2,3-dihydro-1,2-diphenyl-substituted 4H-pyridinone derivatives as new anti flaviviridae inhibitors

Article abstract of DOI:10.1111/j.1747-0285.2011.01102.x

With the aim of identifying novel lead compounds active against emergent human infectious diseases, a series of 2,3-dihydro-4H-pyridinone derivatives has been prepared and evaluated for antiviral activity. Compounds were evaluated in vitro in cell-based assays for cytotoxicity and against a wide spectrum of viruses. In the antiviral screening, several compounds showed to be fairly active against viruses belonging to the Flaviviridae family. The Pestiviruses (bovine viral diarrhoea virus) were inhibited by 4acis (CC₅₀>100μm; EC₅₀ = 14μm), compounds 4ccis and 6a showed a significant activity against Flaviviruses (Yellow Fever Virus) (CC₅₀>100μm; EC₅₀=18μm, CC₅₀>100μm; EC₅₀=10μm). Among these, compound 6a displayed great inhibitory activity against Hepaciviruses (hepatitis C virus) in replicon assay [CC₅₀ > 100μm; EC₅₀(1b)=4μm]. In vitro inhibitory activity against the HCV RNA-dependent RNA polymerase (NS5B) of title compounds is discussed. The antiviral screening of viral strains indicated that compound 6a can be selected as promising tool in novel anti-flaviviruses development.

Full text of DOI:10.1111/j.1747-0285.2011.01102.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01102.x
Chem Biol Drug Des 2011; 77: 441–449
Research Article
2,3-Dihydro-1,2-Diphenyl-substituted 4H-
Pyridinone Derivatives as New Anti Flaviviridae
Inhibitors
Antonella Peduto1, Antonio Massa1,*,
Viruses belonging to the Flaviviridae family have a single-stranded,
positive-sense RNA genome (ssRNA)+ (1–3) and are responsible for
numerous diseases of global importance. This virus family includes
three genera: Flaviviruses, Pestiviruses and Hepaciviruses. The
genus of Flavivirus includes pathogens such as dengue virus, yellow
fever virus (YFV), Japanese encephalitis virus and tick-borne
Antonia Di Mola1, Paolo de Caprariis2,
Paolo La Colla3, Roberta Loddo3,
Sergio Altamura4, Giovanni Maga5 and
Rosanna Filosa2,*
1Dipartimento di Chimica, Universitꢀ di Salerno, 84084 Fisciano
encephalitis virus, many of which are resurging and spreading to
new environments.
(SA), Italy
2Dipartimento di Scienze Farmaceutiche, Universitꢀ di Salerno,
84084 Fisciano (SA), Italy
The genus Pestivirus includes animal pathogens such as bovine viral
diarrhoea virus (BVDV) together with viruses such as the classical
swine fever virus and the border disease virus (4–6). These viruses
cause significant disease in wild and agriculturally important ani-
mals and are studied as a model system for the related human
viruses. BVDV has been considered to be a surrogate virus for hep-
atitis C virus (HCV) (7,8), although robust HCV replicon and HCV cell
culture systems are now available (9–13).
3Dipartimento di Scienze e Tecnologie Biomediche, Universitꢀ di
Cagliari (CA), Italy
4IRBM Science Park, Merck Research Laboratories Rome, 00040
Pomezia, Rome, Italy
5Istituto di Genetica Molecolare, IGM-CNR, Pavia, Italy
*Corresponding authors: Antonio Massa, amassa@unisa.it and
Rosanna Filosa, rfilosa@unisa.it
With the aim of identifying novel lead compounds
active against emergent human infectious diseases,
a series of 2,3-dihydro-4H-pyridinone derivatives
has been prepared and evaluated for antiviral
activity. Compounds were evaluated in vitro in
cell-based assays for cytotoxicity and against a
wide spectrum of viruses. In the antiviral screening,
several compounds showed to be fairly active
against viruses belonging to the Flaviviridae family.
The Pestiviruses (bovine viral diarrhoea virus)
Hepatitis C virus (HCV), the prototype member of the Hepacivirus
genus, currently chronically infects approximately 120 million people
worldwide, or 2% of the world's population. Current standard ther-
apy for chronic hepatitis C consists of pegylated interferon in combi-
nation with ribavirin (14). Unfortunately, this therapy results in a
sustained virological response in only about 50–60% of the patients
treated and is associated with serious side effects, particularly
among patients with genotype 1 (15,16). The relatively low response
rates and the significant side effect burden of current HCV therapies
necessitate the identification of more effective anti-HCV agents,
especially for treatment of patients infected with genotype 1 HCV.
were inhibited by 4a cis (CC50 > 100 lM; EC50
=
14 lM), compounds 4c cis and 6a showed a signifi-
cant activity against Flaviviruses (Yellow Fever
Virus) (CC50 > 100 lM; EC50 = 18 lM, CC50 > 100 lM;
EC50 = 10 lM). Among these, compound 6a dis-
played great inhibitory activity against Hepacivirus-
With the aim of identifying novel leads active against these emer-
gent infectious diseases, we initially screened our 4-pyridinone com-
pound library for inhibitory activity. Structural optimization studies
yielded 2,3-dihydro-4H-pyridinone derivatives 4c cis and 6a having
strong antiviral activity. All compounds were evaluated in vitro in
cell-based assays for cytotoxicity and antiviral activity against
viruses that are representative of two of the three genera of the
Flaviviridae family, i.e. Flaviviruses (YFV) and Pestiviruses (BVDV); as
Hepaciviruses can hardly be used in routine cell-based assays, only
the most interesting compounds were investigated.
es (hepatitis C virus) in replicon assay [CC50
>
100 lM; EC50(1b) = 4 lM]. In vitro inhibitory activity
against the HCV RNA-dependent RNA polymerase
(NS5B) of title compounds is discussed. The antivi-
ral screening of viral strains indicated that com-
pound 6a can be selected as promising tool in novel
anti-flaviviruses development.
