Inhibition of human thymidine phosphorylase by conformationally constrained pyrimidine nucleoside phosphonic acids and their "open-structure" isosteres

Article abstract of DOI:10.1016/j.ejmech.2013.12.026

A series of conformationally constrained uridine-based nucleoside phosphonic acids containing annealed 1,3-dioxolane and 1,4-dioxane rings and their "open-structure" isosteres were synthesized and evaluated as potential multisubstrate-like inhibitors of t

Full text of DOI:10.1016/j.ejmech.2013.12.026

European Journal of Medicinal Chemistry 74 (2014) 145e168
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibition of human thymidine phosphorylase by conformationally
constrained pyrimidine nucleoside phosphonic acids and their
“open-structure” isosteres
1
1
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Ivana Kosiová , Ondrej Simák , Natalya Panova, Milos Budesínský, Magdalena Petrová,
*
ꢀ
Dominik Rejman, Radek Liboska, Ondrej Páv, Ivan Rosenberg
Institute of Organic Chemistry and Biochemistry, Academy of Sciences 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
Article history:
A series of conformationally constrained uridine-based nucleoside phosphonic acids containing annealed
1,3-dioxolane and 1,4-dioxane rings and their “open-structure” isosteres were synthesized and evaluated
as potential multisubstrate-like inhibitors of the human recombinant thymidine phosphorylase (TP, EC
2.4.2.4) and TP obtained from peripheral blood mononuclear cells (PBMC). From a large set of tested
nucleoside phosphonic acids, several potent compounds were identified that exhibited Ki values in the
Received 5 September 2013
Received in revised form
12 December 2013
Accepted 22 December 2013
Available online 3 January 2014
range of 0.048e1 mM. The inhibition potency of the studied compounds strongly depended on the degree
of conformational flexibility of the phosphonate moiety, the stereochemical arrangement of the sugar-
phosphonate component, and the substituent at position 5 of the pyrimidine nucleobase.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Phosphonate
Conformationally constrained nucleotide
analog
Human thymidine phosphorylase
PBMC
Bi-substrate-like inhibitor
Michael addition
1. Introduction
In addition to these mono-substrate-like inhibitors, there is a
class of compounds exists in which both nucleoside and phosphoryl
Thymidine phosphorylase (TP), the enzyme identical to platelet-
derived endothelial cell growth factor [1e5], catalyzes the reversible
moieties are combined in one molecule that can bind simultaneously
to both nucleobase and phosphate binding sites, respectively. Bal-
zarini et al. [36,37] and Votruba et al. [38] reported the inhibition of
Escherichia coli TP with structurally diverse acyclic nucleoside
phosphonic acids (Fig. 2, structures 2e4), and Lequeux et al. [39]
recently published a thorough structural study on the inhibition of
phosphorolysis of thymidine to thymine and 2-deoxy-
a-D-ribofur-
anosylphosphate (Fig. 1).
In contrast to non-neoplastic tissues, the elevated levels of TP
have been found in colorectal, ovarian, pancreatic, and breast tumors
[6,7], and in other hyperproliferative disease states such as rheu-
matoid arthritis [8] and psoriasis [9]. The inhibition of TP may result
in the reduction of tumor growth and metastasis [10e17]. To date, a
number of TP inhibitors of humanTP based on the analogues of uracil
and thymine nucleobases and nucleosides have been reported [18e
32]. The most potent and therapeutically promising inhibitor of
human TP is 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hy-
drochloride (1, TPI [13], Taiho Pharmaceutical Company) with a Ki of
17 nM [33] which, in a combination with the anticancer drug tri-
fluorothymidine, is currently in clinical trials (Fig. 2) [34,35].
the enzyme by various
a,a-difluoromethyl-moiety-containing 1-
thyminylalkylphosphonic acids 4a. Comparative study on the inhi-
bition of E. coli and human TPs with 3-pyrimidinylalkylphosphonic
acids was recently reported by Pomeisl et al. [40]. Irrespective of
the structural and genetic similarities among thymidine phosphor-
ylases from various sources including E. coli and human recombinant
TPs, the sensitivity of the two enzymes toward inhibitors based on
nucleoside phosphonic acids appears to differ. Recently, we
described the potent bi-substrate-like inhibitors 5 and 6aeb of TP
isolated from T-cell lymphomas of the SpragueeDawley rat strain;
the inhibitors were based on pyrrolidine nucleoside phosphonic
acids [41], with IC50 values in the range of 11e45 nM at a thymidine
* Corresponding author. Tel.: þ420 220 183 381.
concentration of 100
mM. Interestingly, these compounds did not
E-mail address: rosenberg@uochb.cas.cz (I. Rosenberg).
1
These authors contributed equally to this paper.
inhibit healthy rat liver, E. coli, or human TPs.
