Studies on enzyme-cleavable dialkoxymethyl-cycloSaligenyl-2′, 3′-dideoxy-2′,3′-didehydrothymidine monophosphates

Article abstract of DOI:10.1021/jm800853p

Recently we reported on conceptually new enzymatically activated cycloSal-pronucleotides. Now, we developed this concept further with new compounds of this type. The basic idea is fast intracellular cleavage of a functionalized group at the cycloSal residue that results in a rapid delivery of the nucleotide and thus an intracellular enrichment of the nucleotide. The introduction of a higher alkylated acylal group, the di-iso-butyryloxymethyl group, to the aromatic ring led to the expected higher stability of these prodrugs against enzymatic cleavage but also entailed surprisingly a decrease in hydrolysis stabilities and solubility problems. For some compounds, a separation of the two diastereomeric forms (R_P or S_P) was achieved. By X-ray structure analysis, the absolute configuration at the P-atom was assigned. For all separated diastereomers the (S_P) form showed better antiviral activity than the (R_P) form.

Full text of DOI:10.1021/jm800853p

6752
J. Med. Chem. 2008, 51, 6752–6760
Studies on Enzyme-Cleavable
Dialkoxymethyl-cycloSaligenyl-2′,3′-dideoxy-2′,3′-didehydrothymidine Monophosphates
Nicolas Gisch,† Jan Balzarini,‡ and Chris Meier*,†
Organic Chemistry, Department of Chemistry, Faculty of Science, UniVersity of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg,
Germany, Rega Institute for Medical Research, Katholieke UniVersiteit LeuVen, Minderbroedersstraat 10, 3000 LeuVen, Belgium
ReceiVed July 11, 2008
Recently we reported on conceptually new enzymatically activated cycloSal-pronucleotides. Now, we
developed this concept further with new compounds of this type. The basic idea is fast intracellular cleavage
of a functionalized group at the cycloSal residue that results in a rapid delivery of the nucleotide and thus
an intracellular enrichment of the nucleotide. The introduction of a higher alkylated acylal group, the di-
iso-butyryloxymethyl group, to the aromatic ring led to the expected higher stability of these prodrugs against
enzymatic cleavage but also entailed surprisingly a decrease in hydrolysis stabilities and solubility problems.
For some compounds, a separation of the two diastereomeric forms (RP or SP) was achieved. By X-ray
structure analysis, the absolute configuration at the P-atom was assigned. For all separated diastereomers
the (SP) form showed better antiviral activity than the (RP) form.
Introduction
However, this antiviral activity was not as good as for some
other cycloSal-d4TMPs (e.g., 3-methyl-cycloSal-d4TMP; t1/2
It has been clearly proven that chiral cycloSala-d4T-mono-
phosphates (cycloSal-d4TMPs) are highly efficient prodrugs for
the delivery of d4TMP 1 (Figure 1) into cells.1 This is of high
interest because the rate-limiting phosphorylation of the ap-
proved anti-HIV drug d4T2 2 (Figure 1) is the formation of the
monophosphate 1 by the cellular enzyme thymidine kinase
(TK).3 Recently, we presented the first compounds of the third
generation of cycloSal-pronucleotides, the 5-diacetoxymethyl-
cycloSal-d4TMPs (5-di-AM-cycloSal-d4TMPs; 3a-c; Figure 1),
as so-called enzymatically activated cycloSal-d4T-monophos-
phates.4
)
17.5 h (PBS, pH ) 7.3); EC50 ) 0.10 ( 0.0 µM5). This might
be due to a lower cellular uptake (transport) and/or to a partial
extracellular cleavage of the diacetoxymethyl function to the
formyl function (step b, Figure 2) in RPMI-1640 culture medium
containing 10% heat-inactivated fetal calf serum (RPMI/
FCS(10%)), which is also used for the cultivation of the CEM
cells in the antiviral testing.
Here, we describe a new series of enzymatically activated
cycloSal-d4T-monophosphates.
Results and Discussion
The concept of enzymatic activation is exemplified shown
for 5-di-AM-3-tBu-cycloSal-d4TMP (3c) in Figure 2. Compound
3c has a hydrolysis half-life of 7.9 h at the physicological pH
value 7.3 (determined in 25 mM phosphate buffer), so it should
be stable enough to pass the cell membrane by passive transport
(step c) without being hydrolyzed to a significant amount in
the extracellular medium (step a). Inside the cell, the weak
electron-withdrawing diacetoxymethyl-group is rapidly con-
verted into a strong acceptor (a formyl group) by enzymatic
cleavage, with the result that the chemical hydrolysis rate
increases dramatically. We determined a half-life of 0.15 h for
the conversion of compound 3c into compound 4c in T-
lymphocyte CEM cell extracts.
The half-life of the released 5-formyl-3-tBu-cycloSal-d4TMP
(4c) is 8-fold lower (1.0 h) as compared to that of 3c. The
hydrolysis of 4c leads rapidly to a charged benzylphosphodiester
(not shown). This prevents an efflux through the cell membrane.
The selective release of d4TMP 1 from 4c has been proven by
31P NMR studies. Compound 3c has an EC50 value of 0.78 (
0.0 µM against HIV-2 in mutant thymidine kinase-deficient
CEM/TK- cells, so an effective delivery of d4TMP 1 can be
assumed.
Chemistry. First, we moved the enzyme-labile diacetoxy-
methyl group from the 5- to the 3-position (compounds 5, Figure
1) to prove the stability of the diacetoxymethyl function in this
position. CycloSal-triesters 5a,b were synthesized starting from
the respective 3-formyl-5-alkyl-cycloSal-d4TMP 6a or 6b. These
compounds have been prepared in a two-step synthesis from
6-formyl-4-alkylsalicyl alcohol 7a,b, respectively. The synthetic
steps performed for the preparation of salicyl alcohols 7a,b are
summarized in Scheme 1. All reactions to yield target structures
5a,b are shown in Scheme 2. 6-Formyl-4-methylsalicyl alcohol
(7a) was synthesized starting from commercially available
5-methylsalicylic acid (8). First, acid 8 was converted into the
methyl ester 9 with conc H2SO4 and CH3OH in 95% yield.
Bromination (Br2 in CHCl3) led to the desired 3-bromo-5-
methyl-methylsalicylate (10; 92% yield). Ester 10 was then
reduced to 6-bromo-4-methylsalicyl alcohol 11a (LiAlH4 in
THF; 91% yield).
6-Bromo-4-tert-butylsalicyl alcohol (11b) was synthesized
starting from 4-tert-butylphenol (12). Ortho-formylation of 12
was carried out almost as described in the literature.6 Bromi-
nation of 5-tert-butylsalicyl aldehyde (13) with Br2 in acetic
acid afforded 3-bromo-5-tert-butylsalicyl aldehyde (14) in 92%
yield.7 Aldehyde 14 was reduced to salicyl alcohol 11b (91%
yield) as it has been done for 10 to yield 11a.
* To whom correspondence should be addressed. Phone: +49-40-42838-
4324. Fax: +49-40-42838-2495. E-mail: chris.meier@chemie.uni-hamburg.de.
†
Organic Chemistry, Department of Chemistry, Faculty of Science,
University of Hamburg.
After protection of salicyl alcohols 11a,b with 2,2-dimethoxy-
propane in acetone as isopropylidene acetals (15a: 74%, 15b:
86% yield),8 the resulting 6-bromo-4-alkylsalicyl alcohol iso-
‡
Rega Institute for Medical Research, Katholieke Universiteit Leuven.
a Abbreviations: di-AM, diacetoxymethyl; di-iBOM, di-iso-butyroxy-
methyl; cycloSal, cyclo-saligenyl; TK, thymidine kinase.
10.1021/jm800853p CCC: $40.75
2008 American Chemical Society
Published on Web 10/04/2008

Dialkoxymethyl-cycloSal-d4TMPs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6753
Figure 1. Structures of d4TMP 1, d4T 2, and cycloSal-d4TMPs 3, 4, and 5.
Figure 2. Concept of enzymatic activation exemplified by compound 3c.
propylidene acetals 15a and 15b were converted by treatment
with n-butyllithium and N,N-dimethylformamide into the cor-
responding carbaldehydes 16a (80% yield) and 16b (76%
yield).9 The cleavage of the isopropylidene group was achieved
by the usage of HCl in a mixture of acetonitrile and water to
yield 6-formyl-4-methylsalicyl alcohol (7a) in 86% and 6-formyl-
4-tert-butylsalicyl alcohol (7b) in 88%, respectively. 3-Formyl-
5-alkyl-cycloSal-d4TMPs 6a and 6b were then synthesized using
our established phosphorus(III)-method via the corresponding
chlorophosphites,10 except that the reaction steps to the chlo-
rophosphites were carried out at -55 °C instead of -20 °C
because, at -20 °C, a side reaction has been observed. 3-Formyl-
5-alkyl-cycloSal-d4TMPs were isolated in 44% yield (6a) and
36% (6b) yield, respectively.
