Discovery of Dihydro-Alkyloxy-Benzyl-Oxopyrimidines as Promising Anti-Influenza Virus Agents

Article abstract of DOI:10.1111/j.1747-0285.2011.01180.x

A series of novel dihydro-alkyloxy-benzyl-oxopyrimidine derivatives were synthesized and evaluated for their activity against influenza virus in Madin-Darby canine kidney cells. Four dihydro-alkyloxy-benzyl-oxopyrimidine derivatives (4a1, 4a2, 4a3, and 4d1) showed potent activity against influenza virus. Among them, compound 4a3 was the most promising lead with broad activity against influenza A (antiviral EC₅₀ values of 9 and 18μm for the A/H1N1 and A/H3N2 subtype, respectively) and influenza B viruses (EC₅₀: 33μm). The antiviral mechanism of action of these dihydro-alkyloxy-benzyl-oxopyrimidine derivatives must be quite different from that of the currently approved anti-influenza virus drugs that target the viral M2 or neuraminidase proteins. The dihydro-alkyloxy-benzyl-oxopyrimidine derivatives represent a new avenue for further optimization and development of novel anti-influenza virus agents. Dihydro-alkyloxy-benzyl-oxopyrimidine (DABO) derivatives were discoveried as new anti-influenza agents. Among them, compound 4a3 was the most promising with broad activity against influenza A (A/H1N1 and A/H3N2 subtype) and influenza B virus.

Full text of DOI:10.1111/j.1747-0285.2011.01180.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01180.x
Chem Biol Drug Des 2011; 78: 596–602
Research Article
Discovery of Dihydro-Alkyloxy-Benzyl-
Oxopyrimidines as Promising Anti-Influenza
Virus Agents
Mingyan Yu1, Ailin Liu2, Guanhua Du2,
Lieve Naesens3, Evelien Vanderlinden3,
Erik De Clercq3 and Xinyong Liu1*
circulating avian A ⁄ H5N1 virus with a mortality of more than 60% in
infected humans, continue to be a major threat to human health.
Influenza viruses are classified into three main types: A, B, and C.
Further subdivision in subtypes (e.g. A ⁄ H1N1 and A ⁄ H3N2) is based
on the structure of the hemagglutinin and neuraminidase (NA) anti-
gens. Currently available drugs for the prophylaxis and treatment of
influenza virus infections are the M2 ion channel blockers amanta-
dine and rimantadine (Figure 1) and the NA inhibitors oseltamivir
and zanamivir (Figure 1) (2,3). These four drugs are effective when
administered early after virus infection or through prophylactic use.
The usefulness of amantadine and rimantadine is limited because of
their neurological side-effects, the lack of activity against influenza
B virus, and the rapid emergence of drug-resistant virus mutants (3).
The NA inhibitors oseltamivir and zanamivir have a more favorable
safety profile and are active against all influenza (A and B) viruses.
Unfortunately, global spread of oseltamivir-resistant A ⁄ H1N1 viruses
has been described during the winter of 2008–2009 (4). Therefore,
there is an urgent need for novel anti-influenza virus agents with a
mechanism of action that differs from that of the existing drugs (5).
1Institute of Medicinal Chemistry, School of Pharmaceutical
Sciences, Shandong University, No. 44 Wenhuaxi Road, Jinan
250012, China
2Institute of Materia Medica, Chinese Academy of Medical
Sciences, Peking Union Medical College, Beijing 100050, China
3Rega Institute for Medical Research, Katholieke Universiteit
Leuven, B-3000 Leuven, Belgium
*Corresponding author: Xinyong Liu, xinyongl@sdu.edu.cn
A series of novel dihydro-alkyloxy-benzyl-oxopyr-
imidine derivatives were synthesized and evalu-
ated for their activity against influenza virus in
Madin–Darby canine kidney cells. Four dihydro-al-
kyloxy-benzyl-oxopyrimidine derivatives (4a1, 4a2,
4a3, and 4d1) showed potent activity against influ-
enza virus. Among them, compound 4a3 was the
most promising lead with broad activity against
influenza A (antiviral EC50 values of 9 and 18 lM
for the A ⁄ H1N1 and A ⁄ H3N2 subtype, respectively)
and influenza B viruses (EC50: 33 lM). The antiviral
mechanism of action of these dihydro-alkyloxy-
benzyl-oxopyrimidine derivatives must be quite
different from that of the currently approved anti-
influenza virus drugs that target the viral M2 or
neuraminidase proteins. The dihydro-alkyloxy-ben-
zyl-oxopyrimidine derivatives represent a new ave-
nue for further optimization and development of
novel anti-influenza virus agents.
Recently, several dihydro-alkyloxy-benzyl-oxopyrimidine (DABO) (Fig-
ure 1) derivatives were reported as potent non-nucleoside reverse
transcriptase inhibitors (NNRTIs) of HIV-1, exerting robust antiviral
activity against both the wild-type HIV-1 and a panel of clinically
relevant HIV-1 mutants bearing mutations in the RT enzyme (6).
