Synthesis and evaluation of double-prodrugs against HIV. Conjugation of D4T with 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil (MKC-442, emivirine)-type reverse transcriptase inhibitors via the SATE prodrug approach

Article abstract of DOI:10.1021/jm040845b

This paper reports the synthesis and the antiviral activities of new double-prodrugs against HIV based on the known mixed SATE (S-acyl-2-thioethyl) prodrug approach. The monophosphate of the nucleoside reverse transcriptase inhibitor (NRTI) d4T was masked

Full text of DOI:10.1021/jm040845b

J. Med. Chem. 2005, 48, 1211-1220
1211
Synthesis and Evaluation of Double-Prodrugs against HIV. Conjugation of D4T
with 6-Benzyl-1-(ethoxymethyl)-5-isopropyluracil (MKC-442, Emivirine)-Type
Reverse Transcriptase Inhibitors via the SATE Prodrug Approach
Lene Petersen,‡ Per T. Jørgensen,‡ Claus Nielsen,§ Thomas H. Hansen,| John Nielsen,| and Erik B. Pedersen*,‡
Nucleic Acid Center,† Department of Chemistry, University of Southern Denmark, Campusvej 55,
DK-5230 Odense M, Denmark, Retrovirus Laboratory, Department of Virology, State Serum Institute,
Artillerivej 5, DK-2300 Copenhagen, Denmark, and Department of Natural Sciences, Bioorganic Chemistry Section,
The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
Received May 13, 2004
This paper reports the synthesis and the antiviral activities of new double-prodrugs against
HIV based on the known mixed SATE (S-acyl-2-thioethyl) prodrug approach. The monophos-
phate of the nucleoside reverse transcriptase inhibitor (NRTI) d4T was masked with one SATE
group and one aromatic group through which a nonnucleoside reverse transcriptase inhibitor
(NNRTI) was linked. Double-prodrug 1 was a hybrid between d4T monophosphate and the
known NNRTI MKC-442, which were linked through a labile p-hydroxybenzoyl protection group
in the N-3 position of MKC-442. Double-prodrugs 2 and 3 were conjugates between d4T
monophosphate and the new NNRTIs 15 and 19 linked through a stable phenolic linker that
was a part of the N-1 substituents of the NNRTIs. The double-prodrugs 1, 2, and 3 all had
good activities against wild-type HIV-1, Y181C mutant, and also against a HIV-2 strain that
was resistant to NNRTIs.
Introduction
transportation into the cell followed by release of the
nucleotide inside the cell. Two phosphorylation steps are
then necessary before the molecule can be recognized
as a substrate for RT.
One of the main targets for the inhibition of the
human immunodeficiency virus (HIV) is the viral en-
zyme reverse transcriptase (RT). There are two major
types of drugs against RT. The nucleoside reverse
transcriptase inhibitors (NRTIs)1 are phosphorylated
inside the cell to give the corresponding triphosphates
that act as substrate analogues toward RT and can be
incorporated into the growing DNA chain. However,
NRTIs do not contain the 3′-hydroxy group, which is
essential for continuation of the DNA chain, and there-
fore they serve as chain terminators resulting in non-
functional viral DNA. The nonnucleoside reverse tran-
scriptase inhibitors (NNRTIs)2 are allosteric inhibitors
of HIV-1 RT. They bind to a hydrophobic pocket close
to but distinct from the active site. Binding of the
inhibitor alters the conformation of the active site and
inactivates the enzyme. As this pocket is only present
in HIV-1 RT, the inhibitors are very specific against
HIV-1 and do not inhibit HIV-2 RT or cellular poly-
merases.
One disadvantage of the NRTIs is the need for
triphosphorylation inside the cell. To circumvent this
problem, a number of prodrugs of NRTIs have been
developed.3 In principle, the first phosphate is added
chemically to the nucleoside, but as a molecule contain-
ing a charged phosphate cannot cross the cell mem-
brane, the phosphate group is masked with different
protecting groups. Ideally the protecting groups assist
The SATE (S-acyl-2-thioethyl) prodrugs were first
introduced in 1993 by Imbach et al.4 The SATE group
was developed as a carboxyesterase labile protection
group for the ddU (2′,3′-dideoxyuridine) nucleotide. The
bis-SATE prodrugs, with two SATE groups masking the
phosphate, have been synthesized for the NRTIs AZT
(3′-azido-3′-deoxythymidine)5 and d4T (3′-deoxy-2′,3′-
didehydrothymidine).6 Remarkable results were ob-
tained especially for d4T, where the bis-SATE prodrug
displayed nearly the same activity in CEM cells lacking
thymidine-kinase (TK- cells) as in normal CEM cells.
Also, mixed SATE prodrugs have been synthesized with
one SATE group and one aromatic moiety masking the
phosphate. This prodrug of AZT was still active al-
though less so as compared to the bis-SATE prodrug.7
The activity of the d4T mixed SATE prodrug was
reported to be in the submicromolar range, without
stating any exact value.8
In our laboratories, we have for some time worked
with analogues of the NNRTI MKC-442 (Figure 1).9 One
series of analogues contained unsaturated and aromatic
moieties as part of the N-1 substituent (Figure 1) and
displayed nanomolar activities against wild-type HIV-
1.10,11
In this paper, we introduce the double prodrug con-
cept based on the known “mixed SATE pronucleotides”
incorporating a SATE chain and an aryl substituent as
biolabile phosphate protection groups.7 Such constructs
have demonstrated their ability to deliver selectively
inside infected cells the corresponding mononucleotide
after esterase activation. D4T was chosen as the NRTI
* To whom correspondence should be addressed. Phone:
+4566502555. Fax: +4566158780. E-mail: ebp@chem.sdu.dk.
† A research center funded by The Danish National Research
Foundation for studies on nucleic acid.
‡ University of Southern Denmark.
§ State Serum Institute.
| The Royal Veterinary and Agricultural University.
10.1021/jm040845b CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/22/2005

1212 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Petersen et al.
Figure 1. Structures of MKC-442 and analogues A-E having
activities against HIV-1 (wt) in the nanomolar range.
as the first phosphorylation is the rate-limiting step for
conversion of d4T to the corresponding triphosphate,12
whereas d4T monophosphate (d4TMP) is easily con-
verted to the di- and triphosphate.13 Through the
aromatic moiety of the mixed SATE prodrug of d4T, we
link a NNRTI resulting in a conjugate that combines
two types of RT inhibitors. These are usually given in
combination treatments, and by combining them the
NNRTI will assist the cellular uptake of the hydrophilic
nucleotide and inside the cell the two fragments will be
released to act at two different sites of RT. Furthermore,
it is believed that cellular uptake of both compounds
might prevent or reduce the building up of reservoirs
with virus mutants.
The target molecule 1 (Figure 2) consists of MKC-442
linked to the double-prodrug through a labile p-hydroxy-
benzoyl group in the N-3 position. The target molecules
2 and 3 are linked through a more stable phenolic linker
that is part of the N-1 substituent of new MKC-442
analogues 15 and 19, which resemble the inhibitors
previously reported (Figure 1).10,11
Figure 2. Structures of the target double-prodrugs 1-3 as
conjugates of a NRTI (d4T) and a NNRTI (MKC-442; 15 or
19).
Results and Discussion
Scheme 1a
Chemistry. For the synthesis of target compound 1
(Figure 2), MKC-442 was protected in the N-3 position
with a p-benzyloxybenzoyl group. The benzyl function
could easily be removed at a later stage to liberate the
phenolic function necessary for double-prodrug forma-
tion.