10% yield) with acetic anhydride and zirconium(IV) chloride.11
The conversion of 6b to 3-di-iso-butyroxymethyl-5-tert-butyl-
cycloSal-d4TMP (3-di-iBOM-5-tBu-cycloSal-d4TMP) with isobu-
tyric anhydride and zirconium(IV) chloride failed. This may
be caused by steric hindrance because even the yields for the
conversion into the 3-diacetoxymethyl-5-alkyl-cycloSal-d4TMPs
5a and 5b were not as good as those obtained in the synthesis
of the 5-diacetoxymethyl counterparts 3a-c.4
Second, two new 5-diisobutyroxymethyl-cycloSal-d4TMPs
(3d,e, Figure 1) were prepared to study whether the acylals have
more beneficial properties against the cleavage in RPMI/
FCS(10%). Compounds 3d and 3e could be synthesized from
4b and 4c using the same method as for the preparation of
diacetoxymethyl acylals in 30% (3d) and 25% (3e) yield,
respectively. Characterization of all triesters was carried out by
means of 1H, 13C, and 31P NMR spectroscopy as well as high-
resolution mass spectrometry.
Finally, triesters 6a and 6b were converted into the acylals
3-diacetoxymethyl-5-methyl-cycloSal-d4TMP (5a, 3-di-AM-5-
Me-cycloSal-d4TMP; 19% yield) and 3-diacetoxymethyl-5-tert-
butyl-cycloSal-d4TMP (5b, 3-di-AM-5-tBu-cycloSal-d4TMP;

6754 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Gisch et al.
Scheme 1. Synthesis of the 6-Formyl-4-alkylsalicyl Alcohols 7a and 7ba
a Reagents and conditions: method A: CH3OH, conc H2SO4, 0 °C to reflux, 2 d; method B: CHCl3, Br2, rt, 1 d; method C: THF, LiAlH4, rt to reflux, 3 h;
method D: HOAc, Br2, 50 °C, 18 h; method E: acetone, 2,2-dimethoxypropane, para-toluenesulfonic acid monohydrate, Na2SO4, 40 °C, 3 d; method F:
THF, n-BuLi, DMF, -78 °C, 3 h; method G: CH3CN/H2O, (cat.) HCl.
Scheme 2. Synthesis of the target cycloSal-d4TMPs 3d, 3e, and 5a
a Reagents and conditions: method H: (i) Et2O or THF, PCl3, pyridine, -55 °C, 4 h; (ii) CH3CN, DIPEA, d4T 1, -20 °C to rt, 3 h; (iii) CH3CN, tBuOOH,
-20 °C to rt, 1 h; method I: CH3CN, acetic anhydride or isobutyric anhydride, ZrCl4, rt, 0.75-3 h.
Because of the nonstereoselective synthesis, cycloSal-pro-
nucleotides were always obtained as mixtures of two diaster-
eomers (RP and SP configuration). For 3c, 3e, and 4c the
diastereomers were separated by preparative RP-HPLC using
water/acetonitrile mixtures as eluent. According to their retention
time, the diastereomers are distinguished into the fast- and the
slow-diastereomers. An unambiguous stereochemical attribution
was made following single-crystal X-ray structure of 4c-slow.
An ORTEP diagram is shown in Figure 3.
Hydrolysis Studies. CycloSal-triesters 3-6 have been studied
for their stability at physicological pH value 7.3 in aqueous 25
mM phosphate buffer. The half-lives refer to the cleavage of
the triesters and are summarized in Table 1. The data for triester
3b and 4b were taken from ref 4.
As expected, 3-formyl-5-alkyl-cycloSal-triester 6a and 6b
showed very short half-lives (0.25 h). These half-lives are similar
to those of 5-formyl-cycloSal-d4TMP 4a (t1/2 ) 0.18 h).4 But
in contrast to the 5-formyl-3-alkyl-cycloSal-phosphate triester
derivatives (t1/2 ) 0.35 h (3-Me, 4b), 1.0 h (3-tBu, 4c)), no
difference in the hydrolysis stability depending on the alkyl
group was detected. For 3-diacetoxymethyl-5-alkyl-cycloSal-
The absolute stereochemistry of the phosphorus atom was
attributed to be (RP), so the (SP) configuration can be assigned
to 4c-fast. In the hydrolysis studies in CEM/0 cell extracts, it
was clearly shown that 4c-slow is formed by enzymatic cleavage
of 3c-slow and 3e-slow, respectively. Thus, these diastereomers
have (RP) configuration, too. According to this, 3c-fast and 3e-
fast must have the (SP) configuration. As an example, the HPLC
chromatograms of the hydrolysis studies of 3c-fast and 3c-slow
in CEM/0 cell extracts after 5 min are shown in Figure 4.
d4TMPs 5a and 5b, the same observation was made (t1/2
)
3.2 h (5a) vs 3.7 h (5b)). Considering the hydrolysis half-lives
of 5-methyl-cycloSal-d4TMP (t1/2 ) 6.2 h)12 and 5-tert-butyl-
cycloSal-d4TMP (t1/2 ) 7.2 h), this has been expected. This
points to the fact that the electron-donating potency of an alkyl
group in the 5-position of the aromatic ring has no significant

Dialkoxymethyl-cycloSal-d4TMPs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6755
Figure 3. ORTEP-plot of the asymmetric unit of 4c-slow, showing
the atom-labeling scheme (atomic ellipsoids represent displacement
parameters at the 50% probability level at approximately -150 °C).
effect on the hydrolysis half-life. But with regard to the concept
of enzymatic activation, the difference between the chemical
stability of acylals 5 in comparison to the chemical stability of
the corresponding 3-formyl-cycloSal-d4TMPs (6a and 6b) is
important. This difference was found to be almost 13- to 15-
fold.
Surprisingly, the chemical hydrolysis half-lives of 5-di-iso-
butyroxymethyl-3-alkyl-cycloSal-d4TMPs 3d-mix (t1/2 ) 1.3 h)
and 3e-mix (t1/2 ) 3.5 h) markedly decreased compared to that
of their 5-diacetoxymethyl-analogues 3b-mix (t1/2 ) 2.3 h) and
3c-mix (t1/2 ) 7.9 h). In hydrolysis studies of the separated
diastereomers, no remarkable difference was found. In all cases,
the hydrolysis half-lives of the fast- and the slow-diastereomers
were found to be almost identical.
The cleavage of the acylal group of the dialkoxymethyl-
cycloSal-d4TMPs 3 and 5 was tested using crude T-lymphocyte
CEM cell extracts (CE; Table 1). In the case of the 5-di-
acetoxymethyl-cycloSal-d4TMPs 3a-c, a very quick cleavage
was found before.4 The same result was obtained for the newly
prepared 3-diacetoxymethyl-cycloSal-d4TMPs 5a and 5b. The
methyl-substituted derivative 5a showed a half-life of 0.25 h,
while the tert-butyl-substituted compound 5b showed a half-
life that was below 0.1 h. Thus, while the tert-butyl group and
the methyl substituent had no influence on the rate of the
chemical hydrolysis, the t-butyl derivative was 3-fold less stable
in cell extracts.
In the case of 5-diacetoxy- and 5-di-iso-butyroxymethyl-3-
alkyl-cycloSal-d4TMPs, the contrary was observed: in the
presence of the tert-butyl group the half-life of the cleavage
decreased in comparison to the half-life of the methyl counter-
part. For 5-di-iso-butyroxymethyl-3-alkyl-cycloSal-d4TMPs half-
lives of 1.0 h (3e-mix) and 0.5 h (3d-mix) were measured in
cell extracts, respectively. Almost the same difference (2-fold)
was found for the 5-di-AM counterparts (0.15 h (3c-mix) vs
0.08 h (3b-mix)). More importantly, it is obvious that the 5-di-
iBOM-cycloSal-d4TMPs 3d-mix and 3e-mix were significantly
more stable against enzymatic cleavage (in both cases ap-
proximately 6-fold in comparison to their 5-di-AM counterparts
3b-mix and 3c-mix).
Figure 4. Top: HPLC chromatograms of the hydrolysis studies in
CEM/0 cell extracts of 3c-fast (line) and 3c-slow (dotted line) after 5
min. Bottom: HPLC chromatograms of the synthesized references of
4c-fast (line) and 4c-slow (dotted line). HPLC method: HPLC analysis
method III.
All acylal substituted compounds 3 and 5 were incubated in
RPMI-1640 culture medium containing 10% heat-inactivated
fetal calf serum (RPMI/FCS (10%)) to investigate their stability
(Table 1). The half-lives of 3-di-AM-cycloSal-d4TMPs were
determined to be t1/2 ) 1.5 h (5a) and t1/2 ) 1.9 h (5b). This is
a 2-fold decrease in half-life compared to those in PBS buffer
(pH 7.3). Nevertheless, this difference is in the same range as
has been found before for 3b. Only minor cleavage of the acylal
function to the formyl function was observed. For compound
3c-mix, the half-life decreased from 7.9 h (chemical hydrolysis)
to 2.8 h (RPMI/FCS(10%)). The separated diastereomers showed
the same result. Maybe the decrease of half-lives could be a
result of residual esterase activity in the fetal calf serum
(although it had been heat-inactivated (30 min, 56 °C)).
