Microwave-assisted synthesis of fluoroquinolones and their nucleosides as inhibitors of HIV integrase

Article abstract of DOI:10.1135/cccc20060978

Six fluoroquinolone ribonucleosides were synthesized by using microwave irradiation starting from fluoroanilines. In most cases the microwave application proved superior in time and yield, especially the one step decarboxylation of the carboxyquinolone esters 3a-3c and the Vorbrueggen glycosylation. The former led to the new type of fluoroquinolone ribosides 8a-8c. Compound 8c in the crystal structure showed C3′-endo and anti conformation. The nucleosides were examined, but found inactive against the replication of HIV-1(IIIB) in cell culture, while they were toxic for the cells at a 50% cytotoxic concentration ranging from 31 to >125 μg/ml. But measurements of the inhibitory effects against HIV-1 integrase enzymatic activity showed an interesting activity for compound 8c.

Full text of DOI:10.1135/cccc20060978

978
Adams, Bats, Nikolaus, Witvrouw, Debyser, Engels:
MICROWAVE-ASSISTED SYNTHESIS OF FLUOROQUINOLONES AND
THEIR NUCLEOSIDES AS INHIBITORS OF HIV INTEGRASE
a1
a2
a3
b1
Martina M. ADAMS , Jan W. BATS , Nadja V. NIKOLAUS , Myriam WITVROUW ,
b2
a4,
Zeger DEBYSER and Joachim W. ENGELS
*
a
Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University,
Marie-Curie-Str. 11, 60439 Frankfurt am Main, Germany;
e-mail: martina.adams@gmx.de, bats@chemie.uni-frankfurt.de,
nikolaus@chemie.uni-frankfurt.de, joachim.engels@chemie.uni-frankfurt.de
Molecular Medicine, Katholieke Universiteit Leuven and IRC Kulak, Kapucijnenvoer 33, B-3000
1
2
3
4
b
1
Leuven, Flanders, Belgium; e-mail: myriam.witvrouw@uz.kuleuven.ac.be,
zeger.debyser@med.kuleuven.ac.be
2
Received February 28, 2006
Accepted May 3, 2006
Dedicated to Professor Antonín Holý on the occasion of his 70th birthday in recognition of his
outstanding contributions to bioactive nucleic acid components.
Six fluoroquin olon e ribon ucleosides were syn th esized by usin g m icrowave irradiation start-
in g from fluoroan ilin es. In m ost cases th e m icrowave application proved superior in tim e
an d yield, especially th e on e step decarboxylation of th e carboxyquin olon e esters 3a–3c an d
th e Vorbrüggen glycosylation . Th e form er led to th e n ew type of fluoroquin olon e ribosides
8a–8c. Com poun d 8c in th e crystal structure sh owed C3′-en do an d an ti con form ation . Th e
n ucleosides were exam in ed, but foun d in active again st th e replication of HIV-1(IIIB) in cell
culture, wh ile th ey were toxic for th e cells at a 50% cytotoxic con cen tration ran gin g from
31 to >125 µg/m l. But m easurem en ts of th e in h ibitory effects again st HIV-1 in tegrase en zy-
m atic activity sh owed an in terestin g activity for com poun d 8c.
Keyw ord s: Nucleosides; Microwaves; Fluoroquin olon es; Glycosidation ; Decarboxylation ;
An ti-HIV activity; HIV in tegrase; An tivirals.
Hum an im m un odeficien cy virus (HIV), a retrovirus wh ich causes th e ac-
quired im m un e deficien cy syn drom e (AIDS), h as a replication cycle ch arac-
terized by th e reverse tran scription of its gen om ic RNA in to double-
stran ded DNA an d by th e in tegration of th is viral DNA copy in to th e h ost
ch rom osom e. In tegrase is an en zym e of particular in terest, sin ce in tegra-
tion is an essen tial step in th e replication cycle of HIV an d n o h um an coun -
terpart of in tegrase h as been foun d so far1–4. Th e on ly in tegrase in h ibitors
with an tiviral effect reported to date are th e diketo acids (DKA) an d th e
pyran opyrim idin es (PDP)5,6
.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 978–990
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20060978

Microwave-Assisted Synthesis of Fluoroquinolones
979
Sin ce several am in oquin olon e derivatives were previously sh own to
exh ibit poten t activity again st replication of HIV-1 in de n ovo-in fected
h um an lym ph oblastoid cells, we decided to work towards n ew syn th etic
quin olon es7,8. Th eir easy an d reliable syn th esis was in troduced by Gould
an d Jacobs9 in 1939. But it was n ot un til 1963 th at n alidixic acid, a sys-
tem ic Gram -n egative an tibacterial agen t, was in troduced as a drug10. Th e
outstan din g an tibacterial an d an tiviral activity of n alidixic acid an d its
n um erous an alogues led to research activities towards n ew syn th etic
quin olon es11–13. Natural n ucleoside an alogues h ave been in vestigated for
an tiviral an d an tican cer th erapy14. Con siderin g th e ph arm acological effects
of both substan ce classes we decided to syn th esize m on ofluorin ated
quin olon e n ucleosides sin ce on ly on e (th e free acid an d eth yl ester of com -
poun d 6) were previously described in literature15. A few groups syn th e-
sized quin olon e n ucleosides, all bearin g a carboxy group in 3 position
durin g th e last decade for biological evaluation but n on e of th em sh owed
con siderable biological effects10,15,16. In th is paper we report th e first full
syn th esis of fluoroquin olon e n ucleosides by m icrowave-in duced ch em istry
with good to excellen t yields, th e an tiviral evaluation of all com poun ds
an d th eir in tegrase in h ibition .
