Stereoselective synthesis and antiviral activity of methyl-substituted cycloSal-pronucleotides

Article abstract of DOI:10.1021/jm3008085

Methyl-substituted cycloSal-pronucleotides of d4TMP were synthesized with high diastereoselectivities in satisfying chemical yields. The individual diastereomers were tested against HIV-1 and HIV-2 infected wild-type CEM/0 and HIV-2 infected thymidine kin

Full text of DOI:10.1021/jm3008085

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Article
Stereoselective Synthesis and Antiviral Activity
of Methyl-Substituted cycloSal-Pronucleotides
Rios Morales H. Edwuin, Jan Balzarini, and Chris Meier
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3008085 • Publication Date (Web): 24 Jul 2012
Downloaded from http://pubs.acs.org on July 26, 2012
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Stereoselective Synthesis and Antiviral Activity of Methyl-Substituted cycloSal-
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Pronucleotides
Edwuin H. Rios Moralesa, Jan Balzarinib and Chris Meier*a
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aOrganic Chemistry, Department of Chemistry, Faculty of Sciences, University of Hamburg,
Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany; Rega Institute for Medical
b
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Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Corresponding author: Prof. Dr. Chris Meier, chris.meier@chemie.uni-hamburg.de, phone:
+49-40-42838-4324
Abstract:
Methylꢀsubstituted cycloSalꢀpronucleotides of d4TMP were synthesized with high
diastereoselectivities in satisfying chemical yields. The individual diastereomers were tested
against HIVꢀ1 and HIVꢀ2 infected wildꢀtype CEM/0 and HIVꢀ2 infected thymidine kinaseꢀ
deficient CEM cells. All diastereomers tested showed significant antiviral activity in CEM/0
and strong activity in CEM/TKꢀ cell cultures. The antiviral activities were strongly depending
on the chirality at the phosphate group and the position of the methylꢀgroup(s) in the cycloSalꢀ
moiety. In CEM/TKꢀ cell cultures the difference in antiviral potency was found to be 7ꢀ to 20ꢀ
fold. The stability of each diastereomer was studied in aqueous phosphate buffer and in
CEM/0 cell extracts. Large differences in the halfꢀlives were found. A comparison of the
relative lipophilicity of the methylꢀsubstituted cycloSalꢀtriesters was performed based on the
retention times obtained by reversed phase HPLC. The results obtained clearly confirm the
importance of a diastereoselective synthesis of cycloSalꢀpronucleotides.
Introduction:
Several nucleoside analogues such as stavudine (d4T), zalcitabine (ddC) or zidovudine (AZT)
are used as potent and selective reverse transcriptase inhibitors to combat human
immunodeficiency virus (HIV) infections. The ultimately bioactive compounds of such
nucleoside analogues are the corresponding 5’ꢀtriphosphates which are formed intracellularly
by three different cellular kinases. Often, the first phosphorylation to the nucleotide is the
rateꢀlimiting step due to the specificity of the involved nucleoside kinases. If certain
dideoxynucleoside analogues, e.g. 2’,3’ꢀdideoxyuridine, are not active against HIV in cell
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culture under conditions in which others are, this is often due to their poor, or lack of,
phosphorylation at the nucleoside kinase level.1 However, the direct administration of the
nucleoside monoꢀ, diꢀ or triphosphate is not possible due to their high polarity which prevents
cell membrane penetration. To bypass this limitation, several pronucleotide strategies2 have
been developed to mask nucleotides and thus enabling their passage through the membrane.
Inside the cells the prodrugs need to undergo chemical or enzymatic transformation to the
phosphorylated metabolite.3 Some of these pronucleotides are Pꢀchiral compounds.4ꢀ6 In those
cases in which the diastereomers could be stereoselectively synthesized4a,b,5d,e or could be
separated by means of HPLC5c,f,g a significant difference in the antiviral activity of the single
diastereomers was observed depending on the configuration of the phosphorus atom.5d,e In the
past, the synthesis, hydrolysis, and antiviral evaluation of a large number of cycloSalꢀ
pronucleotides was reported.4c,g,l They were always synthesized as 1:1 diastereomeric
mixtures and only in a very few cases the individual diastereomers were obtained by means of
semipreparative (RP)ꢀHPLC. Recently, we reported on two approaches for the stereoselective
synthesis of cycloSalꢀpronucleotides using chiral leaving groups.4a,b Here, we report on
stereochemical syntheses, antiviral activity and stability of several methylꢀsubstituted
cycloSalꢀpronucleotides.
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Results and Discussion
Chemistry. Since the stereoselective synthesis of 3ꢀmethyl substituted cycloSalꢀderivatives
failed when using the linear reaction sequence reported before,4b the synthesis of all title
compounds was carried out using a convergent synthesis pathway based on phosphorus (V)
chemistry (Scheme 1). As chiral leaving groups Nꢀcyaniminooxazolidines derived from
Lꢀ
phenylalanine and the nonꢀnatural isomer ꢀphenylalanine were used. The unsubstituted Nꢀ
D
cyaniminothiazolidine was found to be a good leaving group in phosphorylation reactions of
alcohols7 and it was easily prepared according to the procedure of Maienfisch.8 Starting from
salicyl alcohols 1aꢀd, a cyclization with phosphorusoxychloride was performed to obtain the
corresponding racemic cycloSalꢀderivatives racꢀ2aꢀracꢀ2d. These compounds were then
reacted with the chiral leaving groups (R)ꢀ5a and (S)ꢀ5b to give the corresponding 1:1
diastereomeric mixtures (SP)/(RP)ꢀ6aꢀd and (SP)/(RP)ꢀ7aꢀd which could be easily separated by
means of column chromatography into the single diastereomers. Each pure diastereomer was
finally reacted with 3’ꢀdeoxyꢀ2’,3’ꢀdidehydrothymidine (d4T) as nucleoside analogue to give
compounds (SP)ꢀ or (RP)ꢀ8aꢀd. In all cases copper(II)triflate [Cu(OTf)2] in combination with
N,N’ꢀethylenebis(benzaldiimine) (BEN) was used as described before.4a,9 It should be noted,
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that by changing the order of addition of the reagents in the last reaction step each single
diastereomer of (SP)/(RP)ꢀ6aꢀd and (SP)/(RP)ꢀ7aꢀd was converted to the corresponding
cycloSalꢀphosphate triester in good to excellent diastereomeric excess and in dependence on
the solvent. All reactions were stopped after 5 days even if unreacted starting material was
detected by means of thin layer chromatography.
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1
The determination of the obtained diastereospecificity was done by means of H or 31P NMR
spectroscopy or analytical HPLC since not all phosphate triesters showed completely
separated signals in the used analytical methods. However, in those cases, in which the two
phosphate triesters displayed separate signals in all three methods, identical diastereomeric
excesses were determined. Due to the lack of a singleꢀcrystal Xꢀray structure of one of the
title compounds, the absolute assignment of the configuration on the phosphorus atom could
not be made. However, Gisch4k et al. crystallized a similar cycloSalꢀphosphate triester of d4T
and assigned the configuration by Xꢀray analysis, in that case the RP configuration. In that
report, the (RP)ꢀdiastereomer showed a lower antiviral activity against HIVꢀ1 and HIVꢀ2
infected CEM/0 and HIVꢀ2ꢀinfected CEM/TKꢀ cells as compared to its (SP)ꢀcounterpart. This
may be explained by different membrane permeabilities of the single diastereomers. Evidence
for that was recently published by Kortylewicz10 et al. by in vitro uptake kinetic studies of
radiolabeled cyclosaligenyl monophosphates of 5ꢀiodoꢀ2’ꢀdeoxyuridine in LS 174T and
OVCARꢀ3 cancer cell lines. Remarkable differences in the inhibitory potency of both
diastereomers against BChE were also found previously by us as well as in the recent
report.4h,10 According to this, we assigned the (RP)ꢀconfiguration to the less active cycloSalꢀ
phosphate triester. Assuming that the formation of the (RP)ꢀconfigurated compound took place
following an additionꢀelimination mechanism with inversion of configuration11 the
corresponding starting material should have (SP)ꢀconfiguration and vice versa.
