Synthesis and potent anti-HIV-1 activity of novel 6-benzyluracil analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine

Article abstract of DOI:10.1021/jm9600499

Ethyl 2-alkyl-4-aryl-3-oxobutyrates were synthesized from the corresponding arylacetonitriles and 2-brome esters. Condensation of the butyrates with thiourea followed by treatment with chloroacetic acid afforded the 5-alkyl-6-(arylmethyl)uracils. Condensa

Full text of DOI:10.1021/jm9600499

J . Med. Chem. 1996, 39, 2427-2431
2427
Syn th esis a n d P oten t An ti-HIV-1 Activity of Novel 6-Ben zylu r a cil An a logu es of
1-[(2-Hyd r oxyeth oxy)m eth yl]-6-(p h en ylth io)th ym in e
Krzysztof Danel,† Erik Larsen,† Erik B. Pedersen,*,† Bent F. Vestergaard,‡ and Claus Nielsen‡
Department of Chemistry, Odense University, DK-5230 Odense M, Denmark, and Retrovirus Laboratory,
Department of Virology, Statens Seruminstitut, Artillerivej 5, DK-2300 Copenhagen, Denmark
Received J anuary 16, 1996X
Ethyl 2-alkyl-4-aryl-3-oxobutyrates were synthesized from the corresponding arylacetonitriles
and 2-bromo esters. Condensation of the butyrates with thiourea followed by treatment with
chloroacetic acid afforded the 5-alkyl-6-(arylmethyl)uracils. Condensation of the uracils with
acetals using trimethylsilyl triflate (TMS triflate) as a catalyst gave acyclic 5-alkyl-6-
(arylmethyl)uracil derivatives. 6-Benzyl-5-ethyluracil was also condensed with methyl 5-O-
(tert-butyldiphenylsilyl)-2-deoxy-3-O-(phenoxythiocarbonyl)-R,â-D-erythro-pentofuranoside, fol-
lowed by Barton reduction and deprotection, to give the anomers of 6-benzyl-5-ethyl-2′,3′-
dideoxyuridine. Alkylation of the uracils with alkyl chloromethyl sulfides gave new thio
analogues of HEPT. All new N1-substituted uracils were tested for activity against HIV-1,
and the thio analogues were found extremely potent.
The discovery of 1-[(2-hydroxyethoxy)methyl]-6-(phen-
ylthio)thymine (HEPT) as a compound with potent and
selective in vitro activity against human immunodefi-
ciency virus type 1 (HIV-1)1 has led to the synthesis of
many new analogues,2-6 of which 6-benzyl-1-(ethoxy-
methyl)-5-isopropyluracil (MKC-442) has been chosen
as a candidate for clinical trials with AIDS patients.6
Syntheses of the 6-benzyl analogues5-7 have usually
been done by condensation of the sugar moiety with a
5-substituted uracil8,9 followed by lithiation in the
6-position, reaction with benzoyl chloride, subsequent
reduction of the carbonyl group, and eventually depro-
tection of the sugar moiety. The 5- and 6-substituents
of the uracil with respect to HIV-1 inhibition are now
well established,5,6,8-10 but there are still unexplored
possibilities in changing the functionality in the sugar
moiety. The lithiation step can be an obstacle of doing
this. Recently, we have reported a new strategy for the
synthesis of acyclouridine derivatives by condensation
of 6-benzyl-5-ethyluracil with acetals or 1,3-dioxolanes.11
The steric hindrance exerted by the 6-substituent and
the buttressing effect of the 5-substituent were believed
to result in lower yields of the N-1 alkylation product.
Also bulky alkylating reagents were expected to reduce
the yield of this type of product. In particular, we were
interested in synthesizing the N1-(ethylthio)methyl
analog of MKC-442 because such a compound is not
likely to be synthesized by the route of lithiation in the
6-position of 1-(ethylthiomethyl)uracils, because the
most acidic protons here are expected to the ones on the
methylene group next to sulfur which can stabilize the
neighboring anion by its d orbitals.
esters 1a -c were converted by reaction with thiourea
and sodium in ethanol into a 2-thiouracil which was
refluxed with chloroacetic acid overnight to give 5-alkyl-
6-(arylmethyl)uracils 2a -c.11 Silylation12 of the uracils
2a ,c with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) was
done prior to condensation with acetals or 1,3-dioxo-
lanes. The condensation reaction with acetals was
accomplished under the Vorbru¨ggen condition13 using
trimethylsilyl trifluoromethanesulfonate (TMS triflate)
as a Lewis acid catalyst to give high yields of the desired
N1-substituted nucleosides 3 (Scheme 1). When racemic
mixtures were obtained, no attempts were made to
separate the enantiomers. Compounds 3a -c have
previously been prepared by the same method.11 For
compound 3d the method was modified by silylation in
situ of 2b with N,O-bis(trimethylsilyl)acetamide (BSA)
in chloroform followed by reaction with chloromethyl
ethyl ether.
Treatment of 2-deoxy-D-ribose (4) with HCl in metha-
nol,14 followed by selective 5-O-protection with tert-
butylchlorodiphenylsilane in dry pyridine using 4-(N,N-
dimethylamino)pyridine (DMAP) as catalyst14,15 and
finally reaction with phenoxythiocarbonyl chloride16
afforded the methyl 5-O-(tert-butyldiphenylsilyl)-2-deoxy-
3-O-(phenoxythiocarbonyl)-R,â-D-erythro-pentofurano-
side (5). Condensation of compound 5 with silylated
6-benzyl-5-ethyluracil using TMS triflate as catalyst
gave an anomeric mixture of the corresponding nucleo-
side (R/â ) 1/2). After separation of the anomers, each
of them were subjected to a Barton deoxygenation16
followed by deprotection of the silyl group at 5′-O using
tetrabutylammonium fluoride to give the anomers of
6-benzyl-5-ethyl-2′,3′-dideoxyuridine 6a ,b (Scheme 2).
