Synthesis of new MKC-442 analogues containing alkenyl chains or reactive functionalities at C-5

Article abstract of DOI:10.1007/s007060200072

In an effort to obtain more insight into the interaction between HIV-1 reverse transcriptase (RT) and MKC-442 analogues, a new series of compounds was synthesized and evaluated for inhibition of HIV-1 replication. The modifications include bulky alkenyl s

Full text of DOI:10.1007/s007060200072

Monatshefte fuÈr Chemie 133, 1031±1043 ꢀ2002)
Synthesis of New MKC-442 Analogues
Containing Alkenyl Chains or Reactive
Functionalities at C-5
Lene Petersen1, Thomas H. Hansen1, Nagy M. Khalifa1,
Per T. Jùrgensen1, Erik B. Pedersen1;Ã, and Claus Nielsen2
1
Department of Chemistry, University of Southern Denmark, DK-5230 Odense M,
Denmark
2
Retrovirus Laboratory, Department of Virology, State Serum Institute,
DK-2300 Copenhagen, Denmark
Summary. In an effort to obtain more insight into the interaction between HIV-1 reverse trans-
criptase ꢀRT) and MKC-442 analogues, a new series of compounds was synthesized and evaluated
for inhibition of HIV-1 replication. The modi®cations include bulky alkenyl substituents at the C-5
position of the uracil ring. Analogues with reactive centers ꢀaldehyde and epoxide functionalities) at
C-5 were also synthesized in an attempt to develop HIV drugs with improved activity against the
Y181C mutants by forming a covalent bond to the mercapto group in cysteine in the hydrophobic
pocket of the mutated RT. Dif®culties in the syntheses show that the epoxides are chemically
reactive, whereas the aldehydes are more stable. One of the alkenyl analogues showed activity
against HIV-1 in the same range as MKC-442, whereas the reactive analogues were not active
against HIV with the mutation Y181C in RT.
Keywords. HIV-1; Non-nucleoside reverse transcriptase inhibitors; MKC-442 analogues; 5-Alke-
nyluracils; 5-Aldehyde or 5-epoxide substituted uracils.
Introduction
HEPT ꢀ1-ꢀꢀ2-hydroxyethoxy)-methyl)-6-ꢀphenylthio)-thymine, 1, Fig. 1) was
originally synthesized as a nucleoside inhibitor against HIV but was acting as a
non-nucleoside inhibitor by binding to a hydrophobic pocket situated approxi-
Ê
mately 10 A away from the active site in the enzyme reverse transcriptase ꢀRT) [1].
The binding results in a conformational change and inactivation of the enzyme [2].
MKC-442 ꢀemivirine or coactinon, 2, Fig. 1) is an optimized structure of HEPT
and is currently undergoing phase III clinical trials [3]. In an attempt to optimize
this lead structure we have previously introduced a vinyl group at C-5 of the uracil
ring instead of the isopropyl group ꢀ3, Fig. 1) [4]. This compound has shown good
activity against HIV-1 ꢀIC-50 ˆ 0.07 ꢀM) and was almost as active as MKC-442
ꢀIC-50 ˆ 0.02 ꢀM).
Ã
Corresponding author. E-mail: ebp@chem.sdu.dk

1032
L. Petersen et al.
Fig. 1. Non-nucleoside reverse transcriptase inhibitors of HIV-1
When using non-nucleoside analogues as drugs against HIV the major problem
is the fast development of resistance. A very common mutation when using MKC-
442 is the conversion of tyrosine at position 181 in RT to cysteine. We have
previously reported the synthesis of an MKC-442 analogue with a carbaldehyde at
position C-5 [4]. It was hoped that the drug would covalently bind to the hydro-
phobic pocket by formation of a thiohemiacetal between the carbaldehyde and the
mercapto group in cysteine of the mutated virus. This carbaldehyde turned out to
be inactive towards the wild-type as well as the mutant. We also have attempted to
synthesize an epoxide directly attached to C-5. However, this compound turned
out to be too reactive to be isolated, its ring being opened by the m-chloro-
perbenzoic acid formed during epoxidation of the corresponding alkene. In this
paper we describe the introduction of the same reactive groups further away from
the uracil ring. A ¯exible linker of adequate length between the uracil ring and the
reactive groups may improve the chance of a reaction with the mercapto group in
the cysteine in the mutated RT. From the available crystal structure of MKC-442
complexed with RT [5] we deduced a possible appropriate chain length of the
linker. The aldehyde group can be introduced by cleavage of a double bond in
the side chain at C-5, whereas the same double bond can be oxidized to give the
epoxide.
In this paper we also use the intermediate compounds with allyl, 2-butenyl, 1-
methyl-2-propenyl, or allyloxymethyl groups at C-5 of the uracil ring to investigate
the in¯uence of the bulkyness of the C-5 substituent on the activity against HIV-1.
Results and Discussion
Chemistry
The ꢁ-ketoester 6a was synthesized according to the procedure described by Danel
et al. [6] in a Blaise reaction. In this investigation, ethyl 2-bromo-3-methyl-4-
pentenoate ꢀ4) [7] was reacted with Zn and phenylacetonitrile. After hydrolysis of
the formed enamide the desired ꢁ-ketoester was isolated in 60% yield. Compound
6a as well as the other ꢁ-ketoesters 6b,c [8, 9] were used as starting materials for
the uracil rings. These were formed by a ring closure using thiourea and NaOEt.
The formed thiouracils were desulfurized using chloroacetic acid [6, 10] to give the
uracils 7a±c in yields of 24±65% for the two steps. As the starting ꢁ-ketoester 6b
was a mixture of the cis and the trans alkene, 7b was also formed as a stereo-
chemical mixture, and no attempts were made to separate the compounds.

