Antifibrotic and anticancer action of 5-ene amino/iminothiazolidinones

Article abstract of DOI:10.1016/j.ejmech.2016.02.011

Here we describe the synthesis and the antifibrotic and anticancer activity determination of amino(imino)thiazolidinone derivatives. An efficient one-pot three-component reaction which involved [2 + 3]-cyclocondensation and Knoevenagel condensation was us

Full text of DOI:10.1016/j.ejmech.2016.02.011

Accepted Manuscript
Antifibrotic and anticancer action of 5-ene amino/iminothiazolidinones
Danylo Kaminskyy, Gertjan J.M. den Hartog, Magdalena Wojtyra, Maryan Lelyukh,
Andrzej Gzella, Aalt Bast, Roman Lesyk
PII:
S0223-5234(16)30081-2
10.1016/j.ejmech.2016.02.011
EJMECH 8362
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 December 2015
Revised Date: 3 February 2016
Accepted Date: 4 February 2016
Please cite this article as: D. Kaminskyy, G.J.M. den Hartog, M. Wojtyra, M. Lelyukh, A. Gzella, A. Bast,
R. Lesyk, Antifibrotic and anticancer action of 5-ene amino/iminothiazolidinones, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.011.
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ACCEPTED MANUSCRIPT
Antifibrotic and anticancer action of 5-ene amino/iminothiazolidinones
Danylo Kaminskyy, Gertjan J.M. den Hartog, Magdalena Wojtyra, Maryan Lelyukh, Andrzej Gzella, Aalt
Bast, Roman Lesyk
1.2
Me
3
R1
R1
R1
O
O
8
1
0.8
0.6
0.4
0.2
0
O
N
N
R3
R4
N
Ar(Het)
12
13
24
33
34
35
PIRF
N
R
N
N
N
R
S
S
R2
Ar(Het)
S
Ar(Het)
N
O
Pirfenidone
R1
N
O
S
O
R3
R4
R1
Ar(Het)
OH
Ar(Het)
+
Cl
N
+
N
N
R3
R4
S
0
1
2
3
4
H
H
O
Concentration (µM)

ACCEPTED MANUSCRIPT
Antifibrotic and anticancer action of 5-ene amino/iminothiazolidinones
Danylo Kaminskyy1, Gertjan J.M. den Hartog2, Magdalena Wojtyra1, Maryan Lelyukh1, Andrzej
Gzella3, Aalt Bast2, Roman Lesyk1,*
1Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National
Medical University, Pekarska 69, Lviv, 79010, Ukraine;
2Department of Pharmacology & Toxicology, Faculty of Health, Medicine and Life Sciences,
Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands;
3Department of Organic Chemistry, Faculty of Pharmacy, Karol Marcinkowski Poznan University of
Medical Sciences, Grunwaldzka 6, Poznan, 60780, Poland.
Keywords
Amino/amino-thiazolidinones, synthesis, one-pot method, antifibrotic activity, superoxide radicals,
antiproliferative effects
Corresponding author:
dr_r_lesyk@org.lviv.net (R. Lesyk)
tel. +38 0322 75-59-66
fax. +38 0322 75-77-34
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Abstract
Here we describe the synthesis and the antifibrotic and anticancer activity determination of
amino(imino)thiazolidinone derivatives. An efficient one-pot three-component reaction which involved
[2+3]-cyclocondensation and Knoevenagel condensation was used for the synthesis of 5-ene-2-
amino(imino)-4-thiazolidinones. Following amino-imino tautomerism, the compound structures were
confirmed by X-ray analysis. Comparison of SRB assays on fibroblasts and cancer cells revealed that
compounds which significantly reduced the viability of fibroblasts did not possess an anticancer effect.
A series of thiazolidinone derivatives as interesting candidates for further testing has been identified.
Among the tested compounds 2-{3-furan-2-ylmethyl-2-[(2-methyl-3-phenylallylidene)hydrazono]-
thiazolidin-4-one-5-yl}-N-(3-trifluoromethylphenyl)-acetamide (5), N-(2-methoxyphenyl)-2-[5-(4-
oxothiazolidin-2-ylideneamino)[1,3,4]thiadiazol-2-ylsulfanyl]-acetamide
(thiazol-2-ylimino)thiazolidin-5-ylidene]-1,3-dihydroindol-2-one (33),
(12),
3-[3-allyl-4-oxo-2-
and
5(Z)-(thiophen-2-
ylmethylene)-4-(4-chlorophenylamino)thiazol-2(5H)-one (34) possessed high antifibrotic activity levels,
had a similar effect as Pirfenidone, and did not scavenge superoxide radicals. Their antifibrotic potential
was confirmed using the xCelligence system.
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1. Introduction
Structure based approach is an efficient tools for new hit- and lead-compounds design. Utilizing
so-called privileged structures can be considered beneficial in such an approach, and it also greatly
increases the chances of success [1]. 4-Thiazolidinones are prime examples of such compounds.
Achievements in the medical chemistry of the latter significantly increased and are illustrated by the
rapid growth of the number of scientific papers and patents [2-5]. Most of the studies on these
compounds are devoted to the 2,4-thiazolidinedione, rhodanine and 2-alkyl(aryl)-substituted 4-
thiazolidinone derivatives as leads for antimicrobial, antidiabetic, anti-inflammatory and anticancer
agents [5-7]. The variety of thiazolidinones allows to diversify the modification directions of the
mentioned heterocycles and makes them attractive scaffolds in the privileged substructure-based
diversity oriented synthesis [8]. Among the wide range of 4-thiazolidinones two subtypes are of special
interest, namely the 5-ene- and 2-amino(imino)-4-thiazolidinone derivatives [9].
Pharmacological attractiveness of the 5-ene derivatives is reasoned and confirmed by the thesis
about the crucial role of the presence/nature of C5 substituent of a basic core for the biological activity
realization [10-12]. Conjugation of the 5-ene fragment to the C4 carbonyl group renders the compounds
electrophilic and potentially reactive by enabling Michael addition of nucleophilic protein residues to
the exocyclic double bond. This enables for example, reactions with glutathione or other free thiols.
Simultaneously this property may cause 5-ene-4-thiazolidinones to be frequent hitters or pan assay
interference compounds that are useless in drug discovery process because of their insufficient
selectivity [2,13,14]. However, this view has been disproved in many studies and a large number of
lead-compounds belongs to the mentioned 5-ene-4-thiazolidinones. The positive perspective also is
linked to a poly-pharmacological approach, where affinity toward various targets is regarded
advantageously [14,15]. In this context, it is worth mentioning that Michael acceptors are among the
most effective activators of Nrf2, which opens new perspectives in the treatment of inflammation and
cancer [16].
The other promising subtype of 4-thiazolidinone derivatives comprises the 2-amino(imino)-4-
thiazolidinones. These compounds possess antimicrobial [17], anti-inflammatory [18], and anticancer
[19] activities. Members of this group are the 2-arylaminothiazol-4-ones which are effective growth
inhibitors of HT29 adenocarcinoma cells [20] and the human lung cancer cell lines H460 and
H460/TaxR [21,22]. Furthermore, 4-thiazolidinone derivatives are efficient integrin αvβ3 antagonists
[23] and inhibitors of CDK1/cyclin B [24].
Currently, the search for compounds possessing combined types of activities is of special interest,
especially for compounds with simultaneous antiproliferative or cytostatic. Combination of anticancer
and antioxidant, anti-inflammatory [2,3,5], antiprotozoal [25] activities have been discussed. In this field
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the search for new 4-thiazolidinone based compounds with anticancer and antifibrotic activities could be
a promising direction.
Fibroblasts are mesenchymal cells that reside in the pulmonary interstitium and produce collagen
and other extracellular matrix proteins. They play a pivotal role in the normal wound healing or tissue
repair process and excessive stimuli for fibroblasts lead to the tissue remodeling or fibrous tissue that is
observed in bronchial asthma, chronic obstructive pulmonary disease [26] and idiopathic pulmonary
fibrosis. Moreover fibroblasts are associated with cancer cells and cancer progression, and their
structural and functional contributions to this process are discussed [27,28] and are associated with an
array of cytokines produced by fibroblasts. Targeting the so-called cancer-associated fibroblasts
(promoters of tumour growth and progression) is a novel and promising therapeutic strategy against
cancer. In this context, ideal therapeutic molecule will have reduced cancer-promoting properties in
combination with optimized antifibrotic action. Currently, therapeutic options in lung fibrosis are
extremely limited [29]: Pirfenidone, the only registered antifibrotic drug, has proven antifibrotic efficacy
in idiopathic pulmonary fibrosis patients [30,31] but is also associated with side effects viz.
gastrointestinal disturbances and photo toxicity. Key findings in the creation of new antifibrotic agents
are mainly related with inhibition of Wnt- and TGFβ-mediated β-catenin signaling [30], inhibition of the
TGFβ/CTGF axis [33,34] and are considered as attractive direction for the development of new
therapeutics against not only various fibro-proliferative diseases but also cancer. Another argument for
search for anticancer and antifibrotic activities combination is utilization of proven anticancer agents in
the new antifibrotic agents design. For instance, nintedanib (a potent and specific tyrosine kinase
inhibitor) has clinical efficacy in the treatment of pulmonary fibrosis [35]. Thiazolidinone based
compounds with antiproliferative effect were much less toxic to fibroblasts [22,36-38] but possessed
significant antitumor and antifibrotic activities [39]. In addition to the antiproliferative and antifibrotic
activity, superoxide scavenging potency appears to be a beneficial feature to include during the design
of new anticancer and antifibrotic agents. Superoxide anion radicals have been shown to play an
important role in fibrotic disorders by activating fibroblasts to produce collagen and the profibrotic
cytokine transforming growth factor β (TGFβ) and with simultaneous activation of α-smooth muscle
actin [40-42] as well as in cancer growth.
