Inhibitors of Yellow Fever Virus replication based on 1,3,5-triphenyl-4,5-dihydropyrazole scaffold: Design, synthesis and antiviral evaluation

Article abstract of DOI:10.1016/j.ejmech.2017.09.060

By the antiviral screening of an in house library of pyrazoline compounds, 4-(3-(4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (5a) was identified as a promising hit compound for the development of anti- Yellow Fever Virus (YFV)

Full text of DOI:10.1016/j.ejmech.2017.09.060

Accepted Manuscript
Inhibitors of Yellow Fever Virus replication based on 1,3,5-triphenyl-4,5-
dihydropyrazole scaffold: Design, synthesis and antiviral evaluation
Rossella Fioravanti, Nicoletta Desideri, Antonio Carta, Elena Maria Atzori, Ilenia
Delogu, Gabriella Collu, Roberta Loddo
PII:
S0223-5234(17)30775-4
10.1016/j.ejmech.2017.09.060
EJMECH 9775
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 August 2017
Revised Date: 25 September 2017
Accepted Date: 27 September 2017
Please cite this article as: R. Fioravanti, N. Desideri, A. Carta, E.M. Atzori, I. Delogu, G. Collu, R. Loddo,
Inhibitors of Yellow Fever Virus replication based on 1,3,5-triphenyl-4,5-dihydropyrazole scaffold:
Design, synthesis and antiviral evaluation, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.09.060.
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ACCEPTED MANUSCRIPT
Inhibitors of Yellow Fever Virus replication based on 1,3,5-triphenyl-4,5-dihydropyrazole
scaffold: design, synthesis and antiviral evaluation
Rossella Fioravanti, Nicoletta Desideri, Antonio Carta, Elena Maria Atzori, Ilenia Delogu, Gabriella Collu
and Roberta Loddo

ACCEPTED MANUSCRIPT
Inhibitors of Yellow Fever Virus replication based on 1,3,5-triphenyl-4,5-dihydropyrazole
scaffold: design, synthesis and antiviral evaluation
Rossella Fioravantia,*, Nicoletta Desideria, Antonio Cartab, Elena Maria Atzoria, Ilenia Deloguc, Gabriella
Colluc and Roberta Loddoc*
a Dipartimento di Chimica e Tecnologie del Farmaco, Università “La Sapienza” P.le Aldo Moro, 5, 00185 Rome, Italy
b Dipartimento di Chimica e Farmacia, Università di Sassari, via Muroni 23A, 07100 Sassari, Italy
c Dipartimento di Scienze Biomediche, Università di Cagliari, 09042 Monserrato, Cagliari, Italy
ABSTRACT
By the antiviral screening of an in house library of pyrazoline compounds, 4-(3-(4-
phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (5a) was identified as
a promising hit compound for the development of anti- Yellow Fever Virus (YFV) agents.
Structural optimization studies were focused on the development of 5a analogues which retain the
potency as YFV inhibitors and show a reduced cytotoxicity. The synthesized 1-3,5-triphenyl-
pyrazolines (4a-j, 5a-j, 6a-j) were evaluated in cell based assays for cytotoxicity and antiviral
activity against representative viruses of two of the three genera of the Flaviviridae family, i.e.:
Pestivirus (BVDV) and Flavivirus (YFV). These compounds were also tested against a large panel
of different pathogenic RNA and DNA viruses. Most of the new 1-3,5-triphenyl-pyrazolines (4a-j,
5a-j, 6a-j) exhibited a specific activity against YFV, showing EC50 values in the low micromolar
range with almost a 10-fold improvement in potency compared to the reference inhibitor 6-
azauridine. However, the selectivity indexes of the unsubstituted (4a-j) and the phenoxy (5a-j)
analogues were generally modest due to the pronounced cytotoxicity against BHK-21 cells.
Otherwise, the benzyloxy derivatives (6a-j) generally coupled high potency and selectivity. On the
basis of both anti-YFV activity and selectivity index, pyrazolines 6a and 6b were chosen for time of
addition experiments. The selected pyrazolines and the reference inhibitor 6-azauridine displayed
maximal inhibition when added in the pretreatment or during the infection.
Keywords:
4,5-Dihydropyrazole derivatives;
Antiviral activity;
YFV;
BVDV;
RNA and DNA viruses
a,
*
Corresponding author. Tel.: +39 06 49693259; fax: +39 06 49693259.
E.mail address rossella.fioravanti@uniroma1.it
c,*, Corresponding author. Tel.: +39 070 6754202; fax: +39 070 6754210.
E.mail address rloddo@unica.it

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1. Introduction
Viruses belonging to the Flaviviridae family are a major cause of human and veterinary
infectious diseases of global importance. The family consists of four genera: Flavivirus, Pestivirus,
Hepacivirus and Pegivirus. The largest genus Flavivirus comprises more than 50 species of
arthropod-borne viruses that can infect mammalians and birds, usually transmitted to vertebrate by
ticks or mosquitoes, and causing severe diseases and even mortality. Yellow fever virus (YFV) is
the type member of the genus Flavivirus and was the first human pathogenic virus isolated in Ghana
in 1927. West-Nile Virus (WNV), Dengue Virus (DENV), Japanese Encephalitis Virus (JEV) and
Tick-Borne Encephalitis Virus (TBEV) are other human emerging or reemerging pathogens
belonging to Flavivirus genus. The flaviviral infections result in symptoms that range from mild
febrile illness to severe hemorrhagic fever or neurological disease. [1]
The Pestivirus genus comprises three important animal pathogens causing significant
economic losses to the livestock industry: Bovine Viral Diarrhea Virus (BVDV), Classical Swine
Fever Virus (CSFV), and Border Disease Virus (BDV). [2] BVDV, the type member of this genus,
can infect cattle, swine and wild ruminant causing clinical manifestations including severe mucosal
ulceration of mouth, nose and intestine, in addition to immune system dysfunction with
predisposition to secondary infections, abortion and teratogenesis.
The Hepacivirus genus includes the hepatitis C virus (HCV), an important human pathogen
targeting the liver. HCV initially causes an acute infection, generally asymptomatic, the 55-85% of
infected patients develop chronic hepatitis C infection with high risk of cirrhosis or liver cancer.
The WHO estimates that between 130–150 million people worldwide have chronic hepatitis C
infection and that about 700,000 people die each year for the consequence of HCV infection. [3]
Human Pegivirus (HPgV), previously reported as GB virus C (GBV-C) or hepatitis G virus
(HGV), has been recently classified as a member of the species Pegivirus C in Pegivirus genus.
[4] Other members of Pegivirus C and members of Pegivirus A - K can persistently infect a range of
mammalian species. However, their infections are not clearly related with the development of any
disease, with the exception of Theiler’s disease in horses infected with Theiler’s disease associated
virus, Pegivirus D. [5]
To date, human vaccines are available only for YFV, TBEV and JEV. However, their
utilization encounters limitations and difficulties, and many thousands of Flaviviridae infections
continue to occur each year in the endemic areas. In particular, YF vaccine is both highly effective
and relatively safe, conferring a life-long protection against YF disease. Vaccine associated side
effects are usually mild while severe adverse reactions to the liver, kidneys or nervous system are
uncommon (less than 1 case per 100,000 people vaccinated). However, YF vaccine is contro-

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indicated for immune-suppressed individuals, people with severe allergies to egg proteins, infants
under 9 months of age and pregnant women. In 1988, WHO and UNICEF recommended that YF
vaccine be involved in routine immunization program in countries at risk in Africa and Americas.
As of 2016, YF vaccine had been introduced in routine infant immunization programs in 35 of the
42 countries at risk. In these 42 countries, coverage is estimated at 45%. The goal is to achieve a
vaccine coverage > 80% conducting preventive mass vaccination campaigns of the populations at
risk. [6, 7]
The global, social and economic impact due to morbidity and even mortality associated with
these infections, urgently demands effective therapeutic interventions. Success has been recently
achieved with the introduction in therapy of the direct acting antiviral agents (DAAs) for the
treatment of hepatitis C. [3] However, to date no specific drug has been approved to combat
Flavivirus and Pestivirus infections, and patient care remains symptomatic.
By the antiviral screening of an in house library of pyrazoline compounds, 4-(3-(4-
phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (5a) (Figure 1) was
identified as a potent inhibitor of YFV replication (EC50 = 2.1 µM). However, its selectivity index
(SI = 6.2) was modest due to the considerable cytotoxicity (CC50 = 13 µM) (data not published).
As a part of our research on Flaviviridae inhibitors [8, 9], in an effort to identify low
cytotoxic compounds and to retain or to improve the anti-flavivirus activity of the hit compound,
5a, the 4-phenoxy substituent on the phenyl ring at the 3 position of pyrazoline moiety was
removed or replaced by a bulkier benzyloxy substituent in the same position. Moreover, either
electron-donor or withdrawing groups were introduced at the ortho, meta or para position of the 5-
phenyl substituent (Figure 1).

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Figure 1. Structure of 5a based 1,3,5-triphenylpirazolines designed for the development of new
inhibitors of YFV replication.
2. Results and discussion
2.1. Chemistry
The 1,3,5-trisubstituted 4,5-dihydropyrazoles (4a-i, 5a-j, 6a-j) were synthesized according
to Scheme 1. Chalcones 1a-l, 2a-j and 3a-j were obtained by Claisen-Schmidt condensation
between substituted benzaldehydes and suitable acetophenones in basic alcoholic medium. The
subsequent reaction between the appropriate chalcone with 4-hydrazinylbenzenesulfonamide
hydrochloride in basic alcoholic medium provided compounds 4a-i, 5a-j and 6a-j. The reaction
proceeds via hydrazone formation and successive cyclization to pyrazolines. The formation of the
1
pyrazoline derivatives was confirmed in H NMR spectra by the disappearance of signals for
olefinic protons (between 7.2 – 8.2 ppm) and the presence of three double doublets for the two
methylene protons in position 4 (~ 4.0 ppm and ~ 3.1 ppm) and for the proton in position 5 (~ 5.6
ppm).

