Novel isoxazolidine analogues of homonucleosides and homonucleotides

Article abstract of DOI:10.1016/j.tet.2016.10.073

Isoxazolidine analogues of homonucleos(t)ides were synthesized from nucleobase-derived nitrones 20a-20e (uracil, 5-fluorouracil, 5-bromouracil, thymine, adenine) employing 1,3-dipolar cycloadditions with allyl alcohol as well as with alkenylphosphonates (allyl-, allyloxymethyl- and vinyloxymethyl- and vinylphosphonate). Besides reactions with vinylphosphonate the additions proceeded regioselectively to produce mixtures of major cis and minor trans 3,5-disubstituted isoxazolidines (d.e. 28–82%). From vinylphosphonate up to 10% of 3,4-disubstituted isoxazolidines was additionally produced. Vicinal couplings, shielding effects and 2D NOE correlations were employed in configurational assignments as well as in conformational analysis to find out preferred conformations for several isoxazolidines and to observe anomeric effects (pseudoaxial orientation of phosphonylmethoxy groups) for those obtained from vinyloxymethylphosphonate. None of the tested compounds were endowed in vitro with antiviral activity against a variety of DNA and RNA viruses at subtoxic concentrations (up to 250 μM) nor exhibited antiproliferative activity towards L1210, CEM, and HeLa cells (IC₅₀= ≥100 μM).

Full text of DOI:10.1016/j.tet.2016.10.073

Accepted Manuscript
Novel isoxazolidine analogues of homonucleosides and homonucleotides
Dorota G. Piotrowska, Jan Balzarini, Graciela Andrei, Dominique Schols, Robert
Snoeck, Andrzej E. Wróblewski, Joanna Gotkowska
PII:
S0040-4020(16)31132-2
10.1016/j.tet.2016.10.073
TET 28214
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 6 August 2016
Revised Date: 17 October 2016
Accepted Date: 31 October 2016
Please cite this article as: Piotrowska DG, Balzarini J, Andrei G, Schols D, Snoeck R, Wróblewski AE,
Gotkowska J, Novel isoxazolidine analogues of homonucleosides and homonucleotides, Tetrahedron
(2016), doi: 10.1016/j.tet.2016.10.073.
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Novel isoxazolidine analogues of
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homonucleosides and homonucleotides
a,
b
b
b
b
Dorota G. Piotrowska, ∗ Jan Balzarini, Graciela Andrei, Dominique Schols, Robert Snoeck, Andrzej E.
a
a
Wróblewski, and Joanna Gotkowska

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Novel isoxazolidine analogues of
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homonucleosides and homonucleotides
a,
b
b
b
b
Dorota G. Piotrowska, ∗ Jan Balzarini, Graciela Andrei, Dominique Schols, Robert Snoeck, Andrzej E.
a
a
Wróblewski, and Joanna Gotkowska

1
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Tetrahedron
journal homepage: www.elsevier.com
Novel isoxazolidine analogues of homonucleosides and homonucleotides
a,
b
b
b
b
Dorota G. Piotrowska, ∗ Jan Balzarini, Graciela Andrei, Dominique Schols, Robert Snoeck, Andrzej E.
a
a
Wróblewski, and Joanna Gotkowska
a
Bioorganic Chemistry Laboratory, Faculty of Pharmacy, Medical University of Łódź, 90-151 Łódź, Muszyńskiego 1, Poland
KU Leuven, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Isoxazolidine analogues of homonucleos(t)ides were synthesized from nucleobase-derived
nitrones 20a-20e (uracil, 5-fluorouracil, 5-bromouracil, thymine, adenine) employing 1,3-dipolar
cycloadditions with allyl alcohol as well as with alkenylphosphonates (allyl-, allyloxymethyl-
and vinyloxymethyl- and vinylphosphonate). Besides reactions with vinylphosphonate the
additions proceeded regioselectively to produce mixtures of major cis and minor trans 3,5-
disubstituted isoxazolidines (d.e. 28–82%). From vinylphosphonate up to 10% of 3,4-
disubstituted isoxazolidines was additionally produced. Vicinal couplings, shielding effects and
2D NOE correlations were employed in configurational assignments as well as in
conformational analysis to find out preferred conformations for several isoxazolidines and to
observe anomeric effects (pseudoaxial orientation of phosphonylmethoxy groups) for those
obtained from vinyloxymethylphosphonate. None of the tested compounds were endowed in
vitro with antiviral activity against a variety of DNA and RNA viruses at subtoxic
concentrations (up to 250 ꢀM) nor exhibited antiproliferative activity towards L1210, CEM, and
HeLa cells (IC50 = ≥ 100 µM).
Available online
Keywords:
1
,3-Dipolar cycloadditions
Nitrones
Isoxazolidines
Phosphonates
Conformational analysis
Homonucleoside analogues
Homonucleotide analogues
2
009 Elsevier Ltd. All rights reserved.
13
1
. Introduction
transcriptase of different retroviruses, its truncated analogues 3
appeared even more potent exhibiting the inhibitory activity at
concentrations in the nanomolar range. High cytotoxicity
A significant number of antiviral and anticancer drugs can be
14
classified as close structural analogues of nucleosides or
nucleotides. A search for new compounds has resulted in
obtaining many active molecules which showed different levels
toward several cancer cell lines was observed for isoxazolidine
16
nucleosides of the general formula 5. On the other hand, it is
worth mentioning that the biological activity of compounds
containing the isoxazolidine ring is not restricted to anticancer
and antiviral properties, since it was found that they also posses
1–10
of similarities to natural nucleosides.
Modifications of a
nucleoside scaffold are practically unlimited since not only the
sugar and nucleobase units could be altered but also additional
linkers within the structure of the nucleoside can be incorporated.
A list of commonly used ribofuranoside replacers includes 2′,3′-
dideoxyfuranose, cyclopentane, cyclopentene, 1,3-dioxolane, 1,3-
oxathiolane, isoxazolidine rings and also acyclic entities.
17,18
18,21
22–23
antimicrobial,
antioxidant
antifungal,
anti-inflammatory,
24,25
26
and insecticide activity, among others.
Structural modifications of nucleosides may also influence
stereoelectronic effects and contribute to the anomeric effect and
thus control a conformational behavior of the sugar ring and
affect the biological properties of nucleosides. This is
exemplified by a replacement of the ring oxygen atom by a
The idea of incorporating the isoxazolidine ring into a
nucleoside framework as a sugar replacer, first proposed by
11
Tronchet, has been explored to provide several biologically
active compounds (Figure 1). fluorouracil-containing
isoxazolidine 1 was found to induce apoptosis on lymphoid and
6,27,28
carbon atom leading to the formation of carbanucleosides.
A
This modification results in a greater metabolic stability of
nucleoside analogues lacking the natural N-glycoside bond. A
similar increase in stability can be achieved in 1′-
homonucleosides which are formed by insertion of the methylene
12
monocytoid cells and at the same time showed low cytotoxicity.
Antiviral nucleotides were1 also discovered among
phosphonylated isoxazolidines 2 and 3 as well as among their
3
14
group between the nucleobase and the sugar or sugar mimetics as
15
analogues having the 1,2,3-triazole linker 4. While nucleotides
have been found to be potent inhibitors of the reverse
29,30
illustrated by 1′-homoadenosine 6
Moreover, the biological
2
activity of 1′-homonucleosides is also influenced by greater
—
——
∗
Corresponding author. Tel.: +48-42-677-92-35; fax: +48-42-678-83-98; e-mail: dorota.piotrowska@umed.lodz.pl

2
Tetrahedron
conformational flexibility and slightly improved lipophilicity.
Furthermore, 1′-homonucleotides containing
a
non-
ACCEPTED MANUSCRIPT
31,32
Among 1′-homonucleosides
as ribofuranoside mimics several compounds showing
pronounced biological activities were identified 7–13 (Figure
containing five-membered rings
hydrolyzable P–C bond have been also synthesized to obtain
analogues which could be phosphorylated to the active form,
thereby omitting the first and less effective monophosphorylation
33–38
39–45
2
).
In most cases these compounds retain the hydroxymethyl
step as exemplified by compounds 14–19 (Figure 3).
group to allow for their sequential phosphorylation to active
triphosphate metabolites.
Figure 1. Examples of biologically active isoxazolidine nucleoside/nucleotide analogues.
X
X
N
N
N
N
NH2
N
Cl
N
NH2
N
N
N
N
N
N
N
N
Y
N
N
N
HO
N
HO
N
HO
N
O
HO
O
O
O
N
NH3
N
S
OH OH
OH OH
Bn
6
7
8a X=Me
9a X=NH2, Y=H
10
8
b X=Cl
9b X=Cl, Y=NH2
O
N
NH2
NH2
X
NH
N
N
O
N
O
N
N
N
O
HO
HO
O
O
O
OH
O
N
R=
O
R
S
O
HO
HO
HO
HO
O
Ac
1
1
12
13a X=H
3b X=Me
OH
OH
HO
O
1
Figure 2. Examples of structurally diversified 1′-homonucleosides.

3
ACCEPTED MANUSCRIPT
Figure 3. Examples of 1′-homonucleotide analogues.
4
6
Recently, we have reported the synthesis of isoxazolidine-
preliminary study is given and the reactivity of nitrones 20 with
selected alkenylphosphonates 23–26 leading to a new series of
nucleotide analogues cis-27/trans-28 to cis-33/trans-34 is
described together with the results of their antiviral and cytostatic
activities (Scheme 1).
containing analogues of homonucleosides cis-21/trans-22 having
46
a nucleobase (B) at C3 of the isoxazolidine ring. The synthetic
approach relied on the application of the 1,3-dipolar
cycloaddition of allyl alcohol to the nucleobase-derived nitrones
2
0. In this paper, a full account of an already communicated
Scheme 1. Synthetic approach to homonucleosides 21/22 and their homonucleotide analogues cis-27/trans-28 to cis-33/trans-34.
well as cis-21e and trans-22e pairs of diastereoisomers due to
2
. Results and Discussion
almost identical spectral patterns for HC3, H C4 and H5 protons
2
The synthesis of nucleobase-derived nitrones 20 has been
but also for diastereotopic protons in H C–B and H C–OH
2
2
46
1
recently described. The 1,3-dipolar cycloadditions of the
nitrones 20 to allyl alcohol were carried out at 60°C or under
MW irradiation (Scheme 2, Table 1). The reactions were
moieties in the respective H NMR spectra.
regiospecific
and
produced
cis/trans
mixtures
of
diastereoisomeric cycloadducts 21 and 22 in moderate to good
diastereoselectivities (d.e. 82–28%). The cis/trans ratios of the
1
isoxazolidines were calculated from the H NMR spectra of the
reaction mixtures by comparison of integrations of diagnostic
resonances of the H C-4 protons in the isoxazolidine ring as well
2
as the signals of the respective protons of nucleobase moieties.
The relative configurations in homonucleosides cis-21a and
trans-22a have already been established based on 2D NOE
experiments. These assignments have been extended on cis-21b
and trans-22b, cis-21c and trans-22b, cis-21d and trans-22d as
Scheme 2. Reagents and conditions: a) allyl alcohol, 60°C, see
Table 1; b) allyl alcohol MW, 60–85°C, see Table 1.
46

4
Tetrahedron
Table 1. Cycloaddition of the nitrones 20a-20e and allyl
formation of even traces of the expected products. However,
ACCEPTED MANUSCRIPT
alcohol.
cycloadditions of nitrones 20 with alkenes 23–26 were
successfully carried out under microwave irradiation (Scheme 3).
Nitrone
Nucleobase
B
Reaction
time (h)
cis/trans
ratio
Yield (%)
a,b
1
The progress of the reactions was monitored by the H NMR
2
1:22
spectroscopy until the disappearance of the starting nitrone. The
ratios of diastereoisomeric cycloadducts were calculated from the
O
a
a
c
c
c
2
2
0a
0b
24
64:36
69:31
21a – 47 ; 22a – 28
31
NH
N
b
b
c
respective P NMR spectra of the crude reaction mixtures. The
1,3-dipolar cycloadditions of the nitrones 20 with
alkenylphosphonates 23, 24 and 25 (Scheme 3, Table 2) were
regiospecific and gave cis/trans mixtures of diastereoisomeric
cycloadducts cis-27/trans-28, cis-29/trans-30 and cis-31/trans-32
with diastereoselectivities (d.e. 78–40%, Table 2) comparable to
that found for analogous reactions with allyl alcohol (d.e. 82–
2.5
21a – 68 ; 22a – 25
O
F
b
c
c
O
15
69:31
21b – 44 ; 22b – 21
NH
N
O
2
8%, Table 1). In most cases chromatographic removal of the
Br
O
a
c
d
d
2
0c
46
91:9
91:9
21c – 21 ; 21c + 22c – 21
unreacted alkenylphosphonates was difficult and less effective
than distilling-off an excess of allyl alcohol, and thus led to lower
overall yields. In general, longer reaction times were required to
achieve a full conversion of the nitrones 20a with less reactive
dipolarophiles such as 23–25 when compared to an analogous
reaction with allyl alcohol. Moreover, during the reaction of the
adenine-derived nitrone 20e with allylphosphonate 23
decomposition of the starting nitrone was observed and the
unreacted dipolarophile 23 was recovered almost quantitatively.
When the same nitrone 20e was treated with
vinyloxymethylphosphonate formation of a complex reaction
mixture was noticed from which expected pure isoxazolidine
cycloadducts could not be isolated.
b
c
NH
N
5
21c – 26 ; 21c + 22c – 22
O
b
c
d
2
2
0d
0e
O
7.5
74:26
83:17
21d – 37 ; 21d + 22d – 12 ;
c
2
2d – 22
NH
N
O
N
b
c
d
NH2
5
21e – 21 ; 21e + 22e – 26
N
N
N
a
The cycloaddition reaction was conducted at 60°C.
b
The cycloaddition reaction was conducted under MW irradiation.
On the other hand, traces of 5-fluorouracil were found in
crude reaction mixtures when the nitrone 20b was treated with
alkenylphosphonates 23–26 under MW irradiation. To verify the
stability of this nitrone under conditions of the cycloaddition
reaction a solution of 20b in acetonitrile was heated under MW
irradiation and the progress of the reaction was monitored by the
H NMR spectroscopy. Indeed, the formation of 5-fluorouracil
was observed after 7 h (1%) and increased to 6% after an
additional 14 h. The amount of 5-fluorouracil reached 15% after
c
Yield of the pure diastereoisomer
d
Yield of a pure mixture of diastereoisomers
In continuation of our studies on the reactivity of the nitrones
0, allylphosphonate 23, allyloxymethylphosphonate 24,
2
1
vinyloxymethylphosphonate 25 and vinylphosphonate 26 were
selected as dipolarophiles to synthesize 1′-homonucleotide
analogues having non-hydrolyzable P–C bonds separated by
none, one, two or three bonds from C5 in the isoxazolidine ring
in compounds 27/28, 29/30, 31/32 and 33/34, respectively. The
installation of C–O–C–P(O)(OR)2 fragments in the designed
compounds is additionally substantiated by their presence in
nucleoside phosphonate drugs like adefovir, tenofovir and
1
8 h but the solution was contaminated with other unidentified
decomposition products (up to 46%). Similarly, slow
decomposition of the adenine-derived nitrone 20e during MW
1
irradiation of the solution in acetonitrile was observed. H NMR
spectra taken after 12 h revealed decomposition of the nitrone
47,48
cidofovir and several other drug candidates.
2
0e (c.a. 15%), since additional signals appeared in a region
characteristic of adenine protons.
Heating the nitrone 20a with an excess (3 equiv.) of
allylphosphonate 23 at 60–80°C for 24 hr did not result in the
Scheme 3. Reagents and conditions: a) allylphosphonate 23, allyloxymethylphosphonate 24, vinyloxymethylphosphonate 25 or
vinylphosphonate 26, CH CN or dioxane, MW, 65–80°C, 3–30 h.; see Table 2 and Table 3.
3

