Synthesis, hydrolysis and anti-EBV activity of a series of 3′-modified cycloSal-BVDUMP pronucleotides

Article abstract of DOI:10.1081/NCN-100002301

A series of cycloSal-BVDUMP phosphate triesters has been prepared. The prototype compound was 3-methyl-cycloSal-BVDUMP 2. Furthermore, a series of 3′-O-acyl-modified derivatives having carboxylic acids with different lipophilicity or a L-configurated α-am

Full text of DOI:10.1081/NCN-100002301

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SYNTHESIS, HYDROLYSIS AND ANTI-EBV ACTIVITY
OF A SERIES OF 3′-MODIFIED cycloSal-BVDUMP
PRONUCLEOTIDES
C. Meier a , A. Lomp a , A. Meerbach b & P. Wutzler b
a University of Hamburg, Institute of Organic Chemistry , Martin-Luther-King-Platz 6,
Hamburg, D 20146, Germany
b Institute for Antiviral Chemotherapy, Friedrich-Schiller-University Jena , Nordhäuser
Straße 78, Erfurt, D-99089, Germany
Published online: 07 Feb 2007.
To cite this article: C. Meier , A. Lomp , A. Meerbach & P. Wutzler (2001) SYNTHESIS, HYDROLYSIS AND ANTI-EBV ACTIVITY
OF A SERIES OF 3′-MODIFIED cycloSal-BVDUMP PRONUCLEOTIDES, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7,
307-314, DOI: 10.1081/NCN-100002301
To link to this article: http://dx.doi.org/10.1081/NCN-100002301
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 307–314 (2001)
SYNTHESIS, HYDROLYSIS AND ANTI-EBV
ACTIVITY OF A SERIES OF 3ꢀ-MODIFIED
cycloSal-BVDUMP PRONUCLEOTIDES
C. Meier,1,∗ A. Lomp,1 A. Meerbach,2 and P. Wutzler2
1University of Hamburg, Institute of Organic Chemistry,
Martin-Luther-King-Platz 6, D 20146 Hamburg, Germany
2Institute for Antiviral Chemotherapy, Friedrich-Schiller-University
Jena, Nordha¨user Straße 78, D-99089 Erfurt, Germany
ABSTRACT
A series of cycloSal-BVDUMP phosphate triesters has been prepared. The
prototype compound was 3-methyl-cycloSal-BVDUMP 2. Furthermore, a se-
ries of 3ꢀ-O-acyl-modified derivatives having carboxylic acids with different
lipophilicity or a L-configurated α-amino acid (phenylalanine) was prepared.
The hydrolysis properties in phosphate buffer PBS as well as in PBS containing
pig liver esterase (PLE) will be described. Finally, the biological activity against
EBV has been determined.
INTRODUCTION
Nucleosides are extensively used as antiviral or antitumor agents (1,2). The
currently used nucleoside analogues against HIV in the clinic are 3ꢀ-azidothymidine
(AZT), 2ꢀ,3ꢀ-dideoxy-2ꢀ,3ꢀ-didehydrothymidine(d4T), 2ꢀ,3ꢀ-dideoxycytidine(ddC),
2ꢀ,3ꢀ-dideoxyinosine (ddI) as well as 3ꢀ-thiacytidine (3TC) and the recently ap-
proved abacavir (1592U89). The general motif of these nucleoside analogues is a
modified glycon residue as compared to the natural nucleosides. Beside the use
against HIV, nucleosides are also used against herpes virus infections. Examples
are the acyclic guanine bearing nucleoside analogue acyclovir (ACV) which is
∗Corresponding author.
307
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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MEIER ET AL.
used against herpes-simplex virus as well as (E)-5-(2- bromovinyl)-2ꢀ-deoxyuridine
(BVDU) against varicella-zoster virus which is the causative agent of chickenpox
and shingles.
The major problem in the use of nucleoside analogues as antiviral agents is
the prerequisite of being activated into their nucleoside triphosphates by three suc-
cessive phos-phorylation steps. These phosphorylations are achieved by cellular
or viral enzymes. Very often the exact procedure of bioactivation is not known
and therefore one should hope for a newly developed nucleoside analogue that
an efficient metabolism will take place (“black-box” bioactivation). However, for
a few nucleoside analogues the metabolism has been studied in detail and it has
been recognized that often the first conversion of the nucleoside analogue into its
monophosphate is the rate limiting step. This has been shown for d4T as well as
for ddA/ddI. Nevertheless, these nucleoside analogues exert their antiviral activ-
ity although the potential is not used efficiently. In an extreme case the nucleo-
side analogue can not be converted into its monophosphate and consequently no
di- and triphosphate formation takes place and hence the compound is biologically
absolutely inactive (e.g. ddU). In order to circumvent this limitation we and other
have developed the so-called pronucleotide-concept (3). The basic idea is to develop
a lipophilic precursor of the highly polar nucleoside monophosphate (nucleotide)
that is able to penetrate the cell membrane and release the nucleotide efficiently
under specific conditions (enzymatically or chemically) (Fig 1). In this context we
designed the cycloSal-pronucleotides approach (4,5).
This concept has been introduced to deliver the nucleotide by means of chem-
ical hydrolysis (5). The concept has been applied successfully to the intracellu-
lar delivery of d4TMP which lead to the thymidine-kinase(TK)-bypass in TK-
deficient lymphocytes (6,7). Additionally, this concept has been used to achieve
the adenosine-deaminase(ADA)-bypass for the nucleoside analogues ddA (8), d4A
(8,9) and 2ꢀ-F-ara-ddA (10). Interestingly, the cycloSal-masking technique is able
to convert an inactive nucleoside analogue into an active compound. This has been
demonstrated by using 2ꢀ-F-ribo-ddA (10,11). On the other side, the cycloSal-
concept was unable to restore the antiviral activity of AZT in TK-deficient CEM
cells due to the delivery of AZTMP although we have clearly shown that again
Figure 1. Metabolism of nucleosides and the effect of the corresponding pronucleotide.