The work described herein is an extension of our ongoing efforts towards a search for new
therapeutic agents bearing the thiazolidinone frame. The aim of study was to evaluate the potential
antifibrotic and anticancer effects of 4-thiazolidinones and to determine their effects on lung fibroblast
viability and proliferation as well as their superoxide scavenging potency.
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2. Results and discussion
2.1. Chemistry
Synthetic approach to the target compounds was based on the results of our previous studies in the
field of 2-amino(imino)-4-thiazolidinones and their isomeric 4-aminothiazol-2(5H)-ones [4,12,19,43].
The synthetic protocols for compounds 1-11 involved [2+3]-cyclocondensation of S,N-dinucleophiles
(thioureas, thiosemicarbazones) with a row of dielectrophilic synthons [С2]2+ (Schemes 1, 2). The
reactions were performed in glacial acetic acid medium in the presence of anhydrous sodium acetate.
Рresented non-conjugated structures (1-7, 9-11) contain a stereo-center at the C-5 position of the
heterocyclic system and occur in racemic form.
Scheme 1
Compounds 12-16 were obtained by the interaction of the N-heteryl-chloroacetamides with
ammonium thiocyanate in the acetone medium. Following the hypothesis about crucial role of the
presence/nature of C5 position moiety, 5-ene derivatives (23-27, 30, 33) were obtained with high yields
via Knoevenagel reaction based on the CH-acidic site of thiazolidinone core (Scheme 2). For the most
of the compounds (17-22, 28, 29, 31, 32 – bearing simple arylidene, aryl-alkylidene, isatin or
pyrazolone fragments) the efficient one-pot method based on the three-component reaction of S,N-
dinucleophile, electrophilic reagent and oxocompound was used. The latter include the abovementioned
[2+3]-cyclocondensation and Knoevenagel condensation phases. The reaction was performed in the
medium of glacial acetic acid in the presence of sodium acetate (Scheme 2). Mentioned protocol is the
useful approach for the target compounds synthesis based on the various types of thiourea or
thiosemicarbazide derivatives as well as the various oxo-compounds including heterocycles, e.g. isatin
[19,44,45]. Moreover, the cyclic analogues of thiourea also have been successfully utilized in such
approach in the synthesis of compounds bearing 5-ene-4-thiazolidinone fragment [46].
Scheme 2
The regioselectivity of the cyclization is influenced by the reaction conditions, choice of the
haloacetic acid derivative and also by the nature of the two substituents R1, R2 and Ar(Het) of the
thioureas (Scheme 2). Therefore, the products of the reaction may be the mixture of isomers which
differ by the substituents at the positions 2 and 3 [47,48]. Based on the described protocols only one
presented form of compounds was isolated in this study. Synthesized 2-substituted-4-thiazolidinones,
bearing hydrogen atom on an exocyclic nitrogen atom exist in the form of two tautomers differing by
the position of C=N double bond (2-amino- and 2-amino tauthomers); tautomers with exocyclic C=N
double bond exist as mixture of Z- and E-stereoisomers [12,19,49]. Following the aforesaid the large
number of works has been devoted to the investigation of the structure of mentioned compounds. The
latter are represented both in amino and imino forms; predominance of one or another form depends
usually on the nature of substituent in starting thiourea. Presented spectral data are not sufficient
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argument to assert only one form, moreover 1H NMR spectral analysis of the data allows affirming the
presence of two mentioned forms. Main argument for amino form presentation is X-ray data for such
type of compounds [19].
13
The analytical and spectral data (1H NMR, C NMR, LCMS) confirmed the structure and purity
1
of compounds. H NMR spectra of target compounds showed characteristic patterns of protons. In the
most cases there is a doubling of signals corresponding to appropriate tautomeric forms. The location of
СН= group (С5 ylidene fragment compounds 17-27) signal (singlet) at ~7.50-8.50 ppm confirms the
formation of compounds with cis-position of ylidene residue (Z-configuration), similar to other 5-ene-4-
azolidinones [10,12,19,50].
For compounds which include oxo(thio)diazole/thiazole/pyrazole residue (12-16, 23-27, 29, 30,
33) it was shown the existence of imino form in the solid state based on the 13 X-ray data (Fig. 1). This
was the main argument for structures representation (Scheme 2).
Figure 1
The X-ray analysis of 13 geometry showed that the hydrogen atom is attached to N3 which is in
agreement with its localization in tautomer 13-1 (Fig. 1 and 2), containing a secondary amide in the 4-
thiazolidinone system and an exocyclic imine nitrogen. Relevance of this finding is supported by the
values of interatomic distances N3–C4 [1.371(2)] and C2–N3 [1.373(2) Å] as well as C2−N6 [1.298(2)
Å] that are close to the mean values for the single bonds (O=)C–NH[1.369(4)Å] and NH–C(=N)
[1.374(3) Å] as well as double bond C=N [1.280(2) Å], respectively, acquired from the structures
containing 2-imino-1,3-thiazolidine-4-one moiety that are accessible from the Crystal Structure Database
(refcodes: EHITZO, HEGMEN, HEGMIR, HEGMOX, ROMXUN, ULACEQ, VAMPUV, CSD
Cambridge, Version 5.35 , R < 0.07) [51].
Figure 2
A shortening of the distance C4=O20 [1.212(2) Å] and lengthening of the distance C4−N3
[1.371(2) Å], bond in comparison with the typical (HN–)C=O, 1.235(2) Å, and (O=)C–NH, 1.331(2) Å,
bond lengths [52] for the secondary amide group within γ-lactam ring are noteworthy. The difference is
about 8σ for the former and 14σ for the latter bond. This proves that the conjugation of the lone electron
pair on N3 with the C4=O19 carbonyl group is significantly weakened in favor of the conjugation with
the C2=N6 double bond and further with the 1,3,4-oxadiazole ring. The molecule of 13 as a whole is
exactly flat, as it lies on the mirror plane. Concurrently, the S1–C2 and N6–C7 bonds are in Z
configuration and conjugated double bond system C2=N6−N7=C8 is in s-cis conformation. Both torsion
angles S1–C2−N6–C7 and C2−N6−C7−N11 are 0.0º.
The next phase was the synthesis 5-ylidene-4-aminothiazol-2(5H)-ones (34, 35), which are
positional isomers of target compounds. The synthetic protocol included the phase of 4-aminothiazol-
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2(5H)-ones formations followed by Knoevenagel condensation according to the known procedure
[12,50].
Scheme 3
In contrast to the 2-amino(imino)-4-thiazolidinones, compounds 34, 35 exist only in amino form
and possibility of amino-imino tautomerism had been refuted [12]. This thesis is also confirmed by the
35 X-ray data (fig. 3).
Figure 3
The X-ray studies showed that compound 35 exists in the crystal in the tautomeric form 35-1 with
the H atom located on the exocyclic atom N7 (Fig. 3 and 4). This observation is supported by
approximately similar C4−N3 [1.3287(15) Å] and C4−N7 [1.3210(15) Å] bond lengths, a typical feature
for this tautomeric form, as well as by intermolecular N7−H7··O6i and O15–H15···N3ii hydrogen bonds
(Table 1), in which the N7 atom is a hydrogen donor and the N3 atom is a hydrogen acceptor.
Figure 4
Partly double-bond character of the C4−N7 linkage between the thiazolidinone and 2-methyl-4-
hydroxy-5-isopropylphenylamine moieties slows rotation and forces the N3,C4,C5,N7,C8 atoms to
adopt almost a planar conformation (r.m.s.: 0.0443 Å) with atoms N3 (thiazolidinone system) and C8
(2-methyl-4-hydroxy-5-isopropylphenyl residue) synperiplanar to each other [torsion angle:
N3−C4−N7−C8: -9.43(17)°]. The planarity of the N3,C4,C5,N7,C8 framework results in an unfavorable
spatial arrangement of the N7−H7 and C19−H19 groups of atoms which form a short H7···H19 contact
(2.08 Å). The phenyl ring of the p-chlorobenzylidene group and thiazolidinone system are
approximately coplanar [dihedral angle: 4.54(6)°]. Furthermore, the p-chlorophenyl group is in a Z
configuration [torsion angle S1–C5–C19–C20: -1.7(2)°]. The second phenyl ring of the 2-methyl-4-
hydroxy-5-isopropylphenylamine substituent forms with the flat thiazolidinone system a dihedral angle
of 69.39(5)°. Such mutual orientation is stabilized by an intermolecular hydrogen bond O15−H15···N3ii
(Table 1, Supplementary Fig. S1).
