Pyrimidine 2,4-Diones in the design of new HIV RT inhibitors

Article abstract of DOI:10.3390/molecules24091718

The pyrimidine nucleus is a versatile core in the development of antiretroviral agents. Onthis basis, a series of pyrimidine-2,4-diones linked to an isoxazolidine nucleus have been synthesized and tested as nucleoside analogs, endowed with potential anti-

Full text of DOI:10.3390/molecules24091718

molecules
Article
Pyrimidine 2,4-Diones in the Design of New HIV
RT Inhibitors
Roberto Romeo 1,*, Daniela Iannazzo 2 , Lucia Veltri 3 , Bartolo Gabriele 3
,
Beatrice Macchi 4 , Caterina Frezza 4, Francesca Marino-Merlo 5 and Salvatore V. Giofrè 1,
*
1
Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università di Messina,
Via S.S. Annunziata, 98168 Messina, Italy
2
3
4
Dipartimento di Ingegneria, Università di Messina, Contrada Di Dio, 98166 Messina, Italy;
diannazzo@unime.it
Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, Via P. Bucci 12/C,
87036 Arcavacata di Rende, Italy; lucia.veltri@unical.it (L.V.); bartolo.gabriele@unical.it (B.G.)
Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma “Tor Vergata”, 00133 Roma, Italy;
macchi@med.uniroma2.it (B.M.); catafrezza@hotmail.it (C.F.)
IRCCS Centro Neurolesi “Bonino-Pulejo”, 98124 Messina, Italy; fmarino@unime.it
Correspondence: robromeo@unime.it (R.R.); sgiofre@unime.it (S.V.G.);
Tel.: +39-090-676-6565 (R.R.); +39-090-676-6566 (S.V.G.)
5
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Erik De Clercq
Received: 18 February 2019; Accepted: 30 April 2019; Published: 2 May 2019
Abstract: The pyrimidine nucleus is a versatile core in the development of antiretroviral agents. On this
basis, a series of pyrimidine-2,4-diones linked to an isoxazolidine nucleus have been synthesized and
tested as nucleoside analogs, endowed with potential anti-HIV (human immunodeficiency virus)
activity. Compounds 6a–c, characterized by the presence of an ethereal group at C-3, show HIV
reverse transcriptase (RT) inhibitor activity in the nanomolar range as well as HIV-infection inhibitor
activity in the low micromolar with no toxicity. In the same context, compound 7b shows only a
negligible inhibition of RT HIV.
Keywords: reverse nucleosides; Pyrimidine-2,4-dione derivatives; HIV RT inhibitors; biological
activity; molecular docking
1. Introduction
Continuous efforts in the search of new antiviral agents are a consequence of the urgent demand
for new therapeutic agents in which an improved biological activity against viruses is matched with
low toxicity towards host cells. In this context, over recent years the ever-growing development of new
anti-retroviral drugs has turned the prognosis of human immunodeficiency virus (HIV)-1 infection
from a terminal into a chronic disease. In particular, highly active antiretroviral therapy (HAART) has
significantly changed the progression and outcome of infection with HIV-1 [1–5]. HAART comprises
four major classes of drugs: nucleoside reverse transcriptase (RT) inhibitors (NRTIs), non-nucleoside
RT inhibitors (NNRTIs), protease inhibitors (PIs), and integrase INST inhibitors (INSTIs).
In order to overcome some of the limits and side effects of the current treatment regimes, new
therapeutic approaches have been investigated for HIV treatment, such as the use of antagonists of
CCR5, a receptor involved in the virus entry. In this context, the m-Tor inhibitor rapamycin provides a
potential strategy to inhibit HIV, especially in patients with drug resistant HIV strains [6–10].
In spite of the consistent benefits for HIV-1 infected patients undergoing antiretroviral therapy,
a complete immune reconstitution is usually not achieved. Actually, antiretroviral therapy may be
frequently accompanied by immunological unresponsiveness, persistent inflammatory conditions
Molecules 2019, 24, 1718; doi:10.3390/molecules24091718
www.mdpi.com/journal/molecules

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and inefficient cytotoxic T-cell response [11
–
14]. Accordingly, structurally novel HIV inhibitors,
particularly those with a distinct mechanism of action, could add significantly to the existing HAART
repertoire, overcoming several side effects linked to the continuous exposition to antiretroviral drugs
and the higher risk of developing co-morbidities, such as cardiovascular, metabolic and neurological
disease [15–18].
The reverse transcription (RT) of the viral single-stranded (+) RNA genome into double-stranded
DNA plays a basic role in the replication of HIV. Due to this essential step in the viral life cycle, RT
constitutes the target of numerous anti-HIV drugs that are key components of HAART [19–25].
Two classes of drugs, NRTIs and NNRTIs, have been reported as inhibitors of RT [26–31]. NRTIs
act as DNA chain terminators, while NNRTIs bind to a hydrophobic pocket close to the RT active
site and inhibit the enzyme activity by mediating allosteric changes in the RT conformation, thus
causing a distortion in the arrangement of the catalytic active site aspartyl residues [32–37]. However,
in spite of the efficiency of some NRTIs and NNRTIs, the rapid emergence of multidrug-resistant
mutants has promoted the research of new anti-HIV agents with significantly improved drug resistance
profiles [38–44].
In particular, NNRTIs have received a lot of attention because of their favorable potency and low
cytotoxicity. So far, five NNRTIs have been approved for AIDS treatment, and about 50 classes of
structurally diverse NNRTIs are being widely investigated [45–48].
In this research field, the pyrimidine nucleus represents a versatile chemical core in
the design of many antiretroviral agents acting as NNRTIs: Modified pyrimidines constitute
the backbone of many non-nucleoside reverse transcriptase inhibitors. Diaryl-pyrimidines
(DAPYs,
(DABO,
its analogue (TNK-651;
1
[
,
49
53]), 1-[(hydroxyethoxy)-methyl]-6-(phenyl-sulfanyl)thymine (HEPT;
) (Figure 1) are efficient NNRTIs that—through binding at the allosteric,
–
51]) and their derivatives, 2-alkoxy-6-benzyl-3,4-dihydro-4-oxopyrimidine
2
[
52
3 [54,55]) and
4
non-nucleoside binding pocket (NNIBP) of RT—prevent the conformational transition needed for the
formation of a productive polymerase–RNA complex [56].
Recently, we have reported the synthesis and biological activity of the new class of 30-pyrimidinyl
isoxazolidines
5, the truncated reverse isoxazolidinyl nucleosides (TRINs), as HEPT analogues [57].
When tested in vitro for their biological activity, compound
5
showed a nearly complete inhibition of
avian myeloblastosis virus (AMV) RT and HIV RT in the nanomolar range, with weak cytotoxicity
towards human cells.
Figure 1. Modified pyrimidines as non-nucleoside reverse transcriptase inhibitors (NNRTIs).
Docking data indicate that an important role could be played by the substituent at the C-5
position. On this basis, according to the exploration of chemotypes amenable to the construction of
new compounds endowed with significantly improved drug resistance profiles, compared with the
first generation NNRTIs, we have been interested in the evaluation of a series of compounds, amenable
to pyrimidine-2,4-dione scaffold, where structural elaboration, both at the level of the nucleobase and
the isoxazolidine unit, have been performed in order to improve the stabilizing interactions suggested
by docking data.

