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thiazole based hybrids – New class of antitrypanosomal agents
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
Reference:
EJMECH 11287
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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
THIAZOLIDINONE/THIAZOLE BASED HYBRIDS – NEW CLASS OF ANTITRYPANOSOMAL AGENTS Anna Kryshchyshyna, Danylo Kaminskyya, Oleksandr Karpenkob,
O
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Andrzej Gzellac, Phillipe Grellierd, Roman Lesyka,e
R
R N
N
R
S
S
R
N
SC
N
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NH
O H3C
NH
N
EP
N H
Trypanosoma brucei brucei IC50 = 30 nM Trypanosoma brucei gambience IC50 = 110 nM
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N
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S
S
N
N
R
N
N
R
N
R
N N R
N S
ACCEPTED MANUSCRIPT THIAZOLIDINONE/THIAZOLE BASED HYBRIDS – NEW CLASS OF ANTITRYPANOSOMAL AGENTS Anna Kryshchyshyna, Danylo Kaminskyya, Oleksandr Karpenkob,
a
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Andrzej Gzellac, Phillipe Grellierd, Roman Lesyka,e
Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National
Medical University, Pekarska 69, Lviv, 79010, Ukraine Enamine Ltd., Chervonotkatska 78, Kyiv, 02094, Ukraine
c
Department of Organic Chemistry, Karol Marcinkovski Poznan University of Medical
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b
Sciences,Grunwaldzka 6, Poznan, 60-780, Poland d
National Museum of Natural History, UMR 7245 CNRS-MNHN, Team BAMEE, CP 52, 57 Rue Cuvier,
75005, Paris, France e
Department of Public Health, Dietetics and Lifestyle Disorders, Faculty of Medicine, University of
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Information Technology and Management in Rzeszow, Sucharskiego 2, 35-225 Rzeszow, Poland
1
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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
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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
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antitrypanosomal activity towards Trypanosoma brucei brucei and Trypanosoma brucei gambiense. Biological study allowed identifying compounds with the submicromolar levels of IC50, good selectivity
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indexes and relatively low cytotoxicity upon human primary fibroblasts as well as low acute toxicity.
KEYWORDS: thiazoles, thiazolidinones, indoles, imadazothiadiazoles, hybrids, antitrypanosomal
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activity
2
ACCEPTED MANUSCRIPT 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
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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].
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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
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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
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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].
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Quite a lot of different chemical classes of compounds, e.g. thiosemicarbazones, thiazolidines,
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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 [1315]. 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 kinase3
ACCEPTED MANUSCRIPT inhibitors library against Tb allowed identifying a series of active compounds based on 2,4diaminothiazoles 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 reported
to
inhibit
the
activity
of
Tb
dolicholphosphate
mannose
synthase
and
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were
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
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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].
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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
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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
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of computer chemistry too [36], e.g. 2-hydrazolyl-4-thiazolidinones are potent cruzipain inhibitors
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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
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for neglected diseases initiative) one of the new chemical entities to enter clinical development and in 2016 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].
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Screening of over 300000 compounds in ornithinedecarboxylase inhibition high throughput assay discovered four scaffold classes, among which were benzthiazoles and indole derivatives [46]. The
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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 1Hindole 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
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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
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agents [49]. Considering the phthalimide fragment as indole bioisoster, molecules bearing it should also
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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
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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
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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-
Known lead-compounds S R
S
R
N H
N H
R
N H
N H
R
N
O Ar
Ar
N
2
Ar
S
R
R
O
[Ref 25]
N
R
N N
R S
OH B O
R
2
R
B O
1
Me
Me [Ref 45] O O2N
Target compound srtucture
N R
1
N R
[Ref 47]
R R
R
N S
R
R
S
R N
N R
N
N
2
R N
R
N
Me
OH
O
S
N
Cl
H N
Me
O
N H
[Ref 36] F
O
R
N
N
Me SCYX-7158 [Ref 43]
[Ref 26]
O
Cl H N
N
S
AC C
Ar
H N
CF3 O
EP
S 2
HO
H N
N
R
1
Known lead-compounds
N N
N R
S
R
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R
O
R
R
S
H N
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imidazothiadiazole fragment linked by hydrazone moiety (Fig. 1).
N
S
R
N
N
R R
N N R S
N H
N
6
N N R
S
N
ACCEPTED MANUSCRIPT Figure 1. Background of the target compounds design.
RESULTS AND DISCUSSION
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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 reaction of thiosemicarbazones and different [C2]2+ electrophilic synthons (maleimides, maleic anhydride, β-aroylacrylic acids) [16,58]. The reactions were performed in glacial acetic acid medium. For compound
SC
1d, the [C2]2+ reactant was prepared in situ via the reaction of maleic anhydride and appropriate amino
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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
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well as by some aspects of the SAR-analysis of different hydrazono-thiazolidinones [7,33,35].
7
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1
O
R N
N N
S O
S [C2]2+
+
1a-1j
i
HN N 3
R
2
R
1e 1f 1g 1h 1i 1j
4-F-C6H4 4-MeOOC-C6H4 4-NO2-C6H4 4-Br-C6H4 4-Cl-C6H4 4-Me-C6H4
3
R
HN
1
R
O N
N N
S R
O
1k 1l 1m 1n 1o 1p 1q 1r
2
R 3
R
[C2]2+ = O O ;
N R ; O
OH
O O O
O
O
OH
;
+
R
H2N
A
O
B
R2 R3 H furan-2-yl H PhCHCH H 4-Me2N-C6H4 H PhCHCH H Ph H 3-MeO-4-HOOCCH2O-C6H3 H 4-NO2-C6H4CHCCl H 2-NO2-C6H4CHCH R2+ R3 A B
H H Me
Me
C
Me
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O
O
1s 4-F-C6H4 1t 4-F-C6H4
R3 PhCHCH PhCHCH PhCHCH fyran-2-yl
R2+ R3 A A A B B B
H H H H H H
R1 H H allyl H allyl 4-HO-C6H4 4-HO-C6H4 H
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1k-1t
R C C 4-F-C6H4 4-F-C6H4 4-F-C6H4 4-F-C6H4 HO HO
R2 H H H H
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1
R
2
R
R R1 4-F-C6H4 4-OH-C6H4 4-NO2-C6H4 H 3-CF3-C6H4 furfan-2-yl HOOC(CH2)3 H
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HN R
1a 1b 1c 1d
Scheme 1. Synthesis of 4-thiazolidinone-hydrazones. Reagents and conditions: i – substituted thiosemicarbazone (1.0 equiv), appropriate [C2]2+ compound (1.0
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equiv), AcOH, reflux, 3 h; for compound 1d – amino acid (1.0 equiv), maleic anhydride (1.0 equiv),
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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
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ACCEPTED MANUSCRIPT
a
b
hydrogen bonding in the crystal structure of 1fx1.25 H2O.
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Figure 2. (a) ORTEP view of the molecule 1f and H2O showing the atomic labelling scheme. (b) The
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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).
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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
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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
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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
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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
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synthesized according to the known protocols: 1) indole bearing thiosemicarbazones – via Fischer indole synthesis, Vilsmeier-Haack reaction and condensation with thiosemicarbazides; 2) imidazothiadiazole
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bearing compounds were synthesized in the three step synthetic procedure from 5-substituted 2-amino-
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1,3,4-thiadiazole and α-halogenoarylketone [62] (Scheme 2).
10
ACCEPTED MANUSCRIPT O
H ii R
R
H N
+ NH2
R
S
R
R
Me
1
S
2
N H
A
O
N N H2N
N H
O
+
N N H
H
X
=
R
N H 4
R
Hal
O
1
N
S
2a 2b 2c 2d 2e
N H
R H H H Br NO2
N R
R
S
R1 3a 3b 3c 3d 3e
S-CH2CH=CH2 S-CH2COOEt Et Et Et
N H R H H H Br NO2
3 R1
S-CH2CH=CH2 S-CH2COOEt Et Et Et
N
R
Synthesis
1
A5: A6: A7: A8: A9:
of
the
starting
N
R N H R = H R1 = S-CH2CH=CH2 R = H R1 = S-CH2COOEt R = H R1 = Et R = NO2 R1 = Et R = Br R1 = Et
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R
S
phenyl-indole
or
phenyl-imidazothiadiazole
bearing
AC C
thiosemicarbazones.
N H R=H R = Cl R = Br R = NO2
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A1: A2: A3: A4:
2.
ii
R
2
A=
Scheme
N
1
R2 H H 4-HO-C6H4 furfuryl H H 4-HO-C6H4 H 4-HO-C6H4 H H 4-HO-C6H4 H 4-HO-C6H4 H 4-HO-C6H4
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R
i
SC
H N
A A1 A2 A2 A2 A3 A4 A4 A5 A5 A6 A7 A7 A8 A8 A9 A9
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N H
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p
Reagents and conditions: i –mixture of DMF and POCl3 (1/3 vol), stirring 10 min at 0ºС, imidazo[2,1b][1,3,4]thiadiazole, 30 min at 0ºС, 2 h at rt and 2 h at 60ºС, Na2CO3 (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). 11
ACCEPTED MANUSCRIPT
2
OH
1
O
R
O
Cl
N 2
R
N
S
N S
1
R
N
A
N N H
R
X
=
H i
N H 2
R
O O
1
2
43 44 45 46 47
N
R
S
N H
43-47
R2 H Me Et H Me Et Me Et H Me Et H Me Et Me Et H Me Et Et Me
R
M AN U
O
N
R1 H H H H H H H H H H H H H H H H H H H H H
SC
A
5-42
A A1 A1 A1 A2 A2 A2 A3 A3 A4 A4 A4 A5 A5 A5 A6 A6 A7 A7 A7 A8 A9
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
A A9 A2 A2 A2 A2 A2 A2 A4 A4 A5 A5 A5 A7 A7 A7 A9 A9
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R
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
N
N
O
A A1 A1 A1 A1 A2
R1 R2 H Et furfuryl H furfuryl Me furfuryl Et 4-HO-C6H4 H 4-HO-C6H4 Me 4-HO-C6H4 Et 4-HO-C6H4 Me 4-HO-C6H4 Et 4-HO-C6H4 H 4-HO-C6H4 Me 4-HO-C6H4 Et 4-HO-C6H4 H 4-HO-C6H4 Me 4-HO-C6H4 Et 4-HO-C6H4 Me 4-HO-C6H4 Et
R1 R2 H Cl H Br H OH H COOEt H OH
A
Scheme 3. Synthesis of target 4-thiazolidinone-indole/imidazothiadiazole hybrids.
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Reagents and conditions: i – substituted thiosemicarbazone (1.0 equiv), appropriate [C2]2+ compound (1.0 equiv), AcONa (1.0 equiv, for 5-42), AcOH, reflux, 3 h. Structure of fragment A – see scheme 2.
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α-Halogenocarboxylic acids, N-arylmaleimides, maleic anhydride, β-aroylacrylic acids and 2bromobutyrolactone were used as [C2]2+reagents. Utilizing 2-bromobutyrolactone, compounds with free or
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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.
12
ACCEPTED MANUSCRIPT
R
O
Br
O
i
R
1
N
2
O
N
S
N S N H
A
N N H
O
HO O
A
=
R
X
ii O
N H
N
Ar
or O
R
O
H
S O
R
1
N
N
2
O
A
O
R
O
iii
R1 H H H H H H H H H H H furfuryl furfuryl 4-HO-C6H4 4-HO-C6H4 4-HO-C6H4 4-HO-C6H4 4-HO-C6H4
66 67 68 69 70 71 72
A A1 A1 A1 A1 A2 A3 A5
R1 H H H H H H H
R2 Ph 4-F-C6H4 tetralin-6-yl OH OH OH OH
73 74 75
A A1 A1 A1
R1 H H H
R2 HOOCCH2 HOOC(CH2)2 HOOC(CH2)5
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1
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R
A A1 A1 A2 A2 A3 A3 A4 A5 A7 A8 A9 A2 A2 A2 A5 A7 A8 A9
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O
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1
N
O
O
+
OH
H2N (CH2)n
S
2
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R
N H
R2 H Ac H Ac H Ac Ac Ac Ac Ac Ac H Ac Ac Ac Ac Ac Ac
N
N O A
O
Scheme 4. Synthesis of target 4-thiazolidinone-indole/imidazothiadiazoles hybrids.
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Reagents and conditions: i – Method A: thiosemicarbazone (1.0 equiv), α-bromo-γ-butyrolactone (1.0
AC C
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 βaroylacrylic 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.
13
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
R
2
N O Hal
R
A
OH
N
N N H
R1 = 4-HO-C6H4
N
S
H
N
R
X
=
2
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S N H
SC
A
2
R
N
S
R1 = H
R
1
H N
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5) [27,41].
N H
76 77 78 79 80 81 82 83
A A2 A3 A4 A4 A7 A8 A8 A9
84 85 86 87 88 89
A A4 A7 A8 A8 A9 A9
90 91 92 93 94 95
A A2 A4 A5 A7 A8 A9
96 97 98 99 100
A A4 A5 A7 A8 A9
R2 Br NO2 Br NO2 NO2 NO2 Br NO2
R2 NO2 H Br NO2 Br NO2
A
Me
N
R1 = H
Et
O
NH
TE D
S O
N A OH
Et
O
O
Cl
EP
O
Me
R1 = 4-HO-C6H4
AC C
Me
N Et
O S O
N N A
Scheme 5. Synthesis of target 4-thiazole-indole/imidazothiadiazoles hybrids. Reagents and conditions: thiosemicarbazone (1.0 equiv), appropriate [C2]2+ compound (1.0 equiv), AcONa (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 14
ACCEPTED MANUSCRIPT spectral data (1H NMR,
13
C NMR, LCMS) confirmed the structure and purity of compounds. 1H NMR
spectra of target compounds showed characteristic patterns of protons. Additionally, the X-ray analysis
SC
RI PT
was performed for the compound 7 (Fig. 3).
M AN U
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.
TE D
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
EP
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
AC C
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-1,3thiazolidin-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,
15
ACCEPTED MANUSCRIPT C26 from the 5-ethyl-1,3-thiazolidin-4-one fragment. Each of these atoms takes up two alternative
a Figure 4. The hydrogen bonding in 7.
M AN U
SC
RI PT
locations in the crystal structure labelled a and b (Fig. 4)
b
H atoms not involved in hydrogen bonds have been omitted for clarity.
TE D
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
calculated [65,66].
EP
results are presented as growth inhibition percentage. For the active compounds, IC50 values were
activity
AC C
Antitrypanosomal
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.
16
ACCEPTED MANUSCRIPT Table 1. Antitrypanosomal activity of 4-thiazolidinones without indole or imidazothiadiazole fragment towards Trypanosoma brucei brucei Inhibition, %
Inhibition, %
1 µg/mL
1a
-8.23
1.18
1l
1b
56.11
NT
1m
1c
98.91
5.06
1n
1d
10.03
0.06
1o
1e
-4.66
-4.65
1p
1g
44.88
1h
96.98
1i
10 µg/mL
M AN U
10 µg/mL
1 µg/mL
RI PT
Compound
53.24
7.69
14,70
11.14
57.30
38.70
SC
Compound
14.70
11.14
14.49
15.08
1q
25.01
-1.57
NT
1r
24.86
3,28
97.80
14.13
1s
98.53
8.16
1j
29.97
4.18
1t
42.01
36.13
1k
10.91
9.82
TE D
NT
EP
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).
AC C
The minimum activity required for the characterization of the tested compound as a hit was the IC50 ≤ 1.0 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/4thiazolidone 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 IC50 values (43,
17
ACCEPTED MANUSCRIPT 44, 46, 66). The influence of acetamide and tetrahydronaphthalene fragments was similar and caused even more activity decreasing.
H N
O
N N
S R
R
7
Et
IC50, µM
M AN U
Compound
SC
NH
RI PT
Table 2. The IC50 values of 4-thiazolidones with indole fragment against Trypanosoma brucei brucei.
0.03 ± 0.004
4-ClC6H4-NHCOCH2
9.16±1.142
44
4-BrC6H4-NCOCH2
7.86±0.260
46
4-EtCOOC6H4NHCOCH2
6.86±0.261
48
HOCH2CH2
0.17± 0.004
49 66
EP
67
TE D
43
AC C
68
MeCOO(CH2)2
0.06 ± 0.003
Ph-COCH2
7.73±0.310
4-FC6H4-COCH2
10.63±1.810
O
13.13±0.97
74
HOOC(CH2)2NHCOCH2
34.84±0.450
75
HOOC(CH2)5NHCOCH2
14.04±0.830
Pent
0,0014 ± 0.0005
Nf
2,39 ± 0,608
Data are presented as mean ± SD; Pent – Pentamidine, Nf – Nifurtimox
18
ACCEPTED MANUSCRIPT After establishing significant antitrypanosomal properties in the Tb brucei assay, a series of 2-[(2phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-ones was tested towards Tb gambiense (Table 3). The impact of the substituents in the p-position of phenyl ring (R3, Table 5) and N3-position of 4-
RI PT
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
SC
(49) moieties didn’t influence the activity.
M AN U
Table 3. The IC50 values of 4-thiazolidones with indole fragment against Trypanosoma brucei gambiense R
R
2
N
O
N
S R
1
3
N
NH
R1
R2
R3
IC50, µM
5
H
H
H
0.23 ± 0.009
Me
H
H
0.21± 0.014
Et
H
H
0.11± 0.040
H
H
Cl
3.93 ± 0.363
7
AC C
8
EP
6
TE D
Compound
9
Me
H
Cl
2.22 ± 0.071
10
Et
H
Cl
1.42 ± 0.098
11
Me
H
Br
1.35 ± 0.126
12
Et
H
Br
1.50 ± 0.111
14
Me
H
NO2
4.70 ± 0.500
15
Et
H
NO2
6.20 ± 2.200
19
ACCEPTED MANUSCRIPT H
furan-2-ylmethyl
Cl
7.31 ± 1.354
28
Me
furan-2-ylmethyl
Cl
1.71 ± 0.194
29
Et
furan-2-ylmethyl
Cl
1.71 ± 0.161
30
H
4-HO-C6H4
Cl
0.82 ± 0.111
31
Me
4-HO-C6H4
Cl
32
Et
4-HO-C6H4
Cl
34
Et
4-HO-C6H4
NO2
49
MeCOO(CH2)2
H
50
HOCH2CH2
H
51
MeOOCCH2CH2
52
OHCH2CH2
53
MeOOCCH2CH2
59
RI PT
27
1.17 ± 0.126 0.86 ± 0.076
SC
0.14 ± 0.010 0.23± 0.014
Cl
1.49 ± 0.104
M AN U
H
Cl
1.34 ± 0.057
H
Br
3.34 ± 0.534
H
Br
1.36 ± 0.018
HOCH2CH2
furan-2-ylmethyl
Cl
1.40 ± 0.158
60
MeOOCCH2CH2
furan-2-ylmethyl
Cl
1.59 ± 0.084
61
MeOOCCH2CH2
4-HO-C6H4
Cl
0.38 ± 0.080
66
PhCOCH2
H
H
2.82 ± 0.426
4.64 ± 0.725
AC C
Nf
0.0015± 0.0007
EP
Pent
TE D
H
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-3ylmethylene)-hydrazono]-thiazolidin-4-one – IC50 = 9.68±1.8 (Tb brucei), 30.4±8.4 (Tb gambiense), 520
ACCEPTED MANUSCRIPT ethyl-2-[(1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-one – IC50 = 39.51±9.7 (Tb brucei), 73.6±6.3 (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
RI PT
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
SC
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-
M AN U
[(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 IC50 for the derivatives 90, 91 and 96 were much
TE D
higher than those for the unsubstituted analogs.
AC C
EP
Table 4. The IC50 values, µM of thiazoles with indole fragment against Trypanosoma brucei gambiense R
R
1
4
N R
2
R
S
N N
3
N H
Compound
R1
R2
R3
R4
IC50,µM
76
4-Br-C6H4
H
Cl
-
1.52 ±0.400
77
Ph
H
NO2
-
6.00 ±0.600
78
4-BrC6H4
H
NO2
-
2.30 ±0.900
21
ACCEPTED MANUSCRIPT 4-NO2C6H4
H
NO2
4-HO-C6H4
1.50 ± 0.200
90
Me
C2H5OOC
Cl
-
0.53 ±0.205
91
Me
C2H5OOC
NO2
-
0.74±0.068
96
Me
C2H5OOC
NO2
4-HO-C6H4
0.66 ±0.030
Antitrypanosomal
activity
of
RI PT
84
thiazolidinone-hydrazones
with
phenyl-imidazo[2,1-
SC
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
M AN U
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, 4n, 4p was evaluated. SAR data suggested that imidazothiadiazole core itself weakly contributed to the
TE D
activity, but the introduction of thiosemicarbazone fragment improved antitrypanosomal properties.
