Synthesis of N-1-(indanyloxymethyl) and N-1-(4-hydroxybut-2-enyloxymethyl) analogues of the HIV drugs Emivirine and GCA-186

Article abstract of DOI:10.1007/s00706-005-0318-7

A series of Emivirine and GCA-186 analogues substituted at N-1 with indan-1-yloxymethyl (6a-6c) and indan-2-yloxymethyl (6d-6f) were synthesized by reaction of the corresponding bis(indanyloxy)methans with uracils having 5-ethyl or 5-isopropyl and 6-benzy

Full text of DOI:10.1007/s00706-005-0318-7

Monatshefte f€ur Chemie 136, 1247–1254 (2005)
DOI 10.1007/s00706-005-0318-7
Synthesis of N-1-(Indanyloxymethyl)
and N-1-(4-Hydroxybut-2-enyloxymethyl)
Analogues of the HIV Drugs Emivirine
and GCA-186
Nasser R. El-Brollosy1;a, Claus Nielsen2, and Erik B. Pedersen1;ꢀ
Nucleic Acid Centerb, Department of Chemistry, University of Southern Denmark,
1
DK-5230 Odense M, Denmark
2
Retrovirus Laboratory, Department of Virology, State Serum Institute,
DK-2300 Copenhagen, Denmark
Received October 27, 2004; accepted November 16, 2004
Published online July 4, 2005 # Springer-Verlag 2005
Summary. A series of Emivirine and GCA-186 analogues substituted at N-1 with indan-1-yloxy-
methyl (6a–6c) and indan-2-yloxymethyl (6d–6f) were synthesized by reaction of the corresponding
bis(indanyloxy)methans with uracils having 5-ethyl or 5-isopropyl and 6-benzyl or 6-(3,5-dimethyl-
benzyl) substituents. A route to the corresponding N-1 substituted 4-hydroxybut-2-enyloxymethyl
analogue was also devised. All newly synthesized compounds showed potent activity against wild-
type HIV-1, the most active compound being 5-ethyl-1-(indan-1-yloxymethyl)-6-(3,5-dimethyl-
benzyl)uracil (6b), which was 50-fold more active than Emivirine.
Keywords. Bioorganic chemistry; Drug research; HIV; Non-nucleoside reverse transcriptase inhi-
bitors; Emivirine analogues.
Introduction
Effective treatment regimens for the human immunodeficiency virus (HIV-1)
infection have included both HIV protease and reverse transcriptase inhibitors
(RTIs). The non-nucleoside reverse transcriptase inhibitors (NNRTIs) in contrast
to nucleoside reverse transcriptase inhibitors (NRTIs) such as AZT [1], ddC [2],
ddI [3], 3TC [3], are highly specific as their binding site is a hydrophobic
˚
pocket located approximately 10 A from the polymerase active site [4]. NNRTIs
a
Present address: Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt
b
A research center funded by The Danish National Research Foundation for studies on nucleic acid
chemical biology
ꢀ
Corresponding author. E-mail: ebp@chem.sdu.dk

1248
N. R. El-Brollosy et al.
Fig. 1. Chemical structure of HEPT (1), MKC-442, TNK-651, and GCA-186
consist of many classes of compounds [5] and among them the most potent anti-
HIV agents are found. One of the first NNRTIs was 1-[(2-hydroxyethoxy)methyl]-
6-(phenylthio)thymine (HEPT, 1, Fig. 1) [6, 7]. Although HEPT did not show very
high activity against HIV-1, it was considered an interesting lead compound for the
synthesis of new analogues, among them 6-benzyl-1-(ethoxymethyl)-5-isopropy-
luracil (Emivirine, formerly MKC-442, 2a, Fig. 1) [8], the corresponding 1-benzyl-
oxymethyl analogue (TNK-651, 2b, Fig. 1) [4], and the 6-(3,5-dimethylbenzyl)
analogue (GCA-186, 2c, Fig. 1) [9] which all showed high activity against HIV-1.