Alternatively, the more basic pH value of the RPMI-1640
medium (pH 7.6) could also play a role. Therefore, we incubated
the cycloSal-d4TMPs in PBS at pH ) 7.6 (Table 1). The half-
lives of the di-AM-cycloSal-d4TMPs decreased by the same
extent as in the culture medium. But, when comparing the
amount of formed 3- or 5-formyl-cycloSal-d4TMPs at the same
time points in PBS pH ) 7.6 (data taken from the HPLC
chromatograms), the cleavage in RPMI/FCS(10%) (pH ) 7.6)
is more pronounced. So, this points to a cleavage of the
diacetoxymethyl group due to the more basic pH value as well
as to the presence of residual esterase activity of the FCS.
The half-lives of the 3-di-iBOM-cycloSal-d4TMPs 3d and
3e in PBS pH 7.6 were slightly shorter than the half-lives at
pH 7.3 (3d-mix: 1.1 h (pH 7.6) vs 1.3 h (pH 7.3); 3e-mix: 2.7 h
(pH 7.6) vs 3.5 h (pH 7.3)), but more importantly, no significant
cleavage to the corresponding formyl compound (4b or 4c) was

6756 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Gisch et al.
Table 1. Hydrolysis Data and Antiviral Activity of 3b-e, 4b-c, 5a-b, and 6a-b compared to 2
EC50 [µM]b
CEM/0g
CC50[µM]c
t1/2 [h]a
CEM/TK-h
HIV-2
substituent
PBSd pH ) 7.3
CEe
RPMI/FCSf PBSd pH ) 7.6
HIV-1
HIV-2
4b-mix
3b-mix
3d-mix
4c-mix
4c-fast
4c-slow 5-CHO-3-tBu
3c-mix
3c-fast
5-CHO-3-Me
5-di-AM-3-Me
5-di-iBOM-3-Me
5-CHO-3-tBu
5-CHO-3-tBu
0.35
2.3
1.3
1.0
1.3
1.0
7.9
7.9
8.3
3.5
3.3
3.8
0.25
3.2
0.25
3.7
n.a.i
n.d.j
0.08
0.5
0.7
1.5
1.6
1.5
1.7
1.6
2.8
2.9
2.8
5.9
5.7
4.5
0.3
1.5
0.3
1.9
n.a.i
0.35
1.9
1.1
1.1
1.3
0.3
2.6
2.7
2.4
2.7
2.5
2.9
0.3
1.1
0.1
1.2
n.a.i
0.45 ( 0.32
0.50 ( 0.17
1.60 ( 0.57
0.39 ( 0.40
1.27 ( 0.64
0.70 ( 0.14
0.95 ( 0.49
1.0 ( 0.22
4.5 ( 0.7
6.3 ( 2.9
>10
82 ( 20
44 ( 26
20 ( 0.8
33 ( 7.8
27 ( 11
41 ( 1.4
19 ( 0.0
17 ( 2.1
51 ( 21
20 ( 3.5
14 ( 1.4
40 ( 1.4
29 ( 4.9
19 ( 3.5
8.8 ( 1.3
7.9 ( 1.9
182 ( 16
n.d.j
n.d.j
n.d.j
0.15
0.3
15 ( 6.4
2.6 ( 1.9
10 ( 0.0
0.78 ( 0.0
0.29 ( 0.16
3.9 ( 2.7
2.4 ( 2.3
1.4 ( 0.91
3.6 ( 0.42
29 ( 18
6.5 ( 3.1
5.1 ( 1.9
7.3 ( 4.6
62 ( 0.48
0.16 ( 0.071 0.69 ( 0.41
0.81 ( 0.042
0.65 ( 0.35
0.54 ( 0.51
1.1 ( 1.3
0.53 ( 0.45
0.72 ( 0.68
0.89 ( 0.16
3.6 ( 2.2
0.52 ( 0.41
0.99 ( 0.30
0.72 ( 0.007
0.86 ( 0.057
1.4 ( 0.81
0.80 ( 0.13
0.75 ( 0.49
0.84 ( 0.085
2.0 ( 2.3
0.85 ( 0.64
1.9 ( 2.1
1.4 ( 0.78
1.0 ( 0.37
1.0 ( 0.54
1.0 ( 0.57
1.9 ( 1.8
5-di-AM-3-tBu
5-di-AM-3-tBu
3c-slow 5-di-AM-3-tBu
3e-mix
3e-fast
0.15
1.0
1.0
5-di-iBOM-3-tBu
5-di-iBOM-3-tBu
3e-slow 5-di-iBOM-3-tBu
0.8
6a-mix
5a-mix
6b-mix
5b-mix
2
3-CHO-5-Me
3-di-AM-5-Me
3-CHO-5-tBu
3-di-AM-5-tBu
n.d.j
0.25
n.d.j
<0.1
n.a.i
a Hydrolysis half-lives. b Antiviral activity in T-lymphocytes: 50% effective concentration (shown values are means of two to three independent experiments).
c Cytostatic activity: 50% cytostatic concentration. d 25 mM phosphate buffer. e CEM cell extracts (pH ) 6.9). f RPMI/10% heat inactivated fetal calf serum
(FCS), pH 7.6; g Wild-type CEM/0 cells. h Thymidine kinase-deficient CEM TK- cells. i n.a.: not applicable. j n.d.: not determined.
observed. This showed the expected higher stability of the di-
iso-butyroxymethyl group compared to the diacetoxymethyl
group.
The hydrolysis studies of 3d and 3e in RPMI/FCS (10%)
showed increased half-lives compared to those in phosphate
buffer (PBS, pH 7.6). This may be attributed to a poorer
solubility of these lipophilic pronucleotides in RPMI/FCS (10%)
as compared to triesters 3a-c and 4b,c.
Antiviral Evaluation. CycloSal-triesters 3-6 were evaluated
for their anti-HIV activity in vitro (Table 1). All compounds
showed activity against HIV-1 and HIV-2 in wild-type CEM/0
cells and are (except 6a) considerably more active against HIV-2
in CEM/TK- cells when compared to d4T 2.
synthesized successfully. Their complex hydrolytic and antiviral
behavior has been analyzed and was compared with the data
known for the 5-diacetoxymethyl-3-alkyl- (3b,c) and 5-formyl-
3-alkyl-cycloSal-d4TMPs (4b,c). All compounds were proven
to be potent inhibitors of HIV-1 and HIV-2 replication, and
most of them retained strong antiviral potency in TK-deficient
CEM/TK cells.
The di-iso-butyroxymethyl group showed the expected higher
stability against enzymatic cleavage but showed also some
solubility problems due to high lipophilicity. For 3-diacetoxym-
ethyl-5-alkyl-cycloSal-d4TMPs (6a,b) and 3-formyl-5-alkyl-
cycloSal-d4TMPs (5a,b), a significant difference in half-lives
of the chemical hydrolysis was found (13-15 fold), as
conceptually intended.
Both 3-diacetoxymethyl-cycloSal-d4TMPs (5a,b) showed
similar antiviral activity in CEM/TK- cells. This was expected
due to their almost identical hydrolysis behavior in all media.
In contrast, for 3-formyl-5-methyl-cycloSal-d4TMP (6a), a
significant loss of activity in CEM/TK- cells has been observed
(29 ( 18 µM), whereas 3-formyl-5-tert-butyl-cycloSal-d4TMP
(6b) exhibited only a slight activity loss (5.1 ( 1.9 µM).
Although the di-iso-butyroxymethyl group was significantly
more stable against enzymatic cleavage compared to the
diacetoxymethyl group 5-di-iBOM-cycloSal-d4TMPs 3d (X )
Me; EC50 >10 µM) and 3e-mix (X ) tBu; EC50 ) 2.4 ( 2.3
µM) had a lower antiviral activity in CEM/TK- cells than their
5-di-AM counterparts 3b (X ) Me; EC50 ) 6.3 ( 2.9 µM) and
3c-mix (X ) tBu; EC50 ) 0.78 ( 0.0 µM), respectively. One
reason may be again the poorer solubility of the 5-di-iBOM-
cycloSal-d4TMPs in RPMI/FCS(10%). Taking the hydrolysis
half-lives in PBS (pH ) 7.6) into account, at least for 3e, similar
antiviral activities to those of 3c have been expected.
Interestingly, the antiviral data for the separated diastereomers
of 3c,e and 4c showed a clear trend. The (SP)-configurated fast-
eluting diastereomer was always more active than the slow-
diastereomer. In contrast to previous work, the hydrolysis data
of the separated diastereomers were essentially identical here.
Thus, the difference in antiviral activity may be due to a
difference in membrane permeability.
