Anti-hepatitis-C virus activity and QSAR study of certain thiazolidinone and thiazolotriazine derivatives as potential NS5B polymerase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111747

The present study reports on evaluation of anti-HCV activity and QSAR of certain arylidenethiazolidinone derivatives as potential inhibitors of HCV-NS5B polymerase. The pursued compounds involving, 5-aryliden-3-arylacetamidothiazolidin-2,4-diones 4–6(a–f), 5-arylidine-2-(N-arylacetamido)-iminothiazolidin-4-one (10) and their rigid counterparts 5-arylidinethiazolotriazines 13–15(a–f), were synthesized and their structures confirmed by spectral and elemental analyses. The results of NS5B polymerase inhibition assay revealed compound 4e, as the most active inhibitor (IC₅₀ = 0.035 μM), which is four folds greater than that of the reference agent, VCH-759, (IC₅₀ = 0.14 μM). Meanwhile, compounds 4b, 4c, 5a, and 5c, and 13b, 14e and 15c displayed equipotency to 2 folds higher activity than VCH-759 (IC₅₀ values: 0.085, 0.14, 0.14, 0.10, 0.12, 0.09 and 0.07 μM, respectively). Assessment of the anti-HCV activity (GT1a) using human hepatoma cell line (Huh-7.5) illustrates superior activity of 4e (EC₅₀ = 3.80 μM) relative to VCH-759 (EC₅₀ = 5.29 μM). Cytotoxicity evaluation on, Transformed normal cell lines (Human Liver Epithelial-2, THLE-2 and Proximal Tubular Epithelial, RPTEC/TERT1), demonstrate enhanced safety profile of 4e (CC₅₀ = 102.77, 161.37 μM, respectively) compared to VCH-759 (CC₅₀ = 61.83, 81.28 μM, respectively). Molecular docking of the synthesized derivatives to NS5B polymerase allosteric site (PDB: 2HWH) showed similar binding modes to that of the co-crystallized ligand. Moreover, QSAR models were established for the studied thiazolidinones and thiazolotriazines to investigate the molecular characteristics contributing to the observed NS5B polymerase inhibition activity. The obtained results inspire further investigations of thiazolidinones and thiazolotriazine aiming at affording more potent, safe and orally active non-nucleoside NS5B polymerase inhibitors as anti-HCV drug candidates.

Full text of DOI:10.1016/j.ejmech.2019.111747

Journal Pre-proof
Anti-hepatitis-C virus activity and QSAR study of certain thiazolidinone and
thiazolotriazine derivatives as potential NS5B polymerase inhibitors
Ghaneya S. Hassan, Hanan H. Georgey, Esraa Z. Mohammed, Farghaly A. Omar
PII:
S0223-5234(19)30899-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.111747
EJMECH 111747
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 July 2019
Revised Date: 2 September 2019
Accepted Date: 27 September 2019
Please cite this article as: G.S. Hassan, H.H. Georgey, E.Z. Mohammed, F.A. Omar, Anti-hepatitis-C
virus activity and QSAR study of certain thiazolidinone and thiazolotriazine derivatives as potential NS5B
polymerase inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111747.
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Graphical Abstract:
Cl
S
O
O
N
O
HN
The structural formula and 3D representation of bindig modes of 4e,
the most potent inhibitor of HCV-NS5B polymerase (IC50 of 0.035 uM)
and anti-HCV activity (EC50 of 3.80 uM).
.

Anti-Hepatitis-C Virus Activity and QSAR Study of certain Thiazolidinone
and Thiazolotriazine Derivatives as potential NS5B Polymerase Inhibitors
Ghaneya S. Hassana,b, Hanan H. Georgeya*, Esraa Z. Mohammedc, and Farghaly A. Omarc
a
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo,
11562, Egypt.
b
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Badr University, Cairo,
11829, Egypt.
c Pharmaceutical Chemistry Department, Faculty of Pharmacy, October 6 University, Giza,
12585, Egypt.
* Correspoding author; E-mail: hanan.hana@pharma.cu.edu.eg.
Abstract
The present study reports on evaluation of anti-HCV activity and QSAR of certain
arylidenethiazolidinone derivatives as potential inhibitors of HCV-NS5B Polymerase. The
pursued compounds involving, 5-aryliden-3-arylacetamidothiazolidine-2,4-diones 4-6(a-f),
5-arylidine-2-(N-arylacetamido)-iminothiazolidin-4-one (10) and their rigid counterparts 5-
arylidinethiazolotriazines 13-15(a-f), were synthesized and their structures confirmed by
spectral and elemental analysis. The results of NS5B polymerase inhibition assay revealed
compound 4e, as the most active inhibitor (IC50 = 0.035 µM), which is four folds greater
than that of the reference agent, VCH-759, (IC50 = 0.14 µM). Meanwhile, compounds 4b,
4c, 5a, and 5c, and 13b, 14e and 15c displayed equipotency to 2 folds higher activity than
VCH-759 (IC50 values: 0.085, 0.14, 0.14, 0.10, 0.12, 0.09 and 0.07 µM, respectively).
Assessment of the anti-HCV activity (GT1a) using human hepatoma cell line (Huh-7.5)
illustrates superior activity of 4e (EC50 = 3.80 µM) relative to VCH-759 (EC50 = 5.29 µM).
Cytotoxicity evaluation on, Transformed normal cell lines (Human Liver Epithelial-2, THLE-2
and Proximal Tubular Epithelial, RPTEC/TERT1), demonstrate enhanced safety profile of
4e (CC50 = 102.77, 161.37 uM, respectively) compared to VCH-759 (CC50 = 61.83, 81.28
uM, respectively). Molecular docking of the synthesized derivatives to NS5B polymerase
allosteric site (PDB: 2HWH) showed similar binding modes to that of the co-crystallized
ligand. Moreover, QSAR models were established for the studied thiazolidinediones and
thiazolotriazines to investigate the molecular characteristics contributing to the observed
NS5B polymerase inhibition activity. The obtained results inspire further investigations of
thiazolidinediones and thiazolotriazine aiming at affording more potent, safe and orally
active non-nucleoside NS5B polymerase inhibitors as anti-HCV drug candidates.
Keywords
[1]

Thiazolidinones, thiazolotriazines, HCV-NS5B polymerase, Anti-HCV, Molecular docking,
2D QSAR.
1. Introduction
Hepatitis C virus (HCV) infection represents a major global health concern associated with
the development of chronic liver disease. Approximately 1% of the world population are
chronically infected with HCV and at risk of developing liver cirrhosis and hepatocellular
carcinoma [1]. People in developing countries (where HCV infection is prevailing) suffer
from the high cost of treatment. In Egypt where HCV infection threats many of the
population, thus treatment of HCV has become one of the top national main concerns since
2007 [2-4]. The approved standard therapy for HCV infection for more than a decade
depends on the use of pegylated interferon alpha and ribavirin [5]. Recently, a number of
directly acting antivirals (DAA), such as telaprevir, boceprevir, simeprevir and sofosbuvir
[6,7] have been established as part of combination therapies for HCV infection. Despite the
revolutionary progress in HCV therapy, the battle against HCV infections is not fully
terminated, as a result of higher pharmacoeconomical aspects as well as the appearance of
mutant strains resistant to DAA drugs [7-9].
Interestingly, NS5B RNA-dependant RNA-polymerase (RdRp) is the key enzyme
responsible for HCV genome replication, which is responsible for the initial synthesis of a
complementary negative RNA strand as template to produce new positive strand RNA [10,
11]. This biochemical property, which the host mammalian cell lacks, has resulted in
emergence of NS5B RNA-dependant RNA-polymerase as an attractive and validated anti-
HCV target [12-15]. Investigation of NS5B biochemical properties and structural parameters
revealed the classical “right hand” topology of the polymerase family, with the characteristic
fingers, palm, and thumb domains [16-19]. This insight has provided a valuable platform for
developing NS5B polymerase inhibitors, which are broadly categorized into nucleoside and
nonnucleoside inhibitors (NIs and NNIs).
Non-nucleoside inhibitors (NNIs) include diverse set of small molecules that function by
binding NS5B in one of five allosteric pockets located on the thumb and palm domains
resulting in conformational change of the active site [13,15,20]. Several NS5B polymerse
inhibitors bound to thumb II allosteric pocket, encompassing various chemical motifs have
been reported including anthranilic acids, quinazolin-4-ones, thiophene-2-carboxylic acids,
phenylalanines, pyrano-indoles, dihydropyranones, thiazolones or thiazolidine-4-ones
scaffolds [13,14].
[2]

Literature review obviously infers that 4-thiazolidinones inhibit this enzyme and could be
promising candidates for developing new anti-HCV agents. Also, it is observed that 5-
arylidene substituent represents a dominating pharmacophoric element within the 4-
thiazolidinone NS5B polymerase inhibitors [21-25]. A representative example of this class,
compound I (IC50= 2.00 µM), is illustrated in (Figure 1A) [21]. Molecular docking study of
the thiazolone derivative I into NS5B thumb II allosteric binding pocket revealed distinctive
binding modes: i) H-bonding interactions represented by C=O of thiazolone ring with -NH-
of Tyr477, the thiazolone ring nitrogen with –NH- of Ser476, and the sulfonamide oxygen
with -NH2 of Arg501. ii) Hydrophobic contacts between the side chains of Leu419, Leu497
and Trp528 and the furylidine and phenyl ring [21]. Lead optimization through replacement
of furan ring with a large hydrophobic biphenyl moiety and the p-methyl substituent of the
phenylsulfonamido group by an acetyl aminopiperazinyl moiety connected to 2-
methoxyphenyl afforded compound II, (EC50 = 0.79 µM) (Figure 1B). This modification
retained the essential H-bonding interactions and attained enhanced fitting to the
hydrophobic pocket defined within thumb II domain, in addition to adding acidic moiety
which increased the potency [26].
Hydrophobic
Ser476
Tyr477
N
Arg501
N
H
H
N
A
O
B
O
H
N
S
N
O
O
NH
S
O
O
O
S
S
N
H
Hydrophobic
Extention
N
HN
Hydrophobic
N
I
II
(EC = 0.79 uM)
O
(IC = 2 µM)
O
50
50
Hydrophobic
Figure 1: Ligand-target interactions of 5-furylidene-4-thiazolidinone (lead) and HCV-NS5B
Polymerase allosteric site (A), a modified analogue (B).
Motivated by these findings, series of 5-aylidenethiazolidinone derivatives were selected for
study as potential allosteric inhibitors of HCV-NS5B polymerase. The present investigation
encompasses three approaches: the first one represented by the general structure 2A
involves: i) replacement of the thiazolone scaffold of the lead compound (Figure 2.1) by
[3]

