Synthesis and evaluation of new potential HIV-1 non-nucleoside reverse transcriptase inhibitors. New analogues of MKC-442 containing Michael acceptors in the C-6 position

Article abstract of DOI:10.1039/b307800k

Analogues of MKC-442 capable of undergoing Michael addition reactions were synthesised in order to investigate the activity against the HIV-1 mutant (Y181C). An improved activity was postulated on the basis of a possible covalent binding to the mercapto group of Cys181. Lithiation of the C-6 position of l-ethoxymethyl-5-ethyl-1Hpyrimidine-2,4-dione (5) was followed by reaction with α,β-unsaturated aldehydes and oxidation of the alcohols formed to give the alkenoyl analogues 1a-3a. Analogues 1b-3b containing an allyloxymethyl group in the N-l position instead of the ethoxymethyl group could not be synthesised due to isomerisation of the allylic group during the metallation reaction. The NMR data for compounds 1a-3a showed a hindered rotation, which was more pronounced for the 6-cyclohexenylcarbonyl derivative 3a than for the propenyl derivatives la and 2a. Moderate activity against wild type HIV-1 was observed for the alcohol 8 and the ketones 2a-3a. However, no activity was observed against the Y181C mutant.

Full text of DOI:10.1039/b307800k

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Synthesis and evaluation of new potential HIV-1 non-nucleoside
reverse transcriptase inhibitors. New analogues of MKC-442
containing Michael acceptors in the C-6 position
Lene Petersen,a Carsten H. Jessen,a Erik B. Pedersen*a and Claus Nielsenb
a Nucleic Acid Center,† Department of Chemistry, University of Southern Denmark,
Campusvej 55, 5230 Odense M, Denmark.
E-mail: ebp@chem.sdu.dk; Fax: (ϩ45) 66158780; Tel: (ϩ45) 65502555
b Retrovirus Laboratory, Department of Virology, State Serum Institute, Artillerivej 5,
2300 Copenhagen, Denmark
Received 10th July 2003, Accepted 27th August 2003
First published as an Advance Article on the web 15th September 2003
Analogues of MKC-442 capable of undergoing Michael addition reactions were synthesised in order to investigate
the activity against the HIV-1 mutant (Y181C). An improved activity was postulated on the basis of a possible
covalent binding to the mercapto group of Cys181. Lithiation of the C-6 position of 1-ethoxymethyl-5-ethyl-1H-
pyrimidine-2,4-dione (5) was followed by reaction with α,β-unsaturated aldehydes and oxidation of the alcohols
formed to give the alkenoyl analogues 1a–3a. Analogues 1b–3b containing an allyloxymethyl group in the N-1
position instead of the ethoxymethyl group could not be synthesised due to isomerisation of the allylic group
during the metallation reaction. The NMR data for compounds 1a–3a showed a hindered rotation, which was
more pronounced for the 6-cyclohexenylcarbonyl derivative 3a than for the propenyl derivatives 1a and 2a.
Moderate activity against wild type HIV-1 was observed for the alcohol 8 and the ketones 2a–3a. However,
no activity was observed against the Y181C mutant.
A major problem when using MKC-442 is the rapid selection
Introduction
of resistant viruses, mainly harbouring the RT mutation
Y181C.1 Tyr181 is important for the activity of MKC-442 as
the isopropyl group in the C-5 position of MKC-442 induces
a shift of Tyr181, which alters the conformation of the enzyme
and thereby inactivates it. After the shift Tyr181 is positioned
inside a sub-pocket where it can interact with other aromatic
amino acids (Tyr183, Tyr188, Phe227, Trp229) and the C-6
substituent of MKC-442.5
Analogues of MKC-442 have been developed that show
activity against the Y181C mutated virus. TNK-6123 and
GCA-186 (Fig. 1) were developed by Hopkins et al.6 and
showed activities comparable to MKC-442 against wild-type
viruses, whereas they retained some activity against the Y181C
mutated virus in contrast to MKC-442 which lost all its activity.
It was shown from crystal structures of the two inhibitors in
a complex with RT that the two methyl groups of the C-6
substituent of GCA-186 and the flexible C-6 cyclohexyl ring of
TNK-6123 had improved contacts to the sub-pocket where the
C-6 substituent was positioned.