Few studies have been carried out to evaluate the potential activity
of DABO compounds against other viruses than HIV-1, such as
influenza virus. We here report on the chemical synthesis, anti-
influenza A and B virus activity, and structure–activity relationship
of novel DABO derivatives. The potent anti-influenza virus activity
of selected DABO compounds creates an opportunity to design
novel anti-influenza virus agents that could be helpful to combat
influenza virus mutants with resistance to the existing M2 or NA
inhibitors.
Key words: anti-influenza agents, antiviral activity, DABO, influenza
A, influenza B, influenza virus
Received 15 February 2011, revised 2 July 2011 and accepted for publi-
cation 3 July 2011
Human influenza viruses are highly contagious pathogens that affect
the airway epithelia, resulting in acute respiratory symptoms with
potentially severe or life-threatening complications in specific risk
groups (1). It is estimated that, globally, influenza accounts for
approximately 250 000–500 000 deaths during annual influenza epi-
demicsa. Human influenza viruses also cause periodic pandemics such
as seen in 1918, 1957, and 1968, and, more recently, 2009. In addi-
tion, highly pathogenic avian influenza viruses, such as the currently
Materials and Methods
Chemistry
All melting points were determined on a micromelting point appara-
tus and are uncorrected. Infrared spectra (IR) were recorded with a
Nexus 470FT-IR Spectrometer. NMR spectra were obtained on a
Brucker Avance-600 NMR-spectrometer in the indicated solvents.
Chemical shifts are expressed in d units and TMS as internal
596

A Promising Anti-Influenza Virus Agent
Figure 1: Structures of the
existing anti-influenza drugs and
the novel dihydro-alkyloxy-benzyl-
oxopyrimidine derivatives.
reference. Mass spectra were taken on a LC Autosampler Device:
Standard G1313A instrument. Thin layer chromatography (TLC) was
performed on silica gel GF254 for TLC (Merck, Darmstadt, Germany),
and spots were visualized by iodine vapors or by irradiation with
UV light (254 nm). Solvents were reagent grade and, when neces-
sary, were purified and dried by standard methods. Concentration
of the reaction solutions involved the use of rotary evaporator at
reduced pressure. Compounds were prepared using the routes
shown in Scheme 1.
decanted, the residue was washed with THF (150 mL), and the com-
bined THF fractions were treated with aq. HCl (10%, 150 mL) at
room temperature for 45 min. The mixture was concentrated under
reduced pressure, diluted with CH2Cl2 (300 mL), and washed with
saturated NaHCO3 (3 · 350 mL) and brine (3 · 350 mL). The organic
phase was dried over anhydrous Na2SO4, filtered and concentrated
to give the crude products 2a–d, respectively, which were directly
used in the following step without further purification.
General procedure for the preparation of
5-alkyl-6-substituted thiouracil 3a–d
General procedure for the preparation of
b-ketoesters 2a–d
Sodium metal (8.2 g, 356 mmol) was dissolved in absolute ethanol
(50 mL), and thiourea (19 g, 249 mmol) and b-ketoesters 2a–d
(178 mmol) were added to the clear solution at room temperature.
The reaction mixture was refluxed for 6–12 h (checked by TLC)
under a nitrogen atmosphere. The reaction mixture was cooled to
room temperature. Then, solvent was evaporated, and the residues
were dissolved in H2O and were precipitated by addition of conc.
aq. HCl and subsequent acidification to pH 4 with glacial AcOH.
The resulting precipitate was filtered under reduced pressure. The
solid was washed sequentially with H2O, EtOH, and Et2O and then
dried to give 3a–db (7), which is directly used in the next step
without further purification.
The activated zinc dust was prepared by washing zinc dust sequen-
tially with 3 N aq. HCl, distilled water, ethanol, and ether and drying
under vacuum. Activated zinc dust (32.5 g, 500 mmol) was sus-
pended in dry tetrahydrofuran (THF) (200 mL) and refluxed under a
nitrogen atmosphere. A few drops of 2-bromoalkanoates were
added to initiate the reaction. After the appearance of a green color,
arylacetonitriles (100 mmol) were added in one portion followed by
the dropwise addition of 2-bromoalkanoates (260 mmol) over 1 h.
The reaction mixture was refluxed for an additional 2 h, diluted with
THF (600 mL), and quenched with aq K2CO3 (50%, 125 mL). Rapid
stirring for 60 min gave two distinct layers. The THF layer was
Scheme 1: Reagents and conditions: (a) (1) R1CHBrCOOEt, Zn, THF, reflux, (2) 50% K2CO3, (3) 13% HCl; (b) thiourea, EtONa, reflux, 6–
12 h; (c) appropriate N-phenylacetamide halides, K2CO3, DMF, rt, 12 h.
Chem Biol Drug Des 2011; 78: 596–602
597

Yu et al.
