CycloSal-BVDUMP pronucleotides: How to convert an antiviral-inactive nucleoside analogue into a bioactive compound against EBV

Article abstract of DOI:10.1021/jm0209275

Novel cycloSal-BVDUMP triesters 2-4 5-[(E)-2-bromovinyl]-2′-deoxyuridine (BVDU, 1) have been studied with regard to their potential anti-EBV activity. In addition to the 3′-unmodified cycloSal-BVDUMP triesters 2a-f, the 3′-hydroxyl function has been ester

Full text of DOI:10.1021/jm0209275

J . Med. Chem. 2002, 45, 5157-5172
5157
CycloSa l-BVDUMP P r on u cleotid es: How to Con ver t a n An tivir a l-In a ctive
Nu cleosid e An a logu e in to a Bioa ctive Com p ou n d a ga in st EBV
Chris Meier,*,† Andreas Lomp,† Astrid Meerbach,‡ and Peter Wutzler‡
Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany and
†Institute for Antiviral Chemotherapy, Friedrich-Schiller-University J ena, Winzerlaer Strasse 10, D-07745 J ena, Germany
Received May 3, 2002
Novel cycloSal-BVDUMP triesters 2-4 5-[(E)-2-bromovinyl]-2′-deoxyuridine (BVDU, 1) have
been studied with regard to their potential anti-EBV activity. In addition to the 3′-unmodified
cycloSal-BVDUMP triesters 2a -f, the 3′-hydroxyl function has been esterified with different
aliphatic carboxylic acids (3a -g) and R-amino acids having natural and nonnatural CR-
configuration (4a -m ). In addition to the synthesis of these compounds, different physicochem-
ical properties of the new derivatives will be reported, i.e., lipophilicity and hydrolysis behavior.
It could be proven that the monophosphate BVDUMP and not 3′,5′-cyclic BVDUMP was
delivered from most of the compounds by chemical hydrolysis in phosphate buffers at pH 6.8
and 7.3 as well as P3HR-1 cell extracts. Finally, the new compounds were tested for their
anti-EBV activity. As a result, the prototype compounds and particularly triesters 2c,d exhibited
pronounced anti-EBV activity making these compounds promising candidates for further
development. However, the 3′-ester derivatives were devoid of any antiviral activity while the
3′-aminoacyl derivatives showed an antiviral activity dependent upon the amino acid and the
CR-configuration
In tr od u ction
The second generation drug Brivudin (5-[(E)-2-bro-
movinyl]-2′-deoxyuridine, BVDU 1)15 is a potent and
highly selective nucleoside analogue-type inhibitor16 of
the replication of several herpes viruses especially VZV
and HSV-1.17 The mode of action as inhibitor depends
primarily on intracellular conversion of the nucleoside
analogue into the triphosphate form. Brivudin triphos-
phate (BVDUTP) can act either as an inhibitor of the
cellular DNA polymerase or as an alternate substrate
that would lead to the formation of nonsense DNA and
would render the DNA more prone to degradation when
incorporated in DNA.18 As for other known antiherpes
drugs, some limitations are known for the use of BVDU.
There is a lack of activity during virus latency because
viral TK is not expressed, drug resistance of the virus
has been observed, and BVDU is enzymatically de-
graded to the nucleobase 5-[(E)-2-bromovinyl]uracil
within 2-3 h in the bloodstream.17 Additionally, due to
altered or deficient enzymes necessary for the phospho-
rylation to the nucleoside monophosphate and diphos-
phate, this metabolism is often inefficient and thus the
therapeutic activity can be limited. To overcome some
of these limitations, the use of pronucleotides that
release the nucleotide from a lipophilic precursor after
cell entry may be of use.19 We developed the so-called
cycloSal-pronucleotide system.20 The basic idea of the
cycloSal-approach was a release of the nucleotide by a
selective, chemically induced hydrolysis. This concept
has been successfully applied to the intracellular deliv-
ery of a number of anti-HIV active nucleotides21 as well
as to acyclovir (ACV).22
Diseases caused by herpes viruses play an important
role in infections of humans. Acyclovir was the prototype
of the first generation of selective antiviral agents1 and
has been the gold standard for the therapy and sup-
pression of herpes simplex virus (HSV) infections for
more then 20 years.2 It is phosphorylated selectively by
HSV- and VZV-encoded thymidine kinase (TK) in virus-
infected cells to the mono- and diphosphate, respec-
tively, and finally by cellular enzymes to the di- and
triphosphate, which inhibits viral DNA polymerase and/
or causes chain elongation arrest. Today, the second
(brivudin, famciclovir, valaciclovir) and third (cidofovir3)
generations of antiherpes virus drugs are in use.
An emerging area of concern are Epstein-Barr virus
(EBV)-caused viral infections and their role they play
particularly in posttransplant lymphoproliferative dis-
orders. EBV is the causative agent of infectious and
chronic mononucleosis.4 Moreover, it is associated with
the development of several human malignancies5 (e.g.,
gastric carcinomas6) as well as oral hairy leucoplakia.7
Tumors classically linked with EBV are Burkitt’s lym-
phoma8 and nasopharyngeal carcinoma.9 More recently,
associations of EBV and B-cell lymphomas in immuno-
suppressed patients (including AIDS patients),10 certain
rare T-cell lymphomas,1 1lymphoproliferative syn-
dromes,12 and cases of Hodgkin’s lymphomas13 have
been reported. Recently, a successful clinical therapy
of EBV-associated lymphoproliferative deseases with
cidofovir has been reported.14
Here we report on the synthesis, hydrolytic properties,
and biological activities of a series of 3′-unmodified 2a -f
as well as 3′-O-esterified cycloSal-BVDUMPs 3 and 4.
As 3′-modifications, different lipophilic carboxylic acids
(3a -g) as well as R-amino acids (4a -k ) have been
* To whom correspondence should be addressed. Tel: +49-40-42838-
4324; Fax: +49-40-42838-2495; e-mail: chris.meier@chemie.uni-
hamburg.de.
† University of Hamburg.
‡ Friedrich-Schiller-University J ena.
10.1021/jm0209275 CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/08/2002

5158 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Sch em e 1. Synthetic Pathways to CycloSal-Triesters 2-4a
Meier et al.
a
Reaction conditions: (a) TBDMSCl, pyridine, rt, 14 h; (b) carboxylic acids, DMAP, DCC, CH2Cl2, rt, 30 min; (c) TBAF, THF, rt, 4 h;
(d) chlorophosphane, DIPEA, CH3CN, -20 °C, 30 min; (e) TBHP, CH3CN, -20 °C to rt, 1 h; (f) H2NNH2‚H2O, pyridine, CH3COOH, rt, 5
min; (g) N-Boc-amino acids, DMAP, DCC, CH2Cl2, rt, 30 min; (h) TFA, CH3CN, rt, 1 h.
introduced. Although EBV produces its own viral thy-
midine kinase, BVDU 1 is antivirally inactive against
EBV for unknown reasons. One reason may be that
EBV-TK cannot convert BVDU into the monophosphate
and thus does not initiate the required phosphorylation.
So, our aim was to determine whether the cycloSal-
concept is able to broaden the application of BVDU 1
against Epstein-Barr-virus (EBV)-caused infections
through the intracellular delivery of BVDUMP.5 Two
reports on pronucleotides of BVDU 1 have been pub-
lished before but both were unsuccessful.23 In contrast,
the data presented in this report show pronounced anti-
EBV activity for some of the cycloSal-BVDUMP tri-
esters.24
hydrazine hydrate/acetic acid in pyridine to give tri-
esters 2 in 31-50% overall yield. The yields of the latter
route were comparable to those obtained via the first
variant. For the preparation of both series of 3′-esteri-
fied cycloSal-BVDUMP triesters 3 and 4, the acyl group
was first introduced into BVDU 1 to give 3′-acyl-BVDUs
5a -t. So, BVDU 1 was first 5′-O-silylated (TBDMS) in
pyridine (84% yield) and then esterified using carboxylic
acids, N-Boc-protected L- or D-amino acids after DCC/
DMAP activation. Desilylation (2% TBAF in THF)
yielded BVDU derivatives 5a -t (90-97% yield of both
steps). The cycloSal moiety was introduced as mentioned
before to give triesters 3a -g and 7a -m respectively.
Alternatively, the reaction was also possible by using
the corresponding phosphoramidites.25 However, the
yields were again comparable to those of the chloro-
phosphite reaction, and triesters 3a -g were prepared
in 50-60% yield. Finally, the N-Boc protecting group
in 7a -m was cleaved by 5% TFA treatment in CH2Cl2/
MeOH 7:3 to give triesters 4a -m (50-52% yield). The
reaction sequence is outlined in Scheme 1.
Resu lts a n d Discu ssion
Ch em istr y. The prototype cycloSal-triesters 2a -f
were available by our previously reported P(III)-route
using chlorophosphites prepared from appropriate sali-
cyl alcohols and oxidation by t-BuOOH in DMF/THF 1:1
at -40 °C without using protecting groups at the 3′-
position.21b,c,d Alternatively, triesters 2 were prepared
by 5′-O-phosphitylation of 3′-O-levulinyl (Lev)-BVDU 5g
and subsequent oxidation. Cleavage of the 3′-O-Lev
group was achieved by treatment with a solution of
All title compounds 2-4 were isolated as 1:1 diaster-
eomeric mixtures that were inseparable even by means
of preparative HPLC. The compounds were character-
1
ized by H, 13C, and 31P NMR spectroscopy as well as

CycloSal-BVDUMP Pronucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5159
Ta ble 1. Hydrolysis (t1/2) in Phosphate Buffers and log P
Values
phate triester. The degradation pathway has been
proven by different methodologies and has been applied
to the delivery of various nucleosides, e.g. d4T,21a dd-
(4)A,21b F-ara/ribo-ddA,21c CBV/abacavir,21e and ACV.22
The intracellular delivery of the corresponding nucle-
otides has been demonstrated by the observed biological
activity in different cell lines and even in thymidine
kinase-deficient CEM cells or virus strains. However,
all of the above-mentioned nucleoside analogues did not
possess a further nucleophilic group in the vicinity of
the 5′-phosphate group. In BVDU, a 3′-hydroxyl group
is present in the glycon. Assuming an intramolecular
attack at the phosphorus center, this would finally lead
to the formation of 3′,5′-cyclic BVDUMP. Such a situ-
ation has already been observed in the case of the anti-
HSV active penciclovir (PCV): hydrolysis led exclusively
to the formation of the cyclic phosphate diester.27
However, in penciclovir the intramolecular nucleophile
is a primary hydroxyl and thus more reactive than the
secondary 3′-hydroxyl in BVDU. So, the question arose
if the 3′-hydroxyl group is able to compete with the
intermolecular ring opening reaction of the cycloSal-
moiety.
hydrolysisa in phosphate buffer, 37 °C
compd subst X subst R
pH 7.3b
pH 6.8c
logPe
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
3g
4a
4b,c
4d ,e
4f,g
4h ,i
4j,k
4l,m
1
5-Cl
H
H
H
H
H
H
H
0.23
1.52
2.32
6.29
8.61
20.7
5.81
6.30
4.89
13.8
8.10
14.8
4.72
1.76
1.26
3.09
1.40
3.20
1.70
0.32
-
0.33
1.79
2.75
7.79
12.5
33.7
7.60
7.77
9.00
14.6
10.9
16.0
7.02
1.92
1.43
3.40
1.50
3.70
3.50
0.35
-
1.8
1.5
1.6
1.9
2.2
2.9
2.2
2.3
2.4
3.0
2.6
2.8
2.3
5-OMe
3-Me
3,5-diMe
3-tBu
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
-
Ac
Prop
iBu
Piv
Hex
Dec
Lev
Gly
Ala
Val
Leu
Ile
Phe
Pro
H
-0.8
-0.6
-0.1
0.4
-0.02
0.5
-0.4
0.33
-1.6
ACV
-
-
-
-
a
Half-lives (t1/2) were determined from the decreasing peak of
the starting phosphate triester and are the mean of duplicate
The hydrolysis behavior of cycloSal-BVDUMPs 2-4
was examined in aqueous phosphate buffer at two pH-
values, in P3HR-1 cell extract and in human sera.
Hydrolysis products were identified by means of HPLC
using coinjections with independently synthesized refer-
ence compounds, ESI-mass spectrometry, and 31P NMR
spectroscopy. The half-lives were determined by inte-
gration of the decreasing peaks of the triesters versus
time.
b
experiments; values are given in hours (h). 25 mM phosphate
d
buffer, pH 7.3. c 25 mM phosphate buffer, pH 6.8. log P: log of
the partition coefficient determined in n-octanol/water.
mass spectrometry (FAB and ESI) and UV spectroscopy.
As expected, the phosphate triesters displayed two
closely spaced signals in the 31P NMR spectra. The
purity was checked by analytical reversed-phase high-
performance liquid chromatography (RP-HPLC). After
lyophilization, all phosphate triesters 2-4 were ob-
tained as white fluffy solids.
The half-lives were determined in phosphate buffers
and are summarized in Table 1. First of all, all prototype
cycloSal-BVDUMP triesters 2 selectively hydrolyzed in
both buffers to give BVDUMP and the corresponding
salicyl alcohol (Scheme 2). It was unambiguously shown
by means of analytical HPLC, ESI, and 31P NMR that
no 3′,5′-cyclic BVDUMP (cBVDUMP) has been formed.
Therefore, the possible concurrent intramolecular pro-
cess did not take place in contrast to the cycloSal-
PCVMP case mentioned before. This may be attributed
(i) to the lower reactivity of the secondary alcohol
function and (ii) to the attachment of the hydroxyl group
in the ring scaffold. In contrast, the hydroxyl group in
PCV is part of a flexible acyclic structure.
Deter m in a tion of P a r tition Coefficien ts (log P
va lu es). The partition coefficients (log P value) of the
cycloSal-BVDUMPs 2-4 as well as those of the parent
nucleoside analogue 1 and the reference compound
(AZT) were determined in 1-octanol/water by our previ-
ously reported HPLC method (Table 1).21a,21d The parti-
tion coefficients of the prototype cycloSal-BVDUMPs 2
were up to 370-fold and those of the 3′-O-esterified
phosphate triesters 3a -g were up to 470-fold higher as
compared to BVDU 1 (log P ) 0.33, Table 1). Compared
to AZT (log P ) 0.025),21d which enters mammalian cells
by passive, non facilitated diffusion,26 BVDUMP triester
2a -f and 3a -g revealed a pronounced increase in
lipophilicity. However, one should take into consider-
ation that in cases of very high log P values the
formation of micelles, lipid drops or lipid films could not
be excluded. As expected, the lipophilicity was dramati-
cally decreased in the case of the 3′-O-aminoacyl-
modified cycloSal-BVDUMP triesters 4 due to the
protonated amino group. In general, log P values of the
3′-O-aminoacyl-cycloSal-BVDUMPs 4a -m were lower
(up to 12-fold) than the log P value of BVDU with the
consequence that triesters 4 were highly water-soluble.
Kin et ic St u d ies. The cycloSal-pronucleotide ap-
proach has been designed to release the nucleotides and
the masking group selectively by a chemically induced
tandem or cascade reaction. In contrast to other prodrug
concepts based on enzymatically triggered activation,19
our approach involves the successive coupled cleavage
of the phenyl and benzyl esters of the cycloSal phos-
Second, as in the case of the cycloSal-triester of d4T
and dd(4)A, a clear correlation between hydrolytic
stability and the substituents on the aromatic ring was
observed for compounds 2a -f. In both phosphate buff-
ers, the half-lives increased commensurate with the
electron-donating ability of the substituent. In compari-
son, cycloSal-BVDUMPs hydrolyzed about 3-fold faster
as compared to the identically substituted cycloSal-
d4TMP, e. g., 3-Me-cycloSal-d4TMP showed a t1/2 value
of 19h at pH 7.3 while 3-Me-cycloSal-BVDUMP 2d
showed a t1/2 value of 6 h. As expected, the hydrolysis
rate was dependent on the pH-value of the buffer (Table
1). Phosphate triesters 2d -f showed in slightly acidic
(pH 6.8) as well as in slightly basic phosphate buffer
(pH 7.3) stability properties that should be high enough
to allow a sufficient cellular uptake. However, com-
pounds 2a -c hydrolyzed presumably too fast to be

5160 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Meier et al.
Sch em e 2. Major Hydrolysis Pathways of CycloSal-Phosphate Triesters
taken up efficiently. This may be deduced from earlier
work from our laboratory.
triester 2d (data not shown).28 Triester 2d hydrolyzed
with the same rate as in the absence of PLE, indicating
that PLE has no effect on the delivery of BVDUMP. 3′-
Aminoacyl triesters 4 led again first to the formation
of prototype 2d and then to BVDUMP without any
enzymatic contribution.
The hydrolytic behavior of cycloSal-BVDUMP tri-
esters 2-4 was further studied in P3HR-1 cell extracts.
This cell type was used for the antiviral evaluation
against EBV. Hydrolysis studies in the extracts were
done at 37 °C and followed for 8 h only in order to
exclude extract degradation with the risk of changing
the enzyme content. Incubations were stopped by ad-
dition of a solution of acidic methanol, and products
were analyzed by means of HPLC.
Again, a clear correlation between hydrolytic stability
and the substituents on the aromatic ring was observed
for compounds 2. As an example, the half-live of
3-methyl-cycloSal-BVDUMP 2d was determined. The
half-life was found to be 8 h and is therefore in the same
range as found in phosphate buffer, pH 7.3. Conse-
quently, no indication for an enzymatic contribution was
found and so hydrolysis in cell extract is purely chemi-
cally driven. As in the phosphate buffer, triesters 2
hydrolyzed to yield BVDUMP. The latter was subse-
quently dephosphorylated to BVDU 1 by phosphatases/
nucleosidases present in the extracts. This dephospho-
rylation has been proven in a separate assay: BVDUMP
was metabolized to BVDU to an extent of 29% within 4
h. Therefore, one may conclude that BVDU will not arise
directly from the triester. No formation of 5-[(E)-2-
bromovinyl]uracil (BVU) was detected in the extracts.
The hydrolysis of the 3′-O-acyl modified derivatives
3 exhibited a clear difference with respect to the
attached acid. For the 3′-O-Ac-, 3′-O-Hex-, 3′-O-Dec-,
and 3′-O-Lev derivatives 3a , 3e-g, respectively, enzy-
matic deesterification into the prototype triester 2d was
the one hydrolysis product. Triester 2d was then further
cleaved to give BVDUMP. However, also considerable
amounts of 3′-acyl-BVDUMP were formed (Table 2).
Thus, in contrast to the PLE and phosphate buffer
studies, the degradation in cell extracts is not selective.
Moreover, derivatives 3b-d hydrolyzed predominantly
to give 3′-O-acyl-BVDUMP. Interestingly, as in the
studies with PLE, 3′-O-acyl-BVDUMP was not further
metabolized to BVDUMP in the cell extracts.
Interestingly, a marked difference in the hydrolysis
pathway between the 3′-esterified cycloSal-triesters 3
and 4 was observed. As expected, hydrolysis of 3′-O-acyl
modified BVDUMPs 3 led to the cleavage of the
cycloSal-mask releasing 5′-phosphorylated 3′-O-acyl-
BVDUMP (Scheme 2). No cleavage of the 3′-ester to give
the prototype 2d was observed by means of HPLC. The
half-lives of 3′-O-acyl-modified BVDUMPs 3 showed a
correlation between the hydrolysis behavior and the
lipophilicity: the more lipophilic the carboxylic acid the
more stable the phosphate triester. This may be due to
the interaction of the attacking hydroxide with the
highly lipophilic 3′-O-acyl residue or to the formation
of lipid structures such as micelles that limits the
accessibility of the phosphate group. If a further polar
carbonyl functionality is introduced as in the 3′-O-Lev
derivative 3g, the lipophilicity and the half-life de-
creased. It is important to note that the 3′-ester group
in 3′-O-acyl-BVDUMP was stable in all cases to further
hydrolysis to give BVDUMP. So, chemical hydrolysis of
these compounds led to a dead end.
In contrast, the 3′-R-aminoacyl bearing cycloSal-
BVDUMPs 4 displayed markedly lower half-lives. Cy-
cloSal-BVDUMPs 4 hydrolyzed in both phosphate buff-
ers in the range of 0.3-3.7 h. Moreover, in contrast to
the 3′-O-acyl triesters 3, HPLC analysis proved that in
compounds 4 first the aminoacyl residue was cleaved,
leading to the prototype 3-methyl-cycloSal-BVDUMP
2d . In a further hydrolysis step the cycloSal-moiety was
cleaved to give finally BVDUMP and 3-methyl-saligenin.
It should be added that no influence of the CR-config-
uration in the aminoacyl group to the hydrolysis rate
was observed. The formed prototype triester 2d hydro-
lyzed with the same half-life as before. The unexpected
lability of the 3′-aminoacyl group may be attributed to
a protonation of the amino group (until ∼ pH 9) and
thus formation of an ammonium group at pH 6.8 or
7.3. This resulting acceptor group destabilizes the ester
bond by withdrawing electron density from the ester
residue.
Next, triesters 2-4 were studied in phosphate buffer
containing pig liver esterase (PLE). In contrast to the
situation above, it was shown that the 3′-acyl group in
triesters 3 was cleaved rapidly to give the prototype

