Synthesis and antiviral evaluation of 5-(arylazo)salicylaldehyde thiosemicarbazone derivatives as potent anti-bovine viral diarrhea virus agents

Article abstract of DOI:10.1080/00397911.2021.1925298

Thiosemicarbazones which previously characterized as a new class that inhibit anti-bovine viral diarrhea virus (BVDV) prompted us for synthesizing a series of new thiosemicarbazone derivatives. Thus, in this study, we reported the synthesis and antiviral evaluation of a series of 5-(arylazo)salicylaldehyde thiosemicarbazone derivatives for their expected antiviral activity. The desired products were synthesized from the condensation of 5-(arylazo)salicylaldehyde derivatives with N-(4)-substituted thiosemicarbazide derivatives. Antiviral screening was performed to test the anti-bovine viral diarrhea virus properties. From the obtained results, compounds 5, 23 and 24 showed highly selective activity against BVDV by blocking the viral RNA synthesis in cell culture. The BVDV in antiviral drug studies is valuable surrogate for the hepatitis C virus (HCV); so, the above results provided a novel candidate for the development of anti-HCV agents.

Full text of DOI:10.1080/00397911.2021.1925298

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Synthesis and antiviral evaluation of 5-
(arylazo)salicylaldehyde thiosemicarbazone
derivatives as potent anti-bovine viral diarrhea
virus agents
Wahid M. Basyouni, Samir Y. Abbas, Khairy A. M. El-Bayouki, Reham M.
Daawod & Mostafa K. Elawady
To cite this article: Wahid M. Basyouni, Samir Y. Abbas, Khairy A. M. El-Bayouki,
Reham M. Daawod & Mostafa K. Elawady (2021) Synthesis and antiviral evaluation of 5-
(arylazo)salicylaldehyde thiosemicarbazone derivatives as potent anti-bovine viral diarrhea virus
agents, Synthetic Communications, 51:14, 2168-2174, DOI: 10.1080/00397911.2021.1925298
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2021, VOL. 51, NO. 14, 2168–2174
https://doi.org/10.1080/00397911.2021.1925298
Synthesis and antiviral evaluation of
5-(arylazo)salicylaldehyde thiosemicarbazone derivatives as
potent anti-bovine viral diarrhea virus agents
Wahid M. Basyounia, Samir Y. Abbasa, Khairy A. M. El-Bayoukia, Reham M.
Daawodb, and Mostafa K. Elawadyb
aOrganometallic and Organometalloid Chemistry Department, National Research Centre, Cairo, Egypt;
bMicrobial Biotechnology Department, National Research Centre, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 8 March 2021
Thiosemicarbazones which previously characterized as a new class
that inhibit anti-bovine viral diarrhea virus (BVDV) prompted us for
synthesizing a series of new thiosemicarbazone derivatives. Thus, in
this study, we reported the synthesis and antiviral evaluation of a
series of 5-(arylazo)salicylaldehyde thiosemicarbazone derivatives for
their expected antiviral activity. The desired products were synthe-
sized from the condensation of 5-(arylazo)salicylaldehyde derivatives
with N-(4)-substituted thiosemicarbazide derivatives. Antiviral screen-
ing was performed to test the anti-bovine viral diarrhea virus proper-
ties. From the obtained results, compounds 5, 23 and 24 showed
highly selective activity against BVDV by blocking the viral RNA syn-
thesis in cell culture. The BVDV in antiviral drug studies is valuable
surrogate for the hepatitis C virus (HCV); so, the above results pro-
vided a novel candidate for the development of anti-HCV agents.
KEYWORDS
Salicylaldehydes; thiosemi-
carbazides; thiosemicarba-
zones; bovine viral diarrhea
virus; HCV
GRAPHICAL ABSTRACT
Syntheꢀc Methods
Efficient procedure for the synthesis
Biological
Acꢀvity
of
5-(aryldiazo)salicylaldehyde
thiosemicarbazone derivatives was
achieved
Potent anti-bovine viral
diarrhea virus agent
CONTACT Wahid M. Basyouni
w_basyouni2002@yahoo.com; Samir Y. Abbas
samiryoussef98@yahoo.com
Organometallic and Organometalloid Chemistry Department, National Research Centre, Cairo, Egypt.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2021 Taylor & Francis Group, LLC

