Synthesis and anti-HIV activity of thymidine analogues bearing A 4′-cyanovinyl group and some derivatives thereof

Article abstract of DOI:10.1081/NCN-100108323

Treatment of 3′-O-tert-butyldimethylsilyl-2′,5′-dideoxy- 5′-oxothymidine (4) with potassium or magnesium nitromethanide afforded in good yield the resolvable epimeric mixture of the expected blocked nitronucleosides 5 which upon dehydration led to the cor

Full text of DOI:10.1081/NCN-100108323

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SYNTHESIS AND ANTI-HIV ACTIVITY OF THYMIDINE
ANALOGUES BEARING A 4′-CYANOVINYL GROUP AND
SOME DERIVATIVES THEREOF
Jean M.J. Tronchet a , Imre Kovacs a , Pierre Dilda b , Michel Seman b , Graciela Andrei c ,
Robert Snoeck c , Erik De Clercq c & Jan Balzarini c
a Department of Organic Pharmaceutical Chemistry , University of Geneva , Sciences II,
Geneva 4, CH-1211, Switzerland
b Laboratoires Mayoly Spindler, Research Department , 6, Avenue de l'Europe, Chatou,
Cedex, F-78401, France
c Rega Institute for Medical Research , Minderbroedersstraat 10, Leuven, B-3000, Belgium
Published online: 17 Aug 2006.
To cite this article: Jean M.J. Tronchet , Imre Kovacs , Pierre Dilda , Michel Seman , Graciela Andrei , Robert Snoeck , Erik De
Clercq & Jan Balzarini (2001) SYNTHESIS AND ANTI-HIV ACTIVITY OF THYMIDINE ANALOGUES BEARING A 4′-CYANOVINYL GROUP
AND SOME DERIVATIVES THEREOF, Nucleosides, Nucleotides and Nucleic Acids, 20:12, 1927-1939
To link to this article: http://dx.doi.org/10.1081/NCN-100108323
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(12), 1927–1939 (2001)
SYNTHESIS AND ANTI-HIV ACTIVITY
OF THYMIDINE ANALOGUES BEARING
A 40-CYANOVINYL GROUP AND SOME
DERIVATIVES THEREOF
Jean M. J. Tronchet,*,1 Imre Kovacs,1 Pierre Dilda,2
Michel Seman,2 Graciela Andrei,3 RobertSnoeck, 3 Erik De Clercq,3
and Jan Balzarini3
1Department of Organic Pharmaceutical Chemistry, University of
Geneva, Sciences II, CH-1211 Geneva 4, Switzerland
2Laboratoires Mayoly Spindler, Research Department, 6, Avenue
de l’Europe, F-78401 Chatou Cedex, France
3Rega Institute for Medical Research, Minderbroedersstraat 10,
B-3000 Leuven, Belgium
ABSTRACT
Treatment of 30-O-tert-butyldimethylsilyl-20,50-dideoxy-50-oxothymidine
(4) with potassium or magnesium nitromethanide afforded in good yield
the resolvable epimeric mixture of the expected blocked nitronucleosides
5 which upon dehydration led to the corresponding E-nitroenofur-
anosylthymidine 6. Nucleophilic attack of cyanide onto the nitrovinyl
group led to a nucleoside analogue bearing a terminal 1-cyanovinyl group
(7), a soft electrophilic group which, upon reaction with benzeneselenol,
underwent a conjugate addition to the phenylselenonucleoside derivative
9. All these compounds, eventually de-O-silylated, were subject of invitro
biological testing, some exhibiting interesting cytotoxic or antiviral
properties.
*Corresponding author.
1927
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1928
TRONCHET ET AL.
INTRODUCTION
The 1-cyanovinyl group behaves as a soft electrophile. We have shown
(1) that its electrophilicity depends on the structure of the sugar or nucleoside
on which it is fixed and that this property is correlated with the antiviral and
cytotoxic properties of the molecule bearing this group. A number of the
simple sugar derivatives tested bore high biological activities either cytotoxic
or antiviral (2) whereas the corresponding modified uridines (3) were almost
devoid of activity. This led us to extend our interests toward deoxynucleo-
sides and to prepare thymidine analogues bearing this electrophilic group.
RESULTS AND DISCUSSION
Compounds 2–3 (Scheme 1) are known but as they constitute very
important starting materials and since we considerably improved the yields,
we will briefly describe the new procedures used.
Ogilvie’s team (4) using tert-butylchlorodimethylsilane (TBDMSCl) and
imidazole for the di-O-silylation and 80% acetic acid for the mono-de-O-
silylation, prepared 3 in overall yield of 50% from 1. More recently, Wang
and Middleton (5) using slightly different conditions, obtained yields ranging
from 55 to 65%. We ran the di-O-silylation at higher temperature with
TBDMSCl in pyridine and the partial hydrolysis with dilute trifluoroacetic
acid obtaining a 82% overall yield from 1 to 3.
Compound 3 was almost quantitatively (97%) oxidized to 4 using the
Dess-Martin periodinane reagent following the procedure described by
Samano and Robins (6). Oxidation using chromic anhydride-pyridine led to a
much lower yield (70%) (7) whereas the dicyclohexylcarbodiimide=pyridine=
dimethylsulfoxide=trifluroacetic procedure afforded a 84% yield (5).
The aldehyde 4 was submitted to a Henry’s reaction (8) to afford 5 as an
epimeric mixture. We have shown that aldehydosugars adopt a preferred
conformation where the carbonyl group almost eclipses the vicinal C-O single
bond (9) but that the attack by sodium nitromethylide does not proceed from
the most accessible face, but from the opposite face, a preliminary rotation of
the carbonyl group allowing the incipient negative charge on the oxygen
atom to be directed away from the vicinal oxygen atom (9,10). This corre-
sponds to a Felkin-Anh’s stereochemical approach (11) and leads to the
D-ribo epimer as the major compound. On the contrary, in the presence of a
dication like magnesium, the two oxygen atoms are locked together by
chelation and the issue of the reaction is that of a Cram-Kopecky’s (12)
trajectory. Some results are shown in Table 1. The two epimers 5a (b-D-ribo)
and 5b (a-L-lyxo) have been isolated. Their configurations have been assigned
from the results of previous work (9,10). Dehydration (NaOAc=Ac2O) of a
0
0
mixture of 5a and 5b afforded the E-nitroenose derivative 6 (J5 ,6 ¼ 13.5Hz)

