Synthesis of L-analogues of 1-(2',3'-dideoxy-β-D-glycero-pent-2-enofuranosyl)thymine

Article abstract of DOI:10.1211/146080899128734640

β-L(-)-2',3'-Dideoxy-3'-thiacytidine (3TC), its 5-fluoro derivative ((-)-FTC), 2',3'-dideoxycytidine (β-L-ddC), and its 5-fluoro derivative (β-L-FddC) have been reported to have anti-HIV and anti-HBV activity. It was of particular interest therefore to develop a series of β-L-d4T analogues bearing several kinds of amino-linker arms at the C-5 position of the pyrimidine moiety in an attempt to find more potent and less toxic anti-HIV agents. In addition, modification of nucleosides with various functional molecules has been attracting wide interest in biological studies since die primary amino groups could be useful for the attachment of either fluorescent dyes or a non-nucleosidic reverse transcriptase inhibitor. These modified nucleosides were evaluated for antiviral activity against HIV-1(LAI) in CEM-SS cells and HIV-1(IIIB) in MT4 cells. Unfortunately, none of the compounds exhibited significant anti-HIV activity at the doses tested.

Full text of DOI:10.1211/146080899128734640

Pharm. Pharmacol. Commun. 1999, 5: 225±231
# 1999 Pharm. Pharmacol. Commun.
Synthesis of L-Analogues of 1-(20,30-Dideoxy-b-D-glycero-
pent-2-enofuranosyl)thymine
F. CAMARA, A. CIUREA, Z. DELBEDERI, C. FOSSEY, G. FONTAINE, D. GAVRILIU,
J. JOUENNE, D. LADUREE, A. M. AUBERTIN* AND A. KIRN*
Â
Centre d'Etudes et de Recherche sur le Medicament de Normandie, Laboratoire de Pharmacochimie, UFR
Â
Â
3 rue Koeberle, 67000 Strasbourg, France
des Sciences Pharmaceutiques, 1 rue Vaubenard, 14032 Caen Cedex and *INSERM U74,
Abstract
b-L( )-20,30-Dideoxy-30-thiacytidine (3TC), its 5-¯uoro derivative (( )-FTC), 20,30-dide-
oxycytidine (b-L-ddC), and its 5-¯uoro derivative (b-L-FddC) have been reported to have
anti-HIV and anti-HBV activity. It was of particular interest therefore to develop a series of
b-L-d4T analogues bearing several kinds of amino-linker arms at the C-5 position of the
pyrimidine moiety in an attempt to ®nd more potent and less toxic anti-HIV agents. In
addition, modi®cation of nucleosides with various functional molecules has been attracting
wide interest in biological studies since the primary amino groups could be useful for the
attachment of either ¯uorescent dyes or a non-nucleosidic reverse transcriptase inhibitor.
These modi®ed nucleosides were evaluated for antiviral activity against HIV-1LAI in CEM-
SS cells and HIV-1IIIB in MT4 cells. Unfortunately, none of the compounds exhibited
signi®cant anti-HIV activity at the doses tested.
Considerable effort has been directed towards the
search for novel nucleoside structures for use as
antiviral agents. Most of these analogues are syn-
thesized by modi®cation of the naturally occurring
nucleosides and, therefore, possess the b-D-con-
®guration. Recently a number of nucleosides with
L-con®guration have been reported as chemother-
apeutic agents against HIV. The ®rst such com-
pound, b-L( )-20,30-dideoxy-30-thiacytidine (3TC,
Lamivudine) (Belleau et al 1990; Doong et al 1991;
Soudeyns et al 1991) was recently approved as an
anti-HIV agent, and its 5-¯uoro derivative (( )-
FTC) (Furman et al 1992; Schinazi et al 1992) has
also been found to exhibit antiviral activity against
HIV, as well as HBV, in-vitro and in-vivo. 20,30-
Dideoxycytidine (b-L-ddC) (Balzarini et al 1986)
and its 5-¯uoro derivative (b-L-FddC) (Gosselin et
al 1994; Lin et al 1994) are additional L-nucleoside
analogues. Interestingly, while these L-nucleosides
have potent biological activity, some show lower
toxicity pro®les than their D-counterparts. It was of
interest to synthesize the corresponding b-L-enan-
tiomers of b-D-d4T analogues as potential anti-HIV
and anti-HBV agents with resistance to the degra-
dative enzymes.
