z (%): 377 [M]^þ.(100), 359 (0.2), 335 (2.6), 319 (0.2),

304 (41.3), 283 (3.5), 269 (6.9), 238 (18.8), 214 (8.7), 182 (12.3), 166

(7.9), 152 (9.2), 121 (25.1), 91 (11.1), 77 (1.7), 56 (10.1), 39 (1.9); HRMS

(ESI): m/z calculated for C₂₀H₁₉FeNOS þ H^þ[M þ H^þ]: 378.06150.

Found: 378.06154; Anal. Calcd for C₂₀H₁₉FeNOS: C, 63.67%; H, 5.08%;

Fe, 14.80%; N, 3.71%; S, 8.50%. Found: C, 63.51%; H, 5.22%; N, 3.61%; S,

8.64%.

4.1.2.10. 2-Ferrocenyl-3-thenyl-1,3-thiazolidin-4-one (3j). Yield 74%,

yellow oil; IR (neat): n_max2924.1 ((CH₂)_as), 1675.8 (C]O), 1400.2,

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A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

71

Table 5

4.1.2.13. 2-Ferrocenyl-3-(p-tolyl)-1,3-thiazolidin-4-one

(3m).

Yield 48%, light yellow solid, mp 154 ^ꢁC; IR (KBr): n_max3072.5 (arC-

H), 2922.8 ((CH₂)_as), 1672.0 (C]O), 1514.0 (arC ¼ arC), 1456.8

Crystallographic data for crystal structure of 3k.

Empirical formula

Formula weight

Color, crystal shape

Crystal size (mm³)

Temperature (K)

Wavelength (Å)

Crystal system

Space group

C19 H17 Fe N O S

363.25

Orange, prism

(d(CH₂)_scissoring), 1384.6 (d(CH₃)_s), 1366.7, 1303.8, 1006.5, 1025.7,

(

g

(arC-H)); UVeVis (CH₃CN): l_max(log ε) 432 (2.26), 204 (4.85) nm;

0.35 ꢃ 0.19 ꢃ 0.15

¹H NMR (200 MHz, CDCl₃):

d

7.09 (AA'BB', J ¼ 8.2 Hz, 2H, HeC (3⁰''),

293 (2)

HeC (5⁰'')), 6.82 (AA'BB', J ¼ 8.2 Hz, 2H, HeC (2⁰''), HeC (6⁰'')), 5.85

(br. s, 1H, NeCHeS), 4.49 (br. s, 1H, HeC (5⁰)), 4.22 (br. s, 1H, HeC

(4⁰)), 4.17 (s, 5H, HeC (1⁰'), HeC (2⁰'), HeC (3⁰'), HeC (4⁰'), HeC (5⁰')),

4.03 (br. s, 1H, HeC (3⁰)), 3.81 (AA', 2H, SCH₂C]O), 3.73 (br. s, 1H,

0.71073

Monoclinic

P2₁/n

Unit cell dimensions

a (Å)

b (Å)

9.0589 (4)

5.8848 (3)

29.7237 (13)

90

96.106 (4)

90

1575.58 (13)

4

1.531

1.092

2.90 to 29.00

7006

3594, 0.0260

99.9

3594/0/208

1.067

HeC (2⁰)), 2.29 (s, 3H, CH₃); ¹³C NMR (50 MHz, CDCl₃):

d 170.5 (C]

O), 137.8 (C (1⁰'')), 134.5 (C (4⁰'')), 129.7 (C (3⁰''), C (5⁰'')), 127.6 (C (2⁰''),

C (6⁰'')), 85.5 (C (1⁰)), 70.6, 69.4 (C (2⁰), C (5⁰)), 68.9 (C (1⁰'), C (2⁰'), C

(3⁰'), C (4⁰'), C (5⁰')), 68.4, 67.3 (C (3⁰), C (4⁰)), 64.0 (NeCHeS), 33.6

(SCH₂C]O), 21.1 (CH₃); MS (EI, 70 eV) m/z (%): 377 [M]^þ.(100), 359

(0.2), 335 (2.4), 319 (0.2), 304 (35), 283 (2.8), 269 (6.5), 238 (16.5),

214 (9.8), 182 (11.9), 166 (7.4), 152 (10.4), 121 (25.9), 91 (9.4), 77

(1.9), 56 (10.9), 39 (1.8); HRMS (ESI): m/z calculated for

c (Å)

a

b

g

(^ꢁ)

V (Å³)

Z

D_calc(Mg/m³)

m

(mm^ꢀ1

range for data collection (^ꢁ)

)

q

C

₂₀H₁₉FeNOS þ H^þ[M þ H^þ]: 378.06150. Found: 378.06152; Anal.