As such, p-benzyloxybenzoic acid was converted into
the corresponding acid chloride using thionyl chloride
and a catalytical amount of DMF14 and reacted directly
with MKC-44215 in pyridine using diisopropyl ethyl-
amine (DIPEA) as a base to give the benzyl protected
product 4 in 86% yield (Scheme 1). Compound 4 was
easily debenzylated using H2 and Pd/C,16 but TLC
analysis showed decomposition of the compound upon
standing. Compound 4 was therefore deprotected on the
needed scale prior to every reaction.
a
(i) p-Benzyloxybenzoic acid chloride, DIPEA, pyridine, 86%;
(ii) H2, Pd/C; (iii) 5, 1H-tetrazole, CH3CN; (iv) tert-butylhydro-
peroxide, 38% (three steps); (v) (S-pivaloyl-2-thioethyl)-N,N-bis-
(diisopropylamino)phosphine, DIA, 1H-tetrazole, CH3CN, 94%.
(S-Pivaloyl-2-thioethyl)-N,N-bis(diisopropylamino)-
phosphine was prepared according to literature proce-
dures,17 noting that the compound was unstable to any
trace of acid. It was therefore necessary to perform
reactions, TLC analysis and column chromatography,
using an excess of triethylamine (TEA) in all cases.18
The bisamidite was successfully reacted with the de-
benzylated analogue of compound 4 using diisopropyl-
amine (DIA) as a base and tetrazole as an activating

Synthesis of Double-Prodrugs against HIV
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1213
Scheme 2a
a
(i) Bz2O, NaOH, H2O, IPA, 58%; (ii) [1,3]dioxalan-2-ylmethyl-triphenyl-phosphonium bromide, TDA-1, K2CO3; (iii) HCl, H2O, 68%;
(iv) NaBH4, KH2PO4, 78%; (v) CH2O, HCl (g); (vi) ClCH2OCH3, DIPEA, CH2Cl2, 97%; (vii) BCl3.
Scheme 3a
agent. Further reaction of the formed amidite with d4T19
followed by in-situ oxidation by iodine gave a complex
mixture according to TLC analysis, and the 31P NMR
spectra did not show the presence of the expected
product. Instead, (S-pivaloyl-2-thioethyl)-N,N-bis(diiso-
propylamino)phosphine was reacted with d4T under the
same conditions as above to give the amidite 5 in 94%
yield. Compound 4 was deprotected using H2 and Pd/C,
and the product was reacted with the amidite 5 in
CH3CN using 1H-tetrazole as an activating agent,
followed by oxidation with tert-butylhydroperoxide to
give the target compound 1 as a mixture of two dia-
stereomers in 38% overall yield for the three steps
(Scheme 1).
Target compound 2 (Figure 2) was a double-prodrug
between d4T and a new NNRTI resembling structure
D depicted in Figure 1. The phenolic moiety, necessary
for double-prodrug formation, was incorporated as a
part of the linker in the N-1 position of the NNRTI. The
benzoyl group was in this case chosen as the protection
group for the phenolic functionality, as hydrogenolysis
of a benzyl group would not be possible due to the
presence of the double bond in the linker.
For the synthesis of the N-1 substituent, the alcohol
6 was synthesized according to literature procedures by
ethyl esterification of p-hydroxycinnamic acid20 followed
by DIBAL reduction of the ester group.21 Compound 6
was benzoylated using benzoic anhydride and NaOH in
a mixture of H2O and isopropyl alcohol (IPA) to give the
monoprotected compound 7 in 58% yield.22 Alterna-
tively, compound 7 was synthesized from the aldehyde
8,23 which was subjected to the Wittig conditions
described by Daubresse et al.24 using (1,3-dioxolan-2-
yl-methyl)-triphenylphosphonium bromide and TDA-1
(tris[2-(2-methoxyethoxy)ethyl]amine) as a catalyst.
After hydrolysis of the dioxolane, the aldehyde 9 was
obtained in 68% yield as the pure trans-isomer. Com-
pound 9 was reduced with NaBH4 and KH2PO4 to give
the alcohol 7 in 78% yield.24
a
(i) BSA, CHCl3; (ii) 10, TMS-triflate, 38%; (iii) NH3/MeOH,
78%; (iv) 5, 1H-tetrazole; (v) tert-butylhydroperoxide, 62%.
the hydroxyl group of compound 7 was replaced directly
by a chloride. Goff et al.26 obtained similar results when
reacting cinnamylic alcohols with formaldehyde and
HCl(g). Instead, they developed a new method for the
synthesis of this type of chloromethyl ethers where the
corresponding MOM-ether was cleaved with BCl3 to give
the cinnamyl chloromethyl ether. Indeed, the procedure
for cinnamyl alcohol was reproduced to give the crude
chloromethyl ether, which upon reaction with the silyl-
ated base 12 (see structure in Scheme 3) gave the
desired N-1 cinnamyloxymethyl uracil in 45% yield.
Encouraged by these results, the MOM-ether 10 was
synthesized from alcohol 7 by reaction with chloro-
methylmethyl ether and DIPEA as the base to give
compound 10 in 97% yield (Scheme 2). It should be
noted that, due to decomposition of the starting mate-
rial, NaH could not be used as the base for this reaction.
Subjecting the MOM-ether 10 to BCl3 followed by
reaction with the silylated base did not produce the
desired product. Only starting material 10 was re-
isolated, and again we observed the formation of the
undesired chloride directly attached at the allylic posi-
tion.
Attempts were made to synthesize the chloromethyl
ether 11, which would be the ideal compound for N-1
alkylation. However, reaction of alcohol 7 with formal-
dehyde and HCl(g), a procedure that has previously
been used to convert allylic alcohols to the corresponding
chloromethyl ethers,25 only produced the product where

1214 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Petersen et al.
Table 1. Antiviral Activity and Cytotoxicity Data of Novel
Double-Prodrugs (Compounds 1-3) and MKC-442 Analogues
(Compounds 13, 15, 18, 19) in MT-4 Cellsa (MKC-442
(6-Benzyl-1-ethoxymethyl-5-isopropyluracil) and d4T
(3′-Deoxy-2′,3′-didehydrothymidine) Are Used for Comparison)
Scheme 4a
EC50 (µM)b - MT-4 cells
compound
IIIBd
N119e
HIV-2
CC50 (µM)c
d4T
0.5
1.8
79
2.7
1
2.4
3.3
0.3
0.5
0.4
0.5
>10
NDf
NDf
>10
NDf
NDf
>10
4
>100
>100
35
MKC-442
0.03
0.03
0.005
0.02
0.04
0.03
0.003
0.003
1
13
15
2
35
35
35
18
19
3
12
38
35
a See the Experimental Section for a description of the assay.
b EC50: Inhibitory concentration of compound required to achieve
50% inhibition of HIV multiplication in MT-4-infected cells. c CC50
:
Cytotoxic concentration of compound required to reduce the
viability of normal uninfected MT-4 cells by 50%. d Wild-type
HIV-1 strain. e The strain N119 harbors the Y181C mutation that
usually causes resistance toward this type of NNRTI. f Not
determined.
in 70% yield. The methyl sulfide group was then cleaved
using sulfuryl chloride at -78 °C to give the chloro-
methylated compound 17.29 As this compound was
assumed not to be stable, the cold solution was added
directly to a solution of the silylated base 12, which led
to formation of the desired product 18 in 78% yield. The
phenolic benzyl group was removed selectively by hy-
drogenolysis using Pd(OH)2/C as a catalyst to give
compound 19 in 81% yield. Compound 19 was reacted
with (S-pivaloyl-2-thioethyl)-N,N-bis(diisopropylamino)-
phosphine using 1H-tetrazole as an activating agent
followed by tert-butylhydroperoxide oxidation to give the
target compound 3 in 60% yield (Scheme 4).
a
(i) NaH, NaI, ClCH2SMe, DME, 70%; (ii) SO2Cl2, CH2Cl2, -78
°C; (iii) BSA, CHCl3; (iv) 17, CsI, 78%; (v) H2, Pd(OH)2/C, 81%;
(vi) (S-pivaloyl-2-thioethyl)-N,N-bis(diisopropylamino)phosphine,
1H-tetrazole; (vii) tert-butylhydroperoxide, 60%.
