Design, synthesis, antiviral, and cytostatic evaluation of novel isoxazolidine analogs of homonucleotides

Article abstract of DOI:10.1002/ardp.201300382

Moderate diastereoselectivities (d.e. 2-62%) of isoxazolidine homonucleotides were observed for cycloadditions between N-methyl-C- (diethoxyphosphoryl)nitrone and N-allyl nucleobases, with trans-isoxazolidines predominating. The stereochemistry of the substituted isoxazolidines was established based on 2D NOE experiments performed for uracil-containing cycloadducts. The cis- and trans-isoxazolidine phosphonates obtained herein were evaluated in vitro for activity against a broad range of DNA and RNA viruses. None of the compounds were endowed with antiviral activity at subtoxic concentrations, but some of them were found to inhibit the proliferation of L1210 cells with IC₅₀ values in the range of 33-100 μM.

Full text of DOI:10.1002/ardp.201300382

Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
341
Full Paper
Design, Synthesis, Antiviral, and Cytostatic Evaluation of
Novel Isoxazolidine Analogs of Homonucleotides
Magdalena Łysakowska1, Jan Balzarini2, and Dorota G. Piotrowska1
1
ꢀ
ꢀ
Faculty of Pharmacy, Bioorganic Chemistry Laboratory, Medical University of Łódz, Łódz, Poland
Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
2
Moderate diastereoselectivities (d.e. 2–62%) of isoxazolidine homonucleotides were observed for
cycloadditions between N-methyl-C-(diethoxyphosphoryl)nitrone and N-allyl nucleobases, with trans-
isoxazolidines predominating. The stereochemistry of the substituted isoxazolidines was established
based on 2D NOE experiments performed for uracil-containing cycloadducts. The cis- and trans-
isoxazolidine phosphonates obtained herein were evaluated in vitro for activity against a broad range of
DNA and RNA viruses. None of the compounds were endowed with antiviral activity at subtoxic
concentrations, but some of them were found to inhibit the proliferation of L1210 cells with
IC50 values in the range of 33–100 mM.
Keywords: Antiproliferative agents / Cycloaddition / Isoxazolidines
Received: October 9, 2013; Revised: November 20, 2013; Accepted: November 22, 2013
DOI 10.1002/ardp.201300382
Introduction
resistance to enzymatic degradation was also the major
reason to design other nucleoside analogs 5–8 with an
additional methylene group incorporated between the base
and a pseudosugar in so-called homo-N-nucleosides [11–14].
Recently, we succeeded in the synthesis of isoxazolidine
homonucleosides and homonucleotide analogs 9 and 10
(Fig. 2) [15]. In continuation of our efforts at synthesizing
hydrolytically stable isoxazolidine nucleotides, a series of
novel analogs 11 was designed in which the oxygen atom
and a methylamino fragment in the isoxazolidine ring
are relocated as compared with this ring in 9 and 10.
Newly designed structures 11 together with the respective
C-nucleotide 12 [16] and already described mimetics of
nucleotide 13 containing the amide linker [17] form an
introductory series of compounds prepared to recognize the
structural features responsible for antiviral and/or cytostatic
activities.
Despite significant achievements in the synthesis of various
nucleosides and nucleotides as potential antitumoral and
antiviral therapeutics, a search for new compounds continues
and focuses on modifications of sugar and/or nucleobase
moieties [1–5]. Beginning with the Tronchet’s pioneering
work [6], an idea of incorporation of an isoxazolidine
framework instead of the furanose ring has been extensively
exploited and resulted in synthesizing several isoxazolidine
nucleosides or nucleotide analogs with promising biological
activities (Fig. 1). It is assumed that the presence of an
isoxazolidine ring as a pseudosugar assures conformational
similarities of those analogs to natural nucleosides. Moreover,
before incorporation into nucleic acids, for the isoxazolidine
nucleosides 2 containing the hydroxymethyl group [7], in vivo
activation by intracellular phosphorylases is necessary. On the
other hand, phosphonylated isoxazolidines 3 and their
truncated analogs 4 have been designed to avoid the first,
and less effective, step of cellular phosphorylation and at
the same time to achieve greater enzymatic stability due
To synthesize isoxazolidines cis-11/trans-11 the 1,3-dipolar
cycloaddition of the nitrone 14 [18] and N-allylated nucleo-
bases or their mimetics 15 has been employed (Scheme 1).
to the presence the non-hydrolysable P–C bond [8–10]. The Results and discussion
Chemistry
Correspondence: Dr. Dorota G. Piotrowska, Faculty of Pharmacy,
ꢀ
Bioorganic Chemistry Laboratory, Medical University of Łódz, 90-151
All N-allylated nucleobases 15, except for 15r, have already
been described in the literature [11, 19–33]. For the purpose of
this study, they were prepared by allylation of commercially
ꢀ
Łódz, Muszyńskiego 1, Poland.
E-mail: dorota.piotrowska@umed.lodz.pl
Fax: þ48 42 678 83 98
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M. Łysakowska et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Figure 1. Examples of isoxazolidine nucleoside and nucleotide analogs.
Figure 2. Isoxazolidine homonucleosides and homonucleotide analogs 9, 10, 11, 12, and 13.
Scheme 1. Retrosynthesis of homonucleoside analogs 11.
available nucleobases employing four different protocols
(Scheme 2; for the list of applied nucleobases (B) see Table 1).
Compounds 15a and 15b [19], 15c [20] and 15d [21, 22],
15f [11, 23], 15g [24], 15h,i [25], 15n–q [26–28], and 15r were
prepared following the literature procedures using allyl
bromide and potassium carbonate in DMF (Scheme 2, method
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, for 15a–d,
15f–i, 15n–r; (b) N(C4H9)4þIꢀ, NaOH, toluene, for 15j–l; (c) Cs2CO3,
DMF, for 15m; and (d) K2CO3, CH3OH, for 15s.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Isoxazolidine Analogs of Homonucleotides
343
Table 1. Cycloaddition of the nitrone 14 and allylated nucleobase 15.
Entry
Nucleobase
(B)
Reaction
time (h)
cis-11/trans-11 ratio
Yield (%)
a
48
24
40:60
cis-11a (17)a) þ trans-11a (21)a) þ cis-11a and trans-11a (38)b)
b
36:64
trans-11b (11)a) þ cis-11b and trans-11b (79)b)
c
24
48
49:51
45:55
cis-11c and trans-11c (98)b)
d
cis-11d (11)a) þ trans-11d (12)a) þ cis-11d and trans-11d (77)b)
e
f
48
72
48
36:65
37:63
19:81
cis-11e and trans-11e (79)b)
cis-11f (11)a) þ trans-11f (29)a) þ cis-11f and trans-11f (47)b)
cis-11g and trans-11g (98)b)
g
h
i
72
72
44:56
36:64
trans-11h (2.4)a) þ cis-11h and trans-11h (96)b)
trans-11i (5.3)a) þ cis-11i and trans-11i (93)b)
j
48
72
31:69
34:66
cis-11j and trans-11j (78)b)
cis-11k and trans-11k (98)b)
k
l
48
48
30:70
32:68
cis-11l and trans-11l (97)b)
m
cis-11m and trans-11m (83)b)
n
o
p
72
72
72
48:52
20:80
20:80
cis-11n and trans-11n (95)b)
trans-11o (11)a) þ cis-11o and trans-11o (87)b)
trans-11p (19)a) þ cis-11p and trans-11p (34)b)
(Continued)
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Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Table 1. (Continued)
Entry
Nucleobase
(B)
Reaction
time (h)
cis-11/trans-11 ratio
Yield (%)
q
48
46:54
cis-11q (38)a) þ trans-11q (14)a) þ cis-11q and trans-11q (30)b)
72
72
r
20:80
30:70
cis-11r (11)a) þ trans-11r (43)a) þ cis-11r and trans-11r (25)b)
cis-11s (2.4)a) þ trans-11s (39)a) þ cis-11s and trans-11s (56)b)
s
a)
Yield of pure isomer.
Yield of pure mixture of cis- and trans-isomers.
b)
a) [19, 28]. N-Allylpyridinones 15h and 15i were successfully
prepared in the same manner to avoid formation of O-allylated
by-products reported as a major problem in the application of
a PTC approach [25]. On the other hand, it appeared that the
synthesis of imidazoles 15j–l in the presence of tetrabutyl-
ammonium iodide and sodium hydroxide in toluene
(Scheme 2, method b) [34] was even more effective than
employing sodium hydride in THF [29, 30]. To synthesize the
known N-allylated 3-acetylindol 15m [31] and N-allylated
morpholine 15s [32], standard procedures for the alkylation of
indoles [35] and morpholines [36] were successfully applied
(Scheme 2; methods c and d, respectively). Finally, compound
15e [11] was obtained from 15f [33] in boiling methanol.
Reactions of the nitrone 14 with N-allylnucleobases 15a–s
were carried out at 60°C in toluene, toluene–ethanol, or
toluene–chloroform mixtures as solvents, depending on the
solubility of the respective N-allylnucleobase, and afforded
mixtures of diastereoisomeric (3-diethoxyphosphoryl)isoxa-
zolidines cis-11a–s and trans-11a–s (Scheme 3, Table 1). In all
cases, good-to-excellent overall yields of final products were
obtained but only low-to-moderate trans/cis diastereoselectiv-
ities (d.e. 2–62%) of cycloadditions were noticed. The crude
mixtures of the respective cycloadducts were subjected to
column chromatography. We succeeded in isolation of pure
major isomers trans-11a, trans-11d, trans-11f, trans-11h, trans-
11i, trans-11q, trans-11r, and trans-11s and minor isomers
cis-11a, cis-11b, cis-11d, cis-11f, cis-11o, cis-11p, cis-11q, cis-11r,
and cis-11s (Table 1).
To establish relative configurations at C3 and C5 in
isoxazolidines cis-11 and trans-11 2D NOE experiments were
performed for diastereoisomers cis-11a and trans-11a (Fig. 3).
For the isoxazolidine cis-11a, besides the expected NOESY
signals between HC5 and both protons at H2C4 and CH2–Ura, a
diagnostic signal between HC5 and HC3 was observed, which
unequivocally proves the cis arrangement of substituents at
C3 and C5. On the other hand, the NOESY spectrum of trans-
11a lacks the correlation signals between HC5 and HC3 due to
the location of these protons on the opposite sides of the
isoxazolidine ring too far to undergo an efficient relaxation.
The relative configurations of the other isomeric isoxazoli-
dines cis-11b–s and trans-11b–s derived from N-allylated
nucleobases were established taking advantage of already
established configurations for cis-11a and trans-11a, since
similar patterns for the respective isoxazolidine protons were
observed in a series of isoxazolidines cis-11b–s and trans-11b–s
as compared to cis-11a and trans-11a, respectively.
Antiviral and cytostatic evaluation
The pure cis-isoxazolidines cis-11a, cis-11d, cis-11f, cis-11q,
cis-11r, cis-11s, and trans-isoxazolidines trans-11a, trans-11b,
trans-11d, trans-11f, trans-11h, trans-11i, trans-11o, trans-11p,
trans-11q, trans-11r, and trans-11s, as well as diastereoisomeric
mixtures of cis- and trans-isoxazolidines cis-11b/trans-11b (7:3),
cis-11c/trans-11c (4:6), cis-11e/trans-11e (8:2), cis-11e/trans-11e
(1:9), cis-11g/trans-11g (19:81), cis-11h/trans-11h (6:4), cis-11i/
trans-11i (9:1), cis-11j/trans-11j (25:75), cis-11k/trans-11k (3:7),
cis-11l/trans-11l (3:7), cis-11m/trans-11m (1:1), cis-11n/trans-11n
Scheme 3. Reagents and conditions: (a) N-allylated nucleobase
15a–s, toluene or toluene–ethanol mixture or toluene–chloroform
mixture 60°C, 24–72 h, see Table 1.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Isoxazolidine Analogs of Homonucleotides
345
Figure 3. Observed NOEs for cis-11a and trans-11a.
(1:1), and cis-11o/trans-11o (93:7), were evaluated for inhibitory Conclusions
activity against a wide variety of DNA and RNA viruses, using
the following cell-based assays: (a) human embryonic lung
(HEL) cells: herpes simplex virus-1 (KOS), herpes simplex virus-
2 (G), vaccinia virus, vesicular stomatitis virus, and herpes
simplex virus-1 (TKꢀ KOS ACVr); (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, and Punta Toro
virus; (d) MDCK cell cultures: influenza A virus (H1N1 and
H3N2 subtypes) and influenza B virus; (e) CrFK cell cultures:
feline herpes virus (FHV) and feline corona virus (FIPV); and (f)
CEM cell cultures: human immunodeficiency virus type 1
(HIV-1) and HIV-2. Ganciclovir, cidofovir, acyclovir, brivudin,
(S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], oseltamivir car-
boxylate, amantadine, rimantadine, ribavirin, dextran sulfate
(molecular weight 5000, DS-5000), Hippeastrum hybrid agglu-
tinin (HHA), and Urtica dioica agglutinin (UDA) were used as
the reference compounds. The antiviral activity was expressed
as the EC50: the compound concentration required to reduce
virus plaque formation (VZV) by 50% or to reduce virus-
induced cytopathogenicity by 50% (other viruses). Unfortu-
nately, no inhibitory activity against any virus was detected
for the evaluated compounds at 250 mM.
Cycloaddition of N-methyl-C-(diethoxyphosphoryl)nitrone and
N-allylated nucleobases led to the formation of isoxazolidines
in good yields and with moderate trans to cis diastereose-
lectivities (d.e. 2–62%). The relative configurations in the 5-
substituted-3-(diethoxyphosphoryl)isoxazolidines cis-11 and
trans-11 were established based on analysis of the 2D NOE
experiments conducted for uracil-containing isoxazolidines
cis-11a and trans-11a.
All synthesized phosphonates cis-11 and trans-11 were
screened for activity against variety of DNA and RNA viruses
but were found non-toxic at 250 mM. Compounds cis-11l þ
trans-11l (3:7) and cis-11m þ trans-11m (1:1), as well as cis-11r
and trans-11r were found to inhibit proliferation of L1210
with IC50’s in the 33–100 mM range. Moreover, trans-11r
also affected human CEM and HeLa cell proliferation at
an IC50 of 159 and 96 mM, whereas cis-11k þ trans-11k (3:7);
cis-11l þ trans-11l (3:7); and cis-11m þ trans-11m (1:1) as well
as trans-11o and cis-11r inhibited L1210 cell prolifera-
tion selectively. These results encourage us to continue
search for cytostatic compounds in the class of isoxazolidine-
3-phosphonates with special focus on substituted benzimi-
dazoles, indoles, and benzuracil as mimics of nucleobases.
