5′-Epimeric 3′-deoxy-3′,4′-didehydronucleoside- 5′-C-phosphonates: Synthesis and structural assignment by NMR and X-ray analyses

Article abstract of DOI:10.1016/j.tet.2011.04.037

Epimeric 5′-(RS) dialkyl 3′-deoxy-3′,4′-didehydro- 5′-C-phosphonates were prepared by nucleophilic addition of various dialkyl phosphites to 3′-deoxy-3′,4′-didehydronucleoside- 5′-aldehydes. Whereas direct NMR configuration assignment for the C5′ atom bea

Full text of DOI:10.1016/j.tet.2011.04.037

Tetrahedron 67 (2011) 4227e4235
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
50-Epimeric 30-deoxy-30,40-didehydronucleoside-50-C-phosphonates: synthesis
and structural assignment by NMR and X-ray analyses
*
ꢀ
ꢁ
ꢁꢁ
ꢀ
ꢀꢁ
ꢀ
Magdalena Petrova, Milos Budesínsky, Blanka Klepetarova, Ivan Rosenberg
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo 2, 166 10 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Epimeric 50-(RS) dialkyl 30-deoxy-30,40-didehydro-50-C-phosphonates were prepared by nucleophilic
addition of various dialkyl phosphites to 30-deoxy-30,40-didehydronucleoside-50-aldehydes. Whereas
direct NMR configuration assignment for the C50 atom bearing the phosphoryl and hydroxy groups using
the J (P,H40) and J (H50,H40) coupling constants is impossible due to the absence of the H40 atom, suc-
cessful separation, crystallisation and X-ray crystallographic analysis of a pair of epimeric 50-C-phos-
phonates, followed by correlation with a series of NMR parameters, led to efficacious configuration
assignment of individual epimers in the mixtures.
Article history:
Received 22 December 2010
Received in revised form 16 March 2011
Accepted 11 April 2011
Available online 16 April 2011
Keywords:
Abramov reaction
Hydroxy phosphonates
30,40-Didehydronucleoside C-phosphonates
NMR spectra
Ó 2011 Published by Elsevier Ltd.
Allylic coupling constants
g-gauche Interaction
1. Introduction
The recently reported straightforward, high-yielding synthesis
of 30-deoxy-30,40-didehydronucleoside-50-aldehydes35 6 prompted
Nucleoside phosphonic acids containing a PeC bridging bond
instead of the ester PeO linkage represent a significant class of
compounds, among which efficient antiviral agents have been
found.1e12 A number of structurally diverse nucleoside phosphonic
acids have been synthesised and evaluated in our laboratory, e.g.,
compounds 1aee, which differ in the number of bridging atoms of
the phosphoester mimic13e26 (Fig. 1). Some of these compounds
have interesting biological properties.27e29
us to synthesise 50-epimeric 30,40-didehydronucleoside-50-hydroxy
phosphonates 8 (Scheme 2). These compounds combine in their
scaffold both an enzymatically stable hydroxy phosphonate moiety
with bridging PeC bond mimicking the phosphoester group and
a 30,40-unsaturated36 nucleoside motif with the 20-hydroxy group.
Its presence in the molecule allows to consider the compounds 8 as
ribonucleoside-50-phosphate analogues, similarly as the 30-deo-
xynucleotides, which are recognized by RNA-polymerases as
HO
HO
O
HO
HO
A
HO
HO
O
O
HO
HO
O
O
OH
O
P
O
B
P
B
A
O
B
P
P
HO
HO
O
O
O
P
H3C
O
OH H (OH)
1e
OH H (OH)
1a
HO
OH
OH
1d
HO
OH
1c
1b
Fig. 1. Examples of isopolar nucleoside 50-phosphonic acids.
Geminal 50-hydroxy phosphonates 1e, described byWiemer30e32
and our group,20,21,33,34 are isopolar, non-isosteric 50-nucleotide
analogues possessing chirality at the C50 atom. These compounds
have not been fully biologically evaluated, although some bio-
chemical experiments with arabinocytosine 50-hydroxy phospho-
nate have been performed.32
substrates, the chain terminators. Due to the absence of the H40
atom in compounds 8, the previously reported direct NMR assign-
mentof both the C50-R/S configuration and conformation around the
C40eC50 linkage,20 exploiting the characteristic values of the J (P,H40)
and J (H50,H40) coupling constants, was inapplicable, and thus, the
C50-R/S assignment appeared to be a challenging problem.
Abramov nucleophilic addition of dialkyl phosphites to carbonyl
compounds is a straightforward, high-yielding reaction for the
* Corresponding author. E-mail address: rosenberg@uochb.cas.cz (I. Rosenberg).
0040-4020/$ e see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.tet.2011.04.037

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M. Petrova et al. / Tetrahedron 67 (2011) 4227e4235
4228
preparation of geminal hydroxy phosphonates.37 To produce nu-
cleoside 50-hydroxy phosphonates,20,34 20,30-O-protected ribonu-
cleosides were oxidised to 50-aldehydes using a modified Moffat
procedure (DMSO/DCC in the presence of TFA and pyridine), and
these compounds were subjected tothe Et3N-mediated nucleophilic
addition of dialkyl phosphites. As we have already reported,35 the
20,30-O-methoxymethylidene ribonucleoside-50-aldehydes 3, pre-
pared in situ from the respective 20,30-O-methoxymethylidene ri-
bonucleosides 2, underwent the elimination of the orthoester
moiety upon a basic treatment, resulting in the formation of
30-deoxy-30,40-didehydro-50-aldehydes 6 (Scheme 1).
diesters. The obtained diesters 7aee were smoothly deprotected via
-elimination in 8 M methylamine in ethanol at 50 ꢀC (removal of
b
both 2-cyanoethyl groups and the N-protecting groups), and free
acids were converted into sodium salts of 8a,eeh on Dowex 50 (Naþ
form) in highyields (84e90%;Scheme 2). Interestingly, thereactionof
30-deoxy-30,40-didehydronucleoside-50-aldehydes
6 with dialkyl
phosphites under Et3N catalysis also proceeded very well in aqueous
solution. In this case, the crystalline 50R/S diethyl phosphonate 9 was
obtained in 60% yield (Scheme 2).
To improve the cellular uptake of phosphonic acids and thus,
facilitate drug delivery, phospholipase C-cleavable, lipophilic
monoesters were introduced.39 In our case, 3-hexadecyloxypropyl
methyl phosphite38 was selected as the most convenient phos-
phonylating agent. Upon its reaction with 30-deoxy-30,40-didehy-
dronucleoside-50-aldehydes 6 in DMF in the presence of Et3N and
subsequent treatment with 60% aqueous pyridine at 50 ꢀC (removal
of the methyl ester group), followed by treatment with methanolic
ammonia or 8 M methylamine in EtOH to remove N-acyl groups, we
obtained
3-hexadecyloxypropyl
30-deoxy-30,40-didehydro-50-
C-phosphonates 10a,feh (21e62% yields with respect to aldehydes
6aed; Scheme 2). Monoesters 10feh were converted into Naþ salts
in excess of saturated sodium bicarbonate solution upon heating,
followed by desalting on a C18 column in a water/methanol mix-
ture. Monoester 10a was isolated in the form of an Et3NHþ salt.
Since the synthesised 30-deoxy-30,40-didehydronucleoside-50-
C-phosphonates 5a,b, 7aee, 8a,eeh, 9 and 10a,feh were obtained as
50-R and 50-S epimeric mixtures, a methodology for their separation
and identification is highly desirable. In most cases, according to
HPLC and NMR analyses, both epimers were formed in a nearlyequal
ratio. Whereas only partial separation of the 50-R and 50-S epimers of
the bis(2-cyanoethyl) phosphonates of purine nucleosides 7b and
7d was achieved by HPLC on a C18 column in water/acetonitrile,
complete separation of epimers of uracil diethyl phosphonate 9 was
accomplished in water/methanol. Epimers (50S)-9 and (50R)-9 were
successfully crystallised from ethanol and subjected to X-ray anal-
ysis. The crystal structures of both the 50-R and 50-S epimers of 9
were very similar providing us by important findings. Thus, dihy-
drofuran ring is nearly planar, the uracil base adopts the anti ori-
entation and, surprisingly, the phosphorus moiety in both 50-R and
50-S epimers is oriented cis to the uracil nucleobase. The torsion
Scheme 1. Synthesis of phosphonates 4 and 5 from 20,30-O-orthoester 2.