Ch em istr y
The compounds were identified by comparison of
similar NMR data,11,16 1H-1H-COSY, and 1H-nuclear
Overhauser effects (NOE). NOE of 7a proved it to be a
â anomer as irradiation of 4′-H resulted in 2% NOE in
1′-H. N1-Glycosylation was confirmed by NOE in the
CH2Ph group of 7a (CH2, 4%) when 1′-H was irradiated.
The anomeric configuration of the corresponding R
anomer 7b was determined from its precursor 6b. A
decisive feature for the anomeric configuration of 6b was
irradiation of 3′-H which resulted in 3% NOE in 2â′-H
The 3-oxo esters 1a -c were prepared according to the
method of Danel et al.11 by reaction of phenylacetonitrile
with ethyl 2-bromobutyrate or ethyl 2-bromo-3-meth-
ylbutyrate, or by reaction of 3,5-dimethylphenylaceto-
nitrile with ethyl 2-bromobutyrate. The so formed 3-oxo
† Odense University.
‡ Statens Seruminstitut.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(96)00049-0 CCC: $12.00 © 1996 American Chemical Society

2428 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Danel et al.
Sch em e 1
Sch em e 2
Sch em e 3
whereas irradiation of 1′-H gave 5% NOE in 2â′-H. N1-
Glycosylation was confirmed by NOE in the CH2Ph
group of 6b (CH2, 2%) when 1′-H was irradiated. The
R and â anomer assignments of compounds 7a and 7b
were also confirmed by observing 4′-H in the R anomer
1
at lower field in H-NMR than 4′-H in the corresponding
â anomer.17
Alkylation of compounds 2a -c using alkyl chloro-
methyl sulfides and K2CO3 in DMF gave a mixture of
N1- and N3-substituted and disubstituted uracils in a
typical molar ratio of 2:2:5, but only the 1-((alkylthio)-
methyl)uracils 8a -f (Scheme 3) were isolated in a state
of purity and in very low yields (4-10%). Much higher
yields 69-78% were obtained for compounds 8a ,c,e
when synthesized under the Vorbru¨ggen conditions.13
The uracils 2a -c were silylated with HMDS prior to
condensation with methylthiomethyl acetate using TMS
triflate as the catalyst.
442,3,6,7,18,19 E-EBU (6-benzyl-1-(ethoxymethyl)-5-ethyl-
uracil)2,3,7 and E-EBU-dM.1-3,7 Compounds 8a ,d ,f
showed the highest activities and selectivities, but
without showing a clear picture of the structure-
activity relationship. Compound 8a was in our study
even more potent than MKC-442 and E-EBU-dM. It is
surprising to find 8a , with an acyclic (methylthio)methyl
group, as the most active compound since no highly
active compounds with a methoxymethyl counterpart
have yet been reported. We are now doing further
testing of the sulfur compounds 8 against resistant
HIV-1 mutants, and these studies will be reported in
due time.
For compounds 3, we were also testing the effect of
changing R3 ) H in the E-EBU type of compounds into
R3 ) alkyl. The effect was rather dramatic as can be
seen by comparing 3e (E-EBU-dM) with 3g (R3 ) Et)
with a decrease in activity against HIV of about 5
powers. A similar change in activity was observed for
E-EBU with the reported activity ED50 ) 0.041 µM7
Resu lts of th e An ti-HIV Assa y a n d Discu ssion
The HIV-1 strain HTLV-IIIB and MT-4 cells were
used in our assay to investigate the anti-HIV-1 activity
and cytotoxicity of HEPT analogues synthesized in the
present study. The results are summarized in Table 1
together with those of AZT. Besides the commercially
available AZT, we synthesized MKC-442 (3d ) and
E-EBU-dM (6-(3,5-dimethylbenzyl)-1-(ethoxymethyl)-5-
ethyluracil) (3e) and used them as references for the
biological activity. We found that the analogues 8 with
oxygen replaced with sulfur showed comparable activi-
ties and selectivities with those found for MKC-

Anti-HIV-1 Activity of HEPT Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2429
Ta ble 1. Antiviral Activity of HEPT Analogues 3a -g, 7a ,b,
and 8a -f and AZT against HIV-1 in MT-4 Cells
Hz, CH3), 2.43-2.49 (1H, m, CH), 3.32 (1H, d, J ) 9.4 Hz,
CH), 3.78 (2H, s, CH2Ph), 4.12 (2H, q, J ) 7.2 Hz, CH2), 7.17-
7.35 (5H, m, aryl); 13C NMR (CDCl3) δ 14.01 (CH3), 20.22 (CH3),
20.49 (CH3), 28.40 (CH), 49.40 (CH2Ph), 61.08 (OCH2), 65.75
(CH), 127.07, 128.54, 129.62, 133.16 (aryl), 168.83 (C-1), 202.11
(C-3).
a
compd
ED50
,
µM
CD50,b µM
SIc
3a
3b
3c
3d
3e
3f
3g
7a
7b
8a
8b
8c
8d
8e
8f
>100
>100
>100
Eth yl 2-eth yl-4-(3,5-dim eth ylph en yl)-3-oxobu tyr ate (1c):
yield 1.06 g (90%); 1H NMR (CDCl3) δ 0.85 (3H, t, J ) 7.4 Hz,
CH3), 1.24 (3H, t, J ) 7.2 Hz, CH3), 1.86 (2H, m, CH2), 2.28
(6H, s, 2CH3), 3.46 (1 H, t, J ) 7.2 Hz, CH), 3.73 (2H, s,
CH2Ph), 4.15 (2H, q, J ) 7.1 Hz, CH2), 6.80 (2H, s, aryl), 6.89
(1H, s, aryl); 13C NMR (CDCl3) δ 11.6 (CH3), 13.9 (CH3), 21.0
(CH3), 21.4 (2CH3), 48.8 (CH2Ph), 59.3 (OCH2), 61.0 (CH),
127.2, 128.6, 132.9, 138.0 (aryl), 169.4 (C-1), 202.6 (C-3).
