Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4263
145.0 (d, JC,F = 10.2, C-7), 146.6 (C-2), 153.3 (d, JC,F = 249.5,
C-6), 165.1 (CONH), 175.3 (d, JC,F = 2.2, C-4), 175.3
(CONH2). 20: yield 38%; mp 217-218 ꢀC. IR [cm-1]: 826,
1039, 1171, 1309, 1489, 1530, 1664, 1737, 2985, 3258. 1H
NMR (DMSO-d6, δ [ppm], J [Hz]): 1.28 (t, J = 7.1, CH3),
1.54 (t, J = 7.2, CH3), 1.94-2.03 (m, 2 ꢀ piperinyl-H), 2.07-
2.11 (m, 2 ꢀ piperinyl-H), 2.50-2.59 (m, piperinyl-H), 2.92-
2.99 (m, 2 ꢀ piperinyl-H), 3.61-3.64 (m, 2 ꢀ piperinyl-H), 4.18
(d, J = 7.1, CH2), 4.23 (q, J = 7.2, CH2), 4.70 (d, J = 6.0, CH2),
6.80 (d, J = 8.3, benzyl-H), 7.19 (dd, J = 8.3, J = 2.0, benzyl-
H), 7.38 (d, J = 2.0, benzyl-H), 7.40 (d, JF,H = 8.6, H-8), 8.08 (d,
JF,H = 13.1, H-5), 8.70 (H-2), 10.54 (t, J = 6.0, NH). 13C NMR
(DMSO-d6, δ [ppm], J [Hz]): 14.4 (CH3), 14.6 (CH2), 28.1 (2 ꢀ
piperinyl-C), 40.7 (piperinyl-C), 49.3 (CH2), 49.9 (2 ꢀ piperinyl-
C), 60.8 (CH2), 104.0 (d, JC,F = 2.2, C-8), 111.4 (C-3), 113.2 (d,
JC,F = 22.7, C-5), 122.6 (d, JC,F = 6.7, C-4a), 127.3 (benzyl-C),
129.3 (benzyl-C), 130.5 (benzyl-C), 133.5 (benzyl-C), 134.3
113.7 (d, JC,F = 22.7, C-5), 123.4 (d, JC,F = 2.9, C-4a), 127.6
(benzyl-C), 129.7 (benzyl-C), 130.8 (benzyl-C), 133.9 (benzyl-
C), 134.6 (benzyl-C), 135.5 (benzyl-C), 136.9 (C-8a), 145.5 (d,
JC,F = 11.0, C-7), 147.2 (C-2), 153.7 (d, JC,F = 248.8, C-6),
155.9 (NHRCO2R), 165.7 (CONH), 175.8 (d, JC,F = 2.2, C-4).
23: yield 14%; mp 220-222 ꢀC. IR [cm-1]: 1119, 1240, 1562,
1608, 1626, 1666, 1716, 2945, 2981, 3095. 1H NMR (DMSO-d6,
δ [ppm], J [Hz]): 1.16 (b, 2 ꢀ cyclopropyl-H), 1.29 (t, J = 7.1,
CH3), 1.33-1.32 (m, 2 ꢀ cyclopropyl-H), 3.26-3.24 (m, 4 ꢀ
piperazinyl-H), 3.45 (b, cyclopropyl-H), 3.72-3.70 (m, 2 ꢀ
piperazinyl-H), 4.19 (q, J = 7.1, H2), 4.68 (d, J = 6.0, CH2),
7.18 (dd, J = 8.3, J = 2.0, benzyl-H), 7.32 (d, JF,H = 7.1, H-8),
7.37 (d, J = 2.0, benzyl-H), 7.38 (d, J = 8.3, benzyl-H), 8.04 (d,
JF,H = 13.1, H-5), 8.81 (s, H-2), 10.46 (t, J = 6.0, NH). 13C
NMR (DMSO-d6, δ [ppm], J [Hz]): 8.32 (2 ꢀ cyclopropyl-C),
14.8 (CH3), 34.9 (cyclopropyl-C), 40.7 (CH2), 43.6 (2 ꢀ piper-
azinyl-C), 50.0 (2 ꢀ piperazinyl-C), 61.8 (CH2), 105.1 (d, JC,F
=
2.9, C-8), 111.3 (C-3), 113.0 (d, JC,F = 22.7, C-5), 122.3 (d, JC,F
= 3.7, C-4a), 127.3 (benzyl-C), 129.3 (benzyl-C), 130.5 (benzyl-
C), 133.6 (benzyl-C), 134.3 (benzyl-C), 135.1 (benzyl-C), 138.6
(benzyl-C), 135.2 (benzyl-C), 136.7 (C-8a), 145.6 (d, JC,F =
11.0, C-7), 146.8 (C-2), 153.5 (d, JC,F = 249.6, C-6), 165.5 (C-4),
174.5 (CO2Et), 175.5 (d, JC,F = 2.2, C-4).