The analysis of the separated diastereomers of 3c,e and 4c
points to a generally better antiviral activity for the fast-
diastereomers than for the slow-diastereomers. The absolute
configuration at the phosphorus atom of 4c-slow was determined
as (RP) by single-crystal X-ray structure. According to HPLC
studies, the selective release of 4c-slow from 3c-slow and 3e-
slow by enzymatic cleavage was proven. Hence, the better
antiviral activity against HIV-2 in CEM/TK- cells can be
correlated with the (SP)-configuration of the cycloSal-triesters.
Experimental Section
NMR spectra were recorded with a Bruker AMX 400, Bruker AV
1
400, or a Bruker DRX 500 Fourier transform spectrometer. All H
and 13C NMR chemical shifts (δ) are quoted in parts per million (ppm)
downfield from tetramethylsilane and calibrated on solvent signals.
The 31P NMR chemical shifts (proton decoupled) are quoted in ppm
using H3PO4 as the external reference. The spectra were recorded at
room temperature. Mass spectra were obtained with a VG Analytical
VG/70-250 F [FAB, (double focusing), matrix: m-nitrobenzyl
alcohol], ESI mass spectra were recorded with a VG Analytical
Finnigan ThermoQuest MAT 95 XL spectrometer. For thin layer
chromatography (TLC), VWR precoated 60 F254 plates with a 0.2
mm layer of silica gel (VWR no. 5554) were used; sugar-containing
compounds were visualized with sugar spray reagent (0.5 mL
4-methoxybenzaldehyde, 9 mL of EtOH, 0.5 mL of concd sulfuric
acid, and 0.1 mL of glacial acetic acid). All preparative TLCs were
performed on a chromatotron (Harrison Research, model 7924T)
using glass plates coated with 1, 2, or 4 mm layers of VWR 60
PF254 silica gel containing a fluorescent indicator (VWR no. 7749).
Conclusion
In summary, 5-di-iBOM-3-alkyl- (3d,e), 3-di-AM-5-alkyl-
(5a,b), and 3-formyl-5-alkyl-cycloSal-d4TMPs (6a,b) have been

Dialkoxymethyl-cycloSal-d4TMPs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6757
For column chromatography, Merck silica gel 60, 230-400 mesh,
was used. UV spectra were recorded on a Varian Cary 1E
UV-visible spectrophotometer and absorption maximum wave-
lengths λmax are given in nm. UV absorptions of cycloSal nucleotides
were determined from their HPLC data (diode array detector).
Analytical HPLC was performed on a Merck-Hitachi HPLC system
(D-7000) equipped with a LiChroCART 125-3 column containing
reversed phase silica gel Lichrospher 100 RP 18 (5 µM; Merck,
Darmstadt, Germany). Preparative HPLC was carried out on an
HPLC system consisting of a Merck-Hitachi L-6250 Intelligent
Pump, a Merck-Hitachi LaChrom UV detector L-7400, and a Merck
Hitachi D-2500A Chromato-Integrator using a Merck Hibar RT
250-25 column containing reversed phase silica gel Lichrospher
100 RP 18 (5 µM; Merck, Darmstadt, Germany). The flow rate
was 10 mL/min, and detection was performed at a wavelength of
260 nm. The lyophilized products 3-6 did not give useful
microanalytical data, most probably due to incomplete combustion
of the compounds or varying amounts of water, but were found to
be pure by rigorous HPLC analysis. Diethyl ether was dried over
sodium/benzophenone and distilled under nitrogen. THF was dried
over potassium/benzophenone and distilled under nitrogen. Pyridine,
CH2Cl2, and CH3CN were destilled from calcium hydride under
nitrogen. N,N-diisopropylethylamine and triethylamine were distilled
from sodium prior to use. Acetic anhydride, isobutyric anhydride,
and phosphorus(III) chloride were distilled under nitrogen prior to
use. CH3CN for HPLC was obtained from Acros (HPLC grade).
General Procedure D: Deprotection of Salicyl Alcohol Isopro-
pylidene Acetals (16). To a solution of salicyl alcohol isopropylidene
acetals 16 (1.0 equiv) in CH3CN/H2O (7:3), three drops (16a) or 3
mL (16b) of concd hydrochloric acid were added. The reaction
mixture was brought to boil with a heat gun for 30 s. This
procedure was repeated until the deprotection was complete
(monitored via TLC). The phases were separated with CH2Cl2
and water, and the aqueous layer was extracted two times with
CH2Cl2. Then the combined organic layers were dried with
sodium sulfate, and the solvent was removed under reduced
pressure. The products were purified by column chromatography
(CH2Cl2/CH3OH gradient (0-3%)).
General Procedure E: Preparation of 3-Formyl-5-alkyl-cy-
cloSal-d4T-monophosphates (6). The saligenyl chlorophosphites
have been synthesized from the respective 3-formyl-5-alkylsalicyl
alcohols 7 as described in ref 4, but the reactions were carried out
at -55 °C instead of -20 °C. The grade of purity was determined
1
by integration of H NMR signals (single proton Pyr ·HCl vs one
aromatic proton of the saligenyl chlorophosphite).
Under nitrogen, d4T (2, 1.0 equiv) was dissolved in dry CH3CN
and cooled to -20 °C. Then, DIPEA (1.6 equiv) and the respective
saligenyl chlorophosphite (2.0 equiv) in dry CH3CN were added.
The reaction mixture was stirred at room temperature for 3 h.
Subsequently, tert-butyl hydroperoxide (5.5 M in n-nonane; 3.0
equiv) was added at -20 °C. The solution was stirred at room
temperature for 1 h and then poured into a 1 M HOAc/NaOAc
buffer (pH ) 5). The phases were separated with ethyl acetate, the
aqueous layer was extracted with ethyl acetate three times, and the
combined organic layers were dried with sodium sulfate and
concentrated under reduced pressure. The resulting residues were
purified by preparative TLC [Chromatotron; CH2Cl2/CH3OH gradi-
ent ((0-5%; 6a) or (0-2%; 6b)) + 0.1% glacial acetic acid]. The
isolated products were lyophilized from CH3CN/H2O 1:1.
General Procedure F: Preparation of Dialkoxymethyl-cycloSal-
d4T-monophosphates 3 and 5. Under nitrogen, to a solution of the
respective formyl-cycloSal-d4T-monophosphate (4, 6) in dry
CH3CN and freshly distilled acetic or isobutyric anhydride zirco-
nium(IV) chloride (ZrCl4) was added in one portion. The reaction
mixture was stirred at room temperature and monitored via TLC
(CH2Cl2/CH3OH 9:1 v/v (3d, 5a) or ethyl acetate/CH3OH 9:1 v/v
(3e, 5b)). If necessary, additional ZrCl4 was added. Then the mixture
was diluted with phosphate buffer (pH ) 7.3) and ethyl acetate,
and the aqueous layer was extracted three times with ethyl acetate.
The combined organic layers were dried with sodium sulfate, and
the solvent was removed in vacuo. Purification was achieved by
chromatography. The isolated products were lyophilized from
CH3CN/H2O 1:1 or directly from the eluent used for preparative
RP-HPLC.
General Procedure A: Preparation of 6-Bromo-4-alkylsalicyl
Alcohols (11). The 6-bromo-4-alkylsalicyl alcohols 11a and 11b
were synthesized by reduction of a respective 6-bromosalicyl
carbonyl compound (10, 14) with LiAlH4. Therefore, LiAlH4 (1.0
equiv for 14, 2.0 equiv for 10) was suspended under nitrogen in
dry THF and a solution of the respective carbonyl compound (1.0
equiv) in THF was added dropwise within 30-45 min. After stirring
for 2 h at room temperature, the reaction mixture was refluxed for
1 h. At 0 °C, the reaction mixture was acidified to pH 4-5 with 2
N HCl, then diluted with water and the phases were separated with
ethyl acetate. The aqueous layer was extracted with ethyl acetate
four times, and the combined organic layers were dried with sodium
sulfate and concentrated under reduced pressure. The products were
purified by column chromatography (ethyl acetate), whereas in the
case of 11a, an additional purification by preparative TLC [Chro-
matotron; CH2Cl2/CH3OH 19:1 v/v] was necessary.
General Procedure B: Preparation of Salicyl Alcohol Isopro-
pylidene Acetals (15). Salicyl alcohols (11, 1.0 equiv) were
dissolved in acetone and 2,2-dimethoxypropane (5 equiv), para-
toluenesulfonic acid monohydrate (0.1 equiv), and anhydrous
sodium sulfate (3 equiv) were added. The reaction mixture was
stirred for 3 d (15b) or 4 d (15a) at 40 °C and then concentrated
under reduced pressure. The residue was dissolved in water and
ethyl acetate, and the phases were separated. The aqueous layer
was extracted with ethyl acetate, and the combined organic layers
were washed with 1 M NaOH (2×) and with water. After drying
with sodium sulfate, the solvent was removed in vacuo. The
products were purified by preparative TLC [Chromatotron; petro-
leum ether 50-70/CH2Cl2 9:1 v/v (15a) or petroleum ether 50-70/
CH2Cl2 gradient (0-30%, 15b)].