Accordingly, when HP(O)(OMe)2 was added to the in situ pre-
pared 20,30-O-methoxymethylidene ribonucleoside-50-aldehyde 3,
prior to Et3N, in addition to 20,30-O-methoxymethylidene-50-C-
phosphonates 4, 30,40-didehydro derivatives 5 were also formed. On
the contrary, the treatment of the oxidation mixture with Et3N
followed by the addition of HP(O)(OMe)2 after a period of 15 min
lead to exclusive formation of dimethyl 30-deoxy-30,40-didehydro-
50C-phosphono derivatives 5. For practical reasons, the synthesis
of 30,40-unsaturated phosphono derivatives 7e10 was started from
30-deoxy-30,40-didehydronucleoside-50-aldehyde35 6 (Scheme 2).
angle
g
around the C40eC50 linkage is approximately 105ꢀ. The 50-R
and 50-S epimers differ only in the orientation of the 50eOH group
and the H50 proton (see Fig. 2 and Table 1). The found cis position of
the phosphorus moiety and nucleobase underlines the structural
similarity of compounds 8 with natural nucleotides, and thus, the
synthesis and biological evaluation of these compounds as nucleo-
tide analogues seem to be justify.
Scheme 2. Synthesis and isolated yields (%) of phosphono derivatives 7e10.
To obtain free phosphonic acids 8, the dimethyl esters 5a,b were
treated with bromotrimethyl silane in acetonitrile in the presence of
2,6-lutidine. However, during this procedure, substantial cleavage
of the nucleoside bond was observed. Therefore, we used
bis(2-cyanoethyl) phosphite38 as the phosphonylating agent and
conducted the reaction with 30-deoxy-30,40-didehydronucleoside-50-
aldehydes 6aee in DMF in the presence of Et3N. Thus, bis(2-
cyanoethyl) esters 7aee were obtained as 50R/S epimeric mixtures
in excellent yields (70e90%; Scheme 2). The only drawback of this
Fig. 2. Crystal structures of the (50S)-9 (HPLC-slower) and (50R)-9 (HPLC-faster) epi-
mers (thermal ellipsoids at the 50% probability level). For clarity reasons, only the
more occupied parts of the disordered groups are shown.
The NMR spectra of 50-R and 50-S epimer of 9 in both solvents
(DMSO and D2O) were extremely similar. The very small differences
in the chemical shifts and coupling constants (see Table 3 and Table 4)
and the nearly identical NOE contacts observed do not allow
procedure is the susceptibility of one 2-cyanoethyl group to rapid
b-
elimination, which, in some cases, decreased the overall yields of

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M. Petrova et al. / Tetrahedron 67 (2011) 4227e4235
4229
Table 1
Given the similar preferred conformations of the studied
compounds in crystals and in solution (supported by geometry
optimisation calculation using DFT B3LYP 6-311G**), we used the
correlation of chemical shifts of C30 carbon. Applying the obser-
vation in compound 9, in which the C30 carbon of the 50-S epimer
Comparison of the selected torsion angles of the 50-S and 50-R epimers of 9 in the
crystals
Torsion angle
50S
50Ra
50Ra
<
<
<
<
<
<
<
c
(C2eN1eC10eO40)
ꢁ100.7
5.7
ꢁ102.1
5.4
ꢁ101.8
2.8
n0(C40eO40eC10eC20)
n1(O40eC10eC20eC30)
n2(C10eC20eC30eC40)
n3(C20eC30eC40eO40)
n4(C30eC40eO40eC10)
is shifted upfield (probably as the result of the
g-gauche
ꢁ5.4
ꢁ5.7
ꢁ3.9
interaction of 50-OH with C30dsee Fig. 4) relative to the whole
series of prepared compounds, led to the same assignment of the
configuration at C50 as that derived from the allylic couplings
(compare Table 2).
3.5
4.1
3.7
ꢁ0.2
ꢁ1.0
ꢁ2.3
ꢁ3.7
ꢁ2.9
ꢁ0.4
g
(C30eC40eC50eP)
105.1
ꢁ136.0
ꢁ20.4
ꢁ63.1
102.8
ꢁ15.4
ꢁ133.8
ꢁ67.4
102.8
ꢁ19.9
ꢁ139.5
ꢁ67.0
<(C30eC40eC50eH50)
<(C30eC40eC50eO50)
<(C40eC50ePeO)
The phosphorus-31 chemical shifts of the studied compounds
clearly distinguish between phosphonate diesters 5a,b, 9, 7aee
(d
20.41e23.74), monoesters 10a,feh (
d 12.54e14.05) and free
a
The asymmetric unit contains two slightly different independent molecules of
phosphonic acids 8a,eeh ( 12.32e12.57). However, the chemical
d
(50R)-9.
shift differences between the signals of 50-R and 50-S epimer are
either very small (e.g., 0.05 ppm in C-50 epimers of compound 9) or
even zero (see Table 4), and therefore, these differences are not
usable for correlation with the configuration at the C50 carbon atom.
independent direct assignment of the C50 configuration from NMR
spectra. Therefore, we attempted to use the NMR spectra of the 50-R
and 50-S epimers of 9 to find some correlations of their NMR pa-
rameters with those observed in NMR spectra of epimer mixtures
5a,b, 7aee, 8a,eeh and 10a,feh. As we have already mentioned, the
X-ray structures of the 50-R and 50-S epimers differ only in the ori-
entation of the 50eOH group and H50 proton. This difference should
be most sensitively reflected by the H30 proton. The multiplet pat-
terns of H30 in the spectra of the 50-R and 50-S epimers show nearly
identical values of J (30,20)w2.5 Hz (in accordance with nearly iden-
tical conformation of the dihydrofuran ring in solution), but some-
what larger allylic couplings J (30,50) and J (30,P) for the 50-S epimer
(1.2 and 4.2 Hz) than for the 50-Repimer (0.8 and 3.4 Hz), which could
be explained by the larger angle between the planes containing
interacting atoms in the 50-S epimer (w45ꢀ) in comparison with the
50-R epimer (4ꢀ and 12ꢀ found in two conformers in the crystal
structures), leading to largerallylic couplingsinthe 50-S epimerdueto
2. Conclusion
Novel 50-epimeric 30-deoxy-30,40-didehydronucleoside-50-C-
phosphonates 8 and their 3-hexadecyloxypropyl esters 10 as
prodrugs were prepared by Et3N-mediated addition of the corre-
sponding dialkyl phosphites to 30-deoxy-30,40-didehydronucleoside-
50-aldehydes 6. X-ray analysis of the crystals of both the 50-R and 50-S
epimers of diester 9 showed very similar structuresdthe nearly
planar dihydrofuran ring, the anti orientation of the uracil base and
the cis orientation of the phosphorus moiety and nucleobase in both
epimers. Only the orientation of the 50-hydroxy group and the H50
proton was different. The found cis orientation of phosphorus atom
towards nucleobase underlines, together with 20-hydroxyl the
structural similarity of these compounds with natural ribonucleo-
tides. On the base of X-ray structure of pure 50-R and 50-S epimers of 9
we succeeded in the correlation of their NMR parameters with those
observed in NMR spectra of epimer mixtures. Neither free phos-
phonic acids 8a,eeh nor their prodrugs 10a,feh exhibited activity
against HCV in a replicon system. Further biochemical evaluation of
phosphonates 8 in the form of their diphosphoryl derivatives (NTP
analogues) in RNA polymerase assay is underway, as well as the in-
hibition study on nucleoside phosphorylases, and pyrimidine specific
50-nucleotidases.
a higher p
-contribution to the allylic coupling.40 Detailed analysis of
the 1H NMR spectra revealed similar differences in the H30 multiplet
patterns in the whole series of compounds 5a,b, 7aee, 8a,eeh and
10a,feh; this pattern has been used for the assignment of the con-
figuration at the C50 carbon atom (see examples of H30 multiplet
patterns in Fig. 3 and J-values in Table 2). The allylic couplings J (30,P)
reflect differences between the phosphonate diesters (5a,b, 9, 7aee),
monoesters (10a,feh) and free phosphonates (8a,eeh).