Synthesis of MKC-442 Analogues
1033
Scheme 1
Another uracil derivative was prepared starting from the hydroxymethylated
uracil 8 which has previously been synthesized [4]. This was reacted with allyl
alcohol and HCl to give the allyl ether 7d in 70% yield [11].
The compounds 7a, 7b, and 7d were alkylated at N-1 using bis-ꢀtrimethylsilyl)-
acetamide ꢀBSA) and chloromethylethyl ether [6] to give the MKC-442 analogues
9a, 9b, and 9d in 68±85% yield. The C-5 allyl substituted uracil 7c was silylated
under re¯ux in 1,1,1,3,3,3-hexamethyldisilazane ꢀHMDS) [12] and alkylated by
treatment with diethoxymethane, dimethoxymethane, or methylthiomethyl acetate
in the presence of trimethylsilyl tri¯uoromethanesulfonate ꢀTMS tri¯ate) as a Lewis
acid catalyst [13] to give the MKC-442 analogues 9c, 10, and 11 in 83%, 71%, and
57% yield.
A HEPT analogue was synthesized starting from 7b which was silylated using
BSA and alkylated with 2-acetoxyethyl acetoxymethyl ether and SnCl4 [14] to
give 12 in 80% yield. The deprotection was carried out in sodium methoxide in
methanol [14] to give the HEPT analogue 13 in 84% yield.
The epoxide 14b was synthesized to test its activity against the Y181C mutant
of HIV-1 RT. Compound 9b was reacted with m-chloroperoxybenzoic acid
ꢀMCPBA) in order to epoxidize the double bond. After 4 h the desired product 14b
was isolated in 45% yield. It was tested for activity against HIV without further
puri®cation. The product could not be puri®ed using column chromatography as

1034
L. Petersen et al.
Scheme 2
the C-4 carbonyl reacted with the epoxide to give 15b which was the only isolated
product when the reaction time was increased to 48 h ꢀ97% yield).
The C-5 allyl substituted analogue 9c was also reacted with MCPBA for 48 h,
and the product was puri®ed by column chromatography. The desired epoxide 14a
was isolated in only 10% yield, the major product being the compound where the
C-4 carbonyl had reacted with the epoxide to form 15a in 73% yield.
The C-5 allyloxymethyl substituted analogue 9d was reacted overnight with
MCPBA to give the corresponding epoxide 14c which was obtained in 60% yield
after column chromatography. In this case, a reaction between the C-4 carbonyl
and the epoxide is unfavourable. Compounds 14 and 15 were tested for their bio-
logical activity as the racemic mixtures.
The 2-butenyl and the allyloxymethyl analogues 9b,d where reacted with ozone
to cleave the double bonds, and the aldehydes 16a,b were isolated in 53% and 46%
yield, respectively.

Synthesis of MKC-442 Analogues
1035
Scheme 3
Antiviral activity
The anti-HIV activities and cytotoxicities of the synthesized MKC-442 analogues
are summarized in Table 1. The test for activity against HIV-1 was performed
in MT 4 cell cultures infected with either wild type HIV-1 or the HIV-1 strain N119
that harbours a substitution of tyrosine for cysteine at position 181 ꢀY181C). The
expression of HIV-1 was quanti®ed by two different methods, either the HIV-1
antigen detection assay ELISA [15] or indirectly by the MTT assay [16]. As these
methods give different results; the test results for MKC-442 are included as a
reference using both methods. The results for compound 3 are also included for
comparison.
In general, the introduction of an alkenyl group instead of an isopropyl group
at C-5 did not improve the activity against HIV-1 compared to MKC-442. The
most active compound is 9a ꢀIC-50 ˆ 0.04 ꢀM) with a 1-methyl-2-propenyl side
chain at C-5. This compound is almost as active as MKC-442 ꢀIC-50 ˆ 0.02 ꢀM)
and slightly more active than the reference compound 3 ꢀIC-50 ˆ 0.07 ꢀM).

1036
L. Petersen et al.
Table 1. Cytotoxicity and anti-HIV-1 activity of compound 9±16
Wild type
Y181C mutant ꢀN119)
IC-50a=ꢀM
CC-50b=ꢀM
SIc
IC-50a=ꢀM
CC-50b=ꢀM
SIc
9af
0.04
0.52
0.37
43
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>2500
>192
>270
>2
77
>100
30
>100
>100
>1
9be
9ce
>3
9df
>100
>100
>100
>100
>100
24
10e
3.7
>27
>178
>2
11e
0.56
42
12e
13e
32
>3
>312
>4
14ae
0.32
23
>100
>4
14bf
>100
>100
NDd
14cf
>100
>100
28
15ae
±
±
15bf
>100
>100
>3
>3
>100
>100
>100
>100
36
16af
32
16bf
>100
0.005
0.02
0.07
2 ꢀMKC-442)e
2 ꢀMKC-442)f
3f
141
>100
>100
28000
>5000
>1429
>100
>100
>1
>1
>100
a
b
c
50% Inhibitory concentration; 50% cytotoxic concentration; selectivity index, CC-50=IC-50
d
ratio; not determined; quanti®ed by ELISA [15]; quanti®ed by MTT assay [16]
e
f
Interestingly, there is a slight activity against the mutated virus ꢀY181C). The
other C-5 alkenyl substituted compounds 9b±d show more moderate activities
against HIV-1.
The compounds containing reactive epoxides ꢀ14a±c) or aldehydes ꢀ16a,b) in
the side chains at C-5 do not show any improved activity against the wild-type
virus when compared to the C-5 alkenyl substituted starting materials. Also, they
do not show activity against the mutant ꢀY181C). The latter indicates that the
covalent binding of the drug to cysteine in the RT hydrophobic pocket has not
taken place or that this principle is not a suf®cient prerequisite for activity against
the HIV mutant Y181C.
Experimental
1
NMR spectra were recorded on a Varian Gemini 2000 NMR spectrometer at 300 MHz for H and
75 MHz for 13C with TMS as an internal standard. EI mass spectra were recorded on a Finnigan Mat
SSQ 710, FAB mass spectra on a Kratos MS50RF instrument. Melting points were determined on a
Buchi melting point apparatus. Elemental analyses were performed at Atlantic Microlab, Inc.,
Atlanta, Georgia, USA; the found values agreed favourably with the calculated ones. The progress of
reactions was monitored by TLC ꢀanalytical silica gel plates 60 F254). Mercksilica gel ꢀ0.040±
0.063 mm) was used for column chromatography. Solvents for chromatography were bought as
Ê
HPLC grade or distilled prior to use. CHCl3 was dried over 4 A sieves. CH2Cl2 was dried by re¯ux
À3
over P2O5 ꢀ5 g Á dmÀ3) and distilled on 4 A sieves. MeOH was dried by re¯ux over Mg ꢀ5 g Á dm
)
Ê
Ê
and distilled on 3 A sieves. Pyridine was dried over KOH.