ANTI-HIV ACTIVITY OF THYMIDINE ANALOGUES
1929
Scheme 1.

1930
TRONCHET ET AL.
Table 1. Epimeric Distribution of the Henry’s Reaction (4 ! 5) in Different Reaction
Conditions (Quantities Relative to 1mmol of 4)
MeNO2
t-BuOK (mmol)
MeMgCl (mmol)
t (ꢀC)
Yield (%)
D-ribo=L-
lyxo
3
3
4
3
1.3
0
0
0
3
1.3
1.3
À 20
67
71
65
68
2:1
1:3
1:3
1:6
0
0
0
À 78 ! 0
in 76% yield, the L-lyxo epimer reacting approximately 15 times faster than
its epimer.
The first examples of sugar derivatives bearing a 1-cyanovinyl group
have been described by Paulsen (13) then extended to a large variety of sugars
and nucleosides (2,3,10). Treatment of 6 with potassium cyanide in buffered
phase-transfer conditions led in 85% yield to 7 which was de-O-silylated to 8
(94%).
Compounds 7 and 8 are soft electrophiles but as the thyminyl group
bears nucleofugal properties, a number of nucleophilic attacks, particularly
those using phosphorous nucleophiles which were feasible in other series (9),
led to a loss of the thyminyl group probably via the following mechanism
(Scheme 2).
The particularly soft phenylselenol nucleophile was added to 7 to yield
an unresolvable mixture of the two epimers of 9 (79%) which was de-O-
silylated to 10 (93%).
The nucleoside analogues have been tested for the CC50 (cytotoxic
concentration) and IC50 (cytostatic concentration) on CEM cells (Table 2) as
described in the experimental section. Antiproliferative activities of each
compound against human (NCI-H460, HT29, Caki-1, HL60) or murine
(L1210) cancer cell lines and normal cell lines (MRC5) were compared to
evaluate their selectivity (Table 2).
Compound 2, O-silylated at two positions, exhibited more pronounced
cytotoxic and cytostatic properties against CEM cells, than its analogue 3
Scheme 2.

ANTI-HIV ACTIVITY OF THYMIDINE ANALOGUES
1931
Table 2. Cytotoxic (CC50=mM) and Cytostatic (IC50=mM) Activities of Modified Thymidines
Compound Cell line
2
3
5a
5b
6
7
8
9
10a
CEM=CC50)
CEM (IC50)
6.1
5.1
4
nd
2
2
> 50
> 50
> 100 > 100
nd
>.8100 > 100
> 100 > 100
> 100 > 100
> 50
30.5
> 50
35.5
> 100
5.4
3.6
26
1.03
26
24
30
1.214.6 11.6
0.824.9 3.7
31.5 28.3 > 100
2 2
10.6
MRC5 (CC50)
L1210 (CC50)
NCI-H460 (CC50)
HT29 (CC50)
Caki-1 (CC50)
HL-60 (CC50)
3.4
2 3.5
2.6
1217 12
69
> 100
> 100
24
41
5.4
8.7
18.5
50.5
5.7
11
3
33
3.2
47.5 27
22 4.2
100
30.6
100
42.5
45.5 23.5
a2:1 Epimeric mixture.
only silylated at the 30 position. These cytotoxic and antiproliferative (cyto-
static) properties of TBDMS groups on nucleoside analogues have already
been described (14). However, 2 did not show any significant difference
between IC50 and CC50 on CEM cell lines and between CC50 for non-tumoral
(MRC5) and tumoral cells. The two epimers 5a and 5b obtained from 4 by a
Henry’s reaction had very similar cellular activities except on the NCI-H460
cell lines for which the a-L-lyxoepimer (5b) exhibited a better activity than the
b-D-ribo-one (5a). When a mixture of 5a and 5b was dehydrated to afford 6,
an improvement of activities on the cancer cell lines was observed. This was
especially the case with the murine lymphocytic leukemia (L1210) for which
the in vitro selectivity index (ratio between CC50 for non-tumoral (MRC5)
and tumoral cells), was high (13). The most cytostatic nucleoside of this study
was compound 7. However, the in vitro selectivity index observed was low
considering the antiproliferative effect on the non-tumoral cell line
(CC50 ¼ 3.4mM). A decrease of all activities was observed when 7 was de-O-
silylated into 8. An interesting selectivity index on L1210 (12) was restored
with compound 9. The 30-O-desilylation of 9 to afford 10 brought about a
decrease of activity similar to the one observed between compounds 7 and 8.
However 10 retained the antiproliferative properties of 9 on L1210 cells with
a remarkable selectivity index ( > 28).
The compounds were also evaluated against a panel of viruses in cell
culture. None of the compounds showed significant activity at subtoxic
concentrations against herpes simplex virus type 1 and type 2, vaccinia virus
and vesicular stomatitis virus in E6SM cell cultures; vesicular stomatitis virus,
Coxsackie virus B4 and respiratory syncytial virus in HeLa cell cultures; and
parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4 and
Punta Toro virus in Vero cell cultures. However, whereas 9 showed some
marginal inhibitory activity against varicella-zoster virus (strains YS and
YS=R) (EC50: 3.5 mM; CC50: 16mM), compounds 5a, 6, 7, 5b, 9 and 10 were
endowed with anti-cytomegalovirus activity (strains AD-169 and Davis) at