Table 1
In the crystal of 35-1 the solute molecules are connected by hydrogen bonds O15−H15···N3ii [(ii)
1.5-x,0.5-y,1-z] (Table 1) into centrosymmetric dimers which are further linked through three-centre
hydrogen bonds N7−H7···O6i···H19−C19 [(i) 1.5-x,0.5+y,0.5-z] (Table 1, Fig. 3) into two-dimensional
layers growing parallel to the bc plane. The layer thickness is about half of the a parameter length. The
methanol molecules, located in the symmetry centre of the crystal lattice, are connected by three-centre
hydrogen bonds O6···H27−O27···H21–C21iii [(iii) 1.5-x, -0.5+y,0.5-z] with the solute molecules from the
neighbouring layers.
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The synthesis of some compounds (16-18, 20-22, 34) was described in our previous publications
(see experimental part).
2.2. Pharmacology
2.2.1. Fibroblasts viability determined by SRB assay
The need for novel drugs for treatment of idiopathic pulmonary fibrosis [29] prompted the present
search for new compounds that display antifibrotic properties in cultured lung fibroblasts as well as
superoxide scavenging activity. A series of thiazolidinones and Pirfenidone as a referent drug were
evaluated for their effect on fibroblast viability using the SRB assay. In the SRB assay the test
compounds showed a wide range of activities. Based on their activity, the tested compounds were
divided into 3 categories: i) good inhibitors (compounds that inhibit proliferation by at least 50%) for
which the level of inhibition was comparable with Pirfenidone; ii) moderate inhibitors (compounds that
inhibit proliferation by at least 10% but less than 50%) and iii) stimulators (compounds that do not
reduce or stimulate proliferation). The results for these groups are shown in Figure 5. Interestingly, in
the cellular assays Pirfenidone displayed potent antifibrotic activity as evidenced by its inhibition of
fibroblast viability as well as its moderate effect on fibroblast proliferation (see below) which are in line
with clinical findings.
Figure 5
2.2.2. Anticancer activity screening
A series of compounds was screened within Developmental Therapeutic Program (NCI, NIH,
USA; https://dtp.cancer.gov/) according to the procedure described elsewhere [53-55] and involved
several phases. First, tested compounds were evaluated at one-dose primary anticancer assay towards: a
three cell line panel (the human tumor cell lines: MCF7 – breast cancer (BC), NCI-H460 – lung cancer
(NSCLC) and SF-268 CNS cancer (CNSC)) at concentration 10-4M or sixty cell line panel (the human
tumor cell lines were derived from nine different cancer types: leukemia (L), melanoma (M), NSCLC,
colon (CC), CNSC, ovarian (OC), renal (RC), prostate (PC) and BC) at concentration 10-5M. In the
screening assay, each cell line was inoculated and preincubated for 24-48 h. Then tested compounds
were added at the mentioned concentration the culture was incubated for further 48 h. SRB was used as
endpoint in a cytotoxicity assay. Results of screening assay were presented as growth percent (GP) of
the treated cells when compared to the untreated control cells (Tables 2 & 3). Based on 3 cell lines assay
only compounds 17, 20 [19] and 24 were selected for further phases.
Tables 2, 3
In a 60 cancer cell lines assay tested compounds including Pirfenidone did not exhibit significant
activity. Only compound 31 possesses selective action on UO-31 (RC) cell line. This finding is
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consistent with previously data regarding high sensitivity of UO-31 cells to active thiazolidinone
[10,12,50].
At the next phase compounds (4, 17 [19], 18, 20 [19], 30 and 34 [12]) were tested towards a sixty
tumor cell lines panel at 5 different concentrations (100µM, 10µM, 1.0 µM, 0.1µM and 0.01µM) [53-
55]. The same screening protocol was used and GPs were evaluated for each cell lines at each
compounds concentrations. The results were presented as three dose-dependent parameters – GI50, TGI
and LC50 (see experimental part) (Table 4).
Table 4
Among the tested compounds 17 [19], 18 and 20 [19] possessed the prominent activity levels and
inhibited cancer cell lines at concentration less than 1.0 µM. Comparison of SRB assays on fibroblasts
and cancer cell lines revealed that compounds which significant reduced the viability of fibroblasts (Fig.
5a) (13, 24, 33, 34 and Pirfenidone) did not possess a significant anticancer effect. Conversely,
compounds with anticancer activity (18, 20) belong to the group 3 (“stimulators” Fig. 5c) which did not
suppress fibroblast proliferation. Only compound 17, which possesses significant anticancer activity,
displayed moderate antifibrotic action.
2.2.3. Superoxide scavenging
Superoxide scavenging was determined for all compounds by measuring their ability to reduce
superoxide induced reduction of nitroblue tetrazolium (NBT) as described previously [56]. In Figure 6,
the results are shown for the compounds that inhibited NBT reduction. IC50 and Emax values for
inhibition of NBT reduction were calculated as described above. Results from the compounds that did
not inhibit NBT reduction were omitted from this graph for clarity.
Figure 6
Pirfenidone, although being a good inhibitor in the SRB assay, did not scavenge superoxide
radicals. A similar picture emerged for compounds 12 and 34. Compounds that combined good activity
on fibroblast viability as well as superoxide scavenging activity were 13, 24 and 33. While compound 3
was a good inhibitor of fibroblast viability and it did not scavenge any superoxide radicals. This is
remarkable because this compound contains a phenol moiety attached to the thiazolidinone core. Given
the previously recognized importance of superoxide radicals in the pathophysiology of idiopathic
pulmonary fibrosis [40,41] lack of this feature may make this compound probably less interesting for
further testing. Mode of action of such compounds most probably is not related with directed superoxide
inhibition/scavenging. Among the 4-thiazolidinones with anticancer activity only 20 and 24 show
superoxide radicals scavenging. This finding is not consistent with a known approach: search for new
radical scavenging agents as active anticancer agent, including thiazolidonones [57]. While the
anticancer effect of "classical" anticancer drugs (cisplatin and doxorubicin) is accompanied by an
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increase in the intracellular level of ROS that may contribute to their therapeutic effect [58]. Increasing
ROS production represents also an alternative among the chemotherapeutic strategies against cancer
cells [59]. This effect is also associated with the mode of action of related thiazolidinones. It was
reported that ROS levels increased under the action of 5-ene-4-thiazolidinones in HT29 and CEM cells
[60,61]. Moreover, generation of ROS could activate the NF-2E related factor 2/Kelch like ECH-
associated protein 1 (Nrf2/Keap1) pathway, involved to protect cells against oxidative stress [16,62].
2.2.4. Proliferation determined by xCelligence analysis
Using the xCelligence system, the effects of set of thiazolidinones and pirfenidone on human lung
fibroblast proliferation were monitored (Fig. 7).
Figure 7.
Four of the tested compounds (5, 17, 21, and Pirfenidone) concentration-dependently reduced
proliferation over the 150 h time course (P150). Compounds 12, 13, 24, 33 and 34 (belong to group 1 –
Fig. 5a – “good inhibitors”) also reduced P150 but with these compounds no concentration dependency
was observed. Compounds 21 and 34 also concentration dependently reduced the maximum rate of
proliferation (Pdot). Compounds that combined good activity on cell viability as well as superoxide
scavenging activity, namely 13, 24 and 33 also inhibited cell proliferation although this effect did not
show a good correlation with the concentration tested. Testing these compounds in the xCelligence
system confirmed the antifibrotic potential of 5, 12, 13, 24, 33, and 34 which had a similar effect as
Pirfenidone.
3. Conclusion
A series of 2-amino(imino)-4-thiazolidinone derivatives and 4-R-aminothiazol-2(5H)-ones were
synthesized. An efficient one-pot method based on three-component reaction of S,N-dinucleophile,
electrophilic reagent and oxocompound was used as synthetic protocol for 5-ene-2-
amino(imino)thiazolidin-4-ones. Following the amino-imino tautomerism the compound structures were
confirmed by X-ray analysis. Compounds were screened for their antifibrotic and anticancer activities.
Comparison of SRB assays on lung fibroblasts and cancer cells revealed that compounds which
significantly reduced the viability of fibroblasts did not possess antiproliferative or antineoplastic
effects.
Among
the
tested
compounds
2-{3-furan-2-ylmethyl-2-[(2-methyl-3-
phenylallylidene)hydrazono]-thiazolidin-4-one-5-yl}-N-(3-trifluoromethylphenyl)-acetamide (5), N-(2-
methoxyphenyl)-2-[5-(4-oxothiazolidin-2-ylideneamino)[1,3,4]thiadiazol-2-ylsulfanyl]-acetamide (12),
3-[3-allyl-4-oxo-2-(thiazol-2-ylimino)thiazolidin-5-ylidene]-1,3-dihydroindol-2-one (33), and 5(Z)-
(thiophen-2-ylmethylene)-4-(4-chlorophenylamino)thiazol-2(5H)-one (34) possessed high activity
levels, had effects similar to Pirfenidone and did not scavenge superoxide radicals. xCelligence system
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testing confirmed their antifibrotic potential. Thus a range of thiazolidinone derivatives as interesting
candidates for further testing and for design of new antifibrotic agents has been identified.
4. Experimental
4.1. Chemistry
4.1.1. Materials and methods
The starting 4-thioxo-2-thiazolidinone [63], 2-(thiazol-2-ylimino)-thiazolidin-4-one (15) [64] were
obtained by the methods described previously. Melting points were measured in open capillary tubes on
a BŰCHI B-545 melting point apparatus and are uncorrected. The elemental analyses (C, H, N) were
performed using the Perkin-Elmer 2400 CHN analyzer and were within ±0.4% of the theoretical values.