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.026

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I. Kosiová et al. / European Journal of Medicinal Chemistry 74 (2014) 145e168
146
protected 5-chlorouridine 9 which was easily obtained by chlori-
nation of the uridine derivative 8 with N-chlorosuccinimide (NCS)
according to the described procedure [46]. Protected nucleosides 8
and 9 were transformed into the 20,30-O-orthoester derivatives 10
and 11, respectively, by the reaction with trimethyl orthoformate in
the presence of anhydrous HCl in DMF. Subsequent reaction of 10
and 11 with two equivalents of dimethyl chloro phosphite gave,
after removal of the silyl protecting group with TBAF and the
following treatment with bromotrimethylsilane in a CH3CNe2,6-
lutidine mixture (to remove the phosphoester groups), a mixture
of 13a (endo) and 13b (exo) epimers (25:75). The epimers were
separated by RP HPLC. In contrast to the synthesis of 13a,b, the 5-
iodo derivatives 14a and 14b were prepared by a direct iodination
of pure epimers 12a and 12b with iodine in diluted nitric acid [47].
Compounds 12c and 12d were synthesized according to the
method described in the literature [45].
Fig. 1. TP-catalyzed thymidine cleavage.
In contrast to guanine-based pyrrolidine nucleoside phosphonic
acids, recently reported as potent bi-substrate-like inhibitors [42]
of human purine nucleoside phosphorylase, the similar thymine-
based compounds did not inhibit the human TP.
The first nucleoside phosphonic acid inhibiting human TP in the
submicromolar
range,
5-methyl-20,30-O-(2-phosphono-1,1-
2.1.2. Nucleosid-20(30)-O-ylmethanephosphonic acids (Scheme 2)
The regioisomeric 30- (23ae26a) and 20-O-methanephosphonic
acids (23b and 24b), which are conformationally flexible congeners
related to the constrained phosphonates 12 and 13, were prepared
from the corresponding protected nucleosides 15ae18a, 15b and
16b in three steps (Scheme 2). The alkylation of these nucleosides
with methyl tosyloxymethanephosphonate [48] provided phos-
phonate methyl esters 19ae22a, 19b and 20b, respectively. Detri-
tylation of these compounds using 80% acetic acid followed by
removal of the ester groups by bromotrimethylsilane treatment
afforded the final 30- and 20-O-methanephosphonic acids 23ae26a
and 23b, 24b, respectively. The synthesis of 23a was already re-
ported [49].
ethylidene)uridine (50, Fig. 1), a conformationally constrained bi-
substrate-like inhibitor with a Ki value of 236 nM, was reported
by Li [43]. Recently described [44] carba analogues of 50, i.e.,
nucleoside phosphonic acids 7a and 7b, inhibited the human TP in
the high micromolar range.
In this paper, we describe the synthesis of two new series of
pyrimidine nucleoside phosphonic acids, conformationally con-
strained and flexible (“open-structure”), respectively, and describe
their ability to inhibit human TP. The target compounds were
designed to offer a variety of structural types believed to cover a
range of potential candidates that could provide significant data on
the structureeactivity relationship.
2. Results and discussion
2.1.3. 2-(Nucleosid-30(20)-O-yl)-ethanephosphonic acids (Schemes
3 and 4)
2.1. Chemistry
For the synthesis of nucleoside 30- and 20-O-(hydroxyethane)
phosphonic acids (31a and 31b, respectively), and 30- and 20-O-
ethanephosphonic acids (33a and 33b, respectively) which are one
methylene group longer congeners of compounds 23a and 23b with
an additional methylene group, we used the following synthetic
strategy. The O-allylation of 50-O-dimethoxytrityl nucleosides 15a
and15b withallyl bromide [50]affordedtheallyl derivatives 27a and
27b. Treatment of these compounds with osmium tetroxide in the
presence of N-methylmorpholine-N-oxide provided, after silica gel
chromatography, pure O-(2,3-dihydroxypropyl) derivatives 28a and
2.1.1. Nucleosid-20,30-di-O-ylmethanephosphonic acids (Scheme 1)
Conformationally constrained pyrimidine nucleotide analogs
12a,be14a,b, the isopolar phosphonate analogs of pyrimidine
nucleoside 20(30)-phosphates, were synthesized from the appro-
priate nucleosides 8 and 9 by several step syntheses (Scheme 1)
[45]. These compounds were obtained as epimeric mixtures that
provided individual epimers after separation by RP HPLC. 50-
Chlorouridine phosphonates 13a and 13b were prepared from the
Fig. 2. TPI and bi-substrate like nucleoside phosphonic acid-based TP inhibitors.

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147
Scheme 1. Synthesis of nucleosid-20,30-di-O-ylmethanephosphonic acids.
Scheme 2. Synthesis of nucleosid-20(30)-O-ylmethanephosphonic acids.