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We report here the synthesis and biological evaluation of compound
6, where the substituent at C-5
has been replaced with an ethereal unit in order to favor hydrogen bond interactions and to improve
hydrophobic interactions with the hydrophobic residues in the RT pocket [57]. In the same context,
according to our program targeted to the discovery of new compounds able to interfere with viral
replication of HIV, the redesign of N-1 moiety led to phosphonated reverse homo-N,O-nucleosides 7,
characterized by the presence of a phosphonic group at C-40, where a methylene linker is incorporated
between the nucleobase and the isoxazolidine ring: The absence of the natural N-glycosidic bond
should result in a greater metabolic stability of these compounds due to their inertness towards the
hydrolytic activity of cellular phosphorylases (Figure 2). The insertion of the phosphonic group at
C-40 is supported by the consideration that phosphonated N,O-nucleosides should be able to bypass
the initial selective enzymatic monophosphorylation step and overcome the instability of nucleotides
toward phosphodiesterases [58].
Figure 2. New synthesized modified pyrimidines as potential antiviral agents.
Antiviral assays showed that some of the new synthesized pyrimidin-2,4-dione derivatives, in
particular some of those amenable to derivatives 6, inhibited HIV RT activity in the nanomolar range
and HIV-1 infection in the low micromolar range, without cytotoxicity, at concentrations up to 100 µM.
2. Results and Discussion
2.1. Chemistry
The synthesis of isoxazolidinyl pyrimidines 6a
from the route reported for the synthesis of compound
59 60], which was reduced with Cp2Zr(H)Cl to the corresponding azahemiacetal
with Ac2O with the formation of the acetyl derivative 10 (Scheme 1).
–
c
is described in Scheme 1. The approach, adapted
57], starts from the isoxazolidin-3-one
, and then treated
5
[
8
[
,
9
Scheme 1. Synthesis of 30-pyrimidinyl isoxazolidines 6a
–c: (a) ETA, PhOCH2C(O)Cl, DMF, room
temperature; (b) Cp2Zr(H)Cl) (4 equiv), THF, SiO2, 1 h, room temperature; (c) Ac2O, ETA, DCM, room
temperature, 6 h; (d) 5-substituted uracil 11a–c, BSA, TMSOTf, CH3CN, r.t., 4 h.

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The subsequent coupling of 10 with 5-substituted uracils 11a
–
c, prepared by reaction of
5-hydroxymethyluracil with allylic, propargylic and benzylic alcohols, respectively, in conc. HCl at
reflux, afforded the isoxazolidinyl pyrimidines 6a–c in good yields.
According to the data suggested by docking experiments, which indicate that the presence of an
aromatic unit in the ethereal chain at C-5 could improve the potential biological activity as NNRTI of
the synthesized compounds, we also investigated the possible role of the replacement of the phenyl
ring of compound 6b with an heteroaromatic system. For this aim, compound 6c was reacted with
azides 12a–d (Scheme 2). Derivatives 6d–g were easily obtained in very good yields.
Scheme 2. Synthesis of 30-pyrimidinyl isoxazolidines 6d
room temperature, 4 h.
–
g
: (a) CuSO4 5H2O, sodium ascorbate, Et3N,
·
Phosphonated reverse homo-N,O-nucleosides
antitumoral agents [61]. Here, we report a different synthetic route which proceeds with better yields
and allows the obtainment of pure and anomers. The new designed process combines a 1,3-dipolar
7 have been described in literature and tested as
α
β
cycloaddition, leading to the isoxazolidinyl system, and an Arbuzov reaction to insert the phosphonic
group at C-5. Finally, a nucleosidation process introduces the pyrimidine nucleobase at C-3 position.
Thus, C-[(tert-butyldiphenylsilyl)oxy]-N-methylnitrone 13 [62,63], as an isomeric E/Z mixture (1:5),
was reacted with vinyl acetate to give with a good stereoselectivity the epimericisoxazolidines 14a
and 14b, in a relative ratio 1:4.2 (global yield 90%). Compounds 14a and 14b were separated by flash
chromatography; the phosphonate group was then introduced by a Lewis acid catalyzed Arbuzov
reaction which afforded derivatives 15a and 15b (80% yield) (Scheme 3).
Scheme 3. Synthesis of phosphonated reverse homo-N,O-nucleosides 7a
–
d
: (a) vinyl acetate, CH2Cl2,
4 h, room temperature; (b) P(OEt)3, TiCl4, CH2Cl2, 4 h,
−
30 ◦C; (c) TBAF, CBr4, THF, room temperature,
6 h; (d) nucleobase, Cs2CO3, DMF, 95 ◦C, 4 h, room temperature.
Compounds 15a and 15b were treated with TBAF in THF and subsequently converted into the
corresponding bromides 16a and 16b (global yield 65%). The subsequent coupling with fluorouracil,

