Synthesis and biological evaluation of 2-methoxy- and 2-Methylthio-6-[(2'-alkylamino)ethyl]-4(3H)-pyrimidinones with anti-rubella virus activity

Article abstract of DOI:10.1016/S0968-0896(99)00111-X

The synthesis of a new family of antiviral compounds, 2-methoxy-, and 2-methylthio-6-[(2'-alkylamino)ethyl]-4(3H)-pyrimidinones, has been accomplished. The activity of these agents against positive strand (rubella virus and sindbis virus) and negative str

Full text of DOI:10.1016/S0968-0896(99)00111-X

Bioorganic & Medicinal Chemistry 7 (1999) 1925±1931
Synthesis and Biological Evaluation of 2-Methoxy- and
2-Methylthio-6-[(20-alkylamino)ethyl]-4(3H)-pyrimidinones
with Anti-Rubella Virus Activity
Maurizio Botta, a,* Francesca Occhionero, b Rosario Nicoletti, b Paola Mastromarino, c
Cinzia Conti, c Maurizio Magrini c and Ra€aele Saladino d,*
a
Â
Dipartimento Farmaco Chimico Tecnologico, Banchi di Sotto 55, Universita degli Studi, 53100 Siena, Italy
b
Â
Dipartimento di Chimica, Universita ``La Sapienza'', ple. Aldo Moro 5, 00185, Rome, Italy
Â
c
Â
Istituto di Microbiologia, Facolta di Medicina, Universita di Roma ``La Sapienza'', ple. Aldo Moro 5, 00185, Rome, Italy
d
Â
Dipartimento Agrochimico Agrobiologico, Universita degli studi di Viterbo ``La Tuscia'', via San Camillo de Lellis, 01100 Viterbo, Italy
Received 6 November 1998; accepted 26 March 1999
AbstractÐThe synthesis of a new family of antiviral compounds, 2-methoxy-, and 2-methylthio-6-[(20-alkylamino)ethyl]-4(3H)-
pyrimidinones, has been accomplished. The activity of these agents against positive strand (rubella virus and sindbis virus) and
negative strand (vesicular stomatitis virus) RNA viruses is reported. Some of these compounds are ecient and selective inhibitors
of rubella virus. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
derivatives showed selective antitumor9 and antiviral
activity against other type of viruses, as for example,
african swine fever virus (ASFV10) and parain¯uenza 1
virus (sendai virus11), suggests the importance of testing
this family of compounds as broad-spectrum drugs. In
this context, in the course of our studies on the chem-
istry and biological evaluation of antiviral activity of C-
6 substituted uracil and pyrimidinone derivatives,12 we
found that 1,3-dimethyl-6-(10-hydroxy-20-diethylamino)-
uracil 1 posseses a low but selective activity against
vesicular stomatitis virus (VSV).13 We are reporting in
this communication the synthesis of a new class of
antiviral agents, compounds 6a±e, 7a,b, 8, and 11a,b
that are structurally related to 1, together with their
biological evaluation against positive strand (rubella
virus (RV) and sindbis virus (SV)) and negative strand
(vesicular stomatitis virus (VSV)) RNA viruses. Some of
these compounds represent, to the best of our knowl-
edge, the ®rst reported e€ective inhibitors of RV; a virus
that is responsible for important medical and social
problems, for which, at moment, no ecient chemo-
therapy is available.14
Pyrimidine derivatives and their nucleosides have a
great biological signi®cance because they exhibit a wide
range of antiviral and anticancer activities. In contrast
to extensive studies about 5-substituted pyrimidines less
attention has been devoted to 6-substituted isomers,
probably because of their no easy synthetic availability,
and their supposed biological inactivity.1 In the last few
years some uracil and pyrimidinone derivatives sub-
stituted either at C-5 and C-6 position have emerged in
the ®eld of antiviral chemotherapy.2,3 Among the
important 6-substituted uracil derivatives, 1-[(2-hydroxy-
ethyl)methyl]-6-(phenylthio)thymine (HEPT4,5) and its
analogues6,7 (of which 6-benzyl-1-(ethoxymethyl)-5-iso-
propyluracil has been chosen as a candidate for clinical
trials6,7), and 3,4-dihydro-2-alkoxy-6-benzyl-4-oxopyr-
imidines (DABOs8), showed a potent and selective
activity against human immunode®ciency virus type-1
(HIV-1). Thus, the excellent biological activities exhib-
ited by these 6-substituted uracil derivatives provides a
new emphasis to explore chemistry and biological