Synthesis of MKC-442 Analogues
1037
Ethyl 2-!1-methyl-2-propenyl)-3-oxo-4-phenylbutyrate ꢀ6a; C16H20O3)
Activated Zn dust ꢀ18 g, 0.275 mol) was suspended in 125 cm3 dry THF, and the mixture was
re¯uxed. A few drops of ethyl 2-bromo-3-methyl-4-pentenoate ꢀ4) [7] were added. When the colour
of the mixture turned green, 2.40 g phenylacetontrile ꢀ0.020 mol) were added in one portion; then,
4.40 g ethyl 2-bromo-3-methyl-4-pentenoate ꢀ4, 0.020 mol) was added dropwise over 1 h. The mix-
ture was re¯uxed for 15 min, and 60 cm3 50% aq. K2CO3 were added. The mixture was stirred for
45 min to give two phases. The THF phase was decanted, and the H2O phase was washed with THF.
The combined THF phases were reacted with 200 cm3 10% HCl at room temperature for 45 min. The
mixture was evaporated in vacuo, and the residue was dissolved in 150 cm3 CH2Cl2. The organic
phase was washed with sat. aq. NaHCO3 and dried ꢀNa2SO4). The product was puri®ed by silica gel
column chromatography ꢀ20% petroleum ether ꢀ60±80ꢀC) in Et2O) to give 3.11 g 6a ꢀ60%). It was
not possible to separate the diastereoisomers.
1H NMR ꢀCDCl3, ꢂ, 300 MHz): 0.94, 1.06 ꢀ3H, 2d, J ˆ 6.8 Hz, CHCH3), 1.17±1.26 ꢀ3H, m,
CH2CH3), 2.96±3.03 ꢀ1H, m, CHCH3), 3.50 ꢀ1H, d, J ˆ 9.7 Hz, CHCO), 3.80 ꢀ2H, s, CH2Ph),
4.05±4.18 ꢀ2H, q, J ˆ 7.1 Hz, CH2CH3), 4.95±5.09 ꢀ2H, m, CHˆCH2), 5.57±5.77 ꢀ1H, m,
CHˆCH2), 7.07±7.40 ꢀ5H, m, Harom) ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 13.99 ꢀCH2CH3), 17.91,
17.94 ꢀCH3CH), 37.53, 37.77 ꢀCHCH3) 49.57, 49.62 ꢀCH2Ph), 61.25, 61.47 ꢀCHCO), 63.90, 63.97
ꢀCH2CH3), 115.23, 115.49 ꢀCHˆCH2), 127.04, 127.13, 128.47, 128.57, 129.60, 129.68, 132.93,
132.99 ꢀCarom), 139.73, 139.77 ꢀCHˆCH2) 168.16, 168.30 ꢀCOO), 201.39, 201.44 ꢀCO) ppm; MS
‡
ꢀEI): m=z ˆ 260 ꢀM ).
General procedure for preparation of 5,6-substituted-1H-pyrimidine-2,4-diones 7a±c
Na ꢀ25.1 g, 1.1 mol) was dissolved in 500 cm3 absolute EtOH. Thiourea ꢀ58.23 g, 0.77 mol) was
added, and the mixture was heated to re¯ux. The ꢁ-keto ester ꢀ6a±c, 0.051 mol) was added dropwise,
and the mixture was re¯uxed for 3±6 h. EtOH was evaporated in vacuo, and the residue was
dissolved in 400 cm3 H2O. The thiouracil was precipitated by neutralization with conc. HCl. The
mixture was ®ltered, and the precipitate suspended in 500 cm3 10% aq. chloroacetic acid. The
suspension was re¯uxed overnight and ®ltered after cooling. The precipitate was washed with cold
EtOH and dried in vacuo to give 7a±c.
6-Benzyl-5-!1-methyl-2-propenyl)-1H-pyrimidine-2,4-dione ꢀ7a; C15H16N2O2)
White crystals after recrystallization ꢀEtOH=H2O); yield: 24%; m.p.: 188ꢀC ꢀEtOH=H2O); 1H NMR
ꢀCDCl3, ꢂ, 300 MHz): 1.31 ꢀ3H, d, J ˆ 7.4 Hz, CH3), 3.60±3.67 ꢀ1H, m, CH), 3.83 ꢀ2H, s, CH2Ph),
4.92±4.99 ꢀ2H, m, CHˆCH2), 6.09±6.20 ꢀ1H, m, CHˆCH2), 7.19±7.33 ꢀ5H, m, Harom), 9.80 ꢀ2H, s,
2 Â NH) ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 18.26 ꢀCH3), 35.13 ꢀCH2Ph), 36.25 ꢀCH), 113.77
ꢀCHˆCH2), 114.71 ꢀC-5), 127.46, 128.62, 128.98, 134.94 ꢀCarom), 140.85 ꢀCHˆCH2), 149.09,
‡
151.87 ꢀC-2, C-6), 164.22 ꢀC-4) ppm; MS ꢀEI): m=z ˆ 256 ꢀM ).
6-Benzyl-5-but-2-enyl-1H-pyrimidine-2,4-dione ꢀ7b; C15H16N2O2)
White crystals after washing with H2O; yield: 65%; m.p.: 181ꢀC; 1H NMR ꢀDMSO-d6, ꢂ, 300 MHz):
1.51 ꢀ3H, d, J ˆ 3.6 Hz, CH3), 2.88±2.94 ꢀ2H, m, CH2CH), 3.73 ꢀ2H, s, CH2Ph), 5.27±5.30 ꢀ2H, m,
CHˆCH), 7.24±7.35 ꢀ5H, m, Harom), 10.79 ꢀ1H, s, NH), 11.05 ꢀ1H, s, NH) ppm; 13C NMR ꢀDMSO-
d6, ꢂ, 75 MHz): 17.58 ꢀCH3), 26.79 ꢀCH2CH), 35.03 ꢀCH2Ph), 108.60 ꢀC-5), 125.20, 126.93
ꢀCHˆCH) 128.32, 128.48, 128.80, 136.72 ꢀCarom), 149.90, 151.23 ꢀC-2, C-6), 164.77 ꢀC-4) ppm;
‡
MS ꢀEI): m=z ˆ 256 ꢀM ).