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Scheme 1. Synthesis of 4-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamides
(4a-j, 5a-j, 6a-j).
R
R
Ba(OH)2 . 8 H2O,
EtOH, 30 °C, overnight
R1
R1
H
O
O
O
1a-j, 2a-j, 3a-j
.
NHNH2 HCl
KOH, dry EtOH,
120 °C, 24h
H2NO2S
R
R1
N
N
4a-j, 5a-j, 6a-j
SO2NH2
1a, 4a: R = R1 = H
2a, 5a: R = OPh, R1 = H
3a, 6a: R = OBn, R1 = H
3b, 6b: R = OBn, R1 = 2-F
3c, 6c: R = OBn, R1 = 3-F
3d, 6d: R = OBn, R1 = 4-F
3e, 6e: R = OBn, R1 = 2-Cl
3f, 6f: R = OBn, R1 = 3-Cl
3g, 6g: R = OBn, R1 = 4-Cl
3h, 6h: R = OBn, R1 = 2-CH3
31, 6i: R = OBn, R1 = 3-CH3
3j, 6j: R = OBn, R1 = 4-CH3
1b, 4b: R = H, R1 = 2-F
1c, 4c: R = H, R1 = 3-F
1d, 4d: R = H, R1 = 4-F
1e, 4e: R = H, R1 = 2-Cl
1f, 4f: R = H, R1 = 3-Cl
1g, 4g: R = H, R1 = 4-Cl
1h, 4h: R = H, R1 = 2-CH3
11, 4i: R = H, R1 = 3-CH3
1j, 4j: R = H, R1 = 4-CH3
2b, 5b: R = OPh, R1 = 2-F
2c, 5c: R = OPh, R1 = 3-F
2d, 5d: R = OPh, R1 = 4-F
2e, 5e: R = OPh, R1 = 2-Cl
2f, 5f: R = OPh, R1 = 3-Cl
2g, 5g: R = OPh, R1 = 4-Cl
2h, 5h: R = OPh, R1 = 2-CH3
21, 5i: R = OPh, R1 = 3-CH3
2j, 5j: R = OPh, R1 = 4-CH3
2.2. Antiviral tests
Cytotoxicity and antiviral activity of the new compounds (4a-j, 5a-j and 6a-j) and reference
inhibitors are reported in Tables 1 and 2.

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Each compound was initially evaluated in cell based assays for its cytotoxicity and antiviral
activity against Yellow Fever Virus (YFV) and Bovine Viral Diarrhea Virus (BVDV) representative
members of the Flavivirus genus and the Pestivirus genus, respectively. The derivatives able to
interfere with YFV and/or BVDV replication were also tested against two other viruses belonging
to the Flavivirus genus of the Flaviviridae family (Dengue Virus, DENV-2 and West-Nile Virus,
WNV). 6-Azauridine, ribavirin and 2′-C-methyl-guanosin (NM108) were employed as reference
inhibitors of these single-stranded, positive RNA (ssRNA+) viruses.
Moreover, in order to verify the specific inhibition of our pyrazoline derivatives on
Flaviviridae replication, all the analogues were tested against a larger panel of RNA and DNA
viruses. Among ssRNA+ viruses, a retrovirus (Human Immunodeficiency Virus type 1, HIV-1), and
two Picornaviruses (Coxsackie Virus type-5, CVB-5, and Poliovirus type-1, Sabin strain, Sb-1)
were also included. Among single-stranded, negative RNA (ssRNA-) viruses a Paramyxoviridae
(Respiratory Syncytial Virus, RSV) and a Rhabdoviridae (Vesicular Stomatitis Virus, VSV) were
considered. Among double-stranded RNA (dsRNA) viruses, a Reoviridae family member (Reo-1)
was selected. Finally, two representatives of DNA virus families were considered: Herpes Simplex
Virus type-1, HSV-1 (Herpesviridae) and Vaccinia Virus, VV (Poxviridae). 6-Azauridine,
ribavirin, efavirenz (EFV), pleconaril, acyclovir (ACG), and mycophenolic acid (M5255) were used
as reference inhibitors.
As far as the antiviral activity reported in Table 1, the majority of the new 1-3,5-triphenyl-
pyrazolines (4a-j, 5a-j, 6a-j) interfered with YFV replication in the low micromolar concentrations
(EC50 ranging from 1.8 µM to 2.6 µM) providing almost a 10-fold improvement in potency
compared to the reference inhibitor 6-azauridine (EC50 = 20.0 µM). However, the unsubstituted (4a-
j) and the phenoxy (5a-j) analogues were generally endowed with significant cytotoxicity against
BHK-21 cells resulting in compounds with modest selectivity indexes. On the contrary, the
benzyloxy derivatives (6a-j) showed lower cytotoxicity and higher selectivity indexes, as a
consequence. In particular, 6a (R1 = H) and the fluoro substituted derivatives 6b and 6d coupled
high potency and selectivity. In addition, the majority of the benzyloxy derivatives (6b, 6c, 6e-g, 6i,
6j) inhibited also the BVDV replication, generally showing higher activity and selectivity than the
reference compound ribavirin (EC50 = 20.0 µM, SI = 3).
All the analogues were inactive against the other two members of the Flavivirus genus
(WNV and DENV-2) utilized in the antiviral tests (Table 1).
When tested against HIV-1, Reo-1, Sb-1, VV, HSV-1, VSV, and RSV the compounds were
generally devoid of antiviral activity up to the highest concentration tested, although five phenoxy
derivatives (5a-e) and three benzyloxy analogues (6c, 6i, 6j) inhibited CVB-5 replication with EC50

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ranging from 3.5 µM to 13.0 µM (Table 2), while five compounds interfered with HIV-1 replication
showing EC50 ≥ 10 µM and modest selectivity (Table 2).
2.3. Time of addition studies
1,3,5-Triphenyl-pyrazolines 6a and 6b were selected for time of addition experiments
because of their potencies against YFV (EC50 = 2.2 µM and 1.8 µM, respectively) and high
selectivity indexes (SI = 36.4 and 50.0, respectively). Time of addition experiments were performed
to determine the possible step(s) in YFV replication cycle that is inhibited by analogues 6a and 6b.
For this purpose, the test medium containing approximately 10 times higher than the EC50 of
selected compounds 6a and 6b concentration (20 µM) or 4 times higher than the EC50 of 6-
azauridine concentration (90 µM), used as reference inhibitor, were added for two hours before the
infection (pre-treatment), during the two hours of infection of BHK-21 cell cultures with YFV and
every two hours post infection (p.i.) from time 0 to 10 hrs p.i (0 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10
h). 6-Azauridine is a nucleoside analog inhibitor of the orotidine-5’-phosphate decarboxylase
(OMP-decarboxylase). This enzyme is essential in the biosynthesis of the pyrimidine nucleotides, as
it converts the orotidine monophosphate (OMP) in uridine monophosphate (UMP). [10]
After each incubation period, the monolayers were washed two times with maintenance
medium and incubated with fresh medium until 10 hrs post-infection. Then, after 24–36 hrs post-
infection the cytopathogenic effect (CPE) was evaluated and the monolayers were collected,
centrifuged and frozen. The viral titre was determined by a plaque reduction assay.
Data represented in Figures 2 and 3 indicate that both pyrazolines 6a and 6b retained their
inhibitory activity when added in the pretreatment and during the infection, before losing their
antiviral efficacy. Indeed, the virus titre decreases when the compound is added to cells during
pretreatment or during two hours of infection, while the viral titre is very similar to that of the
untreated control when the compound is added in the subsequent phases of the infection. A similar
behavior was observed for the reference inhibitor, at higher concentrations. The comparison of the
curves obtained for our compounds and for the reference inhibitor could suggest a similar
mechanism of action. Like 6-arauridine, both pyrazolines 6a and 6b could act by blocking the entry
of the virus into the cell or interfere with some mechanism during the early stages of the infection.
However, further investigations are necessary for the identification of the anti-YFV target of these
pyrazoline derivatives.

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Time of Addition of 6a against Yellow Fever Virus (YFV)
1,0E+07
1,0E+06
1,0E+05
1,0E+04
1,0E+03
1,0E+02
Untreated control
6-Azauridine (90 µM)
6a (20 µM)
Time (hrs)
Figure 2. Effect of time of addition on anti-YFV activity of derivative 6a (blue). For comparison,
the same test was performed using the reference compound 6-azauridine (black). In red we observe
the untreated control.
Time of Addition of 6b against Yellow Fever Virus (YFV)
1,00E+07
1,00E+06
1,00E+05
Untreated control
6-Azauridine (90 µM)
6b (20 µM)
1,00E+04
1,00E+03
1,00E+02
Time (hrs)

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Figure 3. Effect of time of addition on anti-YFV activity of derivative 6b (blue). For comparison,
the same test was performed using the reference compound 6-azauridine (black). In red we observe
the untreated control.
3.
Conclusion
In conclusion, pursuing our research on antiviral compounds, in this paper we described the
design and synthesis of new series of 1,3,5-triphenyl-4,5-dihydropyrazole derivatives that were
endowed with a noticeable and generally specific anti-YFV activity. In particular, analogues
bearing a bulky 4-benzyloxy substituent on the phenyl ring at the 3 position of pyrazoline moiety,
coupled potency in the low micromolar range with low cytotoxicity resulting in selective
compounds.
4. Experimental section
4.1. Chemistry
Chemicals were purchased from Sigma-Aldrich or AlfaAesar and used without further
purification. Melting points were determined on a Stuar Scientific SMP1 apparatus and are
1
13
uncorrected. H NMR and C NMR spectra were recorded on a Bruker AM-400 spectrometer in
CDCl3 or DMSO-d6, and chemical shifts were reported in ppm (δ). All compounds were routinely
checked by thin-layer chromatography (TLC). TLC was performed on silica gel or aluminum oxide
fluorescent coated plates (Fluka, DC-Alufolien Kieselgel or aluminum oxide F254).
4.1.1. General procedure for the synthesis of chalcones (1a-j, 2a-j and 3a-j).
Barium hydroxide octahydrate (10 mmol) was added to a solution of the suitable
acetophenone (10 mmol) and substituted benzaldehyde (12 mmol) in ethanol (150 mL). The
reaction mixture was stirred overnight at 30°C; then water was added and the mixture was acidified
with 2N HCl, until precipitation was observed. The precipitate was collected by filtration, washed
with water and crystallized. Spectroscopic data of chalcones 1a-j, 2a-j and 3a-j are identical to
those previously reported. [11-16]
4.1.2. General procedure for the synthesis of 4-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamides (4a-j, 5a-j, 6a-j).
A mixture of the appropriate chalcone 1a-j, 2a-j and 3a-j (10 mmol) and 4-
hydrazinylbenzenesulfonamide hydrochloride (20 mmol) in dry ethanol (70 mL) and potassium
hydroxide (20 mmol) was refluxed for 24h with stirring. After cooling, the mixture was poured into