activities of these pyrimidine derivatives. In this context,
the ®nding that C-6 substituted uracil and pyrimidinone
Chemistry
Compounds 6a±e and 7a,b were prepared in a step-wise
manner starting from commercially available acetone
* Corresponding author. Tel.: +39-577-45393; fax: +39-577-270629;
e-mail: botta@unisi.it
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00111-X

1926
M. Botta et al. / Bioorg. Med. Chem. 7 (1999) 1925±1931
dicarboxylic acid diethyl ether 2 and O-methylisourea
hydrogen sulphate 3a or S-methylisourea hydrogen sul-
phate 3b (Scheme 1). Cyclization was performed in the
presence of Ca(OH)2 in ethanol:water mixture (1.0:3.0
v/v) at 25ꢀC for 48 h. In these experimental conditions,
4(3H)-pyrimidinone derivatives 4a and 4b were obtained
in 75 and 78% yields, respectively. The reduction of the
ester moiety in compounds 4a,b was performed, after
many failures, with NaBH4 in re¯uxing isopropanol to
give the corresponding alcohols 5a,b in reproducible
yields of 60 and 57%, respectively. Compounds 5a,b
were treated with a small excess of p-toluenesulfonyl
chloride (TsCl; 1.2 equiv/mol) in the presence of 1.10
equiv/mol of 4-(dimethylamino)pyridine (DMAP) in
CHCl3 at 25ꢀC. After the work up of the reaction the
crude was reacted without further puri®cation with an
excess (2 equiv/mol) of nitrogen nucleophiles (diethyl-
amine, piperidine, morpholine, and piperazine) in
re¯uxing THF to give compounds 6a±e in yields ranging
from 53 to 91%. In the latter two steps, the possible
formation of an O4-tosyl imidate intermediate (formed
by reaction of the amide function with TsCl)15 followed
by undesired C-4 nucleophilic displacement was ruled
out on the basis of the characteristic amide absorption
band, in the range of 1735±1740 cm 1, presents in the
IR spectra of compounds 6a±e. The presence of the C-5
activity. Compound 8 was prepared in 76% yield by
sodium periodate (NaIO4) oxidation of 6b performed in
H2O at 0ꢀC (Scheme 2).
Compounds 11a,b were prepared in a step-wise manner
starting from commercially available ethyl 4-chloro-
acetoacetate 9 and 3b in H2O:MeOH (v/v) at 25ꢀC in
the presence of a large excess of Ca(OH)2 to give 10 in
75% yield (Scheme 3). Chlorine nucleophilic displace-
ment with diethylamine and morpholine, performed in
re¯uxing THF gave compounds 11a,b in 63 and 59%
yields, respectively.
Biology
Con¯uent vero cell monolayers were exposed to com-
pounds 6a±e, 7a,b, 8, and 11a,b for 48 h at 37ꢀC. Cell
morphology, viability and yield were then examined.
Cell viability was assessed on the basis of vital dye
exclusion test, using Trypan Blue, and cell yield was
determined by counting cells with an hemocytometer
after trypsinization. For antiviral assay, the compounds
were tested starting from the highest non-cytotoxic
concentration which did not a€ect any parameter con-
sidered in 100% of the cells. After virus adsorption (1 h,
37ꢀC), the viral inoculum was removed. Cell monolayers
were washed three times with PBS and incubated with
maintenance medium in the presence or absence of
1
proton resonance at ca. 6 ppm in the H NMR spectra,
typical of 4(3H)-pyrimidinones, is also diagnostic.16
Moreover, compounds 6a and 6b were also treated with
H2SO4 (2 N water solution) at 80ꢀC to give the corres-
ponding 6-substituted uracil and 2-thiouracil deriva-
tives, compounds 7a and 7b, in 96 and 94% yields,
respectively (Scheme 1).
Compounds 8 and 11a,b were further synthesized to
evaluate a possible role of both the sulfur oxidation
state and the size of the C-6 side chain on the antiviral
Scheme 2.
Scheme 1.

M. Botta et al. / Bioorg. Med. Chem. 7 (1999) 1925±1931
1927
Scheme 3.
fourfold dilutions of the compounds. Virus yield was
evaluated by plaque assay after a single cycle of virus
multiplication at high multiplicity infection (SNV and
VSV, 8 h; RV, 48 h) and after multiple rounds at low
multiplicity infection (SNV and VSV, 24 h; RV 96 h).
The compounds were tested in two independent experi-
ments conducted in triplicate.
whereas only 8 was active on RV replication in these
experimental conditions.