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3ꢀ-MODIFIED cycloSal-BVDUMP PRONUCLEOTIDES
309
AZTMP was liberated from the lipophilic precursor triester in chemical hydrolysis
studies (12). The reason for this failure has been found in a special metabolism of
AZTMP (13). The first non-HIV directed example was the use of ACV in this con-
cept (14). ACV is not activated by a cellular kinase but by the viral herpes-simplex
thymidine-kinase. By preparing cycloSal-ACVMP compounds the antiviral activ-
ity of ACV could be regained in thymidine-kinase deficient virus lines (14,15).
Here we report on the application of the cycloSal-methodology to the nucleoside
analogue (E)-5-(2-bromovinyl)-2ꢀ-deoxyuridine 1 (BVDU or Brivudin).
BVDU 1 (16) is a potent and highly selective inhibitor of the replication of
several herpes viruses (e.g. HSV-1 and VZV but not HSV-2) (17). BVDU 1 has
been reported to inhibit also EBV infections (18) but these results could not be
reproduced by our laboratories (19). The selectivity as inhibitor primarily depends
upon a specific activation by the virus encoded thymidine-kinase (TK) to the mono-
and diphosphate and finally by cellular enzymes to the triphosphate. In its triphos-
phate BVDU can act either as an inhibitor of the cellular DNA polymerase or
alternate substrate which would render the DNA more prone to degradation when
incorporated in DNA (20). Some limitations for the use of BVDU as antiviral agent
are known. There is a lack of activity during virus latency because of missing
viral TK. Moreover, drug resistant virus strains are known. Additionally, BVDU
will be degraded from the bloodstream within 2–3 hours by pyrimidine nucleoside
phosphorylase to the free pyrimidine nucleobase E-5-(2-bromovinyl)uracil (BVU)
(20). Our aim in this study was to use the cycloSal-masking technology in order to
broaden the application of BVDU 1 into the field of EBV associated lymphoprolif-
erative disorders in immunosuppressed patients (21). It should be added that to our
knowledge only two attempts have been published to develop prodrugs of BVDU
1. Both were not successful (22,23).
RESULTS AND DISCUSSION
Here we report on the preparation of a series of 3ꢀ-unmodified (2) as well as 3ꢀ-
O-modified cycloSal-BVDUMP derivatives (3–6). As 3ꢀ-modification carboxylic
acids with different lipophilicity (3,4) as well as an α-amino acid (L-phenylalanine,
5) have been used. Furthermore, the 3ꢀ-O-methyl ether derivative (6) of the proto-
type triester 2 has been prepared. In all cases we prepared the 3-methyl-substituted
cycloSal-BVDUMP triester because this substituent gave the best antiviral results
so far (6,8–12,14,15). The target phosphate triesters 2–6 are shown in Figure 2.
The preparation of 3-methyl-cycloSal-BVDUMP 2 has been carried out by
direct phosphitylation of BVDU 1 using cyclic 3-methyl-cycloSal-chlorophosphite
in a DMF/THF (2:1) solvent mixture at −20◦C in the presence of diisopropy-
lethylamine (DIPEA) as base. The intermediate phosphite triester was oxidized
in the one-pot reaction procedure using t-butylhydroperoxide (TBHP) as pub-
lished before (6,9,11). Evaporation of the solvent and purification by silica gel
chromatography using the chromatotron technique gave the phosphate triester 2 in

