Synthesis, molecular docking and biological evaluation of N,N-disubstituted 2-aminothiazolines as a new class of butyrylcholinesterase and carboxylesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.01.031

A series of 31 N,N-disubstituted 2-amino-5-halomethyl-2-thiazolines was designed, synthesized, and evaluated for inhibitory potential against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE). The compounds did not inhibit AChE; the most active compounds inhibited BChE and CaE with IC₅₀ values of 0.22-2.3 μM. Pyridine-containing compounds were more selective toward BChE; compounds with the para-OMe substituent in one of the two dibenzyl fragments were more selective toward CaE. Iodinated derivatives were more effective BChE inhibitors than brominated ones, while there was no influence of halogen type on CaE inhibition. Inhibition kinetics for the 9 most active compounds indicated non-competitive inhibition of CaE and varied mechanisms (competitive, non-competitive, or mixed-type) for inhibition of BChE. Docking simulations predicted key binding interactions of compounds with BChE and CaE and revealed that the best docked positions in BChE were at the bottom of the gorge in close proximity to the catalytic residues in the active site. In contrast, the best binding positions for CaE were clustered rather far from the active site at the top of the gorge. Thus, the docking results provided insight into differences in kinetic mechanisms and inhibitor activities of the tested compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (<30 μM), none of the tested compounds significantly affected viability of human fetal mesenchymal stem cells. The results indicate that a new series of N,N-disubstituted 2-aminothiazolines could serve as BChE and CaE inhibitors for potential medicinal applications.

Full text of DOI:10.1016/j.bmc.2016.01.031

Accepted Manuscript
Synthesis, molecular docking and biological evaluation of N,N-disubstituted
2–aminothiazolines as a new class of butyrylcholinesterase and carboxylesterase
inhibitors
Galina F. Makhaeva, Natalia P. Boltneva, Sofya V. Lushchekina, Olga G.
Serebryakova, Tatyana S. Stupina, Alexey A. Terentiev, Igor V. Serkov, Alexey
N. Proshin, Sergey O. Bachurin, Rudy J. Richardson
PII:
S0968-0896(16)30033-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.01.031
BMC 12774
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 November 2015
4 January 2016
17 January 2016
Please cite this article as: Makhaeva, G.F., Boltneva, N.P., Lushchekina, S.V., Serebryakova, O.G., Stupina, T.S.,
Terentiev, A.A., Serkov, I.V., Proshin, A.N., Bachurin, S.O., Richardson, R.J., Synthesis, molecular docking and
biological evaluation of N,N-disubstituted 2–aminothiazolines as a new class of butyrylcholinesterase and
carboxylesterase inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.01.031
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Synthesis, molecular docking and biological evaluation of N,N-
disubstituted 2-aminothiazolines as a new class of butyrylcholinesterase
and carboxylesterase inhibitors
Galina F. Makhaevaa, Natalia P. Boltnevaa, Sofya V. Lushchekinaa,b,
Olga G. Serebryakovaa, Tatyana S. Stupinac, Alexey A. Terentievc, Igor V. Serkova,
Alexey N. Proshina, Sergey O. Bachurina, Rudy J. Richardsond*
a Institute of Physiologically Active Compounds Russian Academy of Sciences,
Chernogolovka, 142432, Russia
b Emanuel Institute of Biochemical Physics Russian Academy of Sciences, Moscow,
119334, Russia
c Institute of Problems of Chemical Physics Russian Academy of Sciences,
Chernogolovka, 142432, Russia
d Department of Environmental Health Sciences and Department of Neurology,
University of Michigan, Ann Arbor, MI 48109 USA
* Corresponding author:
Dr. Rudy J. Richardson
Computational Toxicology Laboratory
Toxicology Program
Department of Environmental Health Sciences
University of Michigan
Ann Arbor, Michigan 48109-2029 USA
E-mail:
Tel:
Fax:
rjrich@umich.edu
+1-734-936-0769
+1-734-763-8095
1

Abstract
A series of 31 N,N-disubstituted 2-amino-5-halomethyl-2-thiazolines was designed,
synthesized, and evaluated for inhibitory potential against acetylcholinesterase (AChE),
butyrylcholinesterase (BChE) and carboxylesterase (CaE). The compounds did not inhibit
AChE; the most active compounds inhibited BChE and CaE with IC50 values of 0.22 to 2.3
µM. Pyridine-containing compounds were more selective toward BChE; compounds with the
para-OMe substituent in one of the two dibenzyl fragments were more selective toward CaE.
Iodinated derivatives were more effective BChE inhibitors than brominated ones, while there
was no influence of halogen type on CaE inhibition. Inhibition kinetics for the 9 most active
compounds indicated non-competitive inhibition of CaE and varied mechanisms
(competitive, non-competitive, or mixed-type) for inhibition of BChE. Docking simulations
predicted key binding interactions of compounds with BChE and CaE and revealed that the
best docked positions in BChE were at the bottom of the gorge in close proximity to the
catalytic residues in the active site. In contrast, the best binding positions for CaE were
clustered rather far from the active site at the top of the gorge. Thus, the docking results
provided insight into differences in kinetic mechanisms and inhibitor activities of the tested
compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (<
30 µM), none of the tested compounds significantly affected viability of human fetal
mesenchymal stem cells. The results indicate that a new series of N,N-disubstituted 2-
aminothiazolines could serve as BChE and CaE inhibitors for potential medicinal
applications.
Keywords
N,N-disubstituted 2-aminothiazolines, acetylcholinesterase, butyrylcholinesterase,
carboxylesterase, molecular docking, Alzheimer’s Disease.
2

1. Introduction
Among functional types of enzymes, serine hydrolases constitute one of the largest and
most varied groups. Of the approximately 240 serine hydrolases found in humans, about half
are serine proteases whereas the remainder carry out metabolic functions. The metabolic
serine hydrolases include diverse lipases, peptidases, esterases, thioesterases, and amidases
that hydrolyze small molecules, peptides, or post-translational (thio)ester protein
modifications 1. Because these enzymes serve important physiological and pathogenic
functions and their mechanisms of action are well understood, they have been popular targets
for drug development 2, 3
.
Acetylcholinesterase (AChE, EC 3.1.1.7) is a metabolic serine hydrolase that catalyzes
the hydrolysis of acetylcholine, thus regulating cholinergic neurotransmission. Therefore, in
disorders such as Alzheimer’s disease (AD), where there is diminished cholinergic activity,
inhibition of AChE has been employed to treat some of the symptoms attributed to decreased
acetylcholine levels 4, 5. Most of the currently available drugs on the market (tacrine,
donepezil, rivastigmine and galanthamine) intended to treat AD are AChE inhibitors 4-6
.
However, during the progression of Alzheimer’s disease, brain AChE levels decline while
butyrylcholinesterase (BChE, EC 3.1.1.8) activity increases, suggesting that acetylcholine
hydrolysis may occur to a greater extent via BChE catalysis 7, 8. In this regard, it has been
reported that highly selective inhibition of BChE is important in raising acetylcholine levels
and improving cognition 8-12. Because selective BChE inhibitors do not exhibit the adverse
cholinergic effects of AChE inhibitors, the search for selective BChE inhibitors is currently a
promising direction in medicinal chemistry research 10, 13-15. Moreover, whereas some of the
anticholinesterase compounds currently employed for AD treatment can inhibit both AChE
and BChE 12, 16, the highly selective AChE inhibitor (-) huperzine A was clinically
ineffective17. Likewise, the selective AChE inhibitor (-) phenserine was found to be
potentially efficacious for mild to moderate AD, but because this drug also inhibits
production of amyloid precursor protein, it is difficult to assess the degree to which its
efficacy stems from AChE inhibition 18. Taken together, these findings suggest that the
development of selective BChE inhibitors could be important for more effective treatment of
AD 19.
In recent years, interest in another group of metabolic hydrolases, the carboxylesterases
(CaE, EC 3.1.1.1), has sharply increased. CaE play major roles in the activation,
detoxification and biodistribution of xenobiotics and numerous classes of drugs including
3

esters, thioesters, carbamates, and amides 20, 21. Representative types of pharmaceutical agents
include angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, antiplatelet
drugs, statins, antivirals, and central nervous system agents 22. Inhibitors of CaE decrease the
rate of hydrolysis of such drugs and thus reduce the rate of conversion of pro-drug to active
drug or, conversely, increase the half-life of the active drug. Consequently, CaE inhibitors
have important therapeutic value 23 and discovery of new CaE inhibitors with high selectivity
is of considerable interest 24.
Recently, N,S-containing heterocyclic compounds, especially derivatives of 2-amino-1,3-
thiazole, have attracted increasing attention due to their chemical and biological properties.
For example, it has become known that many compounds containing the thiazoline (4,5-
dihydro-1,3-thiazole) fragment exhibit antibacterial, anticancer, antifungal, and other types of
biological activities 25-27. However, information about the inhibition of serine esterases by
compounds containing the thiazoline fragment is practically absent in the literature. The
ability to reversibly inhibit cholinesterases was shown for only some 2-substituted thiazolines
and imidazo[2,1-b]thiazole derivatives 28, 29
.
Our preliminary study 30 showed that N,N-disubstituted-2-aminothiazolines could inhibit
BChE and CaE while exhibiting minimal inhibitory activity against AChE. A search of
chemical reactivity of 5-Br-methyl substituted thiazolines for the past 40 years using Reaxys
turned up very few reactions. The alkylation reactions for 5-Br-methyl thiazolines were not
described in the literature. The same results were obtained with SciFinder. Thus, the ability of
2-amino-5-halomethylthiazoline derivatives to alkylate DNA is unlikely. Therefore, we
extended our experimental search for inhibitors of serine esterases with potential biomedical
application in the 2-aminothiazoline series.
Here, we report the synthesis of 31 derivatives of 2-aminothiazolines of general formula
shown in Fig. 1. The syntheses are outlined in Scheme 1 and the structures of the individual
compounds are listed in Table 1. Our strategy for the design of the inhibitors is illustrated in
Fig. 2. We have also performed a biological evaluation of these compounds as inhibitors of
three metabolic serine esterases (AChE, BChE and CaE) along with an optimization of their
structures to yield selective inhibitors of BChE and/or CaE. To gain further insight into the
molecular determinants responsible for the observed ability to inhibit BChE and CaE,
molecular docking of the 9 most active compounds was carried out. In addition, the effect of
the compounds on cell viability was evaluated in human fetal mesenchymal stem cells.
4