1932
TRONCHET ET AL.
Table 3. Anti-Human Cytomegalovirus Activity of Test Compounds in Human Embryonic
Lung Fibroblasts
Anti-CMV
Activity EC50 (mM)a
Cytotoxicity=Cytostatic Activity
Cell Morphologyb
(MCC)
Cell Growthc
(IC50)
AD-169
Davis
Compound
5a
5b
6
7
8
14
17
2
43
4
16
0
31
> 200
50
50
110
92
10
50
42
200
35
161
16
> 200
3.1
50
> 200
20
200
9
3.1
38
10d
30
aInhibitory concentration required to reduce virus plaque formation by 50%. Virus input was
100 plaque forming units (PFU).
bMinimum cytotoxic concentration required to cause a microscopically detectable alteration
in cell morphology.
cCytostatic concentration required to reduce HEL cell growth by 50%.
d2:1 Epimeric mixture.
subtoxic concentrations (Table 3). The highest selectivity (ratio of
CC50=EC50) was performed by 5a (8–27). With regard to their anti-HIV
activity, 9 and 10 inhibited HIV-1- and HIV-2-induced cytopathicity in CEM
cell cultures at 2.5–10mM; and 3.5–5.3mM, respectively, whereas their cyto-
static concentrations were 17 and 34mM when measured in the same assay.
This resulted in a selectivity index of 6–10 for 10.
EXPERIMENTAL
General Synthetic Methods
Melting points (uncorrected) were determined under microscope with a
Mettler FP52melting-point apparatus. Thin layer chromatographies (TLC)
were performed on silica gel HF254 (Merck) with detection by UV light.
Column chromatography was conducted on silica gel 60 (0.04–0.2mm,
Merck). IR spectra were recorded on a Perkin-Elmer Model 1600 spectro-
meter and UV spectra were measured on a Kontron Uvicon 810 spe-
trophotometer. NMR spectra were recorded at 20ꢀC on Bruker WP 200 SY
(1H 200MHz) or Varian Gemini (1H 300MHz) spectrometers (chemical
shifts in ppm from TMS;dunits). Optical rotations were measured with a
Schmidt-Haensch polarimeter. Mass spectra (EIMS, 70eV) were recorded on
a VG-70-70E spectrometer.

ANTI-HIV ACTIVITY OF THYMIDINE ANALOGUES
1933
Antiproliferative Activity
Antiproliferative activities of the compounds expressed at CC50 were
determined after 3 days using five cancer cell lines in exponential grow rate
[L1210 (murine lymphocytic leukemia, ATCC # CCL-219), NCI-H460
(human lung large cell carcinoma, ATCC # HTB-177), HT29 (human colon
adenocarcinoma ATCC # HTB-38), Caki-1 (human kidney carcinoma,
ATCC # HTB-46) and HL60 (human promyelocytic leukemia, ATCC #
CCL-240)]. A normal cell line [MRC5 (human foetal lung fibroblast, ATCC
# CCL-171)] was used as control. With MRC5 cells, antiproliferative effects
of each compound were determined using the MTT assay as previously
described by Park et al(15). Briefly, the MTT test is based on the enzymatic
reduction of the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] in living, metabolically active cells, but not
dead cells. The reaction product, a purple colored formazan soluble in
dimethylsulfoxide, is measured colorimetrically at 550nm, using a multiwell
plate reader. Measurements are, as a rule, duplicated. These results are issued
from two distinct manipulations. Appropriate positive controls were included
CC50 (cytotoxic concentration) and IC50 (cytostatic concentration) of each
compound were determined on the human T lymphoblastoid cell line CEM
(ATCC # CL-119) after 4 days of culture at exponential rate. At the end of
this period, cells were incubated with propidium iodide that stained only
dead cells. The distinction between cytotoxic and cytostatic effects of the
compound was made by using a flow cytometry cell analyzer (Facscalibur
Becton Dickinson). The total cell number and ratio between living and dead
cells were representative of the cytostatic and cytotoxic properties of the
compounds respectively. Measurements are generally duplicated.
Antiviral Assays
The antiviral assays, other than HIV, were based on inhibition of virus-
induced cytopathicity in either E6SM [herpes simplex virus type 1 (HSV-1),
HSV-2, vaccinia virus (VV)], HEL (VZV, CMV) or Vero (VSV, respiratory
syncytial virus, Coxsackie virus, parainfluenza-3 virus, Sindbis virus, Punta
Toro virus, reovirus-1) cell cultures, following previously established proce-
dures. Briefly, confluent cell cultures in microtiter 96-well plates were
inoculated with 100 CCID50 of virus, 1 CCID50 being the virus dose required
to infect 50% of the cell cultures. After a 1 h virus adsorption period, residual
virus was removed, and the cell cultures were incubated in the presence
of varying concentrations (400, 200, 100, . . .mg=mL) of the test compounds.
Viral cytopathicity was recorded as soon as it reached completion in
the control virus-infected cell cultures that were not treated with the test
compounds.