In order to determine the dependence of the diastereomeric excess on the solvent cycloSalꢀ
phosphate triesters were synthesized using the isomerically pure compounds (SP)ꢀ or (RP)ꢀ6aꢀ
d (Figure 1). First, cycloSal triesters (SP)ꢀ and (RP)ꢀ6a were used as model compounds and
reacted with d4T under reaction conditions described above (Scheme 1, step iv). The used
solvents, the observed diastereomeric excess (de) as well as the corresponding yields are
summarized in Table 1.
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Scheme 1. Stereoselective synthesis of cycloSalꢀphosphate triesters
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Y
OH
9
OH
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NH2
CN
X
OH
N
+
1a
X = H, Y = H
X
MeS
SMe
1b
1c
1d
X = CH3, Y = H
X = H, Y = CH3
X = CH3, Y = CH3
3a X = (R)-Bn
4
3b
X = (S)-Bn
(ii)
(i)
NCN
O
NCN
O
Y
O
O
HN
Bn
HN
Bn
*
+
P
O
Cl
X
(R)-5a
(S)-5b
rac-2a
X = H, Y = H
rac-2b
rac-2c
X = CH3, Y = H
X = H, Y = CH3
rac-2d X = CH3, Y = CH3
(iii)
Y
Y
O
P
O
P
NCN
O
NCN
O
O
N
O
N
*
*
O
O
X
X
Bn
Bn
(SP)/(RP)-6a
(SP)/(RP)-6b
(SP)/(RP)-6c
(SP)/(RP)-6d
X = H, Y = H
(SP)/(RP)-7a X = H, Y = H
(SP)/(RP)-7b
X = CH3, Y = H
X = H, Y = CH3
X = CH3, Y = H
(SP)/(RP)-7c X = H, Y = CH3
(SP)/(RP)-7d X = CH3, Y = CH3
X = CH3, Y = CH3
chromatographic separation of the
diastereomeric mixtures
(iv)
O
N
O
Y
O
O
P
Y
O
O
P
NH
NH
O
O
O
O
O
N
O
O
X
O
X
(RP)-8a
(SP)-8a X = H, Y = H
X = H, Y = H
(SP)-8b
(SP)-8c
(RP)-8b
(RP)-8c
(RP)-8d
X = CH3, Y = H
X = H, Y = CH3
X = CH3, Y = H
X = H, Y = CH3
(SP)-8d
X = CH3, Y = CH3
X = CH3, Y = CH3
a Reagents and conditions: (i) POCl3, THF, NEt3, ꢀ70 °C, 1 h, ꢀ50 °C, 1 h, rt, 4 h. (ii) CH3OH, 65 °C, 2 h. (iii)
CH2Cl2, NEt3, rt, 7 h. (iv) 6aꢀd (SP) or (RP), 7aꢀd (SP) or (RP), d4T, Cu(OTf)2, BEN, solvent, NEt3, rt, 5 d.
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Table 1. Yield and solvent dependency on the diastereomeric excess
% dea yieldb (%) product
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starting material
Solvent
THF/CH3CN (1:1)
THF
(RP)ꢀ6a
94
94
92
84
91
90
84
74
26
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(SP)ꢀ8a
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CH3CN
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CH2Cl2
(SP)ꢀ6a
THF/CH3CN (1:1)
THF
(RP)ꢀ8a
CH3CN
CH2Cl2
a % de determined by 1H NMR after purification. b Isolated yields
Since diastereomers (SP)/(RP)ꢀ8a displayed two closely spaced signals in the 31P NMR
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spectrum, the diastereomeric excess was determined by H NMR spectroscopy after column
chromatography. It should be noted that no separation of the diastereomers or enrichment of
one diastereomer by means of column chromatography was possible, so that the determination
1
of the deꢀvalues by H NMR is reliable. Notably both diastereomers (SP)ꢀ and (RP)ꢀ6a led to
the corresponding cycloSal compounds (SP)ꢀ and (RP)ꢀ8a in different deꢀvalues in dependence
of the solvent. Best results were obtained with a 1:1 mixture of THF/CH3CN. The use of this
solvent mixture for stereoselective reactions was previously reported4b,5d but with tertꢀ
butylmagnesium chloride as base. However, the use of this base for the last reaction step
using Nꢀcyaniminooxazolidine as chiral leaving group was found to be unsuitable due to a
loss of diastereomeric excess.4a
No final explanation for the observed isomerization can be given but a plausible
argumentation was done in a previously report.4a In order to prove the suitability of the 1:1
mixture of THF/CH3CN for the last reaction step diastereomers, (SP)ꢀ and (RP)ꢀ8b were
synthesized using both this solvent mixture as well as CH3CN. The reaction in THF was very
slow and a lot of unreacted starting material was detected even after 5 days reaction time.
Again, best deꢀvalues were obtained in the 1:1 mixture of THF/CH3CN (Table 2). Having
found an appropriate solvent for the stereoselective synthesis of cycloSalꢀcompounds, methylꢀ
substituted cycloSalꢀphosphate triesters (SP)ꢀ and (RP)ꢀ8c,d were then synthesized (Table 2).
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Table 2. Diastereomeric excesses of (SP)ꢀ and (RP)ꢀ8b in CH3CN and (SP)ꢀ and (RP)ꢀ8bꢀd in
THF/CH3CN (1:1)
starting material % de Yieldd (%) product
a(RP)ꢀ6b
a(SP)ꢀ6b
(RP)ꢀ6b
(SP)ꢀ6b
(RP)ꢀ6c
(SP)ꢀ6c
(RP)ꢀ6d
(SP)ꢀ6d
91b
82b
95b
94b
95c
92c
94b
95b
n.de
n.de
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(SP)ꢀ8b
(RP)ꢀ8b
(SP)ꢀ8b
(RP)ꢀ8b
(SP)ꢀ8c
(RP)ꢀ8c
(SP)ꢀ8d
(RP)ꢀ8d
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a CH3CN as solvent. b % de determined by 31P NMR spectroscopy of the crude mixture. c % de
determined by 1H NMR after purification. d Isolated yields. e n.d not determined.
In almost all cases very high diastereomeric excesses were obtained in THF/CH3CN (1:1).
Only in the case of (SP)ꢀ6c a slightly higher isomerization was observed.
The title compounds (SP)ꢀ and (RP)ꢀ8aꢀd were also synthesized from the corresponding
isomerically pure diastereomers (SP)ꢀ and (RP)ꢀ7aꢀd which are the mirror image of the used
starting materials (RP)ꢀ and (SP)ꢀ6aꢀd (Table 3).
Table 3. Diastereomeric excesses of (SP)ꢀ and (RP)ꢀ8aꢀd in THF/CH3CN (1:1) with (SP)ꢀ and
(RP)ꢀ7aꢀd as starting materials
starting material % de
yieldc (%) product
(RP)ꢀ7a
(SP)ꢀ7a
(RP)ꢀ7b
(SP)ꢀ7b
(RP)ꢀ7c
(SP)ꢀ7c
(RP)ꢀ7d
(SP)ꢀ7d
94a
96a
90b
96b
88a
94a
86b
97b
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12
33
27
19
24
16
32
(SP)ꢀ8a
(RP)ꢀ8a
(SP)ꢀ8b
(RP)ꢀ8b
(SP)ꢀ8c
(RP)ꢀ8c
(SP)ꢀ8d
(RP)ꢀ8d
a
1
b
% de determined by H NMR after purification.
