1,3-substituted imidazolidine-2,4,5-triones: Synthesis and inhibition of cholinergic enzymes

Article abstract of DOI:10.3390/molecules16097565

A series of novel and highly active acetylcholinesterase and butyrylcholinesterase inhibitors derived from substituted benzothiazoles containing an imidazolidine-2,4,5-trione moiety were synthesized and characterized. The molecular structure of 1-(2,6-dii

Full text of DOI:10.3390/molecules16097565

Molecules 2011, 16, 7565-7582; doi:10.3390/molecules16097565
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
1,3-Substituted Imidazolidine-2,4,5-triones: Synthesis and
Inhibition of Cholinergic Enzymes
Vladimir Pejchal 1, Sarka Stepankova 2, Zdenka Padelkova 3, Ales Imramovsky 1,*
and Josef Jampilek 4,*
1
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology,
University of Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
2
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology,
University of Pardubice, Studentska 573, Pardubice 53210, Czech Republic
3
Department of General and Inorganic Chemistry, Faculty of Chemical Technology,
University of Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
4
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical
Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
* Authors to whom correspondence should be addressed; E-Mails: ales.imramovsky@upce.cz (A.I.);
josef.jampilek@gmail.com (J.J.); Tel.: +42-0466037739 (A.I.); +42-0541562925 (J.J.);
Fax: +42-0466038004 (A.I.); +42-0541240607 (J.J.).
Received: 16 August 2011; in revised form: 30 August 2011 / Accepted: 31 August 2011 /
Published: 5 September 2011
Abstract:
A
series of novel and highly active acetylcholinesterase and
butyrylcholinesterase inhibitors derived from substituted benzothiazoles containing an
imidazolidine-2,4,5-trione moiety were synthesized and characterized. The molecular
structure of 1-(2,6-diisopropyl-phenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-
imidazolidine-2,4,5-trione (3g) was determined by single-crystal X-ray diffraction. Both
optical isomers are present as two independent molecules in the triclinic crystal system.
The lipophilicity of the compounds was determined as the partition coefficient log Kow
using the traditional shake-flask method. The in vitro inhibitory activity on
acetylcholinesterase from electric eel and butyrylcholinesterase isolated from equine serum
was determined. The inhibitory activity on acetylcholinesterase was significantly higher
than that of the standard drug rivastigmine. The discussed compounds are also promising
inhibitors of butyrylcholinesterase, as some of the prepared compounds inhibit
butyrylcholinesterase better than the internal standards rivastigmine and galanthamine. The
highest inhibitory activity (IC50 = 1.66 μmol/L) corresponds to the compound

Molecules 2011, 16
1-(4-isopropylphenyl)-3-[(R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethyl]imidazolidine-2,4,5-trione
7566
(3d). For all the studied compounds, the relationships between the lipophilicity and the
chemical structure as well as their structure-activity relationships are discussed.
Keywords: 1-(aryl)-3-[(R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethyl]imidazolidine-2,4,5-triones;
X-ray diffraction; lipophilicity; in vitro acetylcholinesterase inhibition; in vitro
butyrylcholinesterase inhibition; structure-activity relationships
1. Introduction
Substituted benzothiazoles are heterocyclic systems with a wide range of interesting biological
activities. The substituents of these compounds on the 2-position have been found to be the most
important modulators of the bio-activity of this type of compounds. Diverse biological activities such
as antibacterial [1], fungicidal [2] and anticancer [3,4] were described. Benzothiazoles and
benzoxazoles are also described as potent inhibitors of various enzymes such as 5-lipoxygenase [5],
cyclooxygenase [6], aldose and aldehyde reductase [7], serine hydrolases [8,9] or thrombin inhibitors,
known as coagulation factor II (F2) [10]. Generally it can be noted that benzothiazoles can serve as
unique and versatile scaffolds for experimental drug design.
The group of serine hydrolases includes two important enzymes: Acetylcholinesterase (AChE,
EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) also known as pseudocholinesterase. These
serine hydrolases belong structurally to the class of proteins known as the esterase/lipase family,
within the α/β-hydrolase fold superfamily [11]. The major role of AChE is to catalyze the hydrolysis
of acetylcholine (ACh) in cholinergic synapses, whereas the function of BChE is less clearly defined
because it can hydrolyze ACh as well as other esters [12]. AChE and BChE inhibitors are used in
treatment of various neuromuscular disorders and have provided the first generation of drugs for
treatment of Alzheimer’s disease (AD) [13], which is a progressive physical disorder which causes
increasingly severe impairment in the cognitive and functional ability of individuals suffering from the
disease [14]. It is a degenerative disease of the brain that leads to the conditions collectively called
dementia [15]. The inhibition of AChE and BChE is directly connected with treatment of AD. The
cholinergic hypothesis proposes that AD is caused by reduced synthesis of the neurotransmitter
acetylcholine [16]. The inhibition of the mentioned enzymes causes an increase in the concentration of
acetylcholine in cholinergic synapses, which results in alleviation of the disease. New and potent
AChE inhibitors may be helpful in the treatment of this disease.
The basic 2-substituted-1,3-benzothiazole scaffold is essential for some antimicrobial
compounds [17], herbicides, plant desiccants and defoliant compounds [18]. Isopropyl [(S)-1-[(R)-1-
(6-flourobenzothiazolel-2-yl)ethylcarbamoyl]-2-methylpropyl] carbamate is a commercially used
fungicide which is effective for controlling the oomycete fungal pathogen Plasmopara viticola, which
causes downy mildew in grapevines [19]. Position 2 of benzothiazoles is the most suitable for affecting
physico-chemical properties of these compounds. The aim of this study was the modification of the
amino moiety leading to the synthesis of substituted (6-fluorobenzo[d]thiazol-2-yl)ethanamines with
incorporated 2,4,6-trioxoimidazolidine moieties as potent inhibitors of AChE and BChE. The five-member

