Synthesis and anti-HIV-1 activity of 1,3-phenylene bis-uracil analogues of MKC-442

Article abstract of DOI:10.1002/jhet.5570440216

(Chemical Equation Presented) Reaction of 1,3-phenylenediacetonitrile with the zinc organometallic reagent of ethyl 2-bromobutyrate afforded the 1,3-phenylene-bis(acetoacetate) 2 which was used as the starting material for the synthesis of 1,3-phenylene-bis[6-(2-thiouracil)] 4. Desulphurization of 4 gave the corresponding bis-uracil 6, which after silylation was N-1 alkylated with bis(allyoxy)methane using TMS-triflate as the catalyst or with chloromethyl ethyl ether to give the MKC-442 analogues 7 and 9. The amino-DABO and S-DABO derivatives 11, 12a,b and 14 were also synthesized. The anti-HIV-1 activity test showed that when MKCM42 analogues were constructed with 1,3-phenylene in all cases they were detrimental to have activity against HIV-1.

Full text of DOI:10.1002/jhet.5570440216

Mar-Apr 2007
381
Synthesis and Anti-HIV-1 Activity of 1,3-Phenylene Bis-Uracil
Analogues of MKC-442
Youssef L. Alya and Erik B. Pedersen*
Nucleic Acid Center†, Department of Chemistry and Physics, University of Southern Denmark, Campusvej 55,
DK-5230 Odense M, Denmark
Paolo La Colla and Roberta Loddo
Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e
Biotecnologie Microbiche, Universita di Cagliari, Cittadella Universitaria, 09042 Monserrato, Italy
Received June 28, 2006
O
N
O
N
Et
Et
NH
NH
O
O
RO
OR
Reaction of 1,3-phenylenediacetonitrile with the zinc organometallic reagent of ethyl 2-bromobutyrate
afforded the 1,3-phenylene-bis(acetoacetate) 2 which was used as the starting material for the synthesis of 1,3-
phenylene-bis[6-(2-thiouracil)] 4. Desulphurization of 4 gave the corresponding bis-uracil 6, which after
silylation was N-1 alkylated with bis(allyoxy)methane using TMS-triflate as the catalyst or with chloromethyl
ethyl ether to give the MKC-442 analogues 7 and 9. The amino-DABO and S-DABO derivatives 11, 12a,b
and 14 were also synthesized. The anti-HIV-1 activity test showed that when MKC-442 analogues were
constructed with 1,3-phenylene in all cases they were detrimental to have activity against HIV-1.
J. Heterocyclic Chem., 44, 381 (2007).
HIV-1 strain with RT mutations are shown to emerge
rapidly upon treatment with existing drugs [5]. Therefore,
it is of interest to study whether MKC-442 analogues with
an extra nucleobase of the MKC-442 type attached to the
benzylic part of MKC-442 will show better or comparable
anti HIV-1 activity towards wild-type and Y181C or
Y181C+K103N mutated HIV-1 strains. The perspective
could be that an asymmetric pair of nucleobases
substituted in this type of derivatives could result in
compounds with activity against a broader range of HIV
resistant mutant viruses.
INTRODUCTION
Since the synthesis of 1-[(2-hydroxyethoxy)methyl]-6-
(phenylthio)thymine (HEPT) by Miyasaka et al. [1] in
1989, there has been a growing interest in non-nucleoside
reverse transcriptase inhibitors (NNRTIs). HEPT was
further developed into 6-benzyl-1-(ethoxymethyl)-5-
isopropyluracil (MKC-442, emivirine, Figure 1) [2],
which was brought into phase III clinical trials. However,
Triangle Pharmaceuticals halted development of MKC-
442 in January 2002 when a comparative study showed
emivirine to be less potent than other antiretrovirals [3].
RESULTS AND DISCUSSION
O
In order to synthesis the benzylic base pair at C-6 a
route was chosen in which the β-ketoester 2 would be a
key intermediate as in the synthesis of MKC-442 by
Danel et al. [6]. Commercially available 1,3-phenylene-
diaceto-nitrile (1) was reacted with zinc organometallic
reagent of ethyl 2-bromobutyrate in anhydrous THF to
afford the bis-β-ketoester 2 in 38% yield and the
pyridinone 3 as by-product in 3% yield. Compound 2 was
used as the starting material for the synthesis of the bis-
thiouracil 4 in a ring closure reaction with thiourea and
sodium ethoxide using the method of Danel et al. [6] to
afford this compound as the major product in 42% yield
and 2-thio-pyrimidine-benzylic-β-ketoester 5 as minor
product in 18% yield. Desulphurization of 4 using
chloroacetic acid [7,8] gave the benzylic bis-uracil 6 in
63% yield (Scheme 1).
HN
O
N
O
MKC-442
Figure 1
MKC-442 inhibits reverse transcriptase (RT) of the
HIV-1 virus allosterically, as it binds to the enzyme in a
hydrophobic pocket outside the active site and thereby
changes the conformation of the active site, making RT
inactive. The inhibitor is mainly held in place in the
pocket by hydrogen bonds and hydrophobic bonds to the
nearby amino acids [4]. The importance of finding new
analogues with higher binding affinity is obvious, because

382
Y. L. Aly, E. B. Pedersen, P. La Colla and R. Loddo
Vol 44
Scheme 1
O
O
O
O
O
1. Zn/THF
2. K2CO3
3. 10% HCl
CN
OEt
OH
OEt
Et
CH3CH2CHBrCOOEt
Et
Et
Et
+
+
O
CN
OEt
O
N
H
Et
O
2
1
3
O
O
O
O
OEt
S
EtOH/EtONa
HN
NH
Et
+
+
2
HN
H2N
NH2
S
N
H
N
H
S
S
N
H
4
5
ClCH2COOH
O
O
HN
NH
O
N
H
N
H
O
6
Compound 6 was silylated using bis-(trimethylsilyl)-
acetamide (BSA) and it was subsequently N-1 alkylated
using chloromethyl ethyl ether [7] to give the bis-non-
nucleoside analogues 7 in 23% yield and the N-1 mono-
alkylated by-product 8 in 11% yield. The benzylic bis-
uracils 6 was also reacted with bis(allyloxy)methane [9]
in the presence of trimethylsilyl trifluoromethanesulfonate
(TMS triflate) as a Lewis acid catalyst [10] to give the
bis-non-nucleoside analogue 9 in 34% yield and the N-1
mono-alkylated derivative 10 in 22% yield (Scheme 2).