Molecules 2019, 24, 1718
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in the presence of Cs2CO3 in DMF at 95 ◦C, completed the reaction route, affording the phosphonated
reverse homo-N,O-nucleosides 7a,b (Scheme 4).
Scheme 4. Synthesis of phosphonated reverse homo-N,O-nucleosides 7a
,
b
: (a) vinyl acetate, CH2Cl2,
4 h, room temperature; (b) P(OEt)3, TiCl4, CH2Cl2, 4 h,
−
30 ◦C; (c) TBAF, THF, 6 h, room temperature;
(d) CBr4, THF, room temperature, 6 h; (e) nucleobase, Cs2CO3, DMF, 95 ◦C, 4 h, room temperature.
2.2. Biological Activity
Compounds 6a–g and 7a,b were preliminarily tested in vitro for their cytotoxicity (CC50) towards
MOLT-3 cells, a human T-lymphoblastoid cell line, through the MTS assay. Nevirapine (NVP),
Rilpivirine (RPV) and Efavirenz (EFV) have been used as reference controls. Cytotoxicity against
these cells was not observed with any of the 6a
Conversely, a 50% cytotoxicity was observed for compounds 6d
594 0.8, 552 3, 670 1, 579 M, respectively. Nevirapine (NVP) showed cytotoxicity at
concentrations higher than 1000 µM.
–
c
and
7
analogues at concentrations up to 1000
µM.
–
g
(Table 1) at concentrations of
±
±
±
±
2 µ
Table 1. Inhibitory activities against human immunodeficiency virus (HIV)-1 reverse transcriptase (RT)
of pyrimidinylisoxazolidines and their cytotoxicity.
RT Inhibition Assay 1
Cytotoxicity
MRTIC (µM)
CC50 ± SD (µM) 2
Compound
HIV-RT
MOLT-3
Pearson’s 3
6a
6b
6c
6d
6e
6f
6g
7a
7b
0.1
1
0.01
>100
>100
>100
100
>1000
>1000
>1000
594 ± 0.8
579 ± 2
552 ± 3
670 ± 1
>1000
>1000
>1000
n.d.
0.94
0.95
0.97
-
-
-
-
-
-
n.d.
90 4
NVP
RPV
EVF
0.001
0.038
0.0000094
0.97
n.d.
n.d.
n.d.
1
Minimal inhibitory concentration (MIC) against HIV-RT, defined as the minimum concentration required to
completely inhibit (100%) RT, was evaluated in a cell-free assay. The compounds were tested in two rounds. In the
first round, the concentration range was between 200 and 1 M. In the second round, compounds were tested at
concentrations between 1 and 0.001
M. 2 Half-maximal cytotoxic concentration (CC50), defined as the concentration
required to decrease metabolic activity by 50% standard deviation (SD), was evaluated in MOLT-3 cells using
µ
µ
±
3
an MTS assay. The Pearson product–moment correlation coefficient (Pearson’s r), which reflects the degree and
direction of the linear relationship between two variables, was calculated for each significant CC50 value. After
4
incubation with a crude extract from PBMCs (human peripheral blood mononuclear cells).

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The antiretroviral activity of all the synthesized compounds was tested by a cell-free RT inhibitory
assay, in dose-response fashion [64 66]. In particular, the RT inhibitory activity of the new compounds
–
along with that of the reference antiviral compound, NVP, were tested versus HIV-1 or avian
myeloblastosis virus (AMV) reverse transcriptase. The results reported in Table 1 show that reverse
homo-N,O-nucleosides
7, as well as pyrimidin-2,4-dione analogues containing an heteroaromatic
system (6d ) in the ethereal chain at the C-5 position, did not show any antiretroviral activity in
–
g
the adopted conditions at concentrations up to >1 mM. Conversely, compounds carrying an allyl
or a benzyl unit at C-5 (6a and 6c) showed strong inhibition against HIV RT, but not AMV RT, with
activities in the low nanomolar range. The highest inhibitor activity was evidenced by compound 6c
with an MRTIC value of 0.01 µM, not far from that of nevirapine (NVP). The good anti-HIV RT activity
exerted by compound 6c represents a significant starting point for future in-depth investigations
regarding its in vitro and in vivo anti-HIV activity, toxicity, and oral bioavailability. Moreover, the
reported data further confirm the relevance of pyrimidine nucleus as the chemical core in the design of
new anti-HIV agents with an improved pharmacological profile. It is noteworthy that of the series of
compounds 6a
inhibitory effect.
We then focused our attention on compounds (6a–c) to check whether they directly inhibited
–c, 6b (characterized by the presence of a propargyl group) showed the lowest HIV-1 RT
HIV infection, through a recently developed cell infection assay [65]. For comparison, compound 6g
and NVP were also assayed. The assay consisted of infection with HIV-I infectious molecular clone
NL4-3 (HIVNL4-3) of CEM human lymphoblastoid cells stably transfected with a plasmid of the
green fluorescence protein under the control of HIV-LTR (CEM-GFP). CEM-GFP were unexposed
◦
or exposed to several concentrations of compounds or to NVP for 2 h at 37 C in a humidified
atmosphere supplemented with 5% CO2. Following infection with HIVNL4-3, cells were subjected to
spinoculation and incubated for 3 h at 37 ◦C. After centrifugation, the excess of virus was removed
and the compounds were re-added. Infection was determined by assessing GFP expression through
flow cytometry analysis from 5000 events per sample, after 72 h of incubation. The results in Table 2,
referring to one representative experiment of two performed with similar results, show that 6b was
endowed with a low IC50, not far from that of NVP. Compounds 6a and 6c proved inhibitory in a
range from 10 to 15 µM. Interestingly, toxicity of compound 6b towards uninfected CEM-GFP was
even lower than that of NVP, ensuring a good selectivity index. Thus, the anti-HIV activity exerted by
some of the compounds under investigation represents a significant starting point for future in-depth
investigations regarding their actual antiretroviral potential. Moreover, the reported data further
confirm the relevance of the pyrimidine nucleus as chemical core in the design of new anti-HIV
agents with improved pharmacological profile. Noteworthy, in the series of compounds 6a–c, 6b,
characterized by the presence of a propargyl group, showed the more potent inhibitory effect towards
HIV-1 infection in a cell assay, while 6c, characterized by a benzyl group, exhibited the lowest value of
HIV-RT inhibition in a cell-free assay.
Table 2. Inhibitory activities against human immunodeficiency virus (HIV)-1 infection assayed by flow
cytometry analysis of HIV-1 infected CEM cells harboring a plasmid of the green fluorescence protein
under the control of HIV-LTR (CEM-GFP).
IC50 ± SD (µM) 1
% GFP + cells
Cytotoxicity CC50 ± SD (µM) 2
CEM/GFP
Selectivity Index 3
SI
Compound
6a
6b
6c
6g
NVP
10.1
0.69
15.7
103
747 ± 10.7
1053 ± 8.2
1127 ± 6.6
427 ± 0.1
980 ± 9.9
73.9
1526
71.8
4
0.14
7000
1
Inhibitory concentration 50 (IC50), defined as the compound and or drug concentration required to decrease by
50% HIV-1 infection of CEM-GFP+ cells.2 Half-maximal cytotoxic concentration (CC50), defined as the concentration
required to decrease metabolic activity by 50% standard deviation (SD), as evaluated in uninfected CEM-GFP cells
using an MTS assay.3 Selectivity index: CC50/IC50
±

