Synthesis and anti-HIV-1 evaluation of 1,5-disubstituted pyrimidine-2,4-diones

Article abstract of DOI:10.1002/jhet.5570450434

(Chemical Equation Presented) 1,5-Disubstituted pyrimidine-2,4-diones were synthesized by fusion of 5-bromouracil with an aniline derivative followed by coupling with benzyl halide whereas the opposite synthesis sequence failed. Also 4,6-dichloro-N,N,5-tr

Full text of DOI:10.1002/jhet.5570450434

Jul-Aug 2008
1161
Synthesis and Anti-HIV-1 Evaluation of 1,5-Disubstituted
Pyrimidine-2,4-diones
Yasser M. Loksha, Daniel Globisch and Erik B. Pedersen*
Nucleic Acid Center†, Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55,
DK-5230 Odense M, Denmark
Paolo La Colla, Gabriella Collu and Roberta Loddo
Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e
Biotecnologie Microbiche, Universitá di Cagliari, Cittadella Universitaria, 09042 Monserrato, Italy
Received December 19, 2007
O
N
O
O
N
Br
NH
HN
O
N
H
CN
1,5-Disubstituted pyrimidine-2,4-diones were synthesized by fusion of 5-bromouracil with an aniline
derivative followed by coupling with benzyl halide whereas the opposite synthesis sequence failed. Also
4,6-dichloro-N,N,5-trimethylpyrimidin-2-amine was treated with phenols. The products were tested for
their activity against HIV-1.
J. Heterocyclic Chem., 45, 1161 (2008).
INTRODUCTION
Nair et al. reported that bis-benzylated nucleobase with a
ꢀ-diketo acids are potent HIV integrase inhibitors [12].
In the present work, we found it of interest to synthesize
new analogues of 1,5-disubstituted pyrimidine-2,4-diones
such as target compound 10b with structural resemblances
to the pyrazinone skeleton (Figure 1) for biological
evaluation against HIV-1. Also 1,3-disubstituted and 4,6-
disubstituted pyrimidine derivatives were considered
interesting.
The discovery of novel anti-HIV-1 agents with long-
lasting suppression of viral replication is one of the most
important goals in the field of AIDS research. In addition
to nucleoside analogues, a number of different chemical
classes of compounds have been described that show
potent and selective inhibition of HIV-1 replication by
acting as non-nucleoside reverse transcriptase inhibitors
(NNRTIs). There are at the present time 21 FDA
approved anti-HIV drugs for therapeutic use. Among
these, 11 compounds are RT inhibitors comprising seven
nucleosides (NRTIs), one nucleotide (NtRTI), three non-
nucleoside RT inhibitors (NNRTIs), eight protease
inhibitors, one viral fusion inhibitor, enfuvirtide
(Fuzeon®) [1-4] and one entry inhibitor, Selzentry®, FDA
approved August 2007. The three NNRTIs, nevirapine
(Viramune®) [5,6], delavirdine (Rescriptor®) [7] and
efavirenz (Sustiva®) [8,9] were approved for the treatment
of HIV infection in 1996, 1997 and 1998, respectively.
They became cornerstones of HIV therapy because of
their full potential as a component of the highly-active
anti-retroviral therapy (HAART) [10]. However, one of
the major drawbacks in using NNRTIs in HIV therapy is
due to the rapidly introduction of RT mutations.
Especially the loss of activity against the Y181C and
K103N mutated HIV-1 strains is problematic.
O
N
N
O
N
O
N
HN
O
NH
CN
Pyrazinone anaolgue
CN
10b
Figure 1
RESULTS AND DISCUSSION
Commercially available 5-bromouracil (1) was
alkylated with 3-cyanobenzyl bromide in acetonitrile
using 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) as a
sterically hindered base. Two products were isolated from
the reaction by column chromatography. The major
product is the desired N1-monoalkylated 3-[(5-bromo-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl]benzo-
Recently, Das et al. [11] reported that N-methylated
pyrazinone analogues (Figure 1) showed activity against
both wild-type and NNRTI- and NRTI-resistant virus.

1162
Y. Loksha, D. Globisch, E. Pedersen, P. La Colla, G. Collu and R. Loddo
Vol 45
nitrile (2) and the other one is the N1,N3-bislakylated
product 3 as the minor product. Although compound 2 is
the major product, the yield is low (40%). To increase the
yield of the mono-alkylated product we introduced a
silylation reaction prior to the alkylation step [13].
5-Bromouracil (1) was silylated by N,O-bis(trimethylsil-
yl)acetamide (BSA) followed by refluxing with 2,4,6-tri-
methylbenzyl chloride and only 5-bromo-1-(mesityl-
methyl)pyrimidine-2,4(1H,3H)-dione (4) was isolated as
the sole product without using column chromatography.
