Thiazolidinone/thiazole based hybrids – New class of antitrypanosomal agents

Article abstract of DOI:10.1016/j.ejmech.2019.04.052

Different compounds have been investigated as potent drugs for trypanosomiasis treatment, but no new drug has been marketed in the past 3 decades. 4-Thiazolidinone/thiazole as privileged structures and thiosemicarbazides cyclic analogs are well known scaf

Full text of DOI:10.1016/j.ejmech.2019.04.052

Accepted Manuscript
Thiazolidinone/thiazole based hybrids – New class of antitrypanosomal agents
Anna Kryshchyshyn, Danylo Kaminskyy, Oleksandr Karpenko, Andrzej Gzella,
Phillipe Grellier, Roman Lesyk
PII:
S0223-5234(19)30370-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.052
EJMECH 11287
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 February 2019
Revised Date: 17 April 2019
Accepted Date: 17 April 2019
Please cite this article as: A. Kryshchyshyn, D. Kaminskyy, O. Karpenko, A. Gzella, P. Grellier, R. Lesyk,
Thiazolidinone/thiazole based hybrids – New class of antitrypanosomal agents, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.052.
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ACCEPTED MANUSCRIPT
THIAZOLIDINONE/THIAZOLE BASED HYBRIDS – NEW CLASS OF
ANTITRYPANOSOMAL AGENTS
a
a
b
Anna Kryshchyshyn , Danylo Kaminskyy , Oleksandr Karpenko ,
c
d
a,e
Andrzej Gzella , Phillipe Grellier , Roman Lesyk
R
R
N
O
R
N
N
N
N
R
S
R
N
N
S
N
R
R
S
N
N
N
N
NH
R
S
O
S
H C
3
NH
N
Trypanosoma brucei brucei IC = 30 nM
5
0
N
Trypanosoma brucei gambience IC = 110 nM
5
0
N
H

ACCEPTED MANUSCRIPT
THIAZOLIDINONE/THIAZOLE BASED HYBRIDS – NEW CLASS OF
ANTITRYPANOSOMAL AGENTS
a
a
b
Anna Kryshchyshyn , Danylo Kaminskyy , Oleksandr Karpenko ,
c
d
a,e
Andrzej Gzella , Phillipe Grellier , Roman Lesyk
a
Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National
Medical University, Pekarska 69, Lviv, 79010, Ukraine
b
Enamine Ltd., Chervonotkatska 78, Kyiv, 02094, Ukraine
c
Department of Organic Chemistry, Karol Marcinkovski Poznan University of Medical
Sciences,Grunwaldzka 6, Poznan, 60-780, Poland
d
National Museum of Natural History, UMR 7245 CNRS-MNHN, Team BAMEE, CP 52, 57 Rue Cuvier,
7
5005, Paris, France
e
Department of Public Health, Dietetics and Lifestyle Disorders, Faculty of Medicine, University of
Information Technology and Management in Rzeszow, Sucharskiego 2, 35-225 Rzeszow, Poland
1

ACCEPTED MANUSCRIPT
ABSTRACT: Different compounds have been investigated as potent drugs for trypanosomiasis treatment,
but no new drug has been marketed in the past 3 decades. 4-Thiazolidinone/thiazole as privileged
structures and thiosemirbazides cyclic analogs are well known scaffolds in novel antitrypanosomal agent
design. We present here the design and synthesis of new hybrid molecules bearing thiazolidinone/thiazole
cores linked by the hydrazone group with various molecular fragments. Structure optimisation led to
compounds with phenyl-indole or phenyl-imidazo[2,1-b][1,3,4]thiadiazole moieties showing excelent
antitrypanosomal activity towards Trypanosoma brucei brucei and Trypanosoma brucei gambiense.
Biological study allowed identifying compounds with the submicromolar levels of IC , good selectivity
5
0
indexes and relatively low cytotoxicity upon human primary fibroblasts as well as low acute toxicity.
KEYWORDS: thiazoles, thiazolidinones, indoles, imadazothiadiazoles, hybrids, antitrypanosomal
activity
2

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INTRODUCTION
Trypanosomiasis belongs to so-called global neglected tropical diseases. Human African
Trypanosomiasis (HAT) known as sleeping sickness often occurs in sub-Saharan Africa and is the tsetse
fly transmitted trypanosomiasis caused by two subspecies of Trypanosoma brucei (Tb), namely Tb
gambiense and Tb rhodesiense. Since 2010, the number of cases of HAT has dropped below 10,000
annually, but there are about 20,000 actual cases estimated and about 65 million people are at risk [1].
Problems associated with the treatment of early and late stages of HAT include toxicity, availability of
only parenteral route of administration and generally inadequate effectiveness of the existing drugs, such
as suramin, pentamidine, melarsoprol and eflornithine [2]. American trypanosomiasis or Chagas disease
caused by T cruzi is considered as an important health problem in Latin America with currently estimated
about 7 million people being infected. Nifurtimox and benznidazole are available for the Chagas disease
treatment, although they require prolonged administration and have frequent side-effects that can lead to
discontinuation of the treatment [3]. Whilst the Chagas disease was confined earlier within the region of
Latin America, it has now spread to other continents. Trypanosomiasis affect mostly poor population in
the developing countries and the underinvestment of local governments and pharmaceutical industry in the
antitrypanosomal drugs search resulted in marketing no new drugs in the past 3 decades [4-6].
Quite a lot of different chemical classes of compounds, e.g. thiosemicarbazones, thiazolidines,
triazole and furan based compounds, benzofuran derivatives [7], peptidyl compounds and
peptidomimetics [8], acyl- and arylhydrazones [9], etc. have been investigated as new drug-like molecules
for discovering novel antitrypanosomal agents. Different (thio)ureas/(thio)semicarbazides had shown
high-affinity to the so-called antitrypanosomal validated targets: cruzain and rhodesain [10,11]; cysteine
proteases [12]; some of them were reported as the inhibitors of trypanosome proliferation and growth [13-
1
5]. Thiazole derivatives, especially thiazolidinones are considered as thiourea/thiosemicarbazones’ cyclic
analogs and have been known for good pharmacological profile [7,16-19]. Screening of a focused kinase-
3