CycloSal-BVDUMP Pronucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5161
Ta ble 2. Products after 8 h Incubation Time in P3HR-1 Cell Extract
compd
subst X
subst R
educt
prototype
3′-OR-BVDUMP
BVDUMP
BVDU
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
3g
4a
4b,c
4d ,e
4f,g
4h ,i
4j,k
4l,m
5-Cl
H
H
H
H
H
H
H
Ac
Prop
iBu
Piv
Hex
Dec
Lev
Gly
Ala
Val
Leu
Ile
-
-
-
-
-
-
-
-
-
-
16
14
35
14
3
59
51
42
31
22
9
16
3
7
41
49
24
18
15
<1
10
2
5-OMe
3-Me
3,5-diMe
3-tBu
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
34
51
63
90
29
77
45
82
77
84
19
-
-
-
-
-
29
4
10
2
8
3
<1
5
1
7
5
6
3
2
30
49
44
44
40
43
43
43
11
-
-
-
-
-
-
-
23
32
31
32
34
33
32
30
17
19
25
24
26
24
25
27
-
-
-
-
Phe
Pro
-
-
Ta ble 3. Anti-EBV Activity and Selective Indices of
CycloSal-Triesters 2-4, as well as BVDU 1 and ACV
The situation was significantly different for the tri-
esters 4. As in the phosphate buffers, all triesters are
loosing rapidly the 3′-O-aminoacyl group to yield pro-
totype triester 2d as major product. From the half-lives
and the product distribution it can be deduced that the
3′-O-aminoacyl esters were hydrolyzed chemically. No
significant difference between the natural L-configured
triester and the D-configurated could be detected. In the
case of triesters 4, no trace of 3′-O-aminoacyl-BVDUMP
was found.
To study the stability of the triester 2-4 in human
serum, compounds were incubated in 10% human serum
in phosphate buffer, pH 6.8. As summarized in Table
1, the half-lives of a few representative examples were
found to be in the range as in the pure phosphate
buffers. Again, this clearly points to a chemically driven
cleavage of the triesters. As in the extracts, the main
products of triesters 2 and 4 were again BVDUMP and
BVDU, while triesters 3 led mainly to the formation of
3′-O-acyl-BVDUMP.
An tivir a l Eva lu a tion . The successful thymidine
kinase bypass by cycloSal-d4TMP21 and cycloSal-
ACVMP phosphate triesters22 has been shown before.
These results demonstrated (i) a pronounced structure-
bioactivity correlation with respect to the substituents
on the cycloSal moiety, (ii) the successful membrane
penetration of the pronucleotide, (iii) the efficient in-
tracellular delivery of the nucleotide, and (iv) the
complete independence from TK.29 In sharp contrast,
the corresponding cycloSal-AZTMP derivatives lost
nearly all the antiviral activity observed in wild-type
CEM/O cells21d,30 when tested in TK-deficient CEM
cells.29 The parent nucleoside BVDU 1, as well as the
cycloSal phosphate triesters 2-4 were evaluated for
their ability to inhibit the replication of EBV in human
lymphoblastoid P3HR-1 cells. The antiviral activity of
the clinically used nucleoside analogue acyclovir (ACV)
is given for comparison. The results obtained are
displayed in Table 3.
compd subst X subst R
EC50 (µM)a
CC50 (µM)b SIc
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
4g
4h
4i
5-Cl
H
H
H
H
H
H
H
Ac
Prop
iBu
25.8 ( 7.15
12.3 ( 6.34
3.35 ( 3.76
4.11 ( 0.81
14.4 ( 9.18
32.5 ( 8.58
>89.7
80
92
137
122
143
80
109
282
104
57
>326
>299
152
93
92
38
47
80
45
65
78
37
35
66
36
38
225
392
3.1
7.5
41
30
9.9
2.5
5-OMe
3-Me
3,5-diMe
3-tBu
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
-
>175
-
-
-
-
-
-
>171
Piv
>83.4
Hex
Dec
Lev
Gly
>163
>149
>163
43.5 ( 30.1
35.3 ( 11.6
25.3 ( 13.7
81.8 ( 31.3
>137
2.1
L-Ala
D-Ala
L-Val
D-Val
L-Leu
D-Leu
L-Ile
D-Ile
L-Phe
D-Phe
L-Pro
D-Pro
2.6
1.5
0.6
-
2.0
23.0 ( 9.06
>135
-
>135
-
-
-
>135
4j
4k
4l
4m
1
ACV
g129
46.5 ( 19.3
g138
1.4
-
g138
>300
-
-
6.75 ( 2.62
58
a
50% effective concentration blocking EBV-DNA synthesis.
b 50% cytotoxic concentration. c Selectivity index ) CC50 (µM)/EC50
(µM).
against EBV were 5-methoxy-cycloSal-BVDUMP 2c
(>90-fold more active as compared to BVDU 1) and
3-methyl-cycloSal-BVDUMP 2d (>73-fold more active).
Both compounds are at least as active as the reference
compound ACV. Interestingly, as in our previously
reported results on cycloSal-d4TMP triesters, we noticed
a clear correlation between antiviral activity, the hy-
drolysis rates and the substituents in the cycloSal
moiety.20a
In contrast to triesters 2, all 3′-ester derivatives 3
proved to be antivirally inactive. At least for compounds
3a and 3g this failure in antiviral activity is somewhat
surprising because in the cell extract studies both
compounds released the antivirally active triester 2d
in some amounts as well as BVDUMP. However, the
As expected, the parent nucleoside BVDU 1 was
devoid of any antiviral activity in the EBV infected
P3HR-1 cell system. Most strikingly, the 3′-unmodified
cycloSal-BVDUMPs 2a -f showed pronounced anti-EBV
activity in the cell system. Most active compounds

5162 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Meier et al.
formed BVDUMP amounts from both triesters 3a ,g
were found to be markedly lower as compared to the
prototype triesters 2c,d . In addition, both led also to
the formation of 3′-O-acyl-BVDUMP in (nearly) compa-
rable amounts. Compounds bearing branched or highly
lipophilic ester groups led dominantly to the corre-
sponding 3′-O-acyl-BVDUMP derivatives or proved to
be hydrolytically too stable in order to release BVDUMP
in a reasonable amount (Table 2, <7% BVDUMP in 8
h).
2 provide an efficient method to deliver the nucleoside
monophosphate BVDUMP intracellularly. The delivery
mechanism is the same as reported before, e. g., for
cycloSal-d4TMP,21a and thus proving that an intramo-
lecular concurrent reaction of the secondary alcohol did
not take place. Moreover, as a consequence, the anti-
EBV inactive BVDU 1 was converted into a bioactive
compound. The anti-EBV potency was found to be
comparable or even higher than that of ACV. The point
that the delivery of BVDUMP is sufficient to convert
BVDU into a bioactive compound showed that the
mechanism of action of the cycloSal compounds is based
again on a successful bypass of a cellular or of a EBV
TK. The antiviral evaluation proved that esterification
of the 3′-hydroxy group by simple carboxylic acids
abolished all biological activity for unknown reasons.
Although delivery of BVDUMP from 3′-aminoacyl cy-
cloSal-BVDUMPs 4 was clearly shown in chemical
hydrolysis and cell extract studies, only a few proved
to be antivirally active. The most active compound was
the L-leucine derivative 4f. The advantage of the R-ami-
no acid bearing compounds was their higher solubility
in aqueous media. Finally, to the best of our knowledge,
the work reported here represents the first example of
the application of a pronucleotide approach to a nucleo-
side analogue possessing a 3′-hydroxyl group with the
result of a considerable improvement of antiviral activ-
ity.
Quite intriguing was the behavior of the R-aminoacyl
bearing triesters 4. Although all compounds completely
lost their aminoacyl residue in the cell extracts to yield
the prototype triester 2d , only four triesters showed
some antiviral effect in the assay. The most active
compound was 3-methyl-cycloSal-(3′-Leu)BVDUMP 4f
having the natural L-configuration. However, this com-
pound was found to be 5-fold less active than the
prototype 2d (Table 3). In contrast, the corresponding
D-Leu triester 4g was completely inactive. To some
extend this is also valid for the 3′-Ala triesters 4b and
4c: here both L- or the D-configured triesters showed
some antiviral potential. Moreover, the glycine and the
D-Phe derivatives 4a and 4k showed some anti-EBV
activity although 10-fold lower as compared to triester
2d . It is interesting to note that the L-Phe triester 4j
was devoid of any antiviral potency. The reason for this
unexpected effect of the stereochemistry remains un-
known and cannot be correlated with the results of the
chemical hydrolysis and the cell extract studies. The
same stands for the reasons for the entire inactivity of
both diastereomers of the amino acids Val, Ile, and Pro.
Assuming that the results obtained from the cell extract
studies reflect at least somehow the intracellular me-
dium, one explanation may be a strong impact of the
attached amino acid on the cellular uptake (active
instead of passive?) of triesters 4. Further studies have
to be done in order to shed light on this unexpected
outcome of the antiviral tests. Moreover, it is interesting
to note that the aminoacyl derivatives 4 showed 2- to
3-fold lower CC50 values as compared to the prototype
triesters 2 for some unknown reasons.
Although it is known that EBV has a thymidine
kinase, the above data show that just the delivery of
BVDUMP is sufficient to convert BVDU into an anti-
EBV active drug. Consequently, EBV-TK is obviously
not able to phosphorylate BVDU. It cannot be deduced
from the data, if further metabolism of BVDUMP into
its triphosphate is absolutely necessary for the biological
activity. Possibly, BVDUMP itself is responsible for the
biological effect. Moreover, the complete inactivity of the
3′-ester derivatives in contrast to the prototype triesters
2 demonstrates again that a free 3′-hydroxyl group is
essential for the expression of the antiviral activity. This
conclusion is further confirmed by the complete inactiv-
ity of 3-methyl-cycloSal-(3′-O-methyl)BVDUMP (data
not shown).28 As expected, this triester released the 3′-
O-methyl ether of BVDUMP in the chemical hydrolysis
with a half-life of 6.5 h. However, the ether linkage
proved to be entirely stable.
Exp er im en ta l Section
All experiments involving water-sensitive compounds were
conducted under scrupulously dry conditions (argon atmo-
sphere). Solvents: Anhydrous methylene chloride (CH2Cl2),
anhydrous tetrahydrofurane (THF), and anhydrous acetoni-
trile (CH3CN) were obtained in a Sure/Seal bottle from Fluka
and stored over 4 Å molecular sieves; ethyl acetate, methylene
chloride, and methanol employed in chromatography were
distilled before use. Ethyldiisopropylamine (DIPEA) was dis-
tilled from Na prior to use. The solvents for the HPLC were
obtained from Merck (acetonitrile, HPLC grade). Ion pairing
buffer solution was prepared by mixing 6.6 mL of tetrabuty-
lammonium hydroxide with 1000 mL of water. The pH-value
adjusted to 3.8 by adding concentrated phosphoric acid (buffer
I). To 60 mL of buffer I solution was added 1000 mL of water
(buffer II). Evaporation of solvents was carried out on a rotary
evaporator under reduced pressure or using a high-vacuum
pump. Chromatography: Chromatotron (Harrison Research
7924), silica gel 60Pf (Merck, “gipshaltig”); UV detection at 254
nm. TLC: analytical thin-layer chromatography was per-
formed on Merck precoated aluminum plates 60 F254 with a
0.2-mm layer of silica gel containing a fluorescence indicator;
sugar-containing compounds were visualized with the sugar
spray reagent (0.5 mL of 4-methoxybenzaldehyde, 9 mL of
ethanol, 0.5 mL of concentrated sulfuric acid, and 0.1 mL of
glacial acectic acid) by heating with a fan or a hot plate.
HPLC: (Merck-Hitachi) analytical HPLC, LiChroCART 250-3
with LiChrospher 100 RP-18 endcapped (5 µm), gradient I 12-
80% CH3CN (0-20 min), 12% CH3CN (20-35 min), flow 0.6
mL, UV detection at 250 nm; gradient II 8-100% CH3CN (0-
22 min), 100% CH3CN (22-27 min), 8% CH3CN (27-33 min),
flow 0.6 mL, UV detection at 250 nm; gradient III same as II,
instead of water the ion pairing buffer solution was used. NMR
spectra were recorded using (1H NMR) Bruker AC 250 at 250
MHz, Bruker WM 400 at 400 MHz, Bruker AMX 400 at 400
MHz or Bruker DMX 500 at 500 MHz (CDCl3 or DMSO as
internal standard); (13C NMR) Bruker WM 400 at 101 MHz,
Bruker AMX 400 at 101 MHz or Bruker DMX 500 at 123 MHz
(CDCl3 or DMSO as internal standard); (31P NMR) Bruker
AMX 400 at 162 MHz or Bruker DMX 500 at 202 MHz (H3-
PO4 as external standard). All 1H and 13C NMR chemical shifts
Con clu sion
In summary, from the hydrolytic and antiviral data
disclosed here, the use of prototype cycloSal-BVDUMPs