1934
TRONCHET ET AL.
Inhibition of HIV-1-Induced Cytopathicity in CEM Cell Cultures
The methodology of the anti-HIV assays has been described previously.
Briefly, human CEM ( ꢁ 36105cells mlÀ 1) cells were infected with 100
CCID50 of HIV-1 (IIIB) or HIV-2(ROD) =ml and seeded in 200ml wells of a
microtiter plate, containing appropriate dilutions of the test compounds.
After 4 days of incubation at 37ꢀC, virus-induced CEM giant cell formation
was examined microscopically.
30,50-Bis-(O-tert-butyldimethylsilyl)-20-deoxythymidine (2). To a solu-
tion of thymidine (24.22g, 0.100mol) in anhydrous pyridine (200mL), ter-
butylchlorodimethylsilane (37.67g, 0.250mol) was added. The reaction
mixture was stirred at room temperature for 1h, at 60–70ꢀC for 22h, then
cooled to room temperature. The excess of silylating agent was destroyed by
addition under stirring of water (10mL, 15min). Solvents were evaporated
and coevaporated with toluene (26100mL), the residue dissolved in ice-cold
EtOAc (400mL), washed successively with a cold 1M HCl solution (200mL),
with cold water (26100mL), saturated NaHCO3 aqueous solution, and
aqueous saturated sodium chloride solution (100mL), then dried (Na2SO4),
and concentrated. The residue was crystallized in a mixture of EtOAc (5mL)
and hexane (65mL) giving 2 (39.18g) as a white solid. A second fraction of 2
(4.06g) was obtained after recrystallization in hexane (15mL). Cumulated
yield: 43.23g (92%).
30-O-(tert-Butyldimethylsilyl)-20-deoxythymidine (3). To a solution of 2
(44.2g, 93.9mmol) in CH2Cl2 (510mL) a mixture of trifluoroacetic acid and
water (10:1, v=v, 51mL) was added and the reaction mixture was stirred at
room temperature for 45 min, then in an ice bath for 30min. The pale yellow
solution was diluted with cold CH2Cl2 (100mL), washed with ice-cold water
(450mL), saturated NaHCO3 (200mL) and NaCl (150mL) aqueous solu-
tions, dried (Na2SO4), concentrated, and submitted to a column chromato-
graphy (6:1 ! 4:1 CH2Cl2=acetone) to afford 3 (29.6g, 89%).
1-[3-O-(tert-Butyldimethylsilyl)-2-deoxy-b-D-erythro-pentodialdo-1,4-fur-
anosyl]thymine (4). 3 (7.13g, 20.0mmol) was oxidized with Dess-Martin
periodinane reagent (12.72g, 30.0mmol) following the procedure described
by Samano and Robins (6) affording crude 4 (6,85g, 97%) as a white solid
which was used for the next step without further purification.
1-[3-O-(tert-Butyldimethylsilyl)-2,6-dideoxy-6-nitro-b-D-ribo and a-L-
lyxo-hexofuranosyl]thymine (5a,b). Method A: Under argon atmosphere,
methylmagnesium chloride in THF (6.57mL of a 2.97M solution, 19.5mmol)
was added at 0ꢀC to a stirred solution of nitromethane (2.44mL, 45.0mmol)
in anhydrous THF (75mL) for 10min, and the stirring was continued at that