The 50% inhibitory concentrations of virus yield have
been calculated on a dose±response line obtained by
plotting the percentage of plaque reduction, with respect
to the control plaque count, versus the logarithm of
compound dose. Triplicate wells were utilized for each
concentration tested. The standard deviation was less
than 5% of the mean value. The reduction of viral yield
was found to be concentration-dependent (data not
shown). The CC50 and IC50 of compounds are shown in
Table 2. Analogues 6b and 8 were the most active sub-
stances against all the viruses tested. Among the other
compounds, 6c was also e€ective, although at higher
concentrations, whereas 6e and 11b showed an inhibi-
tory activity only towards SNV and VSV. The other
products showed only a weak e€ect. In order to deter-
mine the selectivity indices (SI) of the products, the ratio
between 50% drug cytotoxicity (CC50) and the con-
centration required to inhibit viral multiplication by
50% was calculated. The data obtained, reported in
Table 2, showed a good SI for compound 6b towards
RV replication. All the other compounds showed low
SIs, indicating a limited selectivity.
Results
The e€ect of compounds 6a±e, 7a,b, 8, and 11a,b on
RV, SV, and VSV replication in Vero cells was studied
under one-step or multi-step multiplication conditions.
After viral adsorption (1 h, 37ꢀC) the drugs or control
diluent were added to the culture medium and viral
yield was determined by plaque assay. As shown in
Table 1, compounds 6b±d were found to strongly inhibit
VSV production, whereas a second set of substances (6e,
7b, 8, and 11b) produced an inhibition of ca. 1 log.
Compounds 6b±d were also e€ective towards the repli-
cation of SNV, whereas 6b, and to a lesser extent 8,
inhibit the multiplication of RV. Furthermore, com-
pounds 6e, 8, and 11b signi®cantly reduced the production
of SNV in multi-round, low multiplicity infection,
Table 1. E€ect of compounds 6a±e, 7a,b, 8, and 11a,b on VSV, SNV, and RV virus multiplicationa
Compound
VSVb
SNVb
RVc
moi 10
moi 0.1
moi 10
moi 0.1
moi 3.0
moi 0.1
6a
6b
6c
6d
6e
7a
7b
8
11a
11b
100
0.03
0.09
1.0
NDd
0.2
0.5
23
7.2
ND
100
9.4
47
27
2.3
8.0
16
29
27
74
90
50
48
66
2.6
12
56
3.4
66
100
2.9
61
78
16
36
100
60
88
71
8.3
69
91
54
1.3
3.7
41
69
58
28
11
100
63
8.7
100
3.5
6.7
100
7.2
11
7.2
Compounds were added at the concentration of 125 mg/mL (6c: 62 mg/mL; 7: 31 mg/mL) to vero cells after viral adsorption (1 h, 37ꢀC) and
maintained throughout the incubation. The values represent the % of plaque forming units relative to to an untreated control. Data represent the
mean of three independent experiments; each experiment was conducted in duplicate. The standard deviations were <5% for all values.
a
b
Titers were determined by plaque assay 8 h post-infection (pi) with SNV and VSV at high multiplicity of infection (moi 10) or 24 h pi using a
low moi (0.1 PFU/cell).
c
The yield of rubella virus was evaluated after a single-round, high multiplicity infection (48 h, moi 3.0) as well as in multi-round, low multiplicity
infection (96 h, moi 0.1).
d
ND, not determined.

1928
M. Botta et al. / Bioorg. Med. Chem. 7 (1999) 1925±1931
Table 2. Antiviral activity of compounds 6a±e, 7a,b, 8, and 11a,b, against VSV, SNV, and RV infection in vero cellsa
b
c
Compound
CC50
IC50
SId
SNV
Ð
5.6
3.1
Ð
15.9
Ð
Ð
3.3
>8
>13.8
RV
125
3.9
38.5
125
>125
125
90
SNV
VSV
RV
VSV
Ð
6a
6b
6c
6d
6e
7a
7b
8
11a
11b
250
250
125
1000
1000
>1000
>1000
62.5
>1000
>1000
>125
>125
29
37
80
81.7
>125
>125
24
2.0
64
2.7
8.0
44.3
40.7
>125
63
>125
>125
19
8.5
3.4
12.5
12.2
Ð
Ð
2.6
Ð
>8.0
>11.1
16
Ð
Ð
3.9
>125
>125
125
72.5
125
83.5
>8
>12
a
Virus yield was determined by plaque assay. The antiviral activity of compounds was evaluated in comparison with ribavirin towards SNV and
VSV (IC50: 100 mg/mL, and 300 mg/mL, respectively).
b
The minimal concentration of compound (mg/mL) which a€ected one cytotoxicity parameter in 50% of cells.