MKC-4421 (also known as Emivirine or Coactinon, Fig. 1)
belongs to the group of HIV-1 non-nucleoside reverse
transcriptase inhibitors (NNRTIs). This group of compounds
inhibits HIV-1 reverse transcriptase (RT) allosterically by
binding to a hydrophobic pocket situated approximately 10 Å
away from the active site.2 MKC-442 was selected for clinical
trials by Triangle Pharmaceuticals,3 but the compound was
withdrawn from clinical phase III trials in January 2002 as it
did not have superior properties compared to Abacavir—
a nucleoside reverse transcriptase inhibitor (NRTI) that was
used as a comparative drug.4
In our group we developed analogues of GCA-186 that
contained unsaturated moieties in the N-1 position (BED-60
and AMB-A10, Fig. 1).7 These analogues showed excellent
activities against wild-type HIV-1 and retained some activity
against the Y181C mutant.
We have previously synthesised analogues of MKC-442
containing reactive groups (epoxides or aldehydes) in the C-5
position.8 The rationale was that the epoxides or aldehydes
should react inside the enzyme forming a covalent bond with
Cys181 introduced by mutation. Unfortunately, the compounds
were not active, either against wild-type or mutant viruses. The
epoxides were chemically very reactive and therefore difficult to
synthesise. The high reactivity was believed to be unfavourable
in the biological perspective under aqueous conditions where
hydrolysis could take place before the drugs were positioned
inside the enzyme.
Fig. 1 Structures of the lead molecules MKC-442, GCA-186, TNK-
6123, BED-60 and AMB-A10.
In this paper we report the synthesis and activities of new
analogues of MKC-442 with potential activity against HIV-1
† A research center founded by The Danish Research Foundation for
studies on nucleic acid chemical biology.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 4 1 – 3 5 4 5
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containing the RT mutation Y181C. We introduced a Michael
acceptor in the C-6 position in the hope that a reaction would
take place between Cys181 of the mutated enzyme and the
Michael acceptor. To study the effect of bulkiness of the C-6
group we chose the targets 1–3 (Fig. 2). In addition to the
possible reaction between the Michael acceptor and Cys181
the hope was that the cyclohexene ring of target 3 would fill out
the RT sub-pocket in analogy with TNK-6123.
Because of the highly conjugated system of 3a it was decided
to verify the quality of the conformations generated using force
field calculations. MP2/6-31g* ab initio optimisations of the two
chosen conformations were performed and the results corre-
sponded excellently to the force field conformations, verifying
that the carbonyl of 3a is placed perpendicular to the uracil ring
in despite of conjugation with the C5–C6 double bond.
5-Ethyl-1H-pyrimidine-2,4-dione (4) was synthesised on a
gram scale in a three step synthesis starting from ethyl butyrate
and ethyl formate.14,15 Compound 4 was silylated using N,O-
bis(trimethylsilyl)acetamide (BSA) and was reacted with
chloromethyl ethyl ether and CsI to give compound 5 in 92%
yield (Scheme 1).
Fig. 2 Target molecules 1–3.
Scheme 1 Reagents and conditions: a) BSA; b) ClCH2OCH2CH3, CsI,
92%.
Results and discussion
To visualise the possibility of a reaction taking place between
the enzyme and the inhibitor, we did modelling studies on com-
pound 3a using MacroModel.9 Because there are no available
crystal structures of MKC-442 with the mutated RT (Y181C)
the study was based on the crystal structure of TNK-651 (an
analogue of MKC-442 where the N-1 ethoxymethyl group was
replaced by a N-1 benzyloxymethyl group) in complex with
the mutated RT (Y181C).10 It was believed that the difference in
the N-1 substituent would not affect the binding conformation
of the C-6 substituent as is the case for the wild-type RT.
A conformational analysis of 3a was performed based on
molecular dynamics11 followed by multiple minimisations of
the generated structures using MMFF.12 The generated con-
formations could be divided into two groups. In both groups
the uracil ring and the plane of the carbonyl group were placed
perpendicular to each other, but for one the carbonyl was
pointing out of the page and for the other the carbonyl
was pointing into the page (Fig. 3).
Lithiation of the C-6 position of compound 5 and reaction
of this intermediate directly with the acid chloride (3-methyl-
but-2-enoyl chloride) to give target compound 2a was
attempted, but only starting material was isolated after
work-up. Instead we used aldehydes as electrophiles. The best
conditions were found to be the use of 4 eq. of LDA and 6 eq.
of the electrophile. Using these conditions we isolated the
alcohols 6–8 in 50%–61% yield.16 The alcohols were oxidised
using CrO3, pyridine and Ac2O and the target compounds 1a,
2a and 3a were isolated in 64%–75% yield, (Scheme 2).17
Scheme 2 Reagents and conditions: a) DIA, n-BuLi; b) R2–CHO, 6
(50%), 7 (56%), 8 (61%); c) CrO3, pyridine, Ac2O, 1a (69%), 2a (64%),
3a (75%). R2 as defined in Fig. 2.