Key words: 2,3-dihydro-1,2-diphenyl-substituted 4H-pyridinone, flavi-
virus
Compounds were also tested against representatives of other virus
families. Among ssRNA+ viruses were a retrovirus [Human Immuno-
deficiency Virus type 1 (HIV-1)], two enteroviruses [Coxsackie Virus
Received 10 July 2010 and accepted for publication 4 February 2011
441

Peduto et al.
type B2 (CVB2), and Poliovirus type-1, Sabin strain (Sb-1)]; among
ssRNA) was a Rhabdoviridae (Vesicular Stomatitis Virus) represen-
tative. Among double-stranded RNA (dsRNA) viruses was a Reoviri-
dae representative (Reo-1). Two representatives of DNA virus
families were also included: Herpes Simplex type 1, (HSV-1; Herpes-
viridae) and Vaccinia Virus (VV; Poxviridae) (data not shown).
After about 15 min, the reaction was quenched with a saturated
solution of NaHCO3, extracted with 50 · 3 mL of CH2Cl2 and dried
over anhydrous Na2SO4. After removing the solvent under reduced
pressure, the crude oil was purified by silica gel flash chromatogra-
phy to afford the pure cyclic adducts.
cis-2,3-dihydro-3,5-dimethyl-1-(2-
Methods and Materials
methoxyphenyl)-2-phenyl-pyridin-4(1H)-one. (3b
cis)
General methods
The title compound was obtained as wax solid in 33% yield. TLC:
Rf 0.41 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
400 MHz) d:1.02 (d, J = 7.0 Hz, 3H, CHCH3), 1.79 (s, 3H, CH3), 3.22
(quint, J = 7.0 Hz, 1H, CHCH3), 3.79 (s, 3H, OCH3), 4.99 (d,
J = 7.0 Hz, 1H, CHCH), 6.87–6.78 (m, 3H, ArH), 7.18–7.21 (m, 7H,
ArH + C=CH). 13C-NMR (100 MHz, CDCl3) d:11.5, 12.9, 43.8, 55.7,
68.1, 105.5, 111.8, 120.8, 126.5, 126.8, 127.3, 127.5, 127.7, 128.0,
128.3, 136.8, 150.4, 194.5. ESY-MS: MS+1, 308. anal. calcd. for
C20H21NO2: C 78.15, H 6.89, N 4.56, found: C 78.26, H 6.79, N 4.70.
All reactions were performed in oven-dried (140 ꢀC) or flame-dried
glassware under an atmosphere of dry nitrogen. All the solvents for
the reactions were of reagent grade and were dried and distilled
immediately before use (dichloromethane from calcium hydride).
Column chromatographic purification of products was carried out
using silica gel 60 (70–230 mesh, Merck). The reagents (Aldrich and
Fluka) were used without further purification. The NMR spectra
were recorded on a Bruker DRX 400 (400 MHz, H; 100 MHz, 13C).
1
Spectra were referenced to residual chloroform (7.26 ppm, 1H,
77.23 ppm, 13C). Chemical shifts are reported in ppm, multiplicities
are indicated by s (singlet), d (doublet), t (triplet), q (quartet), quint
(quintet), m (multiplet) and br (broad). Coupling constants, J, are
reported in Hz. Yields are given for isolated products showing one
spot on a TLC plate and no impurities detectable in the NMR spec-
trum. IR spectra were recorded on a Bruker Vector 22 (frequencies
400–4000 per cm, resolution 2 per cm). Mass spectrometry analysis
was carried out using an electrospray spectrometer Waters 4 micro
quadrupole. Elemental analyses were performed with FLASHEA
1112 series-Thermo Scientific for CHNS-O.
trans-2,3-dihydro-3,5-dimethyl-1-(2-
methoxyphenyl)-2-phenyl-pyridin-4(1H)-one. (3b
trans)
The title compound was obtained as wax solid in 17% yield. TLC:
Rf 0.35 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
400 MHz) d:1.28 (d, J = 7.0 Hz, 3H, CHCH3), 1.78 (s, 3H, CH3), 2.76
(quint, J = 7.0 Hz, 1H, CHCH3), 3.80 (s, 3H, OCH3), 4.78 (d,
J = 7.0 Hz, 1H, CHCH), 6.97–6.80 (m, 3H, ArH), 7.20–7.11 (m, 7H,
ArH + C=CH).
13C-NMR (CDCl3, 100 MHz) d:12.8, 16.3, 46.6, 55.3, 69.2, 105.1,
111.7, 120.7, 126.4, 127.2, 127.3 127.4, 128.1, 134.0, 138.9, 150.2,
153.7, 194.2. ESY-MS: MS+1, 308. anal. calcd. for C20H21NO2: C
78.15, H 6.89, N 4.56, found: C 78.04, H 6.76, N 4.71.
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 viruses were as follows: Madin Darby
Bovine Kidney (MDBK); Baby Hamster Kidney (BHK-21).
cis-2,3-dihydro-3,5-dimethyl-1-(4-nitrobenzyl)- 2-
(4-chlorophenyl)-pyridin-4(1H)-one. (4b cis)
The title compound was obtained as wax solid in 41% yield. TLC:
Rf 0.40 (Petroleum ether ⁄ ethyl acetate = 8 ⁄ 2); 1H-NMR (CDCl3,
400 MHz) d:0.98 (d, J = 7.0 Hz, 3H, CHCH3), 1.77 (s, 3H, CH3), 2.59
(quint, J = 7.0 Hz, 1H, CHCH3), 3.96 (d, J = 7.0 Hz, 1H, CHCH), 4.10
(d, J = 15.0 Hz, 1H, CHH), 4.33 (d, J = 15.0 Hz, 1H, CHH), 7.14–7.10
(m, 3H, ArH + C=CH), 7.34–7.26 (m, 4H, ArH), 7.22 (d, J = 9.0 Hz
1H, ArH). 13C-NMR (CDCl3, 100 MHz) d:11.4, 13.0, 43.6, 56.5, 65.8,
105.8, 124.0, 128.3, 129.0, 129.3, 134.4, 136.7, 143.6, 147.7, 150.5,
193.2. ESY-MS: MS+1, 371. anal. calcd. for C20H19ClN2O3: C 64.78,
H 5.16, N 7.55, found: C 64.89, H 5.02, N 7.65.