Reﬂections collected

Independent reﬂections, R_int

Completeness to

Calcd for C₂₀H₁₉FeNOS: C, 63.67%; H, 5.08%; Fe, 14.80%; N, 3.71%; S,

8.50%. Found: C, 63.38%; H, 5.08%; N, 3.93%; S, 8.62%.

q

¼ 26.00^ꢁ

Data/restraints/parameters

Goodness-of-ﬁt on F²

4.1.3. Crystallographic analysis

Single-crystal diffraction data for compound 3k were collected

at room temperature on an Agilent Gemini S diffractometer with

Final R₁/wR₂indices (I > 2s_I

)

0.0458, 0.0849

0.0651, 0.0921

0.252 and ꢀ0.366

Final R₁/wR₂indices (all data)

Largest diff. peak and hole (e Å^ꢀ3

)

graphite-monochromated MoK

a

radiation (

l

¼ 71073 Å). Data

reduction and empirical absorption corrections were accomplished

using CrysAlisPro [52]. Crystal structure was solved by direct

methods, using SIR2002 [53] and reﬁned using SHELXL program

[54]. All non-H atoms were reﬁned anisotropically to convergence.

All H atoms were placed at geometrically calculated positions with

the CeH distances ﬁxed to 0.93 from Csp²and 0.97 and 0.98 Å from

methylene and methine Csp³, respectively. The corresponding

isotropic displacement parameters of the hydrogen atoms were

equal to 1.2U_eqand 1.5U_eqof the parent Csp²and Csp³, respectively.

The crystallographic data are listed in Table 5. The PARST [55],

PLATON [56] and WinGX [57] programs were used to perform

geometrical calculation. Figures were produced using ORTEP-3 [58]

and MERCURY, Version 2.4 [59].

bulb, whereas the smaller compartment was black and not illu-

minated at all. Each animal was placed at the center of the illumi-

nated compartment, facing away from the round passage. The time

spent in illuminated and dark places, time of the ﬁrst crossing

(transition), as well as the number of entries in each space, was

recorded for 5 min [47].

4.2.3. Open ﬁeld (OF) test

In order to detect any association to immobility in the tests and

changes in motor activity, the OF apparatus was used. The studies

were carried out on mice according to a method previously

described [47]. The ﬂoor of the apparatus was divided into twenty-

ﬁve equal (10 ꢃ 10 cm) squares. Mice were placed individually into

the corner of the arena and allowed to explore it freely. Behavior

scores included the frequency of ambulation (the number of

crossing sector lines with all four paws) and rearing (number of

times mouse stood on its hind limbs).

4.2. Pharmacology

4.2.1. Animals and treatment

Male albino BALB/c mice (4 weeks old) weighing 20e25 g were

used. The animals were kept in cages at room temperature and

allowed access to food and water ad libitum. Fourteen hours before

the start of the experiments the animals were sent to the lab and

were given only water. The experiments were performed, in

accordance with the declaration of Helsinki and European Com-

munity guidelines for the ethical handling of laboratory animals

(EEC Directive of 1986; 86/609/EEC) and the related ethics regula-

tions of our University (01-2857-4). Experimental groups consisted

of 6 animals and all animals were injected intraperitoneally (ip)

with experimental substances (25, 50 and 100 mg/kg) or with

4.2.4. Horizontal wire (HW) test

The HW test was used to assess a compound's effects on the

muscle tone of mice. The test apparatus was based on that

described earlier [47]. The number of animals that were unable to

grasp a horizontal wire, with either the forepaws, or at least with

one hindpaw within 10 s was recorded.