Emivirine was chosen as a drug candidate for clinical trials [10] by Triangle
Pharmaceuticals, but phase III was abandoned when comparative studies showed
Emivirine to be less potent than other HIV inhibitors [11]. It was stated that
Emivirine triggers the liver enzyme Cytochrome P 450, which metabolizes pro-
tease inhibitors [12]. According to structure activity relationship (SAR), studies of
several crystal structures of the reverse transcriptase (RT) complex with inhibitors,
such as HEPT [13], MKC-442 [4], TNK-651 [4], and GCA-186 [9], indicate that
ethyl or isopropyl group at C-5, and benzyl or (3,5-dimethylbenzyl) group at C-6
are the optimal substituents of the uracil ring with respect to HIV-1 inhibition. The
only site where new modifications may be tried is the N-1 position of the uracil
[14]. The substituents at N-1 may have larger size and length, and even bulky N-1
substituents may be accommodated because of the flexibility of the Pro 236 loop
region [9, 14]. Recently, we have synthesized a series of novel active Emivirine
analogues [15–17]. The present work reports a series of novel Emivirine and GCA-
186 analogues modified at N-1 position with larger bulky substituents.
Results and Discussions
Bis(indanyloxy)methanes (4a, 4b) [17, 18] were synthesized according to the
method of Nazaretyan et al. [19] by refluxing the corresponding indanols 3, dibro-
moethane, potassium hydroxide, and tetrabutylammonium bromide in anhydrous
benzene (Scheme 1). 5,6-Disubstituted uracils 5a–5c were prepared according to
the procedure described by Danel et al. [20] from the corresponding 2-alkyl-4-aryl-
3-oxo esters which in turn were prepared by reaction of phenylacetonitrile or (3,5-
dimethylphenyl)acetonitrile with the appropriate ꢀ-bromo esters in THF in the
presence of activated zinc. The so-formed 3-oxo esters were condensed with
thiourea to give the corresponding 2-thiouracils which were desulfurized with
aqueous chloroacetic acid to furnish the required uracils 5a–5c. The uracil deri-
vatives 5a–5c were silylated with N,O-bis-(trimethylsilyl)acetamide (BSA) in

Analogues of the HIV Drugs Emivirine and GCA-186
1249
Scheme 1
acetonitrile and alkylated at the N-1 position with bis(indan-1-yloxy)methane (4a)
and bis(indan-2-yloxy)methane (4b) in the presence of trimethylsilyl trifluoro-
methanesulfonate (TMS triflate) to afford the corresponding Emivirine and GCA-
186 analogues 6a–6f in 51–71% yields (Scheme 1).
Compounds 6a–6c are also regarded as TNK-651 analogues with an ethylene
link between CH2 group of PhCH2O and C-2 of the benzene ring, whereas 6d–6f
are TNK-651 analogues with an extra methylene group.
Another type of acetal for derivatizing the uracils was synthesized by reaction
of cis-but-2-enediol (7) with dibromomethane in the presence of potassium hydrox-
ide and tetrabutylammonium bromide in boiling dry benzene. Treatment of the
product with p-toluoyl chloride in pyridine gave bis(4-p-toluoyloxy-cis-but-2-en-
1-yloxy)methane (8) in 22% overall yield (Scheme 2). The uracils 5a and 5b were
silylated with BSA in acetonitrile and treated with 8 in the presence of TMS triflate
to afford the corresponding N-1-(4-p-toluoyloxy-cis-but-2-en-1-yloxymethyl) deri-
vatives 9a and 9b in 71% and 68% yields (Scheme 2). Compounds 9a and 9b are
interesting new Emivirine analogues with a large N-1 substituent. The N-3 regio-
isomer was neither isolated nor observed.
We found it of importance to study the activity when a bulky group, such as
toluoyl moiety, is located distantly from the N-1 position of the uracil. Also we
found it of interest to prepare an example of the corresponding detoluoylated
analogue. Removal of toluoyl group from 9a was achieved by its treatment with
sodium methoxide in anhydrous methanol to furnish the corresponding 1-(4-
hydroxy-cis-but-2-en-1-yloxymethyl) derivative in 66% yield (Scheme 2). N-1
substitution was proved by the NOE enhancement in the benzyl protons at C-6
when N-1 CH2 was irradiated.