We have also undertaken the synthesis of 20,
30-didehydro-20,30-dideoxy-L-thymidine analogues
bearing several kinds of amino-linker arms at the
C-5 position of the pyrimidine moiety in order to
discover more potent and less toxic anti-HIV
agents. The choice of the attachment site of the
tether on the C-5 position in the pyrimidine ring
was made due to the low interference with base
pairing in DNA. The rationale for the design and
synthesis of such 5-tethered compounds resulted
from preliminary studies (Rong & Soloway 1994;
Rong et al 1995) which demonstrated that the
binding to nucleosides was weak when the exten-
sion of the arms contained four or fewer methylene
Ê
groups. However, when the tether increased to 10 A
by interposing additional methylene groups (6 to
12), there was a substantial increase in the strength
of enzyme binding. In preliminary biochemical
studies, tethering the 5-position by a ¯exible chain
Ê
(10 A) may permit the triphosphates to be generated
and thereby their incorporation in nucleic acids.
Finally, the b-L-nucleosides bearing N-aminoalkyl-
linkers could have useful applications in biological
studies with further derivations of the terminal
Correspondence: D. Laduree, Centre d'Etudes et de
Â
Pharmaceutiques=rue Vaubenard, 1403 Caen, France.
Recherche sur le Medicament de Normandie, UFR de Sciences
Â

226
F. CAMARA ET AL
primary amine with either a ¯uorescent dye or a
non-nucleosidic reverse transcriptase inhibitor.
(KBr) n 3642±3250 (NH2 and OH), 2961 (CH),
1730 (CO ester), 1668 (CO lactame), 1636 (CN),
1553, 1493, 1260, 1098, 1059, 1021; 1H NMR (d6-
DMSO) d (ppm) 7Á83 (s, 1H, H6), 6Á34 (d,
J ˆ 5Á6 Hz, 1H, H10), 5Á89 (d, J ˆ 4Á4 Hz, 1H, OH),
5Á20 (d, J ˆ 5Á6 Hz, 1H, H20), 4Á98 (t, J ˆ 4Á7 Hz,
1H, OH), 4Á37 (broad s, 1H, H30), 4Á08 (broad s, 1H,
H40), 3Á56 (s, 3H, OCH3), 3Á27 (broad s, 2H, 5-
CH2), 3Á27 (s, 2H, H50); 13C NMR (DMSO-d6) d
(ppm) 170Á8 (CO ester and C2), 159Á7 (C4), 134Á6
(C6), 114Á8 (C5), 90Á2 (C10), 89Á1 (C40), 88Á8 (C20),
74Á6 (C30), 60Á7 (C50), 51Á6 (OCH3), 32Á8 (5-CH2).
Materials and Methods
Chemical procedures
Melting points were measured on a Ko¯er appara-
tus and were uncorrected. IR spectra were recorded
on a Unicam Mattson 1000 spectrometer and only
noteworthy absorptions (reciprocal cm) are listed.
1H and13C NMR spectra were obtained on a Jeol
JNM-LA 400 spectrometer using tetramethylsilane
as an internal standard. NH, NH2 and OH signals
appeared as broad singlets exchangeable with D2O
(s ˆ singlet, b ˆ broad, d ˆ doublet, t ˆ triplet,
m ˆ multiplet). Mass spectra were recorded with a
Jeol FX 102 spectrometer with the Fast Atom
Bombardment ionization (FAB Pos.). Thin-layer
chromatography (TLC) was carried out on pre-
coated F254 silica plates and column chromato-
graphy were performed with Merck silica gel 60
(230±400 mesh).
1-(30, 50-Di-O -acetyl-20-bromo-20-deoxy-b-L-
ribofuranosyl)-5-(methoxycarbonylmethyl)-uracil
(4)
Acetyl bromide (5Á4 mL, 75 mmol) was added
dropwise to a boiling suspension of 3 (2Á5 g,
8Á39 mmol) in anhydrous acetonitrile (50 mL). The
reaction mixture was stirred at 80ꢀC for 2 h and
allowed to cool to room temperature. After eva-
poration to dryness under reduced pressure, the
residue was dissolved in dichloromethane (100 mL)
and the solution was washed with water
(3 6 50 mL). The organic layer was separated,
dried (magnesium sulphate), and evaporated in-
vacuo to yield brown crystals (TLC Rf ˆ 0Á5
CH2Cl2:CH3OH 90:10) (51%); mp ˆ 76ꢀC; IR
(KBr) n 1744 (CO ester), 1691 (CO lactame), 1463,
1379, 1226, 1038; 1H NMR (DMSO) d (ppm) 11Á68
(s, 1H, NH), 7Á67 (s, 1H, H6), 6Á15 (d, J ˆ 7Á52 Hz,
1H, H10), 5Á26 (m, 1H, H20), 4Á93 (m, 1H, H30), 4Á25
(m, 3H, H40, H50), 3Á59 (s, 3H, OCH3), 3Á29 (s, 2H,
5-CH2), 2Á13 (s, 3H, COCH3), 2Á05 (s, 3H,
COCH3); 13C NMR (d6-DMSO) d (ppm) 170Á7 (CO
ester), 170Á2 (CO ester), 162Á7 (C4), 150Á4 (C2),
137Á9 (C6), 108Á8 (C5), 88Á6 (C10), 79Á6 (C40), 71Á2
(C30), 62Á9 (C50), 51Á7 (OCH3), 47Á4 (C20), 31Á6 (5-
CH2), 20Á5 (CH3).