Compound 4 was methylated by methyl iodide to afford
5-bromo-1-(mesitylmethyl)-3-methylpyrimidine-2,4(1H,
3H)-dione (5). However, fusion of 5 with 4-cyanophenol
or 4-cyanoaniline could not be acomplished to afford the
desired compounds 6 and 7, respectively (Scheme I).
was irradiated. Also on irradiation of CH3-N in compound
10b at 3.47 ppm, no NOE effect was detected with any
proton while H6 at 5.72 ppm showed 6.5 % NOE when
N-CH2 at 4.82 ppm was irradiated.
Scheme II
NH2
H
CN
O
N
O
Br
HN
NH
1
200o
neat
BSA, CH3CN, reflux
8
H
N
O
N
O
O
O
N
N
CH3I, DBU
CH3CN, rt
NH
NH
Scheme I
NC
Br
NC
H
Br
O
N
O
O
a, 3-CN
b, 4-CN
O
9a,b
10a,b
N
N
N
Br
Br
HN
CN
DBU, CH3CN
O
+
O
N
H
With the 6-(3,5-dimethylbenzyl)-5-ethyl-2,3-dihydro-2-
thioxopyrimidin-4(1H)-one (11) and 4,6-dichloro-N,N-
dimethylpyrimidin-2-amine (15) in our hands from a
previous HIV project we find it of interest to expand the
pyrimidine series using these compounds.
CN
CN
CN
1
2
3
H
N
O
O
O
N
O
Cl
N
Methylation of 6-(3,5-dimethylbenzyl)-5-ethyl-2,3-
dihydro-2-thioxopyrimidin-4(1H)-one (11) [14] with
methyl iodide gave the S-methylated derivative 12 which
by treatment with 3-cyanobenzyl bromide afforded the O-
alkylated derivative (13) and the N3-alkylated derivative
(14) (Scheme III). The 13C nmr spectrum of compound 13
showed the existence of CH2-O at 65.96 ppm and of
compound 14 CH2-N at 46.81 ppm.
N
Br
Br
CH3I, DBU
CH3CN, rt
1
BSA, CH3CN, reflux
4
5
XH
O
X
N
O
N
CN
Scheme III
5
pyridine, reflux
O
or fusion
X = O, NH
O
CN
6: X = O
7: X = NH
HN
MeI
HN
KOH/MeOH
S
N
S
N
H
Instead, 5-bromouracil was fused with 2,4,6-trimethyl-
11
12
aniline at 200° to afford 5-(mesitylamino)-pyrimidine-
2,4(1H,3H)-dione (8) which was silylated with BSA
followed by refluxing with 3-cyano or 4-cyano benzyl
bromides to furnish 3- or 4-{[5-(mesitylamino)-2,4-dioxo-
Br
DBU, CH3CN
CN
CN
3,4-dihydropyrimidin-1(2H)-yl]methyl}
benzo-nitrile
(9a,b). Compounds 9a,b were methylated with methyl
iodide to afford the 3- or 4-{[5-(mesitylamino)-3-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]methyl}benzo-
nitrile 10a,b (Scheme II).
On irradiation of CH3-N in compound 10a at 3.47 ppm,
no NOE effect was detected with any proton while H6 at
5.73 ppm showed 6.5 % NOE when N-CH2 at 4.80 ppm
CN
O
O
N
N
+
S
S
N
N
14
13

Jul-Aug 2008
Synthesis and Anti-HIV-1 Evaluation of 1,5-Disubstituted Pyrimidine-2,4-diones
1163
The 13C nmr spectrum of compound 14 clearly showed
alkylation on N3 and not on the N1 nitrogen atom when
the characteristic chemical shift (157.52 (C6), 159.14
(C2), and 162.74 (C4)) are compared with the
corresponding values of 2,3-dihydro-7H-thiazolo[3,2-a]-
pyrimidin-7-one (144.4, 165.5, and 169.2 corresponding
to C6, C4, and C2, respectively) for the N1-alkylated
derivative versus 2,3-dihydro-5H-thiazolo[3,2-a]pyrimid-
in-5-one (160.3, 161.2, and 161.8 corresponding to C6,
C4, and C2, respectively) for the N3-alkylated derivative
[15].
4-(3,5-Dimethylphenoxy)-6-chloro-N,N,5-trimethyl-py-
rimidin-2-amine (16) was prepared by coupling of 4,6-di-
chloro-N,N,5-trimethylpyrimidin-2-amine (15) [16] with
the sodium salt of 3,5-dimethylphenol in dimethylform-
amide. Refluxing of compound 16 with sodium
methoxide in methanol afforded the methoxy derivative
17. Heating of compound 16 with the sodium salt of
4-cyanophenol in dimethylformamide at 120° furnished
4-(2-(dimethylamino)-6-(3,5-dimethyl-phenoxy)-5-meth-
ylpyrimidin-4-yloxy)benzonitrile (18) (Scheme IV).
Finnigan MAT SSQ 710. MALDI spectra were recorded on a
4.7 T Ultima Fourier transform Mass spectrometer (IonSpec,
Irvine, CA). Melting points were determined in a Büchi melting
point apparatus. The silica gel (0.040–0.063 mm) used for
column chromatography was purchased from Merck.