Molecules 2011, 16
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2,4,6-trioxoimidazolidine ring (a stabilized urea) can be understood as an isostere of a carbamate
moiety [20].
Many low-molecular-weight drugs cross biological membranes through passive transport, which
strongly depends on their lipophilicity, therefore the experimental log P (or log Kow) n-octanol/water
partition coefficients were determined. Structure-activity relationships between the chemical structure,
physical properties and biological activities of the evaluated compounds are discussed.
2. Results and Discussion
2.1. Chemistry
The synthesis of 1-(aryl)-3-[(R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethyl]imidazolidine-2,4,5-triones
3a–k was accomplished according to Scheme 1. The starting compound (R)-1-(6-fluorobenzo[d]
thiazol-2-yl)ethanamine (1) was prepared as the corresponding p-toluenesulfonate salt (PTS) according
to a literature method [21]. Synthesis of the key intermediates disubstituted ureas 2a–k was recently
described [9].
Scheme 1. Synthesis of 1,3-substituted imidazolidine-2,4,5-triones 3a–k.
O
C
N
S
N
S
N
S
a
b
N (CH )
2 n
O
O
+
HN
N
NH
F
F
F
2
HN (CH )
N (CH )
2 n
2 n
PTS
O
R
1
2
3
O
R
R
Reagents and conditions: (a) NaOH/TOL, RT; (b) oxalyl chloride, 0–5 °C, DCM/hexane
(PTS = p-toluenesulfonate).
The desired compounds 3a–k were synthesized in dichloromethane (DCM), where the starting
disubstituted ureas were treated with oxalyl chloride at low temperature (0–5 °C). The final products
were precipitated from the reaction mixture by addition of n-hexane. The purification of the prepared
compounds was realized by dissolving in DCM, and adding n-hexane to induce precipitation of the
1
products in high yields (80%–90%). Products 3a–k were characterized by their melting points, H-,
13C-, 19F-NMR, IR spectroscopy and elemental analyses (CHN).
2.2. Crystallography
The exemplary compound 3g crystallizes in the triclinic crystal system, achiral point group P-1
with two independent molecules (different enantiomers) within the unit cell (Figure 1). The opposite
orientation of the C8 atom seems to be the only remarkable difference between these two molecules.
The interatomic distances and angles including torsion and interplanar angles are similar to the typical
values found in the literature for similar atom combinations [22].
Although cyclic ureas such as, for example, hydantoins or alantoins, are frequently studied, there
are only ten examples of 3,4-dicarbonyl bridged ureas [23-34], and there are only four structures that
are similar to the structure of compound 3g where the diazacyclopentane ring is 3,4-dicarbonyl

Molecules 2011, 16
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substituted and nitrogen atoms are not of the NH type [35,36]. The determined structure of compound
3g is the first asymmetric cyclic urea within the set.
There is a quite extensive π-π stacking network causing the 3D structure formation with interactions
between the heterocyclic rings of 3.372(3)Å and carbonyl groups of 3.121(3)Å, see Figure 2. Although
one might expect very tight organization due to these interactions, remarkable cavities were found
within the crystal lattice of compound 3g, see Figure 1. No solvent can be accommodated in these
cavities, probably because both hydrophilic and hydrophobic parts are present in the molecule of the
mentioned compound. Another reason could be free rotability of the bulky diisopropylphenyl group.
The π-π stacking interactions of aromatic rings and C=O groups in the molecules are shown in Figure 2.
Molecules of compound 3g also form interesting supramolecular architecture, see Figure 3.
Figure 1. Molecular structure (ORTEP 30% probability level) of compound 3g, only one
of two independent molecules is shown. Selected interatomic distances [Å] and bond
angles [°]: O1 C10 1.199(5), O2 C11 1.203(5), O3 C12 1.197(5), N2 C10 1.407(5), C10
N3 1.404(5), N3 C11 1.376(5), C11 C12 1.542(6), C12 N2 1.373(5), S1 C3 1.727(4), C3
C4 1.400(6), C4 N1 1.409(6), N1 C7 1.350(6), C7 S1 1.751(4); O1 C10 N3 126.8(4), O1
C10 N2 126.0(4), O2 C11 N3 127.6(4), O2 C11 C12 126.5(4), O3 C12 C11 127.4(4), O3
C12 N2 128.1(4), N2 C10 N3 107.2(3), C10 N3 C11 110.7(3), N3 C11 C12 105.9(3), C11
C12 N2 104.5(3), C12 N2 C10 111.7(3), C3 S1 C7 88.1(2), S1 C3 C4 111.0(3), C4 N1 C7
106.9(4), N1 C7 S1 117.8(3), N2 C8 C7 108.2(3).

Molecules 2011, 16
Figure 2. π-π Stacking interactions of aromatic rings and C=O groups.
7569
Figure 3. Supramolecular architecture of 3g.
2.3. Lipophilicity
Lipophilicity is a property that has a major effect on the absorption, distribution, metabolism,
excretion and toxicity properties, as well as pharmacological activity, because drugs cross biological
membranes through passive transport, which strongly depends on their lipophilicity. Lipophilicity has

Molecules 2011, 16
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been studied and applied as an important drug property for decades [37]. Hydrophobicities (log P/Clog P)
of the compounds were calculated using the commercially available program ChemOffice Ultra 11.0.
The experimental partition coefficient log Kow (n-octanol/water) was determined using the traditional
shake-flask method. The n-octanol/water partition coefficient P (also referred to as Kow) is a measure
of the propensity of a neutral compound to differentially dissolve in these immiscible phases. It is
usually referred to as the logarithmic ratio, log P. The partition coefficient serves as a quantitative
descriptor of lipophilicity and is one of the key determinants of pharmacokinetic properties. The
partition coefficient log Kow (n-octanol/water) indicates potential for crossing the blood-brain barrier
for direct inhibition of brain cholinesterases [38]. Experimentally it is done by partitioning the
molecule between water and the hydrophobic solvent n-octanol and determining the P value as the
ratio of the concentration of the compound in n-octanol and in water. Log Kow can be used as the
lipophilicity index converted to in silico log P scale. The results are shown in Table 1.
Table 1. Comparison of calculated lipophilicities (log P/Clog P) with determined log Kow
values, Hammett’s σ parameters of prepared substituted imidazolidine-2,4,5-triones and
their AChE and BChE inhibition in comparison with standards rivastigmine (RIV) and
galanthamine (GLT). ChE inhibitions are expressed as mean ± SD (n = 3 experiments),
and log Kow data of the compounds are expressed as mean ± SD (n = 3 experiments).
AChE
BChE
Comp.
R
n
log Kow
log P/Clog P
σ [39]
IC50 [μmol/L]
3a
3b
3c
H
3-CF3
0
0
0
0
0
0
0
0
0
0
1
–
–
21.4 ± 0.19
23.4 ± 0.28
22.4 ± 0.21
16.6 ± 0.29
13.8 ± 0.13
19.1 ± 0.27
15.1 ± 0.29
15.5 ± 0.2
14.5 ± 0.21 1.51 ± 0.03
17.4 ± 0.37 1.12 ± 0.15
13.2 ± 0.22 0.69 ± 0.02
1.66 ± 0.14 1.64 ± 0.03
25.7 ± 0.29 0.41 ± 0.02
10.5 ± 0.18 0.41 ± 0.02
30.2 ± 0.45 0.98 ± 0.08
3.91 / 2.769
4.83 / 3.652
3.78 / 2.688
5.14 / 4.196
4.46 / 3.482
3.94 / 2.202
6.38 / 5.623
4.95 / 3.981
4.88 / 3.767
5.02 / 4.195
3.98 / 3.102
2.36 / 2.099
1.41 / 1.025
0.00
0.43
−0.27
−0.15
0.23
1.00
0.06
0.20
−0.14
0.74
0.00
–
4-OCH3
4-CH(CH3)2
4-Cl
3d
3e
3f
4-CN
3g
2,6-CH(CH3)2
3-Cl-4-CH3
3,5-CH3
3,5-Cl
H
3h
3i
17.0 ± 0.1
0.50 ± 0.02
21.4 ± 0.19
17.0 ± 0.38
18.6 ± 0.16
6.76 ± 0.17 0.58 ± 0.03
29.5 ± 0.39 0.77 ± 0.03
12.6 ± 0.11 1.06 ± 0.09
3j
3k
RIV
GLT
–
501 ± 3.08 19.95 ± 0.31
4.0 ± 0.13 7.96 ± 0.13
–
–
–
–
The results obtained with all the compounds show that the experimentally-determined
lipophilicities (log Kow) of the discussed compounds are in poor accordance with the calculated values
of compounds. This fact suggests significant intramolecular interactions within the whole series of the
compounds. Compounds 3e (4-Cl) and 4f (4-CN) showed the lowest lipophilicity, while compounds
3a (H) and 3d [4-CH(CH3)2] demonstrated the highest. Benzyl derivative 3k showed lower
lipophilicity than phenyl derivative 3a. Therefore it can be assumed that the determined log Kow data
specify lipophilicity within this series of the discussed compounds.