2-Amino-b-L-arabinofurano[10,20:4,5]oxazoline (1)
A mixture of L-arabinose (10 g, 66Á6 mmol), crude
cyanamide (6 g, 0Á14 mol, 2 Eq), methanol (20 mL),
and 6 M NH4OH (3 mL) was heated at 50ꢀC for 3
days. The reaction mixture was cooled to 10ꢀC
and kept at this temperature overnight. The product
was collected with suction, washed with methanol
(2 6 100 mL), diethyl ether (2 6 100 mL), and
dried in-vacuo. White crystals (67%); mp ˆ 228ꢀC;
IR (KBr) n 3414 (NH2), 3189±3148 (OH), 1666
1
(CN), 1608 (CˆC), 1442; H RMN (d6-DMSO) d
(ppm) 6Á32 (s, 2H, NH2), 5Á65 (d, J ˆ 5Á6 Hz, 1H,
H10), 5Á45 (s, 1H, OH), 4Á75 (s, 1H, OH), 4Á52 (d,
J ˆ 5Á4 Hz, 1H, H20), 3Á99 (s, 1H, H30), 3Á63 (m, 1H,
H40), 3Á26 (m, 2H, H50); 13C NMR (d6-DMSO) d
(ppm) 162Á3 (CˆN), 99Á9 (C10), 88Á0 (C20), 84Á6
(C40), 75Á6 (C30), 61Á5 (C50).
1-(50-O-Acetyl-20,30-dideoxy-b- L-glycero-pent-2-
enofuranosyl)-5-(methoxycarbonylmethyl)-uracil
(5)
2,20-Anhydro-1-(b- L-arabinofuranosyl)-5-(methoxy-
carbonylmethyl)-uracil (3)
Zinc dust (11Á2 g) in 1 M NaOH (100 mL) was
treated under irradiation of 35 kHz ultrasound
(150 W) for 3 min and washed with water until
neutralization. Zinc in 50% acetic acid (100 mL)
was then treated under irradiation of ultrasound
for 3 min, washed respectively with water until
neutralization, ethanol (3 6 50 mL), acetone
(3 6 50 mL), diethyl ether (3 6 50 mL), and dried
in-vacuo at 78ꢀC for 2 h.
Dimethyl-a-bromomethylfumarate (2) (4Á6 g, 19Á4
mmol) and triethylamine (2 mL) were added suc-
cessively to a solution of 1 (2 g, 11Á49 mmol) in
methanol (25 mL). The reaction mixture was heated
at 50ꢀC for 1 h, and evaporated in-vacuo. The
residual yellow oil was puri®ed on a silica gel
column prepacked in dichloromethane. Elution of
the column with dichloromethane:methanol (100:0
to 85:15) gave 3 as beige crystals (TLC Rf ˆ 0Á3 in
CH2Cl2:CH3OH 85:15) (81%); mp ˆ 100ꢀC; IR
The freshly activated zinc dust (0Á5 g, 7Á64 mmol,
4 Eq) was added with stirring to a solution of 4 (1 g,

SYNTHESIS OF L-ANALOGUES
227
2Á16 mmol) in anhydrous ethanol (80 mL) and
acetic acid (1 mL). The heterogeneous reaction
mixture was treated under irradiation of 35 kHz
ultrasound (150 W) under an argon atmosphere for
30 min (monitored by TLC CH2Cl2:CH3OH 95:5)
until no starting material remained, ®ltered on
celite and the ®ltrate evaporated to dryness in-
vacuo. The residual yellow oil was puri®ed on a
silica gel column prepacked in dichloromethane.