As we were not successful in the synthesis of com-
pound 11, we instead turned to the MOM-ether 10,
which in principle could function as a reactant for N-1
alkylation. 6-(3,5-Dimethylbenzyl)-5-ethyl-1H-pyrimi-
dine-2,4-dione (12)15 was silylated using BSA (bis-
(trimethylsilyl)acetamide) and reacted with compound
10 and trimethylsilyl trifluoromethanesulfonate27 (TMS-
triflate) (Scheme 3). A mixture of the desired compound
13 and the expected byproducts 6-(3,5-dimethylbenzyl)-
5-ethyl-1-methoxymethyl-1H-pyrimidine-2,4-dione (14)
and the alcohol 7 was formed, necessitating tedious
column chromatography to separate the compounds. A
pure fraction containing compound 13 was isolated as
well as a mixed fraction of compound 13 and 14. The
total yield of compound 13 was estimated to be 38%.
The benzoyl protection group was removed using NH3/
MeOH to give compound 15 in 78% yield. At this stage,
compound 15 could easily be separated from the byprod-
uct 14 by column chromatography. Compound 15 was
reacted with the amidite 5 using 1H-tetrazole as an
activating agent followed by oxidation with tert-butyl-
hydroperoxide. The target compound 2 was isolated in
62% yield (Scheme 3).
Target compound 3 was a conjugate between d4T and
a new NNRTI that also contained an aromatic moiety
as part of the N-1 substituent. This could be used as a
tool to incorporate the phenolic moiety necessary for
double-prodrug formation. The structure of the NNRTI
19 (Scheme 4) resembled that of TNK-651 (6-benzyl-1-
(benzyloxymethyl)-5-isopropyluracil) that has previously
been reported to have activities similar to those of MKC-
442.28
In this case, the benzyl group was a suitable protec-
tion group as there were no reducible functionalities
present in the molecule. For the synthesis of the N-1
substituent, benzyloxybenzyl alcohol was reacted with
chloromethyl methyl sulfide in the presence of NaH and
NaI in dimethoxyethane (DME) to give the product 16
Biological Results and Discussion
For the biological evaluation of the double-prodrugs,
we set up a test system with three different HIV strains.
Strain HTLV-IIIB was the wild-type HIV-1 normally
used for our test experiments. The strain N119 was also
HIV-1 but harbors the mutation Y181C in RT, a
mutation that renders the virus resistant to the NNRTI
MKC-442 and most analogues of this compound. How-
ever, the analogues A-E (Figure 1) actually displayed
activities against this strain at submicromolar lev-
els.10,11 The last strain was a HIV-2 wild-type strain,
which does not contain the hydrophobic pocket in RT
that is the usual binding site for NNRTIs. This strain
is therefore not affected by NNRTIs.
The initial tests were performed in MT-4 cells using
the MTT method for quantification30 (Table 1). In these
tests, d4T showed the expected activities against all
three strains. Also, MKC-442 showed the expected
activities as the compound was active against wild-type
HIV-1 (IIIB) and had no activity against the mutated
strain N119 and HIV-2. The double-prodrug 1 was
active against IIIB and also against N119. This activity
must stem from the d4T part of the molecule; however,
based on these results, it was not clear if the active
component was d4T or d4T monophosphate.
The precursors 13 and 15 for double-prodrug 2 both
had good activities against IIIB and were also active

Synthesis of Double-Prodrugs against HIV
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1215
Table 2. Antiviral Activity and Cytotoxicity Data of Novel Double-Prodrugs (Compounds 1-3) and MKC-442 Analogues (Compounds
15, 19) in CEM Wild Type (wt) or Thymidine-Kinase-Deficient CEM (TK-) Cellsa (MKC-442
(6-Benzyl-1-ethoxymethyl-5-isopropyluracil) and d4T (3′-Deoxy-2′,3′-didehydrothymidine) Are Used for Comparison)
EC50 (µM)b - CEM wt
EC50 (µM)b - CEM TK-
CC50 (µM)c
compound
IIIBd
N119e
HIV-2
IIIB
>10
0.03
N119
HIV-2
CEM wt/TK-
d4T
MKC-442
1
15
2
19
3
0.3
0.8
>100
0.3
0.1
>10
0.02
>10
0.1
>10
0.1
>10
>10
>10
>10
3
>10
4
>10
2
>100
>100
32
0.03
0.03
0.002
0.01
NDf
NDf
0.006
0.006
0.005
NDf
0.3
0.2
0.4
32
0.3
0.2
32
NDf
NDf
NDf
NDf
32
NDf
32
a See the Experimental Section for a description of the assay. b EC50: Inhibitory concentration of compound required to achieve 50%
inhibition of HIV multiplication in MT-4-infected cells. c CC50: Cytotoxic concentration of compound required to reduce the viability of
normal uninfected MT-4 cells by 50%. d Wild-type HIV-1 strain. e The strain N119 harbors theY181C mutation that usually causes resistance
toward this type of NNRTI. f Not determined.
against the mutant N119. However, as expected, com-
pound 15 showed no activity against HIV-2. The double-
prodrug 2 was also active against both IIIB and N119.
Based on these results, it was not possible to determine
which part of the molecule induced the activity.
The precursors 18 and 19 for double-prodrug 3
displayed good activities against both IIIB and N119,
whereas compound 19 was not active against HIV-2 as
expected. Again, the double-prodrug 3 was active against
IIIB and N119, but it was not possible based on these
data to determine from which part of the molecule the
activities stemmed. Interestingly, double-prodrug 3 had
some activity against HIV-2, which must stem from
either d4T or d4T monophosphate (Table 1).
As seen from examination of the results listed in
Table 1, the novel compound 1 was more cytotoxic for
MT-4 cells as d4T and MKC-442, respectively. This
result is in accord with unpublished results from our
group where an N-3 benzoylated derivative of MKC-442
was found more cytotoxic than MKC-442 even though
it was hydrolyzed (within 10 h) to MKC-442 and benzoic
acid in the medium used for biological testing (RPMI
1640) with CC50 ) 31 µM.
To obtain more knowledge about the properties of the
double-prodrugs, we extended the test system to include
two different cell types. One cell type was wild-type
CEM cells (CEM wt), whereas the other type (CEM
TK-) lacked thymidine-kinase (TK) and therefore the
ability to carry out the first phosphorylation of d4T.
Using this cell type made it possible to determine if d4T
was released in the cell as d4T or as the desired d4T-
monophosphate.
Indeed, when d4T was tested (using the ELISA
method31 for quantification, Table 2), the expected
activity against all three strains in CEM wt cells was
observed. When tested in (TK)-deficient CEM cells, all
activity was lost (EC50 > 10 µM). MKC-442 was active
against IIIB in both cell types and lost all activity
against both N119 and HIV-2 in both cell types. Double-
prodrug 1 was active against all three strains in CEM
cells. Based on the results for d4T and MKC-442, the
activity against N119 and HIV-2 must stem from the
d4T part of the molecule. Interestingly, in CEM TK-
cells, good to excellent activities were observed against
all three strains (for HIV-2 the ED50-values in three
independent tests were 3-3-4 µM for compound 1, 3-4-5
µM for compound 2, and 3-2-2 µM for compound 3). As
d4T displayed no activity by itself (the ED50-values in
three independent tests were all above 10 µM), we could
conclude that d4T-monophosphate was indeed released
inside the cell (Table 2).
However, there were two possible routes for the
degradation of double-prodrug 1 (Figure 3). One pos-
sibility was that MKC-442 was hydrolyzed off outside
the cell giving a new type of prodrug 1a and MKC-442
(route A). These two compounds were transported into
the cell where prodrug 1a was hydrolyzed to give d4T-
monophosphate. The other possibility (route B) was
transportation of the double-prodrug into the cell,
followed by hydrolysis in the desired way, releasing d4T-
monophosphate and MKC-442.