28b which were subsequently cleaved with sodium periodate to
provide the aldehydes 29a and 29b, respectively. To obtain alde-
hydes 29a and 29b in one step from the allyl derivatives 27a and 27b,
we also examined the OsO4/NaIO4 system. To our surprise, the
starting nucleosides 15a and 15b were isolated from the reaction
mixture as the main products. We concluded that the formed alde-
hyde 29a,b is enolized, the resulting enol form is hydroxylated, and
the dihydroxy derivative is cleaved with periodate to produce the
unstable 20(30)-O-formyl derivative of nucleosides 15a and 15b.
The prepared aldehydes 29a and 29b were subjected to the
Abramov reaction [51] with diethyl phosphite in the presence of
triethylamine, which provided the respective epimeric hydrox-
yphosphonate derivatives 30a and 30b, respectively. Removal of
the dimethoxytrityl group using 80% acetic acid and subsequent
treatment with bromotrimethylsilane afforded the free phospho-
nates 31a and 31b, respectively. Compounds 30a and 30b were
converted into the respective deoxygenated products 32a and 32b,
respectively, upon treatment with thiocarbonyldiimidazole to
Scheme 3. Synthesis of 2-(thymidin-30-O-yl)-ethanephosphonic acid and 1-hydroxy-2-(thymidin-30-O-yl)-ethanephosphonic acid.

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Scheme 4. Synthesis of 2-(30-deoxy-5-methyluridin-20-O-yl)-ethanephosphonic acid and 2-(30-deoxy-5-methyluridin-20-O-yl)-1-hydroxyethanephosphonic acid.
provide the corresponding imidazoylthiocarbonyl derivatives,
2.1.4. 2-(Nucleosid-20,30-di-O-yl)-ethanephosphonic acid (Scheme
5)
which were subsequently reacted with tributyltin hydride and
AIBN. Subsequent treatment of 32b with bromotrimethylsilane
afforded the free phosphonic acid 33b, whereas the deprotection
of 32a under the same conditions described for 32b led to com-
plete glycosidic bond cleavage. However, the deprotection of 32a
The conformationally constrained nucleotide analogs 45a, 45b,
and 50e53, which are related to compounds 12e14 but contain one
additional methylene group on their side chains, were prepared by
the reaction sequence shown in Scheme 5.
using bromotrimethylsilane in
a
N,O-bis-(trimethylsilyl)acet-
The starting 50-O-silylated nucleosides 34, 8, and 35 were
transformed into the 20,30-O-allylidene derivatives 36e38, respec-
tively, by treatment with acrolein dimethyl acetal in
amideeacetonitrile mixture provided the desired phosphonate
33a.
Scheme 5. Synthesis of 2-(nucleosid-20,30-di-O-yl)-ethanephosphonic acid and 1-hydroxy-2-(nucleosid-20,30-di-O-yl)-ethanephosphonic acid.

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Scheme 6. Synthesis of 3-(5-chlorouridin-20,30-di-O-yl)-propanephosphonic acid.
dichloromethane under hydrogen chloride catalysis. The obtained
allylidene derivatives 36e38 were treated with OsO4 and NaIO4 to
provide the corresponding aldehydes 39e41 which subsequently
afforded the hydroxyphosphonate diesters 42e44, respectively
Subsequent chlorination with NCS and removal of the O-dime-
thoxytrityl and O-silyl protecting groups with 80% acetic acid and
TBAF, respectively, afforded the vinylphosphonate diesters 62a and
62b.
(single epimers on C2 of 1,3-dioxolane ring). The final
a
-hydrox-
These compounds underwent, upon treatment with sodium
methoxide in methanol, an intramolecular Michael addition of the
20- and 30-hydroxyl to the vinylphosphonate moiety resulting in
the formation of the diethyl esters of 63 with anneled 1,4-dioxane
rings. Whereas the cyclization of 62b under sodium methoxide
catalysis provided the epimers 63ba and 63bb in a 3:2 ratio, the
cyclization of 62a proceeded stereoselectively, giving a single
epimer 63aa in a yield greater than 95%. To obtain the second
epimer 63ab, we examined various conditions for the cyclization
reaction of the vinylphosphonate 64 (Scheme 7). The results of the
study are summarized in Table 1. Careful RP HPLC analysis of the
products revealed that the ratio of epimers 66a and 66b could be
dramatically influenced by the solvent and base used. The best
result regarding the content of the desired epimer 66b in the re-
action mixture was achieved with t-BuOHeCs2CO3 and pinacole
Cs2CO3 (Entries 3, 4, and 6). In the latter case, however, the con-
version of the starting vinylphosphonate 64 was only 60%. In
contrast to the successful use of the t-BuOHeCs2CO3 system to
increase the amount of epimer 66b, the application of use DMFe
Cs2CO3 led to an almost exclusive formation of a novel product,
ciseallylphosphonate 65 (Entry 10, 11). Interestingly, when any
reaction mixture (Entry 1e4) was evaporated to dryness and then
analysed by RP HPLC, the presence of w90% of ciseallylphospho-
nate 65 in the mixture was detected. This phenomenon could be
explained by a reverse Michael additionedecyclization of 66a and
66b providing the ciseallylphosphonate 65. The mechanism of the
reaction in this particular case is unclear and will be studied
further. In view of these findings, the workup of the reaction
mixtures (Entries 3 and 4) was performed; desalting under the use
of Dowex 50 (Et3NHþ form) was performed first, and only then
could the solution be safely evaporated to dryness. Mixtures of
epimers 66a and 66b, as well as the cis-compound 65, were suc-
cessfully separated by silica gel chromatography. The desired
epimer 66b was chlorinated by NCS in pyridine, giving the 5-
chlorouracil derivative 67. Its deprotection with TBAF, followed
by bromotrimethylsilane treatment, provided the desired, epi-
merically pure phosphonic acid 63ab.