Elution of the column with dichloromethane±
methanol (100 : 0 to 95:5) gave 5 as yellow crystals
(54%; TLC Rf ˆ 0Á48 in CH2Cl2:CH3OH 95:5) and
5bis as white crystals (20%; TLC Rf ˆ 0Á33 in
CH2Cl2:CH3OH 95:5); mp ˆ 150ꢀC; IR (KBr) 3036
(CH), 1752 (CO ester), 1739 (CO ester), 1697 (CO
lactame), 1682 (CO lactame), 1466, 1344, 1264,
1227, 1085; 1H NMR (d6-DMSO) d (ppm) 11Á46 (s,
1H, NH), 7Á42 (s, 1H, H6), 6Á81 (s, 1H, H10), 6Á42
(d, J ˆ 6Á00 Hz, 1H, H20), 6Á00 (d, J ˆ 6Á00 Hz, 1H,
H30), 4Á96 (s, 1H, H40), 4Á17 (m, 2H, H50), 3Á57 (s,
3H, OCH3), 3Á29 (s, 2H, 5-CH2), 1Á98 (s, 3H,
COCH3); 13C NMR (d6-DMSO) d (ppm) 170Á9
(CO), 170Á2 (CO), 163Á1 (C4), 150Á7 (C2), 138Á3
(C6), 133Á8 (C20), 126Á4 (C30), 108Á0 (C5), 89Á5
(C10), 83Á9 (C40), 67Á7 (C50), 51Á7 (OCH3), 31Á5 (5-
CH2), 20Á5 (CH3).
2H, H50), 3Á35 (s, 2H, 5-CH2), 3Á31 (m, 2H, linker-
CH2OH), 2Á92 (m, 2H, NHCH2-linker), 1Á38±1Á25
(m, 8H, CH2-linker); 13C NMR (d6-DMSO) d
(ppm) 169Á3 (CO amide), 163Á5 (C4), 150Á9 (C2),
139Á0 (C6), 135Á4 (C20), 125Á9 (C30), 108Á7 (C5),
89Á4 (C10), 87Á6 (C40), 62Á6 (C50), 60Á8 (linker-
CH2OH), 32Á4±29Á3±26Á5 (CH2-linker).
5-[N-(6-Aminohexyl)carbamoylmethyl]-1-(20,30-
dideoxy-b-L-glycero-pent-2-enofuranosyl)-uracil
(7a)
The crude residue was crystallized from ethyl
acetate (3 6 50 mL) as brown crystals (50%);
mp ˆ 92ꢀC; IR (KBr) 3600±3114 (NH2 and OH),
2931 (CH), 2858 (CH), 1658 (CO lactame), 1548;
1H NMR (d6-DMSO) d (ppm) 7Á60 (s, 1H, H6),
6Á82 (s, 1H, H10), 6Á40 (d, J ˆ 5Á58 Hz, 1H, H20),
5Á88 (d, J ˆ 5Á58 Hz, 1H, H30), 4Á75 (m, 1H, H40),
3Á56 (m, 2H, H50), 2Á98 (s, 2H, 5-CH2), 2Á56 (m,
4H, NHCH2-linker and linker-CH2NH2), 1Á34±1Á22
(m, 8H, CH2-linker); 13C NMR (d6-DMSO) d
(ppm) 168Á9 (CO amide), 163Á0 (C4), 150Á8 (C2),
138Á6 (C6), 135Á1 (C20), 125Á7 (C30), 108Á6 (C5),
89Á1 (C10), 87Á5 (C40), 62Á5 (C50), 42Á0 (5-CH2),
33Á2±26Á0 (CH2-linker); MS m=z 367, 269, 133
‡
(MH ).
1
5-(Methoxycarbonylmethyl)-uracil (5bis). H NMR
(d6-DMSO) d (ppm) 11Á06 (s, 1H, NH), 7Á40 (s, 1H,
H6), 3Á57 (s, 3H, OCH3), 3Á21 (s, 2H, 5-CH2); 13C
NMR (d6-DMSO) d (ppm) 170Á7 (CO ester), 164Á2
(C4), 151Á4 (C2), 140Á2 (C6), 106Á3 (C5), 57Á7
(OCH3), 31Á4 (5-CH2).
5-[N-(8-Aminooctyl)carbamoylmethyl]-1-(20,30-
dideoxy-b-L-glycero-pent-2-enofuranosyl)-uracil
(7b)
The crude residue was crystallized from ethyl
acetate (3 6 50 mL) as brown crystals (61%);
mp ˆ 96ꢀC; IR (KBr) 3643±3157 (NH2 and OH),
2927 (CH), 2854 (CH), 1697 (CO lactame), 1660
1
General procedure for the synthesis of 5-[N-
(hexan-6-ol)carbamoylmethyl]- and 5-[N-(amino-
alkyl)carbamoylmethyl]-1-(20,30-dideoxy-b- L-gly-
cero-pent-2-enofuranosyl)-uracil (6 and 7a±d,
respectively)
To a solution of 5 (350 mg, 1 mmol) in methanol
(50 mL) was added either 6-aminohexan-1-ol
(1 Eq) or 1,n-diaminoalkane (1 Eq) in the presence
of a catalytic amount of dimethylaminopyridine.