thiazolidine-2,4-dione; ii) switching the furylidine moiety at C-5 by substituted benzylidene
nucleus (aryl A); iii) introduction of an acidic acetamide linker connected to another
substituted phenyl (aryl B) as an additional hydrophobic segment.
R
R
S
A
S
N
N
N
A
O
N
S
O
O
H
N
O
O
N
N
O
HN
B
B
(H; Cl; OCH3)
(H; Cl; OCH3)
2.1
2.3
2.2
Figure 2: General representation of the studied thiazolidinones.
The novelty aspects in this structural manipulation involves the side chain carbonyl group,
an isosteric alternative to S=O in lead compound (Figure 1A), as an extra H-bonding site
augmenting the binding to Arg501 as H-bond donner in the active site. Moreover, the
substitution on the aryl moities (A and B) imparts electronic and lipophilic diversity.
The second approach (Figure 2.2) is bioisosteric replacement of (C=O) in the
thiazolidinedione core structure by (C=NH) to explore the impact of the orientation of the
aryl acetamido side chain on NS5B polymerase inhibitory activity. This will afford 2,3,5-
trisubstituted 4-thiazolidinones, whereby the aryl acetamido side chain has been shifted
from position 3 to 2. Finally, the third approach (Figure 2.3) is mainly directed for
investigation of rigid analogues represented by 3-phenyl thiazolotriazines to explore the
effect of rigidification on the inhibitory activity of the enzyme. This latter approaches has
been recognized in several reports illustrating rigidified bicyclic leads with remarkable NS5B
inhibitory activity. Representatives include benzimidazole derivatives [27], substituted
indoles [28] and thienopyrroles [29].
All the synthesized compounds will be screened for their activity against HCV-NS5B
polymerase and anti-HCV. The most potent compounds will be evaluated for cytotoxicity on
normal cell lines. A molecular docking analysis is also planned to verify the possible binding
modes of the studied compounds within NS5B polymerase allosteric site and identify their
interaction with the enzyme hot spots. Moreover, 2D Quantitative structure activity
relationship (QSAR) study will be carried out to explore the molecular characteristics
controlling the NS5B polymerase inhibitory activity.
[4]

2. Results and Discussion
2.1. Chemistry
The synthetic pathways for preparation of the target compounds are summarized in
schemes 1-3. The core template thiazolidine-2,4-dione (1) was synthesized by reacting
thiourea with monochloroacetic acid followed by acid hydrolysis [30]. It was then condensed
with variably substituted benzaldehyde to give the key intermediates 5-un/substituted
benzylidene thiazolidine-2,4-diones (2a-f) [31]. Treatment of the latter with 2-chloro-N-
un/substituted phenylacetamides (3a-c) in DMF and anhydrous potassium carbonate
[32,33] affords the targeted 2-(5-un/substituted benzylidene-2,4-dioxothiazolidin-3-yl)-N-
un/substituted phenylacetamides 4-6(a-f); Scheme 1.
The structures of the final compounds 4-6(a-f) were confirmed through their spectral and
elemental analyses (Supporting data). The IR spectra characterized by stretching vibrations
of three carbonyl groups at 1658-1757 cm-1, in addition to the amidic NH bands at 3228-
1
3292 cm-1. H NMR spectra revealed singlet signals attributed to CH2 protons of the
acetamido moiety in the range between δ = 4.47- 4.50 ppm. The signals of aromatic protons
also appeared at δ = 6.88-7.89 ppm, in addition to the singlet signals at δ = 7.95-8.10 ppm
corresponding to the methine protons. It is worthy to mention that the 1H NMR of compound
4c showed the characteristic pattern of F/H coupling [34], whereby H-3,5 and H-2,6 protons
of p-flouro substituted benzylidene ring appeared as a triplet signal at δ = 7.40 ppm and a
doublet of doublet signal at δ = 7.73 ppm, respectively. 13C NMR spectra of compounds 4-
6(a-f) showed signals at δ = 44.2-44.6 ppm attributed to CH2, in addition to three carbonyl
signals at δ = 163.6-167.7 ppm. Mass spectra of the desired compounds were matched
with the molecular ion peaks.
[5]

S
S
O
Cl
+
(a)
H2N
NH2
O
O
N
H
OH
(i),(ii)
1
(b)
R
S
R
R
O
(c)
(e)
N
S
N
S
O
O
O
O
O
N
O
H
O
6a-f
HN
2a-f
4a-f
HN
OCH3
(d)
S
R
2a, 4a, 5a, 6a R = H
O
2b, 4b, 5b, 6b R = 4-Cl
2c, 4c, 5c, 6c R = 4-F
N
O
O
2d, 4d, 5d, 6d R = 4-OCH3
2e, 4e, 5e, 6e R = 2-Cl
2f, 4f, 5f, 6f R = 2,4-(Cl)2
HN
5a-f
Cl
Reagents and conditions: (a) (i) H2O, stirr, room temperature, 30 min.; (ii) conc HCl, reflux, 10 h;
o
(b) aromatic aldehyde, anhy. NaOAc, gl. acetic acid, 110 C, 5 h; (c) 2-chloro-N-phenylacetamide
o
(3a), anhy. K2CO3, dry DMF, 100 C, 24 h; (d) 2-chloro-N-(4-chlorophenyl)acetamide (3b), anhy.
o
K2CO3, dry DMF, 100 C, 24 h; (e) 2-chloro-N-(4-methoxyphenyl)acetamide (3c), anhy. K2CO3, dry
DMF, 100 oC, 24 h.
Scheme 1: Synthetic pathway for N-(arylacetamido)-5-arylidenethiazolidin-2,4-diones
4-6(a-f).
The synthesis of the 2,3,5-trisubstituted 4-thiazolidinone 10, (scheme 2) achieved through
cyclization of N-Phenylthiourea (7) with monochloroacetic acid to yield 2-imino-3-
phenylthiazolidin-4-one (8) [35-37]. Subsequent condensation of (8) with benzaldehyde
yielded the key intermediate 5-benzylidene-2-imino-3-phenyl thiazolidin-4-one (9) [38],
which upon treatment with 2-chloro-N-phenylacetamide (3a) affords the targeted derivative
10. The IR spectrum of compound 10 showed signals of the two carbonyl groups at 1672,
1
1718 cm-1, in addition to the NH band at 3273 cm-1. H NMR spectrum showed a singlet
signals at δ = 4.75 ppm corresponding to the aliphatic -CH2- protons, a methine proton at δ
= 7.83 ppm, in addition to the exchangeable NH proton at δ = 10.37 ppm. Mass spectrum of
compound 10 showed the appearance of the molecular ion peak at m/z 413. (Supporting
data)
[6]

S
O
NH
N
S
(b)
(a)
N
N
NH2
S
H
8
7
(c)
S
O
(d)
N
N
S
O
O
N
NH
NH
10
9
Reagents and conditions: (a) NH3, 1 h.; (b) ClCH2COOH, anhy. NaOAc, absolute ethanol, reflux,
o
4 h; (c) benzaldehyde, anhy. NaOAc, gl. acetic acid, 110 C, 5 h; (d) 2-chloro-N-phenylacetamide
(3a), anhy. K2CO3, dry DMF, 100 oC, 24 h.
Scheme 2: Synthetic pathway for 2-(5-Benzylidene-4-oxo-3-phenylthiazolidin-2-
ylideneimino)-N-phenylacetamide (10).
The rigidified thiazolotriazines 13-15(a-f) were prepared analogously as shown in scheme
3. Condensation of 2-iminothiazolidin-4-one (11) [39] with the appropriate aromatic
aldehydes afforded the key arylidenethiazolidinones (12a-f) [26]. Applying mannich reaction
on (12a-f) using the respective aniline derivatives and paraformaldehyde resulted in
compounds 13-15(a-f). The IR spectra of these compounds indicate the appearance C=O
1
signal at 1689-1743 cm-1 and disappearance of NH bands. Also, their H NMR revealed
the appearance of two singlet signals at δ = 4.95-5.07 ppm and 5.30-5.42 ppm
corresponding to two -CH2- groups and the absence of any exchangeable protons. The 13C
NMR spectra revealed two signals at δ = 61.0-62.3 ppm and 65.9-66.7 ppm attributed to
two -CH2- groups. Mass spectra of these compounds accorded their molecular ion peaks.
(Supporting data)
[7]

O
S
S
Cl
(a)
+
H2N
NH2
OH
NH
O
N
H
11
(b)
S
R
R
N
S
R
N
(c)
N
(e)
O
N
N
S
O
N
O
NH
N
H
12a-f
13a-f
15a-f
OCH3
(d)
12a, 13a, 14a, 15a R = H
12b, 13b, 14b, 15b R = 4-Cl
12c, 13c, 14c, 15c R = 4-F
S
R
N
12d, 13d, 14d, 15d R = 4-OCH3
12e, 13e, 14e, 15e R = 2-Cl
12f, 13f, 14f, 15f R = 2,4 (Cl)2
N
O
N
14a-f
Cl
Reagents and conditions: (a) absolute ethanol, anhy. NaOAc, reflux, 4h; (b) aromatic aldehyde,
anhy. NaOAc, gl. acetic acid, 110 oC, 5 h; (c) aniline, 37% formaldehyde solution, dry DMF, 100 oC,
o
6 h; (d) 4-chloroaniline, 37% formaldehyde solution, dry DMF, 100 C, 6h; (e) 4-methoxyaniline,
37% formaldehyde solution, dry DMF, 100 oC, 6 h.
Scheme 3: Synthetic pathway for the thiazolotriazines 13-15(a-f).
2.2. Biological Screening
2.2.1. In vitro HCV-NS5B polymerase inhibitory assay
It is worthy to mention that, although some of the targeted final compounds for the present
investigation are previously reported for restricted biological activities, none of them has
been screened for potential antiviral activity. The previously reported compounds are
4a,b,d,e,f [40], 5b [41], 13a [42], 13b,c [43], 13d, 15a,d [44], 13f, 14f, 15c,f [45] and 15b
[46]. These targeted compounds are prepared by modified procedures, their structure are
confirmed and investigated for potential anti-HCV activity.
The synthesized compounds of 5-arylidine-4-thiazolidinones 4-6(a-f), 9, and 10, as well as
the thiazolotriazines 13-15(a-f) were evaluated in vitro for inhibition of HCV-NS5B
polymerase enzyme. The in vitro RdRp assay was carried out using recombinant NS5B
polymerase, Genotype 1a (GT1a), which contains nucleotide residues 21-371 of the HCV
[8]