1038
L. Petersen et al.
5-Allyl-6-benzyl-1H-pyrimidine-2,4-dione ꢀ7c; C14H14N2O2)
White crystals after recrystallisation ꢀEtOH); yield: 37%; m.p.: 187±189ꢀC ꢀEtOH); 1H NMR
ꢀDMSO-d6, ꢂ, 300 MHz): 2.98 ꢀ2H, d, J ˆ 6.2 Hz, CH2CHˆCH2), 3.71 ꢀ2H, s, CH2Ph), 4.87 ꢀ2H, m,
CHˆCH2), 5.65 ꢀ1H, m, CHˆCH2), 7.18±7.32 ꢀ5H, m, Harom), 10.80 ꢀ1H, s, NH), 11.06 ꢀ1H, s, NH)
ppm; 13C NMR ꢀDMSO-d6, ꢂ, 7.5 MHz): 27.85 ꢀCH2CHˆCH2), 34.92 ꢀCH2Ph), 107.59 ꢀC-5),
115.04 ꢀCHˆCH2), 126.82±128.69 ꢀCarom), 135.75 ꢀCHˆCH2), 136.60 ꢀCarom), 150.10, 151.10 ꢀC-2,
‡
C-6), 164.55 ꢀC-4) ppm; MS ꢀEI): m=z ˆ 242 ꢀM ).
5-Allyloxymethyl-6-benzyl-1H-pyrimidine-2,4-dione ꢀ7d; C15H16N2O3)
6-Benzyl-5-hydroxymethyl-1H-pyrimidine-2,4-dione [4] ꢀ8, 0.93 g, 4 mmol) was added to a solution
of 1 cm3 conc. HCl in 50 cm3 allyl alcohol. The solution was heated to 100ꢀC overnight, cooled
slowly to room temperature, and left to crystallize at 5ꢀC. The precipitate was ®ltered off, washed
with H2O and Et2O, and dried in vacuo to give 0.76 g 7d ꢀ70%) as a white solid. To get an ana-
lytically pure sample it was recrystallized ꢀEtOH=H2O).
1
M.p.: 207±208ꢀC ꢀEtOH=H2O); H NMR ꢀDMSO-d6, ꢂ, 300 MHz): 3.85 ꢀ2H, s, CH2Ph), 3.96
ꢀ2H, d, J ˆ 5.4 Hz, OCH2CHˆCH2), 4.24 ꢀ2H, s, CH2O), 5.15 ꢀ1H, dd, J ˆ 1.0, 10.5 Hz, Htrans), 5.26
ꢀ1H, dd, J ˆ 1.6, 17.3 Hz, Hcis), 5.82±5.95 ꢀ1H, m, Hgem), 7.24±7.36 ꢀ5H, m, Harom), 11.00 ꢀ1H, br s,
NH), 11.17 ꢀ1H, br s, NH) ppm; 13C NMR ꢀDMSO-d6, ꢂ, 75 MHz): 34.97 ꢀCH2Ph), 61.37 ꢀCH2O),
70.30 ꢀOCH2CHˆCH2), 106.76 ꢀC-5), 116.30 ꢀCHˆCH2), 126.71, 128.42, 128.49, 135.14 ꢀCarom),
‡
136.27 ꢀCHˆCH2), 150.84 ꢀC-2), 153.86 ꢀC-6), 164.26 ꢀC-4) ppm; MS ꢀEI): m=z ˆ 272 ꢀM ).
General procedure for the synthesis of 9a, 9b, and 9d
N,O-Bis-ꢀtrimethylsilyl)-acetamide ꢀBSA, 3.37 g, 16.4 mmol) was dissolved in 30 cm3 dry CHCl3.
Compounds 7a, 7b, or 7d ꢀ4.69 mmol) and, after 10 min, 0.55 g chloromethyl ethyl ether
ꢀ5.82 mmol) were added. The solution was stirred overnight at room temperature, quenched with
25 cm3 ice cold sat. aq. NaHCO3, and evaporated to near dryness under reduced pressure. The
product was washed out with Et2O. The combined Et2O phases were dried ꢀNa2SO4) and evaporated
under reduced pressure.
6-Benzyl-1-ethoxymethyl-5-!1-methyl-2-propenyl)-1H-pyrimidine-2,4-dione
ꢀ9a; C18H22N2O3)
1
Puri®ed by preparative TLC ꢀ3% MeOH in CH2Cl2); yield: 68%; H NMR ꢀCDCl3, ꢂ, 300 MHz):
1.18 ꢀ3H, t, J ˆ 7.0 Hz, CH2CH3), 1.34 ꢀ3H, d, J ˆ 7.3 Hz, CHCH3), 3.54±3.65 ꢀ3H, m, CH2CH3,
CHCH3), 4.18, 4.24 ꢀ2H, 2  d, J ˆ 17.5 Hz, CH2Ph), 4.88±5.20 ꢀ4H, m, CH2O, CHˆCH2), 6.14±
6.25 ꢀ1H, m, CHˆCH2), 7.10±7.37 ꢀ5H, m, Harom), 9.43 ꢀ1H, s, NH) ppm; 13C NMR ꢀCDCl3, ꢂ,
75 MHz): 15.02 ꢀCH2CH3), 18.51 ꢀCHCH3), 33.54 ꢀCH2Ph), 36.42 ꢀCHCH3), 65.02 ꢀCH2CH3),
72.75 ꢀCH2O), 113.87 ꢀCHˆCH2), 118.12 ꢀC-5), 127.20, 127.35, 129.13, 135.40 ꢀCarom), 140.81
‡
ꢀCHˆCH2), 149.59, 151.92 ꢀC-2, C-6), 162.51 ꢀC-4) ppm; MS ꢀEI): m=z ˆ 314 ꢀM ).
6-Benzyl-5-but-2-enyl-1-ethoxymethyl-1H-pyrimidine-2,4-dione ꢀ9b; C18H22N2O3)
1
Puri®ed by silica gel column chromatography ꢀ1±3% MeOH in CH2Cl2); yield: 82%; oil; H NMR
ꢀCDCl3, ꢂ, 300 MHz): 1.18 ꢀ3H, t, J ˆ 7.1 Hz, CH2CH3), 1.58±1.60 ꢀ3H, m, CHCH3), 3.10±3.25
ꢀ2H, m, CH2CH), 3.62 ꢀ2H, q, J ˆ 7.0 Hz, CH2CH3), 4.16 ꢀ2H, s, CH2Ph), 5.13 ꢀ2H, s, CH2O), 5.40±
5.44 ꢀ2H, m, CHˆCH) 7.09±7.36 ꢀ5H, m, Harom), 9.97 ꢀ1H, s, NH) ppm; 13C NMR ꢀCDCl3, ꢂ,
75 MHz): 14.85 ꢀCH2CH3), 17.57 ꢀCHCH3), 28.15 ꢀCH2CH), 33.36 ꢀCH2Ph), 64.91 ꢀCH2CH3),