An interesting feature was observed in the NMR spectra of
compounds 1–3. The NCH2O group of the N-1 substituent was
seen as two separate doublets in the spectra of compound 3a
whereas the same CH2 group appeared as a very broad singlet
in the spectra of compounds 1a and 2a (Fig. 4).
Fig. 3 Models of the two main conformations of compound 3a. The
structures were superimposed into the complex between TNK-651 and
the Y181C mutated RT. The distance to Cys181 is illustrated.
The uracil rings of the two main conformations were both
superimposed onto the uracil ring of TNK-651 in complex with
Y181C mutated RT. This was based on the fact that the uracil
rings of different MKC-442 analogues all occupy the same
volume inside wild-type RT.5 Using RasMol13 the orientation
of Cys181 was altered to illustrate the shortest possible distance
between the reactive double-bond and the thiol of the cysteine.
For the conformation having the carbonyl pointing out of the
page (left model, Fig. 3) the distance was measured to 5.8 Å,
which is quite large. However, for the conformation having the
carbonyl pointing into the page the distance was only 3.9 Å,
which made a possible reaction more likely. This structure
also resembled the structure of MKC-442 in complex with
wild-type RT.5
Fig. 4 NMR spectra of compound 3a (upper) and compound 1a
(lower). The NCH2O group is seen as two doublets for compound 3a
whereas the same group is seen as a very broad, flat peak for compound
1a. Vinylic peaks are included for comparison of intensity. The peak at
5.3 (upper spectra) is CH2Cl2 impurity.
These observations could be explained by the calculations
described above for compound 3a where the pyrimidine ring
and the cyclohexenyl ring were shown to be positioned per-
pendicular to each other. We believed that this conformation
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 4 1 – 3 5 4 5
3542

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Table 1
would be more or less locked depending on the size of the C-6
substituent. For a completely locked conformation the two
protons are diastereotopic and can appear as two distinct
doublets as observed for compound 3a. Free rotation around
the bond connecting the pyrimidine ring and the carbonyl
system would make these two protons appear as a singlet. To
prove the hypothesis we heated an NMR sample of compound
1a to increase the degree of rotation in the molecule. We saw a
narrowing of the broad singlet of the CH2 group when heating
to 40 ЊC and at 60 ЊC it appeared as a nice singlet (Fig. 5),
showing a higher degree of flexibility in the structures of
compounds 1a and 2a.
Compound
IC50 a/µM wild type
IC50 a/µM Y181C
CC50 b/µM
6
>100
100
3.8
>100
3.9
NDc
NDc
NDc
>100
>100
>100
>100
>100
>100
>100
30
>100
>100
>100
7
8
1a
2a
3a
3.1
0.031
MKC-442
a 50% Inhibitory concentration. b 50% Cytotoxic concentration.
c Not determined.
against the wild-type virus was observed. The most bulky
alcohol 8 and the more bulky ketones 2a and 3a showed activ-
ities of 3.1–3.9 µM. However, these activities were still a factor
of 100 less than the activity of MKC-442. No activity against
the Y181C mutant was observed for the three ketones 1a, 2a
and 3a. It is therefore unlikely that a reaction has taken place
inside the enzyme between Cys181 and the new Michael
acceptor analogues.
Experimental
General methods
Fig. 5 NMR spectra of compound 1a at 30 ЊC (upper), 40 ЊC (middle)
and 60 ЊC (lower). The NCH2O peak is narrowing upon heating. The
vinylic protons are included for comparison of intensity.
NMR spectra were recorded on a Varian Gemini 2000 NMR
spectrometer. 1H NMR spectra were recorded at 300 MHz and
13C NMR spectra were recorded at 75 MHz. Internal standards
used in 1H NMR spectra were tetramethylsilane (TMS, δ 0.00);
in 13C NMR were CDCl3 (δ 77.00), DMSO-d6 (δ 39.44).
MALDI mass spectra were recorded on a 4.7 Tesla Ultima
(IonSpec, Irvine, CA) Fourier transform ion cyclotron
resonance (FTICR) mass spectrometer. Melting points were
determined on a Büchi melting point apparatus. Elemental
analysis were performed at Mikroanalytisk afdeling, University
of Copenhagen, Copenhagen, Denmark. The progress of
reactions was monitored by TLC (analytical silica gel plates 60
F254). Merck silica gel (0.040–0.063 mm) was used for column
chromatography. Solvents for chromatography were bought as
HPLC grade or distilled prior to use. Reactions were in general
carried out under a N2 or Ar atmosphere. Pyridine was dried
over KOH. CH3CN was dried over 3 Å molecular sieves.
