Phosphonated Carbocyclic 2′-Oxa-3′-azanucleosides as New Antiretroviral Agents
Ugo Chiacchio,
†
Antonio Rescifina,
†
Daniela Iannazzo,
#
Anna Piperno,
#
Roberto Romeo,
#
Luisa Borrello,
†
Maria Teresa Sciortino,
§
Emanuela Balestrieri,
‡
Beatrice Macchi,
‡
Antonio Mastino,
§
and Giovanni Romeo*
,#
Dipartimento Farmaco-Chimico, UniVersita` di Messina, Via SS. Annunziata, Messina 98168, Italy, Dipartimento di Scienze Chimiche,
UniVersita` di Catania, Viale Andrea Doria 6, Catania 95125, Italy, Dipartimento di Neuroscienze, UniVersita` di Roma “Tor Vergata”, Via
Montpellier 1, and IRCCS S. Lucia, Roma 00133, Italy, and Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari, UniVersita` di
Messina, Salita Sperone 31, Messina 98168, Italy
ReceiVed March 12, 2007
Phosphonated carbocyclic 2′-oxa-3′-azanucleosides have been synthesized and tested for their antiretroviral
activity. The obtained results have shown that some of the compounds were as powerful as azydothymidine
in inhibiting the reverse transcriptase activity of the human retrovirus T-cell leukemia/lymphotropic virus
type 1 and in protecting human peripheral blood mononuclear cells against human retrovirus T-cell leukemia/
lymphotropic virus type 1 transmission in vitro. These data indicate that phosphonated carbocyclic 2′-oxa-
3′-azanucleosides possess the necessary requirements to efficiently counteract infections caused by human
retroviruses.
Introduction
Modification of naturally occurring nucleosides is an impor-
tant research area for the development of new antiretroviral
agents.
1
In particular, heterocyclic replacement of the ribose
moiety has resulted in the synthesis of new effective anti-HIV
drugs.
2,3
Recently, we have reported the synthesis of a series
of nucleoside analogues where the furanose ring has been
replaced by a N,O-heterocyclic system.
4
The carbocyclic 2′-
oxa-3′-azanucleosides are endowed with interesting physiologi-
cal activities. In particular, the fluorouracil analogue (ADF,
Figure 1) is characterized by low cytotoxicity and, noteworthy,
has been shown to specifically induce remarkable levels of
apoptosis on lymphoid and monocytoid cells.
4
Most of the nucleoside analogues possessing antiviral activi-
ties rely on phosphorylation by specific kinases; moreover, in
many cases, among the three successive phosphorylation steps,
the first is rate-limiting.
5,6
On this basis, phosphate analogues,
where the phosphate moiety is changed to isosteric and
isoelectronic phosphonates, have been designed; these com-
pounds mimic the nucleoside monophosphates and are able to
bypass the initial selective enzymatic monophosphorylation
step.
7
Accordingly, we have developed the transformation of the
first-generation N,O-nucleosides 1
8
into the phosphonated
nucleosides 2 and 3 (Figure 1) via the 1,3-dipolar cycloaddition
of phosphonated nitrones with suitable dipolarophiles. The
obtained compounds have proven to be potential antiretroviral
agents: pyrimidine N,O-nucleosides exerted a specific inhibitory
activity on two different types of commercial RT,
a
from animal
retroviruses, comparable with that of the well-known AZT,
following incubation with human PBMCs crude extract.
4
Starting from these findings, to acquire more insight into the
physiological properties of this class of compounds and also to
obtain derivatives that could be more potent and more active
against different virus strains, we have extended our studies to
the synthesis and biological evaluation of 5-bromouracil,
adenine, and guanine derivatives of this second generation of
PCOANs. At the same time, a new and feasible synthetic route
toward phosphonated pyrimidine and purine N,O-nucleosides
has been designed.
Moreover, we extended our biological assays to understand
whether PCOANs actually possess the necessary requirements
for efficiently inhibiting the transmission of a human retrovirus.
In this paper we report that PCOANs were able not only to
inhibit the RT enzymatic activity of HTLV-1, the first identified
human retrovirus,
9
but also to efficiently protect human PBMCs
from HTLV-1 infection in culture.
Results and Discussion
In the first attempt, the synthesis of 2d-f and 3d-f was
performed according to the procedure previously reported for
2a-c and 3a-c.
4
Thus, phosphonated nitrone 4 was reacted
* To whom correspondence should be addressed. Phone: +39 090
356230. Fax: +39 090 6766562. E-mail: gromeo@unime.it.
†
Universita` di Catania.
