Synthesis of novel MKC-442 analogues with potent activities against HIV-1

Article abstract of DOI:10.1002/ardp.200300742

Bis(alken-1-yloxy)methanes 2 were synthesized by reacting 2-cyclohexenol, 3-cyclohexenylmethanol, cinnamyl alcohol and its α-methyl analogue with dibromomethane. Condensation of 2 with 5,6-disubstituted uracil derivatives 1 resulted in the desired MKC-442 analogues 3-6. The most active compounds, N-1 cinnamyloxymethyl- and N-1 2-methyl-3-phenylallyloxymethyl substituted 5-ethyl-6-(3,5-dimethylbenzyl)uracils (5b and 6b), showed activity against wild-type HIV-1 in the nanomolar range, and against Y181C and Y181C+K103N, mutant strains known to be resistant to MKC-442, in the micromolar range.

Full text of DOI:10.1002/ardp.200300742

236 El-Brollosy et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 236–241
Nasser R. El-Brollosya,
Erik B. Pedersena,
Claus Nielsenb
Synthesis of Novel MKC-442 Analogues with Potent
Activities against HIV-1
Bis(alken-1-yloxy)methanes 2 were synthesized by reacting 2-cyclohexenol, 3-cy-
clohexenylmethanol, cinnamyl alcohol and its α-methyl analogue with dibro-
momethane.Condensation of 2 with 5,6-disubstituted uracil derivatives 1 resulted in
the desired MKC-442 analogues 3–6. The most active compounds, N-1 cinnamy-
loxymethyl- and N-1 2-methyl-3-phenylallyloxymethyl substituted 5-ethyl-6-(3,5-
dimethylbenzyl)uracils (5b and 6b), showed activity against wild-type HIV-1 in the
nanomolar range, and againstY181C andY181C+K103N, mutant strains known to
be resistant to MKC-442, in the micromolar range.
a
Nucleic Acid Center,
Department of Chemistry,
University of Southern
Denmark, Campusvej 55,
DK-5230 Odense M,
Denmark
b
Retrovirus Laboratory,
Department of Virology,
State Serum Institute,
Artillerivej 5,
Keywords: Non-nucleoside reverse transcriptase inhibitors; MKC-442 analogues;
Human immunodeficiency virus (HIV-1); HIV-1 mutants; Alkenyloxymethyluracils
DK-2300 Copenhagen,
Denmark
Received: November 7, 2002; Accepted: February 13, 2003 [FP742]
DOI 10.1002/ardp.200300742
[18]. Here we describe the structure activity relationship
when the double bond is more heavily substituted.
Introduction
The virally encoded reverse transcriptase (RT) of HIV is
an important target for the development of anti-AIDS
drugs. It plays a multifunctional role in the conversion of
the single-stranded RNA viral genome to double-strand-
ed DNA [1].The non-nucleoside reverse transcriptase in-
hibitors (NNRTIs), in contrast to nucleoside reverse tran-
scriptase inhibitors (NRTIs) such as AZT [2], ddC [3], ddI
[4], 3TC [4], are highly specific since they do not bind to
cellular polymerases.
Chemistry
The 5,6-disubstituted uracil derivatives 1 a–c were pre-
pared from the corresponding 3-oxoester which was it-
self prepared by treating arylacetonitrile with ethyl 2-bro-
mobutyrate or methyl 2-bromo-3-methylbutyrate in THF
in the presence of activated zinc [19]. The di-(substitut-
ed-alkenyloxy)-methane derivatives 2a-d were prepared
from the corresponding alcohols, 2-cyclohexen-1-ol,
3-cyclohexene-1-methanol, trans-cinnamyl alcohol, and
trans-2-methyl-3-phenyl-2-propen-1-ol, in a reaction
with dibromomethane using potassium hydroxide in the
presence of tetrabutylammonium bromide in refluxing
anhydrous benzene according to the method of Nazar-
etyan et al [20]. According to NMR data, compound 2 a
was a (1:1) diastereomeric mixture, whereas 2 b was a
nearly pure diastereomer. 13C-NMR spectra of com-
pounds 2 a–d showed the presence of a O-CH2-O group
at 91–96 ppm.