CH2Cl2 and CHCl3 were dried over 4 Å molecular sieves. THF
was refluxed over Na using benzophenone as an indicator and
distilled immediately before use.
For the synthesis of target compounds 1b, 2b and 3b we
needed to introduce an N-1 allyloxymethyl group. The starting
compound 4 was silylated using BSA and it was alkylated in the
N-1 position using bis(allyloxy)methane7 under Vorbrüggen
conditions.18 Compound 9 was isolated in 93% yield (Scheme 3).
Compound 9 was treated with LDA followed by addition of the
aldehydes, but the expected compounds were not isolated.
Complex mixtures were formed and it was not possible to
identify the main product as it could not be isolated as the pure
compound. For the reaction with cyclohex-1-enecarbaldehyde
as an electrophile the mixture was directly oxidised in an
attempt to isolate a pure compound after this reaction. The
only product isolated in 2% yield after HPLC purification was
identified as compound 1019 (Scheme 3). Apparently the LDA
treatment isomerised the allylic group of compound 9.
1-Ethoxymethyl-5-ethyl-1H-pyrimidine-2,4-dione (5)
BSA (7.12 g, 35 mmol) was dissolved in dry CHCl3 (100 cm3)
and 5-ethyl-1H-pyrimidine-2,4-dione (4, 1.40 g, 10 mmol) was
added. The mixture was stirred for 10 min and then chloro-
methyl ethyl ether (1.42 g, 15 mmol) and CsI (2.60 g, 10 mmol)
were added to the clear solution. The reaction mixture was
stirred for 3 h and then quenched by the addition of sat. aq.
NaHCO3 (100 cm3). The phases were separated and the
aqueous phase was extracted with CH2Cl2 (2 × 100 cm3). The
combined organic phases were dried (MgSO4) and evaporated
under reduced pressure. The product was purified by column
chromatography (silica gel, 5% EtOH–CH2Cl2) to give com-
pound 5 (1.82 g, 92%) as a white powder; mp = 93–95 ЊC (lit.20
mp = 104.8–105.5 ЊC). NMR data were consistent with those
previously reported.20
Scheme 3 Reagents and conditions: a) BSA; b) (CH2᎐᎐CHCH2O)2CH2,
TMS–triflate, 93%; c) DIA, n-BuLi; d) cyclohex-1-enecarbaldehyde;
e) CrO3, pyridine, Ac2O.
General procedure for the synthesis of alcohols 6–8
The test for activity against HIV-1 was performed in MT4
cell cultures infected with either wild-type HIV-1 or the HIV-1
strain N119 harbouring a substitution of cysteine for tyrosine
at position 181 (Table 1). For the alcohols 6 and 7 and the
ketone 1a with the least bulky C-6 substituent, no activity
DIA (1.01 g, 10 mmol) was dissolved in dry THF (30 cm3). The
solution was cooled to Ϫ78 ЊC and n-BuLi (4.44 cm3, 2.25 M
solution in hexanes, 10 mmol) was added. 1-Ethoxymethyl-5-
ethyl-1H-pyrimidine-2,4-dione (5, 0.50 g, 2.5 mmol) was
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 4 1 – 3 5 4 5
3543

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dissolved in dry THF (10 cm3) and added slowly, keeping the
temperature below Ϫ70 ЊC. The mixture was stirred for an
additional 1 h at Ϫ78 ЊC followed by slow addition of the alde-
hyde (15 mmol) in dry THF (5 cm3), still keeping the temper-
ature below Ϫ70 ЊC. The mixture was stirred for 3 h at the same
temperature before the reaction was quenched by addition
of glacial acetic acid (1.5 cm3). The solvent was evaporated
under reduced pressure. The residue was redissolved in CH2Cl2
(80 cm3) and the organic phase was washed with H2O (2 × 50
cm3). The organic phase was dried (MgSO4) and evaporated
under reduced pressure. The product was purified by column
chromatography (silica gel, 2% EtOH–CH2Cl2).
4 h and overnight. Then the mixture was poured into EtOAc
(25 cm3) and the mixture was filtered through silica. The silica
was rinsed with EtOAc until no more UV-activity was
observed. The products were purified by column chromato-
graphy (silica gel, 2% EtOH–CH2Cl2).