General Procedure C: Preparation of 6-Formyl-4-alkylsalicyl
Alcohols (16). Under nitrogen, the 6-bromo-4-alkylsalicyl alcohol
isopropylidene acetal (15a,b, 1.0 equiv) was dissolved in dry THF,
cooled down to -78 °C, and a 1.6 M solution of n-butyl lithium
(2.0 equiv) in hexane was added dropwise. The reaction was stirred
at -78 °C for 2 h and then treated with dry N,N-dimethylformamide
(10.0 equiv) as a 1:1 solution in dry THF. The reaction mixture
was stirred at -78 °C for 0.75 h and warmed slowly up to room
temperature. Then the mixture was diluted with diethyl ether,
washed (3 × H2O, 1 × brine), and dried with magnesium sulfate.
The solvent was removed in vacuo, and the residue was purified
by preparative TLC [Chromatotron; petroleum ether 50-70/CH2Cl2
gradient (0-40%; 15a) or petroleum ether 50-70/CH2Cl2 1:2 v/v
(15b)].
5-Methylsalicylic Acid Methyl Ester (9). 5-Methylsalicylic acid
(8, 5.00 g, 32.9 mmol) was dissolved in 80 mL of CH3OH and
concd sulfuric acid (16 mL, 0.30 mol) was added slowly at 0 °C.
The reaction mixture was heated under reflux for 2 d. After cooling
down to room temperature, the reaction mixture was poured into
250 mL of iced water. The aqueous layer was extracted three times
with CH2Cl2, the combined organic layers were dried with sodium
sulfate, and the solvent was removed in vacuo. Purification was
achieved by preparative TLC [Chromatotron; petroleum ether
50-70/CH2Cl2 1:1 v/v]. Yield: 5.18 g (31.2 mmol, 95%) of a
1
colorless liquid. H NMR (400 MHz, DMSO-d6): δ ) 10.30 (s,
1H, phenol-OH), 7.59-7.56 (m, 1H, aryl-H-6), 7.34 (dd, J ) 8.5,
2.3 Hz, 1H, aryl-H-4), 6.88 (d, J ) 8.5 Hz, 1H, aryl-H-3), 3.88 (s,
3H, OCH3), 2.24 (s, 3H, CH3) ppm.
3-Bromo-5-methyl-methylsalicylate (10). 5-Methylsalicylic acid
methyl ester (9, 6.61 g, 39.7 mmol) was dissolved in 65 mL of
CHCl3, and a solution of Br2 (2.02 mL, 39.5 mmol) in 40 mL of
CHCl3 was added within 45 min at room temperature. The reaction
mixture was stirred for 1 d at room temperature and then washed
with 39% sodium bisulfite solution and water (2×). The organic
layer was dried with sodium sulfate and concentrated under reduced
pressure. The product was purified by recrystallization from
CH3OH. Product remaining in CH3OH was purified by preparative

6758 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Gisch et al.
TLC [Chromatotron; petroleum ether 50-70/CH2Cl2 3:1 v/v]. Yield:
6.04 mL of dry THF. Yield: 1.48 g (5.96 mmol, 76%) of a yellow
solid. 1H NMR (400 MHz, DMSO-d6): δ ) 10.27 (s, 1H, formyl-
H), 7.57 (d, J ) 2.5 Hz, 1H, aryl-H-5), 7.47 (d, J ) 2.5 Hz, 1H,
aryl-H-3), 4.89 (s, 2H, benzyl-H), 1.53 (s, 6H, acetal-CH3), 1.26
(s, 9H, tBu) ppm.
1
8.93 g (36.5 mmol, 92%) of a yellow solid. H NMR (400 MHz,
DMSO-d6): δ ) 10.92 (s, 1H, phenol-OH), 7.72-7.69 (m, 1H, aryl-
H-4), 7.64-7.60 (m, 1H, aryl-H-6), 3.92 (s, 3H, OCH3), 2.25 (s,
3H, CH3) ppm.
3-Bromo-5-tert-butylsalicyl Aldehyde (14). To a stirred solution
of 5-tert-butylsalicyl aldehyde (13, 4.35 g, 24.4 mmol) in 5 mL of
HOAc a solution of Br2 (1.29 mL, 25.2 mmol) in 12 mL of HOAc
was added dropwise within 15 min. The reaction mixture was stirred
for 3 h at 50 °C and was monitored via TLC (petroleum ether
50-70/ethyl acetate 4:1 v/v). As the conversion of 13 was not
complete, 0.43 mL (8.4 mmol) Br2 in 1.7 mL of HOAc were added
dropwise and the reaction solution was stirred for additional 15 h
at 50 °C. Subsequently, the reaction mixture was diluted with
CH2Cl2 and the organic layer was washed with 39% sodium bisulfite
solution, water, saturated aqueous NaHCO3, and brine. After drying
with sodium sulfate, the organic layer was concentrated under
reduced pressure. The product was purified by preparative TLC
[Chromatotron; petroleum ether 50-70]. Yield: 5.79 g (22.5 mmol,
6-Formyl-4-methylsalicyl Alcohol (7a). General procedure D with
6-formyl-4-methylsalicyl alcohol isopropylidene acetal (16a, 1.87 g,
9.06 mmol) dissolved in 50 mL of CH3CN/H2O (7:3 v/v). Yield:
1
1.30 g (7.82 mmol, 86%) of a yellow solid. H NMR (400 MHz,
DMSO-d6): δ ) 10.76 (s, 1H, phenol-OH), 10.00 (s, 1H, formyl-
H), 7.53-7.48 (m, 1H, aryl-H-3), 7.46-7.41 (m, 1H, aryl-H-5),
5.21 (t, J ) 5.5 Hz, 1H, benzyl-OH), 4.53 (d, J ) 5.0 Hz, 2H,
benzyl-H), 2.29 (s, 3H, CH3) ppm.
6-Formyl-4-tert-butylsalicyl Alcohol (7b). General procedure D
with 6-formyl-4-tert-butylsalicyl alcohol isopropylidene acetal (16b,
1.30 g, 5.24 mmol) dissolved in 50 mL of CH3CN/H2O (7:3 v/v).
Yield: 960 mg (4.61 mmol, 88%) of an orange oil. 1H NMR (400
MHz, DMSO-d6): δ ) 10.82 (s, 1H, phenol-OH), 10.05 (s, 1H,
formyl-H), 7.75 (d, J ) 2.5 Hz, 1H, aryl-H-3), 7.64 (d, J ) 2.5
Hz, 1H, aryl-H-5), 5.25 (t, J ) 5.4 Hz, 1H, benzyl-OH), 4.55 (d,
J ) 4.8 Hz, 2H, benzyl-H), 1.29 (s, 9H, tBu) ppm.
3-Formyl-5-methyl-cycloSal-d4T-monophosphate (6a). General
procedure E with 6-formyl-4-methylsalicyl alcohol (7a, 500 mg,
3.01 mmol) dissolved in 18 mL of dry diethyl ether, phosphorus(III)
chloride (0.25 mL, 2.9 mmol), and dry pyridine (0.46 mL, 5.7
mmol) in 2.3 mL of dry diethyl ether. Yield: 415 mg (purity: 96%).
Quantities for cycloSal-d4T-monophosphate synthesis: 410 mg of
crude saligenyl chlorophosphite dissolved in 14 mL of dry CH3CN,
d4T (2, 170 mg, 0.758 mmol) dissolved in 21 mL of dry CH3CN,
DIPEA (0.22 mL, 1.3 mmol), and tert-butyl hydroperoxide (0.43
mL, 5.5 M in n-nonane, 2.4 mmol). Yield: 145 mg (0.332 mmol,
44%) of a diastereomeric mixture (ratio 0.65:1) as a colorless foam.
1H NMR (400 MHz, DMSO-d6): δ ) 11.32 (s, 1H, 1 × NH), 11.30
(s, 1H, 1 × NH), 10.26 (s, 1H, 1 × formyl-H), 10.21 (s, 1H, 1 ×
formyl-H), 7.58-7.55 (m, 2H, 2 × aryl-H-4), 7.42-7.39 (m, 2H,
2 × aryl-H-6), 7.16 (d, J ) 1.3 Hz, 1H, 1 × thymine-H-6), 7.15
(d, J ) 1.5 Hz, 1H, 1 × thymine-H-6), 6.78-6.74 (m, 2H, 2 ×
1′-H), 6.42 (ddd, J ) 6.0, 1.8, 1.5 Hz, 1H, 1 × 3′-H), 6.35 (ddd,
J ) 6.0, 1.8, 1.5 Hz, 1H, 1 × 3′-H), 6.04-5.97 (m, 2H, 2 × 2′-H),
5.61-5.50 (m, 2H, 2 × benzyl-H), 5.48-5.36 (m, 2H, 2 × benzyl-
H), 4.98-4.93 (m, 2H, 2 × 4′-H), 4.42-4.28 (m, 4H, 2 × 5′-H),
2.32 (s, 6H, 2 × CH3), 1.66 (d, J ) 1.0 Hz, 3H, 1 × thymine-
CH3), 1.62 (d, J ) 1.0 Hz, 3H, 1 × thymine-CH3) ppm. 31P NMR
(162 MHz, DMSO-d6): δ ) -9.62, -10.19 ppm.