The cloned gene for HCV NS5B was kindly provided by Dr. Joachim
Jaeger, Astbury Centre, RepliZyme Ltd, UK. Recombinant HCV NS5B
was expressed in E. coli and purified by FPLC chromatography
through DEAE and HyperD Heparin columns.
Synthesis of 2,3-dihydro-4H-pyridinone ring
One-pot-aza-Diels-Alder reaction: general
procedure
In a flame-dried, 2-necked, round-bottom flask, 0.66 mmol of alde-
hyde and 0.70 mmol of aniline were added to 0.7 g of activated
4 ꢀ molecular sieves in 2 mL of dry CH2Cl2 under argon. The reac-
tion was stirred at room temperature for 1 h. Then the mixture was
diluted with 2 mL of dry CH2Cl2 and the temperature was lowered
to )78 ꢀC. At this temperature, 0.066 mmol of the activator (if
required), diisopropylethylamine (0.66 mmol), SiCl4 (0.66 mmol, solu-
tion 1.0 M in CH2Cl2) and 0.71 mmol of Danishefsky's diene were
successively added. At the end of the reaction, the mixture was
treated with a solution of 0.1 mL of trifluoroacetic acid in 1 mL of
CH2Cl2.
trans-2,3-dihydro-3,5-dimethyl-1-(4-nitrobenzyl)-
2-(4-chlorophenyl)-pyridin-4(1H)-one. (4b trans):
The title compound was obtained as wax solid in 37% yield. TLC:
Rf 0.33 (Petroleum ether ⁄ ethyl acetate = 8 ⁄ 2); 1H-NMR (CDCl3,
400 MHz) d:0.88 (d, J = 7.0 Hz, 3H, CHCH3), 1.77 (s, 3H, CH3), 3.02
(quint, J = 7.0 Hz, 1H, CHCH3), 4.19 (m, 2H, CHCH + CHH), 4.35 (d,
J = 15.0 Hz, 1H, CHH), 7.04 (s, 1H, C=CH), 7.13 (d, J = 9.0 Hz, 2H,
442
Chem Biol Drug Des 2011; 77: 441–449

Derivatives as new Anti Flaviviridae inhibitors
ArH) 7.27 (d, J = 9.0 Hz, 2H, ArH), 7.40 (d, J = 7.0 Hz 2H, ArH),
8.22 (d, J = 7.0 Hz 2H, ArH). 13C-NMR (CDCl3, 100 MHz) d:12.8,
15.0, 46.2, 56.6, 67.8, 106.5, 124.2, 128.6, 129.0, 129.7, 134.5,
136.7, 144.1, 147.8, 150.5, 193.4. ESY-MS: MS+1, 371. anal. calcd.
for C20H19ClN2O3: C 64.78, H 5.16, N 7.55, found: C 64.66, H 5.25,
N 7.64.
7.07–7.00 (m, 4H, ArH), 7.20–7.17 (m, 2H, ArH), 7.59 (s, 1H, C=CH),
8.16 (d, J = 7.2 Hz, 2H, ArH) 13C-NMR (CDCl3, 100 MHz) d:12.8,
18.0, 47.8, 67.1, 112.2, 116.0, 116.2 125.6, 127.5, 132.2, 140.6.1,
142.4, 149.9, 161.0, 194.7. ESY-MS: MS+1, 341. anal. calcd. for
C19H17FN2O3: C 67.05, H 5.03, N 8.23, found: C 67.17, H 5.15, N
8.11.
cis-2,3-dihydro-3,5-dimethyl-1-(4-nitrophenyl)-2-
(4-trifluoromethylphenyl)-pyridin-4(1H)-one. (4c
cis)
cis-2,3-dihydro-3,5-dimethyl-1-(2-
methoxyphenyl)- 2-(4-chlorophenyl)-pyridin-
4(1H)-one. (4e cis)
The title compound was obtained as wax solid in 44% yield. TLC:
Rf 0.37 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
400 MHz) d:1.08 (d, J = 6.8 Hz, 3H, CHCH3), 1.90 (s, 3H, CH3), 3.48
(quint, J = 6.8 Hz, 1H, CHCH3), 5.17 (d, J = 6.8 Hz, 1H, CHCH), 7.03
(d, J = 9.2 Hz, 2H, ArH), 7.45 (d, J = 7.9 Hz, 2H, ArH), 7.59 (d,
J = 7.9 Hz, 2H, ArH), 7.64 (s, 1H, C=CH), 8.14 (d, J = 9.2 Hz, 2H,
ArH). 13C-NMR (CDCl3, 100 MHz) d:11.0, 12.8, 43.6, 66.8, 112.8,
115.8, 125.4, 125.5, 125.9, 127.9, 130.6, 139.2, 141.9, 142.2, 149.1,
192.9. ESY-MS: MS+1, 391. anal. calcd. for C20H17F3N2O3: C 61.54,
H 4.39, N 7.18, found: C 61.43, H 4.46, N 7.29.
