Synthesis of 5-alkyl-6-arylmethyl-2-(7-bromo-3,5-dioxaheptylthio)-pyrimidin-4(1H)-ones and 7-oxopyrimidino-1,5,3-oxathiazepines as new S-DABO analogues with anti-HIV activity

Article abstract of DOI:10.1007/s007060050310

New S-DABOs with a long alkylating S-alkyl substituent showing antiretroviral activity against HIV-1 in the micromolar range were prepared from 5,6-disubstituted 4-oxo-2-thiopyrimidines and 1,7-dibromo-3,5-dioxaheptane. The analogues with an ethyl group in position 5 also showed activity in the micromolar range against a Tyr/8/Cys mutant strain of HIV-1. The S-DABO analogues showing activity against the HIV-1 RT mutant strain were transformed to the N-3 and N-1 ring closed 7-oxo-pyrimidino-1,3,5-oxathiazepincs which surprisingly all showed activity against HIV-1 in the micromolar range, as well as against a Tyr/8/Cys mutant strain of HIV-1. Some analogues of S-DABO with a thien-2-ylmethyl residue in position 6 were synthesized and tested against HIV-1 wild type, but they showed less or comparable activities to those of the corresponding 6-benzyl analogues.

Full text of DOI:10.1007/s007060050310

Monatshefte fuÈr Chemie 130, 1499±1512 (1999)
Synthesis of 5-Alkyl-6-arylmethyl-2-(7-bromo-
3,5-dioxaheptylthio)-pyrimidin-4(1H)-ones and
7-Oxopyrimidino-1,5,3-oxathiazepines as New
S-DABO Analogues with Anti-HIV Activity
Ole S. Pedersen1, Lene Petersen1, Malene Brandt1, Claus Nielsen2,
and Erik B. Pedersen1;Ã
1
Department of Chemistry, University of Southern Denmark, Odense University, DK-5230 Odense
M, Denmark
2
Retrovirus Laboratory, Department of Virology, State Serum Institute, DK-2300 Copenhagen,
Denmark
Summary. New S-DABOs with a long alkylating S-alkyl substituent showing antiretroviral activity
against HIV-1 in the micromolar range were prepared from 5,6-disubstituted 4-oxo-2-thiopyrimi-
dines and 1,7-dibromo-3,5-dioxaheptane. The analogues with an ethyl group in position 5 also
showed activity in the micromolar range against a Tyr/8/Cys mutant strain of HIV-1. The S-DABO
analogues showing activity against the HIV-1 RT mutant strain were transformed to the N-3 and N-1
ring closed 7-oxo-pyrimidino-1,3,5-oxathiazepines which surprisingly all showed activity against
HIV-1 in the micromolar range, as well as against a Tyr/8/Cys mutant strain of HIV-1. Some
analogues of S-DABO with a thien-2-ylmethyl residue in position 6 were synthesized and tested
against HIV-1 wild type, but they showed less or comparable activities to those of the corresponding
6-benzyl analogues.
Keywords. 1,7-Dibromo-3,5-dioxaheptane; HIV; 7-Oxopyrimidino-1,5,3-oxathiazepines; S-DABO;
6-Thienylmethyl-2-thiouracils.
Synthese von 5-Alkyl-6-arylmethyl-2-(7-bromo-3,5-dioxaheptylthio)-pyrimidin-4(1H)-onen
und 7-Oxopyrimidino-1,5,3-oxathiazepinen als neue S-DABO-Analoga mit Anti-HIV-Aktivitat
È
Zusammenfassung. Neue S-DABO-Derivate mit langen alkylierten S-Alkylsubstituenten, die
antivirale Eigenschaften gegenuÈber HIV-1 im mikromolaren Bereich zeigen, wurden aus 5,6-
disubstituierten 4-Oxo-2-thiopyrimidinen und 1,7-Dibrom-3,5-dioxaheptan dargestellt. Die Analoga
È
È
mit einer Ethylgruppe in Position 5 zeigten ebenfalls Aktivitat im mikromolaren Bereich gegenuber
È
È
einem Tyr/8/Cys-Mutantenstamm des HIV-1. Die S-DABO-Analoga, die Aktivitat gegenuber dem
HIV-1 RT Mutantenstamm zeigten, wurden zu den uÈber N-3 und N-1 ringverknuÈpften 7-Oxo-
È
pyrimidino-1,3,5-oxathiazepinen zyklisiert, welche uÈberraschenderweise Aktivitat im mikromolaren
Bereich gegenuÈber HIV-1 und dem Tyr/8/Cys-Mutantenstamm des HIV-1 zeigten. Einige S-DABO-
Ã
Corresponding author

1500
O. S. Pedersen et al.
Analoga mit einem Thienyl-2-methylrest in Position 6 wurden ebenfalls dargestellt und gegenuÈber
È
dem HIV-1-Wildtyp getestet. Sie zeigten jedoch geringere als bzw. vergleichbare Aktivitat wie die
entsprechenden 6-Benzylanaloga.
Introduction
The virally coded enzyme reverse transcriptase (RT) is an attractive target in the
search for new antiviral agents against human immunode®ciency virus type 1
(HIV-1). This enzyme is a pivot in the retroviral cycle of HIVas it synthesizes viral
DNA for integration in the host genome with viral RNA as template [1]. Several
speci®c inhibitors of HIV-1 RT have been reported; all of them seem to have their
effect in a mainly hydrophobic pocket located in the proximity of the catalytic site
[2]. Some of these are nevirapine [3], tetrahydroimidazobenzodiazepinethione
(TIBO) derivatives [4], bis-(heteroaryl)-piperazine (BHAP) derivatives [5], 1-((2-
hydroxyethoxy)-methyl)-6-(phenylthio)-thymine (HEPT) derivatives [6], and 3,4-
dihydro-2-alkoxy-6-benzyl-4-oxopyrimidine (DABO) derivatives [7].
One of the HEPT derivatives, 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil
(MKC-442) [8] is an especially promising candidate and is now undergoing phase
III testing. We have been interested in derivatives of the S-DABO class,
characterized by an alkyl thio substituent in place of the alkoxy group in DABO.
The C-6 substituent of S-DABO analogues is an aromatic group, e.g. phenyl or
naphthyl, linked to the pyrimidine ring with a methylene group [9, 10]. In this work
we also introduce the thien-2-ylmethyl residue as a possible C-6 substituent of the
pyrimidine ring and thus expand the range of possible new S-DABO analogues in
the future search for anti-HIV active compounds.
The resistance to MKC-442 for the HIV mutant Tyr181Cys is generally
believed to be caused by a lack of ꢀ-bond interaction between the 6-benzyl
substituent of MKC-442 and Tyr181Cys which anchors the drug inside the pocket
of RT in the case of Tyr181. We are now investigating whether anchoring the drug
can be established by formation of a covalent bond to the S-alkyl group of S-
DABOs. For this purpose we have chosen a bromo substituent on a long 2-alkylthio
substituent assuming no change in antiviral activity when compared to a shorter
substituent [11]. Taking advantage of crystallographic studies [12] we assumed that
proper targets in the RT could be Lys102 and Tyr318 which have an amino group
Ê
or a hydroxy group, respectively, as close as 4±7 A to C-2 of MKC-442. Proposing
Scheme 1