% de determined by 31P NMR
spectroscopy of the crude mixture. c Isolated yields
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As expected, high diastereomeric excesses were also observed using isomerically pure
compounds (SP)ꢀ or (RP)ꢀ7aꢀd. However, the stereospecific synthesis of the title compounds
(SP)ꢀ and (RP)ꢀ8aꢀd could be carried out by using the appropriate isomerically pure
diastereomer (SP)ꢀ and (RP)ꢀ6aꢀd or (SP)ꢀ and (RP)ꢀ7aꢀd as starting material with very high
diastereoselectivities. Interestingly, it was found that the (RP)ꢀcycloSal triesters 8aꢀd were
favorably obtained with higher diastereomeric excesses using (SP)ꢀ7aꢀd as starting materials;
on the other hand (RP)ꢀ6aꢀd isomers led to the (SP)ꢀcycloSal triesters 8aꢀd in higher
diastereoselectivities.
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Lipophilicity. A qualitative measure of the lipophilicity of the unsubstituted and the different
methylꢀsubstituted cycloSalꢀphosphate triesters (SP)ꢀ and (RP)ꢀ8aꢀd and the parent nucleoside
d4T was done by means of reversed phase (RP)ꢀHPLC. The obtained retention times reflect
the relative lipophilicity of the various derivatives (see supporting information). All cycloSal
triesters are markedly more lipophilic than the parent nucleoside d4T. As expected, the
presence of lipophilic groups as a methyl substituent increased the lipophilicity of the
corresponding cycloSal triesters. Therefore, the unsubstituted cycloSal triesters (SP)ꢀ and (RP)ꢀ
8a showed the lowest lipophilicity while the 3,5ꢀdimethyl cycloSal triesters (SP)ꢀ and (RP)ꢀ8d
showed the highest lipophilicity. It was shown that the 5ꢀmethyl substituted cycloSal triesters
(SP)ꢀ and (RP)ꢀ8c were more lipophilic than their 3ꢀmethyl counterpart (SP)ꢀ and (RP)ꢀ8b.
Additionally, in contrast to (SP)ꢀ and (RP)ꢀ8a and (SP)ꢀ and (RP)ꢀ8c, the 3ꢀmethyl substituted
(SP)ꢀ and (RP)ꢀ8b and the 3,5ꢀdimethyl substituted cycloSal triesters (SP)ꢀ and (RP)ꢀ8d showed
good separation of the corresponding isomers. In both cases the slow-eluting diastereomer has
(SP)ꢀconfiguration.
Chemical Hydrolysis Studies. The title compounds (SP)ꢀ and (RP)ꢀ8aꢀd were studied for their
hydrolytic stability in aqueous 25 mM phosphate buffer, pH 7.3 and in CEM cell extracts (pH
= 6.9) as well as evaluated for their antiꢀHIV activity in vitro (Table 4). The final products of
this pHꢀdriven hydrolysis mechanism were exclusively d4TMP and the corresponding salicyl
alcohol.4c
As expected, the unsubstituted cycloSal compounds (SP)ꢀ and (RP)ꢀ8a showed the lowest
hydrolysis stability while the dimethylꢀsubstituted triesters (SP)ꢀ and (RP)ꢀ8d showed the
highest t1/2ꢀvalue, confirming the additional stabilizing effect caused by the methyl
substituents in the aromatic ring due to the electronꢀdonating properties. In addition, the
individual diastereomers of 3ꢀmethylꢀsubstituted cycloSal triesters (SP)ꢀ8b and (RP)ꢀ8b were
found to be more stable against chemical hydrolysis than the 5ꢀmethylꢀsubstitutedꢀ
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counterparts (SP)ꢀ8c and (RP)ꢀ8c showing that a methyl group in the position 3 of the aromatic
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5
ring led to an additional significant increase in hydrolysis halfꢀlive.
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Table 4. Hydrolysis Data and Antiviral Activity of (SP)ꢀ and (RP)ꢀ8aꢀd compared to d4T
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EC50 [ꢁM]b
CEM/0f
CC50 [ꢁM]c
t1/2 [h]a
PBSd pH = 7.3 CEe
CEM/TKꢀg
HIVꢀ2
compd
(SP)ꢀ8a
(RP)ꢀ8a
(SP)ꢀ8b
(RP)ꢀ8b
(SP)ꢀ8c
(RP)ꢀ8c
(SP)ꢀ8d
(RP)ꢀ8d
d4T
HIVꢀ1
HIVꢀ2
0.16 ± 0.028
0.24 ± 0.078
0.09 ± 0.085
0.48 ± 0.35
0.13 ± 0.0
0.34 ± 0.092
1.1 ± 0.29
0.17 ± 0.028
1.1 ± 0.30
0.35 ± 0.26
2.1 ± 0.78
0.22 ± 0.042
1.1 ± 0.17
1.3 ± 0.14
0.35 ± 0.27
2.6 ± 3.1
70 ± 16
140 ± 59
39 ± 2.1
179 ± 69
57 ± 23
143 ± 15
60 ± 3.5
120 ± 3.5
>250
6.8
2.2
9.3
4.2
0.16 ± 0.11
3.2 ± 2.6
22.1
9.2
18
8.7
0.21 ± 0.11
3.1 ± 2.5
11.5
3.3
14.8
10.9
27.6
19.8
n.a.h
0.18 ± 0.071
0.12 ± 0.049
0.40 ± 0.18
0.78 ± 0.16
0.18 ± 0.13
2.7 ± 0.92
150 ± 141
34.3
13.7
n.a.h
a
b
Hydrolysis halfꢀlives. Antiviral activity in Tꢀlymphocytes: 50% effective concentration.
d
e
c cytostatic activity: 50% cytostatic concentration. 25 mM phosphate buffer. CEM cell
f
g
extracts (pH = 6.9). Wildꢀtype CEM/0 cells. Thymidine kinaseꢀdeficient CEM/TKꢀ cells.
h n.a.: Not applicable
Equally substituted diastereomers showed significant differences in the halfꢀlives e.g. t1/2 = 6.8
h for (SP)ꢀ8a and t1/2 = 2.2 h for (RP)ꢀ8a. The (SP)ꢀconfigurated isomer was the more stable one
in all cases. This proves that the chemical stability of the cycloSalꢀtriesters is dependent on
the absolute configuration at the phosphorus atom.
Enzymatic Hydrolysis Studies. In all cases the hydrolysis halfꢀlives in phosphate buffer and
in CEM cell extracts were found more or less comparable which confirmed the initial idea
that the delivery mechanism is relatively independent of enzymatic activation (the correlation
coefficient of the t1/2 values for all eight 8a, 8b, 8c and 8d diastereomers in PBS versus CEM
cell extracts was r = 0.918, supporting information).4c The (SP)ꢀconfigurated isomers were
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invariably more stable as compared to the (RP)ꢀisomers in the studies in both phosphate buffer
pH 7.3 and the CEM cell extracts pH 7.4. The differences in the halfꢀlives of the isomers in
each pair of diastereomers in the CEM cell extracts tended to be somewhat lower than the
differences of the same compounds in the phosphate buffer e.g. t1/2 = 9.3 h for (SP)ꢀ8a and t1/2
= 4.2 h for (RP)ꢀ8a (2ꢀfold difference) versus t1/2 = 6.8 h for (Sp)ꢀ8a and t1/2 = 2.2 h for (Rp)ꢀ
8b (3ꢀfold difference) (Table 4).