Having the bis-β-ketoester 2 readily available as a
starting material, it was straight forward also to synthesis
the corresponding amino-DABO and S-DABO analogues.
These are 2-amino and 2-alkylthio analogues respectively,
of uracils and they are highly active against HIV when
properly substituted [11].
For the synthesis of an amino-DABO derivatives the
-
ketoester 2 was reacted with N,N-dimethylguanidinium
sulphate in EtONa to afford compound 11 in 18% yield
(Scheme 3).
Scheme 2
Scheme 3
O
O
O
N
O
N
NH2
NH
EtOH/EtONa
H2SO4
HN
NH
HN
NH
2
+
N
N
N
O
N
H
N
H
O
11
6
1. BSA/CH3CN
2. ClCH2OCH2CH3
or
1. BSA/CH3CN
2. TMS-triflate,
CH2(OCH=CH2)2
Alkylation of 4 with isopropyl bromide or isobutyl
bromide in the presence of K2CO3 in dry DMF gave S-
alkylated products 12a,b in 23-33% yield and O- and S-
bisalkylated products 13a,b in 12-22% yield. The reaction
of compound 4 with CH3I however, gave different results
depending on the reaction conditions. When 4 was reacted
with CH3I in DMF at room temperature, the expected S-
alkylated product 14 was obtained in 82% yield, while
reaction of 4 with CH3I in DMF in the presence of K2CO3
at room temperature afforded a mixtures of polymethyl-
ated compounds 15-17 in 11-28% yields (Scheme 4).
O
O
HN
NH
O
N
N
O
R1
R2
7: R1 = R2 = !CH2OCH2CH3
7-10
8: R1 = H, R2 = !CH2OCH2CH3
9: R1 = R2 = !CH2OCH2CH=CH2
10: R1 = H, R2 = !CH2OCH2CH=CH2

Mar-Apr 2007 Synthesis and Anti-HIV-1 Activity of 1,3-phenylene-bis uracil Analogues of MKC-442
383
Scheme 4
O
N
O
OR
N
O
N
K2CO3/DMF
HN
NH
N
NH
R!Br
+
+
4
R
R
R
R
S
N
S
S
S
13a,b
12a,b
a: R = !CH(CH3)2
b: R = !CH(CH3)CH2CH3
O
N
O
N
DMF
HN
S
NH
S
14
15
+ CH3I
4
O
N
O
N
OMe
N
OMe
N
K2CO3/DMF
N
N
N
+
+
S
S
S
N
S
16
OMe
N
O
N
N
N
S
S
17
O
N
O
N
O
O
N
HN
NH
HN
NH
+
+
14
N
H
N
N
S
N
N
18
19
Compound 14 was reacted with excess of piperidine to
give the bis-piperidinyl derivative 18 in 32% yield and the
mono-piperidinyl derivative 19 in 26% yield (Scheme 4)
which are additional examples of amino-DABOs.
Table 1
Cytotoxicity and anti-HIV-1 activity of compounds 3-18 in MT-4 cellsa
Compound
No.
CC50
EC50 (µM)c
Y181C
(µM)b
WT
EFVR
K103N+Y181C
Antiviral activity.
3
4
6
7
100±1
>100
>100
>100
>100
>100
>100
>100
36±1
27±2
43±2
>100
>100
>100
80±20
75±25
>100
>100
>100
>100
14±1
47±3
34±2
11±0.1
>100
14±5
>27
>100
>100
>100
>100
>100
>100
>100
>100
>36
>27
>43
>100
>100
>100
>80
>100
>100
>100
>100
>100
>100
>100
>100
>36
>27
>43
>100
>100
>100
>80
>100
>100
>100
>100
>100
>100
>100
>100
>36
>27
>43
>100
>100
>100
>80
The anti-HIV-1 activity and cytotoxicity of the
synthesized MKC-442 analogues 3-18 are summarized in
Table 1. In general, the introduction of a base at the meta-
position of the benzyl group in MKC-442 as well as in
amino-DABO or S-DABO was detrimental to the activity
against HIV-1, when compared with the activity of MKC-
442. Only compounds 7-10, 12a and 16 showed moderate
activity against wild-type HIV-1 but was totally inactive
against resistant mutant viruses.
8
9
10
11
12a
12b
13a
14
15
16
>43
>100
>100
41±11
>80
>75
0.03
17
18
EXPERIMENTAL
>75
100
>80
20
>80
>100
MKC-442
Nmr Spectra were recorded on a Varian Gemini 2000
aData represent mean values of at least two separate experiments;
bCompound dose required to reduce the viability of mock-infected cells
1
spectrometer at 300 MHz for H and 75 MHz for 13C; δ values
c
by 50%, as determined by the MTT method; Compond dose required to
are in ppm relative to tetramethylsilane as an internal standard.
Accurate-ion mass determinations were performed using the 4.7
T Ultima Fourier transform (FT) mass spectrometer (Ion Spec,
achieve 50% protection of MT-4 cells from HIV-1- induced
cytopathogenicity, as determined by the MTT method

384
Y. L. Aly, E. B. Pedersen, P. La Colla and R. Loddo
Vol 44
Irvine, CA). The (M + H+) and (M + Na+) ions were peak
matched using ions derived from the 2,5-dihydroxybenzoic acid
matrix. Thin-layer chromatography (tlc) analyses were
performed on Merck precoated 60 F254 plates. The silica gel
(0.040–-0.063 mm) used for column chromatography was
purchased from Merck. Solvents used for column
chromatography were distilled prior to use, while reagents were
used as purchased.
The β-ketoester 2 (19.89 g, 0.015 mole) was added dropwise and
the mixture was refluxed for 20 hours. Ethanol was evaporated
in vacuo, and the residue was dissolved in 500 ml of water, the
thiouracil 4 and 5 were precipitated by neutralization with HCl.
The precipitate was filtered off, washed with water and
chromatographed on a silica gel column using petroleum ether
(60–80ο):ethyl acetate (1:1, v/v) to give 4 and 5.
