Synthesis of Phosphonated Carbocyclic 2′-Oxa-3′-aza-nucleosides: Novel
Inhibitors of Reverse Transcriptase
Ugo Chiacchio,*
,†
Emanuela Balestrieri,
‡
Beatrice Macchi,
‡
Daniela Iannazzo, Anna Piperno, Antonio Rescifina,
†
Roberto Romeo, Monica Saglimbeni, M. Teresa Sciortino,
§
Vincenza Valveri,
§
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 July 28, 2004
Phosphonated carbocyclic 2′-oxa-3′-aza-nucleosides have been synthesized in good yields by
1,3-dipolar cycloaddition methodology. The cytotoxicity and the reverse transcriptase inhibitory
activity of the obtained compounds have been investigated. Phosphonated carbocyclic 2′-oxa-
3′-aza-nucleosides, while showing low levels of cytotoxicity, exert a specific inhibitor activity
on two different reverse transcriptases, which is comparable with that of AZT, opening new
perspectives on their possible use as therapeutic agents, in anti-retroviral and anti-HBV
chemotherapy.
Introduction
The number of modified nucleosides has grown expo-
nentially in recent years in response to the pressing
need for new treatments against virus infections. In
particular, modified nucleosides have been proved to
efficiently inhibit in vitro and in vivo virus infections
caused by HIV, HBV, and HTLV-1.
1
Many of the
nucleoside analogues proposed against the above-
mentioned viruses present similar mechanism of action
as inhibitors of viral replication: following intracellular
phosphorylation to their 5′-triphosphate forms, they
serve as chain terminators, thus acting as inhibitors in
the viral reverse transcription reaction.
2
However, several problems associated with this kind
of nucleoside analogues, due to their high toxicity and
the appearance of cross-resistance, have led to the
search for different structural solutions which have
afforded new prodrugs comparable in their antiviral
activity to the clinically used nucleoside analogues, but
without their drawbacks.
The biological activity of nucleoside analogues (ddNs)
showing antiviral properties is strictly linked to their
conversion, through cellular enzymes, to the corre-
sponding 5′-mono-, di, and triphosphates, which interact
with viral reverse transcriptase (RT) or interfere with
cell growth, slowing the cell cycle progression. In many
cases, among the three successive phosphorylation
steps, the first is rate-limiting, and further conversions
to di- and triphosphates are catalyzed by less specific
kinases.
3
The known antiviral nucleoside analogues have often
shown inefficiency in the first intracellular phosphory-
lation step by nucleoside kinases. A possibility to
improve the uptake of ddNs might be to bypass the
phosphorylating step; unfortunately, nucleotides, due
to their polar nature, are not able to cross the cell
membrane efficiently. Moreover, they are readily de-
phosphorylated in extracellular fluids and on cell sur-
faces by nonspecific phosphohydrolases.
Several strategies to overcome the problem of the
initial selective and regulated phosphorylation step
could be foreseen. A good delivery system for a nucleo-
side across membranes might be represented by phos-
photriester molecules, which should ensure the absorp-
tion and the transport of the active molecule and should
then be able to deliver intracellular monophosphate
forms.
4
On this basis, recently, the use of nucleotide prodrugs
(pronucleotides) incorporating enzyme-labile transient
phosphate protecting groups has emerged, and a large
number of prodrugs derivatives of nucleoside mono-
phosphates have been prepared.
In this respect, an alternative approach to the dis-
covery of new and potent RT inhibitors involves the
design of phosphate analogues where the phosphate
moiety is changed to isosteric and isoelectronic phos-
phonates.
5
Those enzimatically and chemically stable
phosphonate analogues, which mimic the nucleoside
monophosphates, are able to overcome the instability
of nucleotides toward phosphodiesterases and to en-
hance their cellular uptake by bypassing the initial
enzymatic phosphorylation and could potentially be
effective antiviral agents.
In our laboratory, we have developed the synthesis
of a series of ddNs in which the furanose ring has been
replaced by a N,O-heterocyclic ring.