#
Dipartimento Farmaco-Chimico, Universita` di Messina.
§
Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari,
Universita` di Messina.
‡
Universita` di Roma “Tor Vergata” and IRCCS S. Lucia.
a
Abbreviations: PCOANs, phosphonated carbocyclic 2′-oxa-3′-aza-
nucleosides; RT, reverse transcriptase; HTLV-1, human retrovirus T-cell
leukemia/lymphotropic virus type 1; AZT, azydothymidine; PBMCs,
peripheral blood mononuclear cells; MRTIC, minimal RT inhibitory
concentration; MPC, minimal protective concentration; GIC
25
, growth
inhibitory concentration 25%; CC
20
, cytotoxic concentration 20%; RNIC,
RNA inhibitory concentration.
Figure 1. Nucleoside analogues.
3747J. Med. Chem. 2007, 50, 3747-3750
10.1021/jm070285r CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/20/2007
with vinyl acetate to give a mixture of epimeric isoxazolidines
5 and 6 (Scheme 1) in a 1:2.6 relative ratio (global yield 90%).
The mixture of the two cycloadducts was coupled with
silylated 5-bromouracil, adenine, and 2-N-acetyl-6-O-(diphe-
nylcarbamoyl)guanine in acetonitrile at 55 °C in the presence
of 0.4 equiv of TMSOTf as catalyst. The nucleosidation
proceeded with good yield and complete selectivity, with respect
to the anomeric center, for bromouracil derivative (3d was the
exclusive adduct with 74% yield) and with moderate yields and
poor selectivity for purine derivatives e and f. So with adenine,
R- and β-anomers 2e and 3e have been obtained in a relative
ratio 1:1.5 (total yield 52%), while for guanine, the
1
H NMR
spectrum of the crude reaction mixture showed the presence of
both R- and β-anomers, but only the β-anomer 3f has been
isolated after flash chromatography in very poor yield (15%).
In this last case, nucleosides 2f and 3f appeared to be unstable;
they decomposed easily by cleavage of the glycoside bond and
release of the nucleoside base together with the formation of
decomposition products.
The stereochemistry of the obtained derivatives 2e and 3d-f
was achieved by NOE measurements (see Supporting Informa-
tion).
A successful implementation of the synthetic scheme has been
achieved by the use of the C-[(tert-butyldiphenylsilyl)oxy]-N-
methylnitrone 7:
10
the cycloaddition reaction with vinyl acetate,
in anhydrous ether at room temperature, proceeded with a good
stereoselectivity affording a mixture of epimeric isoxazolidines
8 and 9 (Scheme 2) in a relative ratio 1:4.2 (global yield 90%)
as determined by
1
H NMR analysis.
The stereochemical outcome of the cycloaddition process can
be explained by considering that nitrone 7 has been shown by
1
H NMR and NOE data to be the Z isomer;
10
thus, the major
product 9 could be formed by the Z nitrone reacting in an exo
mode. The crude mixture of isoxazolidines was nucleosidated
with pyrimidine and purine bases. The pyrimidine nucleosidation
was performed with silylated nucleobases in acetonitrile at
50 °C in the presence of 0.4 equiv of TMSOTf as catalyst. The
reaction, carried out with silylated thymine, 5-fluorouracil,
5-bromouracil, and N-acetylcytosine, proceeded with a satisfac-
tory selectivity to give a mixture of R- and β-nucleosides
10a,b,d,g and 11a,b,d,g in a 3:7 relative ratio (global yield 85-
90%, Scheme 2).
Compounds 10a,b,d,g and 11a,b,d,g were separated by flash
chromatography (98:2 chloroform/methanol as eluant), and the
relative configuration was assigned by NOE measurements. In
particular, for β-derivative 11a, the positive NOE effect observed
for H
4′
when irradiating H
1′
is clearly indicative of the cis
configuration of the substituents at C
1′
and C
4′
.
The purine nucleosidation for the adenine derivative was
performed with 6-bromopurine in acetonitrile with DBU at
60 °C in the presence of 4 equiv of TMSOTf as catalyst. The
reaction proceeded with good yield to give, after in situ treatment
with ammoniacal methanol, a mixture of R- and β-nucleosides
10e and 11e in a 2:3 relative ratio (global yield 75%, Scheme
2). Compounds were separated and identified as reported above.
Transformation of the nucleosides 10 and 11 into the
corresponding phosphonates 2 and 3 was finally performed by
desilylation, tosylation, and subsequent Arbuzov reaction with
triethyl phosphite.