1
92%) of a yellow solid. H NMR (400 MHz, CDCl3): δ ) 11.41
(s, 1H, phenol-OH), 9.85 (s, 1H, CHO), 7.81 (d, J ) 2.3 Hz, 1H,
aryl-H-6), 7.51 (d, J ) 2.3 Hz, 1H, aryl-H-4), 1.33 (s, 9H, tBu)
ppm.
6-Bromo-4-methylsalicyl Alcohol (11a). General procedure A
with 3-bromo-5-methyl-methylsalicylate (10, 3.80 g, 15.5 mmol)
dissolved in 50 mL of dry THF and LiAlH4 (1.18 g, 31.1 mmol)
as suspension in 80 mL of dry THF. Yield: 3.06 g (14.1 mmol,
1
91%) of a yellow oil. H NMR (500 MHz, DMSO-d6): δ ) 8.85
(s, 1H, phenol-OH), 7.22-7.18 (m, 1H, aryl-H-5), 7.09-7.03 (m,
1H, aryl-H-3), 5.31 (s, 1H, benzyl-OH), 4.52 (d, J ) 3.3 Hz, 2H,
benzyl-H), 2.20 (s, 3H, CH3) ppm.
6-Bromo-4-tert-butylsalicyl Alcohol (11b). General procedure A
with 3-bromo-5-tert-butylsalicyl aldehyde (14, 5.69 g, 22.1 mmol)
dissolved in 40 mL of dry THF and LiAlH4 (750 mg, 19.8 mmol)
as suspension in 60 mL of dry THF. Yield: 5.25 g (20.3 mmol,
1
92%) of a yellow oil. H NMR (400 MHz, DMSO-d6): δ ) 8.89
(s, 1H, phenol-OH), 7.22-7.18 (m, 2H, aryl-H-3, aryl-H-5), 5.34
(t, J ) 5.4 Hz, 1H, benzyl-OH), 4.55 (d, J ) 4.8 Hz, 2H, benzyl-
H), 1.24 (s, 9H, tBu) ppm.
6-Bromo-4-methylsalicyl Alcohol Isopropylidene Acetal (15a).
General procedure B with 6-bromo-4-methylsalicyl alcohol (11a,
2.16 g, 10.0 mmol) dissolved in 45 mL of acetone, 2,2-dimethoxy-
propane (6.32 mL, 51.0 mmol), para-toluenesulfonic acid mono-
hydrate (191 mg, 1.11 mmol), and anhydrous sodium sulfate (4.3
1
3-Formyl-5-tert-butyl-cycloSal-d4T-monophosphate (6b). Gen-
eral procedure E with 6-formyl-4-tert-butylsalicyl alcohol (7b, 230
mg, 1.10 mmol) dissolved in 8 mL of dry THF, phosphorus(III)
chloride (0.11 mL, 1.3 mmol) and dry pyridine (0.21 mL, 2.6 mmol)
in 1.1 mL of dry THF. Yield: 295 mg (purity: 55%). Quantities
for cycloSal-d4T-monophosphate synthesis: 290 mg of crude
saligenyl chlorophosphite dissolved in 10 mL of dry CH3CN, d4T
(2, 110 mg, 0.495 mmol) dissolved in 15 mL of dry CH3CN, DIPEA
(0.14 mL, 0.80 mmol), and tert-butyl hydroperoxide (0.26 mL, 5.5
M in n-nonane, 1.4 mmol). Yield: 86 mg (0.18 mmol, 36%) of a
diastereomeric mixture (ratio 0.85:1.0) as a colorless foam. 1H NMR
(500 MHz, DMSO-d6): δ ) 11.32 (s, 2H, 2 × NH), 10.27 (s, 1H,
1 × formyl-H), 10.26 (s, 1H, 1 × formyl-H), 7.77-7.72 (m, 2H,
2 × aryl-H-4), 7.71-7.68 (m, 2H, 2 × aryl-H-6), 7.20 (d, J ) 1.0
Hz, 1H, 1 × thymine-H-6), 7.16 (d, J ) 1.0 Hz, 1H, 1 × thymine-
H-6), 6.82-6.77 (m, 2H, 2 × 1′-H), 6.45-6.41 (m, 1H, 1 × 3′-
H), 6.38-6.34 (m, 1H, 1 × 3′-H), 6.05-6.01 (m, 1H, 1 × 2′-H),
6.01-5.97 (m, 1H, 1 × 2′-H), 5.61 (dd, J ) 14.8, 6.0 Hz, 1H, 1 ×
benzyl-H), 5.57 (dd, J ) 15.1, 5.0 Hz, 1H, 1 × benzyl-H),
5.51-5.43 (m, 2H, 2 × benzyl-H), 4.99-4.94 (m, 2H, 2 × 4′-H),
4.43-4.21 (m, 4H, 2 × 5′-H), 1.65 (s, 3H, 1 × thymine-CH3),
1.60 (s, 3H, 1 × thymine-CH3), 1.29 (s, 18H, 2 × tBu) ppm. 31P
NMR (162 MHz, DMSO-d6): δ ) -9.68, -10.03 ppm.
g). Yield: 1.90 g (7.39 mmol, 74%) of a colorless oil. H NMR
(400 MHz, DMSO-d6): δ ) 7.29-7.25 (m, 1H, aryl-H-5),
6.90-6.86 (m, 1H, aryl-H-3), 4.78 (s, 2H, benzyl-H), 2.21 (s, 3H,
CH3), 1.47 (s, 6H, acetal-CH3) ppm.
6-Bromo-4-tert-butylsalicyl Alcohol Isopropylidene Acetal (15b).
General procedure B with 6-bromo-4-tert-butylsalicyl alcohol (11b,
5.00 g, 19.3 mmol) dissolved in 90 mL of acetone, 2,2-dimethoxy-
propane (12.2 mL, 98.4 mmol), para-toluenesulfonic acid mono-
hydrate (370 mg, 1.92 mmol), and anhydrous sodium sulfate (8.4
1
g). Yield: 4.96 g (16.5 mmol, 86%) of a colorless solid. H NMR
(400 MHz, DMSO-d6): δ ) 7.41 (d, J ) 2.5 Hz, 1H, aryl-H-5),
7.12-7.08 (m, 1H, aryl-H-3), 4.82 (s, 2H, benzyl-H), 1.48 (s, 6H,
acetal-CH3), 1.24 (s, 9H, tBu) ppm.
6-Formyl-4-methylsalicyl Alcohol Isopropylidene Acetal (16a).
General procedure C with 6-bromo-4-methylsalicyl alcohol iso-
propylidene acetal (15a, 1.81 g, 7.04 mmol) dissolved in 40 mL of
dry THF, n-butyl lithium (8.80 mL, 1.6 M in hexane, 14.1 mmol),
and N,N-dimethylformamide (5.43 mL, 69.8 mmol) dissolved in
5.43 mL of dry THF. Yield: 1.15 g (5.58 mmol, 80%) of a yellow
oil. 1H NMR (400 MHz, DMSO-d6): δ ) 10.26 (s, 1H, formyl-H),
7.41-7.38 (m, 1H, aryl-H-5), 7.24-7.20 (m, 1H, aryl-H-3), 4.86
(s, 2H, benzyl-H), 2.26 (s, 3H, CH3), 1.54 (s, 6H, acetal-CH3) ppm.
6-Formyl-4-tert-butylsalicyl Alcohol Isopropylidene Acetal (16b).
General procedure C with 6-bromo-4-tert-butylsalicyl alcohol
isopropylidene acetal (15b, 2.35 g, 7.85 mmol) dissolved in 45 mL
of dry THF, n-butyl lithium (9.02 mL, 1.6 M in hexane, 14.4 mmol),
and N,N-dimethylformamide (6.04 mL, 78.7 mmol) dissolved in
3-Diacetoxymethyl-5-methyl-cycloSal-d4T-monophosphate (5a).