Microanalyses were carried out at Chemical Laboratory II at
University of Copenhagen, Denmark.
Synthesis of compounds 2 and 3. DBU (1.8 mL, 12 mmole)
was added to a stirred solution of 5-bromouracil (1.9 g, 10
mmole) in dry acetonitrile (30 mL). 3-Cyanobenzyl bromide
(1.96 g, 10 mmole) was added to the reaction mixture and stirred
for 12 hours, acetic acid (1 mL) was added to the reaction
mixture and the solvents were removed under reduced pressure.
Methylene chloride (50 mL) was added to the residual material
and the solid product formed was collected by filtration, washed
with methylene chloride followed by water and dried to afford
compound 2. The two layers of the filtrate were separated and
the organic layer was evaporated under reduced pressure, ether
(15 mL) was added to the residual material and stirred for 15
min. The solid product formed was collected by filtration and
dried to afford compound 3.
3-[(5-Bromo-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-
1
methyl]benzonitrile (2). Yield 1.2 g (40%); mp 238-240°; H
nmr (DMSO-d6) ꢀ: 4.93 (s, 2H, CH2Ar), 7.58 (t, 1H, J = 7.8 Hz,
Harom), 7.68 (d, 1H, J = 7.8 Hz, Harom), 7.79 (d, 1H, J = 7.8 Hz,
Harom), 7.85 (s, 1H, Harom), 8.38 (s, 1H, H6), 11.84 ppm (s, 1H,
NH); 13C nmr (DMSO-d6) ꢀ: 50.22 (CH2Ar), 95.44 (C5), 118.54
(CN), 111.45, 129.72, 131.09, 131.43, 132.38, 138.01, (Carom),
142.13 (C6), 150.36 (C2), 159.62 ppm (C4); EI MS: m/z 305
(17%, M+, 79Br), 307 (18%, M+, 81Br). Anal. Calcd. for
C12H8BrN3O2 (306.11): C, 47.08; H, 2.63; N, 13.73. Found: C,
47.84; H, 2.54; N, 13.46.
Scheme IV
Cl
N
Cl
N
OH
N
O
NaH,DMF
rt
N
3-{[5-Bromo-3-(3-cyanobenzyl)-2,4-dioxo-3,4-dihydropyr-
imidine-1(2H)-yl]methyl}benzonitrile (3). Yield 600 mg
(14%); mp 133-135°; 1H nmr (CDCl3) ꢀ: 4.98 (s, 2H, CH2), 5.17
(s, 2H, CH2), 7.40-7.76 ppm (m, 8H, Harom); 13C nmr (CDCl3) ꢀ:
45.18 (CH2), 52.22 (CH2), 97.06 (C5), 117.99 (CN), 118.49
(CN), 112.55, 113.34, 129.34, 130.10, 131.46, 131.66, 132.32,
132.40, 132.75, 133.85, 136.18, 137.24 (Carom), 141.49 (C6),
150.71 (C2), 158.82 ppm (C4); EI MS: m/z 420 (37%, M+, 79Br),
422 (34%, M+, 81Br). Anal. Calcd. for C20H13BrN4O2 (421.25): C,
57.02; H, 3.11; N, 13.30. Found: C, 57.28; H, 2.99; N, 13.26.
Synthesis of 5-bromo-1-(mesitylmethyl)pyrimidine-
2,4(1H,3H)-dione (4). Compound 1 (1.9 g, 10 mmole) in dry
acetonitrile (40 mL) was silylated under nitrogen by dropwise
addition of N,O-bis(trimethylsilyl)acetamide (BSA) (8 mL, 33
mmole) and stirring for 15 min. 2,4,6-Trimethylbenzyl chloride
(2.2 g, 13 mmole) was added in one portion to the reaction
mixture and refluxed for 20 hours. The reaction mixture was
cooled to room temperature and the solvent was removed under
reduced pressure, water (50 mL) was added to the residual
material and the solid product formed was collected by filtration,
washed with water and dried to afford compound 4. Yield 3.0 g
(93%); mp 212-214º; 1H nmr (DMSO-d6) ꢀ: 2.23 (s, 6H,
(CH3)2Ar), 2.30 (s, 3H, CH3Ar), 4.84 (s, 2H, CH2Ar), 6.92 (s,
2H, Harom), 7.24 (s, 1H, H6), 11.84 ppm (s, 1H, NH); 13C nmr
(DMSO-d6) ꢀ: 19.37 (2 ꢀ CH3), 20.48 (CH3), 44.99 (CH2), 94.85
(C5), 127.60, 128.35, 129.26, 137.89 (Carom), 142.24 (C6),
150.36 (C2), 159.28 ppm (C4); EI MS: m/z 322 (4%, M+, 79Br),
324 (3%, M+, 81Br).