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As previously reported [57], the RT inhibition exhibited by the tested compounds 6a
–
c
may be
explained by an allosteric mechanism of action. In fact, these compounds were able to inhibit RT
activity without any conversion by phosphorylation into the corresponding triphosphate forms. Thus,
these compounds should not act as competitive inhibitors of RT with respect to the nucleotide substrate,
but inhibition of the polymerase activity would occur before elongation, presumably by preventing the
conformational transition needed to form a productive polymerase–RNA complex.
The biological activity observed for compounds 6a
data [57] (see Supplementary Materials). Starting from the X-ray coordinates of the TNK-651–HIV-1 RT
complex (PDB code: 1RT2), derivatives 6a were docked into the wild-type HIV-1 RT non-nucleoside
binding pocket (NNIBP); the binding free energies ( Gb) are in the range of –7.45 to –8.51 kcal/mol
(Table 3). The binding mode of compounds 6a shows common interactions with the NNBP of RT
(Figure 3). In particular, the aromatic ring of compound 6a was found in close contact with Tyr188
and Trp229, thus allowing type interactions. Additional hydrophobic contacts were established
–c has been rationalized on the basis of docking
–
c
∆
–
c
π-π
with the Leu234, Leu100 and Trp229, while the nucleobase interacted with the Lys101 by the NH
group, which acts as a donor of hydrogen bond, and a polar interaction was exerted by the carbonyl
group at C-2, which was oriented towards the water exposed in proximity to the positively charged
ammonium group of Lys101. Furthermore, the presence of the allyl unit improves hydrophobic and
π
-π
interactions with Pro225, Pro236, Val106 and Phe227. The aromatic ring of compound 6b shows
π
-π
interactions with Phe227, Tyr188 and Trp229; additional hydrophobic contacts are established with
the Leu100, Val 106 and Pro236.
Table 3. ∆G of binding of compounds 6a–c.
Compound
6a
∆G Kcal/mol
−8.51
−7.45
−8.62
6b
6c
Contrary to the effect exerted by the allyl unit in compound 6a, the propargyl group of 6b pushes
the pyrimidine nucleus away from the Lys101 residue, leading to the loss of the hydrogen bond with
pyrimidine NH.
The docking of 6c, the most active compound, confirms that the benzyl ethereal group at C-5
contributes to the stability of the resulting inhibitor–RT complex, leading to a favorable stabilizing
interaction with Tyr318 residue at the allosteric site of RT-HIV, by the formation of a hydrogen bond.
Moreover, the benzyl unit improves π-π interactions with Phe227 and additional hydrophobic contacts
with Val106, Pro236 and Pro225; the nucleobase shows hydrogen bonds with Lys101 and Lys103 by
NH and carbonyl group at C4, respectively.
In contrast with our expectations, the replacement of the phenyl ring in 6c with a triazole unit
(compounds 6d–g) does not afford any positive interaction with aminoacidic residues in the NNRTIs
binding pocket. In particular, docking experiments show that the triazole unit induces a different
binding mode with loss of hydrogen bonds between the nucleobase and the Lys101. Only compound 6g
shows a modest anti-HIV activity.
According to the presence of the phosphonic unit at C-50, compound
7 could be regarded as
monophosphonylated homo-N,O-nucleosides, which could be further explored as possible NRTI chain
terminators after conversion by phosphorylation into the triphosphate form. The cell-free conditions
of the RT inhibition assay employed here did not provide enzymatic phosphorylation. Thus, we
determined their ability to inhibit the reverse transcriptase activity by means of a cell-free assay [36],
after incubation with a crude extract from 1
×
106 PBMCs (human peripheral blood mononuclear cells),
which serves as enzyme supplier for phosphorylation processes. Zidovudine (AZT) (a well-known
nucleoside) and tenofovir (a phosphonated nucleoside actually used in antiviral chemotherapy) were
utilized in the assay as internal positive controls. The obtained results indicate that compound 7b

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exhibits a possible inhibition of RT HIV in the range 85–120
µM: the R-anomer 7a being completely
inactive at the higher concentration tested (200 µM).
The results obtained from RT inhibitory assays and docking studies suggest that phosphonated
reverse homo-N,O-nucleosides 6a represent an interesting class of biologically active molecules,
–
c
worthy of investigation by SAR and in vivo toxicology studies in order to obtain new anti-HIV agents
with a good pharmacological profile.
Figure 3. Docking interactions for compounds 6a, 6b and 6c.

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3. Materials and Methods
3.1. General Information
Solvents and reagents were used as received from commercial sources. Melting points were
determined with a Kofler apparatus. HRMS were determined with a TSQ Quantum XLS Triple
Quadrupole GC-MS/MS (Thermo Scientific, Waltham, MA, USA). NMR spectra (1H-NMR recorded at
500 MHz, 13C-NMR recorded at 125 MHz) were obtained in CDCl3 solution on a Varian instrument
(Agilent Technologies, Palo Alto, CA, USA), and data are reported in ppm relative to TMS as an
internal standard. Thin-layer chromatographic separations were carried out on Merck silica gel 60-F254
precoated aluminum plates (Merck, Darmstadt, Germany). Flash chromatography was carried out
using Merck silica gel (200–400 mesh). Preparative separations were carried out using a Büchi C-601
MPLC instrument (BUCHI Italia S.r.l., Milano, Italy) using Merck silica gel 0.040–0.063 mm, and the
eluting solvents were delivered by a pump at the flow rate of 3.5–7.0 mL/min. All solvents were dried
according to methods in the literature. The identification of samples from different experiments was
secured by mixed melting points and superimposable NMR spectra.
Compounds 8–10 were synthesized as described previously [48]. 5-Substituted uracils 11a
were prepared by reaction of 5-hydroxymethyluracil with allylic, propargylic and benzylic alcohols,
respectively, in conc. HCl at reflux. Azides 12a were synthesized by two different procedures [59]:
Compounds 12a were obtained by treatment of substituted anilines with sodium nitrite and an
equimolar amount of sodium azide in acetonitrile, at 0–5 ◦C for 1 h; and compounds 12c
were prepared
–c
–d
,b
,
d
starting from the corresponding benzyl halides, by treatment with sodium azide and ammonium
chloride [67].
Physical and spectroscopic data for compounds 7a,b are superimposable with that reported in
literature [61].
C-[(tert-butyldiphenylsilyl)oxy]-N-methyl nitrone 13 and isoxazolidines 14 were prepared as
reported previously [62,63].
3.2. General Procedure for the Preparation of 3-Pyrimidinyl-Isoxazolidines 6a–c
A suspension of 5-substituted uracils 11a–c (0.84 mmol) in anhydrous CH3CN (3 mL) was treated
with bis(trimethylsilyl)acetamide (BSA; 2.52 mmol) and was stirred until the solution was clear (15 min).
A solution of benzyl 3-acetoxyisoxazolidine-2-carboxylate (10; 0.56 mmol) in anhydrous CH3CN (3 mL)
and trimethylsilyl triflate (TMSOTf; 0.11 mmol) was then added and the reaction was heated at 70 ◦C
for 5 h. After cooling to 0 ◦C, the solution was carefully neutralized by addition of 5% aq. NaHCO3 and
then concentrated in vacuo. CH2Cl2 (30 mL) was added and the organic phase was separated, washed
with water (2
×
10 mL), dried (Na2SO4), filtered and evaporated to dryness. The residue was purified
by MPLC on a silica gel column (CH2Cl2/MeOH, 98:2) to afford the desired compound (60%–70%).
3.3. General Procedure for the Preparation of 3-Pyrimidinyl-Isoxazolidines 6d–g
To a solution of 6c (1.0 eq.), aryl/alkyl azides 12a
tert-BuOH (40 mL) and H2O (40 mL), CuSO4 5H2O (0.25 eq.) and sodium ascorbate (0.5 eq.) were
added. The mixture was allowed to stir at room temperature for 4 h under N2 and concentrated. The
mixture was then diluted with water and extracted with ethyl acetate (3 30 mL). The organic layer was
–d (1.1 eq.) and triethylamine (1.0 eq.) in
·
×
dried over MgSO4, filtered and concentrated at reduced pressure and the residue was purified by flash
chromatography. (CH2Cl2/MeOH, 98:2). The compounds were crystallized in diethyl ether/CH2Cl2.
4. Conclusions
New pyrimidine-2,4-diones linked to an isoxazolidine nucleus have been synthesized and tested
as compounds endowed with potential anti-HIV activity. Compounds 6a–c, characterized by the
presence of an ethereal substituent at C-3, showed interesting biological features, acting as NNRTIs,
with compound 6c showing the highest HIV-RT inhibitor activity in the nanomolar range, and