6-But-2-enoyl-1-ethoxymethyl-5-ethyl-1H-pyrimidine-2,4-dione
(1a)
Yield: 0.137 g (69%), white powder; mp = 150–153 ЊC (EtOAc–
PE (60–80)); Rf 0.34 (5% EtOH–CH2Cl2); δH(300 MHz; CDCl3;
Me4Si) 1.03 (3 H, t, J 7.4, ArCH2CH3), 1.13 (3 H, t, J 7.0,
OCH2CH3), 2.03 (3 H, dd, J 1.3, 6.8, CHCH3), 2.26 (2 H, m,
ArCH2), 3.52 (2 H, q, J 7.0, OCH2CH3), 4.8–5.5 (2 H, br s,
NCH2O), 6.37 (1 H, dd, J 1.8, 15.9, COCH), 6.89–7.01 (1 H, m,
CHCH3), 9.48 (1 H, s, NH); δC(75 MHz; CDCl3; Me4Si) 13.4
(ArCH2CH3), 14.8 (OCH2CH3), 18.8 (CHCH3), 19.0 (ArCH2),
64.4 (OCH2), 73.2 (NCH2O), 114.3 (C-5), 131.7 (COCH), 146.6
(C-2), 150.7 (CHCH3), 150.8 (C-6), 163.0 (C-4), 189.3 (CO);
m/z (MALDI MS) 289.1 (M ϩ Na)ϩ, 267.1 (M ϩ H)ϩ;
C13H18N2O4; calcd.: C 58.64, H 6.81, N 10.52; found: C 58.39,
H 6.79, N 10.28%.
1-Ethoxymethyl-5-ethyl-6-(1-hydroxybut-2-enyl)-1H-pyrim-
idine-2,4-dione (6)
Yield: 0.34 g (50%), light-brown glace; Rf 0.31 (5% EtOH–
CH2Cl2); δH(300 MHz; CDCl3; Me4Si) 1.09 (3 H, t, J 7.4,
ArCH2CH3), 1.23 (3 H, t, J 7.1, OCH2CH3), 1.78 (3 H, dt, J 6.2,
1.6, CHCH3), 2.48–2.62 (2 H, m, ArCH2), 3.62–3.80 (2 H, m,
OCH CH ), 4.53 (1 H, br s, OH), 5.47–5.96 (5 H, m, CH᎐CH,
᎐
2
3
NCH2O, CHOH), 9.27 (1 H, br s, NH); δC(75 MHz; CDCl3;
Me4Si) 14.3 (ArCH2CH3), 14.9 (OCH2CH3), 17.8 (CHCH3),
18.9 (ArCH2), 65.2 (OCH2), 69.0 (CHOH), 72.5 (NCH2O),
1-Ethoxymethyl-5-ethyl-6-(3-methyl-but-2-enoyl)-1H-pyrim-
117.0 (C-5), 128.3, 130.1 (CH᎐CH), 150.6, 151.9 (C-2, C-6),
idine-2,4-dione (2a)
᎐
163.3 (C-4); m/z (MALDI MS (peak matching)) 291.1315
Yield: 0.135 g (64%), white needles; mp =110–111 ЊC (Et2O–PE
(60–80)); Rf 0.31 (5% EtOH–CH2Cl2); δH(300 MHz; CDCl3;
Me4Si) 1.06 (3 H, t, J 7.4, ArCH2CH3), 1.14 (3 H, t, J 7.0,
OCH2CH3), 2.02 (3 H, d, J 1.1, CH3), 2.28 (3 H, d, J 0.7, CH3),
2.31 (2 H, q, J 7.6, ArCH2), 3.52 (2 H, q, J 6.9, OCH2CH3), 4.9–
5.5 (2 H, br s, NCH2O), 6.24–6.27 (1 H, m, COCH), 9.28 (1 H,
br s, NH); δC(75 MHz; CDCl3; Me4Si) 13.6 (ArCH2CH3), 14.7
(OCH2CH3), 18.7 (CH3-cis), 21.7 (ArCH2), 28.2 (CH3-trans),
64.4 (OCH2CH3), 73.0 (NCH2O), 113.1 (C-5), 123.8 (COCH),
(M ϩ Na)ϩ, calcd. 291.1315.