1
The H-NMR spectra were recorded on Varian Gemini 400 MHz and 13C NMR spectra on Varian
Mercury-400 100 MHz in DMSO-d6 using tetramethylsilane as an internal standard. Chemical shifts are
reported in ppm units with use of δ scale. Mass spectra were obtained using electrospray ionization
(ESI) techniques on an Agilent 1100 Series LCMS.
4.1.2. General procedure for the preparation of 5-unsubstituted and 5-alkyl-2-R-amino(imino)thiazol-
4(5H)-one derivatives (1-16).
Method 1 (compounds 1-11). A mixture of appropriate substituted thiourea or thiosemicarbazone
(10 mmol) and appropriate carboxylic acid derivative (β-aroylacrylic acid, N-arylmaleinimide, γ-
bromobutyrolactone, chloroacetic, 2-bromopropionic and 2-bromobutyric acids) (10 mmol) and sodium
acetate (10 mmol) in 50 mL of acetic acid was refluxed for 3 h. Crystalline precipitate was filtered off,
washed with acetic acid, water, ethanol and diethyl ether and then recrystallized from a mixture DMF :
acetic acid (1:2) or DMF : ethanol (1:2) or glacial acetic acid.
Method 2 (compounds 12-16). A mixture of N-heteryl-2-chloroacetamide (10 mmol) and
ammonium thiocyanate (20 mmol) was refluxed for 3 h in acetone (20 mL). After the reaction was
completed, the mixture was cooled. The precipitate was collected by filtration, washed with water and
recrystallized with glacial acetic acid.
4.1.2.1. 3-(4-Ethoxyphenyl)-2-(4-ethoxyphenylimino)-5-[2-(4-methoxyphenyl)-2-oxoethyl]thiazolidin-4-
о
1
one (1). Yield 69%, mp 147-149 С. H NMR (400 MHz, DMSO-d6): δ 1.29 (t, 3H, J = 7.0 Hz, CH3),
1.35 (t, 3H, J = 6.8 Hz, CH3), 3.80 (dd, 1H, J = 18.0, 9.2 Hz, CHCH2), 3.85 (s, 1H, CH3), 3.96 (m, 3H,
CH2, CHCH2), 4.07 (q, 2H, J = 7.0 Hz, CH2), 4.68 (dd, 1H, J = 9.3, 3.0 Hz, CHCH2), 6.77 (d, 2H, J =
8.8 Hz, arom.), 6.84 (d, 2H, J = 8.8 Hz, arom.), 7.00-7.07 (m, 4H, arom.), 7.38 (d, 2H, J = 8.8 Hz,
arom.), 7.95 (d, 2H, J = 8.2 Hz, arom.). 13C NMR (100 MHz, DMSO-d6): δ 195.3, 165.8, 155.3, 144.3,
11

ACCEPTED MANUSCRIPT
141.2, 140.5, 130.6, 129.6, 128.7, 128.0, 121.9, 114.9, 114.7, 114.1, 63.4, 63.2, 55.7, 44.7, 42.8, 14.8,
14.7. LCMS (ESI) m/z 505 (99.0%, (M+H)+). Calcd. for C28H28N2O5S: C, 66.65; H, 5.59; N 5.55;
Found: C, 66.80; H, 5.70; N, 5.70%.
4.1.2.2. 5-[2-(4-Fluorophenyl)-2-oxoethyl]-2-(4-hydroxyphenylamino)-4,5-dihydrothiazol-4-one (2).
о
1
Yield 76%, mp >230 С. H NMR (400 MHz, DMSO-d6): δ 3.54 (m, 1H, CHCH2), 3.95 (m, 1H,
CHСН2), 4.39 (m, 1H, СНCH2), 6.67 (m, 2H, arom.), 6.89 (d, 1H, J = 8.0 Hz, arom.), 7.30 (m, 2H,
arom.), 7.48 (d, 1H, J = 8.0 Hz, arom.), 8.02-8.12 (m, 2H, arom.), 9.31, 9.33 (s, 1Н, ОН), 10.87, 11.40
(brs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 196.5, 189.4, 176.8, 166.4, 164.4, 154.7, 144.3,
132.6, 131.3 (d, J =9.8 Hz), 130.6, 122.2, 115.9 (d, J = 22 Hz), 115.8, 115.4, 49.2, 42.5. LCMS (ESI)
m/z 345 (100.0%, (M+H)+). Calcd. for C17H13FN2O3S: C, 59.29; H, 3.81; N, 8.13; Found: C, 59.10; H,
3.90, N, 8.00%.
4.1.2.3. N-(4-Bromophenyl)-2-[2-(3-hydroxyphenylamino)-4-oxo-4,5-dihydrothiazol-5-yl]acetamide (3).
о
1
Yield 61%, mp 210-211 С. H NMR (400 MHz, DMSO-d6): δ 2.65-2.75 (m, 1H, CHCH2), 3.30-3.40
(m, 1H, CHCH2), 4.30-4.38 (m, 1H, CHCH2), 6.96-7.02 (m, 2H, arom.), 7.06 (t, 1H, J = 8.1 Hz, arom.),
7.22 (brs, 2H, arom.), 7.42 (brs, 1H, arom.), 7.45-7.50 (m, 2H, arom.), 10.80, 11.50 (brs, 1H, NH), 9.06,
9.18 (s, 1Н, ОН), 9.90, 9.92 (s, 1H, NH)..13C NMR (100 MHz, DMSO-d6): δ 186.7, 173.5, 168.1, 158.1,
154.0, 149.3, 138.1, 131.7, 130.0, 121.1, 116.7, 111.7, 107.8, 44.3, 43.4. LCMS (ESI) m/z 420 (95.9%,
(M+H)+). Calcd. for C17H14BrN3O3S: C, 48.58; H, 3.36;N, 10.00; Found: C, 48.45; H, 3.40; N, 10.20%.
4.1.2.4. N-(4-Bromophenyl)-2-[2-(4-sulfamoylphenylamino)-4-oxo-4,5-dihydrothiazol-5-yl]acetamide
о
1
(4).Yield 80%, mp 222-224 С. HNMR (400 MHz, DMSO-d6): δ 3.54 (m, 1H, CHCH2), 3.90 (m, 1H,
CHСН2), 4.36 (m, 1H, СНCH2), 6.68 (m, 2H, arom.), 6.95 (d, 1H, J = 8.1 Hz, arom.), 7.28 (m, 2H,
arom.), 7.52 (d, 1H, J = 8.0 Hz, arom.), 7.62 (s, 2H, NH2), 8.07-8.14 (m, 2H, arom.), 10.92, 11.38 (brs,
13
1H, NH). C NMR (100 MHz, DMSO-d6): δ 186.7, 165.7, 158.3, 144.3, 139.7, 131.7, 127.2, 121.5,
121.0, 119.2, 115.0, 42.1, 43.7. LCMS (ESI) m/z 483/485 (100.0%, (M+H)+). Calcd. for
C17H15BrN4O4S2: C, 42.24; H, 3.13; N, 11.59; Found: C, 42.30; H, 3.30; N, 11.70%.
4.1.2.5. 2-{3-Furan-2-ylmethyl-2-[(2-methyl-3-phenylallylidene)hydrazono]-thiazolidin-4-one-5-yl}-N-
(3-trifluoromethylphenyl)-acetamide (5). Yield 68%, mp 176-177 оС. 1H NMR (400 MHz, DMSO-d6): δ
2.11 (s, 3H, CH3), 3.03 (dd, 1H, J = 9.0, 16.8 Hz, CHCH2), 3.27-3.33 (m, 1H, CHCH2), 4.56 (dd, 1H, J
= 3.6, 9.2 Hz, CHCH2), 4.93 (s, 2H, CH2), 6.43 (m, 2H, arom.), 7.04 (brs, 1H, arom.), 7.31 (m, 1H,
arom.), 7.41-7.48 (m, 5H, arom.), 7.56 (t, 1H, J = 7.9 Hz, arom.), 7.60 (s, 1H, arom.), 7.72 (d, 1H, J =
8.6 Hz, arom.), 8.07 (s, 1H, arom.), 8.25 (s, 1H, arom.), 10.52 (s, 1H, NH). 13C NMR (100 MHz,
12

ACCEPTED MANUSCRIPT
DMSO-d6): δ 173.6, 168.64, 163.0, 162.5, 148.9, 142.6, 139.5, 139.3, 137.5 (q, J = 121 Hz), 136.3,
134.3, 130.2, 129.6, 128.6, 128.1, 125.2, 122.7, 121.6, 110.7, 108.7, 42.5, 38.9, 34.8, 13.0. LCMS (ESI)
m/z 541 (100.0%, (M+H)+). Calcd. for C27H23F3N4O3S: C, 59.99; H, 4.29; N, 10.36; Found: C, 60.10; H,
4.10; N, 10.50%.
4.1.2.6. 3-Furan-2-ylmethyl-2-(pyridin-3-ylmethylenehydrazono)-thiazolidin-4-one (6). Yield 80%, mp
о
1
156-158 С. H NMR (400 MHz, DMSO-d6): δ 3.96 (s, 2H, CH2), 4.92 (s, 2H, CH2), 6.37 (d, 2H, J =
12.1 Hz, arom.), 7.41-7.44 (m, 1H, arom.), 7.47 (s, 1H, arom.), 8.14 (d, 1H, J = 7.9 Hz, arom.), 8.51 (s,
13
1H, arom.), 8.58 (m, 1H, arom.), 8.87 (s, 1H, CHN). C NMR (100 MHz, DMSO-d6): δ 186.6, 171.9,
164.8, 155.6, 151.5, 148.8, 142.7, 134.2, 129.9, 124.2, 110.8, 109.0, 50.3, 32.2. LCMS (ESI) m/z 301
(98.6%, (M+H)+). Calcd. for C14H12N4O2S: C, 55.99; H, 4.03; N, 18.65; Found: C, 55.80; H, 4.10; N,
18.80%.