yphosphonic acids 45a and 45b were prepared starting from the
uracil compound 43. It was first O-acetylated using acetic anhy-
dride and the obtained acetyl derivative was chlorinated with NCS
in pyridine to give the fully protected 5-chlorouridine derivative
43ab as a mixture of epimers. Their separation on silica gel fol-
lowed by successive treatment with TBAF and ethanolic ammonia
to remove the silyl and acetyl protecting groups, respectively, and
final treatment with bromotrimethylsilane to remove the phos-
phonate ester protecting groups afforded the desired nucleoside
phosphonic acids 45a and 45b.
The hydroxyphosphonates 42e44 were transformed into imi-
dazoylthiocarbonyl derivatives by reaction with 1,10-thio-
carbonyldiimidazole in DCE, and these compounds were reduced
with tributyltin hydride in toluene in the presence of AIBN to afford
the phosphonoethylidene derivatives 46, 47, and 49, respectively, in
good yields. Derivative 48 was prepared from compound 47 by a
direct chlorination with NCS in pyridine. Removal of the silyl pro-
tecting group with TBAF and subsequent treatment with bromo-
trimethylsilane afforded the free phosphonic acids 50e53,
respectively.
2.1.5. 3-(5-Chlorouridin-20,30-di-O-yl)-propanephosphonic acid
(Scheme 6)
Another type of the conformationally constrained nucleoside
phosphonic acids, the derivative 56 was prepared in five steps. The
reaction of protected nucleoside 8 with bromopropionaldehyde
diethylacetal smoothly provided the bromo derivative 54.
The reaction of compound 54 with the sodium salt of diethyl
phosphite in THF, followed by chlorination of the uracil moiety with
NCS, provided the fully protected nucleotide, which was succes-
sively treated with
a mixture of TBAF and BSAebromo-
trimethylsilane to remove the silyl and phosphonate diester
groups, respectively, thus yielding the phosphonate derivative 56
as a single epimer in moderate yield.
2.1.6. Epimeric 2,3-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/2; (30-
O)/3]- and 2,3-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/3; (30-
O)/2]-propanephosphonic acids (Schemes 7 and 8)
The synthesis of the regioisomeric pairs of nucleoside phos-
phonic acids 63aa, 63ab and 63ba, 63bb is depicted in Schemes 7
and 8. The HornereEmmonseWadsworth reaction of aldehydes
60a and 60b, which are easily prepared in three steps from the
regioisomeric 30- and 20-O-DMTr derivatives 57a and 57b,
respectively, with tetraethyl methylenediphosphonate smoothly
provided the fully protected vinylphosphonates 61a and 61b.
2.1.7. Epimeric pairs 1,2-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/1;
(30-O)/2]-ethanephosphonic acids 1,2-(5-chlorouridin-20,30-di-O-
yl)-[(20-O)/2; (30-O)/1]-ethanephosphonic acids (Schemes 9 and
10)
The synthesis of the regioisomeric pairs of these compounds
71aa, 71ab and 71ba, 71bb started from the aldehydes 60a and 60b,
respectively. Removal of the DMTr group followed by acetylation of
the formed hemiacetal hydroxyl led to the acetates 69a and 69b

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Scheme 7. Synthesis of epimeric 2,3-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/2; (30-O)/3]-propanephosphonic acids.
which were chlorinated with NCS in pyridine. Subsequent treat-
ment with diethyl trimethylsilyl phosphite and TMSOTf in aceto-
nitrile provided the fully protected regioisomeric pairs of epimeric
phosphonates 70aa, 70ab and 70ba, 70bb. The two-step depro-
tection of these compounds afforded the phosphonic acids 71aa,
71ab and 71ba, 71bb.
2.2. NMR structure determination
The structures of all important products were determined using
1H and 13C NMR spectra. The NMR data are summarized in Tables 5
and 6 The epimers 13a and 13b were distinguished by the observed
NOE contacts of the >CHeP proton. The compound with >CHeP
Scheme 8. Synthesis of the epimeric 2,3-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/3; (30-O)/2]-propanephosphonic acids.