The stability of double-prodrug 1 was tested by
incubating the compound with the media used for the
biological tests and checking the presence of 1 by LC-
MS analysis at certain time intervals. The results
showed the presence of compound 1 at time 0 and after
20 min. After 40 min, the compound was hardly
detected, and in the subsequent analysis, after 4 h the
compound was not detected.
The results indicate that route A might be the major
route for the breakdown of the double-prodrug 1.
However, we have no information on the time-scale for
cellular uptake, which means that a combination of the
two routes could also apply.
The precursor 15 for double-prodrug 2 showed excel-
lent activities against IIIB and good activities against
the mutant N119 in both cell lines. As expected, no
activity was observed against HIV-2. For double-
prodrug 2, good activities were observed in CEM wt cells
against all three virus types. The activity against HIV-2
must stem from the d4T part of the molecule. In (TK)-
deficient CEM cells, good to excellent activities were
observed against IIIB and N119 (Table 2). These activi-
ties could in principle stem from 15 that was sepa-
rated from the double-prodrug outside the cell. How-
ever, some activity against HIV-2 was observed for
double-prodrug 2, indicating that d4T-monophosphate
was indeed released inside the cell. This conclusion
was supported by stability studies that showed the
presence of double-prodrug 2 even after 6 days in the
test media.
Similar results were obtained for double-prodrug 3
that was also present after 5 days in the media. Even
though no activity of the precursor 19 was found against
HIV-2, double-prodrug 3 was active in both cell lines,
showing that d4T-monophosphate was indeed released
inside the cell.

1216 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Petersen et al.
Figure 3. Possible routes for degradation of double-prodrug 1. Route A: MKC-442 is hydrolyzed off outside the cell followed by
transportation into the cell of MKC-442 and prodrug 1a. Route B: Double-prodrug 1 is transported into the cell and then hydrolyzed
into the active components.
spectra were recorded on a 4.7 T Ultima (IonSpec, Irvine, CA)
Fourier transform ion cyclotron resonance (FTICR) mass
spectrometer. LC-HRMS analysis was performed on a Waters
2795 system with a Waters 996 photodiode array (PDA)
detector using a Waters Symmetry C18 Column (2.1 × 50 mm,
3.5 µm) coupled with a Micromass LCT apparatus with an AP-
ESI probe.using Leu-Enkephalin (556.2771 g/mol) for calibra-
tion (0-95% ACN, 10 min, 0.2 mL/min). Melting points were
determined on a Bu¨chi melting point apparatus. Elemental
analysis were performed at Mikroanalytisk afdeling, Univer-
sity of Copenhagen, Copenhagen, Denmark; found values
agreed favorably with the calculated ones. The progress of
reactions was monitored via 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 sieves. CH2Cl2
was dried over 4 Å sieves.
6-Benzyl-3-(4-benzyloxy-benzoyl)-1-ethoxymethyl-5-
isopropyl-1H-pyrimidine-2,4-dione (4). 4-Benzyloxy-ben-
zoic acid (2.01 g, 8.8 mmol) was dissolved in SOCl2 (30 mL),
and a few drops of DMF were added. The mixture was refluxed
overnight. After the mixture was cooled to room temperature,
the solvent was evaporated under reduced pressure and the
crude 4-benzyloxy-benzoyl chloride was used without further
purification.
DIPEA (1.55 g, 12 mmol) was dissolved in dry pyridine (50
mL). 6-Benzyl-1-ethoxymethyl-5-isopropyl-1H-pyrimidine-2,4-
dione (MKC-442, 1.21 g, 4 mmol) was added, the solution was
cooled to 0 °C, and the crude 4-benzyloxy-benzoyl chloride was
added. The solution was stirred at room temperature over-
night, and then the reaction was quenched with H2O (5 mL)
and the solvent was evaporated under reduced pressure. The
residue was redissolved in CH2Cl2 (200 mL). The organic phase
was washed with H2O (2 × 100 mL), dried (MgSO4), and
evaporated under reduced pressure. The product was purified
by silica column chromatography (50 f 70% CH2Cl2 in
cyclohexane). Yield: 1.77 g (86%), white foam; Rf ) 0.21
(CH2Cl2). 1H NMR (CDCl3): δ 1.18 (3H, t, J ) 7.0 Hz, CH2CH3),
1.29 (6H, d, J ) 6.7 Hz, CH(CH3)2), 2.90 (1H, heptet, J ) 6.9
Hz, CH(CH3)2), 3.62 (2H, q, J ) 7.0 Hz, CH2CH3), 4.24 (2H, s,
CH2Ph), 5.15 (4H, s, PhCH2O, NCH2O), 7.04 (2H, d, J ) 9.0
Hz, H-aryl), 7.15-7.43 (10H, m, H-aryl), 7.90 (2H, d, J ) 9.0
Hz, H-aryl). 13C NMR (CDCl3): δ 15.01 (CH2CH3), 20.37
(CH(CH3)2), 28.54 (CH(CH3)2), 33.59 (CH2Ph), 65.31 (OCH2-
Conclusions
In summary, double-prodrugs based on the SATE
prodrug approach have been developed. Double-prodrug
1 was a hybrid between d4T and the known NNRTI
MKC-442 linked through a cleavable p-hydroxybenzoyl
protection group in the N-3 position of MKC-442. The
double-prodrug had interesting activities against three
virus types in both CEM wt and CEM TK- cells,
showing that d4T monophosphate was indeed released
inside the cell. However, it was not possible to conclude
if the double-prodrug entered the cell as the intact
molecule or if it was hydrolyzed partly outside the cell
to give the new prodrug 1a and MKC-442, which were
then individually transported into the cell.
Double-prodrugs 2 and 3 were hybrids between d4T
and the new NNRTIs 15 and 19 linked through the
phenol containing N-1 substituents of the NNRTIs. The
precursors 15 and 19 both showed excellent activities
against IIIB and even good activities against the mutant
N119, results that correlated well with the activities
previously reported for similar molecules.10,11 Both
double-prodrugs 2 and 3 were active against HIV-2 in
TK-deficient CEM cells, proving release of d4T mono-
phosphate inside the cell. As the possible hydrolysis
pathway of this type of double-prodrug was simple as
compared to double-prodrug 1 and stability studies
proved the compounds to be stable in the test media for
a minimum of 5 days, we conclude that the double-
prodrugs were transported into the cells and hydrolyzed
in the desired way to give d4T monophosphate and the
NNRTIs 15 or 19.
Experimental Section
NMR spectra were recorded on a Varian Gemini 2000 NMR
spectrometer at 300 MHz for 1H and 75 MHz for 13C with
tetramethylsilane as an internal standard. 31P NMR spectra
were recorded at 121.5 MHz using 85% H3PO4 as an external
standard. Chemical shifts are reported in parts per million
(δ), and signals are expressed as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br (broad). MALDI mass

Synthesis of Double-Prodrugs against HIV
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1217
CH3), 70.30, 73.15 (NCH2O, OCH2Ph), 115.38, 119.61, 124.54,
127.25, 127.34, 127.39, 128.31, 128.71, 129.28, 132.89, 135.17,
135.80, 148.43, 150.81, 161.33, 164.20, 167.91 (C-aryl, CO).