Figure 1. General structure of compounds in the present study. X = Br or I; R1 = substituted
or unsubstituted aryl or aralkyl; R2 = H, aryl, or aralkyl. Note that the structure depicts the
hydrohalide salt of the compounds. Syntheses are depicted in Scheme 1 and structures of
individual compounds are listed in Table 1.
Figure 2. Design strategy for the new series of N,N-disubstituted 2-aminothiazolines of
general formula shown in Fig. 1. X = Br or I. Syntheses are depicted in Scheme 1 and
structures of the compounds are presented in Table 1. Proposed interactions of compounds
with active site residues of target enzymes include the following, illustrated with selected
compounds: (a) Halogen and aromatic (benzyl, pyridine) fragments are intended to have
stacking interactions with aromatic Trp, Phe and Tyr residues, primarily the most favorably
oriented Trp82 and Trp231 of BChE; (b) Alkyl fragments are intended to interact with
numerous hydrophobic residues lining the gorge of CaE; (c) Positively charged groups are
intended to interact with Glu of both enzymes (Glu197 of BChE and Glu220 of CaE), and
probably Asp70 of the peripheral anionic site of BChE; (d) Electronegative atoms (N, O) are
expected to interact with the oxyanion hole of both enzymes (Gly116/Gly117/Ala199 for
BChE and Gly142/Gly143/Ala222 for CaE).
5

2. Results and discussion
2.1. Chemistry.
We have synthesized the hydrohalides of 2-amino-substituted 5-halomethyl-2-thiazolines
5a-m and 6a-t by traditional methods (Scheme 1): iodination or bromination of the
corresponding N',N'-derivatives of N-allylthiourea 31 obtained from allylisothiocyanate and
the corresponding amine.
R1
H
a
b
N
N
N
R1R2NH
R2
+
S
S
1
3
2
HX
X
R1
R1
R2
H
S
N
N
X
R2
N
N
X
S
4
5 X = Br;
6 X = I
Scheme 1. Synthesis of N,N-disubstituted 2-aminothiazolines 5,6. Reagents and conditions:
(a) diethyl ether, r.t., 2–5 h, (b) X2, СH3OH, r.t., 24 h.
Previous studies have shown that isomeric six-membered rings (thiazines) are formed in
these reactions as well as thiazolines 32, 33. However, given that these reactions are usually
performed in polar media with heating, only five-membered thiazoline ring compounds are
isolated from the reaction mixture, because under these conditions there is a facile
dihydrothiazine-thiazoline rearrangement 34. The structural features of the synthesized
compounds are shown in Table 1. Most of the compounds were synthesized for the first time
for the present report, but we have previously reported 31 on the synthesis of 7 compounds:
6a, 6b, 6c, 6e, 6f, 6h, 6i, 6k. All of the compounds except 5k, 6k are racemic mixtures.
Compounds 5k, 6k are diastereomeric mixtures.
2.2. Inhibition studies of AChE, BChE and CaE (IC50). Structure-activity relationships.
All 31 compounds were assessed for their potential to inhibit AChE, BChE and CaE by
determining the IC50 – the inhibitor concentration that decreases the enzyme activity by half.
AChE from human erythrocytes (RBCs) was used along with two enzymes of non-human
6

origin, namely BChE from equine serum and CaE from porcine liver because of their lower
cost, high degree of identity with human enzymes, and the exploratory character of this work.
Three drugs: tacrine, donepezil, and benzil (1,2-diphenylethane-1,2-dione) were used as
positive controls 35. The IC50 results are summarized in Table 1.
Table 1
Structures of the synthesized N,N-disubstituted 2-aminothiazolines and their inhibitory
potencies (IC50) toward AChE, BChE, and CaE.a
IC50 (µM)b
Cmpd
R1
R2
X
AChE
(human
RBC)
BChE
(Equine
serum)
CaE
(Porcine
Liver)
H
H
H
H
H
H
H
H
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
H
H
Bn
Bn
Ph
Br
I
Br
I
Br
I
Br
I
Br
I
Br
I
Br
I
Br
I
Br
I
Br
I
Br
I
Br
I
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
29 ± 2
> 100
n.a.
n.a.
> 100
n.a.
> 100
17 ± 1
> 100
> 100
93 ± 5
610 ± 60
32± 3
46 ± 4
5a
6a
5b
6b
5c
6c
5d
6d
5e
6e
5f
Ph
n.a.
pyridin-2-yl
pyridin-2-yl
Bn
110 ± 10
71 ± 7
n.a.
63 ± 6
2.74 ± 0.25
26 ± 1
25 ± 1
38 ± 3
17 ± 1
11 ± 1
4.14 ± 0.39
2.34 ± 0.21
0.77 ± 0.07
34 ± 3
25 ± 1
14 ± 1
> 100
30 ± 1
2.47 ± 0.22
25 ± 2
21 ± 1
44 ± 3
20 ± 2
> 100
Bn
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
> 100
n.a.
n.a.
n.a.
> 100
n.a.
4-FC6H4CH2
4-FC6H4CH2
4-i-PrC6H4CH2
4-i-PrC6H4CH2
4-t-BuC6H4CH2
4-t-BuC6H4CH2
4-MeOC6H4CH2
4-MeOC6H4CH2
CH(Me)Ph
40 ± 4
80 ± 8
6f
2.55 ± 0.23
2.13 ± 0.19
0.32 ± 0.03
0.22 ± 0.02
4.90 ± 0.45
4.66 ± 0.21
> 100
19 ± 2
> 100
18 ± 1
> 100
> 100
117 ± 12
> 100
5g
6g
5h
6h
5i
Bn
Bn
Bn
Bn
6i
5k
6k
5l
6l
5
CH(Me)Ph
4-ClC6H4CH2
4-ClC6H4CH2
2-ClC6H4CH2
2-ClC6H4CH2
Bn
pyridin-3-ylmethyl
pyridin-3-ylmethyl
pyridin-3-ylmethyl
pyridin-3-ylmethyl
2-ClC6H4CH2
3,4-(MeO)2C6H3CH2
3,5-(t-Bu)2-4-OH-
C6H2CH2
6
I
I
I
6n
6o
6p
Bn
Bn
n.a.
Bn
H
furan-2-yl
I
I
n.a.
n.a.
42 ± 4
> 100
131 ± 12
> 100
6q
6r
3,4-
OCH2CH2O)C6H3
6-Me-pyridin-2-yl
dibenzofuran-3-yl
H
H
I
I
n.a.
n.a.
n.a.
> 100
n.a.
> 100
6s
6t
7

Tacrinec
Donepezilc
0.079 ± 0.002
0.022 ± 0.005
> 100
0.034 ± 0.001
(human
BChE)
> 100
(human
CE1)
> 100
(human
CE1)
0.37 ± 0.04
(human
CE1)
7.6 ± 1.3
(human
BChE)
> 100
(human
BChE)
Benzilc,d
n.a. – not active
a General structure of the synthesized compounds is shown in Fig. 1.
b Data are means + SEM from 3 experiments.
c [35]; human CE1 = human liver carboxylesterase 1.
d 1,2-diphenylethane-1,2-dione
Comparing inhibitory potencies of a compound against the three esterase targets yields an
esterase profile, which helps predict the main potential pharmacological effect of the
compound and evaluate its possible side-effects, 36-39 thus helping to define the area(s) of
potential application of the compound. For example, effective inhibitors of AChE and BChE
can be used for AD treatment. However, inhibition of CaE by such anticholinesterase
compounds leads to adverse drug-drug interactions 35. On the other hand, potent and selective
inhibitors of CaE can be used for the regulation of the metabolism and pharmacokinetics of
ester-containing drugs 23, 24. For such CaE inhibitors, anticholinesterase activity can be an
undesirable side effect.
As seen from Table 1, most of the synthesized compounds did not substantially inhibit
AChE, while the degree of BChE and CaE inhibition depended on structural features.
As starting templates for modification, we selected 5-bromomethyl-4,5-dihydro-thiazol-2-
yl-amine (5a) and 5-iodomethyl-4,5-dihydro-thiazol-2-yl-amine (6a) that were not substituted
on the outside nitrogen atom. These compounds were not active inhibitors: they did not
inhibit AChE, BChE and weakly inhibited CaE (5a) (Fig. 1, Table 1).
Introduction of a 2-pyridyl radical on the external nitrogen atom (5d, 6d) resulted in the
appearance of inhibitory activity against all three esterases with preferential inhibition of
BChE. Iodine-containing compound 6d had a higher inhibitory potency toward BChE: IC50 =
2.74 ± 0.25 µM than the corresponding bromine derivative (5d): IC50 = 63 ± 6 µM, while
AСhE and CaE were weakly inhibited by these compounds.
Insertion of a benzyl group in the starting template (5a, 6a) resulted in a noticeable
inhibitory activity toward BChE and CaE: compounds (5b, 6b). Because the benzyl
derivatives (5b, 6b) were active against two enzymes of interest, BChE and CaE, they were
selected for further modification.
8