Table 5. The IC50 values, µM of imidazothiadiazole bearing compounds against Trypanosoma brucei
AC C
EP
gambiense
Compound
R1
2a
S-CH2CHCH2
2b
S-CH2COOEt
2c
Et
3a
S-CH2CH=CH2
2
R
N N
3
R
R2
N
1
R
S
R3
IC50, µM 27.2±1.40
H
H
>30 >40
CHO
22
H
23.4±1.40
ACCEPTED MANUSCRIPT 3c
Et
> 40
4h
S-CH2CH=CH2
4k
Et
H N
H2N
Et
4i
S-CH2CH=CH2
4l
Et
4n
Et
4p
Et
HO
20.9±0.60
NO2
>25
RI PT
4o
H
S N
N H
>25
H
>50
H
2.6±0.50
NO2
14.8±0.90
Br
1.5±0.02
M AN U
N H
Br
SC
Et
6.1±0.30
N
S
4m
H
Table 6. The IC50 values, µM of imidazothiadiazoles’ hydrazones against Trypanosoma brucei gambiense R N
N
TE D
O
R
N
S
3
R
N
N
R2
R3
IC50, µM
H
H
H
3.9 ± 0.70
H
H
Me
4.4± 0.70
18
H
H
Et
20.6± 4.70
35
4-HO-C6H4
H
H
> 20
36
4-HO-C6H4
H
Me
> 20
37
4-HO-C6H4
H
Et
3.6± 1.30
55
H
H
AcOCH2CH2
5.7± 0.60
16
AC C
17
R
EP
Compound
R1
1
S
N
2
R
S
CH2
23
ACCEPTED MANUSCRIPT 4-HO-C6H4
H
AcOCH2CH2
1.00± 0.04
72
H
H
HOOCCH2
>20
19
H
H
Me
3.9± 0.30
20
H
H
Et
9.2± 0.60
21
H
H
22
H
H
23
H
H
24
H
NO2
25
H
Br
26
H
38
4-HO-C6H4
39
4-HO-C6H4
40
4-HO-C6H4
41
O
H
18.0± 2.30
Me
>25
Et
3.7± 0.10
SC
OEt
S
RI PT
62
>20
Me
9.5± 1.30
Br
Et
13.8± 1.40
H
H
9.2± 0.50
H
Me
4.8± 0.20
H
Et
>20
4-HO-C6H4
Br
Me
1.5± 0.03
42
4-HO-C6H4
Br
Et
1.5± 0.04
56
H
H
AcOCH2CH2
11.8± 2.00
H
NO2
AcOCH2CH2
16.2± 0.70
H
Br
AcOCH2CH2
3.2± 0.10
TE D
AC C
58
Et
EP
57
M AN U
Et
63
4-HO-C6H4
H
AcOCH2CH2
2.7± 0.80
64
4-HO-C6H4
NO2
AcOCH2CH2
11.1± 1.60
65
4-HO-C6H4
Br
AcOCH2CH2
1.0± 0.10
R
H N
N
R
S
3
R
N N N
2
24
N S
R
1
ACCEPTED MANUSCRIPT R3
IC50, µM
H
EtOOC
0.652± 0.113
4-NO2-C6H4
H
H
10.3
81
4-NO2-C6H4
NO2
H
4.4
82
4-Br-C6H4
NO2
H
1.2
83
4-NO2-C6H4
H
9.2± 0.80
93
Me
H
EtOOC
2.7 ± 0.90
94
Me
NO2
95
Me
Br
Me
80
S
CH2
Br
Et
SC
92
R1
EtOOC
>20
EtOOC
7.4± 0.40
M AN U
R
RI PT
R2
Compound
OH
R
R
Me
2
N
N N N
R1
S
CH2
Ph
AC C
85
S
TE D
97
EP
R
N
3
R
Compound
N
R
1
S
R2
R3
IC50, µM
H
EtOOC
0.631 ± 0.066
H
H
3.0 ± 0.30
86
4-Br-C6H4
NO2
H
0.594 ± 0.036
87
4-NO2-C6H4
NO2
H
2.6± 0.70
88
4-Br-C6H4
Br
H
0.476 ± 0.066
89
4-NO2-C6H4
Br
H
0.510 ± 0.110
98
Me
H
EtOOC
0.745 ± 0.098
99
Me
NO2
EtOOC
0.639 ± 0.074
Et
25
ACCEPTED MANUSCRIPT 100
Me
Br
EtOOC
1.0 ± 0.10
Allylsulfide fragment negatively influences the imidazothiadiazole derivatives activity. Ethyl
RI PT
fragment in C5 possition of thiazolidone in 18 decreased the activity by an order comparing to 5-methyl4-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
SC
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
M AN U
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-6phenyl-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-(4oxothiazolidin-5-yl)-ethylester (63) with IC50=2.7 µM for both. p-Hydroxyphenyl fragment in the N3 of
TE D
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 86, 88, 89, 100 and 98 showed submicromolar inhibition concentrations. It is worthwhile remarking that
EP
halogen atoms as well as nitro group in the benzene ring can also contribute to the trypanocidal activity of
AC C
the 86, 88, 89, 100. Therefore, the most active compounds of this series appeared to be 2-[(2allylsulfanyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)-hydrazono]-4-methyl-2,3dihydrothiazole-5-carboxylic acid ethyl esters: 92 (IC50=0.652 µM) and 97 (IC50=0.631 µM); and 2-[(2ethyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)-hydrazono]-3-(4-hydroxyphenyl)-4methyl-2,3-dihydrothiazole-5-carboxylic acid ethyl esters 98, 99 and 100 with IC50 within 0.639-1.0 µM, indicating that the presence of 2,3-dihydrothiazole-5-carboxylic acid ethyl ester is crucial for the trypanocidal action of imidazothiadiazoles. 26
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
RI PT
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
SC
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-
M AN U
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
AC C
EP
TE D
structures [53].
27
ACCEPTED MANUSCRIPT
N
S N H
S
N N H X
N N N
H
N
A
R
N H
S
N H
N H
N H
R
R
2
N
N S
favourable for the activity
X Substituents at the phenyl ring
N
S
R
R Alkyl moieties in the C5 are
favourable for the trypanocidal NH activity N
do not greatly contribute to the activity
N
M AN U
R Ester group in the C5 is R
2
SC
O
O
R
A
X
N
1
N N
A
A N H
R
S
H
RI PT
R
X
N N
X
R
N R
3
R
R2 The presence of 4-OH-C6H4 in
R
3
R
2
R2 Aryl fragments in N3-position
N
contriubute to the activity
the N3 possition is controversial R
S
N N
R
TE D
favourable for the activity
2
N
R3 Phenyl fragment is
S
N H
1
N N
X N
R
S
N
N
X
1
N H
S
EP
Activity increasing
AC C
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 IC50 values and no particular impact of different molecular fragments on the toxicity was observed. Nevertheless, 9 compounds possessed very good selectivity indexes (SI > 100) (Tabl. 7).
28
ACCEPTED MANUSCRIPT Table 7. Cytotoxicity upon human primary fribroblast (AB943 cell line) and selectivity indexes IC50, µM
SI
Compound
IC50, µM
SI
5
13.16 ± 2.09
57
65
11.16 ± 0.95
11
6
28.13 ± 3.37
130
78
>96.45
> 40
7
24.83 ± 4.52
220
82
9
5.22 ± 0.19
2
88
30
>100
>130
89
32
85.9 ± 0.43
100
90
34
8.6 ± 0.41
60
92
37
> 90
49
20.92 ± 0.34
50
8.96 ± 2.62
55
19.18 ± 0.76
59 61
RI PT
Compound
>70
>70
> 150
>70
>150
M AN U
SC
>90
20.51 ± 0.91
39
4.33 ± 0.21
7
96
82.9 ± 4.67
127
95
97
> 80
>130
7
98
51.45 ± 8.23
69
3
99
>80
> 130
6.23 ± 0.81
4
Pent
23.20 ± 0.88
5396
>90
> 240
Nf
65.09 ± 2.78
14
TE D
>26
EP
SI = IC50 (AB943) / IC50 (Tbg)
AC C
For derivatives with the high trypanocidal activity, the acute toxicity (mice) was studied and their LD50 were determined. The stock solutions of the compounds used in this study were prepared immediately before usage and increasing amounts of substances (100–1000 mg/kg) were injected intraperitoneally. The LD50 values were calculated according to Litchfield and Wilcoxon. These derivatives showed low acute toxicity in mice with LD50 values within the range of 200–330 mg/kg (Tabl. 8).
Table 8. Acute toxicity of the target compounds 29
ACCEPTED MANUSCRIPT LD50, mg/kg
Compound
LD50, mg/kg
6
220±20.1
32
330±25.3
7
220±13.7
49
200±16.2
8
270±18.2
69
240±20.0
9
310±26.1
70
CONCLUSION
RI PT
Compound
290±19.6
SC
Starting from diverse 4-thiazolidinone-2-hydrazones, new hybrid molecules bearing combination
M AN U
of 4-thiazolidinone or thiazole core and phenyl-indole or 6-phenyl-imidazo[2,1-b][1,3,4]thiadiazole fragment were synthesized. The study of the antitrypanosomal activity towards Trypanosoma brucei brucei and Trypanosoma brucei gambiense strains led to the identification of the hit-compounds with submicromolar IC50 levels, hight selectivity indexes and relatively low cytotoxicity (upon human primary fibroblasts) and acute toxicity (mice). The analysis of structure–activity relationships revealed that
TE D
thiazolidinone/thiazole derivatives were more active than starting thiosemicarbazone analogs; for increase the activity, the C5 substituent of 4-thiazolidinone core should be small and bounded by single bond; the presence of aryl moiety in [6+5] (indole) or [5+5] (imidazothiadiazole) core is essential for
EP
antitrypanosomal activity; the compounds with 2-arylindole fragment are generally more active than 6-
AC C
aryl-imidazo[2,1-b][1,3,4]thiadiazole analogs.
EXPERIMENTAL PART Chemistry
Materials and methods. Melting points of newly synthesized compounds were measured in open capillary tubes on a BUCHI B-545 melting point apparatus and are uncorrected. The elemental analysis (C, H, N) was performed using a Perkin Elmer 2400 CHN analyzer. The analyses indicated by symbols of the elements or functions were within ± 0.4% of the theoretical values. The 1H NMR spectra were recorded on 30
ACCEPTED MANUSCRIPT 13
Varian Gemini 400 MHz and
C NMR spectra on Varian Mercury-400 100 MHz in DMSO-d6 using
tetramethylsilane as an internal standard. Chemical shifts are reported in ppm units by means of δ scale. Mass spectra were obtained using electrospray ionization techniques on an Agilent 1100 Series LCMS.
RI PT
Analytical HPLC was performed on an Agilent 1100 HPLC with Diode Array Detection. Purity of all compounds was determined to be ≥ 95% by the HPLC. The peak purity was checked with the UV spectra.
General procedure for 2-hydrazono-4-thiazolidinones synthesis (1, 5-47, 49, 51, 53-58, 60-72).
SC
The mixture of appropriate thiosemicarbazone (1.0 eq.) and appropriate [C2]2+ synthon (1.0 eq.)
M AN U
(maleic anhydride, N-arylmaleimide, β-aroylacryclic acid, α-halogenocarboxylic acids, α-bromo-γbutyrolactone), sodium acetate (1.0 eq.) in acetic acid was heated under reflux for 2-4 hours. The reaction proceeding was monitored by TLC. After the reaction, the mixture was cooled to the room temperature; the obtained precipitate was filtered off, washed with acetic acid, water and ethanol and recrystallized. For compounds 1d, 73-75: the mixture of maleic anhydride (1.0 eq) and amino acid (1.0 eq) in
TE D
acetic acid was heated under reflux for 1 h. To the reaction mixture thiosemicarbasone (1.0 eq) was added. The reaction mixture was heated under reflux for 3 hours. The reaction proceeding was monitored by
AC C
and recrystallized.
EP
TLC. After the reaction mixture was cooled to room temperature the obtained precipitate was filtered off
N-(4-Fluorophenyl)-2-{3-(4-hydroxyphenyl)-4-oxo-2-[(3-phenyl-allylidene)-hydrazono]-thiazolidin-5-yl}acetamide (1a). Yield 70%, mp 238-240оС. 1Н NMR (400 MHz, DMSO-d6): δ 3.17 (dd, 1H, J = 8.0, 16.4 Hz, CHCH2), 3.28 (dd, 1H, J = 4.0, 16.5 Hz, CHCH2), 4.52 (dd, 1H, J = 3.9, 8.2 Hz, CHCH2), 6.88 (d, 2H, J = 8.2 Hz, arom.), 6.90-6.92 (m, 1H, arom), 7.12 (d, 1H, J = 8.0 Hz, arom.), 7.14-7.20 (m, 5H, arom.), 7.35 (brs, 1H, arom.), 7.60-7.64 (m, 2H, arom.), 8.21 (brs, 2H, arom), 9.10 (s, 1H, OH), 10.24 (s, 1H, NH).13C NMR (100 MHz, DMSO-d6): δ 194.6, 178.2, 176.0, 174.3, 172.6, 167.9, 158.4 (d, J = 210 31
ACCEPTED MANUSCRIPT Hz), 156.2, 135.1, 132.0, 131.3, 129.5, 126.7, 124.2, 116.1 (d, J = 22 Hz), 42.9, 39.4. LCMS (ESI) m/z 489 (M+H)+. Calcd. for C26H21FN4O3S: C, 63.92; H, 4.33; N, 11.47; Found: C, 64.10; H, 4.50; N, 11.30%.
RI PT
N-(4-Nitrophenyl)-2-{4-oxo-2-[(3-phenyl-allylidene)-hydrazono]-thiazolidin-5-yl}-acetamide (1b). Yield 70%, mp 248-250оС. 1Н NMR (400 MHz, DMSO-d6): δ 3.06 (dd, 1H, J = 9.1, 16.8 Hz, CHCH2), 3.25 (dd, 1H, J = 3.8, 16.7 Hz, CHCH2), 4.44 (m, 1H, CHCH2), 7.05 (dd, 1Н, J = 9.3, 15.9 Hz, CH-CH=CH), 7.17 (d, 1Н, J = 6.7 Hz, arom.), 7.30-7.44 (m, 3H, arom), 7.62 (m2H,Hz, arom), 7.82 (d, 2H, J = 8.1 Hz,
SC
arom), 8.18 (m, 3Н, arom.), 8.23 (d, 2H, J = 8.1 Hz, arom.), 11.95 (s, 1H, NH), 10.77 (s, 1H, NH). 13C
M AN U
NMR (100 MHz, DMSO-d6): δ 196.2, 177.7, 166.1, 154.3, 141.6, 136.3, 133.0, 131.7, 129.7, 129.4, 128.1, 125.9, 116.2, 105.5, 43.6, 42.2. LCMS (ESI) m/z 424 (M+H)+ Calcd. for C20H17N5O4S: C, 56.73; H, 4.05; N, 16.54; Found: C, 56.90; H, 4.20; N, 16.20%.
2-{3-Furan-2-ylmethyl-4-oxo-2-[(3-phenyl-allylidene)-hydrazono]-thiazolidin-5-yl}-N-(3-
TE D
trifluoromethylphenyl)-acetamide (1c). Yield 78%, mp 181-183оС. 1НNMR (400 MHz, DMSO-d6): δ 2.82-2.94 (m, 1H, CHCH2), 3.26-3.31 (m, 1H, CHCH2), 4.40 (m, 1H, CHCH2), 4.93 (s, 2H, CH2), 6.32 (t, 1H, J = 6.0 Hz, arom.), 6.42 (d, 1H, J = 6.2 Hz, arom.), 7.00-7.10 (m, 3H, Hz, arom.), 7.26-7.29 (m, 2H,
EP
Hz, arom.), 7.32-7.38 (m, 2H, Hz, arom.), 7.44 (t, 1H, J = 8.1 Hz, arom.), 7.50-7.54 (m, 1H, arom.), 7.72-
AC C
7.75 (m, 1H, arom.), 7.98-8.02 (m, 1H, Hz, arom.), 8.16-8.18 (m, 3H, arom.), 10.55 (s, 1H, NH).
13
C
NMR (100 MHz, DMSO-d6): δ 174.0, 168.9, 162.7, 160.7, 149.3, 143.1, 142.4, 139.8, 137.4 (q, J= 121 Hz), 136.2, 134.3, 130.6, 129.4, 127.9, 125.6, 123.1, 123.1, 115.6, 111.1, 109.2, 43.0, 38.7, 34.6. LCMS (ESI) m/z 527 (M+H)+ Calcd. for C26H21F3N4O2S: C, 59.31; H, 4.02; N, 10.64; Found: C, 59.50; H, 4.20; N, 10.50%.
32
ACCEPTED MANUSCRIPT 4-{2-[2-(Furan-2-ylmethylene-hydrazono)-4-oxothiazolidin-5-yl]-acetylamino}-butyric acid (1d). Yield 64%, mp 211-213оС. 1Н NMR (400 MHz, DMSO-d6): δ 1,62 (m, 2H, CH2), 2.25 (m, 2H, CH2), 2.64 (dd, 1H, J = 9.8, 15.7Hz, CHCH2), 2.90 (dd, 1H, J = 4.4, 15.8 Hz, CHCH2),3.06 (m, 2H, CH2), 4.33 (m, 1H,
RI PT
CHCH2), 6.64 (t, 1H, J = 6.2 Hz, arom.), 6.95 (d, 1H, J = 6.2 Hz,arom.), 7.86 (d, 1H, J = 6.2 Hz, arom.), 8.06 (t, 1H, J = 7.2 Hz, NH), 8.21 (s, 1H, CH=N), 12.00 (brs, 2Н, NН, COOH).
13
C NMR (100 MHz,
DMSO-d6): δ 176.2, 174.7, 169.2, 164.6, 149.9, 146.3, 145.9, 115.8, 112.8, 44.5, 38.6, 38.3, 31.5, 25.0.
SC
LCMS (ESI) m/z 353 (M+H)+. Calcd. for C14H16N4O5S: C, 47.72; H, 4.58; N, 15.90; Found: C, 48.00; H,
M AN U
4.90; N, 15.60%.
2-[2-(Cyclohexylidene-hydrazono)-4-oxothiazolidin-5-yl]-N-(4-fluorophenyl)-acetamide (1e). Yield79%, mp 182-184оС. 1НNMR (400 MHz, DMSO-d6): δ 1.55-1.63 (m, 6H, cyclohex.), 2.21-2.24 (m, 2H, cyclohex.), 2.52-2.57 (m, 2H, cyclohex.), 2.86 (m, 1H, CHCH2), 3.14 (dd, 1H, J = 3.0, 16.5 Hz, CHCH2), 4.31 (dd, 1H, J = 3.0, 9.3 Hz, CH2), 7.13 (t, 2H, J = 8.6 Hz, arom.), 7.55-7.58 (m, 2H, arom.), 10.16 (s,
TE D
1H, NH), 11.66 (s, 1H, NH).13C NMR (100 MHz, DMSO-d6): δ 194.5, 177.8, 168.3, 158.4 (d, J = 239 Hz), 153.4, 150.1, 135.7, 123.2, 121.3 (d, J = 7 Hz), 115.8 (d, J = 22 Hz), 44.5, 43.7, 35.4, 28.5, 27.6, 26.6, 25.9. LCMS (ESI) m/z 363 (M+H)+. Calcd. for C17H19FN4O2S: C, 56.34; H, 5.28; N, 15.46; Found:
AC C
EP
C, 56.50; H, 5.40; N, 15.20%.
For detailed characteristic of compounds 1f-1t see Supplementary Materials.
General procedure for 2-R-6-arylimidazo[2,1-b][1,3,4]thiadiazoles synthesis (2). Mixture of 2-amino-5-R-1,3,4-thiadiazole (1.0 eq.) and 2-bromoacetophenone (1.0 eq.) in ethanol was heated under reflux for 8 h.The reaction mixture was cooled and water solution of sodium carbonate
33
ACCEPTED MANUSCRIPT was added (pH = 9.0). The formed precipitate was filtered off and washed, dried and recrystallized from appropriate solvent [62].
RI PT
2-Allylsulfanyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazole(2a). Yield 73%, mp 111-112оС. 1Н NMR (400 MHz, DMSO-d6): δ 3.98 (d, 2Н, J = 6.8 Hz, СН2), 5.23 (d, 1Н, J = 10.0 Hz, СН2), 5.37 (d, 1Н, J = 16.9 Hz, СН2), 5.92-6.06 (m, 1Н, СН=), 7.28 (t, 1Н, J = 7.4 Hz, arom.), 7.42 (t, 2Н, J = 7.4 Hz, arom.), 7.87 (d, 2Н, J = 7.5 Hz, arom.), 8.68 (s, 1Н, CН).
13
C NMR (100 MHz, DMSO-d6): δ 159.8, 145.4, 145.1,
SC
134.2, 132.9, 129.1, 127.8, 125.1, 120.2, 111.1, 37.1. LCMS (ESI+) m/z 274 (M+H)+. Calcd. for
M AN U
C13H11N3S2: C, 57.12; H, 4.06; N, 15.37; Found: C, 57.30; H, 4.20; N, 15.20%.
(6-Phenylimidazo[2,1-b][1,3,4]thiadiazol-2-ylsulfanyl)-acetic acid ethyl ester (2b). Yield 68%, mp 9091оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.20 (t, 3Н, J = 10.0 Hz, СН3), 4.17 (q, 2Н, J = 7.0 Hz,СН2), 4.28 (s, 2Н,СН2),7.28 (t, 1Н, J = 7.0 Hz, arom.), 7.41 (t, 2Н, J = 7.3 Hz, arom.),7.86 (d, 2Н, J = 7.4 Hz, 13
C NMR (100 MHz, DMSO-d6): δ 168.2, 159.8, 145.4, 145.1, 134.2, 129.1,
TE D
arom.), 8.67 (s, 1Н, CН).
127.8, 125.1, 111.1, 62.1, 35.8, 14.5. LCMS (ESI+) m/z 320 (M+H)+. Calcd. for C14H13N3O2S2: C, 52.65;
EP
H, 4.10; N, 13.16; Found: C, 52.70; H, 4.30; N, 13.00%.