NNRTIs have been found to bind to a specific allosteric
site on HIV-1 RT near the polymerase site. Once bound,
they interfere with reverse transcription by altering either
the conformation or mobility of RT, thereby leading to a
non competitive inhibition of the enzyme [5–7].Since the
original synthesis of 1-[(2-hydroxyethoxy)methyl]-6-
(phenylthio)thymine (HEPT) [8], many analogues of it
have been developed and investigated for their activity
against HIV-1 [9–12]. The analogue 6-benzyl-1-
(ethoxymethyl)-5-isopropyluracil (MKC-442, also known
as Emivirine or Coactinone)[13], showed high activity
against HIV-1 but unfortunately in phase III clinical trials
it was also found to activate a liver enzyme in the 450
family which metabolizes protease inhibitors [14]. For
this reason, and as a part of our continuing interest in the
chemistry of NNRTIs [15–17], we are interested in
whether alternative drug candidates can be found
among the HEPT analogues. Recently, we reported that
MKC-442 analogues with a 1-allyloxymethyl substituent
showed activity against HIV-1 in the picomolar range
The uracil derivatives 1 b, c were silylated with N,O-bis-
(trimethylsilyl)acetamide (BSA) [21] in acetonitrile and
then treated with bis(2-cyclohexen-1-yloxy)methane
(2 a) as described by Vorbrüggen [22].This consisted of
using trimethylsilyl trifluoromethanesulfonate (TMS tri-
flate) as a Lewis acid catalyst to give 1-[(2-cyclohexen-1-
yl)oxy]methyl-6-(3,6-dimethylbenzyl)-5-ethyluracil (3 a)
and 1-[(2-cyclohexen-1-yl)oxy]methyl-6-(3,6-dimethyl-
benzyl)-5-isopropyluracil (3 b) in 87 % and 84 % yields,
respectively. Using bis(3-cyclohexen-1-yl-methoxy)-
methane (2 b) in a reaction with uracils 1 a–c produced
the corresponding N1-(3-cyclohexen-1-yl-methoxy)
methyluracil derivatives 4a-c in 72–79 % yields.
Correspondence: Erik B. Pedersen, Department of Chemis-
try, Nucleic Acid Center, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M, Denmark. Phone:
+45 65502555, Fax: +45 66158780, e-mail: EBP@Chem.sdu.dk.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/04_05/0236 $ 17.50+.50/0

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 236–241
Activity of novel MKC-442 analogues against HIV-1 237
Scheme 1. Synthesis of the MKC-442 analogues 3–6.

238 El-Brollosy et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 236–241
These novel analogues may give improved binding to the
allosteric site of HIV-1 RT due to stacking interactions
between the alkenyl group and the aromatic amino acid
Phe227 in RT.Therefore, we found it of interest to study
some alkenyl substituents having a conjugated phenyl
group. Using the conditions described above, com-
pounds 1 a–c were condensed with bis(trans-cinnamyl-
oxy)methane (2 c) to give the corresponding N1-(trans-
cinnamyloxy-methyl)-5,6-disubstituted uracil derivatives
5 a–c in 55–60 % yields. The corresponding condensa-
tion with bis((E)-2-methyl-3-phenylallyloxy)methane
Figure 1.Chemical structure of MKC-442 and Efavirens.
(2 d)
afforded
5,6-disubstituted-1-((E)-2-methyl-3-
phenylallyloxy)methyl uracils (6 a–c) in 56–59 % yields.
The structures of the synthesized compounds were
determined by comparison to similar NMR data [15–17,
19, 21].
Only compound 5 b, which contains a N-1 cinnamyl sub-
stituent, and compound 6 b, which contains a homolo-
gous substituent, showed marginally higher activity
against HIV-1 than MKC-442 and efavirenz and better
activity against the N119 strain than that observed with
MKC-442. These two compounds also showed activity
against the double mutated strain A17 which was com-
parable to that observed with efavirenz. However, efa-
virenz displayed higher activity against the N119 strain
with one mutation.
Biological properties
Compounds 3–6 were tested for their activity against
HIV-1 in MT-4 cells infected with the wild-type HIV-1
strain IIIB, the NNRTI resistant strain N119, which con-
tains one mutation (Y181C) or with the NNRTI resistant
strain A17, with contains two mutations (K103N +
Y181C) (Table 1).
Table 1. Antiviral activity of compounds 3–6 against HIV-1 in MT-4 cellsa.
Compound
Wild-type HIV-1
CC50 (µM)
N119
A17
SId
(Y181C)
(K103N + Y181C)
b
c
IC50 (µM)
b
b
IC50 (µM)
IC50 (µM)
3 a
3 b
4 a
4 b
4 c
5 a
5 b
5 c
6 a
0.04
0.03
0.30
0.02
0.08
0.01
0.003
0.06
0.04
0.003
0.03
0.02
0.01
31
28
34
24
30
29
32
31
30
30
28
>100
>100
775
930
113
>100
>100
NDe
>100
>100
>100
4
>100
>100
NDe
>100
>100
>100
10
>100
>100
4
1200
375
2900
10667
517
750
10000
933
6
>100
2
6 b
6 c
21
44
0.3
>100
>100
2.7
MKC-442
Efavirenz
5000
>10000
aAll data represent mean values for three separate experiments.
bInhibitory concentration of compound achieving 50 % inhibition of HIV-1 multiplication in MT-4-infected cells.
cCytotoxic concentration of compound required to reduce the viability of normal uninfected MT-4 cells by 50 %.
dSelectivity index: ratio CC50/IC50.
eND = not tested.The symbol (>) indicates that CC50 was not reached at the highest concentration tested.For a descrip-
tion of the assay see the Experimental Section.