CycloSal-BVDUMP Pronucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5163
(δ) are quoted in parts per million (ppm) downfield from
tetramethylsilane, (CD3)(CD2H)SO being set at δH 2.49 as a
reference. 31P NMR chemical shifts are quoted in ppm using
H3PO4 as external reference. The spectra were recorded at
room temperature, and all 13C and 31P NMR were recorded in
proton-decoupled mode. UV spectra were taken with a Varian
Cary 1E UV/Vis spectrometer. Infrared spectra were recorded
with a Perkin-Elmer 1600 Series FT-IR or a ATI Mattson
Genesis Series FT-IR spectrometer in KBr pellets. Mass
spectra were obtained with a Finnigan electrospray MAT 95
Trap XL (ESI) or a VG Analytical VG/70-250 F spectrometer
(FAB, matrix was m-nitrobenzyl alcohol). The test compounds
were isolated as mixtures of diastereomers arising from the
mixed stereochemistry at the phosphate center. The resulting
lyophilized triesters did not gave useful microanalytical data
most probably due to incomplete combustion of the compound,
but were found to be pure by HPLC analysis, high-field
multinuclear NMR spectroscopy, and mass spectroscopy.
5′-O-ter t-Bu t yld im et h ylsilyl-(E)-5-(2-b r om ovin yl)-2‘-
d eoxyu r id in e. To a solution of BVDU (3.00 mmol) in 25 mL
of pyridine was added TBDMSCl (3.75 mmol), and the mixture
was stirred for 14 h at room temperature. Then 2.0 mL MeOH
were added, and the solvent was removed under reduced
pressure. The residue was dissolved in CH2Cl2, washed twice
with water, and dried over sodium sulfate, and again the
solvent was removed under reduced pressure. The residues
were purified by chromatography on silica gel plates on a
chromatotron using a gradient of CH3OH in CH2Cl2. yield:
84%;1H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H, NH); 7.78
(s, 1H, H6); 7.26 (d, 1H, H8); 6.81 (d, 1H, H7); 6.10 (t, 1H,
H1′); 5.30 (d, 1H, OH); 4.19-4.17 (m, 1H, H3′); 3.76-3.74 (m,
3H, H4′, H5′); 2.20-2.09 (m, 2H, H2′); 0.86 (s, 9H, H3-
TBDMS); 0.05 (s, 6H, H1-TBDMS); 13C NMR (101 MHz,
DMSO-d6) δ 161.86 (C4); 149.41 (C2); 139.43 (C6); 130.09 (C7);
109.91 (C5); 107.11 (C8); 87.38 (C4′); 84.98 (C1′); 70.39 (C3′);
63.34 (C2′); 26.01 (C3-TBDMS); 18.27 (C2-TBDMS); -5.15 (C1-
TBDMS); MS (FAB) m/z 447.2 (M + H+); UV (CH3CN) λmax
251.0 nm, 295.0 nm; λmin 216.0 nm, 270.0 nm; Rf value 0.53
(CH2Cl2/MeOH, 9:1).
Gen er a l P r oced u r e for th e P r ep a r a tion of 3′-Ester i-
fied BVDUs 5a -t. To a solution of 5′-TBDMS-BVDU (0.33
mmol), (dimethylamino)pyridine (0.66 mmol), and the car-
boxylic acid (0.37 mmol) in 5 mL of CH2Cl2 was added
dicyclohexylcarbodiimide (0.37 mmol) at room temperature.
The reaction mixture was stirred for 30 min (TLC analysis)
and filtered, and the solvent was removed under reduced
pressure. The residues were purified by chromatography on
silica gel plates on a chromatotron using a gradient of MeOH
in CH2Cl2. Product containing fractions were combined and
evaporated. To a solution of the residue in THF was added
0.5 mL of a 1 M tetrabutylammonium fluoride solution in THF.
The mixture was stirred for 4 h at room temperature. Then,
the solvent was removed under reduced pressure. The residues
were purified by chromatography on silica gel plates on a
chromatotron using a gradient of MeOH in CH2Cl2 to yield
compounds 5a -t.
(E)-5-(2-Br om ovin yl)-3′-O-a cet yl-2′-d eoxyu r id in e 5a :
yield: 87%; 1H NMR (250 MHz, CDCl3) δ 8.52 (s, 1H, NH);
7.90 (s, 1H, H6); 7.38 (d, 1H, H8); 6.68 (d, 1H, H7); 6.32 (dd,
1H, H1′); 5.39-5.33 (m, 1H, H3′); 4.15 (dt, 1H, H4′); 3.99-
3.97 (m, 2H, H5′); 2.49 (ddd, 1H, H2′′); 2.35 (ddd, 1H, H2′);
2.12 (s, 3H, CH3); 13C NMR (63 MHz, CDCl3) 170.69 (C1-Ac);
160.86 (C4); 149.01 (C2); 137.94 (C6); 128.23 (C7); 111.72 (C5);
109.98 (C8); 85.95 (C4′); 85.34 (C1′); 74.54 (C3′); 62.55 (C5′);
38.00 (C2′); 20.97 (C2-Ac); UV (CH3CN) λmax 251.0 nm, 287.0
nm; λmin 214.0 nm; 268.0 nm; Rf value 0.66 (CH2Cl2/MeOH,
9:1).
107.07 (C8); 85.27 (C4′); 84.71 (C1′); 74.63 (C3′); 61.42 (C5′);
37.30 (C2′); 27.03 (C2-Prop); 9.03 (C3-Prop); Rf value 0.59 (CH2-
Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-i-b u t yr yl-2′-d eoxyu r id in e
5c: yield 98%; 1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H, H6);
7.24 (d, 1H, H8); 6.66 (d, 1H, H7); 6.25 (dd, 1H, H1′); 5.24-
5.22 (m, 1H, H3′); 4.01 (dt, H4′); 3.80-3.78 (m, 2H, H5′); 2.49
(sept, 1H, H2-Iso); 2.35 (ddd, 1H, H2′′); 2.23 (ddd, 1H, H2′);
1.10 (d, 6H, CH3); 13C NMR (101 MHz, CDCl3) δ 177.01 (C1-
Iso); 161.81 (C4); 149.49 (C2); 138.07 (C6); 128.34 (C7); 111.53
(C5); 108.94 (C8); 85.69 (C4′); 85.25 (C1′); 74.85 (C3′); 61.73
(C5′); 38.07 (C2′); 33.69 (C2-Iso); 18.52 (C3-Iso); 18.50 (C3-Iso).
Rf value 0.51 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-p iva loyl-2′-d eoxyu r id in e 5d :
yield 77%; 1H NMR (250 MHz, CDCl3) δ 9.12 (s, 1H, NH-BVU);
7.93 (s, 1H, H6); 7.39; 7.33 (d, 1H, H8); 6.71-6.66 (d, 1H, H7);
6.31 (dd, 1H, H1′); 5.37-5.32 (m, 1H, H3′); 4.08 (dt, H4′); 3.98
(m, 2H, H5′); 2.49 (ddd, 1H, H2′′); 2.37 (ddd, 1H, H2′); 1.23 (s,
9H, 3x CH3); 13C NMR (63 MHz, CDCl3) δ 178.44 (C1-Piv);
161.26 (C4); 149.20 (C2); 138.08 (C6); 128.23 (C7); 111.69 (C5);
109.89 (C8); 85.96 (C4′); 85.60 (C1′); 74.32 (C3′); 62.49 (C5′);
38.68 (C2-Piv); 38.00 (C2′); 26.99 (C3-Piv); Rf value 0.68
(CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-h exa n oyl-2′-d eoxyu r id in e
1
5e: yield 65%; H NMR (500 MHz, DMSO-d6) δ 11.60 (s, 1H,
NH-BVU); 8.06 (s, 1H, H6); 7.24 (d, 1H, H8); 6.83 (d, 1H, H7);
6.14 (t, 1H, H1′); 5.22 (t, 2H, H3′, OH); 3.99 (s, 1H, H4′); 3.68-
3.61 (m, 2H, H5′); 2.34-2.26 (m, 4H, H2′, H2-Hex); 1.54 (quin,
2H, H3-Hex); 1.28-1.25 (m, 4H, H4-Hex, H5-Hex); 0.85 (t, 3H,
H6-Hex); 13C NMR (101 MHz, DMSO-d6) δ 172.75 (C1-Hex);
161.80 (C4); 149.47 (C2); 139.33 (C6); 129.96 (C7); 110.21 (C5);
107.00 (C8); 85.19 (C4′); 84.64 (C1′); 74.52 (C3′); 61.37 (C5′);
37.24 (C2′); 33.58 (C2-Hex); 30.79 (C4-Hex); 24.19 (C3-Hex);
21.96 (C5-Hex); 13.97 (C6-Hex); Rf value 0.68 (CH2Cl2/MeOH,
9:1).
(E)-5-(2-Br om ovin yl)-3′-O-d eca n oyl-2‘-d eoxyu r id in e
1
5f: yield 87%; H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H,
NH-BVU); 8.06 (s, 1H, H6); 7.24 (d, 1H, H8); 6.83 (d, 1H, H7);
6.14 (dd, 1H, H1′); 5.25-5.20 (m, 2H, H3′,OH); 3.99 (dt, 1H,
H4′); 3.70-3.57 (m, 2H, H5′); 2.37-2.22 (m, 4H, H2′, H2-Dec);
1.55-1.45 (m, 2H, H3-Dec); 1.30-1.20 (m, 12H, H4-Dec-H9-
Dec); 0.85 (t, 3H, H10-Dec); 13C NMR (101 MHz, DMSO-d6) δ
172.78 (C1-Dec); 161.83 (C4); 149.50 (C2); 139.35 (C6); 129.98
(C7); 110.24 (C5); 107.03 (C8); 85.23 (C4′); 84.67 (C1′); 74.54
(C3′); 61.39 (C5′); 37.26 (C2′); 33.64 (C2-Dec); 31.47 (C3-Dec);
29.04; 28.85; 28.61 (C4-Dec-C8-Dec); 22.31 (C9-Dec); 14.15
(C10-Dec); Rf value 0.69 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-levu lin yl-2′-d eoxyu r id in e
1
5g: yield 88%; H NMR (500 MHz, DMSO-d6) δ 8.06 (s, 1H,
H6); 7.24 (d, 1H, H8); 6.83 (d, 1H, H7); 6.13 (dd, 1H, H1′); 5.22
(s, 1H, OH); 5.19 (dt, 1H, H3′); 3.98 (dt, 1H, H4′); 3.61 (m, 2H,
H5′); 2.72 (t, 2H, H2-Lev); 2.48 (m, 2H, H3-Lev); 2.31 (ddd,
1H, H2′′); 2.25 (ddd, 1H, H2′); 2.09 (s, 3H, H5-Lev); 13C NMR
(101 MHz, DMSO-d6) δ 207.08 (C4-Lev); 172.17 (C1-Lev);
161.81 (C4); 149.49 (C2); 139.35 (C6); 129.95 (C7); 110.21 (C5);
106.99 (C8); 85.09 (C4′); 84.65 (C1′); 74.75 (C3′); 61.36 (C5′);
37.62 (C3-Lev); 37.11 (C2′); 29.70 (C5-Lev); 27.92 (C2-Lev); Rf
value 0.44 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-glycin yl)-2′-d eoxyu r i-
d in e 5h : yield 63%; 1H NMR (400 MHz, DMSO-d6) δ 8.06 (s,
1H, H6); 7.24 (d, 2H, H8, NH-Gly); 6.83 (d, 1H, H7); 6.16 (t,
1H, H1′); 5.35-5.10 (m, 2H, H3′, OH); 4.00 (s, 1H, H4′); 3.75-
3.55 (m, 4H, H5′, H2-Gly); 2.36-2.23 (m, 2H, H2′); 1.38 (s,
9H, H3-Boc); 13C NMR (101 MHz, DMSO-d6) δ 170.21 (C1-
Gly); 161.82 (C4); 156.09 (C1-Boc); 149.48 (C2); 139.32 (C6);
129.97 (C7); 110.25 (C5); 107.03 (C8); 85.12 (C4′); 84.62 (C1′);
78.54 (C2-Boc); 75.21 (C3′); 61.36 (C5′); 42.38 (C2-Gly); 37.16
(C2′); 28.33 (C3-Boc); Rf value 0.48 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-L-a la n in yl)-2′-d eoxy-
u r id in e 5i: yield 69%; 1H NMR (400 MHz, DMSO-d6) δ 11.62
(s, 1H, NH-BVU); 8.06 (s, 1H, H6); 7.37 (d, 1H, NH-Ala); 7.24
(d, 1H, H8); 6.83 (d, 1H, H7); 6.17 (t, 1H, H1′); 5.30-5.20 (m,
2H, H3′, OH); 4.02-3.95 (m, 2H, H4′, H2-Ala); 3.67-3.59 (m,
2H, H5′); 2.36-2.07 (m, 2H, H2′); 1.37 (s, 9H, H3-Boc); 1.24
(E)-5-(2-Br om ovin yl)-3′-O-p r op ion yl-2′-d eoxyu r id in e
1
5b: yield 97%; H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H,
H6); 7.24 (d, 1H, H8); 6.83 (d, 1H, H7); 6.14 (dd, 1H, H1′);
5.35-5.10 (m, 2H, H3′, OH); 4.00 (s, 1H, H4′); 3.66-3.58 (m,
2H, H5′); 2.35-2.27 (m, 4H, H2′, H2-Prop); 1.02 (t, 3H, H3-
Prop); 13C NMR (101 MHz, DMSO-d6) δ 173.52 (C1-Prop);
161.84 (C4); 149.52 (C2); 139.36 (C6); 129.98 (C7); 110.26 (C5);

5164 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Meier et al.
(d, 3H, H3-Ala); 13C NMR (101 MHz, DMSO-d6) δ 172.89 (C1-
Ala); 161.80 (C4); 155.53 (C1-Boc); 149.46 (C2); 139.32 (C6);
129.95 (C7); 110.24 (C5); 107.00 (C8); 85.18 (C4′); 84.54 (C1′);
78.46 (C2-Boc); 75.12 (C3′); 61.38 (C5′); 49.44 (C2-Ala); 37.06
(C2′); 28.31 (C3-Boc); 16.70 (C3-Ala); Rf value 0.55 (CH2Cl2/
MeOH, 9:1).
(E)-5-(2-Bromovinyl)-3′-O-(N-Boc-D-alaninester)-2′-deoxy-
u r id in e 5j: yield 60%; 1H NMR (400 MHz, DMSO-d6) δ 11.62
(s, 1H, NH-BVU); 8.06 (s, 1H, H6); 7.37 (d, 1H, NH-Ala); 7.24
(d, 1H, H8); 6.83 (d, 1H, H7); 6.17 (t, 1H, H1′); 5.26-5.21 (m,
2H, H3′, OH); 4.02-3.96 (m, 2H, H4′, H2-Ala); 3.68-3.59 (m,
2H, H5′); 2.36-2.20 (m, 2H, H2′); 1.37 (s, 9H, H3-Boc); 1.24
(d, 3H, H3-Ala); 13C NMR (101 MHz, DMSO-d6) δ 172.89 (C1-
Ala); 161.80 (C4); 155.53 (C1-Boc); 149.46 (C2); 139.32 (C6);
129.95 (C7); 110.24 (C5); 107.00 (C8); 85.18 (C4′); 84.54 (C1′);
78.46 (C2-Boc); 75.12 (C3′); 61.38 (C5′); 49.44 (C2-Ala); 37.06
(C2′); 28.31 (C3-Boc); 16.70 (C3-Ala); Rf value 0.57 (CH2Cl2/
MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-L-valin yl)-2′-deoxyu r i-
d in e 5k : yield 97%; 1H NMR (500 MHz, DMSO-d6) δ 9.64 (s,
1H, NH-BVU); 7.94 (s, 1H, H6); 7.33 (d, 1H, H8); 6.66 (d, 1H,
H7); 6.28 (dd, 1H, H1′); 5.43 (dt, 2H, H3′); 5.11 (d, 1H, OH);
4.19 (dd, 1H, H2-Val); 4.11 (dt, 1H, H4′); 3.94 (ddd, H5′); 3.93
(ddd, 1H, H5′′); 2.52 (ddd, 1H, H2′); 2.42 (ddd, 1H, H2′′); 2.18-
2.10 (m, 2H, H3-Val); 1.45 (s, 9H, H3-Boc); 0.99 (d, 3H, H4-
Val); 0.91 (d, 3H, H4′-Val); 13C NMR (126 MHz, DMSO-d6) δ
172.21 (C1-Val); 161.62 (C4); 155.81 (C1-Boc); 149.34 (C2);
138.26 (C6); 128.25 (C7); 111.59 (C5); 109.72 (C8); 86.07 (C1′);
85.43 (C4′); 80.23 (C2-Boc); 75.29 (C3′); 62.23 (C5′); 58.39 (C2-
Val); 37.89 (C2′); 30.83 (C3-Val); 28.25 (C3-Boc); 19.09; 17.62
(C4-Val). Rf value 0.56 (CH2Cl2/MeOH, 9:1).
H7); 6.28 (dd, 1H, H1′); 5.42 (m, 2H, H3′); 5.06 (d, 1H, NH-
Ile); 4.23 (dd, 1H, H2-Ile); 4.12 (m, 1H, H4′); 3.95 (m, 2H, H5′);
2.45 (m, 2H, H2′); 1.88 (m, 1H, H3-Ile); 1.45 (s, 9H, H3-Boc);
1.17 (m, 1H, H5-Ile); 0.95 (m, 6H, H4-Ile, H6-Ile); 13C NMR
(101 MHz, CDCl3) δ 172.199 (C1-Ile); 161.36 (C4); 155.68 (C1-
Boc); 149.20 (C2); 138.19 (C6); 128.25 (C7); 111.63 (C5); 109.84
(C8); 86.15 (C1′); 85.34 (C4′); 80.24 (C2-Boc); 75.18 (C3′); 62.30
(C5′); 58.06 (C2-Ile); 37.89 (C2′); 37.53 (C3-Ile); 28.28 (C3-Boc);
25.16 (C5-Ile); 15.69 (C6-Ile); 11.57 (C4-Ile). Rf value 0.60 (CH2-
Cl2/MeOH, 9:1)
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-D-isoleu cin yl)-2′-deoxy-
u r id in e 5p : yield 99%; 1H NMR (400 MHz, CDCl3) δ 9.30 (s,
1H, NH-BVU); 7.94 (s, 1H, H6); 7.35 (d, 1H, H8); 6.67 (d, 1H,
H7); 6.28 (dd, 1H, H1′); 5.42 (m, 2H, H3′); 5.08 (d, 1H, NH-
Ile); 4.25 (dd, 1H, H2-Ile); 4.17 (m, 1H, H4′); 3.97 (m, 2H, H5′);
2.48 (m, 2H, H2′); 1.88 (m, 1H, H3-Ile); 1.45 (s, 9H, H3-Boc);
1.17 (m, 1H, H5-Ile); 0.95 (m, 6H, H4-Ile, H6-Ile); 13C NMR
(101 MHz, CDCl3) δ 172.40 (C1-Ile); 161.44 (C4); 155.70 (C1-
Boc); 149.21 (C2); 138.13 (C6); 128.27 (C7); 111.57 (C5); 109.80
(C8); 85.93 (C1′); 85.26 (C4′); 80.21 (C2-Boc); 74.95 (C3′); 62.16
(C5′); 58.01 (C2-Ile); 38.03 (C2′); 37.63 (C3-Ile); 28.28 (C3-Boc);
25.10 (C5-Ile); 15.65 (C6-Ile); 11.56 (C4-Ile); Rf value 0.60 (CH2-
Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-L-p h en yla la n in yl)-2′-
d eoxyu r id in e 5q: yield 54%; 1H NMR (400 MHz, DMSO-d6)
δ 8.05 (s, 1H, H6); 7.42 (d, 1H, NH-Phe); 7.25 (m, 6H, H8,
H-aryl-Phe); 6.83 (d, 1H, H7); 6.12 (t, 1H, H1′); 5.35-5.10 (m,
2H, H3′, OH); 4.19-4.12 (m, 1H, H2-Phe); 3.80-3.75 (m, 1H,
H4′); 3.58 (ddd, 1H, H5′); 3.59 (ddd, 1H, H5′); 2.32-2.21 (m,
2H, H2′); 1.34 (s, 9H, H3-Boc); 13C NMR (101 MHz, DMSO-
d6) δ 172.21 (C1-Phe); 161.79 (C4); 155.65 (C1-Boc); 149.44
(C2); 139.31 (C6); 137.52 (C4-Phe); 129.95 (C7); 129.31 (C5-
Phe); 128.41 (C4-Phe); 126.70 (C6-Phe); 110.23 (C5); 106.99
(C8); 85.01 (C4′); 84.52 (C1′); 78.64 (C2-Boc); 75.23 (C3′); 61.35
(C5′); 55.68 (C2-Phe); 37.15 (C2′, C3-Phe); 28.26 (C3-Boc); Rf
value 0.66 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-D-valin yl)-2′-deoxyu r i-
1
d in e 5l: yield 97%; H NMR (500 MHz, DMSO-d6) δ 9.00 (s,
1H, NH-BVU); 7.92 (s, 1H, H6); 7.35 (d, 1H, H8); 6.69 (d, 1H,
H7); 6.28 (dd, 1H, H1′); 5.43-5.40 (m, 2H, H3′); 5.07-5.00 (m,
1H, OH); 4.21-4.16 (m, 2H, H4′, H2-Val); 4.01-3.93 (m, 2H,
H5′); 2.48 (ddd, 1H, H2′); 2.41 (ddd, 1H, H2′′); 2.18-2.10 (m,
2H, H3-Val); 1.46 (s, 9H, H3-Boc); 1.00 (d, 3H, H4-Val); 0.93
(d, 3H, H4′-Val); 13C NMR (126 MHz, DMSO-d6) δ 172.40 (C1-
Val); 161.36 (C4); 155.79 (C1-Boc); 149.21 (C2); 138.02 (C6);
128.28 (C7); 111.63 (C5); 109.81 (C8); 85.97 (C1′); 85.29 (C4′);
80.21 (C2-Boc); 74.98 (C3′); 62.17 (C5′); 58.69 (C2-Val); 38.04
(C2′); 30.94 (C3-Val); 28.28 (C3-Boc); 19.09; 17.64 (C4-Val); Rf
value 0.64 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-D-p h en yla la n in yl)-2′-
d eoxyu r id in e 5r : yield 99%; 1H NMR (400 MHz, DMSO-d6)
δ 9.25 (s, 1H, NH-BVU); 7.79 (s, 1H, H6); 7.27 (m, 6H, H8,
H-aryl-Phe); 6.83 (d, 1H, H7); 6.06 (t, 1H, H1′); 5.25-5.15 (m,
2H, H3′); 5.05-4.92 (m, 1H, OH); 4.43 (dt, 1H, H2-Phe); 3.98-
3.95 (m, 1H, H4′); 3.86-3.74 (m, 2H, H5′); 2.25-2.08 (m, 2H,
H2′); 1.32 (s, 9H, H3-Boc); 13C NMR (101 MHz, DMSO-d6) δ
171.92 (C1-Phe); 161.38 (C4); 155.15 (C1-Boc); 149.19 (C2);
138.21 (C6); 135.60 (C4-Phe); 129.18 (C7); 128.73 (C5Ph3);
128.26 (C4-Phe); 127.30 (C6-Phe); 111.59 (C5); 109.80 (C8);
86.04 (C1′); 85.19 (C4′); 80.39 (C2-Boc); 75.22 (C3′); 62.15 (C5′);
54.63 (C2-Phe); 38.24 (C2′); 37.73 (C3-Phe); 28.23 (C3-Boc);
Rf value 0.76 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-L-leu cin yl)-2′-d eoxy-
u r id in e 5m : yield 94%; 1H NMR (500 MHz, DMSO-d6) δ 9.38
(s, 1H, NH-BVU); 7.94 (s, 1H, H6); 7.34 (d, 1H, H8); 6.83 (d,
1H, H7); 6.30 (dd, 1H, H1′); 5.45-5.40 (m, 2H, H3′); 4.98 (s,
1H, OH); 4.43 (dt, 1H, H2-Leu); 4.15-4.05 (m, 1H, H4′); 3.98-
3.87 (m, 2H, H5′); 2.55-2.35 (m, 2H, H2′); 1.80-1.66 (m, 1H,
H4-Leu); 1.65-1.49 (m, 2H, H3-Leu); 1.45 (s, 9H, H3-Boc); 0.96
(d, 6H, H5-Leu); 13C NMR (126 MHz, DMSO-d6) δ 173.29 (C1-
Leu); 161.38 (C4); 155.57 (C1-Boc); 149.28 (C2); 138.25 (C6);
128.26 (C7); 111.65 (C5); 109.78 (C8); 86.09 (C1′); 85.42 (C4′);
80.29 (C2-Boc); 75.29 (C3′); 62.32 (C5′); 52.30 (C2-Leu); 40.98
(C3-Leu); 37.86 (C2′); 28.27 (C3-Boc); 24.84 (C4-Leu); 22.84;
21.67 (C5-Leu). Rf value 0.42 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-D-leu cin yl)-2′-d eoxy-
u r id in e 5n : yield 99%; 1H NMR (500 MHz, DMSO-d6) δ 9.22
(s, 1H, NH-BVU); 7.94 (s, 1H, H6); 7.35 (d, 1H, H8); 6.68 (d,
1H, H7); 6.29 (dd, 1H, H1′); 5.45-5.40 (m, 2H, H3′); 4.98 (d,
1H, OH); 4.27 (m, 1H, H2-Leu); 4.19-4.13 (m, 1H, H4′); 4.00-
3.92 (m, 2H, H5′); 2.49 (ddd, 1H, H2′); 2.39 (ddd, 1H, H2′′);
1.76-1.68 (m, 1H, H4-Leu); 1.64-1.50 (m, 2H, H3-Leu); 1.44
(s, 9H, H3-Boc); 0.97 (d, 6H, H4-Leu); 13C NMR (126 MHz,
DMSO-d6) δ 173.50 (C1-Leu); 161.40 (C4); 155.55 (C1-Boc);
149.22 (C2); 138.14 (C6); 128.27 (C7); 111.61 (C5); 109.81 (C8);
85.97 (C1′); 85.32 (C4′); 80.27 (C2-Boc); 74.97 (C3′); 62.20 (C5′);
52.25 (C2-Leu); 41.07 (C3-Leu); 37.99 (C2′); 28.28 (C3-Boc);
24.86 (C4-Leu); 22.83; 21.73 (C5-Leu); Rf value 0.42 (CH2Cl2/
MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-L-p r olin yl)-2′-d eoxy-
u r id in e 5s: yield 94%; 1H NMR (400 MHz, CDCl3) δ 9.00 (s,
1H, NH-BVU); 7.92 (s, 1H, H6); 7.35 (d, 1H, H8); 6.69 (d, 1H,
H7); 6.30 (m, 1H, H1′); 5.42 (m, 1H, H3′); 4.32 (dd, 1H, H2-
Pro); 4.25 (m, 1H, H4′); 3.95 (m, 2H, H5′); 3.50 (m, 2H, H5-
Pro); 2.45 (m, 2H, H2′); 1.94 (m, 4H, H3-Pro, H4-Pro); 1.46 (s,
9H, H3-Boc); 13C NMR (101 MHz, CDCl3) δ 172.77 (C1-Pro);
161.36 (C4); 154.53 (C1-Boc); 149.21 (C2); 138.25 (C6); 128.26
(C7); 111.63 (C5); 109.75 (C8); 85.91 (C1′); 85.50 (C4′); 80.31
(C2-Boc); 75.27 (C3′); 62.40 (C5′); 58.93 (C2-Pro); 37.93 (C2′);
30.88 (C3-Pro); 29.89 (C5-Pro); 28.36 (C3-Boc); 24.50 (C4-Pro);
Rf value 0.71 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-D-p r olin yl)-2′-d eoxy-
u r id in e, 5t: yield 92%; 1H NMR (400 MHz, CDCl3) δ 9.00 (s,
1H, NH-BVU); 7.92 (s, 1H, H6); 7.35 (d, 1H, H8); 6.69 (d, 1H,
H7); 6.30 (m, 1H, H1′); 5.42 (m, 1H, H3′); 4.30 (dd, 1H, H2-
Pro); 4.25 (m, 1H, H4′); 3.95 (m, 2H, H5′); 3.50 (m, 2H, H5-
Pro); 2.45 (m, 2H, H2′); 1.91 (m, 4H, H3-Pro, H4-Pro); 1.46 (s,
9H, H3-Boc); 13C NMR (101 MHz, CDCl3) δ 172.77 (C1-Pro);
161.36 (C4); 154.53 (C1-Boc); 149.21 (C2); 138.25 (C6); 128.21
(C7); 111.73 (C5); 109.71 (C8); 85.89 (C1′); 85.55 (C4′); 80.31
(C2-Boc); 75.27 (C3′); 62.43 (C5′); 58.93 (C2-Pro); 37.93 (C2′);
30.88 (C3-Pro); 29.89 (C5-Pro); 28.36 (C3-Boc); 24.50 (C4-Pro);
Rf value 0.71 (CH2Cl2/MeOH, 9:1).
(E)-5-(2-Br om ovin yl)-3′-O-(N-Boc-L-isoleu cin yl)-2′-deoxy-
u r id in e 5o: yield 99%; 1H NMR (400 MHz, CDCl3) δ 9.30 (s,
1H, NH-BVU); 7.92 (s, 1H, H6); 7.35 (d, 1H, H8); 6.67 (d, 1H,