New S-DABO Analogues with Anti-HIV Activity
1501
that the S-DABOs will be similarly spatially arranged in the RT pocket of MKC-
442, these amino acids are within reach by using properly chosen bromo
substituted S-alkyl derivatized S-DABOs. Other target amino acids could be
Ser105, Thr107, Ser191, Lys238, and Thr240, although their distances from MKC-
Ê
442 are in the range of 10±12 A.
Results and Discussion
Chemistry
The 2-thiopyrimidines 4a±f were synthesized according to the procedure of Danel
et al. [13]. Ethyl 2-bromo-3-methylbutanoate was synthesized, using the one-pot
procedure of Berry et al. [14] starting from valeric acid which was treated with
thionyl chloride to afford the acid chloride which in turn was brominated with Br2
and esteri®ed with ethanol. The proper bromo ester and arylacetonitrile were
reacted using zinc in THF followed by sequential treatment with aqueous
potassium carbonate and hydrochloric acid to afford the proper ꢁ-keto ester. This
was condensed with thiourea in ethanol and sodium ethoxide to give the 2-
thiopyrimidines 4a±f.
1,7-Dibromo-3,5-dioxaheptane was chosen for S-alkylation of the proper
substituted 2-thiouracils because its ability to serve two purposes. It gives a long
substituent on C-2 in the pyrimidine ring and it contains oxygen in positions that
could be in compliance with one of the hydrophilic regions of the otherwise
hydrophobic pocket of RT [15]. After alkylation, the substituent still contains one
bromo substituent available for formation of covalent bonding to nucleophilic
Scheme 2

1502
O. S. Pedersen et al.
Scheme 3
residues in the hydrophobic pocket. Alkylation of 4 in methanol with methoxide as
base afforded the expected S-alkylated products 5a,b,d,e in 50±75% yield, whereas
5c was obtained in 35% yield only. The products were isolated using column
chromatography followed by recrystallization. Compound 5e was dif®cult to
crystallize and was obtained as an oil which crystallized slowly on standing.
The synthesis of 1,7-dibromo-3,5-dioxaheptane from 2-bromoethanol, for-
maldehyde, and calcium chloride has been described previously [16]. In the present
work, 1,3,5-trioxane was used as substitute of formaldehyde. It was found to be
necessary to replace calcium chloride with sodium sulfate as drying agent in order
to avoid impurities of mono and dichloro analogues which were dif®cult to remove
from the desired dibrominated product by distillation. The structure of compounds
5a±e were con®rmed by NMR spectroscopy. A chemical shift value for C-6 of
approximately 156 ppm indicates S-alkylation at C-2. This value has been observed
in many S-alkylated compounds of the S-DABO type [10, 15, 17].
Compound 4f was alkylated with methyl iodide in methanol with methoxide as
base and afforded 5-isopropyl-2-methylthio-6-(thien-2-ylmethyl)-pyrimidine-
4(3H)-one (6) in 75% yield. Alkylation with allyl bromide using the same base/
solvent mixture afforded 2-allylthio-5-isopropyl-6-(thien-2-ylmethyl)-pyrimidin-
4(3H)-one (7) in 42% yield. In both alkylation reactions the alkylation reagents
were used in 5:1 excess, and as reported by Danel et al. [17] only alkylation at the
sulfur atom was observed. Ring closing reaction of the allylated S-DABO analogue
7 with Br2 in CH2Cl2 according to the procedure of Danel et al. [17] gave 8 with
the thienyl ring additionally brominated at position 5. The structure was deter-
mined by comparing the spectroscopic data with those obtained by Danel et al. The
methylene protons of the thien-2-ylmethyl group were separated characteristically
1
into two doublets in the H NMR spectrum due to the asymmetric position 3.

New S-DABO Analogues with Anti-HIV Activity
1503
Scheme 4
Compounds 5a,b were chosen for a ring closure reaction using a modi®ed
È
Niedballa and Vorbruggen condensation [18, 19]. Compounds 5a, b were silylated
using N,O-bis-trimethylsilylacetamide (BSA) in dry acetonitrile and treated with
trimethylsilyl tri¯uoromethanesulfonate (TMS-tri¯ate) at 40ꢀC to catalyze the
ring closure to give 9 and 10. The product of the reaction seems to be either a
thermic or a dynamic product. For a slow reaction with one equivalent of TMS-
tri¯ate the outcome tends to be a degradation or an N-3 ring closed product (9a,b),
whereas for a fast reaction with two equivalents of TMS-tri¯ate the main product
will be the N-1 ring closed compound 10. The 2 equivalents of TMS-tri¯ate were
added in portions at 40ꢀC until a product appeared on TLC (MeOH/CH2Cl2) at a
lower Rf value than the starting material. During work-up of the N-1 ring closed
product 10 it was necessary to avoid any presence of acid contamination in the
solvent. Using CH2Cl2 untreated with bicarbonate resulted in degradation of the
product during work-up and puri®cation. When 1.2 equivalents TMS-tri¯ate were
added slowly and dropwise at 40ꢀC, no product emerged on TLC within 1±3
hours. The reaction mixture was placed in a 20ꢀC freezer until a product with a
high Rf value appeared on TLC (MeOH/CH2Cl2), the outcome being the N-3 ring
closed product 9b. A similar feature has been observed in the synthesis of N-
glycosides [20]. It has been found that when the optimal reaction time was
exceeded the yield of the N-1 nucleoside decreased in favour of less polar products.
This corresponds well to the formation of the less polar N-3 ring closed products
9a,b with high Rf values compared to the N-1 ring closed product 10 with a lower
Rf value. It has also been suggested that if a reaction is not proceeding according to
TLC, one should add a further amount of catalyst or raise the temperature [18].
This is also in accordance with our observations, just with the addition that the best
result was obtained when additional catalyst was added. The distinction between 9
and 10 was achieved by NOE experiments. For compound 10, irradiation of H-5
showed a 2.5% NOE effect on the benzyl methylene protons. The irradiation of the