AC C
2-Ethyl-6-phenylimidazo[2,1-b][1,3,4]thiadiazole (2c).Yield 75%, mp 143-144оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.34 (t, 3Н, J = 7.5 Hz, СН3), 3.07 (q, 2Н, J = 7.5 Hz, СН2), 7.28 (t, 1Н, J = 7.3 Hz, arom.), 7.41 (t, 2Н, J = 7.3 Hz, arom.), 7.87 (d, 2Н, J = 7.2 Hz, arom.), 8.62 (s, 1Н, CH). 13C NMR (100 MHz, DMSO-d6): δ 166.8, 145.3, 145.1, 134.5, 129.1, 127.7, 125.1, 110.7, 25.5, 13.1. LCMS (ESI+) m/z 230 (M+H)+. Calcd. for C12H11N3S: C, 62.86; H, 4.84; N, 18.32; Found: C, 63.00; H, 5.00; N, 18.10%.
34
ACCEPTED MANUSCRIPT 6-(4-Bromophenyl)-2-ethylimidazo[2,1-b][1,3,4]thiadiazole (2d). Yield 77%, mp 228-229оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.35 (t, 3Н, J = 7.5 Hz, СН3), 3.11 (q, 2Н, J = 7.5 Hz, СН2), 7.67 (d, 2Н, J = 8.6 Hz, arom.), 7.91 (d, 2Н, J = 8.6 Hz, arom.), 8.68 (s, 1Н, CН). 13C NMR (100 MHz, DMSO-d6): δ 167.4,
RI PT
143.8, 132.5, 132.1, 130.9, 127.1, 120.6, 111.3, 25.5, 13.1. LCMS (ESI+) m/z 308/310 (M+H)+. Calcd. for C12H10BrN3S: C, 46.77; H, 3.27; N, 13.63; Found: C, 46.90; H, 3.50; N, 13.50%.
SC
2-Ethyl-6-(4-nitrophenyl)-imidazo[2,1-b][1,3,4]thiadiazole (2e). Yield 80%, mp 193-194оС. 1Н NMR (400 MHz, DMSO-d6 ): δ 1.35 (t, 3Н, J = 7.5 Hz, СН3), 3.08 (q, 2Н, J = 7.5 Hz, СН2), 8.07 (d, 2Н, J = 8.9
M AN U
Hz, arom.), 8.24 (d, 2Н, J = 8.9 Hz, arom.), 8.83 (s, 1Н, CН). 13C NMR (100 MHz, DMSO-d6): δ 168.1, 146.5, 143.1, 141.0, 125.6, 124.7, 113.4, 113.3, 25.5, 13.1. LCMS (ESI+) m/z 275 (M+H)+. Calcd. for C12H10N4O2S: C, 52.55; H, 3.67; N, 20.43; Found: C, 52.70; H, 3.80; N, 20.20%.
General procedure for 2-R-6-arylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde synthesis (3).
TE D
The Vilsmeier-Haack reagent was obtained in the reaction of phosphorus oxychloride with the anhydrous dimethylformamide (1:3 vol) at 0ºС and stirring for 10 min. To the prepared reagent 2-R-6arylimidazo[2,1-b][1,3,4]thiadiazole (1.0 eq) was added and stirred for 30 min at 0ºС, then for 2 h at rt and
EP
for 2 h at 60ºС. Then, to the reaction mixture a solution of sodium bicarbonate was added and the mixture
AC C
was heated to 90 ºС and stirred for 2 h. The reaction mixture was cooled and water was added. The product was extracted with the chloroform; the chloroform phase was dried over sodium sulfate and concentrated. Target compound was recrystallized from the appropriate solvent [62].
2-Allylsulfanyl-6-phenylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3a). Yield 89%, mp 77-78оС. 1
Н NMR (400 MHz, DMSO-d6): δ 4.03 (d, 2Н, J = 6.9 Hz, СН2), 5.25 (d, 1Н, J = 10.0 Hz, СН2=), 5.44
(d, 1Н, J = 17.0 Hz, СН2=), 5.94-6.07 (m, 1Н, СН=), 7.51-7.53 (m, 3Н, arom.), 7.93-7.96 (m, 2Н, arom.), 35
ACCEPTED MANUSCRIPT 9.97 (s, 1Н, СНО).
13
C NMR (100 MHz, DMSO-d6): δ 177.7, 163.2, 154.0, 150.8, 132.7, 132.6, 130.1,
129.3, 129.2, 124.0, 120.7, 37.1. LCMS (ESI+) m/z 302 (M+H)+. Calcd. for C14H11N3OS2: C, 55.79; H,
RI PT
3.68; N, 13.94; Found: C, 55.90; H, 3.80; N, 13.70%.
(5-Formyl-6-phenylimidazo[2,1-b][1,3,4]thiadiazol-2-ylsulfanyl)acetic acid ethyl ester (3b). Yield 88%, mp 98-100оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.18 (t, 3Н, J = 9.3 Hz, СН3), 3.98 (q, 2Н, J = 7.1
Hz, arom.), 10.02 (s, 1Н, CНO).
13
SC
Hz,СН2), 4.12 (s, 2Н,СН2), 7.32 (t, 1Н, J = 7.2 Hz, arom.), 7.45-7.49 (m, 2Н,arom.), 7.84 (d, 2Н, J = 7.3 C NMR (100 MHz, DMSO-d6): δ 176.9, 168.2, 159.6, 145.3, 145.2,
M AN U
134.0, 127.7, 126.6, 125.2, 111.0, 62.2, 35.6, 14.5.LCMS (ESI+) m/z 348 (M+H)+. Calcd. for C15H13N3O3S2: C, 51.86; H, 3.77; N, 12.09; Found: C, 62.00; H, 4.00; N, 11.80%.
2-Ethyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3c). Yield 92%, mp 100-101оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.37 (t, 3Н, J = 7.5 Hz, СН3), 3.16 (q, 2Н, J = 7.5 Hz, СН2), 7.51-7.53 (m,
TE D
3Н, arom.), 7.93-7.96 (m, 2Н, arom.), 9.96 (s, 1Н, СНО).
13
C NMR (100 MHz, DMSO-d6): δ 177.6,
169.7, 154.5, 151.1, 132.8, 130.0, 129.3, 129.2, 123.9, 25.5, 13.5. LCMS (ESI+) m/z 258 (M+H)+. Calcd.
EP
for C13H11N3OS: C, 60.68; H, 4.31; N, 16.33; Found: C, 60.80; H, 4.50; N, 16.20%.
AC C
6-(4-Bromophenyl)-2-ethylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3d). Yield 87%, mp 127128оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.37 (t, 3H, J = 7.4 Hz, CH3) 3.16 (q, 2H, J = 7.4 Hz, CH2), 7.71 (d, 2H, J = 8.4 Hz, arom.), 7.93 (d, 2H, J = 8.4 Hz, arom.), 9.98 (s, 1H, CHO). 13C NMR (100 MHz, DMSO-d6): δ 177.6, 170.0, 152.5, 150.9, 132.1, 132.0, 131.1, 123.9, 123.6, 25.5, 13.4. LCMS (ESI+) m/z 336/338 (M+H)+. Calcd. for C13H10BrN3OS: C, 46.44; H, 3.00; N, 12.50; Found: C, 46.60; H, 3.20; N, 12.40%.
36
ACCEPTED MANUSCRIPT 2-Ethyl-6-(4-nitrophenyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3e). Yield 91%, mp 174175оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.38 (t, 3Н, J = 7.4Hz, СН3), 3.19 (q, 2Н, J = 7.3 Hz, СН2), 8.28 (d, 2Н, J = 8.5 Hz, arom.), 8.35 (d, 2Н, J = 8.5 Hz, arom.), 10.08 (s, 1Н, СНО). 13C NMR (100 MHz,
RI PT
DMSO-d6): δ 177.9, 170.7, 151.0, 150.5, 148.2, 139.0, 130.2, 124.6, 124.3, 25.6, 13.4. LCMS (ESI+) m/z 303 (M+H)+. Calcd. for C13H10N4O3S: C, 51.65; H, 3.33; N, 18.53; Found: C, 51.80; H, 3.50; N, 18.40%.
SC
General procedure for 6-phenylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde thiosemicarbazones and 2-aryl-1H-indole-3-carbaldehyde thiosemicarbazones synthesis (4).
M AN U
The mixture of equimolar quantities of 6-phenylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3) or 2-aryl-1H-indole-3-carbaldehyde (1.0 eq.) and thiosemicarbazide (1.0 eq.) in the acetic acid was heated under reflux for 1 h. After the reaction mixture was cooled to the room temperature, formed precipitate was filtered off, washed with acetic acid, water and ethanol and recrystallized.
TE D
(2-Phenyl-1H-indol-3-yl)-methylenethiosemicarbazone (4a). Yield 91%, mp 227-228оС. 1Н NMR (400 MHz, DMSO-d6): δ 7.16 (t, 1H, J = 7.6 Hz, arom.), 7.24 (t, 1H, J = 7.4 Hz, arom.), 7.37 (brs, 1H, NH2), 7.44 (d, 1H, J = 8.0 Hz, arom.), 7.50 (t, 1H, J = 8.0 Hz, arom.), 7.57 (t, 2H, J = 7.6 Hz, arom.), 7.61 (d,
EP
1H, J = 7.2 Hz, arom.), 8.04 (brs, 1H, NH2), 8.33 (d, 1H, J = 7.7 Hz, arom.), 8.50 (s, 1H, CH), 11.16 (s,
AC C
1H, NH), 11.88 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 177.0 142.7, 141.8, 136.9, 131.4, 129.7, 129.3, 129.2, 125.6, 123.5, 123.1, 121.5, 111.9, 107.9. LCMS (ESI+) m/z 295 (M+H)+. Calcd. for C16H14N4S: C, 65.28; H, 4.79; N, 19.03; Found: C, 65.40; H, 4.90; N, 18.80%.
(2-(4-Chlorophenyl)-1H-indol-3-yl)-methylene-thiosemicarbazone (4b). Yield 93%, mp 234-236оС. 1Н NMR (400 MHz, DMSO-d6): δ 7.18 (t, 1H, J = 7.4 Hz, arom.), 7.28 (t, 1H, J = 7.4 Hz, arom.), 7.43 (brs, 1H, NH2), 7.68 (d, 2H, J = 8.1 Hz, arom.), 7.82 (d, 2H, J = 8.2 Hz, arom.), 8.11 (brs, 1H, NH2), 8.33 (d, 37
ACCEPTED MANUSCRIPT 1H, J = 7.7 Hz, arom.), 8.52 (s, 1H, CH), 11.14 (s, 1H, NH), 11.82 (s, 1H, NH).
13
C NMR (100 MHz,
DMSO-d6): δ 177.2, 142.6, 141.8, 137.0, 131.2, 129.6, 129.4, 129.2, 125.4, 123.9, 123.0, 121.2, 111.6, 108.0. LCMS (ESI+) m/z 329/331 (M+H)+. Calcd. for C16H13ClN4S: C, 58.44; H, 3.98; N, 17.04; Found:
RI PT
C, 58.60; H, 4.10; N, 16.90%.
N1-(2-(4-Chlorophenyl)-1H-indol-3-yl)-methylidene)-N2-(4-hydroxyphenyl)-thiosemicarbazone (4c). Yield
SC
89%, mp 239-241оС. 1Н NMR (400 MHz, DMSO-d6): δ 6.81 (d, 2H, J = 8.2 Hz, arom), 7.12 (t, 1H, J = 7.0 Hz, arom.), 7.21 (t, 1H, J = 7.0 Hz, arom.), 7.42 (d, 2H, J = 8.0 Hz, arom.), 7.74 (d, 2H, J = 8.0 Hz,
M AN U
arom.), 7.87 (d, 2H, J = 8.1 Hz, arom.), 8.05 (brs, 1H, NH), 8.31 (d, 1H, J = 7.2 Hz, arom), 8.52 (s, 1H, CH), 9.42 (s, 1H, OH), 11.82 (s, 1H, NH), 11.86 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 177.1, 143.0, 141.9, 137.6, 137.1, 133.4, 131.4, 131.2, 130.0, 129.6, 129.4, 129.2, 125.4, 123.9, 123.0, 121.4, 112.0, 108.6. LCMS (ESI+) m/z 422/424 (M+H)+. Calcd. for C22H17ClN4OS: C, 62.78; H, 4.07; N, 13.31;
TE D
Found: C, 63.00; H, 4.20; N, 13.00%.
N1-(2-(4-Chlorophenyl)-1H-indol-3-yl)-methylidene)-N2-furfuryl-thiosemicarbazone (4d). Yield 89%, mp 223-225оС. 1Н NMR (400 MHz, DMSO-d6): δ 4.92 (s, 2H, NCH2), 7.18 (t, 1H, J = 7.0 Hz, arom.), 7.22 (t,
EP
1H, J = 7.1 Hz, arom.), 7.41 (d, 1H, J = 6.5 Hz, arom.), 7.45 (d, 2H, J = 8.1 Hz, arom.), 7.64 (m, 3H,
AC C
arom.), 7.82-7.86 (m, 3H, arom), 8.05 (brs, 1H, NH), 8.24 (d, 1H, arom.), 11.78 (brs, 1H, NH), 11.86 (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 177.1, 153.6, 149.4, 143.0, 142.1, 137.0, 134.3, 131.4,
130.2, 129.6, 126.2, 123.9, 122.9, 121.8, 112.30, 111.1, 109.2, 108.9, 44.2. LCMS (ESI+) m/z 410/412 (M+H)+. Calcd. for C21H17ClN4OS: C, 61.68; H, 4.19; N, 13.70; Found: C, 61.90; H, 4.30; N, 13.50%.
(2-(4-Bromophenyl)-1H-indol-3-yl)-methylene-thiosemicarbazone (4e). Yield 91%, mp 241-243оС. 1Н NMR (400 MHz, DMSO-d6): δ 7.21 (t, 1H, J = 7.2 Hz, arom.), 7.26 (t, 1H, J = 7.2 Hz, arom.), 7.40 (brs, 38
ACCEPTED MANUSCRIPT 1H, NH2), 7.70 (d, 2H, J = 8.4 Hz, arom.), 7.85 (d, 2H, J = 8.2 Hz, arom.), 8.05 (brs, 1H, NH2), 8.23 (d, 1H, J = 7.6 Hz, arom.), 8.54 (s, 1H, CH), 11.14 (s, 1H, NH), 11.82 (s, 1H, NH).
13
C NMR (100 MHz,
DMSO-d6): δ 177.0143.0, 141.9, 137.1, 131.3, 130.1, 129.6, 129.4, 125.4, 123.8, 122.0, 121.2, 112.0,
RI PT
109.1. LCMS (ESI+) m/z 374 (M+H)+. Calcd. for C16H13BrN4S: C, 51.48; H, 3.51; N, 15.01; Found: C, 51.60; H, 3.70; N, 14.90%.
SC
For detailed characteristic of compounds 4f-4p see Supplementary Materials.
M AN U
2-[(2-Phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-one (5). Yield 82%, mp 253-255ºC. 1H NMR (400 MHz, DMSO-d6): δ 3.82 (s, 2H, CH2), 7.17 (t, 1H, J = 7.2 Hz, arom.), 7.22 (t, 1H, J = 7.2 Hz, arom.), 7.44-7.48 (m, 2H, arom.), 7.54-7.57 (m, 2H, arom.), 7.65-7.68 (m, 2H, arom.), 8.35 (d, 1H, J = 7.0 Hz, arom.), 8.81 (s, 1H, CH), 11.72 (brs, 1H, NH), 11.77 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 174.2, 152.5, 142.9, 137.0, 131.8, 129.6, 129.1, 128.9, 126.5, 123.1, 121.3, 111.8, 108.9, 33.44. LCMS
N, 16.50%.
TE D
(ESI+) m/z 335 (M+H)+. Calcd. for C18H14N4OS: C, 64.65; H, 4.22; N, 16.75; Found: C, 64.90; H, 4.40;
EP
5-Methyl-2-[(2-phenyl-1H-indol-3-yl-methylene)-hydrazono]-thiazolidin-4-one (6). Yield 76%, mp 266-
AC C
268ºC. 1H NMR (400 MHz, DMSO-d6): δ 1.63 (d, 3H, J = 7.0 Hz, CH3), 4.09 (q, 1H, J = 7.0 Hz, CH), 7.17 (t, 1H, J = 7.2 Hz, arom.), 7.21 (t, 1H, J = 6.9 Hz, arom.), 7.42-7.45 (m, 2H, arom.), 7.56 (t, 2H, J = 7.8 Hz, arom.), 7.66 (d, 2H, J = 7.8 Hz, arom.), 8.33 (d, 1H, J = 7.4 Hz, arom.), 8.59 (s, 1H, CH), 11.65 (brs, 1H, NH), 11.78 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 177.1, 152.6, 152.6, 142.9, 137.1, 131.8, 129.6, 129.1 128.9, 126.5, 123.2, 121.2, 111.8, 108.9, 42.6, 19.6. LCMS (ESI+) m/z 349 (M+H)+. Calcd. for C19H16N4OS: C, 65.50; H, 4.63; N, 16.08; Found: C, 65.70; H, 4.80; N, 15.90%.
39
ACCEPTED MANUSCRIPT 5-Ethyl-2-[(2-phenyl-1H-indol-3-yl-methylene)-hydrazono]-thiazolidin-4-one (7). Yield 74%, mp 247249оС. 1H NMR (400 MHz, DMSO-d6): δ 1.11 (t, 3H, J = 7.2 Hz, CH3), 1.92 (m, 1H, CHCH2), 2.10 (m, 1H, CHCH2), 4.00 (m, 1H, CHCH2), 7.16 (m, 2H, arom.), 7.37-7.43 (t, 2H, J = 8.0 Hz, arom.), 7.52 (t, 2H,
11.61 (s, 1H, NH).
13
RI PT
J = 8.2 Hz, arom.), 7.65 (d, 2H, J = 8.2 Hz, arom.), 8.33 (d, 1H, J = 6.4 Hz, arom.), 8.57 (s, 1H, CH), C NMR (100 MHz, DMSO-d6): δ 172.4, 152.8, 143.3, 137.0, 131.4, 129.7, 129.4,
126.3, 123.6, 122.8, 121.6, 112.1, 108.6, 49.7, 25.9, 10.7. LCMS (ESI+) m/z 363 (M+H)+. Calcd. for
SC
C20H18N4OS: C, 66.28; H, 5.01; N, 15.46; Found: C, 66.50; H, 4.90; N, 15.30%.
M AN U
2-{[2-(4-Chlorophenyl)-1H-indol-3-yl-methylene]-hydrazono}-thiazolidin-4-one (8). Yield 76%, mp 298299оС. 1H NMR (400 MHz, DMSO-d6): δ 3.90 (s, 2H, CH2), 7.20 (t, 1H, J = 7.5 Hz, arom.), 7.26 (t, 1H, J = 7.2 Hz, arom.), 7.47 (d, 1H, J = 8.0 Hz, arom.), 7.64 (d, 2H, J = 8.4 Hz, arom.), 7.68 (d, 2H, J = 8.4 Hz, arom.), 8.32 (d, 1H, J = 7.8 Hz, arom.), 8.53 (s, 1H, CH), 11.84 (s, 1H, NH), 12.04 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 174.3, 152.2, 141.6, 137.0, 134.2, 131.4, 130.3, 129.5, 126.3, 123.8, 122.9,
TE D
121.7, 112.2, 109.0, 33.6. LCMS (ESI+) m/z 369/371 (M+H)+. Calcd. for C18H13ClN4OS: C, 58.62; H,
EP
3.55; N, 15.19; Found: C, 58.80; H, 3.70; N, 15.00%.
2-{[2-(4-Chlorophenyl)-1H-indol-3-yl-methylene]-hydrazono}-5-methylthiazolidin-4-one (9). Yield 78%,
AC C
mp 296-298оС. 1H NMR (400 MHz, DMSO-d6): δ 1.54 (d, 3H, J = 7.2 Hz, CH3), 4.21 (q, 1H, J = 7.2 Hz, CH), 7.21 (t, 1H, J = 8.5 Hz, arom.), 7.28 (t, 1H, J = 7.4 Hz, arom.), 7.47 (d, 1H, J = 7.9 Hz, arom.), 7.64 (d, 2H, J = 8.5 Hz, arom.), 7.68 (d, 2H, J = 8.5 Hz, arom.), 8.32 (d, 1H, J = 7.8 Hz, arom.), 8.54 (s, 1H, CH), 11.76 (s, 1H, NH), 12.00 (s, 1H, NH).
C NMR (100 MHz, DMSO-d6): δ 176.5, 152.4, 141.7,
13
137.0, 134.2, 131.3, 130.2, 129.5, 126.2, 123.8, 122.9, 121.7, 120.2, 112.2, 42.4, 19.4. LCMS (ESI+) m/z 383/385 (M+H)+. Calcd. for C19H15ClN4OS: C, 59.60; H, 3.95; N, 14.63; Found: C, 59.40; H, 3.80; N, 14.80%. 40
ACCEPTED MANUSCRIPT
2-{[2-(4-Chlorophenyl)-1H-indol-3-yl-methylene]-hydrazono}-5-ethylthiazolidin-4-one (10). Yield 78%, mp 290-292оС. 1H NMR (400 MHz, DMSO-d6): δ 0.99 (t, 3H, J = 7.2 Hz, CH3), 1.84-4.89 (m, 1H, CH2),
RI PT
1.98-2.04 (m, 1H, CH2), 4.25-4.27 (m, 1H, CH), 7.23 (t, 1H, J = 7.2 Hz, arom.), 7.27 (t, 1H, J = 7.2 Hz, arom.), 7.48 (d, 1H, J = 7.8 Hz, arom.), 7.66 (d, 2H, J = 8.3 Hz, arom.), 7.69 (d, 2H, J = 8.3 Hz, arom.), 8.33 (d, 1H, J = 7.6 Hz, arom.), 8.55 (s, 1H, CH), 11.83 (s, 1H, NH), 12.05 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 174.2, 152.4, 141.7, 137.0, 134.2, 131.3, 130.2, 129.50, 129.4, 126.3, 123.8, 122.9,
SC
121.8, 112.2, 109.0, 50.0, 25.9, 10.7. LCMS (ESI+) m/z 397/399 (M+H)+. Calcd. for C20H17ClN4OS: C,
M AN U
60.52; H, 4.32; N, 14.12; Found: C, 60.70; H, 4.60; N, 14.00%.