General procedure for the preparation of target
compounds 4a–d
(tC=O), 1271 (tC–N), 1259 (tC–N). ESI-MS: m ⁄ z 464.2 [M+H], 486.2
[M+Na]. C25H22ClN3O2S (463.11).
cCompounds 3a–d (5 mmol) and appropriate N-phenylacetamide
halides (5.5 mmol) were suspended in anhydrous DMF (25 mL) in the
presence of anhydrous potassium carbonate (0.759 g, 5.5 mmol) at
room temperature. The mixtures were irradiated at room tempera-
ture for 12 h. The reaction mixture was poured into cold H2O
(200 mL); the resulting precipitate was collected by filtration under
reduced pressure and washed sequentially with H2O, EtOH, and Et2O
and then dried to give the corresponding crude product, which was
purified by crystallization to give the pure target compounds 4a–d.
2-[(4-bromophenylamino)carbonylmethylthio]-6-
(1-naphthylmethyl)-5-ethylpyrimidin-4(3H)-one
(4b2)
Recrystallized from EtOH-DMF as a white crystal, Yield: 28.7%, mp:
224–226 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.93 (s, 1H, NH),
10.17 (s, 1H, NH), 8.11–7.08 (m, 11H), 4.28 (s, 2H, S-CH2), 3.91 (s,
2H, CH2), 2.38 (q, J = 7.2 Hz, CH2CH3, 2H), 0.83 (t, J = 7.2 Hz,
CH2CH3, 3H). IR (KBr, ⁄ cm): 3253 (tNH), 3043 (tNH), 1652 (tC=O),
1269 (tC–N), 1244 (tC–N). ESI-MS: m ⁄ z 408.1 [M+H], 530.1 [M+Na].
C25H22BrN3O2S (507.06).
2-[(4-chlorophenylamino)carbonylmethylthio]-6-
(1-naphthylmethyl)-5-methylpyrimidin-4(3H)-one
(4a1)
Recrystallized from EtOH-DMF as a white crystal, Yield: 23.6%, mp:
236–238 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.74 (s, 1H, NH),
10.18 (s, 1H, NH), 8.10–7.10 (m, 11H), 4.28 (s, 2H, S-CH2), 3.92 (s,
2H, CH2), 1.87 (s, CH3, 3H). IR (KBr, ⁄ cm): 3267 (tNH), 3117 (tNH),
1663 (tC=O), 1268 (tC–N), 1244 (tC–N). ESI-MS: m ⁄ z 450.5 [M+H],
472.4 [M+Na]. C24H20ClN3O2S (449.1).
2-[(4-methoxyphenylamino)carbonylmethylthio]-
6-(1-naphthylmethyl)-5-ethylpyrimidin-4(3H)-one
(4b3)
Recrystallized from EtOH-DMF as a white crystal, Yield: 22.9%, mp:
237–239 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.95 (s, 1H, NH),
10.09 (s, 1H, NH), 8.14–7.06 (m, 11H), 4.21 (s, 2H, S-CH2), 3.94 (s,
2H, CH2), 3.76 (s, OCH3, 3H), 2.39 (q, J = 7.2 Hz, CH2CH3, 2H), 0.85
(t, J = 7.2 Hz, CH2CH3, 3H). IR (KBr, ⁄ cm): 3255 (tNH), 3130 (tNH),
1650 (tC=O), 1291 (tC–N), 1251 (tC–N).ESI-MS: m ⁄ z 460.2 [M+H],
482.2 [M+Na]. C26H25N3O3S (459.16).
2-[(4-bromophenylamino)carbonylmethylthio]-6-
(1-naphthylmethyl)-5-methylpyrimidin-4(3H)-one
(4a2)
Recrystallized from EtOH-DMF as a white crystal, Yield: 23.9%, mp:
240–242 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.82 (s, 1H, NH),
10.19 (s, 1H, NH), 8.11–7.10 (m, 11H), 4.26 (s, 2H, S-CH2), 3.91 (s,
2H, CH2), 1.87 (s, CH3, 3H). IR (KBr, ⁄ cm): 3259 (tNH), 3045 (tNH),
1651 (tC=O), 1267 (tC–N), 1244 (tC–N). ESI-MS: m ⁄ z 494.1 [M+H],
516.1 [M+Na]. C24H20BrN3O2S (493.05).
2-[(4-chlorophenylamino)carbonylmethylthio]-6-
(2,6-difluorobenzyl)-5-methylpyrimidin-4(3H)-
one (4c1)
Recrystallized from EtOH-DMF as a white crystal, Yield: 27.1%, mp:
1
248–250 ꢀC (dec). H NMR (DMSO-d6, ppm) d: 12.86 (s, 1H, NH),
10.15 (s, 1H, NH), 7.54 (d, J = 9Hz, 2H), 7.37 (d, J = 9Hz, 2H), 7.15–
6.85 (m, 3H), 3.85 (s, 2H, S-CH2), 3.80 (s, 2H, CH2), 2.00 (s, CH3, 3H).
IR (KBr, ⁄ cm): 3262 (tNH), 3044 (tNH), 1658 (tC=O), 1267 (tC–N), 1247
(tC–N). ESI-MS: m ⁄ z 436.4 [M+H]. C20H16ClF2N3O2S (435.06).