MS (MALDI, m/z) ) 535 (M + Na+).
mp 125-127 °C (EtOAc/PE (60-80 °C)); Rf ) 0.38 (EtOH/
CH2Cl2, 5:95). 1H NMR (CDCl3): δ 1.67 (1H, brs, OH), 4.33
(2H, d, J ) 5.1 Hz, CH2), 6.34 (1H, td, J ) 5.6 Hz, 16 Hz,
CHCH2), 6.63 (1H, d, J ) 15.9 Hz, CH-aryl), 7.15-7.21 (2H,
m, H-aryl), 7.40-7.55 (2H, m, H-aryl), 7.60-7.67 (1H, m,
H-aryl), 8.17-0.22 (2H, m, H-aryl). 13C NMR (CDCl3): δ 63.58
(CH2), 121.81, 127.44, 128.55, 128.79, 129.43, 130.04, 130.14,
133.60, 134.53, 150.34 (C-aryl, CHdCH), 165.13 (CO). MS
(MALDI, m/z) calcd 277.0835 (M + Na+), found 277.0837 (M
+ Na+).
2,2-Dimethyl-thiopropionic Acid S-(2-{Diisopropylami-
no-[5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-
yl)-2,5-dihydro-furan-2-ylmethoxy]-phosphanyloxy}-eth-
yl) Ester (5). 1-(5-Hydroxymethyl-2,5-dihydro-furan-2-yl)-5-
methyl-1H-pyrimidine-2,4-dione (d4T, 0.15 g, 0.67 mmol) was
dissolved in dry CH3CN (5 mL) containing 3 Å activated sieves
(0.6 g). The solution was cooled to 0 °C, and a solution of (S-
pivaloyl-2-thioethyl)-N,N-bis(diisopropylamino)phosphine (0.40
g, 1.02 mmol) and DIA (0.14 g, 1.4 mmol) in dry CH3CN (3
mL) was added. A solution of 1H-tetrazole (3.0 mL, 0.45 M in
CH3CN, 1.4 mmol) was then added. The mixture was stirred
for 2 h and then diluted with acid-free EtOAc (20 mL). The
organic phase was washed with brine (2 × 20 mL) and H2O (2
× 20 mL), dried (MgSO4), and evaporated under reduced
pressure. The product was purified by silica column chroma-
tography (cyclohexane/EtOAc/TEA, 50:49:1), yielding the title
compound (0.33 g, 94%) as a clear glace; Rf ) 0.56 (1% TEA/
EtOAc, prerun). 31P NMR (CDCl3/pyridine-d5/H3PO4 exter-
nal): δ 147.99, 149.85. MS (MALDI, m/z) ) 522 (M + Li+).
2,2-Dimethyl-thiopropionic Acid S-(2-{[4-(4-Benzyl-
3-ethoxymethyl-5-isopropyl-2,6-dioxo-3,6-dihydro-2H-
pyrimidine-1-carbonyl)-phenoxy]-[5-(5-methyl-2,4-dioxo-
3,4-dihydro-2H-pyrimidin-1-yl)-2,5-dihydro-furan-2-yl-
methoxy]-phosphoryloxy}-ethyl) Ester (1). 6-Benzyl-3-(4-
benzyloxy-benzoyl)-1-ethoxymethyl-5-isopropyl-1H-pyrimidine-
2,4-dione (4, 0.22 g, 0.43 mmol) and Pd-C (10%, 0.046 g, 0.043
mmol Pd) were suspended in MeOH (25 mL). The atmosphere
was changed for N2 and then H2. The suspension was stirred
1.5 h at room temperature and then filtered through Celite.
The solvent was evaporated under reduced pressure, and the
crude compound was dried in vacuo. To the dried compound
was added 3 Å activated sieves (0.5 g) and dry CH3CN (4 mL).
A solution of 1H-tetrazole (1.9 mL, 0.45 M in CH3CN, 0.86
mmol) was added and then dropwise a solution of the amidite
(5, 0.34 g, 0.66 mmol) in dry CH3CN (4 mL). The mixture was
stirred for 2 h at room temperature followed by addition of a
solution of tert-butyl hydroperoxide (0.15 mL, 5.7 M in decane
(anhydrous, over 4 Å sieves), 0.86 mmol). The mixture was
stirred another 1 h at room temperature and then diluted with
CH2Cl2 (20 mL). The organic phase was washed with 10%
aqueous Na2S2O3 (2 × 20 mL) and H2O (2 × 20 mL), dried
(MgSO4), and evaporated under reduced pressure. The product
was purified by silica column chromatography (0 f 2% EtOH
in CH2Cl2), yielding the title compound (0.14 g, 38%) as a light
yellow foam; Rf ) 0.34 (EtOH/CH2Cl2, 5:95). 1H NMR
(CDCl3): δ 1.15-1.32 (18H, m, OCH2CH3, CH(CH3)2, C(CH3)3),
1.85, 1.89 (3H, 2 × d, J ) 1.2 Hz, CH3-d4T), 2.91 (1H, heptet,
J ) 7.0 Hz, CH(CH3)2), 3.10-3.20 (2H, m, SCH2), 3.60-3.69
(2H, m, OCH2CH3), 4.16-4.48 (6H, m, SCH2CH2, CH2Ph,
POCH2), 5.00-5.06 (1H, m, CH2CHO), 5.10-5.22 (2H, m,
NCH2O), 5.88 (0.5H, dt, J ) 1.9 Hz, 5.6 Hz, CHdCHCHN),
5.93 (0.5H, dt, J ) 1.9 Hz, 6.0 Hz, CHdCHCHN), 6.28-6.35
(1H, m, CH2CHCHdCH), 6.91-6.95 (0.5H, m, OCHN), 6.98-
7.01 (0.5H, m, OCHN), 7.10-7.43, 7.91-7.98 (10H, 2 × m,
H-aryl, H-6), 8.48 (0.5H, s, NH), 8.69 (0.5H, s, NH). 31P NMR
(CDCl3/H3PO4 external): δ -7.08, -6.12. MS (MALDI, m/z)
calcd 875.2698 (M + Na+), found 875.2734 (M + Na+).
Benzoic Acid trans-4-(3-Oxo-propenyl)-phenyl Ester
(9). TDA-1 (1.06 g, 4.67 mmol) was dissolved in a two-phase
system consisting of a saturated aqueous solution of K2CO3
(30 mL) and CH2Cl2 (30 mL). The solution was stirred
vigorously, and [1,3]dioxalan-2-ylmethyl-triphenyl-phospho-
nium bromide (2.91 g, 6.78 mmol) and benzoic acid 4-formyl-
phenyl ester (8, 1.02 g, 4.52 mmol) were added. The mixture
was stirred at room temperature overnight. The phases were
then separated, and the aqueous phase was extracted with
CH2Cl2 (2 × 30 mL). HCl (10% aqueous solution, 30 mL) was
added to the combined organic fractions, and this was stirred
overnight at room temperature. The aqueous layer was diluted
with H2O (30 mL), and the phases were separated and the
aqueous phase extracted with CH2Cl2 (2 × 30 mL). The
combined organic layers were washed with saturated aqueous
solution of NaHCO3 (2 × 30 mL) and brine (2 × 30 mL), dried
(MgSO4), and evaporated under reduced pressure. The product
was purified by silica column chromatography (CH2Cl2), yield-
ing the title compound (0.78 g, 68%) as white needles; mp 103-
105 °C (EtOAc/PE (60-80 °C)); Rf ) 0.49 (EtOAc/CH2Cl2, 1:9).
1H NMR (CDCl3): δ 6.71 (1H, dd, J ) 7.8 Hz, 15.9 Hz,
CHCHO), 7.29-7.34, 7.50-7.56, 7.63-7.70, 8.18-8.23 (10H,
m, H-aryl, CH-aryl), 9.72 (1H, d, J ) 7.4 Hz, CHO). 13C NMR
(CDCl3): δ 122.56, 128.66, 129.07, 129.74, 130.23, 131.76,
133.90, 151.52, 153.14 (C-aryl, CHdCH), 164.76 (COO), 193.49
(CHO). MS (MALDI, m/z) ) 253 (M + H+).
