Design, synthesis, structure elucidation and in vitro antiviral and antimicrobial evaluation

Article abstract of DOI:10.1007/s13738-017-1283-x

In this study, we described the synthesis of the derivatives of thiosemicarbazide, dicarboximide, 1,2,4-triazole-5-thione and 4-oxo-1,3-thiazolidine. Two different dicarboxylic acid anhydrides reacted with 4-substituted-3-thiosemicarbazide, and derivatives of thiosemicarbazide and dicarboximide were obtained. Next, cyclization reaction of dicarboximide derivatives in alkaline media was used to prepare 1,2,4-triazole-5-thione. The 4-oxo-1,3-thiazolidine was synthesized by the reaction of dicarboximide with ethyl bromoacetate. All obtained derivatives were analysed by ¹H and ¹³C NMR spectra, and for one compound, the X-ray crystallography was done. Antimicrobial, antiviral and in vitro evaluations of cytotoxicity were examined. According to the preliminary antiviral screening, compounds 3 and 4 presented the antiviral activity against HSV-1 and CVB3. Additionally, compound 3 shows selective in vitro toxic effect against human epithelial cells FaDu, without affecting normal animal cell line (Vero). The same derivatives 3 and 4 also displayed a wide spectrum of antimicrobial activity against reference microorganisms and indicated both antibacterial and antifungal potential activities.

Full text of DOI:10.1007/s13738-017-1283-x

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-017-1283-x
ORIGINAL PAPER
Design, synthesis, structure elucidation and in vitro antiviral
and antimicrobial evaluation
Anna Pachuta‑Stec1 · Barbara Rajtar2 · Anna Biernasiuk3 · Zbigniew Karczmarzyk4 · Łukasz Świątek2 · Anna Malm3 ·
Waldemar Wysocki4 · Katarzyna Stepaniuk4 · Małgorzata Polz‑Dacewicz2 · Monika Pitucha1
Received: 21 June 2017 / Accepted: 23 December 2017
© Iranian Chemical Society 2018
Abstract
In this study, we described the synthesis of the derivatives of thiosemicarbazide, dicarboximide, 1,2,4-triazole-5-thione and
4-oxo-1,3-thiazolidine. Two diferent dicarboxylic acid anhydrides reacted with 4-substituted-3-thiosemicarbazide, and
derivatives of thiosemicarbazide and dicarboximide were obtained. Next, cyclization reaction of dicarboximide derivatives
in alkaline media was used to prepare 1,2,4-triazole-5-thione. The 4-oxo-1,3-thiazolidine was synthesized by the reaction
of dicarboximide with ethyl bromoacetate. All obtained derivatives were analysed by 1H and 13C NMR spectra, and for one
compound, the X-ray crystallography was done. Antimicrobial, antiviral and in vitro evaluations of cytotoxicity were exam-
ined. According to the preliminary antiviral screening, compounds 3 and 4 presented the antiviral activity against HSV-1 and
CVB3. Additionally, compound 3 shows selective in vitro toxic efect against human epithelial cells FaDu, without afecting
normal animal cell line (Vero). The same derivatives 3 and 4 also displayed a wide spectrum of antimicrobial activity against
reference microorganisms and indicated both antibacterial and antifungal potential activities.
Keywords Thiosemicarbazide derivatives · Dicarboximide derivatives · 1,2,4-Triazole derivatives · 4-Oxo-1,3-thiazolidine
derivatives · Antimicrobial activity · Antiviral activity
Introduction
ganglion [2]. HSV-1 can cause epithelial lesions, especially
orolabial and genital infections, as well as keratitis, encepha-
litis and neonatal infections [1, 2].
Herpes simplex virus type 1 (HSV-1) is an enveloped
dsDNA virus belonging to the Herpesviridae family [1].
It is regarded as one of the most common human viruses,
afecting up to a 90% of worldwide population. Acquisition
of HSV-1 results in a lifelong latent infection of sensory
Mechanisms regulating the latency are not fully under-
stood, but it is suggested that they involve interactions
between immune and nervous system, infected cell signal
transduction, infected cell transcriptional regulation and
infuence of the host’s environment, e.g. stressful situa-
tions. HSV-1 latency can also be modulated by the endo-
crine system [3]. The role of thyroid hormone (T3) in
regulation of herpesvirus replication during reactivation is
believed to be the most signifcant. It may be due to the
fact that T3 infuences the activity of immune system, nerv-
ous system, cell signal transduction and transcriptional
regulation. Thyroid hormone binds to the nuclear receptor
and regulates cell proliferation, diferentiation and apopto-
sis. Moreover, stress may cause fuctuations of T3 [4]. It
was also shown that diferentiated LNCaP cells (human
neuron-like cells) resist HHV-1 replication upon stimu-
lation with T3, probably through active PI3 K (phospho-
inositide 3-kinase) signalling. The treatment with LY294002
* Anna Pachuta-Stec
anna.pachuta@umlub.pl
1
Department of Organic Chemistry, Faculty of Pharmacy,
Medical University of Lublin, 4A Chodźki Street,
20-093 Lublin, Poland
2
Department of Virology, I Faculty of Medicine
with Dentistry Division, Medical University of Lublin, 1
Chodźki Street, 20-093 Lublin, Poland
3
Department of Pharmaceutical Microbiology
with Laboratory for Microbiological Diagnostics, Faculty
of Pharmacy, Medical University, 1 Chodźki Street,
20-093 Lublin, Poland
4
Department of Chemistry, Siedlce University of Natural
Sciences and Humanities, 54 3 Maja Street, 08-110 Siedlce,
Poland
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
(2-morpholin-4-yl-8-phenylchromen-4-one), a PI3 K inhibi-
tor, can reverse T3-mediated repression of viral replication
in LNCaP cells [3].
potential antiviral activity against yellow fever virus (YFV)
and Saint Louis encephalitis virus (SLEV). Based on the
biological study, the authors stated that thiosemicarbazone
derivatives presented antiviral activity against both YFV
and SLEV, while phthalyl-thiazole derivatives were efec-
tive only against SLEV [35].
Most of the drugs used for the management of herpesvi-
rus infections target the DNA polymerase (Pol). Acyclovir
and penciclovir, with their respective prodrugs valacyclovir
and famciclovir, as well as foscarnet, and cidofovir are the
most commonly used drugs [1]. However, the treatment,
especially in case of immunosuppressed individuals, often
leads to development of resistance caused by mutations in
the viral thymidine kinase and/or DNA polymerase [1, 5].
Coxsackie B viruses (CVBs) are single-stranded, posi-
tive-sense RNA viruses belonging to the Picornaviridae fam-
ily. CVBs may be responsible for a variety of diseases, from
mild syndromes to life-threatening conditions such as pan-
creatitis, myocarditis, meningitis and encephalitis. Among
newborns and young infants, the infections are signifcantly
more severe and often lead to long-term sequelae [6, 7].
CVB3 is believed to be the most common cause of virus-
induced myocarditis that may develop into dilated cardio-
myopathy, which is the leading cause of heart failure among
young adults [8].
Our team has been working for many years on the syn-
thesis of derivatives of heterocyclic compounds with the
potential biological activity [36–40].
Recently, we have presented results concerning the syn-
thesis and antimicrobial and antituberculosis evaluation
of N-(substituted-thioureido)aminobicyclo dicarboximide
and 3,4-disubstituted 1,2,4-triazolino-5-thione. It is worth
noting that some of these compounds exhibited strong
bioactivity. They were the most active against clinical iso-
lates of C. albicans. Among them were N4-phenyl-N1-(2-
carboxy-1,2,3,6-tetrahydrobenzoyl)thiosemicarbazide,
N-(phenyl-thioureido)-aminobicyclo(1,2,3,6-tetrahydroph-
thaloyl)-dicarboximide and N-(phenyl-thioureido)-aminobi-
cyclo[2.2.1]hept-5-ene dicarboximide with MIC = 15.6 μg/
mL [41].
In the light of the above-mentioned literature and as an
extension of our previous studies on synthesis and biological
screening [39–43], we designed new analogues of thiosemi-
carbazide, dicarboximide and 1,2,4-triazole-5-thione with
modifcation of their structure. Additionally, we designed
and synthesized 1,2,3,6-tetrahydrophthaloyl-4-oxo-1,3-thia-
zolidine. Then, potential antimicrobial and antiviral activity
of the synthesized compounds were explored.