1504
O. S. Pedersen et al.
corresponding protons in 9a showed no NOE effect. The structure of 9a was
instead con®rmed by selective decoupling experiments of H-5 and the benzyl
protons. C-9 was assigned by decoupling of the phenyl methylene protons.
Decoupling of H-5 affected the coupling patterns of C-7 and C-10a, but not the
coupling pattern of C-9.
Antiviral activity
The test for activity against HIV-1 was performed in MT-4 cell cultures infected
with either wildtype HIV-1 (strain IIIB) or NNRTI resistant HIV-1 (strain N119).
The results are presented in Table 1. The HIV-1 strain N119 harbours a substitution
of cysteine for the tyrosine at position 181 in the reverse transcriptase enzyme,
confering resistance against NNRTI's (Tyr/8/Cys mutant strain) [21].
The benzyl analogues with a dioxaheptane substituent on sulfur in the S-DABO
class of compounds retained anti-HIVactivities showing that long substituents may
be an option in the S-DABO class. Compounds 5a,b,d,e were active in the
micromolar range against wild type HIV-1 with ED50 values ranging from 1 to
10 mM and with CD50 values ranging from 28 to 46 mM. Compounds with an ethyl
substituent in position 5 of the pyrimidine ring as in 5a and 5b also showed activity
against the Tyr/8/Cys mutant strain of HIV-1 with an only 3-and 4-fold decrease
in the ED50 values, respectively. With an isopropyl substituent in position 5 of 5d
and 5e, the activity was increased by a factor 3 compared to 5a and 5b against the
wild type HIV-1 strain; however, no activity against the Tyr/8/Cys strain of HIV-1
was observed. Compound 5c with a 2-thienyl group in place of a phenyl moiety
Table 1. Antiviral activity against HIV-1 in MT-4 cells
ED50 (mM)a
CD50 (mM)b
HIV-1 III
wt
HIV-1 N119
(Tyr/8/Cys)
Compound
MT-4
5a
5b
10
2.7
>37
3.6
1
31.6
10
46
42
5c
5d
>37
>31
>28
NDc
ND
ND
ND
6.3
37
31
5e
6
28
16
100
32
7
2.2
>36
4.9
1.6
0.6
0.04
0.005
8
9a
36
>100
>100
>100
52
9b
10
19
AZT
0.03
4.2
MKC-442
141
a
Effective dose of compound achieving 50% inhibition of HIV-1 antigen produc-
tion in MT-4 cultures; bcytoxic dose of compound required to reduce proliferation
c
of normal uninfected MT-4 cells by 50%; not determined (ND)

New S-DABO Analogues with Anti-HIV Activity
1505
showed no subtoxic activity against the two strains of HIV-1 in the test. The
methylated (6) and allylated (7) S-DABO analogues with 2-thienyl as substitute for
phenyl did show activity against HIV-1 wt in the micromolar range. The antiviral
activity for the 6-thien-2-ylmethyl analogue 7 was comparable with that found for
the 6-benzyl analogue (ED50(7): 2.2 mM and ED50(6-benzyl analogue): 1.5 mM
[17]), but it was more cytotoxic than the benzyl analogue. Compounds where
phenyl is substituted with an aromatic heterocycle may still be an option in the S-
DABO class in the future search for drug candidates, though substitution of benzyl
with thien-2-ylmethyl did not improve the anti-HIV activities. The 6-(thien-2-
ylmethyl) analogues had comparable or less activities relative to the benzyl
analogues, and for compounds 5c and 8 the thien-2-ylmethyl analogues were
devoid of subtoxic anti-HIV activities.
The 7-oxo-pyrimidino-1,5,3-oxathiazepines 9a,b and 10 showed activity in the
micromolar range against HIV-1 wt in MT-4 cells. For compound 10, the ED50
value was as low as 0.6 mM, and the CD50 value was higher than 100 mM which was
the highest concentration in the test. The activity of 9a and 9b is surprising, as
previous N-3 ring closed S-DABO analogues have been without subtoxic activity
against HIV [17]. HEPT and S-DABO analogues additionally alkylated at N-3 or
N-1 have also been found devoid of subtoxic activity [9, 17, 22], probably caused
by the lack of N-3 hydrogen which is a prerequisite for hydrogen bonding to the
Lys101 carbonyl atom of RT in RT-HEPT complexes [12]. Compounds 9b and 10,
representing the N-3 and N-1 ring closed S-DABO analogues with nearly the same
activity against the wild type strain of HIV-1, were also tested against a Tyr/8/Cys
mutant strain. This also gave ED50 values in the micromolar range, surprisingly
with the N-3 ring closed compound 9b as the most active analogue. Compound 10
as the N-1 ring closed analogue and the most potent of these 7-oxopyrimidino-
1,5,3-oxathiazepines showed 100-fold less activity than MKC-442 against HIV-1
wild type but was comparable with MKC-442 in activity against the mutant strain
of HIV-1. The 7-oxopyrimidino-1,5,3-oxathiazepines could serve as new leads in
the search for anti-HIV active compounds as they display moderate anti-HIV
activities, low cytotoxicity, and may be active against mutant HIV-1 strains that
render other NNRTI inactive.
Experimental
1
NMR spectra were recorded on a Varian Gemini 2000 NMR spectrometer at 300 MHz for H and
75 MHz for 13C or on a Bruker AC-250 FT spectrometer at 250 MHz for 1H and at 62.9 MHz for 13
C
with TMS as an internal standard. EI mass spectra were recorded on a Finnigan Mat SSQ 710, FAB
mass spectra on a Kratos MS50RF instrument. IR spectra were recorded on a Perkin Elmer 1720 FT-
IR spectrometer. The progress of the reaction was monitored by TLC (analytical silica gel plates 60
F254). Merck silica gel (0.040±0.063 mm) was used for column chromatography, and Merck silica gel
(0.063±0.200 mm) for preparative thin layer chromatography (PTLC). Elemental analyses were
performed by the Microanalytical Department, Chemical Laboratory II at The University of
Copenhagen, Denmark; the results were in satisfactory agreement with the calculated values.
Typical procedure for the preparation of 3
Zn (10 g), activated by sequential washing with, 3 M HCl, dist. H2O, abs. EtOH, and dry Et2O, was
suspended in dry THF under re¯ux, and 8 drops of ethyl 2-bromo-3-methylbutanoate and a few