+
N
N
Cl
15
16
CN
NaOMe,
MeOH, reflux
HO
N
NaH, DMF,
120o
OMe
N
N
N
N
O
O
N
O
CN
18
17
Antiviral activity. All compounds were evaluated for
their activity against HIV-1 wild-type and its NNRTI-
resistant mutants. Only compound 12 showed moderate
activity against HIV-1 wild-type (EC50 = 2 μM and CC50
>100 μM). All other compounds were inactive at the
maximum concentration tested (100 μM).
EXPERIMENTAL
Nmr Spectra were recorded on a Varian Gemini 2000
1
spectrometer at 300 MHz for H and 75 MHz for 13C with TMS
as an internal standard. EI mass spectra were recorded on a
Synthesis of 5-bromo-1-(mesitylmethyl)-3-methylpyrimid-
ine-2,4(1H,3H)-dione (5). Compound 4 (2.26 g, 7 mmole) was

1164
Y. Loksha, D. Globisch, E. Pedersen, P. La Colla, G. Collu and R. Loddo
Vol 45
added to a stirred solution of potassium hydroxide (0.5 g, 9
mmole) in methanol (30 mL). The solution was stirred for 15
min., then methyl iodide (0.53 mL, 8.4 mmole) was added and
the reaction mixture was stirred for 2 hours. Water (60 mL) was
added to the reaction mixture and the solid product formed was
collected by filtration, washed with water and dried to afford
Harom), 11.64 ppm (s, 1H, NH); 13C nmr (DMSO-d6) ꢀ: 17.50 (2
ꢀ CH3), 20.39 (CH3), 49.48 (CH2), 110.00, 122.92, 127.70,
128.91, 132.39, 134.36, 134.65, 143.12 (Carom), 134.85 (C5),
148.99 (C2), 160.46 (C4); HRms (MALDI, peak matching): m/z
calcd. for C21H21N4O2 (MH+) 361.1651, found 361.1665.
Synthesis of compound 10a,b. Compound 9a,b (0.36 g, 1
mmole) was added to a solution of DBU (0.2 mL, 1.3 mmole) in
dry acetonitrile (10 mL) and stirred for 15 min., then methyl
iodide was added to the reaction mixture at room temperature
and stirred for 3 hours. The solvents were removed under
reduced pressure, water (15 mL) was added to the residual
material and the solid product formed was collected by filtration,
washed with water, and dried to give compounds 10a,b.
3-{[5-(Mesitylamino)-3-methyl-2,4-dioxo-3,4-dihydro-
pyrimidine-1(2H)-yl]methyl}benzonitrile (10a). Yield 320 mg
(86%); mp 124-126°; 1H nmr (CDCl3) ꢀ: 2.11 (s, 6H, (CH3)2Ar),
2.28 (s, 3H, CH3Ar), 3.47 (s, 3H, N-CH3); 4.80 (s, 2H, NCH2),
5.40 (s, 1H, NH), 5.73 (s, 1H, H6), 6.91 (s, 2H, Harom), 7.44-7.61
(m, 4H, Harom); 13C nmr (CDCl3) ꢀ: 17.75 ((CH3)2Ar), 20.81
(CH3Ar), 28.48 (NCH3), 51.56 (NCH2), 113.82 (C6), 118.20
(CN), 130.89 (C5), 112.95, 122.93, 129.45, 129.66, 131.67,
131.90, 133.78, 134.85, 136.04, 137.60 (Carom), 149.68 (C2),
160.21 (C4); HRms (MALDI, peak matching): m/z calcd. for
C22H21N4O2 (MH+) 375.1821, found 375.1785. Anal. Calcd. for
C22H22N4O2·0.9 H2O (390.66): C, 67.64; H, 6.14; N, 14.34.
Found: C, 67.62; H, 5.95; N, 14.37.
4-{[5-(Mesitylamino)-3-methyl-2,4-dioxo-3,4-dihydropyr-
imidine-1(2H)-yl]methyl}benzonitrile (10b). Yield 230 mg
(61%); mp 140-142°; 1H nmr (CDCl3) ꢀ: 2.10 (s, 6H, (CH3)2Ar),
2.28 (s, 3H, CH3Ar), 3.47 (s, 3H, NCH3), 4.82 (s, 2H, NCH2),
5.40 (s, 1H, NH), 5.72 (s, 1H, H6), 6.90 (s, 2H, Harom), 7.28 (d,
2H, J = 8.7 Hz, Harom), 7.62 (d, 2H, J = 8.7 Hz, Harom); 13C nmr
(CDCl3) ꢀ: 17.79 (2 ꢀ CH3), 20.83 (CH3), 28.52 (NCH3), 51.93
(CH2), 113.92 (C6), 118.33 (CN), 122.94 (C5), 111.98, 128.01,
129.45, 132.59, 133.82, 134.90, 136.05, 141.30 (Carom), 149.71
(C2), 160.25 (C4); HRms (MALDI, peak matching): m/z calcd.
for C22H23N4O2 (MH+) 375.1821, found 375.1772. Anal. Calcd.
for C22H22N4O2 (1162.3): C, 69.23; H, 5.46; N, 15.67. Found: C,
69.53; H, 5.85; N, 14.57.