General procedure F with 3-formyl-5-methyl-cycloSal-d4T-mono-
phosphate (6a, 60.0 mg, 138 µmol) dissolved in 0.6 mL of dry
CH3CN, freshly distilled acetic anhydride (0.32 mL, 3.4 mmol),

Dialkoxymethyl-cycloSal-d4TMPs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6759
and ZrCl4 (16 mg, 67 µmol). Reaction time: 45 min. Purification:
preparative TLC [Chromatotron; ethyl acetate/CH3OH 9:1 v/v] and
preparative RP-HPLC (water/CH3CN 3:2 v/v). Yield: 14.4 mg (26.8
µmol, 19%) of a diastereomeric mixture (ratio 0.6:1.0) as a colorless
foam. 1H NMR (400 MHz, DMSO-d6): δ ) 11.33 (s, 2H, 2 ×
NH), 7.78 (s, 1H, 1 × H-8), 7.74 (s, 1H, 1 × H-8), 7.39-7.35 (m,
2H, 2 × aryl-H-4), 7.21-7.16 (m, 4H, 2 × aryl-H-6, 2 × thymine-
H-6), 6.82-6.77 (m, 2H, 2 × 1′-H), 6.41 (ddd, J ) 6.0, 1.8, 1.8
Hz, 1H, 1 × 3′-H), 6.34 (ddd, J ) 6.0, 1.8, 1.8 Hz, 1H, 1 × 3′-H),
6.05-5.98 (m, 2H, 2 × 2′-H), 5.54-5.45 (m, 2H, 2 × benzyl-H),
5.42-5.32 (m, 2H, 2 × benzyl-H), 4.98-4.91 (m, 2H, 2 × 4′-H),
4.35-4.19 (m, 4H, 2 × 5′-H), 2.31 (s, 3H, 1 × CH3), 2.31 (s, 3H,
1 × CH3), 2.12 (s, 3H, 1 × OC(O)CH3), 2.11 (s, 3H, 1 ×
OC(O)CH3), 2.11 (s, 3H, 1 × OC(O)CH3), 2.10 (s, 3H, 1 ×
OC(O)CH3), 1.66 (d, J ) 1.3 Hz, 3H, 1 × thymine-CH3), 1.60 (d,
J ) 1.3 Hz, 3H, 1 × thymine-CH3) ppm. 31P NMR (162 MHz,
DMSO-d6): δ ) -9.56, -9.67 ppm.
3-Diacetoxymethyl-5-tert-butyl-cycloSal-d4T-monophosphate (5b).
General procedure F with 3-formyl-5-tert-butyl-cycloSal-d4T-
monophosphate (6b, 42.4 mg, 89.0 µmol) dissolved in 2 mL of
dry CH3CN, freshly distilled acetic anhydride (0.22 mL, 2.3 mmol),
and ZrCl4 (19 mg, 82 µmol + 15 mg, 84 µmol after 30 min).
Reaction time: 2.5 h. Purification: preparative TLC [Chromatotron;
CH2Cl2/CH3OH gradient (0-2%)] and preparative RP-HPLC
(water/CH3CN 1:1 v/v). Yield: 5.0 mg (8.6 µmol, 10%) of a
diastereomeric mixture (ratio 0.9:1.0) as a colorless foam. 1H NMR
(400 MHz, DMSO-d6): δ ) 11.34 (s, 2H, 2 × NH), 7.79 (s, 1H, 1
× H-8), 7.76 (s, 1H, 1 × H-8), 7.51-7.47 (m, 2H, 2 × aryl-H-4),
7.46-7.42 (m, 2H, 2 × aryl-H-6), 7.19 (d, J ) 1.3 Hz, 1H, 1 ×
thymine-H-6), 7.17 (d, J ) 1.3 Hz, 1H, 1 × thymine-H-6),
6.83-6.79 (m, 2H, 2 × 1′-H), 6.41 (ddd, J ) 6.0, 1.8, 1.8 Hz, 1H,
1 × 3′-H), 6.34 (ddd, J ) 6.0, 1.8, 1.8 Hz, 1H, 1 × 3′-H),
6.07-5.97 (m, 2H, 2 × 2′-H), 5.59-5.48 (m, 2H, 2 × benzyl-H),
5.42 (dd, J ) 14.1, 5.8 Hz, 1H, 1 × benzyl-H), 5.39 (dd, J ) 14.3,
6.5 Hz, 1H, 1 × benzyl-H), 4.99-4.93 (m, 2H, 2 × 4′-H),
4.37-4.22 (m, 4H, 2 × 5′-H), 2.12 (s, 3H, 1 × OC(O)CH3), 2.12
(s, 3H, 1 × OC(O)CH3), 2.11 (s, 3H, 1 × OC(O)CH3), 2.10 (s,
3H, 1 × OC(O)CH3), 1.63 (d, J ) 1.0 Hz, 3H, 1 × thymine-CH3),
1.55 (d, J ) 1.0 Hz, 3H, 1 × thymine-CH3), 1.28 (s, 9H, 1 × tBu),
1.27 (s, 9H, 1 × tBu) ppm. 31P NMR (162 MHz, DMSO-d6): δ )
-9.52, -9.57 ppm.
6H, 2 × OC(O)CH3), 1.59 (d, J ) 1.0 Hz, 3H, thymine-CH3), 1.31
(s, 9H, tBu) ppm. 31P NMR (162 MHz, DMSO-d6): δ ) -9.06
ppm.
Analytical Data of 3c-slow. 1H NMR (400 MHz, DMSO-d6): δ
) 11.31 (s, 1H, NH), 7.52 (s, 1H, H-8), 7.47-7.44 (m, 1H, aryl-
H-4), 7.39-7.36 (m, 1H, aryl-H-6), 7.18 (d, J ) 1.3 Hz, 1H,
thymine-H-6), 6.82-6.79 (m, 1H, 1′-H), 6.42 (ddd, J ) 6.0, 1.8,
1.8 Hz, 1H, 3′-H), 6.06-6.02 (m, 1H, 2′-H), 5.53-5.36 (m, 2H, 2
× benzyl-H), 5.00-4.94 (m, 1H, 4′-H), 4.40-4.28 (m, 2H, 5′-H),
2.11 (s, 6H, 2 × OC(O)CH3), 1.57 (d, J ) 1.3 Hz, 3H, thymine-
CH3), 1.35 (s, 9H, tBu) ppm. 31P NMR (162 MHz, DMSO-d6): δ
) -8.71 ppm.
5-Diisobutyroxymethyl-3-methyl-cycloSal-d4T-monophos-
phate (3d). General procedure F with 5-formyl-3-methyl-cycloSal-
d4T-monophosphate (4b, 51.0 mg, 117 µmol) dissolved in 1 mL
of dry CH3CN, freshly distilled isobutyric anhydride (0.40 mL, 2.5
mmol), and ZrCl4 (30.0 mg, 129 µmol). Reaction time: 3 h.
Purification: preparative TLC [Chromatotron; CH2Cl2/CH3OH
gradient (0-5%)]. Yield: 21.0 mg (35.4 µmol, 30%) of a diaster-
eomeric mixture (ratio 0.8:1.0) as a colorless foam. 1H NMR (500
MHz, DMSO-d6): δ ) 11.35-11.32 (m, 2H, 2 × NH), 7.49 (s,
2H, 2 × H-8), 7.42-7.39 (m, 2H, 2 × aryl-H-4), 7.30-7.28 (m,
2H, 2 × aryl-H-6), 7.21-7.17 (m, 2H, 2 × thymine-H-6),
6.82-6.78 (m, 2H, 2 × 1′-H), 6.43-6.40 (m, 1H, 1 × 3′-H),
6.39-6.35 (m, 1H, 1 × 3′-H), 6.04-5.99 (m, 2H, 2 × 2′-H),
5.56-5.46 (m, 2H, 2 × benzyl-H), 5.45-5.35 (m, 2H, 2 × benzyl-
H), 4.98-4.93 (m, 2H, 2 × 4′-H), 4.34-4.24 (m, 4H, 2 × 5′-H),
2.61 (hept, J ) 6.9 Hz, 4H, 4 × CH(CH3)2), 2.24 (s, 3H, 1 ×
CH3), 2.21 (s, 3H, 1 × CH3), 1.63-1.61 (m, 3H, 1 × thymine-
CH3), 1.61-1.59 (m, 3H, 1 × thymine-CH3), 1.11 (d, J ) 7.1 Hz,
12H, 2 × CH(CH3)2), 1.08 (d, J ) 6.9 Hz, 12H, 2 × CH(CH3)2)
ppm. 31P NMR (162 MHz, DMSO-d6): δ ) -8.73, -8.82 ppm.