The newly synthesized Emivirine and GCA-186 analogues 6a–6f, 9a, 9b, and
10 were tested against wild-type HIV-1 strain IIIB and against the resistant strain
N119 which contain the mutation Y181C in MT-4 cells. As shown in Table 1, all
the tested compounds are active against HIV-1 (wild-type). Compounds 6b, 6c, 6e,

1250
N. R. El-Brollosy et al.
Scheme 2
Table 1. Inhibitory and cytotoxic concentrations against HIV-1 in MT-4 cells
Compd
HIV-1 III (wild-type)
N119 (Y181C)
EC50=ꢁMa
EC50=ꢁMa
CC50=ꢁMb
SIc
d
6a
0.2
24
23
120
–
6b
0.0004
0.003
0.05
57500
11333
520
1.0
6c
34
1.8
d
6d
6e
26
–
0.001
0.002
0.02
31
31000
14000
1800
7.2
e
6f
28
–
e
9a
36
–
e
9b
0.003
0.03
34
11333
>3300
>5000
–
e
10
>100
>100
–
MKC-442
0.02
44
a
Inhibitory concentration of compounds achieving 50% inhibition of HIV multiplication in MT-4
infected cells; b cytotoxic concentration of compound required to reduce the viability of normal un-
infected MT-4 cells by 50%; c selectivity index: ratio CC50=EC50, the symbol (>) indicates that CC50
d
e
was not reached at the highest concentration test; not tested; inactive at subtoxic concentration
6f, and 9b exhibited higher activity than MKC-442. The most active compound is
6b which possessed the highest inhibition activity (EC50 ¼ 0.4 nM). It showed 50
fold higher activity against HIV-1 than MKC-442 and SI was 11-fold higher than
the one observed for MKC-442. Compound 6e was over 20-fold more potent
than MKC-442, whereas compounds 6c, 6f, and 9b were ca. 10-fold more active
than MKC-442. Three compounds 6b, 6c, and 6e showed higher activity than
MKC-442 against the mutant strain N119. The activities for the compounds 6b

Analogues of the HIV Drugs Emivirine and GCA-186
1251
and 6c were close to the one found for Efavirenz (0.3 ꢁM) using the same strain
and assay for the testing [16].
Experimental
1
NMR spectra were recorded on a Varian Gemini 2000 NMR spectrometer at 300 MHz for H and
75MHz for 13C with TMS as an internal standard. Chemical shifts are reported in ppm (ꢂ), and signals
are expressed as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). MALDI mass spectra
were recorded on an IonSpec Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. Melting
€
points were determined on a Buchi melting point apparatus. Elemental analyses were performed at
H. C. Ørsted Institute, University of Copenhagen; the found values agreed favourably with the
calculated ones. The progress of reactions was monitored by TLC (DC-alufolio 60 F254) from Merck.
For column chromatography Merck silica gel (0.040–0.063 mm) was used.
General Procedure for Preparation of 1-(Indanyloxymethyl)uracils 6a–6f
5,6-Disubstituted uracil (5a–5c, 1.0 mmol) was stirred in 15cm3 dry CH3CN under nitrogen and
0.87cm3 of N,O-bis(trimethylsilyl)acetamide (BSA, 3.5 mol) were added. After a clear solution was
obtained, the mixture was cooled to ꢁ50ꢂC and 0.18 cm3 TMS triflate (1.0mmol) were added followed
by the dropwise addition of 0.56g of bis(indan-1-yloxy)methane (4a) or bis(indan-2-yloxy)methane
(4b) (2.0mmol). The reaction mixture was stirred at room temperature for 5–8 h, and the mixture was
quenched by addition of 5 cm3 ice cold sat. aq. NaHCO3 solution, and evaporated under reduced
pressure. The residue was extracted with 3 ꢃ 50cm3 Et2O and the combined organic fractions were
dried (MgSO4), and evaporated under reduced pressure. The products were purified by silica gel
column chromatography (20% Et2O in petroleum ether (60–80ꢂC)) to afford 6a–6f.
