Inhibition of the strand transfer step of HIV-1 integrase by non-natural dinucleotides

Article abstract of DOI:10.1016/j.bmcl.2004.07.050

Remarkable selectivity for inhibition of the strand transfer step of HIV-1 integrase by novel dinucleotides. New, non-natural dinucleotide 5′-monophosphates, with a surrogate isonucleoside component of l-related stereochemistry at the 'terminal' position,

Full text of DOI:10.1016/j.bmcl.2004.07.050

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4815–4817
Inhibition of the strand transfer step of HIV-1 integrase by
non-natural dinucleotides
Guochen Chi,a Nouri Neamatib and Vasu Naira,
*
aDepartment of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602, USA
bDepartment of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Received 19 June 2004; revised 22 July 2004; accepted 23 July 2004
Available online 13 August 2004
Abstract—New, non-natural dinucleotide 50-monophosphates, with a surrogate isonucleoside component of L-related stereochem-
istry at the ÔterminalÕ position, have been synthesized. Structures of 2a–c were confirmed by multinuclear NMR spectra (1H, 13C, 31P,
COSY), UV hypochromicity and FAB HRMS data. These compounds are totally resistant to cleavage by 30- and 50-exonucleases.
The dinucleotides showed remarkable selectivity for inhibition of the strand transfer step of HIV-1 integrase. To the best of our
knowledge, these compounds represent only the second example of selective strand transfer inhibitors of HIV integrase.
Ó 2004 Elsevier Ltd. All rights reserved.
The retroviral enzyme, HIV-1 integrase, incorporates
HIV double helical DNA into host chromosomal
DNA.1–7 This viral enzyme first catalyzes the endonucle-
atic removal of two terminal nucleotides at the 30-end of
each strand of viral DNA (30-processing) leaving re-
cessed ends that terminate with xxCA-OH (Fig. 1). In
the next steps (strand transfer, integration) nucleophilic
attack of the terminal 30-OH of the tailored HIV DNA
on a specific internucleotide phosphodiester functional-
ity results in cleavage of host DNA and this is followed
by integration of the tailored HIV DNA into host
DNA.1–3 The integration process is essential for the rep-
lication of HIV and there is apparently no functional
equivalent of HIV integrase in human cells.
Some oligonucleotides of natural origin are capable of
interfering with the integration process by competing
with viral DNA for binding to HIV integrase.8 Pro-
tein–nucleotide interactions appear to be of importance
in other steps of the replication cycle of HIV such as the
recognition and binding of Tat protein to HIV-1 TAR
RNA.9 However, small oligonucleotides of natural ori-
gin are rapidly cleaved by cellular nuclease activity. In
addition, increasing nuclease resistance by chemical
alteration of the internucleotide phosphate bond results
in decreased integrase activity.8 A non-natural dinucleo-
tide with a conformationally unusual internucleotide
phosphodiester bond that joins a D-deoxynucleoside
and an L-related isodeoxynucleoside,
1 (Fig. 2),
NH2
N
N
NH2
N
N
Endonuclease Activity
N
N
N
N
⇓
O
O
O
O
O
O
LTR (+) 5′-end ACTG…………CAGT
(+) 3′-end
(-) 5′-end
P
P
O
O
P
NH2
N
NH2
N
O
O
O
O
O
O
O
(-) 3′-end TGAC….…..GTCA
O
O
P
N
O
N
O
O
⇑
O
O
Endonuclease Activity
OH
OH
Figure 1. Processing of HIV DNA prior to integration into host DNA.
1
2a
Figure 2. Structure of the anti-HIV integrase compound 1 and its
isomer 2a in which the position of the surrogate isonucleoside is
changed.
Keywords: HIV integrase; Dinucleotides; Strand transfer inhibitors.
*
Corresponding author. Tel.: +1-706-542-6293; fax: +1-706-583-
8283; e-mail: vnair@rx.uga.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.050