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crushed ice. The precipitate was filtered, washed with water and dried. The crude solid was purified
by crystallization from suitable solvent.
4.1.2.1. 4-(3,5-Diphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4a). Yield:
1
69%, m.p. = 212 – 214 °C from EtOH. H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.78 (d, J = 8.5
Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.48 - 7.30 (m, 5H), 7.29 - 7.20 (m, 3H), 7.11 – 7.03 (m, 4H),
5.63 (dd, 1H, CH, J = 12.0, Hz, J = 5.1 Hz), 3.97 (dd, 1H, CH2, Jgem = 17.7 Hz, J = 12.2 Hz), 3.16
(dd, 1H, CH2, Jgem = 17.7 Hz, J = 5.1 Hz). 13C NMR (DMSO-d6, 100 MHz): δ (ppm) 150.2, 146.3,
142.0, 133.3, 132.2, 129.9, 129.6, 129.2, 128.2, 127.6, 126.5, 126.1, 112.5, 62.7, 43.4. MS-ESI:
m/z 378 (M + H+).
4.1.2.2.
4-(5-(2-Fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
(4b). Yield: 27%, m.p. = 260 – 262 °C from MeOH. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.79
(dd, 2H, J = 7.9 Hz, J = 1.3 Hz); 7.61 (d, 2H, J = 9.0), 7.48 - 7.21 (m, 5H), 7.18 – 6.90- (m, 6H),
5.80 (dd, 1H, J = 12.3 Hz, J = 5.1 Hz); 4.00 (dd, 1H, Jgem = 17.7, J = 12.3 Hz); 3.24 (dd, 1H, Jgem
=
17.7, J = 5.1 Hz).13C NMR (DMSO-d6, 100 MHz): δ (ppm) 160.0 (d, J = 244 Hz), 150.5, 146.1,
133.6, 132.0, 130.4 (d, J = 8 Hz), 129.9, 129.2, 128.2 (d, J = 14 Hz), 128.1, 127.7, 126.6, 125.4 (d,
J = 4 Hz), 116.6 (d, J = 20 Hz), 112.3, 57.4, 42.1. MS-ESI: m/z 396 (M + H+).
4.1.2.3. 4-(5-(3-fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4c).
Yield: 20%, m.p. = 158 – 160 °C from C6H6. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.78 (d, 2H,
J = 6.8 Hz); 7.61 (d, 2H, J = 8.8 Hz); 7.50-7.34 (m, 5H), 7.14-7.03 (m, 6H), 5.66 (dd, 1H, J = 12.0
Hz, J = 4.95 Hz), 3.97 (dd, 1H, Jgem = 17.7 Hz, J = 12.2 Hz), 3.21 (dd, Jgem = 17.7 Hz, J = 5.0 Hz,
1H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm) 163.9 (d, J = 244 Hz), 150.3, 146.2, 144.9, (d, J = 7
Hz), 133.6, 132.0, 131.8 (d, J = 8 Hz), 129.9, 129.2, 127.7, 126.6, 122.1, 115.0 (d, J = 21 Hz),
113.2 (d, J = 22 Hz), 112.5, 62.3, 43.2. MS-ESI: m/z 396 (M + H+).
4.1.2.4.
4-(5-(4-Fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
(4d). Yield: 77%, m.p. = 213 – 218 °C from MeOH. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.78
(d, 2H, J = 8.4 Hz); 7.59 (d, J = 8.9 Hz, 2H), 7.47-7.38 (m, 3H); 7.32-7.26 (m, 2H); 7.16 (t, J = 8.9
Hz, 2H); 7.10-7.05 (m, 4H); 5.65 (dd, J = 12.0 Hz, 5.07 Hz, 1H), 3.96 (dd, Jgem = 17.8 Hz, J = 12.1
Hz, 1H); 3.17 (dd, Jgem = 17.8, J = 5.1 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm) 161.9 (d,

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J = 242 Hz), 150.1, 146.3, 138.2 (d, J = 3 Hz), 133.5, 132.1, 129.9, 129.2, 128.3 (d, J = 8 Hz),
127.6, 126.5, 116.4 (d, J = 21 Hz), 112.5, 62.1, 43.3. MS-ESI: m/z 396 (M + H+).
4.1.2.5. 4-(5-(2-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4e).
Yield: 22%, m.p. = 228 – 231 °C from MeOH. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.78 (dd, J
= 8.0 Hz, J = 1.6 Hz, 2H); 7.62 (d, J = 8.9 Hz, 2H); 7.13-7.03 (m, 5H); 7.03-6.97 (m, 6H); 5.91 (dd,
J = 12.2 Hz, J = 5.1 Hz, 1H) 4.16-3.88 (dd, Jgem = 17.8 Hz, J = 12.2 Hz, 1H); 3.24-2.98 (dd, Jgem
=
13
17.8 Hz, J = 5.1 Hz, 1H). C NMR (DMSO-d6, 100 MHz): δ (ppm) 150.4, 146.0, 138.3, 133.6,
132.2, 131.9, 131.7, 130.7, 130.04, 129.9, 129.2, 128.9, 128.8, 128.4, 112.2, 60.43, 42.1. MS-ESI:
m/z 412 (M + H+).
4.1.2.6. 4-(5-(3-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4f).
Yield: 32%, m.p. = 120 – 123 °C from C6H6. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.78 (d, J =
8.0 Hz, 2H), 7.61 (d, J = 8.9 Hz, 2H); 7.48-7.30 (m, 6H); 7.18 (d, J = 7.5 Hz, 1H); 7.11-7.05 (m, 4H);
5.66 (dd, J = 12.2 Hz, J = 5.1 Hz, 1H); 3.96 (dd, Jgem = 17.8 Hz, J = 12.2 Hz, 1H), 3.20 (dd, Jgem
=
17.8 Hz, J = 5.1 Hz, 1H).13C NMR (DMSO-d6, 100 MHz): δ (ppm) 150.3, 146.2, 144.5, 134.1, 133.6,
132.0, 131.6, 130.0, 129.2, 128.2, 127.7, 126.6, 126.1, 124.8, 112.5, 62.2, 43.2. MS-ESI: m/z 412
(M + H+).
4.1.2.7. 4-(5-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4g).
Yield: 37%, m.p. = 203 – 205 °C from C6H6. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.79 (d, J =
6.7 Hz, 2H); 7.61 (d, J = 8.8 Hz, 2H); 7.49-7.39 (m, 5H); 7.28 (d, J = 8.4 Hz, 2H); 7.11-7.02 (m,
4H); 5.68 (dd, J = 12.0 Hz, J = 5.0 Hz, 1H); 3.98 (dd, Jgem = 17.7 Hz, J = 12.1 Hz, 1H); 3.21 (dd,
13
Jgem = 17.7 Hz, J = 5.0 Hz, 1H). C NMR (DMSO-d6, 100 MHz): δ (ppm) 150.2, 146.2, 141.0,
133.7, 132.6, 132.1, 129.9, 129.6, 129.2, 128.2, 127.7, 126.5, 112.5, 62.1, 43.2. MS-ESI: m/z 412
(M + H+).
4.1.2.8. 4-(3-Phenyl-5-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4h). Yield:
23%, m.p. = 204 – 206 °C from C6H6. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.79 (d, J = 7.0 Hz,
2H); 7.60 (d, J = 8.9 Hz, 2H); 7.49-7.35 (m, 4H); 7.22 (t, J = 7.6 Hz, 1H); 7.12-7.01 (m, 6H); 5.58
(dd, J = 12.1 Hz, J = 5.3 Hz, 1H); 3.97 (dd, Jgem = 17.7 Hz, J = 12.2 Hz, 1H); 3.17 (dd, Jgem = 17.7
13
Hz, J = 5.3 Hz, 1H); 2.26 (s, 3H). C NMR (DMSO-d6, 100 MHz): δ (ppm) 150.1, 146.4, 142.2,