Synthesis of MKC-442 Analogues
1039
72.64 ꢀCH2O), 113.81 ꢀC-5), 126.45, 127.22, 127.25, 127.41, 129.17, 135.08 ꢀCarom, CHˆCH),
‡
150.33, 152.15 ꢀC-2, C-6), 163.57 ꢀC-4) ppm; MS ꢀEI): m=z ˆ 314 ꢀM ).
5-Allyloxymethyl-6-benzyl-1-ethoxymethyl-1H-pyrimidine-2,4-dione ꢀ9d; C18H22N2O4)
The product was puri®ed by silica gel column chromatography ꢀ50% EtOAc in petroleum ether
1
ꢀ60±80ꢀC)); yield: 0.280 g ꢀ85%); white solid; m.p.: 99±101ꢀC; H NMR ꢀCDCl3, ꢂ, 300 MHz):
1.17 ꢀ3H, t, J ˆ 7.0 Hz, CH3), 3.61 ꢀ2H, q, J ˆ 7.0 Hz, CH2CH3), 4.03 ꢀ2H, dt, J ˆ 1.3, 5.7 Hz,
CH2CHˆCH2), 4.30 ꢀ2H, s, CH2Ph), 4.39 ꢀ2H, s, CH2O), 5.41 ꢀ2H, s, NCH2O), 5.17 ꢀ1H, m, Htrans),
5.25 ꢀ1H, dq, J ˆ 1.5, 17.3 Hz, Hcis), 5.81±5.94 ꢀ1H, m, Hgem), 7.14±7.36 ꢀ5H, m, Harom), 9.79 ꢀ1H,
s, NH) ppm; 13C NMR ꢀCDCl3, ꢂ, 7.5 MHz): 14.93 ꢀCH3), 33.70 ꢀCH2Ph), 62.25 ꢀArCH2O), 65.14
ꢀCH2CH3), 71.67 ꢀOCH2CH), 72.66 ꢀNCH2O), 111.79 ꢀC-5), 117.51 ꢀCHˆCH2), 127.27, 127.49,
129.15, 134.29 ꢀCarom), 134.96 ꢀCHˆCH2), 151.85 ꢀC-2), 154.97 ꢀC-4), 163.21 ꢀC-6) ppm; MS ꢀEI):
‡
m=z ˆ 330 ꢀM ).
General procedure for the synthesis of 9c, 10, and 11
Compound 7c ꢀ3 mmol) was added to a solution of 10 mg ꢀNH4)2SO4 in 10 cm3 HMDS. The solution
was re¯uxed, and when the silylation was complete, the excess of HMDS was evaporated under
reduced pressure to give the silylated compound as a yellow oil. This was dissolved in dry 10 cm3
dry CH3CN, and the solution was cooled to À35ꢀC. TMS tri¯ate ꢀ0.62 g, 2.79 mmol) was added in
one portion followed by the dropwise addition of 30 mmol dialkyloxymethane or 3.5 mmol methyl-
thiomethyl acetate. The solution was stirred for 3 h at À35ꢀC for 9c and 10 or allowed to warm
slowly to À5ꢀC for 11. The reaction was quenched by addition of 10 cm3 ice cold sat. aq. NaHCO3
and evaporated to near dryness by co-evaporation with 2 Â 50 cm3 EtOH. For 9c and 10, the resulting
solid was suspended in 200 cm3 Et2O, and the mixture was stirred for 1 h. After ®ltration the residue
was extracted with 100 cm3 Et2O, and the combined organic fractions were evaporated under
reduced pressure. For 11, the resulting solid was triturated with CHCl3, and the solvent was dried
ꢀNa2SO4) and evaporated in vacuo. The products were puri®ed by silica gel column chromatography
ꢀ25±30% EtOAc in petroleum ether ꢀ60±80ꢀC)).
5-Allyl-6-benzyl-1-ethoxymethyl-1H-pyrimidine-2,4-dione ꢀ9c; C17H20N2O3)
1
Yield: 0.748 g ꢀ83%); m.p.: 142ꢀC ꢀEtOAc=petroleum ether ꢀ60±80ꢀC)); H NMR ꢀDMSO-d6, ꢂ,
300 MHz): 1.00 ꢀ3H, t, J ˆ 7.1 Hz, CH3), 3.02 ꢀ2H, d, J ˆ 5.9 Hz, CH2CHˆCH2), 3.43 ꢀ2H, q,
J ˆ 7.0 Hz, CH2CH3), 4.04 ꢀ2H, s, CH2Ph), 4.93 ꢀ2H, m, CHˆCH2), 5.14 ꢀ2H, s, CH2O), 5.78 ꢀ1H,
m, CHˆCH2), 7.13±7.33 ꢀ5H, m, Harom), 11.50 ꢀ1H, s, NH) ppm; 13C NMR ꢀDMSO-d6, ꢂ, 75 MHz):
14.72 ꢀCH3), 28.78 ꢀCH2CHˆCH2), 33.07 ꢀCH2Ph), 63.72 ꢀCH2CH3), 72.08 ꢀCH2O), 111.77 ꢀC-5),
115.32 ꢀCHˆCH2), 126.90±129.00 ꢀCarom), 135.42 ꢀCHˆCH2), 135.90 ꢀCarom), 149.90 ꢀC-2),
‡
151.69 ꢀC-6), 163.02 ꢀC-4) ppm; MS ꢀEI): m=z ˆ 300 ꢀM ).
5-Allyl-6-benzyl-1-methoxymethyl-1H-pyrimidine-2,4-dione ꢀ10; C16H18N2O3)
1
Yield: 0.610 g ꢀ71%); m.p.: 136ꢀC ꢀEtOAc in petroleum ether ꢀ60±80ꢀC)); H NMR ꢀDMSO-d6, ꢂ,
300 MHz): 3.03 ꢀ2H, d, J ˆ 5.3 Hz, CH2CHˆCH2), 3.22 ꢀ3H, s, CH3), 4.03 ꢀ2H, s, CH2Ph), 4.87±
4.98 ꢀ4H, m, CH2O, CHˆCH2), 5.65±5.78 ꢀ1H, m, CHˆCH2), 7.14±7.37 ꢀ5H, m, Harom), 11.53 ꢀ1H,
s, NH) ppm; 13C NMR ꢀDMSO-d6, ꢂ, 75 MHz): 28.76 ꢀCH2CHˆCH2), 33.02 ꢀCH2Ph), 55.95 ꢀCH3),
73.54 ꢀCH2O), 111.83 ꢀC-5), 115.35 ꢀCHˆCH2), 126.96, 127.52, 129.05 ꢀCarom), 135.37 ꢀCHˆCH2),
135.82 ꢀCarom), 149.90 ꢀC-2), 151.69 ꢀC-6), 163.02 ꢀC-4) ppm.