6-Benzyl-1-(indan-1-yloxymethyl)-5-isopropyluracil (6a, C24H26N2O3)
White foam; yield 0.22 g (56%); 1H NMR (CDCl3, 300 MHz): ꢂ ¼ 1.29 (d, J ¼ 6.9 Hz, 6H, 2 ꢃ CH3),
2.01–2.07 (m, 1H, 20-H), 2.35–2.42 (m, 1H, 20-H), 2.76–2.91 (m, 2H, 30-H, CH), 3.01–3.09 (m, 1H,
30-H), 4.18 (s, 2H, CH2), 5.14–5.20 (m, 3H, 10-H, CH2), 7.04–7.35 (m, 9Harom), 8.82 (s, 1NH) ppm;
13C NMR (CDCl3, 75 MHz): ꢂ ¼ 20.46 (CH3), 28.35 (CH), 30.15 (CH2), 32.63 (CH2), 33.48 (CH2),
71.85 (CH), 82.32 (CH2), 119.85 (C-5), 124.91, 124.96, 126.56, 127.22, 127.27, 128.70, 129.18,
135.31, 141.96, 143.90 (Carom), 148.66 (C-6), 151.72 (C-2), 162.20 (C-4) ppm; HRMS (MALDI, peak
matching): m=z ¼ [Mþ Na]þ calcd 413.1839, found 413.1836.
5-Ethyl-1-(indan-1-yloxymethyl)-6-(3,5-dimethylbenzyl)uracil (6b, C25H28N2O3)
White foam; yield 0.23g (57%); 1H NMR (CDCl3, 300MHz): ꢂ ¼ 1.06 (t, J ¼ 7.4 Hz, 3H, CH3), 1.98–
2.05 (m, 1H, 20-H), 2.27 (s, 6H, 2 ꢃ CH3), 2.33–2.42 (m, 1H, 20-H), 2.45 (q, J ¼ 7.4 Hz, 2H, CH2), 2.75–
2.83 (m, 1H, 30-H), 3.02–3.09 (m, 1H, 30-H), 4.07 (s, 2H, CH2), 5.12–5.21 (m, 3H, 10-H, CH2), 6.65 (s,
2Harom), 6.88 (s, 1Harom), 7.20–7.36 (m, 4Harom), 8.91 (s, 1NH) ppm; 13C NMR (CDCl3, 75 MHz):
ꢂ ¼ 13.85 (CH3), 19.22 (CH2), 21.29 (CH3), 30.16 (CH2), 32.80 (CH2), 33.24 (CH2), 71.73 (CH), 82.28
(CH2), 116.86 (C-5), 124.91, 124.96, 125.01, 126.54, 128.70, 128.96, 134.88, 138.85, 141.99, 143.88
(Carom), 149.58 (C-6), 151.73 (C-2), 163.13 (C-4) ppm; MS (MALDI): m=z ¼ 427 (Mþ Naþ).
1-(Indan-1-yloxymethyl)-5-isopropyl-6-(3,5-dimethylbenzyl)uracil (6c, C26H30N2O3)
White foam; yield 0.215 g (51%); 1H NMR (CDCl3, 300MHz): ꢂ ¼ 1.29 (d, J ¼ 6.9 Hz, 6H, 2 ꢃ CH3),
2.01–2.08 (m, 1H, 20-H), 2.27 (s, 6H, 2 ꢃ CH3), 2.31–2.39 (m, 1H, 20-H), 2.75–2.91 (m, 2H, CH,
30-H), 3.02–3.08 (m, 1H, 30-H), 4.10 (s, 2H, CH2), 5.14–5.16 (m, 3H, 10-H, CH2), 6.65 (s, 2H, Harom),
6.89 (s, 1H, Harom), 7.22–7.36 (m, 4H, Harom), 8.84 (s, 1NH) ppm; 13C NMR (CDCl3, 75 MHz):
ꢂ ¼ 20.49 (CH3), 21.26 (CH3), 28.34 (CH), 30.13 (CH2), 32.78 (CH2), 33.29 (CH2), 71.84 (CH), 82.23
(CH2), 119.68 (C-5), 124.90, 124.93, 124.99, 126.51, 128.66, 128.82, 134.99, 138.77, 141.99, 143.88

1252
N. R. El-Brollosy et al.
(Carom), 148.92 (C-6), 151.76 (C-2), 162.22 (C-4) ppm; HRMS (MALDI, peak matching): m=z ¼
[Mþ Na]þ calcd 441.2165, found 441.2149.