The title compound was obtained as wax solid in 36% yield. TLC:
Rf 0.42 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
400 MHz) d:1.25 (d, J = 7.0 Hz, 3H, CHCH3), 1.76 (s, 3H, CH3), 2.67
(quint, J = 7.0 Hz, 1H, CHCH3), 3.78 (s, 3H, OCH3), 4.75 (d,
J = 7.0 Hz, 1H, CHCH), 6.84–6.79 (m, 2H, ArH), 6.94 (dd, J = 7.6,
J = 1.6 Hz, ArH), 7.10–7.17 (m, 6H, ArH + C=CH). 13C-NMR (CDCl3,
100 MHz) d:11.8, 15.1, 45.8, 54.4, 67.6, 104.5, 110.8, 119.8, 125.5,
126.6, 127.4, 127.6, 132.2, 132.8, 136.6, 149.0, 152.8, 193.1. ESY-
MS: MS+1, 342. anal. calcd. for C20H20ClNO2: C 70.27, H 5.90, N
4.10, found: C 70.37, H 5.78, N 4.21.
trans-2,3-dihydro-3,5-dimethyl-1-(4-nitrophenyl)-
2-(4-trifluoromethylphenyl)-pyridin-4(1H)-one.
(4c trans)
trans-2,3-dihydro-3,5-dimethyl-1-(2-
methoxyphenyl)- 2-(4-chlorophenyl)-pyridin-
4(1H)-one. (4e trans)
The title compound was obtained as wax solid in 40% yield. TLC:
Rf 0.30 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
400 MHz) d:1.41 (d, J = 7.3 Hz, 3H, CHCH3), 1.82 (s, 3H, CH3), 2.89
(dq, J = 1.9 e 7.3 Hz, 1H, CHCH3), 5.06 (bd, J = 1.9 Hz, 1H, CHCH),
7.05 (d, J = 9.2 Hz, 2H, ArH), 7.36 (d, J = 8.1 Hz, 2H, ArH), 7.61 (m,
3H, ArH + C=CH), 8.19 (d, J = 9.2 Hz, 2H, ArH). 13C-NMR (CDCl3,
100 MHz) d:12.8, 18.1, 47.6, 67.3, 112.5, 116.0, 125.6, 125.7, 126.2,
126.3, 130.4, 140.5, 140.7, 142.5, 149.7, 194.2. ESY-MS: MS+1,
391. anal. calcd. for C20H17F3N2O3: C 61.54, H 4.39, N 7.18, found:
C 61.65, H 4.26, N 7.05.
The title compound was obtained as wax solid in 28% yield. TLC:
Rf 0.36 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
400 MHz) d:1.00 (d, J = 7.0 Hz, 3H, CHCH3), 1.78 (s, 3H, CH3), 3.20
(quint, J = 7.0 Hz, 1H, CHCH3), 3.78 (s, 3H, OCH3), 4.96 (d,
J = 7.0 Hz, 1H, CHCH), 6.87–6.80 (m, 3H, ArH), 7.17–7.10 (m, 6H,
ArH + C=CH). 13C-NMR (CDCl3, 100 MHz) d:10.3, 11.8, 45.6, 54.6,
66.3, 105.2, 110.9, 119.9, 121.5, 125.8, 126.7, 127.2 128.6, 133.1,
134.2, 149.2, 153.0, 193.2. ESY-MS: MS+1, 342. anal. calcd. for
C20H20ClNO2: C 70.27, H 5.90, N 4.10, found: C 70.16, H 5.99, N
4.22.
cis-2,3-dihydro-3,5-dimethyl-1-(4-nitrophenyl)-2-
(4-fluorophenyl)-pyridin-4(1H)-one. 4d cis
The title compound was obtained as wax solid in 30% yield. TLC:
Rf 0.38 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
400 MHz) d:1.07(d, J = 7.0 Hz, 3H, CHCH3), 1.90 (s, 3H, CH3), 3.44
(quint, J = 7.0 Hz, 1H, CHCH3), 5.13 (d, J = 6.8 Hz, 1H, CHCH),
7.03–6.96 (m, 4H, ArH), 7.29–7.23 (m, 2H, ArH), 7.57 (s, 1H, C=CH),
8.12 (d, J = 7.2 Hz, 2H, ArH). 13C-NMR (CDCl3, 100 MHz) d:11.1,
12.9, 43.8, 66.7, 112.7, 115.8, 116.1, 125.5, 129.2, 131.0, 141.8,
149.3, 161.3, 163.7, 193.2. ESY-MS: MS+1, 341. anal. calcd. for
C19H17FN2O3: C 67.05, H 5.03, N 8.23, found: C 67.15, H 5.16, N
8.13.
cis-2,3-dihydro-3,5-dimethyl-1-(4-
methoxyphenyl)- 2-(4-chlorophenyl)-pyridin-
4(1H)-one. (4f cis)
The title compound was obtained as wax solid in 45% yield. TLC:
Rf 0.41 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
400 MHz) d:0.99 (d, J = 7.0 Hz, 3H, CHCH3), 1.80 (s, 3H, CH3), 3.30
(quint, J = 7.0 Hz, 1H, CHCH3), 3.74 (s, 3H, OCH3), 4.91 (d,
J = 7.0 Hz, 1H, CHCH), 6.78 (d, J = 8.9 Hz, 2H, ArH), 6.98 (d,
J = 8.9 Hz, 2H, ArH), 7.22–7.16 (m, 4H, ArH), 7.33 (s, 1H, C=CH).