5'-untranslated E. coli DNA Polymerase I region from GenBank accession number
AF356827, at 0.26 ug/Well concentration. VCH-759 has been selected for comparison, as
positive control since it is reported as one of the NNIs of NS5B polymerase [47].
OH
N
S
O
O
-O
Na+
VCH-759
The obtained dose response curves, were used to calculate IC50 values (µM) that are listed
in Table 1. Figure 3 illustrates graphical representation of the most active compounds
within the thiazolidinones 4-6(a-f) and their rigid analogues of the thiazolotriazines 14-16(a-
f). Analysis of the results of NS5B polymerase inhibitory assay (Table 1) illustrate generally,
that the studied compounds 4-6(a-f), 9, 10, and 13-15(a-f), comprising 5-arylidine-4-
thiazolidinone scaffold, exhibit appreciable HCV-NS5B polymerase inhibition activity.
Compounds 4b, 4e, 5c, 13b, 14e, and 15c are superior inhibitors of HCV-NS5B
polymerase (GT1a) (IC50: 0.085, 0.035, 0.10, 0.12, 0.09, & 0.07 µM, respectively) than
VCH-759, the reference compound (IC50 = 0.14 uM). Additionally, Compounds 4c and 5a
showed equivalent inhibitory activity (IC50: 0.14), whereas, the remaining compounds
display lower activity than the reference agent.
Table 1: NS5B polymerase (GT1a) inhibitory concentrations (IC50 µM) and HCV anti-viral
activity (EC50 µM) values of the tested compounds and VCH-759.
R
A
S
S
N
R
A
N
N
O
O
N
O
O
B
R
B
R
13-15 (a-f)
4-6 (a-f)
[9]

Compds.
No
R (A)
R (B)
IC50
EC50
VCH-759
4a
-
H
4-Cl
4-F
4-OCH3
2-Cl
2,4(Cl)2
H
4-Cl
4-F
4-OCH3
2-Cl
2,4(Cl)2
H
4-Cl
4-F
4-OCH3
2-Cl
2,4(Cl)2
-
-
H
4-Cl
4-F
4-OCH3
2-Cl
2,4(Cl)2
H
4-Cl
4-F
4-OCH3
2-Cl
2,4(Cl)2
H
4-Cl
4-F
-
H
H
H
H
0.14
4.80
0.085
0.14
0.25
0.035
0.44
0.14
0.21
0.10
0.28
2.99
5.12
0.45
0.37
0.39
0.78
1.20
0.32
0.58
0.76
0.62
0.12
2.20
0.20
0.43
1.37
0.49
1.82
0.20
0.99
0.09
0.54
0.75
3.53
0.07
0.54
1.26
0.22
5.29
19.60
11.10
8.40
11.57
3.80
10.78
16.70
7.80
16.92
7.93
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
H
H
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
-
6.47
43.96
10.62
43.89
10.80
19.56
11.02
43.86
11.06
8.98
13.32
10.90
18.72
12.59
30.68
14.45
19.31
10.80
6.58
6f
9
10
-
H
H
H
H
H
H
13a
13b
13c
13d
13e
13f
14a
14b
14c
14d
14e
14f
15a
15b
15c
15d
15e
15f
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
24.31
13.75
9.50
10.80
10.29
40.08
9.54
4-OCH3
2-Cl
2,4(Cl)2
13.53
13.23
[10]

IC50
Figure 3: The NS5B polymerase (GT1a) inhibitory activity of the most active compounds
and VCH-759.
It is noticeable, that the 5-benzylidine-2-imino-3-phenylthiazolidinone 9 and its 2-substituted
imino analogue 10 (IC50: 0.58 and 0.76 µM, respectively) were ~ 4 - 5 folds less active than
the reference compound VCH-759, (IC50 = 0.14 µM). Rigidification of the targeted molecules
13-15(a-f) led to decrease in the enzyme inhibition activity compared to the open chain
analogues 4-6(a-f).
Investigation of the structural aspects within the studied compounds revealed that the
electronic characteristics of substituents on the arylidene moiety (ring A) play an effect on
the IC50 values. The prescence of halogen (Cl) atoms as lipophilic, electron-withdrawing
substituents on position 2 or 4 is more favourable for the enzyme inhibition activity than the
polar, electron-donating methoxy alternative. This can be represented by the halogen-
substituted compounds 4b, e (IC50: 0.085, 0.035 uM) compared to that of the 4-methoxy
analogue 4d (IC50: 0.25 uM). Similarly, (6b, f vs.6d: 0.37, 0.32 > 0.78 uM), (14e vs. 14d:
0.09 > 0.99 uM) and (15f > 15d: 0.22 > 0.54 uM). Explicitly, it is remarkable that the best
results of HCV-NS5B polymerase allosteric inhibition could be attained in case of 2-
chlorobenzylidine moiety on C-5 of the thiazolidindione nucleus (compound: 4e; IC50: 0.035
uM). Moreover, it was observed that mono-halogenation on ring A seems to be more
favourable for enhancing the inhibition activity rather than the di-halogenation. Illustrative
examples are the compounds (4b,c,e vs. 4f: IC50 values 0.085, 0.14, 0.035 > 0.44 uM),
(5b,c vs. 5f: IC50 values 0.21, 0.10 > 5.12 uM), (13b,e vs. 13f: IC50 values 0.12, 0.43 > 1.37
uM) and (14e vs. 14f: IC50 values 0.09 > 0.54 uM). This might imply significant role for
steric parameter within the homologous series. Evidence for such postulation can be stated
through compound 5f, whereby aryl ring A is 2,4-dichlorobenzylidene and aryl ring B is 4-,
[11]

This group, assigned as moderate inhibitors of HCV replication, involves compounds
4a,b,d,f; 5a,c; 6a,c,d,e; 9; 13a,b,c,d,f; 14a,b,e; 15a,e,f. The rest of the tested compounds
exhibited ~ 5 – 9 folds lower anti-HCV activity than that of VCH-759 with EC50 range of
24.31 - 43.96 µM.
2.2.2. Anti-HCV activity
The anti-HCV screening of the studied compounds 4-6(a-f), 9, 10, and 13-15(a-f) was
carried out using a highly permissive subclone of the human hepatoma cell line (Huh-7.5)
[48]. HCV core protein has been determined using the HCV core antigen ELISA kits
(XpressBio Life Science Products) according to the manufacturers instructions (Supporting
data). The effective concentrations that reduce HCV replication by 50% (EC50) of the tested
compounds and the reference agent VCH-759 are summarized in Table 1. Figure 4
illustrates graphical representation of the most active compounds within the thiazolidinones
4-6(a-f) and their rigid analogues of the thiazolotriazines 14-16(a-f).
EC50
Figure 4: Anti-HCV activity of the most active compounds and VCH-759.
The results showed that, the tested compounds reduce HCV replication by 50% at a wide
range of concentrations (3.80 – 43.89 µM). Compound 4e, (the most potent NS5B
polymerase inhibitor: IC50 = 0.035 µM), proved to be the most active suppressor of HCV
replication with EC50 of 3.80 µM (VCH-759 (EC50 = 5.29 µM)). Nine compounds were ~ 1 - 2
folds less active than the reference compound VCH-759 (4c, 5b, 5d, 5e, 10, 14c, 14f, 15b
and 15d: EC50 = 8.40, 7.80, 7.93, 6.47, 8.98, 6.58, 9.50, 10.29 and 9.54 µM, respectively).
Another series encompass 22 compounds showed EC50-values ~ 3 – 4 folds less than that
of VCH-759. This group, assigned as moderate inhibitors of HCV replication, involves
compounds 4a, 4b, 4d, 4f; 5a, 5c; 6a, 6c, 6d, 6e; 9; 13a, 13b, 13c, 13d, 13f; 14a, 14b,
[12]

14e; 15a, 15e, 15f. The rest of the tested compounds exhibited ~ 5 – 9 folds lower anti-
HCV activity than that of VCH-759 with EC50 range of 24.31 - 43.96 µM.
2.2.3. Cytotoxicity study
Compound 4e, which proved to be the most potent inhibitor of both HCV replication and its
NS5B polymerase, was subjected to cytotoxicity study on Transformed Human Liver
Epithelial-2 (THLE-2) and Proximal Tubular Epithelial (RPTEC/TERT1) normal cell lines.
VCH-759 has been used as a reference and the viability of the cells was measured using
MTT assay. The method depends on assessment of cell metabolic activity on basis of color
development resulting from biochemical reaction. The mechanism behind the MTT assay
involves NAD(P)H-dependent cellular oxidoreductase enzyme that converts the yellow
tetrazolium MTT, [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide], into
insoluble formazan, (E,Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan. The formed
formazan can be dissolved with dimethylsulfoxide (DMSO) to give a purple color with
characteristic absorption at 540 nm. The Intensity of purple color is directly proportional to
the cell number and thus indicating the cell viability [49]. Cytotoxic concentration (CC50) of
compound 4e was calculated to assess its safety profile. The results illustrated in Table 2
show that compound 4e exhibited low toxicity on both THLE-2 and RPTEC/TERT1 normal
cell lines compared to the reference compound. Moreover, to assess the selectivity of
compound 4e, Selectivity index (SI) also was calculated indicating a measure of the
selectivity against the infected cells rather than the normal cells (selectivity index = CC50 on
normal cells/EC50 on infected cells). SI also provides information on the therapeutic window
of a compound, the larger the SI, the larger the therapeutic window of the compound [50].
Compound 4e showed selectivity index value larger than VCH-759 reference compound
confirming a tolerable cytotoxicity profile of 4e.
Table 2: Cytotoxicity (CC50, µM)a and Selectivity index (SI)b values of the tested compound
and VCH-759.
Code
THLE-2 cell line
RPTEC/TERT1 cell line
CC50
SI
CC50
SI
VCH-759
4e
61.83
11.69
81.28
15.36
102.77
27.04
161.37
42.47
a: CC50 is the concentration of compounds that reduce the cell viability by 50%.
b: SI: is the ratio of CC50 values on normal cells to EC50 on hepatoma infected cell
lines (Huh7.5).
[13]