1-Ethoxymethyl-5-ethyl-6-(1-hydroxy-3-methylbut-2-enyl)-1H-
pyrimidine-2,4-dione (7)
Yield: 0.40 g (56%), light-brown glace; Rf 0.22 (5% EtOH–
CH2Cl2); δH(300 MHz; CDCl3; Me4Si) 1.10 (3 H, t, J 7.4,
ArCH2CH3), 1.23 (3 H, t, J 7.1, OCH2CH3), 1.78 (3 H, s, CH3),
1.80 (3 H, s, CH3), 2.48–2.70 (2 H, m, ArCH2), 3.71 (2 H, q,
J 7.0, OCH2CH3), 3.64 (1 H, br s, OH), 5.52–5.57, 5.68–5.74 (4
148.8, 150.8, 161.8, 163.4 (CH᎐C, C-2, C-6, C-4), 187.7 (CO);
᎐
H, 2 × m, CH᎐C, NCH O, CHOH), 9.27 (1 H, br s, NH); δ (75
m/z (MALDI MS) 303.1 (M ϩ Na)ϩ, 281.1 (M ϩ H)ϩ;
C14H20N2O4; calcd.: C 59.99, H 7.19, N 9.99; found: C 59.67, H
7.18, N 9.89%.
᎐
2
C
MHz; CDCl3; Me4Si) 13.9 (ArCH2CH3), 14.9 (OCH2CH3),
18.5 (CH3-cis), 19.0 (ArCH2), 26.1 (CH3-trans), 65.2 (CHOH),
66.5 (OCH CH ), 73.0 (NCH O), 116.4 (C-5), 123.4 (CH᎐C),
᎐
2
3
2
139.8 (CH᎐C), 151.5, 151.9 (C-2, C-6), 163.5 (C-4); m/z
6-(Cyclohex-1-enecarbonyl)-1-ethoxymethyl-5-ethyl-1H-pyrim-
idine-2,4-dione (3a)
᎐
(MALDI MS) 305.1 (M ϩ Na)ϩ, 283.2 (M ϩ H)ϩ; C14H22N2O4;
calcd.: C 59.56, H 7.85, N 9.92; found: C 59.72, H 7.91, N
9.57%.
Yield: 0.172 g (75%), light-yellow glace; Rf 0.36 (5% EtOH–
CH2Cl2); δH(300 MHz; CDCl3; Me4Si) 1.02 (3 H, t, J 7.2,
ArCH2CH3), 1.11 (3 H, t, J 7.0, OCH2CH3), 1.62–1.80 (4 H, m,
2 × CH2), 2.07–2.30 (2 H, m, ArCH2CH3), 2.28–2.42 (4 H, m, 2
× CH2), 3.41–3.59 (2 H, m, OCH2CH3), 4.87 (1 H, d, J 10.4,
NCHHO), 5.36 (1 H, d, J 10.4, NCHHO), 6.91 (1 H, t, J 3.9,
6-(Cyclohex-1-enyl-hydroxymethyl)-1-ethoxymethyl-5-ethyl-1H-
pyrimidine-2,4-dione (8)
Yield: 0.47 g (61%), light-yellow foam; Rf 0.29 (5% EtOH–
CH2Cl2); δH(300 MHz; CDCl3; Me4Si) 1.12 (3 H, t, J 7.4,
ArCH2CH3), 1.23 (3 H, t, J 7.1, OCH2CH3), 1.70–2.16 (8 H, m,
4 × CH2), 2.40–2.52, 2.58–2.72 (2 H, 2 × m, ArCH2CH3),
3.60–3.80 (2 H, m, OCH2CH3), 4.76, 5.36 (2 × 1 H, 2 × d, J 9.2,
CHOH), 5.42 (1 H, d, J 11.1, NCHHO), 5.64 (1 H, d, J 11.0,
NCHHO), 5.92–6.00 (1H, m, CH᎐C), 9.49 (1H, s, NH); δ (75
CH᎐C), 9.28 (1 H, s, NH); δ (75 MHz; CDCl ; Me Si) 13.3
᎐
C
3
4
(ArCH2CH3), 14.8 (OCH2CH3), 19.3 (ArCH2), 21.4, 21.5, 22.1,
26.5 (4 × CH2), 64.4 (OCH2CH3), 73.4 (NCH2O), 114.3 (C-5),
139.1 (COC), 146.8 (C-2), 148.9 (C᎐CH), 150.9 (C-6), 162.8
᎐
(C-4), 190.6 (CO); m/z (MALDI MS) 329.1 (M ϩ Na)ϩ, 307.2
(M ϩ H)ϩ; C16H22N2O4, 0.50 H2O; calcd.: C 60.94, H 7.03, N
8.88; found: C 61.19, H 7.01, N 8.76%.