5
Table 2. Cycloaddition of the nitrones 20a-20e and alkenylphosphonates 23-25.
ACCEPTED MANUSCRIPT
a
Nitrone
Nucleobase B
Alkene
23
Reaction time (h)
Cis/trans ratio
Yield [%]
b
c
O
2
0a
30
30
30
80:20
27a – 22 ; 27a + 28a – 24
NH
N
b
c
b
24
72:28
29a – 16 ; 29a + 30a – 32 ; 30a – 9
O
b
c
3
1a – 30 ; 31a + 32a – 13
2
5
89:11
F
b
c
2
0b
O
23
10
16
74:26
79:21
86:14
27b – 3 ; 27b + 28b – 10
b
c
c
NH
N
29b – 13 ; 29b + 30b – 23
2
2
4
5
O
b
1
4
31b – 20 ; 31b + 32b – 26
Br
O
b
c
2
0c
23
24
8
86:14
72:28
70:30
27c – 4 ; 27c + 28c – 15
b
c
NH
N
21
8
29c – 12 ; 29c + 30c – 3.3
b
c
O
31c – 19 ; 31c + 32c – 10
2
5
b
c
2
2
0d
0e
O
23
24
8
10
8
71:29
80:20
84:16
27d – 21 ; 27d + 28d – 15
NH
N
b
c
29d – 5 ; 29d + 30d – 20
O
b
c
b
31d – 27 ; 31d + 32d – 7 ; 32d – 2
2
5
d
N
23
40
26
–
decomposition
NH
2
N
e
2
2
4
5
80:20
69:31
–
N
N
e
2
1
–
a
b
c
d
e
cycloaddition under MW irradiation,
yield of the pure diastereoisomer,
yield of the pure mixture of diastereoisomers,
decomposition of the starting nitrone 20e was observed. The unreacted allylphosphonate 23 was recovered almost quantitatively.
ratio of diastereoisomeric cycloadducts 29e and 30e as well as 31e and 32e were calculated, however pure isomers could not be isolated from the mixture
containing several unidentified products.
The relative configurations in cis-27 and trans-28 as well as in
cis-29 and trans-30 can again be deduced taking into account
1
almost identical H NMR spectral patterns when compared to
those of cis-21 and trans-22. This could be predicted because the
spatial and stereoelectronic influence of the substituents at C3
(
CH –Base) and at C5 (CH –OH in 21/22, CH –P in 27/28 and
2
2
2
CH –OCH P in 29/30) have an indistinguishable impact on the
2
2
preferred conformations of the isoxazolidine rings in the cis and
trans isomers. Although we were unable to unequivocally
establish these conformations in addition to 2D NOE spectral
Figure 4. Relative configurations of cis-21/27/29 and trans-
2/28/30.
2
22
data further support for our configurational assignments comes
from the comparison of the chemical shifts of H-C5 protons in
1
13
1
Although H and C NMR spectra of isoxazolidines cis-31
the cis and trans diastereoisomers Figure 4). Thus, in the H
and trans-32 prepared from vinyloxymethylphosphonate
resembled each other regardless of a nucleobase present they
significantly differed from those of the already discussed cis-
NMR spectra of all trans-configured isoxazolidines (22, 28, 30)
resonances of H-C5 are significantly shifted upfield in
comparison to the cis isomers (21, 27, 29), e.g. 4.12 ppm in 22a
vs. 4.40 ppm in 21a, because the H-C5 protons in the trans
isomers are positioned in the shielding cone of the
heteroaromatic ring. The same phenomenon can be observed for
the Hβ-C4 protons in both the cis and trans isoxazolidines but the
shielding effects are much better pronounced for the cis isomers,
e.g. 1.79 ppm for Hβ-C4 vs. 2.59 ppm for Hα-C4 in 21a and 2.10
ppm for Hβ-C4 vs. 2.31 ppm for Hα-C4 in 22a.
2
1/27/29 and trans-22/28/30 series and for this reason their
relative configurations had to be established independently.
Based on the values of vicinal H-H couplings observed in the
spectrum of cis-31d [J(HC5-HαC4) = 5.2 Hz, J(HC5-HβC4) = 0
49
5
Hz, J(HαC4-HC3) = 8.9 Hz and J(HβC4-HC3) = 2.1 Hz] the E
conformation of the isoxazolidine ring could be unequivocally
assigned (Figure 5, 37). In a similar fashion vicinal couplings
extracted from the spectrum of trans-32d [J(HC5-HαC4) = 1.7
Hz, J(HC5-HβC4) = 5.5 Hz, J(HαC4-HC3) = 7.6 Hz and
J(HβC4-HC3) = 5.5 Hz] were applied to prove the E5
conformation (Figure 5, 38). In both conformations
phosphonylmethoxy substituents at C5 are pseudoaxially oriented
since the anomeric effect operates. These conformational

6
Tetrahedron
ACCEPTED MANUSCRIPT
assignments are further supported by shielding of HβC4 when
compared with HαC4 and lack of shielding of HC5 protons in
both isomers.
Figure 6. Preferred conformations 39, 40 and 41 of cis-33, trans-
4 and cis-35, respectively; observed NOEs marked in blue.
3
Figure 5. Preferred conformations 37 and 38 of cis-31 and trans-
2, respectively; observed NOEs marked in blue.
To establish the trans configuration in 34c it is again worth
noting the meaningful upfield shift (0.3 ppm) of HC5 proton in
3
this isomer (4.18 ppm) in comparison to cis-33c (4.48 ppm) and
NOE correlation peaks between H C-B and HC5 (not detected in
2
On the other hand, when diethyl vinylphosphonate 26 was
applied, in addition to major 3,5-disubstituted isoxazolidines cis-
3a-e and trans-34a-e (Scheme 3, Table 3), the formation of
cis-33c) as well as between H C-B and HβC4 (medium intensity)
2
and H C-B and HαC4 (weak). Furthermore, the preferred
2
3
conformation 40 (Figure 6) of the isoxazolidine ring in trans-34c
can be proposed after analysis of vicinal couplings [J(HC5-
HβC4) = 8.9 Hz, J(HC5-HαC4) = 9.6 Hz and J(P-HβC4) = 7.2
Hz, J(P-HαC4) = 20.1 Hz as well as J(HβC4-HC3) = 1.4 Hz,
J(HαC4-HC3) = 7.8 Hz and J(P-C5-C4-C3) = 6.3 Hz].
minute amounts (less than 10%) of regioisomeric 3,4-
disubstituted products 35a-e and 36a-e was also noticed. Their
presence in the crude products as well as in the fractions obtained
31
after column chromatography was detected by the P NMR
spectroscopy (Table 4) and additionally proved by careful
1
analyses of the H NMR spectra where diagnostic signals of
To unequivocally establish relative configurations in
regioisomers 35 and 36 the attempts at separating them from
major 3,5-disubstituted isomers cis-33 and trans-34 and
eventually isolating at least one pure 3,4-disubstituted isomer (35
or 36) were undertaken. For this purpose, a 22:2:49:27 mixture of
compounds 33c, 34c, 35c and 36c significantly enriched in the
isomer 35c after column chromatography was subjected to
separation on an HPLC column to give minute amounts of pure
nucleobase protons could be assigned to four different
cycloadducts, namely cis-33a-e, trans-34a-e, and 35a-e/36a-e.
1
As observed previously, H NMR spectra of the major (cis-33)
and minor (trans-34) derivatives were also similar within the
series (a-e). In 2D NOE spectrum of cis-33c interactions between
H C-B and HβC4, HαC4 and HC3 as well as HαC4 and HC5
2
protons were noticed thus proving their locations on the same
sides of the isoxazolidine ring. These observations were further
supported by significant shielding of HβC4 (2.23 ppm) when
compared with HαC4 (2.84 ppm). Moreover, based on the
3
5c sufficient enough to perform full characterization by NMR
spectroscopy including the 2D NOE experiment. The cis
configuration of this regioisomer was proved based on the vicinal
H-H, H-P and P-C couplings [J(HC5β-HC4) = 9.2 Hz, J(HC5α-
HC4) = 9.2 Hz and J(P-HβC5) = 9.2 Hz, J(P-HαC5) = 0 Hz as
well as J(HC4-HC3) = 5.5 Hz, J(P-C4C3-H) = 16.1 Hz and J(P-
49
50,51
52,53
analysis of vicinal H-H, H-P
and P-C
couplings [J(HC5-
HβC4) = 6.4 Hz, J(HC5-HαC4) = 10.5 Hz and J(P-HβC4) = 19.4
Hz, J(P-HαC4) = 15.8 Hz as well as J(HβC4-HC3) = 1.3 Hz,
J(HαC4-HC3) = 7.7 Hz and J(P-C5-C4-C3) = 3.3 Hz] extracted
C4C3-CH B) = 8.1 Hz] which allow to establish the preferred
2
1
13
conformation 41 (Figure 6) for this compound. To further support
from H and C NMR spectra of cis-33c one can conclude that
this assignment one should notice that since H-C4 and the CH B
2
the isoxazolidine ring adopts the E conformation 39 (Figure 6).
2
moiety are in a near antiperiplanar orientation neither shielding of
H-C4 (2.57 ppm) by the heteroaromatic ring and a respective
NOE cross peak nor HC5 – HC3 NOE correlation were detected.
Table 3. Isoxazolidines 33, 34, 35 and 36 produced via Scheme 3.
a
Nitrone
Nucleobase B
Reaction time (h)
Ratio of isomers
Yield [%]
3
3:34:35:36
O
2
2
0a
0b
b
3
3a – 14 ;
NH
N
3
62:31:5:2
c
O
33a + 34a + 35a + 36a – 38 ;
F
O
b
3
3b – 33 ;
NH
N
6
3
62:31:6:1
60:32:6:2
c
O
33b + 34b + 35b + 36b –41 ;
Br
O
2
0c
b
33c – 24 ;
NH
N
c
33c + 34c + 35c + 36c – 16 ;
O
b
3
4c – 10
;

7
2
2
0d
0e
O
b
ACCEPTED MANUSCRIP33
T
d – 2 ;
NH
c
N
6
8
50:39:7:4
33d + 34d + 35d + 36d – 8 ;
O
b
3
4d – 1 ;
c
N
N
33e + 34e – 10 ;
3e + 34e + 35e + 36e – 45 ;
NH
2
64:30:5:1
N
c
3
N
a
b
c
cycloaddition under MW irradiation,
yield of the pure diastereoisomer,
yield of the pure mixture of two, three or four diastereoisomers
31
Table 4. P NMR chemical shift values for isoxazolidines 33, 34, 35 and 36.
3
1
δ
P NMR of isoxazolidine phosphonates (ppm)
Nitrone
cis-33
23.10
23.09
22.68
22.72
22.08
trans-34
22.46
22.47
22.05
22.13
21.26
cis-35
27.43
27.29
26.45
27.17
26.77
trans-36
26.56
26.50
26.00
26.27
25.65
2
2
2
2
2
0a
0b
0c
0d
0e
Antiviral and cytostatic evaluation
Antiviral activity
carcinoma HeLa and human dermal microvascular endothelial
cells (HMEC-1). Among all compounds evaluated, marginal, if
any cytostatic activity was observed. Not only compounds
containing adenine, uracil, 5-bromouracil and thymine
substituents as nucleobases were found inactive, but also the
analogues bearing a 5-fluorouracil (5-FU) moiety showed no
significant antiproliferative activity. These findings indicate that
the 5-FU-containing compounds are not efficiently taken-up by
the intact tumor cells and/or do not enzymatically release free 5-
FU and/or do not inhibit thymidylate synthase, one of the most
important target enzymes for 5-fluoro-deoxyuridine-5ʹ-
monophosphate.
Pure nitrones, as well as pure isoxazolidines or respective
mixtures of isoxazolidines were evaluated for their inhibitory
activity against a wide variety of DNA and RNA viruses, using
the following cell-based assays: (a) human embryonic lung
(
HEL) cell cultures: herpes simplex virus-1 (KOS), herpes
simplex virus-2 (G), vaccinia virus, vesicular stomatitis virus,
thymidine kinase deficient (acyclovir-resistant) herpes simplex
–
r
virus-1 (TK KOS ACV ), cytomegalovirus (AD-169 and Davis
+
–
strains), varicella-zoster virus (TK VZV and TK VZV strains);
b) HeLa cell cultures: vesicular stomatitis virus, Coxsackie virus
(
B4 and respiratory syncytial virus; (c) Vero cell cultures: para-
influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4,
Punta Toro virus; (d) CrFK cell cultures: feline corona virus
3
. Conclusion
New nucleobase-derived nitrones 20a-e were efficiently
(
FIPV) and feline herpes virus (FHV), (e) MDCK cell cultures:
applied in the synthesis of isoxazolidine analogues of
homonucleosides and homonucleotides which relied on the 1,3-
dipolar cycloadditions of 20a-e first to allyl alcohol and next to
allyl-, allyloxymethyl-, vinyloxymethyl- and vinylphosphonates.
In general cycloadditions were regioselective and led to the
formation of cis and trans mixtures of 3,5-disubstituted
isoxazolidines with moderate to good diastereoselectivities.
However, in cycloadditions to vinylphosphonate in addition to
major 3,5-disubstituted isoxazolidines also 3,4-disubstituted
isomers were formed (up to 10%).
influenza A virus (H1N1 and H3N2 subtypes) and influenza B
virus and (f) CEM or MT-4 cell cultures: human
immunodeficiency virus-1 (IIIB or NL4.3) and -2 (ROD).
Ganciclovir, cidofovir, acyclovir, brivudin, (S)-9-(2,3-
dihydroxypropyl)adenine [(S)-DHPA], oseltamivir carboxylate,
amantadine, rimantadine, ribavirin, dextran sulfate (molecular
weight 5,000, DS-5000), Hippeastrum hybrid agglutinin (HHA)
and Urtica dioica agglutinin (UDA) were used as the reference
compounds. The antiviral activity was expressed as the EC : the
compound concentration required to reduce virus plaque
formation (VZV) by 50% or to reduce virus-induced
cytopathicity by 50% (other viruses). None of the tested
compounds showed appreciable antiviral activity toward any of
the tested DNA and RNA viruses at the concentration up to 250
50
Relative (cis and trans) configurations of 3,5-disubstituted
isoxazolidines were established based on the detailed analysis of
1
13
H and C NMR spectral data (vicinal couplings, shielding
effects and 2D NOE correlations). Several isoxazolidines exist in
preferred conformations including those obtained from
vinyloxymethylphosphonate in which the phosphonylmethoxy
groups are oriented pseudoaxially due to the anomeric effect.
ꢀM.
Cytostatic activity
The 50% cytostatic concentration (IC ) causing a 50%
All synthesized compounds were evaluated against a broad-
spectrum of DNA and RNA viruses but they were found to be
inactive at concentrations up to 250 ꢀM. Also, the compounds
did not show significant cytostatic activity against murine
50
inhibition of cell proliferation was determined against murine
+
leukemia L1210, human CD T-lymphocyte CEM, human cervix
4