6
Carbocyclic 2′-oxa-
3′-aza-nucleosides 1 are emerging as a new important
class of compounds that are interesting to synthesize
in order to investigate their pharmacological activities.
7
In connection with our synthetic studies on the utility
of nitrones for the synthesis of interesting biologically
nitrogenated compounds,
8
we have devised a synthetic
* To whom correspondence should be addressed. Phone: +39 090
356230. Fax: +39 090 6766562. E-mail: gromeo@unime.it.
†
Universita` di Catania.
‡
Universita` di Roma ‘Tor Vergata’.
§
Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari,
Universita` di Messina.
1389J. Med. Chem. 2005, 48, 1389-1394
10.1021/jm049399i CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/08/2005
route to phoshonated carbocyclic 2′-oxa-3′-aza-nucleo-
sides of type 2 via 1,3-dipolar cycloaddition of phospho-
nated nitrones (Figure 1). The compounds obtained have
been proved to be potential antiviral agents: data on
the biological activity show that 2 are low toxic and
exert a relevant activity as RT inhibitors.
Results and Discussion
The strategy of the synthetic approach is based on
the construction of the phosphonated nitrone 5, which
has been prepared from the commercially available
diethylphosphonoacetaldehyde diethyl acetal (3). Com-
pound 3 has been efficiently hydrolyzed to the corre-
sponding carbonyl derivative 4 by treatment with
Amberlist-15
9
in acetone at room temperature: the
subsequent reaction with N-methylhydroxylamine af-
forded nitrone 5 in good yield (95%) (Scheme 1).
The cycloaddition with vinyl acetate leads to a mix-
ture of epimeric isoxazolidines 6 and 7 (90% overall
yield) in a relative ratio 2.6:1. The crude mixture was
purified by radial chromatography, and two cycload-
ducts were obtained in pure form. Their structure was
confirmed by
1
H NMR experiments; thus, 6, the cis
isomer, shows the H
5
proton as doublet of doublets at δ
6.28, while H
3
proton resonates as a multiplet at δ 2.85.
In compound 7, the same protons give resonances at δ
6.31 (as a doublet) and 3.35 (as a multiplet).
Stereochemical assignments have been performed by
NOE measurements. Thus, in compound 6 irradiation
of the H
5
resonance induces a positive NOE effect on
H
3
proton and on the downfield resonance of methylene
protons at C
4
, indicating a cis relationship between
these protons. On the contrary, irradiation of the same
resonance in compound 7 resulted in the enhancement
of the upfield resonance of the methylene protons at C
4
(Figure 2).
The stereochemical outcome of the cycloaddition
process can be explained by considering that nitrone 5
exists as a mixture of E/Z isomers: NOE data indicated
that the Z form is predominant (Z/E ratio ) 5:1). Thus,
the major product 6 could be formed by the (Z)-nitrone
reacting in an exo mode or by the (E)-nitrone reacting
in an endo mode, in agreement with the results reported
for similar R-alkoxynitrones.
10
AM1 calculations
11
give
theoretical support to the experimental data, indicating
that the (E)-isomer as the more reactive form of nitrone
5: the (E)-endo transition state leading to 6 is about
0.45 kcal/mol more stable than the (E)-exo one leading
to trans stereoisomer 7. These values agree satisfacto-
rily with the observed 2.5:1 cis/trans ratio (Table 1).
Two cycloadducts were independently coupled with
silylated nucleobases, according to the Vorbru¨ eggen
methodology.
12
The condensation with silylated thymine
(8), N-acetylcytosine (9), and 5-fluorouracil (10), per-
formed in acetonitrile at 55 °C in the presence of 0.4
equiv of TMSOTf as catalyst, proceeded with good
yields: a mixture of β- and R-nucleosides 11 and 12 has
been obtained (Scheme 2). The compounds 11d and 12d
were further treated with 5.0% aqueous sodium carbon-
ate to give the deacetylated derivatives 11b and 12b.