2.3. Molecular modeling study
2.3.1. Molecular docking
The observed inhibition of NS5B-polymerase elicited by the studied compounds, as well as
their anti-HCV activity promote investigation of the docking pattern to thumb II pocket of the
enzyme. Identification of the interaction modes with the key amino acids in allosteric binding
site might rationalize their observed activity. Several crystal structures of HCV-NS5B
polymerase co-crystallized with thiazolone inhibitors in thumb II allosteric binding pocket
were identified and reported [51-53]. Based on the structural relevance and the same
mechanism of inhibition to the anticipated compounds, HCV-NS5B polymerase co-
crystallized with compound I, (5-((5-methylfuran-2-yl)-methylenetosylamino)-thiazol-4(5H)-
one), previously mentioned in introductory section was downloaded (PDB code: 2HWH;
http://www.rcsb.org/) [21]. Docking protocol Using MOE 2016.08 molecular modeling
program has been undertaken. A validation step involving redocking of the co-crystallized
ligand into the respective allosteric binding site is initially accomplished. Validation
parameters e.g. RMSD = 0.924 Aº, in addition to similar interaction pattern to that of the co-
crystallized ligand within the allosteric binding site approved the validity of the adopted
docking protocol (Supporting data). This interaction pattern revealed three hydrogen
bonding and two hydrophobic contacts. Specifically, hydrogen bonding of C=O & N of the
thiazolone ring and sulfonamide moiety with backbone –NHs of Tyr477, Ser476, and side
chain –NH2 of Arg501, respectively. In addition to phenyl and furan rings hydrophobic
contacts with the side chains of Leu419, Trp528 and Leu497 [21].
Subsequently, docking procedures have been accomplished for the studied compounds. All
the thiazolidin-2,4-dione derivatives 4-6(a-f) demonstrate comparable binding interactions
(Supporting data). Predominantly, H-bonding of the C=O group at position-2 of thiazolidin-
2,4-dione ring to –NH2 of Arg501, represents a common dominator, and considered as an
important requirement for allosteric inhibition of NS5B polymerase. Moreover, in some
compounds the C=O of the arylacetamido side chain participate also in the interaction with
key amino acid residues Arg501 and Lys533. Hydrophobic contacts with Trp528 and
Lys533 are also apparent. Figure 5 illustrates the docking pattern of the most active
compound 4e (IC50 = 0.035 µM). Its binding to the allosteric site involves H-bonding to –NH2
of Arg501 through the arylacetamido C=O and the C=O at position-2 of thiazolidin-2,4-dione
ring. An additional H-bonding between the C=O group at position-4 of the thiazolidin-2,4-
dione ring with the side chain –NH of Ser476 is observable. Moreover, it demonstrates
hydrophobic contacts through its chlorophenyl moiety with Ile482. Similarily, the 2,3,5-
[14]

trisubstituted 4-thiazolidinone (10) demonstrates H-bonding through its side chain C=O
group to Arg501 amino acid; Figure 6.
(A)
(B)
Figure 5: 2D ligand interaction diagram (A) and 3D representation (B) of compound 4e
(yellow) showing its interaction with NS5B polymerase allosteric site.
(A)
(B)
Figure 6: 2D ligand interaction diagram (A) and 3D representation (B) of compound 10
(yellow) showing its interaction with NS5B polymerase allosteric site.
In case of the thiazolotriazine derivatives 13-15(a-f), H-bonding interactions between
thiaozolidinone C=O and/or C=N of the triazine nucleus to the key amino acid, Arg501 were
also observed. Figure 7A, illustrates the binding modes of compound 15c as a
representative for this class. Additional H-bondings were also detected with Ser476, and
Lys533 amino acids. To facilitate the data interpretation, an overlay complex representation
[15]

of the co-crystallized ligand (red) and its redocked pose (green) in the HCV-NS5B
polymerase allosteric site is shown in Figure 7B.
(A)
(B)
Figure 7: 2D ligand interaction diagram of compound 15c (A) and 2D representation of
superimposition of the co-crystallized ligand (red) and its redocked pose (green) in the
HCV-NS5B polymerase allosteric site (B)
In summary, we found that top-ranked docking poses of almost all the synthesized
compounds displayed similar interaction modes to those of the reported HCV-NS5B
polymerase allosteric inhibitors, indicating that most of the synthesized compounds bind
favorably to HCV-NS5B polymerase thumb II pocket [21].
2.3.2. QSAR study
QSAR is an important tool to search the reliable relationship between chemical structure
and biological activities [54]. In an effort to investigate the most contributing molecular
characteristics of the studied thiazolidnones and thiazolotriazines for the observed NS5B
polymerase inhibitory activity, two QSAR models (1 & 2) were generated using multiple
linear regression (MLR) analysis. Two data sets of the synthesized thiazolidnones 4-6(a-f)
& 10 and thiazolotriaznes 13-15 (a-f) were chosen respectively for generation of QSAR
models. NS5B polymerase inhibitory activities are expressed in logarithmic scale (pIC50 in
Molar concentration) “pIC50 = -log(IC50*10-6)”. Primarily, several trials to generate QSAR
models using MOE 2016.08 based on simple physico-chemical characteristics (e.g. LogP,
molar refractivity, TPSA,……etc) of the studied compounds were investigated.
Unfortunately, no significant models could be attained. Alternatively, a wide range of
descriptors
(total
#:
1097)
available
on
ChemDes
web-based
platform
[16]

(http://www.scbdd.com/chemdes) were calculated for all compounds. In Rapidminer studio
version 9.0.003, forward selection method was used for selection of the significant features.
A MLR protocol was then employed to build up the QSAR models.
Equation (1) represents the best performing QSAR model for the inhibitory activity of
thiazolidinones series against HCV NS5B polymerase.
pIC50 = - 2.372 * bcutp14 - 0.331 * RDFE30 - 11.548 * RDFC20 + 0.185 * RDFU26
+ 0.019 * RPCS + 8.306.
n = 19 r = 0.953
(1)
r2 = 0.908
RMSE = 0.171
Equation (2) represents the best performing QSAR model for the inhibitory activity of
thiazolotriazines against HCV NS5B polymerase.
pIC50 = 5.636 * bcutm3 - 0.943 * ncof + 114.291 * MoRSEC24 - 15.728
r2 = 0.712
RMSE = 0.254
(2)
n = 18
r = 0.844
The two MLR models (1) and (2) exhibited good correlation coefficients (r = 0.953 & 0.844),
squared correlation coefficients (r2 = 0.908 & 0.712), and root mean square errors (RMSE =
0.171 & 0.254), respectively. Cross-validation statistical technique has been applied to
asses the quality with regard to predictive ability of the generated models. Leave-group-out
(leave-10%-out) cross-validation was performed for the generated models resulting in q CV
= 0.900 +/- 0.300 (micro average: 0.879), q2 CV = 0.900 +/- 0.300 (micro average: 0.773)
and RMSE CV = 0.245 +/- 0.139 (micro average: 0.273 +/- 0.000) for model (1). While
model (2) has resulted in q CV = 0.600 +/- 0.490 (micro average: 0.775), q2 CV= 0.600 +/-
0.490 (micro average: 0.601) and RMSE CV = 0.244 +/- 0.186 (micro average: 0.301+/-
0.000). In addition, the low residual value (Supporting data) for each compound in the
datasets outlines the degree of correlation between observed and predicted values and the
models predictive ability. Scattering plots of the observed versus predicted activity values
for the data set of thiazolidinones and thiazolotriazines are represented graphically in
Figures 8 & 9, respectively.
[17]

8.0
7.5
7.0
6.5
6.0
5.5
5.0
5.0
5.5
6.0
6.5
7.0
7.5
8.0
Obs. pIC50
Figure 8: Correlation between observed and predicted pIC50 using MLR model (1) (r2 =
0.908)
8.0
7.5
7.0
6.5
6.0
5.5
5.0
5.0
5.5
6.0
6.5
Obs.pIC50
7.0
7.5
8.0
Figure 9: Correlation between observed and predicted pIC50 using MLR model (2) (r2 =
0.712)
Analysis of QSAR Models
For thiazolidinones model (eq. 1), five descriptors (bcutp14, RDFE30, RDFC20, RDFU2
and RPCS) were selected via forward selection method. bcutp14 is one of the Burden
molecular descriptors based on the polarizability version of the Burden matrix which takes
into account both the connectivity as well as atomic properties of a molecule [55]. Three
[18]