The cytotoxicity of the tested compounds toward the
uninfected host cells was defined as the minimum compound
concentration (MCC) that caused a microscopically detectable
alteration of normal cell morphology. The 50% cytostatic
inhibitory concentration (IC50), causing a 50% inhibition of
cell proliferation was determined against murine leukemia
L1210, human lymphocyte CEM, and human cervix carcino-
ma HeLa cells. None of the tested compounds affected cell
morphology of HEL, HeLa, Vero, MDCK, and CrFK cells at
concentrations up to 100 mM. Instead, a few compounds
proved cytostatic against murine leukemia L1210 cells at an
IC50 lower than 100 mM (i.e., cis-11m þ trans-11m; cis-11l þ
trans-11l; and cis-11r and trans-11r) (Table 2). trans-11r also
affected human CEM and HeLa cell proliferation at an IC50 of
159 and 96 mM, respectively. It would be of interest to further
explore the cytostatic activity of novel structurally closely
related compounds.
Table 2. Inhibitory effect of the tested compounds against the
proliferation of murine leukemia (L1210), human T-lymphocyte
(CEM), and human cervix carcinoma cells (HeLa).
a)
Compound
IC50 (mM)
L1210
CEM
HeLa
cis-11k þ trans-11k (3:7)
cis-11l þ trans-11l (3:7)
cis-11m þ trans-11m (1:1)
trans-11o
cis-11s
trans-11s
152 ꢁ 18
88 ꢁ 9.9
33 ꢁ 3.5
192 ꢁ 11
74 ꢁ 33
70 ꢁ 22
>200
>200
>200
>200
>200
ꢂ159
>200
>200
>200
>200
>200
96 ꢁ 11
a)
Fifty percent inhibitory concentration or compound concen-
tration required to inhibit tumor cell proliferation by 50%.
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ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
C NMR (151 MHz, CDCl ): d ¼ 171.23 (C O), 163.02 (C O), 155.7,
ꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
346
M. Łysakowska et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Experimental
J ¼ 10.5 Hz, J ¼ 5.1 Hz, 1H, CH –CH CH ), 5.33 (ddt, J ¼ 10.5 Hz,
2
2
ꢀꢀ
J ¼ 3.6 Hz, J ¼ 1.2 Hz, 1H, CH –CH CHH), 5.31 (ddt, J ¼ 17.1 Hz,
ꢀꢀ
2
ꢀꢀ
J ¼ 3.6 Hz, J ¼ 1.2 Hz, 1H, CH –CH CHH), 4.80 (dt, J ¼ 5.1 Hz,
ꢀꢀ
2
General
J ¼ 3.6 Hz, 2H, CH –CH CH ); 13C NMR (151 MHz, CDCl3):
1H NMR were taken in CDCl3 on the following spectrometers:
Varian Mercury-300 and Bruker Avance III (600 MHz) with TMS as
an internal standard; chemical shifts d in ppm with respect to
TMS; coupling constants J in Hz. 13C NMR spectra were recorded
for CDCl3 solutions on a Varian Mercury-300 and Bruker Avance
2
2
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
d ¼ 168.61 (C O), 161.09 (C O), 149.23 (C O), 140.52, 134.98,
131.82, 130.89, 130.47, 129.16, 130.89, 129.16, 129.10, 123.50,
118.30 (CH –CH CH ), 115.72, 114.75 (CH –CH CH ), 45.40
ꢀꢀ
2
2
2
2
(CH –CH CH ). Anal. calcd. for C18H14N2O3: C, 70.58; H, 4.61;
2
2
N, 9.15. Found: C, 70.31; H, 4.40; N, 9.21.
III (600 MHz) spectrometer at 75.5 and 151 MHz, respectively. 31
P
NMR spectra were taken in CDCl3 on Varian Mercury-300 and
Bruker Avance III at 121.5 and 243 MHz. IR spectra were measured
on an Infinity MI-60 FT-IR spectrometer. Melting points were
General procedure for the cycloaddition of nitrone 14 to
alkenes 15
Solutions of nitron 14 (1.00 mmol) and the respective alkenes
15a–s (1.00–1.05 mmol) in toluene or toluene–ethanol or
toluene–chloroform mixtures were stirred at 60°C until the
starting nitrone disappeared. Solvents were evaporated in vacuo
and crude products were purified on silica gel columns.
determined on
a Boetius apparatus and are uncorrected.
Elemental analyses were performed by the Microanalytical
Laboratory of this Faculty on Perkin-Elmer PE 2400 CHNS
analyzer. The following adsorbents were used: column chroma-
tography, Merck silica gel 60 (70–230 mesh); analytical TLC,
Merck TLC plastic sheets silica gel 60 F254
.
Diethyl 5-((3,4-dihydro-2,4-dioxopyrimidin-1(2H)-yl)
methyl)-2-methylisoxazolidin-3-yl-3-phosphonates cis-11a
Starting materials
All solvents were dried according to the literature methods.
Nitrone 14 [18] was previously reported.
and trans-11a
From nitrone 14 (0.578 g, 2.96 mmol) and N1-allyluracil 15a
(0.500 g, 3.29 mmol) in toluene–ethanol, pure cis-11a (0.174 g,
17%), trans-11a (0.216 g, 21%), and fractions containing various
mixtures of cis-11a and trans-11a (0.391 g, 38%) were obtained by
column chromatography (chloroform–methanol; from 100:1 to
50:1 v/v).
N4-Acetyl-N1-allylcytosine 15f
A suspension of N4-acetylcytosine (0.500 g, 3.26 mmol), potassium
carbonate (0.520 g, 3.75 mmol), and allyl bromide (0.24 mL,
2.80 mmol) was stirred in anhydrous DMF (7 mL) at room
temperature for 24 h. The resulting suspension was evaporated
in vacuo. The solid was diluted with chloroform and filtered off.
The crude product was purified by column chromatography with
chloroform–methanol mixtures (100:1–50:1) and crystallized
from water to give 15f (0.464 g, 86%) as a white solid; m.p.
Compound cis-11a: White solid (crystallized from mixture of
chloroform–hexane) m.p. 92–93°C. IR (KBr, cmꢀ1) nmax: 3476 nN–H
,
1708 nCꢀꢀO, 1675 nCꢀꢀ , 1252 n O, 1052 nP–O.
1H NMR (300 MHz,
ꢀꢀ
P
ꢀꢀ
O
ꢀꢀ
ꢀꢀ
CDCl3): d ¼ 8.19 (bs, 1H, NH), 7.31 (d, J ¼ 8.1 Hz, 1H), 5.66 (dd,
J ¼ 8.1 Hz, J ¼ 2.4 Hz, 1H), 4.40 (dddd, J ¼ 9.3 Hz, J ¼ 7.8 Hz, J ¼ 3.9
Hz, J ¼ 3.3 Hz, 1H, HC5), 4.25–4.14 (m, 4H, 2 ꢃ CH2OP), 4.08 (dd,
J ¼ 14.1 Hz, J ¼ 3.3 Hz, 1H, HCH), 3.69 (dd, J ¼ 14.1 Hz, J ¼ 9.3 Hz,
1H, HCH), 2.93 (ddd, J ¼ 10.2 Hz, J ¼ 7.5 Hz, J ¼ 2.7 Hz, 1H, HC3),
2.83 (d, J ¼ 0.6 Hz, 3H, CH3N), 2.73 (dddd, J ¼ 12.6 Hz, J ¼ 11.4 Hz,
J ¼ 9.6 Hz, J ¼ 7.8 Hz, 1H, HC4), 2.23 (dddd, J ¼ 18.6 Hz, J ¼ 11.4 Hz,
J ¼ 7.5 Hz, J ¼ 3.9 Hz, 1H, H0C4), 1.37 (t, J ¼ 6.9 Hz, 3H, CH3CH2OP),
1.36 (t, J ¼ 7.2 Hz, 3H, CH3CH2OP); 13C NMR signals of cis-11a were
185–186°C. IR (KBr, cmꢀ1) nmax: 2977 nC–H, 1706 nCꢀꢀO, 1663 nCꢀꢀ
,
O
ꢀꢀ
ꢀꢀ
1569 nCꢀꢀC, 1493 nCꢀꢀ , 999 g
.
1H NMR (300 MHz, CDCl3):
ꢀꢀ
C–H
ꢀꢀ
C
ꢀꢀ
ꢀꢀ
d ¼ 9.72 (s, 1H, NH), 7.58 (d, J ¼ 7.5 Hz, 1H), 7.42 (d, J ¼ 7.5 Hz, 1H),
ꢀꢀ
ꢀꢀ
5.95 (ddt, J ¼ 17.1 Hz, J ¼ 10.2 Hz, J ¼ 5.4 Hz, 1H, CH –CH CH ),
2
2
ꢀꢀ
5.32 (ddt, J ¼ 10.2 Hz, J ¼ 3.0 Hz, J ¼ 1.7 Hz, 1H, CH –CH CHH),
ꢀꢀ
2
ꢀꢀ
5.26 (ddt, J ¼ 17.1 Hz, J ¼ 3.0 Hz, J ¼ 1.7 Hz, 1H, CH –CH CHH),
ꢀꢀ
2
ꢀꢀ
4.51 (dt, J ¼ 5.4 Hz, J ¼ 3.0 Hz, 2H, CH –CH CH ), 2.89 (s, 3H, CH );
ꢀꢀ
2
2
3
13
3
ꢀꢀ
148.07, 131.58, 119.56 (CH –CH CH ), 97.10 (CH –CH CH ),
ꢀꢀ ꢀꢀ
ꢀꢀ
2
2
2
2
extracted from the spectrum of a 9:1 mixture of cis-11a and trans-
13
ꢀ
52.09 (CH –CH CH ), 24.85 (CH ). Anal. calcd. for C H11N3O2
ꢀꢀ
11a, C NMR (75.5 MHz, CDCl ): d ¼ 163.98 (C O), 151.12 (C O),
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀ
2
2
3
9
3
ꢃ 3.5H2O: C, 42.18; H, 7.08; N, 16.40. Found: C, 42.18; H, 7.35; N,
146.29, 101.65, 74.04 (d, J ¼ 6.9 Hz, C5), 63.71 (d, J ¼ 168.8 Hz, C3),
63.03 (d, J ¼ 4.9 Hz, CH2OP), 62.95 (d, J ¼ 6.9 Hz, CH2OP), 50.41
(CH2N), 46.01 (CH3N), 35.35 (C4), 16.83 (d, J ¼ 3.7 Hz, 2 ꢃ
CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 23.65. Anal. calcd.
for C13H22N3O6P ꢃ 2H2O: C, 40.73; H, 6.84; N, 10.96. Found: C,
40.43; H, 6.82; N, 10.76.
16.39.
N1-Allyl-N3-benzoylbenzuracil 15r
To a suspension of N3-benzoylbenzuracil (0.500 g, 1.88 mmol) and
potassium carbonate (0.283 g, 2.05 mmol) in anhydrous DMF
(7 mL), allyl bromide (0.195 mL, 2.25.mmol) was added dropwise.
The mixture was stirred under argon at room temperature for
3 days. Solvent was removed in vacuo, the residue was suspended
in chloroform and the solid was filtered off. The crude product
was purified by column chromatography with chloroform–
methanol mixtures (100:1–50:1) and crystallized from a mixture
of chloroform–ethyl ether to give 15r (0.260 g; 45%) as a white
Compound trans-11a: Colorless oil. IR (film, cmꢀ1) nmax: 3476
nN–H, 1708 nCꢀꢀO, 1684 nCꢀꢀO, 1229 nPꢀO, 1050 nP–O.
1H NMR
(300 MHz, CDCl3): d ¼ 8.74 (bs, 1H, NH), 7.28 (d, J ¼ 7.8 Hz, 1H), 5.69
(dd, J ¼ 7.8 Hz, J ¼ 2.7 Hz, 1H), 4.31–4.22 (m, 1H, HC5), 4.24–4.19
(m, 4H, 2 ꢃ CH2OP), 4.17 (dd, J ¼ 14.7 Hz, J ¼ 3.6 Hz, 1H, HCH), 3.69
(dd, J ¼ 14.7 Hz, J ¼ 6.9 Hz, 1H, HCH), 2.93 (ddd, J ¼ 10.2 Hz,
J ¼ 7.2 Hz, J ¼ 2.4 Hz, 1H, HC3), 2.87 (d, J ¼ 1.2 Hz, 3H, CH3N),
2.63 (dddd, J ¼ 18.6 Hz, J ¼ 12.9 Hz, J ¼ 7.2 Hz, J¼ 7.2 Hz, 1H, H0C4),
2.19 (dddd, J ¼ 12.9 Hz, J ¼ 12.0 Hz, J ¼ 10.2 Hz, J ¼ 8.4 Hz, 1H,
HC4), 1.34 (t, J ¼ 7.2 Hz, 6H, 2 ꢃ CH3CH2OP); 13C NMR (75.5 MHz,
solid; m.p. 125–125.5°C. IR (KBr, cmꢀ1) nmax: 2996 nC–H, 1748 nCꢀꢀ
,
O
ꢀꢀ
1699 nCꢀꢀO, 1669 nCꢀꢀO, 1606 nCꢀꢀ , 990 g
.
1H NMR (300 MHz,
ꢀꢀ
C–H
ꢀꢀ
C
ꢀꢀ
ꢀꢀ
ꢀꢀ
CDCl3): d ¼ 8.25 (dd, J ¼ 8.1 Hz, J ¼ 1.2 Hz, 1H), 8.01–7.95 (m, 2H),
7.75 (dd, J ¼ 7.5 Hz, J ¼ 1.5 Hz, 1H), 7.70 (ddd, J ¼ 8.1 Hz, J ¼ 8.1 Hz,
J ¼ 1.5 Hz, 1H), 7.64–7.67 (m, 1H), 7.55–7.48 (m, 2H), 7.32
(ddd, J ¼ 8.1 Hz, J ¼ 7.5 Hz, J ¼ 1.2 Hz, 1H), 5.95 (ddt, J ¼ 17.1 Hz,
CDCl ): d ¼ 163.74 (C O), 151.14 (C O), 145.30, 102.19, 75.39 (d,
3
J ¼ 7.1 Hz, C5), 64.21 (d, J ¼ 168.1 Hz, C3), 63.53 (d, J ¼ 6.5 Hz,
CH2OP), 62.74 (d, J ¼ 6.9 Hz, CH2OP), 49.41 (CH2N), 46.39 (CH3N),
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Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Isoxazolidine Analogs of Homonucleotides
347
35.20 (C4), 16.83 (d, J ¼ 5.7 Hz, CH3CH2OP), 16.76 (d, J ¼ 5.1 Hz,
CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 22.44. Anal. calcd.
for C13H22N3O6P ꢃ 2H2O: C, 40.73; H, 6.84; N, 10.96. Found: C,
40.76; H, 6.94; N, 11.10.
