Second generation of TSAO compounds directed against HIV-1 TSAO-resistant strains

Article abstract of DOI:10.1081/NCN-100002356

A second generation of TSAO molecules directed against TSAO-resistant strains have been prepared. The presence of two neighboring carbonyl groups at the 4″ position of the 3′-spiro moiety seems to be important for the anti-HIV-1 activity against both wild type and TSAO-resistant strains. NMR conformational studies in solution and theoretical calculations of the novel compounds have also been carried out.

Full text of DOI:10.1081/NCN-100002356

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“SECOND GENERATION” OF TSAO COMPOUNDS
DIRECTED AGAINST HIV-1 TSAO-RESISTANT STRAINS
E. Lobatón a , S. Velázquez a , M. J. Pérez-Pérez a , M. L. Jimeno a , A. San-Félix a , E. De
Clercq b , J. Balzarini b & M. J. Camarasa c
a Instituto de Química Médica (C.S.I.C.) , Madrid, Spain
b Rega Institute for Medical Research, Katholieke Universiteit Leuven , Belgium
c Instituto de Química Médica (C.S.I.C.) , Madrid, Spain
Published online: 07 Feb 2007.
To cite this article: E. Lobatón , S. Velázquez , M. J. Pérez-Pérez , M. L. Jimeno , A. San-Félix , E. De Clercq , J. Balzarini
& M. J. Camarasa (2001) “SECOND GENERATION” OF TSAO COMPOUNDS DIRECTED AGAINST HIV-1 TSAO-RESISTANT STRAINS,
Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 707-710, DOI: 10.1081/NCN-100002356
To link to this article: http://dx.doi.org/10.1081/NCN-100002356
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 707–710 (2001)
“SECOND GENERATION” OF TSAO COMPOUNDS
DIRECTED AGAINST HIV-1 TSAO-RESISTANT
STRAINS
E. Lobato´n,1 S. Vela´zquez,1 M. J. Pe´rez-Pe´rez,1 M. L. Jimeno,1
A. San-Fe´lix,1 E. De Clercq,2 J. Balzarini,2 and M. J. Camarasa1∗
1Instituto de Qu´ımica Me´dica (C.S.I.C.), Madrid, Spain
2Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Belgium
ABSTRACT
A “second generation” of TSAO molecules directed against TSAO-resist-
ant strains have been prepared. The presence of two neighboring carbonyl
groups at the 4ꢀꢀ position of the 3ꢀ-spiro moiety seems to be important for the
anti-HIV-1 activity against both wild type and TSAO-resistant strains. NMR
conformational studies in solution and theoretical calculations of the novel
compounds have also been carried out.
TSAO derivatives represent a particular and peculiar group of potent and
highly specific inhibitors of the human immunodeficiency virus type 1 (HIV-1)
replication that seem to interact at the interface between the p51 and p66 reverse
transcriptase (RT) subunits (1,2). Well-defined aminoacids at both p51 and p66
RT subunits are needed for an optimal interaction of TSAO compounds with the
HIV-1 RT (3). The prototype compound is the thymine derivative named TSAO-T
(1). The most selective compound of this series is TSAO-m3T (2) (Fig. 1). Selec-
tion of at least eight different TSAO-resistant strains in cell culture and molecular
characterization of these strains revealed in all cases a single aminoacid mutation at
position 138 (Glu138Lys). This mutation is important at the level of the p51 subunit
of HIV-1 RT (4). Our experimental data strongly suggest a specific interaction of
∗Corresponding author.
707
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
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REPRINTS
´
LOBATON ET AL.
708
O
CH3
R
N
O
N
SiO
O
H2N 4’’
O OSi
O O
S
(1) R = H (TSAO-T)
(2) R = CH3 (TSAO-m3T)
Figure 1.
the 4ꢀꢀ-amino group of the 3ꢀ-spiro moiety of TSAO molecules with the carboxylic
group of the glutamic acid residue at position 138 (Glu-138) (2).
Based on this hypothesis we now focussed on modifications at the 4ꢀꢀ-amino
group of the spiro moiety of TSAO-m3T in order to obtain a “second generation”
of TSAO molecules directed against TSAO-resistant HIV-1 strains. These novel
compounds contain at this 4ꢀꢀ-position different carbonyl, carboxylic acid and ester
groups that may form H-bonds with NH2 of Lys-138 (TSAO-resistant strains).
The synthesis of the target molecules was carried out by acylation of
TSAO-m3T (2) (Scheme 1), acylation that occurs exclusively on the 4ꢀꢀ-NH2 group
in contrast to the acylation of “classical” enamines where N- and C-acylation has
been reported (5). Thus, reaction of 2 with methyl oxalyl chloride in the presence of
AlCl3 gave the 4ꢀꢀ-N-oxalyl derivative 3 in good yields. The methyl ester group of 3
was further transformed either into the free acid 4 or the amide 5 by treatment with
NaOH 1N or NH3/MeOH, respectively. Treatment of 2 with conveniently function-
alized isocyanates gave the corresponding 4ꢀꢀ-N-alkyl and acyl urea derivatives 6a-f
in moderate to good yields.
The novel TSAO derivatives 3-5 and 6a-f were evaluated against HIV-1 repli-
cation in cell culture. The N-acyl derivatives 3-5, bearing oxalyl substituents at the
4ꢀꢀ position of the spiro moiety, show similar anti-HIV-1 activity than the parent
TSAO-m3T. Moreover, compound 4 also show moderate activity against HIV-1
TSAO-resistant strains. In contrast, introduction of an ureido moiety (6a-f) annihi-
lates activity.
Additionally, a comparative NMR conformational analysis in solution
(acetone-d6) between the 4ꢀꢀ-N-oxalyl derivative 3 (active) and the 4ꢀꢀ-N-urea com-
pound 6e (inactive) has been performed. The possibility of formation of intramolec-
ular hydrogen bonds of the NH protons of the 4ꢀꢀ position of the spiro moiety, that
may stabilize some conformations, has also been studied.
The conformation around the glycosidic bond in compounds 3 and 6e was
3
3
ꢀ
ꢀ
obtained from the vicinal carbon-proton couplings JC2,H1 / JC6,H1 and the corre-
ꢀ
ꢀ
sponding dihedral angles θC2,H1 /θC6,H1 , obtained by modeling studies, using the