Inhibitory concentration of compound (mg/mL) required to inhibity virus yield by 50%.
Selectivity index (CC50/IC50).
c
d
At the moment, compound 6b is a very ecient inhi-
bitor of RV, and it could be used as a reference struc-
ture for the synthesis of more active and selective C-6
substituted pyrimidinone derivatives.
2-Methylthio-6-[(carboxyethyl)methyl]-4(3H)-pyrimidinone
(4b). S-Methyl-2-thiopseudourea sulfate (1.5 g, 5.4 mmol),
diethyl 1,3-acetonedicarboxylate (1 g, 4.9 mmol), and
Ca(OH)2 (0.22 g, 5.9 mmol), were dissolved in H2O
(6 mL) and EtOH (18 mL), and stirred at 25ꢀC for 48 h.
The reaction mixture was neutralized with HCl (2 N
water solution) and extracted with CHCl3. The extract,
washed with NaCl (saturated solution), was dried suc-
cessively over Na2SO4. Evaporation under reduced
pressure gave 2-methylthio-6-[(carboxyethyl)-methyl]-
4(3H)pyrimidinone (4b) (0.89 g, 78%). Mp 112±114ꢀC
Experimental
1H NMR spectra were recorded on a Bruker (200) MHz
spectrometer and are reported in d value. IR spectra
were recorded on a Perkin±Elmer 298 spectrophotometer
using NaCl plates. Microanalyses were performed with a
C. Erba 1106 analyser. MS spectra were recorded on a
VG 70/250S spectrometer with an electron beam of 70
eV. Melting points were obtained on Mettler apparatus
and are uncorrected. All solvents were ACS reagent
grade and when necessary were redistilled and dried
according to standard procedures. Chromatographic
puri®cations were performed on columns packed with
Merck silica gel, 230±400 mesh for ¯ash-technique. TLC
was carried out using Merck platten Kieselgel 60 F254.
1
(EtOAc), IR (CH2Cl2) nmax 1735, 1720, 1660cm 1; H
NMR (CDCl3) d 1.23 (3H, t, J=7.2 Hz, CH3), 2.50 (2H,
s, S-CH3), 3.30 (2H, s, CH2), 4.15 (2H, q, J= 7.2 Hz,
CH2), 6.25 (1H, s, CH); MS: m/e 228 (M+), 213, 185,
181, 157. Anal. calcd for C9H12N2SO3: C, 47.36; H,
5.30; N, 12.27. Found: C, 47.40; H, 5.31; N, 12.35.
2-Methoxy-6-[(20-hydroxy)ethan-10-yl]-4(3H)-pyrimidinone
(5a). 2-Methoxy-6-[(10-carboxyethyl)methan-10-yl]-4(3H)-
pyrimidinone (4a) (1.45 g, 6.8 mmol) and NaBH4 (0.5 g,
13.6 mmol) were dissolved in dry i-PrOH (100 mL)
under nitrogen atmosphere and the mixture was stirred
at 25ꢀC for 48 h. The reaction mixture was treated with
glacial acetic acid (0.1 mL) and successively evapo-
rated under reduced pressure. Flash chromatography
(CHCl3:MeOH, 9.5:0.5) puri®cation of the crude gave
2-methoxy-6-[(20-hydroxy)ethan-10-yl]-4(3H)pyrimidinone
2-Methoxy-6-[(10-carboxyethyl)methan-10-yl]-4(3H)-pyr-
imidinone (4a). O-Methylisourea hydrogen sulfate
(0.93 g, 5.4 mmol), diethyl 1,3-acetonedicarboxylate (1 g,
4.9 mmol), and Ca(OH)2 (0.42 g, 5.4 mmol), were dis-
solved in H2O (6 mL) and EtOH (18 mL), and stirred at
25ꢀC for 48 h. The reaction mixture was neutralized
with HCl (2 N water solution) and extracted with
CHCl3. The extract, washed with NaCl (saturated solu-
tion), was dried successively over Na2SO4. Evaporation
under reduced pressure gave 2-methoxy-6-[(10-carboxy-
ethyl)methan-10-yl]-4(3H)pyrimidinone (4a) (0.78 g,
(5a) (0.68 g, 60%). Mp 137±140ꢀC (MeOH/EtOAc),
1
IR (CH2Cl2) nmax 3200, 1720, 1665 cm
;
1H NMR
(CDCl3) d 2.48 (2H, t, J=6.8 Hz, CH2), 3.68 (2H, t,
J=6.8 Hz, CH2), 4.0 (3H, s, O-CH3), 5.80 (1H, s, CH);
MS: m/e 170 (M+), 152, 139, 127, 99. Anal. calcd for
C7H10N2O3: C, 49.41; H, 5.92; N, 16.46. Found: C,
49.39; H, 5.92; N, 16.50.