5-Alk yl-6-(a r ylm et h yl)u r a cils 2b ,c. Gen er a l P r oce-
d u r e. Sodium (2 g) was dissolved in anhydrous EtOH (45 mL),
and thiourea (4.63 g, 60 mmol) and compounds 1b,c (4.0 mmol)
were added to the clear solution. The reaction mixture was
refluxed for 6 h and evaporated in vacuo at 40-50 °C until
nearly dryness and the residue redissolved in H2O (40 mL).
The 2-thiouracil was precipitated by addition of concentrated
aqueous HCl (7 mL) and subsequent acidification to pH 4 with
glacial acetic acid. The precipitated 2-thiouracil was desul-
furized by suspension in 10% aqueous chloroacetic acid (100
mL) and subsequent reflux overnight. After cooling to room
temperature, the precipitate was filtered off, washed with cold
EtOH and Et2O, and finally dried in vacuo to give compounds
2b,c.
0.005
0.004
2
15
37
0.5
0.002
0.040
0.020
0.006
0.050
0.004
0.040
141
100
100
130
52
1
32
37
37
37
52
68
52
28000
25000
50
8.7
1.4
2
16000
925
1850
6200
1040
17000
1300
AZT
a
Effective dose of compound, achieving 50% inhibition of HIV-1
antigen production in MT-4 cultures. b Cytotoxic dose of compound,
required to reduce the proliferation of normal uninfected MT-4
cells by 50%. c Selectivity index: ratio CD50/ED50. ED50 and CD50
are expressed as the mean values of three independent determina-
tions.
when compared with 3b. In this case a change of R3
from hydrogen to methyl resulted in a completely
inactive compound at 100 µM.
6-Ben zyl-5-isop r op ylu r a cil (2b): yield 701 mg (72%); mp
1
231-233 °C; H NMR (DMSO-d6) δ 1.07 (6H, d, J ) 6.9 Hz,
Compound 7a can be considered as a hybrid between
E-EBU and 2′,3′-dideoxy-5-ethyluridine (D2EtU),20 and
therefore two possible modes of action against HIV were
conceivable, both as a nucleoside and as a non-nucleo-
side HIV reverse transcriptase inhibitor. However, we
failed to find a molecule with such properties since
compound 7a was inactive against HIV-1 in MT-4 cells.
In fact, compound 7a has the same structural feature
at the anomeric center as the inactive compounds 3a -c
and the weakly active compounds 3f,g.
2CH3), 2.49-2.52 (1H, m, CH), 3.78 (2H, s, CH2Ph), 7.18-7.36
(5H, m, aryl), 10.68-10.77 (br s, 2NH); 13C NMR (DMSO-d6)
δ 19.99 (2CH3), 26.29 (CH), 35.21 (CH2Ph), 113.79 (C-5),
126.46, 127.88, 128.46, 136.99 (aryl), 148.45 (C-6), 150.94 (C-
2), 163.77 (C-4). Anal. (C14H16N2O2‚0.5H2O) C, H, N.
6-(3,5-Dim eth ylben zyl)-5-eth ylu r a cil (2c): yield 712 mg
(69%); mp 216-218 °C; 1H NMR (DMSO-d6) δ 0.85 (3H, t, J )
7.3 Hz, CH3), 2.25 (2H, q, J ) 7.1 Hz, CH2), 2.25 (6H, s, 2CH3),
3.67 (2H, s, CH2Ph), 6.86 (3H, s, aryl), 10.64 (1H, s, NH), 10.96
(1H, s, NH); 13C NMR (DMSO-d6) δ 13.32 (CH3), 17.51 (CH2),
20.76 (2CH3), 111.13 (C-5), 125.73, 127.96, 136.54, 137.42
(aryl), 148.66 (C-6), 150.82 (C-2), 164.40 (C-4). Anal.
(C15H18N2O2‚0.25H2O) C, H, N.
Exp er im en ta l Section
Acyclic Ur a cil Der iva tives 3e-g. Gen er a l P r oced u r e.
A mixture of compound 2 (1.0 mmol), HMDS (5 mL), and
(NH4)2SO4 (10 mg) was heated under reflux for 2 h. The
mixture was concentrated at room temperature under reduced
pressure to obtain the silylated base as a pale yellow solid.
Anhydrous CH3CN (10 mL) was added, and the solution was
stirred at -45 °C. TMS triflate (0.24 g, 1.05 mmol) was added
to the mixture followed by dropwise addition of the appropriate
acetal or 1,3-dioxolane (2.0 mmol). The reaction was quenched
after 2-3 h and neutralized by addition of saturated aqueous
NaHCO3 at -45 °C and evaporated in vacuo at room temper-
ature to dryness. The residue was extracted with dry Et2O (2
× 25 mL), and the ether extracts were evaporated under
reduced pressure to give compound 3 after silica column
chromatography with CHCl3. In some cases compound 3 was
further purified by preparative TLC (CHCl3).