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MEIER ET AL.
O
N
Br
HN
U5-BrVinyl
U5-BrVinyl
TBDMSO
HO
b (84%)
c,e (90 - 97%)
d,e (93%)
O
O
O
HO
O
OR
OH
1
7
8-11
OH
L-phenylalanine: 30%
carbox. acids: 50-60%
f,g
a (56%) R = H
O
O
U5-BrVinyl
P
O
O
O
O
O
P
Me
U5-BrVinyl
O
O
O
Me
OR
O
OH
O
O
2
R =
and CH3 (6)
CH3
NH2
Ac (3)
Hex (4)
L
-Phe (5)
a) chlorophosphite, DIPEA, DMF/THF 2:1, 4h, -20°C then TBHP, 30 min, rt; b) TBDMSCl, pyridine, 12h, rt;
c) carboxylic acid (N-Boc protected -phenylalanine), DCC, DMAP, THF, 3h, rt; d) NaH, CH3I, THF, 5h, rt;
L
e) 2% TBAF in THF, 3h, rt; f) chlorophosphite, DIPEA, 2h, rt; then TBHP, 15 min, CH3CN, rt; g) 5% TFA,
CH2Cl2/MeOH 7:3, 30 min, rt
Figure 2. Synthesis of the target 3-methyl-cycloSal-BVDUMP triesters 2–6.
56% yield. However, this reaction procedure was not suitable for the preparation
of the 3ꢀ-O-modified compounds 3–6.
Therefore, BVDU 1 was first protected selectively at the 5ꢀ-O-position by sily-
lation using t-butyldimethylsilylchloride (TBDMS) in pyridine to give 7 in 84%
yield. Next, compound 7 was treated with acetic acid, hexanoic acid and N-Boc
L-phenylalanine in the presence of DCC and DMAP in THF at room temperature
for 3 hours. Additionally, 7 was deprotonated using sodium hydride in THF and
subsequently methylated by addition of methyl iodide to give 5ꢀ-OTBDMS-3ꢀ-O-
methyl-BVDU in 80% yield (24). All derivatives were subsequently desilylated by
treatment with a 2% solution of tetra-n-butylammonium fluoride (TBAF) in THF.
By this reaction sequence the 3ꢀ-O-modified BVDU derivatives 8–11 were obtained
in 90–97% yield. Compounds 8–11 were converted into the target phosphate tri-
esters by 5ꢀ-O-phosphitylation using again 3-methyl-cycloSal-chlorophosphite and
subsequent oxidation with TBHP to yield 3, 4 and the N-Boc protected precursor
of triester 5. Here, the reactions were carried out in acetonitrile because the starting
nucleoside analogues 8–11 were soluable in this solvent in contrast to BVDU 1. In
the case of the L-phenylalanine modified 3-methyl-cycloSal-BVDUMP triester the
N-Boc protecting group was cleaved by treatment with 5% TFA in CH2Cl2/MeOH
(7:3) to give 5. The target phosphate triester 5 was obtained in 30% yield after
purification. Characterization of the triesters 2–6 was done by all means of NMR
spectroscopy as well as MS spectrometry. The purity was checked by HPLC anal-
ysis. The synthetic procedures are summarized in Figure 2.