2-[(4-methoxyphenylamino)carbonylmethylthio]-
6-(1-naphthylmethyl)-5-methylpyrimidin-4(3H)-
one (4a3)
Recrystallized from EtOH-DMF as a white crystal, Yield: 24.2%, mp:
239–241 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.78 (s, 1H, NH),
9.93 (s, 1H, NH), 8.16 (d, J = 8.4Hz, 1H), 7.91 (d, J = 7.8Hz, 1H),
7.75 (d, J = 8.4Hz, 1H), 7.54–7.15 (m, 6H), 6.80 (d, J = 8.4Hz, 2H),
4.30 (s, 2H, S-CH2), 3.90 (s, 2H, CH2), 3.70 (s, OCH3, 3H), 1.89 (s,
CH3, 3H).IR (KBr, ⁄ cm): 3290 (tNH), 3042 (tNH), 1653 (tC=O), 1299
(tC–N), 1246 (tC–N). ESI-MS: m ⁄ z 446.2 [M+H], 468.2 [M+Na].
C25H23N3O3S (445.15).
2-[(4-bromophenylamino)carbonylmethylthio]-6-
(2,6-difluorobenzyl)-5-methylpyrimidin-4(3H)-
one (4c2)
Recrystallized from EtOH-DMF as a white crystal, Yield: 26.6%, mp:
251–253 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.72 (s, 1H, NH),
10.10 (s, 1H, NH), 7.50–6.87 (m, 7H), 3.85 (s, 2H, S-CH2), 3.82 (s,
2H, CH2), 2.00 (s, CH3, 3H). IR (KBr, ⁄ cm): 3261 (tNH), 3041(tNH),
16594(tC=O), 1267 (tC–N), 1247 (tC–N). ESI-MS: m ⁄ z 480.2 [M+H],
502.2 [M+Na]. C20H16BrF2N3O2S (479.01).
2-[(4-chlorophenylamino)carbonylmethylthio]-6-
(1-naphthylmethyl)-5-ethylpyrimidin-4(3H)-one
(4b1)
Recrystallized from EtOH-DMF as a white crystal, Yield: 25.7%, mp:
mp: 217–219 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.78 (s, 1H,
NH), 10.17 (s, 1H, NH), 8.12 (d, J = 7.8Hz, 1H), 7.88 (d, J = 7.2Hz,
1H), 7.71(d, J = 7.8Hz, 1H), 7.53–7.09 (m, 8H), 4.29 (s, 2H, S-CH2),
3.92 (s, 2H, CH2), 2.37 (q, J = 7.2 Hz, CH2CH3, 2H), 0.84 (t,
J = 7.2 Hz, CH2CH3, 3H). IR (KBr, ⁄ cm): 3271 (tNH), 3045 (tNH), 1659
2-[(4-methoxyphenylamino)carbonylmethylthio]-
6-(2,6-difluorobenzyl)-5-methylpyrimidin-4(3H)-
one (4c3)
Recrystallized from EtOH-DMF as a white crystal, Yield: 23.5%, mp:
233–235 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d:12.74 (s, 1H, NH),
9.83 (s, 1H, NH), 7.42–6.87 (m, 7H), 3.87 (s, 2H, S-CH2), 3.77 (s, 2H,
CH2), 3.60 (s, OCH3, 3H), 2.00 (s, CH3, 3H). IR (KBr, ⁄ cm): 3257
598
Chem Biol Drug Des 2011; 78: 596–602

A Promising Anti-Influenza Virus Agent
(tNH), 3050 (tNH), 1656 (tC=O), 1248 (tC–N). ESI-MS: m ⁄ z 432.4
[M+H], 454.3 [M+Na]. C21H19F2N3O3S (431.11).
The following control compounds were included: ribavirin (Virazoleꢁ),
obtained from ICN Pharmaceuticals; amantadine (Sigma);
rimantadine (Sigma); and oseltamivir carboxylate (GS-4071) (a kind
gift from T. Cihlar, Gilead Sciences, Foster City, CA, USA). The test
compounds were evaluated for anti-influenza virus activity by a mul-
ticycle cytopathic effect (CPE) reduction assay. MDCK cells were
seeded into 96-well plates at 7500 cells per well 16 h prior to
infection and incubated at 35 ꢀC. Serial dilutions of the compounds
were added to the cells, together with the influenza virus (multiplic-
ity of infection, fifty 50% cell culture infective doses [CCID50] per
well, which corresponds to 0.0004 PFU per cell). At 3 days postin-
fection (p.i.), microscopy was performed to determine the antiviral
activity, expressed as the compound concentration producing 50%
inhibition of virus-induced CPE [50% effective concentration (EC50)],
as well as the cytotoxicity of the compounds, expressed as the
compound concentration causing minimal changes in cell morphol-
ogy (MCC). The data were confirmed by the formazan-based 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium (MTS) cell viability assay (CellTiter 96 AQueous One
Solution Cell Proliferation Assay from Promega, Madison, WI, USA),
and the spectrophotometric data were used to calculate the EC50
and 50% cytotoxic concentration (CC50).