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Antiviral Evaluation. All diastereomerically pure cycloSal triesters (SP)ꢀ and (RP)ꢀ8aꢀd and
the parent nucleoside d4T were evaluated for their in-vitro antiviral potency against HIVꢀ1
and HIVꢀ2 infected CEM cells. Data for HIVꢀ2 in a mutant cell line (CEM/TKꢀ) was also
included in order to prove the TKꢀbypass (Table 4). Remarkably all (SP)ꢀconfigurated
diastereomers showed a higher antiviral activity against HIVꢀ1 and HIVꢀ2 in wildꢀtype
CEM/0 cells as well as in mutant thymidine kinaseꢀdeficient cells (CEM/TKꢀ) than the (RP)ꢀ
configurated counterparts. Additionally, the (SP)ꢀ and (RP)ꢀcycloSalꢀcompounds 8aꢀd
generally showed higher antiviral activity against HIVꢀ1 in wildꢀtype Tꢀlymphocytic CEM/0
cultures compared to the parent nucleoside analogue d4T. More importantly, all (SP)ꢀ and
(RP)ꢀcycloSal compounds 8aꢀd showed full retention of antiviral activity in CEM/TKꢀ cell
cultures and were found to be much more antivirally active than d4T, which lost its antiviral
potency completely due to the lack of its bioactivating enzyme thymidine kinase. The
efficient release of d4TMP from the corresponding cycloSal triesters was therefore confirmed
in this assay system. In all cases a pronounced difference in the antiviral activity in CEM/TKꢀ
cells between the individual diastereomers (SP)ꢀ8aꢀd and (RP)ꢀ8aꢀd was found (7ꢀfold for (SP)ꢀ
8a and (RP)ꢀ8a, 20ꢀfold for (SP)ꢀ8b and (RP)ꢀ8b, 15ꢀfold for (SP)ꢀ8c and (RP)ꢀ8c and 15ꢀfold
for (SP)ꢀ8d and (RP)ꢀ8d). These results confirm the importance of a diastereoselective
synthesis of cycloSalꢀphosphate triesters. It should be mentioned that in all cases a correlation
of the biological activity with the halfꢀlives in phosphate buffer and in CEM cell extracts was
observed. The (SP)ꢀconfigurated diastereomers showed higher hydrolysis stabilities as well as
higher antiviral activities than the (RP)ꢀconfigurated counterparts. However, it was intriguing
to observe that, although a shorter halfꢀlife correlated with a lower antiviral efficacy, for each
diastereomeric pair of compounds, the absolute t1/2 values of the compounds did not correlate
with their antiviral efficacy (supporting information). For example, (RP)ꢀ8a had the lowest t1/2
and (RP)ꢀ8d the highest t1/2 among the (RP) diastereomers, but they showed virtually identical
antiꢀHIV activities. Likewise, among the (SP) diastereomers, (SP)ꢀ8a had the lowest t1/2 and
(SP)ꢀ8d the highest t1/2, yet their antiviral activities were quite similar. Thus, compound halfꢀ
lives seem to consistently affect the antiviral activity (lower halfꢀlife results in a lower
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antiviral activity) within paired diastereomers but this consistent observation cannot be made
between diastereomers of different compounds. It may be not unlikely that the eventual
antiviral efficacy of the compounds is the complex result of different efficiencies in cellular
uptake of pairs of diastereomeric compounds, combined with different chemical/cellular halfꢀ
lives.
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The antivirally more active (SP) diastereomers were consistently also somewhat more
cytostatic (2ꢀ to 3ꢀfold) than the corresponding (RP) diastereomers.
Differences in the antiviral activities between (SP)ꢀ and (RP)ꢀdiastereomers could also be
observed for (aryloxy)phosphoramidate prodrugs (up to 66ꢀfold),5d,e and both the cycloSalꢀ
and the (aryloxy)phosphoramidateꢀapproach were able to release efficiently d4T
monophosphate from the corresponding prodrug inside the cell.
Conclusion
In summary, three methylꢀsubstituted as well as the unsubstituted cycloSalꢀphosphate triesters
of d4T (SP)ꢀ and (RP)ꢀ8aꢀd were stereospecifically synthesized using a convergent synthetic
route.4a Some isomerization of the configuration at the phosphorous atom of the products was
observed in dependence of the solvent.
The lipophilicity of all cycloSal triesters (SP)ꢀ and (RP)ꢀ8aꢀd was compared with the
lipophilicity of d4T according to the retention times obtained by means of (RP)ꢀHPLC
analysis. 3,5ꢀDimethylꢀsubstituted cycloSal triesters showed the highest lipophilicity. The
diastereomerically pure compounds were studied concerning their stability in phosphate
buffer (pH = 7.3) and in CEM cell extracts. In both hydrolysis media, all (SP)ꢀconfigurated
isomers were found to be more stable than the (RP)ꢀconfigurated counterparts. The cycloSalꢀ
compounds were tested against HIVꢀ1ꢀ and HIVꢀ2ꢀinfected CEM/0 and HIVꢀ2ꢀinfected
CEM/TKꢀ cells. All title compounds proved to be potent inhibitors of HIVꢀ1 and HIVꢀ2
replication in wildꢀtype CEM/0 cell cultures, showing a significant biological activity for all
(SP)ꢀconfigurated isomers which was superior to that found for the corresponding other
diastereomer. The differences in the antiviral potency were found to be between 7ꢀfold and
20ꢀfold. Most important was that whereas the parent nucleoside d4T lost complete antiviral
potency in CEM/TKꢀ cells all cycloSal compounds retained significant antiviral activity.
These results clearly demonstrate the dependence of the biological activity and the halfꢀlives
of cycloSal compounds on the configuration at the phosphorus center and consequently the
importance of diastereospecific access to these compounds. For further studies only the (SP)ꢀ
diastereomers should be used because of the favorable antiviral properties. The (SP)ꢀcycloSalꢀ
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d4TMPs should be prepared starting from (RP)ꢀ6aꢀd in a solvent mixture of CH3CN/THF 1:1
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to give optimal diastereospecificities.
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Experimental Section
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General: All experiments involving waterꢀsensitive compounds were conducted under
scrupulously dry conditions and nitrogen atmosphere. All solvents were dried over an
appropriate drying agent. Triethylamine, dichloromethane and acetonitrile were dried by
heating under reflux over calcium hydride for several days followed by distillation.