ACCEPTED MANUSCRIPT
inhibitors library against Tb allowed identifying a series of active compounds based on 2,4-
diaminothiazoles that were also active against culture of Tb. Some of the above compounds possessed
antitrypanosomal activity at the nanomolar concentrations [20,21]. 5-Arylidenerhodanine-3-acetic acids
were reported to inhibit the activity of Tb dolicholphosphate mannose synthase and
glycosylphosphatidylinositol anchor synthesis as well as characterized by trypanocidal activity against
trypanosomes [22]. Simple 5-ene-2,4-thiazolidinones were proposed as possible scaffolds for the design of
new pteridine reductase 1 inhibitors [23]. On the other hand, fused thiazole/thiazolidinones derivatives,
which can be treated as cyclic mimetics of simple thiazole/thiazolidinones are also of special interest [24].
One of the direction of molecular optimization of the potent antitrypanosomal agents is the
combination of thiazole/thiazolidinone scaffold and hydrazone fragment [7,17,25,26]. For example, a
series of thiazolylhydrazones and 2-iminothiazolidine-4-ones tested in the cruzain inhibition assay and
against cultures of the epimastigote and trypomastigote forms (Trypanosoma (T.) cruzi Y strain) inhibited
cruzain and showed antiproliferative activity at non-cytotoxic concentrations [17, 27-34]. Molecular
hybridization of the thiazole ring with pyridine moiety through hydrazine bridge led to identification of
the selective N-[3-phenyl-3H-thiazol-2-ylidene]-N’-(1-(pyridin-2-yl-ethylidene)-hydrazines inducing
parasite cell death via the apoptotic mechanism [35]. Such data are supported and proved by the methods
of computer chemistry too [36], e.g. 2-hydrazolyl-4-thiazolidinones are potent cruzipain inhibitors
according to the ZINC 5 database analysis (more than 0.5 mln compounds) [37]. The utilization of
thiazole/thiazolidinone bearing scaffolds within hybrid-pharmacophore approach has provided plenty of
biologically active small molecules including antitrypanosomal agents [7,16,38-40]. The combination of
thiazole core with fused [6+5] or [6+6] scaffolds turned out to be especially interesting and resulted in
highly active and selective antitrypanosomal agents. For example, novel 1-indanone thiazolylhydrazones
were obtained by combining the indanone and thiazole moieties in one molecule and showed good
trypanocidal properties against T. cruzi (Tulahuen 2 strain) and low mammal cytotoxicity [41]. Indene
4

ACCEPTED MANUSCRIPT
cycle is presented in the molecule of indatraline, a non-selective monoamine reuptake inhibitor, its
analogs were successfully screened against trypanothione reductase, one of the validated targets in
antitrypanosomal agents search [42]. A benzooxaborole derivative SCYX-7158 became DNDi’s (Drugs
for neglected diseases initiative) one of the new chemical entities to enter clinical development and in
2
016 the PhaseII/III trial started [43,44]. Some of its analogs were also reported as potential
antitrypanosomal agents with micromolar inhibition of trypanosomal leucyl-tRNA synthetase [45].
Screening of over 300000 compounds in ornithinedecarboxylase inhibition high throughput assay
discovered four scaffold classes, among which were benzthiazoles and indole derivatives [46]. The
number of hit-compounds based on 1H-indole and 1H-pyrrolo[2,3-b]pyridine scaffolds were discovered
in the screening of kinase-targeted library involved more than 42,000 compounds [36]. Exactly, the 1H-
indole derivative showed excellent results when testing in the blood stream in vivo experiment in the mice
infected with Tb rhodesiense. Indazole derivatives, namely 3-alkoxy-1-alkylindazoles, 2-disubstituted
indazoline-3-ones with nitrogroup in C5 position and 2,3-substituted indazole-1-oxides, were reported as
antichagasic agents in in vitro studies. Trypanocidal action of 5-nitroindazoles involves inhibition of
trypanothione reductase and possibly involves oxidative stress mechanism [47,48]. 2-Phenyl-1H-indole
was shown to be an attractive scaffold for the design of hydrazine bearing compounds as antimicrobial
agents [49]. Considering the phthalimide fragment as indole bioisoster, molecules bearing it should also
reveal antiparasitic activity and, indeed, some of a series of phthalimido-thiazoles showed tripanocidal
activity (towards T. cruzi) being even more selective than benznidazole [50]. The combination of other
heterocycles with thiazolidine scaffolds, e.g. thiophene, yielded thiophen-2-iminothiazolidine hybrids that
showed tripanocidal activity in in vitro screening against T. cruzi (amastigote and trypomastigote forms)
and cruzain inhibition activity [51]. Research of imidazo[2,1-b][1,3,4]thiadiazoles carried out for the last
two decades proved this class of heterocycles to possess a wide spectrum of biological activities including
antiparasitic [52].
5

ACCEPTED MANUSCRIPT
The high affinity ligands to different validated trypanosomal bio-targets are often not effective in
vivo [7]. Such situation along with low predicted profitability of possible new drugs stimulates small
academic research projects. One of the strategies implemented in such projects involves the exploitation
of privileged scaffolds, discovered in enzyme and growth inhibition assays within structure based design.
The study of antirypanosomal activity of thiazolidinone derivatives resulted in the synthesis and
investigation of various compounds containing thiazole core as well as different molecular fragments that
may be considered as pharmacophores [24,26,53-57]. The efforts were aimed at research of novel
antiparasitic agents: compounds bearing 4-thiazolidone or thiazole core and phenyl-indole or phenyl-
imidazothiadiazole fragment linked by hydrazone moiety (Fig. 1).
Known lead-compounds
Known lead-compounds
S
S
HO
H
H
Cl
R
R
R
N
R
N
N
N
H
N
H
N
N
H
N
N
H
H
N
[
Ref 36]
Cl
O
R
H
N
F
Ar
O
H
N
OH
Ar
N
B
R
R
S
S
N
O
R
CF3
O
S
N
2
R
N
Me
Me
N
SCYX-7158 [Ref 43]
Ar
R
S
1
O
R
OH
O
O
B
[
Ref 25]
O
R
Me
2
1
N
R
R
S
Me
Me
S
[Ref 45]
2
N
R
N
O
N
O N
2
1
Target compound srtucture
R
R
Ar
N
2
[
Ref 26]
R
[
Ref 47]
O
R
R
N
R
N
O
R
R
R
R
N
N
S
N
S
N
R
R
R
S
N
N
N
N
S
N
R
N
R
N
N
R
N
N
N
N
H
H
R
N
R
S
N
S
6