75%). Mp 118±122ꢀC (EtOAc), IR (CH2Cl2) nmax 1730,
1
1715, 1660 cm
;
1H NMR (CDCl3) d 1.25 (3H, t,
J=7.5 Hz, CH3), 3.48 (2H, s, CH2), 3.95 (3H, s, O-
CH3), 4.15 (2H, q, J=7.5 Hz, CH2), 6.08 (1H, s, CH);
13C NMR (CDCl3) d 13.84 (CH3), 43.02 (CH3), 55.12
(CH2), 61.04 (CH2), 157.44 (CH), 162.33 (C), 166.58
(C), 169.30 (C), 171.45; MS: m/e 212 (M+), 197, 181,
169, 141. Anal. calcd for C9H12N2O4: C, 50.94; H, 5.70;
N, 13.20. Found: C, 50.86; H, 5.70; N, 13.11.
2-Methylthio-6-[(20-hydroxy)ethan-10-yl]-4(3H)-pyrimidinone
(5b). 2-Methylthio-6-[(carboxyethyl)methyl]-4(3H)pyr-
imidinone (4b) (0.4 g, 1.75 mmol) and NaBH4 (0.13 g,
3.5 mmol) were dissolved in dry i-PrOH (100 mL) under
nitrogen atmosphere and the mixture was stirred at
25ꢀC for 48 h. The reaction mixture was treated with

M. Botta et al. / Bioorg. Med. Chem. 7 (1999) 1925±1931
1929
glacial acetic acid (0.1 mL) and successively evaporated
under reduced pressure. Flash-chromatography (CHCl3:
MeOH, 9.5:0.5) puri®cation of the crude gave 2-
methylthio-6-[(20-hydroxy)ethan-10-yl]-4(3H)pyrimidinone
(5b) (0.19 g, 57%). Mp 137±140ꢀC (MeOH/EtOAc), IR
(CH2Cl2) nmax 3200,1725, 1660 cm 1; 1H NMR (CDCl3)
(2.30 (3H, s, S-CH3), 2.48 (2H, t, J=6.6 Hz, CH2), 3.68
(2H, t, J=6.6 Hz, CH2), 5.80 (1H, s, CH); MS: m/e 186
(M+), 168, 143, 139, 115. Anal. calcd for C7H10N2SO2:
C, 45.15; H, 5.41; N, 15.04. Found: C, 45.20; H, 5.43; N,
15.05.
6e. (0.23 g, 91%), mp 89±91ꢀC (MeOH:EtOAc), IR
1
(CH2Cl2) nmax 3250, 1735, 1668 cm 1; H NMR (CD3
SOCD3) d 2.46 (3H, s, CH3), 2.72 (2H, t, J=6.4 Hz,
CH2), 2.88 (2H, t, J=6.4 Hz, CH2), 3.60 (4H, m, CH2),
3.91 (4H, m, CH2), 6.10 (1H, s, CH); MS: m/e 254
(M+), 226, 211, 207, 155. Anal. calcd for C11H18N4SO:
C, 51.94; H, 7.13; N, 22.03. Found: C, 51.96; H, 7.12; N,
22.0.
6-[(20-Diethylamino)ethan-13-yl]uracil (7a) and 2-thio-6-
[(20-diethylamino)ethan-10-yl]uracil (7b). General proce-
dure. Compound (6a) (or (6b)) (1 mmol) was dissolved
in H2SO4 (2 N water solution) and the reaction mixture
stirred at 25ꢀC for 48 h. The mixture was carefully neu-
tralized with NaHCO3 (saturated solution) and extrac-
ted with CHCl3. The organic layer was dried under
Na2SO4 and evaporated under reduced pressure. The
crude was crystallised from methanol to give (7a) (or
(7b)).
2-Methoxy-6-[(20 -diethylamino)ethan-10 -yl]-4(3H)-pyr-
imidinone (6a) and 2-methylthio-6-[(20-alkylamino)ethan-
10-yl]-4(3H)-pyrimidinones (6b±e). General procedure.