Table 2
Coupling constants of H30 and chemical shifts of C30 in compounds 5a, 5b, 7aee, 8a,eeh, 9 and 10a,feh and configuration assignment at carbon atom C50
Compd.
Solv.
S/R
H30
J (30, P)
J (30, 20)
J (30, 50)
H30
J (30, P)
J (30, 20)
J (30, 50)
C30
Ratio
S-epimer
R-epimer
S-epi
R-epi
Dd (S/R)
5a
5b
7a
7b
7c
7d
7e
8a
8e
D2O
61:39
50:50
52:48
56:44
44:56
40:60
50:50
50:50
46:54
50:50
53:47
5.584
5.497
5.379
5.549
5.426
5.494
5.384
5.466
5.470
5.505
5.334
5.443
5.309
5.100
5.108
4.994
5.097
4.0
4.0
4.3
4.2
4.3
4.3
4.4
2.8
2.8
2.8
2.8
2.8
4.2
3.7
3.5
3.5
3.3
2.8
2.6
2.6
2.6
2.6
2.6
2.6
2.8
2.8
2.8
2.8
2.8
2.6
2.5
2.6
2.6
2.6
1.0
1.2
1.2
1.2
1.1
1.2
1.2
1.2
1.2
1.2
1.1
1.2
1.2
1.2
1.2
1.3
1.1
5.574
5.488
5.372
5.523
5.411
5.479
5.373
5.445
5.450
5.485
5.304
5.458
5.317
5.145
5.150
5.031
5.128
3.7
3.5
3.6
3.8
3.4
3.7
3.5
2.8
2.7
2.6
2.6
2.5
2.5
2.6
2.6
2.5
2.8
2.7
0.8
0.9
0.9
0.9
0.8
0.9
0.8
0.9
0.8
105.64
102.60
103.07
103.18
103.40
102.84
103.05
101.64
101.61
101.36
101.38
100.79
102.35
99.53
106.20
103.36
104.01
103.68
104.13
103.65
104.17
102.39
102.92
102.50
102.02
102.56
103.41
101.23
99.66
ꢁ0.56
ꢁ0.76
ꢁ0.94
ꢁ0.50
ꢁ0.73
ꢁ0.81
ꢁ1.12
ꢁ0.75
ꢁ1.31
ꢁ1.14
ꢁ0.64
ꢁ1.77
ꢁ1.06
ꢁ1.70
ꢁ1.71
ꢁ1.95
ꢁ1.18
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
D2O
D2O
D2O
D2O
D2O
DMSO
DMSO
DMSO
DMSO
DMSO
8f
2.8
2.8
0.7
0.9
8g
8h
9
10a
10f
10g
10h
2.8
2.8
59:41
w2.5b
3.4
w2.5b
2.5
<<1b
0.8
a
58:42
64:36
52:48
49:51
2.6
2.6
w2.5
2.7
2.6
2.6
w2.5
2.7
0.8
0.8
<<1
1.0
97.95
98.42
98.05
100.37
99.23
a
Data for separated C50 epimers.
Line broadening prevented the determination of more accurate data values.
b

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M. Petrova et al. / Tetrahedron 67 (2011) 4227e4235
4230
Table 3
Proton NMR data of compounds 5a,b, 7aee, 8a,eeh, 9, and 10a,feh at 27 ꢀC. Coupling constants are given in brackets
Compd. (Solvent) Config. H-10
H-20
H-30
H-50
H-2
H-5
H-6
H-8c
(popul.)
5a
5b
7a
7b
7c
7d
7e
8a
8e
8f
50S
(w61%)
50R
(w39%)
50S
(w50%)
50R
(w50)
50S
(w52%) (2.4)
50R
(w48%) (2.7)
50S
(w56%) (2.2)
50R
(w44%) (2.8)
50S
(w44%) (2.0)
50R
(w56%) (2.2)
50S
(w40%) (2.7)
50R
(w60%) (2.2)
50S
(w50%) (2.8)
50R
(w50%) (2.5)
50S
(w50%) (1.7)
50R
6.349d (2.1) 5.084m
6.336 d (2.3) 5.102m
5.584 ddd
(4.0, 2.7, 1.0)
5.574 ddd
(3.7; 2.6; 0.8)
5.497 ddd
(4.0; 2.6; 1.2)
5.01 dt
(16.0; w1; w1)
5.03 dt
(15.0; w1; w1)
4.69 ddt
(15.2; 6.9; 1.1; 1.1)
d
5.871 d (8.1)
7.454 d (8.1)
d
(D2O)
d
5.876 d (8.1)
7.452 d (8.1)
d
6.45 d (2.6)
6.465 d (2.0) 5.25m
6.19 d
5.33m
8.797 s
8.801 s
d
d
d
d
d
8.47 s
8.525 s
d
(DMSO)
(DMSO)
(DMSO)
(DMSO)
(DMSO)
(DMSO)
(D2O)
5.488 ddd (3.5; 2.6; 0.9) 4.65 br dd
(16.0; 7.0; 0.9; <1)
4.71 dddd
4.81m
(5.7; 4.0; 2.4; 0.6) (4.3; 2.6; 1.2)
4.84m 5.372 ddd
(5.8; 4.3; 2.7; 1.2) (3.6; 2.5; 0.9)
5.379 ddd
5.665 dd (8.1; 2.1) 7.275 d (8.1);
(15.6; 6.5; 1.2; 0.6)
4.775 dddd
(15.0; 6.9; 1.2; 0.8)
4.74 ddd
(15.9; 6.8; 1.0; <1)
4.78 ddd
(15.3; 6.9; 0.8)
4.816 br dd
(16.0; 6.6)
4.884 br dd
(15.1; 6.9)
4.705 dddd
(15.8; 6.8; 1.2; 0.5)
4.78 ddt
(15.8; 6.8; 1.0; 1.0)
4.80m
(15.4; 6.9; 1.2)
4.72m
(15.6; 6.7; 0.7)
4.43 dt
(14.9; 1.5; 1.2)
4.47 br d
(14.9; 0.9; <0.5)
4.40 dt
(14.4; 1.4; 1.4)
4.47 br d
(14.8; <1)
4.34 dt
(14.7; 1.4; 1.2)
4.45 br d
(15.1; 0.8)
4.32 dt
(14.3; 1.4; 1.2)
4.35 dd
(14.8; 1.0)
4.33 br d