Pure anomers were isolated by flash chromatography,
and the relative configuration was assigned on the basis
of
1
H NMR and NOE experiments. In particular, for
β-compounds 11, irradiation of H
1′
increased the reso-
nance of H
6
,H
4′
, and H
6′b
(the downfield resonance of
methylene protons at C
6′
). Conversely, when H
6′b
was
irradiated, enhancements of the H
1′
,H
4′
, and H
6′a
signals
were observed, while irradiation of H
6′a
gives rise to a
positive NOE effect for H
6
and H
6′b
. These data unam-
biguously indicate a β-configuration where H
4′
and the
pyrimidine base are in a trans relationship.
Furthermore, the R-configuration for anomers 12 was
supported by the strong NOE effect observed for H
6
when irradiating H
4′
.
Figure 1. Carbocyclic 2′-oxa-3′-aza-nucleosides.
Scheme 1. Synthesis of Isoxazolidines 6 and 7
Figure 2. Relevant NOE effects on compounds 6 and 7.
Table 1. AM1 Calculations Results on Transition State
Structures for Isoxazolidines 6 and 7
percent
transition state
∆H
f
(kcal/mol) calcd obsd
(E)-exo (trans adduct 7) -228.297 31.18 28.57
(E)-endo (cis adduct 6) -228.752 66.87 71.43
(Z)-exo (cis adduct 6) -226.289 1.07
(Z)-endo (trans adduct 7) -226.167 0.88
Scheme 2. Synthesis of Phosphonated Carbocyclic
2′-Oxa-3′-aza-nucleosides 11 and 12
1390 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 Chiacchio et al.
The ratio between R- and β-nucleosides does not
change when the nucleosidation reaction was performed
starting from the crude mixture of isoxazolidines with-
out any preventive separation. These results show that
the coupling reaction of isoxazolidines with silylated
bases occurs without selectivity with respect to the
anomeric center. As previously reported, this is due to
the formation of an intermediate oxonium ion, which
would not be expected to have any significant facial bias,
since the substituents at C
3
and C
4
are not in close
proximity to induce any steric effect upon the incoming
group at C
5
.
13
The anomeric distribution obtained depends on the
attacking nucleobase. With 5-fluorouracil (10), the
β-anomers clearly predominate as nearly exclusive
products, while, in the case of thymine (8) and N-
acetylcytosine (9), a significant amount of the R-nucleo-
side has been obtained. Evidently, the attack on the
intermediate oxonium ion from either the R-orβ-side
is possible,
14
and hence the product distribution is
sensitive to structural changes of the reactants.
Biological Tests. Cytotoxicity Assays. One of the
major limitations in the use of antiviral drugs is their
toxicity to uninfected cells. For this reason, we tested
the cytotoxicity of the newly synthesized compounds
using a conventional viability assay, such as the trypan
blue exclusion test, in cell lines of lymphoid and mono-
cytoid origin. Moreover, considering the pivotal role of
apoptosis as a mechanism, triggered by cellular signals
as well as by a variety of drugs, for the controlled
removal of dead cells, particularly of the immune
system, we assayed the ability of the new compounds
to specifically induce apoptosis in the same cell lines.
Apoptosis was detected by morphological analysis, fol-
lowing staining with a DNA-binding dye, using micros-
copy. Results, expressed as CC
50
(cytotoxic concentration
50, the concentration that causes 50% toxicity by trypan
blue) and AC
50
(apoptotic concentration 50, the concen-
tration required to cause apoptosis in 50% of treated
cells), are reported in Tables 2 and 3, respectively.
No significant toxicity, when using both the classical
trypan blue test and the apoptosis assay, was detected
in 3 days of treatment in the lymphoid (Molt-3) and
monocytoid (U937, HL-60) cell lines assayed, following
treatment with AdC-P (11b) or AdT-P (11a). Conversely,
AdF-P (11c) was shown to cause detectable levels of
toxicity using both the assays. In particular, the mono-
cytoid HL-60 cell line was more sensitive to apoptosis
induced by AdF-P rather than the lymphoid cells.
Nevertheless, CC
50
and AC
50
calculated for AdF-P were
relatively high, showing that all the phosphonate com-
pounds tested were of low toxicity or nontoxic.