The reaction mixture was heated at 50ꢀC for 5 h,
and evaporated to dryness in-vacuo.
(CO lactame), 1458, 1260, 1076; H NMR (d6-
DMSO) d (ppm) 7Á80 (s, 1H, H6), 6Á78 (s, 1H, H10),
6Á37 (d, J ˆ 5Á60 Hz, 1H, H20), 5Á85 (d, J ˆ 5Á60 Hz,
1H, H30), 4Á74 (m, 1H, H40), 3Á64 (m, 2H, H50), 3Á60
(broad s, 3H, NH2, OH), 2Á95 (s, 2H, 5-CH2), 2Á61±
2Á56 (m, 4H, NHCH2-linker and linker-CH2NH2),
1Á32±1Á19 (m, 12H, CH2-linker); 13C NMR (d6-
DMSO) d (ppm) 168Á9 (CO amide), 163Á0 (C4),
150Á8 (C2), 138Á6 (C6), 135Á0 (C20), 125Á7 (C30),
108Á6 (C5), 89Á1 (C10), 87Á4 (C40), 62Á5 (C50), 40Á9
(5-CH2), 31Á9±28Á9±26Á2 (CH2-linker); MS m=z
‡
395, 297 (MH ).
1-(20,30-dideoxy-b-L-glycero-pent-2-enofuranosyl)-
5-[N-(hexan-6-ol)carbamoylmethyl]-uracil (6)
Brown oil (75%); IR (KBr) 3300 (OH), 2930 (CH),
2858 (CH), 1706 (CO lactame), 1674 (CO lac-
5-[N-(10-Aminodecyl)carbamoylmethyl]-1-(20,30-
dideoxy-b-L-glycero-pent-2-enofuranosyl)-uracil
(7c)
The crude residue was crystallized from ethyl
acetate (3 6 50 mL) as brown crystals (66%);
mp ˆ 92ꢀC; IR (KBr) 3642±3357 (NH2 and OH),
2923 (CH), 1705 (CO lactame), 1660 (CO lac-
1
tame), 1605 (CˆC), 1454, 1074, 1056; H NMR
(d6-DMSO) d (ppm) 7Á60 (s, 1H, H6), 6Á82 (s, 1H,
H10), 6Á40 (d, J ˆ 5Á5 Hz, 1H, H20), 5Á90 (d,
J ˆ 5Á5 Hz, 1H, H30), 4Á75 (m, 1H, H40), 3Á56 (m,
1
tame), 1558 (CˆC), 1465, 1081; H NMR (d6-

228
F. CAMARA ET AL
Virology procedures
DMSO) d (ppm) 7Á77 (s, 1H, NH), 7Á57 (s, 1H, H6),
6Á78 (s, 1H, H10), 6Á37 (d, J ˆ 5Á55 Hz, 1H, H20),
5Á86 (d, J ˆ 5Á55 Hz, 1H, H30), 4Á75 (m, 1H, H40),
4Á40 (m, 3H, NH2 and OH), 3Á56 (m, 2H, H50), 2Á98
(s, 2H, 5-CH2), 2Á51±2Á49 (m, 4H, NHCH2-linker
and linker-CH2NH2), 1Á33±1Á21 (m, 16H, CH2-
linker); 13C NMR (d6-DMSO) d (ppm) 168Á9 (CO
amide), 163,4 (C4), 150,8 (C2), 138,6 (C6), 135,1
(C20), 125,7, (C30), 108,6 (C5), 89,1 (C10), 87,4
(C40), 62Á5 (C50), 40Á9 (5-CH2), 31Á9±28Á9±26Á3
The cultures of CEM-SS and MT4 cells were
maintained at 37ꢀC in a 5% CO2 atmosphere in
RPMI-1640 medium supplemented with 10%
complement-depleted foetal bovine serum. The
antiviral HIV-1 activity of a given compound in
CEM-SS cells was measured by quanti®cation of
the reverse transcriptase activity associated with
particles released from HIV-1LAI-infected cells in
the culture medium. CEM-SS cells were infected
with 100 TCDI50 (the virus stock was titrated
under the same experimental conditions); after
30 min adsorption, free virus particles were washed
out and cells were re-suspended in RPMI-1640 plus
10% foetal bovine serum at a ®nal concentration of
‡
(CH2-linker); MS m=z 423, 325, 137 (MH ).