(14.5)
6.17 d
d
5.67 dd (8.1; 2.0)
7.30 d (8.1)
d
d
6.47 d
5.28m
5.549 ddd
(4.2; 2.6; 1.2)
5.523 ddd
(3.8; 2.5; 0.9)
5.426 ddd
(4.3; 2.6; 1.1)
5.411 ddd
8.79 s
8.79 s
d
d
d
8.47 s
8.52 s
d
(5.5; 4.3; 2.2)
5.365m
6.455 d
d
(5.4; 3.7; 2.8)
4.803m
6.283 d
7.350 br d
(w7.5)
7.359 br d
(w7.5)
d
7.964br d (w7.5)
7.936br d (w7.5)
d
6.264 d
4.774m
d
d
(3.4; 2.6; 0.8)
5.494 ddd
6.173 d
5.08 dddd
d
7.94 s
7.99 s
d
(5.8; 4.1; 2.7; 0.5) (4.3; 2.6; 1.2)
5.15 dddd 5.479 ddd
(5.7; 3.6; 2.2; 1.0) (3.7; 2.5; 0.9)
6.187 d
d
d
d
6.18 d
4.83m
5.384 ddd
(4.4; 2.6; 1.2)
5.373 ddd
(3.5; 2.5; 0.8)
5.466 td
d
d
7.16 q (1.2)
7.15 q (1.2)
7.70 d (8.1)
7.82 d (8.1)
7.54 q (1.2)
7.44 q (1.2);
d
(5.9; 2.8; 2.6)
4.78m
6.20 d
d
d
d
(5.8; 2.5; 2.5)
4.985m
6.373
5.944 d (8.1)
d
(2.8; 2.2; 1.7; 1.5) (2.8; 2.8; 1.2)
6.367 d
4.965 ddd
(2.8; 2.0; 1.6)
4.99m
5.445 td
(2.8; 2.8; 0.9)
5.47 td
(2.8; 2.8; 1.2)
5.45 td
(2.7; 2.7; 0.8)
5.505 td
(2.8; 2.8; 1.2)
5.485 td
(2.8; 2.8; 0.7)
5.334 td
(2.8; 2.8; 1.1)
5.304 td
(2.8; 2.8; 0.9)
5.443 td
d
5.940 d (8.1)
d
(w50%) (1.6)
50S
(w46%) (1.9)
50R
6.40 d
d
d
d
(D2O)
6.39 d
4.96m
d
d
d
(w54%) (1.8)
50S
6.460
(w50%) (1.7)
50R
6.467
5.15m
(2.8; 1.7; 1.4)
5.11 dt
8.16 s
8.18 s
d
d
8.21 s
8.25 s
d
(D2O)
d
d
(w50%) (1.6)
50S
(w47%) (1.7)
50R
(w53%) (1.6)
50S
(w59%) (1.8)
50R
(2.8; 1.6; 1.5)
4.79 ddd
8g
8h
9
6.27 d
5.998 d (7.6)
6.001 d (7.5)
d
7.73 d (7.6)
7.59 d (7.5)
d
(D2O)
(4.0; 2.5; 1.7)
4.77 td
6.26 d
d
d
(2.4; 2.2; 1.6)
5.08m
6.27 d
d
7.84 s
7.82 s
d
(D2O)
(2.8; 2.8; 1.2)
5.458 br t
6.26 d
5.05m
4.805m
4.83m
5.08m
4.43 br d
(14.2)
4.52 br d
(16.0)
d
d
d
(w41%) (1.6)
(2.5; 2.5; <1)
5.309 ddd
(4.2; 2.6; 1.2)
5.317 ddd
(3.4; 2.5; 0.8)
5.572 ddd
50S
50R
50S
50R
50S
6.17 d
(2.3)
6.145 d
(2.6)
6.34 d
(2.0)
d
5.62 d (8.1)
5.64 d (8.1)
5.875 d (8.1)
5.87 d (8.1)
5.49 dd (8.0; 2.1)
5.55 dd (8.0; 2.1)
d
7.29 d (8.1)
7.32 d (8.1)
7.47 d (8.1)
7.46 d (8.1)
7.85 d (8.0)
7.57 d (8.0)
d
(DMSO)
4.58 br dd
(15.0; w5.0; 0.8)
4.93 ddd
(16.0; 1.1; 0.6)
4.96 ddd
(15.0; 1.0; 0.8)
4.075 dd
(15.3; 1.0)
4.155 br d
(14.6; <1)
4.02 ddd
(14.9; 5.2; 1.2)
4.04 ddd
(14.3; 5.7; 0.8)
3.98 d
(15.0)
4.06 d
d
d
(D2O)
d
d
(3.9; 2.8; 2.0; 0.6) (4.2; 2.7; 1.1)
5.10m 5.565 ddd
(4.2; 2.6; 2.3; 1.0) (3.6; 2.7; 0.8)
6.33 d
(2.3)
d
d
10a
6.09 d
4.59m
4.655m
4.64m
4.83m
4.40m
4.46m
4.655m
4.923m
5.100 ddd
(3.7; 2.5; 1.2)
5.145 td
(2.7; 2.6; 0.7)
5.108 ddd
(3.5; 2.5; 1.2)
5.150 td
(2.6; 2.6; 0.8)
4.994 ddd
(3.5; 2.6; 1.3)
5.031 br t
(2.5; 2.5; <1)
5.097 ddd
(3.3; 2.5; 1.1)
5.128 td
d
d
(DMSO)
(w58%) (2.2)
50R
(w42%) (1.9)
50S
(w64%) (1.5)
50R
6.10 d
d
d
10fa
(DMSO)
6.25 d
8.14 s
8.155 s
d
8.655 s
8.40 s
d
6.235 d
d
d
(w36%) (2.0)
10ga
(DMSO)
50S
6.115
(w52%) (1.7)
50R
6.155
5.67 d (7.5)
5.62 d (7.5)
d
7.69 d (7.5)
8.02 d (7.5)
d
d
d
(w48%) (1.9)
50S
(w49%) (1.9)
50R
(14.3)
3.985 br t
10ha,b
(DMSO)
6.031 d
d
8.04 s
7.76 s
5.996 d
4.01 br t
d
d
d
(w51%) (2.6)
(2.7; 2.7; 1.0)
a
b
c
The signals of long aliphatic chain could not be structurally assigned.
Measured at 40 ꢀC.
Data for other protons see Table 1S in Supplementary data.

ꢀ
M. Petrova et al. / Tetrahedron 67 (2011) 4227e4235
4231
Table 4
31P and 13C NMR data of compounds 5a,b, 7aee, 8a,eeh, 9, and 10a,feh at 27 ꢀC. Coupling constants J (C,P) are given in brackets
Compd.(Solv.)
Config.(popul.)