Introduction of a second benzyl radical in the molecules (5b, 6b) produced compounds
(5e, 6e) that inhibited BChE and CaE in the absence of anti-AChE activity. Therefore, a
further search for selective and potent inhibitors of esterases was carried out among N,N-
disubstituted 2-aminothiazolines.
Changing one of the benzyl radicals in (5e, 6e) to 3-pyridylmethyl (5l, 6l, 5m, 6m) led to
enhancement of inhibitory activity and selectivity against BChE, which was stronger for 4-Cl
substituted (6l) (IC50 = 2.47 ± 0.22 µM) than for the 2-Cl compound (6m) (IC50 = 21 ± 1
µM). The iodinated analogue (6l) was significantly more active than the brominated one (5l)
(IC50 = 30 ± 1 µM). All 3-pyridylmethyl-containing compounds (5l, 6l, 5m, 6m) were weak
CaE inhibitors and did not have anti-AChE activity.
Further modifications were directed to the introduction of various substituents (alkyl,
methoxy, hydroxyl, fluorine, chlorine) in the aryl moiety of one of the two dibenzyl
fragments: compounds (5f-i, 6f-i).
Introduction of a para-methoxy group (5i, 6i) significantly increased the inhibitory
activity and selectivity of the compounds against CaE: IC50 = 4.90 ± 0.45 µM (5i) and 4.66 ±
0.21 µM (6i), while BChE was inhibited weakly by these compounds (IC50 = 34 ± 3 µM (5i)
and 25 ± 1 µM (6i)).
The presence of an isopropyl radical in the para-position (5g, 6g) further enhanced
activity against CaE: IC50 = 2.55 ± 0.23 µM (5g) and 2.13 ± 0.19 µM (6g). Moreover, in
comparison to the bromine-substituted 5g, the iodine-containing compound 6g also exhibited
relatively strong inhibitory activity against BChE: IC50 = 4.14 ± 0.39 µM.
Benzyl-(4-tert-butyl-benzyl)-(5-halogenomethyl-4,5-dihydro-thiazol-2-yl)-amines (5h,
6h) exhibited the highest inhibitory activity against BChE and CaE. These compounds
contain a tert-Bu group at the para-position of the benzene ring. IC50 values for BChE
inhibition by compounds 5h and 6h were 2.34 ± 0.21 µM and 0.77 ± 0.07 µM, respectively.
For CaE inhibition, IC50 values were 0.32 ± 0.03 µM (5h) and 0.22 ± 0.02 µM (6h).
Compounds (5f-i, 6f-i) did not inhibit AChE.
Thus, analysis of the IC50 values of the synthesized N,N-disubstituted 2-aminothiazolines
presented in Table 1 shows that pyridine-containing compounds 5d, 6d and 5l, 6l are more
selective toward BChE. Modification of the aryl moiety of one of the two dibenzyl fragments
resulted in compounds (5f-i, 6f-i) which did not have anti-AChE activity. Depending on the
structure of the substituents at the outside nitrogen atom of N,N-disubstituted 2-
aminothiazolines, the compounds exhibited preferential inhibitor activity toward CaE
(compounds with a para-OMe substituent 5i, 6i) or both esterases. Iodine derivatives in all
9

cases were more effective BChE inhibitors than bromine derivatives, while there was no
influence of the halogen type on CaE inhibition.
2.3. Kinetic studies of BChE and CaE inhibition.
The 9 most active compounds (6d, 5g-i, 6g-i, 5l-6l) toward BChE and CaE (Table 2)
were selected for inhibition kinetics studies. Lineweaver-Burk plots -- double reciprocal plots
of Michaelis-Menten parameters -- were used to evaluate the type of inhibition.
Table 2
Inhibition constants of the active N,N-disubstituted 2-aminothiazolines toward BChE and
CaEa.
Enzymes
Compounds
BChE (Equine serum)
CaE (Porcine liver)
Ki (µM)
Ki (µM)
Ki (µM)
1.53 ± 0.15
nd
Ki (µM)
nd
nd
6d
5g
6g
5h
6h
5i
nd
3.54 ± 0.34
2.09 ± 0.19
0.44 ± 0.02
0.40 ± 0.03
7.37 ± 0.67
7.81 ± 0.25
nd
1.34 ± 0.12
0.62 ± 0.05
0.27 ± 0.02
nd
7.56 ± 0.15
1.20 ± 0.11
nd
nd
nd
6i
9.51 ± 0.92
0.67 ± 0.07
19.90 ± 1.93
3.60 ± 0.34
nd
nd
5l
nd
6l
a Values for Ki (competitive inhibition constant) and Ki (non-competitive inhibition
constant) were determined from analyses of slopes of 1/V versus 1/S at various inhibitor
concentrations. Values (means ± SEM) are from at least three separate experiments.
nd = not determined.
As seen from IC50 values (Table 1), para-tert-butyl containing compounds 5h (Br
derivative) and 6h (I derivative) were the most potent inhibitors of both BChE and CaE.
Graphical analysis of steady-state inhibition kinetics data for compounds 5h and 6h against
BChE are shown in Fig. 3. For compound 5h, raising the apparent Km of the enzyme for the
substrate did not affect Vmax, which is consistent with competitive inhibition (Fig. 3A). The
derived inhibition Ki was 0.62 ± 0.05 µM. In contrast, binding of 6h to BChE changed both
Vmax and Km values, consistent with mixed-type inhibition (Fig. 3B). The Ki for 6h was 0.27
10

± 0.02 µM (competitive component) and Ki was 1.20 ± 0.11 µM (noncompetitive
component). Thus, replacement of Br (5h) with I (6h) led to a change of mechanism for
BChE inhibition from competitive to mixed and slightly improved the inhibitory potency.
0.030
2 µM
1 µM
0.5 µM
no inhibitor
0.025
0.020
0.015
0.010
0.005
0.000
-1
-5
-4
-3
-2
0
1
2
3
4
5
[S] -1mM -1
A
1 µM
0.5 µM
0.125 µM
no inhibitor
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
-1
-5
-4
-3
-2
0
1
2
3
4
5
6
[S] -1 mM -1
B
Figure 3. Steady state inhibition of BChE by compounds 5h (A) and 6h (B). Lineweaver-
Burk reciprocal plots of initial velocity and substrate concentrations in the presence of
inhibitors 5h, 6h (three concentrations) and their absence are presented. Plot A shows
competitive inhibition and plot B shows mixed-type inhibition.
11

0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.000
0.4 µM
0.2 µM
0.1 µM
no inhibitor
-4
-3
-2
-1
0
1
2
3
[S] -1 mM -1
A
0.3 µM
0.2 µM
0.021
0.1 µM
no inhibitor
0.018
0.015
0.012
0.009
0.006
0.003
0.000
-4
-3
-2
-1
0
1
2
3
4
[S] -1 mM -1
B
Figure 4. Steady state inhibition of CaE by compounds 5h (A) and 6h (B). Lineweaver-Burk
reciprocal plots of initial velocity and substrate concentrations in the presence of inhibitors
5h, 6h (three concentrations) and their absence are presented. Plots A and B show non-
competitive inhibition.
Compounds 5h and 6h were also the most effective CaE inhibitors. Moreover, as shown
in Fig. 4, increasing inhibitor concentrations resulted in a decrease in Vmax while Km
remained unchanged. This is typical of non-competitive inhibition. Both compounds had
12