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compound 6b the highest inhibitory effect on HIV-1 infection with no toxicity. The lack of cytotoxicity
and a possible anti-HIV-1 activity indicates the new compounds 6a–c as interesting starting points for
future investigations.
Conversely, compounds 6d
decrease HIV infection in CEM-GFP cells and exhibited higher levels of cytotoxicity than 6a
Interestingly, compound 6g was the only one of this group of compounds able to inhibit in vitro
infection with HIV-1 of CEM-GFP cells at 103 M concentration. This finding suggests a slight activity
of 6g on HIV replication, presumably through mechanisms other than RT inhibition, although the SI is
too low in comparison with 6a . This might be related to the presence of the alcoholic group. Further
–g did not show any HIV RT inhibitory activity, nor an ability to
–c.
µ
–c
studies are necessary to clarify this point.
Compound 7b, monophosphonylated homo-N,O-nucleoside, where the heterocyclic base is linked
at the C-30 of the isoxazolidine unit by a methylene linker, showed only a slight inhibition of RT HIV,
acting as an NRTI chain terminator.
The obtained data clearly indicate that the pyrimidine-2,4-dione unit deserves further investigation
as a valuable scaffold for extending the current spectrum of antiviral activity of modified nucleosides,
avoiding some unwanted side effects. SAR and in vivo toxicology studies performed on phosphonated
reverse homo-N,O-nucleosides 6a–c could allow the development of new anti-HIV agents with a better
pharmacological profile than that exerted by the currently used anti-HIV agents.
Supplementary Materials: Copies of 1H-NMR spectra of all new compounds; protocols for inhibition assays.
Author Contributions: R.R. and S.V.G. designed the research; B.M. designed the biological assay; F.M.M. and C.F.
performed biological data collection; R.R., S.V.G., D.I., B.G. and L.V. performed chemical synthesis and analyzed
the data; R.R. and S.V.G. wrote the paper. All authors read and approved the final manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors gratefully acknowledge the Italian Ministry of Education, Universities,
and Research (MIUR), the University of Messina (Italy) and the Interuniversity Consortium for Innovative
Methodologies and Processes for Synthesis (CINMPIS).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Weber, R.; Ruppik, M.; Rickenbach, M.; Spoerri, A.; Furrer, H.; Battegay, M.; Cavassini, M.; Calmy, A.;
Bernasconi, E.; Schmid, P.; et al. Decreasing mortality and changing patterns of causes of death in the Swiss
HIV Cohort Study. HIV Med. 2013, 14, 195–207. [CrossRef] [PubMed]
Smith, C.J.; Ryom, L.; Weber, R.; Morlat, P.; Pradier, G.; Reiss, P.; Kowalska, J.; de Wit, S.; Law, M.; El Sadr, W.;
et al. Trends in underlying causes of death in people with HIV from 1999 to 2011 (D:A:D): A multicohort
collaboration. Lancet 2014, 384, 241–248. [CrossRef]
McManus, H.; O’Connor, C.C.; Boyd, M.; Broom, J.; Russell, D.; Watson, K.; Roth, N.; Read, P.J.; Petoumenos, K.;
Law, M.G. Long-Term Survival in HIV Positive Patients with up to 15 Years of Antiretroviral Therapy.
PLoS ONE 2012, 7, e48839. [CrossRef]
4.
5.
6.
Costagliola, D. Demographics of HIV and aging. Curr. Opin. HIV AIDS 2014, 9, 294–301. [CrossRef]
[PubMed]
Pinto, A.N.; Grey, P.; Shaik, A.; Cooper, D.A.; Kelleher, A.D.; Petoumenos, K. Early Treatment of Primary HIV
Infection Is Associated with Decreased Mortality. AIDS Res. Hum. Retrovir. 2018, 34, 936–946. [CrossRef]
Heredia, A.; Le, N.; Gartenhaus, R.B.; Sausville, E.; Medina-Moreno, S.; Zapata, J.C.; Davis, C.; Gallo, R.C.;
Redfield, R.R. Targeting of mTOR catalytic site inhibits multiple steps of the HIV-1 life cycle and suppresses
HIV-1 viremia in humanized mice. Proc. Natl. Acad. Sci. USA 2003, 112, 9412–9417. [CrossRef] [PubMed]
Heredia, A.; Amoroso, A.; Davis, C.; Le, N.; Reardon, E.; Dominique, J.K.; Klingebiel, E.; Gallo, R.C.;
7.
Redfield, R.R. Rapamycin causes down-regulation of CCR5 and accumulation of anti-HIV
β-chemokines:
An approach to suppress R5 strains of HIV-1. Proc. Natl. Acad. Sci. USA 2003, 100, 10411–10416. [CrossRef]