radial distribution function (RDF) (RDFE30, RDFC20 and RDFU26) were also encountered
in model (1). RDF descriptors are based on the distance distribution of the compounds
which can be interpreted as the probability distribution of finding an atom in a spherical
volume of radius r [56]. RDFE30 descriptor assigned for 3D-RDF - signal 30 / weighted by
atomic Sanderson electronegativities. This descriptor suggests a relation between the
biological activity and the 3D molecular distribution of electronegative atoms (electrostatic
environment) calculated at radius of 3.0 Å, from the geometrical center of each molecule
[57]. RDFC20 descriptor is 3D-RDF - signal 20 / weighted by atomic charge. The descriptor
indicates occurrence of some dependence between the biological activity and the 3D
molecular distribution of charge, calculated at radius of 2.0 Å from the geometrical centre of
each molecule. The last RDF descriptor is unweighted (RDFU26) noted for (3D-RDF -
signal 26/unweighted). Finally, RPCS is one of the charged partial surface area (CPSA)
descriptors assigned for the relative positive charge surface area. It represent the solvent-
accessible surface area of the most positive atom divided by the relative positive charge
(RPCG) [58]. The negative coefficients of bcutp14, RDFE30 and RDFC20 mean that
increasing these descriptors leads to decrease in the biological activity for their analogues.
Meanwhile, the positive coefficients of RDFU26 and RPCS indicate that their increase have
a progressive effect on the NS5B polymerase inhibitory activity.
Concerning thiazolotrazines QSAR model (2), the three selected descriptors were bcutm3,
ncof and MoRSEC24. bcutm3 is a Burden descriptors based on a mass version of the
Burden matrix which takes into account both the connectivity as well as atomic properties of
a molecule [55]. Nocf is a constitutional descriptor noted for count of fluorine atoms.
MoRSEC24 is one of 3D molecule representation of Structures based on Electron
diffraction (3D MoRSE) which are derived from Infrared spectra simulation using a
generalized scattering function [59]. It is the 3D-MoRSE - signal 24/weighted by atomic
charge. The positive coefficients of bcutm3 and MoRSEC24 descriptors indicate a
constructive effect on the inhibitory activity of these analogues. Whereas the negative sign
of Ncof descriptor illustrates that the presence of fluorine substituent in thiazolotriazine
derivatives does not affect the NS5B inhibition activity.
3. Conclusion
In this study, thirty-eight compounds were synthesized, structurally elucidated and
evaluated for their NS5B polymerase inhibition and anti-HCV activities. The results revealed
that the compounds exhibited significant enzyme inhibition and anti-HCV activities
[19]

compared to VCH-759, which is a known investigational NS5B polymerase inhibitor [47].
Molecular docking study confirmed the observed activity of the compounds by virtue of their
H-bonding interactions with the key amino acids in the allosteric thumb II pocket of the
enzyme. The study paved the important role of involving the group at position-2 of the
thiazolidindione derivatives for H-bonding with Arg501 in the binding site. QSAR study
ilustrated that bcutp14, RDFE30, RDFC20, RDFU2 and RPCS descriptors might be
important contributors for thiazolidinones activity; Model (1). Meanwhile, bcutm3, ncof and
MoRSEC24 descriptors might be significant for thiazolotriazines inhibitory activity; Model
(2). The study identified compound 4e as the most potent inhibitor for HCV-NS5B
polymerase as well as anti-HCV agent with IC50 of 0.035 uM and EC50 of 3.80 uM,
respectively. Assessment of cytotoxicity of compound 4e indicates a safety profile on THLE-
2 and RPTEC/TERT1 normal cell lines with CC50 values of 102.77, 161.37 uM, respectively.
Optimization of this class of compounds might provide a start point for developing new anti-
HCV agents, and further lead optimization are worth pursuing.
Conflict of interests: The authors declare no conflict of interest.
4. Experimental
4.1. Chemistry
Melting points (°C) were carried out by open capillary tube method using Biocote melting
point apparatus (BIBBY, scientific limited stone, staeeordshire) ST, 150 SA, UK. Elemental
analyses (C, H, and N) were performed on FLASH 2000 CHNS/O analyzer, Thermo
Scientific at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Nasr
City, Cairo. Results of C, H, N were within ± 0.4 of the theoretical values for the given
formulae. Infrared spectra were determined using Shimadzu Fourier-transform infrared
spectroscopy (IR-470, Schimadzu, Kyoto, Japan). All spectra were expressed as υ cm-1,
using potassium bromide discs.1H NMR and 13C NMR spectra were carried out using
Bruker high performance digital FT-NMR spectrometer AVANCE III400 MHz (Bruker
1
Corporation, Germany) at Microanalytical Unit, Faculty of Pharmacy, Cairo University. H
NMR spectra were run at 400 MHz and 13C NMR spectra were run at 100 MHz in DMSO-d6
as a solvent, chemical Shifts are quoted in δ as parts per million (ppm) downfield from
tetramethylsilane (TMS) as internal standard. Mass Spectra were run on Single Quadruple
mass spectrometer ISQ LT, at the Microanalytical Center, Faculty of Science, Cairo
University. Reactions were monitored by thin layer chromatography (TLC) using precoated
[20]

Aluminium sheets, Silica gel Kieselgel 60 F254; (Merck, Darmstadt, Germany). TLC sheets
were visualized at 366, 254 nm by UV VilberLourmat 77202 (Vilber, Marne La Vallee,
France) and methylene chloride was used as eluting solvent.
2-(5-un/substitutedbenzylidene-2,4-dioxothiazolidin-3-yl)-N-un/substitutedphenyl
acetamide 4a-f, 5a-f, 6a-f.
A solution of the appropriate benzylidene thiazolidindione (2a-f) (10 mmol) and anhydrous
potassium carbonate (0.69 g, 5 mmol) in dry DMF (20 mL), was stirred at 100oC for 30 min.
The corresponding 2-chloro N-un/sub phenyl acetamide (3a-c) (10 mmol) was added and
stirring was continued at 100oC for 24 h. The reaction mixture was poured onto crushed ice,
the precipitated product was filtered off, dried and crystallized from ethanol.
2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)-N-phenylacetamide (4a).
Yield: (2.88 g) 85%. m.p. 263-265 °C. Rf: 0.25. IR υ cm-1: 3280 (NH), 1749, 1697, 1662
(C=O). 1H NMR δ ppm: 4.53 (s, 2H, CH2), 7.07 (t, 1H, J = 7.38 Hz, H-4 phenyl), 7.31 (t, 2H,
J = 7.86 Hz, H-3,5 benzylidene), 7.53-7.59 (m, 5H, H-2,4,6 benzylidene, H-3,5 phenyl), 7.67
13
(d, 2H, J = 7.24 Hz, H-2,6 phenyl), 8.01 (s, 1H, C=CH), 10.40 (s, 1H, NH, exch. D2O). C
NMR δ ppm: 44.4 (CH2), 119.6, 121.4, 124.2, 129.3, 129.9, 130.6, 131.3, 133.2, 134.1,
138.7 (C-Ar, C-S, C=CH), 164.2, 165.7, 167.6 (C=O). MS (m/z, %): 338 (M+, 100). Anal.
Calcd. for C18H14N2O3S (338.38): C, 63.89; H, 4.17; N, 8.28. Found: C, 63.78; H, 4.20; N,
8.15.
(5-(4-Chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-phenylacetamide (4b).
Yield: (3.10 g) 83%. m.p. 279-281 °C. Rf: 0.31. IR υ cm-1: 3282 (NH), 1747, 1697, 1666
(C=O), 831 (C-Cl). 1H NMR δ ppm: 4.53 (s, 2H, CH2), 7.07 (t, 1H, J = 7.38 Hz, H-4 phenyl),
7.31 (t, 2H, J = 7.94 Hz, H-3,5 phenyl), 7.55 (d, 2H, J = 7.68 Hz, H-3,5 benzylidene), 7.63
(d, 2H, J = 8.64 Hz, H-2,6 benzylidene), 7.69 (d, 2H, J = 8,60 Hz, H-2,6 phenyl), 8.01 (s,
1H, C=CH), 10.39 (s, 1H, NH, exch. D2O). 13C NMR δ ppm: 44.5 (CH2), 119.6, 122.2,
124.2, 129.3, 129.9, 132.2, 132.3, 132.7, 135.9, 138.7 (C-Ar, C-S, C=CH), 164.1, 165.6,
167.3 (C=O). MS (m/z, %): 372 (M+, 47), 374 (M++2, 19), 93 (100). Anal. Calcd. For
C18H13ClN2O3S (372.83): C, 57.99; H, 3.51; N, 7.51. Found: C, 57.71; H, 3.50; N, 7.33.
2-(5-(4-Fluorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-phenylacetamide (4c).
Yield: (2.60 g) 73%. m.p. 273-275 °C. Rf: 0.28. IR υ cm-1: 3263 (NH), 1751, 1697 (C=O). 1H
NMR δ ppm: 4.53 (s, 2H, CH2), 7.07 (t, 1H, J = 7.38 Hz, H-4 phenyl), 7.31 (t, 2H, J = 7.94
Hz, H-3,5 phenyl), 7.40 (t, 2H, J = 7.86 Hz, H-3,5 benzylidene), 7.55 (d, 2H, J = 7.68 Hz,
H-2,6 phenyl), 7.73 (dd, 2H, J = 8.74, 5.42 Hz, H-2,6 benzylidene), 8.02 (s, 1H, C=CH),
10.39 (s, 1H, NH, exch. D2O). 13C NMR δ ppm: 44.4 (CH2), 116.9, 117.1, 121.1, 124.2,
[21]