CycloSal-BVDUMP Pronucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5165
Gen er a l P r oced u r e for th e P r ep a r a tion of th e cy-
cloSa l-(3′-O-Lev)BVDUMP s 6a -f. To a solution of 3′-O-
levulinyl BVDU 5g (0.28 mmol) in 10 mL of CH3CN was added
diisopropylethylamine (0.56 mmol, DIPEA), and the mixture
was cooled to -20 °C. The chlorophosphanes (0.56 mmol) were
added slowly, and the solution was stirred for 20 min to
complete the reaction (TLC analysis). For the oxidation, tert-
butyl hydroperoxide (0.56 mmol) was added to the reaction
mixture at -20 °C. After being stirred for 0.5 h, the reaction
mixture was warmed to room temperature, and the solvent
was removed under reduced pressure. The reaction mixtures
were purified twice by chromatotron chromatography on silica
gel plates using first a gradient of CH3OH in ethyl acetate
followed by a gradient of CH3OH in CH2Cl2 to yield cycloSal-
3′-O-Lev-BVDUMP triesters 6.
(d, C2-aryl); 143.42 (d, C2-aryl); 137.08 (C4-aryl); 128.09 (C7);
121.47 (d, C1-aryl); 121.32 (d, C1-aryl); 119.39 (C3-aryl); 119.30
(C3-aryl); 111.89 (C8); 111.82 (C8); 110.41 (C5-aryl); 110.29
(C5); 110.16 (C5); 85.27 (C1′), 85.23 (C1′); 83.10 (d, C4′); 83.04
(d, C4′); 74.35 (C3′); 74.22 (C3′), 68.75 (d, C5′); 68.63 (d, C5′);
67.96 (d, C-benzyl); 67.79 (d, C-benzyl); 55.73 (OMe); 37.79
(C2′); 37.72 (C3-Lev); 37.70 (C2′); 29.69 (C5-Lev); 27.76 (C2-
Lev); 31P NMR (202 MHz, CDCl3) δ -7.24; -7.36; Rf value 0.61
(CH2Cl2/MeOH, 9:1).
cyclo(3,5-Dim eth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-
3′-O-levu lin yl-2′-d esoxyu r id in yl)p h osp h a te (3,5-DiMe-
1
cycloSa l-3′-O-Lev-BVDUMP ) 6e: yield 49%; H NMR (400
MHz, CDCl3) δ 9.41 (s, 2H, NH-BVU); 7.75 (s, 1H, H6); 7.74
(s, 1H, H6); 7.45 (d, 1H, H8); 7.43 (d, 1H, H8); 7.00 (s, 2H,
H4-aryl); 6.75 (m, 2H, H6-aryl); 6.72 (d, 1H, H7); 6.71 (d, 1H,
H7); 6.32 (dd, 1H, H1′); 6.30 (dd, 1H, H1′); 5.46-5.19 (m, 6H,
H-benzyl, H3′); 4.53-4.38 (m, 4H, H5′); 4.23 (dt, 1H, H4′); 4.21
(dt, 1H, H4′); 2.83-2.71 (m, 4H, H2-Lev); 2.61-2.55 (m, 4H,
H3-Lev); 2.28 (s, 6H, H5-Lev); 2.23 (s, 6H, CH3-C5-aryl); 2.19
(s, 6H, CH3-C3-aryl); 2.22-2.00 (m, 4H, H2′); 13C NMR (101
MHz, CDCl3) δ 206.37 (C4-Lev); 175.71 (C1-aryl); 172.39 (C1-
Lev); 172.37 (C1-Lev); 161.41 (C4); 149.10 (C2); 136.98 (C6);
136.96 (C6); 134.03 (C5-aryl); 133.97 (C5-aryl); 132.18 (C4-
aryl); 132.14 (C4-aryl); 128.07 (C7); 127.42 (d, C3-aryl); 127.34
(d, C3-aryl); 123.33 (C6-aryl); 120.25 (d, C2-aryl); 120.18 (d,
C2-aryl); 111.99 (C8); 111.92 (C8); 110.37 (C5); 110.28 (C5);
85.32 (C4′), 85.20 (C4′); 83.16 (C1′); 83.10 (C1′); 74.47 (C3′);
74.36 (C3′), 68.87 (d, C5′); 68.81 (d, C5′); 67.89 (d, C-benzyl);
67.67 (d, C-benzyl); 37.80 (C2′); 37.74 (C3-Lev); 37.70 (C2′);
29.70 (C5-Lev); 27.78 (C4-Lev); 20.61 (CH3-C5-aryl); 20.57
(CH3-C5-aryl); 15.31 (CH3-C3-aryl); 15.26 (CH3-C3-aryl); 31P
NMR (202 MHz, CDCl3) δ -5.61; -5.91; Rf value 0.49 (CH2-
Cl2/MeOH, 9:1).
cyclo(3-tBu tylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-3′-O-
levu lin yl-2′-d eoxyu r id in yl)p h osp h a te (3-tBu -cycloSa l-3′-
O-Lev-BVDUMP ) 6f: yield 50%; 1H NMR (400 MHz, CDCl3)
δ 9.98 (s, 2H, NH-BVU); 7.78 (s, 2H, H6); 7.43 (d, 1H, H8);
7.42 (d, 1H, H8); 7.35 (d, 2H, H4-aryl); 7.10 (dd, 1H, H5-aryl);
7.00 (d, 2H, H6-aryl); 6.72 (d, 1H, H7); 6.72 (d, 1H, H7); 6.31
(dd, 1H, H1′); 6.30 (dd, 1H, H1′); 5.46-5.23 (m, 6H, H-benzyl,
H3′); 4.59-4.39 (m, 4H, H5′); 4.22 (m, 2H, H4′); 2.84-2.70 (m,
4H, H2-Lev); 2.61-2.55 (m, 4H, H3-Lev); 2.18 (s, 6H, H5-Lev);
2.22-2.00 (m, 4H, H2′); 1.39 (s, 9H, CH3-tBu); 1.38 (s, 9H, CH3-
tBu); 13C NMR (101 MHz, CDCl3) δ 206.49 (C4-Lev); 206.46
(C4-Lev); 176.36 (C1-aryl); 172.36 (C1-Lev); 161.88 (C4); 161.85
(C4); 149.24 (C2); 139.49 (C); 139.42 (C); 137.17 (C6); 137.11
(C6); 128.02 (C7); 127.89 (C7); 124.46 (C); 124.38 (C); 123.81
(C6-aryl); 123.79 (C6-aryl); 122.10 (d, C2-aryl); 122.01 (d, C2-
aryl); 111.93 (C8); 111.89 (C8); 110.35 (C5); 110.24 (C5); 85.34
(C4′), 85.04 (C4′); 83.11 (C1′); 83.01 (C1′); 74.29 (C3′); 74.23
(C3′), 68.80 (d, C5′); 68.73 (d, C5′); 68.19 (d, C-benzyl); 67.88
(d, C-benzyl); 53.39 (C7-aryl); 37.71 (C2′); 37.70 (C3-Lev); 37.63
(C2′); 29.75 (C8-aryl); 27.74 (C2-Lev); 20.69 (C5-Lev); 31P NMR
(202 MHz, CDCl3) δ -6.67; -7.21; Rf value 0.67 (CH2Cl2/
MeOH, 9:1).
cyclo(5-Ch lor osa ligen yl)-5′-O-(E)-5-(2-b r om ovin yl)-3′-
O-levu lin yl-2′-d eoxyu r id in yl)p h osp h a t e (5-Cl-cycloSa l-
3′-O-Lev-BVDUMP ) 6a : yield 54%; 1H NMR (500 MHz,
CDCl3) δ 9.02 (s, 2H, NH-BVU); 7.72 (s, 2H, H6); 7.45 (d, 1H,
H8); 7.43 (d, 1H, H8); 7.33 (s, 1H, H4-aryl); 7.31 (s, 1H, H4-
aryl); 7.14 (d, 1H, H6-aryl); 7.13 (d, 1H, H6-aryl); 7.04 (d, 1H,
H3-aryl); 7.03 (d, 1H, H3-aryl); 6.75 (d, 1H, H7); 6.71 (d, 1H,
H7); 6.31 (dd, 1H, H1′); 6.29 (dd, 1H, H1′); 5.47-5.27 (m, 6H,
H-benzyl, H3′); 4.56-4.45 (m, 4H, H5′); 4.23 (m, 2H, H4′);
2.84-2.72 (m, 4H, H2-Lev); 2.63-2.51 (m, 4H, H3-Lev); 2.21
(s, 3H, H5-Lev); 2.20 (s, 3H, H5-Lev); 2.14-2.05 (m, 4H, H2′);
13C NMR (101 MHz, CDCl3) δ 206.47 (C4-Lev); 172.41 (C1-
Lev); 172.38 (C1-Lev); 161.67 (C4); 161.63 (C4); 156.27 (C5-
aryl); 156.24 (C5-aryl); 149.18 (C2); 143.50 (C2-aryl); 143.42
(C2-aryl); 137.08 (C4-aryl); 128.09 (C7); 121.47 (C1-aryl);
121.32 (C1-aryl); 119.39 (C3-aryl); 119.30 (C3-aryl); 111.89
(C8); 111.82 (C8); 110.41 (C5aryl); 110.29 (C5); 110.16 (C5);
85.27 (C1′), 85.23 (C1′); 83.10 (C4′); 83.04 (C4′); 74.35 (C3′);
74.22 (C3′), 68.75 (C5′); 68.63 (C5′); 67.96 (d, C-benzyl); 67.79
(d, C-benzyl); 37.79 (C2′); 37.72 (C3-Lev); 37.70 (C2′); 29.69
(C5-Lev); 27.76 (C2-Lev); 31P NMR (202 MHz, CDCl3) δ -7.90;
-7.97; Rf value 0.53 (CH2Cl2/MeOH, 9:1).
cycloSa ligen yl-5′-O-(E)-5-(2-br om ovin yl)-3′-O-levu lin yl-
2′-deoxyu r idin yl)ph osph ate (cycloSal-3′-O-Lev-BVDUMP )
6b: yield 39%; 1H NMR (500 MHz, CDCl3) δ 8.75 (s, 1H, NH-
BVU); 8.74 (s, 1H, NH-BVU); 7.73 (s, 1H, H6); 7.72 (s, 1H,
H6); 7.46 (d, 1H, H8); 7.43 (d, 1H, H8); 7.35 (dd, 2H, H4-aryl);
7.19-7.08 (m, 6H, H3-aryl, H5-aryl, H6-aryl); 6.76 (d, 1H, H7);
6.69 (d, 1H, H7); 6.32 (dd, 1H, H1′); 6.30 (dd, 1H, H1′); 5.49
(dd, 1H, HA-benzyl); 5.45 (dd, 1H, HB-benzyl); 5.36 (dd, 1H,
HA-benzyl); 5.34 (dd, 1H, HB-benzyl); 5.29 (m, 2H, H3′); 4.55-
4.46 (m, 4H, H5′); 4.25-4.22 (m, 2H, H4′); 2.84-2.72 (m, 4H,
H3-Lev); 2.63-2.49 (m, 4H, H2-Lev); 2.21 (s, 3H, H5-Lev); 2.53
(ddd, 2H, H2′′); 2.20 (s, 3H, H5-Lev); 2.08 (ddd, 2H, H2′); 13C
NMR (101 MHz, CDCl3) δ 206.32 (C4-Lev); 172.43 (C1-Lev);
172.40 (C1-Lev); 160.96 (C4); 160.94 (C4); 149.96 (d, C2-aryl);
149.93 (d, C2-aryl); 148.96 (C2); 136.84 (C6); 136.83 (C6);
130.27 (C4-aryl); 128.10 (C7); 125.55 (C5-aryl); 124.81 (C6-
aryl); 124.77 (C6-aryl); 118.63 (C3-aryl); 118.55 (C3-aryl);
112.02 (C8); 111.98 (C8); 110.44 (C5); 110.30 (C5); 85.26 (C1′),
85.15 (C1′); 83.11 (d, C4′); 83.04 (d, C4′); 74.34 (C3′); 74.24
(C3′), 68.71 (d, C5′); 68.62 (d, C5′); 68.04 (d, C-benzyl); 67.88
(d, C-benzyl); 37.82 (C2′); 37.75 (C3-Lev); 29.72 (C5-Lev); 27.79
(C4-Lev); 31P NMR (202 MHz, CDCl3) δ -7.90; -7.97; Rf value
0.51 (CH2Cl2/MeOH, 9:1).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of t h e
cycloSa l-BVDUMP s 2a -f. To a solution of the cycloSal-3′-
O-Lev-BVDUMPs 6 (0.28 mmol) in 10 mL of pyridine was
added a solution of hydrazine hydrate (13.3 mL, H2NNH2*H2O
(24%)/pyridine/CH3COOH 2:4:3), and the reaction mixture was
stirred for 5 min at room temperature. Then the solution was
cooled to 0 °C, and 50 mL of ethyl acetate and 50 mL of water
were added and mixed well. After separation, the organic
phase was washed with 5% sodium hydrogencarbonate solu-
tion and dried with magnesium sulfate, and the solvent was
removed under reduced pressure. The residues were purified
by chromatography on silica gel plates on a chromatotron,
using a gradient of CH3OH in CH2Cl2 to yield the title
compounds 2a -f.
cyclo(5-Meth oxysa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-3′-
O-levu lin yl-2′-deoxyu r idin yl)ph osph ate (5-OMe-cycloSal-
3′-O-Lev-BVDUMP ) 6c: yield 70%; 1H NMR (500 MHz,
CDCl3) δ 9.71 (s, 2H, NH-BVU); 7.73 (s, 1H, H6); 7.72 (s, 1H,
H6); 7.43 (d, 1H, H8); 7.39 (d, 1H, H8); 7.00 (d, 1H, H4-aryl);
6.99 (d, 1H, H4-aryl); 6.85 (d, 1H, H6-aryl); 6.83 (d, 1H, H6-
aryl); 6.75 (d, 1H, H7); 6.69 (d, 1H, H7); 6.62 (d, 1H, H3-aryl);
6.61 (d, 1H, H3-aryl); 6.30 (dd, 1H, H1′); 6.29 (dd, 1H, H1′);
5.46-5.24 (m, 6H, H-benzyl, H3′); 4.52-4.44 (m, 4H, H5′);
4.23-4.20 (m, 2H, H4′); 3.77 (s, 6H, OMe); 2.79-2.75 (m, 4H,
H3-Lev); 2.59-2.48 (m, 6H, H2-Lev, H3′); 2.19 (s, 6H, H5-Lev);
2.12-2.02 (m, 4H, H2′); 13C NMR (101 MHz, CDCl3) δ 206.47
(C4-Lev); 172.41 (C1-Lev); 172.38 (C1-Lev); 161.67 (C4); 161.63
(C4); 156.27 (C5-aryl); 156.24 (C5-aryl); 149.18 (C2); 143.50
cyclo(5-Ch lor sa ligen yl)-5′-O-(E)-5-(2-b r om ovin yl)-2′-
d eoxyu r id in yl)p h osp h a t e (5-Cl-cycloSa l-BVDUMP ) 2a :
yield 91%; 1H NMR (500 MHz, CDCl3) δ 11.57 (s, 2H, NH-
BVU); 7.77 (s, 1H, H6); 7.74 (s, 1H, H6); 7.40-7.37 (m, 4H,
H3-aryl, H6-aryl); 7.28 (d, 1H, H8); 7.27 (d, 1H, H8); 7.14 (dd,