8
Tetrahedron
+
leukemia L1210, human CD4 T-lymphocyte CEM, human
Anal. Calcd. for C H N O : C, 49.79; H, 6.27; N, 17.42. Found:
3 4
ACCEPTED MANUSCRIPT10 15
cervix carcinoma HeLa and human dermal microvascular
endothelial cells.
C, 49.62; H, 6.11; N, 17.61.
Although the tested compounds contained biologically
relevant fragments (nucleobases, the isoxazolidine ring and a
phosphonate) they surprisingly did not show appreciable antiviral
and anticancer activities. Since the isoxazolidine subunit can be
also found in several structures endowed with antibacterial and
antifungal activities we would progress along this line soon to
hopefully discover new therapeutic applications for this class of
compounds.
4
.1.2. trans-1-((5-(Hydroxymethyl)-2-
methylisoxazolidin-3-yl)methyl)pyrimidine-
2
,4(1H,3H)-dione (22a)
Yield: 25% (0.095 g from 1.56 mmol of the nitrone 20a);
-
1
colorless oil; IR (film, cm ) ν : 3430, 3223, 3052, 2960, 2881,
1
7
Hz), 4.12 (dddd, 1H, J = 8.1 Hz, J = 7.8 Hz, J = 5.4 Hz, J = 3.9
Hz, H-C5), 3.88 (dd, 1H, J = 13.8 Hz, J = 5.4 Hz, HCHN), 3.71
max
1
680, 1461, 1386, 1252, 1041; H NMR (300 MHz, CD OD) δ:
3
.91 (brs, 1H, NH), 7.57 (d, 1H, J = 7.8 Hz), 5.63 (d, 1H, J = 7.8
(
dd, 1H, J = 13.8 Hz, J = 7.5 Hz, HCHN), 3.65 (dd, 1H, J = 12.0
4
. Experimental section
Hz, J = 3.9 Hz, HCHOH), 3.56 (dd, 1H, J = 12.0 Hz, J = 5.4 Hz,
HCHOH), 3.25 (dddd, 1H, J = 8.1 Hz, J = 7.5 Hz, J = 5.4 Hz, J =
4
1
H NMR spectra were taken in CDCl , CD OD and D O on
3
3
2
.2 Hz, HC3), 2.66 (s, 3H, CH N), 2.31 (ddd, 1H, J = 12.6 Hz, J
3
the following spectrometers: Varian Gemini 2000BB (200 MHz),
Varian Mercury-300 and Bruker Avance III (600 MHz) with
TMS as internal standard. C NMR spectra were recorded for
CDCl , CD OD and D O solution on the Bruker Avance III at
=
8.1 Hz, J = 8.1 Hz, H -C4), 2.10 (ddd, 1H, J = 12.6 Hz, J = 7.8
a
13
13
Hz, J = 4.2 Hz, H -C4); C NMR (75 MHz, CD OD) δ: 166.8
b
3
(C=O), 153.0 (C=O), 148.2 (C=C), 101.8 (C=C), 80.4 (C5), 67.2
3
3
2
3
1
(C3), 64.8 (CH -OH), 51.0 (CH N), 45.3 (CH N), 34.5 (C4).
Anal. Calcd. for C H N O : C, 49.79; H, 6.27; N, 17.42. Found:
C, 50.03; H, 6.34; N, 17.53.
2
2
3
1
50 MHz and Varian Mercury-300 machine at 75 MHz.
P
10
15
3
4
NMR spectra were performed in CDCl , CD OD and D O
solution on the Varian Gemini 2000BB at 80.0 MHz, Varian
Mercury-300 at 121 MHz or on Bruker Avance III at 242 MHz.
3
3
2
IR spectra were measured on an Infinity MI-60 FT-IR
spectrometer. Melting points were determined on Boetius
apparatus and are uncorrected. Elemental analyses were
performed by the Microanalytical Laboratory of this Faculty on
Perkin-Elmer PE 2400 CHNS analyzer.
4
.1.3. cis-5-Fluoro-1-((5-(hydroxymethyl)-2-
methylisoxazolidin-3-yl)methyl)pyrimidine-
2,4(1H,3H)-dione (21b)
Yield: 44% (0.156 g from 0.74 mmol of the nitrone 20b);
white amorphous solid (crystallized from methanol) mp 162–
1
1
=
3
3
-
1
64°C; IR (KBr, cm ) νmax: 3477, 3171, 3052, 2918, 2828, 1701,
The following adsorbents were used: column chromatography,
Merck silica gel 60 (70–230 mesh); analytical TLC, Merck TLC
1
661, 1245, 1048; H NMR (300 MHz, CD OD) δ: 7.71 (d, 1H, J
3
6.3 Hz), 4.41 (dddd, 1H, J = 8.7 Hz, J = 6.6 Hz, J = 4.6 Hz, J =
.1 Hz, H-C5), 3.81 (dd, 1H, J = 13.9 Hz, J = 4.8 Hz, HCHN),
.71 (dd, 1H, J = 12.1 Hz, J = 3.1 Hz, HCHOH), 3.60 (dd, 1H, J
plastic sheets silica gel 60 F254
.
Preparative HPLC experiment was performed on a Waters
apparatus equipped with Waters 2545 binary gradient module
and Waters 2998 photodiode array detector (190–600 nm).
= 12.1 Hz, J = 4.6 Hz, HCHOH), 3.56 (dd, 1H, J = 13.9 Hz, J =
.1 Hz, HCHN), 3.40 (dddd, 1H, J = 9.1 Hz, J = 8.7 Hz, J = 4.8
Hz, J = 3.3 Hz, H-C3), 2.62 (s, 3H, CH N), 2.60 (ddd, 1H, J =
9
3
1
2.9 Hz, J = 8.7 Hz, J = 8.7 Hz, H -C4), 1.80 (ddd, 1H, J = 12.9
a
13
4
.1. General procedure for the isoxazolidines cis-21 and trans-22
A mixture of nitrone 20 (1.0 mmol) and allyl alcohol (1.0 mL)
Hz, J = 6.6 Hz, J = 3.3 Hz, H -C4); C NMR (75 MHz, CD OD)
b 3
2 1
δ: 159.8 (d, J = 25.8 Hz, C=O), 151.5 (C=O), 140.9 (d, J =
2
2
29.0 Hz, (C=C), 132.8 (d, J = 33.2 Hz, (C=C), 78.3 (C5), 66.8
was stirred at 60°C or irradiated in a Plazmatronika RM800
microwave reactor at 60–85°C for the time shown in Table 1. All
volatiles were removed in vacuo and the crude product was
purified on silica gel column using chloroform–MeOH (10:1, 5:1,
v/v) as the eluent to afford pure isoxazolidines 21 and 22. For
details see Table 1.
(C3), 63.0 (CH OH), 52.2 (CH N), 44.5 (CH N), 33.1 (C4).
2
2
3
Anal. Calcd. for C H FN O : C, 46.33; H, 5.44; N, 16.21.
Found: C, 46.05; H, 5.47; N, 16.27.
10
14
3
4
4
.1.4. trans-5-Fluoro-1-((5-(hydroxymethyl)-2-
methylisoxazolidin-3-yl)methyl)pyrimidine-
2,4(1H,3H)-dione (22b)
4
.1.1. cis-1-((5-(Hydroxymethyl)-2-
methylisoxazolidin-3-yl)methyl)pyrimidine-
2
,4(1H,3H)-dione (21a)
Yield: 21% (0.094 g from 0.74 mmol of the nitrone 20b);
Yield: 68% (0.255 g from 1.56 mmol of the nitrone 20a);
white amorphous solid (crystallized from methanol) mp 158–
-
1
-1
colorless oil; IR (film, cm ) νmax: 3432, 3186, 3048, 2961, 2882,
160°C; IR (KBr, cm ) νmax: 3418, 3063, 2991, 2920, 2825, 1696,
1
1
1
695, 1662, 1462, 1045; H NMR (300 MHz, CD OD) δ: 7.90
1662, 1470, 1227, 1026; H NMR (300 MHz, CD
3
OD) δ: 7.81 (d,
3
(
4
brs, 1H, NH), 7.57 (d, 1H, J = 7.9 Hz), 5.60 (d, 1H, J = 7.9 Hz),
.40 (dddd, 1H, J = 8.7 Hz, J = 6.6 Hz, J = 4.5 Hz, J = 3.0 Hz, H-
C5), 3.83 (dd, 1H, J = 13.5 Hz, J = 4.8 Hz, HCHN), 3.70 (dd,
H, J = 13.3 Hz, J = 3.0 Hz, HCHOH), 3.61 (dd, 1H, J = 13.5
1H, J = 6.3 Hz), 4.13 (dddd, 1H, J = 8.1 Hz, J = 7.5 Hz, J = 5.4
Hz, J = 3.9 Hz, H-C5), 3.84 (dd, 1H, J = 13.8 Hz, J = 5.1 Hz,
HCHN), 3.67 (dd, 1H, J = 13.8 Hz, J = 7.2 Hz, HCHN), 3.65 (dd,
1H, J = 12.0 Hz, J = 3.9 Hz, HCHOH), 3.56 (dd, 1H, J = 12.0
Hz, J = 5.4 Hz, HCHOH), 3.27 (dddd, 1H, J = 8.1 Hz, J = 7.2
1
Hz, J = 9.0 Hz, HCHN), 3.60 (dd, 1H, J = 13.3 Hz, J = 4.5 Hz,
HCHOH), 3.39 (dddd, 1H, J = 9.0 Hz, J = 8.7 Hz, J = 4.8 Hz, J =
3
Hz, J = 5.1 Hz, J = 4.2 Hz, H-C3), 2.67 (s, 3H, CH
(ddd, 1H, J = 12.9 Hz, J = 8.1 Hz, J = 8.1 Hz, H -C4), 2.11 (ddd,
1H, J = 12.9 Hz, J = 7.5 Hz, J = 4.2 Hz, H -C4); C NMR (75
OD) δ: 159.8 (d, J = 25.1 Hz, C=O), 151.8 (C=O),
N), 2.31
3
.3 Hz, H-C3), 2.60 (s, 3H, CH N), 2.59 (ddd, 1H, J = 12.9 Hz, J
a
3
13
=
8.7 Hz, J = 8.7 Hz, H -C4), 1.79 (ddd, 1H, J = 12.9 Hz, J = 6.6
b
a
13
2
Hz, J = 3.3 Hz, H -C4); C NMR (75 MHz, CD OD) δ: 166.8
MHz, CD1
141.2 (d, J = 230.6 Hz, (C=C)), 132.1 (d, J = 32.9 Hz, (C=C),
80.5 (C5), 67.2 (C3), 64.9 (CH OH), 51.0 (CH N), 46.2 (CH N),
3
b
3
2
(
(
C=O), 152.9 (C=O), 148.8 (C=C), 101.3 (C=C), 78.3 (C5), 66.8
C3), 63.1 (CH OH), 52.2 (CH N), 44.6 (CH N), 33.2 (C4).
2
2
3
2
2
3

9
3
1
4.4 (C4). Anal. Calcd. for C H FN O : C, 46.33; H, 5.44; N,
6.21. Found: C, 46.57; H, 5.57; N, 16.26.
(CH ). Anal. Calcd. for C H N O : C, 51.76; H, 6.71; N,
3 11 17 3 4
16.46. Found: C, 51.99; H, 6.77; N, 16.36.
10
14
3
4
ACCEPTED MANU
SCRIPT
4
.1.5. cis-5-Bromo-1-((-5-(hydroxymethyl)-2-
4.1.8. trans-1-((5-(Hydroxymethyl)-2-
methylisoxazolidin-3-yl)methyl)-5-
methylpyrimidine-2,4(1H,3H)-dione (22d)
methylisoxazolidin-3-yl)methyl)pyrimidine-
2
,4(1H,3H)-dione (21c)
Yield: 26% (0.202 g from 2.47 mmol of the nitrone 20c);
Yield: 22% (0.094 g from 1.67 mmol of the nitrone 20d);
-1
white amorphous solid (crystallized from ethyl acetate/hexane)
colorless oil; IR (film, cm ) νmax: 3335, 2923, 2854, 1667, 1441,
-
1
1
mp 180–182°C; IR (KBr, cm ) ν : 3397, 3143, 3060, 3037,
1377, 1261, 1041; H NMR (600 MHz, CD OD) δ: 7.44 (q, J =
max
3
1
2
992, 2825, 1703, 1680, 1621, 1428, 1346, 1115; H NMR (300
1.0 Hz, 1H), 4.17–4.13 (m, 1H, H-C5), 3.87 (dd, 1H, J = 14.1 Hz,
J = 5.6 Hz, HCHN), 3.72 (dd, 1H, J = 14.1 Hz, J = 6.5 Hz,
HCHN), 3.67 (dd, 1H, J = 11.9 Hz, J = 3.9 Hz, HCHOH), 3.58
(dd, 1H, J = 11.9 Hz, J = 5.5 Hz, HCHOH), 3.30–3.24 (m, 1H,
MHz, CD OD) δ: 7.98 (s, 1H), 4.41 (dddd, 1H, J = 8.4 Hz, J =
3
6
.6 Hz, J = 4.8 Hz, J = 2.7 Hz, H-C5), 3.84 (dd, 1H, J = 13.2 Hz,
J = 4.5 Hz, HCHN), 3.72 (dd, 1H, J = 12.3 Hz, J = 2.7 Hz,
HCHOH), 3.61 (dd, 1H, J = 12.3 Hz, J = 4.8 Hz, HCHOH), 3.59
H-C3), 2.69 (s, 3H, CH N), 2.32 (ddd, 1H, J = 12.7 Hz, J = 7.9
3
(
9
dd, 1H, J = 13.2 Hz, J = 9.3 Hz, HCHN), 3.40 (dddd, 1H, J =
.3 Hz, J = 8.4 Hz, J = 4.5 Hz, J = 3.3 Hz, H-C3), 2.61 (s, 3H,
CH N), 2.60 (ddd, 1H, J = 13.2 Hz, J = 8.4 Hz, J = 8.4 Hz, H -
Hz, J = 7.9 Hz, H -C4), 2.13 (ddd, 1H, J = 12.7 Hz, J = 7.7 Hz, J
a
13
= 4.6 Hz, H -C4), 1.89 (d, J = 1.0 Hz, 3H, CH ). C NMR (150
b
3
MHz, CD OD) δ: 165.2 (C=O), 151.7 (C=O), 142.6 (C=C), 109.3
3
a
3
C4), 1.79 (ddd, 1H, J = 13.2 Hz, J = 6.6 Hz, J = 3.3 Hz, H -C4);
(C=C), 78.8 (C5), 65.9 (C3), 63.3 (CH OH), 49.5 (CH N), 42.2
b
2
2
13
C NMR (75 MHz, CD OD) δ: 162.1 (C=O), 152.2 (C=O),
(CH N), 33.2 (C4), 10.9 (CH ). Anal. Calcd. for C H N O : C,
3
3 3 11 17 3 4
1
5
48.3 (C=C), 95.5 (C=C), 78.3 (C5), 66.7 (C3), 63.0 (CH OH),
51.76; H, 6.71; N, 16.46. Found: C, 52.00; H, 6.79; N, 16.35.
2
2.4 (CH N), 44.5 (CH -N), 32.9 (C4). Anal. Calcd. for
2
3
C H BrN O : C, 37.52; H, 4.41; N, 13.13. Found: C, 37.70; H,
10
14
3
4
4
.33; N, 13.10.
4.1.9. cis-3-((6-Amino-9H-purin-9-yl)methyl)-2-
methylisoxazolidin-5-yl)methanol (21e)
Yield: 21% (0.066 g from 1.20 mmol of the nitrone 20e);
4
.1.6. trans-5-Bromo-1-((-5-(hydroxymethyl)-2-
white amorphous solid (crystallized from methanol) mp 225–
-
1
methylisoxazolidin-3-yl)methyl)pyrimidine-
2
2
1
27°C; IR (KBr, cm ) ν : 3400, 3311, 3133, 2937, 2855, 1651,
max
1
,4(1H,3H)-dione (22c)
White amorphous solid; IR (KBr, cm ) νmax: 3450, 3150,
603, 1418, 1299, 1055; H NMR (300 MHz, D O) δ: 7.96 (s,
2
-
1
1H), 7.93 (s, 1H), 4.44–4.36 (m, 1H, H-C5), 4.15 (dd, 1H, J =
14.4 Hz, J = 8.7 Hz, HCHN), 4.02 (dd, 1H, J = 14.4 Hz, J = 5.4
Hz, HCHN), 3.66 (dd, 1H, J = 12.6 Hz, J = 2.7 Hz, HCHOH),
3.50 (dd, 1H, J = 12.6 Hz, J = 6.0 Hz, HCHOH), 3.59–3.49 (m,
1H, H-C3), 2.56 (ddd, 1H, J = 13.2 Hz, J = 8.4 Hz, J = 8.4 Hz,
H -C4), 2.48 (s, 3H, CH N), 1.71 (ddd, 1H J = 13.2 Hz, J = 7.2
3
094, 2972, 2927, 2838, 1686, 1612, 1466, 1432, 1329, 1107;
(signals of trans-22c were extracted from the spectra of a 80:20
1
mixture of trans-22c and cis-21c); H NMR (300 MHz, CD OD)
3
δ: 8.00 (s, 1H), 4.13 (dddd, 1H, J = 8.1 Hz, J = 7.5 Hz, J = 5.4
Hz, J = 4.2 Hz, H-C5), 3.89 (dd, 1H, J = 13.8 Hz, J = 4.8 Hz,
HCHN), 3.70 (dd, 1H, J = 13.8 Hz, J = 8.4 Hz, HCHN), 3.66 (dd,
a
3
13
Hz, J = 4.5 Hz, H -C4); C NMR (75 MHz, D O) δ: 155.3,
b
2
1
H, J = 12.0 Hz, J = 4.2 Hz, HCHOH), 3.56 (dd, 1H, J = 12.0
152.3, 148.6, 142.4, 118.1, 77.3 (C5), 66.2 (C3), 61.9 (CH OH),
2
Hz, J = 5.4 Hz, HCHOH), 3.28 (dddd, 1H, J = 8.4 Hz, J = 7.8
46.3 (CH N), 43.9 (CH N), 32.7 (C4). Anal. Calcd. for
2
3
Hz, J = 4.8 Hz, J = 3.9 Hz, H-C3), 2.67 (s, 3H, CH N), 2.32
C H N O : C, 49.99; H, 6.10; N, 31.80. Found: C, 50.23; H,
11 16 6 2
3
(
1
ddd, 1H, J = 12.6 Hz, J = 8.1 Hz, J = 7.8 Hz, H -C4), 2.11 (ddd,
6.06; N, 32.01.
a
13
H, J = 12.6 Hz, J = 7.8 Hz, J = 3.9 Hz, H -C4); C NMR (75
b
MHz, CD OD) δ: 162.1 (C=O), 152.3 (C=O), 147.6 (C=C), 96.0
3
(
C=C), 80.6 (C5), 67.1 (C3), 65.0 (CH OH), 51.1 (CH N), 46.2
4.1.10. trans-3-((6-Amino-9H-purin-9-yl)methyl)-2-
2
2
(CH N), 34.3 (C4). Anal. Calcd. for C H BrN O : C, 37.52; H,
methylisoxazolidin-5-yl)methanol (22e)
3
10 14
3
4
-
1
4
8
.41; N, 13.13. Found: C, 37.54; H, 4.26; N, 12.97 (obtained on a
0:20 mixture of trans-22c and cis-21c).
Yellowish amorphous solid; IR (KBr, cm ) νmax: 3361, 3301,
3
126, 2923, 2853, 1650, 1601, 1419, 1370, 1211, 1058; (signals
of trans-22e were extracted from the spectra of a 72:28 mixture
1
of trans-22e and cis-21e); H NMR (300 MHz, D O) δ: 8.04 (s,
2
4
.1.7. cis-1-((5-(Hydroxymethyl)-2-
1
H), 8.01 (s, 1H), 4.25–4.04 (m, 3H, CH N, H-C5), 3.67 (dd, 1H,
2
methylisoxazolidin-3-yl)methyl)-5-
methylpyrimidine-2,4(1H,3H)-dione (21d)
J = 12.3 Hz, J = 3.3 Hz, HCHOH), 3.52 (dd, 1H, J = 12.3 Hz, J =
.0 Hz, HCHOH), 3.51–3.40 (m, 1H, H-C3), 2.62 (s, 3H, CH N),
6
3
Yield: 37% (0.156 g from 1.67 mmol of the nitrone 20d);
2.24 (ddd, 1H, J = 12.6 Hz, J = 7.8 Hz, J = 7.8 Hz, H -C4), 2.10
(ddd, 1H, J = 12.6 Hz, J = 7.2 Hz, J = 5.7 Hz, H -C4); C NMR
(150 MHz, CD OD) δ: 155.9, 152.4, 149.5, 142.2, 118.4, 77.0
a
-
1
13
colorless oil; IR (film, cm ) νmax: 3417, 3196, 3062, 2929,
1
b
1
687,1458, 1388, 1052; H NMR (600 MHz, CD OD) δ: 7.44 (q,
3
3
J = 1.2 Hz, 1H), 4.42 (dddd, 1H, J = 8.5 Hz, J = 6.7 Hz, J = 4.7
Hz, J = 3.2 Hz, H-C5), 3.82 (dd, 1H, J = 13.9 Hz, J = 4.9 Hz,
HCHN), 3.72 (dd, 1H, J = 12.2 Hz, J = 3.2 Hz, HCHOH), 3.64
(C5), 66.2 (C3), 63.1 (CH OH), 46.0 (CH N), 43.0 (CH N), 32.1
2
2
3
(C4). Anal. Calcd. for C H N O : C, 49.99; H, 6.10; N, 31.80.
11
16
6
2
Found: C, 50.22; H, 6.01; N, 31.53 (obtained on a 72:28 mixture
of trans-22e and cis-21e).
(
dd, 1H, J = 12.2 Hz, J = 4.7 Hz, HCHOH), 3.63 (dd, 1H, J =
3.9 Hz, J = 9.2 Hz, HCHN), 3.42 (dddd, 1H, J = 9.2 Hz, J = 8.2
Hz, J = 4.9 Hz, J = 3.5 Hz, H-C3), 2.63 (s, 3H, CH N), 2.61
1
3
(
=
3
1
6
ddd, 1H, J = 12.8 Hz, J = 8.5 Hz, J = 8.2 Hz, H -C4), 1.88 (d, J
a
4.2. General procedure for the cycloaddition of nitrone 20 with
alkenylphosphonate 23, 24, 25 and 26
1.2 Hz, 3H, CH ),1.82 (ddd, 1H, J = 12.8 Hz, J = 6.7 Hz, J =
3
13
.5 Hz, H -C4); C NMR (150 MHz, CD OD) δ: 165.5 (C=O),
b
3
A
solution of
a
nitrone 20 (1.0 mmol) and an
51.7 (C=O), 143.4 (C=C), 108.8 (C=C), 76.9 (C5), 65.5 (C3),
1.9 (CH OH), 50.7 (CH N), 43.2 (CH N), 32.0 (C4), 10.9
alkenylphosphonate 23, 24, 25 or 26 (3.0 mmol) in CH CN or
3
2
2
3
dioxane was irradiated in a Plazmatronika RM800 microwave