Benzoic Acid trans-4-(3-Hydroxy-propenyl)-phenyl Es-
ter (7) from Benzoic Acid trans-4-(3-Oxo-propenyl)-
phenyl Ester (9). Benzoic acid trans-4-(3-oxo-propenyl)-
phenyl ester (9, 2.19 g, 8.68 mmol) and KH2PO4 (11.81 g, 86.80
mmol) were dissolved in MeOH (140 mL) and cooled to 0 °C.
NaBH4 (0.82 g, 21.70 mmol) was added in portions. The
mixture was stirred at 0 °C for 1 h, and then the reaction was
quenched by careful addition of H2O (50 mL). The reaction
mixture was acidified to pH ) 3 with aqueous solution of HCl
(4 M). The MeOH phase was evaporated under reduced
pressure, and the remaining aqueous phase was extracted with
CH2Cl2 (3 × 100 mL). The organic phase was washed with
H2O (2 × 100 mL), dried (MgSO4), and evaporated under
reduced pressure. The product was purified by silica column
chromatography (2 f 3% EtOH in CH2Cl2), yielding the title
compound (1.73 g, 78%); data were consistent with those
reported above.
Benzoic Acid trans-4-(3-Methoxymethoxy-propenyl)-
phenyl Ester (10). Benzoic acid trans-4-(3-hydroxy-propenyl)-
phenyl ester (7, 0.25 g, 1 mmol) was dissolved in dry CH2Cl2
(5 mL). The solution was cooled to 0 °C, and DIPEA (0.23 g,
1.8 mmol) and chloro-methoxy-methane (0.12 g, 1.5 mmol)
were added. The solution was stirred overnight at room
temperature. The solvent was evaporated, and the product was
purified by silica column chromatography (cyclohexane/EtOAc,
3:1), yielding the title compound (0.29 g, 97%) as a white solid;
mp 74-75 °C (cyclohexane); Rf ) 0.53 (cyclohexane/EtOAc,
Benzoic Acid 4-trans-(3-Hydroxy-propenyl)-phenyl Es-
ter (7) from trans-4-(3-Hydroxy-propenyl)-phenol (6).
trans-4-(3-Hydroxy-propenyl)-phenol (6, 0.11 g, 0.73 mmol) was
dissolved in isopropyl alcohol (IPA, 5 mL), containing NaOH
(0.080 g, 2 mmol) dissolved in H2O (1 mL). Benzoic anhydride
(0.45 g, 2 mmol) was added, and the solution was stirred at
room temperature for 0.5 h and then the solvent was evapo-
rated under reduced pressure. The residue was partitioned
between EtOAc (25 mL) and H2O (25 mL). The organic phase
was washed with brine (3 × 25 mL), dried (MgSO4), and
evaporated under reduced pressure. The product was purified
by silica column chromatography (0 f 2% EtOH in CH2Cl2),
yielding the title compound (0.11 g, 58%) as white needles;
1
1:1). H NMR (CDCl3): δ 3.41 (3H, s, CH3), 4.24 (2H, d, J )
6.2 Hz, CHCH2), 4.71 (2H, s, OCH2O), 6.28 (1H, td, J ) 6.1
Hz, 16.0 Hz, CHCH2), 6.65 (1H, d, J ) 15.9 Hz, CH-aryl), 7.18
(2H, d, J ) 8.4 Hz, H-aryl), 7.41-7.68 (5H, m, H-aryl), 8.20
(2H, d, J ) 7.5 Hz, H-aryl). 13C NMR (CDCl3): δ 55.33 (CH3),
67.74 (CHCH2), 95.65 (OCH2O), 121.80, 125.87, 127.48, 128.56,
129.48, 130.15, 131.55, 133.59, 134.53, 150.39 (C-aryl, CHdCH),
165.10 (CO). MS (MALDI, m/z) calcd 321.1097 (M + Na+),
found 321.1090 (M + Na+).
Benzoic Acid trans-4-{3-[6-(3,5-Dimethyl-benzyl)-5-
ethyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethoxy]-

1218 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Petersen et al.
propenyl}-phenyl Ester (13). N,O-Bis(trimethylsilyl)acet-
amide (BSA, 1.70 g, 8.38 mmol) was dissolved in dry CH3CN
(25 mL). 6-(3,5-Dimethylbenzyl)-5-ethyl-1H-pyrimidine-2,4-
dione (12, 0.87 g, 3.35 mmol) was added, and the mixture was
stirred 10 min until a clear solution was obtained. The solution
was cooled to -45 °C,and TMS triflate (0.82 g, 3.69 mmol) was
added followed by addition of benzoic acid 4-(3-methoxymethoxy-
propenyl)-phenyl ester (10, 2.00 g, 6.70 mmol) dissolved in dry
CH3CN (10 mL). The mixture was stirred at room temperature
overnight, and then the reaction was quenched with a satu-
rated aqueous solution of NaHCO3 (25 mL). The solvent was
evaporated under reduced pressure, and the residue was
dissolved in CH2Cl2 (150 mL). The organic phase was washed
with H2O (100 mL), brine (100 mL), and H2O (100 mL), dried
(MgSO4), and evaporated under reduced pressure. The product
was partly purified by silica column chromatography first
giving 0.36 g of the desired product 13 (cyclohexane/EtOAc,
1:1) and then purification of mixed fractions yielding 0.71 g of
a mixture between 13 and the byproduct 6-(3,5-dimethyl-
benzyl)-5-ethyl-1-methoxymethyl-1H-pyrimidine-2,4-dione 14
(chromatographed on silica gel with cyclohexane/acetone, 3:1).
From the 1H NMR spectra, it was determined that the desired
product 13 constituted 0.31 g of this mixture. Total yield: 0.67
g (38%) as white foam (pure fraction); Rf ) 0.38 (cyclohexane/
EtOAc, 1:1). 1H NMR (CDCl3): δ 1.08 (3H, t, J ) 7.4 Hz,
CH2CH3), 2.27 (6H, s, 2 × CH3-aryl), 2.48 (2H, q, J ) 7.5 Hz,
CH2CH3), 4.11 (2H, s, CH2-aryl), 4.29 (2H, d, J ) 6.2 Hz,
OCH2CH), 5.20 (2H, s, NCH2O), 6.21 (1H, td, J ) 6.2 Hz, 15.9
Hz, CHCH2), 6.65 (1H, d, J ) 16.0 Hz, CH-aryl), 6.71 (2H, s,
H-aryl), 6.90 (1H, s, H-aryl), 7.15-7.21 (2H, m, H-aryl), 7.40-
7.67 (5H, m, H-aryl), 8.17-8.23 (2H, m, H-aryl), 9.25 (1H, s,
NH). 13C NMR (CDCl3): δ 13.76 (CH2CH3), 19.20 (CH2CH3),
21.27 (2 × CH3-aryl), 33.36 (CH2-aryl), 70.14 (OCH2CH), 72.49,
(NCH2O), 116.87 (C-5), 121.83, 124.94, 124.99, 127.56, 128.55,
128.99, 129.44, 130.14, 132.38, 133.59, 134.21, 134.86, 138.90,
149.30, 150.54, 151.90 (C-aryl, CHdCH, C-2, C-6), 163.20,
165.05 (CO, C-4). MS (MALDI, m/z) calcd 547.2203 (M + Na+),
found 547.2208 (M + Na+).