5166 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Meier et al.
2H, H4-aryl); 6.86 (d, 1H, H7); 6.82 (d, 1H, H7); 6.15 (dd, 1H,
H1′); 6.13 (dd, 1H, H1′); 5.52-5-38 (m, 4H, H-benzyl); 4.40-
4.25 (m, 4H, H5′); 4.23-4.20 (m, 2H, H4′); 3.93-3.91 (m, 2H,
H3′); 2.20-2.11 (m, 4H, H2′); 13C NMR (101 MHz, CDCl3) δ
156.21 (C4); 156.19 (C5-aryl); 149.21 (C2); 143.20 (d, C2-aryl);
143.19 (d, C2-aryl); 137.29 (C4-aryl); 137.18 (C4-aryl); 128.31
(C7); 128.24 (C7); 121.20 (d, C1-aryl); 121.19 (d, C1-aryl);
119.20 (d, C3-aryl); 119.10 (d, C3-aryl); 111.31 (C8); 111.26
(C8); 110.33 (C5-aryl); 110.31 (C5-aryl); 109.40 (C5); 109.33
(C5); 85.30 (C1′), 85.20 (C1′); 84.75 (d, C4′); 84.52 (d, C4′); 69.72
(C3′); 69.35 (C3′), 68.74 (d, C5′); 68.64 (d, C5′); 67.37 (d,
C-benzyl); 67.03 (d, C-benzyl); 40.31 (C2′); 40.19 (C2′); 31P NMR
(202 MHz, CDCl3) δ -8.79; -8.89; MS (FAB) m/z 535.0; 537.0
(M + H+); UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm; λmin
268.8 nm, 224.7 nm; Rf value 0.23 (CH2Cl2/MeOH, 9:1);
analytical HPLC tR 13.36 min (98%, gradient II).
6H, H-benzyl, OH); 4.38-4.22 (m, 6H, H5′, H3′); 3.94-3.90
(m, 2H, H4′); 2.20-2.13 (m, 2H, H2′); 2.20 (s, 3H, CH3-C3-
aryl); 2.18 (s, 3H, CH3-C3-aryl); 13C NMR (101 MHz, DMSO-
d6) δ 161.81 (C4); 161.79 (C4); 149.39 (C2); 148.12 (d, C2-aryl);
148.07 (d, C2-aryl); 139.48 (C6); 139.41 (C6); 131.10 (C4-aryl);
131.08 (C4-aryl); 129.94 (C7); 127.07 (C3-aryl); 126.99 (C3-
aryl); 124.15 (C5-aryl); 123.74 (C6-aryl); 123.71 (C6-aryl);
121.14 (d, C1-aryl); 121.09 (d, C1-aryl); 110.37 (C5); 110.34
(C5); 107.19 (C8); 84.87 (C1′); 84.80 (C1′); 84.67; (d, C4′); 84.63
(d, C4′); 69.89 (C3′); 69.83 (C3′); 68.66 (d, C5′); 68.59 (d, C5′);
67.97 (d, C-benzyl); 67.91 (d, C-benzyl); 15.07 (CH3-C3-aryl);
15.04 (CH3-C3-aryl); 31P NMR (162 MHz, DMSO-d6) δ -8.82;
-8.90; MS (FAB) m/z 515.3 (M); UV (CH3CN) λmax 293.39 nm,
250.10 nm, 196.82 nm; λmin 270.08 nm, 226.79 nm; IR (KBr) ν
3423, 2346, 1701, 1560, 1466, 1364, 1286, 1187, 1086, 1023,
955, 869, 526, 478; Rf value 0.51 (CH2Cl2/MeOH, 9:1); analyti-
cal HPLC tR 12.69 min (>97%, gradient II).
cycloSa ligen yl-5′-O-(E)-5-(2-br om ovin yl)-2′-d eoxyu r id i-
n yl)p h osp h a te (cycloSa l-BVDUMP ) 2b: yield 88%; 1H
NMR (500 MHz, CDCl3/MeOD) δ 7.55 (s, 1H, H6); 7.52 (s, 1H,
H6); 7.39 (d, 1H, H8); 7.34 (d, 1H, H8); 7.37-7.33 (m, 2H, H4-
aryl); 7.21-7.14 (m, 4H, H3-aryl, H6-aryl); 7.10 (t, 2H, H5-
aryl); 6.66 (d, 1H, H7); 6.56 (d, 1H, H7); 6.23 (dd, 4H, H1′);
5.47 (dd, 1H, HA-benzyl); 5.46 (dd, 1H, HA-benzyl); 5.39 (dd,
2H, HB-benzyl); 4.49-4.40 (m, 6H, H5′, H4′); 4.09-4.06 (m,
2H, H3′); 2.43 (ddd, 1H, H2′); 2.40 (ddd, 1H, H2′); 2.14 (ddd,
1H, H2′′); 2.09 (ddd, 1H, H2′′); 13C NMR (101 MHz, CDCl3) δ
161.42 (C4); 149.11 (C2); 149.07 (C2); 146.24 (C2-aryl); 146.18
(C2-aryl); 137.42 (C6); 137.27 (C6); 134.19 (C5-aryl); 134.12
(C5-aryl); 132.27 (C4-aryl); 132.22 (C4-aryl); 128.26 (C7);
128.09 (C7); 127.42 (d, C3-aryl); 127.34 (d, C3-aryl); 123.52
(C6-aryl); 123.39 (C6-aryl); 120.35 (d, C2-aryl); 120.07 (d, C2-
aryl); 111.45 (C8); 111.43 (C8); 110.09 (C5); 110.05 (C5); 85.55
(C1′), 85.28 (C1′); 84.98 (d, C4′); 84.65 (d, C4′); 70.54 (C3′);
69.59 (C3′), 69.17 (d, C5′); 68.88 (d, C5′); 66.64 (d, C-benzyl);
66.65 (d, C-benzyl); 40.62 (C2′); 40.34 (C2′); 31P NMR (202
MHz, CDCl3) δ -2.95; MS (FAB) m/z 501.0; 502.9 (M + H+);
UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm,
224.7 nm; IR (KBr) ν 3471, 3453, 3424, 3311, 3195, 3104, 3070,
2958, 2923, 1714, 1594, 1488, 1459, 1411, 1365, 1292, 1247,
1224, 1191, 1157, 1106, 1058, 1020, 946, 844, 759, 435; Rf value
0.28 (CH2Cl2/MeOH, 9:1); analytical HPLC tR 12.19 min (98%,
gradient II)
cyclo(3,5-Dim eth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-
2′-deoxyu r idin yl)ph osph ate (3,5-DiMe-cycloSal-BVDUMP )
2e: yield 77%; 1H NMR (500 MHz, CDCl3) δ 9.34 (s, 2H, NH-
BVU); 7.59 (s, 1H, H6); 7.57 (s, 1H, H6); 7.38 (d, 1H, H8); 7.36
(d, 1H, H8); 7.00 (d, 2H, H4-aryl); 6.77 (d, 2H, H6-aryl); 6.61
(d, 1H, H7); 6.55 (d, 1H, H7); 6.26 (dd, 1H, H1′); 6.24 (dd, 1H,
H1′); 5.45-5.26 (m, 6H, H-benzyl, H3′); 4.58-4.36 (m, 4H, H5′);
4.14 (m, 1H, H4′); 4.09 (m, 1H, H4′); 2.28 (s, 6H, CH3-C5-aryl);
2.27 (s, 6H, CH3-C3-aryl); 2.22-2.00 (m, 4H, H2′); 13C NMR
(101 MHz, CDCl3) δ 161.42 (C4); 149.11 (C2); 149.07 (C2);
146.24 (C2-aryl); 146.18 (C2-aryl); 137.42 (C6); 137.27 (C6);
134.19 (C5-aryl); 134.12 (C5-aryl); 132.27 (C4-aryl); 132.22 (C4-
aryl); 128.26 (C7); 128.09 (C7); 127.42 (d, C3-aryl); 127.34 (d,
C3-aryl); 123.52 (C6-aryl); 123.39 (C6-aryl); 120.35 (d, C2-aryl);
120.07 (d, C2-aryl); 111.45 (C8); 111.43 (C8); 110.09 (C5);
110.05 (C5); 85.55 (C1′), 85.28 (C1′); 84.98 (d, C4′); 84.65 (d,
C4′); 70.54 (C3′); 69.59 (C3′), 69.17 (d, C5′); 68.88 (d, C5′); 66.64
(d, C-benzyl); 66.65 (d, C-benzyl); 40.62 (C2′); 40.34 (C2′); 20.61
(CH3-C5-aryl); 15.19 (CH3-C3-aryl); 31P NMR (202 MHz,
CDCl3) δ -5.55; -5.98; MS (FAB) m/z 529.1; 531.5 (M + H+);
UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm,
224.7 nm; IR (KBr) ν 3424, 3195, 3102, 3066, 2950, 2923, 2832,
1704, 1594, 1481, 1463, 1363, 1284, 1199, 1149, 1108, 1085,
1025, 995, 950, 856, 802, 665, 532, 430; Rf value 0.49 (CH2-
Cl2/MeOH, 9:1); analytical HPLC tR 13.65 min (98%, gradient
II)
cyclo(5-Meth oxysa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxyu r id in yl)p h osp h a t e (5-OMe-cycloSa l-BVDUMP )
2c: yield 71%; 1H NMR (400 MHz, CDCl3) δ 9.11 (s, 2H, NH-
BVU); 7.58 (s, 1H, H6); 7.56 (s, 1H, H6); 7.40 (d, 1H, H8); 7.36
(d, 1H, H8); 7.02 (d, 1H, H4-aryl); 7.00 (d, 1H, H4-aryl); 6.85
(m, 2H, H3-aryl); 6.65 (d, 1H, H7); 6.64 (d, 1H, H6-aryl); 6.63
(d, 1H, H6-aryl); 6.54 (d, 1H, H7); 6.25 (m, 2H, H1′); 5.46-
5.24 (m, 4H, H-benzyl); 4.57-4.39 (m, 6H, H3′, H5′); 4.13-
4.09 (m, 2H, H4′); 3.79 (s, 3H, OMe); 3.78 (s, 3H, OMe); 2.53-
2.45 (m, 2H, H2′); 2.23-2.12 (m, 2H, H2′); 13C NMR (101 MHz,
CDCl3) δ 156.21 (C4); 156.19 (C5-aryl); 149.21 (C2); 143.20 (d,
C2-aryl); 143.19 (d, C2-aryl); 137.29 (C4-aryl); 137.18 (C4-aryl);
128.31 (C7); 128.24 (C7); 121.20 (d, C1-aryl); 121.19 (d, C1-
aryl); 119.20 (d, C3-aryl); 119.10 (d, C3-aryl); 111.31 (C8);
111.26 (C8); 110.33 (C5-aryl); 110.31 (C5-aryl); 109.40 (C5);
109.33 (C5); 85.30 (C1′), 85.20 (C1′); 84.75 (d, C4′); 84.52 (d,
C4′); 69.72 (C3′); 69.35 (C3′), 68.74 (d, C5′); 68.64 (d, C5′); 67.37
(d, C-benzyl); 67.03 (d, C-benzyl); 55.56 (OMe); 55.53 (OMe);
40.31 (C2′); 40.19 (C2′); 31P NMR (202 MHz, CDCl3) δ -6.40;
-6.54; MS (FAB) m/z 531.4; 533.4 (M + H+); UV (H2O/CH3-
CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm, 224.7 nm; IR
(KBr) ν 3423, 3193, 3102, 3068, 2948, 2836, 1708, 1594, 1496,
1465, 1432, 1363, 1284, 1197, 1159, 1085, 1024, 950, 914, 865,
848, 804, 665, 530, 431; Rf value 0.30 (CH2Cl2/MeOH, 9:1);
analytical HPLC tR 12.32 min (98%, gradient II)
cyclo(3-tBu t ylsa ligen yl)-5′-O-(E)-5-(2-b r om ovin yl)-2′-
d eoxyu r id in yl)p h osp h a te (3-tBu -cycloSa l-BVDUMP ) 2f:
yield 62%; 1H NMR (500 MHz, CDCl3) δ 9.63 (s, 1H, NH-BVU);
9.59 (s, 1H, NH-BVU); 7.63 (s, 1H, H6); 7.59 (s, 1H, H6); 7.41
(d, 1H, H8); 7.36 (d, 1H, H8); 7.36 (d, 2H, H4-aryl); 7.12 (dd,
1H, H5-aryl); 7.11 (dd, 1H, H5-aryl); 7.06 (d, 1H, H6-aryl); 7.01
(d, 1H, H6-aryl); 6.60 (d, 2H, H7); 6.28 (dd, 1H, H1′); 6.25 (dd,
1H, H1′); 5.47-5.28 (m, 4H, H-benzyl); 4.59-4.52 (m, 4H, H5′);
4.48-4.38 (m, 2H, H4′); 2.54-2.45 (m, 2H, H3′); 2.24-2.09 (m,
4H, H2′); 1.43 (s, 9H, CH3-tBu); 1.40 (s, 9H, CH3-tBu); 13C NMR
(101 MHz, CDCl3) δ 161.61 (C4); 149.25 (C2); 149.16 (C2);
149.10 (C3-aryl); 149.01 (C3-aryl); 139.54 (d, C2-aryl); 139.44
(d, C2-aryl); 137.48 (C6); 137.38 (C6); 128.24 (C7); 128.13 (C7);
128.04 (C4-aryl); 127.99 (C4-aryl); 124.65 (C5-aryl); 124.51 (C5-
aryl); 124.05 (C6-aryl); 123.92 (C6-aryl); 122.24 (d, C1-aryl);
122.16 (d, C1-aryl); 111.58 (C8); 111.43 (C8); 110.14 (C5); 85.42
(C1′), 85.27 (C1′); 84.89 (d, C4′); 84.70 (d, C4′); 70.63 (C3′);
69.52 (C3′), 69.10 (d, C5′); 68.77 (d, C5′); 67.84 (d, C-benzyl);
67.05 (d, C-benzyl); 40.65 (C2′); 40.28 (C2′); 34.80 (C7-aryl);
34.78 (C7-aryl); 29.85 (C8-aryl); 29.80 (C8-aryl); 31P NMR (202
MHz, CDCl3) δ -5.68; -6.08; MS (FAB) m/z 557.4 (M + H+);
UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm,
224.7 nm; IR (KBr) ν 3424, 3262, 3249, 3232, 3216, 3205, 3068,
2996, 2960, 2884, 1708, 1693, 1594, 1463, 1440, 1365, 1284,
1214, 1199, 1180, 1145, 1087, 1018, 997, 944, 802, 784, 742,
665, 530, 428; Rf value 0.49 (CH2Cl2/MeOH, 9:1); analytical
HPLC tR 14.77; 14.96 min (98%, gradient II).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxyu r id in yl)p h osp h a te (3-Me-cycloSa l-BVDUMP ) 2d :
yield 99%; 1H NMR (400 MHz, DMSO-d6) δ 11.56 (s, 2H, NH-
BVU); 7.76 (s, 1H, H6); 7.75 (s, 1H, H6); 7.29 (d, 1H, H8); 7.28
(d, 1H, H8); 7.23 (dd, 1H, H5-aryl); 7.22 (dd, 1H, H5-aryl);
7.07-7.05 (m, 4H, H4-aryl, H6-aryl); 6.86 (d, 1H, H7); 6.84
(d, 1H, H7); 6.15 (d, 1H, H1′); 6.14 (d, 1H, H1′); 5.48-5.36 (m,
Gen er a l P r oced u r e for th e P r ep a r a tion of th e cy-
cloSa l-BVDUMP s 3a -g a n d 7a -m . To a solution of 3′-
esterified BVDU 5 (0.28 mmol) in 10 mL of CH3CN was added
diisopropylethylamine (0.56 mmol, DIPEA), and the mixture