13C-NMR (CDCl3, 100 MHz) d:10.2, 11.7, 42.4, 54.4, 66.7, 106.0,
113.5, 120.4, 126.6, 127.7, 128.3, 134.2, 137.7, 146.3.8, 155.5,
192.1. ESY-MS: MS+1, 342. anal. calcd. for C20H20ClNO2: C 70.27,
H 5.90, N 4.10, found: C 70.12, H 5.99, N 4.23.
trans-2,3-dihydro-3,5-dimethyl-1-(4-nitrophenyl)-
2-(4-fluorophenyl)-pyridin-4(1H)-one. (4d trans)
The title compound was obtained as wax solid in 25% yield. TLC:
Rf 0.30 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
400 MHz) d: 1.37(d, J = 7.3 Hz, 3H, CHCH3), 1.80 (s, 3H, CH3), 2.84
(dd, J = 1.9 e 7.3 Hz, 1H, CHCH3), 4.99 (bd, J = 1.9 Hz, 1H, CHCH),
trans-2,3-dihydro-3,5-dimethyl-1-(4-
methoxyphenyl)- 2-(4-chlorophenyl)-pyridin-
4(1H)-one. (4f trans)
The title compound was obtained as wax solid in 35% yield. TLC:
Rf 0.35 (Petroleum ether ⁄ ethyl acetate = 7 ⁄ 3); 1H-NMR (CDCl3,
Chem Biol Drug Des 2011; 77: 441–449
443

Peduto et al.
400 MHz) d:1.32 (d, J = 7.2 Hz, 3H, CHCH3), 1.75 (s, 3H, CH3), 2.67
(dq, J = 1.9 e 7.2 Hz, 1H, CHCH3), 3.76 (s, 3H, OCH3), 4.73 (bd,
J = 1.9 Hz, 1H, CHCH), 6.80 (d, J = 9.0 Hz, 2H, ArH), 6.90 (d,
J = 9.0 Hz, 2H, ArH), 7.15 (d, J = 8.4 Hz, 2H, ArH), 7.26 (d,
J = 9.0 Hz, 2H, ArH), 7.37 (s, 1H, C=CH). 13C-NMR (CDCl3, 100 MHz)
d:11.7, 16.9, 46.3, 54.5, 67.2, 106.1, 113.6, 119.4, 126.8, 127.9,
132.4, 135.9 138.1, 144.4, 155.5, 192.9. ESY-MS: MS+1, 342. anal.
calcd. for C20H20ClNO2: C 70.27, H 5.90, N 4.10, found: C 70.40, H
5.79, N 4.01.
absence or presence of serial dilutions of test compounds. Cell via-
bility was determined after 96 h at 37 ꢀC by the 3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method
(17).
Activity of compounds against YFV was based on inhibition of virus-
induced cytopathogenicity in acutely infected BHK-21 cells. Activity
against BVDV, in infected MDBK cells, was based on inhibition of
virus-induced cytopathogenicity. BHK and MDBK were seeded in 96-
well plates at a density of 5 · 104 and 3 · 104, cells ⁄ well, respec-
tively, and were allowed to form confluent monolayers by incubat-
ing overnight in growth medium at 37 ꢀC in a humidified CO2 (5%)
atmosphere. Cell monolayers were then infected with 50 lL of a
proper virus dilution (in serum-free medium) to give an moi, 0.01.
One hour later, 50 lL of MEM Earle's medium, [supplemented with
inactivated foetal calf serum (FCS)], 1% final concentration, without
or with serial dilutions of test compounds, was added. After a 2–3-
day incubation at 37 ꢀC, cell viability was determined by the MTT
method. Plaques were then counted, and 50% effective concentra-
tions (EC50) were calculated by linear regression technique.
2-(4-chlorophenyl)-4-ethoxy-1,2,3,4-tetrahydro-
3,5-dimethyl-1-(4-nitrophenyl)pyridine (6a)
CeCl3 · 7H2O (0.093 g, 0.25 mmol) was added to a solution of 4a
cis (0.075 g, 0.023 mmol) in dry THF (10 mL) and stirred at room
temperature for 5 min. Then the mixture was cooled at 0 ꢀC and
treated with NaBH4 (0.011 g, 0.30 mmol). After 30 min, the mixture
was slowly quenched with 20 mL of 0.1 M HCl, extracted with
CH2Cl2 and dried over anhydrous Na2SO4. After removing the sol-
vent under reduced pressure, the crude oil was directly reacted
without purification. The crude alcohol 5a was dissolved at room
temperature in 15 mL of ethanol and treated with pyridinium p-tolu-
ene sulfonate (61 mg, 0.24 mmol). The mixture was stirred for
1.5 h and then quenched with 20 mL of saturated NaHCO3 solution.
After removing the ethanol under reduced pressure, the solution
was extracted with CH2Cl2 and dried over anhydrous Na2SO4. Then
the solvent was removed under reduced pressure and the crude oil
was purified by silica gel flash chromatography with petroleum
ether ⁄ Et2O mixture (9:1) to afford the pure cyclic adduct 6a.
Activity of compounds against Hepatitis C virus (HCV) genotype 1b
was measured in HuH-7 cells using a modification of the procedure
described (18). Briefly, cells were plated in presence of 10% FCS in
96-well plates at a density of 7000 cells ⁄ well and incubated for
96 h in presence of compounds at eight point serial concentrations.
Then the inhibition of the HCV replication (EC50) was calculated by
linear regression technique.