1506
O. S. Pedersen et al.
crystals of I2 were added to initiate the reaction. When the mixture turned green, 2.45 g 3,5-
dimethylphenylacetonitrile (16.9 mmol) were added in one portion; subsequently, 9.04 g ethyl 2-
bromo-3-methylbutanoate (43.4 mmol) were added dropwise. The mixture was re¯uxed for further
30 min. After dilution with 150 cm3 THF and cooling to room temperature, the reaction mixture was
stirred with 40 cm3 50% K2CO3 for 30 min. The THF fraction was decanted and the water fraction
was washed with 3Â30 cm3 THF. The combined THF fractions were stirred with 25 cm3 10% HCl
for 30 min. The solvent was evaporated and the residue was redissolved in 150 cm3 CH2Cl2, washed
with NaHCO3, dried over Na2SO4, and evaporated in vacuo to give 3 quantitatively as an oil that was
considered suf®ciently pure for the synthesis of 4.
Ethyl 2-ethyl-3-oxo-4-(2-thienyl)butanoate (3c; C12H16O3S)
Yield: 94%; 1H NMR (CDCl3, ꢂ, 300 MHz): 0.88 (t, 3H, J ˆ 7.3 Hz, CH2CH3), 1.26 (t, 3H,
J ˆ 7.1 Hz, OCH2CH3), 1.90 (quint, 2H, J ˆ 7.4 Hz, CH3CH2CH), 3.51 (t, 1H, J ˆ 7.3 Hz, CH), 4.0
(s, 2H, CH2), 4.17 (q, 2H, J ˆ 7.1, CH2), 6.98±7.24 (m, 3H, H-thiophene) ppm; 13C NMR (CDCl3, ꢂ,
750 MHz): 11.7 (CH3CH2), 13.9 (OCH2CH3), 21.4 (CH3CH2), 42.6 (CH2), 59.2 (CH2), 61.4 (CH),
125.4, 127.0, 127.3 (C-thiophene), 169.6 (COOEt), 201.5 (CH2CO) ppm.
Ethyl 4-(3,5-dimethylphenyl)-2-isopropyl-3-oxobutanoate (3e; C17H24O3)
Rf ˆ 0.62 (10% MeOH in CH2Cl2); 1H NMR (CDCl3, ꢂ, 300 MHz): 0.86 (d, 3H, J ˆ 6.8 Hz,
CHCH3), 0.94 (d, 3H, J ˆ 6.8 Hz, CHCH3), 1.23 (t, 3H, J ˆ 7.1 Hz, CH2CH3), 2.29 (s, 6H, PhCH3),
2.35±2.52 (m, 1H, CH(CH3)2), 3.32 (d, 1H, J ˆ 9.4 Hz, CH), 3.71 (s, 2H, CH2), 4.13 (q, 2H,
J ˆ 7.1 Hz, CH2CH3), 6.80 (s, 2H, H-arom), 6.90 (s, 1H, H-arom) ppm; 13C NMR (CDCl3, ꢂ,
75 MHz): 13.90 (CH2CH3), 20.14, 20.42 (CHCH3), 21.04 (PhCH3), 28.33 (CHCH3)2), 49.33 (CH2),
61.07 (OCH2), 65.60 (CH), 127.50, 128.83, 132.94, 138.17 (C-arom), 169.10 (CHCO2), 202.73
(CH2CO) ppm.
Ethyl 2-isopropyl-3-oxo-4-(2-thienyl)butanoate (3f; C13H18O3S)
Yield: 52% red-brown oil; Rf ˆ 0.49 (CH2Cl2); MS (FAB): m/z ˆ 255 (M‡H); IR (KBr): ꢃ ˆ 1718
1H NMR (CDCl3, ꢂ, 300 MHz): 0.90 (d, 3H, J ˆ 6.8 Hz, CHCH3), 0.97 (d, 3H,
1
(C=O) cm
;
J ˆ 6.7 Hz, CHCH3), 1.25 (t, 3H, J ˆ 7.1 Hz, CH2CH3), 2.37±2.52 (m, 1H, CH(CH3)2), 3.36 (d,
J ˆ 9.4 Hz, 1H, CH), 4.01 (s, 2H, CH2), 4.16 (q, 2H, J ˆ 7.1 Hz, CH2), 6.90 (d, 1H, J ˆ 3.5 Hz, 3-H),
6.97 (dd, 1H, J ˆ 3.3, 5.1 Hz, 4-H), 7.23 (dd, 1H, J ˆ 1.2 Hz, 5.1 Hz, 5-H) ppm; 13C NMR (CDCl3, ꢂ,
75 MHz): 13.92 (CH2CH3), 20.13 (CHCH3), 20.39 (CH3), 28.46 (CHCH3)2), 42.83 (CH2), 61.22
(OCH2), 65.49 (CH), 125.33, 126.95, 127.25, 134.25 (thiophene), 168.91 (CHCO2), 201.14
(CH2CO) ppm.
Typical procedure for the preparation of 4
Na (1.16 g, 50 mg Atom) was dissolved in 25 cm3 abs. EtOH. Ethyl 4-(3,5-dimethylphenyl)-2-
isopropyl-3-oxobutanoate (ꢁ16.9 mmol) and 2.67 g thiourea (34 mmol) were added, and the solution
was re¯uxed for 17 h. The solvent was evaporated and the residue redissolved in 10 cm3 H2O. The
solution was neutralized with HCl and acidi®ed with CH3COOH (pHꢁ3). The solid was ®ltered off
and recrystallized from EtOH to give 4.
2,3-Dihydro-5-ethyl-6-(thien-2-ylmethyl)-2-thioxopyrimidin-4(1H)-one (4c; C11H12N2OS2)
Yield: 24%; Rf ˆ 0.70 (10% EtOH in CH2Cl2); MS (FAB): m/z ˆ 253 (M‡H); 1H NMR (DMSO-d6,
ꢂ, 300 MHz): 0.93 (t, 3H, J ˆ 7.3 Hz, CH3) 2.34 (q, 2H, J ˆ 7.3 Hz, CH2) 4.04 (s, 2H, CH2) 6.98±7.44