ACCEPTED MANUSCRIPT
138.8, 133.5, 132.2, 129.7, 129.5, 129.2, 128.8, 127.6, 126.6, 126.5, 123.2, 112.4, 62.9, 43.5, 21.6.
MS-ESI: m/z 392 (M + H+).
4.1.2.9. 4-(3-Phenyl-5-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4i). Yield:
27%, m.p. = 200 – 203 °C from C6H6. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.78 (d, J = 7.2 Hz,
2H); 7.60 (d, J = 8.9 Hz, 2H); 7.49 – 7.25 (m, 6H); 7.15 – 7.00 (m, 5H); 5.59 (dd, J = 12.1 Hz, J =
5.3 Hz, 1H); 3.95 (dd, Jgem = 17.7 Hz, J = 12.2 Hz, 1H); 3.20 (dd, Jgem = 17.5 Hz, J = 5.3 Hz, 1H);
13
2.24 (s, 3H). C NMR (DMSO-d6, 100 MHz): δ (ppm) 150.1, 146.4, 140.6, 141.2, 133.5, 132.3,
129.8, 129.7, 129.2, 127.6, 126.5, 125.4, 123.9, 122.8, 112.5, 63.0, 43.4, 21.1. MS-ESI: m/z 392 (M
+ H+).
4.1.2.10. 4-(3-Phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4j). Yield:
27%, m.p. = 219 – 223 °C from C6H6. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.79 (d, J = 7.2 Hz,
2H); 7.59 (d, J = 8.8 Hz, 2H); 7.48-7.38 (m, 3H); 7.17-7.11 (m, 4H); 7.11-7.01 (m, 4H); 5.60 (dd, J
= 11.9 Hz, J = 4.9 Hz, 1H); 3.95 (dd, Jgem = 17.7 Hz, J = 12.2 Hz, 1H); 3.16 (dd, Jgem = 17.7, J = 4.9
Hz, 1H); 2.24 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm) 150.1, 146.3, 139.1, 137.3, 133.5,
132.3, 130.1, 129.8, 129.2, 127.6, 126.5, 126.2, 112.5, 62.6, 43.4, 21.1. MS-ESI: m/z 392 (M + H+).
4.1.2.11. 4-(3-(4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
1
(5a). Yield: 36%, m.p. = 175 – 178 °C from EtOH. H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.79
(d, J = 8.8 Hz, 2H); 7.58 (d, J = 8.9 Hz, 2H); 7.45-7.38 (m, 2H); 7.34 (t, J = 7.1 Hz, 2H); 7.28 –
7.22 (m, 3H); 7.18 (t, J = 7.4 Hz, 1H); 7.08-7.01 (m, 8H); 5.61 (dd, J = 12.0 Hz, J = 5.1 Hz, 1H);
13
3.95 (dd, Jgem = 17.7 Hz, J = 12.0 Hz; 1H); 3.14 (dd, Jgem = 17.7 Hz, J = 5.0 Hz, 1H). C NMR
(DMSO-d6, 100 MHz): δ (ppm) 158.2, 156.5, 149.7, 146.4, 142.0, 133.2, 130.7, 129.6, 128.5,
128.1, 127.6, 127.3, 126.2, 124.5, 119.5, 118.9, 112.4, 62.8, 43.5.. MS-ESI: m/z 470 (M + H+).
4.1.2.12.
4-(5-(2-fluorophenyl)-3-(4-phenoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
1
yl)benzenesulfonamide (5b). Yield: 25%, m.p. = 208 – 212 °C from C6H6. H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.80 (d, J = 8.8 Hz, 2H); 7.60 (d, J = 9.0 Hz, 2H); 7.45 – 7.39 (m, 2H); 7.37 -
7.30 (m, 1H); 7.29-7.22 (m, 1H); 7.21-7.16 (m, 1H); 7.15-7.10 (m, 2H); 7.09-7.02 (m, 8H); 5.78
(dd, J = 12.3 Hz, J = 5.1 Hz, 1H), 3.98 (dd, Jgem = 17.6 Hz, J = 12.2 Hz, 1H), 3.22 (dd, Jgem = 17.6
Hz, J = 5.0 Hz, 1H,). 13C NMR (DMSO-d6, 100 MHz): δ (ppm) 160.0 (d, J = 244 Hz), 158.3, 156.4,

ACCEPTED MANUSCRIPT
150.0, 146.2, 133.4, 130.7, 130.4 (d, J = 8 Hz), 128.5, 128.1 (d, J = 3 Hz), 128.2 (d, J = 20 Hz),
127.7, 127.2, 125.4 (d, J = 4 Hz), 124.5, 119.5, 118.9, 116.6 (d, J = 21 Hz), 112.2, 57.4, 42.3. MS-
ESI: m/z 488 (M + H+).
4.1.2.13.
4-(5-(3-fluorophenyl)-3-(4-phenoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (5c). Yield: 54%, m.p. = 180 – 182 °C from MeOH. 1H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.79 (d, J = 8.8 Hz, 2H); 7.60 (d, J = 8.9 Hz, 2H); 7.45 - 7.35 (m, 3H); 7.18 (t, J
= 7.6 Hz, 1H); 7.11-7.02 (m, 11H); 5.65 (dd, J = 12.0 Hz, J = 4.9 Hz, 1H), 3.95 (dd, Jgem = 17.7 Hz,
12.1 Hz, 1H), 3.18 (dd, Jgem = 17.7 Hz, J = 5.0 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm)
162.9 (d, J = 244 Hz), 158.3, 156.4, 149.8, 146.3, 144.9 (d, J = 6 Hz), 133.4, 131.8 (d, J = 8 Hz),
130.7, 128.5, 127.7, 127.2, 124.5, 122.2, 119.5, 118.9, 115.0 (d, J = 21 Hz), 113.2 (d, J = 22 Hz),
112.4, 62.2, 43.3. MS-ESI: m/z 488 (M + H+).
4.1.2.14.
4-(5-(4-fluorophenyl)-3-(4-phenoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (5d). Yield: 56%, m.p. = 184 – 187 °C from MeOH. 1H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.79 (d, J = 8.8, 2H); 7.59 (d, J = 9.0, 2H); 7.45 – 7.39 (m, 2H); 7.31 - 7.25 (m,
2H); 7.21 – 7.13 (m, 3H); 7.08-7.02 (m, 8H); 5.63 (dd, J = 12.0 Hz, J = 4.9 Hz, 1H), 3.94 (dd, Jgem
=
13
17.7 Hz, J = 12.0 Hz, 1H), 3.15 (dd, Jgem = 17.7 Hz, J = 5.0 Hz, 1H). C NMR (DMSO-d6, 100
MHz): δ (ppm) 161.9 (d, J = 243 Hz), 158.3, 156.4, 149.7, 146.3, 138.2 (d, J = 3 Hz), 133.3, 130.7,
128.5, 128.3 (d, J = 8 Hz), 127.6, 127.2, 124.5, 119.5, 118.9, 116.4 (d, J = 21 Hz), 112.4, 62.1,
43.5. MS-ESI: m/z 488 (M + H+).
4.1.2.15.
4-(5-(2-chlorophenyl)-3-(4-phenoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
1
yl)benzenesulfonamide (5e). Yield: 27% m.p. = 131 – 135 °C from MeOH. H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.79 (d, J = 8.7 Hz, 2H); 7.61 (d, J = 8.9 Hz, 2H); 7.56 (d, J = 7.6 Hz, 1H); 7.42
(t, J = 7.9 Hz, 2H); 7.32 (dt, J = 8.0 Hz, J = 1.6 Hz, 1H); 7.25 (t, J = 7.3 Hz, 1H); 7.16 (t, J = 7.4
Hz, 1H); 7.11 – 6.93 (m, 9H); 5.80 (dd, J = 12.1 Hz, J = 5.1 Hz, 1H), 4.04 (dd, Jgem = 17.7 Hz, J =
12.2 Hz, 1H), 3.13 (dd, Jgem = 17.7 Hz, 5.1 Hz, 1H).13C NMR (DMSO-d6, 100 MHz): δ (ppm) 158.4,
156.4, 150.0, 146.1, 138.3, 133.5, 131.7, 130.7, 130.0, 128.6, 128.4, 127.8, 127.1, 124.5, 119.6, 118.9,
112.1, 60.4, 42.2. MS-ESI: m/z 504 (M + H+).

ACCEPTED MANUSCRIPT
4.1.2.16.
4-(5-(3-chlorophenyl)-3-(4-phenoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
1
yl)benzenesulfonamide (5f). Yield: 60%, m.p. = 218 – 220 °C from MeOH. H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.79 (d, J = 8.7 Hz, 2H); 7.60 (d, J = 8.9 Hz, 2H); 7.45 – 7.29 (m, 5H); 7.19 (t,
J = 7.1 Hz, 2H); 7.10 - 7.04 (m, 8H); 5.65 (dd, J = 12.0 Hz, J = 4.9 Hz, 1H), 3.95 (dd, Jgem = 17.8
13
Hz, J = 12.0 Hz, 1H), 3.19 (dd, Jgem = 17.8, J = 4.9 Hz, 1H). C NMR (DMSO-d6, 100 MHz): δ
(ppm) 158.3, 156.4, 149.8, 146.2, 144.5, 134.1, 133.5, 131.6, 130.7, 128.6, 128.2, 127.7, 127.2,
126.1, 124.8, 124.5, 119.5, 118.9, 112.4, 62.1, 43.3. MS-ESI: m/z 504 (M + H+).
4.1.2.17.
4-(5-(4-chlorophenyl)-3-(4-phenoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (5g). Yield: 60%, m.p. = 105 – 107 °C from EtOH. 1H NMR (CDCl3, 400
MHz): δ (ppm) 7.60-7.58 (m, 4H); 7.33 - 7.20 (m, 4H); 7.14-7.04 (m, 3H); 7.00-6.90 (m, 6H); 5.24
(dd, J = 12.1 Hz, J = 5.8 Hz, 1H); 4.28 - 3.90 (bs, 2H); 3.79 (dd, Jgem = 17.3 Hz, J = 12.2 Hz, 1H);
3.06 (dd, Jgem = 17.4 Hz, J = 5.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz): δ (ppm) 158.8, 156.4, 149.0,
147.0, 139.7, 133.8, 130.6, 129.9, 129.6, 128.0, 127.8, 127.1, 126.6, 124.0, 119.4, 118.6, 112.5, 62.9,
43.6. MS-ESI: m/z 504 (M + H+).
4.1.2.18.
4-(3-(4-phenoxyphenyl)-5-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
(5h). Yield: 28%, m.p. = 200 – 204 °C from EtOH. 1H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.80 (d, J
= 8.7 Hz, 2H); 7.60 (d, J = 8.8 Hz, 2H); 7.42 (t, J = 7.8 Hz, 2H); 7.28 (d, J = 7.3 Hz, 1H); 7.21 -
7.13 (m, 2H); 7.11 -7.01 (m, 7H); 6.98 (d, J = 8.8 Hz, 2H); 6.84 (d, J = 7.4 Hz, 1H); 5.70 (dd, J =
12.0 Hz, J = 5.1 Hz, 1H); 4.00 (dd, Jgem = 17.4 Hz, 12.2 Hz, 1H); 3.06 (dd, Jgem= 17.6, J = 4.9 Hz,
1H); 2.47 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm) 158.2, 156.5, 149.8, 146.3, 139.3, 136.2,
135.1, 133.3, 131.6, 130.7, 128.4, 127.9, 127.7, 127.4, 126.8, 124.5, 119.5, 118.9, 112.2, 60.5, 42.2,
19.5. MS-ESI: m/z 484 (M + H+).
4.1.2.19. 4-(3-(4-phenoxyphenyl)-5-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
1
(5i). Yield: 37%, m.p. = 159 – 163 °C from MeOH. H NMR (CDCl3, 400 MHz): δ (ppm) 7.66 -
7.57 (m, 4H); 7.32 - 7.25 (m, 2H); 7.14 (t, J = 7.7 Hz, 1H); 7.07 (t, J = 7.4 Hz, 1H); 7.03 - 6.91 (m,
9H); 5.21 (dd, J = 12.2 Hz, J = 5.9 Hz, 1H); 4.35 -3.87 (bs, 2H); 3.78 (dd, Jgem = 17.3 Hz, J = 12.2
13
Hz, 1H), 3.09 (dd, Jgem = 17.4 Hz, J = 6.0 Hz, 1H); 2.24 (s, 3H). C NMR (CDCl3, 100 MHz): δ
(ppm) 158.6, 156.5, 149.0, 147.4, 141.3, 139.3, 130.2, 129.9, 129.3, 128.8, 128.0, 127.7, 126.9,
126.1, 123.9, 122.7, 119.4, 118.6, 112.4, 63.6, 43.8, 21.5. MS-ESI: m/z 484 (M + H+).