(C-8a), 144.9 (d, JC,F = 11.7, C-7), 147.1 (C-2), 153.6 (d, JC,F
=
General Procedure for the Synthesis of 4-oxo-1,4-Dihydroqui-
noline-3,5-dicarboxylic Acid (210) and 4-oxo-1,4-Dihydroquino-
line-3,7-dicarboxylic Acid (2100). Compounds 3e0/3e00 (10 mmol)
were dissolved in an aqueous sodium hydroxide solution (10%)
and heated at 90 ꢀC for 50 min. The mixture was cooled to room
temperature, and aqueous conc hydrochloride acid solution
added until pH = 2. The precipitate was filtered, washed several
times with water, and dried in vacuo to give 210/2100, a mixture of
regioisomers substituted in the 5- or 7-position. The ratio of the
isomers was determined by NMR spectroscopy. 210/2100(1:2):
yield 97%; mp >350 ꢀC. IR [cm-1]: 809, 907, 1069, 1202, 1385,
1594, 1634, 1645, 1698, 2993, 3063, 3217. 1H NMR (DMSO-d6,
δ [ppm], J [Hz]): 210: 8.00 (t, J = 7.6, H-7), 8.08 (d, J = 7.6, H-6),
8.88 (s, H-2), 8.18-8.21 (m, H-8), 14.16 (b, NH), 15.05 (b,
COOH). 2100: 8.01 (dd, J = 8.6, J = 1.7, H-6), 8.35 (d, J = 8.6,
H-5), 8.45 (d, J = 1.7, H-8), 8.89 (s, H-2), 14.16 (b, NH), 14.67
(b, COOH).
249.6, C-6), 155.5 (NHRCO2R), 165.3 (CONH), 175.6 (C-4).
Cells and Viruses. Vero (African green monkey, ATCC CRL
6318), Vero E6, and MDCK (Madin-Darby canine kidney)
cells were grown in Eagle’s minimal essential medium (MEM,
Gibco) supplemented with 10% fetal calf serum (FCS, Bio-
chrom), 100 U/mL penicillin, and 100 μg/mL streptomycin. The
NiV strain used in this work was isolated from human brain
tissue (kindly provided by Jane Cardosa, Institute of Health and
Community Medicine, University Malaysia Sarawak, Malaysia)
and propagated in Vero E6 cells. Stock virus was harvested when
the cytopathic effect was maximal. For infection, target cells were
incubated for 1 h at 37 ꢀC with NiV and then medium containing
2% FCS was added and the cells were further incubated for 24 h.
NiV G and F Expression Plasmids and Transfection. To obtain
the two expression plasmids pczCFG5-NiVG and pczCFG5-
NiVFtag, DNA fragments comprising the NiV open reading
frames for G and F were cloned into a derivative of the
replication-deficient murine leukemia virus vector pczCFG5
expressing in addition a zeocin resistance gene fused to the
enhanced green fluorescent protein (EGFP) gene.43,44 For tran-
sient expression, cells were transfected using polyethylenimine
(PEI 25 kDa; Polyscience). Briefly, a monolayer of 4 ꢀ 105 Vero
cells per well of a six-well plate were washed with prewarmed
medium, and the mixture of 2 μg DNA in 50 μL of serum-free
medium and 3 μL of PEI (from 1 mg/mL stock in serum-free
medium) in 50 μL of serum-free medium (incubated for 30 min
at RT) was added to the 1 mL medium per well, and subse-
quently compounds were added to the wells. Syncytia were
observed after 24 h incubation at 37 ꢀC.