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Figure 1. Background of the target compounds design.
RESULTS AND DISCUSSION
Chemistry. Firstly, the set of 4-thiazolidinone-hydrazones with hydrazone fragment bearing various
molecular fragments (Scheme 1) was synthesized via known approach based on [2+3]-cyclocondensation
2
+
reaction of thiosemicarbazones and different [C ] electrophilic synthons (maleimides, maleic anhydride,
2
β-aroylacrylic acids) [16,58]. The reactions were performed in glacial acetic acid medium. For compound
2
+
1
d, the [C ] reactant was prepared in situ via the reaction of maleic anhydride and appropriate amino
2
acid in acetic acid. Рresented thiazolidinones contain a stereo-center at the C5 position of the
thiazolidinone core and occur in racemic form. The type of the substituents in C5 position of
thiazolidinone core linked via the single bond was justified by our previous investigations [16,33,58] as
well as by some aspects of the SAR-analysis of different hydrazono-thiazolidinones [7,33,35].
7

ACCEPTED MANUSCRIPT
R
R1
R2
H
R3
PhCHCH
1
1a 4-F-C H4
4-OH-C H4
O
O
R
6
6
1
1
b
c
4-NO -C H
4
H
H
H
H
PhCHCH
PhCHCH
fyran-2-yl
N
2
6
3-CF -C H
4
furfan-2-yl
H
3
6
N
S
2
1d HOOC(CH2)3
R
N
HN
R
1
R
3
2
R + R3
R
HN
1e 4-F-C H4
H
A
A
A
B
B
B
1
a-1j
6
S
i
1f
4-MeOOC-C H4
H
H
H
H
H
6
[
C2]2+
+
HN
1g 4-NO -C H
2 6 4
N
1h 4-Br-C H4
6
3
R
1i
1j
4-Cl-C H4
6
2
R
4-Me-C H4
1
6
O
O
R
N
R
C
C
R1
H
H
R2
H
H
H
H
H
R3
N
1k
furan-2-yl
PhCHCH
4-Me N-C H
PhCHCH
Ph
2
R
S
1
l
N
R
1m 4-F-C
H
allyl
3
6
4
2 6 4
R
1
1
1
1
1
n
4-F-C H4
H
6
1k-1t
o 4-F-C H4
allyl
6
p
q
r
4-F-C H4 4-HO-C H4 H 3-MeO-4-HOOCCH O-C H
6
6
2
6
3
HO
4-HO-C H4
H
H
4-NO -C H CHCCl
6
2
6
4
HO
H
2-NO -C H CHCH
2 6 4
C2]2+
=
2
R + R3
[
1
s
4-F-C H4
H
H
A
B
6
O
O
OH
1t 4-F-C H4
O
6
O
O
OH
O
;
N
R
;
;
O
+
A
B
Me
Me
C
O
O
O
Me
R
O
H N
2
Scheme 1. Synthesis of 4-thiazolidinone-hydrazones.
2
+
Reagents and conditions: i – substituted thiosemicarbazone (1.0 equiv), appropriate [C ] compound (1.0
2
equiv), AcOH, reflux, 3 h; for compound 1d – amino acid (1.0 equiv), maleic anhydride (1.0 equiv),
AcOH, reflux 1 h, thiosemicarbazone (1.0 equiv), AcONa (1.0 equiv), reflux, 3 h.
The amino-imine tautomerism is described for the N3-unsubstituted related compounds. Target
compounds can be presented in the “amino form” (endocyclic С2=N bond) [17] or “imino form”
(
exocyclic double bond) [33,58,59]. However, based on the spectral data, the exocyclic double bond is
prevailing (“imino” form) that, along with the the X-ray analysis data for compound 1f (Fig. 2), become
the argument for compounds presentation.
8

ACCEPTED MANUSCRIPT
a
b
Figure 2. (a) ORTEP view of the molecule 1f and H O showing the atomic labelling scheme. (b) The
2
hydrogen bonding in the crystal structure of 1fx1.25 H O.
2
Non-H atoms are drawn as 30% probability displacement ellipsoids and H atoms are drawn as spheres of
an arbitrary radius. Symmetry codes: (i) 1-x, 1-y, -z; (ii) 1+x, y, z; (iii) 1+x, y, 1+z. H atoms not involved in
hydrogen bonds have been omitted for clarity).
Compound 1f may occur in five tautomeric forms. The X-ray studies revealed that the molecules in a
crystal lattice occur in a one tautomeric form with two secondary amide groups and a hydrazone group
with both N6 and N7 nitrogen atoms in an imine form. Bond lengths N3–C4 and C16–N18 [1.354(2) Å
and 1.351(2) Å] which appear in two secondary amide groups, are close to a literature bond distance
(
O=)C–NH [1.366(2)Å] obtained on the basis of twenty structures with 2-imino-1,3-thiazolidin-4-one
system {refcodes: EHITZO, FOTQEM, HEGMAJ, HEGMEN, HEGMIR, HEGMOX, IKIVIJ, JOMCOF,
LAFJAG, NEBHOU, POSSOH, QAJSEC, RALVEI, ROMXUN, ULACEQ, VAMPUW, WAGVUY,
WOGGOQ, WOGGUW, XUBYOK, Cambridge Structural Database, Version 5.39; [60] R < 0.07}.
Moreover, the lengths of bonds C2−N6 and N7−C8 [1.280(2) and 1.287(2) Ǻ] correspond with a literature
length of a double bond C=N [1.279(1) Å] [61]. A conjugated double bond system C2=N6−N7=C8 in a
molecule shows the s-trans conformation.
9

ACCEPTED MANUSCRIPT
The next stage was the complication of the hydrazone fragment by the introduction of phenyl-indole
or phenyl-imidazothiadiazole fragments into the molecules. Such [6+5] and [5+5] heterocyclic cores
make the molecule less flexible that may positively influence its activity. An additional argument in
favour of the latter is a great number of lead-compounds from different chemical groups, most of which
include [6+5] and phenyl cores that are potential kinase inhibitors and selective Tb growth inhibitors [36].
Starting thiosemicarbazones bearing phenyl-indole or phenyl-imidazothiadiazole fragments were
synthesized according to the known protocols: 1) indole bearing thiosemicarbazones – via Fischer indole
synthesis, Vilsmeier-Haack reaction and condensation with thiosemicarbazides; 2) imidazothiadiazole
bearing compounds were synthesized in the three step synthetic procedure from 5-substituted 2-amino-
1
,3,4-thiadiazole and α-halogenoarylketone [62] (Scheme 2).
1
0