5-[N-(12-aminododecyl)carbamoylmethyl]-1-
(20,30-dideoxy-b- L-glycero-pent-2-enofuranosyl)-
uracil (7d)
1
105 cells mL in the presence of different con-
The crude residue was crystallized from ethyl
acetate (3 6 50 mL) as brown crystals (88%);
mp ˆ 100ꢀC; IR (KBr) 3642±3114 (NH2 and OH),
2920 (CH), 2850, 1706 (CO lactame), 1644 (CO
lactame), 1549, 1466, 1083; 1H NMR (d6-DMSO) d
(ppm) 7Á78 (s, 1H, NH), 7Á60 (s, 1H, H6), 6Á82 (s,
1H, H10), 6Á40 (d, J ˆ 5Á67 Hz, 1H, H20), 5Á89 (d,
J ˆ 5Á67 Hz, 1H, H30), 4Á75 (m, 1H, H40), 4Á50±4Á37
(broad s, 3H, NH2, OH), 3Á56 (m, 2H, H50), 2Á94 (s,
2H, 5-CH2), 2Á52 (m, 4H, NHCH2-linker and lin-
ker-CH2NH2), 1Á33±1Á22 (m, 20H, CH2-linker);
13C NMR (d6-DMSO) d (ppm) 168Á9 (CO amide),
163Á3 (C4), 150Á8 (C2), 138Á5 (C6), 135Á0 (C20)
125Á7 (C30), 108Á6 (C5), 89Á1 (C10), 87Á4 (C40), 62Á5
(C50), 41Á1 (5-CH2), 32Á3±28Á9±26Á3 (CH2-linker);
centrations of test compounds. After 5 days, virus
production was measured by reverse transcriptase
assay as described by Moog et al (1994). The 50%
inhibitory concentration (IC50) was derived from
the computer-generated median effect plot of the
dose±effect data (Chou & Chou 1985). The cyto-
toxicity of the drugs was evaluated in parallel by
incubating uninfected cells in the presence of dif-
ferent concentrations of antiviral products. The cell
viability was determined by a measure of mito-
chondrial dehydrogenase activity, enzymes redu-
cing
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide into formazan (the quantity of
which was measured by the absorbance at 540 nm)
(Mosmann 1983). The 50% cytotoxic concentration
(CC50) is the concentration of drug which reduces
cell viability by 50% and was calculated with the
program used in the determination of the IC50. The
assays using different cells and virus isolates were
done according to previously published protocols;
virus production was quanti®ed by the reverse
transcriptase activity associated to virus particles
released from the cells in the culture medium
(Moog et al 1994). Conditions under which the
inhibitory properties were measured on HIV-1
reverse transcriptase in-vitro, and in-vitro reverse
transcriptase inhibition are described by Moog et al
(1994). The CEM-SS cells were obtained from the
AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH (Bethesda, MD).
‡
MS m=z 451, 353 (MH ).
1-(20,30-Dideoxy-b- L-glycero-pent-2-enofurano-
syl)-5-(methoxycarbonylmethyl)-uracil (8)
Compound 5 (400 mg, 1Á23 mmol) was dissolved in
methanolic sodium methoxide (50 mL, 1 Eq) and
the reaction mixture was stirred for 45 min at room
temperature. The solution was then cooled at 0ꢀC
and neutralized with 12 M hydrochloric acid, and
evaporated to dryness in-vacuo. The oily residue
was puri®ed on a silica gel column prepacked in
dichloromethane. Elution of the column with
dichloromethane:methanol (100:0 to 95:5) afforded
8 as an oil (17%; TLC Rf ˆ 0Á43 in CH2Cl2:CH3OH
90:10); IR (KBr) 3550 (OH), 2961 (CH), 1730 (CO
ester), 1706 (CO lactame), 1681 (CO lactame),
1
1260, 1086, 1037; H NMR (d6-DMSO) d (ppm)
Results and Discussion
11Á46 (s, 1H, NH), 7Á73 (s, 1H, H6), 6Á82 (s, 1H,
H10), 6Á41 (d, J ˆ 6Á09 Hz, 1H, H20), 5Á92 (d,
J ˆ 6Á09 Hz, 1H, H30), 4Á99 (m, 1H, H40), 4Á76 (s,
2H, H50), 3Á57 (m, 3H, OCH3), 3Á21 (s, 2H, 5-CH2);
13C NMR (d6-DMSO) d (ppm) 167Á8 (CO ester),
160Á0 (C4), 147Á6 (C2), 135Á8 (C6), 132Á1 (C20),
122Á7 (C30), 104Á5 (C5), 86Á1 (C10), 84Á4 (C40), 59Á4
(C50), 48Á6 (OCH3), 28Á6 (5-CH2).