Compound (5a) (or (5b)) (1 mmol), p-toluenesulfonyl
chloride (1.1 equiv/mol), and 4-dimethylaminopyridine
(1.1 equiv/mol), were dissolved in dry CHCl3 (70 mL)
under nitrogen atmosphere and the reaction stirred at
25ꢀC for 24 h. The reaction mixture, washed with HCl
(2 N water solution) and NaCl (saturated solution), was
dried over Na2SO4 and succesively evaporated under
vacuum. The crude was dissolved in dry THF (20 mL)
in the presence of the appropriate amine (1.2 equiv/mol)
and the reaction mixture heated at 50ꢀC for 12 h. The
cooled solution was diluted with CHCl3 (100 mL),
washed with NaCl (saturated solution), dried over
Na2SO4 and succesively evaporated under reduced
pressure. Puri®cation of the crude by ¯ash chromato-
graphy (CHCl3:MeOH, 9.5:0.5) gave products (6a) and
(6b±e) in acceptable yields.
7a. (0.20 g, 96%), mp 188±190ꢀC (MeOH:EtOAc), IR
1
(CH2Cl2) nmax 3250, 1735, 1710, 1660 cm 1; H NMR
(CDCl3) d 1.13 (6H, m, CH3), 2.71 (2H, m, CH2), 3.45
(4H, m, CH2), 3.88 (2H, m, CH2), 5.83 (1H, s, CH); MS:
m/e 211 (M+), 196, 168, 140, 125. Anal. calcd for
C10H17N3O2: C, 56.85; H, 8.11; N, 19.89. Found: C,
56.93; H, 8.20; N, 19.90
7b. (0.21 g, 94%), mp 111±113ꢀC (MeOH:EtOAc), IR
1
(CH2Cl2) nmax 3250, 1735, 1660, 1580 cm 1; H NMR
(CDCl3) d 1.13 (6H, m, CH3), 2.69 (2H, m, CH2), 3.43
(4H, m, CH2), 3.88 (2H, m, CH2), 5.88 (1H, s, CH); MS:
m/e 227 (M+), 212, 184, 140, 141. Anal. calcd for
C10H17N3SO: C, 52.84; H, 7.54; N, 18.48. Found: C,
52.90; H, 7.80; N, 18.61.
6a. (0.12 g, 53%), oil, IR (CH2Cl2) nmax 1730, 1715,
1
1660 cm 1; H NMR (CDCl3) d 1.13 (6H, t, J=7.0 Hz,
CH3), 2.69 (2H, t, J=6.8 Hz, CH2), 3.43 (4H, m, CH2),
3.88 (2H, t, J=6.8 Hz, CH2), 4.01 (3H, s, O-CH3), 5.88
(1H, s, CH); MS: m/e 225 (M+), 210, 201, 182. Anal.
calcd for C11H19N3O2: C, 58.65; H, 8.50; N, 18.65.
Found: C, 58.70; H, 8.50; N, 18.70.
2-Methylsulfoxide-6-[(20-alkylamino)ethan-10-yl]-4(3H)-
pyrimidinone (8). 2-Methylthio-6-[(20-diethylamino)-
ethan-10-yl]-4(3H)pyrimidinones (6b) (0.13 g, 0.54 mmol)
and sodium periodate (NaIO4) (0.10 g, 0.54 mmol) were
dissolved in H2O (10 mL) and the reaction mixture was
stirred at 0ꢀC for 16 h. The mixture was ®ltered and
succesively extracted with CHCl3. The organic layer was
dried over Na2SO4 and evaporated under reduced pres-
sure. The crude was puri®ed by preparative TLC
6b. (0.15 g, 64%), oil, IR (CH2Cl2) nmax 1725,
1660 cm 1; 1H NMR (CDCl3) d 1.16 (6H, m, CH3), 2.43
(3H, s, CH3), 2.69 (2H, t, J=6.5 Hz, CH2), 3.45 (4H, m,
CH2), 3.87 (2H, t, J=6.5 Hz, CH2), 5.85 (1H, s, CH);
MS: m/e 241 (M+), 240, 226, 198, 194. Anal. calcd for
C11H19N3SO: C, 54.74; H, 7.93; N, 17.41. Found: C,
54.75; H, 7.93; N, 17.50.
(CHCl3:MeOH, 9.0:1.0) to give (8) (0.11 g, 76%). Oil,
1
IR (CH2Cl2) nmax 1725, 1660, 1060 cm
;
1H NMR
(CDCl3) d 1.19 (6H, m, CH3), 2.60 (3H, s, CH3), 2.71
(2H, t, J=6.5 Hz, CH2), 3.48 (4H, m, CH2), 3.89 (2H, t,
J=6.5 Hz, CH2), 5.90 (1H, s, CH); MS: m/e 257 (M+),
242, 214, 194, 171. Anal. calcd for C11H19N3SO2: C,
51.34; H, 7.44; N, 16.33. Found: C, 51.38; H, 7.44; N,
16.24.