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 236–241
Activity of novel MKC-442 analogues against HIV-1 239
(10 min), the reaction mixture was cooled to –50 °C and tri-
methylsilyl trifluoromethansulfonate (TMS triflate) (0.18 mL,
1 mmol) was added followed by dropwise addition of bis(2-
alkenyloxy)methane (2, 0.42 g, 2 mmol). The reaction mixture
was stirred at room temperature for 3–4 hours. Cold saturated
NaHCO3 solution (5 mL) was added, and the solvent was evap-
orated under reduced pressure at room temperature.The resi-
due was extracted with ether (3 × 50 mL), dried over anhydrous
magnesium sulphate and evaporated under reduced pressure.
The product was chromatographed on a column of silica gel us-
ing CHCl3 for compounds 3 a, b and 4 a–c and Et2O:petroleum
ether (ν:ν = 1:6) for compounds 5 a–c and 6 a–c.
Experimental
NMR spectra were recorded on a Varian Gemini 2000 NMR
spectrophotometer at 300 MHz for 1H and 75 MHz for 13C with
TMS as an internal standard. EI mass spectra were recorded
on a Finnigan MAT SSQ 710. MALDI spectra were recorded on
a 4.7 Tesla Ultima Fourier transform Mass spectrometer
(IonSpec, Irvine, CA). Melting points were determined using 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 the H. C. Ørsted Institute,
Copenhagen, Denmark. Solvents were purified using standard
procedures [23].
1-(2-Cyclohexen-1-yloxymethyl)-6-(3,5-dimethylbenzyl)-5-ethyl-
uracil (3 a)
General Procedure for the Synthesis of Bis(alkenyloxy)-
methane Derivatives 2 a–d
1
Yield 0.31 g (87 %) as a colourless oil. H-NMR (CDCl3): δ
(ppm) = 0.99 (t, 3H, J = 7.3 Hz, CH3), 1.47–1.96 (m, 6 H, 3 ×
CH2), 2.21 (s, 6 H, 2 × CH2), 2.36 (q, 2 H, J = 7.3 Hz, CH2), 4.03
(s, 2 H, CH2), 4.13 (s, 2 H, CH2), 5.08–5.11 (m, 1 H, CH), 5.69–
5.80 (m, 2 H, 2 × CH), 6.63 (s, 2 H, Harom), 6.82 (s, 1 H, Harom),
9.31 (s, 1 H, NH). NMR (CDCl3): δ (ppm) = 13.75 (CH3), 18.89
(CH3), 21.22 (CH2), 24.99 (CH2), 28.57 (CH2), 31.93 (CH2),
33.19 (CH2), 65.43 (CH2), 71.69 (CH), 116.69 (C-5), 124.96,
126.86, 134.99, 138.78 (Carom), 129.83 (CH), 130.44 (CH),
149.48 (C-6), 151.73 (C-2), 163.32 (C-4).HRMS-MALDI:m/z =
391.1992 (M + Na+, C22H28N2NaO3); requires 391.1994.
The appropriate alcohol (100 mmol), dibromomethane (8.79 g,
50.5 mmol) and tetrabutylammonium bromide (1.74 g,
5.35 mmol) were added to potassium hydroxide (5.66 g,
101 mmol) in benzene (30 mL), and the suspension was heat-
ed under reflux for 5 hours. After cooling, water (50 mL) was
added and the resulting solution extracted with ether (3 ×
50 mL).The ether phase was dried with anhydrous magnesium
sulphate and evaporated. The residue was purified by distilla-
tion under reduced pressure (2 a and 2 b) or chromatographed
on a column of silica gel with CHCl3 (2 c and 2 d).
Bis(2-cyclohexen-1-yloxy)methane (2 a)
1-(2-Cyclohexen-1-yloxymethyl)-6-(3,5-dimethylbenzyl)-5-iso-
propyluracil (3 b)
Yield 4.47 g (43 %) as a colourless oil;bp 123 °C/12 mmHg.1H-
NMR (CDCl3): δ (ppm) = 1.53–2.11 (m, 12 H, 6 × CH2), 4.17–
4.20 (m, 2 H, 2 × CH), 4.82 (s, 2 H, CH2), 5.74–5.79 (m, 2 H, 2 ×
CH), 5.8–5.90 (m, 2 H, 2 × CH). 13C-NMR (CDCl3): δ (ppm) =
19.04 (CH2), 25.07 (CH2), 28.69, 28.91 (CH2), 69.81, 70.01
(CH), 91.39, 91.65 (CH2), 127.73, 127.88 (CH), 130.90, 130.96
(CH).