ANTI-HIV ACTIVITY OF THYMIDINE ANALOGUES
1935
temperature for an additional 10min. After cooling to À 78ꢀC, a solution of
4 (5.31g, 15.0mmol) in dry THF (25mL) was added in 15min and the
temperature was allowed to rise to À 23ꢀC in 3h. The reaction mixture was
kept at that temperature for 18h, at 0ꢀC for 9h, diluted with ice-cold EtOAc
(300mL), washed successively with a cold 1M HCl solution (100mL), water
(26100mL), saturated aqueous NaCl (100mL), dried (Na2SO4), evaporated,
and purified by column chromatography (3:2hexane =EtOAc). The two
diastereoisomers were partially separated to afford the less polar product 5a
(0.24g), the slower moving isomer 5b (0.47g), both as white foams, and a
mixture of the two isomers (3.50g), total yield (68%). The ratio of 5a to 5b
1
was 1:6 (from the H NMR spectra of the crude product): 5a (b-D-ribo iso-
26
mer), mp 67.9–69.1ꢀC; RF 0.59 (1:3 hexane=EtOAc); [a]D þ12.7ꢀ (c 1.0,
CHCl3); nKmBaxr 3414 and 3204 (OH, NH), 2955, 2928, and 2859 (CH), 1693
(C ¼ O), 1555 (NO2(as)), 1471, 1382(NO 2(s)), 1125, 1048, and 833cmÀ 1. 1H
NMR (C6D6, 300MHz): d10.20 (s, 1 H, NH), 6.56 (q 1 H, J5,Me-6 1.0Hz, H-
6), 5.63 (dd, 1 H, J1 ,2 a 6.5Hz, J1 ,2 b 7.3Hz, H-10), 4.66 (bd, 1 H, J5 ,5 -OH
0
0
0
0
0
0
6.3Hz, 50-OH), 4.56 (quint., 1 H, J2 a,3 3.1Hz, J2 b,3 6.4Hz, J3 ,4 2.4Hz, H-3 ),
0
0
0
0
0
0
0
4.41 (dd, 1 H, J6 a,6 b 17.5Hz, J5 ,6 b 8.9Hz, Hb-60), 4.40 (m, 1 H, J5 ,6 a 8.3Hz,
0
0
0
0
0
0
0
J4 ,5 ꢁ 1Hz, H-50), 3.96 (dd, 1 H, Ha-60), 3.69 (d, 1 H, J3 ,4 2.4Hz, H-4 ), 2.39
0
0
0
0
(ddd, 1 H, J2 a,2 b 13.4Hz, Hb-20), 1.82( ddd, 1 H, Ha-20), 1.69 (d, 3 H, Me-5),
0.91 (s, 9 H, CMe3), 0.04 and 0.03 (2 s, 6 H, SiMe2). EIMS: m=z (%) 171
(100), 75 (99), 117 (52), 201 (17), 295 (4), and 358 (2, Mꢂþ - t-Bu).
Anal. Calcd for C17H29N3O7Si (415.52): C, 49.14; H, 7.03; N, 10.11.
Found: C, 49.25; H, 7.12; N, 9.96.
0
0
5b (a-L-lyxo isomer) mp 69.7–71.1ꢀC; RF 0.59 (1:3 hexane=EtOAc),
27
[a]D þ21.7ꢀ (c 1.0, CHCl3); nKmBaxr 3400 and 3200 (OH, NH), 2955, 2928, and
2858 (CH), 1689 (C ¼ O), 1556 (NO2(as)), 1470, 1379 (NO2(s)), 1114, 1065,
1
and 836cmÀ 1. H NMR (C6D6, 300MHz, 60ꢀC): d9.10 (bs, 1 H, NH), 6.24
(q, 1 H, JMe-5,6 1.2Hz, H-6), 5.49 (t, 1 H, J1 ,2 a ꢁ J1 ,2 b 6.9Hz, H-10), 4,46
0
0
0
0
(quint., 1 H, J2a ,3 3.3Hz, J2 b,3 6.6Hz, J3 ,4 3.0Hz, H-30), 4.31 (m, 1 H, J4 ,5
0
0
0
0
0
0
0
0
6.0Hz, J5 ,6 a 8.1Hz, J5 ,6 b 3.9Hz, H-50), 4.14 (dd, 1 H, J6 a,6 b 13.2Hz, Hb-60),
0
0
0
0
0
0
4.08 (dd, 1 H, Ha-60), 3.54 (dd, 1 H, H-40), 3.35 (bs, 1 H, 50-OH), 2.27 (quint.,
1 H, J2 a,2 b 13.5Hz, Hb-20), 1.77 (ddd, 1 H, Ha-20), 1.64 (d, 3 H, Me-5), 0.89
(s, 9 H, CMe3), 0.03 and 0.01 (2 s, 6 H, SiMe2). EIMS: m=z(%) 171 (100), 75
(81), 117 (49), 201 (10), 295 (3), and 340 (1, 358 (1, Mꢂþ - t-Bu).
Anal. Calcd for C17H29N3O7Si (415.52): C, 49.14; H, 7.03; N, 10.11.
Found: C, 49.16; H, 7.16; N, 9.88.
0
0
Method B: To a solution of nitromethane (2.75mL, 45.0mmol) in ter-
butanol (45mL), potassium tert-butoxide in tert-butanol (19.5mL of a 1.0M
solution, 19.5mmol) was added in 10min and the mixture, containing the
precipated potassium salt, was stirred for 5min, then 4 (5.21g, 15.0mmol)
was added in one portion. The reaction mixture was stirred at ambient
temperature for 60min, quenched with a saturated aqueous solution of
NH4Cl (50mL) then diluted with EtOAc (200mL) and water (15mL).