For detailed characteristic of compounds 11-47, 49, 51, 53-58, 60-75 see Supplementary Materials.
General procedure for 5-(2-R-ethyl)-2-[(2-phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-
TE D
ones and 5-(2-R-ethyl)-2-[(6-phenyl-2,3,7,7a-tetrahydroimidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)hydrazono]-thiazolidin-4-ones synthesis (48, 50, 52, 59). (Method A) To the appropriate
EP
thiosemicarbazone (1.0 eq) in ethanol α-bromo-γ-butyrolactone (1.0 eq) and triethylamine (1.0 eq,) were added. The reaction mixture was heated under reflux for 5 hours. The reaction proceeding was monitored
AC C
by TLC. After the reaction mixture was cooled to room temperature, the obtained precipitate was filtered off and recrystallized from the ethanol to provide pure title compound.
5-(2-Hydroxyethyl)-2-[(2-phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-one (48). Yield 70%, mp 223-224оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.86-1.90 (m, 1H, СH2), 2.27-2.32 (m, 1H, СH2), 3,633.66 (m, 2H, СH2), 4,16 (dd, 1H, J = 4.0, 9.6 Hz, СH), 4,54 (brs, 1H, OH), 7,17 (t, 1H, J = 7.7 Hz,), 7.417.47 (m, 1H), 7.55 (t, 1H, J = 7.4 Hz, arom.) 7.64 (d, J = 7.4 Hz), 7.64 (d, J = 7.6 Hz, 1H), 8.57 (s, 1H, 41
ACCEPTED MANUSCRIPT CH), 11.63 (brs, 1H, NH), 11.74 (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 172.6, 167.9, 158.2,
143.2, 137.0, 131.4, 129.7, 129.5, 126.3, 123.6, 122.8, 121.7, 112.2, 108.6, 58.9, 40.9, 36.5. LCMS (ESI+) m/z 379 (M+H)+. Calcd. for C20H18N4O2S: C, 63.47; H, 4.79; N, 14.80; Found: C, 63.60; H, 4.90; N,
RI PT
14.70%.
2-{[2-(4-Chlorophenyl)-1H-indol-3-ylmethylene]-hydrazono}-5-(2-hydroxyethyl)-thiazolidin-4-one
(50).
SC
Yield 80%, mp 268-270оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.86-1.91 (m, 1H, СH2), 2.24-2.29 (m, 1H, СH2), 3,57-3.64 (m, 2H, СH2), 4,24 (dd, 1H, J = 3.7, 9.4 Hz, СH), 4,78 (brs, 1H, OH), 7,26 (t, 1H, J =
M AN U
7.6Hz,), 7.27 (t, 1H, J = 7.7 Hz, arom.), 7.47 (d, 1H, J = 7.9Hz, arom.) 7.65 (d, J = 8.5 Hz, 2H, arom.), 7.69 (d, 2H, J = 8.5 Hz, arom.), 8.33 (d, 1H, J = 7.7 Hz, arom.), 8.54 (s, 1H, CH), 11.79 (brs, 1H, NH), 12.05 (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 177.1, 172.7, 152.3, 141.6, 137.0, 134.2, 131.3,
130.3, 129.5, 126.6, 123.8, 122.9, 121.7, 112.2, 109.0, 58.9, 45.8, 36.5. LCMS (ESI+) m/z 413/415
TE D
(M+H)+. Calcd. for C20H17ClN4O2S: C, 58.18; H, 4.15; N, 13.57; Found: C, 58.00; H, 4.00; N, 13.70%.
2-{[2-(4-Bromophenyl)-1H-indol-3-ylmethylene]-hydrazono}-5-(2-hydroxyethyl)-thiazolidin-4-one
(52).
EP
Yield 78%, mp>350оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.84-1.89 (m, 1H, СH2), 2.20-2.25 (m, 1H, СH2), 3.56-3.62 (m, 2H, СH2), 4.22 (dd, 1H, J = 3.7, 9.4 Hz, СH), 4,80 (brs, 1H, OH), 7.25 (t, 1H, J = 7.4
AC C
Hz, arom.), 7.28 (t, 1H, J = 7.4 Hz, arom.), 7.52 (d, 1H, J = 8.0Hz, arom.) 7.70 (d, 2H, J = 8.2 Hz, arom.), 7.75 (d, 2H, J = 8.2 Hz, arom.), 8.36 (m, 1H, arom.), 8.53 (s, 1H, CH), 11.81 (brs, 1H, NH), 11.98 (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 177.0, 162.8, 141.5, 141.2, 137.0, 132.3, 131.7, 130.6, 125.6,
123.83, 123.2, 123.0, 121.6, 112.0, 108.4, 56.9, 36.3, 31.3. LCMS (ESI+) m/z 457/459 (M+H)+. Calcd. for C20H17BrN4O2S: C, 52.52; H, 3.75; N, 12.25; Found: C, 52.70; H, 3.90; N, 12.10%.
42
ACCEPTED MANUSCRIPT 2-{[2-(4-Chlorophenyl)-1H-indol-3-ylmethylene]-hydrazono}-3-(furan-2-ylmethyl)-5-(2-hydroxy-ethyl)thiazolidin-4-one (59). Yield 73%, mp 252-254°С. 1Н NMR (400 MHz, DMSO-d6): δ 1.89-1.94 (m, 1H, CHCH2), 2.25-2.32 (m, 1H, CHCH2), 3.59-3.63 (m, 2H, CH2), 4.37 (dd, J = 3.9, 9.2 Hz, 1H, CH), 4.85 (t,
RI PT
1H, J = 5.1 Hz, OH), 4.91 (s, 2H, CH2), 6.36-6.37 (m, 1H, arom.), 6.39-6.41 (m, 1H, arom.), 7.27-7.31 (m, 2H, arom.), 7.49 (d, 1H, J = 7.2 Hz, arom.), 7.58-7.59 (m, 1H, arom.), 7.69 (brs, 4H, arom.), 8.33 (d, 1H, J = 7.2 Hz, arom.), 8.57 (s, 1H, CH), 12.12 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 174.8, 159.7,
SC
153.7, 149.4, 143.0, 142.2, 137.0, 134.4, 131.4, 130.2, 129.6, 126.2, 123.9, 122.9, 121.8, 112.2, 111.1, 109.0, 108.9, 58.6, 44.8, 40.6,36.4. LCMS (ESI+) m/z 493/495(M+H)+. Calcd. for C25H21ClN4O3S: C,
M AN U
60.91; H, 4.29; N, 11.36; Found: C, 61.00; H, 4.40; N, 11.30%.
General procedure for N-[4-arylthiazol-2-yl]-N'-[2-(arylphenyl)-1H-indol-3-ylmethylene]-hydrazines, 5-methyl-2-(6-arylimidazo[2,1-b]-1,3,4-thiadoazole-5-ylmethylene)hydrazone-4-thiazolidinones
and
ethyl ester of 2-{N'-[2-(arylphenyl)-1H-indol-3-ylmethylene]-hydrazino}-4-methyl-thiazole-5-carboxylic and
4-methyl-2-(6-arylimidazo[2,1-b]-1,3,4-thiadiazole-5-ylmethylidene)hydrazone-2,3-
TE D
acids
dihydrothiazole-5-carboxylic acids synthesis (76-100).
EP
To the appropriate thiosemicarbazone (1.0 eq) in acetic acid bromoacetophenone or ethyl 2chloroacetoacetate (1.1 eq.) and anhydrous sodium acetate (1.0 eq.) were added. The reaction mixture was
AC C
hated under reflux for 3 hours. Then the mixture was cooled to the room temperature and formed precipitate was filtered off, washed with acetic acid, water and ethanol, dried and recrystallized.
(N-[4-(4-Bromophenyl)-thiazol-2-yl]-N'-[2-(4-chlorophenyl)-1H-indol-3-ylmethylene]-hydrazine
(76).
Yield 80%, mp 320-322оС. 1H NMR (400 MHz, DMSO-d6): δ 7.22 (t, 1H, J = 7.2 Hz, arom.), 7.27 (t, 1H, J = 7.0 Hz, arom.), 7.38 (s, 1H, CH), 7.47 (d, 1H, J = 7.7 Hz, arom.), 7.60 (d, 2H, J = 8.6 Hz, arom.), 7.68 (brs, 4H, arom.), 7.81 (d, 2H, J = 8.4 Hz, arom.), 8.33 (d, 1H, J = 7.6 Hz, arom.), 8.38 (s, 1H, CH), 11.91 43
ACCEPTED MANUSCRIPT (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 164.5, 159.4, 147.2, 145.0, 139.0, 138.4, 137.5, 134.2,
131.7, 130.2, 129.8, 128.0, 123.9, 124.0, 122.5, 121.7, 120.8, 119.0, 111.8, 103.8. LCMS (ESI) m/z 507/509 (M+H)+. Calcd. for C26H16BrClN4S: C, 56.76; H, 3.18; N, 11.03; Found: C, 56.90; H, 3.40; N,
RI PT
10.90%.
N-[2-(4-Nitrophenyl)-1H-indol-3-ylmethylene]-N'-(4-phenylthiazol-2-yl)-hydrazine (77). Yield 79%, mp
SC
267-269оС. 1H NMR (400 MHz, DMSO-d6): δ 7.15-7.30 (m, 1H, arom.), 7.31-7.37 (m, 2H, arom.), 7.43 (t, 2H, J = 7.3 Hz, arom.), 7.47-7.54 (m, 2H, arom.), 7.64-7.72 (m, 1H, arom.), 7.88 (d, 1H, J = 7.6 Hz,
M AN U
arom.), 7.96 (d, 2H, J = 8.3 Hz, arom.), 8.13-8.20 (m. 1H, arom.), 8.43 (d, 2H, J = 8.5 Hz, arom.), 8.48 (s, 1H, CH),12.15 (s, 1H, NH).13C NMR (100 MHz, DMSO-d6): δ 168.8, 161.4, 149.9, 147.4, 139.6, 138.1, 137.6, 134.6, 130.5, 129.1, 128.2, 126.1, 125.7, 124.5, 124.5, 122.8, 121.8, 112.5, 110.4, 103.7. LCMS (ESI) m/z 440 (M+H)+. Calcd. for C24H17N5O2S: C, 65.59; H, 3.90; N, 15.93; Found: C, 65.70; H, 4.10; N,
TE D
16.10%.
N-[4-(4-Bromophenyl)-thiazol-2-yl]-N'-[2-(4-nitro-phenyl)-1H-indol-3-ylmethylene]-hydrazine
(78).
Yield 80%, mp 252-254оС. 1H NMR (400 MHz, DMSO-d6): δ 7.22 (t, 1H, J = 7.2 Hz, arom.), 7.27 (t, 1H,
EP
J = 7.0 Hz, arom.), 7.42-7.47 (m, 2H, arom.), 7.58-7.60 (m, 2H, arom.), 7.68 (brs, 4H, arom.), 7.81 (d, 2H, 13
C NMR (100 MHz,
AC C
J = 8.4 Hz, arom.), 8.43 (m, 1H, arom.), 8.98 (s, 1H, CH), 12.13 (s, 1H, NH).
DMSO-d6): δ 164.6, 155.9, 147.4, 144.6, 139.0, 138.1, 137.5, 134.4, 132.0, 130.4, 130.1, 128.0, 124.5, 124.1, 122.8, 121.7, 121.0, 119.3, 112.4, 104.5. LCMS (ESI) m/z 519/520 (M+H)+. Calcd. for C24H16BrN5O2S: C, 65.59; H, 3.90; N, 15.93; Found: C, 55.61; H, 3.11; N, 13.51%.
N-[2-(4-Nitrophenyl)-1H-indol-3-ylmethylene]-N'-[4-(4-nitrophenyl)-thiazol-2-yl]-hydrazine (79). Yield 77%, mp 300-302оС. 1H NMR (400 MHz, DMSO-d6): δ 7.27 (t, 1H, J = 7.2 Hz, arom.), 7.33 (t, 1H, J = 44
ACCEPTED MANUSCRIPT 7.2 Hz, arom.), 7.51 (d, 1H, J = 7.9 Hz, arom.), 7.72 (s, 1H, arom.), 7.94 (d, 2H, J = 8.6 Hz, arom.), 8.12 (d, 2H, J = 8.7 Hz, arom.), 8.28 (d, 2H, J = 8.8 Hz, arom.), 8.37 (d, 1H, J = 7.9 Hz, arom.), 8.43 (d, 2H, J = 8.8 Hz, arom.), 8.45 (s, 1H, CH), 12.13 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 167.3, 166.8,
RI PT
147.4, 146.6, 138.1, 137.5, 130.4, 130.1, 126.8, 126.8, 125.7, 24.9, 124.5, 124.5, 124.1, 121.8, 119.3, 112.4, 110.4, 108.4. LCMS (ESI) m/z 485 (M+H)+. Calcd. for C24H16N6O4S: C, 59.50; H, 3.33; N, 17.35;
SC
Found: C, 59.70; H, 3.50; N, 17.10%.
N-(2-Ethyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)-N'-[4-(4-nitrophenyl)-3H-thiazol-2-
M AN U
ylidene]-hydrazine (80). Yield 78%, mp 246-247оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.42 (t, 3Н, J = 7.2 Hz, СН3), 3.15 (q, 2Н, J = 7.3 Hz, СН2СН3), 7.42 (t, 1Н, J = 7.0 Hz, arom.), 7.52 (t, 2Н, J = 7.2 Hz, arom.), 7.73 (s, 1Н, thiaz), 7.88 (d, 2Н, J = 7.4 Hz, arom.), 8.10 (d, 2Н, J = 8.3 Hz, arom.), 8.27 (d, 2Н, J = 8.0 Hz, arom.), 8.42 (s, 1Н, 5-Н, imidaz), 12.27 (s, 1Н, NH). 13C NMR (100 MHz, DMSO-d6): δ 169.0, 167.4, 149.0, 147.3, 146.7, 146.0, 141.2, 134.1, 131.1, 129.0, 128.6, 126.8, 124.6, 119.6, 112.6, 109.4,
55.70; H, 3.80; N, 20.50%.
TE D
25.7, 13.0. LCMS (ESI) m/z 476 (M+H)+. Calcd. for C22H17N7O2S2: C, 55.57 H, 3.60; N, 20.62; Found: C,
EP
Crystal structure determination of compounds
AC C
Acetic acid 4-{2-[2-(cyclohexylidene-hydrazono)-4-oxothiazolidin-5-yl]-acetylamino}-phenyl ester (1f). Crystal data: C19H22N4O4S, 1.25H2O, Mr = 424.48, triclinic, space group P-1, a = 6.2209(4), b = 13.0177(8), c = 13.7897(5) Å, α = 69.084(5), β = 81.087(5), γ = 80.209(5)°, V = 1022.54(11) Å3, T = 130.0(1) K, Z = 2.
Data collection. A colourless lath (methanol/water) crystal of 0.45 x 0.18 x 0.06 mm was used to record 27043 (Mo Kαradiation, θmax = 29.09°) intensities on an Xcalibur A diffractometer [67]. Accurate unit cell parameters were determined by least-squares techniques from the θ values of 9292 reflections, θ 45
ACCEPTED MANUSCRIPT range 2.68–29.02°. The data were corrected for Lorentz, polarization and for absorption effects [67]. The 5056 total unique reflections (Rint = 0.029) were used for structure determination. The crystallographic data in the CIF form are available as Electronic Supplementary data from the Crystallographic
Data
Centre,
deposition
number
CCDC-1876805;
RI PT
Cambridge
http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre,
SC
12 Union Road, Cambridge, CB2 1EZ, UK; fax: ţ44 1223 336033; e-mail: [email protected]).
5-Ethyl-2-[(2-phenyl-1H-indol-3-yl-methylene)-hydrazono]-thiazolidin-4-one (7).
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Crystal data: C20H18N4OS, Mr = 362.44, monoclinic, space group P21/c, a = 8.7322(3), b = 21.9382(7), c = 8.8824(3) Å, β = 92.867(3)°, V = 1699.47(10) Å3, T = 130.0(1) K, Z = 4. Data collection. A light-yellow block (EtOH) crystal of 0.45 x 0.35 x 0.15 mm was used to record 16730 (Mo Kα radiation, θmax= 27.10°) intensities on an Xcalibur A diffractometer [67]. Accurate unit cell parameters were determined by least-squares techniques from the θ values of 8429 reflections, θ range
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2.29–29.10°. The data were corrected for Lorentz, polarization and for absorption effects [67]. The 3748 total unique reflections (Rint = 0.031) were used for structure determination.
Cambridge
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The crystallographic data in the CIF form are available as Electronic Supplementary data from the Crystallographic
Data
Centre,
deposition
number
CCDC-1876806;
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http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ, UK; fax: ţ44 1223 336033; e-mail: [email protected]).
Biology
Antitrypanosomal activity. Bloodstream forms of Trypanosoma brucei brucei strain 90-13 and Trypanosoma brucei gambiense Feo strain were cultured in HMI9 medium supplemented with 10% FCS at 37°C under an atmosphere of 5% CO2 [65]. In all experiments, log-phase parasite cultures were 46
ACCEPTED MANUSCRIPT harvested by centrifugation at 3000xg and immediately used. Drug assays were based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable cells as previously described [66]. Drug stock solutions were prepared in pure DMSO. T. brucei blood stream forms (105cells/ml) were
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cultured in 96-well plates either in the absence or in the presence of different concentrations of inhibitors in a final volume of 200 µl. After a 72-h incubation, resazurin solution was added in each well at the final concentration of 45µM and fluorescence was measured at 530 nm and 590 nm absorbance after a further
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4-h incubation. The percentage of inhibition of parasite growth rate was calculated by comparing the fluorescence of parasites maintained in the presence of drug to that of in the absence of drug. DMSO was
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used as control. Concentration inhibiting 50% of parasite growth (IC50) was determined from the doseresponse curve with a drug concentrations ranging from10 µg/ml to 0.625 µg/ml. IC50value is the mean +/- the standard deviation of three independent experiments.
In vitro cytotoxicity assay on mammalian cell. Cytotoxicity upon human primary fibroblast (cell line
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AB943). Assays were performed in 96-well plates in RPMI medium containing 25 mM HEPES, pH 7.3, 10% fetal calf serum under 5% CO2 atmosphere, at 37 oC. After trypsin treatment, L-6 cells were seeded at 5000 cells per well in 100 mL. After 24 h incubation, cells were washed and two-fold dilutions of drug
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were added (200 mL perwell). Drug stock solutions were prepared in pure DMSO. The final DMSO
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concentration in the cultures remained below 1%. Control cultures were constituted of cultures treated with pure DMSO instead of drug. The cytotoxicity assay was based on the conversion of a redoxsensitive dye (resazurin) to afluorescent product by viable cells [68]. After 5 days of incubation, resazurin solution was added in each well at thefinal concentration of 45 mM. Fluorescence was measured at 530 nm excitation and 590 nm emission wavelengths after a further 4-h incubation. The percentage of inhibition of cell growth was calculated by comparing thefluorescence of cells maintained in the presence of drug to that of in the absence of drug. 47
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Acute toxicity.The experiments were conducted on white male mice weighing 23-25 g. Compounds were dissolved in saline solution (0.9% NaCl) with l-2 drops of Polysorbate 80 (Tween-80®). After dissolution
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they were administered to mice via intraperitoneal route. The LD50 was evaluated for 4 or 5 different doses each on 6 animals and calculated by the Litchfield-Wilcoxon method [69,70].
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ACKNOWLEDGMENT
This work was partially supported by Ukrainian-France program “DNIPRO” and the Ministry of
SUPPLEMENTARY MATERIALS
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Education and Science of Ukraine (M/188-2015; M/71-2016).
Supplementary materials present the detailed analytical and spectral data of synthesized compounds, X-
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ray data analysis and are avaible at _______.