CycloSal-BVDUMP Pronucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5167
was cooled to -20 °C. Then, chlorophosphanes (0.56 mmol)
were added slowly, and the solution was stirred for 20 min.
For the oxidation of the intermediate cyclic phosphites, tert-
butyl hydroperoxide (0.56 mmol) was added to the reaction
mixture at -20 °C. After being stirred for 0.5 h, the reaction
mixture was warmed to room temperature, and the solvent
was removed under reduced pressure. The residues were
purified twice by chromatography on silica gel plates on a
chromatotron, first using a gradient of CH3OH in ethyl acetate
followed by a gradient of CH3OH in CH2Cl2 to yield the title
compounds 3a -g and 7a -m in 49-73% yield. Triesters 7a -m
were isolated, and the N-Boc group was immediately cleaved
by TFA to give 4a -m .
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-3′-
O-a cetyl-2′-d eoxyu r id in yl)p h osp h a te (3-Me-cycloSa l-3′-
O-Ac-BVDUMP ) 3a : yield 73%; 1H NMR (400 MHz, DMSO-
d6) δ 7.84 (s, 1H, H6); 7.83 (s, 1H, H6); 7.29 (d, 1H, H8); 7.28
(d, 1H, H8); 7.24 (dd, 1H, H5-aryl); 7.23 (dd, 1H, H5-aryl);
7.08-7.05 (m, 4H, H4-aryl, H6-aryl); 6.83 (d, 1H, H7); 6.82
(d, 1H, H7); 6.14 (dd, 1H, H1′); 6.13 (dd, 1H, H1′); 5.20-5.15
(m, 2H, H3′); 5.49-5.38 (m, 4H, H-benzyl); 4.43-4.31 (m, 4H,
H5′); 4.20-4.15 (m, 2H, H4′); 2.43-2.30 (m, 4H, H2′); 2.19 (s,
3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-C3-aryl); 2.04 (s, 3H, CH3-
Ac); 2.03 (s, 3H, CH3-Ac); 13C NMR (101 MHz, DMSO-d6) δ
170.13 (C1-Ac); 161.74 (C4); 161.72 (C4); 149.34 (C2); 148.01
(C2); 148.05 (d, C2-aryl); 139.29 (C6); 139.24 (C6); 131.09 (C4-
aryl); 131.04 (C4-aryl); 129.73 (C7); 127.04 (C3-aryl); 126.96
(C3-aryl); 124.15 (C5-aryl); 123.70 (C6-aryl); 121.12 (d, C1-
aryl); 121.03 (d, C1-aryl); 110.47 (C5); 110.43 (C5); 107.30 (C8);
85.05 (C1′); 84.98 (C1′); 82.08 (d, C4′); 82.02 (d, C4′); 73.36
(C3′); 73.31 (C3′); 68.65 (d, C5′); 68.60 (d, C5′); 67.67 (d,
C-benzyl); 67.62 (d, C-benzyl); 36.18 (C2′); 36.05 (C2′); 20.88
(C2-Ac); 15.02 (CH3-C3-aryl); 14.97 (CH3C3-aryl); 31P NMR
(162 MHz, DMSO-d6) δ -8.95; -9.06; MS (ESI+) m/z 558.9
(M + H); UV (CH3CN) λmax 293.39 nm, 250.10 nm, 196.82 nm;
λmin 268.42 nm, 226.79 nm; IR (KBr) ν 3430, 2346, 1718, 1708,
1654, 1637, 1560, 1474, 1291, 1023, 574, 484, 455; Rf value
0.37 (CH2Cl2/MeOH, 95:5); analytical HPLC tR 17.85 min
(98.63%, gradient I).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-3′-
O-p r op ion yl-2′-d eoxyu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-O-P r op -BVDUMP ) 3b: yield 30%; 1H NMR (500 MHz,
CDCl3) δ 8.85 (s, 2H, NH-BVU); 7.75 (s, 1H, H6), 7.74 (s, 1H,
H6); 7.47 (d, 1H, H8); 7.45 (d, 1H, H8); 7.22 (d, 2H, H4-aryl);
7.08 (dd, 2H, H5-aryl); 6.98 (d, 2H, H3-aryl); 6.75 (d, 1H, H7);
6.73 (d, 1H, H7); 6.34 (dd, 1H, H1′); 6.32 (dd, 1H, H1′); 5.46
(dt, 2H, H3′); 5.35-5.24 (m, 4H, H-benzyl); 4.57-4.41 (m, 4H,
H5′); 4.21 (m, 1H, H4′); 2.52 (dd, 2H, H2′); 2.37 (q, 2H, H2-
Prop); 2.36 (q, 2H, H2-Prop); 2.30 (s, 6H, CH3-C3-aryl); 2.08
(ddd, 2H, H2′′); 1.17 (t, 3H, H3-Prop); 1.16 (t, 3H, H3-Prop);
13C NMR (101 MHz, CDCl3) δ 174.00 (C1-Prop); 160.62 (C4);
148.78 (C2); 136.72 (C6); 131.65 (C4-aryl); 128.02 (C7, C3-aryl);
124.39 (C5-aryl); 123.08 (C6-aryl); 112.11 (C8); 110.54 (C5);
85.25 (C4′), 83.33 (C1′); 83.27 (C1′); 74.09 (C3′); 74.03 (C3′),
68.72 (C5′); 37.92 (C2′); 27.36 (C2-Prop); 15.33 (CH3-C3-aryl);
8.84 (C3-Prop); 31P NMR (202 MHz, CDCl3) δ -6.78; -6.92;
MS (FAB) m/z 571.4 (M); UV (H2O/CH3CN) λmax 289.6 nm,
248.0 nm; λmin 266.6 nm, 224.7 nm; IR (KBr) ν 3417, 3407,
3386, 3361, 3193, 3106, 3070, 2952, 2925, 2854, 1714, 1594,
1465, 1417, 1365, 1294, 1191, 1164, 1110, 1062, 1020, 941, 869,
852, 819, 769, 651, 530, 430; Rf value 0.79 (CH2Cl2/MeOH, 9:1);
analytical HPLC tR 13.60 min (>97%, gradient II).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-3′-
O-ibu tyr yl-2′-d eoxyu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-O-iBu -BVDUMP ) 3c: yield 70%; 1H NMR (500 MHz,
CDCl3) δ 8.72 (s, 2H, NH-BVU); 7.76 (s, 1H, H6), 7.75 (s, 1H,
H6); 7.47 (d, 1H, H8); 7.45 (d, 1H, H8); 7.21 (d, 2H, H4-aryl);
7.07 (dd, 2H, H5-aryl); 6.97 (d, 2H, H3-aryl); 6.73 (d, 1H, H7);
6.72 (d, 1H, H7); 6.33 (dd, 1H, H1′); 6.32 (dd, 1H, H1′); 5.45
(dt, 2H, H3′); 5.35-5.23 (m, 4H, H-benzyl); 4.55-4.42 (m, 4H,
H5′); 4.20-4.16 (m, 2H, H4′); 2.64 (m, 3H, H2′, H2-iBu); 2.29
(s, 6H, CH3-C3-aryl); 2.08 (ddd, 2H, H2′′); 1.19 (d, 3H, H3-
iBu); 1.18 (t, 3H, H3-iBu); 13C NMR (101 MHz, CDCl3) δ 176.74
(C1-iBu); 160.95 (C4); 148.99 (C2); 148.47 (C2-aryl); 148.40
(C2-aryl); 136.80 (C6); 136.76 (C6); 131.63 (C4-aryl); 131.60
(C4-aryl); 128.04 (C7); 127.89 (C3-aryl); 127.80 (C3-aryl);
124.37 (C5-aryl); 124.33 (C5-aryl); 123.07 (C6-aryl); 120.65 (C1-
aryl); 120.57 (C1-aryl); 112.11 (C8); 112.06 (C8); 110.50 (C5);
110.42 (C5); 85.33 (C4′), 85.15 (C4′); 83.40 (C1′); 83.33 (C1′);
74.04 (C3′); 73.99 (C3′), 68.81 (C5′); 68.75 (C5′); 67.95 (C-
benzyl); 67.76 (C-benzyl); 37.89 (C2′); 37.81 (C2′); 33.68 (C2-
iBu); 18.75 (C3-iBu); 15.32 (CH3-C3-aryl); 15.28 (CH3-C3-aryl);
31P NMR (202 MHz, CDCl3) δ -6.79; -6.94; MS (FAB) m/z
585.0; 587.0 (M + H+); UV (H2O/CH3CN) λmax 289.6 nm, 248.0
nm; λmin 266.6 nm, 224.7 nm; IR (KBr) ν 3440, 3424, 3197,
3104, 3070, 2975, 2933, 2881, 1714, 1631, 1594, 1467, 1367,
1294, 1189, 1155, 1116, 1062, 1018, 943, 819, 771, 659, 532,
431; Rf value 0.86 (CH2Cl2/MeOH, 9:1); analytical HPLC tR
13.99 min (>97%, gradient II)
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-3′-
O-p iva loyl-2′-d eoxyu r id in yl)p h osp h a t e (3-Me-cycloSa l-
3′-O-P iv-BVDUMP ) 3d : yield 61%; 1H NMR (400 MHz,
DMSO-d6) δ 11.62 (s, 2H, NH-BVU); 7.84 (s, 1H, H6); 7.83 (s,
1H, H6); 7.29 (d, 1H, H8); 7.28 (d, 1H, H8); 7.23 (dd, 1H, H5-
aryl); 7.22 (dd, 1H, H5-aryl); 7.07-7.05 (m, 4H, H4-aryl, H6-
aryl); 6.83 (d, 1H, H7); 6.82 (d, 1H, H7); 6.16-6.11 (m, 2H,
H1′); 5.49-5.38 (m, 4H, H-benzyl); 5.19-5.14 (m, 2H, H3′);
4.43-4.31 (m, 4H, H5′); 4.15-4.11 (m, 2H, H4′); 2.44-2.27 (m,
4H, H2′); 2.19 (s, 3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-C3-aryl);
1.14 (s, 9H, CH3-Piv); 1.13 (s, 9H, CH3-Piv); 13C NMR (101
MHz, DMSO-d6) δ 177.13 (C1-Piv); 161.72 (C4); 149.34 (C2);
148.06 (d, C2-aryl); 147.99 (d, C2-aryl); 139.37 (C6); 139.26
(C6); 131.08 (C4-aryl); 131.04 (C4-aryl); 129.72 (C7); 127.02
(C3-aryl); 126.95 (C3-aryl); 124.16 (C5-aryl); 123.70 (C6-aryl);
121.08 (d, C1-aryl); 120.52 (d, C1-aryl); 110.49 (C5); 110.45
(C5); 107.33 (C8); 85.13 (C1′); 85.06 (C1′); 82.19 (d, C4′); 82.12
(d, C4′); 73.50 (C3′); 73.45 (C3′); 68.71 (d, C5′); 68.64 (d, C5′);
67.70 (d, C-benzyl); 67.64 (d, C-benzyl); 38.25 (C2-Piv); 36.29
(C2′); 36.15 (C2′); 26.78 (C3-Piv); 15.03 (CH3-C3-aryl); 14.97
(CH3-C3-aryl); 31P NMR (162 MHz, DMSO-d6) δ -8.99; -9.04;
MS (FAB) m/z 598.0 (M - H+); 621.0 (M - H++Na+); UV (CH3-
CN) λmax 291.73 nm, 250.10 nm, 195.15 nm; λmin 268.42 nm,
226.79 nm; IR (KBr) ν 3447, 2346, 1718, 1654, 1560, 1458,
1364, 1281, 1191, 1157, 1017, 940, 776; Rf value 0.86 (CH2-
Cl2/MeOH, 9:1); analytical HPLC tR 20.77 min (>99%, gradient
I).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-3′-
O-h exa n oyl-2′-d eoxyu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-O-Hex-BVDUMP ) 3e: yield 50%; 1H NMR (500 MHz,
CDCl3) δ 8.28 (s, 2H, NH-BVU); 7.76 (s, 1H, H6); 7.75 (s, 1H,
H6); 7.47 (d, 1H, H8); 7.45 (d, 1H, H8); 7.22 (d, 2H, H4-aryl);
7.08 (dd, 2H, H5-aryl); 6.98 (d, 2H, H3-aryl); 6.75 (d, 1H, H7);
6.73 (d, 1H, H7); 6.33 (dd, 1H, H1′); 6.32 (dd, 1H, H1′); 5.46
(dt, 2H, H3′); 5.35-5.22 (m, 4H, H-benzyl); 4.55-4.42 (m, 4H,
H5′); 4.23-4.17 (m, 2H, H4′); 2.53 (dd, 1H, H2′); 2.49 (dd, 1H,
H2′); 2.35 (q, 2H, H2-Hex); 2.34 (q, 2H, H2-Hex); 2.28 (s, 6H,
CH3-C3-aryl); 2.13 (ddd, 1H, H2′′); 2.04 (ddd, 1H, H2′′); 1.68-
1.60 (m, 4H, H3-Hex); 1.38-1.25 (m, 8H, H4-Hex, H5-Hex);
0.97 (t, 6H, H6-Hex); 13C NMR (101 MHz, CDCl3) δ 173.43
(C1-Hex); 173.41 (C1-Hex); 160.69 (C4); 148.83 (C2); 136.77
(C6); 136.73 (C6); 131.65 (C4-aryl); 131.63 (C4-aryl); 128.03
(C7, C3-aryl); 124.38 (C5-aryl); 124.34 (C5-aryl); 123.09 (C6-
aryl); 112.11 (C8); 112.06 (C8); 110.54 (C5); 110.45 (C5); 85.34
(C4′), 85.17 (C4′); 83.35 (C1′); 83.30 (C1′); 74.03 (C3′); 73.98
(C3′), 68.82 (d, C5′); 68.82 (d, C5′); 67.95 (d, C-benzyl); 67.76
(d, C-benzyl); 37.92 (C2′); 37.84 (C2′); 33.97 (C2-Hex); 31.20
(C4-Hex); 24.41 (C3-Hex); 22.25 (C5-Hex); 15.33 (CH3-C3-aryl);
15.28 (CH3-C3-aryl); 13.86 (C6-Hex); 31P NMR (202 MHz,
CDCl3) δ -6.80; -6.93; MS (FAB) m/z 613.5 (M); UV (H2O/
CH3CN) λmax 288.1 nm, 248.0 nm; λmin 266.6 nm, 224.7 nm; IR
(KBr) ν 3447, 2346, 1718, 1654, 1560, 1458, 1364, 1281, 1191,
1157, 1017, 940, 776; Rf value 0.80 (CH2Cl2/MeOH, 9:1);
analytical HPLC tR 16.61 min (>97%, gradient II).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-3′-
O-d eca n oyl-2′-d eoxyu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-O-Dec-BVDUMP ) 3f: yield 56%; 1H NMR (500 MHz,
CDCl3) δ 8.96 (s, 2H, NH-BVU); 7.75 (s, 1H, H6); 7.74 (s, 1H,
H6); 7.47(d, 1H, H8); 7.45 (d, 1H, H8); 7.22 (d, 2H, H4-aryl);

5168 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Meier et al.
7.08 (dd, 2H, H5-aryl); 6.98 (d, 1H, H6-aryl); 6.73 (d, 1H, H7);
6.72 (d, 1H, H7); 6.34 (dd, 1H, H1′); 6.35 (dd, 1H, H1′); 5.46
(dt, 2H, H3′); 5.35-5.22 (m, 4H, H-benzyl); 4.55-4.40 (m, 4H,
H5′); 4.23-4.15 (m, 2H, H4′); 2.54 (dd, 1H, H2′); 2.48 (dd, 1H,
H2′); 2.35 (q, 2H, H2-Dec); 2.34 (q, 2H, H2-Dec); 2.28 (s, 6H,
CH3-C3-aryl); 2.14 (ddd, 1H, H2′′); 2.04 (ddd, 1H, H2′); 1.65-
1.58 (m, 4H, H3-Dec); 1.28 (m, 24H, H4-Dec - H9-Dec); 0.88
(t, 6H, H10-Dec); 13C NMR (126 MHz, CDCl3) δ 173.40 (C1-
Dec); 173.38 (C1-Dec); 161.03 (C4); 161.02 (C4); 149.07 (C2);
148.55 (d, C2-aryl); 148.45 (d, C2-aryl); 136.79 (C6); 136.75
(C6); 131.62 (C4-aryl); 131.60 (C4-aryl); 128.07 (C7); 127.85
(d, C3-aryl); 127.82 (d, C3-aryl); 124.32 (C5-aryl); 124.30 (C5-
aryl); 123.07 (C6-aryl); 112.11 (C5); 112.06 (C5); 110.47 (C8);
110.38 (C8); 85.35 (C4′); 85.18 (C4′); 83.35 (C1′); 83.30 (C1′);
74.03 (C3′), 74.01 (C3′); 67.95 (d, C5′); 67.90 (d, C5′); 37.90
(C2′); 37.82 (C2′); 33.99 (C2-Dec); 31.80 (C4-Dec); 29.34 (C5-
Dec); 29.20 (C7-Dec); 29.18 (C6-Dec); 29.05 (C8-Dec); 24.72 (C3-
Dec); 22.62 (C9-Dec); 15.26 (CH3-C3-aryl); 15.25 (CH3-C3-aryl);
13.85 (C10-Dec); 31P NMR (202 MHz, CDCl3) δ -6.80; -6.94;
MS (FAB) m/z 668.9 (M - H+); UV (H2O/CH3CN) λmax 288.1
nm, 248.0 nm; λmin 266.6 nm, 224.7 nm; IR (KBr) ν 3417, 3407,
3386, 3361, 3193, 3106, 3070, 2952, 2925, 2854, 1714, 1594,
1465, 1417, 1365, 1294, 1191, 1164, 1110, 1062, 1020, 941, 869,
852, 819, 769, 651, 530, 430; Rf value 0.77 (CH2Cl2/MeOH, 9:1);
analytical HPLC tR 21.01 min (>97%, gradient II).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-3′-
O-levu lin yl-2′-d eoxyu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-O-Lev-BVDUMP ) 3g: yield 81%; 1H NMR (400 MHz,
CDCl3) δ 8.93 (s, 2H, NH-BVU); 7.76 (s, 1H, H6); 7.75 (s, 1H,
H6); 7.46 (d, 1H, H8); 7.44 (d, 1H, H8); 7.21 (d, 2H, H4-aryl);
7.08 (dd, 2H, H5-aryl); 6.96 (d, 2H, H6-aryl); 6.73 (d, 1H, H7);
6.71 (d, 1H, H7); 6.33 (dd, 1H, H1′); 6.32 (dd, 1H, H1′); 5.46
(dt, 2H, H3′); 5.35-5.22 (m, 4H, H-benzyl); 4.55-4.40 (m, 4H,
H5′); 4.25-4.17 (m, 2H, H4′); 2.81-2.75 (m, 4H, H3-Lev); 2.23
(s, 6H, CH3-C3-aryl); 2.21 (s, 3H, H5-Lev); 2.20 (s, 3H, H5-
Lev); 2.20-2.00 (m, 4H, H2′); 13C NMR (101 MHz, CDCl3) δ
206.34 (C4-Lev); 172.40 (C1-Lev); 161.03 (C4); 149.03 (C2);
136.81 (C6); 131.62 (C4-aryl); 131.58 (C4-aryl); 128.07 (C7);
127.89 (C3-aryl); 127.78 (C3-aryl); 124.35 (C5-aryl); 124.31 (C5-
aryl); 123.06 (C6-aryl); 112.05 (C8); 112.02 (C8); 110.42 (C5);
110.32 (C5); 85.32 (C4′), 85.14 (C4′); 83.13 (C1′); 83.07 (C1′);
74.39 (C3′); 74.31 (C3′), 68.78 (d, C5′); 68.71 (d, C5′); 67.95 (d,
C-benzyl); 67.76 (d, C-benzyl); 37.75 (C2′, C3-Lev); 29.71 (C5-
Lev); 27.79 (C4-Lev); 15.31 (CH3-C3-aryl); 15.26 (CH3-C3-aryl);
31P NMR (202 MHz, CDCl3) δ -6.80; -6.94; MS (FAB) m/z
611.03 (M - H+); UV (H2O/CH3CN) λmax 288.1 nm, 248.0 nm;
λmin 266.6 nm, 224.7 nm; IR (KBr) ν 3424, 3199, 3104, 3070,
2969, 2927, 1714, 1594, 1467, 1409, 1365, 1294, 1189, 1159,
1122, 1058, 1018, 943, 819, 773, 657, 433; Rf value 0.63 (CH2-
Cl2/MeOH, 9:1); analytical HPLC tR 12.56 min (>97%, gradient
II).
129.74 (C7); 129.72 (C7); 127.06 (C3-aryl); 126.98 (C3-aryl);
124.24 (C5-aryl); 124.22 (C5-aryl); 123.77 (C6-aryl); 123.74 (C6-
aryl); 121.05 (d, C1-aryl); 121.06 (d, C1-aryl); 110.53 (C5);
110.49 (C5); 107.40 (C8); 85.09 (C4′); 85.04 (C1′); 75.02 (C3′);
74.90 (C3′); 68.72 (d, C5′); 68.66 (d, C5′); 67.58 (C-benzyl); 67.38
(C-benzyl); 45.89 (C2-Gly); 35.98 (C2′); 35.83 (C2′); 15.07 (CH3-
C3-aryl); 15.00 (CH3-C3-aryl); 31P NMR (202 MHz, DMSO-d6)
δ -7.76; -7.81; MS (FAB) m/z 572.2; 574.2 (M + H+); UV (CH3-
CN) λmax 291.73 nm, 250.10 nm, 195.15 nm; λmin 268.42 nm,
226.79 nm; IR (KBr) ν 3442, 3421, 3158, 3066, 3031, 2964,
2933, 2854, 2829, 1756, 1681, 1596, 1469, 1432, 1292, 1232,
1199, 1130, 1052, 1024, 1006, 944, 829, 800, 721; Rf value 0.60
(CH2Cl2/MeOH, 8:2); analytical HPLC tR 12.03 min (94%,
gradient III).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxy-3′-O-L-a la n ylu r id in yl)p h osp h a te (3-Me-cycloSa l-3′-
L-Ala -BVDUMP ) 4b: yield 25%; 1H NMR (500 MHz, DMSO-
d6) δ 11.68 (s, 2H, NH-BVU); 8.45 (s, 4H, NH2-Ala); 7.85 (s,
1H, H6); 7.84 (s, 1H, H6); 7.30 (d, 1H, H8); 7.28 (d, 1H, H8);
7.25-7.20 (m, 2H, H5-aryl); 7.12-7.05 (m, 4H, H4-aryl, H6-
aryl); 6.84 (d, 1H, H7); 6.81 (d, 1H, H7); 6.21 (dd, 1H, H1′);
6.19 (dd, 1H, H1′); 5.52-5.36 (m, 4H, H-benzyl); 5.34-5.28 (m,
2H, H3′); 4.48-4.33 (m, 4H, H5′); 4.29-4.23 (m, 2H, H4′); 4.14
(q, 1H, H2-Ala); 4.13 (q, 1H, H2-Ala); 2.52-2.17 (m, 4H, H2′);
2.20 (s, 3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-C3-aryl); 1.38 (d,
3H, H3-Ala); 1.37 (d, 3H, H3-Ala); 13C NMR (126 MHz, DMSO-
d6) δ 169.43 (C1-Ala); 161.73 (C4); 161.71 (C4); 149.35 (C2);
149.34 (C2); 139.27 (C6); 139.18 (C6); 131.12 (C4-aryl); 131.09
(C4-aryl); 129.70 (C7); 129.68 (C7); 126.99 (d, C3-aryl); 127.98
(d, C3-aryl); 124.22 (C5-aryl); 124.21 (C5-aryl); 123.74 (C6-
aryl); 123.72 (C6-aryl); 121.09 (d, C1-aryl); 121.06 (d, C1-aryl);
110.51 (C5); 110.46 (C5); 107.37 (C8); 85.15 (C4′); 85.01 (C1′);
75.29 (C3′); 75.20 (C3′); 68.70 (d, C5′); 68.65 (d, C5′); 48.14
(C2-Ala); 36.14 (C2′); 35.98 (C2′); 15.68 (C3-Ala); 15.04 (CH3-
C3-aryl); 14.96 (CH3-C3-aryl); 31P NMR (162 MHz, DMSO-d6)
δ -7.75; -7.80; MS (FAB) m/z 586.5; 588.6 (M + H+); UV (H2O/
CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm, 224.7 nm; IR
(KBr) ν 3424, 3178, 3075, 3029, 3018, 2956, 2854, 2827, 1752,
1681, 1596, 1465, 1436, 1367, 1290, 1236, 1199, 1132, 1052,
1024, 1002, 956, 946, 831, 800, 775, 721, 659, 532; Rf value
0.15 (CH2Cl2/MeOH, 9:1); analytical HPLC tR 12.03 min (94%,
gradient III).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxy-3′-O-D-a la n ylu r id in yl)p h osp h a t e (3-Me-cycloSa l-
3′-D-Ala -BVDUMP ) 4c: yield 25%; 1H NMR (500 MHz,
DMSO-d6) δ 11.68 (s, 2H, NH-BVU); 8.55 (s, 4H, NH2-Ala);
7.85 (s, 1H, H6); 7.84 (s, 1H, H6); 7.30 (d, 1H, H8); 7.28 (d,
1H, H8); 7.25-7.20 (m, 2H, H5-aryl); 7.08-7.05 (m, 4H, H4-
aryl, H6-aryl); 6.83 (d, 1H, H7); 6.81 (d, 1H, H7); 6.22-6.17
(m, 2H, H1′); 5.51-5.36 (m, 4H, H-benzyl); 5.30-5.26 (m, 2H,
H3′); 4.45-4.33 (m, 4H, H5′); 4.28-4.25 (m, 2H, H4′); 4.10-
4.05 (m, 2H, H2-Ala); 2.52-2.32 (m, 4H, H2′); 2.20 (s, 3H, CH3-
C3-aryl); 2.18 (s, 3H, CH3-C3-aryl); 1.40 (d, 3H, H3-Ala); 1.39
(d, 3H, H3-Ala); 13C NMR (126 MHz, DMSO-d6) δ 169.43 (C1-
Ala); 161.71 (C4); 149.34 (C2); 149.33 (C2); 139.36 (C6); 139.28
(C6); 131.10 (C4-aryl); 131.05 (C4-aryl); 129.70 (C7); 129.69
(C7); 126.98 (d, C3-aryl); 127.97 (d, C3-aryl); 124.19 (C5-aryl);
124.17 (C5-aryl); 124.12 (C6-aryl); 123.72 (C6-aryl); 121.09 (d,
C1-aryl); 121.06 (d, C1-aryl); 110.49 (C5); 110.46 (C5); 107.34
(C8); 85.12 (C4′); 85.05 (C1′); 74.95 (C3′); 74.86 (C3′); 68.70
(d, C5′); 68.63 (d, C5′); 48.07 (C2-Ala); 35.95 (C2′); 35.80 (C2′);
15.95 (C3-Ala); 15.04 (CH3-C3-aryl); 14.97 (CH3-C3-aryl); 31P
NMR (162 MHz, DMSO-d6) δ -7.78; -7.84 (s); MS (FAB) m/z
586.5; 588.6 (M + H+); UV (H2O/CH3CN) λmax 289.6 nm, 248.0
nm; λmin 268.8 nm, 224.7 nm; IR (KBr) ν 3424, 3178, 3075,
3029, 3018, 2956, 2854, 2827, 1752, 1681, 1596, 1465, 1436,
1367, 1290, 1236, 1199, 1132, 1052, 1024, 1002, 956, 946, 831,
800, 775, 721, 659, 532; Rf value 0.51 (CH2Cl2/MeOH, 7:3);
analytical HPLC tR 12.03 min (94%, gradient III).
Gen er a l P r oced u r e for th e P r ep a r a tion of th e cy-
cloSa l-BVDUMP s 4a -m . To cleave the N-Boc-protecting
group, triesters 7a -m , that were prepared according to
triesters 3a -g, were dissolved in 5 mL of CH3CN, mixed with
1 mL of trifluoroacetic acid, and stirred for 1.5 h at room
temperature. The reaction mixtures were diluted with 20 mL
of water and lyophilized. The residues were purified first by
chromatography on silica gel plates on a chromatotron using
a 1:9 mixture of CH3OH in CH2Cl2 and second by gel filtration
on Sephadex LH-20 (Fluka) using CH3OH as eluent.
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxy-3′-O-glycin ylu r id in yl)p h osp h a te (3-Me-cycloSa l-3′-
1
Gly-BVDUMP ) 4a : yield 27%; H NMR (500 MHz, DMSO-
d6) δ 11.65 (s, 2H, NH-BVU); 8.28 (s, 4H, NH2-Gly); 7.84 (s,
1H, H6); 7.83 (s, 1H, H6); 7.30 (d, 1H, H8); 7.29 (d, 1H, H8);
7.22 (m, 2H, H5-aryl); 7.05 (m, 4H, H4-aryl, H6-aryl); 6.84 (d,
1H, H7); 6.82 (d, 1H, H7); 6.19 (dd, 1H, H1′); 6.18 (dd, 1H,
H1′); 5.45 (m, 4H, H-benzyl); 5.33 (m, 2H, H3′); 4.40 (m, 4H,
H5′); 4.26 (m, 2H, H4′); 3.86 (s, 4H, H2-Gly); 2.40 (m, 4H, H2′);
2.20 (s, 3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-C3-aryl); 13C NMR
(101 MHz, DMSO-d6) δ 167.33 (C1-Gly); 161.76 (C4); 161.74
(C4); 149.37 (C2); 148.07 (d, C2-aryl); 148.01 (d, C2-aryl);
139.35 (C6); 139.27 (C6); 131.14 (C4-aryl); 131.11 (C4-aryl);
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxy-3′-O-L-va lin ylu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-L-Va l-BVDUMP ) 4d : yield 32%; 1H NMR (500 MHz,
DMSO-d6) δ 11.68 (s, 2H, NH-BVU); 8.28 (s, 4H, NH2-Val);
7.85 (s, 1H, H6); 7.84 (s, 1H, H6); 7.29 (d, 1H, H8); 7.28 (d,