Compound trans-11c: IR (film, cmꢀ1) nmax: 3264 nN–H, 1672 nCꢀꢀ
,
N
ꢀꢀ
1602 nCꢀꢀ , 1244 n O, 1051 nP–O. NMR signals of trans-11c were
ꢀꢀ
P
C
ꢀꢀ
ꢀꢀ
extracted from the spectra of a 3:7 mixture of cis-11c and trans-11c;
1H NMR (300 MHz, CDCl3): d ¼ 8.36 (s, 1H), 7.94 (s, 1H), 6.01 (s, 2H,
NH2), 4.51–4.30 (m, 3H, HC5 and CH2), 4.23–4.07 (m, 4H,
2 ꢃ CH2OP), 2.97 (ddd, J ¼ 9.6 Hz, J ¼ 8.4 Hz, J ¼ 3.0 Hz, 1H, HC3),
2.86 (d, J ¼ 0.6 Hz, 3H, CH3N), 2.68 (dddd, J ¼ 17.7 Hz, J ¼ 12.6 Hz,
J ¼ 8.4 Hz, J ¼ 7.8 Hz, 1H, H0C4), 2.18 (dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz,
J ¼ 9.6 Hz, J ¼ 7.8 Hz, 1H, HC4), 1.31 (t, J ¼ 6.6 Hz, 3H, CH3CH2OP),
1.29 (t, J ¼ 6.6 Hz, 3H, CH3CH2OP); 13C NMR (75.5 MHz, CDCl3):
d ¼ 155.78, 153.03, 150.13, 141.40, 119.08, 75.40 (d, J ¼ 7.5 Hz, C5),
64.23 (d, J ¼ 169.0 Hz, C3), 63.32 (d, J ¼ 6.9 Hz, CH2OP), 62.60 (d,
J ¼ 6.9 Hz, CH2OP), 46.30 (br s, CH2N), 44.82 (CH3N), 35.11 (br s, C4),
16.73 (d, J ¼ 6.3 Hz, CH3CH2OP), 16.65 (d, J ¼ 5.6 Hz, CH3CH2OP);
31P NMR (121.5 MHz, CDCl3): d ¼ 22.37. Anal. calcd. for
C14H23N6O4P: C, 45.40; H, 6.26; N, 22.69. Found: C, 45.11; H,
6.40; N, 22.53 (obtained on a 3:7 mixture of cis-11c and trans-11c).
Diethyl 5-((3,4-dihydro-2,4-dioxopyrimidin-1(2H)-yl)
methyl)-2-methylisoxazolidin-3-yl-3-phosphonates
cis-11b, trans-11b
From nitrone 14 (0.128 g, 0.657 mmol) and N1-allylthymine 15b
(0.115 g, 0.692 mmol) in toluene–ethanol solution, pure trans-11b
(0.026 g 11%) was obtained followed by fractions containing
mixture of cis-11b and trans-11b (0.187 g, 79%) by column
chromatography (chloroform–methanol; 100:1 to 50:1 v/v).
Compound cis-11b: IR (film, cmꢀ1) nmax: 3473 nN–H, 2984 nC–H
,
1681 nCꢀꢀ , 1231 n O, 1051 nP–O. NMR signals of cis-11b were
ꢀꢀ
P
ꢀꢀ
O
ꢀꢀ
extracted from the spectra of a 7:3 mixture of cis-11b and trans-
1
11b, H NMR (300 MHz, CDCl3): d ¼ 8.43 (bs, 1H, NH2), 7.14 (brq,
ꢀꢀ
J ¼ 1.2 Hz, 1H, CH ), 4.41 (dddd, J ¼ 9.6 Hz, J ¼ 7.8 Hz, J ¼ 7.8 Hz,
ꢀꢀ
Diethyl 5-((6-amino-7H-purin-7-yl)methyl)-2-
methylisoxazolidin-3-yl-3-phosphonates cis-11d and
J ¼ 3.3 Hz, 1H, HC5), 4.26–4.14 (m, 4H, 2 ꢃ CH2OP), 4.07 (dd,
J ¼ 14.4 Hz, J ¼ 3.3 Hz, 1H, HCH), 3.64 (dd, J ¼ 14.4 Hz, J ¼ 9.6 Hz,
1H, HCH), 2.94 (ddd, J ¼ 9.6 Hz, J ¼ 7.8 Hz, J ¼ 3.3 Hz, 1H, HC3), 2.84
(d, J ¼ 0.9 Hz, 3H, CH3N), 2.74 (dddd, J ¼ 18.6 Hz, J ¼ 12.6 Hz,
J ¼ 7.8 Hz, J ¼ 7.8 Hz, 1H, H0C4), 2.23 (dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz,
J ¼ 9.6 Hz, J ¼ 7.8 Hz, 1H, HC4), 1.37 (t, J ¼ 6.9 Hz, 3H, CH3CH2OP),
1.36 (t, J ¼ 6.9 Hz, 3H, CH3CH2OP); 13C NMR (75.5 MHz, CDCl3):
trans-11d
From nitrone 14 (0.134 g, 0.685 mmol) and N7-allyladenine 15d
(0.120 g, 0.685 mmol) in toluene–ethanol, pure cis-11d (0.026 g,
11%), trans-11d (0.029 g, 12%), and fractions containing various
mixtures of cis-11d and trans-11d (0.185 g, 77%) were obtained by
column chromatography (chloroform–methanol; from 100:1 to
50:1 v/v).
ꢀꢀ
ꢀꢀ
ꢀꢀ
d ¼ 164.74 (C O), 151.27 (C O), 142.23, 109.93, 74.14 (d,
ꢀꢀ
J ¼ 6.9 Hz, C5), 62.99 (d, J ¼ 6.4 Hz, CH2OP), 62.90 (d, J ¼ 6.6 Hz,
CH2OP), 63.59 (d, J ¼ 170.3 Hz, C3), 50.25 (CH2N), 45.99 (CH3N),
35.27 (d, J ¼ 1.6 Hz, C4), 16.74 (d, J ¼ 5.7 Hz, CH3CH2OP), 16.70 (d,
J ¼ 5.7 Hz, CH3CH2OP), 12.52 (CH3); 31P NMR (121.5 MHz, CDCl3):
d ¼ 23.66. Anal. calcd. for C14H24N3O6P ꢃ 0.5H2O: C, 45.40; H, 6.80;
N, 11.35. Found: C, 45.49; H, 7.13; N, 11.07 (obtained on a 7:3
mixture of cis-11b and trans-11b).
Compound cis-11d: Colorless oil. IR (film, cmꢀ1) nmax: 3230 nN–H
,
1682 nCꢀꢀN, 1622 nCꢀ , 1410, 1174 n O, 1019 nP–O
.
1H NMR
ꢀꢀ
P
ꢀꢀ
C
ꢀ
ꢀꢀ
(300 MHz, CDCl3): d ¼ 8.10 (s, 1H), 8.04 (s, 1H), 4.70–4.60 (m, 1H,
HC5), 4.62 (dd, J ¼ 14.4 Hz, J ¼ 3.3 Hz, 1H, HCH), 4.46 (dd,
J ¼ 14.4 Hz, J ¼ 9.9 Hz, 1H, HCH), 4.27–4.16 (m, 4H, 2 ꢃ CH2OP),
2.95 (ddd, J ¼ 10.2 Hz, J ¼ 7.2 Hz, J ¼ 2.4 Hz, 1H, HC3), 2.84 (d,
J ¼ 0.6 Hz, 3H, CH3N), 2.90–2.75 (m, 1H, HC4), 2.37 (dddd,
J ¼ 18.0 Hz, J ¼ 12.3 Hz, J ¼ 7.2 Hz, J ¼ 3.3 Hz, 1H, H0C4), 1.39 (t,
J ¼ 6.9 Hz, 6H, 2 ꢃ CH3CH2OP); 13C NMR signals of cis-11d were
extracted from the spectrum of a 6:4 mixture of cis-11d and trans-
Compound trans-11b: Colorless oil. IR (film, cmꢀ1) nmax: 3468
nN–H, 2984 nC–H, 1679 nCꢀꢀO, 1251 nPꢀO, 1052 nP–O.
1H NMR
(300 MHz, CDCl3): d ¼ 8.45 (bs, 1H, NH), 7.10 (brq, J ¼ 1.2 Hz, 1H),
4.26 (dddd, J ¼ 8.1 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz, J ¼ 2.7 Hz, 1H, HC5),
4.22–4.14 (m, 4H, 2 ꢃ CH2OP), 4.14 (dd, J ¼ 14.4 Hz, J ¼ 2.7 Hz, 1H,
HCH), 3.67 (dd, J ¼ 14.4 Hz, J ¼ 7.2 Hz, 1H, HCH), 2.88 (d, J ¼ 1.2 Hz,
3H, CH3N), 2.94–2.85 (m, 1H, HC3), 2.63 (dddd, J ¼ 18.3 Hz,
J ¼ 12.6 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz, 1H, H0C4), 2.20 (dddd, J ¼ 12.6 Hz,
J ¼ 11.7 Hz, J ¼ 10.2 Hz, J ¼ 8.1 Hz, 1H, HC4), 1.92 (d, J ¼ 1.2 Hz, 3H,
CH3), 1.34 (t, J ¼ 7.2 Hz, 6H, 2 ꢃ CH3CH2OP); 13C NMR signals of
11d, 13C NMR (75.5 MHz, CDCl ): d ¼ 154.93, 153.40, 150.01 (C O),
ꢀꢀ
ꢀꢀ
3
143.73, 120.86, 73.36 (d, J ¼ 6.9 Hz, C5), 63.52 (d, J ¼ 169.5 Hz, C3),
62.98 (d, J ¼ 6.9 Hz, CH2OP), 62.94 (d, J ¼ 6.9 Hz, CH2OP), 51.84
(CH2N), 45.81 (CH3N), 35.45 (C4), 16.79 (d, J ¼ 5.4 Hz, CH3CH2OP),
16.64 (d, J ¼ 5.7 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3):
d ¼ 23.62. Anal. calcd. for C14H23N6O4P: C, 45.40; H, 6.26; N, 22.69.
Found: C, 45.32; H, 6.30; N, 22.45.
trans-11b were extracted from the spectrum of a 3:7 mixture of cis-
13
11b and trans-11b, C NMR (75.5 MHz, CDCl ): d ¼ 164.51 (C O),
Compound trans-11d: Solidified, m.p. 171–173°C. IR (KBr, cmꢀ1
)
ꢀꢀ
ꢀꢀ
3
151.34 (C O), 141.11, 110.51, 75.55 (d, J ¼ 7.2 Hz, C5), 63.85 (d,
nmax: 3377 nN–H, 1622 nCꢀꢀ , 1231 n O, 1019 nP–O.
1H NMR
ꢀꢀ
C P
ꢀꢀ ꢀꢀ
ꢀꢀ
ꢀꢀ
J ¼ 170.3 Hz, C3), 63.44 (d, J ¼ 6.4 Hz, CH2OP), 62.66 (d, J ¼ 6.9 Hz,
CH2OP), 49.17 (CH2N), 46.34 (CH3N), 35.15 (C4), 16.75 (d, J ¼ 5.4 Hz,
CH3CH2OP), 16.67 (d, J ¼ 5.4 Hz, CH3CH2OP), 12.53 (CH3); 31P NMR
(121.5 MHz, CDCl3): d ¼ 22.49. Anal. calcd. for C14H24N3O6P ꢃ 0.5
H2O: C, 45.40; H, 6.80; N, 11.35. Found: C, 45.41; H, 7.12; N, 11.21.
(300 MHz, CDCl3): d ¼ 8.12 (s, 1H), 8.04 (s, 1H), 4.76 (dd, J ¼ 14.4 Hz,
J ¼ 2.7 Hz, 1H, HCH), 4.51 (dddd, J ¼ 9.6 Hz, J ¼ 7.2 Hz, J ¼ 6.6 Hz,
J ¼ 2.7 Hz, 1H, HC5), 4.43 (dd, J ¼ 14.4 Hz, J ¼ 6.6 Hz, 1H, HCH),
4.20–4.07 (m, 4H, 2 ꢃ CH2OP), 2.90–2.60 (m, 2H, HC3 and H0C4),
2.85 (d, J ¼ 0.6 Hz, 3H, CH3N), 2.28 (dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz,
J ¼ 9.6 Hz, J ¼ 7.5 Hz, 1H, HC4), 1.31 (t, J ¼ 7.8 Hz, 3H, CH3CH2OP),
1.30 (t, J ¼ 7.2 Hz, 3H, CH3CH2OP); 13C NMR signals of trans-11d
were extracted from the spectrum of a 2:8 mixture of cis-11d and
trans-11d, 13C NMR (75.5 MHz, CDCl3): d ¼ 154.86, 153.52, 150.46,
143.32, 120.72, 73.89 (d, J ¼ 7.4 Hz, C5), 64.53 (d, J ¼ 169.8 Hz, C3),
63.40 (d, J ¼ 6.6 Hz, CH2OP), 62.59 (d, J ¼ 6.9 Hz, CH2OP), 51.21
(CH2N), 46.28 (CH3N), 35.18 (C4), 16.71 (d, J ¼ 5.4 Hz, CH3CH2OP),
16.64 (d, J ¼ 5.4 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3):
d ¼ 22.05. Anal. calcd. for C14H23N6O4P ꢃ 0.5H2O: C, 44.33; H, 6.38;
N, 22.15. Found: C, 44.41; H, 6.09; N, 22.16.
Diethyl 5-((6-amino-9H-purin-9-yl)methyl)-2-
methylisoxazolidin-3-yl-3-phosphonates cis-11c and
trans-11c
From nitrone 14 (0.211 g, 1.08 mmol) and N9-allyladenine 15c
(0.200 g, 1.14 mmol) in toluene–ethanol, inseparable mixture of
cis-11c and trans-11c (0.400 g, 98%) was obtained after purification
on silica gel with chloroform–methanol (from 100:1 to 20:1)
which solidified.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
348
M. Łysakowska et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Diethyl 5-((4-amino-2-oxopyrimidin-1(2H)-yl)methyl)-2-
methylisoxazolidin-3-yl-3-phosphonates cis-11e and
J ¼ 0.9 Hz, 3H, CH3N), 2.74 (dddd, J ¼ 18.6 Hz, J ¼ 12.6 Hz, J ¼ 10.2
Hz, J ¼ 7.8 Hz, 1H, H0C4), 2.25 (dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz,
J ¼ 7.8 Hz, J ¼ 3.9 Hz, 1H, HC4), 2.24 (s, 3H, CH3), 1.37 (t, J ¼ 7.2 Hz,
3H, CH3CH2OP), 1.36 (t, J ¼ 7.2 Hz, 3H, CH3CH2OP); 13C NMR
signals of cis-11f were extracted from the spectrum of a
9:1 mixture of cis-11f and trans-11f, 13C NMR (75.5 MHz,
trans-11e
From nitrone 14 (0.257 g, 1.32 mmol) and N1-allylcytosine
15e (0.200 g, 1.32 mmol) in toluene, inseparable mixture of
cis-11e and trans-11e (0.365 g, 79%) was obtained after purification
on silica gel with chloroform–methanol (from 100:1 to 20:1 v/v).