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C. A.; Bortoluzzi, J. H.; Scotti, M. T.; Scotti, L.; Meneghetti, M. R.; Aquino, T. M.; Araújo-Júnior, J. X. Design, Synthesis, Molecular Docking and Biological Evaluation of Thiophen-2-iminothiazolidine Derivatives for Use Against Trypanosoma cruzi. Bioorg. Med. Chem. 2016, 24, 4228-4240. https://doi.org/10.1016/j.bmc.2016.07.013 [52] Bhongade, B. A.; Talath, S.; Gadad, R. A.; Gadad, A. K. Biological Activities of Imidazo[2,1b][1,3,4]thiadiazole
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[65] Bastos, I.; Motta, F. N.; Charneau, S.; Santana, J. M.; Dubost, L.; Augustyns, K.; Grellier, P. Prolyl
the
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Oligopeptidase of Trypanosoma brucei Hydrolyzes Native Collagen, Peptide Hormones and is Active in Plasma
of
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https://doi.org/10.1016/S0079-6468(08)70386-2
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ACCEPTED MANUSCRIPT
Highlights for the manuscript
“Thiazolidinone/thiazole based hybrids – new class of antitrypanosomal agents”
by Anna Kryshchyshyn, Danylo Kaminskyy, Oleksandr Karpenko,
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Andrzej Gzella, Phillipe Grellier, Roman Lesyk
4-Thiazolidinone/thiazole & phenylindole/phenylimidazothiadiazole hybrids
•
Compounds were highly active toward Trypanosoma brucei brucei and gambiense strains
•
Compounds possessed relatively low cytotoxicity (fibroblasts) and acute toxicity
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•
S0223-5234(19)30370-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.052
Reference:
EJMECH 11287
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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
THIAZOLIDINONE/THIAZOLE BASED HYBRIDS – NEW CLASS OF ANTITRYPANOSOMAL AGENTS Anna Kryshchyshyna, Danylo Kaminskyya, Oleksandr Karpenkob,
O
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Andrzej Gzellac, Phillipe Grellierd, Roman Lesyka,e
R
R N
N
R
S
S
R
N
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N
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NH
O H3C
NH
N
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N H
Trypanosoma brucei brucei IC50 = 30 nM Trypanosoma brucei gambience IC50 = 110 nM
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N
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S
N
N
R
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N
R
N
R
N N R
N S
ACCEPTED MANUSCRIPT THIAZOLIDINONE/THIAZOLE BASED HYBRIDS – NEW CLASS OF ANTITRYPANOSOMAL AGENTS Anna Kryshchyshyna, Danylo Kaminskyya, Oleksandr Karpenkob,
a
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Andrzej Gzellac, Phillipe Grellierd, Roman Lesyka,e
Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National
Medical University, Pekarska 69, Lviv, 79010, Ukraine Enamine Ltd., Chervonotkatska 78, Kyiv, 02094, Ukraine
c
Department of Organic Chemistry, Karol Marcinkovski Poznan University of Medical
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b
Sciences,Grunwaldzka 6, Poznan, 60-780, Poland d
National Museum of Natural History, UMR 7245 CNRS-MNHN, Team BAMEE, CP 52, 57 Rue Cuvier,
75005, Paris, France e
Department of Public Health, Dietetics and Lifestyle Disorders, Faculty of Medicine, University of
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Information Technology and Management in Rzeszow, Sucharskiego 2, 35-225 Rzeszow, Poland
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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
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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
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antitrypanosomal activity towards Trypanosoma brucei brucei and Trypanosoma brucei gambiense. Biological study allowed identifying compounds with the submicromolar levels of IC50, good selectivity
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indexes and relatively low cytotoxicity upon human primary fibroblasts as well as low acute toxicity.
KEYWORDS: thiazoles, thiazolidinones, indoles, imadazothiadiazoles, hybrids, antitrypanosomal
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activity
2
ACCEPTED MANUSCRIPT 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
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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].
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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
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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
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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].
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Quite a lot of different chemical classes of compounds, e.g. thiosemicarbazones, thiazolidines,
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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 [1315]. 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 kinase3
ACCEPTED MANUSCRIPT inhibitors library against Tb allowed identifying a series of active compounds based on 2,4diaminothiazoles 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 reported
to
inhibit
the
activity
of
Tb
dolicholphosphate
mannose
synthase
and
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were
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
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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].
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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
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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
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of computer chemistry too [36], e.g. 2-hydrazolyl-4-thiazolidinones are potent cruzipain inhibitors
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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
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for neglected diseases initiative) one of the new chemical entities to enter clinical development and in 2016 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].
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Screening of over 300000 compounds in ornithinedecarboxylase inhibition high throughput assay discovered four scaffold classes, among which were benzthiazoles and indole derivatives [46]. The
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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 1Hindole 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
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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
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agents [49]. Considering the phthalimide fragment as indole bioisoster, molecules bearing it should also
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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
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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
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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-
Known lead-compounds S R
S
R
N H
N H
R
N H
N H
R
N
O Ar
Ar
N
2
Ar
S
R
R
O
[Ref 25]
N
R
N N
R S
OH B O
R
2
R
B O
1
Me
Me [Ref 45] O O2N
Target compound srtucture
N R
1
N R
[Ref 47]
R R
R
N S
R
R
S
R N
N R
N
N
2
R N
R
N
Me
OH
O
S
N
Cl
H N
Me
O
N H
[Ref 36] F
O
R
N
N
Me SCYX-7158 [Ref 43]
[Ref 26]
O
Cl H N
N
S
AC C
Ar
H N
CF3 O
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S 2
HO
H N
N
R
1
Known lead-compounds
N N
N R
S
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H N
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imidazothiadiazole fragment linked by hydrazone moiety (Fig. 1).
N
S
R
N
N
R R
N N R S
N H
N
6
N N R
S
N
ACCEPTED MANUSCRIPT Figure 1. Background of the target compounds design.
RESULTS AND DISCUSSION
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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 reaction of thiosemicarbazones and different [C2]2+ electrophilic synthons (maleimides, maleic anhydride, β-aroylacrylic acids) [16,58]. The reactions were performed in glacial acetic acid medium. For compound
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1d, the [C2]2+ reactant was prepared in situ via the reaction of maleic anhydride and appropriate amino
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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
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well as by some aspects of the SAR-analysis of different hydrazono-thiazolidinones [7,33,35].
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1
O
R N
N N
S O
S [C2]2+
+
1a-1j
i
HN N 3
R
2
R
1e 1f 1g 1h 1i 1j
4-F-C6H4 4-MeOOC-C6H4 4-NO2-C6H4 4-Br-C6H4 4-Cl-C6H4 4-Me-C6H4
3
R
HN
1
R
O N
N N
S R
O
1k 1l 1m 1n 1o 1p 1q 1r
2
R 3
R
[C2]2+ = O O ;
N R ; O
OH
O O O
O
O
OH
;
+
R
H2N
A
O
B
R2 R3 H furan-2-yl H PhCHCH H 4-Me2N-C6H4 H PhCHCH H Ph H 3-MeO-4-HOOCCH2O-C6H3 H 4-NO2-C6H4CHCCl H 2-NO2-C6H4CHCH R2+ R3 A B
H H Me
Me
C
Me
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O
O
1s 4-F-C6H4 1t 4-F-C6H4
R3 PhCHCH PhCHCH PhCHCH fyran-2-yl
R2+ R3 A A A B B B
H H H H H H
R1 H H allyl H allyl 4-HO-C6H4 4-HO-C6H4 H
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R C C 4-F-C6H4 4-F-C6H4 4-F-C6H4 4-F-C6H4 HO HO
R2 H H H H
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R
2
R
R R1 4-F-C6H4 4-OH-C6H4 4-NO2-C6H4 H 3-CF3-C6H4 furfan-2-yl HOOC(CH2)3 H
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HN R
1a 1b 1c 1d
Scheme 1. Synthesis of 4-thiazolidinone-hydrazones. Reagents and conditions: i – substituted thiosemicarbazone (1.0 equiv), appropriate [C2]2+ compound (1.0
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equiv), AcOH, reflux, 3 h; for compound 1d – amino acid (1.0 equiv), maleic anhydride (1.0 equiv),
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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.
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a
b
hydrogen bonding in the crystal structure of 1fx1.25 H2O.
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Figure 2. (a) ORTEP view of the molecule 1f and H2O showing the atomic labelling scheme. (b) The
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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).
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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
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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
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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.
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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
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synthesized according to the known protocols: 1) indole bearing thiosemicarbazones – via Fischer indole synthesis, Vilsmeier-Haack reaction and condensation with thiosemicarbazides; 2) imidazothiadiazole
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bearing compounds were synthesized in the three step synthetic procedure from 5-substituted 2-amino-
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1,3,4-thiadiazole and α-halogenoarylketone [62] (Scheme 2).
10
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H ii R
R
H N
+ NH2
R
S
R
R
Me
1
S
2
N H
A
O
N N H2N
N H
O
+
N N H
H
X
=
R
N H 4
R
Hal
O
1
N
S
2a 2b 2c 2d 2e
N H
R H H H Br NO2
N R
R
S
R1 3a 3b 3c 3d 3e
S-CH2CH=CH2 S-CH2COOEt Et Et Et
N H R H H H Br NO2
3 R1
S-CH2CH=CH2 S-CH2COOEt Et Et Et
N
R
Synthesis
1
A5: A6: A7: A8: A9:
of
the
starting
N
R N H R = H R1 = S-CH2CH=CH2 R = H R1 = S-CH2COOEt R = H R1 = Et R = NO2 R1 = Et R = Br R1 = Et
TE D
R
S
phenyl-indole
or
phenyl-imidazothiadiazole
bearing
AC C
thiosemicarbazones.
N H R=H R = Cl R = Br R = NO2
EP
A1: A2: A3: A4:
2.
ii
R
2
A=
Scheme
N
1
R2 H H 4-HO-C6H4 furfuryl H H 4-HO-C6H4 H 4-HO-C6H4 H H 4-HO-C6H4 H 4-HO-C6H4 H 4-HO-C6H4
M AN U
R
i
SC
H N
A A1 A2 A2 A2 A3 A4 A4 A5 A5 A6 A7 A7 A8 A8 A9 A9
RI PT
N H
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p
Reagents and conditions: i –mixture of DMF and POCl3 (1/3 vol), stirring 10 min at 0ºС, imidazo[2,1b][1,3,4]thiadiazole, 30 min at 0ºС, 2 h at rt and 2 h at 60ºС, Na2CO3 (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). 11
ACCEPTED MANUSCRIPT
2
OH
1
O
R
O
Cl
N 2
R
N
S
N S
1
R
N
A
N N H
R
X
=
H i
N H 2
R
O O
1
2
43 44 45 46 47
N
R
S
N H
43-47
R2 H Me Et H Me Et Me Et H Me Et H Me Et Me Et H Me Et Et Me
R
M AN U
O
N
R1 H H H H H H H H H H H H H H H H H H H H H
SC
A
5-42
A A1 A1 A1 A2 A2 A2 A3 A3 A4 A4 A4 A5 A5 A5 A6 A6 A7 A7 A7 A8 A9
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
A A9 A2 A2 A2 A2 A2 A2 A4 A4 A5 A5 A5 A7 A7 A7 A9 A9
RI PT
R
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
N
N
O
A A1 A1 A1 A1 A2
R1 R2 H Et furfuryl H furfuryl Me furfuryl Et 4-HO-C6H4 H 4-HO-C6H4 Me 4-HO-C6H4 Et 4-HO-C6H4 Me 4-HO-C6H4 Et 4-HO-C6H4 H 4-HO-C6H4 Me 4-HO-C6H4 Et 4-HO-C6H4 H 4-HO-C6H4 Me 4-HO-C6H4 Et 4-HO-C6H4 Me 4-HO-C6H4 Et
R1 R2 H Cl H Br H OH H COOEt H OH
A
Scheme 3. Synthesis of target 4-thiazolidinone-indole/imidazothiadiazole hybrids.
TE D
Reagents and conditions: i – substituted thiosemicarbazone (1.0 equiv), appropriate [C2]2+ compound (1.0 equiv), AcONa (1.0 equiv, for 5-42), AcOH, reflux, 3 h. Structure of fragment A – see scheme 2.
EP
α-Halogenocarboxylic acids, N-arylmaleimides, maleic anhydride, β-aroylacrylic acids and 2bromobutyrolactone were used as [C2]2+reagents. Utilizing 2-bromobutyrolactone, compounds with free or
AC C
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.
12
ACCEPTED MANUSCRIPT
R
O
Br
O
i
R
1
N
2
O
N
S
N S N H
A
N N H
O
HO O
A
=
R
X
ii O
N H
N
Ar
or O
R
O
H
S O
R
1
N
N
2
O
A
O
R
O
iii
R1 H H H H H H H H H H H furfuryl furfuryl 4-HO-C6H4 4-HO-C6H4 4-HO-C6H4 4-HO-C6H4 4-HO-C6H4
66 67 68 69 70 71 72
A A1 A1 A1 A1 A2 A3 A5
R1 H H H H H H H
R2 Ph 4-F-C6H4 tetralin-6-yl OH OH OH OH
73 74 75
A A1 A1 A1
R1 H H H
R2 HOOCCH2 HOOC(CH2)2 HOOC(CH2)5
SC
1
M AN U
R
A A1 A1 A2 A2 A3 A3 A4 A5 A7 A8 A9 A2 A2 A2 A5 A7 A8 A9
RI PT
O
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1
N
O
O
+
OH
H2N (CH2)n
S
2
TE D
R
N H
R2 H Ac H Ac H Ac Ac Ac Ac Ac Ac H Ac Ac Ac Ac Ac Ac
N
N O A
O
Scheme 4. Synthesis of target 4-thiazolidinone-indole/imidazothiadiazoles hybrids.
EP
Reagents and conditions: i – Method A: thiosemicarbazone (1.0 equiv), α-bromo-γ-butyrolactone (1.0
AC C
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 βaroylacrylic 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.
13
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
R
2
N O Hal
R
A
OH
N
N N H
R1 = 4-HO-C6H4
N
S
H
N
R
X
=
2
M AN U
S N H
SC
A
2
R
N
S
R1 = H
R
1
H N
RI PT
5) [27,41].
N H
76 77 78 79 80 81 82 83
A A2 A3 A4 A4 A7 A8 A8 A9
84 85 86 87 88 89
A A4 A7 A8 A8 A9 A9
90 91 92 93 94 95
A A2 A4 A5 A7 A8 A9
96 97 98 99 100
A A4 A5 A7 A8 A9
R2 Br NO2 Br NO2 NO2 NO2 Br NO2
R2 NO2 H Br NO2 Br NO2
A
Me
N
R1 = H
Et
O
NH
TE D
S O
N A OH
Et
O
O
Cl
EP
O
Me
R1 = 4-HO-C6H4
AC C
Me
N Et
O S O
N N A
Scheme 5. Synthesis of target 4-thiazole-indole/imidazothiadiazoles hybrids. Reagents and conditions: thiosemicarbazone (1.0 equiv), appropriate [C2]2+ compound (1.0 equiv), AcONa (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 14
ACCEPTED MANUSCRIPT spectral data (1H NMR,
13
C NMR, LCMS) confirmed the structure and purity of compounds. 1H NMR
spectra of target compounds showed characteristic patterns of protons. Additionally, the X-ray analysis
SC
RI PT
was performed for the compound 7 (Fig. 3).
M AN U
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.
TE D
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
EP
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
AC C
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-1,3thiazolidin-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,
15
ACCEPTED MANUSCRIPT C26 from the 5-ethyl-1,3-thiazolidin-4-one fragment. Each of these atoms takes up two alternative
a Figure 4. The hydrogen bonding in 7.
M AN U
SC
RI PT
locations in the crystal structure labelled a and b (Fig. 4)
b
H atoms not involved in hydrogen bonds have been omitted for clarity.
TE D
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
calculated [65,66].
EP
results are presented as growth inhibition percentage. For the active compounds, IC50 values were
activity
AC C
Antitrypanosomal
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.
16
ACCEPTED MANUSCRIPT Table 1. Antitrypanosomal activity of 4-thiazolidinones without indole or imidazothiadiazole fragment towards Trypanosoma brucei brucei Inhibition, %
Inhibition, %
1 µg/mL
1a
-8.23
1.18
1l
1b
56.11
NT
1m
1c
98.91
5.06
1n
1d
10.03
0.06
1o
1e
-4.66
-4.65
1p
1g
44.88
1h
96.98
1i
10 µg/mL
M AN U
10 µg/mL
1 µg/mL
RI PT
Compound
53.24
7.69
14,70
11.14
57.30
38.70
SC
Compound
14.70
11.14
14.49
15.08
1q
25.01
-1.57
NT
1r
24.86
3,28
97.80
14.13
1s
98.53
8.16
1j
29.97
4.18
1t
42.01
36.13
1k
10.91
9.82
TE D
NT
EP
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).
AC C
The minimum activity required for the characterization of the tested compound as a hit was the IC50 ≤ 1.0 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/4thiazolidone 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 IC50 values (43,
17
ACCEPTED MANUSCRIPT 44, 46, 66). The influence of acetamide and tetrahydronaphthalene fragments was similar and caused even more activity decreasing.
H N
O
N N
S R
R
7
Et
IC50, µM
M AN U
Compound
SC
NH
RI PT
Table 2. The IC50 values of 4-thiazolidones with indole fragment against Trypanosoma brucei brucei.
0.03 ± 0.004
4-ClC6H4-NHCOCH2
9.16±1.142
44
4-BrC6H4-NCOCH2
7.86±0.260
46
4-EtCOOC6H4NHCOCH2
6.86±0.261
48
HOCH2CH2
0.17± 0.004
49 66
EP
67
TE D
43
AC C
68
MeCOO(CH2)2
0.06 ± 0.003
Ph-COCH2
7.73±0.310
4-FC6H4-COCH2
10.63±1.810
O
13.13±0.97
74
HOOC(CH2)2NHCOCH2
34.84±0.450
75
HOOC(CH2)5NHCOCH2
14.04±0.830
Pent
0,0014 ± 0.0005
Nf
2,39 ± 0,608
Data are presented as mean ± SD; Pent – Pentamidine, Nf – Nifurtimox
18
ACCEPTED MANUSCRIPT After establishing significant antitrypanosomal properties in the Tb brucei assay, a series of 2-[(2phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-ones was tested towards Tb gambiense (Table 3). The impact of the substituents in the p-position of phenyl ring (R3, Table 5) and N3-position of 4-
RI PT
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
SC
(49) moieties didn’t influence the activity.
M AN U
Table 3. The IC50 values of 4-thiazolidones with indole fragment against Trypanosoma brucei gambiense R
R
2
N
O
N
S R
1
3
N
NH
R1
R2
R3
IC50, µM
5
H
H
H
0.23 ± 0.009
Me
H
H
0.21± 0.014
Et
H
H
0.11± 0.040
H
H
Cl
3.93 ± 0.363
7
AC C
8
EP
6
TE D
Compound
9
Me
H
Cl
2.22 ± 0.071
10
Et
H
Cl
1.42 ± 0.098
11
Me
H
Br
1.35 ± 0.126
12
Et
H
Br
1.50 ± 0.111
14
Me
H
NO2
4.70 ± 0.500
15
Et
H
NO2
6.20 ± 2.200
19
ACCEPTED MANUSCRIPT H
furan-2-ylmethyl
Cl
7.31 ± 1.354
28
Me
furan-2-ylmethyl
Cl
1.71 ± 0.194
29
Et
furan-2-ylmethyl
Cl
1.71 ± 0.161
30
H
4-HO-C6H4
Cl
0.82 ± 0.111
31
Me
4-HO-C6H4
Cl
32
Et
4-HO-C6H4
Cl
34
Et
4-HO-C6H4
NO2
49
MeCOO(CH2)2
H
50
HOCH2CH2
H
51
MeOOCCH2CH2
52
OHCH2CH2
53
MeOOCCH2CH2
59
RI PT
27
1.17 ± 0.126 0.86 ± 0.076
SC
0.14 ± 0.010 0.23± 0.014
Cl
1.49 ± 0.104
M AN U
H
Cl
1.34 ± 0.057
H
Br
3.34 ± 0.534
H
Br
1.36 ± 0.018
HOCH2CH2
furan-2-ylmethyl
Cl
1.40 ± 0.158
60
MeOOCCH2CH2
furan-2-ylmethyl
Cl
1.59 ± 0.084
61
MeOOCCH2CH2
4-HO-C6H4
Cl
0.38 ± 0.080
66
PhCOCH2
H
H
2.82 ± 0.426
4.64 ± 0.725
AC C
Nf
0.0015± 0.0007
EP
Pent
TE D
H
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-3ylmethylene)-hydrazono]-thiazolidin-4-one – IC50 = 9.68±1.8 (Tb brucei), 30.4±8.4 (Tb gambiense), 520
ACCEPTED MANUSCRIPT ethyl-2-[(1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-one – IC50 = 39.51±9.7 (Tb brucei), 73.6±6.3 (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
RI PT
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
SC
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-
M AN U
[(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 IC50 for the derivatives 90, 91 and 96 were much
TE D
higher than those for the unsubstituted analogs.
AC C
EP
Table 4. The IC50 values, µM of thiazoles with indole fragment against Trypanosoma brucei gambiense R
R
1
4
N R
2
R
S
N N
3
N H
Compound
R1
R2
R3
R4
IC50,µM
76
4-Br-C6H4
H
Cl
-
1.52 ±0.400
77
Ph
H
NO2
-
6.00 ±0.600
78
4-BrC6H4
H
NO2
-
2.30 ±0.900
21
ACCEPTED MANUSCRIPT 4-NO2C6H4
H
NO2
4-HO-C6H4
1.50 ± 0.200
90
Me
C2H5OOC
Cl
-
0.53 ±0.205
91
Me
C2H5OOC
NO2
-
0.74±0.068
96
Me
C2H5OOC
NO2
4-HO-C6H4
0.66 ±0.030
Antitrypanosomal
activity
of
RI PT
84
thiazolidinone-hydrazones
with
phenyl-imidazo[2,1-
SC
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
M AN U
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, 4n, 4p was evaluated. SAR data suggested that imidazothiadiazole core itself weakly contributed to the
TE D
activity, but the introduction of thiosemicarbazone fragment improved antitrypanosomal properties.