1936
TRONCHET ET AL.
The layers were separated, the organic phase was washed successively with
water (50mL), a saturated aqueous solution of NaCl (50mL), dried (Na2SO4)
and concentrated under reduced pressure. Purification by column chroma-
tography (3:2hexane =EtOAc) afforded 5ab (4,17 g, 67%) containing the
faster and slower moving products in a 2:1 ratio (1H NMR spectra).
1-[(E)-3-O-(tert-Butyldimethylsilyl)-2,5,6-trideoxy-6-nitro-b-D-erythro-
hex-5-enofuranosyl]thymine (6). 5a,b (4.67g, 11.2mmol) was dissolved at
0ꢀC in acetic anhydride (28.0mL) and sodium acetate (5.62g, 68.5mmol) was
added. The mixture was stirred at 0ꢀC for 30min and at room temperature
for 7.5h and the solvent was evaporated (oil pump, bath 35–40ꢀC). The re-
sidue was suspended in EtOAc (240mL), washed with a mixture of saturated
aqueous NaHCO3 and NaCl solutions (50=50mL), with saturated NaCl
(50mL), dried (Na2SO4) and concentrated. The residue was purified by col-
umn chromatography (2:1 hexane=EtOAc) to give compound 6 (3.38g,
76%). An analytical sample was obtained by recrystallization from hex-
24
ane=EtOAc, mp 135.1–136.9ꢀC; RF 0.23 (1:1 hexane=EtOAc); [a]D þ55.1ꢀ
(c 1.0, CHCl3); nKmBaxr 3186 (NH), 2957, 2927, and 2858 (CH), 1705 and 1691
(C ¼ O), 1531 (NO2(as)), 1471, 1352(NO 2(s)), 1119, 1056, and 837cmÀ 1. 1H
0
0
NMR (CDCl3, 200MHz): d9.40 (s, 1 H, NH), 7.31 (dd, 1 H, J5 ,6 13.5Hz,
0
0
0
0
0
0
J4 ,5 4Hz, H-5 ), 7.17 (dd, 1 H, J4 ,6 1Hz, H-6 ), 7.02( q, 1H, J5-Me,6 1.2Hz, H-
6), 6.20 (t, 1 H, J1 ,2 a ꢁ J1 ,2 b ꢁ 6.7Hz, H-10), 4.50–4.33 (m, 2H, J3 ,4 ; 5.5Hz,
0
0
0
0
0
0
J2 a,3 6.4Hz, J2 b,3 5.0Hz, H-30 and H-40), 2.50–2.23 (m, 2H, J
13.6Hz,
0
0
0
0
0
0
2 a,2 b
Hb-20 and Ha-20), 1.97 (d, 3 H, 5-Me), 0.91 (s, 9 H, CMe3), 0.12( s, 6 H,
SiMe2). EIMS: m=z (%) 73 (100), 201 (23), 183 (14), 295 (10), 340 (8, Mꢂþ - t-
Bu).
Anal. Calcd for C17H27N3O6Si (397.51): C, 51.37; H, 6.85; N, 10.57.
Found: C, 51.13; H, 6.84; N, 10.50.
1-[3-O-tert-Butyldimethylsilyl)-2,5-dideoxy-5-C-methylene-b-D-erythro-
hexofuranosylurononitrile]thymine (7). A solution of 6 (600mg, 1.5mmol) in
CH2Cl2 (30mL) was added to a NaOAc=AcOH buffer solution prepared
from NaOAc (2.46g, 30mmol), water (7.5mL), AcOH (1.72mL, 30mmol)
and n-Bu4NHSO4 (100mg, 0.3mmol). To the vigorously stirred biphasic
mixture, KCN (1.37g, 21.0mmol) was added followed by the addition of
further small quantities of KCN (after 2.5h, 0.20g, 3.0mmol; after 5.5h,
0.19g, 3.0mmol; after 25.5h, 0.10, 1.5mmol) where the pH changed from 6.0
to 7.0. The reaction mixture was stirred during an additional 3h, then diluted
with water (10mL). The organic layer was separated, washed with water
(10mL), dried (Na2SO4) and concentrated. The crude product was purified
by column chromatography (8:1 CH2Cl2=EtOAc) to give 7 (480mg, 85%),
25
mp 44.9–47.1ꢀC; RF 0.16 (8:1 CH2Cl2=EtOAc); [a]D þ31.3ꢀ (c 1.0,
CHCl3);nKmBaxr 3186 (NH), 3056, 3028, and 2856 (CH), 2227 (CN), 1697
(C ¼ O), 1472, 1254, 1065, and 837cmÀ 1. 1H NMR (CDCl3, 200MHz):d9.51