4.1.2.7. 5-Ethyl-3-furan-2-ylmethyl-2-(pyridin-3-ylmethylenehydrazono)-thiazolidin-4-one (7). Yield
79%, mp 175-177 оС. 1H NMR (400 MHz, DMSO-d6): δ 1.00 (t, 3H, J = 7.0 Hz, CH3), 1.90 (dd, 1H, J =
13.6, 6.3 Hz, CHCH2), 2.01 (dd, 1H, J= 13.4, 7.3 Hz, CHCH2), 4.27 (m, 1H, CHCH2), 4.93 (s, 2H,
CH2), 6.35 (s, 2H, arom.), 7.44 (m, 2H, arom.), 8.15 (d, 1H, J = 7.6 Hz, arom.), 8.51 (s, 1H, arom.), 8.59
(s, 1H, arom.), 8.88 (s, 1H, arom.). 13C NMR (100 MHz, DMSO-d6): δ 185.3, 172.3, 163.4, 155.0,
152.4, 148.0, 145.2, 133.0, 129.8, 124.6, 113.7, 109.2, 48.6, 32.2, 25.4, 13.0. LCMS (ESI) m/z 329
(97.0%, (M+H)+). Calcd. for C16H16N4O2S: C, 58.52; H, 4.91; N, 17.06; Found: C, 58.70; H, 5.00; N,
16.90%.
4.1.2.8. 2-(N'-Pyridin-3-ylmethylenehydrazono)thiazolidin-4-one (8). Yield 68%, mp >250 оС. 1H NMR
(400 MHz, DMSO-d6): δ 3.81 (s, 3H, CH2), 7.40 (s, 1H, arom.), 8.10 (s, 1H, arom.), 8.41 (s, 1H, arom.),
8.57 (s, 1H, arom.), 8.86 (s, 1H, arom.), 11.88 (brs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.5,
153.9, 153.8, 151.3, 149.2, 134.1, 130.1, 124.1, 33.1.LCMS (ESI) m/z 221 (100.0%, (M+H)+). Calcd.
for C9H8N4OS: C, 49.08; H, 3.66; N, 25.44; Found: C, 47.90; H, 3.80; N, 25.20%.
4.1.2.9. 5-Methyl-2-(N'-pyridin-3-ylmethylenhydrazono)thiazolidin-4-one (9). Yield 59%, mp 241-243
оС.1H NMR (400 MHz, DMSO-d6): δ 1.56 (d, 3H, J = 7.1 Hz, CH3), 4.10 (q, 1H, J = 7.1 Hz, CH), 7.42
(m, 1H, arom.), 8.11 (d, 1H, J = 7.3 Hz, arom.), 8.41 (s, 1H, arom.), 8.57 (m, 1H, arom.), 8.84 (s, 1H,
arom.), 11.93 (brs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.7, 157.3, 153.8, 151.3, 149.2,
134.1, 129.6, 124.1, 33.3, 18.8. LCMS (ESI) m/z 235 (98.4%, (M+H)+). Calcd. for C10H10N4OS: C,
51.27; H, 4.30; N, 23.91; Found: C, 57.40; H, 4.40; N, 24.00%.
13

ACCEPTED MANUSCRIPT
4.1.2.10. 5-Ethyl-2-(N'-pyridin-3-ylmethylenehydrazono)thiazolidin-4-one (10). Yield 61%, mp 192-194
1
оС. H NMR (400 MHz, DMSO-d6): δ 1.04 (t, 3H, J = 7.0 Hz, CH3), 1.87 (m, 1H, CHCH2), 2.04 (m,
1H, CHCH2), 4.10 (m, 1H, CHCH2), 7.43 (m, 1H, arom.), 8.14 (d, 1H, J = 7.9 Hz, arom.), 8.47 (s, 1H,
arom.), 8.57 (m, 1H, arom.), 8.86 (s, 1H, arom.), 11.93 (brs, 1H, NH). 13C NMR (100 MHz, DMSO-d6):
δ 176.3 154.03, 151.1, 149.1, 144.3, 134.3, 130.2, 124.2, 49.4, 25.5, 10.4. LCMS (ESI+) m/z 249
(98.0%, (M+H)+). Calcd. for C11H12N4OS: C, 53.21; H, 4.87; N, 22.56; Found: C, 53.30; H, 5.00; N,
22.60%.
4.1.2.11. 5-(2-Hydroxyethyl)-2-(N'-pyridin-3-ylmethylenehydrazono)thiazolidin-4-one (11). Yield 68%,
о
1
mp 249-251 С. H NMR (400 MHz, DMSO-d6): δ 1.85 (m, 1H, CH2), 2.29 (m, 1H, aliph.), 3.57 (m,
1H, aliph.), 3.65 (m, 1H, aliph.), 4.14 (m, 1H, aliph.), 4.62 (brs, 1H, OH), 7.42 (t, 1H, J = 6.6 Hz,
arom.), 8.13 (d, 1H, J = 7.9 Hz, arom.), 8.41 (s, 1H, CH), 8.84 (s, 1H, arom.), 11.90 (brs, 1H, NH). 13C
NMR (100 MHz, DMSO-d6): δ 185.3, 165.1, 153.9, 151.3, 149.3, 134.1, 130.2, 124.1, 58.5, 45.5, 35.8.
LCMS (ESI) m/z 265 (100.0%, (M+H)+). Calcd. for C11H12N4O2S: C, 49.99; H, 4.58; N, 21.20; Found:
C, 50.10; H, 4.50; N, 21.10%.
4.1.2.12. N-(2-Methoxyphenyl)-2-[5-(4-oxothiazolidin-2-ylideneamino)-[1,3,4]thiadiazol-2-ylsulfanyl]-
acetamide (12). Yield 73%, mp 180-181 оС. 1H NMR (400 MHz, DMSO-d6): δ 3.84 (s, 3H, CH3), 4.11
(s, 2H, CH2), 4.30 (s, 2H, CH2), 6.89-6.93 (m, 1H, arom.), 7.04-7.09 (m, 2H, arom.), 7.99 (d, 1H, J =
8.1 Hz, arom.), 9.64 (s, 1H, NH), 12.35 (brs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.7, 165.8,
163.2, 149.3, 146.4, 138.6, 127.1, 124.7, 121.3, 120.4, 111.3, 55.8, 37.6, 35.8. LCMS (ESI) m/z 396
(95.4%, (M+H)+). Calcd. for C14H13N5O3S3: C, 42.52; H, 3.31; N, 17.71; Found: C, 42.60; H, 3.20; N,
17.90%.
4.1.2.13. 2-[5-(4-Methoxyphenyl)-[1,3,4]oxadiazol-2-ylimino]thiazolidin-4-one (13). Yield 72%, mp
о
1
261-263 С. H NMR (400 MHz, DMSO-d6): δ 3.86 (s, 3H, CH3), 4.12 (s, 2H, CH2), 7.11 (d, 2H, J =
8.2 Hz, arom.), 7.88 (d, 2H, J = 7.9 Hz, arom.), 12.2 (brs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ
186.8, 174.4, 169.8, 164.1, 161.8, 127.9, 116.2, 114.9, 55.6, 36.1. LCMS (ESI) m/z 291 (100.0%,
(M+H)+). Calcd. for C12H10N4O3S: C, 49.65; H, 3.47; N, 19.30; Found: C, 49.80; H, 3.60; N, 19.10%.
4.1.2.14.
N-(4-Chlorophenyl)-2-[5-(4-oxothiazolidin-2-ylideneamino)[1,3,4]thiadiazol-2-ylsulfanyl]-
acetamide (14). Yield 69%, mp 239-241 оС. 1H NMR (400 MHz, DMSO-d6): δ 4.04 (s, 2H. CH2), 4.23
(s, 2H, CH2), 7.13 (d, 2H, J = 8.2 Hz, arom.), 7.47 (d, 2H, J = 8.2 Hz, arom.), 10.28 (s, 1H, NH), 12.25
14

ACCEPTED MANUSCRIPT
(s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.8, 186.7, 165.6, 162.4, 150.7, 137.8, 128.9, 127.3,
120.8, 35.8, 30.9. LCMS (ESI) m/z 400/402 (100.0%, (M+H)+). Calcd. for C13H10ClN5O2S3: C, 39.05;
H, 2.52; N, 17.51;Found: C, 38.90; H, 2.40; N, 17.60%.
4.1.2.15. 2-(6-Methylbenzothiazol-2-ylimino)thiazolidin-4-one (16). Spectral and analytical data are
described [43].
4.1.3. General procedure for the preparation of 5-ene-2-R-amino(imino)thiazol-4(5H)-ones (17-33)
Method 1 (compounds 23-27, 30, 33). A mixture of appropriate 5-unsubstituted thiazolidinone
(compound 12-16) (3 mmol), appropriate oxocompond (4 mmol) and anhydrous sodium acetate (3
mmol) was refluxed for 3 h in glacial acetic acid (30 mL). Powder obtained after cooling was filtered
off, washed with methanol and recrystallized with DMF : ethanol (1:2).