virtually identical inhibitor constants: 0.44 ± 0.02 µM (5h) and 0.40 ± 0.03 µM (6h). Thus, in
contrast to BChE inhibition, replacement of Br with I in the inhibitor did not change the
mechanism or efficiency of CaE inhibition.
Compounds with an iso-Pr group in the para-position (5g, 6g) demonstrated similar
inhibition kinetics (plots not shown) as 5h and 6h while being less potent against both BChE
and CaE. The iodinated derivative 6g was more potent than the brominated compound 5g
against BChE; it was a mixed-type inhibitor with Ki = 1.34±0.12 µM and Ki=7.56±0.15 µM.
CaE inhibition by para-iso-Pr substituted compounds 5g and 6g was described by non-
competitive kinetics, and the inhibition constants were close: Ki = 3.54±0.34 µM (5g) and
2.09±0.19 µM (6g). Thus, the type of halogen (Br or I) had no effect on inhibition
mechanism and little or no effect on inhibitory potency.
Para-OMe containing compounds 5i and 6i demonstrated selectivity toward CaE in
comparison to BChE. Inhibition of CaE occurred by a non-competitive mechanism
(unchanged Km and decreased Vmax with increasing inhibitor concentrations). The Br and I
derivatives had overlapping inhibitor constants: Ki = 7.37 ± 0.67 and 7.81 ± 0.25 µM,
respectively.
Pyridine-containing compounds (6d, 5l, 6l) in general showed selectivity toward BChE in
comparison to CaE. Thus, the active compound containing a 2-pyridinyl fragment 6d (I
derivative) inhibited BChE according to a non-competitive mechanism (Km = constant, Vmax
= decreased with increasing inhibitor concentrations); Ki=1.53 ± 0.15 µM.
3-Pyridinyl-containing compounds 5l (Br-derivative) and 6l (I-derivative) displayed
mixed-type inhibition toward BChE (decreasing Vmax and increasing Km with increasing
inhibitor concentrations). The I-derivative 6l was more potent (Ki = 0.67 ± 0.07, Ki = 3.60 ±
0.34 µM) than the Br-derivative 5l (Ki = 9.51 ± 0.92 µM, Ki = 19.90 ± 1.93 µM).
Thus, inhibition kinetics of N,N-disubstituted 2-aminothiazolines against BChE and CaE
demonstrated that CaE inhibition was in all cases non-competitive, whereas BChE inhibition
was competitive, mixed, or non-competitive depending on the structural features of the
compound.
To gain insight into the various inhibitory mechanisms and differences in inhibitor
potency and selectivity, molecular docking simulations were performed.
2.4. Molecular modeling studies.
13

To help explain the different activities of the target compounds and clarify their
interaction mode in the active sites of BChE and CaE, molecular docking simulations were
performed for compounds 5h, 6h, 5l, 6l, 5i, 6i, 5e, 6e, 5g, 6g, 5d, 6d, 6m using Autodock 4.2
software. The docking results for compounds 5e, 6e, 5g, 6g, 5d, 6d, and 6l-6m comparison
are presented in the supplementary material (Fig. S1-S4).
The modeling results showed that the best docked positions of the studied compounds
inside BChE were within the active site, at the bottom of the gorge, in close proximity to the
catalytic triad residues. In contrast, the best binding positions for CaE were clustered rather
far from the active site, at the top of the gorge, and the ligands were surrounded by several
leucines, isoleucines and methionines. This difference between BChE and CaE in the location
of the inhibitor inside the gorge was observed for the most of studied compounds.
A
B
Figure 5. Binding positions of compounds 5h (carbon atoms are colored grey) and 6h
(carbon atoms are colored blue) inside the gorge of human CaE (A) and BChE (B). Carbon
atoms of the enzyme residues side chains are colored green and the catalytic residues are
depicted in cyan.
Tert-Bu-containing compounds 5h and 6h were the most active inhibitors of both BChE
and CaE. Both compounds were located rather far from the active site of CaE, at the top of
the gorge (Fig. 5A). Positions of the Br-analogue 5h and I-analogue 6h inside CaE were very
similar; no significant difference was observed. This is because their halogen atoms emerge
from the mouth of the gorge and do not interact with protein atoms. The modeling results
agree with the experimentally observed non-competitive mechanism of CaE inhibition by Br-
and I-analogues, 5h and 6h, and their equivalent inhibitor activity against CaE.
14

The best docked positions of 5h and 6h inside BChE were within the active site (Fig. 5B),
at the bottom of the gorge, in close proximity to the catalytic residues. The main contributors
to binding of both 5h and 6h with the BChE active site were electrostatic interactions and
hydrogen bonds of the positively charged thiazoline fragment (amidine group) and the
negatively charged carboxylic group of Glu197. Additionally, 6h had C-I…π interactions
with Trp231. Its Br-containing analogue 5h occupied a mirror-image position, but with the
halogen atom too far from Trp430 and Trp82 to interact with their aromatic rings. This
difference in halogen interactions explains the higher inhibitory activity of the I-derivative 6h
toward BChE and the different mechanisms of BChE inhibition by the Br- and I-derivatives.
In contrast to CaE, the active site of BChE contains numerous aromatic side chains; this
difference furnishes a possible reason for the influence exerted by the halogen atom on the
binding of inhibitors to BChE. Most of I-derivatives tended to interact with aromatic rings, in
particular, the indole ring of tryptophan residues. This observation corresponds well with
recently reported findings that halogens, in particular I, form rather strong halogen bonds,
including C-I…π interactions, comparable with strong hydrogen bonds 40, 41. The indole rings
of Trp82 and Trp231 are aligned parallel to the BChE gorge wall, while the phenyl rings of
Phe329 and Phe398 are oriented in a more perpendicular fashion. This geometrical
arrangement favors interactions of the iodine atoms with the tryptophan residues.
Docking results for compounds with an iso-Pr group in the para-position (5g, 6g) were
similar to those observed for the tert-Bu compounds 5h and 6h (see supplementary data, Fig.
S2), again in agreement with the kinetics studies.
The presence of aromatic groups in the active site of BChE also ensures higher binding
affinities to compounds containing a pyridine ring. The situation is quite different for CaE,
which lacks aromatic residues in the gorge, particularly at the entrance, where most of the
CaE inhibitors bind. The lip of the CaE gorge is mainly lined with methionines and other
aliphatic residues.
Compounds 5l and 6l containing a 3-pyridinyl ring were more effective inhibitors of
BChE than CaE. These are mixed-type BChE inhibitors, and the I-derivative 6l is more active
than the Br-derivative 5l. These compounds bind with BChE very tightly, fully occupying its
active site (Fig. 6A,B). In each compound, the protonated thiazoline fragment forms a strong
hydrogen bond with Glu197. However, the arrangement of other molecular fragments differs
between the two compounds. In 5l, the pyridine ring is oriented with its nitrogen atom in the
oxyanion hole, where it forms hydrogen bonds with the peptide bond nitrogen atoms of
Gly117 and Ala199. For 6l, the p-Cl-phenyl ring is parallel to Trp82 and thus has strong π-π
15

interactions (Fig. 6A). The Br atom of 5l is inserted into a cavity formed by the side chains of
Leu125 and Thr120 and the backbone chains of Gly121 and Trp82 (Fig. 6B). However, the I
atom of 6l has a larger van der Waals radius than Br and cannot fit into this cavity, thus
creating the difference in orientation between these two similar ligands.
A
B
Figure 6. Binding positions of compounds 5l (carbon atoms are colored grey) and 6l (carbon
atoms are colored blue) in the active site of BChE. (A) and (B) are views from different
vantage points. In panel (A) carbon atoms of BChE residues side chains are colored green
and the catalytic residues are rendered in cyan; in panel (B) BChE solvent accessible surface
and van der Waals spheres of the inhibitor halogen atoms are shown.
Compounds with an OMe group in the para-position of one of the two dibenzyl fragments
(5i and 6i) are more selective for CaE than BChE. The position of these two compounds in
CaE is close to the active site, with the methoxy group located near Ser221 in an orientation
favorable for formation of hydrogen bonds both with the catalytic serine and oxyanion hole
(Fig. 7A). Halogen atoms are oriented towards the mouth of the gorge and therefore have no
influence on binding position. In the case of BChE, the orientation of these inhibitors is
determined by interaction of the halogen atoms with Trp231 (Fig. 7B), and binding is
otherwise highly favorable (6i has π-π interactions with Trp82 and forms a hydrogen bond
between the OMe group and Tyr128, whereas the positively charged thiazoline group of 5i
interacts with Glu197). Given that the molecular docking results reveal favorable interactions
of these compounds both the active sites of both CaE and BChE, the reason for the
experimentally determined selectivity of these compounds for CaE must lie elsewhere, such
as differences in transport through the gorge to the active site of the enzymes. Future studies
16

using molecular dynamics simulations might be used to explore the plausibility of this
explanation.
A
B
Figure 7. Binding positions of compounds 5i (carbon atoms are colored grey) and 6i (carbon
atoms are colored blue) inside the gorge of human CaE (A) and BChE (B). Carbon atoms of
the enzyme residues side chains are colored green and the catalytic residues are depicted in
cyan.
2.5. Cytotoxicity studies
To investigate the effect of the test compounds on cell viability, cytotoxicity studies for
these compounds were performed on human fetal mesenchymal stem cells using the
MTT/formazan assay. At the limit of solubility of these compounds (less than 30 µM), none
of the tested compounds resulted in a significant decrease in cell viability (data not shown).
3. Conclusions
In summary, a novel series of N,N-disubstituted 2-aminothiazolines has been designed,
synthesized and evaluated as AChE, BChE, and CaE inhibitors. The results demonstrate that
the tested compounds do not inhibit AChE; accordingly, they would be expected not to cause
unwanted cholinergic side effects if they were employed, e.g., as co-drugs for ester-
containing pharmacological agents. The relative selectivity of the most active compounds
toward BChE and CaE is summarized in Fig. 8. Whereas the compounds did not inhibit
AChE, the level of anti-BChE and anti-CaE activity depended on the structure of the
compounds. Pyridine-containing compounds were more selective toward BChE and
compounds with a para-OMe substituent in one of the two dibenzyl fragments were more
selective toward CaE. Iodinated derivatives were more effective BChE inhibitors than
17