᎐
C
MHz; CDCl3; Me4Si) 14.0 (ArCH2CH3), 14.8 (OCH2CH3),
19.1 (ArCH2), 22.1, 22.4, 24.9, 25.4 (4 × CH2), 65.3 (OCH2-
CH ), 71.6, 71.9 (NCH O, CHOH), 118.0 (C-5), 122.9 (CH᎐C),
1-Allyloxymethyl-5-ethyl-1H-pyrimidine-2,4-dione (9)
᎐
3
2
136.1 (CH᎐C), 150.2, 151.9 (C-2, C-6), 163.2 (C-4); m/z
᎐
BSA (5.09 g, 25 mmol) was dissolved in dry CH3CN (50 cm3). 5-
Ethyl-1H-pyrimidine-2,4-dione (4, 1.40 g, 10 mmol) was added
and the mixture was stirred for 10 min. The resulting clear
solution was cooled to Ϫ45 ЊC. TMS–triflate (2.22 g, 10 mmol)
was added followed by addition of bis(allyloxy)methane (2.56
g, 20 mmol) dissolved in dry CH3CN (2 cm3). The solution was
stirred overnight at rt. The reaction was quenched by the
addition of sat. aq. NaHCO3 (10 cm3). The solvent was evapor-
ated and the residue was partitioned between CH2Cl2 (100 cm3)
and H2O (50 cm3). The aqueous phase was further extracted
with 2 × 100 cm3 CH2Cl2. The combined organic phases were
dried (MgSO4) and evaporated under reduced pressure. The
product was purified by column chromatography (silica gel,
(MALDI MS) 331.2 (M ϩ Na)ϩ, 309.2 (M ϩ H)ϩ; C16H24N2O4,
0.25 H2O; calcd.: C 61.42, H 7.73, N 8.95; found: C 61.51, H
7.76, N 8.70%.
General procedure for synthesis of ketones 1a, 2a and 3a
CrO3 (0.128 g, 1.28 mmol) was pulverised and suspended in dry
CH2Cl2 (4 cm3) and the solution was cooled on ice. Anhydrous
pyridine (0.228 g, 2.88 mmol) and Ac2O (0.150 g, 1.47 mmol)
were dissolved in dry CH2Cl2 (1 cm3) and added slowly to the
cold CrO3 suspension. The mixture was stirred at rt for 45 min.
The alcohols (6–8, 0.75 mmol) were dissolved in dry CH2Cl2
(2 cm3) and added slowly. The solution was stirred at rt between
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 5 4 1 – 3 5 4 5
3544

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3 G. M. Szcech, P. Furman, G. R. Painter, D. W. Barry, K. Borroto-
5% EtOH–CH2Cl2) to give 9 (1.96 g, 93%) as a clear oil which
crystallised on standing. δH(300 MHz; CDCl3; Me4Si) 1.15
(3 H, t, J 7.6, CH3), 2.38 (2 H, dq, J 1.1, 7.5, CH2CH3), 4.10
(2 H, dt, J 1.4, 5.6, OCH2CH), 5.18 (2 H, s, NCH2O), 5.23 (1 H,
dq, J 1.4, 10.4, CH-trans to CH2), 5.33 (1 H, dq, J 1.5, 17.3,
CH-cis to CH2), 5.89 (1 H, ddt, J 5.7, 10.4, 17.5, CH-gem
to CH2), 7.11 (1 H, t, J 1.1, H-6), 9.62 (1 H, br s, NH); δC(75
MHz; CDCl3; Me4Si) 12.6 (CH3), 19.9 (CH2CH3), 70.3, 75.8
Esoda, T. B. Grizzle, M. R. Blum, J-P. Sommadossi, R. Endoh,
T. Niwa, M. Yamamoto and C. Moxham, Antimicrob. Agents
Chemother., 2000, 44, 123–130.
4 Press release: Triangle Pharmaceuticals plans 3rd quarter 2002 NDA
submission for Coviracil® (emtricitabine) and discontinues
development of Coactinon® (emivirine), Durham, NC, USA,
January 17, 2002. http://www.tripharm.com.
5 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 and
D. I. Stuart, J. Med. Chem., 1996, 39, 1589–1600.
6 A. L. Hopkins, J. Ren, H. Tanaka, M. Baba, M. Okamato,
D. I. Stuart and D. K. Stammers, J. Med. Chem., 1999, 42, 4500–
4505.
(OCH CH, NCH O), 117.4 (C-5), 118.2 (CH᎐CH ), 133.2
᎐
2
2
2
(CH᎐CH ), 138.1 (C-6), 151.2 (C-2), 163.9 (C-4); m/z (MALDI
᎐
2
MS (peak matching)) 233.0895 (M ϩ Na)ϩ, calcd. 233.0897.
Viruses and cells
7 N. R. El-Brollosy, P. T. Jørgensen, B. Dahan, A. M. Boel,
E. B. Pedersen and C. Nielsen, J. Med. Chem., 2002, 45, 5721–5726.
8 L. Petersen, E. B. Pedersen and C. Nielsen, Synthesis, 2001, 4,
559–564.