There is no specifc antiviral therapy for CVB3 infec-
tions, and the only possible treatment is symptomatic. In the
past few decades, there had been many reports describing
the molecules that block CVB replication in vitro, infuenc-
ing diferent steps of the viral life-cycle. However, none of
them proved to be efective and safe enough to be introduced
into practical use. Pleconaril which is an inhibitor of viral
RNA uncoating was the most promising drug candidate;
however, due to the considerable side efects, low efciency
and emerging resistance it has not been approved by the
FDA [6].
Experimental
Due to the emergence of drug-resistant mutants of
HSV-1 and lack of specifc antiviral therapy for CVB3-
induced myocarditis, it is crucial to look for new antiviral
compounds.
Materials and methods
Melting points were determined in a Fischer–Johns block
and presented without any corrections. Elemental analysis
was carried out using a PerkinElmer 2400 CHN Analyser.
All results were in good agreement with calculated values.
The error range was 0.4% for each element analysed. 1H
NMR and 13C NMR spectra were recorded on a Bruker
Avance 300 MHz spectrometer in DMSO-d6 and with TMS
as an internal standard. Chemicals were purchased from
Sigma-Aldrich (Munich, Germany) or Merck Co. (Darm-
stadt, Germany) and used without further purifcation.
Five-membered heterocyclic compounds especially
1,2,4-triazole, thiazolidin-4-one and their open-chain coun-
terpart thiosemicarbazide and dicarboximide display a broad
spectrum of biological activities [9–18] including antivi-
ral [19–26] and antimicrobial [27–29] properties. What is
important, derivatives based on this pharmacophore are
already in the market and they have applications as clinically
useful medicines. Among them are thiazolidomycin activity
against Streptomyces species [30], ribavirin—antiviral [31],
fuconazole—antifungal [31], itraconazole—antifungal [31],
voriconazole—antifungal [31], ravuconazole—antifungal
[31], terconazole—antifungal [32], albaconazole—antifun-
gal [33], isavuconazole—antifungal [33] and ritonavir—
antiretroviral [34].
Synthesis and characterization of thiosemicarbazide
derivatives (1–4)
1.52 g (10 mmol) of 1,2,3,6-tetrahydrophtalic anhydride and
10 mmol of appropriate 4-substituted-3-thiosemicarbazide in
dry chloroform were refuxed for 1 h. After cooling, the pre-
cipitate was fltered of and crystallized from ethanol–water.
In 2017 year, C.C. Pacca and colleagues described
series of thiosemicarbazones and phthalyl-thiazoles with a
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Journal of the Iranian Chemical Society
N4‑ethyl‑N1‑(2‑carboxy‑1,2,3,6‑tetrahydrobenzoyl)thio‑
semicarbazide (1)
126.76 (CH=CH), 128.5 (m-Ph2CH), 129.36 (p-PhC-Cl),
138.87 (i-PhC), 173.19 (CONH), 176.41 (COOH), 180.90
(C=S).
CAS Registry Number 1625646-62-9. Yield: 65%; M.p.:
158–159 °C. 1H NMR (DMSO-d6) δ (ppm) = 1.05 (t, 3H,
J = 7.5 Hz, CH3), 2.26 (s, 2H, CH2), 2.49 (s, 2H, CH2), 2.74
(s, 1H, CH), 3.02 (s, 1H, CH), 3.33–3.46 (m, 2H, CH2), 5.62
(s, 2H, CH=CH), 7.38 (s, 1H, NH), 9.15 (s, 1H, NH), 9.71
(s, 1H, NH), 12.48 (s, 1H, COOH); 13C NMR (DMSO-d6)
δ (ppm) = 17.70 (CH3), 25.38 (CH2), 26.70 (CH2), 38.22
(CH-CONH), 38.93 (CH2CH3), 39.86 (CH-COOH), 123.33
(CH=CH), 123.91 (CH=CH), 171.09 (CONH), 174.09
(COOH), 179.91 (C=S).
Synthesis and characterization of N‑(substituted‑thi‑
oureido)aminobicyclo(1,2,3,6‑tetrahydrophth‑
aloyl) dicarboximide (5–7)
The thiosemicarbazide 1–3 (2 mmol) was dissolved in a mix-
ture of glacial acetic acid (4 mL) and concentrated sulphuric
acid (2 mL). The solution was left at room temperature for
1 h, then poured into crushed ice and kept for 2 h at room
temperature. The precipitated solid was fltered of and crys-
tallized from ethanol.
N4‑phenyl‑N1‑(2‑carboxy‑1,2,3,6‑tetrahydrobenzoyl)thio‑
semicarbazide (2)
N‑(ethyl‑thioureido)aminobicyclo(1,2,3,6‑tetrahydrophth‑
aloyl) dicarboximide (5)
CAS Registry Number 1625646-63-0. Yield: 86%; M.p.:
143–145 °C. 1H NMR (DMSO-d6) δ (ppm) = 2.31 (s, 2H,
CH2), 2.49 (s, 2H, CH2), 2.84 (s, 1H, CH), 3.09 (s, 1H, CH),
5.65 (s, 2H, CH=CH), 7.12–7.17 (m, 1H, p-Ph), 7.29–7.35
(m, 2H, m-Ph), 7.53–7.56 (m, 2H, o-Ph), 9.06 (s, 1H, NH),
9.64 (s, 1H, NH), 9.98 (s, 1H, NH), 12.53 (s, 1H, COOH);
13C NMR (DMSO-d6) δ (ppm) = 22.83 (CH2), 26.24 (CH2),
37.82 (CH-CONH), 39.77 (CH-COOH), 124.58 (p-PhCH),
124.86 (o-Ph2CH), 125.31 (CH=CH), 128.11 (m-Ph2CH),
138.92 (i-PhC), 172.67 (CONH), 175.80 (COOH), 180.50
(C=S).
CAS Registry Number 1625646-65-2. Yield: 62%; M.p.
164–166 °C. 1H NMR (DMSO-d6) δ (ppm) = 1.11 (t, 3H,
J = 7.5 Hz, CH3), 2.29–2.50 (m, 4H, 2CH2), 2.58 (s, 1H,
CH), 3.41 (s, 1H, CH), 3.46–3.51 (m, 2H, CH2), 5.93 (s,
2H, CH=CH), 8.28 (s, 1H, NH), 9.70 (s, 1H, NH); 13C
NMR (DMSO-d6) δ (ppm) = 14.29 (CH3), 23.12 (2CH2),
37.20 (CH2CH3), 38.90 (CH-CO), 39.90 (CH-CO), 127.68
(CH=CH), 177.32 (2C=O), 181.38 (C=S).
N4‑(2‑methylphenyl)‑N1‑(2‑carboxy‑1,2,3,6‑tetrahydroben‑
zoyl)thiosemicarbazide (3)
N‑(phenyl‑thioureido)aminobicyclo(1,2,3,6‑tetrahydroph‑
thaloyl) dicarboximide (6)
1
Yield: 60%; M.p.: 100–102 °C. H NMR (DMSO-d6) δ
CAS Registry Number 1625646-67-4. Yield: 82%; M.p.
1
(ppm) = 2.14–2.41 (m, 7H, CH3 + 2CH2), 3.24 (s, 2H,
2CH), 5.88 (s, 2H, CH=CH), 7.09–7.27 (m, 4H, Ph), 7.55
(s, 1H, NH), 9.08 (s, 1H, NH), 9.69 (s, 1H, NH), 10.07
(s, 1H, COOH); 13C NMR (DMSO-d6) δ (ppm) = 17.90
(CH3), 23.38 (CH2), 26.16 (CH2), 37.59 (CH-CONH), 39.16
(CH-COOH), 125.94 (o-PhCH), 127.75 (p-PhCH), 128.20
(m-PhCH), 129.26 (CH=CH), 130.83 (m-PhCH), 133.72
(o-PhC-CH3), 138.42 (i-PhC), 175.04 (CONH), 177.47
(COOH), 180.67 (C=S).
164–166 °C. H NMR (DMSO-d6) δ (ppm) = 2.30–2.39
(m, 4H, 2CH2), 3.40 (s, 2H, 2CH), 5.89 (s, 2H, CH=CH),
7.07–7.22 (m, 1H, p-Ph), 7.30–7.58 (m, 4H, o-Ph + m-Ph),
10.05 (s, 2H, 2NH); 13C NMR (DMSO-d6) δ (ppm) = 22.80
(2CH2), 36.89 (2CH-CO), 124.78 (p-PhCH), 127.37
(o-Ph2CH), 128.48 (m-Ph2CH + CH=CH), 138.51 (i-PhC),
176.79 (2C=O), 180.93 (C=S).