ACCEPTED MANUSCRIPT
O
H
ii
R2
A
R
R
4a
4b
A1
A2
H
H
N
H
N
H
4
c
A2 4-HO-C H
6 4
O
H
2
S
N
+
R
4d
4e
4f
A2
A3
A4
furfuryl
H
H
R
NH2
Me
N
H
N
N
H
H
4
g
A4 4-HO-C H
6 4
4
4i
h
A5
H
X
R
A5 4-HO-C H
6
4
A
=
4j
4k
4l
A6
A7
H
H
N
N
N
O
H
+
1
R
4
A7 4-HO-C H
R
6 4
H N
2
S
Hal
O
4m
A8
H
H
4
n
A8 4-HO-C H
A9
A9 4-HO-C H
6
4
4
N
i
N
S
4o
H
N
1
N
ii
1
4
p
R
R
6
R
R
S
N
H
N
H
3
2
R
R1
R
R1
2
2
2
2
2
a
b
c
d
e
H
H
H
Br
NO2
S-CH CH=CH2
3a
3b
H
H
S-CH CH=CH2
2
2
S-CH COOEt
S-CH COOEt
2
2
Et
Et
Et
3c
3d
H
Br
Et
Et
Et
3e NO2
N
N
1
A =
R
R
R
N
S
N
H
1
H
A1: R = H
A2: R = Cl
A3: R = Br
A4: R = NO2
A5: R = H R = S-CH CH=CH
A6: R = H
A7: R = H R = Et
2
2
1
R
= S-CH COOEt
2
1
1
A8: R = NO R = Et
2
1
A9: R = Br R = Et
Scheme 2. Synthesis of the starting phenyl-indole or phenyl-imidazothiadiazole bearing
thiosemicarbazones.
Reagents and conditions: i –mixture of DMF and POCl (1/3 vol), stirring 10 min at 0ºС, imidazo[2,1-
3
2 3
b][1,3,4]thiadiazole, 30 min at 0ºС, 2 h at rt and 2 h at 60ºС, Na CO (pH = 9.0), heating, 90 ºС, 2 h; ii –
appropriate aldehyde (1.0 equiv), thiosemicarbazide (1.0 equiv), AcOH, reflux, 1 h.
The synthesis of the target thiazolidinone/thiazole-indole/imidazothiadiazole hybrids was performed
based on the above mentioned synthetic protocol (Schemes 3 and 4).
1
1

ACCEPTED MANUSCRIPT
R1
R2
H
Me
Et
H
Me
Et
Me
Et
H
Me
Et
H
Me
Et
Me
Et
H
Me
Et
Et
A
26 A9
27 A2
28 A2
29 A2
R
H
1
R
Et
2
A
A1
A1
A1
A2
A2
A2
A3
A3
A4
5
6
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
furfuryl
furfuryl
furfuryl
H
Me
Et
H
2
R
OH
O
7
8
9
1
1
1
1
1
R
O
Cl
30 A2 4-HO-C H4
N
6
0
1
2
3
31 A2
4-HO-C H4 Me
6
2
R
N
S
32 A2 4-HO-C H4 Et
3 A4 4-HO-C H4 Me
6
N
3
6
5-42
34 A4
A5
4-HO-C H4
Et
1
R
S
14 A4
A4
16 A5
17 A5
6
A
35
4-HO-C H4
H
15
6
N
N
36 A5 4-HO-C H4 Me
37 A5 4-HO-C H4 Et
N
6
H
H
6
1
1
8
9
A5
A6
A6
A7
A7
A7
3
8
9
A7
4-HO-C H4
H
R
6
X
i
3
A7 4-HO-C H4 Me
6
A=
20
N
40 A7 4-HO-C H4 Et
6
21
22
23
H
41
A9
4-HO-C H4 Me
6
42 A9 4-HO-C H4 Et
6
2
R
24 A8
A9
25
Me
1
R
R1
R2
Cl
Br
OH
COOEt
OH
A
O
O
N
O
O
2
R
43 A1
A1
45 A1
H
H
H
H
H
N
4
4
N
S
46
7
A1
A2
N
4
N
H
A
43-47
Scheme 3. Synthesis of target 4-thiazolidinone-indole/imidazothiadiazole hybrids.
Reagents and conditions: i – substituted thiosemicarbazone (1.0 equiv), appropriate [C
2
+
2
] compound (1.0
equiv), AcONa (1.0 equiv, for 5-42), AcOH, reflux, 3 h. Structure of fragment A – see scheme 2.
α-Halogenocarboxylic acids, N-arylmaleimides, maleic anhydride, β-aroylacrylic acids and 2-
2
+
bromobutyrolactone were used as [C ] reagents. Utilizing 2-bromobutyrolactone, compounds with free or
2
acetylated OH group were obtained depending on the reaction medium (Methods A and B) (Scheme 4).
Compounds 73-75 were synthesized in one pot in situ reaction of maleic anhydride, appropriate amino
acid and starting thiosemicarbazones similarly to 1d.
1
2