New S-DABO Analogues with Anti-HIV Activity
1507
(m, 3H, H-thiophene) 12.43 (br s, 2H, 2ÂNH) ppm; 13C NMR (DMSO-d6, ꢂ, 75 MHz): 12.9 (CH3),
17.7 (CH2), 29.3 (CH2), 116.8 (C-5), 125.3, 126.4, 127.1 (C-thiophene), 138.7 (C-2, thiophene),
148.9 (C-6), 161.6 (C-4), 174.5 (C-2) ppm.
2,3-Dihydro-6-(3,5-dimethylphenylmethyl)-5-isopropyl-2-thioxopyrimidin-4(1H)-one
(4e; C16H20N2OS)
Yield: 1.52 g (32%); Rf ˆ 0.57 (6% MeOH in CH2Cl2); m.p.: 183ꢀC; MS (FAB): m/z ˆ 289 (M‡H);
1H NMR (DMSO-d6, ꢂ, 300 MHz): 1.05 (d, 6H, J ˆ 6.9 Hz, CHCH3), 2.21 (s, 6H, CH3), 2.78 (sept.,
1H, J ˆ 6.9 Hz, CH), 3.78 (s, 2H, CH2), 6.79 (s, 2H, arom), 6.84 (s, 1H, arom), 12.11 (s, 1H, NH),
12.18 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, ꢂ, 75 MHz): 19.46 (CHCH3), 20.78 (CH3), 26.55
(CH), 34.49 (CH2), 119.64 (C-5), 125.72, 128.15, 136.69, 137.67 (C-arom), 149.13 (C-6), 160.86
(C-4), 174.28 (C-2) ppm.
2,3-Dihydro-5-isopropyl-6-(thien-2-yl-methyl)-2-thioxopyrimidin-4(1H)-one (4f; C12H14N2OS2)
Yield: 24%; Rf ˆ 0.57 (10% EtOH in CH2Cl2); m.p.: 217±219ꢀC; IR (KBr): ꢃ ˆ 1208 (C=S), 1662
1
(C=O), 3436 (NH) cm 1; H NMR (DMSO-d6, ꢂ, 300 MHz): 1.15 (d, 6H, J ˆ 6.7 Hz, CH(CH3)2),
2.94 (hept, 1H, J ˆ 6.7 Hz, CH), 4.05 (s, 2H, CH2), 6.97±7.0 (m, 2H, 30-H, 40-H), 7.4 (dd, 1H,
J ˆ 1.9 Hz, 4.4 Hz, 50-H), 12.21 (s, 1H, NH), 12.32 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, ꢂ,
75 MHz): 19.55 (2ÂCH3), 26.59 (CH), 29.41 (CH2), 119.36 (C-5), 125.25, 126.31, 127.05, 138.78
(C-thiophene), 148.53 (C-6), 160.81 (C-4), 174.37 (C-2) ppm.
1,7-Dibromo-3,5-dioxaheptane
1,3,5-Trioxane (3.780 g, 42 mmol) was dissolved in 15 cm3 2-bromoethanol (212 mmol). Na2SO4
(3.370 g, 28 mmol) was added, and the mixture was re¯uxed for 17 h. After the temperature had
reached room temperature the mixture was ®ltered and washed sequentially with 2Â10 cm3 H2O,
10 cm3 0.5 M NaOH, 10 cm3 5% NaHSO3, and 10 cm3 0.5 M NaOH. After drying over Na2SO4 the
oil was distilled at 130±136ꢀC/20 mbar (Ref. [23]: b.p.0.04: 39ꢀC) to afford 5.52 g (10%) 1,7-
dibromo-3,5-dioxaheptane as a clear liquid.
1H NMR (CDCl3, ꢂ, 300 MHz): 3.50 (t, 4H, J ˆ 6.1 Hz, CH2Br), 3.90 (t, 4H, J ˆ 6.1 Hz, OCH2),
4.77 (s, 2H, OCH2O) ppm; 13C NMR (CDCl3, ꢂ, 75 MHz): 30.62 (CH2Br), 68.02 (OCH2), 95.38
(OCH2O) ppm.
General procedure for the preparation of 2-(7-bromo-3,5-dioxaheptylthio)-pyrimidin-4(1H)-ones 5
Na (50 mg) was dissolved in 6 cm3 MeOH. 2-Thiouracil (4, 2 mmol) and 1.159g 1,7-dibromo-3,5-
dioxaheptane (4.4mmol) were added. The mixture was stirred at room temperature until all starting
material was consumed according to TLC (6% MeOH in CH2Cl2). H2O (10 cm3) was added, and the
mixture was extracted with 3Â25 cm3 Et2O. The collected organic phases were dried over Na2SO4 and
evaporated. The products 5 were puri®ed on a silica column with EtOAc/petroleum ether (60±80ꢀC).
The products were further puri®ed by recrystallization from ethyl acetate and petroleum ether.
6-Benzyl-2-(7-bromo-3,5-dioxaheptyl)-thio-5-ethylpyrimidin-4(1H)-one (5a; C18H23BrN2O3S)
Yield: 636 mg (74%); Rf ˆ 0.39 (10% MeOH/CH2Cl2); m.p.:79±81ꢀC; MS (FAB): m/z ˆ 429
(M‡H); 1H NMR (CDCl3, ꢂ, 300 MHz): 1.09 (t, 3H, J ˆ 7.5 Hz, CH2CH3), 2.59 (q, 2H, J ˆ 7.5 Hz,
CH2CH3), 3.29 (t, 2H, J ˆ 6.1 Hz, CH2-S), 3.45 (t, 2H, J ˆ 6.1 Hz, CH2-Br), 3.72 (t, 2H, J ˆ 6.1 Hz,
OCH2CH2S), 3.85 (t, 2H, J ˆ 6.1 Hz,OCH2CH2Br), 3.90 (s, 2H, CH2-Ar), 4.68 (s, 2H, O-CH2-O),
7.15±7.35 (m, 5H, arom), 12.7 (s, 1H, NH) ppm; 13C NMR (CDCl3, ꢂ, 75 MHz): 13.04 (CH3CH2),