EXPERIMENTAL
Gen eral
All 1H an d 13C NMR spectra were m easured on a Bruker AM 250 (250 MHz) spectrom eter
or on a Bruker AMX 400 (400 MHz) spectrom eter (δ in ppm , J in Hz). MALDI m ass spec-
tra were recorded on a Fison s VG Tofspec spectrom eter an d ESI m ass spectra on a Fison s VG
Plattform II spectrom eter. Colum n ch rom atograph y was carried out on silica gel 60 (Merck
9385, 34–62 µm ). Meltin g poin ts were m easured on a Büch i apparatus after Totolli an d are
un corrected. Th e m icrowave-supported syn th eses were accom plish ed in equipm en t of th e
com pan y CEM, m odel Discover. All reaction s were carried out in dry flasks un der argon .
Dieth yl [(Fluoroan ilin o)m eth yliden e]m alon ates (2a–2c). Gen eral Procedure
Stoich iom etric am oun ts of a fluoroan ilin e an d dieth yl (eth oxym eth yliden e)m alon ate
(DEMM) were m ixed in a roun d-bottom flask an d m icrowave-irradiated (150 W) at 250 °C
for 2 m in . After coolin g th e obtain ed solid was dried un der reduced pressure.
Diethyl [(2-fluoroanilino)methylidene]malonate (2a). 2-Fluoroan ilin e (1a; 1.11 g, 10 m m ol)
an d dieth yl (eth oxym eth yliden e)m alon ate (DEMM; 2.16 g, 10 m m ol) were reacted accordin g
to th e gen eral procedure. Yield 2.81 g (100%), m .p. 80 °C. 1H NMR (250 MHz, DMSO-d6):
10.89 (d, J = 13.5, NH); 8.47 (d, J = 13.5, H8); 7.63 (t, J = 8.25, 1 H, H5); 7.26 (m , 3 H, H3,
H4, H6); 4.17 (m , 4 H, CH2); 1.27 (m , 6 H, CH3). 13C NMR (63 MHz, DMSO-d6): 167.63
(C=O), 164.32 (C=O), 154.15 (C7), 150.34 (C-F), 127.35 (C1), 125.45 (C5), 117.45 (C4),
116.13 (C3), 115.83 (C6), 94.41 (C8), 59.92 (CH2), 14.16 (CH3). ESI (+), m/z: 282.0 (M + H)+.
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Adams, Bats, Nikolaus, Witvrouw, Debyser, Engels:
Diethyl [(3-fluoroanilino)methylidene]malonate (2b). 3-Fluoroan ilin e (1b; 14.92 g, 0.134 m ol)
an d DEMM (29.03 g, 0.134 m ol) were reacted accordin g to th e gen eral procedure. Yield
37.32 g (99%), m .p. 48 °C. 1H NMR (250 MHz, DMSO-d6): 10.68 (d, J = 13.5, 1 H, NH); 8.39
(d, J = 13.75, 1 H, H8); 7.40 (m , 2 H, H5, H2); 7.22 (m , 1 H, H6); 6.98 (m , 1 H, H4); 4.18 (m ,
4 H, CH2); 1.27 (m , 6 H, CH3). 13C NMR (63 MHz, DMSO-d6): 167.06 (C=O), 164.80 (C=O),
160.65 (C-F), 150.64 (C8), 141.44 (C1), 131.34 (C5), 113.48 (C6), 115.13 (C4), 94.25 (C9),
59.73 (CH2), 14.19 (CH3). ESI (+), m/z: 282.0 (M + H)+.
Diethyl [(4-fluoroanilino)methylidene]malonate (2c). 4-Fluoroan ilin e (1c; 11.70 g, 0.105 m ol)
an d DEMM (22.80 g, 0.105 m ol) were reacted accordin g to th e gen eral procedure. Yield
28.66 g (97%), m .p. 66 °C. 1H NMR (250 MHz, DMSO-d6): 10.71 (d, J = 14, 1 H, NH); 8.34
(d, J = 14, 1 H, H8); 7.43 (m , 2 H, H2, H6); 7.24 (m , 2 H, H3, H5); 4.17 (m , 4 H, CH2); 1.25
(m , 6 H, CH3). 13C NMR (63 MHz, DMSO-d6): 167.25 (C=O), 164.92 (C=O), 161.13 (C-F),
151.66 (C7), 136.12 (C1), 119.78–114.46 (C2, C3, C5, C6), 93.04 (C8), 59.58 (CH2), 14.21
(CH3). ESI (+), m/z: 282.1 (M + H)+.
Eth yl Fluoro-4-oxo-1,4-dih ydroquin olin e-3-carboxylates (3a–3c). Gen eral Procedure
A dieth yl [(fluoroan ilin o)m eth yliden e]m alon ate was suspen ded in diph en yl eth er an d
refluxed for 1 h . After coolin g, a wh ite precipitate was filtered off an d dried in vacuum .
Ethyl 8-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (3a ). Dieth yl [(2-fluoroan ilin o)-
m eth yliden e]m alon ate (2a; 25 g, 89 m m ol) was suspen ded in diph en yl eth er (270 m l) an d it
was proceeded accordin g to th e gen eral procedure. Yield 20.91 g (100%).
Microwave syn th esis. Dieth yl [(2-fluoroan ilin o)m eth yliden e]m alon ate (2a; 5.62 g, 20 m m ol)
was suspen ded in diph en yl eth er (56 m l) an d m icrowave-irradiated (300 W) at 280 °C for
30 m in . After coolin g th e precipitate was filtered off, wash ed with toluen e an d dried un der
reduced pressure. Yield of 3a 1.42 g (30%), m .p. 178 °C. 1H NMR (400 MHz, TFA-d): 9.75 (s,
H2); 8.86 (t, J = 3.04, H5); 8.34 (m , 2 H, H6, H7); 5.08 (m , 2 H, CH2); 1.917 (t, J = 7.12, 3 H,
CH3). ESI (+), m/z: 236.0 (M + H)+. For C12H10NO3F (235.2) calculated: 61.28% C, 4.29% H,
5.96% N; foun d: 61.20% C, 4.05% H, 6.10% N.