5-Ethyl-6-{3-[(5-ethyl-6-oxo-2-thioxo-2,3-dihydropyrimid-
in-4(1H)-yl)methyl]benzyl}-2-thioxo-2,3-dihydropyrimidin-
4(1H)-one (4). Yield 8.84 g (42%); mp 254-256ο; 1H nmr
(deuteriochloroform): δ 0.70 (t, 6H, J = 7.3 Hz, 2 × CH3CH2),
2.15-2.17 (m, 4H, 2 × CH2CH3), 3.74 (s, 4H, 2 × CH2Ar), 6.99-
7.05 (m, 4H, Ar), 12.10, 12.30 (2s, 2H, 2 × NH); 13C nmr
(deuteriochloroform): δ 12.75 (2 × CH3), 17.71 (2 × CH2CH3),
34.41 (2 × CH2Ar), 117.14 (C-5), 126.68, 127.66, 128.90,
137.11 (Carom), 148.92 (C-6), 161.38 (CO), 174.14 (C=S). HRms
(MALDI, peak matching): m/z 415.1266 [C20H22N4O2S2 + H+],
General Procedure for the Preparation of Compounds 2
and 3. Zn dust 25–30 g was activated by stirring with 4 M HCl
(100 ml) for 5 minutes. The zinc dust was filtered and washed
sequentially with water, ethyl alcohol, and ether. Dried by
evaporation under reduced pressure at 80ο for 5 hours and kept
in vacuo overnight. The active Zn was suspended in dry THF
300 ml and heated to reflux. A few drops of ethyl 2-
bromobutyrate were added and the mixture was refluxed until a
green colour appeared. 1,3-Phenylenediacetontrile 10.03 g (66
mmoles) was added in one portion and ethyl 2-bromobutyrate
50.6 g (260 mmoles) in 20 ml THF was added dropwise. After
complete addition the reaction mixture was refluxed for 5 hours,
cooled and diluted with THF (300 ml) and subsequently
quenched by addition of saturated aqueous K2CO3 (100 ml). The
mixture was stirred for 1 hour and the THF layer was decanted
and the residue was washed with THF (3 × 100 ml). The
combined THF fractions were stirred with 10% aqueous HCl (90
ml) for 45 minutes. The solution was concentrated under
reduced pressure and diluted with CH2Cl2 (300 ml). The organic
phase was washed with saturated aqueous NaHCO3 (2 × 100
ml), dried (MgSO4), and evaporated under reduced pressure. The
residue was chromatographed on a silica gel column using
petroleum ether (60–80ο):ethyl acetate (8:2, v/v) to give 2 and 3.
4-[3-(3-Ethoxycarbonyl-2-oxo-pentyl)-phenyl]-2-ethyl-3-
oxo-butyric acid ethyl ester (2). Yield 4.5 g (38%); oil; 1H nmr
(deuteriochloroform): δ 0.84-0.88 (m, 6H, 2 × CH3CH2), 1.24-
1.28 (m, 6H, 2 × CH3CH2O), 1.82-1.89 (m, 4H, 2 × CH2CH3),
3.46 (t, 2H, J = 7.3 Hz, 2 × COCHCO), 3.80 (s, 4H, 2 × ArCH2),
4.12-4.19 (m, 4H, 2 × OCH2CH3), 7.03-7.26 (m, 4H, Ar); 13C
nmr (deuteriochloroform): δ 11.70, 14.01 (4 × CH3), 21.43 (2 ×
CH2CH3), 48.69 (2 × CH2Ar), 59.57 (2 × COCHCO), 61.24 (2 ×
OCH2CH3), 128.40, 128.82, 130.85, 133.64 (Carom), 169.47 (2 ×
COOEt), 202.37 (2 × COCH2Ar). HRms (MALDI, peak
matching): m/z 413.1931 [C22H30O6 + Na+], requires m/z
413.1935.
4-[3-(3,5-Diethyl-4-hydroxy-6-oxo-1,6-dihydro-pyridin-2-
ylmethyl)-phenyl]-2-ethyl-3-oxo-butyric acid ethyl ester (3).
Yield 0.4 g (3%); mp 112-114°; 1H nmr (dimethyl sulfoxide-d6):
δ 0.68-1.09 (m, 12H, 4 × CH3), 1.60-1.71 (m, 2H, CHCH2CH3),
2.28-2.38 (m, 4H, 2 × CH2), 3.54 (t, 1H, J = 7.22 Hz,
COCHCO), 3.71, 3.76 (2s, 4H, 2 × PhCH2), 3.98-4.03 (m, 2H,
OCH2), 6.93-7.18 (m, 4H, Ar); 9.04-9.07 (br s, 1H, OH), 11.08
(br s, 1H, NH); 13C nmr (dimethyl sulfoxide-d6): δ 11.48, 13.05,
13.86, 14.19 (4 × CH3), 16.03, 17.94, 20.87(3 × CH2), 34.98
(CH2Ar), 48.06 (ArCH2CO), 58.80 (COCHCO), 60.69 (OCH2),
110.97, 111.22 (C-3, C-5), 126.50, 127.75, 128.34, 129.32,
134.03, 138.52 (Carom), 139.14 (C-6), 161.55 (C=O), 163.14 (C-
4), 169.09 (CO), 202.84 (CO). HRms (MALDI, peak matching):
m/z 436.2115 [C24H31NO5 + Na+], requires m/z 436.2094. Anal.
Calcd. for (C24H31NO5 × 0.25H2O): C, 68.96; H, 7.60; N, 3.35.
Found: C, 69.13; H, 7.59; N, 3.53.
requires m/z 415.1257. Anal. Calcd. for (C20H22N4O2S2
×
0.25H2O): C, 57.35; H, 5.41; N, 13.37. Found: C, 57.35; H,
5.50; N, 13.43.
4-[3-(5-Ethyl-6-oxo-2-thioxo-2,3-dihydropyrimidin-4(1H)-
ylmethyl)-phenyl]-2-ethyl-3-oxo-butyric acid ethyl ester (5).
1
Yield 1.09 g (18%); semisolid; H nmr (deuteriochloroform): δ
0.85-1.05 (m, 9H, 3 × CH3), 1.55-1.62 (m, 2H, CH2 at C-5),
2.43-2.48 (m, 2H, CH2CH3), 3.58 (t, 1H, J = 7.3 Hz, CHCH2),
3.69, 3.86 (2s, 4H, 2 × CH2Ar), 4.10-4.13 (m, 2H, OCH2), 7.02-
7.28 (m, 4H, Ar), 10.88, 11.20 (2s, 2H, 2 × NH); 13C nmr
(deuteriochloroform): δ 13.10, 13.56, 13.57 (3 × CH3), 17.09,
18.23 (2 × CH2CH3), 35.35, 44.24 (2 × CH2Ar), 49.54
(COCH.CO), 65.99 (OCH2), 118.71 (C-5), 126.58, 128.77,
129.14, 129.79, 134.87, 135.14 (Carom), 148.86 (C-6), 151.76,
161.81 (2 × CO), 173.85 (C=S), 208.47 (CO).