N‑(2‑methylphenyl‑thioureido)aminobicyclo(1,2,3,6‑tet‑
rahydrophthaloyl) dicarboximide (7)
N4‑(4‑chlorophenyl)‑N1‑(2‑carboxy‑1,2,3,6‑tetrahydroben‑
zoyl)thiosemicarbazide (4)
1
Yield: 51%; M.p. 110–112 °C. H NMR (DMSO-d6) δ
1
Yield: 62%; M.p.: 162–164 °C. H NMR (DMSO-d6) δ
(ppm) = 2.14–2.41 (m, 7H, CH3 + 2CH2), 3.24 (s, 2H,
2CH), 5.89 (s, 2H, CH=CH), 7.08–7.27 (m, 4H, Ph), 9.69
(bs, 1H, NH), 10.07 (bs, 1H, NH); 13C NMR (DMSO-d6)
δ (ppm) = 17.90 (CH3), 23.37 (2CH2), 37.54 (2CH-CO),
126.69 (o-PhCH), 127.74 (p-PhCH), 127.99 (m-PhCH),
129.31 (CH=CH), 130.82 (m-PhCH), 136.34 (o-PhC-CH3),
137.50 (i-PhC), 177.46 (2C=O), 181.71 (C=S).
(ppm) = 2.33 (s, 2H, CH2), 2.48 (s, 2H, CH2), 2.87 (s, 1H,
CH), 3.14 (s, 1H, CH), 5.68 (s, 2H, CH=CH), 7.39–7.42 (d,
2H, J = 8.6 Hz, p-Ph), 7.60–7.63 (d, 2H, J = 8.6 Hz, p-Ph),
9.16 (s, 1H, NH), 9.81 (s, 1H, NH), 10.04 (s, 1H, NH), 12.21
(s, 1H, COOH); 13C NMR (DMSO-d6) δ (ppm) = 25.34
(CH2), 26.75 (CH2), 38.33 (CH-CONH), 125.79 (o-Ph2CH),
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Journal of the Iranian Chemical Society
N‑(4‑chlorophenyl‑thioureido)aminobicyclo(1,2,3,6‑tet‑
rahydrophthaloyl) dicarboximide (8)
was fltered of and isolated solution was acidifed with 3 M
HCl. The precipitate was fltered of and crystallized from
ethanol.
1
Yield: 62%; M.p.: 169–171 °C. H NMR (DMSO-d6) δ
(ppm) = 2.26–2.30 (m, 4H, 2CH2), 2.87 (s, 1H, CH), 3.26
(s, 2H, 2CH), 5.89 (s, 2H, CH=CH), 7.34–7.43 (m, 2H, p-
Ph), 7.58–7.61 (m, 2H, p-Ph), 10.06 (s, 1H, NH), 10.19 (bs,
1H, NH); 13C NMR (DMSO-d6) δ (ppm) = 23.34 (2CH2),
37.42 (2CH-CO), 118.81 (o-Ph2CH), 124.90 (p-PhC-Cl),
127.97 (CH=CH), 129.27 (m-Ph2CH), 140.42 (i-PhC),
177.27 (2C=O), 181.90 (C=S).
Method B (11–13)
A solution of N4-substituted-N1-(2-carboxy-1,2,3,6-tetrahy-
drobenzoyl)thiosemicarbazide 1–3 (2 mmol) in 2% sodium
hydroxide (5 mL) was refuxed for 2 h. After cooling, the
reaction mixture was fltered of and isolated solution was
acidifed with 3 M HCl. The precipitate was fltered of and
crystallized from ethanol.
Synthesis and characterization of bis
N‑(substituted‑thioureido)aminobicyclo[2.2.2]
oct‑7‑ene‑2,3,5,6‑tetracarboximide (9–10)
1‑(4‑Ethyl‑1,2,4‑triazol‑5‑thione‑3‑yl)‑cyclohex‑4‑ene‑2‑car‑
boxylic acid (11)
10 mmol of bicyclo[2.2.2]oct-7-ene-2,3,5,6-tetracarboxylic
dianhydride and 20 mmol of appropriate 4-substituted-
3-thiosemicarbazide in dry chloroform were refuxed for 1 h.
After cooling, the precipitate was fltered of and crystallized
from acetonitrile + methanol (1:1).
CAS Registry Number 1625646-73-2. Yield: 58%; M.p.
204–206 °C. 1H NMR (DMSO-d6) δ (ppm) = 1.30 (t, 3H,
J = 7.5 Hz, CH3), 3.02 (s, 1H, CH), 3.39 (s, 4H, 2CH2),
3.62 (s, 1H, CH), 4.07 (q, 2H, J = 7.5 Hz, CH2CH3), 3.44
(s, 2H, 2CH), 5.60 (d, 1H, J = 10.0 Hz, CH=CH), 5.79
(d, 1H, J = 10.0 Hz, CH=CH), 12.36 (bs, 1H, NH), 13.49
(s, 1H, COOH); 13C NMR (DMSO-d6) δ (ppm) = 13.81
(CH3), 25.42 (CH2), 28.12 (CH2), 29.85 (CH-triazole) 38.52
(CH2CH3), 39.95 (CH-COOH) 123.46 (CH=CH), 126.54
(CH=CH), 153.64 (Ctriazole), 166.16 (COOH), 174.55 (C=S).
Bis N‑(ethyl‑thioureido)aminobicyclo[2.2.2]‑oct‑7‑ene‑2,3,5
,6‑tetracarboximide (9)
1
Yield: 63%; M.p. 125–127 °C. H NMR (DMSO-d6) δ
(ppm) = 1.03 (t, 6H, J = 7.0 Hz, 2CH3), 3.36–3.3.41
(m, 10H, 6CH + 2CH2), 6.01 (s, 2H, CH=CH), 8.24 (bs,
2H, 2NH), 9.75 (bs, 2H, 2NH); 13C NMR (DMSO-d6) δ
(ppm) = 14.57 (CH3), 33.69 (2CH), 39.31 (CH2-CH3), 41.49
(4CH-CO), 131.32 (CH=CH), 173.73 (4C=O), 181.50
(2C=S).
1‑(4‑Phenyl‑1,2,4‑tria‑
zol‑5‑thione‑3‑yl)‑cyclohex‑4‑ene‑2‑carboxylic acid (12)
1
Yield: 78%; M.p. 271–273 °C. H NMR (DMSO-d6) δ
(ppm) = 2.16–2.18 (m, 4H, 2CH2), 2.68–2.74 (m, 1H, CH),
3.19 (q, 1H, J = 5.2 Hz, CH), 5.52 (d, 1H, J = 10.1 Hz,
CH=CH), 5.70 (d, 1H, J = 9.8 Hz, CH=CH), 7.37–7.39
(m, 2H, m-Ph), 7.52–7.63 (m, 3H, o-Ph + p-Ph), 12.31 (bs,
1H, NH), 13.65 (s, 1H, COOH); 13C NMR (DMSO-d6) δ
(ppm) = 25.13 (CH2), 26.61 (CH2), 29.97 (CH-triazole),
38.93 (CH-COOH) 123.22 (CH=CH), 125.91 (CH=CH),
128.47 (o-Ph2CH), 129.60 (m-Ph2CH + p-PhCH), 133.95
(i-PhC), 153.38 (Ctriazole), 167.37 (COOH), 174.05 (C=S).
Bis N‑(phenyl‑thioureido)aminobicyclo[2.2.2]‑oct‑7‑ene‑2,3
,5,6‑tetracarboximide (10)
CAS Registry Number 1625646-83-4. Yield: 83%; M.p.
220–222 °C. 1H NMR (DMSO-d6) δ (ppm) = 3.33 (s, 4H,
4CH), 3.47 (s, 2H, 2CH) 6.38 (s, 2H, CH=CH), 7.08–7.36
(m, 4H, m-Ph), 7.44–7.62 (m, 6H, o-Ph + p-Ph), 9.92 (bs,
2H, 2NH), 10.18 (s, 2H, 2NH); 13C NMR (DMSO-d6) δ
(ppm) = 33.23 (2CH), 40.33 (4CH-CO), 125.62 (p-Ph2CH),
128.45 (o-Ph4CH), 130.90 (m-Ph4CH + CH=CH), 138.43
(i-Ph2C), 174.18 (4C=O), 180.69 (2C=S).