2-[(4-chlorophenylamino)carbonylmethylthio]-6-
(2,6-difluorobenzyl)-5-ethylpyrimidin-4(3H)-one
(4d1)
Recrystallized from EtOH-DMF as a white crystal, Yield: 26.6%,
mp: 222–224 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.73 (s, 1H,
NH), 10.10 (s, 1H, NH), 7.54 (d, J = 9Hz, 2H), 7.37 (d, J = 9Hz,
2H), 7.10–6.85 (m, 3H), 3.86 (s, 2H, S-CH2), 3.80 (s, 2H, CH2), 2.51
(q, J = 7.2 Hz, CH2CH3, 2H), 1.01(t, J = 7.2 Hz, CH2CH3, 3H). IR
(KBr, ⁄ cm): 3288 (tNH), 3054 (tNH), 1673 (tC=O), 1650 (tC=O), 1269
(tC–N), 1244 (tC–N). ESI-MS: m ⁄ z 450.3 [M+H]. C21H18ClF2N3O2S
(449.08).
2-[(4-bromophenylamino)carbonylmethylthio]-6-
(2,6-difluorobenzyl)-5-ethylpyrimidin-4(3H)-one
(4d2)
Recrystallized from EtOH-DMF as a white crystal, Yield: 23.1%, mp:
223–225 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.70 (s, 1H, NH),
10.09 (s, 1H, NH), 7.50 (d, J = 9Hz, 2H), 7.47 (d, J = 9Hz, 2H),
7.10–6.85 (m, 3H), 3.86 (s, 2H, S-CH2), 3.80 (s, 2H, CH2), 2.53 (q,
J = 7.2 Hz, CH2CH3, 2H), 1.01(t, J = 7.2 Hz, CH2CH3, 3H). IR (KBr,
⁄ cm): 3284 (tNH), 3056 (tNH), 1657 (tC=O), 1266 (tC–N), 1243 (tC–N).
ESI-MS: m ⁄ z 494.2 [M+H]. C21H18BrF2N3O2S (493.03).
Neuraminidase inhibition assay
Four compounds were evaluated for in vitro inhibitory actions using
the method reported by Guanhua Du and, as the source of NA, the
influenza virus strain A ⁄ H1N1 [strain A ⁄ PR ⁄ 8 ⁄ 34] described by
Laver. This spectrofluorometric assay (excitation wavelength:
355 nm and emission wavelength: 460 nm) uses 2¢-(4-methylumbel-
liferyl)-a-D-acetylneuraminic acid (MUNANA) as the enzyme sub-
strate, which is converted to a fluorescent product upon cleavage
by NA. The intensity of fluorescence can reflect the activity of NA
sensitively.
2-[(4-methoxyphenylamino)carbonylmethylthio]-
6-(2,6-difluorobenzyl)-5-ethylpyrimidin-4(3H)-
one (4d3)
Recrystallized from EtOH-DMF as a white crystal, Yield: 27.1%, mp:
232–234 ꢀC (dec). 1H NMR (DMSO-d6, ppm) d: 12.66(s, 1H, NH),
9.85 (s, 1H, NH), 7.42–6.87 (m, 7H), 3.89 (s, 2H, S-CH2), 3.76 (s, 2H,
CH2), 3.72(s, OCH3, 3H), 2.51 (q, J = 7.2 Hz, CH2CH3, 2H), 1.02(t,
J = 7.2 Hz, CH2CH3, 3H). IR (KBr, ⁄ cm): 3250 (tNH), 3050 (tNH), 1656
(tC=O), 1241 (tC–N). ESI-MS: m ⁄ z 446.3 [M+H], 468.3 [M+Na].
C22H21F2N3O3S (445.13).
In the enzyme reaction system, there were 30 lL of the enzyme in
33 mM MES buffer (pH 3.5), 10 lL of 4 mM CaCl2, 20 lL of 20 lM
MUNANA, and 30 lL water in a 96-well microplate. The terminal
volume was 100 lL. After 10 min at 37 ꢀC, 150 lL of 14 mM NaOH
in 83% ethanol was added to 0.1 mL of the reaction mixture to ter-
minate the reaction. The intensity of the fluorescence was quanti-
tated in Fluostar Galaxy (excitation, 360 nm; emission, 450 nm), and
substrate blanks were subtracted from the sample readings. The
IC50 was calculated by plotting percent inhibition versus the inhibi-
tor concentration, and determination of each point was performed
in duplicate.
Antiviral activities in influenza A ⁄ H1N1,
A ⁄ H3N2, and B virus-infected Madin–Darby
canine kidney (MDCK) cells
Madin–Darby canine kidney cells (8) were grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal
calf serum (FCS; Integro, Zaandam, The Netherlands), 1 mM sodium
pyruvate, and 0.075% sodium bicarbonate. During virus experiments,
the MDCK cells were maintained in Ultra MDCK medium (Lonza,
Basel, Switzerland), supplemented with 0.0225% sodium bicarbon-
ate, 2 mM L-glutamine, and 2 lg ⁄ mL tosylphenylalanylchloromethyl-
keton-treated trypsin (Sigma, St Louis, MO, USA). The human
influenza virus strains A ⁄ PR ⁄ 8 ⁄ 34 (A ⁄ H1N1) and B ⁄ HK ⁄ 5 ⁄ 72 were
purchased from the American Type Culture Collection (ATCC). The
strain A ⁄ HK ⁄ 7 ⁄ 87 (A ⁄ H3N2) was obtained from J. Neyts (Katho-
lieke Universiteit Leuven, Leuven, Belgium). Virus stocks of these
laboratory-adapted strains were prepared by passaging them in
10-day-old embryonated chicken eggs.