Pa
C-10
C-20
C-30
C-40
C-50
C-2
C-4
C-5
C-6
C-8d
5a
50S
(w61%)
50R
(w39%)
50S
(w50)
50R
(w50%)
50S
(w52%)
50R
(w48%)
50S
(w56%)
50R
(w44%)
50S
(w44%)
50S
(w56%)
50S
(w40%)
50R
(w60%)
50S
(w50%)
50R
(w50%)
50S
(w50%)
50R
(w50%)
50S
(w46%)
50R
(w54%)
50S
(w50%)
50R
(w50%)
50S
(w47%)
50R
(w53%)
50S
(w59%)
50R
23.68
95.83
80.52
(<1)
80.63
(1.1)
105.64
(9.4)
106.20
(9.3)
102.60
(8.1)
103.36
(8.3)
103.07
(8.8)
104.01
(9.2)
103.18
(8.7)
103.68
(8.8)
103.40
(8.3)
104.13
(8.1)
102.84
(9.1)
103.65
(8.6)
103.05
(8.9)
104.17
(9.1)
101.64
(6.0)
102.39
(6.8)
160.09
(3.8)
159.25
(3.3)
158.13
(w0)
157.08
(w0)
158.12
(1.4)
156.90
(w0)
157.43
(w0)
156.50
(w0)
157.75
(w0)
156.68
(w0)
156.47
(w0)
155.02
(w0)
156.75
(w0)
158.18
(1.4)
165.60
(w0)
165.34
(2.8)
65.80
(166.3)
65.53
(166.9)
63.88
(163.4)
63.47
(163.7)
64.04
(163.9)
63.69
(164.3)
64.06
(164.0)
63.76
(164.0)
64.00
(163.7)
63.60
(164.2)
64.07
(163.9)
63.72
(163.7)
63.74
(163.5)
64.14
(163.5)
69.90
(139.8)
69.78
(139.2)
70.27
(138.9)
70.27
(138.9)
69.68
(141.8)
69.63
(142.6)
70.02
(137.7)
70.22
(138.1)
70.06
(138.0)
69.78
(w142)
64.11
(164.0)
63.68
(164.5)
66.21
(166.5)
65.87
(167.1)
65.74
(145.2)
65.13
(148.4)
66.31
(136.6)
65.73
153.95
168.86
105.43
144.07
d
(D2O)
23.74
95.88
91.54
91.49
92.50
92.48
91.49
91.65
94.02
93.99
90.96
90.93
92.26
92.34
95.34
95.34
95.31
95.31
93.67
93.70
95.99
95.99
93.09
92.63
92.34
92.23
95.72
95.73
92.41
91.86
91.73
91.14
93.36
92.72
91.02
90.56
153.95
152.31
152.27
150.42
150.44
152.30
152.30
154.40
154.40
157.53
157.53
150.47
150.42
154.18
154.18
154.58
154.59
155.37
155.45
160.08
160.10
161.57
161.43
150.50
150.43
153.98
153.98
150.44
150.45
152.73
152.93
155.01
155.07
153.99
154.32
168.83
152.06
152.00
163.25
163.25
152.03
152.08
163.66
163.66
148.49
148.57
163.93
163.93
169.28
169.23
169.77
169.82
150.96
150.86
169.05
169.06
153.68
153.45
163.31
163.19
168.86
168.84
163.40
163.31
149.78
149.02
165.83
165.90
150.49
150.88
105.54
125.39
125.49
102.91
102.99
125.39
125.34
96.68
144.22
150.69
150.75
140.05
140.28
150.72
150.76
144.55
144.76
148.68
148.69
135.59
135.72
144.31
144.46
140.09
140.24
158.09
158.16
143.32
144.13
156.79
156.70
140.17
140.32
144.11
144.22
140.79
141.26
156.08
156.18
141.54
142.17
156.91
156.88
d
5b
22.57 and 22.71
77.46
142.35
142.02
d
(DMSO)
77.42
77.80
77.80
77.37
77.33
78.37
78.40
77.69
77.65
77.83
77.83
81.55
81.55
81.89
81.89
81.15
81.15
81.91
7a
21.10
(DMSO)
d
7b
20.90
142.06
142.90
d
(DMSO)
21.02
7c
21.03
(DMSO)
21.15
96.80
d
7d
21.04
119.93
120.04
110.74
110.59
104.96
104.74
114.52
114.79
120.96
121.02
98.85
136.64
136.84
d
(DMSO)
21.03
7e
21.28 and 21.24
(DMSO)
d
8a
12.57
d
(D2O)
d
8e
12.52
101.61
(5.5)
102.92
(6.1)
165.52
(2.4)
166.34
(w0)
d
(D2O)
12.50
d
8f
12.39 and 12.36
101.36
165.24
142.10
142.13
d
(D2O)
102.50
164.56
8g
12.35
12.32
12.51
12.49
20.36
20.41
20.63
20.74
14.05
101.38
(5.7)
102.02
(6.6)
100.79
(5.7)
102.56
(7.5)
102.35
(8.5)
103.41
(8.0)
105.32
(9.1)
106.17
(7.2)
99.53
(6.5)
101.23
(6.9)
97.95
(6.2)
99.66
(6.7)
165.68
(1.1)
165.52
(2.8)
165.69
(1.6)
165.70
(1.5)
159.05
(1.4)
157.69
(w0)
160.51
(3.8)
159.43
(3.6)
(D2O)
81.90
(0.8)
81.15
98.65
d
8h
118.30
118.02
102.64
102.80
105.39
105.50
101.80
102.40
118.79
118.67
94.39
139.20
139.00
d
(D2O)
81.11
77.66
77.75
80.45
80.60
78.87
78.60
79.27
78.82
79.50
79.24
78.15
79.13
(w41%)
50S
9
(DMSO)
50R
50S
50R
d
(D2O)
d
d
10a
50S
(w58%)
50R
(w42%)
50S
(w64%)
50R
(w36%)
50S
(w52.5%)
50R
(w47.5%)
50S
(w49%)
50R
162.81
d
(DMSO)
160.99
d
10fb
12.94
12.77
13.43
13.28
12.81
12.54
163.67
(1.9)
161.88
(w0)
139.28
138.86
d
(DMSO)
(139.4)
66.16
(137.9)
65.46
(141.9)
66.18
(137.2)
65.85
10gb
98.42
163.44
(DMSO)
100.37
98.05
99.23
161.65
163.18
161.57
93.85
d
10hb,c
116.30
116.29
135.34
134.74
(DMSO)
(w51%)
(139.5)
a
The phosphorus signals were structurally assigned to C-50 epimers only in case of their different intensities and criteria based on the multiplicity pattern of H-30 difference
between C-30 chemical shifts.
b
The signals of long aliphatic chain could not be structurally assigned.
Measured at 40 ꢀC.
Data for other carbons see Table 2S in Supplementary data.
c
d

ꢀ
M. Petrova et al. / Tetrahedron 67 (2011) 4227e4235
4232
column chromatography, silica gel 40e60 mm (Fluka) was used.
Analytical HPLC was performed on Nucleosil 100e5 C18
(4.6ꢂ150 mm; MachareyeNagel) using a linear gradient of meth-
anol in 0.1 M triethylammonium acetate. Preparative reversed-
phase chromatography was carried out on octadecyl silica
column (25ꢂ250 mm, 20
mm, IOCB Prague); compounds were
eluted with a linear gradient of methanol in water at 15 mL/min.
High resolution FAB mass spectra were recorded on a ZAB-EQ (VG
Analytical) instrument with glycerol and thioglycerol as matrices.
The NMR spectra of compounds 5a,b, 7aee, 8a,eeh, 9 and 10a,feh
were measured on Bruker Avance-600 instrument (1H at
600.13 MHz and 13C at 150.9 MHz) and Bruker Avance-500
instrument (31P at 202.3 MHz) in DMSO or D2O. 1H and 13C
chemical shifts in D2O are referenced to added dioxane (using
d
(H)¼3.75 and
d
(C)¼69.3) and in DMSO to the solvent peak (using
d(H)¼2.50 and
d
(C)¼39.7). Phosphorus chemical shifts are refer-
enced to H3PO4 as the external standard. Structural assignment of
proton and carbon signals was done using the 2D-H,H-COSY, 2D-
H,C-HSQC and 2D-H,C-HMBC spectra. The 1H, 13C and 31P NMR
data are presented in Tables 1S and 2S of Supplementary data.
The X-ray crystallographic analysis of single crystals of (50R)-9
(colourless, 0.04ꢂ0.15ꢂ0.30 mm) and (50S)-9 (colourless,
0.06ꢂ0.14ꢂ0.26 mm) was performed with Xcalibur X-ray diffrac-
tometer with Cu K
a
(
l
¼1.54180 Ǻ), data collected at 150 K. Both
structures were solved by direct methods with SIR9241 and refined
by full-matrix least-squares on F with CRYSTALS.42 All hydrogen
atoms were located in a difference map but those attached to
carbon atoms were later repositioned geometrically and then
refined with riding constraints, while all other atoms were refined
anisotropically in both cases.
3.1.1. Dimethyl
(50RS)-30-deoxy-30,40-didehydrouridine-50-C-phos-
phonate (5a). 20,30-Di-O-methoxymethylidene uridine43 2a (1.43 g,
5.00 mmol) was co-distilled twice with dry DMF and dissolved in
DMF (35 mL) at room temperature under the inert atmosphere of
argon. To the stirred solution, DMSO (2.13 mL, 30.0 mmol) and DCC
(3.10 g, 15.0 mmol) were added, followed by pre-mixed pyridine
Fig. 3. Multiplet pattern (resolution enhanced) of H30 in phosphonate diester 9 (pure
50-S, pure 50-R and their mixture) and 50-epimeric mixtures of free phosphonate 8a and
phosphonate monoester 10a.