Compound cis-11e: IR (film, cmꢀ1) n: 3360 nN–H, 1653 nCꢀꢀO, 1238
CDCl3): d ¼ 171.15 (C O), 163.12 (C O), 155.90, 150.61,
96.54, 74.41 (d, J ¼ 6.9 Hz, C5), 63.65 (d, J ¼ 169.5 Hz, C3), 62.99
(d, J ¼ 6.6 Hz, CH2OP), 62.90 (d, J ¼ 6.9 Hz, CH2OP), 52.41
(CH2N), 45.89 (d, J ¼ 4.9 Hz, CH3N), 35.47 (C4), 25.06 (CH3),
16.79 (d, J ¼ 5.7 Hz, CH3CH2OP), 16.75 (d, J ¼ 5.7 Hz, CH3CH2OP);
31P NMR (121.5 MHz, CDCl3): d ¼ 23.77. Anal. calcd. for
n
O, 1022 nP–O. NMR signals of cis-11e were extracted from the
ꢀꢀ
P
ꢀꢀ
spectra of 8:2 mixture of cis-11e and trans-11e, 1H NMR (300 MHz,
CDCl3): d ¼ 7.38 (d, J ¼ 7.2 Hz, 1H), 6.40–5.40 (vbs, 2H, NH2), 5.68 (d,
J ¼ 6.9 Hz, 1H), 4.53–4.43 (m, 1H, HC5), 4.25–4.12 (m, 5H,
2 ꢃ CH2OP and HCH), 3.62 (dd, J ¼ 13.5 Hz, J ¼ 9.3 Hz, 1H, HCH),
2.94 (ddd, J ¼ 9.6 Hz, J ¼ 8.1 Hz, J ¼ 3.0 Hz, 1H, HC3), 2.81 (s, 3H,
CH3N), 2.72 (dddd, J ¼ 17.1 Hz, J ¼ 12.6 Hz, J ¼ 9.6 Hz, J ¼ 9.3 Hz,
1H, H0C4), 2.21 (dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz, J ¼ 8.1 Hz, J ¼ 4.8 Hz,
1H, HC4), 1.36 (t, J ¼ 6.9 Hz, 6H, 2 ꢃ CH3CH2OP); 13C NMR
ꢀ
C
15H25N4O6P ꢃ 0.5H2O: C, 45.34; H, 6.60; N, 14.10. Found: C,
45.32; H, 6.66; N, 14.21.
Compound trans-11f: Colorless oil. IR (film, cmꢀ1) n: 2985 nC–H
,
1658 nCꢀ , 1283 n O, 1019 nP–O.
1H NMR (300 MHz, CDCl3):
ꢀꢀ
P
ꢀꢀ
O
ꢀ
d ¼ 8.91 (s, 1H, NH), 7.69 (d, J ¼ 7.5 Hz, 1H), 7.38 (d, J ¼ 7.5 Hz, 1H),
4.32 (dddd, J ¼ 8.1 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz, J ¼ 2.4 Hz, 1H, HC5),
4.36 (dd, J ¼ 14.1 Hz, J ¼ 2.4 Hz, 1H, HCH), 4.23–4.10 (m, 4H,
2 ꢃ CH2OP), 3.85 (dd, J ¼ 14.1 Hz, J ¼ 7.2 Hz, 1H, HCH), 2.89 (ddd,
J ¼ 9.9 Hz, J ¼ 7.2 Hz, J ¼ 5.1 Hz, 1H, HC3), 2.85 (d, J ¼ 1.2 Hz, 3H,
CH3N), 2.65 (dddd, J ¼ 18.3 Hz, J ¼ 12.6 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz,
1H, H0C4), 2.25 (s, 3H, CH3), 2.22 (dddd, J ¼ 12.3 Hz, J ¼ 12.3 Hz,
J ¼ 9.9 Hz, J ¼ 8.1 Hz, 1H, HC4), 1.33 (t, J ¼ 7.2 Hz, 6H, 2 ꢃ
CH3CH2OP); 13C NMR signals of trans-11f were extracted from
the spectrum of a 1:9 mixture of cis-11f and trans-11f, 13C NMR
(75.5 MHz, CDCl3): d ¼ 166.32 (C O), 156.79, 146.82, 94.38,
74.16 (d, J ¼ 6.9 Hz, C5), 63.76 (d, J ¼ 170.3 Hz, C3), 62.96 (d,
J ¼ 6.9 Hz, CH2OP), 62.83 (d, J ¼ 7.2 Hz, CH2OP), 51.78 (CH2N), 46.09
(d, J ¼ 5.7 Hz, CH3N), 35.34 (C4), 16.75 (d, J ¼ 5.4 Hz, CH3CH2OP),
16.71 (d, J ¼ 5.7 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3):
d ¼ 23.86. Anal. calcd. for C13H23N4O5P ꢃ 0.5H2O: C, 43.94; H, 6.81;
N, 15.77. Found: C, 44.07; H, 6.55; N, 15.49 (obtained on a 8:2
mixture of cis-11e and trans-11e).
Compound trans-11e: IR (film, cmꢀ1) n: 3348 nN–H, 1613 nCꢀꢀ
,
O
ꢀꢀ
ꢀꢀ
ꢀꢀ
(75.5 MHz, CDCl ): d ¼ 171.33 (C O), 163.21 (C O), 156.02, 149.70,
ꢀꢀ
ꢀꢀ
3
1249 n O, 1052 nP–O. NMR signals of trans-11e were extracted from
ꢀꢀ
P
ꢀꢀ
96.85, 74.80 (d, J ¼ 7.4 Hz, C5), 64.05 (d, J ¼ 170.9 Hz, C3), 62.92 (d,
J ¼ 6.6 Hz, CH2OP), 62.62 (d, J ¼ 6.9 Hz, CH2OP), 51.36 (CH2N), 46.29
(CH3N), 35.11 (C4), 25.00 (CH3), 16.71 (d, J ¼ 5.4 Hz, CH3CH2OP),
16.65 (d, J ¼ 4.6 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3):
d ¼ 22.45. Anal. calcd. for C15H25N4O6P: C, 46.39; H, 6.49; N, 14.43.
Found: C, 46.32; H, 6.46; N, 14.51.
the spectra of a 1:9 mixture of cis-11e and trans-11e, 1H NMR
(300 MHz, CDCl3): d ¼ 7.38 (d, J ¼ 7.2 Hz, 1H), 6.40–5.40 (vbs, 2H,
NH2), 5.72 (d, J ¼ 7.2 Hz, 1H), 4.27 (dddd, J ¼ 10.2 Hz, J ¼ 7.5 Hz,
J ¼ 7.5 Hz, J ¼ 3.0 Hz, 1H, HC5), 4.24 (dd, J ¼ 14.4 Hz, J ¼ 3.0 Hz, 1H,
HCH), 4.23–4.10 (m, 4H, 2 ꢃ CH2OP), 3.77 (dd, J ¼ 14.4 Hz, J ¼ 7.5
Hz, 1H, HCH), 2.85 (d, J ¼ 0.9 Hz, 3H, CH3N), 2.91–2.81 (m, 1H,
HC3), 2.62 (dddd, J ¼ 18.3 Hz, J ¼ 12.6 Hz, J ¼ 7.5 Hz, J ¼ 7.5 Hz, 1H,
H0C4), 2.25 (dddd, J ¼ 12.6 Hz, J ¼ 11.7 Hz, J ¼ 10.2 Hz, J ¼ 7.8 Hz,
1H, HC4), 1.33 (t, J ¼ 6.9 Hz, 6H, 2 ꢃ CH3CH2OP); 13C NMR
ꢀ
Diethyl 5-((4,5-dihydro-3,5-dioxo-1,2,4-triazin-2(3H)-yl)-
methyl)-2-methylisoxazolidin-3-yl-3-phosphonates
(75.5 MHz, CDCl3): d ¼ 166.29 (C O), 156.85, 145.95, 94.83,
cis-11g and trans-11g
75.67 (d, J ¼ 7.2 Hz, C5), 64.01 (d, J ¼ 173.3 Hz, C3), 63.28 (d,
J ¼ 6.6 Hz, CH2OP), 62.64 (d, J ¼ 6.9 Hz, CH2OP), 50.43 (CH2N), 46.40
(CH3N), 35.01 (C4), 16.71 (d, J ¼ 4.3 Hz, CH3CH2OP), 16.65 (d,
J ¼ 5.4 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 22.66.
Anal. calcd. for C13H23N4O5P ꢃ 0.5H2O: C, 43.94; H, 6.81; N, 15.77.
Found: C, 43.87; H, 6.95; N, 15.53 (obtained on a 1:9 mixture of cis-
11e and trans-11e).
From nitrone 14 (0.240 g, 1.24 mmol) and N1-allyl-6-azauracil 15g
(0.200 g, 1.31 mmol) in toluene–ethanol, inseparable mixture of
cis-11g and trans-11g (0.432 g, 98%) was obtained after purification
on silica gel (chloroform–methanol; from 100:1 to 20:1 v/v) as a
colorless oil.
Compound trans-11g: IR (film, cmꢀ1) nmax: 1729 nCꢀꢀO, 1676 nCꢀꢀ
,
O
ꢀꢀ
ꢀꢀ
1223 n O, 1026 nP–O. NMR signals of trans-11g were extracted
ꢀꢀ
P
ꢀꢀ
from the spectra of a 2:8 mixture of cis-11g and trans-11g, 1H NMR
(300 MHz, CDCl3): d ¼ 10.51 (s, 1H, NH), 7.38 (s, 1H), 4.39 (dddd,
J ¼ 7.2 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz, 1H, HC5), 4.25–4.14 (m,
4H, 2 ꢃ CH2OP), 4.14 (dd, J ¼ 13.5 Hz, J ¼ 7.2 Hz, 1H, HCH), 3.97 (dd,
J ¼ 13.5 Hz, J ¼ 4.2 Hz, 1H, HCH), 375 (dd, J ¼ 13.5 Hz, J ¼ 4.2 Hz,
1H, HCH), 3.07 (ddd, J ¼ 9.9 Hz, J ¼ 7.2 Hz, J ¼ 2.4 Hz, 1H, HC3), 2.87
(s, 3H, CH3N), 2.67 (dddd, J ¼ 18.3 Hz, J ¼ 12.6 Hz, J ¼ 7.2 Hz,
J ¼ 7.2 Hz, 1H, H0C4), 2.33 (dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz, J ¼ 9.9 Hz,
J ¼ 7.2 Hz, 1H, HC4), 1.34 (t, J ¼ 7.2 Hz, 6H, 2 ꢃ CH3CH2OP); 13C
N-(1,2-Dihydro-1-((3-diethoxyphosphoryl-2-
methylisoxazolidin-5-yl)methyl)-2-oxopyrimidin-4-yl)-
acetamides cis-11f and trans-11f
From nitrone 14 (0.101 g, 0.520 mmol) and N4-acetyl-N1-allylcyto-
sine 15f (0.100 g, 0.520 mmol) in toluene–ethanol, pure cis-11f
(0.022 g, 11%), trans-11f (0.058 g, 29%), and fractions containing
various mixtures of cis-11f and trans-11f (0.094 g, 47%) were
obtained by column chromatography (chloroform–methanol;
from 100:1 to 50:1 v/v).
ꢀꢀ
ꢀꢀ
ꢀꢀ
NMR (75.5 MHz, CDCl ): d ¼ 156.26 (C O), 149.24 (C O), 134.91,
ꢀꢀ
3
Compound cis-11f: Colorless oil. IR (film, cmꢀ1) nmax: 2986 nC–H
,
74.03 (d, J ¼ 7.0 Hz, C5), 63.76 (d, J ¼ 169.7 Hz, C3), 63.43 (d,
J ¼ 6.9 Hz, CH2OP), 63.05 (d, J ¼ 6.9 Hz, CH2OP), 46.66 (CH2N), 42.44
(CH3N), 36.12 (d, J ¼ 2.3 Hz, C4), 16.75 (d, J ¼ 6.3 Hz, CH3CH2OP),
16.72 (d, J ¼ 5.7 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3):
d ¼ 23.22. Anal. calcd. for C12H21N4O6P ꢃ 0.5H2O: C, 40.34; H, 6.21;
N, 15.68. Found: C, 40.22; H, 6.19; N, 15.53 (obtained on a 2:8
mixture of cis-11g and trans-11g).
1642 nCꢀ , 1197 n O, 1023 nP–O.
1H NMR (300 MHz, CDCl3):
ꢀꢀ
P
ꢀꢀ
O
ꢀ
d ¼ 9.03 (s, 1H, NH), 7.70 (d, J ¼ 7.5 Hz, 1H), 7.36 (d, J ¼ 7.5 Hz, 1H),
4.50 (dddd, J ¼ 9.6 Hz, J ¼ 7.8 Hz, J ¼ 3.9 Hz, J ¼ 2.7 Hz, 1H,
HC5), 4.29 (dd, J ¼ 13.8 Hz, J ¼ 2.7 Hz, 1H, HCH), 4.24–4.14 (m,
4H, 2 ꢃ CH2OP), 3.78 (dd, J ¼ 13.8 Hz, J ¼ 9.6 Hz, 1H, HCH),
2.93 (ddd, J ¼ 10.2 Hz, J ¼ 7.8 Hz, J ¼ 3.0 Hz, 1H, HC3), 2.82 (d,
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

ꢀ
Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Isoxazolidine Analogs of Homonucleotides
349
Diethyl 2-methyl-5-((2-oxopyridin-1(2H)-yl)methyl)-
1H, H0C4), 1.37 (t, J ¼ 6.9 Hz, 3H, CH3CH2OP), 1.36 (t, J ¼ 6.9 Hz, 3H,
CH3CH2OP); 13C NMR signals of cis-11i were extracted from the
spectrum of a 7:3 mixture of cis-11i and trans-11i, 13C NMR
isoxazolidin-3-yl-3-phosphonates cis-11h and trans-11h
From nitrone 14 (0.296 g, 1.52 mmol) and N1-allylpyridin-2-one
15h (0.205 g, 1.52 mmol) in toluene, pure trans-11h (0.012 g, 2.4%)
was obtained after column chromatography (chloroform–metha-
nol; 100:1 to 30:1 v/v) followed by fractions containing various
mixtures of cis-11h and trans-11h (0.481 g, 96%).