brominated ones, while there was no influence of the halogen type on CaE inhibition. The
most potent inhibitors of BChE and CaE were the benzyl-(4-tert-butyl-benzyl)-(5-
halogenomethyl-4,5-dihydro-thiazol-2-yl)-amines. IC50 values for BChE inhibition by 5h
(X=Br) and 6h (X=I) were 2.34 ± 0.21 µM and 0.77 ± 0.07 µM, respectively. For CaE
inhibition, IC50 values were 0.32 ± 0.03 µM (5h) and 0.22 ± 0.02 µM (6h).
The kinetic studies performed for the most active compounds demonstrated that all
studied compounds acted as non-competitive CaE inhibitors and exhibited different
mechanisms (competitive, non-competitive, or mixed-type) of BChE inhibition depending on
the structural features of the compound. Docking simulations showed the best docked
positions of the compounds inside BChE were within the active site, at the bottom of the
gorge, in close proximity to the catalytic residues. In contrast, the best binding positions for
CaE were clustered rather far from the active site, at the top of the gorge. The docking results
help explain differences in the kinetic mechanisms and inhibitor activity of the tested
compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (<
30 µM), none of the tested compounds significantly affected viability of human fetal
mesenchymal stem cells.
Overall, the results indicated that members of a novel series of N,N-disubstituted 2-
aminothiazolines have the potential to be good candidates for BChE and CaE inhibitors for
potential medicinal applications.
4. Experimental section
4.1. Chemistry
All solvents, chemicals, and reagents were obtained commercially and used without
purification. 1H NMR (200 MHz) spectra were recorded on a Bruker СХР-200 NMR
spectrometer using CDCl3, DMSO-D6, acetone-d6, CD3OD, CD3CN, or D2O as solvents with
tetramethylsilane as an internal standard. Chemical shifts, d, are given in parts per million
(ppm), and spin multiplicities are given as s (singlet), br s (broad singlet), d (doublet), t
(triplet), q (quartet) or m (multiplet). Coupling constants, J, are expressed in hertz (Hz).
Melting points were recorded on a Boetius hot-plate apparatus and are uncorrected. Yields
refer to isolated pure products and were not maximized. CHN analysis was performed on a
CHN elemental analyzer vario MICRO cube (elementar GmbH). The MS spectra were
recorded using a Thermo Fisher Exactive instrument with an Orbitrap mass analyzer.
4.2. Synthesis of compounds
4.2.1. A general procedure for the synthesis of compounds
18

A solution of allylisothiocyanate 1 (0.99 g, 0.01 M) in 20 ml of diethyl ether (ether) was
added dropwise to a solution of amine 2 (0.01 M) in 20 ml of ether with intensive stirring.
The reaction mixture was stirred for 2–5 h at room temperature. The thiourea precipitate 3
was filtered, dried and dissolved in 20 ml methanol and a solution of halogen (0.01 M) in 5
ml methanol was added dropwise. The reaction mixture was stirred 24 h at room temperature,
evaporated to 1/3 volume and diluted with 20 ml of ether. The precipitate was filtered,
recrystallized from isopropanol, and the hydrohalogen 5-halomethyl-2-aminothiazolines 5, 6
were obtained with high yields as noted below.
4.2.2. 5-Bromomethyl-4,5-dihydro-thiazol-2-ylamine hydrobromide (5a)
Yield: 86%; mp: 145-147 °C;
1H NMR (200 MHz, D2O):  = 3.74 (dd, J = 2.1, 6.7 Hz, 2H, NCH2), 4.07 (dd, J = 2.1,
5.1 Hz, 2H, BrCH2), 4.45 (m, 1H, CHS);
EI-MS (m/z): 277.0 (M + H+).
Anal. Calcd. for C4H8Br2N2S: C 17.41; H 2.92; N 10.15. Found: C 17.48; H 2.97; N
10.10.
4.2.3. 5-Iodomethyl-4,5-dihydro-thiazol-2-ylamine hydroiodide (6a).
Yield: 82%; mp: 127-129 °C;
1H NMR (200 MHz, DMSO-d6): 3.62 (m, 2H, NCH2), 3.80 (dd, J = 3.7, 12.0 Hz, 1H,
ICHH), 4.00 (dd, J = 7.3, 12.0 Hz, 1H, ICHH), 4.37 (m, 1H, CHS), 9.30 (br s, 3H, NH3);
EI-MS (m/z): 371 (M + H+).
Anal. Calcd. for C4H8I2N2S: C 12.99; H 2.18; N 7.57. Found: C 13.09; H 2.11; N 7.63
4.2.4. Benzyl-(5-bromomethyl-4,5-dihydro-thiazol-2-yl)-amine hydrobromide (5b)
Yield: 87%; mp: 80-82 °C;
1H NMR (200 MHz, CDCl3):  = 3.55 (d, J = 7.3 Hz, 2H, PhCH2), 4.07 (m, 2H,
NCH2CHS), 4.20 (m, 1H, BrCHH), 4.49 (m, 2H, ICHH, CHS), 7.33 (m, 5H, Harom), 10.19 (br
s, 1H, NH), 10.61 (br s, 1H, NH);
EI-MS (m/z): 367 (M + H+).
Anal. Calcd. for C11H14Br2N2S: C 36.09; H 3.85; N 7.65. Found: C 36.01; H 3.88; N 7.72.
4.2.5. Benzyl-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine hydroiodide (6b)
Yield: 77%; mp: 173-175 °C;
1H NMR (200 MHz, DMSO-d6): 3.39 (d, J = 7.0 Hz, 2H, PhCH2), 3.87 (m, 2H,
NCH2CHS), 4.01 (m, 1H, BrCHH), 4.28 (m, 2H, ICHH, CHS), 7.01 (m, 5H, Harom), 9.71 (br
s, 1H, NH), 10.11 (br s, 1H, NH);
19

EI-MS (m/z): 461 (M + H+).
Anal. Calcd. for C11H14I2N2S: C 28.71; H 3.07; N 6.09. Found: C 28.78; H 3.09; N 6.05.
4.2.6. (5-Bromomethyl-4,5-dihydro-thiazol-2-yl)-phenyl-amine hydrobromide (5c)
Yield: 86%; mp: 150-152 °C;
1H NMR (200 MHz, DMSO-d6):  = 3.88 (dd, J = 2.9, 9.3 Hz, 1H, BrCHH), 3.91 (d, J =
6.4 Hz, 2H, NCH2), 4.11 (dd, J = 7.6, 12.0 Hz, 1H, BrCHH), 7.45 (m, 5H, Harom), 11.1 (br s,
1H, NH);
EI-MS (m/z): 353 (M + H+).
Anal. Calcd. for C10H12Br2N2S: C 34.11; H 3.44; N 7.96. Found: C 34.21; H 3.48; N 8.01.
4.2.7. (5-Iodomethyl-4,5-dihydro-thiazol-2-yl)-phenyl-amine hydroiodide (6c)
Yield: 85%; mp: 135-137 °C;
1H NMR (200 MHz, CD3OD): 3.75 (d, J = 7.0 Hz, 2H, NCH2CHS), 4.05 (dd, J = 3.5,
12.1 Hz, 1H, ICHH), 4.24 (dd, J = 7.4, 12.1 Hz, 1H, ICHH), 4.55 (m, 1H, CHS), 7.50 (m, 5H,
Harom);
EI-MS (m/z): 447 (M + H+).
Anal. Calcd. for C10H12I2N2S: C 26.93; H 2.71; N 6.28. Found: C 26.99; H 2.74; N 6.21.
4.2.8. (5-Bromomethyl-4,5-dihydro-thiazol-2-yl)-pyridin-2-yl-amine hydrobromide (5d)
Yield: 83%; mp: 156-158 °C;
1H NMR (200 MHz, DMSO-d6):  = 3.84 (m, 2H, NCH2), 4.16 (m, 2H, BrCH2), 4.43 (m,
1H, CHS), 7.26 (dd, J = 5.1, 6.9 Hz, 1H, Harom), 7.42 (d, J = 8.3 Hz, 1H, Harom), 7.92 (m, 1H,
Harom), 8.41 (d, J = 3.7 Hz, 1H, Harom);
EI-MS (m/z): 354 (M + H+).
Anal. Calcd. for C9H11Br2N3S: C 30.62; H 3.14; N 11.90. Found: C 30.68; H 3.19; N
11.95.
4.2.9. (5-Iodomethyl-4,5-dihydro-thiazol-2-yl)-pyridin-2-yl-amine hydroiodide (6d)
Yield: 76%; mp: 137-139 °C;
1H NMR (200 MHz, DMSO-d6):  = 3.73 (d, J = 7.1 Hz, 2H, NCH2CHS), 4.06 (dd, J =
3.5, 12.0 Hz, 1H, ICHH), 4.22 (dd, J = 7.4, 12.0 Hz, 1H, ICHH), 4.53 (m, 1H, CHS), 7.27 (m,
1H, Harom), 7.41 (d, J = 8.2 Hz, 1H, Harom), 7.95 (m, 1H, Harom), 8.38 (m, 1H, Harom);
EI-MS (m/z): 448 (M + H+).
Anal. Calcd. for C9H11I2N3S: C 24.18; H 2.48; N 9.40. Found: C 24.24; H 2.53; N 9.33.
4.2.10. Benzyl-(5-bromomethyl-4,5-dihydro-thiazol-2-yl)-(4-fluoro-benzyl)-amine
hydrobromide (5e)
20