CycloSal-BVDUMP Pronucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5169
1H, H8); 7.25-7.21 (m, 2H, H5-aryl); 7.08-7.06 (m, 4H, H4-
aryl, H6-aryl); 6.84 (d, 1H, H7); 6.83 (d, 1H, H7); 6.20 (dd, 1H,
H1′); 6.18 (dd, 1H, H1′); 5.52-5.38 (m, 4H, H-benzyl); 5.36-
5.33 (m, 2H, H3′); 4.45-4.33 (m, 4H, H5′); 4.27-4.24 (m, 2H,
H4′); 3.93 (d, 2H, H2-Val); 2.45-2.35 (m, 4H, H2′); 2.20 (s, 3H,
CH3-C3-aryl); 2.18 (s, 3H, CH3-C3-aryl); 2.17-2.10 (m, 2H, H3-
Val); 0.96 (d, 2H, H4-Val); 0.93 (d, 2H, H4-Val); 13C NMR (101
MHz, DMSO-d6) δ 168.49 (C1-Val); 161.74 (C4); 149.37 (C2);
139.32 (C6); 139.23 (C6); 131.14 (C4-aryl); 131.11 (C4-aryl);
129.74 (C7); 127.03 (C3-aryl); 126.95 (C3-aryl); 124.24 (C5-
aryl); 123.74 (C6-aryl); 121.11 (d, C1-aryl); 121.02 (d, C1-aryl);
110.53 (C5); 110.47 (C5); 107.41 (C8); 85.13 (C4′); 85.09 (C1′);
75.28 (C3′); 75.22 (C3′); 68.75 (d, C5′); 68.68 (d, C5′); 67.53 (d,
C-benzyl); 67.31 (d, C-benzyl); 36.25 (C2′); 36.07 (C2′); 29.61
(C3-Val); 29.54 (C3-Val); 18.35 (C4-Val); 17.80 (C4-Val); 15.06
(CH3-C3-aryl); 14.99 (CH3-C3-aryl); 31P NMR (202 MHz,
DMSO-d6) δ -7.77; -7.83; MS (FAB) m/z 614.6; 616.6 (M +
H+); UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm,
224.7 nm; IR (KBr) ν 3432, 3424, 3168, 3068, 2971, 2854, 2829,
1749, 1687, 1631, 1596, 1467, 1432, 1369, 1288, 1199, 1132,
1051, 1024, 1004, 944, 829, 800, 721, 657, 532; Rf value 0.37
(CH2Cl2/MeOH, 9:1); analytical HPLC tR 12.03 min (94%,
gradient III).
2877, 1687, 1596, 1471, 1434, 1369, 1290, 1201, 1135, 1052,
1024, 1006, 944, 833, 798, 721; Rf value 0.27 (CH2Cl2/MeOH,
9:1); analytical HPLC tR 12.03 min (94%, gradient III).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxy-3′-O-D-leu cin ylu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-D-Leu -BVDUMP ) 4g: yield 39%; 1H NMR (500 MHz,
DMSO-d6) δ 8.31 (s, 4H, NH2-Leu); 7.85 (s, 1H, H6); 7.84 (s,
1H, H6); 7.29 (d, 1H, H8); 7.28 (d, 1H, H8); 7.25-7.20 (m, 2H,
H5-aryl); 7.08-7.06 (m, 4H, H4-aryl, H6-aryl); 6.83 (d, 1H, H7);
6.82 (d, 1H, H7); 6.22-6.17 (m, 2H, H1′); 5.52-5.36 (m, 4H,
H-benzyl); 5.33-5.29 (m, 2H, H3′); 4.48-4.32 (m, 4H, H5′);
4.26-4.24 (m, 2H, H4′); 4.00 (t, 2H, H2-Leu); 2.45-2.35 (m,
4H, H2′); 2.19 (s, 3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-C3-aryl);
1.74-1.53 (m, 6H, H3-Leu, H4-Leu); 0.91-0.87 (m, 12H, H5-
Leu); 13C NMR (101 MHz, DMSO-d6) δ 169.47 (C1-Leu); 161.75
(C4); 161.73 (C4); 149.37 (C2); 139.32 (C6); 139.24 (C6); 131.14
(C4-aryl); 131.09 (C4-aryl); 129.69 (C7); 127.04 (C3-aryl);
126.96 (C3-aryl); 124.23 (C5-aryl); 124.21 (C5-aryl); 123.78 (C6-
aryl); 121.04 (C1-aryl); 121.02 (C1-aryl); 110.53 (C5); 110.49
(C5); 107.41 (C8); 85.05 (C4′); 84.99 (C1′); 75.29 (C3′); 75.16
(C3′); 68.75 (d, C5′); 68.63 (d, C5′); 67.53 (C-benzyl); 67.36 (C-
benzyl); 50.79 (C2-Leu); 36.70 (C2′); 35.70 (C2′); 30.87 (C3-
Leu); 24.00 (C4-Leu); 22.21 (C5-Leu); 22.19 (C5-Leu); 15.07
(CH3-C3-aryl); 14.99 (CH3-C3-aryl); 31P NMR (202 MHz,
DMSO-d6) δ -7.78; -7.84; MS (FAB) m/z 628.3; 630.3 (M +
H+); UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm,
224.7 nm; IR (KBr) ν 3436, 3424, 3320, 3168, 3160, 3070, 2964,
2877, 1687, 1596, 1471, 1434, 1369, 1290, 1201, 1135, 1052,
1024, 1006, 944, 833, 798, 721; Rf value 0.27 (CH2Cl2/MeOH,
9:1); analytical HPLC tR 12.03 min (94%, gradient III).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxy-3′-O-D-va lin ylu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-D-Va l-BVDUMP ) 4e: yield 29%; 1H NMR (500 MHz,
DMSO-d6) δ 11.67 (s, 2H, NH-BVU); 8.36 (s, 4H, NH2-Val);
7.87 (s, 1H, H6); 7.86 (s, 1H, H6); 7.31 (d, 1H, H8); 7.30 (d,
1H, H8); 7.26-7.23 (m, 2H, H5-aryl); 7.10-7.08 (m, 4H, H4-
aryl, H6-aryl); 6.85 (d, 1H, H7); 6.84 (d, 1H, H7); 6.22 (dd, 1H,
H1′); 6.21 (dd, 1H, H1′); 5.53-5.40 (m, 4H, H-benzyl); 5.39-
5.34 (m, 2H, H3′); 4.49-4.36 (m, 4H, H5′); 4.29-4.26 (m, 2H,
H4′); 3.95 (d, 1H, H2-Val); 3.94 (d, 1H, H2-Val); 2.45-2.35 (m,
4H, H2′); 2.22 (s, 3H, CH3-C3-aryl); 2.20 (s, 3H, CH3-C3-aryl);
2.19-2.16 (m, 2H, H3-Val); 0.99 (d, 2H, H4-Val); 0.97 (d, 2H,
H4-Val); 13C NMR (101 MHz, DMSO-d6) δ 168.43 (C1-Val);
161.73 (C4); 149.38 (C2); 139.33 (C6); 139.25 (C6); 131.14 (C4-
aryl); 131.10 (C4-aryl); 129.74 (C7); 127.04 (C3-aryl); 126.96
(C3-aryl); 124.24 (C5aryl); 123.74 (C6-aryl); 121.11 (C1-aryl);
121.01 (C1-aryl); 110.53 (C5); 110.49 (C5); 107.41 (C8); 85.04
(C4′); 84.99 (C1′); 75.22 (C3′); 75.12 (C3′); 68.75 (d, C5′); 68.65
(d, C5′); 67.51 (d, C-benzyl); 67.28 (d, C-benzyl); 35.92 (C2′);
35.76 (C2′); 29.56 (C3-Val); 18.34 (C4-Val); 17.78 (C4-Val);
15.07 (CH3-C3-aryl); 15.00 (CH3-C3-aryl); 31P NMR (202 MHz,
DMSO-d6) δ -7.78; -7.85; MS (FAB) m/z 614.6; 616.6 (M +
H+); UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm,
224.7 nm; IR (KBr) ν 3432, 3424, 3168, 3068, 2971, 2854, 2829,
1749, 1687, 1631, 1596, 1467, 1432, 1369, 1288, 1199, 1132,
1051, 1024, 1004, 944, 829, 800, 721, 657, 532; Rf value 0.37
(CH2Cl2/MeOH, 9:1); analytical HPLC tR 12.03 min (94%,
gradient III).
cyclo(3-Me t h ylsa lige n yl)-5′-O-(E )-5-(2-b r om ovin yl)-
2′-d eoxy-3′-O-L-isoleu cin ylu r id in yl)p h osp h a t e
(3-Me-
cycloSa l-3′-L-Ile-BVDUMP ) 4h : yield 25%; 1H NMR (500
MHz, DMSO-d6) δ 11.65 (s, 2H, NH-BVU); 8.01 (s, 4H, NH2-
Ile); 7.85 (s, 1H, H6); 7.84 (s, 1H, H6); 7.29 (d, 1H, H8); 7.28
(d, 1H, H8); 7.25-7.22 (m, 2H, H5-aryl); 7.08-7.06 (m, 4H,
H4-aryl, H6-aryl); 6.84 (d, 1H, H7); 6.83 (d, 1H, H7); 6.19 (dd,
1H, H1′); 6.18 (dd, 1H, H1′); 5.51-5.36 (m, 4H, H-benzyl);
5.35-5.30 (m, 2H, H3′); 4.44-4.33 (m, 4H, H5′); 4.27-4.24 (m,
2H, H4′); 3.94 (d, 2H, H2-Ile); 2.45-2.35 (m, 4H, H2′); 2.20 (s,
3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-C3-aryl); 1.87-1.82 (m, 2H,
H3-Ile); 1.48-1.40 (m, 2H, H4A-Ile); 1.29-1.20 (m, 2H, H4B-
Ile); 0.90-0.86 (m, 12H, H5-Ile, H6-Ile); 13C NMR (101 MHz,
DMSO-d6) δ 168.72 (C1-Ile); 161.72 (C4); 149.33 (C2); 139.30
(C6); 139.20 (C6); 131.10 (C4-aryl); 129.70 (C7); 127.02 (C3-
aryl); 126.96 (C3-aryl); 124.23 (C5-aryl); 124.22 (C5-aryl);
123.74 (C6-aryl); 123.72 (C6-aryl); 121.10 (d, C1-aryl); 121.02
(d, C1-aryl); 110.53 (C5); 110.47 (C5); 107.39 (C8); 85.13 (C4′);
85.09 (C1′); 75.18 (C3′); 75.11 (C3′); 68.75 (d, C5′); 68.71 (d,
C5′); 67.51 (d, C-benzyl); 67.30 (d, C-benzyl); 56.52 (C2-Ile);
36.35 (C2′); 36.24 (C2′); 25.34 (C4-Ile); 22.25 (C5-Leu); 22.15
(C5-Leu); 15.08 (CH3-C3-aryl); 14.99 (CH3-C3-aryl); 14.48 (C4-
Ile); 11.61 (C5-Ile); 31P NMR (162 MHz, DMSO-d6) δ -7.77;
-7.84; MS (FAB)m/z 628.5; 630.5 (M + H+); UV (H2O/CH3-
CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm, 224.7 nm; IR
(KBr) ν 3436, 3261, 3249, 3180, 3068, 2967, 2931, 2884, 2854,
1685, 1639, 1596, 1467, 1284, 1201, 1132, 1056, 1024, 944, 800,
721, 657, 532; Rf value 0.32 (CH2Cl2/MeOH, 9:1); analytical
HPLC tR 12.03 min (94%, gradient III).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxy-3′-O-L-leu cin ylu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-L-Leu -BVDUMP ) 4f: yield 35%; 1H NMR (500 MHz,
DMSO-d6) δ 8.09 (s, 4H, NH2-Leu); 7.84 (s, 1H, H6); 7.83 (s,
1H, H6); 7.29 (d, 1H, H8); 7.28 (d, 1H, H8); 7.25-7.20 (m, 2H,
H5-aryl); 7.08-7.05 (m, 4H, H4-aryl, H6-aryl); 6.83 (d, 1H, H7);
6.82 (d, 1H, H7); 6.19 (dd, 1H, H1′); 6.18 (dd, 1H, H1′); 5.51-
5.36 (m, 4H, H-benzyl); 5.33-5.29 (m, 2H, H3′); 4.45-4.33 (m,
4H, H5′); 4.27-4.24 (m, 2H, H4′); 3.98 (t, 2H, H2-Leu); 2.45-
2.35 (m, 4H, H2′); 2.20 (s, 3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-
C3-aryl); 1.74-1.53 (m, 6H, H3-Leu, H4-Leu); 0.91-0.87 (m,
12H, H5-Leu); 13C NMR (101 MHz, DMSO-d6) δ 168.93 (C1-
Leu); 161.75 (C4); 149.39 (C2); 139.32 (C6); 139.23 (C6); 131.14
(C4-aryl); 129.74 (C7); 127.65 (d, C3-aryl); 127.25 (d, C3-aryl);
124.25 (C5-aryl); 124.23 (C5-aryl); 123.78 (C6-aryl); 123.75 (C6-
aryl); 121.12 (C1-aryl); 110.47 (C5); 107.41 (C8); 85.15 (C4′);
85.12 (C1′); 75.29 (C3′); 75.20 (C3′); 68.79 (C5′); 68.47 (C-
benzyl); 50.89 (C2-Leu); 36.23 (C2′); 35.95 (C2′); 31.38 (C3-
Leu); 24.01 (C4-Leu); 22.25 (C5-Leu); 22.15 (C5-Leu); 15.08
(CH3-C3-aryl); 14.99 (CH3-C3-aryl); 31P NMR (202 MHz,
DMSO-d6) δ -7.77; -7.81; MS (FAB) m/z 628.3; 630.3 (M +
H+); UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm,
224.7 nm; IR (KBr) ν 3436, 3424, 3320, 3168, 3160, 3070, 2964,
cyclo(3-Me t h ylsa lige n yl)-5′-O-(E )-5-(2-b r om ovin yl)-
2′-d eoxy-3′-O-D-isoleu cin ylu r id in yl)p h osp h a t e (3-Me-
cycloSa l-3′-D-Ile-BVDUMP ) 4i: yield 25%; 1H NMR (500
MHz, DMSO-d6) δ 11.65 (s, 2H, NH-BVU); 8.03 (s, 4H, NH2-
Ile); 7.85 (s, 1H, H6); 7.84 (s, 1H, H6); 7.29 (d, 1H, H8); 7.28
(d, 1H, H8); 7.25-7.21 (m, 2H, H5-aryl); 7.08-7.06 (m, 4H,
H4-aryl, H6-aryl); 6.84 (d, 1H, H7); 6.83 (d, 1H, H7); 6.19 (dd,
1H, H1′); 6.18 (dd, 1H, H1′); 5.52-5.38 (m, 4H, H-benzyl);
5.35-5.30 (m, 2H, H3′); 4.47-4.33 (m, 4H, H5′); 4.27-4.24 (m,
2H, H4′); 3.97-3.93 (m, 2H, H2-Ile); 2.45-2.35 (m, 4H, H2′);
2.20 (s, 3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-C3-aryl); 1.86-1.84
(m, 2H, H3-Ile); 1.50-1.40 (m, 2H, H4A-Ile); 1.29-1.20 (m, 2H,
H4B-Ile); 0.91-0.86 (m, 12H, H5-Ile, H6-Ile); 13C NMR (101
MHz, DMSO-d6) δ 168.72 (C1-Ile); 161.72 (C4); 149.33 (C2);
139.30 (C6); 139.20 (C6); 131.10 (C4-aryl); 129.70 (C7); 127.02