Molecules 2011, 16
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2.4. Inhibition of Cholinergic Enzymes
All the prepared carbamate-like compounds were tested for their inhibition of AChE and BChE.
The activities of the compounds were compared with the internal standards rivastigmine (RIV,
Exelon®) and galanthamine (GLT, Reminyl®), see Figure 4. These standards were chosen by reason of
the different structures of both drugs. While rivastigmine is a classical acylating pseudo-reversible
carbamate cholinesterase inhibitor that inhibits both acetylcholinesterase and butyrylcholinesterase,
galanthamine is a non-acylating competitive reversible cholinesterase inhibitor and also an allosteric
ligand at nicotinic acetylcholine receptors. The choice of these reference drugs with different
mechanisms of action can provide relevant results. The results are summarized in Table 1 and
expressed as 50% inhibitory concentration (IC50 [μmol/L]), or the concentration of inhibitor required
for 50% inhibition of the mentioned enzymes.
Figure 4. Structures of the two internal standards used, rivastigmine and galanthamine.
H CO
3
O
N
CH
O
CH
N
H
3
3
H
H C
3
N
O
CH
3
CH
CH
3
3
HO
galanthamine
rivastigmine
The AChE inhibition of the prepared derivatives 3a–k significantly exceeded the AChE inhibition
of rivastigmine and these compounds are in average four-five times less efficient than the other
standard galanthamine. Compound 3e (4-Cl, IC50 = 13.8 μmol/L) showed the highest AChE-inhibiting
activity. High inhibition was also expressed by compounds 3g (2,6-CH(CH3)2, IC50 = 15.1 μmol/L), 3h
(3-Cl-4-CH3, IC50 = 15.5 μmol/L) and 3d [4-CH(CH3)2, IC50 = 16.6 μmol/L]. The BChE inhibition of
the prepared compounds (except compounds 3g, 3j and 3e) also exceeded the BChE inhibition of
rivastigmine. Only compound 3d [4-CH(CH3)2, IC50 = 1.66 μmol/L] showed significantly higher
BChE inhibition than galanthamine.
Based on these facts, it can be concluded that AChE is inhibited by para- and meta-substutited
phenyl rings, while BChE is preferentially inhibited by a para-substutited phenyl ring. These
observations describing steric/positional aspects of substitution on benzene are in agreement with
recently published results by Chiou et al. indicating that that AChE prefers para- and
meta-substitution to ortho-substitution, whereas BChE prefers para-substitution to ortho- and
meta-substitution. These results imply that steric differences in the active sites of both enzymes can be
found [40].
It is noteworthy that compounds with high inhibitory activity possess a branched substituent. For
example, in case of the AChE inhibitors these are compounds 3d [4-CH(CH3)2] and 3g [2,6-CH(CH3)2]
and in case of the BChE inhibitors it is compound 3d [4-CH(CH3)2)].
The dependence of AChE inhibition (log L/IC50 [mol/L]) on log P is illustrated in Figure 5A. The
set of 11 tested compounds can be divided into para-substituted and/or unsubstituted, where a bilinear
dependence can be found, and meta-substituted, where activity sharply increases with a slight
lipophilicity increase. The dependence of BChE inhibition (log L/IC50 [mol/L]) on log P is illustrated

Molecules 2011, 16
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in Figure 5B, and from these relationships it is evident that lipophilicity is only a secondary parameter,
although it seems that BChE inhibition activity decreases with lipophilicity increase within
meta-substituted series.
Figure 5. Dependence of AChE (A) and/or BChE (B) inhibition (log 1/IC50 [mol/L]) on
compound lipophilicity expressed as log P.
5A
4.9
3e
3h
3g
4.8
4.7
3d
3j
3k
3f
3a
3i
3b
3c
4.6
6.0
3.0
3.5
4.0
4.5
5.0
log P
5.5
6.0
6.5
7.0
5B
3d
5.5
5.0
4.5
4.0
3i
3f
3c
3k
3b
3h
3a
3e
3j
3g
3.5
4.0
4.5
5.0
5.5
6.0
6.5
log P
However, inhibition of cholinergic enzymes is also significantly dependent on the electronic
properties of R phenyl substituents expressed as Hammett’s σ parameters. Figure 6A shows an evident
general bilinear trend: with the increase of electron-withdrawing effect of individual substituents to the
value σ = 0.23 (3e, 4-Cl), which is the optimum, AChE-inhibiting activity increases to the value
IC50 = 13.8 μmol/L, and with the following increase of electron-withdrawing effect the activity
decreases. Also other compounds 3g [2,6-CH(CH3)2, σ = 0.06, IC50 = 15.1 μmol/L] and 3h (3-Cl-4-CH3,
σ = 0.20 IC50 = 15.5 μmol/L) with a close value of electron-donor substituent σ showed similar
inhibition activity as 3e.