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151
Table 1
The configuration at the neighboring carbon atom (eCH(OH)eP) in
epimers 45a and 45b could not be reliably determined from the
NMR spectra. The interesting conformation effects were observed
in the series of nucleosides with a six-membered dioxane ring
annealed in positions 20,30 (compounds 63, 67, 69, 70, and 71). In
each of 12 compounds (63aa, 63ab, 63ba, 67, 70aa, 70ab, 70ba,
70bb, 71aa, 71ab, 71ba, and 71bb), the substituent Rp in the
dioxane ring always adopts the equatorial position, as follows from
the vicinal coupling constants of protons J(Ha,Hb) and J(Ha,Hc) (see
Fig. 3 and Table 2) and this equatorial position of Rp in a chair
conformation of the dioxane ring determines the conformation of
the ribofuranose ring either in the 3E form (in compounds 63ab, 67,
70ab, 71ab, 63ba, 70ba and 71ba) or in the 2E form (in compounds
63aa, 70aa, 71aa, 63bb, 70bb, and 71bb), as indicated by the vicinal
coupling constants J(H10,H20), J(H20,H30), and J(H30,H40) (see Fig. 3,
Table 2 and Refs. [52,53]).
Study of the intramolecular Michael addition reaction of vinylphosphonate 64
(Scheme 7).
Entry Solvent
[0.5 mL/
Base
Base
[equiv]
Products [ratio]
Conversion
of 64 [%]
65
66a
66b
5 mmol of 64]
1
2
3
4
5
MeOH
MeOH
t-BuOH
t-BuOH
MeONa
Cs2CO3
6
6
6
1
6
5
5
16
15
5
90
80
39
45
90
5
15
45
40
5
100
100
100
100
30 (and
by-products)
60
100
30 (and
by-products)
20 (and
by-products)
100
100
50 (and
Cyclohexanol
6
7
8
Pinacol
Pyridine
Pyridine
6
6
1
12
40
43
42
18
20
46
42
37
9
2,6-(di-t-Bu)
pyridine
DMF
6
5
90
5
10
11
12
6
6
6
90
90
40
5
5
16
5
5
44
DMF
K2CO3
CsF
t-BuOHa
2.3. Biochemistry
by-products)
13
14
15
16
17
t-BuOH
t-BuOH
t-BuOH
Phenol
DBU
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
The main focus of this study was to assess the inhibitory po-
tential of novel conformationally constrained pyrimidine nucleo-
side phosphonic acids and their corresponding “open-structure”
analogs toward human recombinant thymidine phosphorylases
hrTP-1 (Sigma) and hrTP-2 (kindly provided by Dr. J. Brynda of the
Institute) and isolated enzyme from the human PBMC (buffy coats)
of healthy donors. To achieve this, we (i) optimized a two-step
partial purification of TP to remove a large excess of low-
molecular-weight compounds and the substantial part of ballast
proteins and (ii) performed the inhibition assays to select potent bi-
substrate-like inhibitors of human TP.
Pd(OAc)2 0.1
(PPh3)4Pd 0.1
Cs2CO3
6
6
Decomp.
0
a
Microwave irradiation 81 ꢀC, 200 W, 10 min.
contacts to hydrogens H-10 and H-40 was assigned as endo epimer
13a, whereas the compound with >CHeP contacts to hydrogen H-
20 and H-30 was assigned as exo epimer 13b. The application of the
PSEUROT program [52] for the observed vicinal coupling constants
in the ribofuranose ring conformation gave an equilibrium of two
significantly flattened (max. pucker 20ꢀ) 4E and E4 forms in the ratio
43:57 in 13a and in the ratio 66:34 in 13b. The position of the
substituent in the 30-O-methylphosphonates (23a, 24a, 25a, and
26a) and 20-O-methyl-phosphonates (23b, 24b) is determined by
the splitting of the C-30 or C-30 carbon signal due to 3J(C,P) coupling.
The presence of a CH2 group (manifested by upfield shifts of proton
and carbon signals) in position 20 or 30 of the ribofuranose ring
clearly distinguishes phosphonates 31a, 33a from 31b, 33b.
2.3.1. Partial purification of hpTP from human PBMC
Partially purified TP suitable for kinetic and inhibition studies was
obtained in three steps from a 105 g supernatants of the cell homog-
enates, including (i) ammonium sulfate precipitation, (ii) dialysis, and
(iii) subsequent fast anion-exchange chromatography on a HiTrap Q
column. Protein with TP activity was eluted with 150e200 mM NaCl
with minority of other proteins (purification factor w20).
The R-configuration at the OeCH(R)eO carbon atom was
determined from the observed NOE contacts of OeCH(R)eO to the
H-20 and H-30 hydrogen for compounds 45a, 45b, 50e53, and 56.
2.3.2. Determination of TP activity and kinetic parameters
TP activity and inhibition assays were based on the phospho-
rolysis of 20-deoxy-5-nitrouridine (NdU) to 5-nitrouracil [54]. This
Scheme 9. Synthesis of the epimeric 1,2-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/1; (30-O)/2]-ethanephosphonic acids.