ACCEPTED MANUSCRIPT
4.1.2.20. 4-(3-(4-phenoxyphenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
(5j). Yield: 60%, m.p. = 131 – 135 °C from MeOH. 1H NMR (CDCl3, 400 MHz): δ (ppm) 7.70 (d,
J = 8.9 Hz, 2H); 7.72 (d, J = 8.8 Hz, 2H); 7.39 (t, J = 7.9 Hz, 2H); 7.21 - 7.14 (m, 5H); 7.10 - 7.01
(m, 6H); 5.34 (dd, J = 12.1 Hz, J = 5.8 Hz, 1H); 4.94 - 4.50 (bs, 2H); 3.88 (dd, Jgem = 17.2 Hz, J =
12.2 Hz, 1H), 3.19 (dd, Jgem = 17.2 Hz, J = 5.8 Hz, 1H,); 2.34 (s, 3H). 13C NMR (CDCl3, 100
MHz): δ (ppm) 158.6, 156.5, 149.1, 147.4, 138.2, 137.8, 130.1, 129.9, 128.0, 127.7, 127.4, 126.9,
125.6, 123.9, 119.4, 118.6, 112.4, 63.3, 43.8, 21.1. MS-ESI: m/z 484 (M + H+).
4.1.2.21. 4-(3-(4-(benzyloxy)phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
1
(6a). Yield: 39%, m.p. = 169 – 172 °C from C6H6. H NMR (DMSO-d6, 400 MHz): δ (ppm) 7.71
(d, J = 8.7 Hz, 2H); 7.57 (d, J = 8.8 Hz, 2H); 7.48 - 7.29 (m, 7H); 7.28 - 7.20 (m, 3H); 7.11 - 7.00
(m, 6H); 5.56 (dd, J = 11.9 Hz, J = 4.8 Hz, 1H); 5.14 (s, 2H); 3.91 (dd, Jgem = 17.6 Hz, J = 12.0 Hz,
13
1H); 3.11 (dd, Jgem = 17.6 Hz, J = 4.9 Hz, 1H). C NMR (DMSO-d6, 100 MHz): δ (ppm) 159.8,
150.1, 146.5, 142.1, 137.2, 132.9, 129.6, 128.9, 128.4, 128.2, 128.1, 127.6, 126.2, 124.9, 115.6,
112.2, 69.8, 62.6, 43.6. MS-ESI: m/z 484 (M + H+).
4.1.2.22.
4-(3-(4-(benzyloxy)phenyl)-5-(2-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (6b). Yield: 25%, m.p. = 188 – 192 °C from MeOH. 1H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.75 (d, J = 8.71 Hz, 2H); 7.60 (d, J = 8.79 Hz, 2H); 7.50-7.21 (m, 8H); 7.17-
6.93 (m, 7H); 5.61 (dd, J = 11.90 Hz, J = 4.83 Hz, 1H); 5.15 (s, 2H); 3.89 (dd, Jgem = 17.6 Hz, J =
11.98 Hz, 1H); 3.12 (dd, Jgem = 17.6 Hz, J = 4.89 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz): δ
(ppm) 159.8, 159.7 (d, J = 245 Hz), 150.2, 146.5, 137.2, 133.1, 130.3 (d, J = 10 Hz), 128.9, 128.4,
128.3 (d, J = 14 Hz), 128.2, 128.1, 127.9, 127.6, 125.4 (d, J = 4 Hz), 124.4, 116.7 (d, J = 21 Hz),
115.6, 112.3, 69.8, 61.8, 44.9. MS-ESI: m/z 502 (M + H+).
4.1.2.23.
4-(3-(4-(benzyloxy)phenyl)-5-(3-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-
1
yl)benzenesulfonamide (6c). Yield: 60%, m.p. = 170 – 173 °C from C6H6. H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.72 (d, J = 8.7 Hz, 2H); 7.59 (d, J = 8.8 Hz, 2H); 7.45 (d, J = 7.1 Hz, 2H); 7.42
- 7.30 (m, 4H); 7.12 - 7.00 (m, 9H); 5.61 (dd, J = 11.9 Hz, J = 4.9 Hz, 1H); 5.15 (s, 2H); 3.92 (dd,
Jgem = 17.7 Hz, J = 12.1 Hz, 1H); 3.16 (dd, Jgem = 17.7 Hz, J = 4.8 Hz, 1H).13C NMR (DMSO-d6,
100 MHz): δ (ppm) 162.8 (d, J = 243 Hz), 159.8, 150.2, 146.4, 145.0 (d, J = 6 Hz), 137.2, 133.2,

ACCEPTED MANUSCRIPT
131.8 (d, J = 8 Hz), 128.9, 128.4, 128.2, 128.1, 127.7, 124.8, 122.2, 115.6, 114.9 (d, J = 20 Hz),
113.2 (d, J = 22 Hz), 112.3, 69.8, 62.1, 43.4. MS-ESI: m/z 502 (M + H+).
4.1.2.24.
4-(3-(4-(benzyloxy)phenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (6d). Yield: 45%, m.p. = 136 – 140 °C from MeOH. 1H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.72 (d, J = 8.8 Hz, 2H); 7.58 (d, J = 9.0 Hz, 2H); 7.45 (d, J = 7.0 Hz, 2H); 7.42
- 7.24 (m, 5 H); 7.15 (t, J = 8.8 Hz, 2H); 7.10 - 7.01 (m, 6H); 5.59 (dd, J = 12.0 Hz, J = 5.0 Hz,
1H); 5.15 (s, 2H); 3.91 (dd, Jgem = 17.7 Hz, J = 12.0 Hz, 1H); 3.12 (dd, Jgem = 17.7 Hz, J = 4.9 Hz,
1H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm) 161.9 (d, J = 242 Hz), 159.8, 150.1, 146.4, 138.3 (d,
J = 2 Hz), 137.2, 133.0, 128.9, 128.4, 128.3 (d, J = 8 Hz), 128.2, 128.1, 127.6, 124.9, 116.3 (d, J =
21 Hz), 115.6, 112.3, 69.8, 61.9, 43.5. MS-ESI: m/z 502 (M + H+).
4.1.2.25.
4-(3-(4-(benzyloxy)phenyl)-5-(2-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
1
yl)benzenesulfonamide (6e). Yield: 52%, m.p. = 184 – 188 °C from C6H6. H NMR (DMSO-d6,
400 MHz): δ (ppm) ) 7.71 (d, J = 8.8 Hz, 2H); 7.61 (d, J = 8.9 Hz, 2H); 7.55 (d, J = 7.9 Hz, 1H);
7.44 (d, J = 6.8 Hz, 2H); 7.41 - 7.28 (m, 5H); 7.24 (t, J = 7.2 Hz, 1H); 7.09 - 7.04 (m, 4 H); 7.01 -
6.92 (m, 2H); 5.75 (dd, J = 12.1 Hz, J = 5.1 Hz, 1H); 5.14 (s, 2H); 4.00 (dd, Jgem = 17.6 Hz, J =
12.1 Hz, 1H); 3.10 (dd, Jgem = 17.6 Hz, J = 5.1 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm)
159.8, 150.3, 146.2, 138.4, 137.2, 133.3, 131.7, 130.7, 130.0, 128.9, 128.80, 128.79, 128.4, 128.2,
128.1, 127.8, 124.7, 115.6, 112.0, 69.7, 60.3, 42.3. MS-ESI: m/z 518 (M + H+).
4.1.2.26.
4-(3-(4-(benzyloxy)phenyl)-5-(3-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
1
yl)benzenesulfonamide (6f). Yield: 63%, m.p. = 134 – 136 °C from C6H6. H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.71 (d, J = 8.8 Hz, 2H); 7.59 (d, J = 8.9 Hz, 2H); 7.45 (d, J = 7.1 Hz, 2H);
7.41 - 7.28 (m, 6 H); 7.17 (d, J = 7.5 Hz, 1H) 7.10 - 7.01 (m, 6H); 5.60 (dd, J = 11.9 Hz, J = 4.9 Hz,
1H); 5.15 (s, 2H); 3.91 (dd, Jgem = 17.7 Hz, J = 12.0 Hz, 1H); 3.16 (dd, Jgem = 17.7 Hz, J = 4.8 Hz,
1H). 13C NMR (DMSO-d6, 100 MHz): δ (ppm) 159.8, 150.2, 146.3, 144.6, 137.2, 134.0, 133.2,
131.6, 129.0, 128.4, 128.3, 128.2, 127.7, 126.1, 124.8, 124.7, 115.6, 112.3, 69.7, 62.0, 43.4. MS-
ESI: m/z 518 (M + H+).
4.1.2.27.
4-(3-(4-(benzyloxy)phenyl)-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (6g). Yield: 40%, m.p. = 98 – 102 °C from C6H6. 1H NMR (DMSO-d6): δ
(ppm) 7.71 (d, J = 8.8 Hz, 2H); 7.57 (d, J = 9.0 Hz, 2H); 7.45 (d, J = 7.0 Hz, 2H); 7.42 - 7.30 (m,