Biological Tests. Antiretroviral Activity. For testing the
potential activity of PCOANs against human retroviruses, first
we determined their ability to inhibit HTLV-1 reverse tran-
scriptase activity in vitro, by means of a cell-free assay recently
described by us.
11
The results were expressed as MRTIC.
Identical results were obtained in three different experiments
(Table 1). Most of the newly synthesized compounds, following
activation through preincubation with a crude extract of PHA-
stimulated PBMCs, caused complete inhibition of HTLV-1 RT
Scheme 1. Synthesis of Phosphonated N,O-Nucleosides 2e and 3d-f
Scheme 2. Synthesis of Phosphonated N,O-Nucleosides 2a-e
and 3a-e
a
a
(I) Silylated nucleobase, MeCN, TMSOTf; (II) 6-bromopurine, DBU,
TMSOTF, then NH
3
/MeOH; (III) TBAF, THF; (IV) TSCl, Et
3
N, DCM;
(V) (EtO)
3
P.
3748 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 Brief Articles
activity at 0.6 nM, except for 3d, which was efficient at 8 nM,
and 2e, which was not active. Interestingly, most of the new
compounds showed an activity higher than that of AZT in
inhibiting HTLV-1 RT, following a similar activation.
Different concentrations of PCOANs and of the reference
compound AZT were next tested for their ability to protect
human PBMCs from HTLV-1 infection when added im-
mediately before virus exposure to the cultures using an in vitro
protection assay.
11,12
Differently from HIV, HTLV-1 does not
cause any cytopathic effect following infection but rather
stimulates proliferation of infected cells and eventually im-
mortalizes them. Thus, at 3 weeks in culture after infection,
the presence of HTLV-1 proviral DNA and the expression of
viral RNA were detected by DNA-PCR of a specific HTLV-1
pol gene sequence and by RT-PCR for the Tax/Rex region of
HTLV-1, respectively. The same concentration of the com-
pounds equally inhibited or not inhibited proviral DNA presence
and viral RNA expression. To avoid useless duplication of data,
proviral DNA results are omitted. Results, expressed as the
minimal concentration required to fully protect cell cultures from
HTLV-1 transmission, showed that 3a, 3b, 3c, and AZT fully
inhibited HTLV-1-RNA expression equally at 100, 25, 5, and
1 µM, respectively, while 3e was inhibitory at 100, 25, and 5
µM but not at 1 µM. Conversely, 3d and 2e were not able to
protect against HTLV-1 infection even at the higher concentra-
tions tested. Experiments were repeated three times, using
PBMCs from three different donors, with identical results.
Cell Toxicity. Cell toxicity is a major limit for the utilization
of nucleoside antiviral agents. Thus, freshly separated PBMCs
were assayed for their growth and death during short-term
culture in the presence of the newly synthesized compounds
and of interleukin 2, without additional stimuli. The results
obtained on day 7 in culture, expressed as GIC
25
and CC
20
,
respectively, indicate that most of the examined compounds
were poorly cytostatic and very low or not at all cytotoxic in
comparison with AZT (Table 1). Moreover, no evidence for
induction of apoptosis by PCOANs, at the concentrations tested,
was obtained (data not shown).
Conclusions
Phosphonated N,O-nucleosides have been synthesized in good
yields by the 1,3-dipolar cycloaddition methodology according
to two different routes, which exploit two different nitrones as
starting material. The use of the C-[(tert-butyldiphenylsilyl)-
oxy]-N-methylnitrone 7 has led to better yields with respect to
the approach based on the phosphonated nitrone 4.
Regarding to their antiretroviral action, 2e showed no activity.
Conversely, 3d, although it inhibited RT activity in the cell-
free assays as efficiently as AZT, was unable to protect against
HTLV-1 infection in vitro, suggesting an impairment at cell
entry or at metabolic biotransformation levels. Interestingly, all
the other new compounds tested showed powerful antiretroviral
activity not only in the cell-free assay but also in an experimental
cell model of infection, suggesting that these compounds are
converted properly to the phosphonic acid diphosphate RT
inhibitors by cell kinases. However, in the present report we
have not addressed the exact mechanism by which PCOANs
exert RT inhibition. In particular, we have no information about
whether they act as chain terminators, as it seems plausible.
Future investigations on this point will clarify this aspect. Very
important, toxicity toward lymphoid cells of the new compounds
was remarkably lower than that of AZT. In conclusion, our
results add new perspectives in the development if PCOANs
as possible new pharmacological tools of intervention against
infections sustained by human retroviruses.