6-Benzyl-1-(indan-2-yloxymethyl)-5-isopropyluracil (6d, C24H26N2O3)
White solid; yield 0.265 g (68%); mp 135–136ꢂC; 1H NMR (CDCl3, 300 MHz): ꢂ ¼ 1.28 (d,
J ¼ 7.0 Hz, 6H, 2 ꢃ CH3), 2.84 (hept, J ¼ 7.0 Hz, 1H, CH), 2.91, 3.14 (2m, 4H, 2 ꢃ CH2), 4.16 (s,
2H, CH2), 4.57 (quint, J ¼ 3.4 Hz, 1H, 20-H), 5.21 (s, 2H, CH2), 7.08–7.35 (m, 9Harom), 9.72 (s, 1 NH)
ppm; 13C NMR (CDCl3, 75MHz): ꢂ ¼ 20.39 (CH3), 28.29 (CH), 33.45 (CH2), 39.49 (CH2), 71.57
(CH), 78.91 (CH2), 119.85 (C-5), 124.61, 126.59, 127.20, 129.12, 135.33, 140.47 (Carom), 148.51 (C-
6), 152.05 (C-2), 162.53 (C-4) ppm; MS (MALDI): m=z ¼ 413 (Mþ Naþ).
5-Ethyl-6-(3,5-dimethylbenzyl)-1-(indan-2-y-oxymethyl)uracil (6e, C25H28N2O3)
White solid; yield 0.288 g (71%); mp 132–133ꢂC; 1H NMR (CDCl3, 300 MHz): ꢂ ¼ 1.05 (t, J ¼ 7.4 Hz,
3H, CH3), 2.27 (s, 6H, 2 ꢃ CH3), 2.47 (q, J ¼ 7.4 Hz, 2H, CH2), 2.92, 3.15 (2dd, J ¼ 3.7, 16.3Hz,
J ¼ 6.4, 16.3 Hz, 4H, 2 ꢃ CH2), 4.06 (s, 2H, CH2), 4.57 (quint, J ¼ 3.4 Hz, 1H, 20-H), 5.19 (s, 2H, CH2),
6.68 (s, 2Harom), 6.88 (s, 1Harom), 7.15–7.21 (m, 4H, Harom), 9.78 (s, 1NH) ppm; 13C NMR (CDCl3,
75MHz): ꢂ ¼ 13.79 (CH3), 19.14 (CH2), 21.24 (CH3), 33.18 (CH2), 39.51 (CH2), 71.45 (CH), 78.89
(CH2), 116.90 (C-5), 124.61, 124.95, 126.60, 128.89, 134.91, 138.80, 140.49 (Carom), 149.43 (C-6),
152.06 (C-2), 163.54 (C-4) ppm; MS (MALDI): m=z ¼ 427 (Mþ Naþ).
1-(Indan-2-yloxymethyl)-5-isopropyl-6-(3,5-dimethylbenzyl)uracil (6f, C26H30N2O3)
White foam; yield 0.268 g (64%); 1H NMR (CDCl3, 300MHz): ꢂ ¼ 1.29 (d, J ¼ 7.0 Hz, 6H, 2 ꢃ CH3),
2.28 (s, 6H, 2ꢃCH3), 2.85 (hept, J ¼ 7.0Hz, 1H, CH), 2.92, 3.15 (2dd, J ¼ 3.9, 16.5Hz, J ¼ 6.4,
16.5Hz, 4H, 2ꢃCH2), 4.08 (s, 2H, CH2), 4.58 (quint, J ¼ 3.4 Hz, 1H, 20-H), 5.20 (s, 2H, CH2),
6.68 (s, 2Harom), 6.89 (s, 1Harom), 7.13–7.25 (m, 4Harom), 9.40 (s, 1NH) ppm; 13C NMR (CDCl3,
75MHz): ꢂ ¼ 20.42 (CH3), 21.24 (CH3), 28.32 (CH), 33.28 (CH2), 39.52 (CH2), 71.59 (CH), 78.87
(CH2), 119.70 (C-5), 124.63, 124.96, 126.61, 128.79, 135.02, 138.75, 140.51 (Carom), 148.81 (C-6),
152.01 (C-2), 162.49 (C-4) ppm; MS (MALDI): m=z ¼ 441 (Mþ Naþ).