Table 5. The IC50 values, µM of imidazothiadiazole bearing compounds against Trypanosoma brucei
AC C
EP
gambiense
Compound
R1
2a
S-CH2CHCH2
2b
S-CH2COOEt
2c
Et
3a
S-CH2CH=CH2
2
R
N N
3
R
R2
N
1
R
S
R3
IC50, µM 27.2±1.40
H
H
>30 >40
CHO
22
H
23.4±1.40
ACCEPTED MANUSCRIPT 3c
Et
> 40
4h
S-CH2CH=CH2
4k
Et
H N
H2N
Et
4i
S-CH2CH=CH2
4l
Et
4n
Et
4p
Et
HO
20.9±0.60
NO2
>25
RI PT
4o
H
S N
N H
>25
H
>50
H
2.6±0.50
NO2
14.8±0.90
Br
1.5±0.02
M AN U
N H
Br
SC
Et
6.1±0.30
N
S
4m
H
Table 6. The IC50 values, µM of imidazothiadiazoles’ hydrazones against Trypanosoma brucei gambiense R N
N
TE D
O
R
N
S
3
R
N
N
R2
R3
IC50, µM
H
H
H
3.9 ± 0.70
H
H
Me
4.4± 0.70
18
H
H
Et
20.6± 4.70
35
4-HO-C6H4
H
H
> 20
36
4-HO-C6H4
H
Me
> 20
37
4-HO-C6H4
H
Et
3.6± 1.30
55
H
H
AcOCH2CH2
5.7± 0.60
16
AC C
17
R
EP
Compound
R1
1
S
N
2
R
S
CH2
23
ACCEPTED MANUSCRIPT 4-HO-C6H4
H
AcOCH2CH2
1.00± 0.04
72
H
H
HOOCCH2
>20
19
H
H
Me
3.9± 0.30
20
H
H
Et
9.2± 0.60
21
H
H
22
H
H
23
H
H
24
H
NO2
25
H
Br
26
H
38
4-HO-C6H4
39
4-HO-C6H4
40
4-HO-C6H4
41
O
H
18.0± 2.30
Me
>25
Et
3.7± 0.10
SC
OEt
S
RI PT
62
>20
Me
9.5± 1.30
Br
Et
13.8± 1.40
H
H
9.2± 0.50
H
Me
4.8± 0.20
H
Et
>20
4-HO-C6H4
Br
Me
1.5± 0.03
42
4-HO-C6H4
Br
Et
1.5± 0.04
56
H
H
AcOCH2CH2
11.8± 2.00
H
NO2
AcOCH2CH2
16.2± 0.70
H
Br
AcOCH2CH2
3.2± 0.10
TE D
AC C
58
Et
EP
57
M AN U
Et
63
4-HO-C6H4
H
AcOCH2CH2
2.7± 0.80
64
4-HO-C6H4
NO2
AcOCH2CH2
11.1± 1.60
65
4-HO-C6H4
Br
AcOCH2CH2
1.0± 0.10
R
H N
N
R
S
3
R
N N N
2
24
N S
R
1
ACCEPTED MANUSCRIPT R3
IC50, µM
H
EtOOC
0.652± 0.113
4-NO2-C6H4
H
H
10.3
81
4-NO2-C6H4
NO2
H
4.4
82
4-Br-C6H4
NO2
H
1.2
83
4-NO2-C6H4
H
9.2± 0.80
93
Me
H
EtOOC
2.7 ± 0.90
94
Me
NO2
95
Me
Br
Me
80
S
CH2
Br
Et
SC
92
R1
EtOOC
>20
EtOOC
7.4± 0.40
M AN U
R
RI PT
R2
Compound
OH
R
R
Me
2
N
N N N
R1
S
CH2
Ph
AC C
85
S
TE D
97
EP
R
N
3
R
Compound
N
R
1
S
R2
R3
IC50, µM
H
EtOOC
0.631 ± 0.066
H
H
3.0 ± 0.30
86
4-Br-C6H4
NO2
H
0.594 ± 0.036
87
4-NO2-C6H4
NO2
H
2.6± 0.70
88
4-Br-C6H4
Br
H
0.476 ± 0.066
89
4-NO2-C6H4
Br
H
0.510 ± 0.110
98
Me
H
EtOOC
0.745 ± 0.098
99
Me
NO2
EtOOC
0.639 ± 0.074
Et
25
ACCEPTED MANUSCRIPT 100
Me
Br
EtOOC
1.0 ± 0.10
Allylsulfide fragment negatively influences the imidazothiadiazole derivatives activity. Ethyl
RI PT
fragment in C5 possition of thiazolidone in 18 decreased the activity by an order comparing to 5-methyl4-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
SC
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
M AN U
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-6phenyl-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-(4oxothiazolidin-5-yl)-ethylester (63) with IC50=2.7 µM for both. p-Hydroxyphenyl fragment in the N3 of
TE D
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 86, 88, 89, 100 and 98 showed submicromolar inhibition concentrations. It is worthwhile remarking that
EP
halogen atoms as well as nitro group in the benzene ring can also contribute to the trypanocidal activity of
AC C
the 86, 88, 89, 100. Therefore, the most active compounds of this series appeared to be 2-[(2allylsulfanyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)-hydrazono]-4-methyl-2,3dihydrothiazole-5-carboxylic acid ethyl esters: 92 (IC50=0.652 µM) and 97 (IC50=0.631 µM); and 2-[(2ethyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)-hydrazono]-3-(4-hydroxyphenyl)-4methyl-2,3-dihydrothiazole-5-carboxylic acid ethyl esters 98, 99 and 100 with IC50 within 0.639-1.0 µM, indicating that the presence of 2,3-dihydrothiazole-5-carboxylic acid ethyl ester is crucial for the trypanocidal action of imidazothiadiazoles. 26
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
RI PT
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
SC
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-
M AN U
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
AC C
EP
TE D
structures [53].
27
ACCEPTED MANUSCRIPT
N
S N H
S
N N H X
N N N
H
N
A
R
N H
S
N H
N H
N H
R
R
2
N
N S
favourable for the activity
X Substituents at the phenyl ring
N
S
R
R Alkyl moieties in the C5 are
favourable for the trypanocidal NH activity N
do not greatly contribute to the activity
N
M AN U
R Ester group in the C5 is R
2
SC
O
O
R
A
X
N
1
N N
A
A N H
R
S
H
RI PT
R
X
N N
X
R
N R
3
R
R2 The presence of 4-OH-C6H4 in
R
3
R
2
R2 Aryl fragments in N3-position
N
contriubute to the activity
the N3 possition is controversial R
S
N N
R
TE D
favourable for the activity
2
N
R3 Phenyl fragment is
S
N H
1
N N
X N
R
S
N
N
X
1
N H
S
EP
Activity increasing
AC C
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 IC50 values and no particular impact of different molecular fragments on the toxicity was observed. Nevertheless, 9 compounds possessed very good selectivity indexes (SI > 100) (Tabl. 7).
28
ACCEPTED MANUSCRIPT Table 7. Cytotoxicity upon human primary fribroblast (AB943 cell line) and selectivity indexes IC50, µM
SI
Compound
IC50, µM
SI
5
13.16 ± 2.09
57
65
11.16 ± 0.95
11
6
28.13 ± 3.37
130
78
>96.45
> 40
7
24.83 ± 4.52
220
82
9
5.22 ± 0.19
2
88
30
>100
>130
89
32
85.9 ± 0.43
100
90
34
8.6 ± 0.41
60
92
37
> 90
49
20.92 ± 0.34
50
8.96 ± 2.62
55
19.18 ± 0.76
59 61
RI PT
Compound
>70
>70
> 150
>70
>150
M AN U
SC
>90
20.51 ± 0.91
39
4.33 ± 0.21
7
96
82.9 ± 4.67
127
95
97
> 80
>130
7
98
51.45 ± 8.23
69
3
99
>80
> 130
6.23 ± 0.81
4
Pent
23.20 ± 0.88
5396
>90
> 240
Nf
65.09 ± 2.78
14
TE D
>26
EP
SI = IC50 (AB943) / IC50 (Tbg)
AC C
For derivatives with the high trypanocidal activity, the acute toxicity (mice) was studied and their LD50 were determined. The stock solutions of the compounds used in this study were prepared immediately before usage and increasing amounts of substances (100–1000 mg/kg) were injected intraperitoneally. The LD50 values were calculated according to Litchfield and Wilcoxon. These derivatives showed low acute toxicity in mice with LD50 values within the range of 200–330 mg/kg (Tabl. 8).
Table 8. Acute toxicity of the target compounds 29
ACCEPTED MANUSCRIPT LD50, mg/kg
Compound
LD50, mg/kg
6
220±20.1
32
330±25.3
7
220±13.7
49
200±16.2
8
270±18.2
69
240±20.0
9
310±26.1
70
CONCLUSION
RI PT
Compound
290±19.6
SC
Starting from diverse 4-thiazolidinone-2-hydrazones, new hybrid molecules bearing combination
M AN U
of 4-thiazolidinone or thiazole core and phenyl-indole or 6-phenyl-imidazo[2,1-b][1,3,4]thiadiazole fragment were synthesized. The study of the antitrypanosomal activity towards Trypanosoma brucei brucei and Trypanosoma brucei gambiense strains led to the identification of the hit-compounds with submicromolar IC50 levels, hight selectivity indexes and relatively low cytotoxicity (upon human primary fibroblasts) and acute toxicity (mice). The analysis of structure–activity relationships revealed that
TE D
thiazolidinone/thiazole derivatives were more active than starting thiosemicarbazone analogs; for increase the activity, the C5 substituent of 4-thiazolidinone core should be small and bounded by single bond; the presence of aryl moiety in [6+5] (indole) or [5+5] (imidazothiadiazole) core is essential for
EP
antitrypanosomal activity; the compounds with 2-arylindole fragment are generally more active than 6-
AC C
aryl-imidazo[2,1-b][1,3,4]thiadiazole analogs.
EXPERIMENTAL PART Chemistry
Materials and methods. Melting points of newly synthesized compounds were measured in open capillary tubes on a BUCHI B-545 melting point apparatus and are uncorrected. The elemental analysis (C, H, N) was performed using a Perkin Elmer 2400 CHN analyzer. The analyses indicated by symbols of the elements or functions were within ± 0.4% of the theoretical values. The 1H NMR spectra were recorded on 30
ACCEPTED MANUSCRIPT 13
Varian Gemini 400 MHz and
C NMR spectra on Varian Mercury-400 100 MHz in DMSO-d6 using
tetramethylsilane as an internal standard. Chemical shifts are reported in ppm units by means of δ scale. Mass spectra were obtained using electrospray ionization techniques on an Agilent 1100 Series LCMS.
RI PT
Analytical HPLC was performed on an Agilent 1100 HPLC with Diode Array Detection. Purity of all compounds was determined to be ≥ 95% by the HPLC. The peak purity was checked with the UV spectra.
General procedure for 2-hydrazono-4-thiazolidinones synthesis (1, 5-47, 49, 51, 53-58, 60-72).
SC
The mixture of appropriate thiosemicarbazone (1.0 eq.) and appropriate [C2]2+ synthon (1.0 eq.)
M AN U
(maleic anhydride, N-arylmaleimide, β-aroylacryclic acid, α-halogenocarboxylic acids, α-bromo-γbutyrolactone), sodium acetate (1.0 eq.) in acetic acid was heated under reflux for 2-4 hours. The reaction proceeding was monitored by TLC. After the reaction, the mixture was cooled to the room temperature; the obtained precipitate was filtered off, washed with acetic acid, water and ethanol and recrystallized. For compounds 1d, 73-75: the mixture of maleic anhydride (1.0 eq) and amino acid (1.0 eq) in
TE D
acetic acid was heated under reflux for 1 h. To the reaction mixture thiosemicarbasone (1.0 eq) was added. The reaction mixture was heated under reflux for 3 hours. The reaction proceeding was monitored by
AC C
and recrystallized.
EP
TLC. After the reaction mixture was cooled to room temperature the obtained precipitate was filtered off
N-(4-Fluorophenyl)-2-{3-(4-hydroxyphenyl)-4-oxo-2-[(3-phenyl-allylidene)-hydrazono]-thiazolidin-5-yl}acetamide (1a). Yield 70%, mp 238-240оС. 1Н NMR (400 MHz, DMSO-d6): δ 3.17 (dd, 1H, J = 8.0, 16.4 Hz, CHCH2), 3.28 (dd, 1H, J = 4.0, 16.5 Hz, CHCH2), 4.52 (dd, 1H, J = 3.9, 8.2 Hz, CHCH2), 6.88 (d, 2H, J = 8.2 Hz, arom.), 6.90-6.92 (m, 1H, arom), 7.12 (d, 1H, J = 8.0 Hz, arom.), 7.14-7.20 (m, 5H, arom.), 7.35 (brs, 1H, arom.), 7.60-7.64 (m, 2H, arom.), 8.21 (brs, 2H, arom), 9.10 (s, 1H, OH), 10.24 (s, 1H, NH).13C NMR (100 MHz, DMSO-d6): δ 194.6, 178.2, 176.0, 174.3, 172.6, 167.9, 158.4 (d, J = 210 31
ACCEPTED MANUSCRIPT Hz), 156.2, 135.1, 132.0, 131.3, 129.5, 126.7, 124.2, 116.1 (d, J = 22 Hz), 42.9, 39.4. LCMS (ESI) m/z 489 (M+H)+. Calcd. for C26H21FN4O3S: C, 63.92; H, 4.33; N, 11.47; Found: C, 64.10; H, 4.50; N, 11.30%.
RI PT
N-(4-Nitrophenyl)-2-{4-oxo-2-[(3-phenyl-allylidene)-hydrazono]-thiazolidin-5-yl}-acetamide (1b). Yield 70%, mp 248-250оС. 1Н NMR (400 MHz, DMSO-d6): δ 3.06 (dd, 1H, J = 9.1, 16.8 Hz, CHCH2), 3.25 (dd, 1H, J = 3.8, 16.7 Hz, CHCH2), 4.44 (m, 1H, CHCH2), 7.05 (dd, 1Н, J = 9.3, 15.9 Hz, CH-CH=CH), 7.17 (d, 1Н, J = 6.7 Hz, arom.), 7.30-7.44 (m, 3H, arom), 7.62 (m2H,Hz, arom), 7.82 (d, 2H, J = 8.1 Hz,
SC
arom), 8.18 (m, 3Н, arom.), 8.23 (d, 2H, J = 8.1 Hz, arom.), 11.95 (s, 1H, NH), 10.77 (s, 1H, NH). 13C
M AN U
NMR (100 MHz, DMSO-d6): δ 196.2, 177.7, 166.1, 154.3, 141.6, 136.3, 133.0, 131.7, 129.7, 129.4, 128.1, 125.9, 116.2, 105.5, 43.6, 42.2. LCMS (ESI) m/z 424 (M+H)+ Calcd. for C20H17N5O4S: C, 56.73; H, 4.05; N, 16.54; Found: C, 56.90; H, 4.20; N, 16.20%.
2-{3-Furan-2-ylmethyl-4-oxo-2-[(3-phenyl-allylidene)-hydrazono]-thiazolidin-5-yl}-N-(3-
TE D
trifluoromethylphenyl)-acetamide (1c). Yield 78%, mp 181-183оС. 1НNMR (400 MHz, DMSO-d6): δ 2.82-2.94 (m, 1H, CHCH2), 3.26-3.31 (m, 1H, CHCH2), 4.40 (m, 1H, CHCH2), 4.93 (s, 2H, CH2), 6.32 (t, 1H, J = 6.0 Hz, arom.), 6.42 (d, 1H, J = 6.2 Hz, arom.), 7.00-7.10 (m, 3H, Hz, arom.), 7.26-7.29 (m, 2H,
EP
Hz, arom.), 7.32-7.38 (m, 2H, Hz, arom.), 7.44 (t, 1H, J = 8.1 Hz, arom.), 7.50-7.54 (m, 1H, arom.), 7.72-
AC C
7.75 (m, 1H, arom.), 7.98-8.02 (m, 1H, Hz, arom.), 8.16-8.18 (m, 3H, arom.), 10.55 (s, 1H, NH).
13
C
NMR (100 MHz, DMSO-d6): δ 174.0, 168.9, 162.7, 160.7, 149.3, 143.1, 142.4, 139.8, 137.4 (q, J= 121 Hz), 136.2, 134.3, 130.6, 129.4, 127.9, 125.6, 123.1, 123.1, 115.6, 111.1, 109.2, 43.0, 38.7, 34.6. LCMS (ESI) m/z 527 (M+H)+ Calcd. for C26H21F3N4O2S: C, 59.31; H, 4.02; N, 10.64; Found: C, 59.50; H, 4.20; N, 10.50%.
32
ACCEPTED MANUSCRIPT 4-{2-[2-(Furan-2-ylmethylene-hydrazono)-4-oxothiazolidin-5-yl]-acetylamino}-butyric acid (1d). Yield 64%, mp 211-213оС. 1Н NMR (400 MHz, DMSO-d6): δ 1,62 (m, 2H, CH2), 2.25 (m, 2H, CH2), 2.64 (dd, 1H, J = 9.8, 15.7Hz, CHCH2), 2.90 (dd, 1H, J = 4.4, 15.8 Hz, CHCH2),3.06 (m, 2H, CH2), 4.33 (m, 1H,
RI PT
CHCH2), 6.64 (t, 1H, J = 6.2 Hz, arom.), 6.95 (d, 1H, J = 6.2 Hz,arom.), 7.86 (d, 1H, J = 6.2 Hz, arom.), 8.06 (t, 1H, J = 7.2 Hz, NH), 8.21 (s, 1H, CH=N), 12.00 (brs, 2Н, NН, COOH).
13
C NMR (100 MHz,
DMSO-d6): δ 176.2, 174.7, 169.2, 164.6, 149.9, 146.3, 145.9, 115.8, 112.8, 44.5, 38.6, 38.3, 31.5, 25.0.
SC
LCMS (ESI) m/z 353 (M+H)+. Calcd. for C14H16N4O5S: C, 47.72; H, 4.58; N, 15.90; Found: C, 48.00; H,
M AN U
4.90; N, 15.60%.
2-[2-(Cyclohexylidene-hydrazono)-4-oxothiazolidin-5-yl]-N-(4-fluorophenyl)-acetamide (1e). Yield79%, mp 182-184оС. 1НNMR (400 MHz, DMSO-d6): δ 1.55-1.63 (m, 6H, cyclohex.), 2.21-2.24 (m, 2H, cyclohex.), 2.52-2.57 (m, 2H, cyclohex.), 2.86 (m, 1H, CHCH2), 3.14 (dd, 1H, J = 3.0, 16.5 Hz, CHCH2), 4.31 (dd, 1H, J = 3.0, 9.3 Hz, CH2), 7.13 (t, 2H, J = 8.6 Hz, arom.), 7.55-7.58 (m, 2H, arom.), 10.16 (s,
TE D
1H, NH), 11.66 (s, 1H, NH).13C NMR (100 MHz, DMSO-d6): δ 194.5, 177.8, 168.3, 158.4 (d, J = 239 Hz), 153.4, 150.1, 135.7, 123.2, 121.3 (d, J = 7 Hz), 115.8 (d, J = 22 Hz), 44.5, 43.7, 35.4, 28.5, 27.6, 26.6, 25.9. LCMS (ESI) m/z 363 (M+H)+. Calcd. for C17H19FN4O2S: C, 56.34; H, 5.28; N, 15.46; Found:
AC C
EP
C, 56.50; H, 5.40; N, 15.20%.
For detailed characteristic of compounds 1f-1t see Supplementary Materials.
General procedure for 2-R-6-arylimidazo[2,1-b][1,3,4]thiadiazoles synthesis (2). Mixture of 2-amino-5-R-1,3,4-thiadiazole (1.0 eq.) and 2-bromoacetophenone (1.0 eq.) in ethanol was heated under reflux for 8 h.The reaction mixture was cooled and water solution of sodium carbonate
33
ACCEPTED MANUSCRIPT was added (pH = 9.0). The formed precipitate was filtered off and washed, dried and recrystallized from appropriate solvent [62].
RI PT
2-Allylsulfanyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazole(2a). Yield 73%, mp 111-112оС. 1Н NMR (400 MHz, DMSO-d6): δ 3.98 (d, 2Н, J = 6.8 Hz, СН2), 5.23 (d, 1Н, J = 10.0 Hz, СН2), 5.37 (d, 1Н, J = 16.9 Hz, СН2), 5.92-6.06 (m, 1Н, СН=), 7.28 (t, 1Н, J = 7.4 Hz, arom.), 7.42 (t, 2Н, J = 7.4 Hz, arom.), 7.87 (d, 2Н, J = 7.5 Hz, arom.), 8.68 (s, 1Н, CН).
13
C NMR (100 MHz, DMSO-d6): δ 159.8, 145.4, 145.1,
SC
134.2, 132.9, 129.1, 127.8, 125.1, 120.2, 111.1, 37.1. LCMS (ESI+) m/z 274 (M+H)+. Calcd. for
M AN U
C13H11N3S2: C, 57.12; H, 4.06; N, 15.37; Found: C, 57.30; H, 4.20; N, 15.20%.
(6-Phenylimidazo[2,1-b][1,3,4]thiadiazol-2-ylsulfanyl)-acetic acid ethyl ester (2b). Yield 68%, mp 9091оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.20 (t, 3Н, J = 10.0 Hz, СН3), 4.17 (q, 2Н, J = 7.0 Hz,СН2), 4.28 (s, 2Н,СН2),7.28 (t, 1Н, J = 7.0 Hz, arom.), 7.41 (t, 2Н, J = 7.3 Hz, arom.),7.86 (d, 2Н, J = 7.4 Hz, 13
C NMR (100 MHz, DMSO-d6): δ 168.2, 159.8, 145.4, 145.1, 134.2, 129.1,
TE D
arom.), 8.67 (s, 1Н, CН).
127.8, 125.1, 111.1, 62.1, 35.8, 14.5. LCMS (ESI+) m/z 320 (M+H)+. Calcd. for C14H13N3O2S2: C, 52.65;
EP
H, 4.10; N, 13.16; Found: C, 52.70; H, 4.30; N, 13.00%.