and then dropwise a solution of the amidite (5, 0.184 g, 0.36
mmol) in dry CH3CN (2.5 mL) was added. The mixture was
stirred at room temperature for 2.5 h followed by addition of
a solution of tert-butyl hydroperoxide (0.084 mL, 5.7 M in
decane (anhydrous, over 4 Å sieves), 0.48 mmol). The mixture
was stirred 1 h and then diluted with CH2Cl2 (25 mL). The
organic phase was washed with a saturated aqueous solution
of Na2S2O3 (2 × 20 mL) and H2O (2 × 20 mL), dried (MgSO4),
and evaporated under reduced pressure. The product was
purified by silica column chromatography (EtOH/CH2Cl2, 3:97),
yielding the title compound (0.126 g, 62%) as white foam, Rf
1
) 0.21 (EtOH/CH2Cl2, 5:95). H NMR (CDCl3): δ 1.04-1.12
(3H, m, CH2CH3), 1.22, 1.23 (9H, 2 × s, C(CH3)3), 1.81 (3H, s,
CH3-d4T), 2.27 (6H, s, 2 × ArCH3), 2.40-2.55 (2H, m,
CH2CH3), 3.10-3.20 (2H, m, SCH2), 4.00-4.47 (8H, m, OCH2-
CH, POCH2, CH2-aryl, SCH2CH2), 4.98-5.45 (3H, m, CH2CHO,
NCH2O), 5.82 (1H, m, CHdCHCHN), 6.08-6.20 (1H, m,
CH2CHdCH), 6.27-6.34 (1H, m, CHdCHCHN), 6.54, 6.56
(1H, 2 × d, J ) 15.9 Hz, CH2CHdCH), 6.71 (2H, s, H-aryl),
6.89 (1H, s, H-aryl), 6.97-7.32 (6H, m, OCHN, 4 × H-aryl,
H-6 (d4T)), 9.14, 9.26, 9.41, 9.44 (2H, 4 × s, 2 × NH). 31P NMR
(CDCl3/H3PO4 external): δ -6.26, -5.36. MS (MALDI, m/z)
) 873 (M + Na+).
1-Benzyloxy-4-methylsulfanylmethoxymethyl-ben-
zene (16). NaH (0.80 g, 60% suspension in mineral oil, 20
mmol) was suspended in dimethoxyethane (DME, 10 mL). The
suspension was cooled to 0 °C, and 4-benzyloxybenzyl alcohol
(2.14 g, 10 mmol) was added, followed by addition of NaI (1.50
g, 10 mmol) and chloromethyl methyl sulfide (0.97 g, 10 mmol).
The reaction was stirred 1.5 h at 0 °C and then 4 h at room
temperature. The reaction was quenched by addition of H2O
(20 mL), and the aqueous phase was extracted with Et2O (3
× 50 mL). The organic phase was dried (K2CO3) and evapo-
rated. The product was purified by silica column chromatog-
raphy (10 f 20% Et2O in PE (60-80 °C)), yielding the title
compound (1.91 g, 70%) as a clear oil; Rf ) 0.32 (20% Et2O/PE
(60-80 °C)). 1H NMR (CDCl3): δ 2.17 (3H, s, SCH3), 4.55 (2H,
s, OCH2S), 4.66 (2H, 2, aryl-CH2O), 5.06 (2H, s, aryl-CH2O-
aryl), 6.92-6.98, 7.24-7.45 (9H, m, H-aryl). 13C NMR (CDCl3):
δ 13.87 (CH3S), 69.01 (aryl-CH2O-aryl), 69.99 (aryl-CH2O),
74.07 (OCH2S), 114.82, 127.40, 127.93, 128.55, 129.78, 136.92,
158.50 (C-aryl). MS (MALDI, m/z) ) 297 (M + Na+).
1-(4-Benzyloxy-benzyloxymethyl)-6-(3,5-dimethylben-
zyl)-5-ethyl-1H-pyrimidine-2,4-dione (18). 1-Benzyloxy-4-
methylsulfanylmethoxymethyl-benzene (16, 0.127 g, 0.46 mmol)
was dissolved in dry CH2Cl2 (2 mL) and cooled to -78 °C.
Sulfuryl chloride (0.062 g, 0.46 mmol) was dissolved in CH2Cl2
(2 mL) and added dropwise to the cold solution. The solution
was stirred 30 min at -78 °C. BSA (0.157 g, 0.77 mmol) was
dissolved in dry CHCl3 (2 mL), and 6-(3,5-dimethylbenzyl)-5-
ethyl-1H-pyrimidine-2,4-dione (12, 0.080 g, 0.31 mmol) was
added and the mixture was stirred 10 min to give a clear
solution. CsI (0.081 g, 0.31 mmol) was added, and then the
cold solution from above was transferred. The reaction mixture
was stirred at room temperature overnight. The reaction was
quenched by the addition of a saturated aqueous solution of
NaHCO3 (20 mL). The aqueous phase was extracted with
CH2Cl2 (3 × 20 mL), and the combined organic phases were
dried (MgSO4) and evaporated under reduced pressure. The
product was purified by silica column chromatography (EtOAc/
PE (60-80 °C), 2:1), yielding the title compound (0.117 g, 78%)
as a white foam; Rf ) 0.36 (EtOAc/PE (60-80 °C), 1:1). 1H
NMR (CDCl3): δ 1.07 (3H, t, J ) 7.3 Hz, CH2CH3), 2.26 (6H,
s, 2 × CH3), 2.46 (2H, q, J ) 7.3 Hz, CH2CH3), 4.05 (2H, s,
aryl-CH2-aryl), 4.58 (2H, s, OCH2-aryl), 5.06 (2H, s, OCH2-
aryl), 5.17 (2H, s, NCH2O), 6.63 (2H, s, H-aryl), 6.88 (1H, s,
H-aryl), 6.95 (2H, d, J ) 8.0 Hz, H-aryl), 7.22-7.46 (7H, m,
H-aryl), 9.18 (1H, s, NH). 13C NMR (CDCl3): δ 13.74 (CH2CH3),
19.14 (CH2CH3), 21.24 (2 × CH3), 33.21 (CH2-aryl), 70.00
(OCH2-aryl), 71.28 (OCH2-aryl), 72.53 (NCH2O), 114.79 (C-
aryl), 116.72 (C-5), 124.97, 127.41, 127.94, 128.55, 128.91,
129.47, 129.65, 134.83, 136.83, 138.81 (C-aryl), 149.29, 151.84
(C-2, C-6), 158.64 (C-aryl), 163.18 (C4). MS (MALDI, m/z) )
507 (M + Na+).
6-(3,5-Dimethylbenzyl)-5-ethyl-1-[trans-3-(4-hydroxy-
phenyl)-allyloxymethyl]-1H-pyrimidine-2,4-dione (15).
Benzoic acid trans-4-{3-[6-(3,5-dimethylbenzyl)-5-ethyl-2,4-
dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethoxy]-propenyl}-phen-
yl ester (13, 0.32 g, 0.61 mmol) was dissolved in a saturated
solution of NH3 in MeOH (10 mL). The solution was stirred
overnight at room temperature, and then the solvent was
evaporated under reduced pressure. The residue was redis-
solved in CH2Cl2 (50 mL), and the organic phase was washed
with H2O (25 mL), dried (MgSO4), and evaporated under
reduced pressure. The product was purified by silica column
chromatography (EtOH/CH2Cl2, 2:98), yielding the title com-
pound (0.20 g, 78%) as white foam; Rf ) 0.24 (EtOH/CH2Cl2,
1
5:95). H NMR (CDCl3): δ 1.06 (3H, t, J ) 7.4 Hz, CH2CH3),
2.26 (6H, s, 2 × CH3-aryl), 2.46 (2H, q, J ) 7.3 Hz, CH2CH3),
4.10 (2H, s, CH2-aryl), 4.24 (2H, d, J ) 6.2 Hz, OCH2CH), 5.18
(2H, s, NCH2O), 6.05 (1H, td, J ) 6.4 Hz, 15.8 Hz, CHCH2),
6.53 (1H, d, J ) 15.6 Hz, CH-aryl), 6.70 (2H, s, H-aryl), 6.79
(2H, d, J ) 8.9 Hz, H-aryl), 6.89 (1H, s, H-aryl), 7.22 (2H, d,
J ) 8.6 Hz, H-aryl), 9.23 (1H, s, NH). 13C NMR (CDCl3): δ
13.74 (CH2CH3), 19.19 (CH2CH3), 21.25 (2 × CH3-aryl), 33.35
(CH2-aryl), 70.53 (OCH2CH), 72.47 (NCH2O), 115.56 (C-aryl),
116.86 (C-5), 122.00 (C-aryl), 125.01 (CH-aryl), 127.36, 127.95,
128.86, 133.31, 134.83 (C-aryl), 138.91 (CH2CH), 149.59 (C-
2), 151.91, 155.96 (C-aryl, C-6), 163.34 (C-4). MS (MALDI, m/z)
) 443 (M + Na+).