1‑[4‑(2‑Methylphenyl)‑1,2,4‑tria‑
zol‑5‑thione‑3‑yl]‑cyclohex‑4‑ene‑2‑carboxylic acid (13)
1
Synthesis and characterization of 3,4‑disubstituted
1,2,4‑triazolino‑5‑thione (11–15)
Yield: 55%; M.p. 242–244 °C. H NMR (DMSO-d6) δ
(ppm) = 1.96–2.38 (m, 7H, CH3 + 2CH2), 2.55–3.15 (m,
2H, 2CH), 5.52 (d, 1H, J = 9.0 Hz, CH=CH), 5.69 (d, 1H,
J = 9.0 Hz, CH=CH), 7.19–7.49 (m, 4H, Ph), 12.25 (bs,
1H, NH), 13.68 (s, 1H, COOH); 13C NMR (DMSO-d6) δ
(ppm) = 17.96 (CH3), 25.67 (CH2), 27.45 (CH2), 30.79
(CH-triazole), 39.43 (CH-COOH), 126.61 (o-PhCH), 127.77
(p-PhCH), 128.89 (m-PhCH), 129.41 (CH=CH), 130.49
Method A (11–15)
A solution of N-(substituted-thioureido)aminobicyclo dicar-
boximide 5–9 (2 mmol) in 2% sodium hydroxide (5 mL)
was refuxed for 2 h. After cooling, the reaction mixture
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Journal of the Iranian Chemical Society
(m-PhCH), 136.81 (o-PhC-CH3), 137.33 (i-PhC), 153.65
N‑[(4‑oxo‑3‑phenyl‑1,3‑thiazoli‑
din‑2‑ylimine)]‑bicyclo(1,2,3,6‑tetrahydrophthaloyl)
dicarboximide (17)
(Ctriazole), 167.26 (COOH), 174.64 (C=S).
2,6‑[Bis‑(4‑ethyl‑1,2,4‑triazol‑5‑thione‑3‑yl)]‑bicyclo[2.2.2]
oct‑7‑ene‑3,5‑dicarboxylic acid (14)
1
Yield: 85%; M.p. 232–234 °C. H NMR (DMSO-d6) δ
(ppm) = 2.16–2.25 (m, 2H, CH2), 2.39–2.52 (m, 2H,
CH2), 3.21 (s, 2H, 2CH), 4.34 (s, 2H, CH2), 5.95 (s, 2H,
CH=CH), 7.24–7.58 (m, 5H, Ph). 13C NMR (DMSO-
d6) δ (ppm) = 23.82 (2CH2), 33.65 (CH2thiazole), 38.17
(2CH-CO), 128.35 (CH=CH), 128.49 (o-Ph2CH), 129.38
(p-PhCH), 129.63 (m-Ph2CH), 134.72 (i-PhC), 170.72
(Cthiazole), 171.99 (C=O), 179.26 (2C=O).
1
Yield: 51%; M.p. 262–264 °C. H NMR (DMSO-d6) δ
(ppm) = 1.24 (t, 6H, J = 7.5 Hz, 2CH3), 3.05–3.35 (m, 4H,
4CH), 3.56–3.67 (m, 2H, 2CH), 3.88–3.99 (m, 4H, 2CH2),
6.22 (s, 2H, CH=CH), 10.72 (bs, 2H, 2NH), 12.66 (s, 2H,
2COOH); 13C NMR (DMSO-d6) δ (ppm) = 13.89 (2CH3),
36.76 (2CH), 37.57 (2CH-triazole), 38.21 (2CH2-CH3),
50.42 (2CH-COOH), 131.59 (CH=CH), 154.75 (2Ctriazole),
165.24 (2COOH), 174.02 (2C=S).
N‑[(4‑oxo‑3‑(2‑methylphenyl)‑1,3‑thiazoli‑
din‑2‑ylimine)]‑bicyclo(1,2,3,6‑tetrahydrophthaloyl)
dicarboximide (18)
2,6‑[Bis‑(4‑phenyl‑1,2,4‑triazol‑5‑thione‑3‑yl)]‑bicy‑
clo[2.2.2]oct‑7‑ene‑3,5‑dicarboxylic acid (15)
1
CAS Registry Number 1625646-85-6. Yield: 57%; M.p.
Yield: 72%; M.p. 250–252 °C. H NMR (DMSO-d6) δ
1
287–289 °C. H NMR (DMSO-d6) δ (ppm) = 2.71–3.24
(ppm) = 2.20–2.41 (m, 5H, CH3 + CH2), 2.49 (s, 1H, CH),
3.19 (s, 2H, CH2), 3.31 (s, 1H, CH), 4.29 (s, 2H, CH2),
5.91 (s, 2H, CH=CH), 7.25–7.36 (m, 4H, Ph). 13C NMR
(DMSO-d6) δ (ppm) = 17.42 (CH3), 23.82 (2CH2), 33.65
(CH2thiazole), 38.18 (2CH-CO), 127.44 (o-PhCH), 128.35
(CH=CH), 129.04 (p-PhCH), 130.00 (m-PhCH), 131.29
(m-PhCH), 133.98 (o-PhC-CH3), 136.45 (i-PhC), 169.78
(Cthiazole), 171.88 (C=O), 175.27 (2C=O).
(m, 6H, 6CH), 6.21–6.30 (m, 1H, CH=CH), 6.36–6.41
(m, 1H, CH=CH), 7.28–7.30 (m, 4H, m-Ph), 7.45–7.59
(m, 6H, o-Ph + p-Ph), 12.08 (bs, 2H, 2NH), 13.50 (s, 1H,
COOH), 13.54 (s, 1H, COOH); 13C NMR (DMSO-d6) δ
(ppm) = 34.59 (2CH), 37.75 (2CH-triazole), 47.29 (2CH-
COOH), 128.76 (p-Ph2CH), 129.52 (o-Ph4CH), 131.47
(m-Ph4CH + CH=CH), 133.76 (i-Ph2C), 152.90 (2Ctriazole),
166.90 (2COOH), 174.34 (2C=S).
Synthesis and characterization of N‑[(4‑oxo‑3‑sub‑
stituted‑1,3‑thiazolidin‑2‑ylimine)]‑bicyc‑
lo(2,3,5,6‑tetrahydrophthaloyl) dicarboximide
(16–20)
Bis N‑[(4‑oxo‑3‑ethyl‑1,3‑thiazolidin‑2‑ylimine)]‑bicyclo[2.2
.2]‑oct‑7‑ene‑2,3,5,6‑tetracarboximide (19)
1
Yield: 77%; M.p. 316–318 °C. H NMR (DMSO-d6) δ
(ppm) = 1.16 (t, 6H, J = 7.0 Hz, 2CH3), 3.34 (s, 4H,
4CH), 3.46 (s, 2H, 2CH), 3.72 (q, 4H, J = 7.0, 14.0 Hz
2CH2CH3), 4.12 (s, 4H, 2CH2), 6.27 (d, 1H, J = 3.0 Hz,
CH=CH), 6.28 (d, 1H, J = 3.0 Hz, CH=CH),). 13C NMR
(DMSO-d6) δ (ppm) = 12.31 (2CH3), 33.60 (2CH2thiazole),
34.37 (2CH), 38.37 (2CH2CH3), 41.93 (4CH-CO), 131.95
(CH=CH), 169.77 (2Cthiazole), 172.09 (2C=O), 172.73
(4C=O).
To a suspension of N-(substituted-thioureido)aminobicy-
clo dicarboximide (2.5 mmol) in absolute ethanol (25 mL),
anhydrous sodium acetate (10 mmol) and ethyl bromoacetate
(2.5 mmol) were added and mixture was refuxed for 4 h.
After cooling, the solution was allowed to stand overnight.
The precipitate was fltered of and crystallized from ethanol.
N‑[(4‑oxo‑3‑ethyl‑1,3‑thiazoli‑
din‑2‑ylimine)]‑bicyclo(1,2,3,6‑tetrahydrophthaloyl)
dicarboximide (16)
Bis N‑[(4‑oxo‑3‑phenyl‑1,3‑thiazolidin‑2‑ylimine)]‑bicyclo[2
.2.2]‑oct‑7‑ene‑2,3,5,6‑tetracarboximide (20)
1
Yield: 76%; M.p. 162–164 °C. H NMR (DMSO-d6) δ
1
(ppm) = 1.15 (t, 3H, J = 6.0 Hz, CH3), 2.17–2.25 (m, 2H,
CH2), 2.39–2.45 (m, 2H, CH2), 3.24–3.26 (m, 2H, 2CH),
3.74 (q, 2H, J = 6.0, 15.0 Hz CH2CH3), 4.10 (s, 2H, CH2),
5.92–5.94 (m, 2H, CH=CH). 13C NMR (DMSO-d6) δ
(ppm) = 12.29 (CH3), 23.82 (2CH2), 33.42 (CH2thiazole),
38.19 (CH2-CH3), 38.33 (2CH-CO), 128.36 (CH=CH),
169.58 (Cthiazole), 172.10 (C=O), 175.40 (2C=O).