1
0
Tetrahedron
reactor at 65–80°C for the time shown in Tables 2 and 3. All
9.7 Hz, J = 7.9 Hz, J = 3.8 Hz, J = 3.0 Hz, H-C3), 3.30 (dd, 1H, J
ACCEPTED MANUSCRIPT
volatiles were removed in vacuo and the crude product was
purified on silica gel column using chloroform–MeOH (10:1, 5:1,
v/v) as the eluent to afford pure isoxazolidines 21/22, 27/28,
= 13.5 Hz, J = 9.7 Hz, HCHN), 2.75 (ddd, 1H, J = 13.3 Hz, J =
7.9 Hz, J = 7.9 Hz, H -C4), 2.64 (s, 3H, CH N), 2.25 (ddd, 1H, J
a
3
= 19.6 Hz, J = 14.9 Hz, J = 4.6 Hz, HCHP), 1.99 (ddd, 1H, J =
18.2 Hz, J = 14.9 Hz, J = 8.6 Hz, HCHP), 1.76 (ddd, 1H, J = 13.3
2
9/30, 31/32 or 33/34. For details see Table 2 and 3.
Hz, J = 7.9 Hz, J = 3.8 Hz, H -C4), 1.37 (t, 6H, J = 7.0 Hz,
b
13
2
CH CH OP); C NMR (150 MHz, CDCl ) δ: 157.0 (d, J = 26.4
Hz, C=O), 149.3 (C=O), 139.8 (d, J = 234.3 Hz, C=C), 130.8 (d,
J = 32.9 Hz, C=C), 71.0 (C5), 66.30 (C3), 62.0 (d, J = 6.8 Hz,
3
2
3
1
4
.2.1. Diethyl cis-((3-((2,4-dioxo-3,4-
2
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)methyl)phosphonate (27a)
CH OP), 62.0 (d, J = 6.8 Hz, CH OP), 51.6 (CH N), 44.5
2
2
2
Yield: 22% (0.130 g from 1.63 mmol of the nitrone 20a);
(CH
3
N), 37.6 (d, J = 4.5 Hz, C4), 30.8 (d, J = 139.9 Hz, CP),
31
-
1
1
6.4 (d, J = 5.8 Hz, 2 × CH CH OP); P NMR (242 MHz,
colorless oil; IR (film, cm ) ν : 3454, 3164, 3051, 2984, 2872,
3 2
max
1
2
823, 1689, 1632, 1455, 1250, 1025. H NMR (300 MHz,
CDCl ) δ: 26.24. Anal. Calcd. for C14H23FN O P: C, 44.33; H,
6.11; N, 11.08. Found: C, 44.43; H, 6.18; N, 11.05.
3
3
6
CD OD) δ: 7.58 (d, 1H, J = 7.9 Hz), 5.62 (d, 1H, J = 7.9 Hz),
3
4
=
.55 (ddddd, 1H, J = 7.9 Hz, J = 7.4 Hz, J = 7.2 Hz, J = 6.9 Hz, J
6.0 Hz, H-C5), 4.22–4.12 (m, 4H, 2 × POCH CH ), 3.88 (dd,
2
3
1
H, J = 13.9 Hz, J = 4.6 Hz, HCHN), 3.60 (dd, 1H, J = 13.9 Hz,
J = 8.5 Hz, HCHN), 3.36 (dddd, 1H, J = 8.5 Hz, J = 7.9 Hz, J =
.6 Hz, J = 4.2 Hz, H-C3), 2.75 (ddd, 1H, J = 13.5 Hz, J = 7.9
Hz, J = 7.9 Hz, H -C4), 2.62 (s, 3H, CH ), 2.22 (ddd, 1H, J =
4.2.4. Diethyl trans-((3-((5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)methyl)phosphonate (28b)
4
-
1
Colorless oil; IR (film, cm ) νmax: 3399, 3194, 3071, 2988,
a
3
2
2.2 Hz, J = 15.0 Hz, J = 6.0 Hz, HCHP), 2.17 (ddd, 1H, J = 22.2
2925, 2851, 2821, 1696, 1663, 1470, 1443, 1242, 1050, 968;
Hz, J = 15.0 Hz, J = 7.2 Hz, HCHP), 1.78 (ddd, 1H, J = 13.5 Hz,
(signals of trans-28b were extracted from the spectra of a 2:8
1
J = 6.9 Hz, J = 4.2 Hz, H -C4), 1.33 (t, 6H, J = 7.0 Hz, 2 ×
mixture of trans-28b and cis-27b); H NMR (600 MHz, CDCl )
b
3
13
POCH CH ). C NMR (75 MHz, CD OD) δ: 166.6 (C=O), 152.8
δ: 9.20 (brs, 1H, NH), 7.49 (d, 1H, J = 5.7 Hz), 4.35–4.31 (m,
2
3
3
(C=O), 148.5 (C=C), 101.5 (C=C), 72.3 (C5), 67.5 (C3), 63.7 (d,
1H, H-C5), 4.20–4.11 (m, 4H, 2 × CH OP), 3.95 (dd, 1H, J =
2
J = 6.3 Hz, POCH ), 63.5 (d, J = 6.6 Hz, POCH ), 52.1 (CH N),
13.9 Hz, J = 3.7 Hz, HCHN), 3.48 (dd, 1H, J = 13.9 Hz, J = 8.6
Hz, HCHN), 3.38–3.34 (m, 1H, H-C3), 2.87–2.75 (m, 1H, Ha-
C4), 2.69 (s, 3H, CH N), 2.38–2.29 (m, 2H, HCHP, H -C4),
2
2
2
4
4.8 (NCH ), 38.9 (d, J = 7.7 Hz, C4), 31.5 (d, J = 139.7 Hz,
3
31
CH P), 16.9 (d, J = 6.0 Hz, 2 × POCH CH ). P NMR (121.5
2
2
3
3
b
MHz, CD OD) δ: 29.11. Anal. Calcd. for C H N O P: C, 46.54;
H, 6.70; N, 11.63. Found: C, 46.40; H, 6.90; N, 11.61.
2.08–2.03 (m, 1H, HCHP), 1.37 (t, 6H, J = 7.0 Hz, CH CH OP);
3
14 24
3
6
3
2
13
C NMR (150 MHz, CDCl ) δ: 157.2 (d, J = 26.3 Hz, C=O),
3
1
49.6 (C=O), 139.8 (d, J = 226.4 Hz, C=C), 130.4 (d, J = 32.9
Hz, C=C), 71.6 (C5), 66.0 (C3), 62.0 (d, J = 6.4 Hz, CH OP),
2
4
.2.2. Diethyl trans-((3-((2,4-dioxo-3,4-
61.9 (d, J = 6.1 Hz, CH
OP), 51.2 (CH N), 46.3 (CH N), 38.0 (d,
2 2 3
dihydropyrimidin-1(2H)-yl)methyl)-2-
J = 3.4 Hz, C4), 33.2 (d, J = 137.7 Hz, CP), 16.4 (d, J = 5.8 Hz, 2
31
methylisoxazolidin-5-yl)methyl)phosphonate (28a)
× CH CH OP); P NMR (242 MHz, CDCl ) δ: 25.87. Anal.
3
2
3
-
1
Colorless oil; IR (film, cm ) νmax: 3459, 3173, 3051, 2985,
911, 2823, 1679, 1631, 1453, 1228, 1023. (signals of trans-28a
Calcd. for C14
H23FN
3
O
6
P: C, 44.33; H, 6.11; N, 11.08. Found: C,
4
4.47; H, 5.98; N, 11.17 (obtained on a 2:8 mixture of trans-28b
2
and cis-27b).
were extracted from the spectra of a 6:4 mixture of trans-28a and
1
cis-27a); H NMR (600 MHz, CD OD) δ: 7.59 (d, 1H, J = 7.9
3
Hz), 5.66 (d, 1H, J = 7.9 Hz), 4.32–4.26 (brsx, 1H, J ≈ 7.0 Hz, H-
C5), 4.18–4.09 (m, 4H, 2 × CH OP), 3.89 (dd, 1H, J = 14.1 Hz, J
4.2.5. Diethyl cis-((3-((5-bromo-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)methyl)phosphonate (27c)
2
=
5.2 Hz, HCHN), 3.78 (dd, 1H, J = 14.1 Hz, J = 7.0 Hz,
HCHN), 3.28 (brqu, 1H, J ≈ 7.0 Hz, H-C3), 2.70 (s, 3H, CH N),
3
2
1
.31 (dd, 2H, J = 7.4 Hz, J = 6.7 Hz, H C4), 2.28 (ddd, 1H, J =
8.5 Hz, J = 15.2 Hz, J = 6.3 Hz, HCHP), 2.18 (ddd, 1H, J = 18.9
Yield: 4% (0.018 g from 1.03 mmol of the nitrone 20c);
2
-
1
yellow oil; IR (film, cm ) ν : 3475, 3158, 3090, 2988, 2913,
max
1
Hz, J = 15.2 Hz, J = 7.2 Hz, HCHP), 1.34 (t, 6H, J = 7.1 Hz, 2 ×
CH CH OP). C NMR (150 MHz, CD OD) δ: 165.3 (C=O),
2833, 1682, 1617, 1443, 1431, 1251, 1226, 1022, 963; H NMR
13
(600 MHz, CDCl ) δ: 9.18 (brs, 1H, NH), 7.77 (s, 1H), 4.59
3
2
3
3
1
6
51.6 (C=O), 146.8 (C=C), 100.4 (C=C), 72.5 (C5), 65.9 (C3),
(ddddd, 1H, J = 8.7 Hz, J = 7.8 Hz, J = 7.8 Hz, J = 7.5 Hz, J =
2.2 (d, J = 6.5 Hz, POCH ), 62.0 (d, J = 6.5 Hz, POCH ), 49.5
4.7 Hz, H-C5), 4.19–4.11 (m, 4H, 2 × CH OP), 4.09–4.03 (m,
2
2
2
(
CH N), 48.5 (NCH ), 38.1 (d, J = 8.1 Hz, C4), 31.4 (d, J = 139.7
1H, HCHN), 3.38–3.33 (m, 2H, HCHN, H-C3), 2.88–2.73 (m,
1H, H -C4), 2.63 (s, 3H, CH N), 2.26 (ddd, 1H, J = 17.6 Hz, J =
2
3
31
Hz, CH P), 15.3 (d, J = 6.9 Hz, 2 × POCH CH ). P NMR (242
2
2
3
a
3
MHz, CD OD) δ: 27.59. Anal. Calcd. for C H N O P: C, 46.54;
H, 6.70; N, 11.63. Found: C, 46.32; H, 6.75; N, 11.53 (obtained
on a 6:4 mixture of trans-28a and cis-27a).
14.9 Hz, J = 4.7 Hz, HCHP), 1.99 (ddd, 1H, J = 18.3 Hz, J = 14.9
Hz, J = 8.7. Hz, HCHP), 1.75 (ddd, 1H, J = 13.4 Hz, J = 7.5 Hz,
J = 3.4 Hz, H -C4), 1.37 (t, 3H, J = 7.1 Hz, CH CH OP), 1.35 (t,
3
14 24
3
6
b
3
2
13
3
1
H, J = 7.1 Hz, CH CH OP); C NMR (150 MHz, CDCl ) δ:
3
2
3
59.5 (C=O), 150.2 (C=O), 145.8 (C=C), 95.5 (C=C), 71.0 (C5),
OP), 62.0 (d, J = 7.2 Hz,
N), 37.6 (d, J = 4.4 Hz, C4),
30.8 (d, J = 140.8 Hz, CP), 16.4 (d, J = 5.9 Hz, 2 × CH CH OP);
4
.2.3. Diethyl cis-((3-((5-fluoro-2,4-dioxo-3,4-
66.1 (C3), 62.0 (d, J = 7.3 Hz, CH
CH OP), 51.8 (CH N), 44.5 (CH
2
dihydropyrimidin-1(2H)-yl)methyl)-2-
2
2
3
methylisoxazolidin-5-yl)methyl)phosphonate (27b)
3
2
31
P NMR (242 MHz, CDCl ) δ: 26.19. Anal. Calcd. for
Yield: 3% (0.011 g from 0.88 mmol of the nitrone 20b);
3
-
1
colorless oil; IR (film, cm ) ν : 3410, 3185, 3064, 2985, 2962,
2
C H23BrN O P: C, 38.20; H, 5.27; N, 9.55. Found: C, 38.05; H,
14 3 6
max
1
922, 2851, 2820, 1698, 1664, 1466, 1444, 1376, 1098, 966; H
5.04; N, 9.69.
NMR (600 MHz, CDCl ) δ: 8.84 (brs, 1H, NH), 7.57 (d, 1H, J =
3
5
7
.8 Hz), 4.59 (ddddd, 1H, J = 9.0 Hz, J = 8.6 Hz, J = 7.9 Hz, J =
.9 Hz, J = 4.6 Hz, H-C5), 4.21–4.11 (m, 4H, 2 × CH OP), 4.02
2
(dd, 1H, J = 13.5 Hz, J = 3.0 Hz, HCHN), 3.36 (dddd, 1H, J =