Molecules 2019, 24, 1718
11 of 14
8.
Nicoletti, F.; Lapenta, C.; Donati, S.; Spada, M.; Ranazzi, A.; Cacopardo, B.; Mangano, K.; Belardelli, F.;
Perno, C.; Aquaro, S. Inhibition of human immunodeficiency virus (HIV-1) infection in human peripheral
blood leucocytes-SCID reconstituted mice by rapamycin. Clin. Exp. Immunol. 2009, 155, 28–34. [CrossRef]
[PubMed]
9.
Donia, M.; McCubrey, J.A.; Bendtzen, K.; Nicoletti, F. Potential use of rapamycin in HIV infection. Br. J.
Clin. Pharmacol. 2010, 70, 784–793. [CrossRef] [PubMed]
10. Nicoletti, F.; Fagone, P.; Meroni, P.; McCubrey, J.; Bendtzen, K. mTOR as a multifunctional therapeutic target
in HIV infection. Drug Discov. Today 2011, 16, 715–721. [CrossRef]
11. Agnello, S.; Brand, M.; Chellat, M.F.; Gazzola, S.; Riedl, R. A structural view on medicinal chemistry strategy
against drug resistance. Angew. Chem. Int. Ed. 2019, 53, 521–538. [CrossRef] [PubMed]
12. Matteucci, C.; Grelli, S.; Balestrieri, E.; Minutolo, A.; Argaw-Denboba, A.; Macchi, B.; Sinibaldi-Vallebona, P.;
Perno, C.F.; Mastino, A.; Garaci, E. Thymosin alpha 1 and HIV-1: Recent advances and future perspectives.
Future Microbiol. 2017, 12, 141–155. [CrossRef] [PubMed]
13. Beyrer, C.; Pozniak, A. HIV drug resistance—An emerging threat to epidemic control. N. Engl. J. Med. 2017
377, 1605–1607. [CrossRef]
,
14. Lehman, D.A.; Wamelwa, D.C.; McCoy, C.O.; Matsen, F.A.; Langat, A.; Chohan, B.H.; Benki-Nugent, S.;
Custers-Allen, R.; Bushman, F.D.; John-Stewart, G.C.; et al. Low-frequency Nevirapine resistance at multiple
sites may predict treatment failure in infants on nevirapine-based treatment. J. Acq. Immun. Def. Synd. 2012
60, 225–233. [CrossRef] [PubMed]
,
15. Geronikaki, A.; Eleftheriou, P.; Poroikov, V. Anti-HIV Agents: Current status and recent trends.
In Communicable Diseases of the Developing World; Springer: Berlin, Germany, 2018; pp. 37–95.
16. D’Ettorre, G.; Ceccarelli, G.; Pavone, P.; Vittozzi, P.; De Girolamo, G.; Schietroma, I.; Serafino, S.; Giustini, N.;
Vullo, V. What happens to cardiovascular system behind the undetectablelevel of HIV viremia? AIDS Res. Ther.
2016, 13, 21. [CrossRef] [PubMed]
17. Bhatia, N.S.; Chow, F.C. Neurologic Complications in Treated HIV-1 Infection. Curr. Neurol. Neurosci. Rep.
2016, 16, 62. [CrossRef]
18. Haas, D.W.; Tarr, P.E. Perspectives on pharmacogenomics of antiretroviral medications and HIV-associated
comorbidities. Curr. Opin. HIV AIDS 2015, 10, 116–122. [CrossRef]
19. De Clercq, E. A 40-year journey in search of selective antiviral chemotherapy. Annu. Rev. Pharmacol. Toxicol.
2011, 51, 1–24. [CrossRef]
20. Ghosh, R.K.; Ghosh, S.M.; Chawla, S. Recent advances in antiretroviral drugs. Expert Opin. Pharmacother.
2011, 12, 31–46. [CrossRef] [PubMed]
21. Jain, V.; Deeks, S.G. When to Start Antiretroviral Therapy. Curr. Hiv Aids Rep. 2010, 7, 60–68. [CrossRef]
[PubMed]
22. De Clercq, E. Antiviral drug discovery: Ten more compounds, and ten more stories (part B). Med. Res. Rev.
2009, 29, 611–645. [CrossRef]
23. De Clercq, E. The discovery of antiviral agents: Ten different compounds, ten different stories. Med. Res. Rev.
2008, 28, 929–953. [CrossRef]
24. Dal Pozzo, F.; Andrei, G.; Lebeau, I.; Beadle, J.R.; Hostetler, K.Y.; De Clercq, E.; Snoeck, R. In vitro evaluation
of the anti-orf virus activity of alkoxyalkyl esters of CDV, cCDV and (S)-HPMPA. Antivir. Res. 2007, 75,
52–57. [CrossRef] [PubMed]
25. Holy, A. Antiviral acyclic nucleoside phosphonates structure activity studies. Antivir. Res. 2006, 71, 248–253.
[CrossRef] [PubMed]
26. Dash, C.; Ahmadibeni, Y.; Hanley, M.J.; Pandhare, J.; Gotte, M.; Le Grice, S.F.J.; Parang, K. Inhibition of
multi-drug resistant HIV-1 reverse transcriptase by nucleoside β-triphosphates. Bioorg. Med. Chem. Lett.
2011, 21, 3519–3522. [CrossRef] [PubMed]
27. Chung, S.; Himmel, D.M.; Jiang, J.-K.; Wojtak, K.; Bauman, J.D.; Rausch, J.W.; Wilson, J.A.; Beutler, J.A.;
Thomas, C.J.; Arnold, E.; et al. Synthesis, Activity, and Structural Analysis of Novel -Hydroxytropolone
α
Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease, H. J. Med. Chem.
2011, 54, 4462–4473. [CrossRef] [PubMed]
28. Koczor, C.A.; Lewis, W. Nucleoside reverse transcriptase inhibitor toxicity and mitochondrial DNA. Exp. Opin.
Drug Metab. Toxicol. 2010, 6, 1493–1504. [CrossRef]