5170 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Meier et al.
(C3-aryl); 126.96 (C3-aryl); 124.23 (C5-aryl); 124.22 (C5-aryl);
123.74 (C6-aryl); 123.72 (C6-aryl); 121.10 (d, C1-aryl); 121.02
(d, C1-aryl); 110.53 (C5); 110.47 (C5); 107.39 (C8); 85.13 (C4′);
85.09 (C1′); 75.18 (C3′); 75.11 (C3′); 68.75 (d, C5′); 68.71 (d,
C5′); 67.51 (d, C-benzyl); 67.30 (d, C-benzyl); 56.52 (C2-Ile);
36.35 (C2′); 36.24 (C2′); 25.34 (C4-Ile); 15.08 (CH3-C3-aryl);
14.99 (CH3-C3-aryl); 14.48 (C4-Ile); 11.61 (C5-Ile); 31P NMR
(162 MHz, DMSO-d6) δ -7.79; -7.87; MS (FAB) m/z 628.5;
630.5 (M + H+); UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm;
λmin 268.8 nm, 224.7 nm; IR (KBr) ν 3436, 3261, 3249, 3180,
3068, 2967, 2931, 2884, 2854, 1685, 1639, 1596, 1467, 1284,
1201, 1132, 1056, 1024, 944, 800, 721, 657, 532; Rf value 0.32
(CH2Cl2/MeOH, 9:1); analytical HPLC tR 12.03 min (94%,
gradient III).
5.30 (m, 2H, H3′); 4.47-4.33 (m, 6H, H5′, H2-Pro); 4.27-4.24
(m, 2H, H4′); 3.25-3.19 (m, 4H, H5-Pro); 2.45-2.39 (m, 4H,
H2′); 2.28-2.17 (m, 4H, H3-Pro); 2.20 (s, 3H, CH3-C3-aryl);
2.18 (s, 3H, CH3-C3-aryl); 1.93-1.86 (m, 2H, H4-Pro); 13C NMR
(126 MHz, DMSO-d6) δ 168.40 (C1-Pro); 161.70 (C4); 149.33
(C2); 139.31 (C6); 139.21 (C6); 131.12 (C4-aryl); 131.08 (C4-
aryl); 129.67 (C7); 127.03 (C3-aryl); 127.00 (C3-aryl); 124.21
(C5-aryl); 123.73 (C6-aryl); 123.71 (C6-aryl); 121.11 (d, C1-
aryl); 121.04 (d, C1-aryl); 110.50 (C5); 110.44 (C5); 107.37 (C8);
85.19 (C4′); 85.15 (C4′); 81.82 (C1′); 81.70 (C1′); 75.55 (C3′);
75.45 (C3′); 68.72 (d, C5′); 68.63 (d, C5′); 67.64 (d, C-benzyl);
67.46 (d, C-benzyl); 58.83 (C2-Pro); 45.85 (C5-Pro); 36.07 (C2′);
35.90 (C2′); 27.76 (C3-Pro); 23.18 (C4-Pro); 15.08 (CH3-C3-
aryl); 14.99 (CH3-C3-aryl); 31P NMR (162 MHz, DMSO-d6) δ
-7.77; -7.80; MS (FAB) m/z 612.1; 614.1 (M + H+); UV (H2O/
CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm, 224.7 nm; IR
(KBr) ν 3424, 3178, 3066, 3029, 2992, 2962, 2823, 1749, 1687,
1594, 1467, 1367, 1292, 1232, 1197, 1130, 1052,1024, 1006,
944, 827, 798, 773, 719, 659, 430; Rf value 0.70 (CH2Cl2/MeOH
9:1); analytical HPLC tR 12.03 min (94%, gradient III).
cyclo(3-Me t h ylsa lige n yl)-5′-O-(E )-5-(2-b r om ovin yl)-
2′-d eoxy-3′-O-L-p h en yla la n ylu r id in yl)p h osp h a te (3-Me-
cycloSa l-3′-L-P h e-BVDUMP ) 4j: yield 61%; 1H NMR (500
MHz, DMSO-d6) δ 7.85 (s, 1H, H6); 7.84 (s, 1H, H6); 7.30-7.0
(m, 18H, H8, H4-aryl, H5-aryl,H6-aryl, H-aryl-Phe); 6.83 (d,
1H, H7); 6.81 (d, 1H, H7); 6.07-6.03 (m, 2H, H1′); 5.51-5.32
(m, 4H, H-benzyl); 5.14-5.08 (m, 2H, H3′); 4.32-4.22 (m, 4H,
H5′); 3.91-3.88 (m, 2H, H2-Phe); 3.77-3.73 (m, 2H, H4′);
2.92-2.83 (m, 4H, H3-Phe); 2.41-2.23 (m, 4H, H2′); 2.20 (s,
3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-C3-aryl); 13C NMR (126
MHz, DMSO-d6) δ 168.15 (C1-Phe); 160.96 (C4); 148.23 (C2);
139.26 (C6); 139.12 (C6); 131.14 (C4-aryl); 131.08 (C4-aryl);
130.12 (C4-Phe); 129.73 (C7); 129.42 (C5-Phe); 128.43 (C6-
Phe); 126.98 (C3-aryl); 126.76 (C3-aryl); 124.21 (C5-aryl);
123.73 (C6-aryl); 121.16 (C1-aryl); 110.47 (C5); 110.43 (C5);
107.33 (C8); 84.97 (C4′, C1′); 73.69 (C3′); 68.83 (C5′); 68.71
(C5′); 49.02 (C2-Phe); 35.67 (C2′); 30.78 (C3-Phe); 15.08 (CH3-
C3-aryl); 31P NMR (162 MHz, DMSO-d6) δ -7.83, -7.85; MS
(FAB) m/z 662.7; 630.7 (M + H+); UV (H2O/CH3CN) λmax 289.6
nm, 248.0 nm; λmin: 268.8 nm, 224.7 nm; IR (KBr) ν 3434,
3261, 3228, 3208, 3197, 3185, 3170, 3062, 3029, 2985, 2964,
2937, 2854, 2832, 1752, 1687, 1631, 1596, 1467, 1286, 1230,
1199, 1132, 1054, 1024, 1004, 944, 800, 759, 721, 659, 532; Rf
value 0.51 (CH2Cl2/MeOH, 7:3); analytical HPLC tR 12.03 min
(94%, gradient III).
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxy-3′-O-D-p r olin ylu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-D-P r o-BVDUMP ) 4m : yield 43%; 1H NMR (500 MHz,
DMSO-d6) δ 11.65 (s, 2H, NH-BVU); 9.25 (s, 2H, NH2-Pro);
7.86 (s, 1H, H6); 7.85 (s, 1H, H6); 7.29 (d, 1H, H8); 7.28 (d,
1H, H8); 7.25-7.21 (m, 2H, H5-aryl); 7.08-7.06 (m, 4H, H4-
aryl, H6-aryl); 6.83 (d, 1H, H7); 6.81 (d, 1H, H7); 6.20-6.16
(m, 2H, H1′); 5.52-5.35 (m, 4H, H-benzyl); 5.32-5.27 (m, 2H,
H3′); 4.47-4.33 (m, 6H, H5′, H2-Pro); 4.27-4.24 (m, 2H, H4′);
3.25-3.16 (m, 4H, H5-Pro); 2.45-2.39 (m, 4H, H2′); 2.28-2.17
(m, 4H, H3-Pro); 2.20 (s, 3H, CH3-C3-aryl); 2.18 (s, 3H, CH3-
C3-aryl); 1.93-1.86 (m, 2H, H4-Pro); 13C NMR (126 MHz,
DMSO-d6) δ 168.53 (C1-Pro); 161.78 (C4); 161.75 (C4); 149.41
(C2); 149.37 (C2); 148.05 (d, C2-aryl); 147.90 (d, C2-aryl);
139.35 (C6); 139.27 (C6); 131.13 (C4-aryl); 131.09 (C4-aryl);
129.70 (C7); 127.05 (C3-aryl); 126.97 (C3-aryl); 124.22 (C5-
aryl); 123.75 (C6-aryl); 123.73 (C6-aryl); 121.14 (d, C1-aryl);
121.03 (d, C1-aryl); 110.53 (C5); 110.48 (C5); 107.41 (C8); 85.10
(C4′); 85.05 (C4′); 81.82 (C1′); 81.70 (C1′); 75.45 (C3′); 75.37
(C3′); 69.79 (C5′); 68.67 (C-benzyl); 68.60 (C-benzyl); 58.87 (C2-
Pro); 58.81 (C2-Pro); 45.88 (C5-Pro); 45.74 (C5-Pro); 35.95
(C2′); 27.86 (C3-Pro); 27.82 (C3-Pro); 23.26 (C4-Pro); 15.07
(CH3-C3-aryl); 14.98 (CH3-C3-aryl); 31P NMR (162 MHz,
DMSO-d6) δ -7.89; -7.83; (FAB) m/z 612.1, 614.1 (M + H+);
UV (H2O/CH3CN) λmax 289.6 nm, 248.0 nm; λmin 268.8 nm,
224.7 nm; IR (KBr) ν 3424, 3178, 3066, 3029, 2992, 2962, 2823,
1749, 1687, 1594, 1467, 1367, 1292, 1232, 1197, 1130, 1052,
1024, 1006, 944, 827, 798, 773, 719, 659, 430; Rf value 0.70
(CH2Cl2/MeOH, 9:1); analytical HPLC tR 12.03 min (94%,
gradient III).
Deter m in a tion of th e P a r tition Coefficien ts (log P
Va lu es). Log P values were determined as follows: a sample
of the compounds 2, 3, or 4 was dissolved in 0.3 mL of
1-octanol. To this solution were added 0.3 mL of water. This
mixture was vigorously vortexed for 10 min, and the two
phases were separated by centrifugation (2 min at 13400 rpm).
Aliquots were analyzed by analytical HPLC (Merck LiChro-
CART column, LiChrospher 100 reversed-phase silica gel RP-
18 endcapped (5 µm), gradient 0-100% CH3CN in water (0-
20 min), 100% CH3CN (20-22 min), 0% CH3CN in water
(22.1-35 min), flow 0.5 mL, UV detection at 250 nm). The P
values were calculated by integration of the peaks of the
aqueous and organic phase.
cyclo(3-Me t h ylsa lige n yl)-5′-O-(E )-5-(2-b r om ovin yl)-
2′-d eoxy-3′-O-D-p h en yla la n ylu r id in yl)p h osp h a te (3-Me-
1
cycloSa l-3′-D-P h e-BVDUMP ) 4k : yield 50%; H NMR (500
MHz, DMSO-d6) δ 11.65 (s, 2H, NH-BVU); 9.24 (s, 4H, NH2-
Phe); 7.80 (s, 2H, H6); 7.30-7.0 (m, 18H, H8, H4-aryl, H5-
aryl, H6-aryl, H-aryl-Phe); 6.82 (d, 1H, H7); 6.80 (d, 1H, H7);
6.05 (dd, 1H, H1′); 6.04 (dd, 1H, H1′); 5.43 (m, 4H, H-benzyl);
5.19 (m, 2H, H3′); 4.32 (m, 4H, H5′); 4.27 (m, 2H, H4′); 4.11
(s, 2H, H2-Phe); 3.61 (m, 4H, H3-Phe); 2.25 (m, 4H, H2′); 2.19
(s, 3H, CH3-C3-aryl); 2.17 (s, 3H, CH3-C3-aryl); 13C NMR (101
MHz, DMSO-d6) δ 168.15 (C1-Phe); 161.77 (C4); 149.34 (C2);
139.34 (C6); 139.23 (C6); 131.15 (C4-aryl); 131.10 (C4-aryl);
129.74 (C4-Phe); 129.73 (C7); 129.74 (C5-Phe); 128.82 (C6-
Phe); 127.06 (C3-aryl); 126.98 (C3-aryl); 124.22 (C5-aryl);
123.75 (C6-aryl); 121.12 (C1-aryl); 110.53 (C5); 110.49 (C5);
107.40 (C8); 85.02 (C4′); 84.95 (C1′); 75.08 (C-benzyl); 74.83
(C-benzyl); 73.50 (C3′); 73.18 (C3′); 68.81 (C5′); 68.68 (C5′);
53.69 (C2-Phe); 53.45 (C2-Phe); 36.17 (C2′); 35.71 (C2′); 35.68
(C3-Phe); 35.53 (C3-Phe); 15.08 (CH3-C3-aryl); 15.00 (CH3-C3-
aryl); 31P NMR (202 MHz, DMSO-d6) δ -7.84; -7.91; MS
(FAB) m/z 662.7; 630.7 (M + H+); UV (H2O/CH3CN) λmax 289.6
nm, 248.0 nm; λmin 268.8 nm, 224.7 nm; IR (KBr) ν 3434, 3261,
3228, 3208, 3197, 3185, 3170, 3062, 3029, 2985, 2964, 2937,
2854, 2832, 1752, 1687, 1631, 1596, 1467, 1286, 1230, 1199,
1132, 1054, 1024, 1004, 944, 800, 759, 721, 659, 532; Rf value
0.51 (CH2Cl2/MeOH, 9:1); analytical HPLC tR 12.03 min (94%,
gradient III).
Kin etic Da ta . (a ) Aqu eou s Bu ffer s. 12 µL of DMSO stock
solutions (50 mM) of the triesters were diluted in 300 µL of
water or water/DMSO (c ) 2.0 mM). 0.3 mL of this solution
were added to 0.3 mL of aqueous buffer (50 mM phosphate
buffer, pH 7.3 or 50 mM phosphate buffer, pH 6.8) containing
5 µL of an aqueous AZT solution (AZT as internal standard)
at 37 °C. The final concentrations were 0.96 mM for the
triesters and 25 mM for the aqueous buffer. Aliquots of 60 µL
of the hydrolysis mixture were taken, and the hydrolysis was
stopped by addition of 5 µL of glacial acetic acid and frozen in
liquid air. After being thawed, samples were analyzed by
cyclo(3-Meth ylsa ligen yl)-5′-O-(E)-5-(2-br om ovin yl)-2′-
d eoxy-3′-O-L-p r olin ylu r id in yl)p h osp h a te (3-Me-cycloSa l-
3′-L-P r o-BVDUMP ) 4l: yield 43%; 1H NMR (500 MHz,
DMSO-d6) δ 11.65 (s, 2H, NH-BVU); 9.25 (s, 2H, NH2-Pro);
7.85 (s, 1H, H6); 7.84 (s, 1H, H6); 7.29 (d, 1H, H8); 7.28 (d,
1H, H8); 7.25-7.21 (m, 2H, H5-aryl); 7.08-7.06 (m, 4H, H4-
aryl, H6-aryl); 6.83 (d, 1H, H7); 6.81 (d, 1H, H7); 6.19 (dd, 1H,
H1′); 6.17 (dd, 1H, H1′); 5.53-5.35 (m, 4H, H-benzyl); 5.32-

CycloSal-BVDUMP Pronucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5171
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analytical HPLC (Merck LiChroCART column, LiChrospher
100 reversed-phase silica gel RP-18 endcapped (5 µm); UV
detection at 250 nm). The hydrolysis of the compounds 2-4
was followed by integration of the peak areas in the HPLC
chromatograms. The rate constants k were determined from
slope of the logarithmic degradation curve. The half-lives (t1/2
were calculated by using the rate constants k.
)
(b) P 3HR-1 Cell Extr a ct. 1.5 mM stock solution of the
triesters in DMSO were prepared. 20 µL of this stock solution
was mixed with 100 µL of cell extract and 20 µL of a 70 mM
magnesium chloride solution. The hydrolysis process was
stopped after 8 h by addition of 300 µL of acidic methanol and
storage for 5 min at 0 °C. The mixtures were centrifuged by
13000 rpm for 10 min, and the supernatant was analyzed as
mentioned above.
(c) Hu m a n Ser a : The studies were performed as described
in (b) but instead of cell extracts 10% of human serum in
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An ti-EBV Eva lu a tion . (a ) Cell Cu ltu r es. The EBV
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were grown in RPMI-1640 medium supplemented with 10%
heat-inactivated FCS, L-glutamine and antibiotics at 36.5 °C
in a humidified 5% CO2-containing atmosphere. The latter
three cell lines served only as controls in the hybridization
technique. Ramos is an EBV-negative cell line. Namalwa is
an EBV genome carrying cell line with two EBV genome copies
per cell. Raji cells are also EBV positive with about 30-60
EBV genome copies per cell. After hybridization with an EBV
specific probe and detection Ramos cells give no signal,
Namalwa cells give a very weak signal and Raji cells give a
moderate signal.
(b) Exp osu r e of P 3HR-1 Cells to Dr u gs. Exponentially
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(c) DNA Isola tion . Control and drug treated cells were
pelleted, washed twice with PBS, and resuspended in 200 µL
of PBS. DNA was extracted using a DNA extraction kit
(QIAamp DNA Blood Minikit, Qiagen). DNA concentration was
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(d ) Slot Blot Hybr id iza tion . Ten micrograms of total
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the Bam H1-W-fragment of the EBV genome. After hybrid-
ization chemiluminescence detection was carried out followed
by 30 min exposition to a Kodak film. The amount of EBV DNA
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and nontreated P3HR-1 cells and the 50% effective concentra-
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(e) Deter m in a tion of CC50 for Cell Gr ow th . P3HR-1 cells
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mL in 96-well plates. Cell numbers were determined using a
Coulter Z-2 particle counter and 50% inhibitory concentrations
for cell growth (CC50) were calculated.
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J M0209275