Bis(4-p-toluoyloxy-cis-2-buten-1-yloxy)methane (8, C25H28O6)
A mixture of 5.66g KOH (0.101mol), 8.88g of cis-2-butene-1,4-diol (7, 0.10mol), 3.54cm3 of
dibromomethane (0.0505 mol), and 1.74 g of tetrabutylammonium bromide (5.35 mmol) was heated
under reflux in 50cm3 of anhydrous benzene for 4 h. The reaction mixture was left to cool and 100cm3
H2O were added. The mixture was extracted with 3ꢃ100 cm3 Et2O, and the combined Et2O extracts
were dried (MgSO4), and evaporated under reduced pressure to give 2.75 g (29%) of bis(4-hydroxy-cis-
2-buten-1-yloxy)methane, which was stirred in 20cm3 of anhydrous pyridine at 0ꢂC, when 4.67g of p-
toluoyl chloride (0.0302 mol) were added slowly. The reaction mixture was stirred at 0ꢂC for 2 h, and
left at room temperature for overnight. The solvent was removed under reduced pressure and the
residue was extracted with 3ꢃ100 cm3 Et2O. The Et2O fractions were collected, dried (MgSO4), and
evaporated under reduced pressure to give compound 8 as a colourless oil in 22% (4.7g) overall yield,
which was used in the next step without further purification.
5,6-Disubstituted 1-(4-p-Toluoyloxy-cis-2-buten-1-yloxymethyl)uracils 9a and 9b
5,6-Disubstituted uracils 5a and 5b (1.0mmol) were stirred in 15cm3 dry CH3CN under N2, and
0.87cm3 BSA (3.5mmol) were added. After a clear solution was obtained (10 min), the mixture was
cooled to ꢁ50ꢂC and 0.18cm3 TMS triflate (1.0mmol) was added followed by addition of 0.85g 8
(2.0mmol). The reaction mixture was stirred under N2 at room temperature for 6 h. Cold aq. NaHCO3
solution (5cm3) was added, and the solvent was evaporated under reduced pressure. The residue was
extracted with 3ꢃ50 cm3 Et2O. The Et2O extracts were dried (MgSO4), evaporated under reduced
pressure, and the residue was purified by chromatography on a silica gel column (20% Et2O in
petroleum ether (60–80ꢂC)) to afford 9a and 9b.

Analogues of the HIV Drugs Emivirine and GCA-186
1253
6-Benzyl-5-isopropyl-1-(4-p-toluoyloxy-cis-2-buten-1-yloxymethyl)uracil
(9a, C27H30N2NaO5)
1
Obtained as an oil; yield 0.328 g (71%); H NMR (CDCl3, 300 MHz): ꢂ ¼ 1.28 (d, J ¼ 7.0 Hz, 6H,
2ꢃCH3), 2.40 (s, 3H, CH3), 2.82 (hept, J ¼ 7.0 Hz, 1H, CH), 4.29 (s, 2H, CH2), 4.31 (d, J ¼ 6.3 Hz,
2H, CH2), 4.85 (d, J ¼ 6.0 Hz, 2H, CH2), 5.16 (s, 2H, CH2), 5.70–5.87 (m, 2H, 2ꢃCH), 7.10–7.36 (m,
7Harom), 7.90 (d, J ¼ 8.2 Hz, 2Harom), 9.13 (brs, 1NH) ppm; 13C NMR (CDCl3, 75MHz): ꢂ ¼ 20.36
(CH3), 21.61 (CH3), 28.34 (CH), 33.53 (CH2), 60.47 (CH2), 65.27 (CH2), 72.83 (CH2), 119.85 (C-5),
127.23, 127.61, 129.04, 129.20, 129.56, 129.61, 135.25, 143.64 (Carom, CH), 148.34 (C-6), 151.83
(C-2), 162.36 (C-4), 166.30 (CO) ppm; HRMS (MALDI, peak matching): m=z ¼ [Mþ Na]þ calcd
485.2033, found 485.2047.