ꢁ

control substances (diazepam (Hemofarm, Vrsac, Serbia), 2 mg/kg,

4.2.5. Involvement of the GABA receptor complex in anxiolytic

activity of compound 3g

or olive oil, 10 mL/kg), 1 h before the commencement of each

experiment.

The involvement of GABA_A-receptor complexes was evaluated

by experiments that included the application of a competitive

antagonist Flumazenil (FLU; SigmaeAldrich, St. Louis, Missouri,

USA). Three groups of mice (6 per group) were given FLU (15 min

before other substances) as an ip injection (3 mg/kg) and after-

wards the animals were treated as follows: group I (negative con-

trol group) received vehicle (olive oil) in a dose of 10 mL/kg, group II

(positive control group) received diazepam in a dose of 1 mg/kg and

group III (experimental) received compound 3g in a dose of 50 mg/

4.2.2. Light/dark (LD) test

The light/dark transition (the apparatus was a box of the

following dimensions: 40 cm ꢃ 60 cm ꢃ 20 cm) was used as the test

of unconditioned anxiety. The apparatus had two chambers con-

nected by a round opening (7.0 cm) located at ﬂoor level, in the

center of the dividing wall, by which mice could cross between the

chambers. A larger chamber was white and illuminated by a 60 W

ꢀ

A. Pejovic et al. / European Journal of Medicinal Chemistry 83 (2014) 57e73

72

kg [45]. After the treatments the animals were submitted to a LD

test.

by AutoDock Vina ability to accurately predict the diazepam-

binding site, consistent with the available experimental data

[23,49]. Autodock Tools version 1.5.6 was used to convert the ligand

and receptor molecules to proper ﬁle formats (pdbqt) for AutoDock

Vina docking. The same program was used for the visualization of

docking results [63]. The output pdbqt ﬁles for the preferred BZD-

docking poses of the most active compound (3g, in vivo experi-

ments) are given in Supporting Information. All in silico experi-

ments were run using Intel^®Core™ i7-3930K 3.20 GHz Six core

unlocked CPU Processor.

A second set of experiments served to evaluate the interaction of

compound 3g with the picrotoxin GABA_A-receptor-binding site. An

ip dose of a non-competitive antagonist picrotoxin (PIC, 1 mg/kg;

Tokyo Chemical Industry, Tokyo, Japan) administered 15 min before

other substances was used in this sense [60,61]. The treatments of

positive and negative controls, as well as the experimental groups,

were same as described in the previous paragraph. After the

treatments the animals were submitted to a LD test.

4.2.6. Diazepam-induced sleep

Transparency declarations

Sleep inducing or potentiating effects of the synthesized library

compounds were investigated in experiments where a 20 mg/kg

dose (ip) of diazepam was used to induce sleep in mice. The time

required to induce loss of the righting reﬂex was deﬁned as sleep

latency, while the time that elapsed between the loss and recovery

of the righting reﬂex was considered sleeping time [47].

The authors declare no conﬂict of interest.

Acknowledgments

The authors from Serbia acknowledge the ﬁnancial support

(Projects No. 172034 and 172061) of the Ministry of Education,

Science and Technological Development of the Republic of Serbia.

4.2.7. Anticonvulsant activity

The inﬂuence of 3g on convulsions induced by pentylenetetrazol

(PTZ; Alfa Aesar GmbH & Co KG, Karlsruhe, Germany) and isoniazid

(ISN; Tokyo Chemical Industry, Tokyo, Japan) was studied [48]. One

hour before PTZ (70 mg/kg) or INH (250 mg/kg) injections, the

animals were ip treated with vehicle (olive oil, 10 mL/kg), 3g

(50 mg/kg) or diazepam (1 mg/kg). After the PTZ application the

onset of seizures (sec) and occurrence of hind-limb tonic exten-

sions (sec) were recorded, whereas in ISN treated animals the

seizure onset (min) and % of living mice in 30 min were monitored.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2014.05.062.

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Article Doi

Discovery of anxiolytic 2-ferrocenyl-1,3-thiazolidin-4-ones exerting GABAA receptor interaction via the benzodiazepine-binding site

DOI: 10.1016/j.ejmech.2014.05.062

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Full text of DOI:10.1016/j.ejmech.2014.05.062

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