1
Yield 0.32 g (84 %) as a colourless oil. H-NMR (CDCl3): δ
(ppm) = 1.28 (d, 6 H, J = 7.0 Hz, 2 × CH3), 1.59–2.02 (m, 6 H, 3 ×
CH2), 2.28 (s, 6 H, 2 × CH3), 2.64 (heptet, 1 H, J = 7.0 Hz, CH),
4.13 (s, 4 H, 2 × CH2), 5.20–5.24 (m, 1 H, CH), 5.82–5.91 (m,
2 H, 2 × CH), 6.71 (s, 2 H.Harom), 6.89 (s, 1 H, Harom), 9.55 (s, 1 H,
NH). 13C-NMR (CDCl3): δ (ppm) = 18.90 (CH3), 20.40 (CH3),
21.23 (CH2), 24.99 (CH2), 28.28 (CH), 28.57 (CH2), 33.27
(CH2), 65.43 (CH2), 71.66 (CH), 119.54 (C-5), 124.97, 126.91,
135.13, 138.71 (Carom), 128.73 (CH), 131.70 (CH), 148.88
(C-6), 151.91 (C-2), 162.63 (C-4). HRMS-MALDI: m/z =
405.2149 (M + Na+, C23H30N2NaO3); requires 405.2152.
Bis(3-cyclohexen-1-ylmethyloxy)methane (2 b)
Yield 5.44 g (46 %) as a colourless oil;bp 135 °C/12 mmHg.1H-
NMR (CDCl3): δ (ppm) = 1.23–1.36 (m, 2 H, 6a-H, 6Јa-H),
1.70–2.16 (m, 12 H, 4 × CH2, 2 × CH, 6b-H, 6Ј-H), 3.42 (m, 4 H,
2 × CH2), 4.68 (s, 2 H, CH2), 5.62–5.70 (m, 4 H, 4 × CH). 13C-
NMR (CDCl3):δ (ppm) = 24.57 (CH2), 25.70 (CH2), 28.53 (CH2),
33.90 (CH), 72.60 (CH2), 95.52 (CH2), 125.91 (CH), 127.03
(CH).
6-Benzyl-1-(3-cyclohexen-1-ylmethoxymethyl)-5-isopropyluracil
(4 a)
Yield: 0.29 g (79 %) as a white solid; mp 130–131°C. 1H-NMR
(CDCl3): δ (ppm) = 1.28 (d, 6 H, J = 6.9 Hz, 2 × CH3), 1.73–1.78
(m, 4 H, 2 × CH2), 2.03–2.06 (m, 3 H, CH, CH2), 2.88 (heptet,
1 H, J = 6.9 Hz, CH), 3.44 (d, 2 H, J = 6.4 Hz, CH2), 4.20 (s, 2 H,
CH2), 5.15 (s, 2 H, CH2), 5.63–5.66 (m, 2 H, 2 × CH), 7.11–7.37
(m, 5 H, Harom), 9.50 (s, 1 H, NH). 13C-NMR (CDCl3): δ (ppm) =
20.39 (CH3), 24.45 (CH2), 25.37 (CH2), 28.28 (CH), 33.35 (CH),
33.74 (CH2), 73.24 (CH2), 74.10 (CH2), 119.76 (C-5), 125.68
(CH), 127.01, 127.25, 129.16, 135.39 (Carom), 127.18 (CH),
148.53 (C-6), 151.96 (C-2), 162.50 (C-4). EI MS: m/z = 368
(M+). Anal. Calcd for C22H28N2O3 (368.47): C, 71.71; H, 7.66; N,
7.60. Found: C, 71.62; H, 7.66; N, 7.38 %.
Bis((E)-cinnamyloxy)methane (2 c)
Yield 7.4 g (53 %) as a colourless oil. 1H-NMR (CDCl3): δ (ppm)
= 4.27 (dd, 4 H, J = 1.4, 6.1 Hz, 2 × CH2), 4.82 (s, 2 H, CH2), 6.26
(td, 2 H, J = 6.1, 15.9 Hz, 2 × CH), 6.61 (d, 2 H, J = 15.9 Hz, 2 ×
CH), 7.20–7.39 (m, 10 H, Harom). 13C-NMR (CDCl3): δ (ppm) =
68.04 (CH2), 93.75 (CH2), 125.53 (CH), 126.44, 127.65,
128.50, 136.60 (Carom), 132.60 (CH).
Bis((E)-2-methyl-3-phenylallyloxy)methane (2 d)
Yield 5.9 g (59 %) as a yellow oil. 1H-NMR (CDCl3): δ (ppm) =
1.92 (s, 6 H, 2 × CH3), 4.18 (s, 4 H, 2 × CH2), 4.82 (s, 2 H, CH2),
6.56 (s, 2 H, 2 × CH), 7.19–7.35 (m, 10 H, Harom). 13C-NMR
(CDCl3): δ (ppm) = 15.64 (CH3), 73.58 (CH2), 93.64 (CH2),
127.11 (CH), 126.44, 128.06, 128.88, 134.60 (Carom), 137.45
(CH).