5-Ethyl-6-(3,5-dimethylbenzyl)-1-(4-p-toluoyloxy-cis-2-buten-1-yl-oxymethyl)uracil
(9b, C28H32N2O5)
White solid; yield 0.322 g (68%); mp 132–134ꢂC; 1H NMR (CDCl3, 300 MHz): ꢂ ¼ 1.05 (t,
J ¼ 7.3 Hz, 3H, CH3), 2.28 (s, 6H, 2ꢃCH3), 2.39 (s, 3H, CH3), 2.44 (q, J ¼ 7.3 Hz, 2H, CH2), 4.08
(s, 2H, CH2), 4.30 (d, J ¼ 5.6 Hz, 2H, CH2), 4.86 (d, J ¼ 5.7 Hz, 2H, CH2), 5.16 (s, 2H, CH2), 5.74–
5.85 (m, 2H, 2ꢃCH), 6.71 (s, 2Harom), 6.88 (s, 1Harom), 7.20 (d, J ¼ 8.0 Hz, 2Harom), 7.90 (d,
J ¼ 8.0 Hz, 2Harom), 9.90 (brs, 1NH) ppm; 13C NMR (CDCl3, 75MHz): ꢂ ¼ 13.68 (CH3), 19.10
(CH2), 21.20 (CH3), 21.55 (CH3), 33.23 (CH2), 60.44 (CH2), 65.25 (CH2), 72.65 (CH2), 116.89 (C-
5), 124.94, 127.22, 128.90, 129.56, 129.60, 134.82, 143.55 (Carom), 127.53 (CH), 138.80 (CH), 149.15
(C-6), 152.04 (C-2), 163.51 (C-4), 166.23 (CO) ppm; MS (EI): m=z ¼ 476 (Mþ).
6-Benzyl-1-(4-hydroxy-but-2-en-1-yloxymethyl)-5-isopropyluracil (10, C19H24N2O4)
Compound 9a (0.462 g, 1.0 mmol) was dissolved in a solution prepared from 0.026 g Na (1.1mmol)
and 15 cm3 anhydrous CH3OH. The mixture was stirred at room temperature for 2 h. The solvent was
evaporated under reduced pressure and the residue was purified by chromatography on a silica gel
1
column with CHCl3 to afford compound 10 as a colourless oil in 66% (0.227g) yield. H NMR
(CDCl3, 300 MHz): ꢂ ¼ 1.28 (d, J ¼ 6.8 Hz, 6H, 2ꢃCH3), 2.86 (hept, J ¼ 6.8 Hz, 1H, CH), 3.17 (brs,
1OH), 4.16–4.26 (m, 6H, 3ꢃCH2), 5.15 (s, 2H, CH2), 5.50–5.58 (m, 1H, CH), 5.80–5.89 (m, 1H, CH),
7.10–7.37 (m, 5Harom), 9.94 (s, 1NH) ppm; 13C NMR (CDCl3, 75MHz): ꢂ ¼ 20.35 (CH3), 28.30 (CH),
33.48 (CH2), 57.91 (CH2), 64.98 (CH2), 72.21 (CH2), 120.30 (C-5), 127.17 (CH), 127.22, 129.19,
135.20 (Carom), 133.22 (CH), 148.40 (C-6), 152.60 (C-2), 162.40 (C-4) ppm; HRMS (MALDI, peak
matching): m=z ¼ [Mþ Na]þ calcd 367.1630, found 367.1623.
Viruses and Cells
The inhibitory activity against HIV-1 infection was evaluated using MT-4 cells [21] as target cells and
the HIV-1 strain HTLV-IIIB [22] and the NNRTI resistant strain N119 [23] as infectious virus. The
virus was propagated in H9 [21] cells at 37ꢂC, 5% CO2 using RPMI 1640 with 10% heat-inactivated
fetal calf serum (FCS) and antibiotics (growth medium). Culture supernatant was filtered (0.45 nm),
aliquoted, and stored at ꢁ80ꢂC until use. Both HIV-1 strains were obtained from the NIH AIDS
Research and Reference Program.
Inhibition of HIV-1 Replication
Compounds were examined for possible antiviral activity against both strains of HIV-1 using MT-4
cells as target cells. MT-4 cells were incubated with virus (0.005 MOI) and growth medium containing
the test dilutions of compounds for six days in parallel with virus-infected and uninfected control
cultures without compound added. Expression of HIV in the cultures was indirectly quantified using
the MTT assay [24]. Compounds mediating less than 30% reduction of HIV expression were con-
sidered without biological activity. Compounds were tested in parallel for cytotoxic effect in uninfected
MT-4 culture containing the test dilutions of compound as described above. A 30% inhibition of cell
growth relative to control cultures was considered significant. The 50% inhibitory concentration (EC50)

1254
N. R. El-Brollosy et al.: Analogues of the HIV Drugs Emivirine and GCA-186
and 50% cytotoxic concentration (CC50) were determined by interpolation from the plots of percent
inhibition versus concentration of compound.
Acknowledgements
The Royal Danish Ministry of Foreign Affairs is gratefully acknowledged for financial support.
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