ꢀꢀ
(75.5 MHz, CDCl ): d ¼ 178.90 (C O), 140.67, 118.36, 75.28 (d,
ꢀꢀ
3
J ¼ 6.9 Hz, C5), 63.45 (d, J ¼ 170.0 Hz, C3), 63.04 (d, J ¼ 6.9 Hz,
CH2OP), 62.99 (d, J ¼ 6.9 Hz, CH2OP), 58.30 (CH2N), 45.95 (CH3N),
35.26 (C4), 16.75 (d, J ¼ 5.7 Hz, CH3CH2OP), 16.68 (d, J ¼ 4.3 Hz,
CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 23.48. Anal. calcd.
for C14H23N2O5P ꢃ 2H2O: C, 45.90; H, 7.43; N, 7.65. Found: C,
45.94; H, 7.57; N, 7.63 (obtained on a 7:3 mixture of cis-11i and
trans-11i).
Compound cis-11h: IR (film, cmꢀ1) nmax: 2983 nC–H, 1658 nCꢀꢀ
,
O
ꢀꢀ
1580 nCꢀ , 1232 n O, 1023 nP–O. NMR signals of cis-11h were
ꢀꢀ
C
ꢀ
P
ꢀꢀ
extracted from the spectra of a 6:4 mixture of cis-11h and trans-
1
11h, H NMR (300 MHz, CDCl3): d ¼ 7.40–7.30 (m, 2H), 6.59–6.54
Compound trans-11i: Colorless oil. IR (film, cmꢀ1) nmax: 2920 nC–H
1640 nCꢀꢀO, 1552 nCꢀꢀ , 1196 n O, 1023 nP–O
1H NMR (300 MHz,
,
(m, 1H), 6.18–6.13 (m, 1H), 4.50 (dddd, J ¼ 9.0 Hz, J ¼ 7.8 Hz,
J ¼ 4.8 Hz, J ¼ 2.7 Hz, 1H, HC5), 4.42 (dd, J ¼ 13.2 Hz, J ¼ 2.7 Hz, 1H,
HCH), 4.23–4.14 (m, 4H, 2 ꢃ CH2OP), 3.76 (dd, J ¼ 13.2 Hz, J ¼ 9.0
Hz, 1H, HCH), 2.94 (ddd, 1H, J ¼ 9.6 Hz, J ¼ 7.8 Hz, J ¼ 2.7 Hz, HC3),
2.83 (d, J ¼ 0.9 Hz, 3H, CH3N), 2.77 (dddd, J ¼ 17.4 Hz, J ¼ 12.6 Hz,
J ¼ 9.6 Hz, J ¼ 7.8 Hz, 1H, H0C4), 2.27 (dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz,
J ¼ 7.8 Hz, J ¼ 4.8 Hz, 1H, HC4), 1.36 (t, J ¼ 7.2 Hz, 6H, 2 ꢃ
ꢀ
.
ꢀꢀ
P
ꢀꢀ
C
ꢀꢀ
ꢀꢀ
CDCl3): d ¼ 7.33–7.29 (m, 2H), 6.41–6.37 (m, 2H), 4.29 (dddd,
J ¼ 8.4 Hz, J ¼ 7.5 Hz, J ¼ 7.5 Hz, J ¼ 2.7 Hz, 1H, HC5), 4.23–4.12 (m,
4H, 2 ꢃ CH2OP), 4.03 (dd, J ¼ 14.7 Hz, J ¼ 2.7 Hz, 1H, HCH), 3.80 (dd,
J ¼ 14.7 Hz, J ¼ 7.5 Hz, 1H, HCH), 2.99–2.80 (m, 1H, HC3), 2.86 (d,
J ¼ 1.2 Hz, 3H, CH3N), 2.63 (dddd, J ¼ 18.3 Hz, J ¼ 12.3 Hz, J ¼ 7.5
Hz, J ¼ 7.2 Hz, 1H, H0C4), 2.15 (dddd, J ¼ 12.3 Hz, J ¼ 12.0 Hz,
J ¼ 10.2 Hz, J ¼ 8.4 Hz, 1H, HC4), 1.34 (t, J ¼ 7.2 Hz, 6H, 2 ꢃ
CH3CH2OP); 13C NMR signals of trans-11i were extracted from
the spectrum of a 3:7 mixture of cis-11i and trans-11i, 13C NMR
CH3CH2OP); 13C NMR (75.5 MHz, CDCl3): d ¼ 162.56 (C O),
139.92, 139.43, 120.28, 105.65, 73.88 (d, J ¼ 7.4 Hz, C5), 63.70 (d,
J ¼ 169.8 Hz, C3), 62.70 (d, J ¼ 6.9 Hz, CH2OP), 62.48 (d, J ¼ 7.2 Hz,
CH2OP), 51.74 (CH2N), 45.90 (CH3N), 35.61 (d, J ¼ 1.7 Hz, C4), 16.72
(d, J ¼ 5.7 Hz, CH3CH2OP), 16.66 (d, J ¼ 5.1 Hz, CH3CH2OP); 31P NMR
(121.5 MHz, CDCl3): d ¼ 23.80. Anal. calcd. for C14H23N2O5P ꢃ H2O:
C, 48.27; H, 7.23; N, 8.04. Found: C, 48.26; H, 7.46; N, 8.21
(obtained on a 6:4 mixture of cis-11h and trans-11h).
ꢀꢀ
(75.5 MHz, CDCl ): d ¼ 178.71 (C O), 140.54, 118.36, 75.59 (d,
ꢀꢀ
3
J ¼ 7.4 Hz, C5), 63.36 (d, J ¼ 170.0 Hz, C3), 63.19 (d, J ¼ 6.6 Hz,
CH2OP), 62.69 (d, J ¼ 6.9 Hz, CH2OP), 57.92 (CH2N), 46.27 (CH3N),
34.80 (C4), 16.66 (d, J ¼ 4.6 Hz, CH3CH2OP), 16.60 (d, J ¼ 5.7 Hz,
CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 22.30. Anal. calcd.
for C14H23N2O5P ꢃ 2H2O: C, 45.90; H, 7.43; N, 7.65. Found: C,
45.90; H, 7.45; N, 7.72.
Compound trans-11h: Colorless oil. IR (film, cmꢀ1) nmax: 2982
nC–H, 1659 nCꢀꢀO, 1582 nCꢀꢀC, 1233 nPꢀO, 1023 nP–O.
1H NMR
(300 MHz, CDCl3): d ¼ 7.38–7.30 (m, 2H), 6.58–6.55 (m, 1H), 6.18–
6.13 (m, 1H); 4.40 (dd, J ¼ 13.8 Hz, J ¼ 3.0 Hz, 1H, HCH), 4.33 (dddd,
J ¼ 10.5 Hz, J ¼ 7.8 Hz, J ¼ 6.6 Hz, J ¼ 3.0 Hz, 1H, HC5), 4.23–4.10 (m,
4H, 2 ꢃ CH2OP), 3.93 (dd, J ¼ 13.8 Hz, J ¼ 6.6 Hz, 1H, HCH), 2.91–
2.78 (m, 1H, HC3), 2.85 (d, J ¼ 1.2 Hz, 3H, CH3N), 2.64 (dddd,
J ¼ 18.3 Hz, J ¼ 13.2 Hz, J ¼ 7.8 Hz, J ¼ 7.8 Hz, H0C4), 2.25 (dddd,
J ¼ 13.2 Hz, J ¼ 12.6 Hz, J ¼ 10.5 Hz, J ¼ 8.4 Hz, 1H, HC4), 1.33 (t,
J ¼ 7.2 Hz, 6H, 2 ꢃ CH3CH2OP); 13C NMR (75.5 MHz, CDCl3):
Diethyl 5-((1H-imidazol-1-yl)methyl)-2-methylisoxazolidin-
3-yl-3-phosphonates cis-11j and trans-11j
From nitrone 14 (0.180 g, 0.920 mmol) and N1-allylimidazole 15j
(0.100 g, 0.920 mmol) in toluene, inseparable mixture of cis-11j
and trans-11j (0.218 g, 78%) was obtained after purification on
silica gel with chloroform–methanol (100:1 to 50:1 v/v) as
yellowish oil.
ꢀ
d ¼ 162.73 (C O), 139.92, 138.76, 120.84, 105.82, 75.59 (d,
ꢀ
Compound trans-11j: IR (film, cmꢀ1) nmax: 1665 nCꢀꢀC, 1511 nCꢀꢀ
,
J ¼ 7.2 Hz, C5), 64.32 (d, J ¼ 167.8 Hz, C3), 63.48 (d, J ¼ 6.3 Hz,
CH2OP), 62.57 (d, J ¼ 6.9 Hz, CH2OP), 50.27 (CH2N), 46.41 (CH3N),
35.44 (C4), 16.82 (d, J ¼ 5.4 Hz, CH3CH2OP), 16.75 (d, J ¼ 5.2 Hz,
CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 22.71. Anal. calcd.
for C14H23N2O5P ꢃ H2O: C, 48.27; H, 7.23; N, 8.04. Found: C, 48.35;
H, 7.35; N, 8.18.
C
ꢀꢀ
ꢀꢀ
1232 nPꢀO, 1024 nP–O. NMR signals of trans-11j were extracted from
the spectra of a 3:7 mixture of cis-11j and trans-11j; 1H NMR
(300 MHz, CDCl3): d ¼ 7.51 (s, 1H), 7.05 (m, 1H), 6.99–6.97 (m, 1H),
4.30 (dddd, J ¼ 7.2 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz, J ¼ 5.4 Hz, 1H, HC5),
4.19 (dd, J ¼ 15.0 Hz, J ¼ 7.2 Hz, 1H, HCH), 4.22–4.09 (m, 4H,
2 ꢃ CH2OP), 4.01 (dd, J ¼ 15.0 Hz, J ¼ 5.4 Hz, 1H, HCH), 2.84 (d,
J ¼ 0.6 Hz, 3H, CH3N), 2.72 (ddd, J ¼ 9.9 Hz, J ¼ 7.2 Hz, J ¼ 2.4 Hz,
1H, HC3), 2.57 (dddd, J ¼ 18.0 Hz, J ¼ 12.6 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz,
1H, H0C4), 2.08 (dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz, J ¼ 9.9 Hz, J ¼ 7.2 Hz,
1H, HC4), 1.32 (t, J ¼ 7.2 Hz, 3H, CH3CH2OP), 1.31 (t, J ¼ 7.2 Hz, 3H,
CH3CH2OP); 13C NMR (75.5 MHz, CDCl3): d ¼ 137.83, 129.26,
120.10, 75.58 (d, J ¼ 8.1 Hz, C5), 64.08 (d, J ¼ 168.4 Hz, C3), 63.27
(d, J ¼ 6.0 Hz, CH2OP), 62.70 (d, J ¼ 6.9 Hz, CH2OP), 48.57 (CH2N),
46.22 (CH3N), 34.96 (d, J ¼ 2.9 Hz, C4), 16.73 (d, J ¼ 4.2 Hz,
CH3CH2OP); 16.65 (d, J ¼ 4.0 Hz, CH3CH2OP); 31P NMR
(121.5 MHz, CDCl3): d ¼ 21.90. Anal. calcd. for C12H22N3O4P ꢃ 2
H2O: C, 44.86; H, 7.53; N, 13.08. Found: C, 44.67; H, 7.69; N, 12.82
(obtained on a 3:7 mixture of cis-11j and trans-11j).
Diethyl 2-methyl-5-((4-oxopyridin-1(4H)-yl)methyl)-
isoxazolidin-3-yl-3-phosphonates cis-11i and trans-11i
From nitrone 14 (0.302 g, 1.55 mmol) and N1-allylpyridin-4-one 15i
(0.210 g, 1.55 mmol) in toluene–ethanol, pure trans-11i (0.027 g,
5.3%) was obtained after column chromatography (chloroform–
methanol; 100:1 to 20:1 v/v) followed by fractions containing various
mixtures of cis-11i and trans-11i (0.475 g, 93%) as a colorless oil.
Compound cis-11i: IR (film, cmꢀ1) nmax: 2986 nC–H, 1615 nCꢀꢀ
,
O
ꢀꢀ
1554 nCꢀꢀ , 1197 n O, 1023 nP–O.
1H NMR signals of cis-11i were
ꢀꢀ
C
ꢀꢀ
P
ꢀꢀ
extracted from the spectrum of a 9:1 mixture of cis-11i and trans-
11i, 1H NMR (300 MHz, CDCl3): d ¼ 7.38–7.36 (m, 2H), 6.43–6.38
(m, 2H), 4.34 (dddd, J ¼ 7.8 Hz, J ¼ 7.2 Hz, J ¼ 3.9 Hz, J ¼ 3.0 Hz, 1H,
HC5), 4.19 (dd, J ¼ 14.1 Hz, J ¼ 7.8 Hz, 1H, HCH), 4.25–4.10 (m, 4H,
2 ꢃ CH2OP), 3.71 (dd, J ¼ 14.1 Hz, J ¼ 3.0 Hz, 1H, HCH), 2.96 (ddd,
J ¼ 10.2 Hz, J ¼ 7.5 Hz, J ¼ 3.0 Hz, 1H, HC3), 2.85 (d, J ¼ 0.6 Hz, 3H,
CH3N), 2.78 (dddd, J ¼ 12.6 Hz, J ¼ 10.2 Hz, J ¼ 9.9 Hz, J ¼ 7.2 Hz,
1H, HC4), 2.26 (dddd, J ¼ 18.6 Hz, J ¼ 12.6 Hz, J ¼ 7.5 Hz, J ¼ 3.9 Hz,
Diethyl 5-((1H-benzo[d]imidazol-1-yl)methyl)-2-
methylisoxazolidin-3-yl-3-phosphonates cis-11k, trans-11k
From nitrone 14 (0.128 g, 0.660 mmol) and N1-allylbenzo[d]-
imidazole 15k (0.104 g, 0.660 mmol), in toluene, inseparable
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

350
M. Łysakowska et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
mixture of cis-11k and trans-11k (0.23 g, 98%) was obtained after
purification on silica gel with chloroform–methanol (from 100:1
to 50:1 v/v) as a colorless oil.