The title compound was obtained as wax solid in 62% yield for
two steps. TLC: Rf 0.42 (Petroleum ether ⁄ ethylacetate = 7 ⁄ 3); 1H-
NMR (CDCl3, 400 MHz) d:1.01 (d, J = 6.9 Hz, 3H, CHCH3), 1.20 (t,
J = 7.0 Hz, 3H, CH2CH3), 1.86 (s, 3H, CH3), 2.47 (m, 1H, CHCH3),
3.37 (d, J = 10.0 Hz, 1H, OCHCH), 3.46 (quint, J = 7.0 Hz, 1H,
CHHCH3), 3.57 (quint, J = 7.0 Hz, 1H, CHHCH3), 4.70 (d, J = 4.9 Hz,
1H, CHCH), 6.65 (s, 1H, C=CH), 6.75 (d, J = 7.3 Hz, 2H, ArH) 7.21 (d,
J = 9.0 Hz, 2H, ArH), 7.29 (d, J = 9.0 Hz 2H, ArH), 8.01 (d,
J = 7.0 Hz 2H, ArH). 13C-NMR (CDCl3, 100 MHz) d:14.9, 15.7, 17.0,
29.7, 37.5, 63.9, 65.8, 113.2, 115.9, 124.6, 125.8, 128.8, 128.9,
133.8, 136.1, 139.6, 149.8. ESY-MS: MS+1, 387. anal. calcd. for
C21H23ClN2O3: C 65.20, H 5.99, N 7.24, found: C 65.33, H 5.88, N
7.16.
A daughter plate was assembled in the same conditions described
earlier and cell viability (CC50) was determined by the 3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method.
Enzymatic assays
Assays were performed as follows: NS5B (25–50 nM) was incubated
in a final volume of 25 Rl in the presence of 50 mM Tris–HCl
pH8.0, 1 mM DTT, 0.25 mg ⁄ mL BSA, 2.5 RM (3¢-OH ends) of the
RNA ⁄ RNA homopolymeric template (rA)40 ⁄ (rU)20, 0.25 mM MnCl2
and 2 RM [3H]-UTP (37Ci ⁄ mmol). Samples were incubated 40 min
at 25¢XC. Twenty Rl aliquots were spotted on DE-81 filters (Wath-
man), which were washed twice in 0.5 M K2HPO4, once in water
and once in acetone.
Cell-based assays–antiviral assay
Bound radioactivity was detected by liquid scintillation counter (Mi-
crobeta Trilux, Perkin-Elmer).
Activity against YFV and BVDV was based on inhibition of virus-
induced cytopathogenicity in acutely infected BHK-21 and MDBK
cells, respectively. Compounds were dissolved in 100 lM DMSO
and then diluted in culture medium.
As a standard compound we used, in all our antienzymatic assay,
the molecule ATA (Aurintricarboxylic acid) which reproducibly
showed, under our assay conditions, an ID50 of 1.5 € 0.5 lM (19).
For cytotoxicity tests, run in parallel with antiviral assays, MDBK,
BHK, cells were resuspended in 96-multiwell plates at an initial
density of 6 · 105 and 1 · 106, cells ⁄ mL, respectively, in mainte-
nance medium, without or with serial dilutions of test compounds.
Cell viability was determined after 48–120 h at 37 ꢀC in a humidi-
fied CO2 (5%) atmosphere by the MTT method. Cell cultures were
then incubated at 37 ꢀC in a humidified, 5% CO2 atmosphere in the
Results and Discussion
Chemistry
The first step of our synthetic approach required the synthesis of
2,3-dihydro-1,2-diphenyl-4H-pyridinone ring. In an efficient one-pot
444
Chem Biol Drug Des 2011; 77: 441–449

Derivatives as new Anti Flaviviridae inhibitors
O
procedure, the condensation of aromatic aldehydes with several
substituted amines provided imine intermediates that were submit-
ted to aza-Diels Alder reaction promoted by SiCl4 ⁄ Lewis base sys-
tem (Scheme 1). A good number of derivatives were obtained in
good to high yield as racemic mixture.
O
H2
CH3
H2
H1
CH3
N
N
H1
H
Very interestingly in the presence of alkylated Danishefsky's diene
b, this procedure allowed the synthesis of a series of new tetra-
substituted dihydropyridinones even in the presence of imines with
electron-rich and electron-withdrawing groups. The synthesis of di-
and tetra-substituted dihydropyridinones 1, 1a–e, 2a–d, 3a and
4a, whose biological activity is also discussed in this paper, is
reported in our previous work (20). In this work, we focused on
reactivity of alkylated Danishefsky's diene b for the synthesis of a
series of new tetra-substituted dihydropyridinones whose yield, as
mixture of diastereomers, is reported in parentheses as follows: 3b
(50%), 4b (78%), 4c (88%), 4d (55%), 4e (64%) and 4f (80%). It
is worthy to note that different reaction conditions were tried in
order to improve the yield of 3b and 4d, but unfortunately these
attempts revealed unsuccessful.
O2N
O2N
CF3
CF3
cis
trans
Figure 1: Roesy correlations between the protons of the aro-
matic group and the protons of the methyl group.
OEt
H3
For all the tetra-substituted dihydropyridinones, the diastereoselec-
tivity was rather low, but the diastereomers were easily separated
by chromatography and characterized by NMR and MS spectros-
copy.
H4
CH3
N
H2
H
H
In particular, the stereochemistry was assigned on the basis of the
1H-NMR coupling constants for the protons H1-H2 (Jtrans = 1.9 Hz,
Jcis = 6.8 Hz) in comparison with literature data (21).
O2N
Cl
Roesy experiments on 4c cis and trans confirmed this stereochem-
istry in which for both the diastereomers, the aromatic ring is in a
pseudo-axial position (Figure 1).
Figure 2: Roesy correlations of the aromatic protons with H4
and the protons of the methyl group.
Scheme 1: Synthesis of 2,3-dihydro-1,2-diphenyl-substituted 4H-pyridinone derivatives.
Chem Biol Drug Des 2011; 77: 441–449
445

Peduto et al.
Roesy correlations were evident between the protons of the aro-
matic group and the protons of the methyl group of the cis isomer,
while no correlation is observed between the protons of this aro-
matic group respectively with H2 of the cis isomer and with the
protons of methyl group of the trans isomer.
Moreover in order to examine the influence of methylene group on
the biological activity, the dihydropyridinone 4b, in which the p-
nitro benzylamine was incorporated in the aza-D.A. adduct, was
synthesized using the same one-pot procedure.