Results and Discussion
Chemistry
Compounds were prepared using the routes shown in Scheme 1.
The b-ketoesters (2a–d) were prepared by a simple and high-yield-
ing method of Hannick and Kishi, through the reaction of arylaceto-
nitriles (1a–b) with activated zinc dust and 2-bromoalkanoates
(9,10). Subsequent condensation of b-ketoesters (2a–d) with thio-
urea in the presence of EtONa in refluxing ethanol gave the key
intermediates 5-alkyl-6-substituted thiouracil 3a–d (11). Next,
Chem Biol Drug Des 2011; 78: 596–602
599

Yu et al.
selective S-alkylation of 3a–d with the appropriate N-phenylaceta-
mide halides (1:1.1) in the presence of K2CO3 in anhydrous DMF
afforded the DABO compounds 4a–d. The analytical and spectral
data of the all compounds were in full agreement with the pro-
posed structures.
tration), representing the compound concentration at which MCC
were observed (12,13). In all experiments, oseltamivir carboxylate,
ribavirin, amantadine, and rimantadine were used as the reference
compounds. The data obtained are shown in Table 1.
Four DABO compounds exhibited good activity against influenza
virus. The most active and selective DABO derivative was compound
4a3, which had broad anti-influenza virus activity, with antiviral
EC50 values (i.e. average values from both the CPE and MTS assay)
of 9, 18, and 33 lM, for influenza A ⁄ H1N1, A ⁄ H3N2, and influenza
B virus, respectively. Moreover, compound 4a3 displayed no cyto-
toxicity at the highest concentration tested (224 lM), thus yielding
a selectivity index (defined as the ratio of MCC to EC50) of at least
7 (for influenza B virus) or at least 12–25 (for influenza A ⁄ H1N1
and A ⁄ H3N2). In this respect, 4a3 was superior to the reference
compound ribavirin, which had a lower selectivity index (ꢀ11) than
4a3. Interestingly, 4a3 showed favorable activity against the
A ⁄ PR ⁄ 8 ⁄ 34 (A ⁄ H1N1) strain, which carries two amantadine resis-
tance mutations in the M2 protein, as reflected in its high EC50 val-
Antiviral activities in influenza A ⁄ H1N1,
A ⁄ H3N2, and B virus-infected MDCK cells
The compounds were evaluated for antiviral activity against human
influenza viruses of the A ⁄ H1N1 (strain A ⁄ PR ⁄ 8 ⁄ 34), A ⁄ H3N2
(strain A ⁄ HK ⁄ 7 ⁄ 87), and B (strain B ⁄ HK ⁄ 5 ⁄ 72) (sub)type, using a
cell-culture-based assay in MDCK cells, as previously described
(12,13). Antiviral activity was expressed as the 50% antivirally
effective concentration (EC50) or compound concentration producing
50% inhibition of virus-induced CPE, as determined by microscopy
or MTS assay. Cytotoxicity was expressed as the 50% cytotoxic
concentration (CC50), or compound concentration causing 50% cyto-
toxicity in the MTS assay, and the MCC (minimal cytotoxic concen-
Table 1: Anti-influenza virus activity of dihydro-alkyloxy-benzyl-oxopyrimidine derivatives in Madin–Darby canine kidney cells
O
R1
HN
O
S
N
NH
Ar
R2
a
Antiviral EC50 (lM)
Cytotoxicity (lM)
Influenza
A ⁄ H1N1
Influenza
A ⁄ H3N2
Influenza B
CPE
MCCc
b
Cpd No.
Ar
R1
R2
CPE
MTS
CPE
MTS
MTS
CC50
4a1
4a2
4a3
4b1
4b2
4b3
4c1
4c2
4c3
4d1
4d2
4d3
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH3
CH3
CH3
Cl
Br
OCH3
Cl
Br
OCH3
Cl
Br
OCH3
Cl
Br
>222
>202
9.0
>216
>197
>218
>229
>209
>232
133
>202
>224
2.6
10
134
>222
>202
9.0
21
55
26
22
44
9.7
>222
>202
‡45
>222
>202
‡22
>222
>202
>224
11
133
‡20
>224
‡8.6
7.9
‡8.7
>229
‡42
‡46
‡44
‡40
45
>216
>197
>218
>229
>209
>232
‡22
>202
>224
4.7
>216
>197
>218
>229
>209
>232
27
>202
>224
1.7
>216
>197
>218
>229
>209
>232
16
>202
>224
2.4
>216
>197
>218
>229
>209
>232
>222
>202
>224
>100
9
>216
>197
>218
>229
>209
>232
>222
>202
>224
>100
8.4
18
>218
>229
>209
>232
>222
>202
>224
>100
>100
>500
258
CH2CH3
CH2CH3
CH2CH3
OCH3
Oseltamivir carboxylate
Ribavirin
Amantadine
>100
‡100
>500
500
9.7
149
34
10
2.4
0.15
9
3.9
0.05
>500
>500
>500
>500
Rimantadine
32
MCC, minimal changes in cell morphology.
aAntiviral activity was expressed as the EC50 value, defined as the compound concentration producing 50% inhibition of virus replication, as estimated by
microscopic scoring of the cytopathic effect (CPE), or by measuring cell viability in the formazan-based MTS assay. Values are the mean of duplicate experi-
ments.
b50% cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay.
cMinimum compound concentration that causes a microscopically detectable alteration of normal cell morphology.