ACCEPTED MANUSCRIPT
A
R1
R2
4
4
5
5
5
5
5
5
8
9
0
1
2
3
4
5
A1
A1
A2
A2
A3
A3
A4
A5
H
H
H
H
H
H
H
H
H
H
Ac
H
Ac
H
Ac
Ac
Ac
Ac
Ac
Ac
H
O
56 A7
57 A8
58 A9
1
O
R
H
H
Br
O
2
N
i
R
59
A2
furfuryl
furfuryl
O
N
60 A2
Ac
S
6
6
6
6
1
2
3
4
A2
4-HO-C H4 Ac
N
6
A5 4-HO-C H4 Ac
6
1
S
A
A7
4-HO-C H4 Ac
6
R
A8 4-HO-C H4 Ac
6
N
HO
O
1
N
H
N
R
65 A9 4-HO-C H4 Ac
6
O
H
H
O
Ar
N
or
R
ii
R1
H
R2
Ph
X
A
66 A1
67 A1
N
=
S
A
O
O
O
N
N
2
H
4-F-C H4
6
H
R
O
68
69
70
71
72
A1
A1
A2
A3
A5
H
H
H
H
H
tetralin-6-yl
OH
A
OH
OH
OH
1
O
O
R
iii
R1
H
R2
N
A
A1
7
3
HOOCCH2
O
O
O
N
S
74 A1
75 A1
H
HOOC(CH
)
2 2
2
R
N
H
HOOC(CH2)5
+
OH
O
N
H
H N (CH
)
2
2
n
A
Scheme 4. Synthesis of target 4-thiazolidinone-indole/imidazothiadiazoles hybrids.
Reagents and conditions: i – Method A: thiosemicarbazone (1.0 equiv), α-bromo-γ-butyrolactone (1.0
equiv), and triethylamine (1.0 equiv), EtOH, reflux, 5 h (for compounds with free OH group); Method B:
thiosemicarbazone (1.0 equiv), α-bromo-γ-butyrolactone (1.0 equiv), AcONa (1.0 equiv), AcOH, reflux, 3
h (for compounds with acethylated OH group); ii - thiosemicarbazone (1.0 equiv), maleic anhydride or β-
aroylacrylic acid (1.0 equiv), AcOH, reflux, 3 h; iii –maleic anhydride (1.0 equiv), aminoacid (1.0 equiv),
AcOH, reflux, 1 h, thiosemicarbazone (1.0 equiv), reflux 3 h. Structure of fragment A – see scheme 2.
1
3

ACCEPTED MANUSCRIPT
Considering the similarity of 4-thiazolidinone and thiazole core, the series of thiazole-hydrazone
hybrids were synthesized within substructure-based diversity oriented synthesis [63]. Thiazole-hydrazones
were synthesized in the reaction of corresponding thiosemicarbazones and α-halogenated ketones (Scheme
5
) [27,41].
2
R2
Br
NO2
R
A
A2
A3
7
7
6
7
N
S
H
N
78 A4
A4
80 A7
Br
NO2
79
O
Hal
N
1
NO2
R = H
A
8
8
1
2
A8
A8
NO2
Br
2
83 A9
NO2
2
R
R
OH
A
R2
84
A4 NO2
85 A7
A8
87 A8 NO2
1
S
H
Br
R
N
86
N
H
R
1
= 4-HO-C H
6
4
N
N
N
H
S
H
8
8
8
9
A9
A9 NO2
Br
N
R
X
A
=
A
N
Me
H
N
A
1
R = H
O
90 A2
Et
NH
N
9
9
9
9
9
1
2
3
4
5
A4
A5
A7
A8
A9
S
O
A
Et
OH
O
O
Cl
A
O
R
1
= 4-HO-C H
Me
6
4
9
9
9
6
7
8
A4
A5
A7
Me
N
O
Et
N
S
99 A8
100 A9
N
O
A
Scheme 5. Synthesis of target 4-thiazole-indole/imidazothiadiazoles hybrids.
2+
Reagents and conditions: thiosemicarbazone (1.0 equiv), appropriate [C ] compound (1.0 equiv), AcONa
2
(
1.0 equiv), AcOH, reflux, 3 h. Structure of fragment A – see scheme 2.
Geometrical isomers (E and Z) around imine double bound are possible for thiosemicarbazones and
corresponding thiazoles/thiazolidinones [27,64] and were previously described [59]. The analytical and
1
4

ACCEPTED MANUSCRIPT
1
13
1
spectral data ( H NMR, C NMR, LCMS) confirmed the structure and purity of compounds. H NMR
spectra of target compounds showed characteristic patterns of protons. Additionally, the X-ray analysis
was performed for the compound 7 (Fig. 3).
Figure 3. ORTEP view of the molecule 7 showing the atomic labelling scheme.
Non-H atoms are drawn as 30% probability displacement ellipsoids and H atoms are drawn as spheres of
an arbitrary radius.
The X-ray crystal structure analysis of compound 7 showed that the amidine hydrogen atom was attached
to N3 atom that complied with the structure containing a secondary amide group in the thiazolidinone
system. Meanwhile, the N6 and N7 atoms from a hydrazone group are imine nitrogen atoms. Relevance of
this finding is supported by the values of interatomic distances N3–C4 [1.372(2) Å] and C2–N3
[
1.3705(18) Å], C2=N6 [1.2883(18) Å] and N7=C8 [1.2858(18) Å] that are close to the mean values for the
single bonds (O=)C–NH [1.357(3) Å] and HN–C(=N) [1.376(3) Å], as well as double bond C=N
1.281(3) Å] respectively. These values are acquired from the structures containing N-(4-oxo-1,3-
[
thiazolidin-2-ylidene)hydrazone moiety based on the Crystal Structure Database, Version 5.39 (refcodes:
NEBHOU, DIQHIW, FOTQEM, IKIVIJ, QAJSEC, ROMXUN, SOHHIH, ULACEQ, WOGGOQ,
XUBYOK, R < 0.075) [60]. Performed studies revealed that some atoms in the crystal structure of 7 were
disordered. This observation concerns the part of the molecule that includes atoms S1, C4, C5, O24, C25,
1
5

ACCEPTED MANUSCRIPT
C26 from the 5-ethyl-1,3-thiazolidin-4-one fragment. Each of these atoms takes up two alternative
locations in the crystal structure labelled a and b (Fig. 4)
a
b
Figure 4. The hydrogen bonding in 7.
H atoms not involved in hydrogen bonds have been omitted for clarity.
Antitrypanosomal activity. The antiparasitic activity of newly synthesized compounds was
screened on Trypanosoma brucei gambience and Trypanosoma brucei brucei. Firstly, the compounds
were tested at two concentrations of 10 and 1µg/mL and parasite growth inhibition was measured, the
results are presented as growth inhibition percentage. For the active compounds, IC50 values were
calculated [65,66].
Antitrypanosomal activity of thiazolidinone-hydrazones (1a-1t) without indole or
imidazothiadiazole fragments. For compounds of the first series (Scheme 1, Table 1) without indole or
imidazothiadiazole fragments, high inhibition rates were not observed (1µg/mL concentration). The
derivatives with norbornane moiety 1h, 1i as well as 1c inhibited growth of more than 90% of the
parasites, but at the same time they were not active at lower concentration.
1
6