Molecules 2011, 16
Figure 6. Dependence of AChE (A) and/or BChE (B) inhibition (log 1/IC50 [mol/L]) on
7573
substituent electron Hammett's σ parameters.
6A
4.9
3e
3g
3h
4.8
3d
3j
3k
3f
4.7
3i
3a
3c
3b
4.6
-0.3
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
σ
6B
6.0
5.5
5.0
4.5
4.0
3d
3i
3k
3a
3f
3c
3b
3h
3e
3j
3g
-0.3
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
σ
In case of the BChE inhibitors the optimal value seems to be σ = −0.15 [3d, 4-CH(CH3)2,
IC50 = 1.66 μmol/L] and with the following increase of electron-withdrawing effect the BChE-inhibiting
activity decreases and is influenced minimally, see Figure 6B. The evident trend can be observed only
within the meta-substituted series, where activity decreases with an increase of electron-withdrawing
effect. Another compound with an electron-donor σ value similar to σ of compound 3d, σ = −0.14 (3i,
3,5-CH3, IC50 = 6.76 μmol/L) is an inhibitor comparable with galanthamine. Also compounds with σ
values close to σ = −0.15 (the Hammett’s parameter of the most effective compound 3d) showed slightly
lower BChE-inhibiting activity, see 3c (σ = −0.27, IC50 = 13.2 μmol/L), 3a (σ = 0.00, IC50 = 14.5 μmol/L),
3k (σ = 0.00, IC50 = 12.6 μmol/L). On the other hand, the bulky disubstituted derivative 3g
[2,6-CH(CH3)2, σ = 0.06, IC50 = 30.2 μmol/L] possesses four times less inhibitory activity than the
most active compound 3d.

Molecules 2011, 16
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3. Experimental
3.1. General
All reagents and solvents were purchased from commercial sources (Sigma-Aldrich, Merck, Acros
Organics, Lach-Ner CZ). Commercial grade reagents were used without further purification. The
reactions were monitored and the purity of the products was checked by thin-layer chromatography
plates coated with 0.2 mm silica gel 60 F254 (Merck, Darmstadt, Germany). TLC plates were visualized
by UV irradiation (254 nm). All the melting points were determined on a Melting Point B-545
apparatus (Buchi, Germany) and are uncorrected. Infrared spectra (ZnSe ATR experiments) were
recorded on a FT-IR spectrometer (Perkin Elmer, USA) in the range of 600–4000 cm−1. The NMR
spectra were measured in DMSO-d6 solutions at ambient temperature on a Bruker Avance III 400 MHz
1
13
spectrometer (Karlsruhe, Bruker, Germany, 400 MHz for H, 100 MHz for C and 376.5 MHz for
19F). Proton chemical shifts in DMSO-d6 are related to the middle of the solvent multiplet (δ = 2.50).
13C-NMR spectra were measured using APT pulse sequences. Carbon chemical shifts are referenced to
19
the middle of the solvent multiplet (δ = 39.5 in DMSO-d6). F-NMR spectra were measured using
waltz-16 proton decoupling and were standardised against fluorobenzene as the secondary external
standard (δ = −113.1 against CFCl3 as the primary standard [41], td 64k zero filled to 128k). Chemical
shifts for AB systems were calculated as weighed average of the line positions weighted by the
peak intensities.
3.2. Synthesis
General Procedure for the Synthesis of Compounds 3a–k
The appropriate 1-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-3-substituted phenyl urea 2a–k
(5 mmol) was added to a cooled solution (0–5 °C) of oxalyl chloride (7 mmol) in dichloromethane
(20 mL) and mixture was stirred at temperature 0–5 °C for 1 h. The mixture was left to warm up to the
ambient temperature and stirred for another 2 h. The reaction mixture was filtered and concentrated
under reduced pressure. The addition of n-hexane caused precipitation of products 3a–k. The
precipitated solid was collected by filtration, dried in vacuum to give the title compounds in 80–85%
yields as light yellow crystalline solids (unless stated otherwise).
1-[(1R)-1-(6-Fluoro-1,3-benzothiazol-2-yl)ethyl]-3-phenylimidazolidine-2,4,5-trione (3a). Yield: 85%;
m.p. 156–157 °C; IR (cm−1): 3060, 2949, 1731, 1657, 1599, 1563, 1501,1453, 1404,1348, 1313, 1291,
1
1247, 1198, 1172, 1131, 1003, 947, 912, 836, 828, 757, 740, 686; H-NMR δ: 1.95 (d, 3H, 1-H,
J = 7.2 Hz) CH3, 5.88 (q, 1H, 2-H, J = 7.2 Hz) CH, 7.41 (dt, 1H, 4-H, J = 2.7 Hz, J = 9.1 Hz,
3J(19F, 1H) = 9.1 Hz), 7.45–7.57 (m, 5H, 2´-H, 3´-H, 4´-H, 5´-H, 6´-H), 8.05 (dd, 1H, 6-H, J = 3.4 Hz,
1
3J(19F, H) = 9.0 Hz), 8.07 (d, 1H, 3-H, J = 8.9 Hz); 13C-NMR δ: 17.1, 49.1, 108.7 (d,
2J(19F, 13C) = 27.4 Hz), 115.0 (d, 2J(19F, 13C) = 24.9 Hz), 124.3 (d, 3J(19F, 13C) = 9.7 Hz), 126.7, 128.9,
129.2, 130.5, 136.5 (d, 3J(19F, 13C) = 11.9 Hz), 148.8, 152.5, 156.0, 156.4, 159.8 (d,
4
1J(19F, 13C) = 243.1 Hz), 169.3 (d, J(19F, 13C) = 3.3 Hz); 19F-NMR δ: −115.67; Anal. Calcd. for
C18H12FN3O3S (369.37): 58.53% C, 3.27% H, 11.38% N; found: 58.28% C, 3.38% H, 11.32%N.

