Novel cycloSal nucleotides with reduced inhibitory potency toward human butyrylcholinesterase

Article abstract of DOI:10.1081/NCN-200061791

Two novel cycloSal-d4T monophosphates (d4TMPs) with increased stem demand have been synthesized via a new synthetic route. While 3-cyclohexyl-cycloSal d4TMP did not show a significantly reduced inhibitory potency toward human butyrylcholinesterase, the opposite was the case for the second novel pronucleotide, bis-(cycloSal-d4TMP). Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200061791

This article was downloaded by: [Michigan State University]
On: 26 December 2014, At: 22:18
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
NOVEL CYCLOSAL NUCLEOTIDES WITH REDUCED
INHIBITORY POTENCY TOWARD HUMAN
BUTYRYLCHOLINESTERASE
C. Ducho a , S. Jessel a , N. Gisch a , J. Balzarini b & C. Meier a
a Institute of Organic Chemistry, University of Hamburg , Hamburg, Germany
b Rega Institute for Medical Research, Katholieke Universiteit , Leuven, Belgium
Published online: 15 Nov 2011.
To cite this article: C. Ducho , S. Jessel , N. Gisch , J. Balzarini & C. Meier (2005) NOVEL CYCLOSAL NUCLEOTIDES WITH
REDUCED INHIBITORY POTENCY TOWARD HUMAN BUTYRYLCHOLINESTERASE, Nucleosides, Nucleotides and Nucleic Acids,
24:5-7, 519-522, DOI: 10.1081/NCN-200061791
To link to this article: http://dx.doi.org/10.1081/NCN-200061791
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):519–522, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200061791
NOVEL CYCLOSAL NUCLEOTIDES WITH REDUCED INHIBITORY
POTENCY TOWARD HUMAN BUTYRYLCHOLINESTERASE
5
C. Ducho, S. Jessel, and N. Gisch
Institute of Organic Chemistry, University of
Hamburg, Hamburg, Germany
5
J. Balzarini
Rega Institute for Medical Research, Katholieke Universiteit, Leuven,
Belgium
5
C. Meier
Institute of Organic Chemistry, University of Hamburg, Hamburg, Germany
5
Two novel cycloSal-d4T monophosphates (d4TMPs) with increased steric demand have been
synthesized via a new synthetic route. While 3-cyclohexyl-cycloSal d4TMP did not show a significantly
reduced inhibitory potency toward human butyrylcholinesterase, the opposite was the case for the second
novel pronucleotide, bis-( cycloSal-d4TMP).
Keywords Pronucleotides, cycloSal, Butyrylcholinesterase, Antiviral Activity
INTRODUCTION
The cycloSal pronucleotide system has been developed for an intracellular
delivery of therapeutically active nucleoside monophosphates (NMPs) and has
already been applied to different nucleoside analogues successfully, e.g., the anti-
HIV active 3’-deoxy-2’,3’-didehydrothymidine (d4T) 1.[1] Recently, the interaction
of cholinesterases with cycloSal nucleotides has been reported. While no inhibition
of the physiologically essential acetylcholinesterase (AChE, E.C. 3.1.1.7) has been
observed,[2] a structure-activity relationship has been obtained for butyrylcholines-
terase (BChE, E.C. 3.1.1.8).[3] As the inhibition of BChE is an unwanted effect and
could become a hurdle in the application of cycloSal derivatives in antiviral chemo-
therapy, ways to overcome this effect are needed. One promising approach is
based on the thought that bulky substituents in the aryl moiety of the cycloSal system
could prevent the pronucleotides from binding in the active site of BChE due to
steric repulsion. Hence, novel cycloSal nucleotides with increased steric demand
were prepared. We already reported on 3,5-bis-tertbutyl-6-fluoro-cycloSal-d4TMP 2,
Address correspondence to C. Ducho, Institute of Organic Chemistry, University of Hamburg, Martin-
Luther-King-Platz 6, Hamburg D-20146, Germany.
519
Order reprints of this article at www.copyright.rightslink.com

520
C. Ducho et al.
FIGURE 1 Compound 2, a derivative of d4T 1, the two target structures 3 and 4, and the reference compounds
5–7.
a pronucleotide with strongly reduced inhibitor activity toward human BChE.[4]
Here, we present two novel cycloSal nucleotides, 3-cyclohexyl-cycloSal-d4TMP 3 and
bis-(cycloSal-d4TMP) 4 and compare them to the known reference compounds 5–
7[1,3] (Figure 1).
RESULTS
For the synthesis of 3 and 4, a new synthetic route via 3-bromo salicyl alcohol
isopropylidene acetal 8 as the key intermediate has been established (Figure 2).
Compound 8 was synthesized starting from 2-bromophenol 9, which was
converted to 3-bromo salicyl alcohol 10 in a two-step procedure with an overall
yield of 53%. Alcohol 10 was protected as isopropylidene acetal to afford 8 in 99%
yield. For the synthesis of 3, intermediate 8 was reacted in a Grignard cross-
FIGURE 2 Synthesis of target compounds 3 and 4.