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Introduction
Thiosemicarbazones are versatile building blocks in the synthesis of densely substituted
heterocycles.[1–3] Thiosemicarbazones have been reported as first antiviral compounds
recognized to have a broad spectral antiviral activities against range of DNA and RNA
viruses.[4] Modified aromatic and substituted thiosemicarbazones were investigated as
broad spectra of antiviral activities including Hepatitis C Virus (HCV) subgenomic
RNA replicon inhibitors and human immunodeficiency.[5–8] The N-methylisatin-
b-thiosemicarbazone (methisazone & marboran) (Figure 1) was demonstrated as an
effective antiviral drug in the chemoprophylaxis of small pox in South India.[9] Also,
thiosemicarbazones and their metal complexes represent an essential structural unit
with a wide range of biological and pharmacological properties such as anticancer and
antimicrobial activities. Owing to their versatile chemistry, research has been progressed
to design and synthesize broad spectra of thiosemicarbazone derivatives to verify their
antibacterial, antifungal, antitumor and antiviral properties.[10–15]
Therefore, the present study was aimed to synthesize several newer thiosemicarbazone
derivatives with screening for their antiviral properties hoping to obtain potent antiviral
thiosemicarbazone derivatives with fewer side effects, rapid clearance rate or less inci-
dence of relapse.
Results and discussion
The 5-(arylazo)salicylaldehyde precursors I-V were prepared as shown in Scheme 1 from
reacting salicylaldehyde with the appropriate 4-substituted-aryldiazonium chloride accord-
ing to the procedure described previously.[16–18] Hydroxyl group has positive resonance
effect (þR) which is the main factor responsible for the high nucleophilicity of position-5
at salicylaldehyde. So, according to the effect of hydroxyl group, the coupling reaction will
orient to position-5 which is para to hydroxyl group. Formyl group at salicylaldehyde has
-R effect. So, the formyl group will orient the coupling reaction to meta position of formyl
group (position-5 at salicylaldehyde). Moreover, Hammett r constants supported these
results. The Hammett values revealed that the position-5 at salicylaldehyde is more electron
rich site. According to the both effects of hydroxyl and formyl groups at salicylaldehyde,
the coupling reaction will orient to position-5 which is para to hydroxyl group and meta
to formyl group to produce 5-(arylazo)salicylaldehyde precursors I-V as clean cut product.
The thiosemicarbazone derivatives 1-25 were synthesized in high yields through
two different synthetic methodologies (Scheme 2). The first approach (Method A)
was
a multicomponent coupling reaction (one-pot synthesis, catalyst-free) of
CH3
N
S
NH2
N
O
S
H3C
S
N
NH2
H
N
N
N
N
NH2
N
NH2
N
H
H
CH3
Triapine
(anticancer)
Tiacetazone
(turberculostatic)
Maroban®
Antiviral drug
Figure 1. Representative bioactive thiosemicarbazones.

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W. M. BASYOUNI ET AL.
OH
OH
N
X
N Cl
NH2
HCl
N
N
CHO
NaNO2
CHO
X
I
IV,
X = NO2
,
X = F
X= Br
III, X= CH3
II,
V,
X = SO2NH2
X
Scheme 1. Synthesis of 5-(4-substitutedphenylazo)salicylaldehyde derivatives I–V.
OH
CHO
N
N
X
I, X = F
II, X= Br
III, X= CH3
IV, X = NO2
V, X = SO2NH2
S
OH
S
H2N
N
NHR
N
N
H
N
N
H
NHR
or N2H4.H2O & R-N=C=S
1-25
X
1 X = F R = H
6
X = Br R = H
11 X = CH3 R = H
2 X = F R = CH3
3 X = F R = C2H5
4 X = F R = C6H5
7
X = Br R = CH3
X = Br R = C2H5
X = Br R = C6H5
12 X = CH3 R = CH3
13 X = CH3 R = C2H5
14 X = CH3 R = C6H5
8
9
5
10
15
X = Br R = C6H5OCH3-4
X = F R = C6H4OCH3-4
X = CH3 R = C6H4OCH3-4
21
X = SO2NH2 R = H
16
X = NO2 R = H
17 X = NO2 R = CH3
18 X = NO2 R = C2H5
19 X = NO2 R = C6H5
22 X = SO2NH2 R = CH3
23 X = SO2NH2 R = C2H5
24 X = SO2NH2 R = C6H5
20 X = NO2 R = C6H4OCH3-4 25 X = NSO2NH2 R = C6H4OCH3-4
Scheme 2. Synthesis of 5-(4-substituted-phenylazo)salicylaldehyde thiosemicarbazones 1–25.
5-(arylazo)- salicylaldehydes I-V, isothiocyanates and hydrazine hydrate, in short reac-
tion times and high yields. The second one (Method B, Scheme 2) involved a procedure
comprising two steps: the first one, is the synthesis of thiosemicarbazide derivatives
from the reaction of the corresponding isothiocyanates with hydrazine hydrate, followed
by reacting them with the 5-(arylazo)salicylaldehydes I-V to afford the desired thiosemi-
carbazone derivatives 1-25 in good yields. The synthesized thiosemicarbazone deriva-
tives were characterized by bearing various substituents at arylazo and N-(4)-substituted
thiosemicarbazone moieties. Structure of the synthesized N-(4)-substituted thiosemicar-
bazones 1-25 was inferred from their correct elemental analyses and careful studying of
their spectral data. The infrared spectra the obtained products were characterized by the
presence of intense bands due phenolic group ꢀ(OH) in the region 3468–3409 cmꢀ1
The spectra showed also other bands in the regions 3300–3100 cmꢀ1, 1618–1614 cmꢀ1
.
,
and 1281–1223 cmꢀ1 attributed to NH, CH¼N and C¼S diagnostic groups for thiose-
1
micarbazones with disappearance of carbonyl group bands. H NMR spectra of products
1–25 were characterized, generally, by the presence of broad signals integrated as one