Yield: 80%; M.p. 318–320 °C. H NMR (DMSO-d6) δ
(ppm) = 3.29 (s, 6H, 6CH), 4.26 (s, 4H, 2CH2), 6.24 (d, 1H,
J = 3.0 Hz, CH=CH), 6.27 (d, 1H, J = 3.0 Hz, CH=CH),
7.34–7.39 (m, 4H, m-Ph), 7.46–7.57 (m, 6H, o-Ph + p-
Ph). 13C NMR (DMSO-d6) δ (ppm) = 33.76 (2CH2thiazole),
34.34 (2CH), 41.86 (4CH-CO), 128.49 (p-Ph2CH), 129.38
(o-Ph4CH), 129.62 (m-Ph4CH), 131.89 (CH=CH), 134.75
1 3

Journal of the Iranian Chemical Society
(i-Ph2C), 170.82 (2Cthiazole), 171.89 (2C=O), 172.43
were propagated in the Vero cell culture. Virus stocks were
stored at − 70 °C until used.
(4C=O).
X‑ray crystallography
Cytotoxicity assay
X-ray data of compound 17 were collected on the Kuma
KM-4 difractometer: crystal size 0.60 × 0.60 × 0.40 mm,
MoKα (λ = 0.71073 Å) radiation, ω/2θ scans, T = 293(2)
K, absorption correction: Xabs2 [44], Tmin/Tmax of
0.4542/2.0561. The structure was solved by direct meth-
ods using SHELXS-2013/1 [45] and refned by full-matrix
least-squares with SHELXL-2014/7 [45]. All H atoms were
positioned geometrically, treated as riding on their parent C
atoms with C–H distances of 0.98 Å (CH), 0.97 Å (CH2) and
0.93 Å (aromatic) and refned with isotropic displacement
parameters taken as 1.5 times of those of the respective par-
ent atoms. All calculations were performed using WINGX
version 2014.1 package [46]. CCDC-1556158 contains the
supplementary crystallographic data for this paper. These
data can be obtained free of charge at www.ccdc.cam.ac.uk/
conts/retrieving.html [or from the Cambridge Crystallo-
graphic Data Centre (CCDC), 12 Union Road, Cambridge
CB2 1EZ, UK; fax: +44(0) 1223 336 033; email: deposit@
ccdc.cam.ac.uk].
Compounds were dissolved in dimethyl sulphoxide—DMSO
(POCH, Gliwice, Poland) in the concentration of 50 mg/mL
and further diluted with a complete test medium. 100 µL of
the cell culture prepared was seeded into 96-well plastic plates
(Becton Dickinson and Company, Franklin Lakes, NJ USA)
with a cell density of 1.5 × 104 (Vero) and 2 × 104 (FaDu)
cells per well. After a 24 h incubation at 37 °C, the media were
removed and the cells were treated with a solution of the exam-
ined substance diluted in the media with 2% of the serum. The
cells were submitted to a series of compound concentrations,
from 1000 to 1.9 µg/mL. Twofold serial dilutions of com-
pounds were added to the cells in triplicates. The cell cultures
were incubated for 72 h at 37 °C in the 5% CO2 atmosphere.
Cytotoxicity of the tested compounds was estimated with
the use of the MTT method and described by Takenouchi and
Munekata, with modifcations [47, 48]. The MTT method is
a quantitative colorimetric toxicity test, based on the transfor-
mation of yellow, soluble tetrazolium salts (3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyltetrazolium bromide) to purple–blue
insoluble formazan. This process occurs naturally in mitochon-
dria of living cells. After a 72-h incubation with compounds,
cell cultures were supplemented with 10 µL per well of 5 mg/
mL MTT (Sigma-Aldrich, Saint Louis, MO, USA) stock in
PBS (BIOMED, Lublin, Poland) and the incubation was con-
tinued for 4 h at 37 °C. Then, 100 µL of aqueous solution con-
taining 50% dimethylformamide (POCH, Gliwice, Poland) and
20% SDS (Sigma-Aldrich, Saint Louis, MO, USA) was added
to solubilize the insoluble formazan precipitates produced by
MTT. After the all-night incubation, the absorbance was meas-
ured by the Epoch plate reader (BioTek, Winooski, Vermont,
USA) at two wavelengths—540 and 620 nm. On the basis of
the test results, the cytotoxic concentration (CC50), which is
the amount of tested substance that is required to reduce the
number of viable cells by 50% compared to the control cul-
ture, was determined and calculated using the Gen 5 2.01.14
software (BioTek, Winooski, Vermont, USA). Cell viability
(%) was calculated as (A540/620 of the treated/A540/620 of the
control) × 100. When the CC50 value was calculated, nontoxic
concentrations of the examined compounds were selected to
assess their antiviral activity. The investigation was carried
out in triplicate.
Crystal data of 17: C17H15N3O3S, M = 341.38, tri-
clinic, space group P 1, a = 9.959(2), b = 12.280(2)(3),
c = 13.481(2) Å, α = 91.51(2), β = 96.55(2), γ = 103.50(2)°,
V = 1590.3(5) Å3, Z = 4, dcalc = 1.426 Mg m−3
,
F(000) = 712, μ(Mo Kα) = 0.225 mm−1, T = 293(2) K, 9304
measured refections (θ range 2.12–30.07), 9304 unique
refections, fnal R = 0.069, wR = 0.199, S = 1.085 for 5047
refections with I > 2σ(I).
Antiviral activity
Cells and viruses
The Vero cell culture (ECACC No. 84113001—established
from a kidney of a normal adult African Green monkey)
and the FaDu cell culture (ATCC HTB-43 established from
a punch biopsy of an hypopharyngeal tumour removed from
a Hindu patient) were used in the experiment. The media in
the culture (Dulbecco’s modifed Eagle medium—DMEM
and modifed Eagle medium—MEM, Sigma-Aldrich, Saint
Louis, MO, USA) were supplemented with 10% foetal
bovine serum—FBS (Sigma-Aldrich, Saint Louis, MO,
USA), 100 U/mL of penicillin and 0.1 mg/mL of strepto-
mycin (Polfa Tarchomin, Warsaw, Poland). The cell culture
was incubated at 37 °C in the 5% CO2 atmosphere.
For antiviral activity of the examined compounds, the
Herpes simplex virus type l—HSV-1 (ATCC No. VR-260)
and coxsackievirus B3—CVB3 (ATCC No. VR-30) from the
American Type Culture Collection were used. The viruses
1 3

Journal of the Iranian Chemical Society
ATCC came from American Type Culture Collection, rou-
tinely used for the evaluation of antimicrobials. All the used
microbial cultures were frst subcultured on nutrient agar or
Sabouraud agar for bacteria and fungi, respectively.
Antiviral activity assay
Screening of antiviral activity by cytopathic efect
reduction assay
The surface of Mueller–Hinton agar (for bacteria) and
RPMI 1640 with MOPS (for fungi) were inoculated with
the suspensions of bacterial or fungal species. Microbial
suspensions were prepared in sterile saline (0.85% NaCl)
with an optical density of McFarland standard scale 0.5.
Samples containing examined compounds were dissolved
in 1 mL dimethyl sulphoxide (DMSO). Furthermore, bacte-
rial and fungal suspensions were put onto Petri dishes with
solid media containing 2000 µg/mL of the tested compounds
followed incubation. The inhibition of microbial growth
was judged by comparison with a control culture prepared
without any sample tested. Ciprofoxacin or fuconazole
(Sigma) were used as reference antibacterial or antifungal
compounds, respectively.
The Vero cells, after a 24 h incubation, were infected with
viruses in the dose of 100 TCID50/mL. After 1 h incubation
at 37 °C, the suspension of the viruses was removed and the
media with 2% of serum together with the tested compounds
in the nontoxic concentration were added to the cell cultures.
The viruses diluted in the culture media without the tested
compounds were used as a control. Acyclovir and ribavirin
(Sigma-Aldrich, Saint Louis, MO, USA) were used as refer-
ence compounds. After a 48–72 h incubation at 37 °C, the
cytopathic efect (CPE) of the viruses was examined by a
light microscope. The antiviral activity of compounds was
evaluated by direct observation of CPE reduction.