Yield: 77%; mp: 178-180 °C;
1H NMR (200 MHz, DMSO-d6):  = 3.88 (d, J = 6.4 Hz, 2H, NCH2CHS), 4.05 (dd, J =
3.2, 12.5 Hz, 1H, BrCHH), 4.20 (dd, J = 7.3, 12.5 Hz, 1H, BrCHH), 4.57 (m, 1H, CHS), 4.71
(s, 4H, CH2NCH2), 7.31 (m, 10H, Harom);
EI-MS (m/z): 457 (M + H+).
Anal. Calcd. for C18H20Br2N2S: C 47.39; H 4.42; N 6.14. Found: C 47.47; H 4.47; N 6.08.
4.2.11. Dibenzyl-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine hydroiodide (6e)
Yield: 81%; mp: 165-167 °C;
1H NMR (200 MHz, DMSO-d6):  = 3.68 (d, J = 6.4 Hz, 2H, NCH2CHS), 4.05 (dd, J =
3.2, 12.0 Hz, 1H, BrCHH), 4.21 (dd, J = 7.2, 12.0 Hz, 1H, BrCHH), 4.48 (m, 1H, CHS), 4.71
(s, 4H, CH2NCH2), 7.38 (m, 10H, Harom), 10.50 (br s, 1H, NH);
EI-MS (m/z): 551 (M + H+).
Anal. Calcd. for C18H20I2N2S: C 39.29; H 3.66; N 5.09. Found: C 39.33; H 3.66; N 5.13.
4.2.12. Benzyl-(4-fluoro-benzyl)-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine
hydrobromide (5f)
Yield: 78%; mph: 178-180 °C;
1H NMR (200 MHz, CDCl3):  = 3.72 (d, J = 6.3 Hz, 2H, NCH2CHS), 4.33 (m, 2H,
BrCH2), 4.43 (m, 1H, CHS), 4.50 (c, 2H, CH2NCH2), 5.10 (c, 2H, CH2NCH2), 7.09 (m, 4H,
Harom), 7.38 (m, 5H, Harom), 11.59 (c, 1H, NH);
EI-MS (m/z): 475 (M + H+).
Anal. Calcd. for C18H19Br2FN2S: C 45.59; H 4.04; N 5.91. Found: C 45.51; H 4.11; N
5.88.
4.2.13. Benzyl-(4-fluoro-benzyl)-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine hydroiodide
(6f)
Yield: 88%; mp: 163-165 °C;
1H NMR (200 MHz, CD3CN):  = 3.72 (m, 2H, NCH2CHS), 4.11 (m, 2H, ICH2), 4.50
(m, 1H, CHS), 4.73 (m, 4H, CH2NCH2), 7.04 (m, 2H, Harom), 7.20 (m, 2H, Harom), 7.35 (m,
2H, Harom), 7.47 (m, 3H, Harom), 8.90 (br s, 1H, HI);
EI-MS (m/z): 569(M + H+).
Anal. Calcd. for C18H19FI2N2S: C 38.05; H 3.37; N 4.93. Found: C18H19FI2N2S: C 38.00;
H 3.33; N 4.96
4.2.14. Benzyl-(5-bromomethyl-4,5-dihydro-thiazol-2-yl)-(4-isopropyl-benzyl)-amine
hydrobromide (5g)
21

Yield: 82%; mp: 118-120 °C;
1H NMR (200 MHz, CDCl3):  = 1.25 (d, J = 6.8 Hz, 6H, CH(CH3)2), 2.92 (m, 1H,
CH(CH3)2), 3.53 (d, J = 6.0 Hz, 2H, NCH2CHS), 4.34 (m, 3H, CHS, BrCH2), 4.55 (br. s, 2H,
CH2NCH2), 5.02 (br. s, 2H, CH2NCH2), 7.25 (m, 6H, Harom) , 7.40 (m, 3H, Harom);
EI-MS (m/z): 499 (M + H+).
Anal. Calcd. for C21H26Br2N2S: C 50.62; H 5.26; N 5.62. Found: C 50.60; H 5.21; N 5.64.
4.2.15. Benzyl-(4-isopropyl-benzyl)-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine
hydroiodide (6g)
Yield: 84%; mp: 143-145 °C;
1H NMR (200 MHz, CDCl3):  = 1.24 (d, J = 6.8 Hz, 6H, CH(CH3)2), 2.92 (m, 1H,
CH(CH3)2), 3.53 (d, J = 5.9 Hz, 2H, NCH2CHS), 4.27 (m, 2H, ICH2), 4.45 (m, 1H, CHS),
4.77 (m, 4H, CH2NCH2), 7.21 (m, 6H, Harom), 7.39 (m, 3H, Harom);
EI-MS (m/z): 593(M + H+).
Anal. Calcd. for C21H26I2N2S: C 42.58; H 4.42; N 4.73. Found: C 42.52; H 4.40; N 4.77.
4.2.16. Benzyl-(5-bromomethyl-4,5-dihydro-thiazol-2-yl)-(4-tert-butyl-benzyl)-amine
hydrobromide (5h)
Yield: 87%; mp: 138-140 °C;
1H NMR (200 MHz, DMSO-d6):  = 1.29 (s, 9H, C(CH3)3), 3.89 (d, J = 6.4 Hz, 2H,
NCH2CHS), 4.05 (dd, J = 3.2, 12.0 Hz, 1H, BrCHH), 4.21 (dd, J = 7.3, 12.0 Hz, 1H,
BrCHH), 4.58 (m, 1H, CHS), 4.64 (br. s, 4H, CH2NCH2), 6.87 (d, J = 8.8 Hz, 2H, Harom),
7.27 (m, 7H, Harom);
EI-MS (m/z): 513 (M + H+).
Anal. Calcd. for C22H28Br2N2S: C 51.57; H 5.51; N 5.47. Found: C 51.68; H 5.57; N 5.41.
4.2.17. Benzyl-(4-tert-butyl-benzyl)-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine
hydroiodide (6h)
Yield: 83%; mp: 161-163 °C;
1H NMR (200 MHz, CDCl3):  = 1.30 (s, 9H, C(CH3)3), 3.53 (d, J = 7.1 Hz, 2H,
NCH2CHS), 4.24 (m, 2H, ICH2), 4.50 (m, 1H, CHS), 4.53 (br s, 2H, CH2NCH2), 5.05 (br s,
2H, CH2NCH2), 7.15 (m, 4H, Harom), 7.45 (m, 5H, Harom), 10.40 (br s, 1H, HI);
EI-MS (m/z): 607 (M + H+).
Anal. Calcd. for C22H28I2N2S: C 43.58; H 4.65; N 4.62. Found: C 43.52; H 4.66; N 4.66.
4.2.18. Benzyl-(5-bromomethyl-4,5-dihydro-thiazol-2-yl)-(4-methoxy-benzyl)-amine
hydrobromide (5i)
22

Yield: 90%; mp: 112-114 °C;
1H NMR (200 MHz, DMSO-d6):  = 3.77 (s, 3H, OCH3), 3.88 (m, 2H, NCH2CHS), 4.05
(dd, J = 3.2, 12.0 Hz, 1H, BrCHH), 4.21 (dd, J = 7.3, 12.0 Hz, 1H, BrCHH), 4.64 (m, 5H,
CHS, CH2NCH2), 6.87 (d, J = 8.8 Hz, 2H, Harom), 7.27 (m, 7H, Harom);
EI-MS (m/z): 487 (M + H+).
Anal. Calcd. for C19H22Br2N2OS: C 46.93; H 4.56; N 5.76. Found: C 46.99; H 4.62; N
5.70.
4.2.19. Benzyl-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-(4-methoxy-benzyl)-amine
hydroiodide (6i)
Yield: 82%; mp: 150-152 °C;
1H NMR (200 MHz, CD3CN):  = 3.68 (m, 2H, NCH2CHS), 3.85 (s, 3H, OCH3), 4.10
(m, 2H, ICH2), 4.45 (m, 1H, CHS), 4.75 (m, 4H, 2xPhCH2), 7.17 (d, J = 8.8 Hz, 2H, Harom),
7.26 (d, J = 8.8 Hz, 2H, Harom), 7.33 (m, 2H, Harom), 7.45 (m, 3H, Harom), 8.95 (br s, 1H, HI);
EI-MS (m/z): 581 (M + H+).
Anal. Calcd. for C19H22I2N2OS: C 39.33; H 3.82; N 4.83. Found: C 39.31; H 3.84; N
4.85.
4.2.20. Benzyl-(5-bromomethyl-4,5-dihydro-thiazol-2-yl)-(1-phenyl-ethyl)-amine
hydrobromide (5k)
Yield: 85%; mp: 167-169 °C;
1H NMR (200 MHz, DMSO-d6):  = 1.60 (d, J = 6.5 Hz, 3H, CH3), 3.91 (d, J = 6.5 Hz,
NCH2CHS), 4.01 (d, J = 12.0 Hz, 1H, BrCHH), 4.21 (dd, J = 7.5, 12.0 Hz, 1H, BrCHH), 4.56
(m, 2H, CHS, PhCHH), 4.81 (m, 1H, PhCHH), 5.47 (m, 1H, CHMe), 7.11 (m, 2H, Harom),
7.28 (m, 3H, Harom), 7.39 (m, 5H, Harom);
EI-MS (m/z): 471 (M + H+).
Anal. Calcd. for C19H22Br2N2S: C 48.53; H 4.72; N 5.96. Found: C 48.59; H 4.772; N
6.02.
4.2.21. Benzyl-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-(1-phenyl-ethyl)-amine hydroiodide
(6k)
Yield: 76%; mp: 155-157 °C.
1H NMR (200 MHz, CD3CN):  = 1.72 (d, J = 6.8 Hz, 3H, CH3), 3.69 (m, 2H,
NCH2CHS), 4.05 (m, 1H, ICHH), 4.10 (m, 1H, ICHH), 4.50 (m, 1H, CHS), 4.74 (m, 2H,
PhCH2), 5.63 (m, 1H, CHMe), 7.18 (m, 2H, Harom), 7.43 (m, 7H, Harom), 8.85 (br s, 1H, HI);
23