(404 mL, 5.00 mmol) and TFA (191 mL, 2.50 mmol) in DMF (5 mL).
After completion of oxidation (checked by TLC in 10% EtOH in
CHCl3; within 3 h), triethylamine (2.80 mL, 20.0 mmol) was added.
After elimination was completed (TLC in 10% EtOH in CHCl3; within
5 min), dimethyl phosphite (1.83 mL, 20.0 mmol) was added and
the mixture was left for 5 min. After TLC (10% EtOH in CHCl3)
revealed the complete conversion, the excess of oxidation agent
was decomposed by oxalic acid (1.26 g, 10.0 mmol). The mixture
was concentrated in vacuo, MeOH was added to the residue and the
mixture was filtered through Celite, concentrated in vacuo and
purified by chromatography on silica gel (elution with gradient of
20% MeOH in CHCl3), on reverse phase (linear gradient of MeOH in
water) and subsequently freeze-dried from water. Yield 0.73 g, 44%
(off-white lyophylisate).
Fig. 4. Crystal structures of diester 9 showing the different orientations of the 50-OH
HRMS (ESI): for C11H15N2NaO8P (MþNa)þ calcd: 357.0458,
group to C30. The -gauche interaction of the 50-OH with C30 in the 50-S epimer could
g
found: 357.0462; 0.9816 ppm.
be responsible for the upfield position of its carbon C30.
nmax (KBr) 2959m, 1186m (PeOMe); 1715s, 1690vs, 1633m,
1462m, 1422m (base); 1660s (C¼C); 1105m, 1082m (CeOH); 1256m
(P¼O); 1040s, 755m, 548m (POC).
3. Experimental
3.1. General
1H, 13C and 31P NMR data see Tables 3 and 4.
3.1.2. Dimethyl (50RS)-6-N-benzoyl-30-deoxy-30,40-didehydroadenosine-
50-C-phosphonate (5b). 20,30-Di-O-methoxymethylidene-6-N-benzoy-
ladenosine44 2b (1.57 g, 3.80 mmol) was co-distilled twice with dry
toluene and dissolved in DMSO (11 mL) at room temperature under
the inert atmosphere of argon. To the stirred solution, DCC (2.35 g,
Solvents were evaporated at 40 ꢀC and 2 kPa, and products
were dried over phosphorus pentoxide at 13 Pa. The course of the
reactions was checked by TLC (Merck) whereby the products were
detected by UV monitoring and by spraying with 1% ethanolic
solution of 4-(4-nitrobenzyl)pyridine followed by heating and
treating with gaseous ammonia (blue colour of diesters). For flash
11.4 mmol), followed by pre-mixed pyridine (307
TFA (145 L, 1.90 mmol) in DMSO (4 mL) were added. After comple-
tion of oxidation (checked by TLC in 10% EtOH in CHCl3; within 2 h),
mL, 3.80 mmol) and
m

ꢀ
M. Petrova et al. / Tetrahedron 67 (2011) 4227e4235
4233
triethylamine (2.10 mL, 15.0 mmol) was added and after 15 min
(TLC in 10% EtOH in CHCl3), dimethyl phosphite (1.38 mL, 15.0 mmol)
was added and the mixture was stirred for further 15 min (TLC in 10%
EtOH in CHCl3). The reaction mixture was freeze-dried to remove
DMSO, the residue was dissolved in MeOH/water mixture (1:1) and
filtered through Celite. The filtrate was co-distilled with ethanol. The
crude product was purified by chromatography on silica gel (elution
with gradient of 0e20% MeOH in CHCl3) and subsequent chroma-
tography on reverse phase (elution with linear gradient of MeOH in
water) and subsequently freeze-dried from water. Yield 1.23 g 70%
(white lyophylisate).
HRMS (ESI): for C20H24N7NaO8P (MþNa)þ calcd: 544.1316,
found: 544.1313; ꢁ0.6550 ppm.
nmax (KBr) 3211m (OH, NH); 2256w (CN); 1715s, 1566s, 1376w
(NH-iBu); 1683vs, 1608s, 1534w, 1476m, 1408m, 785m, 721w
(base); 1254m (P¼O); 1080s, 1050s, 1036s, 1004m (POCC).
1H, 13C and 31P NMR data see Tables 3 and 4.
3.2.5. Bis-(2-cyanoethyl) (50RS)-30-deoxy-30,40-didehydro-5-methyl-
uridine-50-C-phosphonate (7e). Prepared from 50-aldehyde 6e (Et3N
36.0 mL, 0.26 mmol); yield 133 mg, 74% (white foam).
HRMS (ESI): for C16H19N4NaO8P (MþNa)þ calcd: 449.08327,
found: 449.08326; ꢁ0.01753 ppm.
HRMS (ESI): for C19H20N5NaO7P (MþNa)þ calcd: 484.0993,
found: 484.0988; ꢁ0.8620 ppm.
nmax (KBr) 3270m (OH, NH); 2256w (CN); 1697s, 1654m, 1635m,
1377w, 784w (base); 1260w, 1228w (P¼O); 1079m,1054m (CeOH);
1035m, 1003m (POCC).
nmax (KBr) 3418vs, 3272m, 3110m (OH, NH); 2957w, 1177m,
1048s, 1038s, 753w, 555m (PeOMe); 1700m, 1512m, 1073s, 711m
(NHBz); 1661w (C¼C); 1612s, 1583m, 1487m, 1337m, 1285m, 1222s,
797w, 642m (base); 1249s (P¼O).
1H, 13C and 31P NMR data see Tables 3 and 4.
1H, 13C and 31P NMR data see Tables 3 and 4.
3.2. Preparation of bis(cyanoethyl) 30-deoxy-30,40-didehydronu
3.3. Preparation of 30-deoxy-30,40-didehydronucleoside-50-C-
phosphonic acids 8a,eeh
cleoside-50-C-phosphonates 7aee
To a solution of aldehyde35 6aee (1.00 mmol) in dried DMF
(8 mL), freshly prepared bis(2-cyanoethyl) phosphite38 (0.75 g,
4.00 mmol) was added at room temperature, followed by Et3N (up
to 0.06 mL, 0.45 mmol). The reaction was completed usually within
15e30 min (checked by TLC in 20% MeOH in CHCl3). The mixture
was concentrated in vacuo and the residue was purified by chro-
matography on silica gel (elution with gradient of 20% MeOH in
CHCl3).
To bis-(2-cyanoethyl) phosphonate 7aee (1 mmol), 8 M MeNH2
in absolute EtOH (40 mL) was added, and the mixture was heated to
50 ꢀC overnight. TLC (H1: ethyl acetate/acetone/EtOH/water
4:1:1:1; and IPAW: 2-propanol/concentrated aqueous ammonia/
water 7:1:2) revealed formation of a free phosphonic acid. The
mixture was concentrated in vacuo, co-distilled three times with
EtOH and purified on reverse phase (linear gradient of MeOH in
water). The product was transformed to its Na salt on Dowex 50
(Naþ) and freeze-dried from water.
3.2.1. Bis-(2-cyanoethyl) (50RS)-30-deoxy-30,40-didehydrouridine-50-
C-phosphonate (7a). Prepared from 50-aldehyde 6a (Et3N 21.0
mL,
3.3.1. (50RS) 30-Deoxy-30,40-didehydrouridine-50-C-phosphonic acid
(8a). Prepared from bis-(2-cyanoethyl) phosphonate 7a; yield
100 mg, 85% (white lyophylisate).
0.15 mmol); yield 130 mg, 70% (off-white foam).
HRMS (ESI): for C15H17N4NaO8P (MþNa)þ calcd: 435.0676,
found: 435.0676; ꢁ0.1018 ppm.
HRMS (ESI): for C9H10N2O8P (MþH)þ calcd: 305.0180, found:
nmax (CHCl3) 3386m, 3276m (OH, NH); 2256w (CN); 1720s,
1687vs, 1474w, 1412w, 1389w (base); 1261m (P¼O); 1081s, 1050s,
1003m (POCC).