Inhibition of Reverse Transcriptase Activity in
Vitro. The ability of the newly synthesized compounds
to inhibit retroviral RNA-dependent DNA polymerase
(reverse transcriptase) activity was determined by
means of a novel, cell-free assay, originally developed
by us for a preliminary screening of potential inhibitors
of reverse transcriptase (RT). In this assay, which is
described here for the first time, RNA isolated from
stable transfectants expressing constitutively the gly-
coprotein D (gD) of HSV-1 was used as a template for
the RT activity of commercial avian myeloblastosis virus
RT (AMV-RT) and moloney murine leukemia virus RT
(MLV-RT). These two RTs were chosen on the basis of
their well-known characteristic of being extensively
utilized commercial sources for routine RT-PCR, with
high activity. The RT activity was revealed by directly
detecting specific HSV-1-gD DNA following a successive
polymerase chain reaction (PCR) amplification using
specific primers. Zidovudine (AZT), a well-known nu-
cleoside RT inhibitor, and (5′S)-5-fluoro-1-isoxazolidin-
5-yl-1H-pyrimidine-2,4-dione (ADF), a nucleoside com-
pound showing biological activity
6
but lacking RT-
inhibitory activity in preliminary tests, as expected by
the absence of a hydroxymethyl group in the furanose
ring, were utilized in the assay as internal positive and
negative controls, respectively. Before being assayed,
the new compounds and AZT were incubated with a
crude extract from human peripheral blood mononuclear
cells (PBMCs) stimulated with PHA for 72 h. Preincu-
bation with the PBMC crude extract served to supply
the necessary enzymes to transform the prodrugs from
esters to mono-, di-, and triphosphate or phosphonate
forms for effective inhibition of RT activity and cDNA
elongation. Results obtained using AMV-RT are shown
Table 2. Evaluation of Cytotoxicity of Phosphonates in Lymphoid and Monocytoid Cell Lines
a
24 h 48 h 72 h
cell line AdT-P AdC-P AdF-P AdT-P AdC-P AdF-P AdT-P AdC-P AdF-P
Molt-3 >1000 >1000 247 ( 37 >1000 >1000 197
b
>1000 >1000 141 ( 25
U937 >1000 >1000 ND
c
>1000 >1000 ND
c
>1000 >1000 ND
c
HL-60 >1000 >1000 >1000 >1000 >1000 457 ( 53 >1000 >1000 408
b
a
Cells were exposed under optimal culture conditions to concentrations of AdT-phosphonate (AdT-P, 11a), AdC-phosphonate (AdC-P,
11b), and AdF-phosphonate (AdF-P, 11c), ranging from 16 to 1000 µM, or control medium for the times indicated. Values indicate the
CC
50
, calculated as the concentrations of the drug required to cause 50% toxicity, detected by trypan blue exclusion test. Each value is
determined by three or more experiments.
b
Mean of two values.
c
Not done.
Table 3. Evaluation of Apoptosis Induced by Phosphonates in Lymphoid and Monocytoid Cell Lines
a
24 h 48 h 72 h
cell line AdT-P AdC-P AdF-P AdT-P AdC-P AdF-P AdT-P AdC-P AdF-P
Molt-3 >1000 >1000 457 ( 31 >1000 >1000 277
b
>1000 >1000 170 ( 30
U937 >1000 >1000 ND
c
>1000 >1000 ND
c
>1000 >1000 ND
c
HL-60 >1000 >1000 250 ( 32 >1000 >1000 151 ( 22 >1000 >1000 ND
c
a
Cells were exposed under optimal culture conditions to concentrations of AdT-phosphonate (AdT-P, 11a), AdC-phosphonate (AdC-P,
11b) and AdF-phosphonate (AdF-P, 11c), ranging from 16 to 1000 µM, or control medium for the times indicated. Values indicate the
AC
50
, calculated as the concentrations of the drug required to cause 50% apoptosis, detected by microscopy analysis following staining
with acridine orange. Each value is determined by three or more experiments.
b
Mean of two values.
c
Not done.