1040
L. Petersen et al.
5-Allyl-6-benzyl-1-methylthiomethyl-1H-pyrimidine-2,4-dione ꢀ11; C16H18N2O2S)
Yield: 0.517 g ꢀ57%); m.p.: 110±112ꢀC ꢀEtOAc in petroleum ether ꢀ60±80ꢀC)); 1H NMR ꢀCDCl3, ꢂ,
300 MHz): 1.92 ꢀ3H, s, CH3), 3.23 ꢀ2H, d, J ˆ 5.1 Hz, CH2CHˆCH2), 4.00 ꢀ2H, s, CH2Ph), 4.97±
5.03 ꢀ2H, m, CHˆCH2), 5.69 ꢀ2H, s, CH2O), 5.70±5.90 ꢀ1H, m, CHˆCH2), 7.11±7.35 ꢀ5H, m,
H
arom) ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 20.38 ꢀCH3), 29.60 ꢀCH2CHˆCH2), 33.81 ꢀCH2Ph),
67.27 ꢀCH2S), 113.32 ꢀC-5), 115.61 ꢀCHˆCH2), 127.53, 129.27, 134.64 ꢀCarom), 134.88 ꢀCHˆCH2),
149.20 ꢀC-2), 152.87 ꢀC-6), 169.97 ꢀC-4) ppm.
1-!!2-Acetoxyethoxy)-methyl)-6-benzyl-5-!2-butenyl)-1H-pyrimidine-2,4-dione
ꢀ12; C20H24N2O5)
Compound 7b ꢀ918 mg, 3.59 mmol) and 949 mg 2-acetoxyethyl acetoxymethyl ether ꢀ5.39 mmol)
were dissolved in 10 cm3 dry CH2Cl2. BSA ꢀ1.6 cm3, 6.54 mmol) was added dropwise under N2, and
the solution was stirred overnight. Then the solution was cooled to 0ꢀC, and 0.52 g SnCl4 ꢀ2 mmol)
were added. The mixture was allowed to warm to room temperature, stirred overnight, and then
quenched with 25 cm3 cold sat. aq. NaHCO3. The product was extracted with 3 Â 25 cm3 CHCl3. The
combined organic fractions were dried ꢀNa2SO4) and evaporated under reduced pressure to give a
yellow gel which was puri®ed on a silica gel chromatotron ꢀ5% MeOH=CH2Cl2).
1
Yield: 0.734 g ꢀ80%); white gel; H NMR ꢀCDCl3, ꢂ, 300 MHz): 1.56±1.59 ꢀ3H, m, CHCH3),
2.05 ꢀ3H, s, CH3CO), 3.12±3.16 ꢀ2H, m, CH2CH), 3.79±3.83 ꢀ2H, m, CH2CH2), 4.14±4.20 ꢀ4H, m,
CH2Ph, CH2CH2), 5.19 ꢀ2H, s, NCH2O), 5.40±5.45 ꢀ2H, m, CHˆCH), 7.09±7.38 ꢀ5H, m, Harom),
10.42 ꢀ1H, s, NH) ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 17.43 ꢀCHCH3), 20.51 ꢀCH3CO), 28.01
ꢀCH2CH), 33.16 ꢀCH2Ph), 62.97 ꢀCH2CH2), 67.23 ꢀCH2CH2), 72.79 ꢀNCH2O), 113.87 ꢀC-5),
126.34, 127.07, 127.18, 127.27, 129.07, 134.76 ꢀCarom, CHˆCH), 149.97, 152.22 ꢀC-2, C-6), 163.55
‡
ꢀC-4), 170.78 ꢀCO) ppm; MS ꢀEI): m=z ˆ 372 ꢀM ).
6-Benzyl-5-!2-butenyl)-1-!!2-hydroxyethoxy)-methyl)-1H-pyrimidine-2,4-dione
ꢀ13; C18H22N2O4)
1-ꢀꢀ2-Acetoxyethoxy)-methyl)-6-benzyl-5-ꢀ2-butenyl)-1H-pyrimidine-2,4-dione ꢀ12, 0.565 g,
1.51 mmol) was dissolved in MeOH. NaOMe in 2 cm3 MeOH ꢀ1 M, 2 mmol) was added, and the
solution was stirred overnight at room temperature. Then the pH was adjusted to 4 by addition of 1 M
HCl. After stirring for 20 min, sat. aq. NaHCO3 was added. Unreacted starting material precipitated
and was ®ltered off; the product was extracted with 3 Â 50 cm3 CHCl3. The organic phase was dried
ꢀNa2SO4) and evaporated under reduced pressure.
1
Yield: 0.320 g ꢀ84%); white gel; H NMR ꢀCDCl3, ꢂ, 300 MHz): 1.57±1.62 ꢀ3H, m, CHCH3),
3.10±3.15 ꢀ2H, m, CH2), 3.69 ꢀ4H, s, CH2CH2), 4.13 ꢀ2H, s, CH2Ph), 5.18 ꢀ2H, s, NCH2O), 5.40±
5.44 ꢀ2H, m, CHˆCH), 7.10±7.37 ꢀ5H, m, Harom), 8.02 ꢀ1H, s, NH) ppm; 13C NMR ꢀCDCl3, ꢂ,
75 MHz): 17.71 ꢀCHCH3), 28.34 ꢀCH2CH), 33.65 ꢀCH2Ph), 61.59 ꢀCH2OH), 70.75 ꢀOCH2CH2),
73.15 ꢀNCH2O), 114.00 ꢀC-5), 126.60, 127.00 ꢀCHˆCH), 127.36, 129.19, 134.84 ꢀCarom), 149.99,
‡
152.06 ꢀC-2, C-6), 163.16 ꢀC-4) ppm; MS ꢀEI): m=z ˆ 330 ꢀM ).
Synthesis of compounds 14a and 15a
5-Allyl-6-benzyl-1-ethoxymethyl-1H-pyrimidine-2,4-dione ꢀ9c, 1.25 mmol) and 0.235 g MCPBA
ꢀ1.5 mmol) were dissolved in 20 cm3 CHCl3 and stirred at room temperature for 48 h. Then the
mixture was diluted with 100 cm3 CHCl3 and washed with 10% aq. NaHSO3, 10% aq. Na2S2O3, sat.
aq. NaHCO3, and H2O. The organic phase was evaporated under reduced pressure. The product was
puri®ed by silica gel column chromatography.