1
1
4
.2.6. Diethyl trans-((3-((5-bromo-2,4-dioxo-3,4-
(C=O), 147.9 (C=C), 101.4 (C=C), 75.6 (C5), 73.2 (CH O),
2
ACCEPTED MANUSCRIPT
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)methyl)phosphonate (28c)
65.3 (C3), 64.3 (d, J = 6.6 Hz, 2 × CH OP), 63.9 (d, J = 162.7
Hz, CH P), 51.1 (CH N), 43.8 (CH N), 32.3 (C4), 15.9 (d, J =
2
2
2
3
-
1
31
Yellow oil; IR (film, cm ) νmax: 3443, 3175, 2985, 2925,
853, 2822, 1688, 1620, 1442, 1247, 1025, 965; (signals of trans
6.3 Hz, 2 × CH
3
CH
2
OP); P NMR (121 MHz, D
Anal. Calcd. for C15H N O P: C, 46.04; H, 6.70; N, 10.74.
26 3 7
2
O) δ: 24.95.
2
were extracted from the spectra of a 20:80 mixture of trans and
Found: C, 45.89; H, 6.83; N, 10.61.
1
cis); H NMR (600 MHz, CDCl ) δ: 9.02 (brs, 1H, NH), 7.73 (s,
3
1
4
H), 4.38–4.33 (m, 1H, H-C5), 4.21–4.12 (m, 4H, 2 × CH OP),
.09–4.03 (m, 1H, HCHN), 3.54 (dd, 1H, J = 13.5 Hz, J = 9.1
2
4.2.9. Diethyl trans-(((3-((2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-
Hz, HCHN), 3.37–3.34 (m, 1H, H-C3), 2.71 (s, 3H, CH N),
2
1
3
.42–2.30 (m, 3H, H C4, HCHP), 2.08–2.03 (m, 1H, HCHP),
2
13
.38–1.35 (m, 6H, 2 × CH CH OP); C NMR (150 MHz,
yl)methoxy)methyl)phosphonate (30a)
3
2
CDCl ) δ: 159.4 (C=O), 150.3 (C=O), 145.4 (C=C), 95.8 (C=C),
Yield: 9% (0.030 g from 0.85 mmol of the nitrone 20a);
3
-
1
7
3.4 (C5), 65.8 (C3), 61.9 (d, J = 6.4 Hz, 2 × CH OP), 50.7
colorless oil; IR (film, cm ) ν : 3472, 3173, 3051, 2984, 2929,
2
max
1
(
CH N), 46.3 (CH N), 38.0 (d, J = 5.9 Hz, C4), 33.2 (d, J = 138.7
1681, 1631, 1454, 1370, 1245, 1023; H NMR (600 MHz,
2
3
31
Hz, CP), 16.4 (d, J = 5.8 Hz, 2 × CH CH OP); P NMR (242
CD OD) δ: 7.57 (d, 1H, J = 7.8 Hz), 5.65 (d, 1H, J = 7.8 Hz),
3
2
3
MHz, CDCl ) δ: 25.71. Anal. Calcd. for C H BrN O P: C,
4.27–4.22 (m, 1H, H-C5), 4.21–4.14 (m, 4H, CH OP), 3.94 (d,
3
14 23
3
6
2
3
8.20; H, 5.27; N, 9.55. Found: C, 38.05; H, 5.02; N, 9.64
2H, J = 8.3 Hz, CH P), 3.89 (dd, 1H, J = 14.0 Hz, J = 5.4 Hz,
2
(obtained on a 2:8 mixture of trans-28c and cis-27c).
HCHN), 3.74 (dd, 1H, J = 14.0 Hz, J = 6.8 Hz, HCHN), 3.73 (dd,
1
H, J = 10.7 Hz, J = 3.5 Hz, HCHO), 3.66 (dd, 1H, J = 10.7 Hz,
J = 5.5 Hz, HCHO), 3.32–3.26 (m, 1H, H-C3), 2.68 (s, 3H,
CH N), 2.36 (ddd, 1H, J = 12.8 Hz, J = 8.0 Hz, J = 7.9 Hz, H
C4), 2.16 (ddd, 1H, J = 12.8 Hz, J = 7.7 Hz, J = 4.6 Hz, H -C4),
4
.2.7. Diethyl cis-((2-methyl-3-((5-methyl-2,4-
3
-
b
dioxo-3,4-dihydropyrimidin-1(2H)-
yl)methyl)isoxazolidin-5-yl)methyl)phosphonate
b
13
1.36 (t, 6H, J = 7.0 Hz, CH CH OP); C NMR (150 MHz,
3 2
(
27d)
CD OD) δ: 165.6 (C=O), 151.8 (C=O), 146.7 (C=C), 100.4
3
Yield: 21% (0.102 g from 1.27 mmol of the nitrone 20d);
(C=C), 77.1 (C5), 74.4 (CH
CH P), 62.7 (d, J = 6.5 Hz, 2 × CH
(CH N), 33.3 (C4), 15.4 (d, J = 5.6 Hz, 2 × CH
NMR (242 MHz, CD
P: C, 46.04; H, 6.70; N, 10.74. Found: C, 45.81; H,
2
O), 65.6 (C3), 64.6 (d, J = 166.2 Hz,
-
1
yellow oil; IR (film, cm ) ν : 3406, 3163, 3042, 2984, 2928,
2
2
OP), 49.5 (CH
CH
2
N), 44.6
max
1
31
2
816, 1689, 1466, 1369, 1250, 1024; H NMR (300 MHz,
3
3
2
OP);
P
CD OD) δ: 7.42 (q, J = 1.2 Hz, 1H), 4.55 (ddddd, 1H, J = 7.9 Hz,
3
OD) δ: 21.77. Anal. Calcd. for
3
J = 7.4 Hz, J = 7.1 Hz, J = 7.0 Hz, J = 6.3 Hz, H-C5), 4.18–4.07
C H N O
15 26 3 7
(
m, 4H, 2 × CH OP), 3.85 (dd, 1H, J = 13.9 Hz, J = 5.0 Hz,
6.79; N, 10.56.
2
HCHN), 3.59 (dd, 1H, J = 13.9 Hz, J = 8.2 Hz, HCHN), 3.37
dddd, 1H, J = 8.2 Hz, J = 7.9 Hz, J = 5.0 Hz, J = 4.6 Hz, H-C3),
.74 (ddd, 1H, J = 13.0 Hz, J = 7.9 Hz, J = 7.9 Hz, H -C4), 2.62
(
2
a
4.2.10. Diethyl cis-(((3-((5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-
(
s, 3H, CH N), 2.25 (ddd, 1H, J = 21.4 Hz, J = 15.3 Hz, J = 6.3
3
Hz, HCHP), 2.14 (ddd, 1H, J = 22.4 Hz, J = 15.3 Hz, J = 7.1 Hz,
HCHP), 1.86 (d, 3H, J = 1.2 Hz, CH ), 1.78 (ddd, 1H, J = 13.0
yl)methoxy)methyl)phosphonate (29b)
3
Hz, J = 7.0 Hz, J = 4.6 Hz, H -C4), 1.34 (t, 6H, J = 6.9 Hz, 2 ×
Yield: 13% (0.026 g from 0.49 mmol of the nitrone 20b);
b
13
-1
CH CH OP); C NMR (75 MHz, CD OD) δ: 166.8 (C=O),
yellow oil; IR (film, cm ) ν : 3466, 3172, 3063, 2985, 2918,
3
2
3
max
1
1
52.9 (C=O), 144.3 (C=C), 110.4 (C=C), 72.3 (d, J = 9.9 Hz,
2849, 2822, 1697, 1464, 1444, 1241, 1049, 972; H NMR (600
C5), 67.6 (C3), 63.7 (d, J = 6.4 Hz, CH OP), 63.5 (d, J = 6.4 Hz,
MHz, CDCl ) δ: 9.10 (brs, 1H, NH), 7.62 (d, 1H, J = 5.9 Hz),
2
3
CH OP), 52.0 (CH N), 44.9 (CH N), 39.0 (d, J = 7.4 Hz, CH ),
4.52–4.49 (m, 1H, H-C5), 4.24–4.16 (m, 4H, CH OP), 3.94–3.77
2
2
3
2
2
3
1.6 (d, J = 138.9 Hz, CP), 16.9 (d, J = 6.0 Hz, CH CH OP), 16.9
(m, 5H, HCHN, CH P, CH O), 3.55 (dd, 1H, J = 13.8 Hz, J = 9.8
3
2
2
2
31
(d, J = 6.0 Hz, CH CH OP), 12.4 (CH ); P NMR (121 MHz,
Hz, HCHN), 3.44–3.41 (m, 1H, H-C3), 2.62 (s, 3H, CH N),
3
2
3
3
CD OD) δ: 29.13. Anal. Calcd. for C H N O P: C, 48.00; H,
2.64–2.58 (m, 1H, H -C4), 1.88 (ddd, 1H, J = 13.0 Hz, J = 6.5
3
15 26
3
6
a
6
.98; N, 11.19. Found: C, 48.25; H, 6.93; N, 11.23.
Hz, J = 3.1 Hz, H -C4), 1.37 (t, 6H, J = 7.0 Hz, 2 × CH CH OP);
b
3
2
13
C NMR (150 MHz, CDCl ) δ: 157.3 (d, J = 26.2 Hz, C=O),
3
1
49.6 (C=O), 139.7 (d, J = 233.8 Hz, C=C), 131.5 (d, J = 32.7
4
.2.8. Diethyl cis-(((3-((2,4-dioxo-3,4-
Hz, C=C), 75.4 (C5), 72.5 (d, J = 10.9 Hz, CH
2
O), 65.7 (d, J =
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-
yl)methoxy)methyl)phosphonate (29a)
166.1 Hz, CH P), 65.2 (C3), 62.5 (d, J = 6.7 Hz, CH OP), 62.4
2
2
(d, J = 6.7 Hz, CH OP), 51.3 (CH N), 44.0 (CH N), 31.9 (C4),
2
2
3
31
16.5 (d, J = 5.6 Hz, 2 × CH CH OP); P NMR (242 MHz,
3 2
Yield: 16% (0.055 g from 0.85 mmol of the nitrone 20a);
CDCl ) δ: 20.95. Anal. Calcd. for C15H25FN O P: C, 44.01; H,
3 3 7
6.16; N, 10.27. Found: C, 44.25; H, 5.96; N, 10.22.
-
1
colorless oil; IR (film, cm ) ν : 3472, 3169, 3050, 2983, 2929,
max
1
1
679, 1631, 1453, 1372, 1249, 1027; H NMR (300 MHz,
CD OD) δ: 7.54 (d, 1H, J = 7.8 Hz), 5.61 (d, 1H, J = 7.8 Hz),
3
4
.50 (dddd, 1H, J = 8.6 Hz, J = 6.5 Hz, J = 4.8 Hz, J = 3.5 Hz, H-
4.2.11. Diethyl cis-(((3-((5-bromo-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-
C5), 4.22–4.12 (m, 4H, CH OP), 3.94 (d, 2H, J = 8.4 Hz, CH P),
3
JAB = 11.0 Hz, J = 3.5 Hz, HCHO), 3.70 (dAB, 1H, JAB = 11.0
Hz, J = 4.8 Hz, HCHO), 3.64 (dd, 1H, J = 13.9 Hz, J = 8.8 Hz,
HCHN), 3.39 (dddd, 1H, J = 8.8 Hz, J = 8.7 Hz, J = 4.9 Hz, J =
2
2
.81 (dd, 1H, J = 13.9 Hz, J = 4.9 Hz, HCHN), 3.72 (dAB, 1H,
yl)methoxy)methyl)phosphonate (29c)
Yield: 12% (0.047 g from 0.85 mmol of the nitrone 20c); light
-
1
yellow oil; IR (film, cm ) ν : 3425, 3189, 3059, 2985, 2960,
max
1
3
.2 Hz, H-C3), 2.63 (ddd, 1H, J = 13.0 Hz, J = 8.6 Hz, J = 8.6
2925, 2854, 2820, 1695, 1622, 1445, 1338, 1247, 1023, 970; H
Hz, H -C4), 2.60 (s, 3H, CH N), 1.82 (ddd, 1H, J = 13.0 Hz, J =
NMR (600 MHz, CDCl ) δ: 9.38 (brs, 1H, NH), 7.80 (s, 1H),
a
3
3
6
.5 Hz, J = 3.2 Hz, H -C4), 1.34 (t, 6H, J = 7.0 Hz, 2 ×
4.52–4.49 (m, 1H, H-C5), 4.22–4.17 (m, 4H, CH OP), 3.93 (dd,
b
2
13
CH CH OP); C NMR (75 MHz, D O) δ: 166.8 (C=O), 152.3
1H, J = 13.9 Hz, J = 4.1 Hz, HCHN), 3.91–3.87 (m, 2H, CH P),
3
2
2
2