Method 2 (compounds 17-22, 28, 29, 31, 32). A mixture of the appropriate thiourea derivative (10
mmol), chloroacetic acid (10 mmol) and appropriate oxocompound (10 mmol), and sodium acetate (10
mmol) in 100 mL of acetic acid was heated under reflux for 3 h. Crystalline precipitate was filtered off,
washed with acetic acid, water, ethanol and diethyl ether and then recrystallized from a mixture DMF :
acetic acid (1:2) or acetic acid.
4.1.3.1. (Z)-5-(4-Chlorobenzylidene)-2-(4-hydroxyphenylamino)thiazol-4(5H)-one (17). Spectral and
analytical data are described [19].
4.1.3.2. (Z)-5-Benzylidene-2-(4-hydroxyphenylamino)thiazol-4(5H)-one (18). Spectral and analytical
data are described [65].
4.1.3.3. (Z)-5-(3-Phenylallylidene)-2-(4-hydroxyphenylamino)thiazol-4(5H)-one (19). Yield 78%, mp
о
1
276-278 С. H NMR (400 MHz, DMSO-d6): δ 6.78, 6.88, 7.58 (d, 4H, J = 7.6 Hz, 4-OH-C6H4), 6.80-
6.65, 7.05-7.16, 7.25-7.43, 7.47-7.63 (m, 8H, C6H5CH=CH-CH=), 9.48, 9.53 (s, 1H, OH), 11.34, 11.91
(s, 1H, NH).13C NMR (100 MHz, DMSO-d6): δ 179.9, 169.1, 162.4, 154.9, 141.6, 136.0, 129.3, 129.0,
127.7, 125.5, 124.1, 122.3, 116.0, 115.5. LCMS (ESI) m/z 323 (100.0%, (M+H)+). Calcd. for
C18H14N2O2S: C, 67.06; H, 4.38; N, 8.69; Found: C, 67.20; H, 4.30; N, 8.80%.
4.1.3.4.
(Z)-5-[2-Chloro-3-(4-nitrophenyl)-allylidene]-2-[(4-hydroxyphenyl)-methylamino]thiazol-
4(5H)-one (20).Spectral and analytical data are described [19].
15

ACCEPTED MANUSCRIPT
4.1.3.5. (Z)-5-(2-Methyl-3-phenylallylidene)-2-[(4-hydroxyphenyl)methylamino]-thiazol-4(5H)-one (21).
Spectral and analytical data are described [65].
4.1.3.6. (Z)-5-(3-Hydroxybenzylidene)-2-(4-hydroxyphenylamino)thiazol-4(5H)-one (22). Spectral and
analytical data are described [65].
4.1.3.7. (Z)-5-(3-Ethoxy-4-hydroxybenzylidene)-2-(6-methylbenzothiazol-2-ylamino)thiazol-4(5H)-one
(23).Yield 71%, mp 202-204 оС. 1H NMR (400 MHz, DMSO-d6): δ 1.42 (t, 3H, J = 6.9 Hz, CH3), 2.42
(s, 3H, CH3), 4.15 (q, 2H, J = 6.8 Hz, CH2), 6.96 (d, 1H, J = 8.2 Hz, arom.), 7.17 (d, 1H, J = 8.2 Hz,
13
arom.), 7.30 (m, 2H, arom.), 7.67-7.74 (m, 3H, arom.), 9.93 (s, 1H, OH), 12.73 (s, 1H, NH). C NMR
(100 MHz, DMSO-d6): δ 186.6, 167.5, 167.3, 158.6, 149.8, 148.9, 147.2, 134.1, 133.6, 127.9, 124.8,
124.6, 121.8, 121.2, 120.2, 116.4, 115.5, 64.0, 21.1, 14.8. LCMS (ESI) m/z 412 (98.3%, (M+H)+).
Calcd. for C20H17N3O3S2: C, 58.38; H, 4.16; N, 10.21; Found: C, 58.50; H, 4.10; N, 10.40%.
4.1.3.8. 5-(4-Chlorobenzylidene)-2-(thiazol-2-ylimino)-thiazolidin-4-one (24). Yield 68%, mp 256-258
о
1
оС, lit 257.5-258.5 С [66]. H NMR (400 MHz, DMSO-d6): δ 7.40 (d. 1H, J = 3.9 Hz, thiazol.), 7.55-
7.60 (m, 2H, arom.), 7.63 (brs, 1H, arom.), 7.64-7.66 (m, 2H, arom.), 7.68 (s, 1H, arom.), 12.63 (brs,
1H, NH). Calcd. for C13H8ClN3OS2: C, 48.52; H, 2.51; N, 13.06; Found: C, 48.40; H, 2.60; N, 12.90%.
4.1.3.9.
2-{5-[5(Z)-(4-Chlorobenzylidene)-4-oxothiazolidin-2-ylideneamino][1,3,4]thiadiazol-2-
о
1
ylsulfanyl}-N-(2-methoxyphenyl)acetamide (25). Yield 63%, mp 245-246 С. H NMR (400 MHz,
DMSO-d6): δ 3.86 (s, 3H, CH3), 4.33 (brs, 2H, CH2), 6.93 (d, 1H, J = 8.0 Hz, arom.), 7.05-7.11 (m, 2H,
arom.), 7.66 (brs, 4H. arom.), 7.79 (s, 1H, arom.), 8.00 (d, 1H, J = 7.5 Hz, arom.), 9.65 (s, 1H, arom.),
13.02 (brs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.8, 182.8, 163.2, 131.9, 129.6, 129.6,
124.7, 121.2, 120.4, 112.6, 112.6, 111.3, 55.8, 38.5. LCMS (ESI) m/z 518/520 (96.4%, (M+H)+). Calcd.
for C21H16ClN5O3S3: C, 48.96; H, 3.11; N, 13.52; Found: C, 49.10; H, 3.00; N, 13.40%.
4.1.3.10.
Z-5-(4-Dimethylaminobenzylidene)-2-[5-(4-methoxyphenyl)[1,3,4]oxadiazol-2-ylimino]-
о
1
thiazolidin-4-one (26). Yield 74%, mp 291-292 С. H NMR (400 MHz, DMSO-d6): δ 3.05 (brs, 6H,
2*CH3), 3.85 (s, 3H, CH3), 6.88 (brs, 2H, arom.), 7.12 (brs, 2H, arom.), 7.50 (brs, 2H, arom.), 7.66 (brs.
1H, arom.), 7.90 (brs, 2H, arom.). LCMS (ESI) m/z 412 (97.4%, (M+H)+). Calcd. for C21H19N5O3S: C,
59.84; H, 4.54; N, 16.62; Found: C, 60.00; H, 4.50; N,16.50%.
4.1.3.11.
N-(4-Chlorophenyl)-2-{5-[5(Z)-(4-methoxybenzylidene)-4-oxothiazolidin-2-
о
1
ylideneamino][1,3,4]thiadiazol-2-ylsulfanyl}-acetamide (27). Yield 59%, mp 238-239 С. H NMR
16

ACCEPTED MANUSCRIPT
(400 MHz, DMSO-d6): δ 3.84 (s, 3H, CH3), 4.10 (s, 2H, CH2), 7.15 (d, 2H, J = 8.3 Hz, arom.), 7.39 (d,
2H, J = 8.3 Hz, arom.), 7.60-7.64 (m, 4H, arom.), 7.73 (s, 1H, =CH-Ar), 10.54 (s, 1H, NH), 12.35 (s,
13
1H, NH). C NMR (100 MHz, DMSO-d6): δ 174.0, 170.2, 167.2, 165.6, 165.5, 161.2, 160.5, 158.7,
137.8, 133.2, 132.4, 128.9, 127.3, 120.8, 115.2, 55.6, 37.8. LCMS (ESI) m/z 516/518 (95.1%, (M-H)+).
Calcd. for C21H16ClN5O3S3: C, 48.69; H, 3.11; N, 13.52; Found: C, 48.60; H, 3.20; N, 13.70%.
4.1.3.12. 3-[2-(4-Hydroxyphenylamino)-4-oxo-4H-thiazol-5-ylidene]-1,3-dihydroindol-2-one (28). Yield
о
1
70%, mp >260 С. H NMR (400 MHz, DMSO-d6): δ 6.79, 6.82, 7.62 (d, 4Н, J = 8.3 Hz, arom), 6.89,
6.93 (d, 1H, J = 7.5 Hz, arom.), 6.95-7.07, 7.28-7.35 (m, 2Н, arom.), 8.89, 8.97 (d, 1H, J = 7.9 Hz,
arom.), 9.45 (s, 1H, ОН), 11.01, 11.04 (s, 1H, NН), 11.40, 12.10 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6): δ 186.7, 180.4, 172.7, 169.0, 155.2, 143.0, 136.9, 131.6, 130.2, 128.0, 124.9, 122.6, 120.6,
115.6, 110.2. LCMS (ESI) m/z 338 (95.6%, (M+H)+). Calcd. for C17H11N3O3S: C, 60.53; H, 3.29; N,
12.46; Found: C, 60.70; H, 3.40; N,12.60%.
4.1.3.13.