Dichlormethane was stored over activated 4ꢀÅ molecular sieves and acetonitrile over 3ꢀÅ
molecular sieves. THF was dried by heating under reflux over potassium and benzophenone
followed by distillation. Ethyl acetate, petroleum ether 50ꢀ70, dichloromethane and methanol
for chromatography were distilled before used. Evaporation of solvents was carried out on a
rotary evaporator under reduced pressure or using a highꢀvacuum pump. Column
chromatography was performed by using Merck silica gel 60, 230ꢀ400 mesh. Analytical thinꢀ
layer chromatography was performed on Merck precoated aluminium plates 60 F254 with a
0.2ꢀmm layer of silica gel containing a fluorescent indicator; sugarꢀcontaining compounds
were visualized with the sugar spray reagent (0.5 mL of 4ꢀmethoxybenzaldehyde, 9 mL of
ethanol, 0.1 mL of glacial acetic acid and 0.5 mL of concentrated sulphuric acid) by heating
with a fan. The coupling product of the cycloSalꢀmask and the leaving groups was visualised
with a solution of potassium permanganate and potassium carbonate in sodium hydroxide
(1.5 g KMnO4, 10 g K2CO3, 1.5 mL 10% NaOH and 200 mL H2O). NMR Spectra were
recorded on a 400 or 500 MHz spectrometer (Bruker AMX 400, Bruker AV 400, or a Bruker
1
DRX 500). All H and 13C NMR chemical shifts are quoted in ppm and were calibrated on
solvent signals. 31PꢀNMR chemical shifts are quoted in ppm using H3PO4 as external
reference. All 13C and 31P NMR spectra were recorded in the protonꢀdecoupled mode. High
resolution mass spectra were obtained with a VG Analytical VG/70ꢀ250F spectrometer (FAB,
matrix was mꢀnitrobenzyl alcohol). HRꢀESI spectra were obtained with an Agilent
Technologies ESIꢀTOF 6224 spectrometer. Analytical HPLC was performed on a VWRꢀ
Hitachi LaChromElite HPLC System consisting of a VWRꢀHitachi Lꢀ2130 pump,
autosampler, and a VWRꢀHitachi UV detector Lꢀ2455. The columns used were a
LiChroCART 125ꢀ3 column containing reversed phase silica gel LiChrospher® 100 RPꢀ18e
(5 ꢁm, Merck, Darmstadt, Germany) and a Nucleodur C18 Isis, 5 ꢁm (MachereyꢀNagel).
Elution was performed using a water/acetonitrile (SigmaꢀAldrich, HPLC grade) eluent:
Method I (for the determination of the halfꢀlives): 5ꢀ100% CH3CN (0ꢀ25 min), 100ꢀ5%
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CH3CN (25ꢀ30 min), 5% CH3CN (30ꢀ35 min), a flow rate 0.5 mL/min, and UV detection at
265 nm. Method II (for the determination of the lipophilicity): 5ꢀ100% CH3CN (0ꢀ50 min),
100ꢀ5% CH3CN (50ꢀ55 min), 5% CH3CN (55ꢀ60 min), a flow rate 0.5 mL/min, and UV
detection at 265 nm. The purity of cycloSalꢀphosphate triesters (SP)ꢀ and (RP)ꢀ8aꢀd was
checked by means of HPLC and was in all cases ≥ 95%.
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General Procedure A: Preparation of 2-chloro-4H-1,3,2-benzodioxaphosphorin-2-oxide
derivatives racꢀ2
A solution of the corresponding salicyl alcohol 1 (1.0 equiv) and triethylamine (2.1 equiv) in
THF was added dropwise within 1 h to a stirred solution of P(O)Cl3 (1.1 equiv) in THF at
ꢀ70 °C. The reaction was allowed to warmꢀup to ꢀ50 °C and stirred for 1 h. The reaction
mixture was then allowed to warmꢀup to room temperature and stirred for an additional 4 h.
The triethylammonium chloride was filtered. The solvent was removed under reduced
pressure using a highꢀvacuum pump. The crude product was purified by column
chromatography on silica gel (petroleum ether 50ꢀ70/ethyl acetate 1:1).
General Procedure B: Preparation of the leaving groups (R)ꢀ5a, (S)ꢀ5b
The respective 2ꢀamino alcohol (R)ꢀ3a or (S)ꢀ3b (1.0 equiv) was added to a solution of
dimethylcyanodithioiminocarbonate 4 (1.0 equiv) in methanol. The reaction mixture was
heated 3 h under reflux und stirred at room temperature for 15 h. The originating methanethiol
was oxidized to methanesulfonic acid by using nitric acid. The product was filtered, washed
with cold petroleum ether, and dried under reduced pressure.
General Procedure C: Preparation of the diastereomeric mixtures (SP)/(RP)ꢀ6a-6d,
(SP)/(RP)ꢀ7a-7d
To a suspension of the leaving group (R)ꢀ5a or (S)ꢀ5b (1.0 equiv) and triethylamine (1.1
equiv) in dichloromethane was added a solution of 2ꢀchloroꢀ4Hꢀ1,3,2ꢀbenzodioxaphosphorinꢀ
2ꢀoxid derivative racꢀ2a, racꢀ2b, racꢀ2c or racꢀ2d (1.0 ꢀ 1.5 equiv) in dichloromethane at
room temperature. The reaction mixture was stirred 5ꢀ10 h at room temperature. The solvent
was removed under reduced pressure using a highꢀvacuum pump. Ethyl acetate was added,
the reaction mixture was stirred for 30 min at room temperature and stored 2 h at 0 °C. The
precipitated salt was filtered, the solvent removed and the crude product was purified by
column chromatography on silica gel (petroleum ether 50ꢀ70/ethyl acetate 1:2).
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General Procedure D: Preparation of the cycloSal-phosphotriesters (SP)/(RP)ꢀ8a-8d
Copper(II)triflate (1.0 equiv), BEN (1.0 equiv) and the isomerically pure diastereomer (1.0
equiv) were put in a flask under nitrogen atmosphere and solved in the corresponding solvent.
The solution was then stirred for 30 min and added to a solution of d4T (2.5 equiv) and
triethylamine (2.5 equiv) in the same solvent as before at room temperature. The reaction
mixture was stirred for five days and quenched with saturated ammonium chloride solution
and extracted with dichloromethane three times. The combined organic layers were dried over
sodium sulphate and concentrated under reduced pressure. The crude product was purified by
column chromatography on silica gel (dichloromethane/methanol 19:1).
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(RP,4’RC)-
and
(SP,4’RC)-2-(4’-benzyl-2’-N-cyanimino-oxazolidin-3’-yl)-4H-1,3,2-
benzodioxaphosphorin-2-oxide (SP)/(RP)ꢀ6a. General procedure C with (R)ꢀ4ꢀbenzylꢀ2ꢀ(Nꢀ
cyanimino)oxazolidine (R)ꢀ5a (0.35 g, 1.74 mmol) in 20 mL dichloromethane, 2ꢀchloroꢀ4Hꢀ
1,3,2ꢀbenzodioxaphosphorinꢀ2ꢀoxide (rac-2a, 0.53 g, 2.61 mmol) in 20 mL dichloromethane
and 0.27 mL (0.19 g, 1.9 mmol) triethylamine. The product (SP)/(RP)ꢀ6a (0.405 g, 64%) was
obtained as a colourless foam as a diastereomeric mixture which was then separated by
column chromatography.
1
(RP)ꢀ6a: HꢀNMR: (400 MHz, CDCl3): δ = 7.41ꢀ7.26 (Hꢀaromat.), 7.25ꢀ7.09 (Hꢀaromat.),
5.79 (Hꢀ4), 5.47 (Hꢀ4’), 4.81ꢀ4.71 (Hꢀ13), 4.58ꢀ4.50 (Hꢀ12), 4.50ꢀ4.45 (Hꢀ12’), 3.50 (Hꢀ14),
3.01 (Hꢀ14’); 31PꢀNMR: (162 MHz, CDCl3): δ = ꢀ14.35 ppm.
1
(SP)ꢀ6a: HꢀNMR: (400 MHz, CDCl3): 7.39ꢀ7.23 (Hꢀaromat.), 7.23ꢀ7.17 (Hꢀaromat.), 7.16ꢀ
7.11 (Hꢀaromat.), 7.10ꢀ7.05 (Hꢀaromat.), 5.87 (Hꢀ4), 5.43 (Hꢀ4’), 4.83ꢀ4.74 (Hꢀ13), 4.60ꢀ4.52
(Hꢀ12), 4.52ꢀ4.45 (Hꢀ12’), 3.40 (Hꢀ14), 2.99 (Hꢀ14’); 31PꢀNMR: (162 MHz, CDCl3): δ =
ꢀ14.17 ppm.