EI-MS (m/z): 565(M + H+).
Anal. Calcd. for C19H22I2N2S: C 40.44; H 3.93; N 4.96. Found: C 40.22; H 3.98; N 4.92.
4.2.22. (5-Bromomethyl-4,5-dihydro-thiazol-2-yl)-(4-chloro-benzyl)-pyridin-3-ylmethyl-
amine hydrobromide (5l)
Yield: 88%; mp: 188-190 °C;
1H NMR (200 MHz, DMSO-d6):  = 3.86 (m, 2H, NCH2CHS), 4.14 (m, 2H, BrCH2) ,
4.51 (m, 1H, CHS), 4.85 (br. s, 2H, CH2NCH2), 5.07 (br. s, 2H, CH2NCH2), 7.36 (m, 4H,
Harom), 7.88 (dd, J = 5.4, 8.4 Hz, 1H, Harom), 7.83 (d, J = 8.4 Hz, 1H, Harom), 8.49 (d, J = 5.4
Hz, 1H, Harom), 8.91 (s, 1H, Harom);
EI-MS (m/z): 492 (M + H+).
Anal. Calcd. for C17H18Br2ClN3S: C 41.59; H 3.61; N 8.62. Found: C 41.22; H 3.98; N
8.92.
4.2.23. (4-Chloro-benzyl)-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-pyridin-3-ylmethyl-amine
hydroiodide (6l)
Yield: 85%; mp: 100-102 °C;
1H NMR (200 MHz, DMSO-d6): 3.47 (m, 2H, NCH2CHS), 3.69 (dd, J = 2.7, 12.2 Hz,
1H, ICHH), 3.93 (dd, J = 7.1, 12.2 Hz, 1H, ICHH), 4.29 (m, 1H, CHS), 4.60 (c, 2H,
CH2NCH2), 4.73 (c, 2H, CH2NCH2), 7.19 (dd, J = 8.3, 19.0 Hz, 4H, Harom), 7.74 (dd, J = 5.8,
7.8 Hz, 1H, Harom), 8.16 (d, J = 7.8 Hz, 1H, Harom), 8.61 (c, 1H, Harom), 8.63 (d, J = 5.8 Hz,
1H, Harom);
EI-MS (m/z): 586 (M + H+).
Anal. Calcd. for C17H18ClI2N3S: C 34.86; H 3.10; N 7.17. Found: C 34.91; H 3.15; N
7.11.
4.2.24. (5-Bromomethyl-4,5-dihydro-thiazol-2-yl)-(2-chloro-benzyl)-pyridin-3-ylmethyl-
amine hydrobromide (5m)
Yield: 88%; mp: 173-175 °C;
1H NMR (200 MHz, CDCl3): 3.83 (m, 2H, NCH2CHS), 4.08 (dd, J = 3.2, 12.7 Hz, 1H,
BrCHH), 4.22 (dd, J = 7.3, 12.7 Hz, 1H, BrCHH), 4.53 (m, 1H, CHS), 4.80 (c, 2H,
CH2NCH2), 4.86 (c, 2H, CH2NCH2), 7.35 (m, 4H, Harom), 7.41 (m, 1H, Harom), 7.84 (m, 1H,
Harom), 8.51 (m, 1H, Harom);
EI-MS (m/z): 492 (M + H+).
Anal. Calcd. for C17H18Br2ClN3S: C 41.53; H 3.69; N 8.55. C 41.44; H 3.62; N 8.59.
24

4.2.25. (2-Chloro-benzyl)-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-pyridin-3-ylmethyl-amine
hydroiodide (6m)
Yield: 79%; mp: 180-182 °C;
1H NMR (200 MHz, DMSO-d6): 3.70 (m, 2H, NCH2CHS), 4.01 (dd, J = 2.9, 12.0 Hz,
1H, ICHH), 4.23 (dd, J = 7.1, 12.0 Hz, 1H, ICHH), 4.54 (m, 1H, CHS), 4.81 (c, 4H,
CH2NCH2), 7.39 (m, 5H, Harom), 7.85 (d, J = 7.3 Hz, 1H, Harom), 8.54 (m, 2H, Harom);
EI-MS (m/z): 586 (M + H+).
Anal. Calcd. for C17H18ClI2N3S: C 34.86; H 3.10; N 7.17. Found: C 34.80; H 3.18; N
7.22.
4.2.26. Benzyl-(2-chloro-benzyl)-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine hydroiodide
(6n)
Yield: 74%; mp: 186-188 °C;
1H NMR (200 MHz, DMSO-d6): 3.73 (d, J = 6.8 Hz, 2H, NCH2CHS), 4.02 (dd, J = 2.9,
12.2 Hz, 1H, ICHH), 4.24 (dd, J = 7.6, 12.2 Hz, 1H, ICHH), 4.57 (m, 1H, CHS), 4.72 (c, 2H,
CH2NCH2), 4.76 (c, 2H, CH2NCH2), 7.31 (m, 9H, Harom), 10.55 (br. s. 1H, NH);
EI-MS (m/z): 585 (M + H+).
Anal. Calcd. for C18H19ClI2N2S: C 36.98; H 3.28; N 4.79. Found: C 37.07; H 3.33; N
4.84.
4.2.27. Benzyl-(3,4-dimethoxy-benzyl)-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine
hydroiodide (6o)
Yield: 81%; mp: 128-130 °C;
1H NMR (200 MHz, CDCl3): 3.56 (d, J = 6.8 Hz, 2H, NCH2CHS), 3.85 (c, 3H, OCH3),
3.89 (c, 3H, OCH3), 4.20 (dd, J = 3.9, 11.7 Hz, 1H, ICHH), 4.37 (dd, J = 7.3, 11.7 Hz, 1H,
ICHH), 4.45 (m, 1H, CHS), 4.55 (br. s, 2H, CH2NCH2), 4.92 (br. s, 2H, CH2NCH2), 6.83 (m,
3H, Harom), 7.23 (m, 2H, Harom), 7.38 (m, 3H, Harom);
EI-MS (m/z): 611 (M + H+).
Anal. Calcd. for C20H24I2N2O2S: C 39.36; H 3.96; N 4.59. Found: C 39.42; H 4.10; N
4.66.
4.2.28. 4-{[Benzyl-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amino]-methyl}-2,6-di-tert-butyl-
phenol hydroiodide (6p)
Yield: 78%; mp: 168-170 °C;
1H NMR (200 MHz, DMSO-d6):  = 1.41 (s, 18H, 2C(CH3)3), 3.92 (d, J = 6.6 Hz, 2H,
NCH2CHS), 4.22 (m, 2H, ICH2), 4.63 (m, 5H, CH2NCH2, CHS), 6.86 (c, 1H, OH), 6.96 (c,
25