129.3, 129.9, 133.0, 133.1, 138.7, 162.3 (C-Ar, C-S, C=CH), 164.2, 165.7, 167.5 (C=O). MS
(m/z, %): 356 (M+, 100). Anal. Calcd. for C18H13FN2O3S (356.37): C, 60.67; H, 3.68; N,
7.86. Found: C, 60.39; H, 3.33; N, 7.72.
2-(5-(4-Methoxybenzylidene)-2,4-dioxothiazolidin-3-yl)-N-phenylacetamide (4d).
Yield: (3.42 g) 88%. m.p. 255-257 °C. Rf: 0.23. IR υ cm-1: 3302 (NH), 1741, 1691 (C=O). 1H
NMR δ ppm: 3.85 (s, 3H, OCH3), 4.51 (s, 2H, CH2), 7.06 (t, 1H, J = 7.38 Hz, H-4 phenyl),
7.13 (d, 2H, J = 8.84 Hz, H-3,5 benzylidene), 7.31 (t, 2H, J = 7.92 Hz, H-3,5 phenyl), 7.55
(d, 2H, J = 7.72 Hz, H-2,6 benzylidene), 7.63 (d, 2H, J = 8.80 Hz, H-2,6 phenyl), 7.96 (s,
1H, C=CH), 10.39 (s, 1H, NH, exch. D2O). 13C NMR δ ppm: 44.3 (CH2), 56.0 (OCH3),
115.5, 118.1, 119.6, 124.3, 125.7, 129.3, 132.8, 134.1, 138.7, 161.8 (C-Ar, C-S, C=CH),
164.3, 165.9, 167.7 (C=O). MS (m/z, %): 368 (M+, 100). Anal. Calcd. for C19H16N2O4S
(368.41): C, 61.94; H, 4.38; N, 7.60. Found: C, 62.35; H, 4.34; N, 7.45.
2-(5-(2-Chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-phenylacetamide (4e).
Yield: (3.36 g) 90%. m.p. 227-229 °C. Rf: 0.31. IR υ cm-1: 3271 (NH), 1751, 1697, 1666
(C=O), 763 (C-Cl). 1H NMR δ ppm: 4.54 (s, 2H, CH2), 7.07 (t, 1H, J = 7.40 Hz, H-4 phenyl),
7.31 (t, 2H, J = 7.94 Hz, H-3,5 phenyl), 7.54-7.58 (m, 4H, H-3,4,5,6 benzylidene), 7.64-7.69
(m, 2H, H-2,6 phenyl), 8.10 (s, 1H, C=CH), 10.41 (s, 1H, NH, exch. D2O). 13C NMR δ ppm:
44.5 (CH2), 119.6, 124.2, 125.2, 128.6, 129.2, 129.3, 129.5, 130.8, 131.3, 132.6, 134.9,
138.7 (C-Ar, C-S, C=CH), 164.1, 165.3, 167.3 (C=O). MS (m/z, %): 372 (M+, 76), 374
(M++2, 30), 93 (100). Anal. Calcd. for C18H13ClN2O3S (372.83): C, 57.99; H, 3.51; N, 7.51.
Found: C, 58.23; H, 3.48; N, 7.37.
2-(5-(2,4-Dichlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-phenylacetamide (4f).
Yield: (3.79 g) 93%. m.p. 220-222 °C. Rf: 0.37. IR υ cm-1: 3265 (NH), 1743, 1701, 1664
(C=O), 744 (C-Cl). 1H NMR δ ppm: 4.54 (s, 2H, CH2), 7.07 (t, 1H, J = 7.38 Hz, H-4 phenyl),
7.31 (t, 2H, J = 7.94 Hz, H-3,5 phenyl), 7.55-7.60 (m, 2H, H-5,6 benzylidene), 7.62-7.69
(m, 2H, H-2,6 phenyl), 7.88 (s, 1H, H-3 benzylidene), 8.02 (s, 1H, C=CH), 10.41 (s, 1H, NH,
exch. D2O). 13C NMR δ ppm: 44.5 (CH2), 119.6, 121.6, 124.2, 125.7, 128.0, 128.8, 129.3,
130.4, 130.6, 135.9, 136.3, 138.7 (C-Ar, C-S, C=CH), 164.0, 165.3, 167.1 (C=O). MS (m/z,
%): 406 (M+, 40), 408 (M++2, 28), 410 (M++4, 6), 371 (100). Anal. Calcd. For
C18H12Cl2N2O3S (407.27): C, 53.08; H, 2.97; N, 6.88. Found: C, 53.08; H, 2.68; N, 6.65.
2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)-N-(4-chlorophenyl) acetamide (5a).
Yield: (3.18 g) 85%. m.p. 252-254 °C. Rf: 0.32. IR υ cm-1: 3278 (NH), 1747, 1693, 1660
1
(C=O), 829 (C-Cl). H NMR δ ppm: 4.53 (s, 2H, CH2), 7.38 (d, 2H, J = 8.88 Hz, H-3,5
phenyl), 7.52-7.60 (m, 5H, H-2,3,4,5,6 benzylidene), 7.66 (d, 2H, J = 7.12 Hz, H-2,6
[22]

phenyl), 8.00 (s, 1H, C=CH), 10.54 (s, 1H, NH, exch. D2O). 13C NMR δ ppm: 44.4 (CH2),
121.2, 121.3, 127.8, 129.2, 129.8, 130.6, 131.3, 133.2, 134.1, 137.7 (C-Ar, C-S, C=CH),
164.4, 165.7, 167.6 (C=O). MS (m/z, %): 372 (M+, 100), 374 (M++2, 39). Anal. Calcd. for
C18H13ClN2O3S (372.83): C, 57.99; H, 3.51; N, 7.51. Found: C, 57.67; H, 3.30; N, 7.65.
2-(5-(4-Chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-chlorophenyl)acetamide
(5b).
Yield: (3.67 g) 90%. m.p. 285-287 °C. Rf: 0.36. IR υ cm-1: 3259 (NH), 1747, 1691, 1672
1
(C=O), 825 (C-Cl). H NMR δ ppm: 4.53 (s, 2H, CH2), 7.38 (d, 2H, J = 8.84 Hz, H-3,5
benzylidene), 7.57 (d, 2H, J = 8.88 Hz, H-2,6 benzylidene), 7.63 (d, 2H, J = 8.64 Hz, H-3,5
phenyl), 7.69 (d, 2H, J = 8.64 Hz, H-2,6 phenyl), 8.01 (s, 1H, C=CH), 10.54 (s, 1H, NH,
exch. D2O). 13C NMR δ ppm: 44.4 (CH2), 121.2, 122.1, 127.8, 129.2, 129.9, 132.1, 132.3,
132.8, 135.9, 137.6 (C-Ar, C-S, C=CH), 164.4, 165.6, 167.4 (C=O). MS (m/z, %): 406 (M+,
80), 408 (M++2, 58), 410 (M++4, 12), 252 (100). Anal. Calcd. for C18H12Cl2N2O3S (407.27):
C, 53.08; H, 2.97; N, 6.88. Found: C, 53.23; H, 2.72; N, 6.88.
2-(5-(4-Fluorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-chlorophenyl)acetamide
(5c).
Yield: (3.09 g) 79%. m.p. 281-283 °C. Rf: 0.33. IR υ cm-1: 3255 (NH), 1745, 1693, 1666
1
(C=O), 833 (C-Cl). H NMR δ ppm: 4.53 (s, 2H, CH2), 7.38-7.44 (m, 4H, H-3,5 phenyl, H-
3,5 benzylidene), 7.57 (d, 2H, J = 8.88 Hz, H-2,6 phenyl), 7.73 (dd, 2H, J = 8.74, 5.42 Hz,
13
H-2,6 benzylidene), 8.02 (s, 1H, C=CH), 10.54 (s, 1H, NH, exch. D2O). C NMR δ ppm:
44.4 (CH2), 116.9, 117.2, 121.2, 127.8, 129.2, 129.9, 133.1, 133.2, 137.6, 162.3 (C-Ar, C-S,
C=CH), 164.4, 165.7, 167.5 (C=O). MS (m/z, %): 390 (M+, 85), 392 (M++2, 33), 236 (100).
Anal. Calcd. for C18H12ClFN2O3S (390.82): C, 55.32; H, 3.09; N, 7.17. Found: C, 55.09; H,
3.01; N, 6.91.
2-(5-(4-Methoxybenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-chlorophenyl)acetamide
(5d).
Yield: (3.59 g) 89%. m.p. 259-261 °C. Rf: 0.30. IR υ cm-1: 3292 (NH), 1741, 1687, 1672
(C=O), 827 (C-Cl). 1H NMR δ ppm: 3.85 (s, 3H, OCH3), 4.52 (s, 2H, CH2), 7.12 (d, 2H, J =
8.84 Hz, H-3,5 benzylidene), 7.37 (d, 2H, J = 8.84 Hz, H-2,6 benzylidene), 7.58 (d, 2H, J =
8.84 Hz, H-3,5 phenyl), 7.63 (d, 2H, J = 8.84 Hz, H-2,6 phenyl), 7.95 (s, 1H, C=CH), 10.54
13
(s, 1H, NH, exch. D2O). C NMR δ ppm: 44.4 (CH2), 56.0 (OCH3), 115.5, 118.0, 121.2,
125.7, 127.8, 129.2, 132.8, 134.1, 137.7, 161.8 (C-Ar, C-S, C=CH), 164.5, 165.8, 167.7
(C=O). MS (m/z, %): 402 (M+, 100), 404 (M++2, 38), Anal. Calcd. for C19H15ClN2O4S
(402.85): C, 56.65; H, 3.75; N, 6.95. Found: C, 56.41; H, 3.39; N, 6.95.
[23]