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Table 1. Screening of anti BVDV activity of the synthesized thiosemicarbazone derivatives.
Compound No.
10 mg/ml
Notes
4
5
10
14
15
19
23
24
Toxic effect
2 ꢁ 10-4.6 PFU
Toxic effect
Toxic effect
Toxic effect
Toxic effect
2 ꢁ 10-4.6 PFU
2 ꢁ 10-4.6 PFU
Toxic effect
85 ꢁ 10-4.6 PFU
–
Active
–
–
–
–
Active
Active
–
25
Positive control
–
hydrogen at d ¼ 10.32–11.21 ppm region (D2O-exchangeable) assigned to the phenolic
OH protons. The spectra of the 4-amino derivatives 1, 6, 11, 16, and 21 revealed NH2
proton signals as two signals at d ¼ 8.09–8.12 ppm and 8.17–8.19 ppm regions (D2O-
exchangeable). The spectra of N4-substituted derivatives were characterized by the pres-
ence of NH-alkyl and NH-aryl proton signals at d ¼ 8.56–8.65 ppm and
10.14–10.24 ppm regions (D2O-exchangeable), respectively. The spectra revealed also
signals due to NH–N ¼ protons at d ¼ 11.46–11.87 ppm region (D2O-exchangeable)
beside the other characteristic signals. 13C NMR spectral assignments were based on
characteristic signal positions of the functional groups. In 13C NMR spectra the signals
resonated in the deshielded region of d about 177 ppm assigned to C ¼ S.
Anti-viral screening of the new synthesized compounds
Anti-viral screening of selected examples from the synthesized products was carried out on
the Bovine Viral Diarrhea Virus (BVDV), which is a single stranded positive RNA virus
classified as a member of the same family as Hepatitis C Virus (HCV) i.e. Flaviviridae.
The synthesized compounds were tested in vitro for their antiviral activity. Viral infectivity
assay was carried out using the plaque formation method.[19] A plaque is a localized focus
of virus (BVDV)-infected (Madin-Darby Bovine Kidney, MDBK) cells which under optimal
conditions, the autophagy induced cell death originates from a single infectious virus par-
ticle. Counting of these foci for a serial dilution of virus suspension is a precise method
for quantification of viral infectivity. Under these conditions, reduction in virus plaque
counts provides a sensitive mean for measuring antiviral activity. The results of the plaque
reduction assay are summarized in Table 1. The antiviral effect obtained for the synthe-
sized compounds suggested that the tested thiosemicarbazone derivatives (4, 10, 14, 15, 19
and 25) have no antiviral activity or have a toxic effect on the cells. On the other hand,
cell cultures containing thiosemicarbazone derivatives (5, 23, and 24) displayed >40 fold
decrease in viral infectivity (2ꢁ 10ꢀ4.6 versus 85 ꢁ 10ꢀ4.6 PFU) when compared with mock
BVDV infected MDBK cells in absence of tested thiosemicarbazone derivatives.
Structure activity relationship (SAR)
From the results obtained from Table 1: It was noticed that: Compounds 5, 23, and 24
have a strong antiviral activity. Compounds 4, 10, 14, 15, 19 and 25 were of toxic effect on
the cells. Using the general structures provided in Figure 2, certain aspects of the structure