1
compound 5. Yield 1.9 g (81%); mp 176-178º; H nmr (CDCl3)
ꢀ: 2.24 (s, 6H, (CH3)2Ar), 2.32 (s, 3H, CH3Ar), 3.46 (s, 3H,
NCH3), 4.94 (s, 2H, N-CH2), 6.93 (s, 1H, H6), 6.96 (s, 2H,
Harom); 13C nmr (CDCl3) ꢀ: 19.68 ((CH3)2Ar), 21.01 (CH3Ar),
29.27 (NCH3), 46.21 (N-CH2), 95.95 (C5), 125.68, 129.94,
138.25, 138.53 (Carom), 139.47 (C6), 151.28 (C2), 159.16 (C4);
HRms (MALDI, peak matching): m/z calcd. for
C15H17BrNaN2O2 (MNa+) 359.0366, found 359.0375. Anal.
Calcd. for C15H17BrN2O2 (337.21): C, 53.43; H, 5.08; N, 8.31.
Found: C, 53.69; H, 4.94; N, 8.25.
5-(Mesitylamino)pyrimidine-2,4(1H,3H)-dione (8). Under a
stream of nitrogen, compound 1 (1.9 g, 10 mmole) was fused
with 2,4,6-trimethylaniline (1.48 g, 11 mmole) at 200° for 3
hours. The reaction mixture was left to reach 70°, then ethanol
(10 mL) was added and the mixture was stirred with a spatula
until getting a suspension. The solid product formed was
collected by filtration, dried and chromatographed on a column
of silica gel using CH2Cl2:EtOAc (1:1, v/v) as an eluent to give
compound 12.
Yield 910 mg (37%); mp > 250°; 1H nmr (DMSO-d6) ꢀ: 2.08
(s, 6H, (CH3)2Ar), 2.21 (s, 3H, CH3Ar), 5.54 (s, 1H, NH), 5.89
(s, 1H, H6), 6.89 (s, 2H, Harom), 10.00 (bs, 1H, NH), 11.27 (bs,
1H, NH); 13C nmr (DMSO-d6) ꢀ: 17.46 (2 ꢀ CH3), 20.44 (CH3),
113.05 (C6), 122.03, 128.94, 134.81, 135.50 (Carom), 134.50
(C5), 149.43 (C2), 161.05 (C4); HRms (MALDI, peak
matching): m/z calcd. for C13H15NaN3O2 (MNa+) 268.1057,
found 268.1057.
Synthesis of compound 9a,b. Compound 8 (300 mg, 1.2
mmole) in dry acetonitrile (15 mL) was silylated under nitrogen
by dropwise addition of N,O-bis(trimethylsilyl)acetamide (BSA)
(1.3 mL, 5.3 mmole) and stirring for 15 min. 3-Cyano or 4-
cyanobenzyl bromide (1.6 mmole) was added in one portion to
the reaction mixture and refluxed for 14 hours. The reaction
mixture was cooled to room temperature and the solvent was
removed under reduced pressure, water (20 mL) was added to
the residual material and the solid product formed was collected
by filtration, washed with water and dried to afford compounds
9a,b.
6-(3,5-Dimethylbenzyl)-5-ethyl-2-(methylthio)-pyrimidine-
4(1H)-one (12). Compound 11 (1.1 g, 4 mmole) was added to a
stirred solution of potassium hydroxide (0.25 g, 4.4 mmole) in
methanol (15 mL). The solution was stirred for 15 min., then
methyl iodide (0.27 mL, 4.4 mmole) was added and the reaction
mixture was stirred for 1 hour. Water (30 mL) was added to the
reaction mixture and the solid product formed was collected by
filtration, washed with water and dried to afford compound 12.
3-{[5-(Mesitylamino)-2,4-dioxo-3,4-dihydropyrimidine-
1(2H)-yl]methyl}benzonitrile (9a). Yield 350 mg (81%); mp
184-186°; 1H nmr (DMSO-d6) ꢀ: 2.05 (s, 6H, (CH3)2Ar), 2.20 (s,
3H, CH3Ar), 4.82 (s, 2H, CH2), 5.94 (s, 1H, H6), 6.10 (bs, 1H,
NH), 6.86 (s, 2H, Harom), 7.50-7.57 (m, 2H, Harom), 7.62 (s, 1H,
Harom), 7.74 (dt, 1H, J = 7.5, 1.5 Hz, Harom), 11.51 (s, 1H, NH);
13C nmr (DMSO-d6) ꢀ: 17.46 (2 ꢀ CH3), 20.40 (CH3), 48.93
(CH2), 116.15 (C6), 118.48 (CN), 122.96 (C5), 111.34, 128.92,
129.75, 130.65, 131.10, 131.94, 134.35, 134.62, 134.89, 138.98
(Carom), 149.00 (C2), 160.46 (C4); HRms (MALDI, peak
matching): m/z calcd. for C21H21N4O2 (MH+) 361.1665, found
361.1649. Anal. Calcd. for C21H20N4O2 (360.41): C, 69.98; H,
5.59; N, 15.55. Found: C, 70.11; H, 5.61; N, 15.36.