5-Ethyl-6-{3-[(5-ethyl-2,6-dioxopyrimidin-4-(1H,3H)-yl)-
methyl]benzyl}pyrimidine-2,4(1H,3H)-dione (6). Thiouracil 4
(3.82 g, 9.2 mmoles) was suspended in 300 ml 10% aqueous
chloroacetic acid. The suspension was refluxed overnight and
filtered after cooling. The precipitate was washed with cold
ethanol and dried in vacuo to give compound 6. Yield 2.2 g
1
(63%); mp 335-337ο; H nmr (deuteriochloroform): δ 0.70 (t,
6H, J = 7.27 Hz, CH3CH2), 2.09-2.17 (m, 4H, CH2CH3), 3.74 (s,
4H, CH2Ar), 7.04-7.23 (m, 4H, Ar), 12.10, 12.30 (2s, 2H, 2 ×
NH); 13C nmr (deuteriochloroform): δ 13.34 (2 × CH3), 17.58 (2
× CH2CH3), 34.89 (2 × CH2Ar), 111.34 (C-5), 126.65, 127.66,
128.80, 137.23 (Carom), 148.43 (C-6), 150.88, 164.44 (2 × CO).
HRms (MALDI, peak matching): m/z 383.1728 [C20H22N4O4 +
H+], requires m/z 383.1714. Anal. Calcd. for (C20H22N4O4
×
0.25H2O): C, 62.08; H, 5.86; N, 14.48. Found: C, 62.31; H,
5.68; N, 14.43.
General Procedure for the Synthesis of Compounds 7 and
8. N,O-Bis-(trimethylsilyl)-acetamide (BSA, 6.2 ml, 2.5
mmoles) was dissolved in 30 ml dry MeCN under nitrogen and
compound 6 (3.82 g, 10 mmoles) was added. After 10 minutes
the reaction mixture become a clear solution and chloromethyl
ethyl ether (3 ml, 30 mmoles) was added. The reaction mixture
was stirred overnight at room temperature under nitrogen,
quenched with 25 ml ice cold saturated aqueous NaHCO3 and
extracted with CH2Cl2 (2 × 50 ml). The organic layer was dried
(MgSO4), and evaporated under reduced pressure. The residue
was purified by silica gel column chromatography using
chloroform:methanol (9:1, v/v) to give compounds 7 and 8.
5-Ethyl-1-ethoxymethyl-6-{3-[(5-ethyl-1-ethoxymethyl-2,6-
dioxopyrimidin-4(1H,3H)-yl)methyl]benzyl}pyrimidine-
Synthesis of Compounds 4 and 5. Na (50.2 g, 2.2 moles)
was dissolved in 800 ml absolute ethanol. Thiourea 116.46 g
(1.54 moles) was added, and the mixture was heated to reflux.

Mar-Apr 2007 Synthesis and Anti-HIV-1 Activity of 1,3-phenylene-bis uracil Analogues of MKC-442
385
1
2,4(1H,3H)-dione (7). Yield 1.15 g (23%); white foam; H nmr
(deuteriochloro-form): δ 1.03 (t, 6H, J = 7.3 Hz, 2 × CH3 at C-
5), 1.15 (t, 6H, J = 7.0 Hz, 2 × CH3CH2), 2.40-2.45 (m, 4H, 2 ×
CH2CH3 at C-5), 3.50-3.63 (m, 4H, 2 × OCH2CH3), 4.14 (s, 4H,
2 × CH2Ar), 5.09 (s, 4H, NCH2O), 6.90-7.30 (m, 4H, Ar), 10.06
(s, 2H, 2 × NH); 13C nmr (deuteriochloroform): δ 13.69 (2 × CH3
at C-5), 14.95 (2 × CH3CH2), 19.08 (2 × CH2CH3), 33.18 (2 ×
CH2Ar), 64.94 (2 × OCH2CH3), 72.61 (NCH2O), 117.05 (C-5),
126.08, 126.36, 130.02, 136.48 (Carom), 148.53 (2 × C-6), 151.96
(2 × CO), 163.41(2 × CO). Anal. Calcd. for (C26H34N4O6 ×
0.5H2O): C, 61.52; H, 6.95; N, 11.12. Found: C, 61.79; H, 6.95;
N, 10.97.
(2 × CH3), 18.25, 19.02 (2 × CH2CH3), 33.27, 35.81 (2 ×
CH2Ar), 70.36 (OCH2), 72.52 (NCH2O), 113.56, 117.03 (2 × C-
5), 117.66 (CH2=CH), 133.51 (CH=CH2), 126.56, 129.70,
136.05, 136.71 (Carom), 148.25, 148.79 (2 × C-6), 152.08,
152.30, 163.75, 164.77 (4 × CO). Anal. Calcd. for (C24H28N4O5):
C, 63.70; H, 6.24; N, 12.38, Found: C, 63.45; H, 6.20; N, 12.11.
6-(3-((2-(dimethylamino)-5-ethyl-1,6-dihydro-6-oxopyrim-
idin-4-yl)methyl)benzyl)-2-(dimethylamino)-5-ethylpyrimid-
in-4(3H)-one (11). The β-ketoester 2 (1.23 g, 3.15 mmoles) was
dissolved in EtONa (Na, 0.28g, 12.6 mmoles in 50 ml absolute
ethanol). N,N-Dimethylguanidinium sulphate (2.34 g, 8.6
mmoles) was added and the reaction mixture was refluxed for 3
days, then cooled and the mixture was filtered, and ethanol was
removed to half volume in vacuo. The residue was poured onto
water and extracted with chloroform. The organic layer was
washed with water (2 × 50 ml), dried over MgSO4 and
evaporated in vacuo. The residue was purified by silica gel
column chromatography using chloroform:methanol (95:5, v/v)
5-Ethyl-1-ethoxymethyl-6-{3-[(5-ethyl-2,6-dioxopyrimidin-
4(1H,3H)-yl)methyl]benzyl}pyrimidine-2,4(1H,3H)-dione (8).