6c. (0.2 g, 79%), mp 90±92ꢀC (EtOAc), IR (CH2Cl2)
1
nmax 1735, 1668 cm 1; H NMR (CDCl3) d 1.49±1.60
(6H, m, CH2), 2.41 (3H, s, CH3), 2.68 (2H, t, J=6.5 Hz,
CH2), 3.54 (4H, m, CH2), 3.87 (2H, t, J=6.4 Hz, CH2),
6.0 (1H, s, CH); MS: m/e 253 (M+), 225, 210, 206, 225,
155. Anal. calcd for C12H19N3SO: C, 56.89; H, 7.56; N,
16.58. Found: C, 56.90; H, 7.63; N, 16.50.
2-Methylthio-6-chloromethyl-4(3H)-pyrimidinone (10). 2-
Methyl-2-thiopseudourea sulfate (1 g, 3.6 mmol), ethyl
4-chloroacetoacetate (0.64 g, 3.9 mmol), and Ca(OH)2
(0.3 g, 4 mmol), were dissolved in H2O (6 mL) and
EtOH (18 mL), and stirred at 25ꢀC for 48 h. The reac-
tion mixture was neutralized with HCl (2 N water solu-
tion) and extracted with EtOAc. The extract, washed
with NaCl (saturated solution), was dried successively
over Na2SO4 and evaporated under reduced pressure.
6d. (0.18 g, 71%), mp 97±99ꢀC (MeOH:EtOAc), IR
(CH2Cl2) nmax 1740, 1668 cm 1; 1H NMR (CD3SOCD3)
d 2.48 (3H, s, CH3), 2.67 (2H, t, J=6.4 Hz, CH2), 2.79
(2H, t, J=6.4 Hz, CH2), 2.85 (4H, m, CH2), 3.63 (4H,
m, CH2), 5.99 (1H, s, CH); MS: m/e 255 (M+), 227, 212,
208, 155. Anal. calcd for C11H17N3SO2: C, 51.74; H,
6.71; N, 16.46. Found: C, 51.80; H, 7.70; N, 16.40.

1930
M. Botta et al. / Bioorg. Med. Chem. 7 (1999) 1925±1931
The crude was puri®ed by ¯ash chromatography
(CHCl3:MeOH, 9.0:1.0) to give (10) as the only recov-
ered product (0.56 g, 75%). Mp 155±157ꢀC (MeOH:
Plaque assay. Serial tenfold dilutions of RV, SV, and VSV
were inoculated on to con¯uent vero cell monolayers.
After a 1 h adsorption period at 37ꢀC, the inoculum was
removed and cells were washed three times with phos-
phate-bu€ered saline (PBS) before being overlaid with
MEM containing 0.4% (w/v) agar (Oxoid). After two
days (SNV, VSV) or ®ve days (RV) incubation at 37ꢀC,
plaques were stained with 0.1% crystal violet solution.
1
EtOAc), IR (CH2Cl2) nmax 1725, 1661 cm 1; H NMR
(CDCl3) d 2.55 (3H, s, CH3), 4.33 (2H, s, CH2), 6.40
(1H, s, CH); MS: m/e 190 (M+), 175, 162, 147, 143.
Anal. calcd for C6H7ClN2SO: C, 37.80; H, 3.70; N,
14.69. Found: C, 37.85; H, 3.70; N, 14.75.
2-Methylthio-6-[(10-alkylamino)methan-10-yl]-4(3H)-pyr-
imidinones (11a,b). General procedure. Compound 10
(1 mmol) and the appropriate amine (diethyl amine or
morpholine) (1 equiv/mol) were dissolved in dry THF
(20 mL) and the reaction mixture heated at 90ꢀC for
12 h. The cooled solution was diluted with CHCl3
(100 mL), washed with NaCl (saturated solution), dried
over Na2SO4 and successively evaporated under reduced
pressure. Puri®cation of the crude by ¯ash chromato-
graphy (CHCl3:MeOH, 9.2:0.8) gave products (11a,b) in
acceptable yields.
Antiviral assay. For antiviral assays, con¯uent mono-
layers of vero cells grown in 24-well plates were inocu-
lated with SNV and VSV (10 or 0.1 PFU/cell) and with
RV (3 or 0.1 PFU/cell). After virus adsorption (1 h,
37ꢀC), the viral inoculum was removed. Cell monolayers
were washed three times with PBS and incubated with
maintenance medium in the presence or absence of the
compounds. Virus yield was evaluated by plaque assay
after a single cycle of virus multiplication at high multi-
plicity infection (SNV and VSV, 24 h; RV, 96 h).