C. Ducho et al.
521
TABLE 1 Properties of Target Compounds 3, 4 Compared to Reference cycloSal-d4TMPs
Anti-HIV activityc
(EC50, mM)
Compound
Hydrolysis half-lifea (h)
BChE inhibitionb (IC50, mM)
CEM/O
CEM/TKÀ
5
7
4.4
5.1
8.2
3.2
8.5
17
0.77
0.35
40
0.13
0.27
0.27
n.d.
n.d.
0.08
n.d.
0.19
0.30
0.15
2.3
n.d.
n.d.
0.08
n.d.
22
4 mix
4 fast
4 slow
6
14
>50
1.2
3.1
–
3
25
–
d4T 1
aHydrolytic stability in phosphate buffer, pH 7.3, 37°C.
bCholinesterase assay using human serum as source of BChE activity, procedure as published before. (From
Ref. [3].)
cEffective concentration to protect CEM wild-type or CEM thymidine kinase (TK)-deficient cells against the
cytopathogenicity of HIV-2 by 50%; n.d. = not determined yet.
coupling reaction to yield, after deprotection, 3-cyclohexyl salicyl alcohol 11 (34%
over two steps). Compound 11 was subsequently converted to target compound 3
(mixture of two diasteromers) using chlorophosphite chemistry as described
before[1,3,4] (30% yield). For the synthesis of 4, intermediate 8 was converted in a
homo-coupling reaction. After deprotection, alcohol 12 was isolated in 24% yield.
Again, target compound 4 could be obtained from 12 using the chlorophosphite
approach. Due to difficult purification, 4 (mixture of three diastereomers) could
only be isolated in 8% yield. Partial separation of the diastereomeric mixture of 4
was possible using preparative RP-HPLC to afford a fast fraction (one
diastereomer) and a slow fraction (mixture of two diastereomers).
The fast and the slow fraction of 4 displayed significantly different hydrolytic
stabilities (Table 1). While 4 fast (t1/2 = 3.2 h) was less stable than the unsubstituted
cycloSal-d4TMP 5 (t1/2 = 4.4 h), 4 slow was about 2-fold more stable (t1/2 = 8.5 h).
Both the fast and the slow fraction showed a significantly reduced inhibitory
potency toward human BChE as compared to phosphate triester 5, fast about 18-
fold, and slow more than 65-fold lower. In comparison to 3-phenyl-cycloSal-d4TMP
7, this effect was even more pronounced with IC50 values about 40-fold lower for
4 fast and more than 140-fold for 4 slow. Consequently, 4 slow actually is a non-
inhibitor of BChE. The diastereomeric mixture 4 mix displayed an anti-HIV activity
in wild-type CEM cells comparable to that of d4T and the unsubstituted prototype,
but in thymidine kinase (TK)-deficient CEM cells it turned out to lose activity about
9-fold in comparison to the data obtained in the wild-type cells. This points to an
insufficient TK bypass, possibly a result of bad membrane penetration due to the
highly increased size of the molecule and/or its polarity. On the other hand, the
antiviral activity of dimer 4 in the CEM/TKÀ cells still was about 10-fold better than
that of d4T 1, which proves a partial intracellular delivery of d4TMP.

522
C. Ducho et al.
In contrast to 4, derivative 3 only showed a 4-fold reduction of inhibitor activity
toward BChE as compared to the unsubstituted prototype 5 and 2.5-fold as
compared to the 3-methyl derivative 6, while its hydrolysis half-life (t1/2 = 25 h) is in
the same range than that of 3-methyl-cycloSal-d4TMP 6 (t1/2 = 17 h). On the other
hand, 3 turned out to be about 9-fold less inhibitory to BChE than the structural
similar 3-phenyl derivative 7.
In conclusion, bis-cycloSal nucleotides like 4 may become an interesting new
class of cycloSal prodrugs as they display significantly reduced inhibitory activity
toward human BChE and have a mask–drug ratio of 1:2. Further work has to be
done in order to optimize the antiviral properties of 4, eventually by increasing its
hydrolytic stability.
REFERENCES
1. Meier, C. CycloSal-pronucleotides—design of chemical Trojan horses. Mini Rev. Med. Chem. 2002, 2, 219–
234.
2. Ducho, C.; Balzarini, J.; Meier, C. Non-inhibition of acetylcholinesterase by cycloSal nucleotides. Nucleosides
Nucleotides 2003, 22, 841–843.
3. Meier, C.; Ducho, C.; Go¨rbig, U.; Esnouf, R.; Balzarini, J. Interaction of cycloSal-pronucleotides with
cholinesterases from different origins. A structure-activity relationship. J. Med. Chem. 2004, 47, 2839–2852.
4. Ducho, C.; Wendicke, S.; Go¨rbig, U.; Balzarini, J.; Meier, C. 3,5-Di-( tert-butyl)-6-fluoro-cycloSal-d4TMP—a
pronucleotide with a considerably improved masking group. Eur. J. Org. Chem. 2003, 4786–4791.