Novel Inhibitors of Reverse Transcriptase Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1391
in Figure 3. The quality and the specificity of the assay
are illustrated in Figure 3a, where RT activity was
assayed in the absence of the nucleoside compounds. No
difference was observed with or without the addition of
the crude extract to the complete reaction mixture in
the appearance of a clear band of amplified DNA. No
amplification product was detected when isolated RNA,
primers, or AMV-RT was singly subtracted from the
reaction mixture. No inhibitory effect was exerted by
AdC-P, AdT-P, or AdF-P at 100 nM concentration, as
well as at lower concentrations (not shown), in the
absence of crude extract activation (Figure 3b,c). Con-
versely, following exposure of the nucleosides to the
crude extract from (PBMCs), all phosphonates com-
pletely inhibited the formation of amplified products at
10 nM concentration, with the exception of ADF, which
was completely inactive. The same results were obtained
when a concentration of 100 nM was used (not shown).
No RT-inhibitory activity was observed for all the
compounds at 1 nM concentration. As shown in Figure
3d, the activity of AZT overlapped with that of phos-
phonates. Similar results were obtained when MLV-RT
was utilized. These results demonstrate that the phos-
phonate compounds exert a specific inhibitory action on
RT from at least two different retroviruses, which is
comparable with that of AZT.
Conclusions
Phosphonated N,O-nucleosides, containing thymine,
fluorouracil, and cytosine, have been synthesized in good
yields by 1,3-dipolar cycloaddition methodology.
The cytotoxicity and the RT-inhibitory activity of the
obtained compounds have been investigated. The results
indicate that the synthesized phosphonate nucleosides
presented low levels of cytotoxicity assessed by a
conventional assay to detect viability, such as the trypan
blue exclusion test, as well as by detection of cell death
by apoptosis. More importantly, the obtained phospho-
nate nucleosides seem to act as RT inhibitors, when
tested in a simple cell-free assay, as powerfully as AZT
is, opening new perspectives in future investigations on
their possible use as therapeutic agents, particularly in
antiretroviral and anti-HBV chemotherapy.
Experimental Section
General. Melting points were determined with a Kofler
apparatus and are uncorrected. Elemental analyses were
performed with a Perkin-Elmer elemental analyzer. NMR
spectra were recorded on a Varian instrument at 500 MHz
(
1
H) and at 125 MHz (
13
C) using deuteriochloroform or deu-
terated methanol as solvent; chemical shifts are given in ppm
from TMS as internal standard. NOE experiments were
performed by a modified cyclenoe sequence, implemented in
Darmstadt, which does alternate scan subtraction of two FIDs
in which the saturation frequency is moved on-resonance and
off-resonance. Power may be reduced from ordinary NOE
experiments because the irradiation is cycled through the lines
of the multiplet; it is a steady-state NOE in which a single
resonance is saturated at low power for approximately 5 ×
T1 (5 s in our case) before acquiring the FID. Thin-layer
chromatographic separations were performed on Merck silica
gel 60-F
254
precoated aluminum plates. Preparative separa-
tions were carried out by flash chromatography using Merck
silica gel 0.035-0.070 mm. Preparative centrifugally acceler-
ated radial thin-layer chromatography (PCAR-TLC) was per-
formed with a Chromatotron Model 7924 T (Harrison Re-
search, Palo Alto, CA); the rotors (1 or 2 mm layer thickness)
were coated with silica gel Merck grade type 7749, TLC grade,
with binder and fluorescence indicator (Aldrich 34,644-6) and
the eluting solvents were delivered by the pump at a flow rate
of 0.5-1.5 mL/min.
The identification of samples from different experiments was
secured by mixed melting point and superimposable NMR
spectra.
Starting Materials. Diethylphosphonoacetaldehyde diethyl
acetal (3), thymine (8), N-acetylcytosine (9), and 5-fluorouracil
(10) were purchased from Aldrich Co. All solvents were dried
according to literature methods.