(RP,4’RC)- and (SP,4’RC)-8-Methyl-2-(4’-benzyl-2’-N-cyanimino-oxazolidin-3’-yl)-4H-
1,3,2-benzodioxaphosphorin-2-oxide (SP)/(RP)ꢀ6b. General procedure C with (R)ꢀ4ꢀbenzylꢀ
2ꢀ(Nꢀcyanimino)oxazolidine (R)ꢀ5a (0.50 g, 2.48 mmol) in 20 mL dichloromethane, 2ꢀchloroꢀ
8ꢀmethylꢀ4Hꢀ1,3,2ꢀbenzodioxaphosphorinꢀ2ꢀoxide (rac-2b, 0.810 g, 3.72 mmol) in 20 mL
dichloromethane and 0.38 mL (0.276 g, 2.73 mmol) triethylamine. The product (SP)/(RP)ꢀ6b
(0.430 g, 45%) was obtained as a colourless foam as a diastereomeric mixture which was then
separated by column chromatography.
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(RP)ꢀ6b: 1HꢀNMR: (400 MHz, CDCl3): δ = 7.40ꢀ7.28 (Hꢀaromat.) 7.23 (Hꢀ7), 7.09 (Hꢀ6), 6.97
(Hꢀ5), 5.75 (Hꢀ4), 5.43 (Hꢀ4’), 4.82ꢀ4.74 (Hꢀ14), 4.55 (Hꢀ13), 4.52ꢀ4.47 (Hꢀ13’), 3.43 (Hꢀ15),
3.09 (Hꢀ15’), 2.34 (Hꢀ9) ppm; 31PꢀNMR: (162 MHz, CDCl3): δ = ꢀ13.05 ppm.
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(SP)ꢀ6b: HꢀNMR: (400 MHz, CDCl3): 7.40ꢀ7.24 (Hꢀaromat.), 7.22 (Hꢀ7), 7.09 (Hꢀ6), 6.97
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(Hꢀ5), 5.85 (Hꢀ4), 5.39 (Hꢀ4’), 4.86ꢀ4.77 (Hꢀ14), 4.58 (Hꢀ13), 4.53ꢀ4.47 (Hꢀ13’), 3.43 (Hꢀ15),
2.99 (Hꢀ15’), 2.30 (Hꢀ9) ppm; 31PꢀNMR: (162 MHz, CDCl3): δ = ꢀ12.87 ppm.
(RP,4’RC)- and (SP,4’RC)-6-Methyl-2-(4’-benzyl-2’-N-cyanimino-oxazolidin-3’-yl)-4H-
1,3,2-benzodioxaphosphorin-2-oxide (SP)/(RP)ꢀ6c. General procedure C with (R)ꢀ4ꢀbenzylꢀ
2ꢀ(Nꢀcyanimino)oxazolidine (R)ꢀ5a (0.50 g, 2.48 mmol) in 20 mL dichloromethane, 2ꢀchloroꢀ
6ꢀmethylꢀ4Hꢀ1,3,2ꢀbenzodioxaphosphorinꢀ2ꢀoxide (rac-2c, 0.810 g, 3.72 mmol) in 20 mL
dichloromethane and 0.38 mL (0.276 g, 2.73 mmol) triethylamine. The product (SP)/(RP)ꢀ6c
(0.503 g, 53%) was obtained as a colourless foam as a diastereomeric mixture which was then
separated by column chromatography.
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(RP)ꢀ6c: HꢀNMR: (400 MHz, CDCl3): δ = 7.40ꢀ7.25 (Hꢀaromat.), 7.15 (Hꢀ7), 7.00 (Hꢀ8),
6.92 (Hꢀ5), 5.75 (Hꢀ4), 5.42 (Hꢀ4’), 4.79ꢀ4.70 (Hꢀ14), 4.52 (Hꢀ13), 4.50ꢀ4.44 (Hꢀ13’), 3.49
(Hꢀ15), 3.00 (Hꢀ15’), 2.35 (Hꢀ9) ppm; 31PꢀNMR: (162 MHz, CDCl3): δ = ꢀ14.23 ppm.
(SP)ꢀ6c: 1HꢀNMR: (400 MHz, CDCl3): δ = 7.40ꢀ7.21 (Hꢀaromat.), 7.14 (Hꢀ7), 7.00 (Hꢀ8), 6.92
(Hꢀ5), 5.83 (Hꢀ4), 5.38 (Hꢀ4’), 4.82ꢀ4.73 (Hꢀ14), 4.55 (Hꢀ13), 4.52ꢀ4.46 (Hꢀ13’), 3.41 (Hꢀ15),
3.40 (Hꢀ15’), 2.34 (Hꢀ9) ppm; 31PꢀNMR: (162 MHz, CDCl3): δ = ꢀ14.04 ppm.
(RP,4’RC)- and (SP,4’RC)-6,8-Dimethyl-2-(4’-benzyl-2’-N-cyanimino-oxazolidin-3’-yl)-4H-
1,3,2-benzodioxaphosphorin-2-oxide (SP)/(RP)ꢀ6d. General procedure C with (R)ꢀ4ꢀbenzylꢀ
2ꢀ(Nꢀcyanimino)oxazolidine (R)ꢀ5a (0.30 g, 1.49 mmol) in 12 mL dichloromethane, 2ꢀchloroꢀ
6,8ꢀdimethylꢀ4Hꢀ1,3,2ꢀbenzodioxaphosphorinꢀ2ꢀoxide (rac-2d, 0.52 g, 2.24 mmol) in 12 mL
dichloromethane and 0.23 mL (0.16 g, 1.63 mmol) triethylamine. The product (SP)/(RP)ꢀ6d
(0.386 g, 65%) was obtained as a colourless foam as a diastereomeric mixture which was then
separated by column chromatography.
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(RP)ꢀ6d: HꢀNMR: (400 MHz, CDCl3): δ = 7.40ꢀ7.26 (Hꢀaromat.), 7.02 (Hꢀ7), 6.75 (Hꢀ5),
5.70 (Hꢀ4), 5.38 (Hꢀ4’), 4.81ꢀ4.72 (Hꢀ13), 4.54 (Hꢀ12), 4.51ꢀ4.45 (Hꢀ12’), 3.43 (Hꢀ14), 3.07
(Hꢀ14’), 2.30 (Hꢀ20, Hꢀ21) ppm; 31PꢀNMR: (162 MHz, CDCl3): δ = ꢀ12.97 ppm.
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(SP)ꢀ6d: HꢀNMR: (400 MHz, CDCl3): δ = 7.40ꢀ7.21 (Hꢀaromat.), 7.01 (Hꢀ7), 6.75 (Hꢀ5),
5.80 (Hꢀ4), 5.33 (Hꢀ4’), 4.84ꢀ4.75 (Hꢀ13), 4.57 (Hꢀ12), 4.53ꢀ4.47 (Hꢀ12’), 3.42 (Hꢀ14), 2.98
(Hꢀ14’), 2.30 (Hꢀ20), 2.25 (Hꢀ21) ppm; 31PꢀNMR: (162 MHz, CDCl3): δ = ꢀ12.75 ppm.