Ethyl 7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (3b ). Dieth yl [(3-fluoroan ilin o)-
m eth yliden e]m alon ate (2b; 11.24 g, 40 m m ol) was suspen ded in diph en yl eth er (110 m l)
an d proceeded after th e gen eral procedure. Yield 6.23 g (66%).
Microwave syn th esis. Dieth yl [(3-fluoroan ilin o)m eth yliden e]m alon ate (2b; 5.62 g, 20 m m ol)
was suspen ded in diph en yl eth er (56 m l) an d m icrowave-irradiated (300 W) at 280 °C for
30 m in . After coolin g th e precipitate was filtered off, wash ed with toluen e an d dried un der
vacuum . Yield of 3b 4.21 g (90%), m .p. 296 °C. 1H NMR (400 MHz, TFA-d): 9.69 (s, 1 H,
H2); 9.09 (m , 1 H, H8); 8.18 (m , 1 H, H5); 8.07 (m , 1 H, H6); 5.05 (q, J = 7.09, 2 H, CH2)
1.90 (t, J = 7.09, 3 H, CH3). ESI (+), m/z: 235.8 (M + H)+. For C12H10NO3F (235.2) calculated:
61.28% C, 4.29% H, 5.96% N; foun d: 61.05% C, 4.29% H, 6.14% N.
Ethyl 6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (3c). Dieth yl [(4-fluoroan ilin o)-
m eth yliden e]m alon ate (2c; 3.03 g, 10.8 m m ol) was suspen ded in diph en yl eth er (56 m l) an d
proceeded after th e gen eral procedure. Yield 2.22 g (88%).
Microwave syn th esis. Dieth yl [(4-fluoroan ilin o)m eth yliden e]m alon ate (2c; 4 g, 14.2 m m ol)
was suspen ded in diph en yl eth er (35 g) an d m icrowave-irradiated (300 W) at 280 °C for
15 m in . After coolin g th e precipitate was filtered off, wash ed with toluen e an d dried un der
reduced pressure. Yield of 3c 2.57 g (77%), m .p. 299 °C. 1H NMR (250 MHz, TFA-d): 9.17 (s,
1 H, CH); 8.12 (m , 2 H, H5, H7); 7.79 (m , 1 H, H8); 4.59 (q, J = 6.5, 2 H, CH2); 1.35 (t, J =
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Microwave-Assisted Synthesis of Fluoroquinolones
981
6.5, 3 H, CH3). 13C NMR (100.62 MHz, TFA-d): 175.35 (C4), 169.00 (C11), 158.62 (C-F),
147.04 (C9), 138.64 (C2), 131.92 (C7), 126.4 (C10), 120.95 (C5), 118.10 (C8), 107.48 (C3),
67.24 (C12), 14.50 (C13). ESI (+), m/z: 235.8 (M + H)+.
Fluoroquin olin -4(1H)-on es (4a–4c). Gen eral Procedure
An eth yl fluoro-4-oxo-1,4-dih ydroquin olin e-3-carboxylate was m ixed with diph en yl eth er
an d a catalytic am oun t of water in a pressure vial with m agn etic stirrer. Th e suspen sion was
m icrowave-irradiated (300 W) at 20 bar an d 260 °C for 20 m in . After coolin g th e precipitate
was filtered off, wash ed with toluen e an d dried in vacuo.
8-Fluoroquinolin-4(1H)-one (4a). Eth yl 8-fluoro-4-oxo-1,4-dih ydroquin olin e-3-carboxylate
(3a; 0.35 g, 1.5 m m ol) was suspen ded in diph en yl eth er (6.2 m l) an d water (6.5 µl, 0.36 m m ol)
was added. Yield 0.235 g (96%), m .p. 262 °C. 1H NMR (250 MHz, DMSO-d6): 7.88 (m , 2 H,
H2, H5); 7.53 (m , 1 H, H6); 7.26 (m , 1 H, H7); 6.09 (d, J = 7.25, 1 H, H3). 13C NMR
(63 MHz, DMSO-d6): 175.67 (C4), 154.06 (C-F), 150.12 (C10), 140.26 (C2), 130.20 (C9),
127.30 (C6), 120.61 (C7), 116.01 (C5), 109.37 (C3). ESI (+), m/z: 163.8 (M + H)+.
7-Fluoroquinolin-4(1H)-one (4b). Eth yl 7-fluoro-4-oxo-1,4-dih ydroquin olin e-3-carboxylate
(3b; 0.35 g, 1.5 m m ol) was suspen ded in diph en yl eth er (6.2 m l) an d water (6.5 µl, 0.36 m m ol)
was added. Yield 0.240 g (98%), m .p. 207 °C. 1H NMR (250 MHz, DMSO-d6): 12.80 (bs, 1 H,
NH); 8.14 (m , 1 H, H8); 7.92 (d, J = 7.5, 1 H, H2); 7.36 (m , 2 H, H5, H6); 6.04 (d, J = 7.25,
1 H, H3). 13C NMR (63 MHz, DMSO-d6): 176.24 (C4), 165.67 (C-F), 141.46 (C2), 139.91 (C9),
128.33 (C8), 122.83 (C10), 112.04 (C5), 111.66 (C6), 108.98 (C3). ESI (+), m/z: 163.7 (M + H)+.