305.0184; 1.0731 ppm.
nmax (KBr) 3423s, 3255s, 3110m, 2813w (OH, NH); 1697s, 1637m,
1473w, 1271m, 766w (base); 1091s, 973s, 583m, 486m (PO32ꢁ).
1H, 13C and 31P NMR data see Tables 3 and 4.
1H, 13C and 31P NMR data see Tables 3 and 4.
3.2.2. Bis-(2-cyanoethyl) (50RS)-6-N-benzoyl-30-deoxy-30,40-didehy-
3.3.2. (50RS) 30-Deoxy-30,40-didehydro-5-methyluridine-50-C-phos-
phonic acid (8e). Prepared from bis-(2-cyanoethyl) phosphonate
7e; yield 340 mg, 90% (white lyophylisate).
droadenosine-50-C-phosphonate (7b). Prepared from 50-aldehyde
6b (Et3N 21.0 mL, 0.15 mmol); yield 128 mg, 85% (white foam).
HRMS (ESI): for C23H21N7O7P (MꢁH)ꢁ calcd: 538.1246, found:
538.1243; ꢁ0.5431 ppm.
HRMS (ESI): for C10H12N2O8P (MþH)þ calcd: 319.0337, found:
319.0342; 1.4914 ppm.
nmax (CHCl3) 3425m, 3321m, 3126m (OH, NH); 2253w (CN);
1717s, 1703m, 1688m, 1527m, 1511m, 712m, 704m (NHBz); 1666m
(C¼C); 1614vs, 1591m, 1481m, 1412m, 1334m, 797m (base); 1250vs
(P¼O); 1080vs, 1059s, 1050vs, 1034vs, 1005s (POCC).
1H, 13C and 31P NMR data see Tables 3 and 4.
nmax (KBr) 3422s, 3260m, 2756m (OH, NH); 1699vs, 1656m,
1519w, 1476m, 1391w, 1271m, 790w, 767w, 723w (base); 1088s,
971s, 581m, 484w (PO32ꢁ).
1H, 13C and 31P NMR data see Tables 3 and 4.
3.3.3. (50RS)
30-Deoxy-30,40-didehydroadenosine-50-C-phosphonic
3.2.3. Bis-(2-cyanoethyl) (50RS)-4-N-benzoyl-30-deoxy-30,40-didehy-
drocytidine-50-C-phosphonate (7c). Prepared from 50-aldehyde 6c
acid (8f). Prepared from bis-(2-cyanoethyl) phosphonate 7b; yield
147 mg, 88% (white lyophylisate).
(Et3N 63.0
m
L, 0.45 mmol); yield 140 mg, 90% (white foam).
HRMS (ESI): for C10H11N5O6P (MþH)þ calcd: 328.0452, found:
328.0445; ꢁ2.3776 ppm.
HRMS (ESI): for C22H22N5NaO8P (MþNa)þ calcd: 538.1098,
found: 538.1097; ꢁ0.3008 ppm.
nmax (KBr) 3364vs, 3222s (OH, NH); 1654s,1648s,1605m,1578m,
1476m, 1421m, 1335m, 1209m, 797m, 645m (base); 1088vs, 1050
versus, 970m, 580m, 487m (PO32ꢁ).
nmax (KBr) 3364m, 3212m (NH, OH); 2256w (CN); 1697s, 1681s,
1563m, 1603m, 1582w, 1501m, 1074s, 687m (NHBz); 1681s, 1666s,
1650m, 1619s, 1487vs, 1374m, 781m (base); 1254vs (P¼O); 1085s,
1055s, 1037s, 1006s (POCC).
1H, 13C and 31P NMR data see Tables 3 and 4.
1H, 13C and 31P NMR data see Tables 3 and 4.
3.3.4. (50RS) 30-Deoxy-30,40-didehydrocytidine-50-C-phosphonic acid
(8g). Prepared from bis-(2-cyanoethyl) phosphonate 7c; yield
108 mg, 84% (white lyophylisate).
3.2.4. Bis-(2-cyanoethyl) (50RS)-30-deoxy-30,40-didehydro-2-N-iso-
butyrylguanosine-50-C-phosphonate (7d). Prepared from 50-alde-
hyde 6d (Et3N 63.0 mL, 0.45 mmol); yield 121 mg, 74% (white foam).
HRMS (ESI): for C9H11N3O7P (MþH)þ calcd: 304.0340, found:
304.0339; ꢁ0.2770 ppm.

ꢀ
M. Petrova et al. / Tetrahedron 67 (2011) 4227e4235
4234
nmax (KBr) 3330s, 3187s, 3095s (OH, NH); 1655vs, 1610m, 1524m,
3.4. Preparation of 3-hexadecyloxypropyl 30-deoxy-30,40-dideh
1492m, 1399m, 1289m, 784m, 600w (base); 1086s, 968s, 582m,
ydronucleoside-50-C-phosphonates 10a,feh
486w (PO32ꢁ).
1H, 13C and 31P NMR data see Tables 3 and 4.
To a solution of aldehyde35 6aed (1 mmol) in dried DMF
(8 mL), freshly prepared methyl 3-hexadecyloxypropyl phos-
phite38 (0.45 g, 1.20 mmol) was added, followed by Et3N (0.14 mL,
1.00 mmol), and the mixture was left aside at room temperature
overnight. The course of the reaction was checked by TLC in 10%
EtOH in CHCl3. The mixture was concentrated in vacuo and the
residue was purified by chromatography on silica gel (elution
with gradient of EtOH in ethyl acetate). The methyl group was
removed upon heating in 60% aqueous pyridine (20 mL) at 50 ꢀC
overnight (TLC H1: ethyl acetate/acetone/EtOH/water 4:1:1:1),
then the mixture was concentrated in vacuo and co-distilled
three times with EtOH. Removal of N-benzoyl groups for ade-
nine and cytosine compounds was achieved by concentrated
methanolic ammonia (40 mL); the N-isobutyryl group from
guanine compound was removed by 8 M MeNH2 in absolute
EtOH (20 mL). The course of the reaction was checked by TLC
(H1). The solvent was then removed in vacuo and the resulting
monoester was purified by chromatography on silica gel (elution
with gradient of H-1 in ethyl acetate). Treatment with saturated
sodium bicarbonate solution upon heating, provided sodium salt
of the desired monoester, which was purified on reverse phase
(linear gradient of MeOH in water), and subsequently freeze-
dried from water.
3.3.5. (50RS)
30-Deoxy-30,40-didehydroguanosine-50-C-phosphonic
acid (8h). Prepared from bis-(2-cyanoethyl) phosphonate 7d; yield
127 mg, 87% (white lyophylisate).
HRMS (ESI): for C10H11N5O7P (MþH)þ calcd: 344.0402, found:
344.0405; 1.0396 ppm.
nmax (KBr) 3385m, 3122s, 2756m (OH, NH); 1698vs, 1635m,
1609m, 1578w, 1533m, 1483w, 1376w, 779w, 729w, 640w (base);
1088s, 972m, 578m, 487w (PO32ꢁ).
1H, 13C and 31P NMR data see Tables 3 and 4.
3.3.6. Diethyl (50RS)-30-deoxy-30,40-didehydrouridine-50-C-phospho-
nate (9). To a solution of 50-aldehyde35 6a (225 mg, 1.00 mmol) in
water (8 mL), diethyl phosphite (0.52 mL, 4.02 mmol) and Et3N
(10.0 mL, 0.07 mmol) were added at room temperature. After TLC
(20% MeOH in CHCl3) revealed total conversion (20 min), the
mixture was concentrated in vacuo and co-distilled with EtOH. The
crystal residue gave 220 mg (60%) of the phosphonate 9, mp 130 ꢀC
decomp.; [
a
]
20 ꢁ167.3 (c 0.518, H2O).