1508
O. S. Pedersen et al.
18.60 (CH3CH2), 30.19 (SCH2), 30.65 (BrCH2), 40.26 (CH2Ph), 66.43 (OCH2CH2S), 67.97
(OCH2CH2Br), 95.35 (OCH2O), 122.47 (C-5), 126.46, 128.42, 129.04, 138.34 (C-arom), 156.11 (C-
6), 161.69 (C-4), 165.24 (C-2) ppm.
2-(7-Bromo-3,5-dioxaheptyl)-thio-6-(3,5-dimethylbenzyl)-5-ethylpyrimidin-4(1H)-one
(5b; C20H27BrN2O3S)
Yield: 687 mg (75%); Rf ˆ 0.30 (6% MeOH/CH2Cl2); m.p.: 116±117ꢀC; MS (FAB): m/z ˆ 457
1
(M‡H); H NMR (CDCl3, ꢂ, 300 MHz): 1.09 (t, 3H, J ˆ 7.5 Hz, CH2CH3), 2.57 (s, 6H, Ph-CH3),
2.59 (q, 2H, J ˆ 7.5 Hz, CH2CH3), 3.32 (t, 2H, J ˆ 6.1 Hz, CH2-S), 3.45 (t, 2H, J ˆ 6.1 Hz, CH2-Br),
3.75 (t, 2H, J ˆ 6.1 Hz, OCH2CH2S), 3.82 (s, 2H, CH2-Ar), 3.85 (t, 2H, J ˆ 6.1 Hz, OCH2CH2Br),
4.7 (s, 2H, O-CH2-O), 6.84 (m, 3H, arom), 12.7 (s, 1H, NH) ppm; 13C NMR (CDCl3, ꢂ, 75 Hz): 13.04
(CH3CH2), 18.62 (CH3CH2), 21.13 (CH3Ph), 30.27 (SCH2), 30.61 (BrCH2), 40.13 (CH2Ph), 66.41
(OCH2CH2S), 67.96 (OCH2CH2Br), 95.33 (OCH2O), 122.40 (C-5), 126.82, 128.07, 137.89, 138.11
(C-arom), 156.00 (C-6), 161.91 (C-4), 165.30 (C-2) ppm.
2-(7-Bromo-3,5-dioxaheptyl)-thio-5-ethyl-6-(thien-2-ylmethyl)-pyrimidin-4(1H)-one
(5c; C16H21BrN2O3S2)
Yield: 300 mg (35%); Rf ˆ 0.38 (6% MeOH in CH2Cl2); m.p.: 80±81ꢀC; MS (FAB): m/z ˆ 435
(M‡H); 1H NMR (CDCl3, ꢂ, 300 MHz): 1.12 (t, 3H, J ˆ 7.6 Hz, CH2CH3), 2.60 (q, 2H, J ˆ 7.5 Hz,
CH2CH3), 3.39 (t, 2H, J ˆ 6.1 Hz, CH2-S), 3.47 (t, 2H, J ˆ 6.1 Hz, CH2-Br), 3.82 (t, 2H, J ˆ 6.1 Hz,
OCH2CH2S), 3.88 (t, 2H, J ˆ 6.1 Hz, OCH2CH2Br), 4.06 (s, 2H, CH2-Ar), 4.73 (s, 2H, OCH2O),
6.8±7.2 (m, 3H, arom), 12.71 (s, 1H, NH) ppm; 13C NMR (CDCl3, ꢂ, 75 MHz): 13.15 (CH3CH2),
18.50 (CH3CH2), 30.25 (CH2S), 30.67 (CH2Br), 34.71 (CH2), 66.53 (OCH2CH2S), 67.98
(OCH2CH2Br), 95.38 (OCH2O), 122.11 (C-5), 124.41, 125.72, 126.61, 140.03 (C-arom), 156.58
(C-6), 160.61 (C-4), 165.21 (C-2) ppm.
6-Benzyl-2-(7-bromo-3,5-dioxaheptyl)-thio-5-isopropylpyrimidin-4(1H)-one (5d; C19H25BrN2O3S)
Yield: 508 mg (58%); Rf ˆ 0.64 (6% MeOH/CH2Cl2); m.p.: 75±76.5ꢀC; MS (FAB): m/z ˆ 443
(M‡H); 1H NMR (CDCl3, ꢂ, 300 MHz): 1.29 (d, 6H, J ˆ 7.0 Hz, CH CH3), 3.10 (sept, 1H,
J ˆ 7.0 Hz, CHCH3), 3.30 (t, 2H, J ˆ 6.1 Hz, CH2-S), 3.45 (t, 2H, J ˆ 6.1 Hz, CH2-Br), 3.73 (t, 2H,
J ˆ 6.1 Hz, OCH2CH2S), 3.85 (t, 2H, J ˆ 6.1 Hz, OCH2CH2Br), 3.94 (s, 2H, CH2-Ar), 4.69 (s, 2H,
O-CH2-O), 7.15±7.35 (m, 5H, arom), 12.88 (s, 1H, NH) ppm; 13C NMR (CDCl3, ꢂ, 75 MHz): 19.59
(CH3CH), 27.82 (CH3CH), 30.06 (SCH2), 30.66 (BrCH2), 40.85 (CH2Ph), 66.47 (OCH2CH2S),
67.94 (OCH2CH2Br), 95.34 (OCH2O), 125.16 (C-5), 126.37, 128.44, 128.83, 138.63 (C-arom),
156.24 (C-6), 161.31 (C-4), 164.80 (C-2) ppm.
2-(7-Bromo-3,5-dioxaheptyl)-thio-6-(3,5-dimethylbenzyl)-5-isopropylpyrimidin-4(1H)-one
(5e; C21H29BrN2O3S)
Yield: 466 mg (50%); Rf ˆ 0.64 (6% MeOH/CH2Cl2); m.p.: 47±48ꢀC; MS (FAB): m/z ˆ 469, 471
(M‡H); 1H NMR (CDCl3, ꢂ, 300 MHz): 1.29 (d, 6H, J ˆ 6.9 Hz, 2ÂCHCH3), 2.27 (s, 6H, PhCH3),
3.09 (sept, 1H, J ˆ 7.0 Hz, CH3CH), 3.32 (t, 2H, J ˆ 6.1 Hz, CH2), 3.44 (t, 2H, J ˆ 6.1 Hz, CH2), 3.76
(t, 2H, J ˆ 6.1 Hz, CH2), 3.85 (t, 3H, J ˆ 6.1 Hz, OCH2), 3.86 (s, 2H, CH2Ph), 4.69 (s, 2H, OCH2O),
6.81 (s, 2H, arom), 6.84 (s, 1H, arom), 12.93 (s, 1H, NH) ppm; 13C NMR (CDCl3, ꢂ, 75 MHz): 19.57
(CH3CH), 21.13 (CH3Ph), 27.84 (CH3CH), 30.11 (SCH2), 30.63 (BrCH2), 40.70 (CH2Ph), 66.42
(OCH2CH2S), 67.92 (OCH2CH2Br), 95.30 (OCH2O), 125.08 (C-5), 126.58, 127.95, 137.88, 138.34
(C-arom), 156.14 (C-6), 161.51 (C-4), 164.85 (C-2) ppm.