6-Fluoroquinolin-4(1H)-one (4c). Eth yl 6-fluoro-4-oxo-1,4-dih ydroquin olin e-3-carboxylate
(3c; 0.235 g, 1 m m ol) was suspen ded in diph en yl eth er (4.1 m l) an d water (4.3 µl, 0.24 m m ol)
was added. Yield 0.163 g (100%), m .p. 197 °C. 1H NMR (250 MHz, DMSO-d6): 11.91 (s, 1 H,
NH); 7.93 (d, J = 7.25, 1 H, H2); 7.62 (m , 3 H, H5, H7, H8); 6.03 (d, J = 7.25, 1 H, H3).
13C NMR (63 MHz, DMSO-d6): 175.89 (C4), 160.14 (C-F), 139.61 (C2), 136.77 (C9), 126.86
(C10), 121.13 (C7), 120.66 (C5), 109.01 (C8), 107.76 (C3). ESI (+), m/z: 163.7 (M + H)+.
Syn th esis of Meth yl Fluoro-4-oxo-1,4-dih ydroquin olin e-3-carboxylate Nucleosides (7a–7c).
Gen eral Procedure
An eth yl fluoro-4-oxo-1,4-dih ydroquin olin e-3-carboxylate (3a–3c; 1.18 g, 5 m m ol) an d
1,2,3,5-O-tetraacetyl-β-D-ribofuran ose (2.07 g, 1.3 equiv., 6.5 m m ol) were suspen ded in dry
aceton itrile (100 m l) an d N,O-bis(trim eth ylsilyl)acetam id (1.32 g, 1.3 equiv., 6.5 m m ol) was
added. Th e colorless solution was refluxed for 30 m in . After coolin g to room tem perature
trim eth ylsilyl triflate (1.44 g, 1.3 equiv., 6.5 m m ol) was added an d th e solution refluxed for
an oth er 3.5 h . After coolin g th e solution was quen ch ed with saturated NaHCO3 solution ,
th e precipitate filtered off an d iden tified as un reacted n ucleobase. Th e solution was ex-
tracted with m eth ylen e ch loride th ree tim es, dried over an h ydrous MgSO4 an d th e solven t
was evaporated in vacuo.
8-Fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-1,4-dihydroquinoline-3-carboxylate (5a).
Th e brown oil was n ot purified. Yield 1.03 g (42%). ESI (+), m/z: 494.1 (M)+.
7-Fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-1,4-dihydroquinoline-3-carboxylate (5b).
Th e brown oil was purified by colum n ch rom atograph y (CH2Cl2–MeOH 95:5). Yield 0.93 g
(38%). RF 0.32 (CH2Cl2–MeOH 95:5). ESI (+), m/z: 494.2 (M)+.
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982
Adams, Bats, Nikolaus, Witvrouw, Debyser, Engels:
6-Fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-1,4-dihydroquinoline-3-carboxylate (5c).
Th e oran ge foam was n ot purified. Yield 1.58 g (64%). RF 0.63 (CH2Cl2–MeOH 9:1). ESI (+),
m/z: 494.0 (M)+; 516.0 (M + Na)+.
Deprotection of Acetylated Nucleosides. Gen eral Procedure
An eth yl fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuran osyl)-1,4-dih ydroquin olin e-3-carboxyl-
ate (5a–5b) was dissolved in dry m eth an ol an d a 5.4 M sodium m eth oxide solution was
added. Th e solution was stirred for 1 h , n eutralized with Dowex 50WX8, filtered, wash ed
with m eth an ol an d th e solven t evaporated in vacuo. Th e product was reprecipitated from
m eth an ol.
Methyl 8-fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-1,4-dihydroquinoline-3-carboxylate
(7a). 8-Fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuran osyl)-1,4-dih ydroquin olin e-3-carboxylate
(5a; 0.5 g, 1 m m ol), dry m eth an ol (9 m l) an d th e sodium m eth oxide solution (0.06 m l) were
reacted accordin g to th e gen eral procedure. Yield 0.26 g (76%). RF 0.75 (CH2Cl2–MeOH 4:1).
1H NMR (250 MHz, DMSO-d6): 9.21 (m , 1 H, H2); 8.40 (m , 1 H, H5); 8.31 (m , 1 H, H6); 8.27
(m , 1 H, H7); 5.69 (m , 1 H, H1′); 4.69 (m , 1 H, H2′); 4.21 (m , 2 H, H3′, H4′); 3.87 (m , 2 H,
H5′a, H5′b); 3.59 (s, 3 H, CH3). MALDI (+), m/z: 353.93 (M + H)+.
Methyl 7-fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-1,4-dihydroquinoline-3-carboxylate
(7b). 7-Fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuran osyl)-1,4-dih ydroquin olin e-3-carboxylate
(5b; 0.59 g, 1.2 m m ol), dry m eth an ol (9 m l) an d th e sodium m eth oxide solution (0.06 m l)
were reacted accordin g to th e gen eral procedure. Yield 0.24 g (60%). RF 0.25 (CH2Cl2–MeOH
9:1). 1H NMR (250 MHz, DMSO-d6): 9.35 (m , 1 H, H2); 8.31 (m , 1 H, H5); 7.61 (d, J = 11,