Diethyl (2E)- and (2Z)-2-[Methyl(oxido)imino]eth-
ylphosphonate (5). A solution of diethyl 2-oxoethylphospho-
nate
9
(4) (20 mmol), N-methylhydroxylamine hydrochloride (20
mmol), and triethylamine (20 mmol) in toluene (70 mL) was
stirred, at room temperature, for 3 h. The reaction mixture
was filtered, the solvent was evaporated under reduced pres-
sure, and the residue was purified by silica gel flash chroma-
Figure 3. Inhibition of reverse transcriptase (RT) activity of
compounds 11a-c, in comparison with ADF (negative control)
and AZT (reference control), evaluated by a cell-free assay
based on a RT-PCR reaction. RNA template from glycoprotein
D of HSV-1 was reversely transcribed by AMV-RT into cDNA
in the presence of the compounds. DNA was then amplified
and visualized on ethidium bromide containing agarose gel.
(a) Control runs in which RT activity was assayed, in the
absence of the nucleoside compounds, with or without the
addition of a crude extract from stimulated PBMCs and with
single subtraction, from the reaction mixture, of isolated RNA,
primers, or AMV-RT. (b) Experimental runs in which RT
activity was assayed in the presence of AdC-P or AdT-P at
the concentrations of 100, 10, or 1 nM, with or without
preincubation with the crude extract. (c) Experimental runs
in which RT activity was assayed in the presence of AdF-P or
ADF at the concentrations of 100, 10, or 1 nM, with or without
preincubation with the crude extract. (d) Experimental runs
in which RT activity was assayed in the presence of AZT at
the concentrations of 100, 10, or 1 nM, with or without
preincubation with the crude extract.
1392 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 Chiacchio et al.
tography (CHCl
3
/CH
3
OH 95:5) to give nitrone 5 as colorless
oil. Yield 95% (Z/E ) 5/1).
Synthesis of Isoxazolidines 6 and 7. A solution of nitrone
5 (5.7 mmol) in vinyl acetate (30 mL) was stirred at 60 °C for
24 h. After this period, the reaction mixture was evaporated
under reduced pressure and the residue purified by radial
chromatography (CHCl
3
/CH
3
OH 99.5:0.5; spot evidenced with
cerium molibdic reagent) to give the pure isoxazolidines 6 and
7.
(3RS,5SR)-3-[(Diethoxyphosphoryl)methyl]-2-methyl-
isoxazolidin-5-yl acetate (6): yield 65%, light yellow oil.
(3RS,5RS)-3-[(Diethoxyphosphoryl)methyl]-2-methyl-
isoxazolidin-5-yl acetate (7): yield 25%, light yellow oil.
General Procedure for the Preparation of Nucleo-
sides 11 and 12. A suspension of bases 8-10 (0.62 mmol) in
dry acetonitrile (3 mL) was treated with bis(trimethylsilyl)-
acetamide (BSA) (2.54 mmol) and refluxed for 15 min under
stirring. To the clear solution obtained were added a solution
of the epimeric isoxazolidines 6/7 (0.52 mmol) in dry acetoni-
trile (3 mL) and trimethylsilyltriflate (TMSOTf) (0.4 mmol)
dropwise, and the reaction mixture was stirred at 55 °C for 6
h. After being cooled at 0 °C, the solution was neutralized by
careful addition of aqueous 5% sodium bicarbonate and then
concentrated in vacuo. After the addition of dichloromethane
(8 mL), the organic phase was separated, washed with water
(2 × 10 mL), dried over sodium sulfate, filtered, and evapo-
rated to dryness. The residue was purified by flash chroma-
tography (CHCl
3
/MeOH 95:5) to give β-nucleosides 11a,c,d and
R-nucleosides 12a,d.
Diethyl {(1′SR,4′RS)-1′-[[(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)]-3′-methyl-2′-oxa-3′-azacyclopent-
4′-yl]}methylphosphonate (11a): yield 68%, sticky oil.
Diethyl {(1′RS,4′RS)-1′-[[(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)]-3′-methyl-2′-oxa-3′-azacyclopent-
4′-yl]}methylphosphonate (12a): yield 12%, sticky oil.