3-[2-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylimino)-4-oxothiazolidin-5-
ylidene]-1,3-dihydroindol-2-one (29). Yield 68%, mp >260 оС. 1H NMR (400 MHz, DMSO-d6): δ 2.41
(s, 3H, CH3), 3.20 (s, 3H, CH3), 6.88 (d, 2H, J = 8.4 Hz, arom.), 6.90-7.00 (m, 4H, arom.), 7.24-7.32 (m,
2H, arom.), 9.00 (d, 1H, J = 7.0 Hz, arom.), 10.90 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ
186.3, 180.1, 169.0, 168.4, 164.4, 153.8, 144.2, 134.3, 133.1, 129.4, 129.1, 128.1, 127.8, 127.4, 124.9,
122.2, 119.9, 110.7, 35.2, 10.5. LCMS (ESI) m/z 432 (96.2%, (M+H)+). Calcd. for C22H17N5O3S: C,
61.24; H, 3.97; N, 16.23; Found: C, 61.40; H, 3.80, N, 16.50%.
4.1.3.14. 5-Bromo-3-[4-oxo-2-(thiazol-2-ylimino)thiazolidin-5-ylidene]-1,3-dihydroindol-2-one (30).
о
Yield 71%, mp >250 С. 1H NMR (400 MHz, DMSO-d6): δ 6.88 (d, 1H, J = 8.1 Hz, arom.), 7.48-7.53
(m, 2H, arom.), 7.73 (brs, 1H, arom.), 9.00, 9.17 (s, 1H, arom.), 11.12, 11.21 (s, 1H, NH), 12.95 (brs,
13
1H, NH). C NMR (100 MHz, DMSO-d6): δ 168.3, 162.3, 142.6, 140.8, 136.5, 134.2, 130.2, 125.1,
122.1, 118.2, 113.5, 112.1, 35.9, 30.9. LCMS (ESI) m/z 407/409 (98.0%, (M+H)+). Calcd. for
C14H7BrN4O2S2: C, 41.29; H, 1.73; N, 13.76; Found: C, 41.10; H, 1.80. N, 13.90%.
4.1.3.15. 3-{2-[(4-Hydroxyphenyl)-methylamino]-4-oxo-4H-thiazol-5-ylidene}-1,3-dihydroindol-2-one
(31). Yield 70%, mp >300 оС. 1H NMR (400 MHz, DMSO-d6): δ 3.65 (s, 3H, CH3), 6.85 (d, 2H, J = 8.8
Hz, arom.), 7.00 (d, 2H, J = 8.8 Hz, arom.), 6.80-7.00 (m, 2H, arom.), 7.24-7.26 (m, 1H, arom.), 9.00 (d,
1H, J = 7.0 Hz, arom.), 9.65(s, 1H, OH), 10.80 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.6,
179.5, 179.0, 168.9, 158.5, 143.1, 138.9, 132.6, 131.6, 128.6, 128.1, 125.3, 121.9, 120.5, 116.4, 110.1,
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42.1. LCMS (ESI) m/z 352 (100.0%, (M+H)+). Calcd. for C18H13N3O3S: C, 61.53; H, 3.73; N, 11.96;
Found: C, 61.60; H, 3.60; N, 12.10%.
4.1.3.16.
3-{2-[(4-Hydroxyphenyl)-methylamino]-4-oxo-4H-thiazol-5-ylidene}-5-methyl-1,3-
dihydroindol-2-one (32). Yield 63%, mp >300 оС. 1H NMR (400 MHz, DMSO-d6): δ 2.30 (s, 3H, CH3),
3.60 (s, 3H, CH3), 6.91 (d, 2H, J = 8.2 Hz, arom.), 7.31 (d, 2H, J = 8.2 Hz, arom.), 6.75 (d, 1Н, J = 8.8
Hz, arom.), 7.05 (d, 1Н, J = 8.8 Hz, arom.), 8.80 (s, 1Н, arom.), 9.80 (s, 1H, OH), 10.65 (s, 1H, NH).
13C NMR (100 MHz, DMSO-d6): δ 186.7, 168.9, 158.4, 140.9, 138.6, 132.6, 132.1, 130.5, 128.7, 128.5,
125.4, 122.0, 120.5, 116.3, 109.8, 42.1, 21.1. LCMS (ESI) m/z 366 (99.0%, (M+H)+). Calcd. for
C19H15N3O3S: C, 62.45; H, 4.14; N, 11.50; Found: C, 62.60; H, 4.20; N, 11.30%.
4.1.3.17. 3-[3-Allyl-4-oxo-2-(thiazol-2-ylimino)thiazolidin-5-ylidene]-1,3-dihydroindol-2-one (33).
Yield 71%, mp >230 оС. 1H NMR (400 MHz, DMSO-d6): δ 4.98 (d, 2H, J = 4.8 Hz, CH2), 5.23 (q, 2H,
J = 10.3 Hz, CH), 5.81 (m, 1H, CH), 6.88 (d, 1H, J = 8.1 Hz, arom.), 7.48-7.53 (m, 2H, arom.), 7.62 (m,
1H, arom.), 7.73 (m, 1H, arom.), 8.92 (s, 1H, arom.), 11.12 (s, 1H, NH). 13C NMR (100 MHz, DMSO-
d6): δ 168.7, 168.4, 165.3, 156.8, 143.8, 140.9, 132.5, 132.2, 131.4, 128.2, 127.2, 122.0, 120.3, 118.6,
117.4, 110.4, 44.6. LCMS (ESI) m/z 369 (100.0%, (M+H)+). Calcd. for C17H12N4O2S2: C, 55.42; H,
3.28; N, 15.21; Found: C, 55.50; H, 3.40; N, 15.30%.
4.1.4. General procedure for synthesis of 5-aryliden-4-amino-1,3-thiazol-2(5H)-ones (34, 35)
A mixture of 4-thioxo-2-thiazolidinone (10 mmol) and appropriate amine (10 mmol) was refluxed
for 1 h in 25 mL of ethanol. After cooling to the room temperature formed precipitate (4-R-amino-
thiazol-2(5H)-one) was filtered off, washed with methanol and recrystallized with appropriate solvent.
A mixture of appropriate 4-R-aminothiazol-2(5H)-one (3 mmol), aldehyde (3.3 mmol), and
anhydrous sodium acetate (3 mmol) was refluxed for 3 h in glacial acetic acid (10 mL). Obtained
precipitate was filtered off, washed with acetic acid, water and methanol and recrystallized with DMF :
ethanol or DMF : acetic acid (1:2) mixtures.
4.1.4.1. (Z)-5-(Thiophen-2-ylmethylene)-4-(4-chlorophenylamino)thiazol-2(5H)-one (34). Spectral and
analytical data described [12].
4.1.4.2. (Z)-5-(4-Chlorobenzylidene)-4-(4-hydroxy-5-isopropyl-2-methylphenylamino)thiazol-2(5H)-one
(35). Yield 71%, mp 242-245оС. 1H NMR (400 MHz, DMSO-d6): δ 1.20 (d, 6Н, J = 7.0 Hz, 2*СН3),
2.08 (s, 3Н, СН3), 3.18 (m, 1Н, СН), 6.71 (s, 1H, arom.), 7.01 (s, 1H, arom.), 7.59 (d, 2H, J = 8.7 Hz,
13
arom.), 7.62 (d, 2H, J = 8.7 Hz, arom.), 8.02 (s, 1Н, CH=), 9.45 (s, 1H, OH), 10.67 (s, 1H, NH). C
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NMR (100 MHz, DMSO-d6): δ 176.9, 172.9, 153.8, 134.5, 133.2, 132.4, 132.2, 131.3, 129.8, 129.5,
127.9, 126.7, 124.3, 116.6, 26.3, 22.6, 17.6. LCMS (ESI) m/z 387/389 (99.8%, (M+H)+). Calcd. for
C20H19ClN2O2S: C, 62.09; H, 4.95; N, 7.24; Found: C, 62.20; H, 4.80; N, 7.30%.
4.1.5. Crystal structure determination
4.1.5.1.
ylimino]thiazolidin-4-one (13).
Crystal data: C12H10N4O3S, Mr = 290.30, monoclinic, space group P21/m, a = 7.23203(19), b =
Crystal
structure
determination
of
2-[5-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-
6.35516(16), c = 13.4288(3) Å,
mm-1, T = 130(2) K.
β
= 104.275(3)º, V = 598.14(3) Å3, Z = 2, Dcalc = 1.612 g/cm3, ꢀ = 0.285
Data collection: A yellow lath crystal (DMF) of 0.23×0.13×0.03 mm was used to record 5141
(MoKα-radiation, θmax= 29.10°) intensities on an Xcalibur Atlas diffractometer (Agilent (2011).
CrysAlis PRO. Oxford Diffraction Ltd, Yarnton, England). Intensity data collection employed the
ω
−scans mode with Enhance (Mo) X-ray Source. The data were corrected for Lorentz and polarization
effects. Multi-scan absorption correction has been applied too. The minimum and maximum
transmissions were 0.9844 and 1.0000. Data reduction and analysis were carried out with the Oxford
Diffraction programs (Agilent (2011). CrysAlis PRO. Oxford Diffraction Ltd, Yarnton, England). The
1567 total unique reflections (R(int) = 0.019) were used for further calculations. H atom bonded to N
atom was located from difference map and refined freely. All H atoms of CH groups were placed in
calculated positions. As the molecule lies on the mirror plane in the crystal, symmetry-independent
methylene and methyl H atoms were refined with the atomic displacement parameters fixed at 1.2Ueq(C)
and 1.5Ueq(C), respectively. The remaining H atoms were refined as riding on their parent C atoms, with
C–H = 0.93 Å and Uiso(H) = 1.2Ueq(C).