600
Chem Biol Drug Des 2011; 78: 596–602

A Promising Anti-Influenza Virus Agent
ues for the M2 inhibitors amantadine and rimantadine (Table 1).
Finally, 4a3 was also found to have more consistent anti-influenza
virus activity than oseltamivir carboxylate, an NA inhibitor for which
the outcome in cell-culture-based antiviral assays is prone to some
variation (14).
Table 2: Evaluation of the dihydro-alkyloxy-benzyl-oxopyrimidine
derivatives for inhibitory effect on influenza virus neuraminidasea
b
Cpd. No.
Concentration
Inhibition ratio (%)
IC50
4a1
4a2
89 lM
81 lM
90 lM
89 lM
12 nM
16
11
7
6
99
–
–
–
–
4a3
4d1
Two related compounds, 4a1 and 4a2, had moderate activity
against influenza A ⁄ H3N2 (EC50: 22 and 50 lM, respectively). Com-
pound 4d1 also showed activity against both influenza A ⁄ H1N1
and A ⁄ H3N2 [average EC50 values: 78 lM (A ⁄ H1N1) and 22 lM
(A ⁄ H3N2)]. For these four active compounds, the 50% cytotoxic con-
centrations determined by MTS assay were higher than ꢀ200 lM
(the highest concentration tested). Microscopical evaluation demon-
strated that 4a1, 4a2, 4a3, and 4d1 caused MCC at compound
concentrations of 133, 20, >224, and 44 lM, respectively.
Zanamivir
0.29 nM
aInfluenza virus strain used: strain A ⁄ PR ⁄ 8 ⁄ 34.
b''–'' means that the IC50 could not be calculated.
Conclusions
In conclusion, a series of novel DABO derivatives were synthesized
and found to have broad anti-influenza virus activity. The action
mechanism of DABO derivatives appears to be quite different from
that of currently approved anti-influenza virus drugs. These DABO
inhibitors represent a new avenue for future research and develop-
ment of novel anti-influenza drugs. Further studies on molecular
modification and specific target identification of these DABO deriva-
tives are underway in our laboratories.
Structure-activity relationship (SAR) analysis on this series of com-
pounds demonstrates that the anti-influenza virus activity of these
DABO derivatives is influenced by the substituent at the C-6 posi-
tion of the pyrimidine ring. Compounds with the large side chain
(1-naphthylmethyl at C-6) showed better inhibitory activity than
those with small ones (2,6-difluorobenzyl analogs), and these results
indicate that steric factors influence the inhibitory activity. The nat-
ure of the R2 substituent at the phenyl ring of the C-2 side chain
also influenced the anti-influenza virus activity of these DABO deriv-
atives, the order being (from high to low activity) 4-OCH3 > 4-Cl >
4-Br, thus emphasizing the importance of having an electron-donat-
ing group at the phenyl moiety. Replacement of the R1 methyl at
the C-5 position of the pyrimidine ring by an ethyl group leads to
compounds devoid of anti-influenza virus activity. The only exception
to this limited SAR analysis was 4d1, which showed activity
against influenza A ⁄ H3N2. From the above, the antiviral activity of
DABO derivatives appears to depend on different factors: electronic
properties [compound 4a3 with an electron-donating group on the
C-2 side chain phenyl is clearly more active than the corresponding
derivatives 4a1 and 4a2 with an electron-withdrawing substituent]
and steric bulkiness [6-(1-naphthylmethyl) analogs are more active
than the 6-(2,6-difluorobenzyl) analogs].
Acknowledgments
We are grateful to Leentje Persoons and Wim van Dam for excel-
lent technical assistance. We gratefully acknowledge the financial
support from Graduate Independent Innovation Foundation of Shan-
dong University (GIIFSDU No. 21310070613090), Joint Research
Fund for Overseas Chinese Young Scholars of Natural Science Foun-
dation of China (NSFC No.30928032), the National Natural Science
Foundation of China (NSFC No.30873133, No.30772629,
No.30371686), Key Project of NSFC for International Cooperation
(No.30910103908), the Research Fund for the Doctoral Program of
Higher Education of China (No. 20070422083), the Fonds voor Wet-
enschappelijk Onderzoek Vlaanderen (FWO No. 9.0188.07), and the
Geconcerteerde Onderzoeksacties (GOA ⁄ 10 ⁄ 014).
Neuraminidase inhibition assay
References
With regard to the antiviral mechanism of action, we are currently
trying to identify the target of the four active DABO compounds.