Experimental Section
Compounds 2a,g and 3a-c,g were previously reported.
4
Com-
pounds 2e and 3d-f described in Scheme 1 have been synthesized
according to the same procedure.
4
Compounds 2a-e and 3a-e have
also been prepared starting from nitrone 7, according to Scheme 2
(see Supporting Information).
Diethyl [(1′RS,4′RS)-1′-(6-Amino-9H-purin-9-yl)-3′-methyl-
2′-oxa-3′-azacyclopent-4′-yl]methylphosphonate (2e). 2e was the
second eluted product. Yield 20.8%, sticky oil.
1
H NMR (500 MHz,
CDCl
3
): δ 1.35 (dt, 6H, J ) 3.5 and 7.1 Hz), 1.95 (ddd, 1H, J )
10.3, 15.0, 18.1 Hz, H
4′′a
), 2.15 (ddd, 1H, J ) 3.7, 15.0, 20.8 Hz,
H
4′′b
), 2.81 (s, 3H, N-CH
3
), 2.87 (m, 1H, H
5′
a), 3.13 (m, 1H, H
5′b
),
3.54 (m, 1H, H
4′
), 4.18 (m, 4H), 5.96 (bs, 2H, NH
2
), 6.32(m, 1H,
H
1′
), 8.04 (s, 1H, H
8
), 8.34 (s, 1H, H
3
).
13
C NMR (125 Hz,
CDCl
3
): δ 16.4, 16.45, 26.6, 29.3, 41.8, 43.7, 61.9, 62.1, 63.3,
81.9, 119.9, 138.9, 149.5, 153.0. HRMS (EI) calcd for (M
+
)
C
14
H
23
N
6
O
4
P, 370.1518. Anal. (C
14
H
23
N
6
O
4
P) C, H, N.
Diethyl [(1′SR,4′RS)-1′-(5-Bromo-2,4-dioxo-3,4-dihydropyrimid-
1(2H)-yl-3′-methyl-2′-oxa-3′-azacyclopent-4′-yl]methylphospho-
nate (3d). Yield 74%, sticky oil.
1
H NMR (500 MHz, CDCl
3
): δ
1.34 (dt, 3H, J ) 4.9 and 7.2 Hz), 1.35 (dt, 3H, J ) 4.9 and 7.2
Hz), 1.90 (ddd, 1H, J ) 9.9, 14.9, and 18.2 Hz, H
4′′a
), 2.09 (m,
1H, H
4′′b
), 2.32 (ddd, 1H, J ) 4.2, 9.5, and 14.0 Hz, H
5′a
), 2.77 (s,
3H, N-CH
3
), 2.91 (ddd, 1H, J ) 7.8, 9.5, and 9.9 Hz, H
4′
), 3.21
(dt, 1H, J ) 7.8 and 14.0 Hz, H
5′b
), 4.12 (m, 4H), 6.09 (dd, 1H, J
) 4.2 and 7.8 Hz, H
1′
), 8.16 (s, 1H, H
6
), 9.61 (bs, 1H, NH).
13
C
NMR (125 Hz, CDCl
3
): δ 16.4, 16.5, 29.30, 42.7, 44.7, 61.9, 62.1,
63.3, 83.0, 96.4, 140.0, 149.7, 159.4. HRMS (EI) Calcd for (M
+
)
C
13
H
21
BrN
3
O
6
P, 425.0351. Anal. (C
13
H
21
BrN
3
O
6
P) C, H, N.
Diethyl [(1′SR,4′RS)-1′-(6-Amino-9H-purin-9-yl)-3′-methyl-
2′-oxa-3′-azacyclopent-4′-yl]methylphosphonate (3e). 3e was the
first eluted product. Yield 31.2%, sticky oil.
1
H NMR (500 MHz,
CDCl
3
): δ 1.34 (dt, 6H, J ) 1.5 and 7.1 Hz), 2.0 (ddd, 1H, J )
10.1, 15.0, and 18.1 Hz, H
4′′a
), 2.13 (ddd, 1H, J ) 3.0, 15.0, and
20.5 Hz, H
4′′b
), 2.67 (ddd, 1H, J ) 3.6, 9.3, and 14.0 Hz, H
5′a
),
2.77 (s, 3H, N-CH
3
), 2.99 (dddd, 1H, J ) 3.0, 7.9, 9.3, and 10.1
Hz, H
4′
), 3.28 (ddd, 1H, J ) 7.9, 8.1, 14.0 Hz, H
5′b
), 4.09 (m, 4H),
6.14 (bs, 2H, NH
2
), 6.43 (dd, 1H, J ) 3.6 and 8.1 Hz, H
1′
), 8.31
(s, 1H, H
8
), 8.32 (s, 1H, H
3
).