ACCEPTED MANUSCRIPT
Table 1. Antitrypanosomal activity of 4-thiazolidinones without indole or imidazothiadiazole fragment
towards Trypanosoma brucei brucei
Inhibition, %
Inhibition, %
Compound
Compound
1
0 µg/mL
1 µg/mL
1.18
NT
10 µg/mL
1 µg/mL
7.69
1
a
-8.23
56.11
98.91
10.03
-4.66
44.88
96.98
97.80
29.97
10.91
1l
1m
1n
1o
1p
1q
1r
1s
53.24
14,70
57.30
14.70
14.49
25.01
24.86
98.53
42.01
1
b
11.14
38.70
11.14
15.08
-1.57
3,28
1c
5.06
0.06
-4.65
NT
1
d
1e
1
g
1
h
NT
1i
14.13
4.18
9.82
8.16
1j
1t
36.13
1
k
Antitrypanosomal activity of thiazolidinone-hydrazones with phenyl-indole fragment. Screening of
the set of indole-containing compounds explored some substitution patterns on thiazolidine ring (Table 2).
The minimum activity required for the characterization of the tested compound as a hit was the IC ≤ 1.0
5
0
µM. The lowest IC50 values were observed for 2-[(2-phenyl-1H-indol-3-ylmethylene)-hydrazono]-
thiazolidin-4-ones with different alkyl moieties in the C5 of thiazolidone core (compounds 7, 48 and 49).
Ethyl-, hydroxyethyl- as well as methoxypropionyl substituents in the C5 position of thiazole/4-
thiazolidone core turned out to be favourable for the trypanocidal activity. On the other hand, the
introduction of the aryl fragments at the same position caused three fold increasing of the IC values (43,
5
0
1
7

ACCEPTED MANUSCRIPT
4
4, 46, 66). The influence of acetamide and tetrahydronaphthalene fragments was similar and caused even
more activity decreasing.
Table 2. The IC50 values of 4-thiazolidones with indole fragment against Trypanosoma brucei brucei.
H
N
O
N
N
S
R
NH
Compound
7
R
IC µM
50,
Et
-NHCOCH
-NCOCH
NHCOCH
0.03 ± 0.004
9.16±1.142
7.86±0.260
6.86±0.261
0.17± 0.004
0.06 ± 0.003
7.73±0.310
10.63±1.810
43
44
46
48
49
66
67
4-ClC
4-BrC
4-EtCOOC
6
H
4
2
6
H
4
2
6
H
4
2
HOCH CH2
2
MeCOO(CH2)2
Ph-COCH
2
4-FC
H
4
-COCH
2
6
O
68
13.13±0.97
7
4
5
HOOC(CH
2
2
)
2
NHCOCH
2
34.84±0.450
14.04±0.830
7
HOOC(CH
)
5
NHCOCH
2
Pent
Nf
0,0014 ± 0.0005
2,39 ± 0,608
Data are presented as mean ± SD; Pent – Pentamidine, Nf – Nifurtimox
1
8

ACCEPTED MANUSCRIPT
After establishing significant antitrypanosomal properties in the Tb brucei assay, a series of 2-[(2-
phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-ones was tested towards Tb gambiense (Table
3
3
). The impact of the substituents in the p-position of phenyl ring (R , Table 5) and N3-position of 4-
thiazolidone ring was exploited. The most active compounds 7, 49 and 66 discovered in the previous
assay (Table 4) inhibited growth of the parasite at the same levels of concentrations. Interestingly, that the
replacement of the hydrogen in C5 of thiazolidone core (5) by methyl (6), ethyl (7) or methoxopropionyl
(
49) moieties didn’t influence the activity.
Table 3. The IC values of 4-thiazolidones with indole fragment against Trypanosoma brucei gambiense
5
0
3
R
2
R
N
O
N
N
S
1
R
NH
1
2
3
Compound
R
R
R
IC , µM
50
5
6
7
8
9
H
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
0.23 ± 0.009
0.21± 0.014
0.11± 0.040
3.93 ± 0.363
2.22 ± 0.071
1.42 ± 0.098
1.35 ± 0.126
1.50 ± 0.111
4.70 ± 0.500
6.20 ± 2.200
H
H
Cl
Cl
Cl
Br
Br
NO
NO
Me
Et
10
11
12
14
15
Me
Et
Me
Et
2
2
1
9

ACCEPTED MANUSCRIPT
2
2
2
3
3
3
3
4
5
5
5
5
5
6
6
6
7
8
9
0
1
2
4
9
0
1
2
3
9
0
1
6
H
furan-2-ylmethyl
furan-2-ylmethyl
furan-2-ylmethyl
Cl
7.31 ± 1.354
1.71 ± 0.194
1.71 ± 0.161
0.82 ± 0.111
1.17 ± 0.126
0.86 ± 0.076
0.14 ± 0.010
0.23± 0.014
1.49 ± 0.104
1.34 ± 0.057
3.34 ± 0.534
1.36 ± 0.018
1.40 ± 0.158
1.59 ± 0.084
0.38 ± 0.080
2.82 ± 0.426
0.0015± 0.0007
4.64 ± 0.725
Me
Cl
Cl
Cl
Cl
Cl
NO
H
Et
H
4-HO-C
6
6
6
6
H
H
H
H
4
4
4
4
Me
4-HO-C
Et
Et
4-HO-C
4-HO-C
2
MeCOO(CH )
2 2
H
H
H
H
H
HOCH
MeOOCCH
OHCH CH
MeOOCCH
HOCH
CH
Cl
Cl
Br
Br
Cl
Cl
Cl
H
2
2
2
CH
2
2
2
2
2
CH
CH
furan-2-ylmethyl
furan-2-ylmethyl
2
2
MeOOCCH
CH
2
2
2
MeOOCCH
CH
4-HO-C
H
H
4
2
6
PhCOCH
2
Pent
Nf
Data are presented as mean ± SD; Pent – Pentamidine, Nf – Nifurtimox
The latter argued the necessity of phenyl-indole and thiazolidone fragments in the molecules for the
trypanocidal properties. For compounds without phenyl ring attached to indole fragment, significant
anyitrypanosomal activity was not found (e.g. 2-[(1H-indol-3-ylmethylene)-hydrazono]-5-
methylthiazolidin-4-one – IC50 = 21.7±3.4 (Tb brucei), 77.8±9.1 (Tb gambiense); 2-[(1H-indol-3-
ylmethylene)-hydrazono]-thiazolidin-4-one – IC50 = 9.68±1.8 (Tb brucei), 30.4±8.4 (Tb gambiense), 5-
2
0