1 H, H6); 7.23 (t, J = 7.75, 1 H, H8); 5.80 (d, J = 3.5, 1 H, H1′); 4.62 (m , 1 H, H2′); 4.26 (m ,
2 H, H3′, H4′); 3.89 (m , 2 H, H5′a, H5′b); 3.68 (s, 3 H, CH3). ESI (–), m/z: 352.0 (M – H)–.
Methyl 6-fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-1,4-dihydroquinoline-3-carboxylate
(7c). 6-Fluoro-4-oxo-1-(2,3,5-tri-O-acetyl-β-D-ribofuran osyl)-1,4-dih ydroquin olin e-3-carboxylate
(5c; 1.58 g, 3.2 m m ol), dry m eth an ol (24 m l) an d th e sodium m eth oxide solution (0.18 m l)
were reacted accordin g to th e gen eral procedure. Yield 0.68 g (67%). RF 0.2 (CH2Cl2–MeOH
9:1). 1H NMR (250 MHz, DMSO-d6): 8.95 (s, 1 H, H2); 7.91 (m , 3 H, H5, H7, H8); 6.07 (m ,
1 H, H1′); 4.19 (m , 1 H, H2′); 4.06 (m , 2 H, H4′, H3′); 3.68 (m , 2 H, H5′a, H5′b); 3.58 (s, 3 H,
CH3). 13C NMR (63 MHz, DMSO-d6): 169.32 (C4), 169.18 (C-F), 123.2 (C7), 118.08 (C5),
109.99 (C8), 108.04 (C3), 103.76 (C5), 83.56 (C4′), 74.19 (C2′), 63.15 (C3′), 60.06 (C5′).
MALDI (+), m/z: 354.95 (M + H)+.
Syn th esis of Fluoroquin olin -4(1H)-on e Nucleosides (6a–6c). Gen eral Procedure
Th e fluoroquin olin -4(1H)-on es (4a–4c) an d 1,2,3,5-O-tetraacetyl-β-D-ribofuran ose were sus-
pen ded in dry aceton itrile an d N,O-bis(trim eth ylsilyl)acetam ide was added. Th e solution was
refluxed for 30 m in . After coolin g to room tem perature, trim eth ylsilyl triflate was added an d
th e solution refluxed for 3 h . After coolin g th e solution s were quen ch ed with saturated
NaHCO3 solution , extracted with m eth ylen ech loride th ree tim es, dried over an h ydrous
MgSO4 an d th e solven t evaporated in vacuo. Purification was don e as stated for th e differen t
substan ces.
8-Fluoro-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)quinolin-4(1H)-one (6a). 8-Fluoroquin olin -
4(1H)-on e (4a; 0.82 g, 5 m m ol), 1,2,3,5-O-tetraacetyl-β-D-ribofuran ose (2.07 g, 1.3 equiv.,
6.5 m m ol), aceton itrile (100 m l), N,O-bis(trim eth ylsilyl)acetam ide (1.32 g, 1.3 equiv., 6.5 m m ol)
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Microwave-Assisted Synthesis of Fluoroquinolones
983
an d trim eth ylsilyl triflate (1.44 g, 1.3 equiv., 6.5 m m ol) were reacted accordin g to th e gen -
eral procedure. Th e product was deprotected with out furth er purification . Yield 0.81 g (39%).
Microwave syn th esis. Th e reaction was carried out in a dry flask un der argon . 8-Fluoro-
quin olin -4(1H)-on e (4a ; 0.816 g, 5.0 m m ol) an d 1,2,3,5-O-tetraacetyl-β-D-ribofuran ose
(1.91 g, 6 m m ol) were suspen ded in dry aceton itrile (50 m l) an d N,O-bis(trim eth ylsilyl)-
acetam ide (1.22 g, 1.1 equiv., 6 m m ol) was added. Th e solution was m icrowave-irradiated
(150 W) at 80 °C for 15 m in . After coolin g to room tem perature, trim eth ylsilyl triflate
(1.33 g, 1.1 equiv., 6 m m ol) was added an d th e solution m icrowave-irradiated (150 W) at
80 °C for 50 m in . After coolin g th e solution was quen ch ed with saturated NaHCO3 solution ,
extracted with m eth ylen e ch loride th ree tim es, dried over an h ydrous MgSO4 an d th e solven t
evaporated in vacuo. Th e oil was purified by colum n ch rom atograph y (CH2Cl2–MeOH 95:5).
Yield of 6a 1.69 g (80%). ESI (+), m/z: 422.0 (M + H)+.
7-Fluoro-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)quinolin-4(1H)-one (6b ). 7-Fluoroquin olin -
4(1H)-on e (4b; 1.63 g, 10 m m ol), 1,2,3,5-O-tetraacetyl-β-D-ribofuran ose (4.14 g, 1.3 equiv.,
13 m m ol), aceton itrile (100 m l), N,O-bis(trim eth ylsilyl)acetam ide (2.64 g, 1.3 equiv., 13 m m ol)
an d trim eth ylsilyl triflate (2.88 g, 1.3 equiv., 13 m m ol) were reacted accordin g to th e gen -
eral procedure. Th e foam was deprotected with out furth er purification . ESI (+), m/z: 422.0
(M – H)+.
6-Fluoro-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)quinolin-4(1H)-one (6c). 6-Fluoroquin olin -
4(1H)-on e (4c; 0.82 g, 5 m m ol), 1,2,3,5-O-tetraacetyl-β-D-ribofuran ose (2.07 g, 1.3 equiv.,
6.5 m m ol), aceton itrile (100 m l), N,O-bis(trim eth ylsilyl)acetam ide (1.32 g, 1.3 equiv.,
6.5 m m ol) an d trim eth ylsilyl triflate (1.44 g, 1.3 equiv., 6.5 m m ol) were reacted accordin g to
th e gen eral procedure. Th e product was purified by colum n ch rom atograph y (CH2Cl2–MeOH
95:5). Yield 1.34 g (64%).