13
C NMR (125 Hz, CDCl
3
): δ 16.4,
16.5, 26.6, 29.6, 42.6, 43.6, 61.9, 62.1, 63.3, 80.2, 119.1, 149.5,
152.5, 155.2. HRMS (EI) calcd for (M
+
)C
14
H
23
N
6
O
4
P, 370.1518.
Anal. (C
14
H
23
N
6
O
4
P) C, H, N.
Diethyl [(1′RS,4′RS)-1′-(2-Acetamido-9H-purin-6-yldiphenyl-
carbamate)-9-yl-3′-methyl-2′-oxa-3′-azacyclopent-4′-yl]meth-
Table 1. Inhibitory Activity and Cytotoxicity for Compounds 2e and
3a-e
compd
HTLV-1-RT
a
MRTIC, nM
anti-HTLV-1 assay
b
MPC, µM
GIC
25
,
c
µM
(Pearson’s r)CC
20
,
d
µM
3a 0.6 1 817 (0.88) >2048
3b 0.6 1 119 (0.99) >2048
3c 0.6 1 56 (0.98) >2048
3d 8 >100 215 (0.94) >2048
3e 0.6 5 39 (0.92) >2048
2e >10
4
>100 121 (0.83) >2048
AZT 4 1 15 (0.91) 1153
a
MRTIC toward HTLV-1-RT, determined as the minimal concentration
required to completely inhibit reverse transcription evaluated in a cell-free
assay by a RT-PCR assay through an amplification program of 20 cycles.
b
MPC determined as the minimal concentration of the compounds required
to completely protect PBMCs from HTLV-1 transmission, as revealed by
inhibition of Tax/Rex viral mRNA expression, evaluated by liquid
hybridization assay, at 3 weeks after exposure to the virus in the presence
or the absence of PCOANs.
c
Compound concentration required to cause
GIC
25
, evaluated by living cell count using trypan blue exclusion test in
uninfected PBMCs after 1 week of culture.
d
CC
20
evaluated by trypan blue
exclusion test in uninfected PBMC after 1 week of culture.
Brief Articles Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3749
ylphosphonate (3f). Yield 15%, sticky oil.
1
H NMR (500 MHz,
CDCl
3
): δ 1.34 (dt, 6H, J ) 2.6 and 7.0 Hz), 1.96 (ddd, 1H, J )
10.2, 14.7, and 18.0 Hz, H
4′′a
), 2.11 (ddd, 1H, J ) 2.9, 14.7, and
20.5 Hz, H
4′′b
), 2.54 (s, 3H, C(O)CH
3
), 2.65 (ddd, 1H, J ) 3.7,
9.0, and 14.0 Hz, H
5′a
), 2.75 (s, 3H, N-CH
3
), 2.98 (dddd, 1H, J )
2.9, 8.3, 9.0, and 10.2 Hz, H
4′
), 3.30 (ddd, 1H, J ) 7.9, 8.3, 14.0
Hz, H
5′b
), 4.10(dq, 4H, J ) 7.0 and 10.8 Hz), 6.35 (dd, 1H, J )
3.7 and 8.3 Hz, H
1′
), 7.23-7.44 (m, 10H), 7.45 (bs, 1H, NH), 8.07-
(s, 1H), 8.43 (s, 1H,).
13
C NMR (125 Hz, CDCl
3
): δ 16.3, 16.3,
24.0, 26.4, 43.2, 44.6, 49.2, 62.9, 63.1, 81.4, 116.1, 121.9, 127.0,
129.5, 140.6, 141.9, 144.8, 152.5, 153.9, 153.70, 170.0. HRMS (EI)
calcd for (M
+
), C
29
H
34
N
7
O
7
P 623.2257. Anal. (C
29
H
34
N
7
O
7
P) C,
H, N.
Acknowledgment. This work was partially supported by
M.I.U.R. (Progetto P.R.I.N. 2005, 2006).
Supporting Information Available: Experimental details on
the synthesis of the compounds described in this paper, spectral
data for all relevant compounds, elemental analysis data of all
compounds, and details of all biological methods. This material is
available free of charge via the Internet at http://pubs.acs.org.
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3750 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 Brief Articles