ACCEPTED MANUSCRIPT
ethyl-2-[(1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-one – IC = 39.51±9.7 (Tb brucei), 73.6±6.3
5
0
(
Tb gambiense); the synthesis of compounds is not presented). The comparison of the activities of
compounds with furan-2-ylmethyl (27-29) and p-hydroxyphenyl (30-32) substituents in the N3 position of
thiazolidone core and their unsubstituted analogs (5-7) showed that the substitution of the N3 position is
not essential. However, p-hydroxyphenyl fragment is more advantageous than furan-2-ylmethyl. The
introduction of halogen atoms as well as nitro group on the phenyl ring does not contribute to the
antitrypanosomal activity, except the derivative 34 that showed excellent growth inhibition, reaffirming
the benefits of ethyl group in C5 and p-hydroxyphenyl fragment in N3 of thiazolidone core. Generally, 2-
[
(2-phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-ones showed very good trypanocidal
properties inhibiting growth of the Tb gambiense at micro- and submicromolar concentrations.
Subsequently, we exploited the impact of 4-thiazolidone ring replacing it with thiazole core (Table 4).
Overall IC50 values were at the same level. Carboxyethyl group in C5 of thiazole ring appeared to be
important for the trypanosomatide growth inhibition and IC for the derivatives 90, 91 and 96 were much
5
0
higher than those for the unsubstituted analogs.
Table 4. The IC values, µM of thiazoles with indole fragment against Trypanosoma brucei gambiense
5
0
4
1
R
R
N
2
N
R
S
N
3
R
N
H
1
2
3
4
Compound
R
R
R
R
IC ,µM
50
7
7
7
6
7
8
4-Br-C
Ph
6
H
4
H
H
H
Cl
NO
NO
-
-
-
1.52 ±0.400
6.00 ±0.600
2.30 ±0.900
2
2
4-BrC H
6 4
2
1

ACCEPTED MANUSCRIPT
8
9
9
9
4
0
1
6
4-NO
2
C
6
H
4
H
NO
2
4-HO-C
6
H
4
1.50 ± 0.200
0.53 ±0.205
0.74±0.068
0.66 ±0.030
Me
Me
Me
C
C
C
H
5
H
5
H
5
OOC
Cl
-
-
2
2
2
OOC NO
2
2
OOC NO
4-HO-C
6
H
4
Antitrypanosomal
b][1,3,4]thiadiazoles fragment. The next step was the screening of imidazo[2,1-b][1,3,4]thiadiazoles
Tabl. 5, 6): the first set consisted of thiosemicarbazone bearing compounds and the second set consisted
activity
of
thiazolidinone-hydrazones
with
phenyl-imidazo[2,1-
(
of thiazolidinone/thiazole-imidazothiadiazoles hybrids as thiosemicarbazone cyclic analogs. To estimate
the impact of thiazole and 4-thiazolidinone rings on trypanocidal action, the activity of corresponding
intermediates 2a-2c, 3a, 3c as well as thiosemicarbazones 4h, 4k, 4m, 4o and thiosemicarbazides 4i, 4l,
4
n, 4p was evaluated. SAR data suggested that imidazothiadiazole core itself weakly contributed to the
activity, but the introduction of thiosemicarbazone fragment improved antitrypanosomal properties.
Table 5. The IC values, µM of imidazothiadiazole bearing compounds against Trypanosoma brucei
5
0
gambiense
2
R
N
1
R
N
S
3
N
R
1
2
3
Compound
R
R
R
IC , µM
50
2
a
S-CH
2
CHCH
COOEt
Et
CH=CH
2
27.2±1.40
>30
2
b
S-CH
2
H
H
2
c
>40
3
a
S-CH
2
2
CHO
2
H
23.4±1.40
2

ACCEPTED MANUSCRIPT
3
c
Et
> 40
6.1±0.30
20.9±0.60
>25
4
4
h
k
S-CH
2
CH=CH
2
H
H
Et
H
N
H N
2
N
S
4
m
Et
NO
Br
H
2
4
o
Et
>25
4
i
S-CH
2
CH=CH
2
>50
HO
4
l
Et
Et
Et
H
2.6±0.50
14.8±0.90
1.5±0.02
S
N
N
N
4
n
p
H
H
NO
2
4
Br
Table 6. The IC values, µM of imidazothiadiazoles’ hydrazones against Trypanosoma brucei gambiense
5
0
R
N
O
N
N
N
1
S
N
R
3
R
S
2
N
R
1
2
3
Compound
R
H
R
R
R
IC , µM
50
16
17
18
35
36
37
55
H
H
H
H
H
H
H
H
Me
Et
3.9 ± 0.70
4.4± 0.70
20.6± 4.70
> 20
H
H
S
4-HO-C
4-HO-C
4-HO-C
H
H
4
H
4
H
4
CH2
H
6
6
6
Me
Et
> 20
3.6± 1.30
5.7± 0.60
AcOCH CH
2 2
2
3

ACCEPTED MANUSCRIPT
62
72
19
20
21
22
23
24
25
26
38
39
40
41
42
56
57
58
63
64
65
4-HO-C
6
H
4
H
H
AcOCH
2
CH
2
1.00± 0.04
>20
H
HOOCCH
2
H
OEt
H
Me
Et
3.9± 0.30
9.2± 0.60
18.0± 2.30
>25
S
O
H
H
H
H
H
H
H
Me
Et
H
H
3.7± 0.10
>20
H
NO
Br
Br
H
Et
2
H
Me
Et
9.5± 1.30
13.8± 1.40
9.2± 0.50
4.8± 0.20
>20
H
4-HO-C
4-HO-C
4-HO-C
4-HO-C
4-HO-C
H
H
4
H
4
H
4
H
4
H
4
H
6
6
6
6
6
H
Me
Et
Et
H
Br
Br
H
Me
Et
1.5± 0.03
1.5± 0.04
11.8± 2.00
16.2± 0.70
3.2± 0.10
2.7± 0.80
11.1± 1.60
1.0± 0.10
AcOCH
AcOCH
AcOCH
AcOCH
AcOCH
AcOCH
2
2
2
2
2
2
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
H
NO
Br
H
2
H
4-HO-C
4-HO-C
4-HO-C
H
4
H
4
H
4
6
6
6
NO
Br
2
N
H
R
N
N
N
1
S
N
R
3
R
S
2
N
R
2
4