1
2
Tetrahedron
ACCEPTED MANUSCRIPT
3
.79 (dAB, 1H, JAB = 10.9 Hz, J = 2.8 Hz, HCHO), 3.76
(dAB, 1H, JAB = 10.9 Hz, J = 3.8 Hz, HCHO), 3.58 (dd, 1H, J =
4
.2.14. Diethyl trans-(((2-methyl-3-((5-methyl-2,4-
1
2
3.9 Hz, J = 10.1 Hz, HCHN), 3.43–3.39 (m, 1H, H-C3), 2.64–
.59 (m, 1H, H -C4), 2.61 (s, 3H, CH N), 1.84 (ddd, 1H, J = 13.0
dioxo-3,4-dihydropyrimidin-1(2H)-
yl)methyl)isoxazolidin-5-
a
3
Hz, J = 6.4 Hz, J = 2.3 Hz, H -C4), 1.35 (t, 6H, J = 7.1 Hz, 2 ×
CH CH OP); (signals of cis-29c were extracted from the
NMR spectrum of a 65:35 mixture of cis-29c and trans-30c);
NMR (150 MHz, CDCl ) δ: 159.7 (C=O), 150.4 (C=O), 146.5
b
yl)methoxy)methyl)phosphonate (30d)
1
1
3
3
C
C
-1
3
2
Yellowish oil; IR (film, cm ) νmax: 3488, 3183, 3061, 2986,
2
929, 1679, 1455, 1370, 1244, 1211, 1047, 1023; (signals of
3
trans-30d were extracted from the spectra of a 32:68 mixture of
(C=C), 95.2 (C=C), 75.3 (C5), 72.5 (d, J = 10.6 Hz, CH O), 65.7
1
2
trans-30d and cis-29d); H NMR (600 MHz, CDCl ) δ: 9.16 (brs,
3
(d, J = 161.5 Hz, CP), 65.1 (C3), 62.5 (d, J = 6.6 Hz, CH OP),
2
1
H, NH), 7.14 (s, 1H), 4.26–4.23 (m, 1H, H-C5), 4.21–4.16 (m,
6
2.5 (d, J = 6.6 Hz, CH OP), 51.4 (CH N), 44.1 (CH N), 31.9
2 2 3
31
4H, CH OP), 3.94–3.84 (m, 3H, CH P, HCHN), 3.74 (dAB, 1H,
JAB = 10.6 Hz, J = 4.4 Hz, HCHO), 3.69 (dAB, 1H, JAB = 10.6
Hz, J = 5.5 Hz, HCHO), 3.53 (dd, 1H, J = 12.6 Hz, J = 8.3 Hz,
2
2
(C4), 16.5 (d, J = 5.3 Hz, 2 × CH CH OP); P NMR (80 MHz,
3 2
CDCl ) δ: 21.91. Anal. Calcd. for C H BrN O P: C, 38.31; H,
3
15 25
3
7
5
.36; N, 8.94. Found: C, 38.36; H, 5.57; N, 8.86.
HCHN), 3.33–3.28 (m, 1H, H-C3), 2.68 (s, 3H, CH N), 2.37
3
(
ddd, 1H, J = 11.6 Hz, J = 8.2 Hz, J = 8.2 Hz, H -C4), 2.10 (ddd,
a
1
H, J = 11.6 Hz, J = 7.6 Hz, J = 3.7 Hz, H -C4), 1.92 (s, 3H,
b
13
4
.2.12. Diethyl trans-(((3-((5-bromo-2,4-dioxo-3,4-
CH ), 1.36 (t, 6H, J = 7.1 Hz, 2 × CH CH OP); C NMR (150
MHz, CDCl ) δ: 164.3 (C=O), 151.2 (C=O), 142.0 (C=C), 110.0
3
3
2
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-
yl)methoxy)methyl)phosphonate (30c)
3
(C=C), 75.3 (C5), 74.7 (d, J = 9.6 Hz, CH O), 65.7 (C3), 65.6 (d,
2
-
1
J = 163.5 Hz, CP), 62.4 (d, J = 6.1 Hz, CH
Hz, CH OP), 50.2 (CH N), 46.0 (CH N), 33.8 (C4), 16.5 (d, J =
5
CDCl ) δ: 20.89. Anal. Calcd. for C16H N O P: C, 47.41; H,
6
3
OP), 62.4 (d, J = 6.5
2
Yellowish oil; IR (film, cm ) νmax: 3174, 3059, 2983, 2932,
2
2
3
2
819, 1699, 1679, 1621, 1439, 1227, 1057; 754; (signals of
31
.5 Hz, 2 × CH CH OP), 12.2 (CH ); P NMR (242 MHz,
3 2 3
trans-30c were extracted from the spectra of a 65:35 mixture of
cis-29c and trans-30c); H NMR (600 MHz, CDCl ) δ: 9.50 (brs,
1
4
3
1
3
2
8
3
7
3
.96; N, 10.37. Found: C, 47.22; H, 7.20; N, 10.35 (obtained on a
2:68 mixture of trans-30d and cis-29d).
H, NH), 7.70 (s, 1H), 4.26–4.21 (m, 1H, H-C5), 4.22–4.17 (m,
H, CH OP), 3.98 (dd, 1H, J = 13.9 Hz, J = 3.9 Hz, HCHN),
2
.93–3.83 (m, 2H, CH P), 3.78 (dAB, 1H, J = 10.6 Hz, J = 4.0
2
AB
Hz, HCHO), 3.70 (dAB, 1H, JAB = 10.6 Hz, J = 5.4 Hz, HCHO),
4
.2.15. Diisopropyl cis-(((3-((2,4-dioxo-3,4-
3
1
8
.50 (dd, 1H, J = 13.9 Hz, J = 8.7 Hz, HCHN), 3.33–3.29 (m,
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)oxy)methyl)phosphonate
H, H-C3), 2.68 (s, 3H, CH N), 2.41 (ddd, 1H, J = 12.9 Hz, J =
3
.4 Hz, J = 8.2 Hz, H -C4), 2.10 (ddd, 1H, J = 12.9 Hz, J = 7.6
a
(
31a)
Hz, J = 2.9 Hz, H -C4), 1.35 (t, 6H, J = 7.1 Hz, 2 × CH CH OP);
b
3
2
13
Yield: 30% (0.013 g from 0.906 mmol of the nitrone 20a);
C NMR (150 MHz, CDCl ) δ: 159.5 (C=O), 150.4 (C=O),
-1
3
white amorphous solid; IR (film, cm ) ν : 3432, 3113, 2984,
2
CDCl ) δ: 9.95 (brs, 1H, NH), 7.55 (d, 1H, J = 7.9 Hz, C=C),
5.64 (d, 1H, J = 7.9 Hz, C=C), 5.29 (d, 1H, J = 5.1 Hz, H-C5),
4
1
max
1
6
45.6 (C=C), 95.7 (C=C), 77.6 (C5), 74.8 (d, J = 9.0 Hz, CH O),
5.6 (d, J = 165.1 Hz, CP), 65.5 (C3), 62.5 (d, J = 6.6 Hz,
1
2
930, 2854, 1714, 1680, 1454, 1236, 1027; H NMR (300 MHz,
3
CH OP), 62.5 (d, J = 6.6 Hz, CH OP), 50.2 (CH N), 46.1
2
2
2
31
(
CH N), 33.3 (C4), 16.5 (d, J = 5.3 Hz, 2 × CH CH OP);
P
3
3
2
.79–4.67 (m, 2H, POCH(CH ) ), 3.98 (dd, 1H, J = 13.4 Hz, J =
3 2
NMR (80 MHz, CDCl3) δ: 21.80. Anal. Calcd. for
0.4 Hz, HCHP), 3.95 (dAB, 1H, JAB = 13.7 Hz, J = 9.4 Hz,
C H BrN O P: C, 38.31; H, 5.36; N, 8.94. Found: C, 38.36; H,
15
25
3
7
HCHN), 3.93 (dAB, 1H, JAB = 13.7 Hz, J = 5.3 Hz, HCHN), 3.62
dd, 1H, J = 13.4 Hz, J = 8.7 Hz, HCHP), 3.44–3.36 (m, 1H, H-
C3), 2.58 (ddd, 1H, J = 13.7 Hz, J = 8.7 Hz, J = 5.1 Hz, H -C4),
5
3
.56; N, 8.88 (obtained on a 65:35 mixture of cis-29c and trans-
0c).
(
a
2
.60 (s, 3H, CH N), 2.06 (dd, 1H, J = 13.7 Hz, J = 1.3 Hz, H -
3 b
13
C4), 1.32–1.23 (m, 12H, POCH(CH ) ); C NMR (75 MHz,
3
2
4
.2.13. Diethyl cis-(((2-methyl-3-((5-methyl-2,4-
CDCl ) δ: 164.4 (C=O), 151.3 (C=O), 147.1 (C=C), 102.7 (d, J =
3
dioxo-3,4-dihydropyrimidin-1(2H)-
yl)methyl)isoxazolidin-5-
yl)methoxy)methyl)phosphonate (29d)
1
7
2.3 Hz, C5), 101.3 (C=C), 71.4 (d, J = 5.5 Hz, POCH(CH ) ),
3 2
1.3 (d, J = 5.9 Hz, POCH(CH ) ), 63.2 (C3), 61.1 (d, J = 171.1
3
2
Hz, CP), 51.7 (CH N), 47.1 (CH N), 37.6 (C4), 24.4 (d, J = 4.6
2
3
Yield: 5% (0.022 g from 1.03 mmol of the nitrone 20d);
-
1
Hz, POCH(CH
= 4.7 Hz, POCH(CH
3
)
2
), 24.3 (d, J = 3.6 Hz, POCH(CH
), 24.2 (d, J = 4.5 Hz, POCH(CH ) ); P
3 2
) ), 24.3 (d, J
3 2
31
yellowish oil; IR (film, cm ) ν : 3476, 3175, 3053, 2984, 2928,
1
8
H-C5), 4.23–4.16 (m, 4H, CH OP), 3.93–3.85 (m, 2H, CH P),
3
max
1
3
)
2
681, 1466, 1369, 1249, 1026; H NMR (600 MHz, CDCl ) δ:
3
NMR (121 MHz, CDCl3) δ: 20.84. Anal. Calcd. for
.68 (brs, 1H, NH), 7.24 (q, 1H, J = 1.0 Hz,), 4.51–4.48 (m, 1H,
C H N O P: C, 47.41; H, 6.96; N, 10.37. Found: C, 47.63; H,
16
28
3
7
2
2
6
.86; N, 10.48.
.88 (dd, 1H, J = 13.6 Hz, J = 4.0 Hz, HCHN), 3.79 (dAB, 1H,
JAB = 10.9 Hz, J = 2.9 Hz, HCHO), 3.74 (dAB, 1H, JAB = 10.9
Hz, J = 4.4 Hz, HCHO), 3.49 (dd, 1H, J = 13.6 Hz, J = 9.7 Hz,
4
.2.16. Diisopropyl trans-(((3-((2,4-dioxo-3,4-
HCHN), 3.44–3.40 (m, 1H, H-C3), 2.61 (s, 3H, CH N), 2.63–
3
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)oxy)methyl)phosphonate
2
1
7
.58 (m, 1H, H -C4), 1.93 (d, 3H, J = 1.0 Hz, CH ), 1.84 (ddd,
a
3
H, J = 13.0 Hz, J = 6.5 Hz, J = 2.8 Hz, H -C4), 1.35 (t, 6H, J =
b
(
32a)
13
.1 Hz, 2 × CH CH OP); C NMR (150 MHz, CDCl ) δ: 164.3
-1
3
2
3
White amorphous solid; IR (film, cm ) νmax: 3386, 3192,
(C=O), 151.1 (C=O), 142.9 (C=C), 109.4 (C=C), 75.3 (C5), 72.9
3
056, 2926, 2855, 1685, 1458, 1385, 1248, 1099; (signals of
(
d, J = 10.5 Hz, CH O), 65.7 (d, J = 166.4 Hz, CP), 65.4 (C3),
2
trans-32a were extracted from the spectra of a 46:54 mixture of
trans-32a and cis-31a); H NMR (600 MHz, CDCl ) δ: 8.53 (brs,
1H, NH), 7.34 (d, 1H, J = 7.9 Hz), 5.67 (d, 1H, J = 7.9 Hz), 5.33
6
2.6 (d, J = 5.9 Hz, CH OP), 62.4 (d, J = 6.1 Hz, CH OP), 51.4
1
2
2
3
(
CH N), 44.2 (CH N), 32.5 (C4), 16.5 (d, J = 5.6 Hz, 2 ×
2 3
31
CH CH OP), 12.1 (CH ); P NMR (242 MHz, CDCl ) δ: 20.95.
3
2
3
3
(t, 1H, J = 5.3 Hz, H-C5), 4.80–4.75 (m, 2H, POCH(CH ) ), 3.99
3 2
Anal. Calcd. for C H N O P: C, 47.41; H, 6.96; N, 10.37.
16
28
3
7
(dd, 1H, J = 13.5 Hz, J = 3.7 Hz, HCHN), 3.97 (dd, 1H, J = 13.5
Found: C, 47.56; H, 7.04; N, 10.25.

1
3
Hz, J = 9.4 Hz, HCHP), 3.72 (dd, 1H, J = 13.5 Hz, J = 9.0 Hz,
ACCEPTED MANUSCRIPT
HCHP), 3.53–3.46 (m, 1H, H-C3), 3.33 (dd, 1H, J = 13.5 Hz, J =
.3 Hz, HCHN), 2.86 (s, 3H. CH N), 2.49 (ddd, 1H, J = 13.7 Hz,
4
3
.2.19. Diisopropyl cis-(((3-((5-bromo-2,4-dioxo-
,4-dihydropyrimidin-1(2H)-yl)methyl)-2-
9
3
J = 7.6 Hz, J = 1.5 Hz, H -C4), 2.19 (ddd, 1H, J = 13.7 Hz, J =
a
methylisoxazolidin-5-yl)oxy)methyl)phosphonate
31c)
Yield: 19% (0.081 g from 0.89 mmol of the nitrone 20c);
colorless oil; IR (film, cm ) ν : 3174, 3059, 2983, 2932, 2819,
699, 1679, 1621, 1439, 1227, 1057, 999; H NMR (600 MHz,
CDCl ) δ: 9.64 (brs, 1H, NH), 7.96 (s, 1H), 5.34 (d, 1H, J = 5.2
Hz, H-C5), 4.80–4.74 (m, 2H, CH(CH ) ), 4.00 (dd, 1H, J = 13.6
Hz, J = 10.3 Hz, HCHP), 3.99 (dAB, 1H, JAB = 13.6 Hz, J = 9.9
Hz, HCHN), 3.97 (dAB, 1H, JAB = 13.6 Hz, J = 5.0 Hz, HCHN),
.68 (dd, 1H, J = 13.6 Hz, J = 8.4 Hz, HCHP), 3.41–3.38 (m, 1H,
5
.3 Hz, J = 5.3 Hz, H -C4), 1.37–1.34 (m, 12H, POCH(CH ) );
b
3
2
(
1
3
C NMR (150 MHz, CDCl ) δ: 163.3 (C=O), 150.7 (C=O),
3
1
46.1 (C=C), 105.5 (d, J = 12.3 Hz, C5), 101.1 (C=C), 71.3 (d, J
-1
max
=
6.6 Hz, POCH(CH ) ), 71.2 (d, J = 6.6 Hz, POCH(CH ) ), 64.5
1
3
2
3 2
1
(
C3), 61.6 (d, J = 171.6 Hz, CH P), 51.6 (CH N), 48.1 (CH N),
2 2 3
3
3
9.6 (C4), 24.1 (d, J = 4.1 Hz, POCH(CH ) ), 24.0 (d, J = 4.6 Hz,
3 2
3
2
POCH(CH ) ), 24.0 (d, J = 4.2 Hz, POCH(CH ) ), 23.9 (d, J =
3
2
3 2
31
4
.9 Hz, POCH(CH ) ); P NMR (242 MHz, CDCl ) δ: 18.92.
3
2
3
Anal. Calcd. for C H N O P: C, 47.41; H, 6.96; N, 10.37.
16
28
3
7
3
Found: C, 47.54; H, 6.75; N, 10.21 (obtained on a 46:54 mixture
of trans-32a and cis-31a).
H-C3), 2.64 (s, 3H, CH N), 2.60 (ddd, 1H, J = 13.9 Hz, J = 8.9
Hz, J = 5.2 Hz, H -C4), 2.09 (dd, 1H, J = 13.9 Hz, J = 1.5 Hz,
H -C4), 1.37–1.32 (m, 12H, 2 × CH(CH ) ); C NMR (150
3
a
13
b
3 2
MHz, CDCl ) δ: 159.7 (C=O), 150.6 (C=O), 146.2 (C=C), 102.6
3
4
3
.2.17. Diisopropyl cis-(((3-((5-fluoro-2,4-dioxo-
,4-dihydropyrimidin-1(2H)-yl)methyl)-2-
(
d, J = 10.7 Hz, C5), 95.5 (C=C), 71.3 (d, J = 6.3 Hz, CH(CH ) ),
3 2
7
1.2 (d, J = 6.8 Hz, CH(CH ) ), 63.1 (C3), 61.0 (d, J = 170.2 Hz,
3 2
methylisoxazolidin-5-yl)oxy)methyl)phosphonate
31b)
Yield: 20% (0.050 g from 0.60 mmol of the nitrone 20b);
CP), 51.8 (CH N), 46.9 (CH N), 37.4 (C4), 24.1 (d, J = 4.3 Hz,
2
3
(
31
CH(CH ) ), 24.0 (d, J = 4.2 Hz, CH(CH ) ); P NMR (242 MHz,
3
2
3 2
-
1
CDCl
) δ: 19.73. Anal. Calcd. for C16H27BrN O P: C, 39.68; H,
3 3 7
colorless oil; IR (film, cm ) ν : 3423, 3195, 3064, 2983, 2928,
2
max
1
5.62; N, 8.68. Found: C, 39.71; H, 5.68; N, 8.86.
852, 2823, 1702, 1665, 1467, 1376, 1242, 1131, 990; H NMR
(
600 MHz, CDCl ) δ: 8.69 (brs, 1H, NH), 7.85 (d, 1H, J = 5.8
3
Hz), 5.34 (d, 1H, J = 5.1 Hz, H-C5), 4.83–4.74 (m, 2H,
CH(CH ) ), 4.01 (dd, 1H, J = 13.4 Hz, J = 10.6 Hz, HCHP), 3.98
4
2
.2.20. Diisopropyl cis-(((2-methyl-3-((5-methyl-
,4-dioxo-3,4-dihydropyrimidin-1(2H)-
3
2
(dd, 1H, J = 13.8 Hz, J = 9.0 Hz, HCHN), 3.91 (dd, 1H, J = 13.8
yl)methyl)isoxazolidin-5-yl)oxy)methyl)phosphonate
31d)
Yield: 27% (0.110 g from 0.98 mmol of the nitrone 20d);
Hz, J = 5.3 Hz, HCHN), 3.67 (dd, 1H, J = 13.4 Hz, J = 8.6 Hz,
HCHP), 3.46 (dddd, 1H, J = 8.9 Hz, J = 8.6 Hz, J = 5.3 Hz, J =
(
1
.4 Hz, H-C3), 2.66 (s, 3H, CH N), 2.61 (ddd, 1H, J = 13.8 Hz, J
-1
3
yellow oil; IR (film, cm ) ν : 3479, 3176, 3055, 2980, 2932,
2820, 1713, 1466, 1373, 1248, 1101, 1015; H NMR (600 MHz,
CDCl ) δ: 9.07 (brs, 1H, NH), 7.29 (q, 1H, J = 1.0 Hz), 5.33 (d,
1
1
max
=
8.6 Hz, J = 5.3 Hz, H -C4), 2.11 (dd, 1H, J = 13.8 Hz, J = 1.4
1
a
13
Hz, H -C4), 1.38–1.36 (m, 12H, 2 × CH(CH ) ); C NMR (150
b
3 2
3
MHz, CDCl ) δ: 157.4 (d, J = 26.2 Hz, C=O), 149.8 (C=O),
3
H, J = 5.2 Hz, H-C5), 4.80–4.74 (m, 2H, CH(CH ) ), 4.02 (dd,
3 2
1
39.7 (d, J = 233.9 Hz, C=C), 131.6 (d, J = 32.9 Hz, C=C), 102.6
H, J = 13.6 Hz, J = 9.9 Hz, HCHP), 3.91 (dAB, 1H, J = 13.8
(
(
(
d, J = 12.1 Hz, C5), 71.3 (d, J = 6.4 Hz, 2 × CH(CH ) ), 63.0
3
2
Hz, J = 8.3 Hz, HCHN), 3.89 (dAB, 1H, J = 13.8 Hz, J = 5.3 Hz,
HCHN), 3.68 (dd, 1H, J = 13.6 Hz, J = 8.9 Hz, HCHP), 3.44
C3), 60.9 (d, J = 170.6 Hz, CP), 51.6 (CH N), 47.0 (CH N), 37.2
2
3
31
C4), 24.1 (d, J = 4.1 Hz, 2 × CH(CH ) ); P NMR (242 MHz,
3
2
(dddd, J = 8.9 Hz, J = 8.3 Hz, J = 5.3 Hz, J = 2.1 Hz, 1H, H-C3),
CDCl ) δ: 19.72. Anal. Calcd. for C H FN O P: C, 45.39; H,
3
16 27
3
7
2
5
1
.64 (s, 3H, CH N), 2.61 (ddd, 1H, J = 13.9 Hz, J = 8.9 Hz, J =
3
6
.43; N, 9.93. Found: C, 45.47; H, 6.57; N, 10.11.
.2 Hz, H -C4), 2.10 (dd, 1H, J = 13.9 Hz, J = 2.1 Hz, H -C4),
a
b
.94 (d, 3H, J = 1.0 Hz, CH ), 1.37–1.33 (m, 12H, 2 ×
3
13
CH(CH ) ); C NMR (75 MHz, CDCl ) δ: 164.2 (C=O), 151.0
3
2
3
4
3
.2.18. Diisopropyl trans-(((3-((5-fluoro-2,4-dioxo-
,4-dihydropyrimidin-1(2H)-yl)methyl)-2-
(C=O), 142.8 (C=C), 109.7 (C=C), 102.7 (d, J = 12.0 Hz, C5),
7
1.1 (d, J = 5.5 Hz, 2 × CH(CH ) ), 63.4 (C3), 61.2 (d, J = 170.7
3
2
methylisoxazolidin-5-yl)oxy)methyl)phosphonate
Hz, CP), 51.6 (CH N), 46.8 (CH N), 37.6 (C4), 24.1 (d, J = 4.1
Hz, 2 × CH(CH
1
1
2
3
(
32b)
31
-
1
3
)
2
), 12.2 (CH ); P NMR (242 MHz, CDCl ) δ:
3
3
Colorless oil; IR (film, cm ) νmax: 3412, 3180, 3055, 2983,
9.57. Anal. Calcd. for C H N O P: C, 48.68; H, 7.21; N,
17 30 3 7
2
932, 2879, 2821, 1698, 1665, 1465, 1335, 1241, 1102, 990;
0.02. Found: C, 48.43; H, 7.07; N, 9.98.
(
signals of trans-32b were extracted from the spectra of a 36:64
1
mixture of trans-32b and cis-31b); H NMR (600 MHz, CDCl )
3
δ: 9.28 (brs, 1H, NH), 7.49 (d, 1H, J = 5.8 Hz), 5.34 (d, 1H, J =
4
2
.2.21. Diisopropyl trans-(((2-methyl-3-((5-methyl-
,4-dioxo-3,4-dihydropyrimidin-1(2H)-
5
1
.2 Hz, H-C5), 4.82–4.74 (m, 2H, CH(CH ) ), 3.98 (dd, 1H, J =
3.8 Hz, J = 3.7 Hz, HCHN), 3.98 (dd, 1H, J = 13.8 Hz, J = 9.6
3 2
yl)methyl)isoxazolidin-5-yl)oxy)methyl)phosphonate
32d)
Yield: 2% (0.008 g from 0.98 mmol of the nitrone 20d);
Hz, HCHP), 3.73 (dd, 1H, J = 13.8 Hz, J = 8.9 Hz, HCHP), 3.56–
.50 (m, 1H, H-C3), 3.28 (dd, 1H, J = 13.8 Hz, J = 9.2 Hz,
(
3
HCHN), 2.87 (s, 3H, CH N), 2.71 (ddd, 1H, J = 13.9 Hz, J = 7.7
-1
3
colorless oil; IR (film, cm ) ν : 3429, 3180, 3055, 2980, 2926,
max
Hz, J = 1.9 Hz, H -C4), 2.19 (ddd, 1H, J = 13.9 Hz, J = 5.6 Hz, J
1
a
13
2853, 1683, 1465, 1247, 1101, 993; H NMR (600 MHz, CDCl
δ: 8.51 (brs, 1H, NH), 7.17 (q, 1H, J = 1.0 Hz), 5.32 (dd, 1H, J =
.5 Hz, J = 1.7 Hz, H-C5), 4.80–4.74 (m, 2H, CH(CH ) ), 3.98
3
)
=
4.9 Hz, H -C4), 1.38–1.34 (m, 12H, 2 × CH(CH ) ); C NMR
b
3
2
(
150 MHz, CDCl ) δ: 157.2 (d, J = 26.9 Hz, C=O), 149.6 (C=O),
3
5
3
2
1
39.8 (d, J = 235.0 Hz, C=C), 130.5 (d, J = 32.8 Hz, C=C), 105.7
(dd, 1H, J = 13.6 Hz, J = 9.4 Hz, HCHP), 3.97 (dd, 1H, J = 13.8
(
(
(
d, J = 12.1 Hz, C5), 71.2 (d, J = 6.7 Hz, 2 × CH(CH ) ), 64.6
3
2
Hz, J = 3.7 Hz, HCHN), 3.72 (dd, 1H, J = 13.6 Hz, J = 8.9 Hz,
HCHP), 3.59–3.53 (m, 1H, H-C3), 3.34 (dd, 1H, J = 13.8 Hz, J =
9
J = 7.6 Hz, J = 1.7 Hz, H -C4), 2.20 (ddd, 1H, J = 13.7 Hz, J =
5
C3), 62.0 (d, J = 169.8 Hz, CP), 51.0 (CH N), 48.0 (CH N), 39.4
2
3
31
C4), 24.1 (d, J = 4.2 Hz, 2 × CH(CH ) ); P NMR (242 MHz,
3
2
.1 Hz, HCHN), 2.86 (s, 3H, CH N), 2.69 (ddd, 1H, J = 13.7 Hz,
3
CDCl ) δ: 18.88. Anal. Calcd. for C H FN O P: C, 45.39; H,
3
16 27
3
7
a
6
3
.43; N, 9.93. Found: C, 45.23; H, 6.33; N, 9.78 (obtained on a
6:64 mixture of trans-32b and cis-31b).
.5 Hz, J = 5.5 Hz, H -C4), 1.94 (d, 3H, J = 1.0 Hz, CH ), 1.38–
b
3