Chemistry
We report a simple synthetic method for 5-sub-
stituted uracil nucleosides, useful for attachment of
various functional groups. The synthesis used L-
arabinose as the starting material which on reaction
with cyanamide in aqueous methanolic ammonia

SYNTHESIS OF L-ANALOGUES
229
gave 2-amino-b- L-arabinofurano[10,20:4,5]oxazo-
line (1) (Figure 1) (Holy 1972). The key step
involved the addition reaction of dimethyl a-bro-
momethylfumarate to arabinoaminooxazoline fol-
lowed by a subsequent cyclization in methanol
under re¯ux in the presence of triethylamine in a
one-¯ask reaction which afforded a crude product.
Puri®cation was achieved by column chromato-
graphy on silica gel using 15% methanol in
dichloromethane as eluent to afford puri®ed
2,20 - anhydro-1-(b-L-arabinofuranosyl)-5-(methoxy-
carbonyl)-uracil (3) in 81% yield. The b-anomer
was exclusively formed in this reaction. The ring
opening of the 2,20-anhydro linkage with a con-
comitant introduction of bromine atom at C-20
seemed to be an attractive process (Marumoto &
Honjo 1974). The reaction of 3 with acetyl bromide
in anhydrous acetonitrile under re¯ux afforded
crystalline 1-(30,50-di-O-acetyl-20-bromo-20-de-oxy-
b- L-ribofuranosyl)-5-(methoxycarbonylmethyl)-ura-
cil (4) in 51% yield. This reaction of ®ssionof the ether
linkage of 2,20-anhydro nucleoside was realised with a
concomitantesteri®cationof thehydroxylgroupson30
and 50 positions. The subsequent reductive b-elimina-
tion proceeded smoothly by adding freshly activated
zinc powder in anhydrous ethanol in the presence of a
catalytic amount of acetic acid to compound 4 (Shir-
agami et al 1996). Theheterogeneous reaction mixture
was submitted to ultrasoundirradiation under an argon
atmosphere at room temperature for 30 min and the
reductive b-elimination reaction gave the expected
ole®nic nucleoside 5 (54%) with the nucleoside bond
cleavage leading to the substituted uracil as the major
by-product (5bis) (20%) puri®ed after chromato-
graphy.
Various linker arms containing amino groups
were then easily introduced to the 5 position of 5
via amide linkages by ester-amide exchange reac-
tions of 5-carbonylmethyl esters (Shinozuka 1998).
Thus the reaction of 5 with either 6-aminohexan-1-
Figure 1. Synthesis of compounds 6, 7a±d and 8. Reagents: i. NH2CN, 6 M NH4OH, CH3OH; ii. 2, triethylamine, methanol; iii.
CH3COBr, anhydrous CH3CN; iv. zinc dust, anhydrous ethanol, acetic acid; v. 6-aminohexan-1-ol or 1,n-diaminoalkane,
dimethylaminopyridine, methanol; vi. CH3O Na , methanol.
‡

230
F. CAMARA ET AL
Table 1. Inhibitory effect of b-L-d4T analogues against HIV-1LAI replication in CEM-SS and HIV-1IIIB in MT4 cells.
Compound
Number of CH2 units
HIV-1LAI in CEM-SS cells
HIV-1IIIB in MT4 cells
IC50 (mM)
CC50 (mM)
CC50/IC50
IC50 (mM)
CC50 (mM)
CC50/IC50
d4T
7a
7b
7c
0Á059Æ 0Á016
>100
>10
>100
86
>1695
0Á28Æ 0Á08
>10
>100
>100
>100
>100
>10
>100
>100
42
>357
6
8
10
12
>10
>100
25
3Á44
8Á18
7d
4Á4
36
>0Á42
IC50 is the drug concentration required to inhibit HIV-1 multiplication by 50%. CC50 is the drug concentration which causes
50% cytotoxicity to uninfected cells. All data represent the meanÆ s.d of three separate experiments.
Chou, J., Chou, T. C. (1985) Dose-effect analysis with micro-
computers: quantitation of ED50, LD50, synergism, antagon-
ism, low-dose risk, receptor binding and enzyme kinetics.