11m were extracted from the spectra of a 2:8 mixture of cis-11m
1
and trans-11m; H NMR (300 MHz, CDCl3): d ¼ 8.43–8.37 (m, 1H),
7.84 (m, 1H), 7.43–7.28 (m, 3H), 4.45 (dddd, J ¼ 8.1 Hz, J ¼ 7.5 Hz,
J ¼ 6.6 Hz, J ¼ 3.3 Hz, 1H, HC5), 4.33 (dd, J ¼ 17.1 Hz, J ¼ 3.3 Hz, 1H,
HCH), 4.28–4.07 (m, 5H, 2 ꢃ CH2OP, HCH), 2.85 (d, J ¼ 1.2 Hz, 3H,
CH3N), 2.75 (ddd, J ¼ 10.2 Hz, J ¼ 6.6 Hz, J ¼ 2.7 Hz, 1H, HC3),
2.55 (s, 3H,CH3), 2.62 (dddd, J ¼ 18.0 Hz, J ¼ 12.3 Hz, J ¼ 6.6 Hz,
J ¼ 6.6 Hz, 1H, H0C4), 2.16 (dddd, J ¼ 12.3 Hz, J ¼ 12.3 Hz,
J ¼ 10.2 Hz, J ¼ 8.1 Hz, 1H, HC4), 1.31 (t, J ¼ 6.3 Hz, 3H, CH3CH2OP),
1.29 (t, J ¼ 6.6 Hz, 3H, CH3CH2OP); 13C NMR (75.5 MHz, CDCl3):
Compound trans-11k: IR (film, cmꢀ1) nmax: 1672 nCꢀꢀ , 1235 n
,
ꢀꢀ
P O
C
ꢀꢀ
ꢀꢀ
1025 nP–O. NMR signals of trans-11k were extracted from the
spectra of a 3:7 mixture of cis-11k and trans-11k; 1H NMR (300 MHz,
CDCl3): d ¼ 7.98 (s, 1H), 7.84–7.79 (m, 1H), 7.48–7.39 (m, 1H), 7.34–
7.25 (m, 2H), 4.43 (dddd, J ¼ 7.5 Hz, J ¼ 7.5 Hz, J ¼ 6.3 Hz, J ¼ 3.3 Hz,
1H, HC5), 4.30–4.06 (m, 6H, HCH and 2 ꢃ CH2OP), 2.82 (d, J ¼ 1.2 Hz,
3H, CH3N), 2.73 (ddd, J ¼ 9.3 Hz, J ¼ 7.2 Hz, J ¼ 2.7 Hz, 1H, HC3), 2.59
(dddd, J ¼ 18.0 Hz, J ¼ 12.6 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz, 1H, H0C4), 2.14
(dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz, J ¼ 9.3 Hz, J ¼ 7.2 Hz, 1H, HC4), 1.30
ꢀꢀ
d ¼ 193.12 (C O), 137.29, 135.82, 126.03, 123.45, 122.64, 117.39,
ꢀꢀ
109.76, 75.58 (d, J ¼ 7.2 Hz, C5), 64.00 (d, J ¼ 169.8 Hz, C3), 63.26
(d, J ¼ 6.9 Hz, CH2OP), 62.75 (d, J ¼ 6.9 Hz, CH2OP), 48.20 (CH2N),
46.36 (CH3N), 35.21 (C4), 27.80 (CH3), 16.66 (d, J ¼ 6.6 Hz,
2 ꢃ CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 21.86. Anal.
calcd. for C19H27N2O5P ꢃ H2O: C, 55.33; H, 7.09; N, 6.79. Found: C,
55.55; H, 6.94; N, 6.76 (obtained on a 2:8 mixture of cis-11m
and trans-11m).
(t, J ¼ 6.9 Hz, 3H, CH3CH2OP), 1.29 (t, J ¼ 6.9 Hz, 3H, CH3CH2OP); 13
C
NMR (75.5 MHz, CDCl3): d ¼ 143.68, 143.30, 134.18, 123.10, 122.27,
120.34, 109.7, 75.36 (d, J ¼ 7.5 Hz, C5), 64.04 (d, J ¼ 168.6 Hz, C3),
63.25 (d, J ¼ 6.6 Hz, CH2OP), 62.70 (d, J ¼ 6.8Hz, CH2OP), 47.44
(CH2N), 46.30 (CH3N), 35.11 (d, J ¼ 2.2 Hz, C4), 16.69 (d, J ¼ 5.3 Hz,
CH3CH2OP), 16.62 (d, J ¼ 5.5 Hz, CH3CH2OP); 31P NMR (121.5 MHz,
CDCl3): d ¼ 22.52. Anal. calcd. for C16H24N3O4P ꢃ H2O: C, 51.75; H,
7.06; N, 11.31. Found: C, 51.49; H, 7.06; N, 11.23 (obtained on a 3:7
mixture of cis-11k and trans-11k).
Diethyl 5-((1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxopurin-
7-yl)methyl)-2-methylisoxazolidin-3-yl-3-phosphonates
cis-11n and trans-11n
From nitrone 14 (0.098 g, 0.50 mmol) and N5-allyl-1,3-dimethyl-
xanthine 15n (0.100 g, 0.500 mmol) in toluene, inseparable
mixture of cis-11n and trans-11n (0.180 g, 95%) was obtained after
purification on silica gel with chloroform–methanol (50:1 v/v) as
amorphous solid.
Diethyl 2-methyl-5-((5,6-dimethyl-1H-benzo[d]imidazol-1-
yl)methyl)isoxazolidin-3-yl-3-phosphonates cis-11l and
trans-11l
From nitrone 14 (0.105 g, 0.540 mmol) and N1-allyl-5,6-
dimethylbenzo[d]imidazole 15l (0.100 g, 0.540 mmol) in toluene,
inseparable mixture of cis-11l and trans-11l (0.199 g, 97%) was
obtained after purification on silica gel with chloroform–
methanol (from 100:1 to 20:1 v/v) as a colorless oil.
Compound cis-11n: IR (film, cmꢀ1) nmax: 2983 nC–H, 2961 nC–H
,
1710 nCꢀꢀO, 1663 nCꢀꢀO, 1244 nPꢀO, 1024 nP–O. 1H NMR signals of cis-
11n were extracted from the spectrum of a 7:3 mixture of cis-11n
and trans-11n, 1H NMR (300 MHz, CDCl3): d ¼ 7.66 (s, 1H), 4.66 (dd,
J ¼ 14.4 Hz, J ¼ 4.5 Hz, 1H, HCH), 4.40 (dd, J ¼ 14.4 Hz, J ¼ 9.3 Hz,
1H, HCH), 4.54–4.34 (m, 1H, HC5), 4.26–4.13 (m, 4H, 2 ꢃ CH2OP),
3.60 (s, 3H, CH3), 3.40 (s, 3H, CH3), 2.94 (ddd, J ¼ 9.9 Hz, J ¼ 7.8 Hz,
J ¼ 3.0 Hz, 1H, HC3), 2.82 (d, J ¼ 0.9 Hz, 3H, CH3N), 2.73 (dddd,
J ¼ 18.0 Hz, J ¼ 12.6 Hz, J ¼ 7.8 Hz, J ¼ 5.4 Hz, 1H, H0C4), 2.26 (dddd,
J ¼ 12.6 Hz, J ¼ 12.6 Hz, J ¼ 9.9 Hz, J ¼ 7.2 Hz, 1H, HC4), 1.37 (t,
J ¼ 7.2 Hz, 6H, 2 ꢃ CH3CH2OP); 13C NMR signals of cis-11n were
extracted from the spectrum of a 1:1 mixture of cis-11n and trans-
Compound trans-11l: IR (film, cmꢀ1) nmax: 2979 nC–H, 1499 nCꢀꢀ
,
C
ꢀꢀ
1443 nCꢀꢀC, 1232 nPꢀO, 1024 nP–O. NMR signals of trans-11l were
extracted from the spectra of a 3:7 mixture of cis-11l and trans-11l;
1H NMR (600 MHz, CDCl3): d ¼ 7.88 (s, 1H), 7.58 (s, 1H), 7.21–7.18
(m, 1H), 4.45 (dddd, J ¼ 8.1 Hz, J ¼ 8.1 Hz, J ¼ 8.1 Hz, J ¼ 3.5 Hz, 1H,
HC5), 4.35 (dd, J ¼ 14.0 Hz, J ¼ 3.5 Hz, 1H, HCH), 4.20–4.11 (m, 4H,
2 ꢃ CH2OP), 3.75 (dd, J ¼ 14.0 Hz, J ¼ 8.1 Hz, 1H, HCH), 2.99 (ddd,
J ¼ 9.8 Hz, J ¼ 8.1 Hz, J ¼ 3.1 Hz, 1H, HC3), 2.85 (d, J ¼ 0.8 Hz, 3H,
CH3N), 2.60 (dddd, J ¼ 18.2 Hz, J ¼ 12.6 Hz, J ¼ 8.1 Hz, J ¼ 7.3 Hz,
1H, H0C4), 2.42 (s, 3H, CH3), 2.40 (s, 3H, CH3), 2.16 (dddd,
J ¼ 12.6 Hz, J ¼ 12.6 Hz, J ¼ 9.8 Hz, J ¼ 8.1 Hz, 1H, HC4), 1.33 (t,
J ¼ 7.1 Hz, 3H, CH3CH2OP), 1.31 (t, J ¼ 7.1 Hz, 3H, CH3CH2OP); 13C
NMR (75.5 MHz, CDCl3): d ¼ 142.92, 141.73, 132.74, 132.41, 131.30,
120.29, 109.93, 75.39 (d, J ¼ 7.4 Hz, C5), 64.12 (d, J ¼ 168.0 Hz, C3),
63.27 (d, J ¼ 6.5 Hz, CH2OP), 62.71 (d, J ¼ 6.9 Hz, CH2OP), 47.63
(CH2N), 46.33 (CH3N), 35.18 (d, J ¼ 2.3 Hz, C4), 20.49 (2 ꢃ CH3), 16.75
(d, J ¼ 5.8 Hz, CH3CH2OP), 16.68 (d, J ¼ 5.2 Hz, CH3CH2OP); 31P NMR
(243 MHz, CDCl3): d ¼ 21.49. Anal. calcd. for C18H28N3O4P ꢃ H2O:
C, 54.13; H, 7.57; N, 10.52. Found: C, 54.37; H, 7.37; N, 10.42
(obtained on a 3:7 mixture of cis-11l and trans-11l).
11n, 13C NMR (75.5 MHz, CDCl ): d ¼ 155.34 (C O), 151.53 (C O),
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
3
148.85, 142.69, 106.43, 74.54 (d, J ¼ 6.9 Hz, C5), 63.54 (d,
J ¼ 169.5 Hz, C3), 63.22 (d, J ¼ 6.6 Hz, CH2OP), 62.84 (d, J ¼ 6.9 Hz,
CH2OP), 49.04 (CH2N), 45.86 (CH3N), 35.35 (C4), 30.00 (CH3), 28.15
(CH3), 16.72 (d, J ¼ 5.8 Hz, 2 ꢃ CH3CH2OP); 31P NMR (121.5 MHz,
CDCl3): d ¼ 23.66. Anal. calcd. for C16H26N5O6P: C, 46.26; H, 6.31;
N, 16.86. Found: C, 46.50; H, 6.54; N, 16.74 (obtained on a 7:3
mixture of cis-11n and trans-11n).
Compound trans-11n: IR (film, cmꢀ1) nmax: 2984 nC–H, 2918 nC–H
,
1706 nCꢀꢀO, 1661 nCꢀO, 1257 nPꢀO, 1023 nP–O. NMR signals of trans-
11n were extracted from the spectra of a 15:85 mixture of cis-11n
and trans-11n; 1H NMR (600 MHz, CDCl3): d ¼ 7.70 (s, 1H), 4.68 (dd,
J ¼ 14.3 Hz, J ¼ 2.5 Hz, 1H, HCH), 4.47 (dd, J ¼ 14.3 Hz, J ¼ 6.8 Hz,
1H, HCH), 4.41 (dddd, J ¼ 9.9 Hz, J ¼ 7.4 Hz, J ¼ 7.1 Hz, J ¼ 2.5 Hz,
1H, HC5), 4.25–4.14 (m, 4H, 2 ꢃ CH2OP), 3.63 (s, 3H, CH3), 3.43 (s,
3H, CH3), 2.91–2.76 (m, 1H, HC3), 2.87 (d, J ¼ 0.3 Hz, 3H, CH3N),
2.70 (dddd, J ¼ 18.1 Hz, J ¼ 12.6 Hz, J ¼ 7.4 Hz, J ¼ 7.4 Hz, 1H, H0C4),
2.21 (dddd, J ¼ 12.6 Hz, J ¼ 10.9 Hz, J ¼ 9.9 Hz, J ¼ 7.4 Hz, 1H, HC4),
1.36 (t, J ¼ 7.1 Hz, 3H, CH3CH2OP), 1.35 (t, J ¼ 7.1 Hz, 3H,
1-(1-((3-Diethoxyphosphoryl-2-methylisoxazolidin-5-yl)-
methyl)-1H-indol-3-yl)ethanones cis-11m and trans-11m
From nitrone 14 (0.098 g, 0.50 mmol) and N-allyl-3-acetylindole
15m (0.100 g, 0.500 mmol) in toluene, inseparable mixture of cis-
11m and trans-11m (0.164 g, 83%) was obtained after purification
on silica gel with chloroform–methanol (100:1 v/v) as an yellowish
oil.
CH3CH2OP); 13C NMR (75.5 MHz, CDCl3): d ¼ 155.42 (C O),
ꢀ
ꢀ
Compound trans-11m: IR (film, cmꢀ1) nmax: 2982 nC–H, 1642
nCꢀꢀO, 1529 nCꢀꢀC, 1391, 1234 nPꢀO, 1023 nP–O. NMR signals of trans-
ꢀꢀ
151.60 (C O), 148.60, 142.39, 106.89, 75.15 (d, J ¼ 7.7 Hz, C5),
ꢀꢀ
64.25 (d, J ¼ 169.2 Hz, C3), 63.44 (d, J ¼ 6.6 Hz, CH2OP), 62.71
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Isoxazolidine Analogs of Homonucleotides
351
(d, J ¼ 7.2 Hz, CH2OP), 48.12 (CH2N), 46.29 (CH3N), 34.97 (C4), 30.07
(CH3), 28.22 (CH3), 16.72 (d, J ¼ 5.8 Hz, CH3CH2OP), 16.66 (d,
J ¼ 5.7 Hz, CH3CH2OP); 31P NMR (243 MHz, CDCl3): d ¼ 22.34. Anal.
calcd. for C16H26N5O6P: C, 46.26; H, 6.31; N, 16.86. Found: C, 46.01;
H, 6.50; N, 16.93 (obtained on a 15:85 mixture of cis-11n and trans-
11n).