2-(5-(2-Chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-chlorophenyl)acetamide
(5e).
Yield: (3.71 g) 91%. m.p. 227-229 °C. Rf: 0.36. IR υ cm-1: 3261 (NH), 1755, 1693, 1668
1
(C=O), 748 (C-Cl). H NMR δ ppm: 4.54 (s, 2H, CH2), 7.38-7.40 (m, 2H, H-4,5
benzylidene), 7.54-7.60 (m, 4H, H-3,5 phenyl, H-3,6 benzylidene), 7.64-7.69 (m, 2H, H-2,6
13
phenyl), 8.10 (s, 1H, C=CH), 10.55 (s, 1H, NH, exch. D2O). C NMR δ ppm: 44.5 (CH2),
121.2, 125.1, 127.8, 128.6, 129.2, 129.5, 130.8, 131.2, 132.7, 134.9, 137.6 (C-Ar, C-S,
C=CH ), 164.3, 165.3, 167.3 (C=O). MS (m/z, %): 406 (M+, 90), 408 (M++2, 64), 410 (M++4,
13), 252 (100). Anal. Calcd. for C18H12Cl2N2O3S (407.27): C, 53.08; H, 2.97; N, 6.88.
Found: C, 52.92; H, 2.90; N, 6.63.
2-(5-(2,4-Dichlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-chlorophenyl)acetamide
(5f).
Yield: (3.67 g) 83%. m.p. 244-246 °C. Rf: 0.38. IR υ cm-1: 3259 (NH), 1755, 1691, 1668
1
(C=O), 727 (C-Cl). H NMR δ ppm: 4.54 (s, 2H, CH2), 7.38-7.40 (m, 2H, H-5,6
benzylidene), 7.57-7.60 (m, 2H, H-3,5 phenyl), 7.65 (m, 2H, H-2,6 phenyl), 7.89 (s, 1H, H-3
benzylidene), 8.03 (s, 1H, C=CH), 10.55 (s, 1H, NH, exch. D2O). 13C NMR δ ppm: 44.6
(CH2), 121.3, 125.7, 127.9, 128.1, 128.9, 129.2, 130.3, 130.4, 130.6, 135.9, 136.3, 137.6
(C-Ar, C-S, C=CH), 164.3, 165.2, 167.1 (C=O). MS (m/z, %): 440 (M+, 53), 442 (M++2, 55),
444 (M++4, 20), 56 (100). Anal. Calcd. for C18H11Cl3N2O3S (441.72): C, 48.94; H, 2.51; N,
6.34. Found: C, 48.92; H, 2.18; N, 6.55.
2-(5-Benzylidene-2,4-dioxothiazolidin-3-yl)-N-(4-methoxyphenyl) acetamide (6a).
Yield: (2.77 g) 75%. m.p. 262-264 °C. Rf: 0.24. IR υ cm-1: 3280 (NH), 1745, 1695, 1658
(C=O). 1H NMR δ ppm: 3.73 (s, 3H, OCH3), 4.49 (s, 2H, CH2), 6.89 (d, 2H, J = 9.04 Hz, H-
3,5 phenyl), 7.45-7.48 (m, 3H, H-3,4,5 benzylidene), 7.51-7.60 (m, 2H, H-2,6 benzylidene),
7.67 (d, 2H, J = 7.16 Hz, H-2,6 phenyl), 8.00 (s, 1H, C=CH), 10.24 (s, 1H, NH, exch. D2O).
13C NMR δ ppm: 44.3 (CH2), 55.6 (OCH3), 114.4, 121.2, 121.4, 129.9, 130.6, 131.3, 131.8,
133.2, 134.0, 155.9 (C-Ar, C-S, C=CH), 163.7, 165.8, 167.6 (C=O). MS (m/z, %): 368 (M+,
100). Anal. Calcd. for C19H16N2O4S (368.41): C, 61.94; H, 4.38; N, 7.60. Found: C, 62.19;
H, 4.67; N, 7.32.
2-(5-(4-Chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-methoxyphenyl)acetamide
(6b).
Yield: (3.67 g) 91%. m.p. 278-280 °C. Rf: 0.29. IR υ cm-1: 3265 (NH), 1745, 1695, 1664
(C=O), 825 (C-Cl). 1H NMR δ ppm: 3.72 (s, 3H, OCH3), 4.49 (s, 2H, CH2), 6.89 (d, 2H, J = 9
Hz, H-3,5 phenyl), 7.45 (d, 2H, J = 9.04 Hz, H-3,5 benzylidene), 7.63 (d, 2H, J = 8.64 Hz,
[24]

H-2,6 benzylidene), 7.69 (d, 2H, J = 8.64 Hz, H-2,6 phenyl), 8.01 (s, 1H, C=CH), 10.24 (s,
1H, NH, exch. D2O). 13C NMR δ ppm: 44.4 (CH2), 55.6 (OCH3), 114.4, 121.2, 122.2, 129.9,
131.8, 132.2, 132.3, 132.7, 135.9, 155.9 (C-Ar, C-S, C=CH), 163.6, 165.6, 167.4 (C=O).
MS (m/z, %): 402 (M+, 100), 404 (M++2, 39). Anal. Calcd. for C19H15ClN2O4S (402.85): C,
56.65; H, 3.75; N, 6.95. Found: C, 56.91; H, 3.94; N, 6.95.
2-(5-(4-Fluorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-methoxyphenyl)acetamide
(6c).
Yield: (2.98 g) 77%. m.p. 268-270 °C. Rf: 0.27. IR υ cm-1: 3255 (NH), 1743, 1691, 1662
(C=O). 1H NMR δ ppm: 3.72 (s, 3H, OCH3). 4.49 (s, 2H, CH2), 6.89 (d, 2H, J = 9.04, H-3,5
phenyl), 7.40-7.48 (m, 4H, H-3,5 benzylidene, H-2,6 phenyl), 7.47 (dd, 2H, J = 5.42, 8.78
Hz, H-2,6 benzylidene), 8.02 (s, 1H, C=CH), 10.24 (s, 1H, NH, exch. D2O). 13C NMR δ ppm:
44.4 (CH2), 55.6 (OCH3), 114.4, 116.9, 117.2, 121.2, 130.0, 131.9, 132.9, 133.1, 133.2,
155.9 (C-Ar, C-S, C=CH), 163.7, 165.7, 167.5 (C=O). MS (m/z, %): 386 (M+, 100). Anal.
Calcd. for C19H15FN2O4S (386.4): C, 59.06; H, 3.91; N, 7.25. Found: C, 59.13; H, 3.96; N,
6.93.
2-(5-(4-Methoxybenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-methoxyphenyl)
acetamide (6d).
Yield: (3.23 g) 81%. m.p. 263-265 °C. Rf: 0.22. IR υ cm-1: 3269 (NH), 1739, 1687, 1666
(C=O). 1H NMR δ ppm: 3.72 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.47 (s, 2H, CH2), 6.89 (d,
2H, J = 9.04 Hz, H-3,5 benzylidene), 7.13 (d, 2H, J = 8.84 Hz, H-3,5 phenyl), 7.45 (d, 2H, J
= 9 Hz, H-2,6 benzylidene), 7.63 (d, 2H, J = 8.84 Hz, H-2,6 phenyl), 7.95 (s, 1H, C=CH),
10.23 (s, 1H, NH, exch. D2O). 13C NMR δ ppm: 44.2 (CH2), 55.6 (OCH3), 56.0 (OCH3),
114.4, 115.5, 118.1, 121.2, 125.7, 131.9, 132.8, 134.0, 155.9, 161.7 (C-Ar, C-S, C=CH),
163.8, 165.9, 167.7 (C=O). MS (m/z, %): 398 (M+, 100). Anal. Calcd. for C20H18N2O5S
(398.43): C, 60.29; H, 4.55; N, 7.03. Found: C, 60.47; H, 4.49; N, 6.92.
2-(5-(2-Chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-methoxyphenyl)acetamide
(6e).
Yield: (3.39 g) 84%. m.p. 230-232 °C. Rf: 0.29 (elution system: methylene chloride). IR υ
cm-1: 3271 (NH), 1751, 1701, 1666 (C=O), 829 (C-Cl). 1H NMR δ ppm: 3.73 (s, 3H, OCH3),
4.50 (s, 2H, CH2), 6.88 (d, 2H, J = 9, H-3,5 phenyl), 7.46-7.49 (m, 2H, H-4,5 benzylidene),
7.53-7.58 (m, 2H, H-3,6 benzylidene), 7.64-7.69 (m, 2H, H-2,6 phenyl), 8.10 (s, 1H, C=CH),
10.25 (s, 1H, NH, exch. D2O). 13C NMR δ ppm: 44.4 (CH2), 55.6 (OCH3), 114.4, 121.2,
125.2, 128.7, 129.1, 129.5, 130.8, 131.3, 131.8, 132.7, 134.9, 156.0 (C-Ar, C-S, C=CH),
[25]

163.6, 165.4, 167.4 (C=O). MS (m/z, %): 402 (M+, 100), 404 (M++2, 40). Anal. Calcd. for
C19H15ClN2O4S (402.85): C, 56.65; H, 3.75; N, 6.95. Found: C, 56.45; H, 3.71; N, 6.99.
2-(5-(2,4-Dichlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(4-methoxyphenyl)
acetamide (6f).
Yield: (3.76 g) 86%. m.p. 235-237 °C. Rf: 0.35. IR υ cm-1: 3228 (NH), 1743, 1691, 1658
(C=O), 829 (C-Cl). 1H NMR δ ppm: 3.73 (s, 3H, OCH3), 4.50 (s, 2H, CH2), 6.89 (d, 2H, J =
9.08 Hz, H-3,5 phenyl), 7.45 (d, 2H, J = 9.04 Hz, H-2,6 phenyl), 7.64-7.65 (m, 2H, H-5,6
benzylidene), 7.88 (s, 1H, H-3 benzylidene ), 8.02 (s, 1H, C=CH), 10.25 (s, 1H, NH, exch.
D2O) . 13C NMR δ ppm: 44.4 (CH2), 55.6 (OCH3), 114.4, 121.2, 125.8, 128.0, 128.8, 130.3,
130.4, 130.6, 131.8, 135.9, 136.3, 156.0 (C-Ar, C-S, C=CH ), 163.6, 165.3, 167.1 (C=O).
MS (m/z, %): 436 (M+, 100), 438 (M++2, 72), 440 (M++4, 15). Anal. Calcd. for
C19H14Cl2N2O4S (437.3): C, 52.19; H, 3.23; N, 6.41. Found: C, 52.46; H, 3.17; N, 6.28.
2-(5-Benzylidene-4-oxo-3-phenylthiazolidin-2-ylideneimino)-N-phenylacetamide (10).
To a stirred solution of compound 9 (2.8 g, 10 mmol) in dry DMF (20 mL), anhydrous
potassium carbonate (0.69 g, 5 mmol) was added and the mixture was heated at 100oC for
30 min. The corresponding 2-chloro-N-phenylacetamide (1.69 g, 10 mmol) was added and
stirring was continued at 100oC for 24 h. The cooled reaction mixture was poured onto
crushed ice. The precipitate was filtered off, dried and crystallized from ethanol.
Yield: (3.60 g) 87%. m.p. 291-293 °C. Rf: 0.44. IR υ cm-1: 3273 (NH), 1718, 1672 (C=O). 1H
NMR δ ppm: 4.75 (s, 2H, CH2), 6.97 (d, 2H, J = 7.36 Hz, H-3,5 phenyl), 7.06 (t, 1H, J =
7.36 Hz, H-4 phenyl), 7.18 (t, 1H, J = 7.42, H-4 anilino), 7.32 (t, 2H, J = 7.92, H-3,5
benzylidene), 7.40-7.53 (m, 5H, H-2,4,6 benzylidene, H-3,5 anilino), 7.57-7.69 (m, 4H, H-
2,6 phenyl, H-2,6 anilino), 7.83 (s, 1H, C=CH), 10.37 (s, 1H, NH, exch. D2O).13C NMR δ
ppm: 44.8 (CH2), 119.7, 121.4, 124.1, 125.5, 129.8, 130.0, 130.2, 133.6, 138.9, 147.8 (C-
Ar, C-S, C=CH), 150.1 (C=N), 164.6, 166.2 (C=O). MS (m/z, %): 413 (M+, 7), 321 (100).
Anal. Calcd. for C24H19N3O2S (413.49): C, 69.71; H, 4.63; N, 10.16. Found: C, 69.58; H,
4.37; N, 9.87.
General Procedure for synthesis of 2H-thiazolo[3,2-a][1,3,5]triazin-6(7H)-ones 13a-f,
14a-f, 15a-f
A mixture of formaldehyde solution (37%, 1 mL) and the appropriate aromatic amine (5
mmol) in dry DMF (2 mL) was stirred for 30 min at room temperature. The mixture was then
added dropwise with stirring to a solution of the respective benzylidene derivative 12a-f (5
mmol) in dry DMF (10 mL) and heated at 100 ºC for 6 h. After cooling, it was poured onto
[26]