1
The H-NMR spectra were recorded on a Bruker A 250 FT
NMR spectrometer with tetramethylsilane as an internal
standard. Chemical shifts are reported in parts per million
(δ), and signals are expressed as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br (broad). Silica gel
(0.040-0.063 mm) and analytical silica gel TLC plates 60 F254
were purchased from Merck. Tetrahydrofuran (THF) was
distilled from sodium/benzophenone prior to use.
3-Oxo Ester s 1b,c. Gen er a l P r oced u r e. Activated zinc
dust (18 g, 275 mmol) was suspended in dry THF (125 mL) at
reflux, and a few drops of ethyl 2-bromo ester were added to
initiate the reaction. After the appearance of a green color
(ca. 45 min), the arylacetonitrile (4.50 mmol) was added in one
portion followed by dropwise addition of ethyl 2-bromo ester
(10 mmol) over a period of 1 h. The reaction mixture was
refluxed for an additional 10 min, diluted with THF (375 mL),
and quenched with aqueous K2CO3 (50%, 54 mL). Rapid
stirring for 45 min gave two distinct layers. The THF layer
was decanted, the residue was washed with THF (2 × 100 mL),
and the combined THF fractions were treated with aqueous
HCl (10%, 50 mL) at room temperature for 45 min. The
mixture was concentrated in vacuo, diluted with CH2Cl2 (300
mL), and washed with saturated aqueous NaHCO3 (200 mL).
The organic phase was dried (Na2SO4) and evaporated in vacuo
to give an oily residue, which was used without further
purification for the synthesis of compound 2b,c. Further
purification of compounds 1b,c could be achieved by chroma-
tography on silica gel (200 g) with petroleum ether (bp 60-80
°C)/Et2O (95:5).
6-(3,5-Dim e t h ylb e n zyl)-1-(1-e t h oxym e t h yl)-5-e t h yl-
u r a cil (3e): yield 345 mg (94%); mp 159-61 °C (lit.5 mp 160-
160.7 °C).
6-(3,5-Dim e t h ylb e n zyl)-5-e t h yl-1-(1-m e t h oxye t h yl)-
u r a cil (3f): yield 320 mg (87%); mp 153-154 °C dec; 1H NMR
(DMSO-d6) δ 0.85 (3H, t, J ) 7.2 Hz, CH3), 1.35 (3H, d, J )
6.3 Hz, CH3), 2.18-2.23 (8H, m, CH2, 2CH3), 3.09 (3H, s,
OCH3), 4.09 (1H, d, J ) 17.2 Hz, CH2Ph), 4.24 (1H, d, J )
17.2 Hz, CH2Ph), 5.88 (1H, d, J ) 5.1 Hz, CH), 6.76 (2H, s,
aryl), 6.85 (1H, s, aryl), 11.32 (1H, s, NH); 13C NMR (DMSO-
d6) δ 12.99 (CH3), 18.34 (CH2), 20.57 (CH3), 20.75 (2CH3), 33.16
(CH2Ph), 55.73 (OCH2), 84.80 (CH), 115.93 (C-5), 124.91,
127.73, 136.91, 137.49 (aryl), 148.66 (C-6), 151.12 (C-2), 162.72
(C-4). Anal. (C18H24N2O3) C, H, N.
Eth yl 2-isop r op yl-3-oxo-4-p h en ylbu tyr a te (1b): yield
1.03 g (92%) as an oil; 1H NMR (CDCl3) δ 0.85 (3H, d, J ) 6.6
Hz, CH3), 0.94 (3H, d, J ) 6.7 Hz, CH3), 1.22 (3H, t, J ) 7.1
6-(3,5-Dim e t h ylb e n zyl)-1-(1-e t h oxyp r op yl)-5-e t h yl-
u r a cil (3g): yield 248 mg (72%); mp 131-133 °C; 1H NMR
(CDCl3) δ 0.84 (3H, t, J ) 7.3 Hz, CH3), 0.94 (3H, t, J ) 7.0

2430 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Danel et al.
Hz, CH3), 1.65-1.73 (2H, m, CH2), 2.26 (6H, s, 2CH3), 3.41
(2H, q, J ) 7.0 Hz, OCH2), 4.13 (1H, d, J ) 16.7 Hz, CH2Ph),
4.33 (1H, d, J ) 16.8 Hz, CH2Ph), 6.07 (1H, br s, NCHO), 6.66
(2H, s, aryl), 6.84 (1H, s, aryl), 9.98 (1H, br s, NH); 13C NMR
(CDCl3) δ 9.80 (CH3), 13.12 (CH3), 14.33 (CH3), 18.76 (CH2),
21.20 (2CH3), 28.53 (CH2), 33.76 (CH2Ph), 65.35 (OCH2), 89.09
(CH), 117.83 (C-5), 124.93, 128.14, 136.47, 138.19 (aryl), 149.77
(C-6), 152.30 (C-2), 163.49 (C-4). Anal. (C20H28N2O3‚0.5H2O)
C, H, N.
6-Ben zyl-1-(eth oxym eth yl)-5-isopr opylu r acil (3d). Com-
pound 2b (0.2 g, 0.8 mmol) was suspended in CHCl3 (10 mL),
and to this suspension was added bis(trimethylsilyl)acetamide
(BSA) (0.35 g, 1.72 mmol). The mixture became clear after
stirring at room temperature for 10 min. To this solution was
added chloromethyl ethyl ether (0.15 g, 1.16 mmol) and stirring
continued until no change in the amount of the starting
material could be noticed on TLC. After evaporation of the
solvent in vacuo the resulting syrup was chromatographed on
silica gel with CHCl3 to afford 200 mg (80%) as crystals; mp
108-110 °C (lit.18 mp 109-110 °C).