Molecules 2011, 16
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1-(3-Trifluoromethylphenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]imidazolidine-2,4,5-trione
(3b). Yield: 80%; m.p. 145–146 °C; IR (cm−1): 3120, 2752, 1720, 1603, 1567, 1519, 1495, 1455, 1401,
1325, 1255, 1199, 1178, 1126, 1089, 1070, 1006, 953, 880, 913, 861, 829, 815, 791, 756, 695, 672, 658;
1H-NMR δ: 1.96 (d, 3H, 1-H, J = 7.2 Hz), 5.91 (q, 1H, 2-H, J = 7.2 Hz), 7.41 (dt, 1H, 4-H, J = 2.5 Hz,
3
1
J = 9.0 Hz, J(19F, H) = 9.0 Hz), 7.82 (m, 4H, 2´-H, 4´-H, 5´-H,6´-H), 8.04 (dd, 1H, 6-H, J = 3.3 Hz,
3J(19F, H) = 8.8 Hz), 8.07 (d, 1H,3-H, J = 8.8 Hz); 13C-NMR δ: 17.1, 49.1, 108.6 (d,
1
13
2
13
3
13
2J(19F, C) = 27.1 Hz), 114.9 (d, J(19F, C) = 24.9 Hz), 123.2 (q, J(19F, C) = 4.0 Hz), 123.6 (q,
13
3
13
3
13
1J(19F, C) = 273.6 Hz), 124.2 (d, J(19F, C) = 9.3 Hz), 125.6 (q, J(19F, C) = 3.5 Hz), 129.8 (q,
2J(19F, 13C) = 32.4 Hz), 130.7, 130.8, 131.2, 136.5 (d, J(19F, 13C) = 11.7 Hz), 149.8 (d,
3
5J(19F, 13C) = 1.4 Hz), 152.1, 155.6, 156.1, 159.8 (d, J(19F, 13C) = 243.2 Hz), 169.0 (d,
1
4J(19F, C) = 3.2 Hz); F-NMR δ: −61.37 (s, 3F), −115.61 (s. 1F); Anal. Calcd. for C19H11F4N3O3S
13
19
(437.37): 52.18% C, 2.54% H, 9.61% N; found: 52.29% C, 2.48% H, 9.66% N.
1-(4-Methoxyphenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]imidazolidine-2,4,5-trione (3c).
Yield: 83%, m.p. 79–80 °C; IR (cm−1): 3075, 2934, 1735, 1604, 1566, 1514, 1454, 1398, 1331, 1303,
1257, 1197, 1172, 1131, 1024, 938, 911, 824, 753, 701; 1H-NMR δ: 1.94 (d, 3H, 1-H, J = 7.2 Hz), 3.80
(s, 3H) OCH3, 5.87 (q, 1H, 2-H, J = 7.2 Hz), 7.08 (d, 2H, 2´-H, 6´-H, J = 8.8 Hz), 7.34 (d, 2H, 3´-H,
5´-H, J = 8.8 Hz), 7.41 (dt, 1H, 4-H, J = 2.8 Hz, J = 9.2 Hz, J(19F, H) = 9.2 Hz), 8.05 (d, 1H, 3-H,
3
1
3
1
13
J = 8.9 Hz) 8.07 (dd, 1H, 6-H, J = 3.3 Hz, J(19F, H) = 8.7 Hz); C-NMR δ: 17.1, 49.0, 55.4, 108.7
2
13
2
13
3
13
(d, J(19F, C) = 27.2 Hz), 114.4, 114.9 (d, J(19F, C) = 24.9 Hz), 122.9, 124.2 (d, J(19F, C) = 9.7
Hz), 128.2, 136.5 (d, J(19F, 13C) = 11.9 Hz), 148.8, 152.8, 156.2, 156.5, 159.3, 159.8 (d,
3
1J(19F, 13C) = 243.2 Hz), 169.3 (d, J(19F, 13C) = 3.2 Hz); 19F-NMR δ: −115.67; Anal. Calcd. for
4
C19H14FN3O4S (399.40): 57.14% C, 3.53% H, 10.52% N; found: 57.25% C, 3.49% H, 10.60% N.
1-(4-Isopropylphenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]imidazolidine-2,4,5-trione (3d).
Yield: 80%; m.p. 70–71 °C; IR (cm−1): 3061, 2955, 1734, 1602, 1567, 1515, 1454, 1397, 1342, 1248,
1199, 1134, 1019, 937, 912, 840, 816, 754, 654; 1H-NMR δ: 1.22 (d, 6H, CH3-isopropyl, J = 6.8 Hz),
1.96 (d, 3H, 1-H, J = 7.2 Hz), 2.94 (sept., 1H, CH-isopropyl, J = 7.2 Hz), 5.87 (q, 1H, 2-H, J = 7.2 Hz),
7.34 (d, 2H, 2´-H, 6´-H, J = 8.3 Hz), 7.40 (d, 2H, 3´-H, 5´-H, J = 8.3 Hz), 7.41 (dt, 1H, 4-H, J = 2.8 Hz,
3
1
J = 9.2 Hz, J(19F, H) = 9.2 Hz), 8.05 (d, 1H, 3-H, J = 8.9 Hz), 8.07 (dd, 1H, 6-H, J = 2.6 Hz,
3J(19F, H) = 8.7 Hz); C-NMR δ: 17.0, 23.7, 33.2, 49.0, 108.6 (d, J(19F, C) = 27.3 Hz), 114.9 (d,
1
13
2
13
2J(19F, 13C) = 24.9 Hz), 124.2 (d, J(19F, 13C) = 9.4 Hz), 126.6, 127.0, 128.0, 136.5 (d,
3
3J(19F, 13C) = 11.8 Hz), 148.8, 149.3,152.6, 156.0, 156.4, 159.8 (d, 1J(19F, 13C) = 243.3 Hz), 169.3 (d,
4J(19F, 13C) = 3.4 Hz); 19F-NMR δ: −115.73; Anal. Calcd. for C21H18FN3O3S (411,45): 61.30% C,
4.41% H, 10.21% N; found: 61.55% C, 4.33% H, 10.33% N.
1-(4-Chlorophenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]imidazolidine-2,4,5-trione (3e).
Yield: 85%; m.p. 149–150 °C; IR (cm−1): 3060, 2938, 1737, 1600, 1564, 1516, 1494, 1455, 1401,
1350, 1311, 1290, 1248, 1198, 1176, 1128, 1091, 1017, 1002, 950, 911, 838, 849, 821, 750, 664, 714,
699, 655; 1H-NMR δ: 1.93 (d, 3H, 1-H, J = 7.2 Hz), 5.86 (q, 1H, 2-H, J = 7.2 Hz), 7.41 (dt, 1H, 4-H,
J = 2.8 Hz, J = 9.2 Hz, 3J(19F, 1H) = 9.2 Hz), 7.46 (d, 2H, 2´-H, 6´-H, J = 9.2 Hz), 7.62 (d, 2H, 3´-H,
3
1
5´-H, J = 9.0 Hz), 8.04 (dd, 1H, 6-H, J = 2.8 Hz, J(19F, H) = 8.6 Hz), 8.06 (d, 1H, 3-H, J = 8.8 Hz);
13C-NMR δ: 17.0, 48.9, 108.6 (d, J(19F, C) = 27.2 Hz), 114.9 (d, J(19F, C) = 24.8 Hz), 124.2 (d,
2
13
2
13