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Scheme 10. Synthesis of epimeric 1,2-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/2; (30-O)/1]-ethanephosphonic acids.
conversion resulted in a significant spectrophotometric shift from
340 nm to 314 nm (
D
ε340 ¼ 9200 Mꢁ1 cmꢁ1 at pH 6.5). The Km and V
values for 20-deoxy-5-nitrouridine (NdU) and inorganic phosphate
were determined with human TPs of different origins. The cleavage
was measured for 5e7 min in 96-well plates at 37 ꢀC using an
Infinite IF500 reader (Tecan), and the Km and V values were
calculated using GraphPad Prism 4 (average of three measure-
ments). Michalis-Menten and Lineweaver-Burk plots are available
in the Supporting Information. All data are summarized in Table 3.
Interestingly, there is one order of magnitude difference between
PiKm values for hrTP-1 (Sigma) and hpTP (and hrTP-2).
2.3.3. Inhibition assays e determination of Ki values using 20-
deoxy-5-nitrouridine (NdU) as a substrate
The initial inhibition experiments with compounds depicted in
Fig. 4 were conducted with human recombinant hrTP-1 (Sigma) at
concentrations of 50, 10, and 1
m
M for the inhibitor (NdU ¼ 300
mM;
Pi ¼ 5 mM) to select the most potent inhibitors for determining Ki
values. When a compound exhibited no inhibition (vi/v0) at an in-
hibitor concentration of 50 mM (6:1 NdUeinhibitor ratio), further
evaluation was not performed. The compounds that demonstrated
modest and high inhibitory activities were subjected to the deter-
mination of Ki values from Dixon and Hanes plots (available in the
Supporting Information) using the SigmaPlot software. The Ki values
were calculated from the equations of linear regressions on the basis
Fig. 3. Preferred conformations of compounds with an annealed 1,4-dioxane ring.
Table 2
Selected vicinal proton couplings in ribofuranose and annealed 1,4-dioxane ring.
Compound
Solvent
J (H10, H20)
J (H20, H30)
J (H30, H40)
Ribofuranose conformation
J (Ha, Hb)
J (Ha, Hc)
63ab
67
D2O
<1
1.2
1.0
<1
7.9
8.2
8.3
8.2
8.4
8.2
1.2
2.8
0.5
4.5
4.6
4.7
a
4.5
4.4
4.3
4.4
4.3
4.4
4.5
4.7
4.6
9.6
8.8
9.1
9.0
1.2
<1
<1
<1
<1
0.6
9.0
7.0
9.6
3E
3E
3E
3E
2E
2E
2E
2E
2E
2E
3E
3E
3E
10.2
9.6
10.7
11.3
9.9
2.6
2.7
3.1
2.7
2.6
2.8
2.7
CDCl3
CDCl3
D2O
D2O
D2O
CDCl3
D2O
CDCl3
D2O
D2O
CDCl3
D2O
70ab
71ab
63aa
71aa
70aa
63bb
70bb
71bb
63ba
70ba
71ba
11.2
11.3
10.2
2.6
a
a
11.5
2.6
9.5
2.7
a
a
11.1
2.8
The superscript “a” denotes the J-values that could not be determined due to the overlap of signals.

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153
Table 3
The first series consists of the derivatives 12e14 with an
annealed 1,3-dioxolane ring substituted in the C2 position with a
phosphoryl group. This substitution resulted in chirality on the C2-
atom and, thus, resulted in the formation of pairs of endo and exo
epimers (12ae12b, 12ce12d, 13ae13b, and 14ae14b). Whereas the
endo epimers were found to be low micromolar and submicromolar
inhibitors that exhibited a decrease in Ki values in the order 12a,
12c, 13a, 14a (B ¼ U z T » U5eCl z U5eI) with a single order of
magnitude Ki increase between 12c (B ¼ T) and 13a (B ¼ U5eCl), the
exo epimers 12b and 12d (B ¼ U and T) were inactive. The com-
pound 13b (B ¼ U5eCl) showed, surprisingly, a Ki value only three
times lower than its endo congener 13a, which suggests that a
different binding mode between the endo and exo epimers led to a
similar efficiency. Moreover, the introduction of an electron-
withdrawing substituent at the C5 position (Cl, I) of the uracil
nucleobase led to a significant increase in the inhibitory effect.
These data are in agreement with those reported previously and
suggest that the 5-halo group may improve the active siteeligand
interaction in the lipophilic pocket of the enzyme [55] and may
Comparison of Km and V values for NdU and inorganic phosphate (Pi) for three TPs.
TP
NdUKm
[m
M]
NdUV [nmol/min]
PiKm
[m
M]
PiV [nmol/min]
hrTP-1a
hpTPb
53 ꢂ 7
1.10 ꢂ 0.05
0.55 ꢂ 0.01
0.92 ꢂ 0.05
140 ꢂ 10
1330 ꢂ 100
1500 ꢂ 300
1.50 ꢂ 0.01
0.52 ꢂ 0.01
0.80 ꢂ 0.07
80 ꢂ 20
85 ꢂ 20
hrTP-2c
a
Human recombinant TP expressed in Chinese hamster ovarian cells (Sigma; the
enzyme is not further available).
b
Partially purified TP from human PBMC (buffy coat).