1
Yield 0.48 g (11%); oil; H nmr (deuteriochloroform): δ 1.21-
1.28 (m, 9H, 3 × CH3), 2.03-2.10 (m, 4H, 2 × CH2CH3 at C-5),
3.53, 3.56 (2s, 4H, 2 × CH2Ar), 3.57-3.60 (m, 2H, OCH2CH3),
4.69 (s, 2H, NCH2O), 7.06-7.31 (m, 4H, Ar), 9.82, 10.08, 10.16
(3s, 3H, 3 × NH); 13C nmr (deuteriochloroform): δ 13.55, 13.62,
14.94 (3 × CH3), 18.14, 18.99 (2 × CH2CH3), 33.13, 35.70 (2 ×
CH2Ph), 63.73 (OCH2CH3), 69.71 (NCH2O), 113.45, 116.87 (2 ×
C-5), 126.40, 127.20, 127.39, 129.69, 136.11, 136.52 (Carom),
147.99, 148.75 (2 × C-6), 151.94, 163.54, 164.58, 170.90 (4 ×
CO).
General Procedure for the Synthesis of Compounds 9 and
10. Compound 6 (0.38 g, 1.0 mmole) was dissolved in 30 ml dry
CH3CN under nitrogen and BSA (1.74 ml, 3.5 mmoles) was
added. The reaction was cooled to −30ο and TMS-triflate
(trimethylsilyl trifluoromethanesulfonate, 0.18 ml, 1.0 mmole)
was added dropwise followed by addition of bis(allyloxy)-
methane (0.76 ml, 4.0 mmoles). The reaction mixture was stirred
at room temperature for 2 days and quenched by a saturated
solution of NaHCO3 (10 ml). CH3CN was evaporated under
reduced pressure and the residue was extracted with chloroform
(2 × 50 ml) and dried over MgSO4. The solvent was evaporated
under reduced pressure and the residue was purified by silica gel
column chromatography using chloroform:methanol (9:1, v/v) to
give compounds 9 and 10.
1
to give compound 11. Yield 0.25 g (18%); mp: 286-288ο; H
nmr (dimethyl sulfoxide-d6): δ 0.78 (t, 6H, J = 7.1 Hz, 2 ×
CH3CH2), 2.21-2.37 (m, 4H, 2 × CH2CH3), 2.87 (s, 12H, 4 ×
NCH3), 3.58 (s, 4H, 2 × CH2Ar), 6.96-7.16 (m, 4H, Ar), 11.10
(s, 2H, 2 × NH); 13C nmr (dimethyl sulfoxide-d6): δ 13.76 (2 ×
CH3), 17.94 (2 × CH2CH3), 36.77 (4 × NCH3), 43.35 (2 ×
CH2Ph), 111.61 (C-5), 126.43, 127.83, 130.43, 138.84 (Carom),
152.56 (2 × C-6), 161.67 (2 × CO), 164.09 (2 × C-2). HRms
(MALDI, peak matching): m/z 437.2642 [C24H32N6O2 + H+],
requires m/z 437.2660. Anal. Calcd. for (C24H32N6O2
×
0.25H2O): C, 65.36; H, 7.43; N, 19.05. Found: C, 65.32, H, 7.31;
N, 18.63.
General Procedure for the Synthesis of Compounds 12a,b
and 13a,b. Compound 4 (0.81 g, 2 mmoles) was dissolved in 20
ml dry DMF, K2CO3 (0.55 g, 4 mmoles) and isopropyl bromide
or isobutyl bromide (4.8 mmoles) were added, the reaction
mixture was stirred at room temperature for 3 days. The DMF
was evaporated under reduced pressure, and the residue was
chromatographed using ethyl acetate:petroleum ether (60-80ο)
(1:1, v/v) to afford 12a,b and 13a,b.
5-Ethyl-1-allyloxymethyl-6-{3-[(5-ethyl-3-allyloxymethyl-
2,6-dioxopyrimidin-4(1H,3H)-yl)methyl]benzyl}pyrimidine-
2,4(1H,3H)-dione (9). Yield 0.18 g (34%); white foam; H nmr
5-Ethyl-2-isopropylsulfanyl-6-{3-[(5-ethyl-2-isopropylsulf-
anyl-pyrimidin-4(1H,3H)-yl)methyl]benzyl}pyrimidine-2,4-
1
1
(1H,3H)-one (12a). Yield 0.3 g (33%); mp 198-200ο; H nmr
(deuteriochloro-form): δ 1.04 (t, 6H, J = 7.3 Hz, 2 × CH3CH2),
2.43-2.48 (m, 4H, 2 × CH2CH3), 4.09 (s, 4H, 2 × CH2Ar), 4.10-
4,13 (m, 4 H, 2 × OCH2), 5.12 (s, 4H, NCH2O), 5.15 (m, 2H,
CH2=CH), 5.19 (m, 2H, CH2=CH), 5.82-5.88 (m, 2H, 2 ×
CH=CH2), 7.02-7.31 (m, 4H, Ar), 10.16 (s, 2H, 2 × NH); 13C
nmr (deuteriochloroform): δ 13.71 (2 × CH3), 19.08 (2 ×
CH2CH3), 33.23 (2 × CH2Ar), 70.45 (OCH2), 72.41 (NCH2O),
117.76 (C-5), 117.76 (CH2=CH), 133.45 (CH=CH2), 126.13,
126.39, 130.05, 136.41 (Carom), 148.40 (C-6), 151.99, 163.41 (2
× CO). HRms (MALDI, peak matching): m/z 545.2364
[C28H34N4O6 + Na+], requires m/z 545.2371. Anal. Calcd. for
(C28H34N4O6 × 0.25H2O): C, 63.80; H, 6.60; N, 10.63. Found: C,
63.60; H, 6.47; N, 10.57.
(dimethyl sulfoxide-d6): δ 0.93 (t, 6H, J = 7.2 Hz, 2 × CH3CH2),
1.22 (d, 12H, J = 6.7 Hz, 4 × CH3[Pri]), 2.41-2.48 (m, 4H, 2 ×
CH2CH3), 3.69-3.76 (m, 2H, 2 × SCH), 3.80 (s, 4H, 2 × CH2Ar),
7.09-7.20 (m, 4H, Ar), 12.43 (s, 2H, 2 × NH); 13C (dimethyl
sulfoxide-d6): δ 13.02 (2 × CH3), 18.07 (2 × CH2CH3), 22.34 (4
× CH3[Pri]), 35.42 (2 × SCH), 40.77 (2 × CH2Ar), 120.80 (C-5),
126.82, 128.00, 129.28, 138.38 (Carom), 156.73 (2 × C-6),
159.99, 162.71 (C-2 and C-4). HRms (MALDI, peak matching):
m/z 521.1994 [C26H34N4O2S2 + Na+], requires m/z = 521.2015.