Further chemical modifications were introduced at the position 4.
Substituents on the aromatic groups in 1 and 2 position were easily
introduced at this stage by using different aldehydes and several
substituted amines.
The modification of dihydropyridinone ring was realized in order to
evaluate the influence of the ketone group on the outcome of bio-
logical investigations.
Table 1: Cytotoxicity and antiviral activity of compounds against Pestiviruses and Flaviviruses. n = 0, R=H, R=CH3,Dn = 1
A
B
C
Compounds
R1
R2
Pestivirus
Flavivirus
O
R
R
N
n
R2
CC50 MDBKa
EC50 BVDVb
CC50 BHK-21c
EC50 YFVd
R1
1A,B
H
H
>100
>100
>100
>100
>100
>100
78
>100
>100
>100
>100
>100
>100
78
>100
>100
>100
>100
>100
>100
38
>100
>100
>100
>100
>100
>100
38
1aA,B
H
H
H
4-NO2
4- CF3
4-OCH3
4-OCH3
4-OCH3
H
1bA,B
1cA,B
1dA,B
2-OCH3
4-NO2
H
1eA,B
2 cisA,C
2 transA,C
2a cisA,C
2a transA,C
2b cisA,C
2b transA,C
2c cisA,C
2c transA,C
2d cisA,C
2d transA,C
2e cisA,C
2e transA,C
3a cisA,C
3a transA,C
3b cisA,C
3b transA,C
4a cisA,C
4a transA,C
4b cisC,D
4b transC,D
4c cisA,C
4c transA,C
4d cisA,C
4d transA,C
4e cisA,C
4e transA,C
4f cisA,C
4f transA,C
6a
H
H
H
H
H
H
H
H
H
61
61
44
96
84
62
‡100
22
37
44
96
84
62
‡100
22
37
4-NO2
4-NO2
2-NO2
2-NO2
2-Cl
2-Cl
4-Cl
4-Cl
4-CN
4-CN
H
H
H
H
4-Cl
4-Cl
4-Cl
4-Cl
4-CF3
4-CF3
4-F
100
100
57
‡100
15
26
23
>100
>100
>100
22
100
100
57
‡100
15
26
23
>100
>100
>100
22
34
34
H
H
H
>100
>100
>100
15
38
22
>100
>100
>100
15
38
22
42
43
>100
>100
>100
18
4-NO2
4-NO2
2-OCH3
2-OCH3
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
2-OCH3
2-OCH3
4-OCH3
4-OCH3
80
80
80
80
>100
14
>100
>100
>100
>100
>100
>100
>100
>100
>100
9
42
43
>100
>100
>100
>100
>100
>100
>100
9
21
35
11
>100
>100
>100
>100
>100
>100
>100
>100
28
30
37
35
>100
>100
>100
>100
>100
30
37
35
10
4-F
4-Cl
4-Cl
4-Cl
4-Cl
21
35
11
>100
a–dCC50 and EC50 (lM): Data represent mean values for three independent determinations. Variation among duplicate samples was <15%. Interesting antiviral
activity values have been highlighted in bold. aCompound concentration (lM) required to reduce the viability of mock-infected MDBK (Bovine normal kidney)
cells by 50%, as determined by the MTT method. bCompound concentration (lM) required to achieve 50% protection of MDBK cells from the BVDV (Bovine
Viral Diarrhoea Virus) induced cytopathogenicity, as determined by the MTT method. cCompound concentration (lM) required to reduce the viability of mock-
infected BHK (Hamster normal kidney fibroblast) monolayers by 50%, as determined by the MTT method. dCompound concentration (lM) required to achieve
50% protection of BHK (Kidney fibroblast) cells from the YFV (Yellow Fever Virus)-induced cytopathogenicity, as determined by the MTT method.
BHK, Baby Hamster Kidney; MDBK, Madin Darby Bovine Kidney.
446
Chem Biol Drug Des 2011; 77: 441–449

Derivatives as new Anti Flaviviridae inhibitors
In particular, the effect of a further stereogenic centre was taken
into account as well as the presence of a more hindered ether
group.
Different substituents were placed on the aromatic rings. A differ-
ent behaviour was shown by compounds both with electron-with-
drawing groups and with electron-releasing groups on the
aromatic ring B. The presence of a chlorine atom in position 4 or
2 resulted in increased activity as seen for derivatives 2c and 2d
comparatively to the analogues 2e and 2a–b endowed with elec-
tron withdrawing substituents like nitrile and nitro group. To fur-
ther investigate SAR of the phenyl ring moiety A, we designed
and synthesized analogues bearing the 2-methoxy and 4-nitro sub-
stituents (3a and 3b). They showed significant activities against
Pesti and Flavivirus representative, but were also cytotoxic. Inter-
esting results were exhibited among analogues simultaneously
substituted on both phenyl rings; derivative 4a cis with 4¢-NO2 on
the ring A and 4¢¢-Cl on ring B displayed increased activity
against BVDV (14 lM) and no cytotoxic effect on the uninfected
host cell. When 4¢¢-Cl was replaced by 4¢¢-CF3 (4c cis) there was
a shift of activity, it was active against YFV (18 lM) but not
against BVDV. Replacement of NO2 group by methoxy in position
4¢ or 2¢, led to compounds 4e and 4f that showed a stronger
anti-BVDV and anti-YFV activity but demonstrated cytotoxic effect
on the uninfected host cells. The introduction of a methylene
spacer between dihydropyridinone and phenyl ring A caused a
loss of activity against BVDV (4b cis).
Therefore, compound 4a cis was chemoselectively reduced in the
presence of CeCl3 and NaBH4 in anhydrous THF at room tempera-
ture (22).