Microwave syn th esis. Th e reaction was carried out in a dry flask un der argon . 6-Fluoro-
quin olin -4(1H)-on e (4c; 0.90 g, 5.5 m m ol) an d 1,2,3,5-O-tetraacetyl-β-D-ribofuran ose (1.91 g,
6 m m ol) were suspen ded in dry aceton itrile (50 m l) an d N,O-bis(trim eth ylsilyl)acetam ide
(1.22 g, 1.1 equiv., 6 m m ol) was added. Th e solution was m icrowave-irradiated (150 W) at
80 °C for 15 m in . After coolin g to room tem perature, trim eth ylsilyl triflate (1.33 g,
1.1 equiv., 6 m m ol) was added an d th e solution m icrowave-irradiated (150 W) at 80 °C for
50 m in . After coolin g th e solution was quen ch ed with saturated NaHCO3 solution , extracted
with m eth ylen ech loride th ree tim es, dried over an h ydrous MgSO4 an d th e solven t evapo-
rated in vacuo. Th e oil was purified by colom n ch rom atograph y (CH2Cl2–MeOH 95:5). Yield
of 6c 2.23 g (96%). RF 0.66 (CH2Cl2–MeOH 9:1). 1H NMR (250 MHz, DMSO-d6): 8.15 (d, J =
8, 1 H, H2); 7.88 (m , 1 H, H5); 7.76 (m , 1 H, H7); 7.60 (m , 1 H, H8); 6.41 (d, J = 5.5, 1 H,
H1′); 6.15 (d, J = 8, 1 H, H3); 5.45 (m , 1 H, H2′); 5.29 (m , 1 H, H3′); 4.36 (m , 3 H, H4′, H5′a,
H5′b); 2.06 (s, 3 H, C2′-Ac); 2.02 (s, 3 H, C3′-Ac); 1.99 (s, 3 H, C5′-Ac). 13C NMR (100.61 MHz,
DMSO-d6): 175.75 (C4), 170.00 (C=O, acetyl), 169.41 (C=O, acetyl), 169.16 (C=O, acetyl),
159.17 (C-F), 139.28 (C9), 136.14 (C2), 127.79 (C7), 120.80 (C10), 120.50 (C5), 119.10 (C8),
109.20 (C3), 89.39 (C1′), 79.06 (C4′), 72.20 (C2′), 69.01 (C3′), 62.65 (C5′), 20.51 (C-Ac).
ESI (+), m/z: 422.1 (M + H)+.
Deprotection of Nucleosides 6a–6c. Gen eral Procedure
A fluoro-1-(2,3,5-tri-O-acetyl-β-D-ribofuran osyl)quin olin -4(1H)-on e (6a–6c) was dissolved in
dry m eth an ol an d a 5.4 M sodium m eth oxide solution was added. Th e solution was stirred
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 978–990

984
Adams, Bats, Nikolaus, Witvrouw, Debyser, Engels:
for 1 h , n eutralized with Dowex 50WX8, filtered, wash ed with m eth an ol an d th e solven t
was evaporated in vacuo. Th e products were reprecipitated from m eth an ol.
8-Fluoro-1-(β-D-ribofuranosyl)quinolin-4(1H)-one (8a). 8-Fluoro-1-(2,3,5-tri-O-acetyl-β-D-ribo-
furan osyl)quin olin -4(1H)-on e (6a; 0.5 g, 1.19 m m ol), dry m eth an ol (9 m l) an d th e sodium
m eth oxide solution (0.06 m l) were reacted accordin g to th e gen eral procedure. Yield 0.24 g
(68%). RF 0.48 (CH2Cl2–MeOH 4:1). 1H NMR (250 MHz, DMSO-d6): 8.32 (d, J = 8.25, 1 H,
H2); 8.08 (m , 1 H, H7); 7.59 (m , 1 H, H5); 7.36 (m , 1 H, H6); 6.10 (d, J = 9.25, 1 H, H3);
5.70 (m , 2 H, H1′, 2′-OH); 5.22 (d, J = 5.25, 1 H, 3′-OH); 5.18 (d, J = 5, 1 H 5′-OH); 4.11 (m ,
1 H, H2′); 3.90 (m , 1 H, H3′); 3.84 (m , 1 H, H4′); 3.70 (m , 2 H, H5′a, H5′b). 13C NMR
(63 MHz, DMSO-d6): 175.61 (C4), 169.71 (C-F), 169.18 (C9), 140.71 (C2), 128.91 (C7),
123.21 (C10), 110.55 (C6), 107.96 (C3), 103.43 (C5), 91.33 (C1′), 85.29 (C4′), 73.66 (C2′),
62.07 (C3′), 60.06 (C5′). ESI (–), m/z: 292.9 (M – 2 H)2–
.
7-Fluoro-1-(β-D-ribofuranosyl)quinolin-4(1H)-one (8b). 7-Fluoro-1-(2,3,5-tri-O-acetyl-β-D-ribo-
furan osyl)quin olin -4(1H)-on e (6b; 5.9 g, im pure), dry m eth an ol (100 m l) an d th e sodium
m eth oxide solution (0.79 m l) were reacted accordin g to th e gen eral procedure. Th e product
was purified by colum n ch rom atograph y an d reprecipitated from m eth an ol. Yield 1.2 g
(43%). RF 0.47 (CH2Cl2–MeOH 4:1). 1H NMR (250 MHz, DMSO-d6): 8.45 (d, J = 8.25, 1 H,
H2); 8.22 (m , 1 H, H8); 7.66 (m , 1 H, H5); 7.27 (m , 1 H, H6); 6.10 (d, J = 8.25, 1 H, H3);
5.94 (d, J = 4.25, 1 H, H1′); 5,72 (d, J = 6.0, 1 H, 2′-OH); 5.24 (m , 2 H, 3′-OH, 5′-OH); 4.13
(m , 1 H, H2′); 4.02 (m , 2 H, H3′, H4′); 3.66 (m , 2 H, H5′a, H5′b). 13C NMR (63 MHz,
DMSO-d6): 175.95 (C4), 162.61 (C-F), 141.05 (C9), 138.96 (C2), 129.03 (C8), 123.18 (C10),
112.20 (C6), 109.25 (C3), 102.26 (C5), 91.54 (C1′), 85.06 (C4′), 74.59 (C2′), 62.28 (C3′),
60.13 (C5′). MALDI (+), m/z: 296.5 (M + H)+.