ANTI-HIV ACTIVITY OF THYMIDINE ANALOGUES
1937
0
0
0
0
(s, 1 H, NH), 7.41 (q, 1 H, J5-Me,6, 1Hz, H-6), 6.37 (t, 1 H, J1 ,2 a ꢁ J1 ,2 b ꢁ 6.5
0
0
0
0
0
0
Hz, H-1 ), 6.12and 6.08 (2 s, 2H, H 2C ¼ C5 ), 4.40 (dt, 1 H, J3 ,4 4.5Hz, J2 b,3
4.5Hz, J2 a,3 6.5Hz, H-30), 4.23 (d, 1 H, H-40), 2.37 (ddd, 1 H, J2 a,2 b 14Hz,
Hb-20), 2.25 (dt, 1 H, Ha-20), 1.95 (d, 3 H, 5-Me), 0.89 (s, 9 H, CMe3), 0.09
and 0.08 (2 s, 6 H, SiMe2). EIMS: m=z (%) 320 (100, Mꢂþ - t-Bu), 168 (78), 73
(76), 252 (22), and 377 (0.2, Mꢂþ).
0
0
0
0
Anal. calcd for C18H27N3O4Si (377.52): C, 57.27; H, 7.21; N, 11.13.
Found: C, 56.92; H, 7.29; N, 10.87.
1-[2,5-Dideoxy-5-C-methylene-b-D-erythro-hexofuranosylurononitrile]-
thymine (8). A solution of 7 (570mg, 1.50mmol) in a mixture of tri-
fluoroacetic acid and water (10:1, 12.5mL) was stirred at room temperature
for 5h, evaporated and coevaporated with toluene (3615mL). The crude
product was purified by column chromatography (7:3 CH2Cl2=acetone) to
give compound 8 (370mg, 94%), mp 145.1–148.1ꢀC; RF 0.18 (2:1
KBr
max
CH2Cl2=acetone); a2D8 þ 26:8 ꢀ (c 1.0, MeOH);n
3372 and 3223 (OH and
1
NH), 2224 (CN), 1721 and 1656 (C ¼ O), 1477, 1272, and 1055cmÀ 1. H
NMR (DMSO-d6, 200MHz, 40ꢀC):d11.00 (s, 1 H, NH), 7.42( q, 1 H, J5-Me,6
0
0
0
1Hz, H-6), 6.27 (d, 1 H, J4 ,Ha 0.3Hz, HaHbC ¼ C5 ), 6.23 (d, 1 H, J4 ,Hb
0.7Hz, HaHbC ¼ C5 ), 6.23 (t, 1 H, J1 ,2 a ꢁ J1 ,2 b ꢁ 6.6Hz, H-10), 5.58 (d, 1 H,
0
0
0
0
0
J3 ,4 5Hz, H-40), 4.35 (m, 1 H, J2 a,3 5.0Hz, J2 b,3 6.9 Hz, J3 ,3 -OH ꢁ 5Hz, H-
0
0
0
0
0
0
0
0
30), 4.27 (d, 1 H, 30-OH), 2.35 (quint., 1 H, J2 a,2 b 13.5Hz, Hb-20), 2.19 (ddd, 1
H, Ha-20), and 1.78 (d, 3 H, 5-Me). EIMS: m=z (%) 126 (100, 55 (81), 94 (71),
138 (46), 263 (9, Mꢂþ).
0
0
Anal. Calcd for C12H13N3O4 (263.26): C, 54.75; H, 4.98; N, 15.96.
Found: C, 54.59; H, 4.93; N, 15.89.
1-[3-O-(tert-Butyldimethylsilyl)-2,5-dideoxy-5-C-phenylselenomethyl-b-
D-ribo- and a-L-lyxo-hexofuranosylurononitrile]thymine (9a,b). A mixture of
7 (566mg, 1.5mmol), benzeneselenol (0.95mL, 9.0mmol), and triethylamine
(2.09mL, 0.15mmol) was stirred at room temperature under argon atmo-
sphere for 1h, and diluted with EtOAc (5mL) and hexane (6mL). Air was
bubbled through the solution during 1h and the mixture was submitted to
column chromatography (13:7 hexane=EtOAc to give 9a,b (630mg, 79%) as
a colorless solid, mp 44.6–46.5ꢀC; RF 0.18 (3:2hexane =EtOAc);nmKBaxr 3186
(NH), 3054 (CH arom.), 2955, 2927, and 2855 (CH alph.), 2244 (CN), 1695
1
(C ¼ O), 1472, 1363, 1258, 1226, 1084, and 836cmÀ 1. H NMR (CDCl3,
200MHz), the ratio of the two isomers was about 2:1, major component:
d9.23 (s, 1 H, NH), 7.68–7.20 (m, 6 H, Ph and H-6), 6.37 (t, 1 H,
J1 ,2 a ꢁ J1 ,2 b ꢁ 6.7Hz, H-10), 4.32( dt, 1 H, J2 a,3 5 Hz, J2 b,3 6.5Hz, J3 ,4
0
0
0
0
0
0
0
0
0
0
5Hz, H-30), 3.98 (dd, 1 H, J4 ,5 2Hz, H-40), 3.27 (dd, 1 H, J5 a,5 b 13Hz, J5 ,5 b
0
0
00
00
0
00
7.7Hz, Hb-500), 3.12( dd, 1 H, J5 ,5 a 8Hz, Ha-500), 2.88 (td, 1 H, H-50), 2.45-
0
00
0
2.13 (m, 2H, Ha-2 , Hb-20), 1.95 (d, 3 H, JMe-5,6 1Hz, Me-5), 0.84 (s, 9 H,
CMe3), 0.04 and À 0.02(2 s, 6 H, SiMe2). Minor isomer: d9.26 (s, 1 H, NH),