New S-DABO Analogues with Anti-HIV Activity
1509
5-Isopropyl-2-methylthio-6-(thien-2-ylmethyl)-pyrimidine-4(3H)-one (6; C13H16N2OS2)
4f (266 mg, 1 mmol) was suspended in 6 cm3 dry MeOH, and sodium methoxide (59 mg, 1.1 mmol)
was added. Methyl iodide (710 mg, 5 mmol) was added, and the reaction was monitored by TLC by
acidifying a small sample. After 20 min the reaction was stopped by addition 5 cm3 H2O to the white
suspension. The solid was ®ltered off, dried and recrystallized from EtOAc and petroleum ether (b.p.:
60±80ꢀC) to give 210 mg (75%) 6.
Rf ˆ 0.31 (30% EtOAc in petroleu mether (60±80ꢀC)); m.p.: 177±179ꢀC (EtOAc in petroleum
1
ether (60±80ꢀC)); MS (FAB): m/z ˆ 281 (M‡H); IR (KBr): ꢃ ˆ 1646 (C=O), 3436 (NH) cm 1; H
NMR (DMSO-d6, ꢂ, 300 MHz): 1.21 (d, 6H, J ˆ 6.8 Hz, CH(CH3)2), 2.49 (s, 3H, SCH3), 3.13 (hept,
1H, J ˆ 6.9 Hz, CH), 4.07 (s, 2H, CH2), 6.93±6.96 (m, 2H, thiophene), 7.34 (dd, 1H, J ˆ 1.7 Hz,
3.1 Hz, thiophene), 12.5 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, ꢂ, 75 MHz): 12.52 (SCH3), 19.63
(CH(CH3)2), 26.89 (CH), 34.62 (CH2), 124.73, 125.64, 126.69, 140.54 (C-thiophene) ppm.
2-Allylthio-5-isopropyl-6-(thien-2-ylmethyl)-pyrimidin-4(3H)-one (7; C15H18N2OS2)
4f (266 mg, 1 mmol) was suspended in 6 cm3 dry MeOH. Sodium methoxide (59 mg, 1.1 mmol) and
605 mg allyl bromide (5 mmol) were added. After 16.5 h 10 cm3 H2O was added, and the mixture
was extracted with Et2O (3Â25 cm3). The organic fractions were dried over Na2SO4 and evaporated.
The residue was recrystallized from EtOAc and petroleum ether (b.p.: 60±80ꢀC) and then from Et2O
and petroleum ether (b.p.: 60±80ꢀC) to give 130 mg (42%) of 7.
Rf ˆ 0.37 (30% EtOAc in petroleum ether (60±80ꢀC)); m.p.: 99±101ꢀC; MS (FAB): m/z ˆ 307
1
(M‡H); IR (KBr): ꢃ ˆ 1646 (C=O), 3437 (NH) cm 1; H NMR (DMSO-d6, ꢂ, 300 MHz): 1.18 (d,
6H, J ˆ 7.0 Hz, CH(CH3)2), 3.10 (hept, 1H, J ˆ 6.8 Hz, CH(CH3)2), 3.76 (d, 2H, J ˆ 6.9 Hz, SCH2),
4.04 (s, 2H, CH2), 5.05 (dd, 1H, J ˆ 9.9 Hz, 1.1 Hz, CH=CHH), 5.22 (dd, 1H, J ˆ 15.7 Hz, 1.1 Hz,
CH=CHH), 5.86 (m, 1H, CH=CH2), 6.87±6.93 (m, 2H, thiophene), 7.30 (dd, 1H, J ˆ 1.2 Hz, 3.7 Hz,
thiophene), 12.44 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, ꢂ, 75 MHz): 19.59 (CH(CH3)2), 26.90
(CH(CH3)2), 32.10 (CH2), 34.70 (CH2), 118.21 (CH=CH2), 123.54 (C-5), 124.69, 125.67, 126.71 (C-
thiophene), 133.64 (CH=CH2), 140.57 (C-thiophene), 157 (C-6), 159.17 (C-2), 162.54 (C-4) ppm.
3-Bromomethyl-5-((5-bromothien-2-yl)-methyl)-2,3-dihydro-6-isopropyl-7H-
thiazolo[3,2-a]pyrimidin-7-one (8; C15H16Br2N2OS2)
7 (239 mg, 0.78 mmol) was dissolved in 5 cm3 dry CH2Cl2. Bis-(trimethylsilyl)-acetamide (160 mg,
0.78 mmol, 0.2 cm3) was added in one portion, and 310 mg Br2 (1.94 mmol, 0.1 cm3) dissolved in
5 cm3 dry CH2Cl2 was added dropwise. During the reaction time the colour of the reaction mixture
changed from red-brown to dark green. After 4 h the mixture was evaporated and puri®ed by column
chromatography (0.5±1% MeOH in CHCl3) to give 172 mg (47%) of 8.
Rf ˆ 0.20 (5% MeOH in CH2Cl2); m.p.: 157±168ꢀC; MS (FAB): m/z ˆ 465 (M‡H); IR (KBr):
1
ꢃ ˆ 1608 (C=O) cm 1; H NMR (CDCl3, ꢂ, 300 MHz): 1.33 (d, 3H, J ˆ 6.7 Hz, CH3), 1.37 (d, 3H,
J ˆ 6.9 Hz, CH3), 2.98 (m, 1H, CH(CH3)2), 3.23 (m, 1H, CHHBr), 3.42 (d, 1H, J ˆ 11.9 Hz, SCHH),
3.59 (m, 1H, SCHH), 3.71 (t, 1H, J ˆ 10.6 Hz, CHHBr), 3.91 (d, 1H, J ˆ 17.2 Hz,CHH-thiophene),
4.32 (d, 1H, J ˆ 16.9 Hz, CHH-thiophene), 4.84 (m, 1H, CHCH2Br), 6.66 (d, 1H, J ˆ 3.7 Hz,
thiophene), 6.97 (d, 1H, J ˆ 3.8 Hz, thiophene) ppm; 13C NMR (CDCl3, ꢂ, 75 MHz): 19.35 (CH3),
20.24 (CH3), 28.33, 28.40 (CH(CH3)2, CHBr), 29.82 (CH2-thiophene), 30.44 (C-2), 63.98 (C-3),
112.04 (C-thiophene), 126.04 (C-6), 126.37, 130.36, 138.67 (C-thiophene), 142.28 (C-5), 164.90,
168.28 (C-7, C-8a) ppm.
9-Benzyl-8-ethyl-7-oxo-2,3,5-trihydropyrimidino[3,2-c]-1,5,3-oxathiazepine (9a; C16H18N2O2S)
5a (407 mg, 0.95 mmol) was suspended in 8 cm3 dry CH3CN. Bis-(trimethylsilyl)-acetamide
(0.50 cm3, 2.43 mmol) was added, and the mixture was stirred for 4 h at room temperature. The