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cycloSal-3’-deoxy-2’,3’-didehydrothymidine monophosphate (SP)ꢀ8a. General procedure
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D
with (4’RC)ꢀ2ꢀ(4’ꢀbenzylꢀ2’ꢀNꢀcyaniminoꢀoxazolidinꢀ3’ꢀyl)ꢀ4Hꢀ1,3,2ꢀbenzodioxaphosꢀ
phorinꢀ2ꢀoxide (RP)ꢀ6a (50 mg, 0.135 mmol), BEN (32 mg, 0.135 mmol), copper(II)triflate
(49 mg, 0.135 mmol) in 4 mL THF/CH3CN (1:1 v/v) and d4T (76 mg, 0.34 mmol),
triethylamine (47 ꢁL, 34 mg, 0.34 mmol) in 4 mL THF/CH3CN (1:1 v/v). The product (SP)ꢀ6a
1
(13.6 mg, 26%) was obtained as a colourless foam. HꢀNMR: (400 MHz, DMSOꢀd6): δ =
11.34 (NH), 7.39ꢀ7.34 (Hꢀ12), 7.28 (Hꢀ14), 7.21 (Hꢀ13), 7.19ꢀ7.15 (Hꢀ6), 7.11 (Hꢀ11), 6.80ꢀ
6.76 (Hꢀ1’), 6.38ꢀ6.34 (Hꢀ3’), 6.03ꢀ5.97 (Hꢀ2’), 5.55ꢀ5.34 (Hꢀ8), 4.99ꢀ4.92 (Hꢀ4’), 4.37ꢀ4.22
(Hꢀ5’), 1.62 (Hꢀ7) ppm; 31PꢀNMR: (162 MHz, DMSOꢀd6): δ = ꢀ9.37 ppm.
cycloSal-3’-deoxy-2’,3’-didehydrothymidine monophosphate (RP)ꢀ8a. General procedure
D
with
(4’RC)ꢀ2ꢀ(4’ꢀbenzylꢀ2’ꢀNꢀcyaniminoꢀoxazolidinꢀ3’ꢀyl)ꢀ4Hꢀ1,3,2ꢀ
benzodioxaphosphorinꢀ2ꢀoxide (SP)ꢀ6a (50 mg, 0.135 mmol), BEN (32 mg, 0.135 mmol),
copper(II)triflate (49 mg, 0.135 mmol) in 4 mL THF/CH3CN (1:1 v/v) and d4T (76 mg, 0.34
mmol), triethylamine (47 ꢁL, 34 mg, 0.34 mmol) in 4 mL THF/CH3CN (1:1 v/v). The product
(RP)ꢀ8a (9.8 mg, 19%) was obtained as a colourless foam. 1HꢀNMR: (400 MHz, DMSOꢀd6): δ
= 11.35 (NH), 7.42ꢀ7.36 (Hꢀ12), 7.29 (Hꢀ14), 7.23ꢀ718 (Hꢀ13, Hꢀ6), 7.14 (Hꢀ11), 6.82ꢀ6.79
(Hꢀ1’), 6.45ꢀ6.41 (Hꢀ3’), 6.05ꢀ6.00 (Hꢀ2’), 5.55ꢀ5.35 (Hꢀ8), 4.99ꢀ4.92 (Hꢀ4’), 4.33ꢀ4.28 (Hꢀ
5’), 1.68 (Hꢀ7) ppm; 31PꢀNMR: (162 MHz, DMSOꢀd6): δ = ꢀ9.34 ppm.
3-Methyl-cycloSal-3’-deoxy-2’,3’-didehydrothymidine monophosphate (SP)ꢀ8b. General
procedure D with (4’RC)ꢀ8ꢀMethylꢀ2ꢀ(4’ꢀbenzylꢀ2’ꢀNꢀcyaniminoꢀoxazolidinꢀ3’ꢀyl)ꢀ4Hꢀ1,3,2ꢀ
benzodioxaphosphorinꢀ2ꢀoxide (RP)ꢀ6b (50 mg, 0.13 mmol), BEN (31 mg, 0.13 mmol),
copper(II)triflate (47 mg, 0.13 mmol) in 4 mL THF/CH3CN (1:1 v/v) and d4T (73 mg, 0.33
mmol), triethylamine (46 ꢁL, 33 mg, 0.33 mmol) in 4 mL THF/CH3CN (1:1 v/v). The product
1
(SP)ꢀ8b (11.6 mg, 23%) was obtained as a colourless foam. HꢀNMR: (400 MHz, DMSOꢀd6):
δ = 11.35 (NH), 7.25 (Hꢀ12), 7.21ꢀ7.17 (Hꢀ6), 7.12ꢀ7.05 (Hꢀ13, Hꢀ14), 6.82ꢀ6.77 (Hꢀ1’), 6.38ꢀ
6.33 (Hꢀ3’), 6.03ꢀ5.99 (Hꢀ2’), 5.47 (Hꢀ8a), 5.35 (Hꢀ8b), 4.97ꢀ4.91 (Hꢀ4’), 4.33ꢀ4.19 (Hꢀ5’),
2.18 (Hꢀ15), 1.62 (Hꢀ7) ppm; 31PꢀNMR: (162 MHz, DMSOꢀd6): δ = ꢀ8.77 ppm.
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3-Methyl-cycloSal-3’-deoxy-2’,3’-didehydrothymidine monophosphate (RP)ꢀ8b. General
procedure D with (4’RC)ꢀ8ꢀMethylꢀ2ꢀ(4’ꢀbenzylꢀ2’ꢀNꢀcyaniminoꢀoxazolidinꢀ3’ꢀyl)ꢀ4Hꢀ1,3,2ꢀ
benzodioxaphosphorinꢀ2ꢀoxide (SP)ꢀ6b. (50 mg, 0.13 mmol), BEN (31 mg, 0.13 mmol),
copper(II)triflate (47 mg, 0.13 mmol) in 4 mL THF/CH3CN (1:1 v/v)and d4T (73 mg, 0.33
mmol), triethylamine (46 ꢁL, 33 mg, 0.33 mmol) in 4 mL THF/CH3CN (1:1 v/v). The product
(RP)ꢀ8b (10.6 mg, 20%) was obtained as a colourless foam. 1HꢀNMR: (400 MHz, DMSOꢀd6):
δ = 11.33 (NH), 7.25 (Hꢀ12), 7.20ꢀ7.17 (Hꢀ6), 7.11ꢀ7.05 (Hꢀ13, Hꢀ14), 6.82ꢀ6.77 (Hꢀ1’), 6.43ꢀ
6.39 (Hꢀ3’), 6.03ꢀ5.98 (Hꢀ2’), 5.45 (Hꢀ8a), 5.35 (Hꢀ8b), 4.98ꢀ4.91 (Hꢀ4’), 4.31ꢀ4.24 (Hꢀ5’),
2.21 (Hꢀ15), 1.65 (Hꢀ7) ppm; 31PꢀNMR: (162 MHz, DMSOꢀd6): δ = ꢀ8.57 ppm.
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5-Methyl-cycloSal-3’-deoxy-2’,3’-didehydrothymidine monophosphate (SP)ꢀ8c. General
procedure D with (4’RC)ꢀ6ꢀMethylꢀ2ꢀ(4’ꢀbenzylꢀ2’ꢀNꢀcyaniminoꢀoxazolidinꢀ3’ꢀyl)ꢀ4Hꢀ1,3,2ꢀ
benzodioxaphosphorinꢀ2ꢀoxide (RP)ꢀ6c (50 mg, 0.13 mmol), BEN (31 mg, 0.13 mmol),
copper(II)triflate (47 mg, 0.13 mmol) in 4 mL THF/CH3CN (1:1 v/v) and d4T (73 mg, 0.33
mmol), triethylamine (46 ꢁL, 33 mg, 0.33 mmol) in 4 mL THF/CH3CN (1:1 v/v). The product
(SP)ꢀ8c (13.8 mg, 26%) was obtained as a colourless foam.1HꢀNMR: (400 MHz, DMSOꢀd6): δ
= 11.32 (NH), 7.17ꢀ7.14 (Hꢀ6), 7.17ꢀ7.12 (Hꢀ12), 7.07 (Hꢀ14), 6.98 (Hꢀ11), 6.79ꢀ6.75 (Hꢀ1’),
6.38ꢀ6.33 (Hꢀ3’), 6.03ꢀ5.97 (Hꢀ2’), 5.44 (Hꢀ8a), 5.35 (Hꢀ8b), 4.98ꢀ4.91 (Hꢀ4’), 4.36ꢀ4.28 (Hꢀ
5’), 2.26 (Hꢀ15), 1.62 (Hꢀ7) ppm; 31PꢀNMR: (162 MHz, DMSOꢀd6): δ = ꢀ9.32 ppm.