6-Fluoro-1-(β-D-ribofuranosyl)quinolin-4(1H)-one (8c). 6-Fluoro-1-(2,3,5-tri-O-acetyl-β-D-ribo-
furan osyl)quin olin -4(1H)-on e (6c; 1.899 g, 4.506 m m ol), dry m eth an ol (35 m l) an d th e so-
dium m eth oxide solution (0.30 m l) were reacted accordin g to th e gen eral procedure. Th e
product was recrystallized from m eth an ol. Yield 1.293 g (97%). RF 0.125 (CH2Cl2–MeOH
9:1). 1H NMR (250 MHz, DMSO-d6): 8.50 (d, J = 8.75, 1 H, H2); 7.84 (m , 2 H, H5, H7); 7.65
(m , 1 H, H8); 6.11 (d, J = 7.75, 1 H, H3); 6.01 (d, J = 4, 1 H, H1′); 4.19 (m , 1 H, H2′); 4.02
(m , 2 H, H4′, H3′); 3.67 (m , 2 H, H5′a, H5′b). 13C NMR (63 MHz, DMSO-d6): 174.87 (C4),
168.89 (C-F), 138.54 (C9), 128.93 (C7), 122.32 (C10), 117.47 (C5), 109.29 (C8), 77.91 (C4′),
71.10 (C2′), 67.82 (C3′), 61.05 (C5′). ESI (+), m/z: 296.0 (M + H)+.
X-ray an alysis of 8c: C14H14FNO5·0.5CH3OH·0.25H2O. Crystal system : m on oclin ic, space
group: P21↑, crystal color: colorless, un it cell param eters: a = 8.0476(12) Å, b = 43.884(7) Å,
c = 8.1096(12) Å, β = 90.432(6)°, V = 2863.9(8) Å3, Z = 8, R = 0.102, GOF = 1.03. A sin gle
crystal was m easured on a Siem en s Sm art CCD diffractom eter, radiation MoKα at tem pera-
ture of ca. –114 °C. Th e structure was determ in ed by direct m eth ods usin g program SHELXS.
CCDC 600092 con tain s th e supplem en tary crystallograph ic data for th is paper. Th ese data
can be obtain ed free of ch arge via www.ccdc.cam .ac.uk/con ts/retrievin g.h tm l (or from th e
Cam bridge Crystallograph ic Data Cen tre, 12, Un ion Road, Cam bridge, CB2 1EZ, UK; fax:
+44 1223 336033; or deposit@ccdc.cam .ac.uk).
In vitro An ti-HIV Assay an d Drug Susceptibility Assay
Th e an tiviral activity of th ese com poun ds on th e HIV-in duced CPE in h um an lym ph ocyte
MT-4 cell culture was determ in ed by th e MT-4/MTT-assay17. All com poun ds proved to be in -
active again st HIV-1 replication at subtoxic con cen tration s.
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Microwave-Assisted Synthesis of Fluoroquinolones
985
The overall integration assay. Th e en zym atic overall in tegration reaction was carried out as
described previously with m in or m odification s18,19. In th e overall in tegration assay, bin din g
of in tegrase to DNA substrate, 3′-processin g an d DNA stran d tran sfer can be detected. Th e fi-
n al reaction m ixture for th is assay was 20 m m ol in HEPES, pH 7.5, 5 m m ol in dith ioth reitol
(DTT), 10 m m ol in MgCl2, 75 m m ol in NaCl, 15% (v/v) in poly(eth ylen e glycol) 8000,
30 n m ol in th e oligon ucleotide substrate, an d 760 n m ol in th e His-tag IN (10 µl was th e
fin al volum e). Reaction s were started by th e addition of th e en zym e. In h ibitors were in cu-
bated sh ortly with th e reaction com pon en ts before th e addition of IN. Reaction s were
allowed to proceed at 37 °C, stopped by th e addition of form am ide loadin g buffer
(95% form am ide, 30 m M EDTA, 0.1% Xylen e Cyan ole, 0.1% Brom oph en ol Blue, 0.1% SDS).
Th e products were separated in a 15% den aturin g polyacrylam ide/urea gel. Quan tification of
th e results was perform ed usin g th e Ph osph orIm ager (Molecular Dyn am ics, Sun n yvale (CA),
U.S.A.). Specific activities of differen t en zym e preparation s (1 µM) were determ in ed in th is
assay. Th e exten t of 3′-processin g or DNA stran d tran sfer were based on th e am oun ts of
–2 ban ds or stran d tran sfer products relative to th e in ten sity of th e total radioactivity
presen t in th e lan e as determ in ed usin g th e software OptiQuan t Acquisition an d An alysis
(Perkin Elm er Corporate, Frem on t (CA), U.S.A.).
In vitro anti-HIV and drug susceptibility assays. Th e in h ibitory effect of an tiviral drugs
on th e HIV-in duced CPE in h um an lym ph ocyte MT-4 cell culture was determ in ed by th e
MT-4/MTT-assay17. Th is assay is based on th e reduction of th e yellow 3-(4,5-dim eth yl-
th iazol-2-yl)-2,5-diph en yl-2H-tetrazolium brom ide (MTT) by m itoch on drial deh ydrogen ase
of m etabolically active cells to a blue form azan e derivative, wh ich can be m easured spectro-
ph otom etrically. Th e 50% cell culture in fective dose (CCID50) of differen t HIV strain s was
determ in ed by titration of th e virus stock usin g MT-4 cells. For th e drug-susceptibility
assays, MT-4 cells were in fected with 100–300 CCID50 of th e virus stock in th e presen ce of
five-fold serial dilution s of th e an tiviral drugs. Th e con cen tration of various com poun ds pro-
vidin g 50% protection again st th e CPE of differen t HIV strain s, (EC50) was determ in ed.