1
Yield 750 mg (65%); mp 213-215°; H nmr (CDCl3) ꢀ: 1.09 (t,
3H, J = 7.5 Hz, CH3CH2), 2.27 (s, 6H, (CH3)2Ar), 2.50 (s, 3H,
CH3S), 2.58 (q, 2H, J = 7.5 Hz, CH3CH2), 3.84 (s, 2H, CH2Ar);
13C nmr (CDCl3) ꢀ: 13.17 (CH3CH2), 18.80 (CH3CH2), 21.28
(CH3S), 40.32 (CH2Ar), 122.08 (C5), 126.77, 127.99, 137.77,
138.04 (Carom), 156.85 (C2), 161.81 (C4), 165.03 (165.03); EI
MS: m/z 288 (100%, M+). Anal. Calcd.. for C16H20N2OS
(288.41): C, 66.63; H, 6.99; N, 9.71. Found: C, 66.26; H, 6.91;
N, 9.59.
Synthesis of compounds 13 and 14. Compound 12 (0.29 g, 1
mmole) was added to a stirred solution of DBU (0.2 mL, 1.3
mmole) in dry acetonitrile (10 mL) and stirred for 15 min., then
3-cyanobenzyl bromide (0.22 g, 1.1 mmole) was added to the
reaction mixture at room temperature, stirred for 3 hours and
then refluxed for 5 hours. The solvent was removed under
4-{[5-(Mesitylamino)-2,4-dioxo-3,4-dihydropyrimidine-
1(2H)-yl]methyl}benzonitrile (9b). Yield 300 mg (70%); mp
202-204°; 1H nmr (DMSO-d6) ꢀ: 2.05 (s, 6H, (CH3)2Ar), 2.20 (s,
3H, CH3Ar), 4.86 (s, 2H, CH2), 5.93 (s, 1H, H6), 6.86 (s, 2H,
Harom), 7.32 (d, 2H, J = 8.3 Hz, Harom), 7.79 (d, 2H, J = 8.3 Hz,

Jul-Aug 2008
Synthesis and Anti-HIV-1 Evaluation of 1,5-Disubstituted Pyrimidine-2,4-diones
1165
reduced pressure, water (15 mL) was added to the residual
material and the solid product formed was collected by filtration,
dried and chromatographed on a column of silica gel using
petroleum ether:EtOAc (11:1, v/v) as an eluent to give
compounds 13 and 14.
3-{[6-(3,5-Dimethylbenzyl)-5-ethyl-2-(methylthio)-pyrim-
idine-4-yloxy]methyl}benzonitrile (13). Yield 73 mg (18 %);
mp 95-97°; 1H nmr (CDCl3) ꢀ: 0.94 (t, 3H, J = 7.4 Hz, CH3CH2),
2.18 (s, 6H, (CH3)2Ar), 2.43 (s, 3H, CH3S), 2.52 (q, 2H, J = 7.4
Hz, CH3CH2), 3.88 (s, 2H, CH2Ar), 5.38 (s, 2H, CH2O), 6.74,
6.77 (2 ꢀ s, 3H, Harom), 7.41-7.63 ppm (m, 4H, Harom); 13C nmr
(CDCl3) ꢀ: 12.77 (CH3CH2), 13.45 (CH3CH2), 17.60 (CH3S),
20.63 ((CH3)2Ar), 39.45 (CH2Ar), 65.96 (CH2O), 115.80 (CN),
117.98 (C5), 111.83, 125.94, 127.39, 128.85, 130.32, 130.91,
131.37, 131.12, 137.36, 137.84 (Carom), 165.89 (C4), 166.00
(C2), 166.69 (C6); HRms (MALDI, peak matching): m/z calcd.
for C24H25NaN3OS (MNa+) 426.1611, found 426.1612.
3-{[4-(3,5-Dimethylbenzyl)-5-ethyl-2-(methylthio)-6-oxopy-
rimidine-1(6H)-yl]methyl}benzonitrile (14). Yield 80 mg
(20%); mp 128-130°; 1H nmr (CDCl3) ꢀ: 1.04 (t, 3H, J = 7.4 Hz,
CH3CH2), 2.21 (s, 6H, (CH3)2Ar), 2.41 (s, 3H, CH3S), 2.56 (q, 2H,
J = 7.4 Hz, CH3CH2), 3.75 (s, 2H, CH2Ar), 5.14 (s, 2H, CH2N),
6.78, 6.85 (2 ꢀ s, 3H, Harom), 7.31-7.56 ppm (m, 4H, Harom); 13C
nmr (CDCl3) ꢀ: 13.16 (CH3CH2), 14.98 (CH3CH2), 19.52 (CH3S),
21.26 ((CH3)2Ar), 40.15 (CH2Ar), 46.81 (CH2N), 118.55 (CN),
121.258 (C5), 112.62, 127.04, 128.06, 129.32, 131.41, 131.53,
132.60, 136.95, 137.74, 137.86 (Carom), 157.52 (C6), 159.14 (C2),
162.74 (C4); HRms (MALDI, peak matching): m/z calcd. for
C24H25NaN3OS (MH+) 426.1611, found 426.1597.