Meth yl 5-O-(ter t-Bu tyld ip h en ylsilyl)-2-d eoxy-3-O-(p h e-
n oxyt h ioca r b on yl)-r,â-D-er yth r o-p en t ofu r a n osid e (5).
2-Deoxy-D-ribose (4, 2.5 g, 18.7 mmol) was dissolved in MeOH
(25 mL), and a solution of 1 mol % of HCl in MeOH (50 mL)
was added. After 20 min the reaction mixture was quenched
with pyridine (2 mL) and evaporated in vacuo. The oily
residue was dissolved in dry pyridine (20 mL), DMAP (244
mg, 2 mmol) and tert-butylchlorodiphenylsilane (5.50 g, 20
mmol) were added, and the reaction was mixture stirred for 2
h. Then phenoxythiocarbonyl chloride (3.45 g, 20 mmol) was
added in small portions, and the reaction mixture was stirred
for an additional 12 h, quenched by addition of MeOH (1 mL),
and evaporated in vacuo. The residue was partitioned between
CHCl3 (50 mL) and H2O (50 mL). The organic phase was dried
(Na2SO4) and evaporated in vacuo. After chromatographic
workup on silica gel (100 g) using the gradient 0-25% Et2O
in petroleum ether (bp 60-80 °C), compound 5 was obtained
as a colorless oil (7.50 g, 77%). 1H and 13C NMR data are in
accordance with those in ref 16.
6-Ben zyl-5-et h yl-2′,3′-d id eoxyu r id in e (7a ) a n d It s r
An om er 7b. Condensation of compound 2a (465 mg, 2.0
mmol) with compound 5 (1.04 g, 2.0 mmol) was done using
the same method as for the acyclic uracil derivatives 3e-g.
The anomeric mixture (R/â ) 1/2) was separated with prepara-
tive TLC using the gradient 5-30% Et2O in petroleum ether
(bp 60-80 °C) to give 430 mg of â anomer 6a and 234 mg of R
anomer 6b. Each of the anomers (0.3 mmol) and R,R′-
azoisobutyronitrile (AIBN, 23 mg, 0.15 mmol) were dissolved
with stirring in anhydrous toluene (15 mL) under nitrogen.
Tributyltin hydride (0.25 mL, 0.94 mmol) was added, and
stirring was continued for 3 h at 80 °C. The solvent was
evaporated, and the residue was dissolved in CHCl3 (25 mL),
washed with saturated aqueous NaHCO3 (2 × 25 mL) and
water (2 × 25 mL), and dried over Na2SO4. The solvent was
evaporated in vacuo and the resulting residue dissolved in 1.1
M tetrabutylammonium fluoride in THF (10 mL). The reaction
mixture was stirred for 2 h at room temperature and evapo-
rated in vacuo and the residue dissolved in CHCl3 (25 mL).
The organic phase was washed with H2O (2 × 50 mL), dried
(Na2SO4), and evaporated in vacuo. The â anomer 7a and the
R anomer 7b were obtained, respectively, after preparative
TLC using MeOH/CH2Cl2 (2:98) as eluent.
2′R-H, 3′R-H), 2.11-2.60 (4H, m, 2′â-H, 3′â-H, CH2), 3.39 (1H,
dd, J ) 5.5, 11.9 Hz, 5′a-H), 3.67 (1H, dd, J ) 3.1, 11.8 Hz,
5′b-H), 3.93 (1H, d, J ) 17.2 Hz, CH2Ph), 4.20 (1H, d, J )
17.2 Hz, CH2Ph), 4.61-4.71 (1H, m, 4′-H), 5.73-5.79 (1H, m,
1′-H), 7.11-7.39 (5H, m, aryl); 13C NMR (CDCl3) δ 13.78 (CH3),
19.29 (CH2), 27.48 (C-3′), 29.71 (C-2′), 34.74 (CH2Ph), 64.75
(C-5′), 82.72 (C-4′), 89.01 (C-1′), 116.32 (C-5), 127.40, 127.45,
129.25, 135.13 (aryl), 148.93 (C-6), 150.57 (C-2), 163.33 (C-4);
FAB MS (CHCl3, 3-nitrobenzyl alcohol) m/ z ) 331 (M + H+).
1-((Alk ylth io)m eth yl)u r a cils 8a -f: P r oced u r e A. To a
suspension of the uracil 2 (2 mmol) and K2CO3 (0.55 g, 4 mmol)
in anhydrous DMF (10 mL) was added chloromethyl alkyl
sulfide (2 mmol) and the mixture was stirred vigorously for 4
days. The solvent was removed in vacuo and the residue
coevaporated with toluene (2 × 10 mL). The residue was
extracted with CHCl3 (2 × 10 mL) and the CHCl3 phase
evaporated in vacuo to give compound 8 after being chromato-
graphed using preparative TLC with CHCl3.
P r oced u r e B. The uracil 2 (2 mmol) was refluxed for 2 h
in HMDS (10 mL) in the presence of (NH4)SO4 (10 mL)
followed by evaporation in vacuo. The resulting residue was
dissolved in MeCN (10 mL), the solution cooled to -40 °C, and
TMS triflate (2 mmol) added. The mixture was cooled to -60
°C, and (methylthio)methyl acetate (6 mmol) was added. The
temperature of the mixture was allowed to increase slowly.
When the temperature had reached 4 °C after 15 h, the
mixture was quenched by addition of a saturated aqueous
NaHCO3 (5 mL) and evaporated in vacuo. The resulting
residue was purified using preparative TLC with CHCl3 as
eluent to give compounds 8 in 69-78% yield.