Subsequently MIC (minimal inhibitory concentration) of
the compounds was examined by the microdilution broth
method, using their twofold dilutions in Mueller–Hinton
broth (for bacteria) and RPMI 1640 broth with MOPS (for
fungi) prepared in 96-well polystyrene plates. Final concen-
trations of the compounds ranged from 1000 to 0.488 µg/
mL. Microbial suspensions were prepared in sterile saline
(0.85% NaCl) with an optical density of 0.5 McFarland
standard. Next bacterial or fungal suspension was added
per each well containing 200 µL broth and various con-
centrations of the examined compounds. After incubation,
the MIC was assessed spectrophotometrically as the lowest
concentration of the samples showing complete bacterial or
fungal growth inhibition. Appropriate DMSO, growth and
sterile controls were carried out. The medium with no tested
substances was used as control.
Determination of TCID50 by the virus titre reduction method
After the evaluation of the antiviral activity of compounds,
the cells were frozen, and after thawing, the viruses were
titrated in 96-well plastic plates with the Vero cell culture.
The titration of the viruses was performed using end-point
titration technique. Vero cells were cultivated for 24 h at
37 °C in 96-well fat-bottomed plates. Growth medium was
removed and tenfold dilutions of the virus in the absence of
the samples (virus control) and compound-treated infected
cells were added in quadruplicate and plates were incubated
at 37 °C until typical cytopathic efect (CPE) was visible.
The titres of the virus were estimated according to the
Reed–Muench method, expressed as 50% tissue infectious
dose (TCID50) per mL [49].
The MBC (minimal bactericidal concentration) or MFC
(minimal fungicidal concentration) is defned as the lowest
concentration of the compounds that is required to kill a
particular bacterial or fungal species. MBC or MFC was
determined by removing the culture used for MIC determi-
nations from each well and spotting onto appropriate agar
medium. The plates were incubated. The lowest compound
concentrations with no visible growth observed was assessed
as a bactericidal or fungicidal concentration. All the experi-
ments were repeated three times and representative data are
presented [43, 52, 53].
In vitro antimicrobial assay
The examined compounds: 1–20 were screened in vitro
for antibacterial and antifungal activities using the broth
microdilution method according to European Committee
on Antimicrobial Susceptibility Testing (EUCAST) [50]
and Clinical and Laboratory Standards Institute guidelines
[51] against a panel of reference strains of microorgan-
isms, including Gram-positive bacteria (Staphylococcus
aureus ATCC 25923, Staphylococcus aureus ATCC 6538,
Staphylococcus epidermidis ATCC 12228, Bacillus subtilis
ATCC 6633, Bacillus cereus ATCC 10876, Micrococcus
luteus ATCC 10240), Gram-negative bacteria (Bordetella
bronchiseptica ATCC 4617, Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Proteus mirabi-
lis ATCC 12453, Salmonella typhimurium ATCC 14028,
Pseudomonas aeruginosa ATCC 9027) and fungi belonging
to yeasts (Candida albicans ATCC 10231, Candida par-
apsilosis ATCC 22019). The microorganisms belonging to
In this study, no bioactivity was defined as a
MIC > 1000 µg/mL, mild bioactivity as a MIC in the range
501–1000 µg/mL, moderate bioactivity with MIC from
126 to 500 µg/mL, good bioactivity as a MIC in the range
26–125 µg/mL, strong bioactivity with MIC between 10 and
25 µg/mL and very strong bioactivity as a MIC < 10 µg/
mL. The MBC/MIC or MFC/MIC ratios were calculated in
order to determine bactericidal/fungicidal (MBC/MIC ≤ 4,
1 3

Journal of the Iranian Chemical Society
MFC/MIC ≤ 4) or bacteriostatic/fungistatic (MBC/MIC > 4,
thiosemicarbazide, di- and tetracarboximide derivatives
in alkaline media. Reaction of N-(substituted-thioureido)
aminobicyclo di- and tetracarboximides with ethyl bromoac-
etate in the presence of sodium acetate in absolute etha-
nol led to the formation of 4-oxo-1,3-thiazolidine (16–20).
This preparation is based on the most applied method—
Hantzsch synthesis—in which α-halocarbonyl compounds
react with thioamides [31, 54, 55]. Probably, sulphur atom
of thioureido moiety attacked halogen atom of ethyl bro-
moacetate and then elimination of ethanol and hydrogen
bromide took place and 1,3-thiazolidine was formed without
selected intermediate (Fig. 1) [54]. Derivatives with thio-
ureido group possess two positions for nucleophilic attacks,
so two diferent structural isomers A and B can be obtained
(Fig. 1) [56]. According to the crystal structure of 17, it
can be speculated that only one structural isomer A (Fig. 1)
that is N-[(4-oxo-3-substituted-1,3-thiazolidin-2-ylimine)]-
bicyclo(2,3,5,6-tetrahydrophthaloyl) dicarboximide has
been synthesized. This is consistent with the description in
the literature [57]. Unfortunately, in the case of N4-(4-chl
orophenyl)-N1-(2-carboxy-1,2,3,6-tetrahydrobenzoyl)thio-
semicarbazide, cyclization reaction failed and triazole and
1,3-thiazolidine were not obtained. The chemical structure
of all obtained compounds was confrmed and proved by 1H
and 13C NMR spectra.
MFC/MIC > 4) efect of the tested compounds [52].
Results and discussion
Chemistry
The main goal of this investigation was synthesis, identif-
cation and in vitro antimicrobial and antiviral screening of
thiosemicarbazide, di- and tetracarboximide, 1,2,4-triazole-
5-thione and 4-oxo-1,3-thiazolidine. The synthetic pathway
is presented in Schemes 1 and 2. Derivatives 3, 4, 7, 8, 9, 13,
14, 16–20 are unknown in scientifc literature.
Selected dicarboxylic acid anhydrides were used as an
initial compound. Linear derivatives of thiosemicarbazide
(1–4) and tetracarboximide (9–10) were prepared by heat-
ing anhydride with 4-substituted-3-thiosemicarbazide in
dry chloroform. Dicarboximides (5–8) were obtained in the
cyclization reactions of linear thiosemicarbazides (1–4) in
a mixture of concentrated sulphuric acid and glacial ace-
tic acid. In the case of bicyclo[2.2.2]oct-7-ene-2,3,5,6-
tetracarboxylic anhydride, the attempts to obtain the linear
thiosemicarbazides failed and bis N-(substituted-thioureido)
aminobicyclo tetracarboximides were prepared directly. Next
new 3,4-disubstituted 1,2,4-triazolino-5-thione (11–15) were
synthesized by the cyclization reaction of corresponding
The 1H NMR spectra of compounds 1–4 showed
three peaks due to the presence of the three NH groups
O
O
S
R
O
NH2
HN
C
NH
CH3Cl
reflux
OH
NH
S
CH3COOH
H2SO4, r.t.
O
N
NH
+
S
R
5 - 8
HN
NH
NH
O
O
1 - 4
R
O
2% NaOH
reflux
2% NaOH
reflux
O
O
OH
N
N
N
R
N
NH
O
S
11 - 13
N
O
16 - 18
R
S
Compound No
1, 5, 11, 16
2, 6, 12, 17
3, 7, 13, 18
4, 8
R
C2H5
C6H5
2-CH3C6H4
4-ClC6H4
Scheme 1 Synthesis of compounds 1–8, 11–13 and 16–18
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Journal of the Iranian Chemical Society
NH2
HN
O
O
R
O
O
S
S
R
CHCl 3
reflux
NH
NH
S
O
O
+
NH N
N
NH
HN
R
O
O
9 - 10
O
O
OH
OH
2% NaOH
2% NaOH
reflux
reflux
O
N
O
N
O
O
NH
HN
N
N
N
N
S
R
R
N
N
N
N
R
R
S
S
O
O
S
14 - 15
19 - 20
O
O
Compound No
9, 14, 19
R
C2H5
C6H5
10, 15, 20
Scheme 2 Synthesis of compounds 9–10, 14–15 and 19–20
Fig. 1 Probably mechanism
leading to the formation of
1,3-thiazolidine
S
R
HS
C
R
HS
N
R
Br
O C2H5
C
NH
N
C
NH
+
O
R1 NH
R1 NH
R1
R
R1
N
R1
R
N
NH C2H5
NH C2H5
O
O
S
S
O
O
R1
N
R
N
R
N
R1
N
O
O
S
S
B
A
O
N
R1 =
O
between δ 7.38–10.04 ppm. In the 13C NMR spectra of
these compounds, corresponding two signals for C=O
group and C=S group were displayed at 171.09–173.19
of 8.28–10.19 ppm. In the 13C NMR spectra for com-
pounds 5–10, typical signals corresponding to C=O groups
of cyclic carboximides and for C=S group of linear chain
were observed. They resonated between 174.09–177.32 ppm
and 180.69–181.38 ppm, respectively. 1H NMR spectra for
cyclic 1,2,4-triazole-5-thione 11–15 showed typical proton
1
and 179.91–180.90 ppm, respectively, whereas H NMR
spectra for the carboximides (5–10) displayed one or two
typical proton signals of the two NH groups in the region
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Journal of the Iranian Chemical Society
signal of the NH group in the range of 10.72–12.36 ppm.