6H, (CH3)2N)), 3.92 (s, 3H, OCH3), 6.75 (s, 3H, Harom); 13C nmr
(CDCl3): ꢀ 6.96 (CH3), 21.23 ((CH3)2Ar), 36.47 ((CH3)2N),
53.41 (OCH3), 88.48 (C5), 118.65, 125.38, 138.45, 154.10
(Carom), 159.21 (C2), 167.69 (C4), 169.52 (C6), HRms (MALDI,
peak matching): m/z calcd. for C16H22N3O2 (MH+) 288.1707,
found 288.1713.
4-(2-(Dimethylamino)-6-(3,5-dimethylphenoxy)-5-methyl-
pyrimidine-4-yloxy)benzonitrile (18). To
a
solution of
compound 16 (0.29 g, 1 mmole) and 4-hydroxybenzonitrile
(0.13 g, 1.1 mmole) in dry dimethylformamide (9 mL) was
added sodium hydride (55% suspension in paraffin oil, 0.052 g,
1.2 mmole) portionwise. After refluxing at 120° for 70 hours,
the reaction mixture was quenched by dropwise addition of
water (2 mL). After addition of water (35 mL) and ether (25
mL), the two layers were separated and the water layer was
extracted two times with ether (25 mL). The ether extracts were
collected, dried over magnesium sulfate, filtered and evaporated
under reduced pressure. The residual material was
chromatographed on a silica gel column using petroleum
ether/ether (5:1, v/v) as an eluent to afford compound 18. Yield:
0.11 g (29%) as a white solid; mp 105-107°; 1H nmr (CDCl3): ꢀ
2.13 (s, 3H, CH3), 2.32 (s, 6H, (CH3)2Ar), 2.80 (s, 6H, (CH3)2N),
6.80 (s, 3H, Harom), 7.27 (d, 2H, J = 8.9 Hz, Harom), 7.65 (d, 2H, J
= 8.9 Hz, Harom); 13C nmr (CDCl3): ꢀ 7.22 (CH3), 21.22
((CH3)2Ar), 36.31 ((CH3)2N), 89.69 (C5), 107.54 (C-CN),
118.80 (CN), 119.09, 122.10, 126.05, 133.23, 138.60, 153.40,
157.53 (Carom), 158.76 (C2), 167.45 (C4), 169.15 (C6); HRms
(MALDI, peak matching): m/z calcd. for C22H23N4O2 (MH+),
375.1816, found 375.1805.
4-Chloro-6-(3,5-dimethylphenoxy)-N,N,5-trimethylpyrim-
idine-2-amine (16). To
Cells and Viruses. MT-4, C8166, and H9/IIIB cells were
grown at 37° in a 5% CO2 atmosphere in RPMI 1640 medium
supplemented with 10% fetal calf serum (FCS), 100 IU/mL
penicillin G, and 100 μg/mL streptomycin. Cell cultures were
checked periodically for the absence of mycoplasma
a solution of 4,6-dichloro-N,N,5-
trimethylpyrimidin-2-amine (15) (2.06 g, 10 mmole) and 3,5-
dimethylphenol (1.34 g, 11 mmole) in dry dimethylformamide
(20 mL) was added sodium hydride (55% suspension in paraffin
oil, 0.52 g, 12 mmole) portionwise. After stirring for 2 hours
under dry conditions at room temperature, the reaction mixture
was quenched by addition of water (2 mL) dropwise followed by
40 mL water in one portion, pure compound 16 was precipitated
then collected by filtration and dried. Yield: 2.85 g (98%), as a
white solid; mp 111-113°; 1H nmr (CDCl3): ꢀ 2.19 (s, 3H, CH3),
2.31 (s, 6H, (CH3)2Ar)), 2.95 (s, 6H, (CH3)2N), 6.77 (s, 2H,
Harom), 6.82 (s, 1H, Harom); 13C nmr (CDCl3): ꢀ 10.97 (CH3),
21.20 ((CH3)2Ar), 36.63 ((CH3)2N), 101.95 (C5), 119.21, 126.36,
138.68, 153.00 (Carom), 159.09 (C6), 160.58 (C2), 168.02 (C4);
HRms (MALDI, peak matching): m/z calcd. for C15H19N3OCl
(MH+) 292.1211, found 292.1213.