AC C
2-Ethyl-6-phenylimidazo[2,1-b][1,3,4]thiadiazole (2c).Yield 75%, mp 143-144оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.34 (t, 3Н, J = 7.5 Hz, СН3), 3.07 (q, 2Н, J = 7.5 Hz, СН2), 7.28 (t, 1Н, J = 7.3 Hz, arom.), 7.41 (t, 2Н, J = 7.3 Hz, arom.), 7.87 (d, 2Н, J = 7.2 Hz, arom.), 8.62 (s, 1Н, CH). 13C NMR (100 MHz, DMSO-d6): δ 166.8, 145.3, 145.1, 134.5, 129.1, 127.7, 125.1, 110.7, 25.5, 13.1. LCMS (ESI+) m/z 230 (M+H)+. Calcd. for C12H11N3S: C, 62.86; H, 4.84; N, 18.32; Found: C, 63.00; H, 5.00; N, 18.10%.
34
ACCEPTED MANUSCRIPT 6-(4-Bromophenyl)-2-ethylimidazo[2,1-b][1,3,4]thiadiazole (2d). Yield 77%, mp 228-229оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.35 (t, 3Н, J = 7.5 Hz, СН3), 3.11 (q, 2Н, J = 7.5 Hz, СН2), 7.67 (d, 2Н, J = 8.6 Hz, arom.), 7.91 (d, 2Н, J = 8.6 Hz, arom.), 8.68 (s, 1Н, CН). 13C NMR (100 MHz, DMSO-d6): δ 167.4,
RI PT
143.8, 132.5, 132.1, 130.9, 127.1, 120.6, 111.3, 25.5, 13.1. LCMS (ESI+) m/z 308/310 (M+H)+. Calcd. for C12H10BrN3S: C, 46.77; H, 3.27; N, 13.63; Found: C, 46.90; H, 3.50; N, 13.50%.
SC
2-Ethyl-6-(4-nitrophenyl)-imidazo[2,1-b][1,3,4]thiadiazole (2e). Yield 80%, mp 193-194оС. 1Н NMR (400 MHz, DMSO-d6 ): δ 1.35 (t, 3Н, J = 7.5 Hz, СН3), 3.08 (q, 2Н, J = 7.5 Hz, СН2), 8.07 (d, 2Н, J = 8.9
M AN U
Hz, arom.), 8.24 (d, 2Н, J = 8.9 Hz, arom.), 8.83 (s, 1Н, CН). 13C NMR (100 MHz, DMSO-d6): δ 168.1, 146.5, 143.1, 141.0, 125.6, 124.7, 113.4, 113.3, 25.5, 13.1. LCMS (ESI+) m/z 275 (M+H)+. Calcd. for C12H10N4O2S: C, 52.55; H, 3.67; N, 20.43; Found: C, 52.70; H, 3.80; N, 20.20%.
General procedure for 2-R-6-arylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde synthesis (3).
TE D
The Vilsmeier-Haack reagent was obtained in the reaction of phosphorus oxychloride with the anhydrous dimethylformamide (1:3 vol) at 0ºС and stirring for 10 min. To the prepared reagent 2-R-6arylimidazo[2,1-b][1,3,4]thiadiazole (1.0 eq) was added and stirred for 30 min at 0ºС, then for 2 h at rt and
EP
for 2 h at 60ºС. Then, to the reaction mixture a solution of sodium bicarbonate was added and the mixture
AC C
was heated to 90 ºС and stirred for 2 h. The reaction mixture was cooled and water was added. The product was extracted with the chloroform; the chloroform phase was dried over sodium sulfate and concentrated. Target compound was recrystallized from the appropriate solvent [62].
2-Allylsulfanyl-6-phenylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3a). Yield 89%, mp 77-78оС. 1
Н NMR (400 MHz, DMSO-d6): δ 4.03 (d, 2Н, J = 6.9 Hz, СН2), 5.25 (d, 1Н, J = 10.0 Hz, СН2=), 5.44
(d, 1Н, J = 17.0 Hz, СН2=), 5.94-6.07 (m, 1Н, СН=), 7.51-7.53 (m, 3Н, arom.), 7.93-7.96 (m, 2Н, arom.), 35
ACCEPTED MANUSCRIPT 9.97 (s, 1Н, СНО).
13
C NMR (100 MHz, DMSO-d6): δ 177.7, 163.2, 154.0, 150.8, 132.7, 132.6, 130.1,
129.3, 129.2, 124.0, 120.7, 37.1. LCMS (ESI+) m/z 302 (M+H)+. Calcd. for C14H11N3OS2: C, 55.79; H,
RI PT
3.68; N, 13.94; Found: C, 55.90; H, 3.80; N, 13.70%.
(5-Formyl-6-phenylimidazo[2,1-b][1,3,4]thiadiazol-2-ylsulfanyl)acetic acid ethyl ester (3b). Yield 88%, mp 98-100оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.18 (t, 3Н, J = 9.3 Hz, СН3), 3.98 (q, 2Н, J = 7.1
Hz, arom.), 10.02 (s, 1Н, CНO).
13
SC
Hz,СН2), 4.12 (s, 2Н,СН2), 7.32 (t, 1Н, J = 7.2 Hz, arom.), 7.45-7.49 (m, 2Н,arom.), 7.84 (d, 2Н, J = 7.3 C NMR (100 MHz, DMSO-d6): δ 176.9, 168.2, 159.6, 145.3, 145.2,
M AN U
134.0, 127.7, 126.6, 125.2, 111.0, 62.2, 35.6, 14.5.LCMS (ESI+) m/z 348 (M+H)+. Calcd. for C15H13N3O3S2: C, 51.86; H, 3.77; N, 12.09; Found: C, 62.00; H, 4.00; N, 11.80%.
2-Ethyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3c). Yield 92%, mp 100-101оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.37 (t, 3Н, J = 7.5 Hz, СН3), 3.16 (q, 2Н, J = 7.5 Hz, СН2), 7.51-7.53 (m,
TE D
3Н, arom.), 7.93-7.96 (m, 2Н, arom.), 9.96 (s, 1Н, СНО).
13
C NMR (100 MHz, DMSO-d6): δ 177.6,
169.7, 154.5, 151.1, 132.8, 130.0, 129.3, 129.2, 123.9, 25.5, 13.5. LCMS (ESI+) m/z 258 (M+H)+. Calcd.
EP
for C13H11N3OS: C, 60.68; H, 4.31; N, 16.33; Found: C, 60.80; H, 4.50; N, 16.20%.
AC C
6-(4-Bromophenyl)-2-ethylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3d). Yield 87%, mp 127128оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.37 (t, 3H, J = 7.4 Hz, CH3) 3.16 (q, 2H, J = 7.4 Hz, CH2), 7.71 (d, 2H, J = 8.4 Hz, arom.), 7.93 (d, 2H, J = 8.4 Hz, arom.), 9.98 (s, 1H, CHO). 13C NMR (100 MHz, DMSO-d6): δ 177.6, 170.0, 152.5, 150.9, 132.1, 132.0, 131.1, 123.9, 123.6, 25.5, 13.4. LCMS (ESI+) m/z 336/338 (M+H)+. Calcd. for C13H10BrN3OS: C, 46.44; H, 3.00; N, 12.50; Found: C, 46.60; H, 3.20; N, 12.40%.
36
ACCEPTED MANUSCRIPT 2-Ethyl-6-(4-nitrophenyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3e). Yield 91%, mp 174175оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.38 (t, 3Н, J = 7.4Hz, СН3), 3.19 (q, 2Н, J = 7.3 Hz, СН2), 8.28 (d, 2Н, J = 8.5 Hz, arom.), 8.35 (d, 2Н, J = 8.5 Hz, arom.), 10.08 (s, 1Н, СНО). 13C NMR (100 MHz,
RI PT
DMSO-d6): δ 177.9, 170.7, 151.0, 150.5, 148.2, 139.0, 130.2, 124.6, 124.3, 25.6, 13.4. LCMS (ESI+) m/z 303 (M+H)+. Calcd. for C13H10N4O3S: C, 51.65; H, 3.33; N, 18.53; Found: C, 51.80; H, 3.50; N, 18.40%.
SC
General procedure for 6-phenylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde thiosemicarbazones and 2-aryl-1H-indole-3-carbaldehyde thiosemicarbazones synthesis (4).
M AN U
The mixture of equimolar quantities of 6-phenylimidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (3) or 2-aryl-1H-indole-3-carbaldehyde (1.0 eq.) and thiosemicarbazide (1.0 eq.) in the acetic acid was heated under reflux for 1 h. After the reaction mixture was cooled to the room temperature, formed precipitate was filtered off, washed with acetic acid, water and ethanol and recrystallized.
TE D
(2-Phenyl-1H-indol-3-yl)-methylenethiosemicarbazone (4a). Yield 91%, mp 227-228оС. 1Н NMR (400 MHz, DMSO-d6): δ 7.16 (t, 1H, J = 7.6 Hz, arom.), 7.24 (t, 1H, J = 7.4 Hz, arom.), 7.37 (brs, 1H, NH2), 7.44 (d, 1H, J = 8.0 Hz, arom.), 7.50 (t, 1H, J = 8.0 Hz, arom.), 7.57 (t, 2H, J = 7.6 Hz, arom.), 7.61 (d,
EP
1H, J = 7.2 Hz, arom.), 8.04 (brs, 1H, NH2), 8.33 (d, 1H, J = 7.7 Hz, arom.), 8.50 (s, 1H, CH), 11.16 (s,
AC C
1H, NH), 11.88 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 177.0 142.7, 141.8, 136.9, 131.4, 129.7, 129.3, 129.2, 125.6, 123.5, 123.1, 121.5, 111.9, 107.9. LCMS (ESI+) m/z 295 (M+H)+. Calcd. for C16H14N4S: C, 65.28; H, 4.79; N, 19.03; Found: C, 65.40; H, 4.90; N, 18.80%.
(2-(4-Chlorophenyl)-1H-indol-3-yl)-methylene-thiosemicarbazone (4b). Yield 93%, mp 234-236оС. 1Н NMR (400 MHz, DMSO-d6): δ 7.18 (t, 1H, J = 7.4 Hz, arom.), 7.28 (t, 1H, J = 7.4 Hz, arom.), 7.43 (brs, 1H, NH2), 7.68 (d, 2H, J = 8.1 Hz, arom.), 7.82 (d, 2H, J = 8.2 Hz, arom.), 8.11 (brs, 1H, NH2), 8.33 (d, 37
ACCEPTED MANUSCRIPT 1H, J = 7.7 Hz, arom.), 8.52 (s, 1H, CH), 11.14 (s, 1H, NH), 11.82 (s, 1H, NH).
13
C NMR (100 MHz,
DMSO-d6): δ 177.2, 142.6, 141.8, 137.0, 131.2, 129.6, 129.4, 129.2, 125.4, 123.9, 123.0, 121.2, 111.6, 108.0. LCMS (ESI+) m/z 329/331 (M+H)+. Calcd. for C16H13ClN4S: C, 58.44; H, 3.98; N, 17.04; Found:
RI PT
C, 58.60; H, 4.10; N, 16.90%.
N1-(2-(4-Chlorophenyl)-1H-indol-3-yl)-methylidene)-N2-(4-hydroxyphenyl)-thiosemicarbazone (4c). Yield
SC
89%, mp 239-241оС. 1Н NMR (400 MHz, DMSO-d6): δ 6.81 (d, 2H, J = 8.2 Hz, arom), 7.12 (t, 1H, J = 7.0 Hz, arom.), 7.21 (t, 1H, J = 7.0 Hz, arom.), 7.42 (d, 2H, J = 8.0 Hz, arom.), 7.74 (d, 2H, J = 8.0 Hz,
M AN U
arom.), 7.87 (d, 2H, J = 8.1 Hz, arom.), 8.05 (brs, 1H, NH), 8.31 (d, 1H, J = 7.2 Hz, arom), 8.52 (s, 1H, CH), 9.42 (s, 1H, OH), 11.82 (s, 1H, NH), 11.86 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 177.1, 143.0, 141.9, 137.6, 137.1, 133.4, 131.4, 131.2, 130.0, 129.6, 129.4, 129.2, 125.4, 123.9, 123.0, 121.4, 112.0, 108.6. LCMS (ESI+) m/z 422/424 (M+H)+. Calcd. for C22H17ClN4OS: C, 62.78; H, 4.07; N, 13.31;
TE D
Found: C, 63.00; H, 4.20; N, 13.00%.
N1-(2-(4-Chlorophenyl)-1H-indol-3-yl)-methylidene)-N2-furfuryl-thiosemicarbazone (4d). Yield 89%, mp 223-225оС. 1Н NMR (400 MHz, DMSO-d6): δ 4.92 (s, 2H, NCH2), 7.18 (t, 1H, J = 7.0 Hz, arom.), 7.22 (t,
EP
1H, J = 7.1 Hz, arom.), 7.41 (d, 1H, J = 6.5 Hz, arom.), 7.45 (d, 2H, J = 8.1 Hz, arom.), 7.64 (m, 3H,
AC C
arom.), 7.82-7.86 (m, 3H, arom), 8.05 (brs, 1H, NH), 8.24 (d, 1H, arom.), 11.78 (brs, 1H, NH), 11.86 (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 177.1, 153.6, 149.4, 143.0, 142.1, 137.0, 134.3, 131.4,
130.2, 129.6, 126.2, 123.9, 122.9, 121.8, 112.30, 111.1, 109.2, 108.9, 44.2. LCMS (ESI+) m/z 410/412 (M+H)+. Calcd. for C21H17ClN4OS: C, 61.68; H, 4.19; N, 13.70; Found: C, 61.90; H, 4.30; N, 13.50%.
(2-(4-Bromophenyl)-1H-indol-3-yl)-methylene-thiosemicarbazone (4e). Yield 91%, mp 241-243оС. 1Н NMR (400 MHz, DMSO-d6): δ 7.21 (t, 1H, J = 7.2 Hz, arom.), 7.26 (t, 1H, J = 7.2 Hz, arom.), 7.40 (brs, 38
ACCEPTED MANUSCRIPT 1H, NH2), 7.70 (d, 2H, J = 8.4 Hz, arom.), 7.85 (d, 2H, J = 8.2 Hz, arom.), 8.05 (brs, 1H, NH2), 8.23 (d, 1H, J = 7.6 Hz, arom.), 8.54 (s, 1H, CH), 11.14 (s, 1H, NH), 11.82 (s, 1H, NH).
13
C NMR (100 MHz,
DMSO-d6): δ 177.0143.0, 141.9, 137.1, 131.3, 130.1, 129.6, 129.4, 125.4, 123.8, 122.0, 121.2, 112.0,
RI PT
109.1. LCMS (ESI+) m/z 374 (M+H)+. Calcd. for C16H13BrN4S: C, 51.48; H, 3.51; N, 15.01; Found: C, 51.60; H, 3.70; N, 14.90%.
SC
For detailed characteristic of compounds 4f-4p see Supplementary Materials.
M AN U
2-[(2-Phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-one (5). Yield 82%, mp 253-255ºC. 1H NMR (400 MHz, DMSO-d6): δ 3.82 (s, 2H, CH2), 7.17 (t, 1H, J = 7.2 Hz, arom.), 7.22 (t, 1H, J = 7.2 Hz, arom.), 7.44-7.48 (m, 2H, arom.), 7.54-7.57 (m, 2H, arom.), 7.65-7.68 (m, 2H, arom.), 8.35 (d, 1H, J = 7.0 Hz, arom.), 8.81 (s, 1H, CH), 11.72 (brs, 1H, NH), 11.77 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 174.2, 152.5, 142.9, 137.0, 131.8, 129.6, 129.1, 128.9, 126.5, 123.1, 121.3, 111.8, 108.9, 33.44. LCMS
N, 16.50%.
TE D
(ESI+) m/z 335 (M+H)+. Calcd. for C18H14N4OS: C, 64.65; H, 4.22; N, 16.75; Found: C, 64.90; H, 4.40;
EP
5-Methyl-2-[(2-phenyl-1H-indol-3-yl-methylene)-hydrazono]-thiazolidin-4-one (6). Yield 76%, mp 266-
AC C
268ºC. 1H NMR (400 MHz, DMSO-d6): δ 1.63 (d, 3H, J = 7.0 Hz, CH3), 4.09 (q, 1H, J = 7.0 Hz, CH), 7.17 (t, 1H, J = 7.2 Hz, arom.), 7.21 (t, 1H, J = 6.9 Hz, arom.), 7.42-7.45 (m, 2H, arom.), 7.56 (t, 2H, J = 7.8 Hz, arom.), 7.66 (d, 2H, J = 7.8 Hz, arom.), 8.33 (d, 1H, J = 7.4 Hz, arom.), 8.59 (s, 1H, CH), 11.65 (brs, 1H, NH), 11.78 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 177.1, 152.6, 152.6, 142.9, 137.1, 131.8, 129.6, 129.1 128.9, 126.5, 123.2, 121.2, 111.8, 108.9, 42.6, 19.6. LCMS (ESI+) m/z 349 (M+H)+. Calcd. for C19H16N4OS: C, 65.50; H, 4.63; N, 16.08; Found: C, 65.70; H, 4.80; N, 15.90%.
39
ACCEPTED MANUSCRIPT 5-Ethyl-2-[(2-phenyl-1H-indol-3-yl-methylene)-hydrazono]-thiazolidin-4-one (7). Yield 74%, mp 247249оС. 1H NMR (400 MHz, DMSO-d6): δ 1.11 (t, 3H, J = 7.2 Hz, CH3), 1.92 (m, 1H, CHCH2), 2.10 (m, 1H, CHCH2), 4.00 (m, 1H, CHCH2), 7.16 (m, 2H, arom.), 7.37-7.43 (t, 2H, J = 8.0 Hz, arom.), 7.52 (t, 2H,
11.61 (s, 1H, NH).
13
RI PT
J = 8.2 Hz, arom.), 7.65 (d, 2H, J = 8.2 Hz, arom.), 8.33 (d, 1H, J = 6.4 Hz, arom.), 8.57 (s, 1H, CH), C NMR (100 MHz, DMSO-d6): δ 172.4, 152.8, 143.3, 137.0, 131.4, 129.7, 129.4,
126.3, 123.6, 122.8, 121.6, 112.1, 108.6, 49.7, 25.9, 10.7. LCMS (ESI+) m/z 363 (M+H)+. Calcd. for
SC
C20H18N4OS: C, 66.28; H, 5.01; N, 15.46; Found: C, 66.50; H, 4.90; N, 15.30%.
M AN U
2-{[2-(4-Chlorophenyl)-1H-indol-3-yl-methylene]-hydrazono}-thiazolidin-4-one (8). Yield 76%, mp 298299оС. 1H NMR (400 MHz, DMSO-d6): δ 3.90 (s, 2H, CH2), 7.20 (t, 1H, J = 7.5 Hz, arom.), 7.26 (t, 1H, J = 7.2 Hz, arom.), 7.47 (d, 1H, J = 8.0 Hz, arom.), 7.64 (d, 2H, J = 8.4 Hz, arom.), 7.68 (d, 2H, J = 8.4 Hz, arom.), 8.32 (d, 1H, J = 7.8 Hz, arom.), 8.53 (s, 1H, CH), 11.84 (s, 1H, NH), 12.04 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 174.3, 152.2, 141.6, 137.0, 134.2, 131.4, 130.3, 129.5, 126.3, 123.8, 122.9,
TE D
121.7, 112.2, 109.0, 33.6. LCMS (ESI+) m/z 369/371 (M+H)+. Calcd. for C18H13ClN4OS: C, 58.62; H,
EP
3.55; N, 15.19; Found: C, 58.80; H, 3.70; N, 15.00%.
2-{[2-(4-Chlorophenyl)-1H-indol-3-yl-methylene]-hydrazono}-5-methylthiazolidin-4-one (9). Yield 78%,
AC C
mp 296-298оС. 1H NMR (400 MHz, DMSO-d6): δ 1.54 (d, 3H, J = 7.2 Hz, CH3), 4.21 (q, 1H, J = 7.2 Hz, CH), 7.21 (t, 1H, J = 8.5 Hz, arom.), 7.28 (t, 1H, J = 7.4 Hz, arom.), 7.47 (d, 1H, J = 7.9 Hz, arom.), 7.64 (d, 2H, J = 8.5 Hz, arom.), 7.68 (d, 2H, J = 8.5 Hz, arom.), 8.32 (d, 1H, J = 7.8 Hz, arom.), 8.54 (s, 1H, CH), 11.76 (s, 1H, NH), 12.00 (s, 1H, NH).
C NMR (100 MHz, DMSO-d6): δ 176.5, 152.4, 141.7,
13
137.0, 134.2, 131.3, 130.2, 129.5, 126.2, 123.8, 122.9, 121.7, 120.2, 112.2, 42.4, 19.4. LCMS (ESI+) m/z 383/385 (M+H)+. Calcd. for C19H15ClN4OS: C, 59.60; H, 3.95; N, 14.63; Found: C, 59.40; H, 3.80; N, 14.80%. 40
ACCEPTED MANUSCRIPT
2-{[2-(4-Chlorophenyl)-1H-indol-3-yl-methylene]-hydrazono}-5-ethylthiazolidin-4-one (10). Yield 78%, mp 290-292оС. 1H NMR (400 MHz, DMSO-d6): δ 0.99 (t, 3H, J = 7.2 Hz, CH3), 1.84-4.89 (m, 1H, CH2),
RI PT
1.98-2.04 (m, 1H, CH2), 4.25-4.27 (m, 1H, CH), 7.23 (t, 1H, J = 7.2 Hz, arom.), 7.27 (t, 1H, J = 7.2 Hz, arom.), 7.48 (d, 1H, J = 7.8 Hz, arom.), 7.66 (d, 2H, J = 8.3 Hz, arom.), 7.69 (d, 2H, J = 8.3 Hz, arom.), 8.33 (d, 1H, J = 7.6 Hz, arom.), 8.55 (s, 1H, CH), 11.83 (s, 1H, NH), 12.05 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 174.2, 152.4, 141.7, 137.0, 134.2, 131.3, 130.2, 129.50, 129.4, 126.3, 123.8, 122.9,
SC
121.8, 112.2, 109.0, 50.0, 25.9, 10.7. LCMS (ESI+) m/z 397/399 (M+H)+. Calcd. for C20H17ClN4OS: C,
M AN U
60.52; H, 4.32; N, 14.12; Found: C, 60.70; H, 4.60; N, 14.00%.