Molecules 2011, 16
7576
3J(19F, 13C) = 9.8 Hz), 128.6, 129.4, 129.8, 133.5 136.6 (d, 3J(19F, 13C) = 11.9 Hz), 148.9, 152.4, 155.9,
156.4, 159.5 (d, 1J(19F, 13C) = 243.0 Hz), 169.3 (d, 4J(19F, 13C) = 3.3 Hz); 19F-NMR δ: −115.55; Anal.
Calcd. for C18H11ClFN3O3S (403.81): 53.54% C, 2.75% H, 10.41% N; found: 53.22% C, 3.80% H,
10.50% N.
1-(4-Cyanophenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]imidazolidine-2,4,5-trione (3f). A
white crystalline compound; Yield: 80%; m.p. 172–173 °C; IR (cm−1): 3071, 1739, 1653, 1602, 1564,
1508, 1455, 1397, 1355, 1314, 1249, 1207, 1136, 1064, 996, 940, 913, 842, 817, 750, 691, 667; 1H-NMR
δ: 1.94 (d, 3H, 1-H, J = 7.2 Hz), 5.90 (q, 1H, 2-H, J = 7.2 Hz), 7.41 (dt, 1H, 4-H, J = 2.8 Hz, J = 9.2 Hz,
3J(19F, 1H) = 9.2 Hz), 7.65 (d, 2H, 2´-H, 6´-H, J = 8.4 Hz), 8.00 (d, 2H, 3´-H, 5´-H, J = 8.4 Hz), 8.04
(dd, 1H, 6-H, J = 2.9 Hz, 3J(19F, 1H) = 8.8 Hz), 8.06 (d, 1H, 3-H, J = 8.9 Hz); 13C-NMR δ: 17.0, 49.2,
2
2
108.7 (d, J(19F, 13C) = 27.3 Hz), 111.2, 115.0 (d, J(19F, 13C) = 24.9 Hz), 118.2, 124.3 (d,
3J(19F, 13C) = 9.6 Hz), 126.9, 133.4, 134.5, 136.5 (d, J(19F, 13C) = 11.8 Hz), 148.8, 151.9, 155.3,
3
156.0, 159.8 (d, 1J(19F, 13C) = 243.0 Hz), 169.0 (d, 4J(19F, 13C) = 3.2 Hz); 19F-NMR δ: −115.61; Anal.
Calcd. for C19H11FN4O3S (394,38): 57.86% C, 2.81% H, 14.21% N; found: 57.68% C, 2.90% H,
14.08% N.
1-(2,6-Diisopropylphenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]imidazolidine-2,4,5-trione
(3g). Yield: 81%; m.p. 165–166 °C; IR (cm−1): 3060, 2951, 2809, 1739, 1602, 1567, 1458, 1395, 1364,
1
1295, 1251, 1216, 1194, 1139, 1034, 1005, 960, 911, 853, 846, 811, 793, 755, 741, 710; H-NMR δ:
1.04 (b, 3H, CH3-isopropyl, J = 6.7 Hz), 1.09 (d, 3H, CH3-isopropyl, J = 6.7 Hz), 1.14 (d, 3H,
CH3-isopropyl, J = 6.7 Hz), 1.16 (d, 3H, CH3-isopropyl, J = 6.7 Hz), 2.01 (d, 3H, 1-H, J = 7.2 Hz),
3.00 (m, 2H, CH-isopropyl, J = 6.7 Hz), 5.89 (q, 1H, 2-H, J = 7.2 Hz), 7.33–7.36 (m, 3´-H, 5´-H), 7.42
3
1
(dt, 1H, 4-H, J = 2.7 Hz, J = 9.2 Hz, J(19F, H) = 9.2 Hz), 7.97 (dd, 1H, 6-H, J = 4.8 Hz,
3J(19F, 1H) = 9.0 Hz), 8.08 (dd, 4-H, J = 9.2 Hz, 4J(19F, 1H) = 2.8 Hz); 13C-NMR δ: 17.0, 23.7, 23.8, 23.9,
2
2
24.0, 28.1, 49.3, 108.8 (d, J(19F, 13C) = 27.6 Hz), 115.2 (d, J(19F, 13C) = 25.3 Hz), 124.0 (d,
3J(19F, 13C) = 9.2 Hz), 124.3, 125.5, 130.8, 136.3 (d, J(19F, 13C) = 11.8 Hz), 147.1 (d,
3
5J(19F, 13C) = 1.5 Hz), 148.8, 153.0, 156.5, 156.8, 159.8 (d, J(19F, 13C) = 242.9 Hz), 169.8 (d,
1
4J(19F, 13C) = 2.6 Hz); 19F-NMR δ: −115.63; Anal. Calcd. for C24H24FN3O3S (453.53): 63.56% C,
5.33% H, 9.27% N; found: 63.33 C, 5.40% H, 9.20% N.
1-(3-Chloro-4-methylphenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]imidazolidine-2,4,5-trione
(3h). Yield: 85%; m.p. 161–162 °C; IR (cm−1): 3029, 1724, 1600, 1565, 1498, 1457, 1382, 1412, 1396,
1339, 1246, 1217, 1195, 1142, 1096, 1057, 1008, 960, 890, 847, 816, 757, 696, 681, 661; 1H-NMR δ:
1.95 (d, 3H, 1-H, J = 7.5 Hz), 2.38 (s, 3H, CH3), 5.89 (q, 1H, 2-H, J = 7.2 Hz), 7.34 (dd, 1H, 6´-H,
J = 2.0 Hz, J = 8.0 Hz), 7.40 (dt, 1H, 4-H, J = 2.4 Hz, J = 9.2 Hz, 3J(19F, 1H) = 9.2 Hz), 7.51 (d, 1H, 2´-H,
J = 2.4 Hz), 7.53 (d, 1H, 5´-H, J = 8.0 Hz), 8.04 (d, 1H, 3-H, J = 8.8 Hz), 8.06 (dd, 1H, 6-H, J = 3.2 Hz,
1
2
3J(19F, H) = 8.8 Hz); 13C-NMR δ: 17.0, 19.3, 49.1, 108.6 (d, J(19F, 13C) = 27.2 Hz), 114.9 (d,
2J(19F, C) = 24.8 Hz), 124.2 (d, J(19F, C) = 9.7 Hz), 125.4, 126.7, 129.2, 131.6, 133.2, 135.5 (d,
13
3
13
3J(19F, 13C) = 11.8 Hz), 136.6, 148.8 (d, J(19F, 13C) = 2.0 Hz), 152.2, 155.7, 156.2, 159.8 (d,
5
1J(19F, 13C) = 243.1 Hz), 169.1 (d, J(19F, 13C) = 3.0 Hz); 19F-NMR δ: −115.59; Anal. Calcd. for
4
C19H13ClFN3O3S (417.84): 54.61% C, 3.14% H, 10.06% N; found: 54.75% C, 3.08% H, 10.11% N.