This reaction gave a mixture of two epimers in 6 ⁄ 4 ratio that was
treated, without purification, with pyridinium p-toluene sulfonate in
ethanol to afford 6a as the main isomer with trans- cis configura-
tion in 62% yield for two steps.
The stereochemistry was assigned on the basis of the of the 1H-
NMR coupling constant of the proton H4 (d = 3.37, doublet with
J = 10.0 Hz).
Roesy experiments confirmed this stereochemistry in which the aro-
matic ring is still in a pseudo-axial position (correlations of the aro-
matic protons with H4 and the protons of the methyl group; no
correlation between H3 and the protons of the aromatic ring (Fig-
ure 2).
Biological studies
Selective reduction of compound 4a cis and introduction of an ethyl
ether moiety afforded compound 6a which was almost fourfold
more potent against YFV (EC50 10 lM) virus (Scheme 2).
Some of these derivatives exhibited moderated activity against
BVDV effects in bovine MDBK cells and against YFV effects in BHK
kidney fibroblast.
In view of these observations, the most active compounds 4a cis,
4c cis and 6a have been selected for a preliminary antiviral study
against Hepaciviruses replicon assay. The aim of this assay is to
select agents which specifically affect the replication on the HCV
virus. The system is based on a RNA replicon (containing HCV non
structural proteins from NS3 to NS5B).
From the data obtained in parallel cell-based assays for cytotoxicity
and antiviral activity, several compounds exhibited cytotoxicity in
different cell lines. As far as the antiviral activity is concerned, title
dihydropyridinones can be divided into derivatives devoid of (1,
1a–e) or characterized by the 3,5-dimethyl substituents (2, 2a–e,
3a–b, 4a–f, 6a) (Table 1).
The HCV NS5B protein is a virally encoded RNA-dependent RNA
polymerase (RdRp), the activity of which is critical for the replica-
tion of the virus (23). The HCV RNA-dependent RNA polymerase
(RdRp, NS5B) is an attractive target for the development of novel
HCV treatments (24–26). The activity of this virally encoded
enzyme is essential for HCV replication and many nucleoside and
non-nucleoside NS5B inhibitors have been described in the litera-
ture (27).
The formers are completely inactive against tested virus, whereas
the introduction of two methyl groups led to an increase of antiviral
activity in particular for compounds with stereochemistry cis.
Starting from the lead compound 2, analogues 2a–e, 3a–b, 4a–e
were first synthesized to investigate the structure–activity relation-
ship (SAR) of the N1 and C2 positions.
OH
O
O
4
3
2
NaBH4, CeCl3
EtOH
N
N
N
dry THF
pyridinium p-toluene sulfonate
Cl
Cl
Cl
6
NO2
5a
NO2
6a
NO2
4a cis
Scheme 2: Synthesis of compound 6a.
Chem Biol Drug Des 2011; 77: 441–449
447

Peduto et al.
Table 2: Anti-HCV activity and cytotoxic activity of compounds
4a cis, 4c cis and 6a compared with ATA
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Compound
CC50 (lM)a
EC50 HCV (lM)b
SIc
ID50(lM)d
4a cis
4c cis
6a
>50
>50
22.7 € 2.2
23.2 € 3.3
4.0 0.7
0.075e
0.45
0.46
>25
175 € 20
‡200
140 20
>100
ATA
>10e
>133
1.5 0.5
aCompound concentration (lM) required to reduce the viability of HuH-7 cells
determined by the MTT method.
bCompound concentration (lM) required to achieve 50% protection of HuH-7
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tion for four independent experiments.
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cIn vitro selectivity index(CC50 ⁄ EC50).
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eData from Reference (19).
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virus as a surrogate model of hepatitis C virus for the evalua-
tion of antiviral agents. Antiviral Res;60:1–15.
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K., Colonno R., Gao M. (2005) Development of a cell-based
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To highlight the possible interactions, investigation of HCV NS5B
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for their ability to suppress the in vitro activity of recombinant HCV
RNA polymerase NS5B (Table 2).
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for the hepatitis C virus. Antiviral Res;52:1–17.
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Both compounds 6a and 4a cis showed a weak antienzymatic
activity, with 6a (50% inhibitory potency, ID50 = 140 € 20 lM),
slightly more potent than 4a cis (ID50 = 175 € 20). Compound 4c
cis was inactive (ID50 = ‡200). While there were substantial differ-
ences between the ID50 and EC50 values, which could be due, at
least partially, to the differences between in vitro enzymatic assay
conditions and the cellular replicon system, these results might sug-
gest that compound 6a can potentially target the HCV NS5B pro-
tein.
12. Wakita T., Pietschmann T., Kato T., Date T., Miyamoto M., Zhao
Z., Murthy K., Habermann A., Krausslich H.G., Mizokami M.,
Bartenschlager R., Liang T.J. (2005) Production of infectious hep-
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However, the fact that compounds 4a cis and 6a showed similar
antienzymatic activities against NS5B in vitro, but had different an-
tiviral selectivity in cellular systems, does not allow drawing any
definitive conclusion, at this stage, about the actual in vivo molecu-
lar target of our compounds. Further biological investigations should
be carried out to understand in detail the mechanism underlying an-
tiviral activity of these compounds.
14. Zhong J., Gastaminza P., Cheng G., Kapadia S., Kato T., Burton
D.R., Wieland S.F., Uprichard S.L., Wakita T., Chisari F.V. (2005)
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In conclusion, we have described the identification of a novel
series of dihydropyridinones derivatives, and the obtained preli-
minary results suggest that some of these compounds might
serve as potential candidates for antiviral agents. Initial optimiza-
tion of the series has resulted in compound 6a with significant
antiviral activity in replicon assays in the Huh-7 hepatoma cell
line harbouring a self-replicating HCV subgenomic replicon of
genotype 1b.
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