Diethyl {(1′SR,4′RS)-1′-[4-(acetylamino)-2-oxopyrimi-
din-1(2H)-yl]-3′-methyl-2′-oxa-3′-azacyclopent-4′-yl}-
methylphosphonate (11d): yield 56%, sticky oil.
Diethyl {(1′RS,4′RS)-1′-[4-(acetylamino)-2-oxopyrimi-
din-1(2H)-yl]-3′-methyl-2′-oxa-3′-azacyclopent-4′-yl}-
methylphosphonate (12d): yield 14%, sticky oil.
Diethyl [(1′SR,4′RS)-1′-(5-fluoro-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)-3′-methyl-2′-oxa-3′-azacyclopent-4′-
yl]methylphosphonate (11c): yield 75%, sticky oil.
Synthesis of Diethyl [(1′SR,4′RS)-1′-(4-amino-2-oxopy-
rimidin-1(2H)-yl)-3′-methyl-2′-oxa-3′-azacyclopent-4′-yl-
]methylphosphonate (11b). A solution of 11d (100 mg) in a
mixture of aqueous K
2
CO
3
(5%, 5 mL) and methanol (5 mL)
was left to stir for 4 h; solvent was then evaporated under
reduced pressure, and the residue was purified by flash
chromatography (CHCl
3
/CH
3
OH 95:5) to give the deacetylated
compound 11b: yield 90%, colorless sticky oil.
Evaluation of Toxicity and Apoptosis. Toxicity was
evaluated by a standard viability assay, using the trypan blue
exclusion test. Apoptosis was evaluated by morphological
analysis of the cells, performed following staining with acridine
orange as previously described.
15
Briefly, over 600 cells,
including those showing typical apoptotic characteristics, were
counted using a fluorescence microscope. The identification of
apoptotic cells was based on the presence of uniformly stained
nuclei showing chromatin condensation and nuclear fragmen-
tation.
Reverse Transcriptase Inhibition Assay. The capacity
of the described compounds to inhibit RT activity was inves-
tigated by evaluating their activity toward cDNA generation
from an RNA template using a cell-free RT reaction assay,
based on routinely adopted RT-PCR procedures. This included
RNA from stable transfectants expressing gD of HSV-1, as a
template. The gD-expressing transfectants have been de-
scribed elsewhere.
16
Transfectants were grown in D-MEM plus
12% fetal bovine serum (FBS), 400 µg/mL G418 (Gibco-
Invitrogen Co., Paisley, Scotland, UK), and 30 µg/mL BrdU
(Sigma-Aldrich Co., St. Louis, MO). The choice of a nonretro-
viral, ectopic cellular system of expression as a source of RNA
template was motivated to avoid problems with retroviral or
human DNA, possibly present in reagents utilized during the
execution of the entire assay. Cells underwent passage every
2 days and at each passage cell growth was monitored by
evaluating living cells, using the trypan blue dye exclusion
test. The new synthesized compounds and the nucleosides
AdFU
6
or AZT (Sigma-Aldrich Co.) were activated in vitro
through incubation with a crude extract from 1 × 10
6
PBMCs,
which served as enzyme supplier for phosphorylation pro-
cesses. PBMCs, separated by density gradient from peripheral
blood of healthy donors, negative for HIV-1/2, HTLV-1/2, and
B/C hepatitis, were previously stimulated with PHA (2 µg/mL)
and IL-2 (20 U/mL), for 72 h in RPMI plus 20% FBS. For the
preparation of the crude extract, PBMCs were rinsed three
times in cold PBS and then solubilized in lysis buffer (50 mM
Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA pH 7.4, 0.05%
Triton-X, NaCl 150 mM, 0.25% sodium deoxycholate, 0.1% NP-
40, and, freshly added, 1 mM PMSF, 15 µM DTT, 5 µg/mL