6-Ben zyl-5-eth yl-1-((m eth ylth io)m eth yl)u r acil (8a): pro-
cedure A, yield 41 mg (7%); procedure B, yield 452 mg (78%);
1
mp 185-188 °C; H NMR (CDCl3) δ 1.08 (3H, t, J ) 7.4 Hz,
CH3), 2.28 (3H, s, SCH3), 2.50 (2H, q, J ) 7.4 Hz, CH2), 4.17
(2H, s, CH2Ph), 4.79 (2H, s, NCH2S), 7.10-7.38 (5H, m, aryl),
9.76 (1H, s, NH); 13C NMR (CDCl3) δ 13.87 (CH3), 15.72
(SCH3), 19.30 (CH2), 34.12 (CH2Ph), 46.90 (NCH2S), 117.32
(C-5), 127.32, 127.54, 129.37, 134.58 (aryl), 148.60 (C-6), 151.78
(C-2), 163.11 (C-4). Anal. Calcd (C15H18N2O2S‚0.25H2O): C,
61.10; H, 6.32; N, 9.50. Found: C, 61.60; H, 6.26; N, 9.16.
6-Ben zyl-5-eth yl-1-((eth ylth io)m eth yl)u r a cil (8b): pro-
cedure A, yield 60 mg (10%); yellow oil; 1H NMR (CDCl3) δ
1.08 (3H, t, J ) 7.4 Hz, CH3), 1.27 (3H, t, J ) 7.4 Hz, CH3),
2.50 (2H, q, J ) 7.4 Hz, CH2), 2.76 (2H, q, J ) 7.4 Hz, CH2),
4.16 (2H, s, CH2Ph), 4.81 (2H, s, NCH2S), 7.10-7.38 (5H, m,
aryl), 9.93 (1H, s, NH); 13C NMR (CDCl3) δ 13.88 (CH3), 14.94
(SCH3), 19.28 (CH2), 26.55 (SCH2), 34.21 (CH2), 45.05 (NCH2S),
117.27 (C-5), 127.29, 127.50, 129.34, 134.61 (aryl), 148.63 (C-
6), 151.72 (C-2), 163.24 (C-4).
6-Ben zyl-5-isopr opyl-1-((m eth ylth io)m eth yl)u r acil (8c):
procedure A, yield 25 mg (4%); procedure B, yield 456 mg
(75%); mp 134-136 °C; 1H NMR (CDCl3) δ 1.3 (6H, d, J ) 6.9
Hz, 2CH3), 2.29 (3H, s, SCH3), 2.86-2.97 (1H, m, CH), 4.19
(2H, s, CH2Ph), 4.81 (2H, s, NCH2S), 7.10-7.39 (5H, m, aryl),
9.40 (1H, s, NH); 13C NMR (CDCl3) δ 15.77 (SCH3), 20.51
(2CH3), 28.44 (CH), 34.17 (CH2Ph), 47.10 (NCH2S), 120.18 (C-
5), 127.29, 127.48, 129.35, 134.74 (aryl), 148.01 (C-6), 151.75
(C-2), 162.14 (C-4). Anal. Calcd (C16H20N2O2S‚0.25H2O): C,
62.21; H, 6.69; N, 9.07. Found: C, 62.75; H, 6.74; N, 8.75.
6-Ben zyl-1-((eth ylth io)m eth yl)-5-isop r op ylu r a cil (8d ):
procedure A, yield 38 mg (6%); mp 136-138 °C; 1H NMR
(CDCl3) δ 1.27 (3H, t, J ) 7.4 Hz, CH3), 1.30 (6H, d, J ) 7.0
Hz, 2CH3), 2.78 (2H, q, J ) 7.4 Hz, CH2), 2.86-2.97 (1H, m,
CH), 4.18 (2H, s, CH2Ph), 4.83 (2H, s, NCH2S), 7.10-7.39 (5H,
m, aryl), 9.49 (1H, s, NH); 13C NMR (CDCl3) δ 14.98 (CH3),
20.52 (2CH3), 26.65 (CH), 28.43 (SCH2), 34.28 (CH2Ph), 45.31
(NCH2S), 120.15 (C-5), 127.28, 127.46, 129.34, 134.76 (aryl),
Com p ou n d 7a : yield 32 mg (10%) as a foam; 1H NMR
(CDCl3) δ 1.04 (3H, t, J ) 7.4 Hz, CH3), 1.45-1.57 (1H, m,
2′R-H), 1.67-1.79 (1H, m, 3′R-H), 2.22-2.64 (4H, m, 2′â-H, 3′â-
H, CH2), 3.61 (1H, dd, J ) 3.3, 12.0 Hz, 5′a-H), 3.85 (1H, d, J
) 11.2 Hz, 5′b-H), 3.97 (1H, d, J ) 17.2 Hz, CH2Ph), 4.03-
4.09 (1H, m, 4′-H), 4.24 (1H, d, J ) 17.2 Hz, CH2Ph), 5.63 (1H,
dd, J ) 5.3, 7.8 Hz, 1′-H), 7.11-7.40 (5H, m, aryl), 9.69 (1H,
br s, NH); 13C NMR (CDCl3) δ 13.69 (CH3), 19.33 (CH2), 25.48
(C-3′), 29.08 (C-2′), 34.80 (CH2), 63.99 (C-5′), 81.10 (C-4′), 88.65
(C-1′), 116.85 (C-5), 127.33, 127.42, 129.99, 135.13 (aryl),
148.71 (C-6), 150.70 (C-2), 163.11 (C-4); FAB MS (CHCl3,
3-nitrobenzyl alcohol) m/ z ) 331 (M + H+).