Molecules 2011, 16
7577
1-(3,5-Dimethylphenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]urea (3i). Yield: 83%; m.p.
122–123 °C; IR (cm−1): 3004, 1731, 1604, 1566, 1520, 1455, 1400, 1379, 1345, 1279, 1256, 1206,
1
1161, 1098, 1010, 963, 919, 905, 858, 844, 825, 811, 756, 683, 654; H-NMR δ: 1.94 (d, 3H, 1-H,
J = 7.2 Hz), 2.32 (s, 6H, CH3), 5.86 (q, 1H, 2-H, J = 7.2 Hz), 7.03 (s, 2H, 2´-H, 6´-H), 7.11 (s, 1H, 4´-H),
3
1
7.41 (dt, 1H, 4-H, J = 2.8 Hz, J = 9.2 Hz, J(19F, H) = 9.2 Hz), 8.04 (dd, 1H, 6-H, J = 3.2 Hz,
1
3J(19F, H) = 8.8 Hz), 8.07 (d, 1H, 3-H, J = 8.9 Hz); 13C-NMR δ: 17.0, 20.7, 49.0, 108.7 (d,
2
3
2J(19F, 13C) = 27.3 Hz), 114.9 (d, J(19F, 13C)) = 24.9 Hz), 124.2, 124.3 (d, J(19F, 13C) = 9.5 Hz),
130.2, 130.3, 136.5 (d, J(19F, 13C) = 11.7 Hz), 138.5, 148.8, 152.5, 156.0, 1156.4, 159.8 (d,
1J(19F, 13C) = 243.0 Hz), 169.4 (d, J(19F, 13C) = 3.0 Hz); 19F-NMR δ: −115.67; Anal. Calcd. for
3
4
C20H16FN3O3S (397.42): 60.44% C, 4.06% H, 10.57% N; found: 60.56% C, 3.96% H, 10.59% N.
1-(3,5-Dichlorophenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]imidazolidine-2,4,5-trione (3j).
Yield: 83%; m.p. 190–191 °C; IR (cm−1): 3130, 2950, 1727, 1566, 1573, 1590, 1519, 1452, 1432,
1406, 1381, 1342, 12454, 1205, 1196, 1173, 1129, 1105, 1010, 966, 916, 857, 828, 817, 757, 726, 674,
1
664; H-NMR δ: 1.94 (d, 3H, 1-H, J = 6.8 Hz), 5.89 (q, 1H, 2-H, J = 6.8 Hz), 7.41 (dt, 1H, 4-H,
3
1
J = 2.4 Hz, J = 9.2 Hz, J(19F, H) = 9.2 Hz), 7.52 (d, 2H, 2´-H, 6´-H, J = 2.0 Hz), 7.80 (t, 1H, 4´-H,
J = 2.0 Hz), 8.04 (d, 1H, 3-H, J = 8.4 Hz), 8.06 (dd, 1H, 6-H, J = 3.2 Hz, J(19F, H) = 8.8 Hz);
3
1
13
2
13
13C-NMR δ: 17.0, 49.2, 108.7 (d, J(19F, C) = 27.2 Hz), 115.0 (d, J(19F, C) = 24.8 Hz), 124.3 (d,
3J(19F, 13C) = 9.8 Hz), 125.3, 128.7, 132.5, 134.4, 136.5 (d, J(19F, 13C) = 11.6 Hz), 148.8, 151.8,
3
155.2, 155.9, 159.5 (d, 1J(19F, 13C) = 243.1 Hz), 168.9 (d, 4J(19F, 13C) = 3.3 Hz); 19F-NMR δ: −115.55;
Anal. Calcd. for C18H10Cl2FN3O3S (438.26): 49.33% C, 2.30% H, 9.59% N; found: 49.49% C, 2.26%
H, 9.66% N.
1-Benzyl-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]imidazolidine-2,4,5-trione (3k). Yield: 84%;
m.p. 193–195 °C; IR (cm−1): 3035, 2950, 1721, 1603, 1566, 1493, 1456, 1440, 1413, 1397, 1349,
1305, 1251, 1201, 1191, 1133, 1073, 1025, 858, 818, 755, 700, 680, 665; 1H-NMR δ: 1.90 (d, 3H, 1-H,
J = 7.2 Hz), 4.73 (s, 2H, CH2-benzyl), 5.80 (q, 1H, 2-H, J = 7.2 Hz), 5.6 (t, 1H, 4´-H, J = 6.0 Hz),
7.31-7.38 (m, 5H, 2´-H, 3´-H, 4´-H, 5´-H, 6´-H), 7.40 (dt, 1H, 4-H, J = 2.8 Hz, J = 8.8 Hz,
1
3
1
3J(19F, H) = 8.8 Hz), 8.04 (dd, 1H, 6-H, J = 3.3 Hz, J(19F, H) = 8.9 Hz), 8.07 (d, 1H, 3-H,
J = 8.8 Hz); 13C-NMR δ: 17.1, 42.0, 48.9, 108.7 (d, J(19F, 13C) = 27.2 Hz), 114.9 (d,
2
2J(19F, 13C) = 24.9 Hz), 124.2 (d, J(19F, 13C) = 9.7 Hz), 126.7,127.7, 128.6, 135.3 136.4 (d,
3
3J(19F, 13C) = 11.6 Hz), 148.8, 153.4, 156.7, 156.9, 159.8 (d, J(19F, 13C) = 243.0 Hz), 169.5 (d,
1
4J(19F, 13C) = 3.9 Hz); 19F-NMR δ: −115.71; Anal. Calcd. for C19H14FN3O3S (383.40): 59.52% C,
3.68% H, 10.96% N; found: 59.65% C, 3.60% H, 11.02% N.
3.3. Determination of Crystallography
The X-Ray data for colourless crystals of compound 3g were obtained at 150 K using Oxford
Cryostream low-temperature device on a Nonius KappaCCD diffractometer with MoKα radiation
(λ = 0.71073 Å), a graphite monochromator and the φ and χ scan mode. Data reductions were
performed with DENZO-SMN [42]. The absorption was corrected by integration methods [43].
Structures were solved by direct methods (Sir92) [44] and refined by full matrix least-square based on
F2 (SHELXL97) [45]. Hydrogen atoms were mostly localized on a difference Fourier map, however to