Compound trans-11p: White solid (crystallized from mixture of
chloroform–hexane) m.p. 150–151°C. IR (KBr, cmꢀ1) nmax: 2991
nC–H, 1707 nCꢀꢀO, 1667 nCꢀꢀ , 1232 n O, 1033 nP–O.
1H NMR
ꢀꢀ
P
ꢀꢀ
O
ꢀꢀ
ꢀꢀ
(300 MHz, CDCl3): d ¼ 7.52 (s, 1H), 4.35 (dddd, J ¼ 7.5 Hz, J ¼ 7.2 Hz,
J ¼ 7.2 Hz, J ¼ 4.8 Hz, 1H, HC5), 4.33 (dd, J ¼ 13.2 Hz, J ¼ 7.5 Hz, 1H,
HCH), 4.23–4.11 (m, 4H, 2 ꢃ CH2OP), 4.08 (dd, J ¼ 13.2 Hz, J ¼ 4.8
Hz, 1H, HCH), 3.98 (s, 3H CH3), 3.57 (s, 3H CH3), 3.07 (ddd,
J ¼ 9.6 Hz, J ¼ 7.2 Hz, J ¼ 2.4 Hz, 1H, HC3), 2.89 (d, J ¼ 0.9 Hz, 3H,
CH3N), 2.62 (dddd, J ¼ 18.3 Hz, J ¼ 12.3 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz,
1H, H0C4), 2.37 (dddd, J ¼ 12.3 Hz, J ¼ 12.3 Hz, J ¼ 9.6 Hz, J ¼ 7.2 Hz,
1H, HC4), 1.34 (t, J ¼ 7.2 Hz, 3H, CH3CH2OP), 1.33 (t, J ¼ 7.2 Hz, 3H,
Diethyl 5-((8-chloro-1,2,3,6-tetrahydro-1,3-dimethyl-2,6-
dioxopurin-7-yl)methyl)-2-methylisoxazolidin-3-yl-3-
phosphonates cis-11o and trans-11o
From nitrone 14 (0.077 g, 0.39 mmol) and N5-allyl-6-chloro-
1,3-dimethylxanthine 15o (0.100 g, 0.390 mmol) in toluene–
ethanol, pure trans-11o (0.018 g, 11%) was obtained followed by
fractions containing various mixtures of cis-11o and trans-11o
(0.154 g, 87%) by column chromatography (chloroform–metha-
nol; 50:1 v/v).
CH3CH2OP); 13C NMR (75.5 MHz, CDCl ): d ¼ 154.94 (C O), 151.34
ꢀ
ꢀ
3
ꢀꢀ
ꢀꢀ
(C O), 148.76, 141.62, 107.48, 74.74 (d, J ¼ 7.4 Hz, C5), 63.81 (d,
J ¼ 168.3 Hz, C3), 63.17 (d, J ¼ 6.6 Hz, CH2OP), 62.43 (d, J ¼ 6.9 Hz,
CH2OP), 46.59 (d, J ¼ 4.9 Hz), 43.60, 36.38 (d, J ¼ 2.3 Hz, C4), 33.72
(CH3), 29.86 (CH3), 16.68 (d, J ¼ 4.3 Hz, CH3CH2OP), 16.62 (d,
J ¼ 5.7 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 23.38.
Anal. calcd. for C16H26N5O6P: C, 46.26; H, 6.31; N, 16.86. Found: C,
46.36; H, 6.41; N, 16.90.
Compound cis-11o: IR (film, cmꢀ1) nmax: 2923 nC–H, 1703 nCꢀꢀ
,
O
ꢀꢀ
1655 nCꢀꢀO, 1256 nPꢀO, 1018 nP–O. NMR signals of cis-11o were
extracted from the spectrum of a 93:7 mixture of cis-11o and trans-
1
11o; H NMR (600 MHz, CDCl3): d ¼ 4.62–4.53 (m, 1H, HC5), 4.57
3-Benzoyl-1-[3-(diethoxyphosphoryl)-2-methyl-
isoxazolidin-5-ylmethyl]-1H-pyrimidine-2,4-diones cis-11q
(dd, J ¼ 12.7 Hz, J ¼ 3.2 Hz, 1H, HCH), 4.35–4.19 (m, 5H, HCH and
2 ꢃ CH2OP), 3.58 (s, 3H, CH3), 3.42 (s, 3H, CH3), 2.92 (ddd, J ¼ 9.5 Hz,
J ¼ 8.3 Hz, J ¼ 2.2 Hz, 1H, HC3), 2.84 (s, 3H, CH3N), 2.81 (dddd,
J ¼ 17.2 Hz, J ¼ 12.8 Hz, J ¼ 9.5 Hz, J ¼ 7.6 Hz, 1H, H0C4), 2.38 (dddd,
J ¼ 12.8 Hz, J ¼ 11.9 Hz, J ¼ 8.3 Hz, J ¼ 3.5 Hz, 1H, HC4), 1.42 (t,
J ¼ 7.0 Hz, 3H, CH3CH2OP), 1.39 (t, J ¼ 7.2 Hz, 3H, CH3CH2OP); 13C
and trans-11q
From nitrone 14 (0.076 g, 0.39 mmol) and N1-allyl-N3-benzoylur-
acil 15q (0.100 g, 0.45 mmol) in toluene, pure cis-11q (0.066 g,
38%), trans-11q (0.025 g, 14%) and fractions containing various
mixtures of cis-11q and trans-11q (0.052 g, 30%) were obtained
after column chromatography (chloroform–methanol from 200:1
to 50:1 v/v).
ꢀꢀ
ꢀꢀ
ꢀꢀ
NMR (75.5 MHz, CDCl ): d ¼ 154.47 (C O), 151.32 (C O), 147.54,
ꢀꢀ
3
140.20, 107.40, 75.09 (d, J ¼ 7.7 Hz, C5), 63.59 (d, J ¼ 168.0 Hz, C3),
63.22 (d, J ¼ 6.3 Hz, CH2OP), 62.75 (d, J ¼ 6.3 Hz, CH2OP), 48.65
(CH2N), 45.40 (CH3N), 35.80 (C4), 30.15 (CH3), 28.32 (CH3), 16.87 (d,
J ¼ 8.0 Hz, CH3CH2OP); 16.78 (d, J ¼ 6.0 Hz, CH3CH2OP); 31P NMR
(121.5 MHz, CDCl3): d ¼ 23.24. Anal. calcd. for C16H25ClN5O6P ꢃ
H2O: C, 41.08; H, 5.82; N, 14.97. Found: C, 40.99; H, 5.83; N, 14.80
(obtained on a 9:1 mixture of cis-11o and trans-11).
Compound cis-11q: Colorless oil. IR (film, cmꢀ1) nmax: 2982 nC–H
,
1748 nC¼O, 1704 nC¼O, 1661 nC¼C, 1441 nC¼C, 1247 nP¼O, 1025 nP–O
.
1H NMR (300 MHz, CDCl3): d ¼ 7.96–7.93 (m, 2H), 7.68–7.60 (m,
2H), 7.53–7.47 (m, 2H), 7.45 (d, J ¼ 7.8 Hz, 1H), 5.78 (dd, J ¼ 7.8 Hz,
1H), 4.45 (dddd, J ¼ 9.6 Hz, J ¼ 8.1 Hz, J ¼ 3.6 Hz, J ¼ 3.3 Hz, 1H,
HC5), 4.26–4.16 (m, 4H, 2 ꢃ CH2OP); 4.10 (dd, J ¼ 14.4 Hz, J ¼ 3.3
Hz, 1H, HCH), 3.79 (dd, J ¼ 14.4 Hz, J ¼ 9.6 Hz, 1H, HCH), 2.97 (ddd,
J ¼ 11.4 Hz, J ¼ 7.5 Hz, J ¼ 3.3 Hz, 1H, HC3), 2.88 (d, J ¼ 0.9 Hz, 3H,
CH3N), 2.74 (dddd, J ¼ 12.9 Hz, J ¼ 11.4 Hz, J ¼ 11.4 Hz, J ¼ 8.1 Hz,
1H, HC4), 2.25 (dddd, J ¼ 18.3 Hz, J ¼ 11.4 Hz, J ¼ 7.5 Hz, J ¼ 3.6 Hz,
1H, H’C4), 1.37 (t, J ¼ 7.2 Hz, 6H, 2 ꢃ CH3CH2OP); 13C NMR
(75.5 MHz, CDCl3): d¼168.6 (C¼O), 162.58 (C¼O), 149.93 (C¼O),
146.08, 135.17, 131.51, 130.57, 129.24, 101.54, 74.00 (d, J ¼ 6.9 Hz,
C5), 63.59 (d, J ¼ 170.0 Hz, C3), 63.03 (d, J ¼ 6.9 Hz, CH2OP); 62.55 (d,
J ¼ 6.8 Hz, CH2OP), 50.57 (CH2N), 45.98 (CH3N), 35.29 (C4), 16.88 (d,
J ¼ 4.6 Hz, CH3CH2OP), 16.80 (d, J ¼ 4.5 Hz, CH3CH2OP); 31P NMR
(121.5 MHz, CDCl3): d¼23.25. Anal. calcd. for C20H26N3O7P: C,
53.21; H, 5.81; N, 9.31. Found: C, 53.26; H, 5.90; N, 9.10.
Compound trans-11o: Colorless oil. IR (film, cmꢀ1) nmax: 2920
nC–H, 1708 nCꢀꢀO, 1675 nCꢀꢀ , 1231 n O, 1022 nP–O.
1H NMR
ꢀꢀ
P
ꢀꢀ
O
ꢀꢀ
ꢀꢀ
(300 MHz, CDCl3): d ¼ 4.64–4.54 (m, 1H, HCH), 4.40 (dd, J ¼ 14.7 Hz,
J ¼ 7.5 Hz, 1H, HCH), 4.49–4.35 (m, 1H, HC5), 4.23–4.12 (m, 4H,
2 ꢃ CH2OP), 3.56 (s, 3H, CH3), 3.40 (s, 3H, CH3), 3,00 (ddd, J ¼ 9.6 Hz,
J ¼ 7.2 Hz, J ¼ 2.7 Hz, 1H, HC3), 2.85 (d, J ¼ 0.6 Hz, 3H, CH3N), 2.69
(dddd, J ¼ 18.0 Hz, J ¼ 12.6 Hz, J ¼ 7.2 Hz, J ¼ 7.2 Hz, 1H, H0C4), 2.33
(dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz, J ¼ 9.6 Hz, J ¼ 7.2 Hz, 1H, HC4), 1.35
(t, J ¼ 7.2 Hz, 3H, CH3CH2OP), 1.34 (t, J ¼ 7.2 Hz, 3H, CH3CH2OP);
13
ꢀꢀ
ꢀꢀ
ꢀꢀ
C NMR (75.5 MHz, CDCl ): d ¼ 154.39 (C O), 151.25 (C O),
ꢀꢀ
3
147.34, 139.46, 107.80, 75.26 (d, J ¼ 7.7 Hz, C5), 64.02 (d,
J ¼ 168.3 Hz, C3), 63.42 (d, J ¼ 6.6 Hz, CH2OP), 62.71 (d, J ¼ 7.2 Hz,
CH2OP), 48.65 (CH2N), 46.45 (CH3N), 35.58 (C4), 30.15 (CH3), 28.30
(CH3), 16.78 (d, J ¼ 6.0 Hz, CH3CH2OP), 16.74 (d, J ¼ 5.7 Hz,
CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 22.41. Anal. calcd.
for C16H25ClN5O6P ꢃ H2O: C, 41.08; H, 5.82; N, 14.97. Found: C,
41.04; H, 5.93; N, 14.82.
Compound trans-15q: Colorless oil. IR (film, cmꢀ1) nmax: 2927 nC–
H, 1706 nC¼O, 1654 nC¼O, 1437 nC¼C, 1234 nP¼O, 1025 nP–O. 1H NMR
(300 MHz, CDCl3): d ¼ 7.95–7.91 (m, 2H), 7.69–7.63 (m, 2H), 7.53–
7.47 (m, 2H), 7.38 (d, J ¼ 8.1Hz, 1H), 5.81 (dd, J ¼ 8.1 Hz, 1H), 4.32–
4.23 (m, 1H, HC5), 4.22–4.11 (m, 4H, 2 ꢃ CH2OP), 4.17 (dd, J ¼ 14.4
Hz, J ¼ 3.0 Hz, 1H, HCH), 3.67 (dd, J ¼ 14.4 Hz, J ¼ 7.5 Hz, 1H, HCH),
2.91 (ddd J ¼ 10.5 Hz, J ¼ 6.9 Hz, J ¼ 2.1 Hz, 1H, HC3), 2.90 (d, J ¼ 0.9
Hz, 3H, CH3N), 2.62 (dddd, J ¼ 18.0 Hz, J ¼ 12.6 Hz, J ¼ 6.9 Hz,
J ¼ 6.9 Hz, 1H, H’C4), 2.19 (dddd, J ¼ 12.6 Hz, J ¼ 12.6 Hz, J ¼ 10.5
Diethyl 5-((2,3-dihydro-3,7-dimethyl-2,6-dioxo-6H-purin-
1(7H)-yl)methyl)-2-methylisoxazolidin-3-yl-3-phosphonates
cis-11p and trans-11p
From nitrone 14 (0.089 g, 0.45 mmol) and N-allylteobromine 15p
(0.100 g, 0.45 mmol), in toluene–ethanol, pure trans-11p (0.034 g,
19%) was obtained followed by fractions containing various
mixtures of cis-11p and trans-11p (0.063 g, 34%) after column
chromatography (chloroform–methanol; 100:1–20:1 v/v).
Hz, J ¼ 7.8 Hz, 1H, HC4); 1.33 (t, J ¼ 6.9 Hz, 6H, 2 ꢃ CH3CH2OP); 13
C
NMR signals of cis-11q were extracted from the spectrum of a 2:8
mixture of cis-11q and trans-11q, 13C NMR (151 MHz, CDCl3):
d ¼ 168.66 (C¼O), 162.32 (C¼O), 145.01 (C¼O), 130.46, 131.39,
129.21, 75.14 (d, J ¼ 6.9 Hz, C5), 64.00 (d, J ¼ 167.6 Hz, C3), 63.25
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

ꢀ
ꢀ
ꢀ
ꢀ
352
M. Łysakowska et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
(d, J ¼ 6.4 Hz, CH2OP), 62.54 (d, J ¼ 6.2 Hz, CH2OP), 49.75 (CH2N),
46.26 (CH3N), 35.14 (C4), 16.53 (d, J ¼ 5.4 Hz, CH3CH2OP), 16.49 (d,
J ¼ 5.6 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 22.37.