5-Diisobutyroxymethyl-3-tert-butyl-cycloSal-d4T-monophos-
phate (3e). General procedure F with 5-formyl-3-tert-butyl-cycloSal-
d4T-monophosphate (4c, 97.5 mg, 0.205 mmol) dissolved in 2 mL
of dry CH3CN, freshly distilled isobutyric anhydride (0.85 mL, 5.3
mmol) and ZrCl4 (23.9 mg, 103 µmol + 23.9 mg, 103 µmol after
1 h). Reaction time: 2.5 h. Purification: preparative TLC [Chro-
matotron; CH2Cl2/CH3OH gradient (0-5%)] and preparative RP-
HPLC (water/CH3CN 2:5 v/v or water/CH3CN 2:3 v/v, respec-
tively). Yield: 32.4 mg (51.1 µmol, 25%); 15.3 mg (24.1 µmol) of
3e-mix (ratio 0.8:1.0), 7.7 mg (12 µmol) of 3e-fast, and 9.4 mg
(15 µmol) of 3e-slow as colorless foams. 1H NMR (400 MHz,
DMSO-d6): δ ) 11.35 (s, 2H, 2 × NH), 7.54 (s, 2H, 2 × H-8),
7.44 (s, 2H, 2 × aryl-H-4), 7.36 (d, J ) 2.0 Hz, 2H, 2 × aryl-H-
6), 7.21 (d, J ) 1.3 Hz, 1H, 1 × thymine-H-6), 7.18 (d, J ) 1.0
Hz, 1H, 1 × thymine-H-6), 6.83-6.79 (m, 2H, 2 × 1′-H), 6.42
(ddd, J ) 6.0, 1.8, 1.5 Hz, 1H, 1 × 3′-H), 6.40 (ddd, J ) 6.0, 1.8,
1.8 Hz, 1H, 1 × 3′-H), 6.05-6.01 (m, 2H, 2 × 2′-H), 5.53-5.38
(m, 4H, 4 × benzyl-H), 5.00-4.96 (m, 2H, 2 × 4′-H), 4.41-4.29
(m, 4H, 2 × 5′-H), 2.62 (hept, J ) 7.0 Hz, 4H, 4 × CH(CH3)2),
1.57 (d, J ) 1.3 Hz, 3H, 1 × thymine-CH3), 1.54 (d, J ) 1.0 Hz,
3H, 1 × thymine-CH3), 1.34 (s, 9H, 1 × tBu), 1.31 (s, 9H, 1 ×
tBu), 1.11 (d, J ) 7.0 Hz, 12H, 2 × CH(CH3)2), 1.09 (d, J ) 7.0
Hz, 12H, 2 × CH(CH3)2) ppm. 31P NMR (162 MHz, DMSO-d6):
δ ) -8.57, -8.96 ppm.
The synthesis of compounds 3c, 4b, and 4c has already been
published in ref 4. The separation of the diastereomers was achieved
by preparative RP-HPLC with water/CH3CN 2:1 v/v (3c) and water/
CH3CN 2:1 v/v containing 0.5% HOAc (4c) as eluent.
5-Formyl-3-tert-butyl-cycloSal-d4T-monophosphate (4c). Ana-
lytical Data of 4c-fast. 1H NMR (400 MHz, DMSO-d6): δ ) 11.35
(s, 1H, NH), 9.96 (s, 1H, formyl-H), 7.92 (s, 1H, aryl-H-4),
7.79-7.75 (m, 1H, aryl-H-6), 7.22 (d, J ) 1.0 Hz, 1H, thymine-
H-6), 6.84-6.80 (m, 1H, 1′-H), 6.44-6.40 (m, 1H, 3′-H), 6.05-6.01
(m, 1H, 2′-H), 5.61-5.45 (m, 2H, 2 × benzyl-H), 5.00-4.96 (m,
1H, 4′-H), 4.40-4.31 (m, 2H, 5′-H), 1.61 (s, 3H, thymine-CH3),
1.35 (s, 9H, tBu) ppm. 31P NMR (162 MHz, DMSO-d6): δ ) -9.43
ppm.
Analytical Data of 4c-slow. 1H NMR (400 MHz, DMSO-d6): δ
) 11.33 (s, 1H, NH), 9.97 (s, 1H, formyl-H), 7.93 (s, 1H, aryl-H-
4), 7.79-7.76 (m, 1H, aryl-H-6), 7.20 (d, J ) 1.0 Hz, 1H, thymine-
H-6), 6.82-6.79 (m, 1H, 1′-H), 6.45-6.41 (m, 1H, 3′-H), 6.07-6.03
(m, 1H, 2′-H), 5.62-5.40 (m, 2H, 2 × benzyl-H), 5.01-4.97 (m,
1H, 4′-H), 4.41-4.34 (m, 2H, 5′-H), 1.58 (d, J ) 1.0 Hz, 3H,
thymine-CH3), 1.39 (s, 9H, tBu) ppm. 31P NMR (162 MHz, DMSO-
d6): δ ) -9.06 ppm.
1
Analytical Data of 3e-fast. H NMR (400 MHz, DMSO-d6): δ
) 11.34 (s, 1H, NH), 7.54 (s, 1H, H-8), 7.44 (s, 1H, aryl-H-4),
7.36 (d, J ) 1.6 Hz, 1H, aryl-H-6), 7.21 (d, J ) 1.3 Hz, 1H,
thymine-H-6), 6.83-6.80 (m, 1H, 1′-H), 6.42 (ddd, J ) 6.0, 1.6,
1.6 Hz, 1H, 3′-H), 6.04-6.01 (m, 1H, 2′-H), 5.53-5.39 (m, 2H, 2
× benzyl-H), 5.00-4.96 (m, 1H, 4′-H), 4.37-4.31 (m, 2H, 5′-H),
2.62 (hept, J ) 6.9 Hz, 2H, 2 × CH(CH3)2), 1.57 (d, J ) 1.0 Hz,
3H, thymine-CH3), 1.31 (s, 9H, tBu), 1.11 (d, J ) 7.3 Hz, 6H, 1 ×
CH(CH3)2), 1.10 (d, J ) 7.0 Hz, 6H, 1 × CH(CH3)2) ppm. 31P
NMR (162 MHz, DMSO-d6): δ ) -8.96 ppm.
5-Diacetoxymethyl-3-tert-butyl-cycloSal-d4T-monophosphate (3c).
1
Analytical Data of 3c-fast. H NMR (400 MHz, DMSO-d6): δ )
Analytical Data of 3e-slow. 1H NMR (400 MHz, DMSO-d6): δ
) 11.32 (s, 1H, NH), 7.54 (s, 1H, H-8), 7.44 (s, 1H, aryl-H-4),
7.35 (d, J ) 1.6 Hz, 1H, aryl-H-6), 7.18 (d, J ) 1.0 Hz, 1H,
thymine-H-6), 6.82-6.79 (m, 1H, 1′-H), 6.40 (ddd, J ) 6.0, 1.9,
1.6 Hz, 1H, 3′-H), 6.05-6.02 (m, 1H, 2′-H), 5.53-5.38 (m, 2H,
11.35 (s, 1H, NH), 7.52 (s, 1H, H-8), 7.47-7.44 (m, 1H, aryl-H-
4), 7.39-7.36 (m, 1H, aryl-H-6), 7.22 (d, J ) 1.0 Hz, 1H, thymine-
H-6), 6.84-6.78 (m, 1H, 1′-H), 6.42 (ddd, J ) 6.0, 1.8, 1.5 Hz,
1H, 3′-H), 6.05-6.01 (m, 1H, 2′-H), 5.53-5.38 (m, 2H, 2 × benzyl-
H), 4.99-4.94 (m, 1H, 4′-H), 4.38-4.28 (m, 2H, 5′-H), 2.11 (s,

6760 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Gisch et al.
2 × benzyl-H), 5.00-4.96 (m, 1H, 4′-H), 4.40-4.30 (m, 2H, 5′-
H), 2.62 (hept, J ) 6.9 Hz, 2H, 2 × CH(CH3)2), 1.55 (d, J ) 1.0
Hz, 3H, thymine-CH3), 1.34 (s, 9H, tBu), 1.11 (d, J ) 7.3 Hz, 6H,
1 × CH(CH3)2), 1.10 (d, J ) 7.0 Hz, 6H, 1 × CH(CH3)2) ppm.
31P NMR (162 MHz, DMSO-d6): δ ) -8.57 ppm.
References
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Single-Crystal X-ray Diffraction Analysis of 4c-slow.13 C22H25-
N2O8P, FW: 476.41, monoclinic space group P21, a ) 8.3061(11)
Å, b ) 15.864(2) Å, c ) 9.0212(12) Å, R ) 90°, ꢀ ) 110.58°, γ
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eÅ-3
.
Hydrolysis Studies of cycloSal Phosphate Triesters. Hydrolysis
studies of cycloSal nucleotides (phosphate buffer, pH ) 7.3 and
pH 7.6) by HPLC analysis (method I) have been described
before.17,18 Studies in cell extracts were performed as reported in
ref 8 with different incubation times but without using acetic acid
to stop the reaction for cycloSal nucleotides with acid-sensitive
substituents. Studies in RPMI/FCS(10%) were carried out in the
same way using culture medium instead of cell extracts.
Antiretroviral Evaluation. The method of antiviral evaluation
has already been described in ref 4.
Acknowledgment. We thank the University of Hamburg,
the “Geconcerteerde Onderzoeksacties (GOA no. 05/19) and
the Fonds voor Wetenschappelijk Onderzoek (FWO)sVlaanderen
for financial support. We also thank Leen Ingels and Ria Van
Berwaer for excellent technical assistance and Dr. Frank
Hoffmann for the help in solving the X-ray structure.
Supporting Information Available: 13C NMR and UV spec-
troscopic data, mass spectrometric data, Rf values, melting points,
analytical HPLC data of new cycloSal compounds; methods for
HPLC analysis; general nomenclature of salicyl alcohols and
cycloSal-d4TMPs. This material is available free of charge via the
Internet at http://pubs.acs.org.
JM800853P