148.05 (C-6), 151.67 (C-2), 162.24 (C-4). Anal. (C17H22
-
N2O2S‚0.25H2O) C, H, N.
6-(3,5-Dim et h ylb en zyl)-5-et h yl-1-((m et h ylt h io)m et h -
yl)u r a cil (8e): procedure A, yield 37 mg (6%); procedure B,
1
yield 438 mg (69%); mp 112-114 °C; H NMR (CDCl3) δ 1.08
(3H, t, J ) 7.4 Hz, CH3), 2.28 (9H, s, SCH3, 2CH3), 2.48 (2H,
q, J ) 7.4 Hz, CH2), 4.08 (2H, s, CH2), 4.83 (2H, s, NCH2S),
6.69 (2H, s, aryl), 6.90 (1H, s, aryl), 9.78 (1H, s, NH); 13C NMR
(CDCl3) δ 13.89 (CH3), 15.75 (CH3), 19.30 (CH2), 21.24 (2CH3),
Com p ou n d 7b: yield 23 mg (7%) as a foam; 1H NMR
(CDCl3) δ 1.04 (3H, t, J ) 7.4 Hz, CH3), 1.43-1.68 (2H, m,

Anti-HIV-1 Activity of HEPT Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2431
(7) Tanaka, H.; Takashima, H.; Ubasawa, M.; Sekiya, K.; Inouye,
N.; Baba, M.; Shigeta, S.; Walker, R. T.; De Clercq, E.; Miyasaka,
T. Synthesis and antiviral activity of 6-benzyl analogs of 1-[(2-
hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as potent
and selective anti-HIV-1 agents. J . Med. Chem. 1995, 38, 2860-
2865.
(8) Tanaka, H.; Takashima, H.; Ubasawa, M.; Sekiya, K.; Nitta, I.;
Baba, M.; Shigeta, S.; Walker, R. T.; De Clercq, E.; Miyasaka,
T. Synthesis and antiviral activity of deoxy analogs of 1-[(2-
hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as potent
and selective anti-HIV-1 agents. J . Med. Chem. 1992, 35, 4713-
4719.
(9) Tanaka, H.; Baba, M.; Hayakawa, H.; Sakamaki, T.; Miyasaka,
T.; Ubasawa, M.; Takashima, H.; Sekiya, K.; Nitta, I.; Shigeta,
S.; Walker, R. T.; Balzarini, J .; De Clercq, E. A new class of HIV-
1-specific 6-substituted acyclouridine derivatives: Synthesis and
anti-HIV-1 activity of 5- or 6-substituted analogues of 1-[(2-
hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT). J . Med.
Chem. 1991, 34, 349-357.
(10) Pan, B. C.; Chen, Z.-H.; Piras, G.; Dutschman, G. E.; Rowe, E.
C.; Cheng, Y. C.; Chu, S. H. Synthesis and anti-HIV-1 activities
of 6-arylthio and 6-arylselenoacyclonucleosides. J . Heterocycl.
Chem. 1994, 31, 177-185.
(11) Danel, K.; Larsen, E.; Pedersen, E. B. An easy synthesis of 5,6-
disubstituted acyclouridine derivatives. Synthesis 1995, 934-
936.
(12) Wittenburg, E. A new synthesis of nucleosides. Z. Chem. 1964,
4, 303-304.
(13) Vorbru¨ggen, H.; Krolikiewiecz, K.; Bennua, B. Nucleoside syn-
thesis with trimethylsilyl triflate and perchlorate as catalysts.
Chem. Ber. 1981, 114, 1234-1255.
33.98 (CH2Ph), 46.95 (NCH2S), 117.18 (C-5), 125.02, 129.18,
134.37, 139.06 (aryl), 148.92 (C-6), 151.87 (C-2), 163.23 (C-4).
6-(3,5-Dim et h ylben zyl)-5-et h yl-1-((et h ylt h io)m et h yl)-
u r a cil (8f): procedure A, yield 46 mg (7%); mp 145-147 °C;
1H NMR (CDCl3) δ 1.08 (3H, t, J ) 7.4 Hz, CH3), 1.28 (3H. t,
J ) 7.4 Hz, CH3), 2.28 (6H, s, 2CH3), 2.49 (2H, q, J ) 7.4 Hz,
CH2), 2.77 (2H, q, J ) 7.4 Hz, CH2), 4.08 (2H, s, CH2Ph), 4.83
(2H, s, NCH2S), 6.69 (2H, s, aryl), 6.90 (1H, s, aryl), 9.82 (1H,
s, NH); 13C NMR (CDCl3) δ 13.89 (CH3), 14.96 (CH3), 19.29
(CH2), 21.23 (2CH3), 26.57 (SCH2), 34.07 (CH2Ph), 45.12
(NCH2S), 117.13 (C-5), 124.99, 129.16, 134.39, 139.02 (aryl),
148.96 (C-6), 151.76 (C-2), 163.31 (C-4). Anal. (C18H24N2O2S)
C, H, N.
Vir u s a n d Cells. The HIV-1 strain HTLV-IIIB21 was
propagated in H9 cells22 at 37 °C, 5% CO2 using RPMI 1640
with 10% heat-inactivated fetal calf serum (FCS) and antibiot-
ics (growth medium). Culture supernatant was filtered (0.45
nm), aliquotted, and stored at -80 °C until use.