5-Ethyl-2-isopropylsulfanyl-4-isopropyloxy-6-{3-[(5-ethyl-
2-isopropylsulfanyl-4-isopropyloxy-pyrimidin-4(1H,3H)-yl)-
methyl]benzyl}pyrimidine-2,4(1H,3H)-one (13a). Yield 0.2 g
1
(19%); oil; H nmr (deuteriochloroform): δ 0.90-1.14 (m, 6H, 2
5-Ethyl-6-{3-[(5-ethyl-1-allyloxymethyl-2,6-dioxopyrimid-
in-4(1H,3H)-yl)methyl]benzyl}pyrimidine-2,4(1H,3H)-dione
(10). Yield 0.1 g (22%); foam; H nmr (deuteriochloroform): δ
× CH3CH2), 1.22-1.38 (m, 18H, 6 × CH3[Pri]), 2.44-2.59 (m, 4H,
2 × CH2CH3), 3.79-3.87 (m, 2H, 2 × SCH) 3.94 (s, 4H, 2 ×
CH2Ar), 5.30-5.38 (m, 1H, OCH), 7.05-7.26 (m, 4H, Ar), 12.10
(s, 1H, NH); 13C nmr (deuteriochloroform): δ 13.12, 13.16 (2 ×
CH3), 18.23, 18,67 (2 × CH2CH3), 21.88, 22.72, 23.03 (6 ×
CH3[Pri]), 35.56, 36.51 (2 × SCH), 40.33, 40.44 (2 × CH2Ar),
68.98 (OCH), 116.55, 122.01 (2 × C-5), 126.80, 126.92, 128.23,
129.54, 138.35, 138.60 (Carom), 156.46, 161.67 (2 × C-6),
1
1.11-1.66 (m, 6H, 2 × CH3CH2), 2.34-2.38 (m, 4H, 2 ×
CH2CH3), 3.98, 4.09 (2s, 4H, 2 × CH2Ar), 4.13-4.18 (m, 2H,
OCH2CH), 5.20 (s, 2H, NCH2O), 5.33-5.36 (m, 2H, CH2=CH),
5.75-5.86 (m, 1H, CH=CH2), 7.05-7.32 (m, 4H, Ar), 9.96, 10.38
(2s, 2H, 2 × NH); 13C nmr (deuteriochloroform): δ 13.63, 13.70

386
Y. L. Aly, E. B. Pedersen, P. La Colla and R. Loddo
Vol 44
164.80, 165.42, 166.88, 167.09 (C-2 and C-4). HRms (MALDI,
peak matching): m/z 563.2480 [C29H40N4O2S2 + Na+], requires
m/z 563.2485.
temperature for 48 hours. The DMF was evaporated under
reduced pressure. The products 15, 16 and 17 were isolated after
column chromatography using chloroform:petroleum ether (60-
80°) (7:3, v/v).
5-Ethyl-2-secbutylsulfanyl-6-{3-[(5-ethyl-2-secbutylsulfan-
yl-pyrimidin-4(1H,3H)-yl)methyl]benzyl}pyrimidine-2,4-
5-Ethyl-2-methylthio-3-methyl-6-{3-[(5-ethyl-2-methylthio-
3-methyl-pyrimidin-4(1H,3H)-yl)methyl]benzyl}pyrimidine-
1
(1H,3H)-one (12b). Yield 0.23 g (23%); mp 181-183ο; H nmr
1
2,4(1H,3H)-one (15). Yield 0.2 g (28%); mp 175-177ο; H nmr
(dimethyl sulfoxide-d6): δ 0.84 (t, 6H, J = 7.2 Hz, 2 × CH3CH2 at
C-5), 0.93 (t, 6H, J = 7.4 Hz, 2 × CH3CH2[sec-Bu]), 1.22 (d, 6H,
(deuteriochloro-form): δ 1.04 (t, 6H, J = 7.2 Hz, 2 × CH3CH2),
2.41 (s, 6H, 2 × SCH3), 2.53-2.60 (m, 4H, 2 × CH2CH3), 3.46 (s,
6H, 2 × NCH3), 3.84 (s, 4H, 2 × CH2Ar), 7.11-7.26 (m, 4H, Ar);
13C nmr (deuteriochloroform): δ 13.09 (2 × CH3), 14..64 (2 ×
SCH3), 19.46 (2 × CH2CH3), 30.30 (2 × NCH3), 40.22 (2 ×
CH2Ar), 120.76 (C-5), 127.06, 128.12, 129.62, 138.47 (Carom),
158.17 (C-6), 158.37, 162.73 (C-2 and C-4). HRms (MALDI,
peak matching): m/z 471.1872 [C24H30N4O2S2 + H+], requires
m/z 471.1883. Anal. Calcd. for (C24H30N4O2S2 × 0.25H2O): C,
60.67; H, 6.47; N, 11.79. Found: C, 60.65; H, 5.94; N, 11.51.
5-Ethyl-2-methylthio-4-methoxy-6-{3-[(5-ethyl-2-methyl-
thio-4-methoxy-pyrimidin-4(1H,3H)-yl)methyl]benzyl}pyrim-
idine-2,4(1H,3H) (16). Yield 0.1 g (11%); oil; 1H nmr
(deuteriochloroform): δ 0.88-0.97 (m, 6H, 2 × CH3CH2), 2.50 (s,
6H, 2 × SCH3), 2.52-2.54 (m, 4H, 2 × CH2CH3), 3.95 (s, 6H, 2 ×
OCH3), 3.96 (s, 4H, 2 × CH2Ar), 7.05-7.25 (m, 4H, Ar); 13C nmr
(deuteriochloroform): δ 13.19 (2 × CH3), 14.06 (2 × SCH3),
18.18 (2 × CH2CH3), 40.35 (2 × CH2Ar), 53.81 (2 × OCH3),
116.52 (2 × C-5), 126.75, 128.34, 129.25, 138.56 (Carom), 158.70
(2 × C-6), 165.46, 167.73 (C-2 and C-4). HRms (MALDI, peak
matching): m/z 471.1897 [C24H30N4O2S2 + H+], requires m/z
471.1883.