1938
TRONCHET ET AL.
7.68–7.20 (m, 6 H, Ph and H-6), 6.25 (t, 1 H, J1 ,2 a ꢁ J1 ,2 b ꢁ 7.0Hz, H-10),
0
0
0
0
4.50 (quint., 1 H, J2 a,3 3.5Hz, J2 b,3 6.5Hz, J3 ,4 3.8Hz, H-30), 4.05 (dd, 1 H,
0
0
0
0
0
0
J4 ,5 5 Hz, H-40), 3.30–3.00 (m, 3 H, H2C-500 and H-50), 2.45–2.13 (m, 2 H,
Ha-20, Hb-20), 1.92( d, 3 H, JMe-5,6 1Hz, Me-5), 0.90 (s, 9 H, CMe3), 0.13 and
0.11 (2 s, 6 H, SiMe2). EIMS: m=z (%) 73 (100), 478 (50, Mꢂþþ1 - t-Bu), 168
(37), 320 (23, Mꢂþ - t-Bu - PhSeH), 201 (22), 352 (5), and 410 (3).
Anal. Calcd for C24H33N3O4SeSi (534.59): C, 53.92; H, 6.22; N, 7.86.
Found: C, 54.02; H, 6.30; N, 7.73.
0
0
1-(2,5-dideoxy-5-C-phenylselenomethyl-b-D-ribo and a-L-lyxo-hexo-fur-
anosylurononitrile]thymine (10a,b). Compound 10a,b was prepared from
9a,b (630mg, 1.18mmol) as described for 8. The crude product was purified
by column chromatography (3:1 CH2Cl2=acetone) to give 10a,b (460mg,
93%) as a pale yellow solid. Mp 66.3–67.4ꢀC; RF 0.16 (3:1 CH2Cl2=acetone);
KBr
n
3430 and 3200 (OH and NH), 3052 (CH arom.), 2924 (CH aliph.), 2243
max
(CN), 1690 (C ¼ O), 1476, 1273, 1076, and 739cmÀ 1. H NMR (DMSO-d6,
300MHz), the ratio of the two isomers was about 2:1; major component:
d11.16 (s, 1 H, NH), 7.42( q, 1 H, JMe-5,6 1Hz, H-6), 7.60–7.20 (m, 5 H, 0PhSe),
1
6.18 (t, 1 H, J1 ,2 a ꢁ J1 ,2 b 6.8 Hz, H-10), 5.54 (d, 1 H, J3 ,3 -OH 4.5Hz, 3 -OH),
0
0
0
0
0
0
4.26 (m, 1 H, J2 a,3 5.1Hz, J2 b,3 6.5Hz, J3 ,4 4.5Hz, H-30), 3.84 (t, 1 H, J4 ,5
0
0
0
0
0
0
0
0
4.5Hz, H-40), 3.42( m, 1 H, J5 ,5 a 10.0Hz, J5 ,5 b 5.2Hz, H-50), 3.31 (dd, 1 H,
0
00
0
00
J5 a,5 b 14Hz, Hb-500), 3.22 (dd, 1 H, Ha-500), 2.26 (m, 1 H, J2 a,2 b 14Hz, Hb-
20), 2.13 (ddd, 1 H, Ha-20), and 1.75 (d, 3 H, Me-5); minor isomer: d11.16 (s, 1
H, NH), 7.42( q, 1 H, JMe-5,6 1Hz, H-6), 7.60–7.20 (m, 5 H, PhSe), 6.19 (dd, 1
00
00
0
0
H, J1 ,2 a 6.3Hz, J1 ,2 b 7.8Hz, H-10), 5.59 (d, 1 H, J3 ,3 -OH 4.8Hz, 30-OH), 4.37
0
0
0
0
0
0
(m, 1 H, J2 a,3 3.2Hz, J2 b,3 6.6Hz, J3 ,4 3.3Hz, H-30), 3.89 (dd, 1 H, J4 ,5
0
0
0
0
0
0
0
0
6.9Hz, H-40), 3.50 (m, 1 H, J5 ,5 a 10.2Hz, J5 .5 b 4.5Hz, H-50), 3.29 (dd, 1 H,
0
00
0
00
J5 a,5 b 14Hz, Hb-500), 3.18 (dd, 1 H, Ha-500), 2.33 (dd, 1 H, J2 a,2 b 14Hz, Hb-
20), 2.08 (ddd, 1 H, Ha-20), 1.75 (d, 3 H, Me-5). EIMS m=z (%) 78 (100), 126
(73), 158 (57), 55 (53), 314 (3), 251 (2), and 421 (1, Mꢂþþ1).
Anal. Calcd for C18H19N3O4Se (420.33): C, 51.44; H, 4.56; N, 10.00.
Found: C, 51.74; H, 4.90; N, 9.74.
00
00
0
0
ACKNOWLEDGEMENTS
The authors thank Dr Eder for the elemental analyses and Prof.
F. Gulacar for the mass spectra. The excellent technical assistance in the
virological experiments by Mrs. Ann Absillis, Mrs. Frieda De Meyer,
Mrs. Anita Camps, Mrs. Lies Vandenheurck and Mrs. Anita van Lierde
was highly appreciated. This work was generally supported by the Swiss
National Research Foundation (grant # 20.50827-97), by the European
Commission, and by the Geconcerteerde Onderzoeksacties Vlaanderen,
Kredit Nr 00=12.

ANTI-HIV ACTIVITY OF THYMIDINE ANALOGUES
1939
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Received May 30, 2001
Accepted September 5, 2001