5-Methyl-cycloSal-3’-deoxy-2’,3’-didehydrothymidine monophosphate (RP)ꢀ8c. General
procedure D with (4’RC)ꢀ6ꢀMethylꢀ2ꢀ(4’ꢀbenzylꢀ2’ꢀNꢀcyaniminoꢀoxazolidinꢀ3’ꢀyl)ꢀ4Hꢀ1,3,2ꢀ
benzodioxaphosphorinꢀ2ꢀoxide (SP)ꢀ6c (32 mg, 0.084 mmol), BEN (20 mg, 0.084 mmol),
copper(II)triflate (30 mg, 0.084 mmol) in 4 mL THF/CH3CN (1:1 v/v) and d4T (47 mg, 0.21
mmol), triethylamine (29 ꢁL, 21 mg, 0.21 mmol) in 4 mL THF/CH3CN (1:1 v/v). The product
(RP)ꢀ8c (7.1 mg, 21%) was obtained as a colourless foam. 1HꢀNMR: (400 MHz, DMSOꢀd6): δ
= 11.34 (NH), 7.20ꢀ7.17 (Hꢀ6), 7.19ꢀ7.15 (Hꢀ12), 7.07 (Hꢀ14), 7.01 (Hꢀ11), 6.81ꢀ6.77 (Hꢀ1’),
6.44ꢀ6.39 (Hꢀ3’), 6.04ꢀ5.98 (Hꢀ2’), 5.43 (Hꢀ8a), 5.33 (Hꢀ8b), 4.97ꢀ4.91 (Hꢀ4’), 4.30ꢀ4.25 (Hꢀ
5’), 2.27 (Hꢀ15), 1.68 (Hꢀ7) ppm; 31PꢀNMR: (162 MHz, DMSOꢀd6): δ = ꢀ9.25 ppm.
3,5-Dimethyl-cycloSal-3’-deoxy-2’,3’-didehydrothymidine
monophosphate
(SP)ꢀ8d.
General procedure D with (4’RC)ꢀ6,8ꢀDimethylꢀ2ꢀ(4’ꢀbenzylꢀ2’ꢀNꢀcyaniminoꢀoxazolidinꢀ3’ꢀ
yl)ꢀ4Hꢀ1,3,2ꢀbenzodioxaphosphorinꢀ2ꢀoxide (RP)ꢀ6d (80 mg, 0.20 mmol), BEN (47 mg, 0.20
mmol), copper(II)triflate (72 mg, 0.20 mmol) in 4 mL THF/CH3CN (1:1 v/v) and d4T (0.11
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g, 0.50 mmol), triethylamine (69 ꢁL, 50 mg, 0.50 mmol) in 4 mL THF/CH3CN (1:1 v/v). The
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1
product (SP)ꢀ8d (17.2 mg, 21%) was obtained as a colourless foam. HꢀNMR: (500 MHz,
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DMSOꢀd6): δ = 11.34 (NH), 7.20ꢀ7.18 (Hꢀ6), 7.04 (Hꢀ12), 6.89 (Hꢀ14), 6.81ꢀ6.77 (Hꢀ1’),
6.38ꢀ6.33 (Hꢀ3’), 6.03ꢀ5.98 (Hꢀ2’), 5.41 (Hꢀ8a), 5.31 (Hꢀ8b), 4.97ꢀ4.91(Hꢀ4’), 4.31ꢀ4.16 (Hꢀ
5’), 2.22 (Hꢀ15), 2.14 (Hꢀ16), 1.62 (Hꢀ7) ppm; 31PꢀNMR: (162 MHz, DMSOꢀd6): δ = ꢀ8.75
ppm.
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3,5-Dimethyl-cycloSal-3’-deoxy-2’,3’-didehydrothymidine
monophosphate
(RP)ꢀ8d.
General procedure D with (4’RC)ꢀ6,8ꢀDimethylꢀ2ꢀ(4’ꢀbenzylꢀ2’ꢀNꢀcyaniminoꢀoxazolidinꢀ3’ꢀ
yl)ꢀ4Hꢀ1,3,2ꢀbenzodioxaphosphorinꢀ2ꢀoxide (SP)ꢀ6d. (32 mg, 0.081 mmol), BEN (19 mg,
0.081 mmol), copper(II)triflate (29 mg, 0.081 mmol) in 4 mL THF/CH3CN (1:1 v/v)and d4T
(45 mg, 0.20 mmol), triethylamine (28 ꢁL, 20 mg, 0.20 mmol) in 4 mL THF/CH3CN (1:1
1
v/v). The product (RP)ꢀ8d (9.7 mg, 29%) was obtained as a colourless foam. HꢀNMR:
(500 MHz, DMSOꢀd6): δ = 11.33 (NH), 7.18ꢀ7.16 (Hꢀ6), 7.05 (Hꢀ12), 6.88 (Hꢀ14), 6.80ꢀ6.76
(Hꢀ1’), 6.43ꢀ6.38 (Hꢀ3’), 6.03ꢀ5.99 (Hꢀ2’), 5.40 (Hꢀ8a), 5.28 (Hꢀ8b), 4.97ꢀ4.91 (Hꢀ4’), 4.28ꢀ
4.22 (Hꢀ5’), 2.22 (Hꢀ15), 2.17 (Hꢀ16), 1.65 (Hꢀ7) ppm; 31PꢀNMR: (162 MHz, DMSOꢀd6): δ =
ꢀ8.43 ppm.
Hydrolysis studies of cycloSal-phosphate triesters. Hydrolysis studies of cycloSal triesters
(phosphate buffer, pH = 7.3) by reversed phase HPLC analysis have been done as described
in ref 12. Studies in cell extracts were also performed as reported in ref 12 but with a 3.0 mM
solution of the cycloSal phosphate triester in DMSO and with different incubation times. The
HPLC analysis was performed in both cases using the Method I described above.
Antiretroviral Evaluation. The method of antiviral evaluation has already been described in
ref 4a, and was based on the microscopical examination of virusꢀinduced cytopathicity (giant
cell formation) in CEM cell cultures after 4 days of virus and drug exposure.
Acknowledgements: CM is grateful to the University of Hamburg and the Deutsche
Forschungsgemeinschaft (DFG) for finacial support (Me1161/9ꢀ1). Moreover, the authors are
grateful to Dr. Ulf Görbig, Macherey & Nagel for continious support in receiving
chromatography materials. J.B. thanks the KU Leuven for financial support (GOA 10/014),
and Mrs. Leen Ingels and Mrs. Lizette van Berckelaer for excellent technical assistance.
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Supporting Information Available: HPLCꢀchromatogramms of compounds 8 and detailed
analytical data for compounds 2 and 8 as well as correlations of stability data in different
media and antiꢀHIVꢀactivity is provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
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2’ꢀβꢀCꢀmethyluridine Nucleotide Prodrug (PSIꢀ7977) for the Treatment of Hepatitis C
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