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3ꢀ-MODIFIED cycloSal-BVDUMP PRONUCLEOTIDES
311
O
N
Br
HN
O
P
O
P
O
O
O
U5-BrVinyl
a) R = H
O
O
O
O
O
HO
Me
b)
OH
a)
H2O/OH-
OR
2-5
Me
OH
c) R = acyl
3Me-cycloSal-BVDUMP 2
spontaneous
reaction in aq. sol.
a)
a)
R = H
b)
b)
5’-BVDUMP 12
+
U5-BrVinyl
U5-BrVinyl
O
OH
O
O
O
O
b)
O
+
P
P
OH
Me
O
O
spontaneous
reaction
O
O
OH
Me
3’,5’-cBVDUMP 13
Figure 3. Hydrolysis pathways possible for the title phosphate triesters 2–5.
Next, we conducted a series of hydrolysis studies in order to prove the selective
delivery of BVDUMP 12 from the title phosphate triesters (pathway a, Fig 3).
In contrast to our work published before on the corresponding triesters of d4T,
ddA/d4A and 2ꢀ-F- ara/ribo-ddA (6, 9, 11), one should take into account that at least
in the case of the 3ꢀ-O-unmodified 3-methyl-cycloSal-BVDUMP 2 a nucleophilic
attack of the 3ꢀ-hydroxyl group at the phosphorus center may occur (pathway b,
Fig 3). This would lead to the formation of undesired 3ꢀ,5ꢀ-cyclic BVDUMP 13
(cBVDUMP). We observed such a formation of an cyclic phosphate diester before
in the case of the acyclic anti-herpetic agent penciclovir (25). This was also the
reason why we decided to acylate the 3ꢀ-hydroxyl group in BVDU 1. However,
in the case of a rapid deesterification by cellular (carboxy)esterases in the cells
this would lead back to triester 2 (pathway c, Fig 3), such a cyclisation reaction
to cBVDUMP should again be possible in addition to the BVDUMP formation.
Only triester 6 bearing the 3ꢀ-O-methyl ether should hydrolize only to yield the
corresponding 3ꢀ-OMe-BVDUMP. The hydrolysis pathway leading to BVDUMP
12 as well as the mentioned possible concurrence reaction are depicted in Figure 3.
First, we hydrolyzed triesters 2–6 in a 25 mM phosphate buffer, pH 7.3.
The reactions were followed by means of HPLC analysis. The hydrolysis reac-
tions in these studies followed clearly pseudo-first-order-kinetics. The common
and most important result of all reactions was that in all cases only BVDUMP 12
or the corresponding 3ꢀ-O-esterified BVDUMPs could be detected in the HPLC-
chromatograms. There was definately no formation of the mentioned cBVDUMP
13. This has been proven by separate preparation of 13 and subsequent coelution
with the hydrolysis mixture of 3-methyl-cycloSal-BVDUMP 2 as well as by mass

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MEIER ET AL.
spectrometry using an ESI-MS installation possessing an ion-trap, which allows
tandem-MS analysis.
Theprototypecompound3-methyl-cycloSal-BVDUMP2displayedahalf-life
of 6.7 h whereas the 3ꢀ-esterified triesters bearing a carboxylic acid showed half-
livesintherangeof11to27h. Thereseemstobeaclearrecognizabletrend:themore
lipophilic the carboxylic acid the more stable the phosphate triester. This may be due
to the interaction of the attacking hydroxide nucleophile with the highly lipophilic
3ꢀ-O-acyl residue which result in a kind of protection of the phosphorus atom
to nucleophilic attack. In contrast, the α-amino acid bearing cycloSal-BVDUMP
triester 5 displayed a clearly lower t1/2 value reflecting a lower hydrolytic stability in
the slightly basic phosphate buffer. This may be due to the unprotected amino group
present in the L-phenylalanine residue that may create a more polar surrounding
in the vicinity of the phosphate group which makes a nucleophilic attack more
probable. The half-lives are summarized in Table 1.
In order to study possible enzymatic contributions caused by intracellularly
present(carboxy)esterasesthetitlecompounds2–6weretreatedinphosphatebuffer,
pH 7.3 with 50 units of pig liver esterase (PLE) as a reasonable model for contribut-
ing enzymes. Again, the hydrolyses were followed by means of HPLC analysis.
The observed half-lives are also given in Table 1. Interestingly, the half-lives were
found to be considerably lower as compared to the studies in phosphate buffer
without PLE. More importantly, the hydrolysis product was in all cases the 3ꢀ-O-
deesterified prototype triester 2 which points to an efficient enzymatically driven
process. In the case of 3ꢀ-OAc-triester 3 we observed the formation of BVDUMP
12 from the initial product 3-methyl-cycloSal-BVDUMP 2. In all other hydrolyses
we were unable to detected the co-formation of the corresponding 3ꢀ-O-esterified
BVDUMP.
Table 1. LogP Values, Hydrolysis Data and Antiviral Activity of the Title Triesters
Hydrolysis Half-Life
EC50 [µg/ml]
LogP
Value
In Phosphate
Buffer pH 7.3 [h]
With 50 U PLE
[h]
DNA
Synthesis
Compound
CC50 [µg/ml]
2
3
4
5
6
0.67
1.16
>2.11
1.66
6.7a
11.3a
27.6a
1.2a
n.d.d
3.6b,c
2.1b
2.1
>50
>100
31.5
>50
63.0
61.0
>200
16.0
2.1b
1.43
8.0a
n.d.d
>100
ACV
BVDU 1
n.a.e
n.a.e
1.5
>100
88.3
75.0
0.33
n.a.e
n.a.e
EC50 : concentration required to reduce EBV DNA synthesis or VCA expression by 50%;
CC50: concentration required to reduce the growth of exponentially growing P3HR-1 cells by
50%; a) corresponding monophosphate; b) 3-Me-cycloSal-BVDUMP 2; c) BVDUMP 12; d) not
determined; e) not available.