leupeptin, 5 µg/mL pepstatin, 5 µg/mL aprotinin, 1 mM Na
3
-
VO
4
,20mMNa
3
F, all from Sigma) on ice and centrifuged at
10 000 rpm. The compounds were incubated with the crude
extract from stimulated PBMCs, for1honice. The crude
extract was then inactivated for 5 min at 95 °C. RNA isolation
from 5 × 10
6
gD-expressing transfectants was performed using
Trizol (Gibco-Invitrogen Co.), according to the manufacturer’s
instructions. Total RNA (1 µg) was reverse transcribed using
a reverse primer (5′-TGT CGT CAT AGT GGG CCT CCA T-3′)
(0.5 µM) specific for a sequence of the HSV-1 US6 gene that
codes for gD, in a reaction mix containing RT buffer (1×),
RNase inhibitor (100 U), dNTP (4 mM), DTT (10 mM), and 30
U of avian myeloblastosis virus RT (AMV-RT) or 40 U of
moloney murine leukemia virus RT (MLV-RT) (all from
Promega Co., Madison WI). The reactions were performed in
the presence or in the absence of the activated compounds, at
the concentration of 100, 10, and 1 nM, for1hat37°C.After
incubation at 95 °C for 5 min, 5 µL of the RT reaction product
was used for DNA PCR in a reaction mix containing 1× Taq
Gold buffer (Promega Co.), 0.5 µM primers (US6 reverse 5′-
TGT CGT CAT AGT GGG CCT CCA T-3′ and US6 forward
5′-AGA CTT GTT GTA GGA GCA TTC G-3′), 0.3 mM dNTP,
5 mM MgCl
2
, and 1.25 U Taq Gold (Promega Co.), for 30 cycles
(30 s at 95 °C, 30 s at 60 °C, and 45 s at 72 °C) on a Cetus
DNA thermal cycler 2400 (Perkin-Elmer, Norwalk, CT). Fol-
lowing the final cycle, samples were incubated at 72 °C for 20
min to ensure the completion of the final extension step.
Amplified DNA (350 bp) was visualized on 1% agarose gel
containing 10 µg/mL ethidium bromide in 1× TAE buffer.
Acknowledgment. This work was partially sup-
ported by M.I.U.R. (progetto P.R.I.N. 2002 and F.I.R.B.).
The C.I.N.M.P.I.S. (Italy) is also acknowledged for a
fellowship to M.S.
Supporting Information Available: Spectroscopic data
(
1
H and
13
C NMR) and elemental analyses data of compounds
5-7, 11a-d, 12a, and 12d. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) (a) Richman, D. D. HIV Chemoterapy. Nature 2001, 410, 995-
1001. (b) De Clercq, E. New Developments in Anti-HIV Chemo-
therapy. Biochim. Biophys. Acta 2002, 1587, 258-275. (c) De
Clercq, E. New Anti-HIV Agents and Targets. Med. Res. Rev.
2002, 22, 531-565. (d) Macchi, B.; Balestrieri, E.; Mastino, A.
Effects of Nucleoside-Based Anti-Retroviral Chemotherapy on
Human T-Cell Leukaemia/Lymphotropic Virus Type-1 (HTLV-
1). J. Antimicrob. Chemother. 2003, 51, 1327-1330. (e) De
Clercq, E. Antiviral Drugs in Current Clinical Use. J. Clin. Virol.
2004, 30, 115-133.
(2) (a) Mitsuya, H.; Yarchoan, R.; Broder, S. Molecular Targets for
Aids Therapy. Science 1990, 249, 1533-1544. (b) De Clercq, E.
HIV Resistance to Reverse Transcriptase Inhibitors. Biochem.
Pharmacol. 1994, 47, 155-169. (c) Katz, R. A.; Skalka, A. M.
The Retroviral Enzymes. Annu. Rev. Biochem. 1994, 63, 133-
173. (d) Turner, B. G.; Summers, M. F. Structural Biology of
HIV. J. Mol. Biol. 1999, 285,1-32. (e) Jonckheere, H.; Anne,
J.; De Clercq, E. The HIV-1 Reverse Transcription (RT) Process
as Target for RT Inhibitors. Med. Res. Rev. 2000, 20, 129-154.
Novel Inhibitors of Reverse Transcriptase Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1393