1
4
Tetrahedron
1
1
.34 (m, 12H, 2 × CH(CH ) ); (signals of trans-32d were
1048, 981; H NMR (600 MHz, CDCl ) δ: 9.78 (brs, 1H, NH),
3
2
ACCEPTED MANUSCRIPT
3
13
extracted from the C NMR spectrum of a 63:37 mixture of
trans-32d and cis-31d) C NMR (150 MHz, CDCl ) δ: 164.0
7.64 (d, 1H, J = 5.9 Hz), 4.45 (dd, 1H, J = 10.4 Hz, J = 6.4 Hz,
13
H-C5), 4.27–4.17 (m, 4H, CH OP), 3.94–3.88 (M part of ABM
3
2
(
1
C=O), 150.8 (C=O), 142.0 (C=C), 109.9 (C=C), 105.5 (d, J =
system, 1H, HCHN) and 3.65–3.57 (AB part of ABM system,
2H, HCHN and H-C3), 2.79 (dddd, 1H, J = 15.4 Hz, J = 13.4 Hz,
J = 10.5 Hz, J = 7.6 Hz, H -C4), 2.59 (s, 3H, CH N), 2.21 (dddd,
2.2 Hz, C5), 71.2 (d, J = 6.6 Hz, CH(CH ) ), 71.1 (d, J = 6.5 Hz,
3
2
CH(CH ) ), 64.7 (C3), 61.9 (d, J = 170.7 Hz, CP), 51.0 (CH N),
3
2
2
a
3
4
8.1 (CH N), 39.7 (C4), 24.0 (d, J = 4.0 Hz, 2 × CH(CH ) ), 12.2
1H, J = 19.5 Hz, J = 13.4 Hz, J = 6.4 Hz, J = 1.1 Hz, H -C4),
3
3 2
b
31
(CH3); P NMR (242 MHz, CDCl ) δ: 18.51. Anal. Calcd. for
1.39 (t, 3H, J = 7.0 Hz, CH CH OP) 1.38 (t, 3H, J = 7.0 Hz,
3
3
2
13
C H N O P: C, 48.68; H, 7.21; N, 10.02. Found: C, 48.88; H,
CH CH OP); C NMR (150 MHz, CDCl ) δ: 157.5 (d, J = 26.2
17
30
3
7
3
2
3
7
.15; N, 9.99.
Hz, C=O), 149.9 (C=O), 139.7 (d, J = 234.1 Hz, C=C), 131.4 (d,
J = 32.8 Hz, C=C), 70.2 (d, J = 172.4 Hz, C5), 65.2 (d, J = 3.7
Hz, C3), 63.5 (d, J = 6.5 Hz, CH OP), 62.6 (d, J = 6.7 Hz,
2
4
.2.22. Diethyl cis-(3-((2,4-dioxo-3,4-
CH
Hz, CH
(242 MHz, CDCl ) δ: 22.23. Anal. Calcd. for C H FN O P: C,
2
OP), 50.9 (CH
N), 43.4 (CH N), 32.2 (C4), 16.5 (d, J = 5.6
2 3
31
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)phosphonate (33a)
3
CH OP), 16.5 (d, J = 5.6 Hz, CH
2
CH OP); P NMR
3 2
3
13 21
3
6
Yield: 14% (0.073 g from 1.69 mmol of the nitrone 20a);
white amorphous solid; mp 114–115°C; IR (KBr, cm ) νmax:
42.74; H, 5.79; N, 11.50. Found: C, 42.93; H, 5.90; N, 11.34.
-
1
3
1
445, 3153, 3069, 3045, 2993, 2926, 1704, 1669, 1468, 1418,
1
240, 1015, 976; H NMR (200 MHz, CDCl ) δ: 8.50 (brs, 1H,
4.2.25. Diethyl trans-(3-((5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)-2-
3
NH), 7.44 (d, 1H, J = 7.9 Hz), 5.63 (dd, 1H, J = 7.9 Hz, J = 2.3
Hz), 4.43 (dd, 1H, J = 10.4 Hz, J = 6.4 Hz, H-C5), 4.30–4.10 (m,
4
methylisoxazolidin-5-yl)phosphonate (34b)
-
1
H, CH OP), 3.97–3.80 (M part of ABM system, 1H, HCHN)
Colorless oil; IR (KBr, cm ) νmax: 3398, 3158, 2995, 2963,
2
and 3.69–3.51 (AB part of ABM system, 2H, HCHN, H-C3),
.89–2.64 (m, 1H, H -C4), 2.57 (s, 3H, CH -N), 2.19 (dddd, 1H,
2906, 2821, 1700, 1667, 1444, 1233, 1048, 980; (signals of
2
trans-34b were extracted from the spectra of a 15:70:15 mixture
a
3
1
J = 19.8 Hz, J = 13.4 Hz, J = 6.4 Hz, J = 1.2 Hz, H -C4), 1.37 (t,
of cis-33b, trans-34b, 35b/36b); H NMR (600 MHz, CDCl ) δ:
b
3
3
H, J = 7.0 Hz, CH CH OP), 1.36 (t, 3H, J = 7.1 Hz,
9.75 (brs, 1H, NH), 7.45 (d, 1H, J = 5.6 Hz), 4.28–4.15 (m, 5H,
3
2
13
CH CH OP); C NMR (150 MHz, CDCl ) δ: 163.9 (C=O),
H-C5, CH OP), 4.01 (dd, 1H, J = 13.8 Hz, J = 3.5 Hz, HCHN),
3
2
3
2
1
51.1 (C=O), 147.0 (C=C), 101.1 (C=C), 70.2 (d, J = 171.8 Hz,
3.52–3.45 (m, 1H, H-C3), 3.28 (dd, 1H, J = 13.8 Hz, J = 9.4 Hz,
HCHN), 2.89–2.81 (m, 1H, H -C4), 2.74 (s, 3H, CH N), 2.33
C5), 65.2 (d, J = 3.9 Hz, C3), 63.4 (d, J = 6.5 Hz, CH OP), 62.5
2
a
3
(
1
d, J = 7.1 Hz, CH OP), 50.9 (CH N), 43.5 (CH N), 32.3 (C4),
(ddd, 1H, J = 12.8 Hz, J = 8.2 Hz, J = 8.2 Hz, H -C4), 1.40–1.34
b
13
2
2
3
6.5 (d, J = 5.4 Hz, CH CH OP), 16.5 (d, J = 5.4 Hz,
(m, 6H, 2 × CH CH OP); C NMR (150 MHz, CDCl ) δ: 157.3
3 2 3
3
2
31
CH CH OP); P NMR (80 MHz, CDCl ) δ: 23.10. Anal. Calcd.
for C H N O P: C, 44.96; H, 6.39; N, 12.10. Found: C, 44.87;
(d, J = 25.9 Hz, C=O), 149.8 (C=O), 139.9 (d, J = 235.1 Hz,
C=C), 130.5 (d, J = 32.9 Hz, C=C), 73.2 (d, J = 167.8 Hz, C5),
3
2
3
13
22
3
6
H, 6.36; N, 11.94.
65.8 (d, J = 6.2 Hz, C3), 63.4 (d, J = 6.6 Hz, CH OP), 62.6 (d, J
2
=
7.1 Hz, CH O), 49.7 (CH N), 46.1 (CH N), 32.5 (C4), 16.5 (d,
2 2 3
31
J = 5.5 Hz, CH CH OP), 16.5 (d, J = 5.3 Hz, CH CH OP);
P
3
2
3
2
4
.2.23. Diethyl trans-(3-((2,4-dioxo-3,4-
NMR (242 MHz, CDCl
P: C, 42.74; H, 5.79; N, 11.50. Found: C,42.59; H,
5.68; N, 11.21 (obtained on a 15:70:15 mixture of cis-33b, trans-
4b, 35b/36b).
3
) δ: 21.58. Anal. Calcd. for
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)phosphonate (34a)
C H21FN O
13 3 6
-
1
3
Yellowish oil; IR (film, cm ) νmax: 3422, 3055, 2984, 2920,
682, 1458, 1392, 1234, 1023; (signals of trans-34a were
1
extracted from the spectra of a 4:86:10 mixture of cis-33a, trans-
1
3
4a, 35a/36a); H NMR (200 MHz, CDCl ) δ: 8.94 (brs, 1H,
4.2.26. Diethyl cis-(3-((5-bromo-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)phosphonate (33c)
3
NH), 7.28 (d, 1H, J = 7.9 Hz), 5.66 (dd, 1H, J = 7.9 Hz, J = 1.9
Hz), 4.29–4.09 (m, 5H, 2 × CH CH OP, HC5), 4.00 (dd, 1H, J =
3
2
1
1
3.5 Hz, J = 3.5 Hz, HCHN), 3.54–3.42 (m, 1H, H-C3), 3.29 (dd,
H, J = 13.5 Hz, J = 9.3 Hz, HCHN), 2.83 (dddd, 1H, J = 20.9
Yield: 24% (0.094 g from 0.92 mmol of the nitrone 20c);
colorless crystalline solid (crystallized from ethyl acetate/hexane)
-
1
Hz, J = 13.0 Hz, J = 10.1 Hz, J = 7.3 Hz, H -C4), 2.71 (s, 3H,
mp 209-213°C with decomposition; IR (KBr, cm ) νmax: 3369,
a
CH N), 2.30 (dddd, 1H, J = 13.0 Hz, J = 8.2 Hz, J = 8.2 Hz, J =
3155, 3046, 2993, 2904, 2859, 2818, 1698, 1625, 1447, 1416,
3
1
1
.9 Hz, H -C4), 1.36 (t, 3H, J = 7.0 Hz, CH CH OP), 1.34 (t, 3H,
1334, 1229, 1042, 983; H NMR (300 MHz, CDCl ) δ: 9.06 (brs,
b
3
2
3
13
J = 7.0 Hz, CH CH OP); C NMR (150 MHz, CDCl ) δ: 163.6
1H, NH), 7.88 (s, 1H), 4.48 (dd, 1H, J = 10.5 Hz, J = 6.4 Hz, H-
3
2
3
(
1
C=O), 151.0 (C=O), 146.1 (C=C), 101.5 (C=C), 73.1 (d, J =
69.3 Hz, C5), 65.9 (d, J = 6.3 Hz, C3), 63.3 (d, J = 6.7 Hz,
CH OP), 62.6 (d, J = 6.8 Hz, CH OP), 49.7 (CH N), 46.1
C5), 4.33–4.18 (m, 4H, 2 × CH OP), 3.96 (dd, 1H, J = 13.9 Hz, J
2
= 5.0 Hz, HCHN), 3.70 (dd, 1H, J = 13.9 Hz, J = 9.5 Hz,
HCHN), 3.63–3.59 (m, 1H, H-C3), 2.84 (dddd, 1H, J = 15.8 Hz,
J = 13.4 Hz, J = 10.5 Hz, J = 7.7 Hz, H -C4), 2.61 (s, 3H, CH N),
2
2
2
3
1
(CH N), 32.7 (C4), 16.5 (d, J = 5.3 Hz, CH CH OP); P NMR
3
3
2
a
3
(
4
80 MHz, CDCl ) δ: 22.50. Anal. Calcd. for C H N O P: C,
4.96; H, 6.39; N, 12.10. Found: C, 45.10; H, 6.60; N, 11.93
2.23 (dddd, 1H, J = 19.4 Hz, J = 13.4 Hz, J = 6.4 Hz, J = 1.3 Hz,
H -C4), 1.42 (d, 3H, J = 7.0 Hz, CH CH OP), 1.41 (d, 3H, J =
3
13 22
3
6
b
3
2
13
(obtained on a 4:86:10 mixture of cis-33a, trans-34a, 35a/36a).
7.0 Hz, CH CH OP); C NMR (150 MHz, CDCl ) δ: 159.4
3
2
3
(C=O), 150.2, (C=O), 146.0 (C=C), 95.1 (C=C), 69.9 (d, J =
1
76.1 Hz, C5), 64.8 (C3), 63.2 (d, J = 6.3 Hz, CH OP), 62.4 (d, J
2
4
.2.24. Diethyl cis-(3-((5-fluoro-2,4-dioxo-3,4-
= 6.9 Hz, CH
(d, J = 5.4 Hz, CH
NMR (121 MHz, CDCl3) δ: 22.68. Anal. Calcd. for
P: C, 36.64; H, 4.97; N, 9.86. Found: C, 36.68; H,
2
OP), 50.8 (CH
N), 43.2 (CH N), 32.0 (C4), 16.4
2 3
31
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)phosphonate (33b)
3
CH OP), 16.3 (d, J = 5.4 Hz, CH
2
CH OP); P
3 2
Yield: 33% (0.106 g from 0.89 mmol of the nitrone 20b);
C H21BrN O
13 3 6
-
1
white amorphous solid, mp 126–127°C; IR (KBr, cm ) νmax:
403, 3157, 3057, 2994, 2906, 2821,1700, 1667, 1420, 1232,
4.69; N, 9.80.
3