Acknowledgements
11a. (0.14 g, 63%), oil, IR (CH2Cl2) nmax 1732,
1
1655 cm 1; H NMR (CDCl3) d 1.0 (6H, t, J=7.2 Hz,
Supported by Ministero Sanita, Istituto Superiore Sanita,
Progetto AIDS 1997. Financial support from Italian
MURST is acknowledged. One of us (M.B.) wishes to
express thanks for a grant from the Merck Research
Laboratories (Academic Development Program in
Chemistry). Thanks are due to Professor S. Spadari,
Istituto di Genetica Biochimica ed Evoluzionistica del
CNR, Pavia, Italy, for the HIV-1 RT assays of some of
the synthesized compounds.
CH3), 2.52 (7H, m, CH3 and CH2), 3.42 (2H, s, CH2),
6.41 (1H, s, CH); MS: m/e 227 (M+), 212, 199, 184, 141.
Anal. calcd for C10H17N3SO: C, 52.83; H, 7.53; N,
18.58. Found: C, 52.90; H, 7.53; N, 18.65.
11b. (0.14 g, 59%), mp 95±97ꢀC (MeOH:EtOAc), IR
1
(CH2Cl2) nmax 1745, 1660 cm 1; H NMR (CDCl3) d
2.48 (3H, s, CH3), 2.67 (2H, t, J=6.8 Hz, CH2), 2.79
(2H, t, J=6.8 Hz, CH2), 2.85 (4H, m, CH2), 3.65 (4H,
m, CH2), 5.99 (1H, s, CH); MS: m/e 241 (M+), 226, 198,
194. Anal. calcd for C10H15N3SO2: C, 49.77; H, 6.26; N,
17.41. Found: C, 49.80; H, 6.26; N, 17.50.
References
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Tetrahedron: Asymmetry 1990, 1, 441.
Cells. Vero cells were cultured at 37ꢀC in a 5% CO2
atmosphere in Eagle's minimum essential medium
(MEM) containing 1.2 mg/mL NaHCO3 and supple-
mented with 6% (v/v) fetal bovine serum (FBS), 2 mM
glutamine, 100 IU/mL penicillin and 100 mg/mL strep-
tomycin. For cell maintenance the serum concentration
was lowered to 2% (v/v).
Viruses. RV (Therien strain), SNV (AR 339 strain) and
VSV (Indiana strain) were grown in vero cells in Eagle's
MEM supplemented with 2% FBS. Semicon¯uent
monolayers were inoculated with viruses at a multi-
plicity of infection of 0.1 PFU/cell and incubated at
37ꢀC for 24 h (SNV, VSV) or 72 h (RV). After infection,
supernatants were collected, centrifuged at 1000 g for
10 min to remove cellular debris, and then stored in
small aliquots at 80ꢀC.
Cytotoxicity assays. Cytotoxicity was evaluated by
incubating twofold serial dilutions of the compounds in
maintenance medium with con¯uent vero cell mono-
layers grown in 96-well plates (Falcon). Cell morphol-
ogy, viability and yield were examined after 48 h
incubation at 37ꢀC. Cell viability was assessed on the
basis of vital dye exclusion test, using Trypan Blue, and
cell yield determined by counting cells with an hemo-
cytometer trypsinization.
11. Saladino, R.; Danti, M. C.; Mincione, E.; Crestini, C.;
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M. Botta et al. / Bioorg. Med. Chem. 7 (1999) 1925±1931
1931
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of the corresponding crude. Tosylate from 5a: 1H NMR (CDCl3)
d 2.43 (3H, s, CH3), 2.88 (2H, t, J=7.0 Hz, CH2), 3.95 (2H, t, J=
7.0 Hz, CH2), 4.01 (3H, s, CH3), 6.58 (1H, s, CH), 7.33 (2H, d,
J=9.0 Hz, CH), 7.93 (2H, d, J= 9.0 Hz, CH). Tosylate from 5b:
1H NMR (CDCl3) d 2.32 (3H, s, CH3), 2.43 (3H, s, CH3), 2.88
(2H, t, J=7.1 Hz, CH2), 3.95 (2H, t, J=7.1 Hz, CH2), 6.58 (1H,
s, CH), 7.33 (2H, d, J=9.0 Hz, CH), 7.90 (2H, d, J=9.0 Hz, CH).
14. Terly, K. F. Advances in Virus Res. 1994, 44, 69.
15. Hayakawa, Y.; Hirose, M.; Noyori, R. J. Org. Chem. 1993,
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1
16. H NMR spectra of the tosylates produced from 5a and 5b
were obtained by analytical TLC puri®cation of a small amount