Anal. calcd. for C20H26N3O7P: C, 53.21; H, 5.81; N, 9.31. Found: C,
52.97; H, 6.08; N, 9.35.
(0.013 g, 2.4%), trans-11s (0.206 g, 39%), and fractions containing
various mixtures of cis-11s and trans-11s (0.299 g, 56%) were
obtained after column chromatography (chloroform–methanol;
from 200:1 to 20:1 v/v).
Compound cis-11s: Colorless oil. IR (film, cmꢀ1) nmax: 2980 nC–H
,
1226 nPꢀO, 1024 nP–O
.
1H NMR (300 MHz, CDCl3): d ¼ 4.35 (dddd,
J ¼ 11.1 Hz, J ¼ 7.5 Hz, J ¼ 7.5 Hz, J ¼ 3.9 Hz, 1H, HC5), 4.25–4.14 (m,
4H, 2 ꢃ CH2OP), 3.71 (t, J ¼ 4.5 Hz, 4H, 2 ꢃ CH2O), 3.00 (ddd,
J ¼ 8.7 Hz, J ¼ 8.7 Hz, J ¼ 3.9 Hz, 1H, HC3), 2.81 (d, J ¼ 1.2 Hz, 3H,
CH3N), 2.71–2.45 (m, 7H, HC4, CH2N and 2 ꢃ CH2N), 2.24 (dddd,
J ¼ 18.6 Hz, J ¼ 12.9 Hz, J ¼ 8.7 Hz, J ¼ 7.5 Hz, 1H, H0C4), 1.34 (t,
J ¼ 7.5 Hz, 6H, 2 ꢃ CH3CH2OP); 13C NMR signals of cis-11s were
extracted from the spectrum of a 8:2 mixture of cis-11s and trans-
11s, 13C NMR (151 MHz, CDCl3): d ¼ 74.19 (d, J ¼ 7.5 Hz, C5), 66.87
(2 ꢃ CH2O), 64.06 (d, J ¼ 171.8 Hz, C3), 62.94 (d, J ¼ 6.6 Hz, CH2OP),
62.51 (d, J ¼ 7.2 Hz, CH2OP), 61.89, 54.19, 46.62 (d, J ¼ 8.4 Hz), 36.96
(C4), 16.50 (d, J ¼ 5.4 Hz, CH3CH2OP), 16.48 (d, J ¼ 5.4 Hz,
CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 23.92. Anal. calcd.
for C13H27N2O5P ꢃ H2O: C, 45.88; H, 8.59; N, 8.23. Found: C, 45.91;
H, 8.71; N, 8.34.
3-Benzoyl-1-[3-(diethoxyphosphoryl)-2-methyl-
isoxazolidin-5-ylmethyl]-1H-quinazoline-2,4-diones
cis-11r and trans-11r
From nitrone 14 (0.073 g, 0.37 mmol) and N1-allyl-N3-benzoylbenz-
uracil 15r (0.114 g, 0.370 mmol) in toluene, pure cis-11r (0.020 g,
11%), trans-11r (0.075 g, 43%), and fractions containing various
mixtures of cis-11r and trans-11r (0.043, 25%) were obtained after
column chromatography (chloroform–methanol from 200:1 to
50:1 v/v).
Compound cis-11r: Colorless oil. IR (film, cmꢀ1) nmax: 2978 nC–H
,
1700 nCꢀꢀO, 1662 nCꢀꢀO, 1607 nCꢀꢀC, 1478 nCꢀꢀ , 1240 n O, 1012 nP–O
.
ꢀꢀ
P
ꢀꢀ
C
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
1H NMR (300 MHz, CDCl3): d ¼ 8.19 (dd, J ¼ 8.1 Hz, J ¼ 1.3 Hz), 8.00–
7.96 (m, 2H), 7.84 (brd, J ¼ 8.1 Hz), 7.73 (ddd, J ¼ 8.1 Hz, J ¼ 6.9 Hz,
J ¼ 1.5 Hz), 7.52–7.47 (m, 2H), 7.29 (dt, J ¼ 8.1 Hz, J ¼ 1.3 Hz), 4.59
(dddd, J ¼ 9.9 Hz, J ¼ 8.4 Hz, J ¼ 3.6 Hz, J ¼ 2.9 Hz, 1H, HC5), 4.32
(dd, J ¼ 14.4 Hz, J ¼ 9.9 Hz, 1H, HCH), 4.28–4.18 (m, 4H, 2 ꢃ CH2OP),
4.18 (dd, J ¼ 14.4 Hz, J ¼ 2.9 Hz, 1H, HCH), 2.94 (ddd, J ¼ 9.9 Hz,
J ¼ 7.8 Hz, J ¼ 2.4 Hz, 1H, HC3), 2.83 (d, J ¼ 1.2 Hz, 3H, CH3N), 2.79
(dddd, J ¼ 12.6 Hz, J ¼ 9.9 Hz, J ¼ 8.4 Hz, J ¼ 8.4 Hz, 1H, H0C4), 2.36
(dddd, J ¼ 18.6 Hz, J ¼ 12.6 Hz, J ¼ 7.8 Hz, J ¼ 3.6 Hz, 1H, HC4), 1.39
(t, J ¼ 7.2 Hz, 3H, CH3CH2OP), 1.38 (t, J ¼ 7.2 Hz, 3H, CH3CH2OP);
13C NMR signals of cis-11r were extracted from the spectrum of a
1:1 mixture of cis-11r and trans-11r, 13C NMR (75.5 MHz, CDCl3):
Compound trans-11s: Colorless oil. IR (film, cmꢀ1) nmax: 2855
nC–H, 1236 n O, 1024 nP–O
.
1H NMR (300 MHz, CDCl3): d ¼ 4.25–
ꢀꢀ
P
ꢀꢀ
4.13 (m, 5H, 2 ꢃ CH2OP, HC5), 3.71 (t, J ¼ 4.8 Hz, 4H, 2 ꢃ CH2O)
2.92–2.87 (m, 1H, HC3), 2.87 (d, J ¼ 0.9 Hz, 3H, CH3N), 2.64–2.48 (m,
7H, H0C4, CH2N and 2 ꢃ CH2N), 2.18 (dddd, J ¼ 12.3 Hz, J ¼ 12.3 Hz,
J ¼ 10.5 Hz, J ¼ 8.4 Hz, 1H, HC4), 1.35 (t, J ¼ 7.2 Hz, 6H, 2 ꢃ
CH3CH2OP); 13C NMR (75.5 MHz, CDCl3): d ¼ 75.03 (d, J ¼ 6.9 Hz,
C5), 66.80 (2 ꢃ CH2O), 63.59 (d, J ¼ 167.8 Hz, C3), 63.07 (d,
J ¼ 6.6 Hz, CH2OP), 62.43 (d, J ¼ 6.9 Hz, CH2OP), 61.51, 54.16,
46.20, 36.94 (C4), 16.66 (d, J ¼ 4.0 Hz, CH3CH2OP), 16.58 (d,
J ¼ 4.0 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3): d ¼ 23.82.
Anal. calcd. for C13H27N2O5P ꢃ H2O: C, 45.88; H, 8.59; N, 8.23.
Found: C, 45.79; H, 8.70; N, 8.25.
ꢀꢀ
ꢀꢀ
ꢀꢀ
d ¼ 168.74 (C O), 161.28 (C O), 149.65 (C O), 140.76, 135.68,
ꢀꢀ
ꢀꢀ
ꢀꢀ
135.07, 131.77, 130.53, 128.98, 128.46, 123.54, 116.23, 115.38,
75.17 (d, J ¼ 7.3 Hz, C5), 64.65 (d, J ¼ 170.7 Hz, C3), 63.37 (d,
J ¼ 6.1 Hz, CH2OP), 63.05 (d, J ¼ 6.9 Hz, CH2OP), 46.71 (CH2N), 45.46
(CH3N), 35.73 (d, J ¼ 1.7 Hz, C4), 16.80 (d, J ¼ 6.1 Hz, CH3CH2OP),
16.72 (d, J ¼ 5.9 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3):
d ¼ 23.55. Anal. calcd. for C24H28N3O7P ꢃ 0.5H2O: C, 56.47; H, 5.73;
N, 8.23. Found: C, 56.47; H, 5.48; N, 8.29.
Antiviral activity assays
The compounds were evaluated against the following viruses:
herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-
deficient (TKꢀ) HSV-1 KOS strain resistant to ACV (ACVr), herpes
simplex virus type 2 (HSV-2) strains Lyons and G, varicella-zoster
virus (VZV) strain Oka, TKꢀ VZV strain 07–1, human cytomegalo-
virus (HCMV) strains AD-169 and Davis, vaccinia virus Lederle
strain, respiratory syncytial virus (RSV) strain Long, vesicular
stomatitis virus (VSV), Coxsackie B4, parainfluenza 3, influenza
virus A (subtypes H1N1, H3N2), influenza virus B, Reovirus-1,
Sindbis, Reovirus-1, Punta Toro, human immunodeficiency virus
type 1 strain IIIB, and human immunodeficiency virus type 2
strain ROD. The antiviral, other than anti-HIV, assays were based
on inhibition of virus-induced cytopathicity or plaque formation
in human embryonic lung (HEL) fibroblasts, African green
monkey cells (Vero), human epithelial cells (HeLa), or Madin-
Darby canine kidney cells (MDCK). Confluent cell cultures in
microtiter 96-well plates were inoculated with 100 CCID50 of virus
(1 CCID50 being the virus dose to infect 50% of the cell cultures)
or with 20 plaque-forming units (PFU) (VZV) in the presence
of varying concentrations of the test compounds. Viral cytopa-
thicity or plaque formation was recorded as soon as it reached
completion in the control virus-infected cell cultures that were
not treated with the test compounds. Antiviral activity was
expressed as the EC50 or compound concentration required to
reduce virus-induced cytopathogenicity or viral plaque formation
by 50%.
Compound trans-11r: White solid (crystallized from mixture of
ethyl ether–hexane) m.p. 105–106°C. IR (KBr, cmꢀ1) nmax: 2979
nC–H, 1702 nCꢀꢀO, 1664 nCꢀꢀ , 1239 n O, 1051 nP–O.
1H NMR
ꢀꢀ
P
ꢀꢀ
O
ꢀꢀ
ꢀꢀ
(300 MHz, CDCl3): d ¼ 8.24 (dd, J ¼ 8.1 Hz, J ¼ 1.8 Hz), 8.00–7.98 (m,
2H), 7.76 (brdd, J ¼ 1.5 Hz, J ¼ 8.1 Hz), 7.67 (brt, J ¼ 8.1 Hz); 7.54–
7.49 (m, 2H), 7.34 (brt, J ¼ 8.1 Hz), 4.50 (dd, J ¼ 14.4 Hz, J ¼ 3.6 Hz,
1H, HCH), 4.32–4.23 (m, 1H, HC5), 4.23–4.12 (m, 4H, 2 ꢃ CH2OP),
4.15 (dd, J ¼ 14.4 Hz, J ¼ 7.2 Hz, 1H, HCH), 3.08–2.98 (m, 1H, HC3),
2.88 (s, 3H, CH3N), 2.70 (dddd, J ¼ 19.5 Hz, J ¼ 12.9 Hz, J ¼ 6.9 Hz,
J ¼ 6.9 Hz, 1H, H0C4), 2.43 (dddd, J ¼ 12.9 Hz, J ¼ 12.9 Hz, J ¼ 10.5
Hz, J ¼ 8.1 Hz, 1H, HC4), 1.33 (t, J ¼ 7.2 Hz, 6H, 2 ꢃ CH3CH2OP); 13
C
NMR (75.5 MHz, CDCl ): d ¼ 168.52 (C O), 160.98 (C O), 149.72
3
ꢀꢀ
(C O), 140.70, 135.84, 135.16, 131.61, 130.56, 129.25, 128.99,
ꢀꢀ
123.81, 115.64, 115.26, 75.35 (d, J ¼ 7.5 Hz, C5), 64. 65 (d,
J ¼ 167.1 Hz, C3), 62.81 (d, J ¼ 6.9 Hz, CH2OP); 62.68 (d, J ¼ 6.9 Hz,
CH2OP), 46.50, 45.65, 36.18 (C4), 16.82 (d, J ¼ 5.7 Hz, CH3CH2OP),
16.74 (d, J ¼ 5.7 Hz, CH3CH2OP); 31P NMR (121.5 MHz, CDCl3):
d ¼ 22.64. Anal. calcd. for C24H28N3O7P ꢃ 0.5H2O: C, 56.47; H, 5.73;
N, 8.23. Found: C, 56.66; H, 5.65; N, 8.04.
Diethyl 2-methyl-5-(morpholinomethyl)isoxazolidin-3-yl-3-
phosphonates cis-11s and trans-11s
From nitrone 14 (0.322 g, 1.65 mmol) and N-allylmorpholine 15s
(0.210 g, 1.65 mmol) in toluene, pure isoxazolidines cis-11s
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 341–353
Isoxazolidine Analogs of Homonucleotides
353
Anti-HIV activity assays
[11] U. Chiacchio, F. Genovese, D. Iannazzo, V. Librando,
P. Merino, A. Rescifina, R. Romeo, A. Procopio, G. Romeo,
Tetrahedron 2004, 60, 441–448.
Inhibition of HIV-1(IIIB)- and HIV-2(ROD)-induced cytopathicity in
CEM cell cultures was measured in microtiter 96-well plates
containing ꢄ3 ꢃ 105 CEM cells/mL infected with 100 CCID50 of
HIV per milliliter and containing appropriate dilutions of
the test compounds. After 4–5 days of incubation at 37°C in a
CO2-controlled humidified atmosphere, CEM giant (syncytium)
cell formation was examined microscopically. The 50% effective
concentration (EC50) was defined as the compound concentration
required to inhibit HIV-induced giant cell formation by 50%.
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Cytostatic activity assays
All assays were performed in 96-well microtiter plates. To each
well, (5–7.5) ꢃ 104 tumor cells and a given amount of the test
compound were added. The cells were allowed to proliferate for
48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic
CEM and human cervix carcinoma HeLa cells) at 37°C in a
humidified CO2-controlled atmosphere. At the end of the
incubation period, the cells were counted in a Coulter counter.
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inhibited cell proliferation by 50%.
_
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The synthetic part of this work was supported by the Medical University
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ꢀ
of Łódz internal funds (503/3-014-1/503-06-300 and 502-03-/3-014-01/502-
4642.
34-030). The financial support of KU Leuven (GOA 10/014) is gratefully
acknowledged. The authors thank Leentje Persoons, Frieda De Meyer,
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The authors have declared no conflict of interest.
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ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com