D
HRMS (ESI): for C13H19N2NaO8P (MþNa)þ calcd: 385.0771,
found: 385.0770; ꢁ0.2976 ppm.
nmax (KBr) 3422m, 3364m, 3316m, 3163m (OH, NH); 1721vs,
1699vs, 1687vs, 1672vs, 1455m, 1420m, 1382m, 1262s, 829m, 757w
(base); 1246m, 1211s (P¼O); 1076s, 1051vs (CeOH); 1162s, 1108m
(PeOEt); 1051vs, 1018vs, 970s, 561m (POCC).
3.4.1. 3-Hexadecyloxypropyl (50RS)-30-deoxy-30,40-didehydrouridineþ-
50-C-phosphonate (10a). Prepared from 50-aldehyde 6a as Et3NH
salt; yield 222 mg, 38% (white lyophylisate).
1H and 13C NMR data see Tables 3 and 4.
50-R and 50-S epimers were separated by preparative HPLC using
gradient of MeOH in water and crystallised from EtOH.
HRMS (ESI): for C28H48N2O9P (MþH)þ calcd: 587.3103, found:
587.3106; 0.5904 ppm.
nmax (KBr) 3404m, 3226m (OH, NH); 2920vs, 2851s (CH2);
2679w, 2491w,1434w,1183m,1161m, 836w, 819w (Et3NHþ); 1713s,
1693vs, 1633w, 1467m, 1256m, 763w, 722w (base); 1209m, 1081m,
1055s, 991w, 971w (PO2ꢁ).
3.3.7. Diethyl (50R)-30-deoxy-30,40-didehydrouridine-50-C-phospho-
20
nate (50R-9). Colourless crystals, mp 130 ꢀC decomp.; [
(c 0.237, H2O).
a
]
ꢁ152.3
D
HRMS (ESI): (1) for C13H20O8N2P (MþH)þ calcd: 363.09518,
found: 363.09503; ꢁ0.41193 ppm; (2) for C13H19O8N2NaP (MþNa)þ
calcd: 385.07712, found: 385.07678; ꢁ0.90350 ppm.
1H, 13C and 31P NMR data see Tables 3 and 4.
3.4.2. 3-Hexadecyloxypropyl (50RS)-30-deoxy-30,40-didehydroadeno-
sine-50-C-phosphonate (10f). Prepared from 50-aldehyde 6b; yield
353 mg, 58% (white lyophylisate).
Crystal data: C13H19N2O8P, triclinic, space group P1, a¼9.4279
(18) A, b¼9.4286 (15) A, c¼10.8221 (15) A,
a
¼79.198 (12)ꢀ,
¼81.553
b
ꢂ
ꢂ
ꢂ
3
ꢂ
(14)ꢀ,
g
¼61.700 (18) , V¼830.1 (3) A , Z¼2, M¼362.27, 27,324 re-
HRMS (ESI): for C29H51N5O7P (MþH)þ calcd: 612.3521, found:
612.3519; ꢁ0.2173 ppm.
ꢀ
flections measured, 6402 independent reflections. Final R¼0.0685,
wR¼0.1129, GoF¼0.11 for 4784 reflections with I>2
s
(I) and 452
nmax (KBr) 3331m, 3264m, 3186m, 3120m (OH, NH); 2924vs,
2853s (CH2); 1645s, 1601m, 1579w, 1469m, 1420w, 1334w, 1294w,
parameters. The asymmetric unit contains two independent mole-
cules of 50R-9, which are not significantly different. In both of these
molecules, the terminal carbon of one of the ethyl groups is disor-
dered over two sites with their site occupation factors being 0.56 and
0.44 (Fig. 2: only one of the two sites is represented). CCDC 773860.
1H, 13C and 31P NMR data see Tables 3 and 4.
798w, 721w, 647w (base); 1218s, 1087s, 1058s, 993w, 967w (PO2
ꢁ).
1H, 13C and 31P NMR data see Tables 3 and 4.
3.4.3. 3-Hexadecyloxypropyl (50RS)-30-deoxy-30,40-didehydrocytid-
ine-50-C-phosphonate (10g). Prepared from 50-aldehyde 6c; yield
364 mg, 62% (white lyophylisate).
3.3.8. Diethyl (50S)-30-deoxy-30,40-didehydrouridine-50-C-phospho-
HRMS (ESI): for C28H51N3O8P (MþH)þ calcd: 588.3408, found:
588.3408; ꢁ0.0024 ppm.
20
nate (50S-9). Colourless crystals, mp 130 ꢀC decomp.; [
(c 0.251, H2O).
a]
ꢁ172.5
D
nmax (KBr) 3400m, 3341m, 3198m (OH, NH); 2924vs, 2853s,
146m (CH2); 1651vs, 1613m, 1525w, 1493m, 1398w, 1287w, 787w,
721w, 600w (base); 1378w (CH3); 1219m, 1087m, 1058s, 1000w,
965w (PO2ꢁ).
HRMS (ESI): for C13H20O8N2P (MþH)þ calcd: 363.09518, found:
363.09496; ꢁ0.60026 ppm.
Crystal data: C13H19N2O8P, monoclinic, space group C2,
ꢂ
ꢂ
ꢂ
b
a¼1ꢀ5.958 (3) A, b¼9.5771 (11) A, c¼10.9485 (18) A,
¼104.436
1H, 13C and 31P NMR data see Tables 3 and 4.
3
ꢂ
(18) , V¼1620.4 (5) A , Z¼4, M¼1620.4 (5), 20,741 reflections
measured, 3349 independent reflections. Final R¼0.1016,
3.4.4. 3-Hexadecyloxypropyl
(50RS)-30-deoxy-30,40-didehy-
wR¼0.1226, GoF¼1.14 for 2432 reflections with I>2
s
(I) and 254
droguanosine-50-C-phosphonate (10h). Prepared from 50-aldehyde
6d; yield 129 mg, 21% (white lyophylisate).
parameters. Both ethyl groups are disordered over two sites with
their site occupation factors being 0.55 and 0.45 (Fig. 2: only one of
the two sites is represented). Several restrains were used to regu-
larise the thermal motions of these groups. CCDC 773861.
1H, 13C and 31P NMR data see Tables 3 and 4.
HRMS (ESI): for C29H49N5O8P (MþH)þ calcd: 626.3324, found:
626.3332; 1.1931 ppm.
nmax (KBr) 3487m, 3413m, 3125m (OH, NH); 2924vs, 2853s,
1468w (CH2); 1693vs, 1632m, 1610m, 1532w, 1483w, 1412w, 1179m,

ꢀ
M. Petrova et al. / Tetrahedron 67 (2011) 4227e4235
4235
ꢀ
ꢀ
14. Otmar, M.; Rosenberg, I.; Masojídkova, M.; Holy, A. Collect. Czech. Chem.
Commun. 1993, 58, 2159e2179.
782w, 722w, 641w (base); 1376m (CH3); 1220m, 1085m, 1057s,
1027m, 992w, 970w (PO2ꢁ).
ꢀ
ꢀ
15. Otmar, M.; Rosenberg, I.; Masojídkova, M.; Holy, A. Collect. Czech. Chem.
1H, 13C and 31P NMR data see Tables 3 and 4.
Commun. 1993, 58, 2180e2196.
ꢀ
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Financial support provided by the grants 203/09/1919, 203/09/
0820, and 202/09/0193 (Czech Science Foundation) and by
Research Centers KAN200520801 (Acad. Sci. CR) and LC06077
(Ministry of Education, CR), under the Institute research project
Z40550506, is gratefully acknowledged. The authors are indebted
to Dr. Tomas Cihlar and Dr. Richard Mackman (Gilead Sci., Ltd., CA)
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Tocík for valuable discussion, Eva Zborníkova, MSc. for excellent
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Dr. Pavel Fiedler for the measurement and interpretation of the IR
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2011.04.037.
biochem 2008, 9, 2883e2888.
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