Synthesis of MKC-442 Analogues
1041
6-Benzyl-1-ethoxymethyl-5-oxiranylmethyl-1H-pyrimidine-2,4-dione
ꢀ14a; C17H20N2O4)
Eluted from the silica gel column with EtOAc=petroleum ether ꢀ60±80ꢀC); yield: 10%; m.p.: 122ꢀC;
1H NMR ꢀCDCl3, ꢂ, 300 MHz): 1.16 ꢀ3H, t, J ˆ 7.0 Hz, CH3), 2.50 ꢀ2H, d, J ˆ 5.0 Hz, ArCH2CH),
2.55 ꢀ2H, d, J ˆ 6.0 Hz, CH2CH), 3.10 ꢀ2H, q, CH2CH3), 3.64 ꢀ1H, m, CH), 4.22 ꢀ2H, m, CH2Ph),
5.05 ꢀ2H, m, NCH2O), 7.08±7.35 ꢀ5H, m, Harom), 9.82 ꢀ1H, s, NH) ppm; 13C NMR ꢀCDCl3, ꢂ,
75 MHz): 14.84 ꢀCH3), 28.28 ꢀArCH2CH), 33.81 ꢀCH2Ph), 46.81 ꢀCH2CH), 50.97 ꢀCH2CHCH2),
65.06 ꢀCH2CH3), 72.93 ꢀNCH2O), 110.64 ꢀC-5), 127.39±129.63, 135.90 ꢀCarom), 151.99, 152.28
‡
ꢀC-2, C-6), 163.75 ꢀC-4) ppm; MS ꢀEI): m=z ˆ 316 ꢀM ).
4-Benzyl-3-ethoxymethyl-6-hydroxymethyl-5,6-dihydro-3H-furo[2,3-d]pyrimidin-2-one
ꢀ15a; C17H20N2O4)
1
Eluted from the silica gel column with MeOH; yield: 73%; m.p.: 186ꢀC; H NMR ꢀCDCl3, ꢂ,
300 MHz): 1.16 ꢀ3H, bs, CH3), 2.98 ꢀ2H, d, J ˆ 6.4 Hz, H-5), 3.56±3.74, 3.82±3.94 ꢀ4H, 2 m,
CH2CH3, CH2OH), 4.12 ꢀ2H, s, CH2Ph), 4.98 ꢀ1H, bs, H-6), 5.28 ꢀ2H, s, NCH2O), 7.04±7.40 ꢀ5H,
m, Harom) ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 14.32 ꢀCH3), 25.81 ꢀC-5), 34.88 ꢀCH2Ph), 62.48
ꢀCH2OH), 64.17 ꢀCH2CH3), 72.83 ꢀNCH2O), 83.38 ꢀC-6), 105.38 ꢀC-4a), 126.88, 127.40, 128.64,
‡
133.88 ꢀCarom), 151.27, 158.21 ꢀC-2, C-4), 176.48 ꢀC-7a) ppm; MS ꢀEI): m=z ˆ 315 ꢀM À1).
Synthesis of compounds 14b and 15b
6-Benzyl-5-ꢀtrans-2-butenyl)-1-ethoxymethyl-1H-pyrimidine-2,4-dione ꢀ9b, 0.100 g, 0.32 mmol)
was dissolved in 15 cm3 dry CH2Cl2 and cooled to 0ꢀC. MCPBA ꢀ0.100 g, 0.58 mmol) was added,
and the solution was allowed to warm to room temperature. After 4 h or 48 h the mixture was diluted
with Et2O and washed with 20% aq. Na2S2O3, sat. aq. NaHCO3, and brine. The organic phase was
dried ꢀMgSO4) and evaporated under reduced pressure.
6-Benzyl-1-ethoxymethyl-5-!3-methyloxiranylmethyl)-1H-pyrimidine-2,4-dione
ꢀ14b; C18H22N2O4)
1
After reaction with MCPBA for 4 h; yield: 45%; oil; H NMR ꢀCDCl3, ꢂ, 300 MHz): 1.17 ꢀ3H, t,
J ˆ 7.1 Hz, CH2CH3), 1.23 ꢀ3H, d, J ˆ 5.1 Hz, CHCH3), 2.48±2.55, 2.74±3.14 ꢀ4H, m, CH2CHCH),
3.55±3.67 ꢀ2H, m, CH2CH3), 4.11±4.33 ꢀ2H, m, CH2Ph), 4.99±5.43 ꢀ2H, m, CH2O), 7.09±7.38 ꢀ5H,
m, Harom), 10.04 ꢀ1H, s, NH) ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 14.91 ꢀCH2CH3), 17.32 ꢀCHCH3),
28.07 ꢀCH2CH), 33.85 ꢀCH2Ph), 54.67, 58.21 ꢀCHCH), 65.03 ꢀCH2CH3), 72.69 ꢀCH2O), 110.85
ꢀC-5), 127.19, 129.12, 129.17, 134.96 ꢀCarom) 151.88, 152.00 ꢀC-2, C-6), 163.65 ꢀC-4) ppm; MS
‡
ꢀEI): m=z ˆ 329 ꢀM À1).
4-Benzyl-3-ethoxymethyl-6-!1-hydroxyethyl)-5,6-dihydro-3H-furo[2,3-d]pyrimidin-2-one
ꢀ15b; C18H22N2O4)
Stirred with MCPBA for 48 h; yield: 97%; 1H NMR ꢀCDCl3, ꢂ, 300 MHz): 1.11±1.29 ꢀ6H, m,
CHCH3, CH2CH3), 2.84 ꢀ1H, dd, J ˆ 9.1, 15.6 Hz, H-5), 3.07 ꢀ1H, dd, J ˆ 6.4, 15.2 Hz, H-5), 3.63
ꢀ2H, q, J ˆ 7.0 Hz, CH2CH3), 4.08 ꢀ1H, d, J ˆ 16.2 Hz, CHHPh), 4.15 ꢀ1H, d, J ˆ 16.3 Hz, CHHPh),
4.20±4.29 ꢀ1H, m, CHOH), 4.74±4.81 ꢀ1H, m, H-6), 5.28 ꢀ2H, s, CH2O), 7.12±7.38 ꢀ5H, m, Harom
)
ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 14.95 ꢀCH2CH3), 17.62 ꢀCHCH3), 24.34 ꢀC-5), 35.55 ꢀCH2Ph),
65.08 ꢀCHOH), 67.08 ꢀCH2CH3), 73.62 ꢀCH2O), 86.85 ꢀC-6), 105.84 ꢀC-4a), 127.58, 127.97, 129.34,
134.34 ꢀCarom), 152.17 ꢀC-4), 158.76 ꢀC-2), 176.86 ꢀC-7a) ppm.