9 See http://www.schrodinger.com or: F. Mohamadi, N. G. J.
Richards, W. C. Guida, R. Liskamp, M. Lipton, C. Caufield,
G. Chang, T. Hendrickson and W. C. Still, J. Comp. Chem., 1990, 11,
440–467.
10 (a) J. Ren, C. Nichols, L. Bird, P. Chamberlain, K. Weaver, S. Short,
D. I. Stuart and D. K. Stammers, J. Mol. Biol., 2001, 312, 795–805;
(b) Structures were downloaded from the Protein Data Bank (PDB)
http://www.rcsb.org/pdb/.
The inhibitory activity against HIV-1 infection was evaluated
using MT-4 cells21 as target cells and either the wild-type HIV-1
strain HTLV-IIIB22 or the Y181C mutated strain N11923
as infectious viruses. The virus was propagated in H9 cells21 at
37 ЊC, 5% CO2 using RPMI 1640 with 10% heat-inactivated
fetal calf serum (FCS) and antibiotics (growth medium).
Culture supernatant was filtered (0.45 nm), separated into
aliquots, and stored at Ϫ80 ЊC until use.
11 M. P. Allen, D. J. Tildesley, Computer simulation of liquids,
Clarendon Press, Oxford, 1987.
12 T. A. Halgren, J. Comp. Chem., 1996, 17, 490–519.
13 RasMol version 2.6 was downloaded from http://www.umass.edu/
microbio/rasmol/getras.htm.
14 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
and D. I. Stuart, J. Med. Chem., 1996, 39, 1589–1600; (a) S. S. Klioze
and F. P. Darmory, J. Org. Chem., 1975, 40, 1588–1592.
15 J. H. Burckhalter and H. C. Scarborough, J. Am. Pharm. Assoc.,
1955, 44, 545–550.
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.
MT4 cells were incubated with virus (0.005 MOI) and growth
medium containing the test dilutions of compound for 6 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.22 Compounds
mediating less than 30% reduction of HIV expression were
considered without biological activity. Compounds were tested
in parallel for cytotoxic effect in uninfected MT4 cultures
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 (IC50)
and the 50% cytotoxic concentration (CC50) were determined
by interpolation from the plots of percent inhibition versus
concentration of compound.
16 H. Tanaka, H. Hayakawa and T. Miyasaka, Tetrahedron, 1982, 38,
2635–2642.
17 P. J. Garegg and B. Samuelsson, Carbohydr. Res., 1978, 67,
267–270.
18 H. Vorbrüggen, K. Krolikiewicz and B. Bennua, Chem. Ber., 1981,
114, 1234–1255.
1
19 NMR data for compound 10: H NMR (CDCl3): δ = 1.01 (3H, t,
J = 7.3 Hz, CH2CH3), 1.47 (3H, dd, J = 1.6 Hz, 6.8 Hz, CHCH3),
1.63–1.78 (4H, m, 2 × CH2), 2.08–2.28 (2H, m, CH2CH3), 2.28–2.42
(4H, m, 2 × CH2), 4.49 (1H, quintet, J = 6.6 Hz, CHCH3), 4.99 (1H,
d, J = 11.0 Hz, NCHHO), 5.65 (1H, d, J = 11.0 Hz, NCHHO), 6.14
(1H, dd, J = 1.6 Hz, 6.4 Hz, OCH), 6.95 (1H, t, J = 3.8 Hz, CH), 9.18
(1H, s, NH). 13C NMR (CDCl3): δ = 9.16 (CHCH3), 13.18
(CH2CH3), 19.42, 21.30, 21.45, 22.08, 26.65 (5 × CH2), 73.30
References
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᎐
142.88 (CH᎐C), 146.56, 150.71 (C-2, C-4), 150.38 (OCH), 162.64
᎐
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20 D-K. Kim, Y-W. Kim, J. Gam, G. Kim, J. Lim, N. Lee, H-T. Kim
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2 For recent reviews on NNRTI’s see: (a) R. W. Buckheit, Expert
Opin. Invest. Drugs., 2001, 10, 1423–1442; (b) M. Hogberg and
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22 T. Mosmann, J. Immunol. Methods, 1983, 65, 55–63.
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S. Goff and J. Griffin, Proc. Natl. Acad. Sci. U.S.A., 1991, 88,
11241–11245; (b) H. Hara, T. Fujihashi, T. Sakata, A. Kaji and
H. Kaji, AIDS Res. Hum. Retroviruses, 1997, 13, 6695–6705.
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