1-(3-Cyclohexen-1-ylmethoxymethyl)-6-(3,5-dimethylbenzyl)-5-
ethyluracil (4 b)
1
Yield 0.28 g (74 %) as a colourless oil. H-NMR (CDCl3): δ
(ppm) = 1.05 (t, 3 H, J = 7.4 Hz, CH3), 1.74–1.78 (m, 4 H, 2 ×
CH2), 1.81–1.84 (m, 3 H, CH, CH2), 2.29 (s, 6 H, 2 × CH3), 2.44
(q, 2 H, J = 7.4 Hz, CH2), 3.43 (d, 2 H, J = 6.2 Hz, CH2), 4.09 (s,
2 H, CH2), 5.13 (s, 2 H, CH2), 5.64–5.66 (m, 2 H, 2 × CH), 6.70
(s, 2 H, Harom), 6.90 (s, 1 H, Harom), 9.41 (s, 1 H, NH). 13C-NMR
(CDCl3): δ (ppm) = 13.76 (CH3), 19.11 (CH2), 21.26 (CH3),
24.45 (CH2), 25.38 (CH2), 28.29 (CH), 33.13 (CH), 33.75 (CH2),
General Procedure for the Synthesis of 1-(2-alkenyloxy-
methyl)uracils 3 a, b, 4 a–c, 5 a–c and 6 a–c
Uracil (1, 1 mmol) was stirred in anhydrous CH3CN (15 mL) un-
der N2 and N,O-bis-(trimethylsilyl)acetamide (BSA) (0.87 mL,
3.5 mmol) was added. After a clear solution was obtained

240 El-Brollosy et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 236–241
73.10 (CH2), 74.09 (CH2), 116.78 (C-5), 124.99, 127.01,
134.94, 138.84 (Carom), 125.68 (CH), 128.91 (CH), 149.43 (C-
6), 151.88 (C-2), 163.37 (C-4). HRMS-MALDI: m/z = 405.2149
(M + Na+, C23H30N2NaO3); requires 405.2152.
6-Benzyl-5-isopropyl-1-((E)-2-methyl-3-phenylallyloxymethyl)-
uracil (6 a)
1
Yield: 0.23 g (57 %) as a colourless oil. H-NMR (CDCl3): δ
(ppm) = 1.27 (d, 6 H, J = 7.0 Hz, 2 × CH3), 1.86 (s, 3 H, CH3),
2.84 (heptet, 1 H, J = 7.0 Hz, CH), 4.18 (s, 2 H, CH2), 4.23 (s,
2 H, CH2), 5.22 (s, 2 H, CH2), 6.51 (s, 1 H, CH), 7.12–7.37 (m,
10 H, Harom), 9.40 (s, 1 H, NH). 13C-NMR (CDCl3): δ (ppm) =
15.49 (CH3), 20.38 (CH3), 28.32 (CH), 33.51 (CH2), 72.87
(CH2), 75.91 (CH2), 119.80 (C-5), 126.58, 127.26, 128.07,
128.86, 129.21, 133.96, 135.31 (Carom), 127.53 (CH), 137.14
(C(Me)=), 148.43 (C-6), 151.91 (C-2), 162.38 (C-4). HRMS-
MALDI: m/z = 427.1992 (M + Na+, C25H28N2NaO3); requires
427.2000.
1-(3-Cyclohexen-1-ylmethoxymethyl)-6-(3,5-dimethylbenzyl)-
5-isopropyluracil (4 c)
Yield: 0.29 g (72 %) as a white solid; mp 149–150 °C. 1H-NMR
(CDCl3):δ (ppm) = 1.29 (d, 6 H, J = 7.0 Hz, CH3), 1.59–1.93 (m,
4H, 2 × CH2), 2.03–2.17 (m, 3 H, CH, CH2), 2.23 (s, 6 H, 2 ×
CH3), 2.83 (heptet, 1 H, J = 7.0 Hz, CH), 3.44 (d, 2 H, J = 6.4 Hz,
CH2), 4.11 (s, 2 H, CH2), 5.15 (s, 2 H, CH2), 5.64–5.66 (m, 2 H,
2 × CH), 6.71 (s, 2 H, Harom), 6.90 (s, 1 H, Harom), 9.42 (s, 1 H,
NH). 13C-NMR (CDCl3): δ (ppm) = 20.40 (CH3), 21.25 (CH3),
24.47 (CH2), 25.39 (CH2), 28.30 (CH), 33.17 (CH), 33.76 (CH2),
73.26 (CH2), 74.09 (CH2), 119.52 (C-5), 124.99, 127.02,
135.09, 138.77 (Carom), 125.70 (CH), 128.79 (CH), 148.81
(C-6), 151.99 (C-2), 162.54 (C-4). EI MS: m/z = 396 (M+). Anal.
Calcd for C24H32N2O3 (396.52): C, 72.70; H, 8.13; N, 7.06.
Found: C, 72.69; H, 8.18; N, 6.82 %.