1042
L. Petersen et al.
6-Benzyl-1-ethoxymethyl-5-oxiranylmethoxymethyl-1H-pyrimidine-2,4-dione
ꢀ14c; C18H22N2O5)
5-Allyloxymethyl-6-benzyl-1-ethoxymethyl-1H-pyrimidine-2,4-dione ꢀ9d, 0.248 g, 0.75 mmol) was
dissolved in 10 cm3 CH2Cl2 and cooled to 0ꢀC. MCPBA was added ꢀ0.311 g ꢀ50±90%), 0.9±
1.6 mmol), and the solution was stirred overnight at room temperature under N2. Then 20 cm3 Et2O
were added, and the organic phase was washed with 2 Â 20 cm3 sat. aq. Na2S2O3, 2 Â 20 cm3 sat. aq.
NaHCO3, and 2 Â 2 cm3 brine, dried ꢀMgSO4), and evaporated under reduced pressure. The product
14c was isolated after column chromatography ꢀEtOAc:petroleum etherꢀ60±80ꢀC) ˆ 1:1).
Yield: 0.157 g ꢀ60%); white solid; m.p.: ˆ 99±102ꢀC; 1H NMR ꢀCDCl3, ꢂ, 300 MHz): 1.18 ꢀ3H,
t, J ˆ 7.0 Hz, CH3), 2.56 ꢀ1H, dd, J ˆ 2.7 Hz, 5.0 Hz, CHCH2), 2.74 ꢀ1H, t, J ˆ 4.6 Hz, CHCH2),
3.07±3.12 ꢀ1H, m, epoxide-CH), 3.45 ꢀ1H, dd, J ˆ 5.8 Hz, 11.4 Hz, OCH2CH), 3.61 ꢀ2H, q,
J ˆ 7.0 Hz, CH2CH3), 3.79 ꢀ1H, dd, J ˆ 3.2 Hz, 11.6 Hz, OCH2CH), 4.30 ꢀ2H, s, CH2Ph), 4.42, 4.48
ꢀ2H, 2  d, J ˆ 11.2 Hz, ArCH2O), 5.14 ꢀ2H, s, NCH2O), 7.14±7.37 ꢀ5H, m, Harom), 9.72 ꢀ1H, br s,
NH) ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 14.95 ꢀCH3), 33.72 ꢀCH2Ph), 44.24 ꢀCHCH2), 50.61
ꢀCH2CH), 63.44 ꢀArCH2O), 65.18 ꢀOCH2CH3), 71.20 ꢀOCH2CH), 72.70 ꢀNCH2O), 111.50 ꢀC-5),
127.31, 127.47, 129.16, 134.88 ꢀCarom), 151.76 ꢀC-2), 155.18 ꢀC-4), 163.18 ꢀC-6) ppm; MS ꢀEI):
‡
m=z ˆ 346 ꢀM ).
General procedure for the synthesis of 16a and 16b
Compounds 9b or 9d ꢀ0.75 mmol) was dissolved in 15 cm3 dry CH2Cl2 and cooled to À78ꢀC. O3 was
bubbled through the solution until a blue colour appeared ꢀca. 2 min). Then O2 was bubbled through
until the blue colour disappeared. Dimethyl sul®de ꢀ0.28 g, 4.5 mmol) was then added at À78ꢀC, and
the solution was allowed to warm to room temperature and stirred overnight. Then the solvent was
evaporated under reduced pressure.
!6-Benzyl-1-ethoxymethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-acetaldehyde
ꢀ16a; C16H18N2O4)
1
Puri®ed by preparative TLC ꢀEtOAc); yield: 53%; H NMR ꢀCDCl3, ꢂ, 300 MHz): 1.17 ꢀ3H, t,
J ˆ 7.5 Hz, CH2CH3), 3.57 ꢀ2H, s, CH2CHO), 3.62 ꢀ2H, q, J ˆ 6.7 Hz, CH2CH3), 4.09 ꢀ2H, s,
CH2Ph), 5.17 ꢀ2H, s, NCH2O), 7.10±7.36 ꢀ5H, m, Harom), 9.64 ꢀ1H, s, CHO), 10.50 ꢀ1H, s, NH)
ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 14.81 ꢀCH2CH3), 33.92 ꢀCH2Ph), 39.99 ꢀCH2CHO), 64.97
ꢀCH2CH3), 72.81 ꢀNCH2O), 107.42 ꢀC-5), 127.13, 127.33, 129.14, 134.13 ꢀCarom) 151.79, 152.57
‡
ꢀC-2, C-6), 163.36 ꢀC-4), 197.53 ꢀCHO) ppm; MS ꢀEI): m=z ˆ 302 ꢀM ).
!6-Benzyl-1-ethoxymethyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrimidin-5-ylmethoxy)-acetaldehyde
ꢀ16b; C17H20N2O5)
Puri®ed by silica gel column chromatography ꢀCH2Cl2 ± 50% CH2Cl2 in EtOAc); clear glace; yield:
0.114 g ꢀ46%); 1H NMR ꢀCDCl3, ꢂ, 300 MHz): 1.11 ꢀ3H, t, J ˆ 7.0 Hz, CH3), 3.54 ꢀ2H, q, J ˆ 7.0 Hz,
CH2CH3), 4.12 ꢀ2H, s, CH2Ph), 4.30 ꢀ2H, s, ArCH2O), 4.42 ꢀ2H, s, OCH2CHO), 5.08 ꢀ2H, s,
NCH2O), 7.07±7.29 ꢀ5H, m, Harom), 9.55 ꢀ1H, s, CHO) ppm; 13C NMR ꢀCDCl3, ꢂ, 75 MHz): 14.96
ꢀCH3), 33.78 ꢀCH2Ph), 63.82 ꢀArCH2O), 65.26 ꢀCH2CH3), 72.83 ꢀNCH2O), 75.95 ꢀOCH2CHO),
110.95 ꢀC-5), 127.41, 129.23, 134.77 ꢀCarom), 151.62 ꢀC-2), 155.76 ꢀC-4), 163.21 ꢀC-6), 199.82
‡
ꢀCHO) ppm; MS ꢀFAB, peakmatching): m=z ˆ 355.1271 ꢀM ‡ Na ; calcd.: 355.1270).
Viruses and cells
The HIV-1 strains HTLV-IIIB [17] and the NNRTI resistant strain N119 [18] were propagated in H9
cells [19] at 37ꢀC, 5% CO2 using RPMI 1640 with 10% heat-inactivated fetal calf serum ꢀFCS) and

Synthesis of MKC-442 Analogues
1043
antibiotics ꢀgrowth medium). The culture supernatant was ®ltered ꢀ0.45 nm), aliquoted, and stored at
À80ꢀC 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 MT4
cells as target cells. MT4 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 quantitated by the
HIV-1 antigen detection assay ELISA [15] or indirectly quanti®ed using the MTT assay [16].
Compounds mediating less than 30% reduction of HIV expression were considered without bio-
logical 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 signi®cant. The 50% inhibitory concentration ꢀIC-50) and
the 50% cytotoxic concentration ꢀCC-50) were determined by interpolation from the plots of percent
inhibition vs. concentration of compound.
Acknowledgements
The Royal Danish Ministry of Foreign Affairs is gratefully acknowledged for ®nancial support.
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Received October 15, 2001. Accepted November 19, 2001