Human recombinant TP expressed in E. coli.
c
of three independent measurements. These data for the most potent
compounds are summarized in Table 4 and are also depicted in Fig. 4.
2.4. Inhibitory properties of prepared compounds
Four series of conformationally constrained nucleoside phos-
phonic acids derived from uridine and 5-substituted uridines were
synthesized (Table 4, Fig. 4).
Fig. 4. Overview of the prepared nucleoside phosphonic acids as potential TP inhibitors (for inhibitory data see Table 4); compounds with underlined numbers exhibited Ki values
equal or below 15 M; remaining compounds did not pass the selection assay (no inhibition at ratio of substrate to inhibitor 6:1).
m

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154
Table 4
The Ki values for the most potent inhibitors of human thymidine phosphorylase hrTP-1 and hpTP.
Inhibitor
B
Structure
hrTP-1a
Ki [ M]
hpTPb
m
Ki/hrTP-1Km
Ki [
mM]
Ki/hpTPK
m
12a
U
11.0
0.207
nd
nd
12cc
13a
14a
T
8.80
0.160
0.018
0.014
nd
1.12
nd
nd
0.0140
nd
U5eCl
U5eI
0.95 (0.77)g
0.75
13b
26a
33a
45a
U5eCl
U5eCl
T
2.90
11.0
nd
0.054
0.207
nd
nd
nd
8.68
0.109
0.049
0.0038
3.90e
U5eCl
nd
nd
0.306
45b
50d
U5eCl
nd
nd
1.67
0.0210
0.0019
T
0.201
0.0038
0.148
51
52
53
U
0.893e
0.066
0.460f
0.0169
0.0012
0.0086
0.840
0.0105
0.0006
0.0035
U5eCl
U5eF
0.048 (0.030)g
0.277
56
U5eCl
14.5f
0.270
18.2
0.22
a
Human recombinant TP expressed in Chinese hamster ovarian cells (Sigma; the enzyme supply was disconnected).
Partially purified TP from human PBMC (buffy coat).
Ref. [45].
Ref. [43,44].
b
c
d
e
The Ki values for 33a and 51 were calculated from IC50 values according to the equation Ki ¼ (IC50/(1 þ [S]/Km which is valid for competitive inhibition (Km ¼ 53
mM,
[S] ¼ 125
m
M, IC50 ¼ 10 and 3
m
M, respectively).
f
Human recombinant TP expressed in E. coli (hrTP-2).
g
Ki regarding to inorganic phosphate; nd e not determined.; Ki values represent the average value of three experiments; standard deviation varied from 7 to 10%.

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155
increase the acidity of the 3-NH moiety [56] to strengthen the
hydrogen bonding between the uracil nucleobase and serine 217. In
addition, the presence of an electronegative substituent at the C5
position of the uracil nucleobase may increase the electropositivity
of the O40eC10 atoms; thus, such a compound should more effec-
tively mimic the transition state of the substrate / product
conversion.
On the basis of these results, we synthesized conformationally
flexible, regioisomeric 30-O- and 20-O-methylphosphonic acids
23ae26a and 23be24b, respectively, that are isosteric with their
cyclic congeners 12e14, to achieve better conformational accom-
modation in TP binding sites. These “open-structures”, however,
were inactive, with the exception of 30-O-methanephosphonic acid
26a (B ¼ U5eCl), which exhibited a Ki value identical to that of cyclic
compound 12a (B ¼ U) but more than one order of magnitude
greater than that of 13a (B ¼ U5eCl). This result suggests that 26a
possesses a higher binding entropy of due to the flexibility of the
phosphonate chain.
and 63bae63bb. The enlargement of the anneled ring from a 1,3-
dioxolane to a 1,4-dioxane ring led to a loss of inhibitory activity
of compounds 63. An NMR spectroscopy-based conformational
study of compounds 63 revealed (similar to the case of 71) inter-
esting conformational changes of the pentofuranose part of the
molecule, which is dependent on the configuration of the carbon
atom bearing the phosphomethyl residue (for more details, see
chapter NMR structure determination). The conformational changes,
the spatial requirements of compounds 63, and the positions of
nucleobase and phosphoryl groups may explain the inactivity of
these compounds.
The fourth series consists of two epimeric pairs, 71aae71ab and
71bae71bb (all four compounds with B ¼ U5eCl), which are isos-
teric to 13a. Similar to the case of compounds 63, we did not
observe any inhibitory activity, most likely due to the inappropriate
stereochemical arrangement of the molecule.