ACCEPTED MANUSCRIPT
5H); 7.25 (d, J = 8.6 Hz, 2H); 7.10 - 7.00 (m, 6 H); 5.60 (dd, J = 12.0 Hz, J = 5.0 Hz, 1H); 5.15 (s,
13
2H); 3.91 (dd, Jgem = 17.6 Hz, J = 12.0 Hz, 1H); 3.13 (dd, Jgem = 17.6 Hz, J = 5.0 Hz, 1H). C
NMR (DMSO-d6, 100 MHz): δ (ppm) 159.8, 150.2, 146.3, 141.1, 137.2, 133.1, 132.6, 129.5,
128.9, 128.4, 128.21, 128.17, 127.6, 124.8, 115.6, 112.3, 69.8, 62.0, 43.4. MS-ESI: m/z 518 (M +
H+).
4.1.2.28. 4-(3-(4-(benzyloxy)phenyl)-5-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
1
(6h). Yield: 33%, m.p. = 230 – 234 °C from CH3CN. H NMR (DMSO-d6, 400 MHz): δ (ppm)
7.71 (d, J = 8.8 Hz, 2H); 7.58 (d, J = 9.0 Hz, 2H); 7.44 (d, J = 8.4 Hz, 2H); 7.41 - 7.30 (m, 3H);
7.26 (d, J = 7.5 Hz, 1H); 7.15 (t, J = 7.5 Hz, 1H); 7.09 - 7.02 ( m, 5H); 6.95 (d, J = 8.8 Hz, 2H);
6.82 (d, J = 6.4 Hz, 1H); 5.63 (dd, J = 12.1 Hz, J = 5.3 Hz, 1H) 5.13 (s, 2H); 3.96 (dd, Jgem = 17.5
Hz, J = 12.1 Hz, 1H); 3.00 (dd, Jgem = 17.5 Hz, J = 5.1 Hz, 1H); 2.44 (s, 3H). 13C NMR (DMSO-d6,
100 MHz): δ (ppm) 159.8, 150.0, 146.5, 139.2, 137.3, 133.1, 131.3, 130.1, 130.0, 129.0, 128.9,
128.2, 127.6, 126.2, 125.1, 115.6, 112.3, 69.8, 62.5, 43.6, 21.1. MS-ESI: m/z 498 (M + H+).
4.1.2.29.
4-(3-(4-(benzyloxy)phenyl)-5-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-
1
yl)benzenesulfonamide (6i). Yield: 29%, m.p. = 131 – 134 °C from EtOH. H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.71 (d, J = 8.7 Hz, 2H); 7.57 (d, J = 9.0 Hz, 2H); 7.47 - 7.29 (m 5H); 7.21 (t, J
= 7.7 Hz, 1H); 7.10 - 6.98 (m, 9H); 5.49 (dd, J = 12.0 Hz, J = 5.3 Hz, 1H); 5.14 (s, 2H); 3.90 (dd,
J
gem = 17.7 Hz, J = 12.0 Hz, 1H); 3.09 (dd, Jgem = 17.7 Hz, J = 5.2 Hz, 1H); 2.23 (s, 3H). 13C NMR
(DMSO-d6, 100 MHz): δ (ppm) 159.7, 150.0, 146.6, 142.3, 138.8, 137.2, 132.9, 129.5, 128.9,
128.8, 128.4, 128.3, 128.2, 127.6, 126.6, 124.9, 123.2, 115.6, 112.2, 69.8, 62.7, 43.6, 21.5. MS-ESI:
m/z 498 (M + H+).
4.1.2.30.
4-(3-(4-(benzyloxy)phenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (6j). Yield: 39%, m.p. = 135 – 137 °C from MeOH. 1H NMR (DMSO-d6,
400 MHz): δ (ppm) 7.73 (d, J = 8.7 Hz, 2H); 7.58 (d, J = 8.8 Hz, 2H); 7.49 - 7.37 (m, 5H); 7.15 -
7.00 (m, 10H); 5.54 (dd, J = 11.9 Hz, J = 5.0 Hz, 1H); 5.17 (s, 2H); 3.91 (dd, Jgem = 17.5 Hz, J =
13
12.0 Hz, 1H); 3.12 (dd, Jgem = 17.5 Hz, J = 5.0 Hz, 1H); 2.25 (s, 3H). C NMR (DMSO-d6, 100
MHz): δ (ppm) 159.8, 150.0, 146.5, 139.2, 137.3, 133.1, 131.3, 130.1, 130.0, 129.0, 128.9, 128.2,
127.6, 126.2, 125.1, 115.6, 112.3, 69.8, 62.5, 43.6, 21.1. MS-ESI: m/z 498 (M + H+).
4.2. Biology

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4.2.1. Test compounds
Compounds were solubilized in DMSO at a concentration of 100 mM, and subsequently
subjected to a serial dilutions in the culture medium. The final concentrations of test compound,
diluted with ratio 1: 5, are: 100, 20, 4 and 0.8 µM. The first dilution, with ratio 1:50, diluted the
compounds from 100 mM to a concentration of 2 mM, reducing the percentage of DMSO from
100% to 2%. The second dilution, with ratio 1:20, leads the compounds from 2 mM to a
concentration of 100 µM and reduces the concentration of DMSO from 2% to 0.1%, a non-toxic
concentration for cells. Next dilutions, performed with ratio 1: 5, result in a further decrease in the
percentage of DMSO in contact with the cells.
6-Azauridine, 2′-C-methyl-guanosin (NM108), ribavirin, efavirenz (EFV), acyclovir (ACG),
pleconaril and mycophenolic acid (M5255) were employed as reference inhibitors and were
solubilized in DMSO at a concentration of 100 mM, and subsequently subjected to serial dilutions
in the culture medium. The final concentrations of 6-azauridine, ribavirin and ACG are: 100, 20, 4
and 0.8 µM. The final concentrations of NM108, EFV, pleconaril and M5255 diluted are: 100, 20,
4, 0.8, 0.16, 0.032, 0.0064 and 0.00128 µM.
4.2.2. Cells and Viruses
Cell lines were purchased from American Type Culture Collection (ATCC). The absence of
mycoplasma contamination was checked periodically by the Hoechst staining method. Cell lines
supporting the multiplication of RNA and DNA viruses were the following: CD4+ human T-cells
containing an integrated HTLV-1 genome (MT-4); Madin Darby Bovine Kidney (MDBK) [ATCC
CCL22 (NBL-1) Bos Taurus], Baby Hamster Kidney (BHK-21) [ATCC CCL10 (C-13)
Mesocricetus auratus] and Monkey kidney (Vero 76) [ATCCCRL 1587 Cercopithecus Aethiops].
Viruses were purchased from American Type Culture Collection (ATCC), with the exception of
Yellow Fever Virus (YFV), Dengue virus type 2 (DENV-2), West Nile virus (WNV) and Human
Immunodeficiency Virus type-1 (HIV-1). Viruses representative of positive-sense single stranded
RNAs (ssRNA+) were: (i) Retroviridae: the IIIB laboratory strain of HIV-1, obtained from the
supernatant of the persistently infected H9/IIIB cells (NIH 1983); (ii) Flaviviridae: yellow fever
virus (YFV) [strain 17-D vaccine (Stamaril Pasteur J07B01)], Dengue virus type 2 (DENV-2)
[clinical isolate], West Nile virus (WNV) [clinical isolate] and bovine viral diarrhoea virus (BVDV)
[strain NADL (ATCC VR-534)]; (iii) Picornaviridae: human enterovirus B [coxsackie type B5
(CV-B5), strain Faulkner, (ATCC VR-185)], and human enterovirus C [poliovirus type-1 (Sb-1),
Sabin strain Chat (ATCC VR-1562)]. Viruses representative of negative-sense, single-stranded
RNAs (ssRNA-) were: (iv) Paramyxoviridae: human respiratory syncytial virus (RSV) [strain A2
(ATCC VR-1540)]; (v) Rhabdoviridae: vesicular stomatitis virus (VSV) [lab strain Indiana (ATCC

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VR 158)]. The virus representative of double-stranded RNAs (dsRNA) Reoviridae was reovirus
type-1 (Reo-1) [simian virus 12, strain 3651 (ATCC VR- 214)]. DNA virus representatives were:
(vi) Poxviridae: vaccinia virus (VV) [strain Elstree (Lister Vaccine) (ATCC VR-1549)]; and (vii)
Herpesviridae: human herpesvirus 1 (HSV-1) [strain KOS (ATCC VR- 1493)].
4.2.3. Cytotoxicity Assays
Cytotoxicity assays were run in parallel with antiviral assays. Exponentially growing MT-4
cells were seeded at an initial density of 1x105 cells/mL in 96-well plates in RPMI-1640 medium,
supplemented with 10% foetal bovine serum (FBS), 100 units/mL penicillin G and 100 mg/mL
streptomycin. Cell cultures were then incubated at 37 °C in a humidified, 5% CO2 atmosphere, in
the absence or presence of serial dilutions of test compounds. Cell viability was determined after 96
h at 37 °C by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method.
[17] MDBK and BHK cells were seeded in 96-well plates at an initial density of 6x105 and 1x106
cells/mL, respectively, in Minimum Essential Medium with Earle’s salts (MEM-E), L glutamine, 1
mM sodium pyruvate and 25 mg/L kanamycin, supplemented with 10% horse serum (MDBK) or
10% foetal bovine serum (FBS) (BHK). Cell cultures were then incubated at 37 °C in a humidified,
5% CO2 atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability
was determined after 72 hrs at 37 °C by the MTT method. Vero76 cells were seeded in 96-well
plates at an initial density of 5x105 cells/mL, in Dulbecco’s Modified Eagle Medium (D-MEM)
with L-glutamine and 25 mg/L kanamycin, supplemented with 10% FBS. Cell cultures were then
incubated at 37 °C in a humidified, 5% CO2 atmosphere in the absence or presence of serial
dilutions of test compounds. Cell viability was determined after 48-96 h at 37 °C by the MTT
method. [17]
4.2.4. Antiviral assays
Antiviral activity against HIV-1 was based on inhibition of virus-induced cytopathogenicity
in MT-4 cells acutely infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 µL of
RPMI containing 1x104 MT-4 cells were added to each well of flat bottom microtitre trays,
containing 50 µL of RPMI without or with serial dilutions of test compounds. Then, 20 µL of a
HIV-1 suspension containing 100 CCID50 were added. After 4-day of incubation at 37 °C, cell
viability was determined by the MTT method. [15] Antiviral activity against YFV, DENV-2, WNV
and Reo-1 was based on inhibition of virus-induced cytopathogenicity in BHK-21 cells acutely
infected with a m.o.i. of 0.01. Activity of compounds activity against BVDV was based on