Structure solution and refinement. The structure was solved by the direct methods using the
program SHELXS-97 [67], and refinement was done against F2 for all data using SHELXL-97 [67]. The
final refinement converged with R = 0.0302 (for 1352 data with F2> 4σ(F2)), wR = 0.0852 (on F2 for all
data), and S = 1.039 (on F2 for all data). The largest difference peak and hole were 0.367 and -0.232 eÅ-
3. The molecular illustrations were drawn using ORTEP-3 for Windows [68]. Software used to prepare
material for publication was WINGX [68], OLEX [69] and PLATON [70].
The supplementary crystallographic data have been deposited at the Cambridge Crystallographic
Data Centre (CCDC), 12 Union ROAD, Cambridge CB2 1EZ (UK), Tel.: (+44) 1223/336-408, Fax:
(+44) 1223/336-033, E-mail: deposit@ccdc.cam.ac.uk, http://www.ccdc.cam.ac.uk (deposition No.
CCDC 992833).
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4.1.5.2. Crystal structure determination of (Z)-5-(4-chlorobenzylidene)-4-(4-hydroxy-5-isopropyl-2-
methylphenylamino)-5H-thiazol-2-one methanol semisolvate (35).
Crystal data: C20H19ClN2O2S, ½(CH4O), Mr = 402.90, monoclinic, space group C2/c, a =
20.6265(18), b = 13.6576(6), c = 17.8624(16) Å,
1.328 g/cm3, = 0.314 mm-1, T = 130(2) K.
β =
= 126.789(14)°, V = 4029.8(5) Å3, Z = 8, Dcalc
µ
Data collection: A yellow block crystal (0.55 x 0.43 x 0.30 mm) was used to record 9665 (MoKα
radiation, θmax = 29.09º) intensities on an Agilent Xcalibur Atlas diffractometer (Agilent (2011).
CrysAlis PRO. Oxford Diffraction Ltd, Yarnton, England)). The data collection routine, unit cell
refinement, and data processing were carried out with the program CrysAlisPro (Agilent (2011).
CrysAlis PRO. Oxford Diffraction Ltd, Yarnton, England). The intensities were collected in the
ω scan
mode with Enhance (Mo) X-ray Source. The data were corrected for Lorentz, polarization effects.
Multi-scan absorption corrections have been applied too. The minimum and maximum transmissions
were 0.9611 and 1.0000. The 4678 unique reflections (R(int) = 0.016) were used for further calculations.
The position of the H atom bonded to N and O atoms in solute molecule were obtained from difference
Fourier map and was refined freely. The remaining H atoms were positioned geometrically and were
refined within the riding model approximation: Cmethyl−H = 0.96 Å, Cmethine−H = 0.98 Å, C(sp2)−H =
0.93 Å and Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C) for methyl H atoms. The methyl groups were refined as
rigid groups, which were allowed to rotate.
Structure solution and refinement: The structure was solved by the direct methods using the
program SHELXS-97 [67], and refinement was done against F2 for all data using SHELXL-97 [67]. The
final refinement converged with R = 0.0303 (for 3939 data with F2> 4σ(F2)), wR = 0.0840 (on F2 for all
data), and S = 1.092 (on F2 for all data). The largest difference peak and hole were 0.339 and -0.224 eÅ-
3. The molecular illustration was drawn using ORTEP-3 for Windows [68]. Software used to prepare
material for publication was WINGX [68], OLEX2 [69] and PLATON [70].
The supplementary crystallographic data have been deposited at the Cambridge Crystallographic
Data Centre (CCDC), 12 Union ROAD, Cambridge CB2 1EZ (UK), Tel.: (+44) 1223/336-408, Fax:
(+44) 1223/336-033, E-mail: deposit@ccdc.cam.ac.uk, World Wide Web: http://www.ccdc.cam.ac.uk
(deposition No. CCDC 992832).
4.2. Pharmacology
4.2.1. Cell culture
Human fetal lung fibroblast (HFL-1) cells were obtained from the American Type Culture
Collection (Rockville, MD). The cells were cultured in tissue culture flasks (Falcon; Becton–Dickinson
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Labware, Lincoln Park, NJ) with DMEM supplemented with 10% FCS, 100 mg/mL penicillin and 250
ꢀg/mL streptomycin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and
passaged every 4–5 days at a 1:4 ratio. HFL-1 cells were used between the 14th and 18th passages.
4.2.2. Sulphorhodamine B assay
SRB assay was performed according to Jacobs et al. [71] in 96-well plates in triplicate after 48 h
treatment. In brief, cells were seeded at a density of 8000 cells/well, allowed to attach overnight, and
treated with the various concentrations of compound. Afterwards, 100ꢀL of 20% trichloroacetic acid
was added to each well and refrigerated at 4 ºC for 3 h, then the supernatant was discarded and the plate
was washed for 5 times with water and air dried. 100 µL of SRB solution 0.4% (w/v) in 1% acetic acid
was added to each well and incubated for 30 min at room temperature. Unbound SRB was removed and
the plates were air dried. Bound SRB was solubilized with 150 µL of 10 mM Tris-HCl and then the
plates were shaken for 5 min. The optical density at 570 nm wavelength was measured and the ratio of
cell proliferation to control group was calculated. IC50 and Emax values for inhibition of cell viability
were calculated by 3 parameter curve fitting using Prism 5 for Windows (GraphPad Software Inc.).
4.2.3. Superoxide scavenging
The ability of the compounds to scavenge superoxide radicals was determined in a system
consisting of xanthine (1 mM), xanthine oxidase (0.11 U/mL) and nitro blue tetrazolium (50 µM). Test
compounds (dissolved in DMSO) were added at concentrations up to 10 µM. The DMSO concentration
was kept constant at 0.1%. The NBT reduction was monitored spectrophotometrically for 3 min at 560
nm, the rate (dA560/dt) was determined from the linear part of the absorption vs. time plot. IC50 and
Emax values for inhibition of NBT reduction were calculated by 3 parameter curve fitting as described
above.
4.2.4. xCelligence
The effect of the test compounds on cell proliferation was determined by real time monitoring
using the xCelligence system (Roche GmbH, Darmstadt) as described by Ke et al. [72]. In short, HLF-1
were inoculated in the 96 well plate at a density of 20,000 cells per well. Cell attachment was maximal
after approximately 30 hours. Test compounds or the vehicle (DMSO) were then added to the cells and
proliferation was monitored for another 150 h. The difference in signal between t = 30 h and t = 180 h
was calculated and represents the 150 hour proliferation (P150). The maximum rate of proliferation Pdot
was calculated by determining the maximum slope of the curve.
4.2.5. Anticancer activity screening
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A primary anticancer assay was performed for three and an approximately 60 human tumor cell
line panels derived from nine neoplastic diseases, in accordance with the protocol of the Drug
Evaluation Branch, National Cancer Institute, Bethesda, MD [53-55]. Tested compounds were added to
the culture at a single concentration (10-4 and 10−5M), and the cultures were incubated for 48 h. End
point determinations were made with a protein binding SRB. The results for each tested compound were
reported as the growth percentage of the treated cells when compared to that of the untreated control
cells. The percentage growth was evaluated spectrophotometrically versus controls not treated with test
agents. The cytotoxic and/or growth inhibitory effects of the most active selected compounds were
tested in vitro against the full panel of about 60 human tumor cell lines at 10-fold dilutions of five
concentrations ranging from 10−4 to 10−8M. A 48 h continuous drug exposure protocol was followed,
and an SRB protein assay was used to estimate cell viability or growth. By use of the seven absorbance
measurements [time zero, (Tz), control growth in the absence of drug (C), and test growth in the
presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at each of
the drug concentrations levels. Percentage growth inhibition was calculated as:
[(Ti − Tz) / (C − Tz)] × 100 for concentrations for which Ti ≥ Tz;
[(Ti − Tz ) / Tz] × 100 for concentrations for which Ti < Tz.
Three dose response parameters (GI50, TGI, LC50) were calculated for each compound. Growth
inhibition of 50% (GI50) was calculated from [(Ti − Tz) / (C − Tz)] × 100 = 50, which was the drug
concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB
staining) compared to the net protein increase seen in the control cells. The drug concentration resulting
in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of drug resulting
in a 50% reduction in the measured protein at the end of the drug treatment compared to that at the
beginning) indicating a net loss of cells following treatment was calculated from [(Ti − Tz) / Tz] × 100 =
−50. Values were calculated for each of these three parameters if the level of activity was reached;
however, if the effect was not reached or was exceeded, the value for that parameter was expressed as
more or less for the maximum or minimum concentration tested. The lowest values are obtained with
the most sensitive cell lies. The compounds having GI50≤100 ꢀM were considered active
(https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm) [53-55].
5. Acknowledgement
The authors support all people of good will currently struggling for liberty and justice in Ukraine.
We are grateful to Dr. V.L. Narayanan from Drug Synthesis and Chemistry Branch, National Cancer
Institute, Bethesda, MD, USA, for in vitro evaluation of anticancer activity.
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