In h ibition of HIV-1 Rep lica tion . Compounds were ex-
amined for possible antiviral activity against HIV-1 using
MT-4 cells as target cells. For screening studies, 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 2 h. Cultures were maintained with
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 HIV-1 antigen
detection ELISA.23 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
(14) Hansen, P.; Pedersen, E. B. A simple synthetic route to silylated
methyl 3-azido-2,3-dideoxy-R,â-D-erythro-pentofuranoside. Acta
Chem. Scand. 1990, 44, 522-523.
(15) Chaudhary, S. K.; Hernandez, O. 4-Dimethylaminopyridine: An
efficient and selective catalyst for the silylation of alcohols.
Tetrahedron Lett. 1979, 99-102.
(16) Motawia, M. S.; Pedersen, E. B. A new route to 2′,3′-dideoxy-
cytidine. Liebigs Ann. Chem. 1990, 599-602.
(17) Okabe, M.; Sun, R.-C.; Tam, S. Y.-K.; Todaro, L. J .; Coffen, D.
L. Synthesis of the dideoxynucleosides ddC and CNT from
glutamic acid, ribonolactone, and pyrinidine bases. J . Org. Chem.
1988, 53, 4780-4786.
cytotoxicity using the MTT assay as previously described.24
A
30% inhibition of cell growth relative to control cultures was
considered significant.
(18) Baba, M.; Shigeta, S.; Yuasa, S.; Takashima, H.; Sekiya, K.;
Ubasawa, M.; Tanaka, H.; Miyasaka, T.; Walker, R. T.; De
Clercq, E. Preclinical evaluation of MKC-442, a highly potent
and specific inhibitor of human immunodeficiency virus type 1
in vitro. Antimicrob. Agents Chemother. 1994, 38, 688-692.
(19) Yuasa, S.; Sadakata, Y.; Takashima, H.; Sekiya, K.; Inoue, N.;
Ubasawa, M.; Baba, M. Selective and synergistic inhibition of
human immunodeficiency virus type 1 reverse transcriptase by
a non-nucleoside inhibitor, MKC-442. Mol. Pharmacol. 1993, 44,
895-900.
(20) Chu, C. K.; Shinazi, R. F.; Arnold, B. H.; Cannon, D. L.;
Doboszewski, B.; Bhadti, V. B.; Gu, Z. Comparative activity of
2′,3′-saturated and unsaturated pyrimidine and purine nucleo-
sides against human immunodeficiency virus type 1 in periph-
eral blood mononuclear cells. Biochem. Pharmacol. 1988, 37,
3543-3548.
(21) Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C.
Detection, isolation and continuous production of cytophathic
retroviruses (HTLV-III) from patients with AIDS and pre-AIDS.
Science 1984, 224, 497-500.
(22) Harada, S.; Koyanagi, Y.; Yamamoto, N. Infection of HTLV-III/
LAV in HTLV-I-carrying cells MT-2 and MT-4 and application
in a plaque assay. Science 1985, 229, 563-566.
Refer en ces
(1) Baba, M.; De Clercq, E.; Tanaka, H.; Ubasawa, M.; Takashima,
H.; Sekiya, K.; Nitta, I.; Umezu, K.; Walker, R. T.; Mori, S.; Ito,
M.; Shigeta, S.; Miyasaka, T. Highly potent and selective
inhibition of human immunodeficiency virus type 1 by a novel
series of 6-substituted acyclouridine derivatives. Mol. Pharma-
col. 1991, 39, 805-810.
(2) Balzarini, J .; Karlsson, A.; De Clercq, E. Human immunodefi-
ciency virus type 1 drug-resistance patterns with different 1-[(2-
hydroxyethoxy)methyl-6-(phenylthio)-thymine derivatives. Mol.
Pharmacol. 1993, 44, 694-701.
(3) Baba, M.; Yuasa, S.; Niwa, T.; Yamamoto, M.; Yabuuchi, S.;
Takashima, H.; Ubasawa, M.; Tanaka, H.; Miyasaka, T.; Walker,
R. T.; Balzarini, J .; De Clercq, E.; Shigeta, S. Effect of human
serum on the in vitro anti-HIV-1 activity of 1-[(2-hydroxyethoxy)-
methyl]-6-(phenylthio)thymine (HEPT) derivatives as related to
their lipophilicity and serum protein binding. Biochem. Phar-
macol. 1993, 45, 2507-2512.
(4) Miyasaka, T.; Tanaka, H.; De Clercq, E.; Baba, M.; Walker, R.
T.; Ubasawa, M. Preparation of antiviral pyrimidine nucleosides.
European Patent 449726 (October 2, 1991); Chem. Abstr. 1991,
116, 41986.
(5) Miyasaka, T.; Tanaka, H.; De Clercq, E.; Baba, M.; Walker, R.
T.; Ubasawa, M. Preparation of 6-substituted acyclopyrimidine
nucleoside derivatives as virucides. European Patent Application
420763 (April 3, 1991); Chem. Abstr. 1991, 115, 158838.
(6) Baba, M.; Tanaka, H.; Miyasaka, T.; Yuasa, S.; Ubasawa, M.;
Walker, R. T.; De Clercq, E. HEPT derivatives: 6-Benzyl-
ethoxymethyl-5-isopropyluracil (MKC-442). Nucleosides Nucleo-
tides 1995, 14, 575-583.
(23) Nielsen, C. M.; Bygbjerg, I. C.; Vestergaard, B. F. Detection of
HIV antigens in eluates from whole blood collected on filter
paper (Letter). Lancet 1987, No. 1, 566-567.
(24) Mosmann, T. Rapid colorimetic assay for cellular growth and
survival. Application to proliferation and cytotoxicity assays. J .
Immunol. Methods 1983, 65, 55-63.
J M9600499