6-(3,5-Dimethylbenzyl)-5-ethyl-1-((E)-2-methyl-3-phenyl-
allyloxymethyl)uracil (6 b)
Yield: 0.25 g (59 %) as a white solid; mp 111–113 °C. 1H-NMR
(CDCl3): δ (ppm) = 1.04 (t, 3 H, J = 7.3 Hz, CH3), 1.87 (s, 3 H,
CH3), 2.27 (s, 6 H, 2 × CH3), 2.44 (q, 2 H, J = 7.3 Hz, CH2), 4.12
(s, 2 H, CH2), 4.17 (s, 2 H, CH2), 5.19 (s, 2 H, CH2), 6.50 (s, 1 H,
CH), 6.71 (s, 2 H, Harom), 6.90 (s, 1 H, Harom), 7.21–7.35 (m, 5 H,
H
arom), 8.99 (s, 1 H, NH). 13C-NMR (CDCl3): δ (ppm) = 13.75
6-Benzyl-1-((E)-cinnamyloxymethyl)-5-isopropyluracil (5 a)
(CH3), 15.52 (CH3), 19.18 (CH2), 21.27 (CH3), 33.30 (CH2),
72.78 (CH2), 75.93 (CH2), 116.79 (C-5), 126.62 (CH), 125.00,
127.51, 128.11, 128.87, 129.00, 134.00, 134.88, 138.92
(Carom), 137.15 (C(Me)=), 149.34 (C-6), 151.73 (C-2), 163.13
(C-4). EI MS: m/z = 418 (M+). Anal. Calcd for C26H30N2O3
(418.53):C, 74.61;H, 7.22;N, 6.69.Found:C, 74.46;H, 7.19;N,
6.66 %.
1
Yield 0.21 g (55 %) as a colourless oil. H-NMR (CDCl3): δ
(ppm) = 1.28 (d, 6 H, J = 6.8 Hz, 2 × CH3), 2.85 (heptet, 1 H, J =
6.8 Hz, CH), 4.21 (s, 2 H, CH2), 4.28 (dd, 2 H, J = 1.2, 6.2 Hz,
CH2), 5.21 (s, 2 H, CH2), 6.19 (td, 1 H, J = 6.2, 15.9 Hz, CH),
6.60 (d, 1 H, J = 15.9 Hz, CH), 7.10–7.38 (m, 10 H, Harom), 9.51
(s, 1 H, NH). 13C-NMR (CDCl3): δ (ppm) = 20.37 (CH3), 28.33
(CH), 33.56 (CH2), 70.23 (CH2), 72.59 (CH2), 119.85 (C-5),
124.61 (CH), 126.51, 127.21, 127.24, 127.84, 128.50, 129.17,
135.30, 136.33 (Carom), 133.38 (CH), 148.36 (C-6), 152.04
(C-2), 162.39 (C-4). HRMS-MALDI: m/z = 413.1836 (M + Na+,
C24H26N2NaO3); requires 413.1856.
6-(3,5-Dimethylbenzyl)-5-isopropyl-1-((E)-2-methyl-3-phenyl-
allyloxymethyl)uracil (6 c)
1
Yield: 0.24 g (59 %) as a colourless oil. H-NMR (CDCl3): δ
(ppm) = 1.28 (d, 6 H, J = 6.9 Hz, 2 × CH3), 1.87 (s, 3 H, CH3),
2.28 (s, 6 H, 2 × CH3), 2.83 (heptet, 1 H, J = 6.9 Hz, CH), 4.15 (s,
2 H, CH2), 4.19 (s, 2 H, CH2), 5.22 (s, 2 H, CH2), 6.51 (s, 1 H,
CH), 6.72 (s, 2 H, Harom), 6.90 (s, 1 H, Harom), 7.24–7.33 (m, 5 H,
1-((E)-Cinnamyloxymethyl)-6-(3,5-dimethylbenzyl)-5-ethyluracil
(5 b)
arom), 9.54 (s, 1 H, NH). 13C-NMR (CDCl3): δ (ppm) = 15.49
Yield: 0.23 g (57 %) as a white solid; mp 131–132 °C. 1H-NMR
(CDCl3):δ (ppm) = 1.04 (t, 3H, J = 7.3 Hz, CH3), 2.26 (s, 6 H, 2 ×
CH3), 2.43 (q, 2 H, J = 7.3 Hz, CH2), 4.10 (s, 2 H, CH2), 4.26 (dd,
2 H, J = 1.2, 6.2 Hz, CH2), 5.18 (s, 2 H, CH2), 6.19 (td, 1 H, J =
6.2, 15.9 Hz, CH), 6.60 (d, 1 H, J = 15.9 Hz, CH), 6.70 (s, 2 H,
Harom), 6.89 (s, 1 H, Harom), 7.26–7.39 (m, 5H, Harom), 8.89 (s,
1 H, NH). 13C-NMR (CDCl3): δ (ppm) = 13.75 (CH3), 19.20
(CH2), 21.26 (CH3), 33.35 (CH2), 70.27 (CH2), 72.53 (CH2),
116.83 (C-5), 124.64 (CH), 124.99, 126.53, 127.91, 128.56,
128.99, 134.86, 136.33, 138.91 (Carom), 133.38 (CH), 149.31
(C-6), 151.77 (C-2), 163.05 (C-4). EI MS: m/z = 404 (M+). Anal.