5-Ethyl-2-methylthio-3-methyl-6-{3-[(5-ethyl-2-methylthio-
4-methoxy-pyrimidin-4(1H,3H)-yl)methyl]benzyl}pyrimidine-
2,4(1H,3H)-one (17). Yield 0.15 g (18%); oil; 1H nmr
(deuteriochloroform): δ 0.89-1.08 (m, 6H, 2 × CH3CH2), 2.40,
2.51 (2s, 6H, 2 × SCH3), 2.52-2.57 (m, 4H, 2 × CH2CH3), 3.46
(s, 3H, NCH3), 3.82, 3.99 (2s, 4H, 2 × CH2Ph), 3.95 (s, 3H,
OCH3), 7.06-7.26 (m, 4H, Ar); 13C nmr (deuteriochloroform): δ
13.07, 13.13 (2 × CH3), 13.99, 14.69 (2 × SCH3), 18.51, 19.43 (2
× CH2CH3), 30.25 (NCH3), 40.18, 40.34 (2 × CH2Ar), 53.78
(OCH3), 116.40, 120.71 (2 × C-5), 126.75, 127.00, 128.21,
129.39, 138.44, 138.53 (Carom), 158.13, 158.36 (2 × C-6),
162.73, 165.41 (2 × C-2), 167.41, 167.66 (2 × C-4). HRms
(MALDI, peak matching): m/z 471.1892 [C24H30N4O2S2 + H+],
requires m/z 471.1883.
J = 6.8, 2 × CH3CH[sec-Bu]), 1.50-1.60 (m, 4H, 2
×
CH2CH3[sec-Bu]), 2.40-2.51 (m, 4H, 2 × CH2CH3 at C-5), 3.60-
3.66 (m, 2H, 2 × SCH), 3.79 (s, 4H, 2 × CH2.Ar), 7.08-7.20 (m,
4H, Ar), 12.43 (s, 1H, NH); 13C nmr (dimethyl sulfoxide-d6): δ
11.01 (2 × CH3 at C-5), 13.03 (2 × CH3[sec-Bu]), 18.09 (2 ×
CH2.CH3 at C-5), 19.91 (2 × CH3CH[sec-Bu]), 28.59 (2 ×
CH2CH3[sec-Bu]), 39.52 (2 × SCH), 40.57 (2 × CH2Ar), 120.86
(2 × C-5), 126.82, 127.95, 129.32. 138.40 (Carom), 156.77 (2 × C-
6), 159.91, 162.72 (C-2 and C-4). HRms (MALDI, peak
matching): m/z 549.2329 [C28H38N4O2S2 + Na+], requires m/z
549.2328. Anal. Calcd. for (C28H38N4O2S2): C, 63.84; H, 7.27; N,
10.64. Found: C, 63.59; H, 7.37, N, 10.40.
5-Ethyl-2-secbutylsulfanyl-4-secbutyloxy-6-{3-[(5-ethyl-2-
secbutylsulfanyl-4-secbutyloxy-pyrimidin-4(1H,3H)-yl)meth-
yl]benzyl}pyrimidine-2,4(1H,3H)-one (13b). Yield 0.26 g
(22%); oil; 1H nmr (deuteriochloroform): δ 0.90-1.07 (m, 15H, 5
× CH3), 1.27, 1.36 (2d, 9H, J = 7.8Hz, 3 × CH3CH[sec-Bu]),
1.53-1.80 (m, 6H, 3 × CH2CH[sec-Bu]), 2.45-2.59 (m, 4H, 2 ×
CH2CH3 at C-5), 3.67-3.80 (m, 2H, 2 × SCH[sec-But]), 3.84,
3.95 (2s, 2 × CH2Ar), 4.08-4.15 (m, 1H, OCH[sec-But]), 7.06-
7.20 (m, 4H, Ar), 11.99 (s, 1H, NH); 13C nmr (CDCl3): δ 9.66,
11.32 (2 × CH3 at C-5), 11.53, 13.19, 13.23 (3 × CH3[sec-But]),
18.28, 19.25 (2 × CH2CH3 at C-5), 20.35, 20.44, 20.55 (3 ×
CH3CH[sec-But]), 28.87, 29.24, 29.46 (3 × CH2CH[sec-But]),
40.37, 40.49 (2 × SCH[sec-But]), 41.87, 42.80 (2 × CH2Ar),
73.56 (OCH), 116.55, 122.07 (2 × C-5), 126.85, 126.95, 128.26,
129.59, 138.42, 138.65 (Carom), 156.62, 161.60 (2 × C-6),
164.76, 165.46, 167.12, 167.24 (C-2 and C-4). HRms (MALDI,
peak matching): m/z 605.2965 [C32H46N4O2S2 + Na+], requires
m/z 605.2954.
5-Ethyl-2-methylthio-6-{3-[(5-ethyl-2-methylthio-pyrimid-
in-4(1H,3H)-yl)methyl]benzyl}pyrimidine-2,4(1H,3H)-one
(14). Compound 4 (0.8 g, 2 mmoles) was dissolved in 30 ml dry
DMF under nitrogen and CH3I (0.56 g, 0.26 ml, 4 mmoles) was
added. The reaction mixture was stirred at room temperature for
24 hours and poured onto cold water. The product was collected
by filtration, washed three times with water and
chromatographed using ethyl acetate:petroleum ether (60-80ο)
(1:1, v/v) to give 14.
Synthesis of Compounds 18 and 19. Compound 14 (2.2 g,
5 mmoles) was dissolved in piperidine (4.2 g, 50 mmoles).
The reaction mixture was refluxed for 24 hours, cooled and
poured onto cold water. The solid formed was collected by
filtration and washed with water. The products 18 and 19
were isolated after column chromatography using
chloroform:methanol (95:5, v/v).
1
Yield 0.7 g (82%); mp 266-268ο; H nmr (deuteriochloro-
form): δ 0.80 (t, 6H, J = 7.3 Hz, 2 × CH3CH2), 2.29 (s, 6H, 2 ×
SCH3), 2.31-2.36 (m, 4H, 2 × CH2CH3), 3.72 (s, 4H, 2 ×
CH2Ar), 6.02-7.14 (m, 4H, Ar), 12.41 (s, 2H, 2 NH); 13C nmr
(deuteriochloroform): δ 12.50 (2 × CH3), 12.98 (2 × SCH3),
18.13 (2 × CH2CH3), 39.62 (2 × CH2Ar), 120.79 (C-5), 126.79,
128.14, 129.06, 138.43 (Carom), 157.62 (C-6), 159.91, 162.74 (C-
2 and C-4). HRms (MALDI, peak matching): m/z 465.1372
[C22H26N4O2S2 + Na+], requires m/z 465.1389. Anal. Calcd. for
(C22H26N4O2S2 × 0.5H2O): C, 58.51; H, 6.03; N, 12.41. Found:
C, 58.85; H, 6.33; N, 12.15.