1510
O. S. Pedersen et al.
mixture was cooled to 45ꢀC, and 0.18 cm3 TMS-tri¯ate (0.95 mmol) dissolved in 4 cm3 dry CH3CN
were added dropwise. The temperature was allowed to raise slowly to room temperature while
monitored by TLC (10% MeOH in CH2Cl2). After 3 weeks at room temperature the mixture was
heated to 40ꢀC for 2 h and then diluted with 50 cm3 CH2Cl2 and washed with sat. NaHCO3
(3Â10 cm3) and H2O (3Â10 cm3). The organic phase was dried over Na2SO4 and evaporated under
reduced pressure to give a yellow oil which was chromatographed on silica (1% MeOH in CH2Cl2)
to give 54 mg (19%) of 9a as a foam.
Rf ˆ 0.79 (10% MeOH in CH2Cl2); MS (FAB): m/z ˆ 303 (M‡H); 1H NMR (CDCl3, ꢂ,
300 MHz): 1.04 (t, 3H, J ˆ 7.6 Hz, CH2CH3), 2.56 (q, 2H, J ˆ 7.6 Hz, CH2CH3), 3.09 (t, 2H,
CH2CH2S), 3.90 (s, 2H, CH2-Ph), 4.13 (m, 2H, OCH2CH2), 5.80 (s, 2H, H-5), 7.16±7.32 (m, 5H,
arom) ppm; 13C NMR (CDCl3, ꢂ, 75 MHz): 12.54 (CH2CH3), 19.74 (CH2CH3), 34.14 (C-2), 40.15
(CH2-Ph), 71.18 (C-3), 77.03 (C-5), 125.80 (C-8), 126.61, 128.57, 128.78, 137.87 (C-arom), 157.20,
163.00 (C-7, C-10a), 158.93 (C-9) ppm.
9-(3,5-Dimethylbenzyl)-8-ethyl-7-oxo-2,3,5-trihydropyrimidino[3,2-c]-1,5,3-oxathiazepine
(9b; C18H22N2OS2)
5b (248 mg, 0.6 mmol) was suspended in 8 cm3 dry CH3CN, 0.31 cm3 bis-(trimethylsilyl)-acetamide
(1.5 mmol) were added slowly, and the solution was stirred at room temperature for 4 h. The mixture
was placed under N2 and cooled to 20ꢀC. 0.12 cm3 TMS-tri¯ate (0.7 mmol) were added dropwise,
and the mixture was placed in the freezer ( 20ꢀC) for 2 weeks while it was monitored on TLC (10%
MeOH in CH2Cl2). The mixture was diluted with 50 cm3 CH2Cl2 and washed with sat. NaHCO3
(3Â10 cm3) and H2O (3Â10 cm3). The organic phase was dried over Na2SO4 and evaporated under
reduced pressure. The residue was puri®ed by silica column chromatography (25±50% petroleum
ether in EtOAc) to give 35 mg (18%) of 9b as a white solid.
Rf ˆ 0.78 (10% MeOH in CH2Cl2); MS (FAB): m/z ˆ 331 (M‡H); 1H NMR (CDCl3, ꢂ,
300 MHz): 1.06 (t, 3H, J ˆ 7.4 Hz, CH2CH3), 2.27 (s, 6H, 2ÂPhCH3), 2.58 (q, 2H, J ˆ 7.4 Hz,
CH2CH3), 3.10 (t, 2H, J ˆ 4.6 Hz, CH2S), 3.83 (s, 2H, CH2Ph), 4.14 (t, 2H, J ˆ 4.6 Hz, CH2O), 5.81
(s, 2H, OCH2N), 6.84 (s, 3H, arom) ppm; 13C NMR (CDCl3, ꢂ, 75 MHz): 12.52 (CH2CH3), 19.78
(CH2CH3), 21.11 (CH3Ph), 34.12 (C-2), 40.04 (CH2-Ph), 71.19 (C-3), 77.02 (C-5), 125.73 (C-8),
126.55, 128.27, 137.63, 138.07 (C-arom), 157.14 (C-9), 159.19 (C-10a), 163.05 (C-7) ppm.
7-(3,5-Dimethylbenzyl)-8-ethyl-2,3,5-trihydro-9-oxo-pyrimidino[1,2-c]-1,5,3-oxathiazepine
(10; C18H22N2OS2)
5b (304 mg, 0.66 mmol) was suspended in 8 cm3 dry CH3CN, 0.34 cm3 bis-(trimethylsilyl)-
acetamide (1.65 mmol) were added, and the mixture was heated to 50ꢀC overnight protected by a
drying tube. The mixture was cooled to 40ꢀC, and 0.24 cm3 TMS-tri¯ate (1.32 mmol) were added
in small portions. The temperature was allowed to raise to 20ꢀC, and the mixture was left in a
20ꢀC freezer for two days. After dilution with 50 cm3 CH2Cl2 (washed with NaHCO3 and dried
over K2CO3) and washing with 3Â20 cm3 sat. NaHCO3 and 3 x 20 cm3 H2O, the organic phase was
dried over Na2SO4 and evaporated under reduced pressure to afford a clear oily residue which was
puri®ed by silica column chromatography (25±50% petroleum ether in EtOAc) to give 110 mg (50%)
of 10 as a white solid.
1
Rf ˆ 0.45 (10% MeOH in CH2Cl2); m.p.: 137±140ꢀC; MS (FAB): m/z ˆ 331 (M‡H); H NMR
(CDCl3, ꢂ, 300 MHz): 1.04 (t, 3H, J ˆ 7.5 Hz, CH2CH3), 2.24 (s, 6H, 2ÂPhCH3), 2.49 (q, 2H,
J ˆ 7.5 Hz, CH2CH3), 3.11 (t, 2H, J ˆ 4.6 Hz, CH2S), 4.01 (s, 2H, CH2Ph), 4.07 (t, 2H, J ˆ 4.6 Hz,
CH2O), 5.31 (s, 2H, OCH2N), 6.66 (s, 2H, arom), 6.87 (s, 1H, arom) ppm; 13C NMR (CDCl3, ꢂ,
75 MHz): 12.52 (CH2CH3), 19.69 (CH2CH3), 21.08 (CH3Ph), 33.96, 34.18 (C-2, CH2-Ph), 70.57
(C-3), 79.84 (C-5), 125.06 (C-arom), 125.58 (C-8), 129.12, 134.75, 139.07 (C-arom), 147.26 (C-7),
163.75 (C-10a), 168.32 (C-9) ppm.

New S-DABO Analogues with Anti-HIV Activity
1511
Viruses and cells
The HIV-1 strain HTLV-IIIB [24] and the NNRTI resistant strain N119 [21] were propagated in H9
cells [25] at 37ꢀC, 5% CO2 using RPMI 1640 with 10% heat-inactivated fetal calf serum (FCS) and
antibiotics (growth medium). The culture supernatant was ®ltered (0.45 nm), aliquoted, and stored at
80ꢀC until use. Both HIV-1 strains were obtained from the NIH AIDS Research and Reference
Program.
Inhibition of HIV-1 replication
Compounds were examined for possible antiviral activity against both strains of HIV-1 using MT-4
cells as Target cells. MT-4 cells were incubated with virus (0.005 MOI) for 2 h, washed, and
thereafter added in a proportion of 1:10 to uninfected cells which had been preincubated in growth
medium containing the test compound for 6 days in parallel with virus-infected control cultures
without compound added. Expression of HIV in the culture medium was quantitated by the HIV-1
antigen detection assay ELISA [26]. Compounds mediating less than 30% reduction of antigen
expression were considered without biological activity. Compounds mediating a reduction of 30% or
more were examined for cytotoxic effect using concentration dependent inhibition of MT-4 cell
proliferation as measure of cytotoxicity using the MTT assay as previously described [27]. A 30%
inhibition of cell growth relative to control cultures was considered signi®cant.
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Received May 7, 1999. Accepted June 11, 1999