In: Computer Software for Apple II Series and IBM-PC and
ol or 1, n-diaminoalkanes (n ˆ 6, 8, 10 and 12) in
methanol in the presence of dimethylaminopyridine
gave the expected 5-[N-(hexan-6-ol)carbamoyl-
methyl]- and 5-[N-(aminoalkyl)carbamoylmethyl]-
1-(20,30-dideoxy-b-L-glycero-pent-2-enofuranosyl)-
uracil (6 and 7a±d, respectively).
Finally, the selective deprotection of the 50-acetyl
group of 5 was realised using methanolic sodium
methoxide at room temperature yielding the 1-
(20,30-Dideoxy-b-L-glycero-pent-2-enofuranosyl)-
5-(methoxycarbonylmethyl)-uracil (8) as an oily
compound.
Instruction Manual. Elsevier-Biosoft, Elsevier Science Pub-
lishers, Cambridge, pp 19±28
Doong, S.-L., Tsai, C. H., Schinazi, R. F., Liotta, D. C., Cheng,
Y. -C. (1991) Inhibition of the replication of hepatitis B
virus in vitro by 20,30-dideoxy-30-thiacytidine and related
analogues. Proc. Natl Acad. Sci. USA 88: 8495±8499
Furman, P. A., Davis, M., Liotta, D. C., Paff, M., Frick, L. W.,
Nelson, D. J., Dornsife, R. E., Wurster, J. A., Wilson, L. J.,
Fyfe, J. A., Tuttle, J. V., Miller, W. H., Condreay, L.,
Averette, D. R., Schinazi, R. F., Painter, G. R. (1992) The
anti-hepatitis B virus activities, cytotoxicities, and anabolic
pro®les of the ( ) and (‡) enantiomers of cis-5-¯uoro-1-[2-
(hydroxymethyl)-1,3-oxathiolan-5-yl]-cytosine. Antimicrob.
Agents Chemother. 36: 2686±2692
Antiviral activity
The b- L-d4T analogues 7a±d were evaluated for
inhibition of HIV-1 multiplication in cells of the
lymphocytic lineage (CEM-SS and MT4). The lack
of anti-HIV activity of 7a±d is in accordance with
previously published results (Van Draanen et al
1992) (Table 1). We conclude that compounds 7a±
d are devoid of antiviral activity because they are
not ef®ciently phosphorylated intracellularly in
CEM-SS and MT4 cells.
Gosselin, G., Schinazi, R. F., Sommadossi, J.-P., Mathe, C.,
Bergogne, M.-C., Aubertin, A.-M., Kirn, A., Imbach, J.-L.
(1994) Anti-human immunode®ciency virus activities of the
b- L-enantiomer of 20,30-dideoxycytidine and its 5-¯uoro
derivative in vitro. Antimicrob. Agents Chemother. 38:
1292±1297
Holy, A. (1972) Nucleic acid components and their analogues.
Preparation of 20-deoxy-L-ribonucleosides of the pyrimidine
series. Collection Czechoslov. Chem. Commun. 37: 4072±
4087
Lin, T. S., Luo, M. Z., Pai, S. B., Dutschuman, G. E., Cheng,
Y.-C. (1994) Synthesis and biological evaluation of 20,30-
dideoxy-L-pyrimidine nucleosides as potential antiviral
agents against human immunode®ciency virus (HIV) and
hepatitis B virus (HBV). J. Med. Chem. 37: 798±803
Marumoto, R., Honjo, M. (1974) One-step halogenation at the
20 position of uridine and related reaction of cytidine and N4
acetyl-cytidine. Chemical Pharmaceutical Bulletin 22: 128±
134
Acknowledgements
We are grateful to S. Schmidt and D. Albrecht for
excellent technical assistance in the antiviral and
cytotoxic assays. We thank the Fondation pour la
Â
Recherche Medicale (Sidaction grant) for ®nancial
support of this work.
Moog, C., Wick, A., Le Ber, P., Kirn, A., Aubertin, A.-M.
(1994) Bicyclic imidazo derivatives, a new class of highly
selective inhibitors for the human immunode®ciency virus
type 1. Antiviral Res. 24: 275±288
Mosmann, T. (1983) Rapid colorimetric assay for cellular
growth and survival: application to proliferation and cyto-
toxicity assays. J. Immunol. Methods 65: 55±63
Rong, F. G., Soloway, A. H. (1994) Synthesis of 5-tethered
carborane-containing pyrimidine nucleosides as potential
agents for DNA incorporation. Nucleosides Nucleotides
13: 2021±2034
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