1
5
3
1
5
.4 Hz, CH CH OP), 12.1 (CH ); P NMR (121 MHz,
3 2 3
ACCEPTED MANUSCR
.2.27. Diethyl trans-(3-((5-bromo-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)-2-
IPT
CDCl ) δ: 22.11. Anal. Calcd. for C H N O P: C, 46.54; H,
3
14 24
3
6
4
6
.70; N, 11.63. Found: C, 46.45; H, 6.98; N, 11.82.
methylisoxazolidin-5-yl)phosphonate (34c)
Yield: 10% (0.039 g from 0.92 mmol of the nitrone 20c);
-
1
4.2.30. Diethyl cis-(3-((6-amino-9H-purin-9-
yl)methyl)-2-methylisoxazolidin-5-yl)phosphonate
yellowish amorphous solid; IR (KBr, cm ) ν : 3418, 3158,
max
1
2
(
(
992, 2906, 2819, 1698, 1622, 1446, 1230, 1046, 981; H NMR
(
33e)
300 MHz, CDCl ) δ: 9.65 (brs, 1H, NH), 7.66 (s, 1H), 4.28–4.18
-1
3
Yellow oil; IR (film, cm ) νmax: 3323, 3177, 2984, 1650,
599, 1476, 1416, 1327, 1300, 1242, 1047, 970; (signals of cis-
m, 4H, 2 × CH OP), 4.18 (dd, 1H, J = 9.6 Hz, J = 8.9 Hz, H-
2
1
C5), 4.03 (dd, 1H, J = 13.8 Hz, J = 3.6 Hz, HCHN), 3.51–3.45
m, 1H, H-C3), 3.29 (dd, 1H, J = 13.6 Hz, J = 9.6 Hz, HCHN),
.81 (dddd, 1H, J = 20.1 Hz, J = 12.8 Hz, J = 9.6 Hz, J = 7.8 Hz,
H -C4), 2.72 (s, 3H, CH N), 2.30 (dddd, 1H, J = 12.8 Hz, J = 8.9
3
3e were extracted from the spectra of a 65:35 mixture of cis-33e
(
2
1
and trans-34e); H NMR (600 MHz, CDCl ) δ: 8.36 (s, 1H), 8.08
3
(
s, 1H), 5.78 (brs, 2H, NH ), 4.49 (dd, 1H, J = 10.1 Hz, J = 6.7
2
a
3
Hz, H-C5), 4.31–4.15 (m, 6H, CH N, 2 × CH OP), 3.74–3.68 (m,
2
2
Hz, J = 7.2 Hz, J = 1.4 Hz, H -C4), 1.37 (d, 3H, J = 7.0 Hz,
b
13
1H, H-C3), 2.84–2.72 (m, 1H, H
dddd, 1H, J = 19.9 Hz, J = 13.2 Hz, J = 6.7 Hz, J = 1.9 Hz, Hb-
C4), 1.41 (t, 3H, J = 6.7 Hz, CH CH OP), 1.39 (t, 3H, J = 6.7 Hz,
a
-C4), 2.56 (s, 3H, CH N), 2.29
3
CH CH OP), 1.35 (d, 3H, J = 7.0 Hz, CH CH OP); C NMR
3
2
3
2
(
(
9
6
(
5
150 MHz, CDCl ) δ: 159.4 (C=O), 150.4 (C=O), 145.5 (C=C),
3
3
2
5.8 (C=C), 73.2 (d, J = 167.9 Hz, C5), 65.7 (C3), 63.4 (d, J =
.7 Hz, CH OP), 62.6 (d, J = 7.0 Hz, CH OP), 49.9 (CH N), 46.2
13
CH CH OP); C NMR (150 MHz, CDCl ) δ: 155.6 (CNH ),
3
2
3
2
2
2
2
1
52.8, 149.9, 142.2, 119.5, 70.4 (d, J = 170.4 Hz, C5), 66.1 (C3),
CH N), 32.5 (C4), 16.5 (d, J = 5.6 Hz, CH CH OP), 16.5 (d, J =
3
3
2
31
63.4 (d, J = 6.5 Hz, CH
2
OP), 62.6 (d, J = 6.0 Hz, CH OP), 46.2
2
.6 Hz, CH CH OP); P NMR (121 MHz, CDCl ) δ: 22.05.
3
2
3
(
CH N), 43.6 (CH N), 32.5 (C4), 16.5 (d, J = 4.9 Hz,
2
3
Anal. Calcd. for C H BrN O P: C, 36.64; H, 4.97; N, 9.86.
31
13
21
3
6
CH CH OP), 16.4 (d, J = 5.3 Hz, CH CH OP); P NMR (242
3
2
3
2
Found: C, 36.68; H, 4.64; N, 9.56.
MHz, CDCl ) δ: 22.08. Anal. Calcd. for C H N O P: C, 45.40;
3
14 23
6
4
H, 6.26; N, 22.69. Found: C, 45.63; H, 6.08; N, 22.50 (obtained
on a 65:35 mixture of cis-33e and trans-34e).
4
.2.28. Diethyl cis-(2-methyl-3-((5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-
yl)methyl)isoxazolidin-5-yl)phosphonate (33d)
4
.2.31. Diethyl trans-(3-((6-amino-9H-purin-9-
Yield: 2% (0.009 g from 1.11 mmol of the nitrone 20d);
yl)methyl)-2-methylisoxazolidin-5-yl)phosphonate
-
1
colorless oil; IR (film, cm ) ν : 3481, 3171, 3054, 2985,
2
max
(
34e)
1
931,2818, 1688, 1468, 1239, 1048; H NMR (300 MHz, CDCl )
-1
3
Yellow oil; IR (film, cm ) νmax: 3323, 3177, 2984, 1650,
δ: 8.81 (brs, 1H, NH), 7.27 (q, 1H, J = 1.2 Hz), 4.44 (dd, 1H, J =
1
599, 1476, 1416, 1327, 1300, 1242, 1047, 970; (signals of
1
3
0.3 Hz, J = 6.6 Hz, H-C5), 4.29–4.15 (m, 4H, 2 × CH OP),
.92–3.83 (M part of ABM system, 1H, HCHN) and 3.63–3.54
2
trans-34e were extracted from the spectra of a 65:35 mixture of
cis-33e and trans-34e); H NMR (600 MHz, CDCl ) δ: 8.37 (s,
1
2
H -C4), 2.70 (s, 3H, CH -N), 2.35 (dddd, 1H, J = 11.9 Hz, J =
9.1 Hz, J = 9.1 Hz, J = 2.9 Hz, H
CH CH OP), 1.35 (t, 3H, J = 6.7 Hz, CH CH OP); C NMR
1
3
(AB part of ABM system, 2H, HCHN and H-C3), 2.87–2.71 (m,
H), 7.94 (s, 1H), 5.78 (brs, 2H, NH ), 4.31–4.10 (m, 7H, CH N,
2
2
1
1
H, H -C4), 2.57 (s, 3H, CH N), 2.19 (ddd, 1H, J = 19.6 Hz, J =
a
3
× CH OP, H-C5), 3.56–3.50 (m, 1H, H-C3), 2.84–2.72 (m, 1H,
2
2.8 Hz, J = 6.6 Hz, H -C4), 1.91 (d, 3H, J = 1.2 Hz, CH ), 1.38
b
3
a
3
(
t, 3H, J = 7.1 Hz, CH CH OP) 1.37 (t, 3H, J = 7.1 Hz,
3 2
13
-C4), 1.37 (t, 3H, J = 6.7 Hz,
b
13
CH CH OP); C NMR (150 MHz, CDCl ) δ: 164.3 (C=O),
3
2
3
3
2
3
2
1
51.0 (C=O), 143.0 (C=C), 109.4 (C=C), 70.2 (d, J = 171.9 Hz,
(150 MHz, CDCl ) δ: 155.7 (CNH ), 153.0, 150.0, 141.4, 119.3,
3
2
C5), 65.4 (d, J = 3.7 Hz, C3), 63.3 (d, J = 6.4 Hz, CH OP), 62.5
2
7
6
(CH
5
2.7 (d, J = 161.9 Hz, C5), 66.5 (d, J = 5.7 Hz, C3), 63.2 (d, J =
.6 Hz, CH OP), 62.6 (d, J = 6.8 Hz, CH OP), 45.8 (CH N), 43.9
(
d, J = 6.7 Hz, CH OP), 51.0 (CH N), 43.6 (CH N), 32.4 (C4),
2 2 3
2
2
2
1
6.6 (d, J = 5.5 Hz, CH CH OP), 16.5 (d, J = 5.4 Hz,
3 2
31
N), 33.3 (C4), 16.5 (d, J = 4.9 Hz, CH
CH
OP), 16.4 (d, J =
3
3
2
CH CH OP), 12.1 (CH ); P NMR (121 MHz, CDCl ) δ: 22.73.
31
3
2
3
3
.3 Hz, CH CH OP); P NMR (242 MHz, CDCl ) δ: 21.25.
3
2
3
Anal. Calcd. for C H N O P: C, 46.54; H, 6.70; N, 11.63.
14
24
3
6
Anal. Calcd. for C H N O P: C, 45.40; H, 6.26; N, 22.69.
Found: C, 45.31; H, 6.05; N, 22.58 (obtained on a 65:35 mixture
14
23
6
4
Found: C, 46.32; H, 6.73; N, 11.58.
of cis-33e and trans-34e).
4
.2.29. Diethyl trans-(2-methyl-3-((5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-
yl)methyl)isoxazolidin-5-yl)phosphonate (34d)
4
.2.32. Diethyl cis-(4-((5-bromo-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-5-yl)phosphonate (35c)
Yield: 1% (0.005 g from 1.11 mmol of the nitrone 20d);
-
1
colorless oil; IR (film, cm ) ν : 3463, 3178, 3058, 2985, 2930,
2
8
max
1
A 22:2:49:27 mixture of isoxazolidines 33c, 34c, 35c and 36c
830, 1687, 1468, 1234, 1024; H NMR (300 MHz, CDCl ) δ:
.39 (brs, 1H, NH), 7.10 (q, 1H, J = 1.0 Hz), 4.27–4.12 (m, 5H, 2
3
(0.030 g) was subjected to the separation on a X Bridge Prep,
C18, 5 ꢀm, OBD, 19×100 mm column using water/methanol
×
CH OP, H-C5), 3.95 (dd, 1H, J = 13.7 Hz, J = 3.9 Hz, HCHN),
2
(
(
(
70:30, v/v) to provide cis-35c (0.002 g) as a colorless oil
retention time = 6.50 min). H NMR (600 MHz, CDCl ) δ: 7.71
s, 1H), 4.28–4.10 (m, 4H, 2 × CH OP), 4.16 (ddd, 2H, J = 9.2
3
.53–3.41 (m, 1H, H-C3), 3.30 (dd, 1H, J = 13.7 Hz, J = 9.1 Hz,
HCHN), 2.79 (dddd, 1H, J = 20.7 Hz, J = 12.9 Hz, J = 9.6 Hz, J
7.3 Hz, H -C4), 2.72 (s, 3H, CH N), 2.31 (dddd, 1H, J = 12.9
1
3
2
=
a
3
Hz, J = 9.2 Hz, J = 9.2 Hz, H C5), 4.08 (dd, 1H, J = 13.3 Hz, J =
2
Hz, J = 8.2 Hz, J = 8.2 Hz, J = 2.1 Hz, H -C4), 1.95 (d, 3H, J =
b
2
.5 Hz, HCHN), 3.49 (dddd, J = 16.1 Hz, J = 9.0 Hz, J = 5.5 Hz,
J = 2.5 Hz, H-C3), 3.44 (dd, J = 13.4 Hz, J = 9.0 Hz, HCHN),
.67 (s, 3H, CH N), 2.57 (dddd, J = 15.5 Hz, J = 9.2 Hz, J = 9.2
1
=
.0 Hz, CH ), 1.37 (t, 3H, J = 7.0 Hz, CH CH OP), 1.35 (t, 3H, J
3 3 2
13
7.0 Hz, CH CH OP); C NMR (150 MHz, CDCl ) δ: 163.9
3 2 3
2
3
(
C=O), 150.8 (C=O), 141.9 (C=C), 110.1 (C=C), 73.2 (d, J =
69.1 Hz, C5), 66.0 (d, J = 5.8 Hz, C3), 63.3 (d, J = 6.8 Hz,
CH OP), 62.6 (d, J = 6.8 Hz, CH OP), 49.7 (CH N), 46.0
Hz, J = 5.5 Hz, H-C4), 1.36 (d, J = 7.0 Hz, 3H, CH CH OP),
1
CDCl ) δ: 2 signals of C=O not detected due to very low
concentration, 146.0 (C=C), 96.2 (C=C), 67.1 (C5), 65.1 (C3),
3
2
1
13
.35 (d, J = 7.0 Hz, 3H, CH CH OP); C NMR (150 MHz,
3 2
2
2
2
3
(
CH N), 32.9 (C4), 16.5 (d, J = 5.4 Hz, CH CH OP), 16.5 (d, J =
3
3
2

1
6
Tetrahedron
6
2.8 (d, J = 6.7 Hz, CH OP), 62.7 (d, J = 6.7 Hz, CH OP),
containing different concentrations of the test compounds was
added. After 2-4 days of further incubation at 37 °C, the cell
number was determined with a Coulter counter. The cytostatic
2
ACCEPTED MANUSCRIPT
2
5
1.9 (d, J = 8.1 Hz, CH N), 44.3 (d, J = 149.5 Hz, C4), 44.0
2
(
CH N), 16.5 (d, J = 5.6 Hz, CH CH OP), 16.4 (d, J = 5.6 Hz,
3 3 2
31
CH CH OP); P NMR (121 MHz, CDCl ) δ: 26.45.
concentration was calculated as the CC , or the compound
3
2
3
50
concentration required to inhibit cell proliferation by 50%
relative to the number of cells in the untreated controls.
Alternatively, cytotoxicity of the test compounds in confluent
4
.3. Antiviral Activity Assays
The compounds were evaluated against different herpes
(HEL, Vero, HeLa and CRFK) cell cultures (used for the
antiviral assays) was expressed as the minimum cytotoxic
concentration (MCC) or the compound concentration that caused
a microscopically detectable alteration of cell morphology.
viruses, including herpes simplex virus type 1 (HSV-1) strain
KOS, thymidine kinase-deficient (TK ) HSV-1 KOS strain
resistant to ACV (ACV ), herpes simplex virus type 2 (HSV-2)
-
r
strain G, varicella-zoster virus (VZV) strains Oka and YS, TK-
VZV strains 07-1 and YS-R, human cytomegalovirus (HCMV)
strains AD-169 and Davis as well as feline herpes virus (FHV),
the poxvirus vaccinia virus (Lederle strain), para-influenza-3
virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro
virus, respiratory syncytial virus (RSV), feline coronovirus
Acknowledgments
The authors wish to express their gratitude to Mrs. Leentje
Persoons, Mrs. Frieda De Meyer, Mrs. Leen Ingels, Mrs. Evelyne
Van Kerckhove, Mrs. Sandra Claes and Mrs. Lizette van
Berckelaer for excellent technical assistance. The synthetic part
of this work was supported by the Medical University of Lodz
internal funds (503/3-014-1/503-31-001 and 502-03/3-014-
(
(
FIPV) and influenza A virus subtypes H1N1 (A/PR/8), H3N2
A/HK/7/87) and influenza B virus (B/HK/5/72) and human
immunodeficiency virus (HIV-1/IIIB and HIV-2/ROD). The
antiviral assays, other than HIV, were based on inhibition of
virus-induced cytopathicity or plaque formation (for VZV) in
human embryonic lung (HEL) fibroblasts, African green monkey
kidney cells (Vero), human epithelial cervix carcinoma cells
0
1/502-34-068). The biological part of this work was supported
by the KU Leuven (GOA 15/19 TBA).
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