Cell Vitality Assay. The tetrazolium salt 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT; Sigma) was
incorporated in living cells and converted into a violet formazan
product by mitochondrial dehydrogenases. Only living, vital
cells and cells in the very early phase of apoptosis can transform
MTT to crystals. Vero cells (1 ꢀ 105) in a six-well plate were
preincubated for 16 h with chemical compounds. Medium was
removed and cells incubated for 2 h with 1 mL/well fresh
medium (MEM 5% FCS). After removal of the medium, cells
were incubated for 2 h at 37 ꢀC with 750 μL of MTT solution per
well. Subsequently, the solution was removed and cells incu-
bated for 45 min at RT with 750 μL of extraction solution. Then
12 ꢀ 50 μL aliquots were transferred into a 96-well plate and
evaluated using an ELISA reader measuring the absorbance at
570 nm. Results (mean values) were presented as % vitality in
relation to untreated healthy cells.
General Procedure for the Synthesis of Ethyl 4-[3-(2,4-dichlor-
obenzylcarbamoyl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-
7-yl)]piperazine-1-carboxylate (22) and Ethyl 4-[1-cyclopropyl-3-
(2,4-dichlorobenzylcarbamoyl)-6-fluoro-4-oxo-1,4-dihydroquino-
line-7-yl)]piperazine-1-carboxylate (23). Norfloxacin and cipro-
floxacin (1 equiv, 3 mmol) and 6 equiv (18 mmol) of NEt3 were
dissolved in abs DMF (40 mL). Ethyl chloroformate (5 equiv,
15 mmol) was added under stirring at 0 ꢀC for 1 h and sub-
sequently stirred at room temperature for 4 h. Then 50 mL of
distilled H2O were added, the solution was extracted with ethyl
acetate (4 ꢀ 50 mL), and the combined organic layers were dried
over anhydrous Na2SO4. The solvent was removed in vacuo,
and the resulting residue dissolved in a mixture of 15 mL of
DMF and 3 mL of NEt3. Ethyl chloroformate (5 equiv,
15 mmol) of was added under stirring at 0 ꢀC for 1 h, and
3 equiv (9 mmol) of 2,4-dichlorobenzylamine was added and
stirred at room temperature for 3 d. After 50 mL of distilled H2O
were added, the solution was extracted with ethyl acetate (3 ꢀ
70 mL) and the combined organic layers dried over anhydrous
Na2SO4. The solvent was removed in vacuo and the residue
purified by column chromatography on silica gel (eluent: ethyl
acetate/ethanol 30:1) to give 22 and 23, respectively. 22: yield
10%; mp 237-239 ꢀC. IR [cm-1]: 827, 1119, 1238, 1529, 1664,
1
1720, 2879, 2989, 3103, 3267. H NMR (DMSO-d6, δ [ppm],
J [Hz]): 1.29 (t, J = 7.2, CH3), 1.54 (t, J = 7.0, CH3), 3.24-3.23
(m, 4 ꢀ piperazinyl-H), 3.72-3.69 (m, 4 ꢀ piperazinyl-H), 4.19
(q, J = 7.2, CH2), 4.26 (q, J = 7.0, H2), 4.70 (d, J = 5.6, CH2),
6.80 (d, JF,H = 6.8, H-8), 7.19 (dd, J = 8.5, J = 2.0, benzyl-H),
7.38 (d, J = 2.0, benzyl-H), 7.39 (d, J = 8.5, benzyl-H), 8.09 (d,
JF,H = 13.1, H-5), 8.71 (s, H-2), 10.50 (t, J = 5.6, NH). 13C
NMR (DMSO-d6, δ [ppm], J [Hz]): 14.9 (CH3), 15.1 (CH3), 41.1
(CH2), 44.0 (2 ꢀ piperazinyl-C), 50.0 (CH2), 50.4 (2 ꢀ piper-
azinyl-C), 62.2 (CH2), 104.4 (d, JC,F = 2.9, C-8), 111.8 (C-3),
Molecular Modeling. The protein structure of the NiV post-
fusion coiled coil trimer was downloaded from the PDB
(1WP720). The structure was preprocessed using the Schrodin-
ger Protein Preparation Guide:45 hydrogens were added to the