4816
G. Chi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4815–4817
Table 1. Anti-HIV-1 integrase data for dinucleotides
BaseBz
BaseBz
HO
RO
P
N
O
Compounds
30-Processing,
Strand transfer,
IC50 (lM)
O
O
i
IC50 (lM)
R1
3a-c
R1
2a
2b
2c
1
>1000
>1000
>1000
19
65
41
4
ABz
ABz
114
25
HO
O
DMTrO
O
O
P
O
5
OH
ii
iii
O
OR
BaseBz
O
stacking in the preferred conformations. Base stacking
implies the presence of conformationally unusual inter-
nucleotide phosphate bonds because of the spatial
arrangement of the two sugar rings that is necessary to
accommodate this assembly of the bases (Fig. 2).
R1
6a-c
O
O
P
OR
ABz
A
-O
P
O
O
RO
O
O
O-
O
P
O
O
P
O
iv
Integrase inhibition assays were conducted with purified
recombinant HIV-1 integrase using a 21-mer oligonucleo-
tide substrate.15 The data (Table 1) clearly showed that
compounds 2a–c have strand transfer inhibitory activity
against wild-type HIV-1 integrase but do not exhibit
inhibition of the 30-processing step. This is in sharp con-
trast to compound 1 (pIsodApdC), which showed strong
inhibition of both key steps of the integrase mechanism
of action. The major structural difference between
dinucleotide 1 and its counterpart 2a (or the related
compound 2b) is the position of the surrogate
isonucleoside component. The inhibition of integrase
by 1 is likely the result of base recognition and binding
by the viral enzyme. Thus, it is remarkable that this
apparently small structural change in the counterpart
of 1 can produce such a major impact on the mode of
inhibition of integrase. The only reported selective
inhibitor of strand transfer is a class of diketo contain-
ing compounds,16 and, to the best of our knowledge,
the compounds described in this communication repre-
sent the second examples. Finally, compounds 2a–c
are resistant to cleavage of the internucleotide phospho-
diester linkage by mammalian 30- and 50-exonucleases.
O
O-
O
OR
BaseBz
Base
O
O
R2
2a-c
R1
7a-c
R1
Base
2a, R2 = OH, Base = C
2b, R2 = OH, Base = A
2c, R2 = H, Base = A
a
b
c
OBz
OBz
H
R = 2-cyanoethyl
C
A
A
Scheme 1. Reagents and conditions: [i] (i-Pr2N)2POCH2CH2CN, 1H-
tetrazole, CH2Cl2, rt, 1h; [ii] (1) 1H-tetrazole, 3h, (2) I2/THF/H2O/Py;
(3) 2% CHCl2COOH in CH2Cl2; [iii] (1) i-Pr2NP(OCH2CH2CN)2, 1H-
tetrazole, CH2Cl2, 1h; (2) I2/THF/H2O/Py; [iv] concd NH4OH, rt, 24h.
previously synthesized by us,10 exhibits resistance to
mammalian 30- and 50-exonucleases.11 This compound
is an inhibitor of wild-type HIV-1 integrase activity,
inhibiting both the 30-processing and strand transfer
steps.10 We report here some new results that suggest
that changing the position of the surrogate nucleoside
component of the dinucleotide (e.g., compound 2a)
can dramatically change the mode of inhibitory activity
from both key steps of integrase action to just the strand
transfer step.
Acknowledgements
The isonucleosides were prepared from ribitol cyclic
sulfate as the precursor12 and then were appropriately
tailored for the coupling steps. Dinucleotides were
synthesized through the phosphoramidite method
Scheme 1).10 Thus, isodeoxynucleoside 3 with a free
50-hydroxyl group was condensed with the phosphoryl-
ating reagent [(i-Pr2N)2POCH2CH2CN] in the presence
of 1H-tetrazole to give the phosphoramidite 4, which
was immediately coupled through the free 30-hydroxyl
group of deoxynucleoside 5. Iodine oxidation and selec-
tive removal of the trityl group using dichloroacetic
acid10 provided the phosphotriester 6. Phosphorylation
of dinucleotide 6 was performed using di(2-cyano-
ethyl)N,N-diisopropyl-phosphoramidite as the phos-
phorylating reagent.13 After deprotection, the final
products were purified by HPLC. The structures of
2a–c were confirmed by multinuclear NMR data (1H,
13C, 31P, COSY) and FAB HRMS data.14 The observed
hypochromicity (12–18%) from the quantitative UV
data [2a, kmax 262 (e 19,900); 2b, kmax 259 (e 26,400);
2c, kmax 259 (e 24,500)] suggested the presence of base
The project described was supported by Grant Number
RO1 AI 43181 (to V.N.) from the National Institutes of
Health. Its contents are solely the responsibility of the
authors and do not necessarily represent the official
views of the NIH. One of us (V.N.) thanks the Georgia
Research Alliance for an award toward the purchase of
the 500MHz NMR spectrometer used in this project.
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1H), 4.47 (m, 1H), 4.21–4.28 (m, 3H), 3.90–4.04 (m, 5H),
2.63–2.66 (m, 1H), 2.55–2.58 (m, 1H). 13C NMR (D2O):
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2c,1H NMR (D2O): 8.39 (s, 1H), 8.34 (s, 1H), 8.19 (s, 1H),
8.16 (s, 1H), 6.30 (t, 1H, J = 6.5), 5.21 (m, br, 1H), 4.82 (s,
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3.98–4.09 (m, 3H), 3.89–3.93 (m, 3H), 3.81 (m, 1H), 2.71
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64.8, 55.7, 38.9, 33.6. 31P NMR (D2O): 1.00, 0.25. FAB-
HRMS: [M+1]+ calcd for C20H27N10O10P2 629.1387,
found 629.1412. UV (H2O): kmax 259 (e 24,500).
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8.16 (s, 1H), 6.29 (t, 1H, J = 7.0), 4.98 (m, 1H), 4.80 (s, br,