4-(3,5-Dimethylphenoxy)-6-methoxy-N,N,5-trimethyl-pyr-
imidin-2-amine (17). Sodium (0.93 g, 40.4 mmole) was
dissolved in dry methanol (80 mL), followed by addition of 4-
chloro-6-(3,5-dimethylphenoxy)-N,N,5-trimethylpyrimidin-2-
amine (16) (1.4 g, 4.8 mmole). After refluxing at 80° for 65
hours, the solvent was removed under reduced pressure and
water (40 mL) was added to the residue, followed by
neutralization by 4 M hydrochloric acid. Ether (30 mL) was
added and the two layers were separated. The water layer was
washed two times with ether (15 mL) and the ether extracts were
collected, dried over magnesium sulfate, filtered and evaporated
under reduced pressure. The residual material was
chromatographed on a column of silica gel using petroleum
ether/ether (16:1, v/v) as an eluent to afford compound 17.
Yield: 0.33 g (24%) as a white solid; mp 66-68°; 1H nmr
(CDCl3): ꢀ 1.98 (s, 3H, CH3), 2.29 (s, 6H, (CH3)2Ar)), 2.97 (s,
contamination with
a
MycoTect Kit (Gibco). Human
immunodeficiency viruses type 1 (HIV-1, IIIB strain) was
obtained from supernatants of persistently infected H9/IIIB
cells. The HIV-1 stock solutions had titers of 4.5 ꢀ 106 50% cell
culture infectious dose (CCID50)/mL. The K103R+V179D+
P225H mutant (EFVR) was derived from an IIIB strain passaged
in MT-4 cells in the presence of efavirenz (up to 2 μM). The
Y181C mutant (NIH N119) was derived from an AZT-sensitive
clinical isolate passaged initially in CEM and then in MT-4 cells
in the presence of nevirapine (10 μM). The double mutant
K103N+Y181C (NIH A17) was derived from the IIIB strain
passaged in H9 cells in the presence of BI-RG 587 (1 μM).
EFVR, N119, and A17 stock solutions had titers of 4.0 ꢀ 107, 1.2
ꢀ 108, and 2.1 ꢀ 107 CCID50/mL, respectively.
HIV Titration. Titration of HIV was performed in C8166
cells by the standard limiting dilution method (dilution 1:2, four
replica wells per dilution) in 96-well plates. The infectious virus
titer was determined by light microscope scoring of syncytia
after 4 days of incubation. Virus titers were expressed as
CCID50/mL.
Anti-HIV Assays. The activity of test compounds against
multiplication of wild type HIV-1, EFVR, N119, and A17 in
acutely infected cells was based on inhibition of virus-induced
cytopathogenicity in MT-4 cells. Briefly, an amount of 50 μL of
culture medium containing 1 ꢀ 104 cells was added to each well
of flat-bottom microtiter trays containing 50 μL of culture
medium with or without various concentrations of test
compounds. Then, an amount of 20 μL of HIV suspensions

1166
Y. Loksha, D. Globisch, E. Pedersen, P. La Colla, G. Collu and R. Loddo
Vol 45
Struct. Biol., 1995, 2, 293.
(containing the appropriate amount of CCID50 to cause complete
cytopathogenicity at day 4) was added. After incubation at 37ꢀ,
cell viability was determined by the 3-(4,5-dimethyl-thiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) method [17]. The
cytotoxicity of test compounds was evaluated in parallel with
their antiviral activity and was based on the viability of mock-
infected cells, as monitored by the MTT method.
[7] Esnouf, R. M.; Ren, J.; Hopkins, A. L.; Ross, C. K.; Jones, E.
Y.; Stammers, D. K.; Stuart, D. I. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
3984.
[8] Ren, J.; Milton, J.; Weaver, K. L.; Short, S. A.; Stuart, D. I.;
Stammers, D. K. Struct. Fold Des., 2000, 8, 1089.
[9] Lindberg, J.; Sigurdsson, S.; Lowgren, S.; Andersson, H. O.;
Sahlberg, C.; Noréen, R.; Fridborg, K.; Zhang, H.; Unge, T. Eur. J.
Biochem., 2002, 269, 1670.
[10] Staszewski, S.; Morales-Ramirez, J.; Tashima K. T.; Rachlis,
A.; Skiest, D.; Stanford, J.; Stryker, R.; Johnson, P.; Labriola, D. F.;
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M. R.; Koymans, L. M. H.; Vinkers, M.; Daeyaert, F.; Ludovici, D. W.
M.; Kukla, J.; De Corte, B.; Kavash, R. W.; Ho, C. Y.; Ye, H.;
Lichtenstein, M. A.; Andries, K.; Pauwels, R.; De Béthune, M.-P.;
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Acknowledgement. This work received funding from the
European Community’s Sixth Framework Programme under
contract number LSHP-CT-2004-503162 (Selection and
development of microbicides for mucosal use to prevent sexual
HIV transmission/acquisition) [18].
REFERENCES AND NOTES
[†] A research center funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
[*] To whom correspondence should be addressed. Fax: +45
66158780; e-mail: ebp@ifk.sdu.dk.
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[18] The information in this document is provided as is and no
guarantee or warranty is given that the information is fit for any
particular purpose. The user thereof uses the information as its sole risk
and liability.
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