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inhibition of virus-induced cytopathogenicity in MDBK cells acutely infected with a m.o.i. of 0.01.
Briefly, BHK and MDBK cells were seeded in 96-well plates at a density of 5x104 and 3x104
cells/well, respectively, and were allowed to form confluent monolayers by incubating overnight in
growth medium at 37 °C in a humidified CO2 (5%) atmosphere. Cell monolayers were then infected
with 50 µL of a proper virus dilution in maintenance medium [MEM-Earl with L-glutamine, 1 mM
sodium pyruvate and 0.025 g/L kanamycin, supplemented with 0.5% inactivated FBS] to give an
m.o.i of 0.01. After 2 hrs, 50 µL of maintenance medium, without or with serial dilutions of test
compounds, were added. After a 3-/4-day incubation at 37 °C, cell viability was determined by the
MTT method. [17]
Antiviral activity against CV-B5, Sb-1, VV, HSV-1, VSV and RSV was determined by
plaque reduction assays in infected cell monolayers. To this end, Vero 76-cells were seeded in 24-
well plates at a density of 2x105 cells/well and were allowed to form confluent monolayers by
incubating overnight in growth medium [Dulbecco’s Modified Eagle Medium (D-MEM) with L-
glutamine and 4500 mg/L D-glucose and 0.025 g/L kanamycin, supplemented with 10% FBS] at 37
°C in a humidified CO2 (5%) atmosphere. Then, monolayers were infected for 2 h with 250 µL of
proper virus dilutions to give 50 to 100 PFU/well. Following removal of unadsorbed virus, 500 µL
of maintenance medium [D-MEM with L-glutamine and 4500 mg/L Dglucose, supplemented with
1% inactivated FBS] containing 0.75% methylecellulose, without or with serial dilutions of test
compounds, were added. Cultures were incubated at 37 °C for 2 (Sb-1 and VSV), 3 (CVB-5, VV
and HSV-1) or 5 days (RSV) and then fixed with PBS containing 50% ethanol and 0.8% crystal
violet, washed and air-dried. Plaques were then counted.
4.2.5. Time of addition assay
A time-of-addition experiment was carried out with BHK-21 cells. The confluent
monolayers of BHK-21 cells, seed in 96-well tissue culture plates were inoculated at room
temperature with 50000 PFU of YFV, corresponding to a multiplicity of infection of 1 PFU/cell.
After adsorption for 60 min, the monolayers were washed two times with maintenance medium in
the presence of FBS inactivated and incubated with the same medium at 5% CO2 and 37 °C. The
test medium containing 10 × EC50 compound 6a and 6b concentration or 4 × EC50 6-azauridine
concentration was added before the infection (pre-treatment), during the two hours of infection of
BHK-21 cell cultures with YFV and every two hours post infection (p.i.) from time 0 to 10 hrs p.i
(0 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10).
After each incubation period, the monolayers were washed two times with maintenance
medium and incubated with fresh medium until 10 hrs post-infection. Then, after 24–36 hrs post-

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infection the CPE was evaluated and the monolayers were collected, centrifuged and frozen at -80
°C. The viral titre was determined by a plaque reduction assay.
4.2.6. Linear regression analysis
The extent of cell growth/viability and viral multiplication, at each drug concentration
tested, were expressed as percentage of untreated controls. Concentrations resulting in 50%
inhibition (CC50 or EC50) were determined by linear regression analysis.
Acknowledgments
Financial support from Italian Fondazione Banco di Sardegna and Progetti di Ateneo 2014
n. C 26A1429N Università ‘La Sapienza’ is gratefully acknowledged.
References
1. E.A. Gould, T. Soloman, Pathogenic flaviviruses, Lancet. 371(2008) 500–509.
2. N. Tautz, B.A. Tews, G. Meyers, The molecular biology of pestiviruses, Adv Virus Res. 93
(2015) 47–160.
3. L. Guangdi, E. De Clercq, Current therapy for chronic hepatitis C: The role of direct-acting
antivirals, Antiv. Res. 142 (2017) 83–122.
4. J.T. Stapleton, S. Foung, A. Scott Muerhoff, J. Bukh, P. Simmonds, The GB viruses: a review
and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within
the family Flaviviridae, J.Gen. Virol. 92 (2011) 233–246
.
5. S. Chandriani, P. Skewes-Cox, W. Zhong, D.E. Ganem, T.J. Divers, A.J. Van Blaricum, B.C.
Tennant, A.L. Kistler, Identification of a previously undescribed divergent virus from the
Flaviviridae family in an outbreak of equine serum hepatitis, Proc. Natl. Acad. Sci. USA 110
(2013) E1407–15.
6. http://www.who.int/mediacentre/factsheets/fs100/en/
7. http://www.who.int/mediacentre/factsheets/fs378/en/
8. R. Fioravanti, N. Desideri, M. Biava, P. Droghini, E.M. Atzori, C. Ibba, G. Collu, G. Sanna, I.
Delogu, R. Loddo, N-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)anilines: A novel class of anti-
RSV agents, Bioorg. Med. Chem. Lett. 25 (2015) 2401-2404.
9. A. Carta, I. Briguglio, S. Piras, P. Corona, R. Ibba, E. Laurini, M. Fermeglia, S. Pricl, N.
Desideri, E.M. Atzori, P. La Colla, G. Collu, I. Delogu, R. Loddo, A combined in silico/in vitro

ACCEPTED MANUSCRIPT
approach unveils common molecular requirements for efficient BVDV RdRp binding of linear
aromatic N-polycyclic systems, Eur. J. Med. Chem. 117 (2016) 321-334.
10. J. Neyts, A. Meerbach, P. McKenna, E. De Clercq, Use of the yellow fever virus vaccine strain
17D for the study of strategies for the treatment of yellow fever virus infections, Antiv. Res. 30
(1996) 125-32.
11. Q. Jiang, J. Jia, B. Xu, A. Zhao, C. Guo, Iron-Facilitated Oxidative Radical Decarboxylative
Cross-Coupling between α Oxocarboxylic Acids and Acrylic Acids: An Approach to α,β-
Unsaturated Carbonyls, J. Org. Chem. 80 (2015) 3586-3596.
12. V.A. Larionov, E.P. Markelova, A.F. Smol'yakov, T.F. Savel'yeva, V.I. Maleev, Y.N. Belokon,
Chiral octahedral complexes of Co(III) as catalysts for asymmetric epoxidation of chalcones
under phase transfer conditions, RSC Advances. 5 (2015) 72764-72771.
13. S.J. Robinson, J.P. Petzer, G. Terre'Blanche, A. Petzer, M.M. van der Walt, J.J. Bergh, A.C.U.
Lourens, 2-Aminopyrimidines as dual adenosine A1/A2A antagonists, Eur. J. Med. Chem. 104
(2015) 177-188.
14. D. Huang, J.X. Wang, Y. Hu, Y. Zhang, J. Tang, A new solvent-free synthesis of α,β-
unsaturated ketones from acetals with aryl ketones under microwave irradiadion, Synthetic
Commun. (2002) 32, 971-979.
15. A. Wilhelm, L.A. Lopez-Garcia, K. Busschots, W. Fröhner, F. Maurer, S. Boettcher, H. Zhang,
J.O. Schulze,R.M. Biondi, M. Engel, 2-(3-Oxo-1,3-diphenylpropyl)malonic acids as potent
allosteric ligands of the PIF pocket of Phosphoinositide-Dependent Kinase-1: development and
prodrug concept, J. Med. Chem. 55 (2012) 9817-9830.
16. Zhu Ji, Srikanth Natarajan, Ng Siu-Choon, Kon Oi-Lian, Sim Keng-Yoow. Synthesis of 2-(4-
halogenobenzyl)-3-arylbenzo[b]thiophenes
and
a
2-(4-fluorobenzyl)-3-
arylbenzo[b]selenophene as selective ligands for antiestrogen-binding sites, J. Chem. Res.
Miniprint. 3 (1994) 672-682.
17. R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn, J. Desmyter, E. De
Clercq, Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV
compounds, J. Virol. Methods, 20 (1988) 309-321.