2,2-Dimethyl-thiopropionic Acid S-(2-trans-{[4-{3-[6-
(3,5-Dimethylbenzyl)-5-ethyl-2,4-dioxo-3,4-dihydro-2H-
pyrimidin-1-ylmethoxy]-propenyl}-phenoxy]-[5-(5-methyl-
2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-2,5-dihydro-
furan-2-ylmethoxy]-phosphoryloxy}-ethyl) Ester (2). 6-(3,5-
Dimethylbenzyl)-5-ethyl-1-[trans-3-(4-hydroxy-phenyl)-
allyloxymethyl]-1H-pyrimidine-2,4-dione (15, 0.100 g, 0.24
mmol) was dissolved in dry CH3CN (2.5 mL) in a flame-dried
bottle containing 3 Å molecular sieves (0.25 g). 1H-Tetrazole
solution (1.06 mL, 0.45 M in CH3CN, 0.48 mmol) was added,

Synthesis of Double-Prodrugs against HIV
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1219
6-(3,5-Dimethyl-benzyl)-5-ethyl-1-(4-hydroxy-benzyl-
oxymethyl)-1H-pyrimidine-2,4-dione (19). 1-(4-Benzyloxy-
benzyloxymethyl)-6-(3,5-dimethylbenzyl)-5-ethyl-1H-pyrimidine-
2,4-dione (18, 1.00 g, 2.06 mmol) and catalyst (0.15 g, 20 wt %
Pd (dry basis) on carbon, wet) were suspended in CH2Cl2/
MeOH (50 mL, 1:1). The atmosphere was exchanged for H2,
and the mixture was stirred 1.5 h at room temperature.
Another portion of catalyst was added (0.07 g, 20 wt % Pd (dry
basis) on carbon, wet), and the mixture was stirred under H2
overnight. Another portion of catalyst (0.07 g, 20 wt % Pd (dry
basis) on carbon, wet) was added, and the reaction mixture
was stirred another 2 h under H2. The mixture was then
filtered through Celite that was washed with additional
portions of MeOH and CH2Cl2. The combined filtrates were
evaporated under reduced pressure, and the product was
purified by silica column chromatography (EtOAc/PE (60-80
°C), 1:1), yielding the title compound (0.659 g, 81%) as a white
foam; Rf ) 0.36 (EtOAc/PE (60-80 °C), 1:1). 1H NMR
(CDCl3): δ 1.05 (3H, t, J ) 7.4 Hz, CH2CH3), 2.26 (6H, s, 2 ×
CH3), 2.44 (2H, q, J ) 7.5 Hz, CH2CH3), 4.05 (2H, s, aryl-CH2-
aryl), 4.55 (2H, s, OCH2-aryl), 5.17 (2H, s, NCH2O), 6.63 (2H,
s, H-aryl), 6.81 (2H, d, J ) 8.5 Hz, H-aryl), 6.87 (1H, s, H-aryl),
7.17 (2H, d, J ) 8.5 Hz, H-aryl), 9.50 (1H, s, NH). 13C NMR
(CDCl3): δ 13.71 (CH2CH3), 19.14 (CH2CH3), 21.25 (2 × CH3),
33.21 (CH2-aryl), 71.61 (OCH2-aryl), 72.68 (NCH2O), 115.48
(C-aryl), 116.80 (C5), 125.00, 128.96, 129.30, 129.58, 134.78,
138.86 (C-aryl), 149.66, 151.96 (C-2, C-6), 155.99 (C-aryl),
163.60 (C-4). MS (MALDI, m/z) ) 417 (M + Na+).
using MT-4, CEM, and CEM TK- cells as target cells. 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 MT-4 cultures was
indirectly quantified using the MTT assay as previously
described.30 Expression of HIV in CEM and CEM TK- cultures
was quantified by HIV-1 and HIV-2 antigen detection ELISA.31
Compounds mediating less than 30% reduction of HIV expres-
sion were considered without biological activity. Compounds
were tested in parallel for cytotoxic effect in uninfected
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 con-
centration (IC50) and the 50% cytotoxic concentration (CC50)
were determined by interpolation from the plots of percent
inhibition versus concentration of compound.
Stability Studies. Double-prodrugs 1, 2, or 3 (1.5 mg) were
dissolved in DMSO (15 mL), and 30 µL of this solution was
added to growth medium to obtain a concentration of 0.117
mM. Samples (50 µL) were taken at certain time intervals and
analyzed directly by LC-MS.
Supporting Information Available: Elemental analysis
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
2,2-Dimethyl-thiopropionic Acid S-(2-{{4-[6-(3,5-Di-
methylbenzyl)-5-ethyl-2,4-dioxo-3,4-dihydro-2H-pyrim-
idin-1-ylmethoxymethyl]-phenoxy}-[5-(5-methyl-2,4-dioxo-
3,4-dihydro-2H-pyrimidin-1-yl)-2,5-dihydro-furan-2-
ylmethoxy]-phosphoryloxy}-ethyl) Ester (3). 6-(3,5-Di-
methylbenzyl)-5-ethyl-1-(4-hydroxy-benzyloxymethyl)-1H-
pyrimidine-2, 4-dione (19, 0.094 g, 0.24 mmol) was dissolved
in dry CH3CN (2.5 mL) in a flame-dried bottle containing 3 Å
molecular sieves (0.25 g). 1H-Tetrazole solution (1.06 mL, 0.45
M in CH3CN, 0.48 mmol) was added, and then dropwise was
added a solution of the amidite (5, 0.184 g, 0.36 mmol) in dry
CH3CN (2.5 mL). The mixture was stirred at room temperature
for 2.5 h followed by addition of a solution of tert-butyl
hydroperoxide (0.084 mL, 5.7 M in decane (anhydrous, over 4
Å sieves), 0.48 mmol). The mixture was stirred 1 h and then
diluted with CH2Cl2 (25 mL). The organic phase was washed
with a saturated aqueous solution of Na2S2O3 (2 × 20 mL) and
H2O (2 × 20 mL), dried (MgSO4), and evaporated under
reduced pressure. The product was purified by silica column
chromatography (EtOH/CH2Cl2, 3:97), yielding the title com-
pound (0.118 g, 60%) as a white foam; Rf ) 0.16 (EtOH/CH2Cl2,
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1
5:95). H NMR (CDCl3): δ 1.07 (3H, t, J ) 7.5 Hz, CH2CH3),
1.22, 1.23 (9H, 2 × s, C(CH3)3), 1.79, 1.86 (3H, 2 × d, J ) 1.1
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(CDCl3) δ 1.20 (3H, t, J ) 7.0 Hz, CH2CH3), 1.31 (6H, d, J ) 6.5
Hz, CH(CH3)2), 2.88-3.01 (1H, m, CH), 3.64 (2H, q, J ) 7.0 Hz,
CH2CH3), 4.26 (2H, s, CH2Ph), 5.16 (2H, brs, NCH2O), 6.59 (2H,
d, J ) 8.7 Hz, H-aryl), 7.14-7.45 (6H, m, H-aryl, OH), 7.64 (2H,
d, J ) 8.5 Hz, Ha-aryl); 13C NMR (CDCl3) δ 14.98 (CH2CH3),
20.35 (CH(CH3)2), 28.52 (CH), 33.72 (CH2Ph), 65.41 (OCH2CH3),
JM040845B