RESULTS
Synthesis
Con ven tion al m eth ods for syn th esizin g fluoroquin olon es were sligh tly var-
ied after Koga et al. (Sch em e 1)20. Sin ce h igh tem peratures are n eeded in th e
syn th esis of th e h eterocycle, we decided to m ake use of a m icrowave appli-
cator. Therefore we m odified the procedure of Kidwai et al.21 for the first step.
2-, 3- an d 4-Fluoroan ilin e were N-substituted with dieth yl (eth oxym eth yl-
iden e)m alon ate to give dieth yl [(fluoroan ilin o)m eth yliden e]m alon ates
(2a–2c). Th e electroph ilic rin g closure was perform ed in diph en yl eth er fol-
lowin g Koga’s procedure. Th e syn th esis of quin olon es (4a–4c) was reduced
from a two-step syn th esis by h ydrolysis an d decarboxylation to a sin gle-
step, on e-pot reaction , m odified after Curran et al.22 Usin g th is m eth od we
were able to sh orten th e syn th esis by on e step an d to obtain very good
yields (96–100%).
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986
Adams, Bats, Nikolaus, Witvrouw, Debyser, Engels:
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 978–990

Microwave-Assisted Synthesis of Fluoroquinolones
987
Several attem pts to syn th esize th e n ucleoside failed due to th e lack of sol-
ubility of th e quin olon es in th e Vorbrüggen reaction . A sligh tly m odified
Vorbrüggen m eth od of Moore et al.23 worked best for th e gen eral proce-
dure, but we obtain ed excellen t results (96% yields) by usin g m icrowave ir-
radiation in th e syn th esis of 6c in stead of 64% an d 80% for 6a in stead of
39% for th e stan dard procedure. We do explain th ese results by a com bin a-
tion of optim al solubility an d h eat distribution due to th e m icrowave irra-
diation . Deprotection was carried out usin g stan dard procedures with
sodium m eth oxide in m eth an ol. Nucleosides (7a–7c an d 8a–8c) were ob-
tain ed in acceptable yields (20–69% in 4 steps or 5 steps for decarboxylated
n ucleobases).
We were able to crystallize com poun d (8c) from m eth an ol. Th e crystal
structure (Fig. 1) sh ows th at in th e solid state th e n ucleobase is an ti to th e
sugar an d th e sugar con form ation is C3′-en do-en velope. Th e plan ar
quin olon e rin gs of th e n ucleosides are stacked but flipped by 180° so th at
th e sugar m oieties avoid steric in terferen ce.
Th e fluorin e m olecules do n ot sh ow h ydrogen bon din g in th e crystal
cluster (Fig. 2). Hydrogen bon din g can be observed on ly between th e car-
bon yl group an d th e h ydroxy groups of th e sugar. Th e crystal cluster con -
tain s h ydroph ilic an d h ydroph obic layers. Hydroph ilic layers are form ed by
sugar m oieties wh ereas h ydroph obic layers by relatively n on polar quin ol-
on e rin gs.
O5
C8
C10
C9
C7
F′
C11
C6
C14
N1
C1
C12
O3
O4
C13
C5
C2
O1
C4
C3
O2
FIG. 1
Crystal structure of 6-fluoro-1-(β-D-ribofuran osyl)quin olin -4(1H)-on e (8c)
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988
Inhibition of HIV-1 IN Activity
Adams, Bats, Nikolaus, Witvrouw, Debyser, Engels:
Com poun ds (7a–7c an d 8a–8c) were tested in IN in h ibition assays wh ich
h ave been recen tly reviewed18,24. Activity below 100 µg/m l was displayed
by com poun d (8c) with IC50 = 63.4 ± 24.0 µg/m l. Th is biological activity
is strikin g sin ce com poun d (8c) lacks th e carboxyl group at C-3. Non -
n ucleosidic quin olon es tested for HIV-1-IN activity possessed th e C-3
carboxyl m oiety8,25. Th erefore it is even m ore surprisin g th at th e carboxyl-
ated com poun ds did n ot sh ow an y con siderable biological activity. Th ese
data are very prom isin g in com parison with oth er un m odified din ucleo-
sides, wh ile m on on ucleosides h ave n o activity at th is con cen tration 26. Th e
in tegrase of HIV is an attractive target for an tiviral th erapy sin ce th ere is n o
kn own fun ction al h om olog in h um an cells27.
DISCUSSION
Com poun d 8c is th e first quin olon e n ucleoside with an in terestin g an d
prom isin g biological activity. Con siderin g th is activity, it sh ould be a good
substan ce for structure-activity relation sh ip tests an d an in terestin g precur-
sor of oth er m odified n ucleosides aim in g at a h igh er HIV-in tegrase activity.
FIG. 2
Crystal cluster of 6-fluoro-1-(β-D-ribofuran osyl)quin olin -4(1H)-on e (8c)
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 7, pp. 978–990

Microwave-Assisted Synthesis of Fluoroquinolones
989
Th is is surprisin g con siderin g th at carboxylated n ucleosides did n ot sh ow
an y activity alth ough th is fun ction al group is crucial for th e an tibacterial
activity of fluoroquin olon es. Com poun d 8c is on e of th e m ost active an ti-
in tegrase n ucleosides kn own sofar an d could be a prom isin g precursor of a
n ew ran ge of an tiretroviral structures.
The authors acknowledge the financial support by the EU FP6 program TRIoH, Contract Number:
LSHB-CT-2003-503480.
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