ORDER
REPRINTS
“SECOND GENERATION” TSAO COMPOUNDS
709
O
N
O
CH3
CH3
H3C
H3C
N
O
N
O
O
O
N
SiO
HN
SiO
HN
O
O
NaOH 1N or
NH3/MeOH
ClCOCOOCH3
AlCl3
R
H3CO
O
O
O OSi
O OSi
S
S
O
O
O
O
4 R=OH (70%)
5 R=NH2 (54%)
3 (60%)
TSAO-m3T
O
2
CH3
H3C
N
O
O
N
R ’N=C=O
a H (40%)
SiO
b COPh (90%)
a
NH
HN
c COOCH2CH3 (80%)
d COC(CH3)=CH2 (75%)
e CH2CH3 (50%)
R’ =
R’
O
O OSi
S
O
O
f CH2COOCH2CH3 (30%)
6a-f
Scheme 1.
modified Karplus relations (6). Although both compounds showed a syn/anti equi-
librium, for compound 6e the equilibrium is mainly shifted to the syn conformer
(Psyn = 83%, Panti = 17%) while for compound 3 a more equal distribution of
syn/anti forms is observed (Psyn = 56%, Panti = 44%). The syn conformer of 6e
can be stabilized by an intramolecular hydrogen bond between the amido group
(NHa) directly attached to the spiro moiety (see Scheme 1) and the C-2 carbonyl
of the thymine. The syn/anti populations of compounds 3 and 6e were confirmed
by n.O.e experiments.
Hydrogen bonds were studied by temperature dependent chemical shift NMR
experiments (7) using acetone-d6 as solvent. The temperature coefficients ꢀδ/ꢀT
(ppb/K) values indicate that only the NHa of compound 6e is involved in intramolec-
ular hydrogen bonds, which is in agreement with the conformational analysis results
described above.
Finally, semiempirical quantum mechanical AM1 calculations for syn and
anti conformers of nucleosides 3 and 6e were carried out. These studies indicate
that the NHa...CO-2 bond distance in the syn conformer of compound 6e is suitable
for hydrogen bond formation which is in agreement with the results obtained by
NMR studies in solution.
In conclusion, the presence of two neighboring carbonyl groups at the 4ꢀꢀ
position of the spiro moiety is important for the activity. Compound 4 is the first
example of a “second generation” of TSAO derivatives that is active against TSAO-
resistant strains. NMR and theoretical studies show significant differences between
4ꢀꢀ-N-oxalyland4ꢀꢀ-N-ureaTSAOderivatives3and6eintheirabilityofformationof
intramolecular H-bonds that may compromise the interaction of these compounds
with HIV-1 RT and therefore the observed activity.

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LOBATON ET AL.
710
ACKNOWLEDGMENT
We thank the Spanish CICYT (Project SAF2000-0153-C02-01), the Spanish
Comunidad de Madrid (Project 08.2/0031.1/1999) and the European Commission
(Project QLRT-1999-30291) for financial support. Esther Lobato´n thanks Janssen-
Cylag S.A. for an award to young researchers for the present work.
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