crushed ice and the resulting solid was filtered, washed with water and crystallized from
methanol.
7-Benzylidene-3,4-dihydro-3-phenyl-2H-thiazolo[3,2-a][1,3,5]triazin-6(7H)-one (13a).
Yield: (1.49 g) 93%. m.p. 188-190 °C. Rf: 0.43. IR υ cm-1: 1703 (C=O). 1H NMR δ ppm: 5.06
(s, 2H, CH2), 5.41 (s, 2H, CH2), 6.95 (t, 1H, J = 7.32 Hz, H-4 phenyl), 7.09 (d, 2H, J = 7.88
Hz, H-2,6 phenyl), 7.28 (t, 2H, J = 7.96 Hz, H-3,5 phenyl), 7.42 (t, 1H, J = 7.18, H-4
benzylidene), 7.49 (t, 2H, J = 7.46, H-3,5 benzylidene), 7.56 (d, 2H, J = 7.40, H-2,6
13
benzylidene), 7.70 (s, 1H, C=CH). C NMR δ ppm: 61.1 (C-4 triazine), 66.1 (C-2 triazine),
118.8, 120.2, 122.5, 129.7, 129.9, 130.1, 130.3, 130.4, 133.7, 147.6 (C-Ar, C-S, C=CH),
148.5 (C=N), 164.2 (C=O). MS (m/z, %): 321 (M+, 12), 105 (100). Anal. Calcd. For
C18H15N3OS (321.4): C, 67.27; H, 4.70; N, 13.07. Found: C, 67.49; H, 4.58; N, 13.32.
7-(4-Chlorobenzylidene)-3,4-dihydro-3-phenyl-2H-thiazolo[3,2-a][1,3,5]triazin-6(7H)-
one (13b).
Yield: (1.66 g) 93%. m.p. 189-191 °C. Rf: 0.49. IR υ cm-1: 1703 (C=O), 769 (C-Cl). 1H NMR
δ ppm: 5.06 (s, 2H, CH2), 5.40 (s, 2H, CH2), 6.95 (t, 1H, J = 7.32 Hz, H-4 phenyl), 7.08 (d,
2H, J = 7.80 Hz, H-2,6 phenyl), 7.27 (t, 2H, J = 8 Hz, H-3,5 phenyl), 7.56-7.61 (m, 4H, H-
13
2,3,5,6 benzylidene), 7.70 (s, 1H, C=CH). C NMR δ ppm: 61.1 (C-4 triazine), 66.1 (C-2
triazine), 118.8, 121.0, 122.5, 129.0, 129.7, 129.9, 131.8, 132.7, 134.9, 147.6 (C-Ar, C-S,
C=CH), 148.3 (C=N), 164.1 (C=O). MS (m/z, %): 355 (M+, 32), 357 (M++2, 13), 105 (100).
Anal. Calcd. For C18H14ClN3OS (355.84): C, 60.76; H, 3.97; N, 11.81. Found: C, 60.89; H,
4.01; N, 11.97.
7-(4-Fluorobenzylidene)-3,4-dihydro-3-phenyl-2H-thiazolo[3,2-a][1,3,5]triazin-6(7H)-
one (13c).
Yield: (1.58 g) 93%. m.p. 186-188 °C. Rf: 0.46. IR υ cm-1: 1712 (C=O). 1H NMR δ ppm: 5.06
(s, 2H, CH2), 5.40 (s, 2H, CH2), 6.95 (t, 1H, J = 7.30 Hz, H-4 phenyl), 7.08 (d, 2H, J = 7.92
Hz, H-2,6 phenyl), 7.27 (t, 2H, J = 7.98 Hz, H-3,5 phenyl), 7.34 (t, 2H, J = 8.8, H-3,5
13
benzylidene), 7.61-7.65 (m, 2H, H-2,6 benzylidene), 7.72 (s, 1H, C=CH). C NMR δ ppm:
61.1 (C-4 triazine), 66.1 (C-2 triazine), 112.9, 116.8, 118.8, 122.5, 128.4, 129.9, 132.1,
132.5, 147.6, 161.6 (C-Ar, C-S, C=CH), 164.2 (C=N), 180.8 (C=O). MS (m/z, %): 339 (M+,
47), 105 (100). Anal. Calcd. For C18H14FN3OS (339.39): C, 63.70; H, 4.16; N, 12.38. Found:
C, 63.42; H, 4.04; N, 12.67.
7-(4-Methoxybenzylidene)-3,4-dihydro-3-phenyl-2H-thiazolo[3,2-a][1,3,5]triazin-6(7H)-
one (13d).
[27]

Yield: (1.55 g) 88%. m.p. 131-133 °C. Rf: 0.41. IR υ cm-1: 1693 (C=O). 1H NMR δ ppm: 3.81
(s, 3H, OCH3), 5.05 (s, 2H, CH2), 5.39 (s, 2H, CH2), 6.94 (t, 1H, J = 7.28 Hz, H-4 phenyl),
7.05-7.10 (m, 4H, H-2,6 phenyl, H-3,5 benzylidene), 7.27 (t, 2H, J = 8 Hz, H-3,5 phenyl),
7.51 d, 2H, J = 8.8 H-2,6 benzylidene), 7.66 (s, 1H, C=CH). 13C NMR δ ppm: 55.89 (OCH3),
61.0 (C-4 triazine), 66.1 (C-2 triazine), 115.2, 117.0, 118.8, 122.4, 126.2, 129.9, 130.3,
132.1, 147.7, 148.7 (C-Ar, C-S, C=CH), 161.0 (C=N), 164.4 (C=O). MS (m/z, %): 351 (M+,
97), 77 (100). Anal. Calcd. For C19H17N3O2S (351.42): C, 64.94; H, 4.88; N, 11.96. Found:
C, 65.16; H, 4.73; N, 11.78.
7-(2-Chlorobenzylidene)-3,4-dihydro-3-phenyl-2H-thiazolo[3,2-a][1,3,5]triazin-6(7H)-
one (13e).
Yield: (1.29 g) 72%. m.p. 178-180 °C. Rf: 0.49. IR υ cm-1: 1700 (C=O), 756 (C-Cl). 1H NMR
δ ppm: 5.06 (s, 2H, CH2), 5.42 (s, 2H, CH2), 6.96 (t, 1H, J = 7.30 Hz, H-4 phenyl), 7.09 (d,
2H, J = 7.88 Hz, H-2,6 phenyl), 7.28 (t, 2H, J = 7.46 Hz, H-3,5 phenyl), 7.46-7.52 (m, 2H,
H-4,5 benzylidene), 7.55 (dd, 1H, J = 7.54, 1.75 Hz, H-3 benzylidene), 7.60 (dd, 1H, J =
13
7.62, 1.46 Hz, H-6 benzylidene), 7.84 (s, 1H, C=CH). C NMR δ ppm: 61.2 (C-4 triazine),
66.1 (C-2 triazine), 118.8, 122.6, 123.9, 125.3, 128.5, 129.1, 129.9, 130.7, 131.6, 131.9,
134.5, 147.5 (C-Ar, C-S, C=CH) , 148.24 (C=N), 163.8 (C=O). MS (m/z, %): 355 (M+, 53),
357 (M++2, 20), 105 (100). Anal. Calcd. For C18H14ClN3OS (355.84): C, 60.76; H, 3.97; N,
11.81. Found: C, 60.98; H, 3.97; N, 12.04.
7-(2,4-Dichlorobenzylidene)-3,4-dihydro-3-phenyl-2H-thiazolo[3,2-a][1,3,5]triazin-
6(7H)-one (13f).
Yield: (1.39 g) 69%. m.p. 192-194 °C. Rf: 0.54. IR υ cm-1: 1743 (C=O), 771 (C-Cl). 1H NMR
δ ppm: 5.07 (s, 2H, CH2), 5.42 (s, 2H, CH2), 6.96 (t, 1H, J = 7.30 Hz, H-4 phenyl), 7.09 (d,
2H, J = 8 Hz, H-2,6 phenyl), 7.28 ( t, 2H, J = 7.94 Hz, H-3,5 phenyl), 7.56-7.61 (m, 2H, H-
5,6 benzylidene), 7.77 (s, 1H, C=CH), 7.82 (s, 1H, H-3 benzylidene). 13C NMR δ ppm: 61.3
(C-4 triazine), 66.1 (C-2 triazine), 118.8, 122.6, 124.2, 124.6, 128.7, 129.9, 130.3, 130.7,
132.2, 135.4, 143.1, 147.5 (C-Ar, C-S, C=CH), 148.0 (C=N), 163.6 (C=O). MS (m/z, %):
389 (M+, 31), 391 (M++2, 22), 393 (M++4, 4), 105 (100). Anal. Calcd. For C18H13Cl2N3OS
(390.29): C, 55.39; H, 3.36; N, 10.77. Found: C, 55.18; H, 3.21; N, 11.08.
7-Benzylidene-3-(4-chlorophenyl)-3,4-dihydro-2H-thiazolo[3,2-a][1,3,5]triazin-6(7H)-
one (14a).
Yield: (1.27 g) 71%. m.p. 182-184 °C. Rf: 0.49. IR υ cm-1: 1705 (C=O), 831 (C-Cl). 1H NMR
δ ppm: 5.05 (s, 2H, CH2), 5.40 (s, 2H, CH2), 7.10-7.14 (m, 2H, H-2,6 phenyl), 7.32-7.36 (d,
2H, H-3,5 phenyl), 7.43 (t, 1H, J = 7.16 Hz, H-4 benzylidene), 7.50 (t, 2H, J = 7.46, H-3,5
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