Molecules 2019, 24, 1718
12 of 14
29. Martin, J.C.; Hitchcock, M.J.M.; De Clercq, E.; Prusoff, W.H. Early nucleoside reverse transcriptase inhibitors
for the treatment of HIV: A brief history of stavudine (D4T) and its comparison with other dideoxynucleosides.
Antivir. Res. 2010, 85, 34–38. [CrossRef] [PubMed]
30. De Clercq, E. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs): Past, Present, and Future.
Chem. Biodivers. 2004, 1, 44–64. [CrossRef]
31. De Clercq, E. The role of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the therapy of HIV-1
infection. Antivir. Res. 1998, 38, 153–179. [CrossRef]
32. Ding, J.; Das, K.; Moereels, H.; Koymans, L.; Andries, K.; Janssen, P.A.; Hughes, S.H.; Arnold, E. Structure
of HIV-1 RT/TIBO R 86183 complex reveals similarity in the binding of diverse nonnucleoside inhibitors.
Nat. Struct. Biol. 1995, 2, 407–415. [CrossRef] [PubMed]
33. Das, K.; Lewi, P.; Hughes, S.H.; Arnold, E. Crystallography and the design of anti-AIDS drugs: Conformational
flexibility and positional adaptability are important in the design of non-nucleoside HIV-1 reverse transcriptase
inhibitors. Prog. Biophys. Mol. Biol. 2005, 88, 209–231. [CrossRef]
34. Romeo, G.; Chiacchio, U.; Corsaro, A.; Merino, P. ChemicalSynthesis of Heterocyclic
−
Sugar Nucleoside
Analogues. Chem. Rev. 2010, 110, 3337–3370. [CrossRef] [PubMed]
35. Romeo, R.; Carnovale, C.; Giofrè, S.V.; Macchi, B.; Frezza, A.; Marino-Merlo, C.; Pistarà, V.; Chiacchio, U.
Truncatedphosphonated C-10-branched N,O-nucleosides: A new class of antiviral agents. Bioorg. Med. Chem.
2012, 20, 3652–3657. [CrossRef]
36. Balestrieri, E.; Matteucci, C.; Ascolani, A.; Piperno, A.; Romeo, R.; Romeo, G.; Chiacchio, U.; Mastino, A.;
Macchi, B. Effect of Phosphonated Carbocyclic 20-Oxa-30-Aza-Nucleoside on Human T-Cell Leukemia Virus
Type 1 Infection In Vitro. Antimicrob. Agents Chemother. 2008, 52, 54–64. [CrossRef]
37. Chiacchio, U.; Iannazzo, D.; Piperno, A.; Romeo, R.; Romeo, G.; Rescifina, A.; Saglimbeni, M. Synthesis and
biological evaluation of phosphonated carbocyclic 20-oxa-30-aza-nucleosides. Bioorg. Med. Chem. 2006, 14,
955–959. [CrossRef] [PubMed]
38. Guillemont, G.; Pasquier, E.; Palandjian, P.; Vernier, D.; Gaurrand, S.; Lewi, P.J.; Heeres, J.; de Jonge, L.M.;
Koymans, F.F.; Daeyaert, M.H.; et al. Synthesis of Novel Diarylpyrimidine Analogues and Their Antiviral
Activity against Human Immunodeficiency Virus Type 1. J. Med. Chem. 2005, 48, 2072–2079. [CrossRef]
[PubMed]
39. Chen, X.; Zhan, P.; Li, D.; De Clercq, E.; Liu, X. Recent advances in DAPYs and related analogues as HIV-1
NNRTIs. Curr. Med. Chem. 2011, 18, 359–376. [CrossRef]
40. Schrijvers, R. Etravirine for the treatment of HIV/AIDS. Expert Opin. Pharmacother. 2013, 2014, 1087–1096.
[CrossRef]
41. Minudo, J.J.; Haubrich, R. Etravirine: A second-generation NNRTI for treatment-experienced adults with
resistant HIV-1 infection. Futur. HIV Ther. 2008, 2, 525–537. [CrossRef] [PubMed]
42. Zhan, P.; Pannecouque, C.; De Clercq, E.; Liu, X. Anti-HIV Drug Discovery and Development: Current
Innovations and Future Trends. J. Med. Chem. 2016, 59, 2849–2878. [CrossRef] [PubMed]
43. Li, D.; Zhan, P.; De Clercq, E.; Liu, X. Strategies for the Design of HIV-1 Non-Nucleoside Reverse Transcriptase
Inhibitors: Lessons from the Development of Seven Representative Paradigms. J. Med. Chem. 2012, 55,
3595–3613. [CrossRef]
44. Marino-Merlo, F.; Macchi, B.; Armenia, D.; Bellocchi, M.C.; Ceccherini-Silberstein, F.; Mastino, A.; Grelli, S.
Focus on recently developed assays for detection of resistance/sensitivity to reverse transcriptase inhibitors.
Appl. Microbiol. Biotechnol. 2018, 102, 9925–9936. [CrossRef] [PubMed]
45. Li, X.; Gao, P.; Huang, B.; Zhou, Z.; Yu, Z.; Yuan, Z.; Liu, H.; Pannecouque, C.; Daelemans, D.; De Clercq, E.; et al.
Discovery of novel piperidine-substituted indolylarylsulfones as potent HIV NNRTIs via structure-guided
scaffold morphing and fragment rearrangement. Eur. J. Med. Chem. 2017, 126, 190–201. [CrossRef]
46. Li, X.; Zhang, L.; Tien, Y.; Song, Y.; Zhan, P.; Liu, X. Novel HIV-1 non-nucleoside reverse transcriptaseinhibitors:
A patentreview (2011–2014). Expert Opin. Ther. Pat. 2014, 24, 1199–1227. [CrossRef]
47. Zhan, P.; Chen, X.; Li, D.; Fang, Z.; De Clercq, E.; Liu, X. HIV NNRTIs: Structural diversity, pharmacophore
similarity, and implications for drug design. Med. Res. Rev. 2013, 33, E1–E72. [CrossRef]
48. De Bethune, M.P. Non-nucleoside reverse transcriptase inhibitors (NNRTIs), their discovery, development,
and use in treatment of HIV infections: A review of the last 20 years (1989–2009). Antivir. Res. 2010, 85,
75–90. [CrossRef]