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Table 1. Cytotoxicity and antiviral activity of 4-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamides (4a-j, 5a-j, 6a-j) against ssRNA+ (YFV, DENV-2, WNV, BVDV) viruses.
cSI
CC50
BHK-21/
EC50YFV
-
hSI
CC50
MDBK/
EC50 BVDV
BHK-21
aCC50
(µM)
YFV
bEC50
(µM)
DENV-2
dEC50
(µM)
WNV
eEC50
(µM)
MDBK
fCC50
(µM)
BVDV
gEC50
(µM)
Compds
R
R1
4a
4b
H
H
H
2-F
15
21
>15
>21
-
-
-
-
16
19
>16
>19
-
-
-
4c
H
3-F
15
≥15
-
-
-
20
>20
-
4d
4e
4f
4g
4h
4i
4j
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
6a
6b
6c
6d
6e
6f
6g
H
H
H
H
H
H
H
4-F
13±2
14±2
9±1
32
14±0.001
25
2.6±0.4
2.4±0.4
2.5±0.1
>32
2.2±0.2
>25
5.0
5.8
3.6
-
6.4
-
3.6
6.2
17.9
5.6
5.0
5.0
4.5
4.2
3.6
5.0
5.0
36.4
50.0
5.5
31.6
6.3
22.5
7.9
12.2
5.0
>13
>14
>9
>13
>14
>9
18
9
2
11
18
23
32
5
36
12
6
5
6
19
16
6
>18
>9
>2
>11
>18
>23
>32
>5
>36
>12
>6
>5
>6
>19
>16
>6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2-Cl
3-Cl
4-Cl
2-CH3
3-CH3
4-CH3
H
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-CH3
3-CH3
4-CH3
H
2-F
3-F
4-F
-
-
>14
-
>8
>13
>34
>10
>9
>9
>10
>8
>8
>9
>14
-
>8
>13
>34
>10
>9
>9
>10
>8
>8
>9
8±1
2.2±0.2
2.1±0.1
1.9±0.1
1.8±0.2
1.8±0.2
1.8±0.2
2.2±0.2
1.9±0.1
2.2±0.2
1.8±0.1
1.8±0.1
2.2±0.2
1.8±0.2
2.0±0.05
1.9±0.1
1.9±0.1
2.0±0.05
1.9±0.1
1.8±0.2
1.8±0.2
1.8±0.2
OPh
OPh
OPh
OPh
OPh
OPh
OPh
OPh
OPh
OPh
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
13±1
34±3
10±2
9±0.5
9±1
10±0.3
8±0.8
8±1
9±1
9±0.8
80±4
90±5
11±1
60±8
12±0.5
45±3
15±1
22±3
9±0.3
36±4
>9
>9
21
72
>21
>72
>80
>90
>11
>60
>12
>45
>15
>22
>9
>80
>90
>11
>60
>12
>45
>15
>22
>9
-
>100
15±2
>100
52±5
40±5
16±1
>100
56±5
80±7
52.0±8
10.0±1
>100
7.5±2
5.5±0.5
4.5±1.5
>100
12.5±4
2.5±0.5
>1.9
1.5
-
6.9
7.3
3.6
-
2-Cl
3-Cl
4-Cl
2-CH3
3-CH3
4-CH3
6h
6i
6j
4.5
32
20.0
>36
>36
Ref. Compds.
6-Azauridine
NM 108
>100
48±6
20.0±5
>5
1.7±0.2
0.8±0.1
Ribavirin
62.0±1
20.0±2
3
Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%.
aCompound concentration required to reduce the viability of mock-infected BHK (Hamster normal kidney fibroblast) monolayers by 50%, as
b
determined by the MTT method. Compound concentration required to reduce the viability of mock-infected BHK cells from the YFV-induced
c
cytopathogenicity, as determined by the MTT method. Selectivity index (SI) was the ratio between CC50 against BHK-21 and EC50 against YFV.
dCompound concentration required to achieve 50% protection of BHK cells from DENV-2 induced cytopathogenicity, as determined by the MTT
method. eCompound concentration required to achieve 50% protection of BHK cells from WNV induced cytopathogenicity, as determined by the
f
MTT method. Compound concentration required to reduce the viability of mock-infected MDBK (Bovine normal kidney) cells by 50%, as
g
determined by the MTT method. Compound concentration required to achieve 50% protection of MDBK cells from the BVDV-induced
cytopathogenicity, as determined by the MTT method. hSelectivity index (SI) was the ratio between CC50 against MDBK and EC50 against BVDV.
The sign ‘>’ indicates that the EC50 is higher than the CC50 for the corresponding host cell line or beyond the highest tested concentration.

ACCEPTED MANUSCRIPT
Table 2. Cytotoxicity and antiviral activity of 4-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamides (4a-j, 5a-j, 6a-j) against ssRNA+ (HIV-1, CVB-5, Sb-1), ssRNA- (VSV,
RSV), dsRNA (Reo-1) and DNA (VV, HSV-1) viruses.
gSI
CC50
Vero76/
EC50
Sb-1
VV
HSV-1 VSV
RSV
BHK-
21
MT-4 HIV-1
aCC50 bEC50
Reo-1 Vero76
CVB-5
fEC50
(µM)
Compds
R
R1
dEC50
(µM)
eCC50
(µM)
hEC50
(µM)
cCC50
(µM)
(µM)
(µM)
CVB-5
4a
4b
H
H
H
42
42
>42
>42
13.0±0
.5
15
21
>15
>21
22
28
> 22
> 28
-
>22
>28
>22
>28
>22
>28
>22
>28
>22
>28
2-F
-
-
4c
H
3-F
44±1
15
>15
24
> 24
>24
>24
>24
>24
>24
4d
4e
H
H
4-F
45
44
>45
>44
12
13
>12
>13
23
26
> 23
> 26
-
-
>23
>26
>23
>26
>23
>26
>23
>26
>23
>26
2-Cl
10.0±0
.8
4f
H
3-Cl 41±0.1
8
>8
21
> 21
-
>21
>21
>21
>21
>21
4g
4h
4i
H
H
4-Cl
2-CH3
3-CH3
4-CH3
H
36
43
37
40
>36
>43
>37
>40
32
14
25
7
>32
>14
>25
>7
21
24
> 21
> 24
-
>21
>24
>25
>20
>85
>96
>28
>25
>29
>88
>88
>90
>81
>86
>100
>100
>90
>90
>92
>85
>82
>100
>100
>100
>21
>24
>25
>20
>85
>96
>28
>25
>29
>88
>88
>90
>81
>86
>100
>100
>90
>90
>92
>85
>82
>100
>100
>100
>21
>24
>25
>20
>85
>96
>28
>25
>29
>88
>88
>90
>81
>86
>100
>100
>90
>90
>92
>85
>82
>100
>100
>100
>21
>24
>25
>20
>85
>96
>28
>25
>29
>88
>88
>90
>81
>86
>21
>24
>25
>20
>35
>96
>28
>25
>29
>88
>88
>14
>14
>20
-
H
25
> 25
-
4j
H
20
> 20
-
5a
5b
5c
5d
5e
5f
OPh
OPh
OPh
OPh
42±2 13.0±1
11
32
11
8
>11
>32
>11
>8
85±5/35
96±1
28±4
25±2
29±3
88
3.5±0.5
7.0±1
4.2±0.2
6.0±1
10.5±0.5
> 88
24.3
2-F
36
25
33
40
8
>36
>25
>33
>40
>8
13.7
3-F
6.7
4-F
4.2
OPh 2-Cl
OPh 3-Cl
OPh 4-Cl
8
>8
2.8
9
>9
-
5g
5h
5i
9
>9
7
>7
88
> 88
-
OPh 2-CH3 36±4 13.0±0.6
7
>7
90/14
81/14
86/20
> 100
> 100
90±5/25
90
> 90
-
OPh 3-CH3
OPh 4-CH3
35
7.2
>35
>7.2
>100
>39
8
>8
> 81
-
5j
8
>8
> 86
-
6a
6b
6c
6d
6e
6f
OBn
OBn
OBn
OBn
H
>100
39
84
92
9
>84
>92
>9
> 100
> 100
8.5±2.5
> 90
-
>100 >100
>100 >100
2-F
3-F
4-F
-
65
>65
10.6
>90
>90
>92
>85
>82
>25
>90
>92
>85
>82
>100
26
>100
>26
69
12
47
16
20
8
>69
>12
>47
>16
>20
>8
-
OBn 2-Cl
OBn 3-Cl
OBn 4-Cl
92
> 92
-
36±4 11.0±1
85
> 85
-
-
6g
6h
6i
70
>70
>100
>44
82
> 82
OBn 2-CH3 ≥100
100
> 100
8.0±2
13±4.5
-
>100 >100
>100 >100
>100 >100
OBn 3-CH3
OBn 4-CH3
44
35
100±0.1
100±0.2
12.5
7.7
6j
>35
32
>32
Ref. Compds.
0.002±
0.001
EFV
40±2
Ribavirin
6-Azauridine
ACG
>100
16±2
>100
83±3
9±1
40.0±5
1.2±0.2
>100
18.0±2
1.8±0.5
Pleconaril
M5255
0.005±0.002
16000 2.7±0.6
1.5±0.2
Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%.
aCompound concentration required to reduce the viability of mock-infected MT-4 (cd4+ Human T-cells containing an integrated HTL V-1 genome)
b
cells by 50%, as determined by the MTT method. Compound concentration required to achieve 50% protection of MT-4 cells from the HIV-1
c
induced cytioathigenicity, as determined by the MTT method. Compound concentration required to reduce the viability of mock-infected BHK
d
(hamster normal kidney fibroblast) monolayers by 50%, as determined by the MTT method. Compound concentration required to achieve 50%
e
protection of BHK cells from the Reo (Reovirus 1), induced cytopathogenicity, as determined by the MTT method. Compound concentration
required to reduce the viability of mock-infected VERO76 (monkey normal kidney) monolayers by 50%. The two values of cytotoxicity reported in
some cases for the cytotoxicity of Vero 76 cell lines are due to the fact that for the RSV the compound remains in contact with the cells for 5 days
(data reported in red) instead of 2 days for Sb-1 and VSV and 3 days for CVB-5, VV and HSV-1 (data reported in black), therefore the cytotoxicity is
f
g
higher. Compound concentration required to reduce the plaque number of CVB-5 (Coxsackievirus B5) by 50% in Vero76 monolayers. Selectivity
index (SI) was the ratio between CC50 against Vero 76 cells (data reported in black) and EC50 against CVB-5. hCompound concentration required to
reduce the plaque number of Sb-1 (Poliovirus 1), VV (Vaccina virus), HSV-1 (Herpesvirus 1), VSV (Vescicular Stomatitis Virus) and RSV
(Respiratory Syncytial Virus) by 50% in Vero76 monolayers.
The sign ‘>’ indicates that the EC50 is higher than the CC50 for the corresponding host cell line or beyond the highest tested concentration.

ACCEPTED MANUSCRIPT
•
A series of 1,3,5-triphenyl-4,5-dihydropyrazole derivatives were designed and synthesized as
YFV inhibitors
•
•
•
All compounds were tested against a large panel of RNA and DNA viruses
Most of the analogues exhibited a potent and specific activity against YFV
The benzyloxy derivatives (6a-j) generally coupled potency in the low micromolar range and
high selectivity.