5-Ethyl-2-piperidinyl-6-{3-[(5-ethyl-2-piperidinyl-pyrimid-
in-4(1H,3H)-yl)methyl]benzyl}pyrimidine-2,4(1H,3H)-one
1
(18). Yield 32%; mp 263-265ο; H nmr (deuteriochloroform): δ
0.83-0.91 (m, 6H, 2 × CH3CH2), 1.44-1.56 (m, 12H, piperidino),
2.29-2.41 (m, 4H, 2 × CH2CH3), 3.45-3.49 (m, 8H, piperidino),
3.66 (s, 4H, 2 × CH2Ar), 7.08-7.18 (m, 4H, Ar), 11.32 (s, 2H, 2
× NH); 13C nmr (deuteriochloroform): δ 13.69 (2 × CH3), 17.95
(2 × CH2CH3), 23.90, 24.92, 44.98 (piperidino), 30.60 (2 ×
CH2Ar), 112.07 (2 × C-5), 126.41, 127.53, 128.95, 138.82
(Carom), 152.10 (2 × C-6), 158.18, 164.37 (C-2 and C-4). HRms
(MALDI, peak matching): m/z 539.3092 [C30H40N6O2 + Na+],
requires m/z 539.3105.
General Procedure for the Synthesis of Compounds 15, 16
and 17. Compound 4 (0.81 g, 2 mmoles) was dissolved in 20 ml
dry DMF, and K2CO3 (0.54 g, 4 mmoles) and CH3I (0.62 g, 4
mmoles) were added. The reaction mixture was stirred at room

Mar-Apr 2007 Synthesis and Anti-HIV-1 Activity of 1,3-phenylene-bis uracil Analogues of MKC-442
387
5-Ethyl-2-methylthio-6-{3-[(5-ethyl-2-piperidinyl-pyrimid-
in-4(1H,3H)-yl)methyl]benzyl}pyrimidine-2,4(1H,3H)-one
(19). Yield 26%; mp 248-250ο; 1H nmr (dimethyl sulfoxide-d6): δ
0.80-0.85 (m, 6H, 2 × CH3CH2), 1.39-1.57 (m, 6H, piperidino),
2.29-2.35 (m, 4H, 2 × CH2CH3), 2.29 (s, 3H, SCH3), 3.43-3.61 (m,
4H, piperidino), 3.71 (s, 4H, 2 × CH2Ar), 7.02-7.12 (m, 4H, Ar),
7.85, 8.16 (2s, 2H, 2 × NH); 13C nmr (dimethyl sulfoxide-d6): δ
12.66, 13.71 (2 × CH3), 13.14 (SCH3), 18.01, 18.32 (2 × CH2CH3),
22.61, 24.62, 45.02 (piperidino), 39.62, 43.63 (2 × CH2Ar),
112.22, 119.95 (2 × C-5), 126.61, 126.52, 128.04, 129.09, 138.72
(Carom), 152.17 (2 × C-6), 159.36, 159.73, 164.53, 165.27 (C-2 and
C-4). HRms (MALDI, peak matching): m/z 480.2415
[C26H33N5O2S+ H+], requires m/z 480.2428.
Acknowledgement. This work received funding from the
European Community’s Sixth Framework Programme under
contract number LSHP-CT-2004-503162 (Selection and
development of microbicides for mucosal use to prevent sexual
HIV transmission/acquisition) [13].
REFERENCES AND NOTES
[†] A research center funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
[a] On leave from chemistry department, faculty of education
Kafr El-Sheikh branch, Tanta University, Kafr El-Sheikh, Egypt.
[*] To whom correspondence should be addressed. Fax: +45
66158780; e-mail: ebp@chem.sdu.dk.
Cell-based Assays
Compounds. Compounds were dissolved in DMSO at 100
mM and then diluted in culture medium.
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Walker, J. Balzarini and E. De Clercq, J. Med. Chem., 32, 2507 (1989).
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[3] Press statement from Triangle Pharmaceuticals Inc. 18
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Cells and Viruses. Cell lines were purchased from American
Type Culture Collection (ATCC). The absence of mycoplasma
contamination was checked periodically by the Hoechst staining
method. Cell lines supporting the multiplication of RNA viruses
were the CD4+ human T-cells containing an integrated HTLV-1
genome (MT-4).
Cytotoxicity Assays. For cytotoxicity evaluations,
exponentially growing cells derived from human haematological
tumors [CD4+ human T-cells containing an integrated HTLV-1
genome (MT-4)] were seeded at an initial density of 1x105
cells/ml in 96 well plates in RPMI-1640 medium supplemented
with 10% fetal calf serum (FCS), 100 units/mL penicillin G and
100 µg/mL streptomycin. Cell cultures were then incubated at
37 °C in a humidified, 5% CO2 atmosphere in the absence or
presence of serial dilutions of test compounds. Cell viability was
determined after 96 hrs at 37 °C by the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-tetrazolium bromide (MTT) method [12].
Antiviral Assays. Activity of compounds against Human
Immunodeficiency virus type-1 (HIV-1) was based on inhibition
of virus-induced cytopathogenicity in MT-4 cells acutely
infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly,
50 µL of RPMI containing 1x104 MT-4 were added to each well
of flat-bottom microtitre trays containing 50 µL of RPMI,
without or with serial dilutions of test compounds. Then, 20 µL
of an HIV-1 suspension containing 100 CCID50 were added.
After a 4-day incubation, cell viability was determined by the
MTT method.
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B. Pedersen and C. Nielsen, J. Med. Chem., 45, 5721 (2002).
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[11] M. Artico, Drugs of the Future, 27, 159 (2002).
[12] R. Pawels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P.
Herdewijn, J. Desmyster and E. De Clercq, J. Virol. Methods., 20, 309,
(1998).
[13] The information in this document is provided as is and no
guarantee or warranty is given that the information is fit for any
particular purpose. The user thereof uses the information as its sole risk
and liability.