Molecules 2011, 16
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ensure uniformity of the treatment of the crystal, all hydrogen atoms were recalculated into idealized
positions (riding model) and assigned temperature factors Hiso(H) = 1.2 Ueq(pivot atom) or of 1.5 Ueq
for the methyl moiety with C–H = 0.96, 0.98 and 0.93 Å for methyl, methine and hydrogen atoms in
the aromatic rings, respectively.
Selected crystallographic data for compound 3g: C24H24FN3O3S, M = 453.52, triclinic, P-1,
a = 10.1760(7), b = 10.7150(6), c = 21.5609(18)Å, α = 99.229(6), β = 100.256(8), γ = 91.188(6), Z = 4,
V = 2280.5(3) Å3, Dc = 1.321 g/cm3, μ = 0.181 mm−1, Tmin = 0.959, Tmax = 0.986; 41644 reflections
measured (θmax = 27.5°), 10323 independent (Rint = 0.0630), 6104 with I > 2σ(I), 577 parameters,
S = 1.079, R1(obs. data) = 0.0915, wR2(all data) = 0.1922; max., min. residual electron density = 1.295,
2
2
2
½
−0.538eǺ−3. Rint = Σ |Fo -Fo,mean2| / Σ Fo , GOF = [Σ (w(Fo − Fc2)2) / (Ndiffrs − Nparams)] for all data,
R(F) = Σ| |Fo| − |Fc| | / Σ |Fo| for observed data, wR(F2) = [Σ (w(Fo − Fc2)2) / (Σ w(Fo ) )] for all data.
Crystallographic data for structural analysis have been deposited with the Cambridge
Crystallographic Data Centre (deposition number CCDC 837618). Copies of this information may be
obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge CB2 1EY, UK
(fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
2
2 2
½
3.4. Determination of Partition Coefficient Kow
Before the partition coefficient is determined, the two solvents are mutually saturated at the
temperature of the experiment. To do this, it is practical to shake two large stock bottles, one
containing n-octanol and a sufficient quantity of water, and the other containing water and a sufficient
quantity of n-octanol, for 24 hours on a mechanical shaker and then to let them stand long enough to
allow the phases to separate [46].
The procedure of determination was the following: n-Octanol (2 mL) was placed in a test tube.
Then an octanol solution of the chosen inhibitor (15 μL, 0.01 M) was added. Mixture was intensively
shaken for 15 min. This mixture (1 mL) was placed into the cell, and its absorbance at the absorption
maximum wavelength was measured. The reference solution was n-octanol. The value of absorbance
corresponding to 100% of the chosen inhibitor in n-octanol was obtained. An n-octanol solution of the
chosen inhibitor (0.01 M, 15 μL) was added into the mixture of n-octanol and water (1:1, total volume
4 mL). The mixture was intensively shaken for 15 min and then centrifuged (3,000 rpm, 10 min).
One mL of the n-octanol layer was put into the cell, and its absorbance at the wavelength of absorption
maximum was measured. The comparative solution was n-octanol again. The percentage content of
chosen inhibitor in the octanol layer (%) was obtained. The n-octanol/water partition coefficient is
defined as Pow = c1/c2, where c1 and c2 are molar concentrations of tested compounds in n-octanol and
water. For each compound, at least three determinations were performed. The log Kow values of the
individual compounds are shown in Table 1.
3.5. Lipophilicity Calculations
Log P, i.e., the logarithm of the partition coefficient for n-octanol/water, was calculated using the
program CS ChemOffice Ultra ver. 11.0 (CambridgeSoft, Cambridge, MA, USA). Clog P values (the
logarithm of n-octanol/water partition coefficient based on established chemical interactions) were
generated by means of the same software. The results are shown in Table 1.

Molecules 2011, 16
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3.6. In Vitro Evaluation of AChE- and BChE-Inhibiting Activity
The ability of all tested compounds to inhibit acetylcholinesterase from electric eel (Electrophorus
electricus L.) and butyrylcholinesterase from equine serum (both purchased from Sigma) was tested.
The effectiveness of the inhibitor could be described by the 50% inhibitory concentration IC50. The
IC50, or the half maximal inhibitory concentration, represents the concentration of an inhibitor that is
required for 50% inhibition of the enzyme (sometimes it is referred to as the negative logarithm of the
molar concentration inhibiting the enzyme activity by 50%, pI50 = log 1/IC50). The IC50 values were
determined by the spectrophotometric Ellman’s method.
The Ellman’s method is a simple, rapid and direct method to determine the SH and –S–S– group
content in proteins [47]. This method is widely used for measuring of cholinesterase activity and
effectivity of cholinesterase inhibitors. Cholinesterase activity is measured indirectly by quantifying
the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol
reagent 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e.,
acetylthiocholine, ATCh) hydrolysis by the cholinesterase [48]. All tested compounds were dissolved
in dioxane (concentration 0.01 M) and then diluted in demineralized water (concentration 0.001 M and
0.0001 M). The procedure of determination of IC50 is in detail described in [49]. For determination of
IC50 values the inhibition was determined at 12 different compound concentrations with three
replicates. The obtained results are summarized in Table 1.
4. Conclusions
A series of eleven original 2-substituted benzothiazole derivates incorporating 1,3-disubstituted
imidazolidine-2,4,5-triones were synthesized and characterized. Their octanol/water partition
coefficients were determined experimentally as a basic property of compounds crossing biological
membranes through passive transport. Their ability to inhibit cholinergic enzymes (AChE and BChE)
was tested using Ellman’s method. The determined IC50 for each prepared compound was also
compared with the inhibitory activity of the internal standards rivastigmine and galanthamine. All the
discussed carbamate-like compounds expressed significantly higher AChE inhibitory activity than the
standard rivastigmin and slightly lower inhibitory activity than the standard galanthamine. Almost half
of the prepared compounds are stronger BChE inhibitors than both the internal standards used,
galanthamine and rivastigmine. 1-(4-chlorophenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-
imidazolidine-2,4,5-trione (3e, IC50 = 13.8 μmol/L) showed the highest AChE-inhibiting activity
within the series and 1-(4-isopropylphenyl)-3-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-
imidazolidine-2,4,5-trione (3d, IC50 = 1.66 μmol/L) expressed the highest BChE-inhibiting activity,
which is almost five times higher the that activity of galanthamine. Substitution of phenyl in the
position para is advantageous for inhibitory activity, especially in case of BChE inhibitors. It was also
found that inhibition of both cholinergic enzymes is more connected with electron properties of
individual substituents on phenyl than with lipophilicity of the discussed compounds. A branched
substituent on the phenyl ring positively influenced cholinergic inhibitory activity.

Molecules 2011, 16
7580
Acknowledgements
This study was supported by the Ministry of Education of the Czech Republic (MSM 0021627501
and MSM 0021627502).
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