For detailed characteristic of compounds 11-47, 49, 51, 53-58, 60-75 see Supplementary Materials.
General procedure for 5-(2-R-ethyl)-2-[(2-phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-
TE D
ones and 5-(2-R-ethyl)-2-[(6-phenyl-2,3,7,7a-tetrahydroimidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)hydrazono]-thiazolidin-4-ones synthesis (48, 50, 52, 59). (Method A) To the appropriate
EP
thiosemicarbazone (1.0 eq) in ethanol α-bromo-γ-butyrolactone (1.0 eq) and triethylamine (1.0 eq,) were added. The reaction mixture was heated under reflux for 5 hours. The reaction proceeding was monitored
AC C
by TLC. After the reaction mixture was cooled to room temperature, the obtained precipitate was filtered off and recrystallized from the ethanol to provide pure title compound.
5-(2-Hydroxyethyl)-2-[(2-phenyl-1H-indol-3-ylmethylene)-hydrazono]-thiazolidin-4-one (48). Yield 70%, mp 223-224оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.86-1.90 (m, 1H, СH2), 2.27-2.32 (m, 1H, СH2), 3,633.66 (m, 2H, СH2), 4,16 (dd, 1H, J = 4.0, 9.6 Hz, СH), 4,54 (brs, 1H, OH), 7,17 (t, 1H, J = 7.7 Hz,), 7.417.47 (m, 1H), 7.55 (t, 1H, J = 7.4 Hz, arom.) 7.64 (d, J = 7.4 Hz), 7.64 (d, J = 7.6 Hz, 1H), 8.57 (s, 1H, 41
ACCEPTED MANUSCRIPT CH), 11.63 (brs, 1H, NH), 11.74 (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 172.6, 167.9, 158.2,
143.2, 137.0, 131.4, 129.7, 129.5, 126.3, 123.6, 122.8, 121.7, 112.2, 108.6, 58.9, 40.9, 36.5. LCMS (ESI+) m/z 379 (M+H)+. Calcd. for C20H18N4O2S: C, 63.47; H, 4.79; N, 14.80; Found: C, 63.60; H, 4.90; N,
RI PT
14.70%.
2-{[2-(4-Chlorophenyl)-1H-indol-3-ylmethylene]-hydrazono}-5-(2-hydroxyethyl)-thiazolidin-4-one
(50).
SC
Yield 80%, mp 268-270оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.86-1.91 (m, 1H, СH2), 2.24-2.29 (m, 1H, СH2), 3,57-3.64 (m, 2H, СH2), 4,24 (dd, 1H, J = 3.7, 9.4 Hz, СH), 4,78 (brs, 1H, OH), 7,26 (t, 1H, J =
M AN U
7.6Hz,), 7.27 (t, 1H, J = 7.7 Hz, arom.), 7.47 (d, 1H, J = 7.9Hz, arom.) 7.65 (d, J = 8.5 Hz, 2H, arom.), 7.69 (d, 2H, J = 8.5 Hz, arom.), 8.33 (d, 1H, J = 7.7 Hz, arom.), 8.54 (s, 1H, CH), 11.79 (brs, 1H, NH), 12.05 (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 177.1, 172.7, 152.3, 141.6, 137.0, 134.2, 131.3,
130.3, 129.5, 126.6, 123.8, 122.9, 121.7, 112.2, 109.0, 58.9, 45.8, 36.5. LCMS (ESI+) m/z 413/415
TE D
(M+H)+. Calcd. for C20H17ClN4O2S: C, 58.18; H, 4.15; N, 13.57; Found: C, 58.00; H, 4.00; N, 13.70%.
2-{[2-(4-Bromophenyl)-1H-indol-3-ylmethylene]-hydrazono}-5-(2-hydroxyethyl)-thiazolidin-4-one
(52).
EP
Yield 78%, mp>350оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.84-1.89 (m, 1H, СH2), 2.20-2.25 (m, 1H, СH2), 3.56-3.62 (m, 2H, СH2), 4.22 (dd, 1H, J = 3.7, 9.4 Hz, СH), 4,80 (brs, 1H, OH), 7.25 (t, 1H, J = 7.4
AC C
Hz, arom.), 7.28 (t, 1H, J = 7.4 Hz, arom.), 7.52 (d, 1H, J = 8.0Hz, arom.) 7.70 (d, 2H, J = 8.2 Hz, arom.), 7.75 (d, 2H, J = 8.2 Hz, arom.), 8.36 (m, 1H, arom.), 8.53 (s, 1H, CH), 11.81 (brs, 1H, NH), 11.98 (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 177.0, 162.8, 141.5, 141.2, 137.0, 132.3, 131.7, 130.6, 125.6,
123.83, 123.2, 123.0, 121.6, 112.0, 108.4, 56.9, 36.3, 31.3. LCMS (ESI+) m/z 457/459 (M+H)+. Calcd. for C20H17BrN4O2S: C, 52.52; H, 3.75; N, 12.25; Found: C, 52.70; H, 3.90; N, 12.10%.
42
ACCEPTED MANUSCRIPT 2-{[2-(4-Chlorophenyl)-1H-indol-3-ylmethylene]-hydrazono}-3-(furan-2-ylmethyl)-5-(2-hydroxy-ethyl)thiazolidin-4-one (59). Yield 73%, mp 252-254°С. 1Н NMR (400 MHz, DMSO-d6): δ 1.89-1.94 (m, 1H, CHCH2), 2.25-2.32 (m, 1H, CHCH2), 3.59-3.63 (m, 2H, CH2), 4.37 (dd, J = 3.9, 9.2 Hz, 1H, CH), 4.85 (t,
RI PT
1H, J = 5.1 Hz, OH), 4.91 (s, 2H, CH2), 6.36-6.37 (m, 1H, arom.), 6.39-6.41 (m, 1H, arom.), 7.27-7.31 (m, 2H, arom.), 7.49 (d, 1H, J = 7.2 Hz, arom.), 7.58-7.59 (m, 1H, arom.), 7.69 (brs, 4H, arom.), 8.33 (d, 1H, J = 7.2 Hz, arom.), 8.57 (s, 1H, CH), 12.12 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 174.8, 159.7,
SC
153.7, 149.4, 143.0, 142.2, 137.0, 134.4, 131.4, 130.2, 129.6, 126.2, 123.9, 122.9, 121.8, 112.2, 111.1, 109.0, 108.9, 58.6, 44.8, 40.6,36.4. LCMS (ESI+) m/z 493/495(M+H)+. Calcd. for C25H21ClN4O3S: C,
M AN U
60.91; H, 4.29; N, 11.36; Found: C, 61.00; H, 4.40; N, 11.30%.
General procedure for N-[4-arylthiazol-2-yl]-N'-[2-(arylphenyl)-1H-indol-3-ylmethylene]-hydrazines, 5-methyl-2-(6-arylimidazo[2,1-b]-1,3,4-thiadoazole-5-ylmethylene)hydrazone-4-thiazolidinones
and
ethyl ester of 2-{N'-[2-(arylphenyl)-1H-indol-3-ylmethylene]-hydrazino}-4-methyl-thiazole-5-carboxylic and
4-methyl-2-(6-arylimidazo[2,1-b]-1,3,4-thiadiazole-5-ylmethylidene)hydrazone-2,3-
TE D
acids
dihydrothiazole-5-carboxylic acids synthesis (76-100).
EP
To the appropriate thiosemicarbazone (1.0 eq) in acetic acid bromoacetophenone or ethyl 2chloroacetoacetate (1.1 eq.) and anhydrous sodium acetate (1.0 eq.) were added. The reaction mixture was
AC C
hated under reflux for 3 hours. Then the mixture was cooled to the room temperature and formed precipitate was filtered off, washed with acetic acid, water and ethanol, dried and recrystallized.
(N-[4-(4-Bromophenyl)-thiazol-2-yl]-N'-[2-(4-chlorophenyl)-1H-indol-3-ylmethylene]-hydrazine
(76).
Yield 80%, mp 320-322оС. 1H NMR (400 MHz, DMSO-d6): δ 7.22 (t, 1H, J = 7.2 Hz, arom.), 7.27 (t, 1H, J = 7.0 Hz, arom.), 7.38 (s, 1H, CH), 7.47 (d, 1H, J = 7.7 Hz, arom.), 7.60 (d, 2H, J = 8.6 Hz, arom.), 7.68 (brs, 4H, arom.), 7.81 (d, 2H, J = 8.4 Hz, arom.), 8.33 (d, 1H, J = 7.6 Hz, arom.), 8.38 (s, 1H, CH), 11.91 43
ACCEPTED MANUSCRIPT (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ 164.5, 159.4, 147.2, 145.0, 139.0, 138.4, 137.5, 134.2,
131.7, 130.2, 129.8, 128.0, 123.9, 124.0, 122.5, 121.7, 120.8, 119.0, 111.8, 103.8. LCMS (ESI) m/z 507/509 (M+H)+. Calcd. for C26H16BrClN4S: C, 56.76; H, 3.18; N, 11.03; Found: C, 56.90; H, 3.40; N,
RI PT
10.90%.
N-[2-(4-Nitrophenyl)-1H-indol-3-ylmethylene]-N'-(4-phenylthiazol-2-yl)-hydrazine (77). Yield 79%, mp
SC
267-269оС. 1H NMR (400 MHz, DMSO-d6): δ 7.15-7.30 (m, 1H, arom.), 7.31-7.37 (m, 2H, arom.), 7.43 (t, 2H, J = 7.3 Hz, arom.), 7.47-7.54 (m, 2H, arom.), 7.64-7.72 (m, 1H, arom.), 7.88 (d, 1H, J = 7.6 Hz,
M AN U
arom.), 7.96 (d, 2H, J = 8.3 Hz, arom.), 8.13-8.20 (m. 1H, arom.), 8.43 (d, 2H, J = 8.5 Hz, arom.), 8.48 (s, 1H, CH),12.15 (s, 1H, NH).13C NMR (100 MHz, DMSO-d6): δ 168.8, 161.4, 149.9, 147.4, 139.6, 138.1, 137.6, 134.6, 130.5, 129.1, 128.2, 126.1, 125.7, 124.5, 124.5, 122.8, 121.8, 112.5, 110.4, 103.7. LCMS (ESI) m/z 440 (M+H)+. Calcd. for C24H17N5O2S: C, 65.59; H, 3.90; N, 15.93; Found: C, 65.70; H, 4.10; N,
TE D
16.10%.
N-[4-(4-Bromophenyl)-thiazol-2-yl]-N'-[2-(4-nitro-phenyl)-1H-indol-3-ylmethylene]-hydrazine
(78).
Yield 80%, mp 252-254оС. 1H NMR (400 MHz, DMSO-d6): δ 7.22 (t, 1H, J = 7.2 Hz, arom.), 7.27 (t, 1H,
EP
J = 7.0 Hz, arom.), 7.42-7.47 (m, 2H, arom.), 7.58-7.60 (m, 2H, arom.), 7.68 (brs, 4H, arom.), 7.81 (d, 2H, 13
C NMR (100 MHz,
AC C
J = 8.4 Hz, arom.), 8.43 (m, 1H, arom.), 8.98 (s, 1H, CH), 12.13 (s, 1H, NH).
DMSO-d6): δ 164.6, 155.9, 147.4, 144.6, 139.0, 138.1, 137.5, 134.4, 132.0, 130.4, 130.1, 128.0, 124.5, 124.1, 122.8, 121.7, 121.0, 119.3, 112.4, 104.5. LCMS (ESI) m/z 519/520 (M+H)+. Calcd. for C24H16BrN5O2S: C, 65.59; H, 3.90; N, 15.93; Found: C, 55.61; H, 3.11; N, 13.51%.
N-[2-(4-Nitrophenyl)-1H-indol-3-ylmethylene]-N'-[4-(4-nitrophenyl)-thiazol-2-yl]-hydrazine (79). Yield 77%, mp 300-302оС. 1H NMR (400 MHz, DMSO-d6): δ 7.27 (t, 1H, J = 7.2 Hz, arom.), 7.33 (t, 1H, J = 44
ACCEPTED MANUSCRIPT 7.2 Hz, arom.), 7.51 (d, 1H, J = 7.9 Hz, arom.), 7.72 (s, 1H, arom.), 7.94 (d, 2H, J = 8.6 Hz, arom.), 8.12 (d, 2H, J = 8.7 Hz, arom.), 8.28 (d, 2H, J = 8.8 Hz, arom.), 8.37 (d, 1H, J = 7.9 Hz, arom.), 8.43 (d, 2H, J = 8.8 Hz, arom.), 8.45 (s, 1H, CH), 12.13 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 167.3, 166.8,
RI PT
147.4, 146.6, 138.1, 137.5, 130.4, 130.1, 126.8, 126.8, 125.7, 24.9, 124.5, 124.5, 124.1, 121.8, 119.3, 112.4, 110.4, 108.4. LCMS (ESI) m/z 485 (M+H)+. Calcd. for C24H16N6O4S: C, 59.50; H, 3.33; N, 17.35;
SC
Found: C, 59.70; H, 3.50; N, 17.10%.
N-(2-Ethyl-6-phenyl-imidazo[2,1-b][1,3,4]thiadiazol-5-ylmethylene)-N'-[4-(4-nitrophenyl)-3H-thiazol-2-
M AN U
ylidene]-hydrazine (80). Yield 78%, mp 246-247оС. 1Н NMR (400 MHz, DMSO-d6): δ 1.42 (t, 3Н, J = 7.2 Hz, СН3), 3.15 (q, 2Н, J = 7.3 Hz, СН2СН3), 7.42 (t, 1Н, J = 7.0 Hz, arom.), 7.52 (t, 2Н, J = 7.2 Hz, arom.), 7.73 (s, 1Н, thiaz), 7.88 (d, 2Н, J = 7.4 Hz, arom.), 8.10 (d, 2Н, J = 8.3 Hz, arom.), 8.27 (d, 2Н, J = 8.0 Hz, arom.), 8.42 (s, 1Н, 5-Н, imidaz), 12.27 (s, 1Н, NH). 13C NMR (100 MHz, DMSO-d6): δ 169.0, 167.4, 149.0, 147.3, 146.7, 146.0, 141.2, 134.1, 131.1, 129.0, 128.6, 126.8, 124.6, 119.6, 112.6, 109.4,
55.70; H, 3.80; N, 20.50%.
TE D
25.7, 13.0. LCMS (ESI) m/z 476 (M+H)+. Calcd. for C22H17N7O2S2: C, 55.57 H, 3.60; N, 20.62; Found: C,
EP
Crystal structure determination of compounds
AC C
Acetic acid 4-{2-[2-(cyclohexylidene-hydrazono)-4-oxothiazolidin-5-yl]-acetylamino}-phenyl ester (1f). Crystal data: C19H22N4O4S, 1.25H2O, Mr = 424.48, triclinic, space group P-1, a = 6.2209(4), b = 13.0177(8), c = 13.7897(5) Å, α = 69.084(5), β = 81.087(5), γ = 80.209(5)°, V = 1022.54(11) Å3, T = 130.0(1) K, Z = 2.
Data collection. A colourless lath (methanol/water) crystal of 0.45 x 0.18 x 0.06 mm was used to record 27043 (Mo Kαradiation, θmax = 29.09°) intensities on an Xcalibur A diffractometer [67]. Accurate unit cell parameters were determined by least-squares techniques from the θ values of 9292 reflections, θ 45
ACCEPTED MANUSCRIPT range 2.68–29.02°. The data were corrected for Lorentz, polarization and for absorption effects [67]. The 5056 total unique reflections (Rint = 0.029) were used for structure determination. The crystallographic data in the CIF form are available as Electronic Supplementary data from the Crystallographic
Data
Centre,
deposition
number
CCDC-1876805;
RI PT
Cambridge
http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre,
SC
12 Union Road, Cambridge, CB2 1EZ, UK; fax: ţ44 1223 336033; e-mail: [email protected]).
5-Ethyl-2-[(2-phenyl-1H-indol-3-yl-methylene)-hydrazono]-thiazolidin-4-one (7).
M AN U
Crystal data: C20H18N4OS, Mr = 362.44, monoclinic, space group P21/c, a = 8.7322(3), b = 21.9382(7), c = 8.8824(3) Å, β = 92.867(3)°, V = 1699.47(10) Å3, T = 130.0(1) K, Z = 4. Data collection. A light-yellow block (EtOH) crystal of 0.45 x 0.35 x 0.15 mm was used to record 16730 (Mo Kα radiation, θmax= 27.10°) intensities on an Xcalibur A diffractometer [67]. Accurate unit cell parameters were determined by least-squares techniques from the θ values of 8429 reflections, θ range
TE D
2.29–29.10°. The data were corrected for Lorentz, polarization and for absorption effects [67]. The 3748 total unique reflections (Rint = 0.031) were used for structure determination.
Cambridge
EP
The crystallographic data in the CIF form are available as Electronic Supplementary data from the Crystallographic
Data
Centre,
deposition
number
CCDC-1876806;
AC C
http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ, UK; fax: ţ44 1223 336033; e-mail: [email protected]).
Biology
Antitrypanosomal activity. Bloodstream forms of Trypanosoma brucei brucei strain 90-13 and Trypanosoma brucei gambiense Feo strain were cultured in HMI9 medium supplemented with 10% FCS at 37°C under an atmosphere of 5% CO2 [65]. In all experiments, log-phase parasite cultures were 46
ACCEPTED MANUSCRIPT harvested by centrifugation at 3000xg and immediately used. Drug assays were based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable cells as previously described [66]. Drug stock solutions were prepared in pure DMSO. T. brucei blood stream forms (105cells/ml) were
RI PT
cultured in 96-well plates either in the absence or in the presence of different concentrations of inhibitors in a final volume of 200 µl. After a 72-h incubation, resazurin solution was added in each well at the final concentration of 45µM and fluorescence was measured at 530 nm and 590 nm absorbance after a further
SC
4-h incubation. The percentage of inhibition of parasite growth rate was calculated by comparing the fluorescence of parasites maintained in the presence of drug to that of in the absence of drug. DMSO was
M AN U
used as control. Concentration inhibiting 50% of parasite growth (IC50) was determined from the doseresponse curve with a drug concentrations ranging from10 µg/ml to 0.625 µg/ml. IC50value is the mean +/- the standard deviation of three independent experiments.
In vitro cytotoxicity assay on mammalian cell. Cytotoxicity upon human primary fibroblast (cell line
TE D
AB943). Assays were performed in 96-well plates in RPMI medium containing 25 mM HEPES, pH 7.3, 10% fetal calf serum under 5% CO2 atmosphere, at 37 oC. After trypsin treatment, L-6 cells were seeded at 5000 cells per well in 100 mL. After 24 h incubation, cells were washed and two-fold dilutions of drug
EP
were added (200 mL perwell). Drug stock solutions were prepared in pure DMSO. The final DMSO
AC C
concentration in the cultures remained below 1%. Control cultures were constituted of cultures treated with pure DMSO instead of drug. The cytotoxicity assay was based on the conversion of a redoxsensitive dye (resazurin) to afluorescent product by viable cells [68]. After 5 days of incubation, resazurin solution was added in each well at thefinal concentration of 45 mM. Fluorescence was measured at 530 nm excitation and 590 nm emission wavelengths after a further 4-h incubation. The percentage of inhibition of cell growth was calculated by comparing thefluorescence of cells maintained in the presence of drug to that of in the absence of drug. 47
ACCEPTED MANUSCRIPT
Acute toxicity.The experiments were conducted on white male mice weighing 23-25 g. Compounds were dissolved in saline solution (0.9% NaCl) with l-2 drops of Polysorbate 80 (Tween-80®). After dissolution
RI PT
they were administered to mice via intraperitoneal route. The LD50 was evaluated for 4 or 5 different doses each on 6 animals and calculated by the Litchfield-Wilcoxon method [69,70].
SC
ACKNOWLEDGMENT
This work was partially supported by Ukrainian-France program “DNIPRO” and the Ministry of
SUPPLEMENTARY MATERIALS
M AN U
Education and Science of Ukraine (M/188-2015; M/71-2016).
Supplementary materials present the detailed analytical and spectral data of synthesized compounds, X-
TE D
ray data analysis and are avaible at _______.
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Highlights for the manuscript
“Thiazolidinone/thiazole based hybrids – new class of antitrypanosomal agents”
by Anna Kryshchyshyn, Danylo Kaminskyy, Oleksandr Karpenko,
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Andrzej Gzella, Phillipe Grellier, Roman Lesyk
4-Thiazolidinone/thiazole & phenylindole/phenylimidazothiadiazole hybrids
•
Compounds were highly active toward Trypanosoma brucei brucei and gambiense strains
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Compounds possessed relatively low cytotoxicity (fibroblasts) and acute toxicity
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•