In the 13C NMR spectra for these compounds, carbon of
triazole ring resonates in the range of 152.90–154.75 ppm,
whereas carbon corresponds to C=S in the range of
174.02–174.64 ppm. The 4-oxo-1,3-thiazolidine 16–20 in
1H NMR spectra showed characteristic signal for CH2 group
at δ 4.10–4.34 ppm. Beside this, in 13C NMR spectra for
these derivatives a signal for C=O group was observed at
δ 171.88–172.89 ppm. All other signals for aliphatic, aro-
matic, rings of 1,2,3,6-tetrahydrobenzoyl, 1,2,3,6-tetrahy-
drophthaloyl and bicyclo[2.2.2]-oct-7-ene were observed in
usual regions.
group P 1 with two independent molecules in the asym-
metric part of the unit cell, denoted as A and B in Fig. 2, in
the same arbitrarily chosen absolute confguration S at C8
atom and R at C9 atom.
The bond distances and angles in molecules A and B
of 17 are in normal ranges [58] and are comparable to the
corresponding values observed in closely related struc-
tures with thiazolidin-4-one system, e.g. 2-imino-3-(2-
nitrophenyl)-1,3-thiazolidin-4-one [59], (Z)-3-phenyl-2-
(propan-2-yl-idene-hydrazono)thiazolidin-4-one [60] and
2-methyl-3-(4-oxo-3-phenyl-thiazolidin-2-ylidenamino)-
4-(3H)-chinazolinon [61]. The thiazolidine ring is planar to
within 0.043(4) Å in A and 0.050(4) Å in B. The molecules
A and B difer in conformation of the phenyl substituent in
relation to the thiazolidine system, as shown in Fig. 3. This
The crystal structure determination of 17 was undertaken
in order to confrm the synthesis pathway and its assumed
molecular structure. The X-ray structure analysis revealed
that compound 17 crystallizes in centrosymmetric space
Fig. 2 ORTEP drawing of 17 at 50% probability
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Journal of the Iranian Chemical Society
Biological activity
The obtained compounds 1–20 have been studied as poten-
tial antiviral agents against Herpes simplex virus type l and
coxsackievirus B3, and revealed a high efcacy as inhibitors
of virus reproduction. Acyclovir was used as a reference
compound for HSV-1 and ribavirin for CVB3. The infuence
of 1–20 on the Vero and FaDu cell cultures after incubation
for 48–72 h is presented in Table 2.
The CC50 of tested substances 1–7 and 10 ranged from
27.02 to 509.57 µg/mL in Vero and 8.85–275.87 µg/mL
in FaDu cell lines. The lowest cytotoxicity against Vero
was observed for compounds 1, 2, 5, 7 (> 500 µg/mL)
and against FaDu for compound 7 (275.87 µg/mL). The
highest cytotoxicity was noted for compound 4 in both
Vero and FaDu cell lines (CC50 = 27.02 and 8.85 µg/mL,
respectively). On the other hand, the most interesting com-
pound was N4-(2-methylphenyl)-N1-(2-carboxy-1,2,3,6-
tetrahydrobenzoyl)-thiosemicarbazide (3). This derivative
displayed high toxicity against human epithelial cells FaDu
in comparison with small toxicity against normal animal
cell line (Vero).
Fig. 3 Overlay of X-ray molecules A and B of compound 17 by least-
squares ftting of the atoms of thiazolidine systems (RMS = 0.0324
Å)
conformation is described by the torsion angle C12-N16-
C17-C18 of 96.3(4) and 77.6(5)° in A and B, respectively.
The cis-gauche position of the 3a,4,7,7a-tetrahydroisoin-
dole-1,3-dione system with respect to the thiazolidine ring
is very similar in both molecules as shown by the torsion
angles S13-C12-N11-N1 of 4.6(5)° in A and 5.7(5)° in B
and C12-N11-N1-C2 of 99.4(4)° in A and 88.3(5)° in B.
The 3a,4,7,7a-tetrahydroisoindole-1,3-dione fused system
of 17 comprises a pyrolidine ring and partially saturated
cyclohexene ring. The pyrolidine ring is planar to within
0.014(4) Å in A and 0.061(5) Å in B. The cyclohexene ring
adopts the boat conformation in both molecules A and B
with asymmetry parameters ΔCC3 of 2.3(5) and 10.4(4)° and
Compounds from group of triazoles (11–15) demonstrated
higher cytotoxicity against the Vero line (21.75–344.50 µg/
mL) compared to the FaDu line (336.54–473.25 µg/mL).
In group 4-oxo-1,3-thiazolidine (16–20), the highest
Table 2 Cytotoxicity of compounds 1–20
Compounds
Cell line/CC50 (µg/mL)a
Vero
FaDu
1
>500
153.51 34.99
262.37 53.26
10.37 1.73
2
509.57 24.21
466.80 29.79
27.02 4.18
>500
486.33 23.67
>500
208.63 50.82
21.75 6.86
344.50 20.51
187.10 9.33
31.98 1.03
81.30 3.12
225.80 32.27
101.60 32.53
284.55 51.41
7.47 0.67
ΔCC4,C5 of 2.7(6) and 10.1(5)° iSn A and B, respectively [62].
3
S
4
8.85 1.98
In the crystal structure of 17, molecules form a three-
dimensional network through the weak C–H…X (X = S, O)
intermolecular hydrogen bonds (Table 1). The inversion-
related molecules A(x, y, z) and B(1 − x, 1 − y, 1-z) and mol-
ecules A(x, y, z) and B(1 − x, 1 − y, 1 − z) form molecular
trimers via C14A-H…S13B and C14B-H…O7B hydrogen
bonds. These trimers are linked into molecular chains par-
allel to X crystallographic axis through C14A–H…O15B
(− 1 + x, y, z) hydrogen bond.
5
240.60 13.29
270.35 33.42
275.87 40.77
99.58 31.60
473.25 37.83
336.54 48.45
396.74 14.37
341.06 32.46
461.00 55.15
330.15 2.19
274.20 9.05
500.00 0.00
23.98 3.15
6
7
10
11
12
13
14
15
16
17
18
19
20
Table 1 Hydrogen-bond geometry (Å, °)
D–H…A
D–H
H…A
D…A
D–H…A
C14A–H14A…S13Bi
C14B–H14D…O7Bi
C14A–H14B…O151Bii
0.97
0.97
0.97
2.73
2.33
2.40
3.636(5)
3.135(6)
3.256(6)
155
140
147
44.05 12.03
38.43 11.08
The CC50 is the cytotoxic concentration required to reduce the num-
ber of viable cells by 50%
aMean SD values come from three independent experiments
(i) = 1 − x, 1 − y, 1 − z; (ii) = − 1 + x, y, z
1 3

Journal of the Iranian Chemical Society
cytotoxicity was found in compound 19, both for Vero and
FaDu lines, and CC50 were 7.47 and 23.98 µg/mL, respec-
tively. Compound 18 was the least toxic compound in this
group. CC50 was 284.55 µg/mL for Vero and 500 µg/mL
for FaDu.
TCID50 by the virus titre reduction method demonstrated
that compound 3 in the concentration of 125 and 250 µg/
mL influenced the HSV-1 replication by reducing the
virus replication level by 3.45 log and 4.29 log, which
resulted in reducing the titre by 40.5 and 50.04%, respec-
tively. Compound 4 in all tested concentrations (15.62 and
31.25 µg/mL) demonstrated the antiviral activity, too. This
derivative caused the decrease in the titre of HSV-1 by
3.03 log and 3.95 log, respectively (which corresponds
to the following percentage levels of inhibition: 33.6 and
46.4%).
DMSO used as an eluent for the examined compounds in
the tested concentration had no toxic efect on cell cultures.
All compounds were evaluated for their cytotoxicity on cell
line by a standard MTT assay.
Compounds were tested for in vitro antiviral activity
using the cytopathic efect (CPE) inhibitory assay and by
the virus titre reduction method. The results are presented
in Table 3.