2172
W. M. BASYOUNI ET AL.
OH
S
R
N
N
N
N
H
N
H
X
Figure 2. General formula of the synthesized compounds 1–25.
activity relationships for these compounds can be more clearly highlighted. As anticipated, a
clear difference in antiviral activity is noted between compounds 1–25 within and between
each series, pointing to the reinforcing and opposing effects of X and R groups, where X
were alkyl, halides, nitro and sulfonamide moieties; R were H, alkyl and aryl moieties.
From the obtained antiviral results it was noticed that, activity of thiosemicarbazone
derivatives 5, 23, and 24 can be attributed to the presence of the 5-(arylazo) salicylalde-
hyde moiety and dependence on the other substituents. Thus, the results indicated that the
presence of 4-fluoro- and 4-methoxyphenyl- substituents at arylazo and N-(4)-thiosemicar-
bazone moieties respectively (compound 5) showed a strong antiviral activity. Moreover,
the presence of sulphonamido- at arylazo and ethyl-23, phenyl-24 at N-(4)-thiosemicarba-
zone moieties revealed a strong antiviral activity. Therefore, this study showed a discovery
of a new lead molecule to design more potent anti-viral agents. Further studies for the
active compounds 5, 23, and 24 as an anti-HCV candidate is currently in progress.
Conclusion
We have synthesized a series of new 5-(arylazo) salicylaldehyde-thiosemicarbazone deriva-
tives bearing various substituents at arylazo and N-(4)-substituted thiosemicarbazone moi-
eties. The expected anti-BVDV property of the synthesized derivatives was tested. The
results indicated that introduction of fluoro- and 4-methoxyphenyl- substituents at arylazo
and N-(4)-thiosemicarbazone moieties respectively (i.e. compound 5) showed a strong
anti-BVDV activity. Also, the presence of sulphonamido- at arylazo and ethyl- (23), phe-
nyl- (24) at N-(4)-thiosemicarbazone moieties exhibited a potent anti- BVDV activity. We,
therefore have report on a lead molecule to design more potent anti-viral agents.
Experimental part
All melting points are recorded on digital Gallen Kamp MFB-595 instrument and may
be uncorrected. The IR spectra (KBr) (cmꢀ1) were measured on a JASCO spectropho-
1
tometer. H NMR spectra were recorded on Joel spectrometers (at 500 MHz) and are
reported relative to deuterated solvent signals in deuterated dimethylsulfoxide (DMSO-
d6). 13C NMR spectra were recorded on Joel Spectrometers (at 125 MHz) in deuterated
dimethylsulfoxide (DMSO-d6).
Synthesis of the 5-(arylazo) salicylaldehydethiosemicarbazone derivatives 1-25
Method A
A mixture of salicylaldehyde derivatives I–V (0.01 mol), hydrazine hydrate (0.012 mol)
and isothiocyanate (0.01 mol) in ethanol (25 mL) was heated under reflux for 20 min.,

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then left to cool. The solid product obtained was filtered off and crystallized from the
proper solvent to give the thiosemicarbazones 1–25.
Method B
A mixture of salicylaldehyde derivatives I–V (0.01 mol) and the desired thiosemicarba-
zide derivatives (namely thiosemicarbazide, N-(methyl) thiosemicarbazide, N-(ethyl) thi-
osemicarbazide, N-(phenyl) thiosemicarbazide) (0.01 mol) in ethanol (25 mL) was heated
under reflux for 30 min., then left to cool. The solid product obtained was filtered off
and crystallized from the proper solvent to give the thiosemicarbazones 1–25.
2-(5-((4-Fluorophenyl)diazenyl)-2-hydroxybenzylidene)hydrazinecarbothioamide (1):
1
Yield 82%; m.p. 268–270ꢂC; IR: ꢀ/cmꢀ1 ¼ 3425, 3244, 3153 (NH2 NH), 1602 (C¼N); H
NMR (500 MHz, DMSO): d/ppm: 7.01 (d, 1H, J ¼ 8.7 Hz, Ar-H), 7.37 (m, 2H, Ar-H),
7.72 (d, 1H, J ¼ 6.5 Hz, Ar-H), 7.83 ꢀ 7.92 (m, 2H, Ar-H), 8.09, 8.18 (2 s, 2H, NH2), 8.41
(s, 1H, Ar-H), 8.54 (s, 1H, CH¼N), 10.82 (br, 1H, OH), 11.48 (s, 1H, NH); 13C NMR
(126MHz, DMSO): 116.7, 116.9, 117.4, 123.7, 124.6, 124.9, 125.0, 139.1, 145.8, 149.3,
160.0, 162.9, 164.8, 178.3 (C¼S); MS, m/z (%): 317 (Mþ; 49.4); Anal. Calcd. for
C14H12FN5OS (317.3): C, 52.99; H, 3.81; N, 22.07; Found: C, 53.14; H, 3.78; N, 21.97%.
Funding
Financial support was provided by the Science and Technology Development Fund in Egypt
(STDF) through the project No. 14993.
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