Calcd for C25H28N2O3 (404.50): C, 74.23; H, 6.98; N, 6.93.
Found: C, 74.02; H, 6.91; N, 6.86 %.
H
(CH3), 20.39 (CH3), 21.24 (CH3), 28.32 (CH), 33.33 (CH2),
72.90 (CH2), 75.88 (CH2), 119.66 (C-5), 126.55 (CH), 125.00,
127.42, 128.06, 128.86, 134.03, 135.02, 128.81 (Carom),
137.17 (C(Me)=), 148.69 (C-6), 152.02 (C-2), 162.54 (C-
4).HRMS-MALDI: m/z = 455.2311 (M + Na+, C27H32N2NaO3);
requires 455.2305.
Virus and cells
The inhibitory activity of the analogues against HIV-1 infection
was evaluated using MT-4 cells [24] as target cells and the
HIV-1 strain HTLV-IIIB [25] as infectious virus. The virus was
propagated in H9 cells [24] grown at 37 °C, 5 % CO2 in RPMI
1640 media containing 10 % heat-inactivated fetal calf serum
(FCS) and antibiotics. Culture supernatant was filtered
(0.45 nm), aliquoted, and stored at –80 °C until use.
1-((E)-Cinnamyloxymethyl)-6-(3,5-dimethylbenzyl)-5-iso-
propyluracil (5 c)
1
Yield: 0.25 g (60 %) as a colourless oil. H-NMR (CDCl3): δ
Inhibition of HIV-1 replication
(ppm) = 1.29 (d, 6 H, J = 6.8 Hz, 2 × CH3), 2.27 (s, 6 H, 2 × CH3),
2.86 (heptet, 1 H, J = 6.8 Hz, CH), 4.13 (s, 2 H, CH2), 4.28 (dd,
2 H, J = 1.1, 6.1 Hz, CH2), 5.21 (s, 2 H, CH2), 6.23 (td, 1 H, J =
6.1, 15.8 Hz, CH), 6.61 (d, 1 H, J = 15.8 Hz, CH), 6.71 (s, 2 H,
Compounds were evaluated for possible antiviral activity
against both strains of HIV-1 using MT4 cells as target cells.
MT4 cells were incubated with virus (0.005 MOI) and growth
medium containing the test dilutions of compound for six days.
Uninfected control and virus infected cultures without com-
pound added were grown in parallel. Expression of HIV in the
cultures was quantified indirectly using the MTT assay [26].
Compounds mediating less than 30 % reduction of HIV expres-
sion were considered to be biologically inactive. Compounds
were tested in parallel for cytotoxic effect in uninfected MT-4
H
arom), 6.89 (s, 1 H, Harom), 7.22–7.39 (m, 5H, Harom), 9.66 (s,
1 H, NH). 13C-NMR (CDCl3): δ (ppm) = 20.38 (CH3), 21.23
(CH3), 28.34 (CH), 33.39 (CH2), 70.22 (CH2), 72.63 (CH2),
119.70 (C-5), 124.67 (CH), 124.98, 126.51, 127.83, 128.51,
128.83, 134.99, 136.35, 138.78 (Carom), 133.31 (CH), 148.65
(C-6), 152.13 (C-2), 162.55 (C-4). HRMS-MALDI: m/z =
441.2149 (M + Na+, C26H30N2NaO3); requires 441.2157.

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 236–241
Activity of novel MKC-442 analogues against HIV-1 241
cultures containing the test dilutions of compound as described
above. A 30 % inhibition of cell growth relative to control cul-
tures was considered significant. The 50 % inhibitory concen-
tration (IC50) and the 50 % cytotoxic concentration (CC50) were
determined by interpolation from the plots of percent inhibition
versus concentration of compound.The test for activity against
HIV-1 was performed in MT-4 cell cultures infected with either
wild-type HIV-1 (strain IIIB [26]) or NNRTI resistant HIV-1
(strain N119 [27], strain A17 [28, 29].
[13] H. Tanaka, H. Takashima, M. Ubasawa, K. Sekiya, N. In-
ouye, M. Baba, S. Shigeta, R. T. Walker, E. De Clercq, T.
Miyasaka, J. Med. Chem. 1995, 38, 2860–2865.
[14] G. M. Szczech, P. Furman, G. R. Painter, D. W. Barry, K.
Borroto-Esoda, T. B. Grizzle, M. R. Blum, J.-P. Somma-
dossi, R. Endoh, T. Niwa, M.Yamamoto, C. Moxham, Anti-
microb. Agents. Chemother. 2000, 44, 123–130.