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313
It is worth mentioning that the 3ꢀ-O-α-amino acid containing triester 5 was
cleaved to the same extent as compared to the 3ꢀ-O-carboxylic acid bearing deriva-
tives. It seems that PLE has no preference to cleave α-amino acid esters as compared
to esters formed from “normal” carboxylic acids. As a conclusion from these studies
it should be possible to cleave all 3ꢀ-O-ester groups readily by enzymatic deester-
ification as a main degradation pathway to yield the prototype 3ꢀ-O-unmodified
triester 2.
Inadditiontothehydrolyticpropertieswedeterminedthepartitioncoefficients
(logP) of the triesters in a n-octanol/water mixture (Table 1). As can be seen all
compounds 2–6 displayed considerably higher logP values as compared to the
parent nucleoside 1. Particularly, the logP of the 3ꢀ-OHex modified derivative 4 was
extremely high (logP > 2.11) so that one can not exclude micelle formation or lipid
film formation in aqueous solution. Such formation will influence the hydrolytic
behavior of the triester markedly.
The described phosphate triesters 2–6 were tested for their antiviral activity
against EBV in human lymphoblastoid P3HR-1 cells. The EC50 values as well as
the CC50 values are listed in Table 1. For comparison, the values of BVDU 1 and
ACV are given. From the data it can be seen that only the prototype compound 3-
methyl-cycloSal-BVDUMP 2 showed considerable antiviral activity against EBV.
When compared with the parent nucleoside BVDU 1, compound 2 was about a
100-fold more active (it should be added that 1 devoid of any antiviral activity
below 100 mg/ml). The antiviral potency of triester 2 was in the same order of
magnitude of the reference compound acyclovir (ACV). There was a striking loss in
activity observed for the 3ꢀ-O-L-phenylalanine derivative 5 (15-fold). The other 3ꢀ-
O-esterified compounds were completely inactive in the test assay. This observation
was somewhat surprising because during the above described hydrolysis studies no
general complication in the degradation has been observed. Therefore, the reasons
forthepronounced differencesinantiviral activityremainansecret. Moreover, these
results clearly show that in this presented study the use of chemical hydrolysis and
PLE catalyzed deesterification did not provide sufficient data in order to explain the
behavior of the compounds in cellular media. As a consequence, hydrolysis studies
in P3HR-1 cell extracts should be carried out. The data found in such experiments
may give a more realistic “inside” view of the cell and therefore the degradation of
the cyclosal-BVDUMP described herein.
Nevertheless, the encouraging of the presented study is that the antiviral data
of the prototype derivative 2 clearly demonstrate that by using the cycloSal-masking
technology an antivirally inactive nucleoside analogue like BVDU 1 can be con-
verted into a bioactive derivative against EBV. This will initiate further studies in
our laboratories with respect to new α-amino acid bearing cycloSal-BVDUMP tri-
esters as well as further substitution pattern in the cycloSal-masking group. Finally,
it should be mentioned that to the best of our knowledge the work presented here
represents the first example of a successful application of a pronucleotide strategy
to a nucleoside analogue that contains a free 3ꢀ-hydroxyl group.

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