Compounds 3 and 4 only slightly reduced the CVB3
replication.
Noncytotoxic concentrations of 3.90, 15.62, 31.25, 62.50,
125 and 250 µg/mL were used for testing the antiviral activ-
ity of compounds.
Other compounds did not demonstrate any signifcant
antiviral activity. Thus, there is a need of further research
to estimate their infuence on diferent viruses. Prospec-
tively, study of the mechanisms underlying their antiviral
activity is necessary for the development of antiviral thera-
peutics to treat patients infected with HSV-1 and CVB3.
The preliminary screening revealed the antiviral activ-
ity of compounds 3 and 4 from thiosemicarbazide group,
both against HSV-1 and CVB3. Further determination of
Table 3 Antiviral activity of the
Compounds
Concentration
(µg/mL)
HSV-1
CVB3
compounds against HSV-1 and
CVB3
Antiviral
activity
(TCID50/mL)a
Antiviral
activity
(TCID50/mL)a
1
2
3
250
250
125
250
15.62
31.25
250
250
250
−
−
+
+
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
ND
+
ND
ND
5.07 0.41
4.23 1.50
5.49 2.12
4.57 0.76
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
−
−
+
+
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
ND
+
+
ND
ND
9.24 0.71
8.72 0.88
8.20 1.41
8.50 0.00
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4
5
6
7
10
250
15.62
250
11
12
13
125
14
31.25
62.50
125
125
250
3.90
31.25
250
125
0
15
16
17
18
ND
ND
ND
0
ND
8.52 1.67
ND
ND
ND
ND
19
20
Acyclovir
Ribavirin
Virus control
0
9.06 1.69
Data represent the mean SD of at least 2–3 independent experiments
“−” negative antiviral activity in the screening assay
“+” positive antiviral activity in the screening assay
ND not determined
aThe virus titres are shown in log. TCID50—50% tissue culture infectious dose
1 3

Journal of the Iranian Chemical Society
Using methods outlined in the previous section, inhibi-
tion of growth by examined compounds: 2–7 and 14 for the
reference strains of bacteria and fungi belonging to Can-
dida spp. was shown. The results of our study presented
in Table 4 indicated that compound 4 showed both the
highest efect and the widest spectrum of activity against
all Gram-positive bacteria, Bordetella spp. belonging to
Gram-negative bacteria and fungi. This compound indi-
cated especially very strong activity against S. epidermidis
ATCC 12228 with MIC = 7.81 µg/mL and strong activ-
ity towards S. aureus ATCC 43300. The bioactivity against
remaining reference staphylococci and micrococci was good
(MIC = 31.25–62.5 µg/mL).
bacteria was also sensitive to 4 (MIC = 500 µg/mL and
MBC > 1000 µg/mL). The activity towards all these bac-
teria was bacteriostatic, except micrococci (bactericidal
effect with MBC/MIC = 2). Besides, this compounds
exhibited fungicidal activity towards Candida spp. ATTC
(MIC = 500 µg/mL and MFC = 500–1000 µg/mL, MFC/
MIC = 1–2).
On the basis of minimal inhibitory concentration val-
ues, it was shown that other compound 3 had also wide
spectrum of antimicrobial activity against Gram-positive
bacteria and yeasts. The bactericidal efect of this sub-
stance was good against staphylococci, micrococci and
Bacillus subtilis ATTC 6633 (MIC = 62.5–125 µg/mL and
MBC = 62.5–250 µg/mL, MBC/MIC = 1–4) and moder-
ate towards Bacillus cereus ATTC 10876 (MIC = 500 µg/
mL and MBC = 1000 µg/mL, MBC/MIC = 2). Compound
Moreover, substance 4 exhibited moderate or mild activ-
ity (MIC = 125–250 µg/mL) towards Bacillus spp. ATTC.
Bordetella bronchiseptica ATTC 4617 from Gram-negative
Table 4 Activity data expressed as MIC (MBC/MFC) (µg/mL) against the reference strains of microorganisms
Species/compound MIC (MBC/MFC) (µg/mL) of the tested compounds
CIP/FLUa
2
3
4
5
6
7
14
Gram-positive
bacteria
Staphylococcus
aureus ATCC
25923
Staphylococcus
aureus ATCC
6538
Staphylococcus
aureus ATCC
43300
–
–
–
–
62.5 (125) 62.5 (> 1000)
125 (500) 31.25 (> 1000)
–
–
–
–
–
–
–
–
–
–
–
–
–
0.488
0.244
0.244
0.122
62.5 (125) 15.62 (> 1000) 1000 (> 1000)
Staphylococcus
62.5 (62.5) 7.81 (> 1000) 1000 (> 1000) 1000 (> 1000)
epidermidis
ATCC 12228
Micrococcus luteus 1000 (> 1000) 62.5 (62.5) 31.25 (62.5)
1000 (> 1000) 1000 (> 1000) 100 (> 1000) 1000 (> 1000) 0.976
ATCC 10240
Bacillus subtilis
1000 (> 1000) 125 (250) 250 (> 1000)
1000 (> 1000) 1000 (> 1000)
–
–
–
–
0.030
0.061
ATCC 6633
Bacillus cereus
ATCC 10876
–
–
500 (1000) 125 (> 1000)
–
–
Gram-negative
bacteria
Bordetella bron-
chiseptica ATCC
4617
–
500 (> 1000)
–
–
–
–
0.976
Fungi
Candida albicans 1000 (> 1000) 250 (500) 500 (1000)
–
–
500 (1000)
1000 (> 1000)
–
–
–
0.976a
1.953a
ATTC 10231
Candida parapsilo- 1000 (> 1000) 250 (1000) 500 (500)
1000 (1000)
sis ATTC 22019
Bold values indicate compounds with signifcant activity
The standard chemotherapeutics agents: ciprofoxacin (CIT) or fuconazole (FLUa) used as positive control for bacteria and yeasts, respectively
No bioactivity − MIC > 1000 µg/mL; mild bioactivity − MIC = 501–1000 µg/mL; moderate bioactivity − MIC = 126–500 µg/mL; good bioac-
tivity − MIC = 26–125 µg/mL; strong bioactivity − MIC = 10–25 µg/mL; very strong bioactivity − MIC < 10 µg/mL
1 3

Journal of the Iranian Chemical Society
3 exhibited also fungicidal activity towards Candida spp.
ATTC (MIC = 250 µg/mL and MFC = 500–1000 µg/mL,
MFC/MIC = 1–2) (Table 4).
thiosemicarbazide, is the most interesting due to its specifc
distinction between carcinoma and normal cells.
The obtained study can be applied in practice. On
this basis, we are able to design novel biological active
compounds which may be further modifed and used as
lead molecules for future investigation. Furthermore, this
in turn will give hope for medicinal chemists as well as
pharmacologists in the prediction of increased activity and
allow to synthesize hitherto unknown substances exhibit-
ing better antibacterial, antifungal and antiviral activities
and then those reported in the above study. In this context,
it is concluded that the research results make a great con-
tribution to information on biological active compounds.
In conclusion, these studies set the direction of further
research.
The remaining synthesized compounds 2, 5–7 and
14 had low activity on the single bacteria and fungi
(MIC = 500–1000 µg/mL and MBC or MFC ≥ 1000 µg/
mL) or had no inhibitory efect on the growth of microor-
ganisms (Table 4).
In turn, compounds 1, 8–13 and 15–20 indicated inactiv-
ity towards reference strains of bacteria and fungi.
Structure–activity relationship
It can be stated from the antiviral study that among obtained
compounds, only two linear thiosemicarbazides (3,4) bear-
ing 2-methylphenyl and 4-chlorophenyl groups attached to
thiosemicarbazide at 4th position demonstrated the best
activity against HSV-1 and CVB3. The antiviral activity
in these derivatives is probably associated with the pres-
ence of a substituent attached to the phenyl ring at 2nd or
4th position, respectively, regardless of the character of
substituent (electron donating or withdrawing). Other thio-
semicarbazides with aliphatic or unsubstituted phenyl group
were absolutely inactive. The dicarboximide and cyclic
1,2,4-triazole-5-thione and 4-oxo-1,3-thiazolidine had no
any antiviral activity too. The cyclization reaction did not
improve this activity. Similar results were obtained also for
antimicrobial study. Compounds 3 and 4 presented the best
antimicrobial activity too. According to this, it can be specu-
lated that introduction of substituted phenyl group but only
to thiosemicarbazide was the most benefcial in terms of
both antiviral and antimicrobial activities.
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