ACCEPTED MANUSCRIPT
1
2
3
Compound
R
R
R
R
IC , µM
50
S
92
Me
CH2
H
EtOOC
0.652± 0.113
80
81
82
83
93
94
95
4-NO
2
2
-C
-C
6
6
H
4
4
H
NO
NO
Br
H
H
10.3
4.4
4-NO
H
2
2
4-Br-C
H
H
1.2
6
4
4-NO -C
H
4
Et
H
9.2± 0.80
2.7 ± 0.90
>20
2
6
Me
Me
Me
H
EtOOC
EtOOC
EtOOC
NO
Br
2
7.4± 0.40
OH
R
N
N
3
R
S
N
2
R
N
N
1
R
N
S
1
2
3
Compound
R
R
R
R
IC , µM
5
0
S
97
Me
CH2
H
EtOOC
0.631 ± 0.066
85
86
87
88
89
98
99
Ph
H
NO
NO
Br
H
3.0 ± 0.30
0.594 ± 0.036
2.6± 0.70
4-Br-C
H
H
H
6
4
2
2
4-NO
4-Br-C
4-NO -C
2
-C
6
H
4
6
H
4
Et
H
0.476 ± 0.066
0.510 ± 0.110
0.745 ± 0.098
0.639 ± 0.074
H
Br
H
2
6
4
Me
Me
H
EtOOC
EtOOC
NO
2
2
5

ACCEPTED MANUSCRIPT
1
00
Me
Br
EtOOC
1.0 ± 0.10
Allylsulfide fragment negatively influences the imidazothiadiazole derivatives activity. Ethyl
fragment in C5 possition of thiazolidone in 18 decreased the activity by an order comparing to 5-methyl-
4
-thiazolidinone in 17. 4-Oxothiazolidin-5-yl-acetic acid 72 turned out to be one order less active than its
methyl ester 55. The derivatives 55, 58, 62, 63 with acethylated etanole fragment in the C5, aryl
substituents in the N3 of thiazolidinone and para-substituted phenyl ring attached to imidazothiadiazole
core inhibited growth of the parasite at the same range of concentrations within 1.0-5.7 µM. On the other
hand, loss of the activity of structurally similar derivatives (57, 64) was due either to the presence of
nitrogroup in the phenyl ring or ethyl fragment in the C2 of imidazothiadiazole core. Other 5-methyl-6-
phenyl-imidazo[2,1-b][1,3,4]thiadiazoles did not show significant levels of antitrypanosomal activity
except those containing thiazole-5-carboxylic acid ethyl ester fragment (93) or acetic acid 2-(4-
oxothiazolidin-5-yl)-ethylester (63) with IC =2.7 µM for both. p-Hydroxyphenyl fragment in the N3 of
5
0
thiazolidinone ring did not improve the activity (35-37), although its presence along with ester group in
the C5 or aryl moiety in the C3 of thiazolidinone or thiazole rings is controversial as such compounds as
8
6, 88, 89, 100 and 98 showed submicromolar inhibition concentrations. It is worthwhile remarking that
halogen atoms as well as nitro group in the benzene ring can also contribute to the trypanocidal activity of
the 86, 88, 89, 100. Therefore, the most active compounds of this series appeared to be 2-[(2-
allylsulfanyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)-hydrazono]-4-methyl-2,3-
dihydrothiazole-5-carboxylic acid ethyl esters: 92 (IC =0.652 µM) and 97 (IC =0.631 µM); and 2-[(2-
5
0
50
ethyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)-hydrazono]-3-(4-hydroxyphenyl)-4-
methyl-2,3-dihydrothiazole-5-carboxylic acid ethyl esters 98, 99 and 100 with IC within 0.639-1.0 µM,
5
0
indicating that the presence of 2,3-dihydrothiazole-5-carboxylic acid ethyl ester is crucial for the
trypanocidal action of imidazothiadiazoles.
2
6

ACCEPTED MANUSCRIPT
Relatively low antitrypanosomal activity of the compounds 1 (Table 1) along with the low IC50
values for structurally relative thiazolidinones bearing phenyl-indole or phenyl-imidazothiadiazole
fragments in molecules indicate that the presence of menthioned moieties highly influence the
antiparasitic properties of this class of compounds. Additionally, some SAR outcomes can be outlined: i)
the presence of phenyl ring (attached to indole or imidazothiadiazole core) is essential; ii)
thiazolidinone/thiazole derivatives are more active than thiosemicarbazone analogs; iii) compounds with
indole fragment are more active than imidazothiadiazole analogs; iv) small substituent linked by single
bond at C5 position of thiazolidinone core is preferred; v) significant differences between thiazole and 4-
thiazolidinone derivatives were not observed (Fig. 5). Such outcomes had been supported by in silico
investigations of these series of compounds and are usefull for further optimization of the hit-compounds
structures [53].
2
7

ACCEPTED MANUSCRIPT
S
N
R
N
S
N
N
N
N
S
H
N
N
H
H
N
H
R
X
X
R
A
A
A
N
H
N
H
N
N
1
R
A
S
N
N
H
N
H
H
2
R
O
R
R
Alkyl moieties in the C5 are
2
N
O
R
favourable for the trypanocidal
activity
N
NH
S
X
Substituents at the phenyl ring
do not greatly contribute to the activity
R
Ester group in the C5 is R
favourable for the activity
N
N
S
N
X
N
X
N
1
N
R
H
N
S
3
2
R
R
3
2
R2
Aryl fragments in N3-position
contriubute to the activity
R
R
R2
R3 Phenyl fragment is
favourable for the activity
N
The presence of 4-OH-C H in
6 4
N
the N3 possition is controversial
N
R
N
S
R
X
S
N
N
X
N
N
1
R
N
N
S
H
Activity increasing
Figure 5. Some structure-activity relationships findings
Cytotoxicity upon human primary fribroblast cell line. For the identified hit-compounds the
cytotoxicity upon human primary fibroblast (AB943 cell line) was evaluated. The tested substances
possessed relatively low toxicity IC values and no particular impact of different molecular fragments on
5
0
the toxicity was observed. Nevertheless, 9 compounds possessed very good selectivity indexes (SI > 100)
Tabl. 7).
(
2
8