[15] E. B. Pedersen, K. Danel, L. Bruun, C. Nielsen, Russ. J.
HIV/AIDS Relat. Problems 1998, 2, 73–77.
[16] J. S. Larsen, L. Christensen, G. Ludvig, P.T. Jorgensen, E.
B. Pedersen, C. Nielsen, J. Chem. Soc. Perkin Trans. I
2000, 18, 3035–3038.
References
[17] L. Petersen, E. B. Pedersen, C. Nielsen, Synthesis 2001,
[1] H. Mitsuya, R. Yarchoan, S. Broder, Science 1990, 249,
559–564.
1533–1544.
[18] N. R. El-Brollosy, P.T. Jørgensen, B. Dahan, A. M. Boel, E.
B. Pedersen, C. Nielsen, J. Med. Chem. 2002, 45, 5721–
5726.
[2] H. Mitsuya, K. J.Weinhold, P. A. Furman, M. H. St Clair, S.
N.Lehrman, R.C.Gallo, D.Bolognesi, D.W.Barry, S.Brod-
er, Proc. Natl. Acad. U.S.A. 1985, 82, 7096–7100.
[19] K. Danel, E. Larsen, E. B. Pedersen, Sythesis 1995, 8,
[3] H. Mitsuya, S. Broder, Proc. Natl. Acad. U.S.A. 1986, 83,
934–936.
1911–1915.
[20] A. Kh. Nazaretyan, G. O Torosyan, A.T. Babayan, J. Appl.
Chem. USSR 1985, 58, 2396–2400.
[4] R.Yarchoan, H. Mitsuya, R. V. Thomas, J. M. Pluda, N. R.
Hartman, C. F. Perno, K. S. Marczyk, J. P. Allain, D. G.
Johns, S. Broder, Science 1989, 245, 412–415.
[21] K. Danel, E. Larsen, E. B. Pedersen, B. F. Vestergaard, C.
Nielsen, J. Med. Chem. 1996, 39, 2427–2431.
[5] L. A. Kohlstaedt, J. Wang, J. M. Friedman, P. A. Rice, T. A.
Steitz, Science 1992, 256, 1783–1790.
[22] H. Vorbrüggen, K. Krolikiewiecz, B. Bennua, Chem. Ber.
1981, 114, 1234–1255.
[6] J. S. Ren, R. Esnouf,. E. Garman, Y. Jones, D. Somers, C.
Ross, I.Kirby, J.Keeling, G.Darby, D.Stuart, D.Stammers,
Nature Struct. Biol. 1995, 2, 293–302.
[23] J. Leonard, B. Lygo, G. Procter, Advanced Practical Or-
ganic Chemistry 2nd edition. The Alden Press, Osney
Mead, Oxford, 1995.
[7] A. L. Hopkins, J. Ren, R. M. Esnouf, B. E. Willcox, E. Y.
Jones, C. Ross, T. Miyasaka, R.T.Walker, H.Tanaka, D. K.
Stammers, D. I. Stuart, J. Med. Chem. 1996, 39, 1589–
1600.
[24] S. Harada,Y. Koyanagi, N.Yamamoto, Science 1985, 229,
563–566.
[25] M.Popovic, M.G.Sarngadharan, E.Read, R.C.Gallo, Sci-
ence 1984, 224, 497–500.
[8] T.Miyasaka, H.Tanaka, M.Baba, H.Hayakawa, R.T.Walk-
er, J. Balzarini, E. De Clercq, J. Med. Chem. 1989, 32,
2507–2509.
[26] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.
[27] D. Richman, C. K. Shih, I. Lowy, J. Rose, P. Prodanovich, S
Goff, J. Griffin, Proc. Natl. Acad. Sci. USA 1991, 88,
11241–11245.
[9] J. Balzarini, A. Karlsson, E. De Clercq, Mol. Pharmacol.
1993, 44, 694–701.
[10] T. Miyasaka, H.Tanaka, E. De Clercq, M. Baba, R.T.Walk-
[28] H. Hara, T. Fujihashi, T. Sakata, A. Kaji, H. Kaji, AIDS Res.
Hum. Retroviruses 1997, 13, 695–705.
er, M. Ubasawa, EP449726, 1991, CA: 1991, 116, 41986.
[11] T. Miyasaka, H.Tanaka, E. De Clercq, M. Baba, R.T.Walk-
er, M. Ubasawa, EP420763, 1991, CA: 1991, 115,
158838.
[29] J. H. Nunberg, W. A. Schleif, E. J. Boots, J. A. O’Brien, J.
Virol. 1991, 65, 4887–4892.
[12] M. Baba, H. Tanaka, T. Miyasaka, S.Yuasa, M. Ubasawa,
R.T.Walker, E.De Clercq, Nucleosides Nucleotides 1995,
14, 575–583.