Molecules 2019, 24, 1718
13 of 14
49. Mordant, C.; Schmitt, B.; Pasquier, E.; Demestre, C.; Queguiner, L.; Masungi, C.; Peeters, A.; Smeulders, L.;
Bettern, E.; Hertogs, K.; et al. Synthesis of novel diarylpyrimidine analogues of TMC278 and their antiviral
activity against HIV-1 wild-type and mutant strains. Eur. J. Med. Chem. 2007, 42, 567–579. [CrossRef]
[PubMed]
50. Feng, X.-Q.; Liang, Y.-H.; Zeng, Z.-S.; Chen, F.-E.; Balzarini, J.; Pannecouque, C.; De Clercq, E. Structural
modifications of DAPY analogues with potent HIV activity. Chem. Med. Chem. 2009, 4, 219–224. [CrossRef]
51. Liang, Y.-H.; He, Q.-Q.; Zeng, Z.-S.; Liu, Z.-Q.; Feng, X.-Q.; Chen, F.-E.; Pannecouque, C.; De Clercq, E.
Synthesis and anti-HIV activity of 2-naphtyl substituted DAPY analogues as non-nucleoside reverse
transcriptase inhibitors. Bioorg. Med. Chem. 2010, 18, 4601–4605. [CrossRef] [PubMed]
52. Rotili, D.; Tarantino, D.; Artico, M.; Nawrozkij, M.B.; Gonzalez-Ortega, E.; Clotet, B.; Samuele, A.; EstØ, J.A.;
Maga, G.; Mai, A. Diarylpyrimidine-dihydrobenzyloxopyrimidine hybrids: New, wide-spectrumanti-HIV-1
agents active at (sub)nanomolar level. J. Med. Chem. 2011, 54, 3091–3096. [CrossRef] [PubMed]
53. Nawrozkij, M.B.; Rotili, D.; Tarantino, D.; Botta, G.; Eremiychuk, A.S.; Musmuca, I.; Ragno, R.; Samuele, A.;
Zanoli, S.; Armand-Ugòn, M.; et al. 5-Alkyl-6-benzyl-2-(2-oxo-2-phenylethylsulfonyl)pyrimidine-4(3H)-ones,
a series of anti-HIV-1 agents of the dihydro-alkoxy-benzyl-oxopyrimidine family with peculiar
structure-activity relationship profile. J. Med. Chem. 2008, 51, 4641–4652. [CrossRef]
54. Lu, X.; Chen, Y.; Guo, Y.; Liu, Z.; Shi, X.; Xu, Y.; Wang, X.; Zhang, Z.; Liu, J. The design and synthesis of
N-1-alkylated-5-aminoarylalkylsubstituted-6-methyluracils as potential non-nucleoside HIV-1 RT inhibitors.
Bioorg. Med. Chem. 2007, 15, 7399–7407. [CrossRef]
55. Danel, K.; Larsen, L.M.; Pedersen, E.B.; Sanna, G.; La Colla, P.; Loddo, R. Synthesis and antiviral activity of
new dimeric inhibitors against HIV-1. Bioorg. Med. Chem. 2008, 16, 511–517. [CrossRef]
56. Campiani, G.; Ramunno, A.; Maga, G.; Nacci, V.; Fattorusso, C.; Catalanotti, B.; Morelli, E.; Novellino, E.
Non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors: Past, present, and future perspectives.
Curr. Pharm. Des. 2002, 8, 615–657. [CrossRef]
57. Romeo, R.; Giofrè, S.V.; Macchi, B.; Balestrieri, E.; Mastino, A.; Merino, P.; Romeo, G.; Chiacchio, U.
Truncated reverse isoxazolidinyl nucleosides: A new class of allosteric HIV-1 reverse transcriptase inhibitors.
Chem. Med. Chem. 2012, 7, 565–569. [CrossRef] [PubMed]
58. Macchi, B.; Romeo, G.; Chiacchio, U.; Frezza, C.; Marino Merlo, F.; Mastino, A. Phosphonated nucleoside
analoguesasantiviral agents. In Topics in Medicinal Chemistry; Springer: Berlin/Heidelberg, Germany, 2015;
Volume 9, pp. 53–92.
59. Reddy, A.S.; Kumar, M.S.; Reddy, G.R. A convenient method for the preparation of hydroxamic acids.
Tetrahedron Lett. 2000, 41, 6285–6288. [CrossRef]
60. Piperno, A.; Chiacchio, U.; Iannazzo, D.; Giofrè, S.V.; Romeo, G.; Romeo, R. First example of direct
RuO4-catalyzed oxidation of isoxazolidines to 3-oxazolidones. J. Org. Chem. 2007, 72, 3958–3960. [CrossRef]
61. Piotrowska, D.G.; Balzarini, J.; Andrei, G.; Schols, D.; Snoeck, R.; Wroblewski, A.E.; Gatkowska, J. Novel
isoxazolidine analogues of homonuclemosides and homonucleotides. Tetrahedron 2016, 72, 8294–8308.
[CrossRef]
62. Romeo, R.; Giofr
5-alkynyl isoxazolidinyl nucleosides. Eur. J. Org. Chem. 2011, 28, 5690–5695. [CrossRef]
63. Romeo, R.; Giofr , S.V.; Garozzo, A.; Bisignano, B.; Corsaro, A.; Chiacchio, M.A. Synthesis and biological
è, S.V.; Iaria, D.; Sciortino, M.T.; Ronsisvalle, S.; Chiacchio, M.A.; Scala, A. Synthesis of
è
evaluation of furopyrimidine N,O-nucleosides. Bioorg. Chem. Med. 2013, 21, 5688–5693. [CrossRef] [PubMed]
64. Frezza, C.; Balestrieri, E.; Marino-Merlo, F.; Mastino, A.; Macchi, B. A novel, cell-free PCR-based assay for
evaluating the inhibitor activity of antiretroviral compounds against HIV reverse transcriptase. J. Med. Virol.
2014, 86, 1–7. [CrossRef] [PubMed]
65. Frezza, C.; Grelli, S.; Federico, M.; Marino-Merlo, F.; Mastino, A.; Macchi, B. Testing anti-HIV
activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with
transfection-derived HIV-1 NL4-3. J. Med. Virol. 2016, 88, 979–986. [CrossRef] [PubMed]
66. Medici, M.A.; Sciortino, M.T.; Perri, D.; Amici, C.; Avitabile, E.; Ciotti, M.; Balestrieri, E.; De Smaele, E.;
Franzoso, G.; Mastino, A. Protection by herpes simplex virus glycoprotein D against Fas-mediated apoptosis:
Role of nuclear factor kappaB. J. Biol. Chem. 2003, 19, 36059–36067. [CrossRef]

Molecules 2019, 24, 1718
14 of 14
67. Romeo, R.; Giofr
è
, S.V.; Carnovale, C.; Chiacchio, M.A.; Campisi, A.; Mancuso, R.; Cirmi, S.; Navarra, M.
Synthesis and Biological Activity of Triazole-Appended N,O-Nucleosides. Eur. J. Org. Chem. 2014, 25,
5442–5447. [CrossRef]
Sample Availability: Samples of the compounds 6a–g and 7a,b are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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