They are effective against both influenza A and B type viruses,
implying that their mechanism of action differs from that of the M2
ion channel blockers amantadine and rimantadine, which are known
to be inactive against influenza B virus.
1. Lewis D.B. (2006) Avian flu to human influenza. Annu Rev
Med;57:139–154.
2. De Clercq E. (2006) Antiviral agents active against influenza A
viruses. Nat Rev Drug Discov;5:1015–1025.
3. Moscona A. (2008) Medical management of influenza infection.
Annu Rev Med;59:397–413.
4. Moscona A. (2009) Global transmission of oseltamivir-resistant
influenza. New Engl J Med;360:953–956.
5. Weinstock D.M., Zuccotti G. (2009) The evolution of influenza
resistance and treatment. J Am Med Assoc;301:1066–1069.
6. Mugnaini C., Alongi M., Togninelli A., Gevariya H., Brizzi A., Ma-
netti F., Bernardini C. et al. (2007) Dihydro-alkylthio-benzyl-oxo-
pyrimidines as inhibitors of reverse transcriptase: synthesis and
rationalization of the biological data on both wild-type enzyme
and relevant clinical mutants. J Med Chem;50:6580–6595.
In addition, we verified whether these DABO derivatives may be
acting on the viral NA. The enzymatic NA inhibitory assay was
performed using the method reported by Guanhua Du (15) and, as
the source of NA, the influenza virus strain A ⁄ H1N1 [strain
A ⁄ PR ⁄ 8 ⁄ 34] described by Laver (16). The compound concentration
producing 50% inhibition of NA activity (IC50) was calculated by
plotting the percentage inhibition versus the inhibitor concentra-
tion, with each data point performed in duplicate. The results
showed that the DABO compounds had no inhibitory effect on NA
(Table 2).
Chem Biol Drug Des 2011; 78: 596–602
601

Yu et al.
7. Mai A., Artico M., Sbardella G., Massa S., Novellino E., Greco
G., Loi A.G., Tramontano E., Marongiu M.E., La Colla P. (1999) 5-
Alkyl-2-(alkylthio)-6-(2,6-dihalophenylmethyl)-3, 4- dihydropyrimi-
din- 4(3H)-ones: novel potent and selective dihydro-alkoxy-ben-
zyl-oxopyrimidine derivatives. J Med Chem;42:619–627.
8. Matrosovich M., Matrosovich T., Carr J., Roberts N.A., Klenk
H.D. (2003) Overexpression of the alpha-2,6-sialyltransferase in
MDCK cells increases influenza virus sensitivity to neuramini-
dase inhibitors. J Virol;77:8418–8425.
ship of aglycoristocetin derivatives with cyclobutenedione carry-
ing hydrophobic chains. Antiviral Res;82:89–94.
13. Vanderlinden E., Gçktas F., Cesur Z., Froeyen M., Reed M.L.,
Russell C.J., Cesur N., Naesens L. (2010) Novel inhibitors of
influenza virus fusion: structure-activity relationship and interac-
tion with the viral hemagglutinin. J Virol;84:4277–4288.
14. Tisdale M. (2000) Monitoring of viral susceptibility: new chal-
lenges with the development of influenza NA inhibitors. Rev
Med Virol;10:45–55.
9. Hannick S.M., Kishi Y. (1983) An improved procedure for the
Blaise reaction: a short, practical route to the key intermediates
of the saxitoxin synthesis. J Org Chem;48:3833–3835.
15. Morphy R., Rankovic Z. (2005) Designed multiple ligands. An
emerging drug discovery paradigm. J Med Chem;48:6523–6543.
16. Laver W.G., Coleman P.M., Webster R.G., Hinshaw V.S., Air G.M.
(1984) Influenza virus neuraminidase with hemagglutinin activity.
Virology;137:314–323.
10. Danel K., Larsen E., Pedersen E.B. (1995) Easy synthesis of 5,6-
disubstituted acyclouridine derivatives. Synthesis;8:934–936.
11. Radi M., Falciani C., Contemori L., Petricci E., Maga G., Samuele
A., Zanoli S., Terrazas M., Castria M., Togninelli A., Estꢀ J.A.,
Clotet-Codina I., Armand-Ugꢁn M., Botta M. (2008) A multidisci-
plinary approach for the identification of novel HIV-1 non-nucleo-
side reverse transcriptase inhibitors: S-DABOCs and DAVPs.
ChemMedChem;3:573–593.
Notes
aWorld Health Organization reported on 24 February 2010. Available
from: http://www.who.int/.
b3a, Mp 218–220 ꢀC; 3b, Mp 201–203 ꢀC; 3c, Mp 279–281 ꢀC
(dec); 3d, Mp 248–250 ꢀC (dec).
12. Naesens L., Vanderlinden E., Roth E., Jeko J., Andrei G., Snoeck
R., Pannecouque C., Illyꢀs E., Batta G., Herczegh P., Sztaricskai F.
(2009) Anti-influenza virus activity and structure-activity relation-
602
Chem Biol Drug Des 2011; 78: 596–602