2H, Harom), 6.92 (c, 1H, Harom), 7.25 (m, 2H, Harom), 7.34 (m, 3H, Harom), 10.82 (br. s, 1H,
NH);
EI-MS (m/z): 679 (M + H+).
Anal. Calcd. for C26H36I2N2OS: C 46.03; H 5.35; N 4.13. Found: C 46.08; H 5.31; N
4.17.
4.2.29. Benzyl-furan-2-ylmethyl-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine
hydroiodide(6q)
Yield: 80%; mp: 132-135 °C;
1H NMR (200 MHz, CDCl3): 3.67 (d, J = 6.8 Hz, 2H, NCH2CHS), 3.98 (dd, J = 3.2, 12.2
Hz, 1H, ICHH), 4.17 (dd, J = 7.1, 12.2 Hz, 1H, ICHH), 4.52 (m, 1H, CHS), 4.67 (c, 2H,
CH2NCH2), 4.73 (c, 2H, CH2NCH2), 6.37 (dd, J = 2.0, 3.2 Hz, 1H, Harom), 6.47 (d, J = 3.2 Hz,
1H, Harom), 7.23 (d, J = 2.0 Hz, 1H, Harom), 7.30 (m, 5H, Harom);
EI-MS (m/z): 541 (M + H+).
Anal. Calcd. for C16H18I2N2OS: C 35.57; H 3.36; N 5.19. Found: C 35.52; H 3.40; N
5.12.
4.2.30. (2,3-Dihydro-benzo[1,4]dioxin-6-yl)-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine
hydroiodide (6r)
Yield: 78%; mp: 171-173 °C;
1H NMR (200 MHz, DMSO-d6): 3.50 (m, 2H, NCH2), 3.86 (dd, J = 2.9, 11.7 Hz, 1H,
ICHH), 4.00 (dd, J = 7.3, 11.7 Hz, 1H, ICHH), 4.20 (c, 4H, CH2CH2), 4.34 (m, 1H, CHS),
6.65 (dd, J = 2.4, 8.6 Hz, 1H, Harom), 6.74 (d, J = 2.4 Hz, 1H, Harom), 6.82 (d, J = 8.6 Hz, 1H,
Harom);
EI-MS (m/z): 505 (M + H+).
Anal. Calcd. for C12H14I2N2O2S: C 28.59; H 2.80; N 5.56. Found: C 28.65; H 2.85; N
5.55.
4.2.31. (5-Iodomethyl-4,5-dihydro-thiazol-2-yl)-(6-methyl-pyridin-2-yl)-amine hydroiodide
(6s)
Yield: 78%; mp: 215-217 °C;
1H NMR (200 MHz, DMSO-d6): 2.58 (c, 3H, CH3), 3.67 (m, 2H, NCH2CHS), 4.13 (m,
2H, ICH2), 4.38 (m, 1H, CHS), 7.09 (t, J = 8.5 Hz, 2H, Harom), 7.80 (t, J = 7.8 Hz, 2H, Harom);
EI-MS (m/z): 462 (M + H+).
Anal. Calcd. for C10H13I2N3S: C 26.05; H 2.84; N 9.11. Found: C 26.15; H 2.77; N 9.05.
4.2.32. Dibenzofuran-3-yl-(5-iodomethyl-4,5-dihydro-thiazol-2-yl)-amine hydroiodide (6t)
Yield: 80%; mp: 200-202 °C;
26

1H NMR (200 MHz, DMSO-d6):  = 3.55 (dd, J = 2.2, 6.8 Hz, 2H, NCH2CHS), 3.92 (dd,
J = 3.4, 12.0 Hz, 1H, ICHH), 4.09 (dd, J = 7.3, 12.0 Hz, 1H, ICHH), 4.38 (m, 1H, CHS), 7.23
(dd, J = 2.0, 8.6 Hz, 1H, Harom), 7.33 (dt, J = 1.2, 7.8 Hz, 1H, Harom), 7.45 (dt, J = 1.2, 7.3 Hz,
1H, Harom), 7.52 (m, 2H, Harom), 7.94 (d, J = 7.8 Hz, 1H, Harom), 8.03 (d, J = 8.3 Hz, 1H,
Harom);
EI-MS (m/z): 537 (M + H+).
Anal. Calcd. for C16H14I2N2OS: C 35.84; H 2.63; N 5.22. Found: C 35.89; H 2.71; N
5.19.
4.3. Biological assay
4.3.1. Enzymatic assays
4.3.1.1. In vitro AChE, BChE and CaE inhibition
Acetylcholinesterase (AChE, EC 3.1.1.7, from human erythrocyte, C0663 Sigma),
butyrylcholinesterase (BChE, EC 3.1.1.8, from equine serum, С4290 Sigma),
carboxylesterase (CaE, EC 3.1.1.1, from porcine liver, E2884 Sigma), acetylthiocholine
iodide (ATCh), butyrylthiocholine iodide (BTCh), 5,5´-dithiobis-(2-nitrobenzoic acid)
(DTNB), 4-nitrophenol acetate (4-NPA),were purchased from Sigma-Aldrich (Germany).
AChE and BChE activities were measured by the method of Ellman et al. 42. The assay
solution consisted of 0.1 M potassium phosphate buffer pH 7.5, 25°C with the addition of
0.33 mM DTNB, 0.02 unit/mL of AChE or BChE and 1 mM of substrate (acetylthiocholine
iodide or butyrylthiocholine iodide, respectively). Assays were carried out with a blank
containing all components except ATCh and BTCh in order to account for non-enzymatic
reaction.
The activity of CaE was determined spectrophotometrically by the release of 4-
nitrophenol at 405 nm 43. The assay solution consisted of 0.1 M potassium phosphate buffer
pH 8.0, 25°C with the addition of 1 mM 4-nitrophenyl acetate and 0.02 unit/mL of CaE.
Assays were carried out with a blank containing all components except CaE.
The tested compounds were dissolved in DMSO; the incubation mixture contained 2%
(v/v) of the solvent. Eight different concentrations of the test compounds in the range 10-12-
10-4 M were selected in order to obtain inhibition of AChE and BChE activity between 20%
and 80%. The test compounds were added to the assay solution and preincubated at 25oC
with the enzymes for 10 min followed by the addition of substrate. A parallel control was
made for the assay solution with no inhibitor. Measurements were performed in a BioRad
27

Benchmark Plus microplate spectrophotometer (France). Each experiment was performed in
triplicate. The results were expressed as the mean ± SEM. The reaction rates in the presence
and absence of inhibitor were compared, and the percent of residual enzyme activity due to
the presence of test compounds was calculated. IC50 (the concentration of inhibitor required
to decrease the enzyme activity by 50%) values were determined graphically from inhibition
curves (log inhibitor concentration vs percent residual enzyme activity) using Origin 6.1
software.
4.3.1.2. Kinetic study of BChE and CaE inhibition. Determination of steady-state inhibition
constants.
To elucidate the inhibition mechanisms for the 9 most active compounds, BChE and CaE
residual activities were determined in the presence of 3 increasing concentrations of the test
compounds and 5-8 decreasing concentrations of the substrates. The test compounds were
preincubated with the enzymes at 25°C for 10 min, followed by the addition of the substrates.
A parallel control was made for an assay of the rate of hydrolysis of the same concentrations
of substrates in the solutions with no inhibitor. The kinetic parameters of substrate hydrolysis
were determined. Measurements were performed in a BioRad Benchmark Plus microplate
spectrophotometer (France). Each experiment was performed in triplicate. Results were fitted
to Lineweaver-Burk double-reciprocal kinetic plots of 1/V versus 1/[S] and values of
inhibition constants Ki (competitive component) and αKi (noncompetitive component) were
calculated using the program Origin 6.1.
4.3.2. Cytotoxicity studies
Human fetal mesenchymal stem cells (FetMSC), nonimmortalized fibroblast-like cells
obtained from bone marrow of 5- to 6-week-old fetuses 44, were maintained in F12/DMEM
medium supplemented with 10% FBS, l-glutamine (3 mM), penicillin 50 IU/ml and
streptomycin 50 µg/ml. The FetMSC human fetal mesenchymal stem cell line is a certified
culture available from the Russian collection of vertebrate cell cultures of the Institute of
Cytology, Russian Academy of Sciences.
To investigate the cytotoxicity of synthetic compounds in FetMSC cells, the 3-(4,5-
dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay was performed. For
the МТТ cell viability assay 45, cells were plated in 96-well plates (6 × 103 cells/well) and
28

allowed to adhere for 24 h. Thereafter, the medium was replaced with fresh medium
containing test compounds at different concentrations. Control cells were treated with 0.1%
(v/v) DMSO. After 72 h exposure, 0.5 mg/ml of (MTT) was added to each well and the cells
were incubated for 4 h. Then, culture medium was aspirated, and MTT-formazan was
dissolved in 100% DMSO. MTT staining intensity was read at 570 nm. The MTT staining of
control cells was taken as 100%.
4.3.3. Molecular modeling studies
The crystal structure of human BChE (PDB ID 1P0I) 46 was used. Resolution of the
structure was 2.00 Å, but it lacked some amino acid residues – Asp378, Asp379 and Gln455,
and the Gln486 side chain. All the gaps were located on the protein surface far from the gorge
entrance; nevertheless, they were reconstructed according to the procedure outlined by
Masson et al 47.
The crystal structure of human CaE (PDB ID 2H7C) 48 was used. Resolution was 2.00Å,
but the structure lacked the Glu99 side chain. Although this residue is located rather far from
the active site, it was reconstructed manually. This crystal structure contains Coenzyme A in
the active site and the gorge, which slightly expands it, making it more favorable for docking
of bulky inhibitors.
For both protein structures, all crystallographic waters were removed, along with ligands,
ions, and sugars. Hydrogen atoms were added with Reduce 49 software, which protonates
histidine rings with regard to the surrounding hydrogen-bonding network.
Marvin Sketch with Calculator Plugins was used for characterizing ligand structures
(generating all stereoisomers where possible) and prediction of pKa of inhibitors (Marvin
Sketch 6.0.4, 2013, ChemAxon, http://www.chemaxon.com). Additionally, pKa values of the
most interesting (leading) compounds were predicted by means of ACD/I-Lab
(https://ilab.acdlabs.com/iLab2/). The values predicted by the two programs differed
significantly; in some cases the pKa value predicted by one program was more than 7.40 (pH
of the experimental measurements), and less than 7.40 by the second one for the same
compound (see Supplementary data, Fig. S5). Due to this uncertainty of protonation states of
the compounds, both protonation forms of the amidine group of amino-thiazolines were used.
With all possible stereoisomers (see Supplementary data, Fig. S5) and protonation states, the
library of ligand compounds used in this study included 130 structures.
29