3. Conclusions
The second series consists of derivatives 45a, 45b, and 50e53
with an annealed 1,3-dioxolane ring substituted with a phospho-
nomethyl moiety in the 2-position. The synthesis of compound 50
We synthesized a series of new conformationally constrained
uracil-based nucleoside phosphonic acids containing annealed 1,3-
dioxolane and 1,4-dioxane rings bearing a phosphonate moiety and
their “open-structure” isosters. These compounds were designed to
provide a variety of structural types believed to cover a range of
potential candidates that could provide sufficient data on the
structureeactivity relationship. Inhibition study with human re-
combinant and native TPs revealed that compounds with an
annealed ring are much more potent inhibitors of TPs than their
flexible isosters. Of 33 tested nucleoside phosphonic acids, we
selected 6 compounds that exhibited the Ki values in the range of
(B
¼
T) and its submicromolar inhibition effect on hrTP-1
(Ki ¼ 236 nM) have been previously reported [43,44]; however,
we were unable to reproduce the published synthesis of 50.
Therefore, we devised a new synthetic route that provided 50 as a
single epimer in good yield and found, for hrTP-1 and hpTP, Ki
values of 201 and 148 nM, respectively. On the basis of these results,
we synthesized additional compounds 51e53, which differ in the
nucleobase (B ¼ U, U5eCl, or U5eF, respectively); among these
compounds, we selected the nucleoside phosphonic acid 52
(B ¼ U5eCl) as the most potent, potentially bi-substrate-like inhib-
itor of hrTP-1 and hpTP developed thus far, with Ki values of 66 and
48 nM, respectively. Our assumption that 52 could be a bi-
substrate-like inhibitor was supported by the determination of
the Ki value relative to inorganic phosphate as the second substrate
of TP; the Ki values were determined with various concentrations of
inorganic phosphate (from 1 to 5 mM). The determined value of
30 nM (the Dixon plot is available in the Supporting Information)
strongly supported the hypothesis that 52 is a bi-substrate-like
inhibitor of human TP. Further extension of the phosphonoalkyl
chain (phosphonate 56) led to a decrease in the affinity to hrTP-1
and hpTP by factors of 220 and 380, respectively.
0.048e1
mM. These compounds exhibited a competitive type of
inhibition relative to NdU as obvious from DixoneWebb plots (see
Supporting Information). The most potent inhibitor 52 exhibited Ki
values 48 and 30 nM with 20-deoxy-5-nitrouridine and inorganic
phosphate as substrates, respectively. These values suggest that the
phosphonic acid 52 is a bi-substrate-like inhibitor of human TP. The
bi-substrate-like binding mode of 52 to human TP should be
confirmed unambiguously through resolution of the crystal struc-
ture of human TP with inhibitor 52. Interestingly, no crystal
structure of human TP with a nucleoside phosphonic acid has thus
far been reported.
In general, bi-substrate-like inhibitors can be expected to
exhibit lower binding entropies than either of the individual sub-
strates. In addition, bi-substrate-like inhibitors may also improve
the binding enthalpy through an additional interaction in the
binding site. Following this idea, we synthesized epimeric com-
pounds 45a and 45b (B ¼ U5eCl in both cases) bearing an additional
hydroxy group in the alpha position to the phosphoryl group. The
hydroxyl could serve both as the donor and the acceptor of the
hydrogen bond in the TP binding site. However, in the case of
compounds 45a and 45b, their affinity to hpTP was decreased by
factors of 6 and 35, respectively, in comparison with that of 52. The
“open-structures”, i.e., the regioisomeric 30-O- (31a and 33a) and
20-O-(2-phosphonoethyl) (31b and 33b) derivatives, did not exert
any significant inhibition effect. Only compound 33a (B ¼ T)
exhibited a weak activity (decreasing the affinity to hpTP by a factor
of 81) compared to that of 52.
The third series consists of conformationally constrained com-
pounds, the vicinal 20,30-O-phosphonopropylidene derivatives 63
(all compounds with B ]B ¼ U5eCl) with annealed 1,4-dioxane
ring, related to 52. In fact, the phosphonic acids 63 are isosteric
to that of 52. Two possible size-positionings of the phosphono-
methyl moiety on the 1,4-dioxane ring gave rise to two
regioisomers and each of them in the form of epimers 63aae63ab
4. Experimental
4.1. General
The course of the reactions was followed by TLC on Merck Silica
gel 60 F254 aluminum sheets and the products were visualized
either by UV monitoring (254 nm), or by spraying with 1% ethanolic
solution of 4-(4-nitrobenzyl)pyridine (PNBP) which, after short
heating and exposing the sheet to ammonia vapors, showed the
phosphonate esters as blue spots, or by spraying with 5% ethanolic
solution of sulfuric acid and subsequent heating (detection of O-
DMTr derivatives, deep orange spots). Preparative column chro-
matography (PLC) was performed on silica gel (40e60 mm, Fluka),
with elution at the rate of 40 mL/min. PLC and TLC were carried out
with the following solvent systems (v/v): chloroformeethanol 9:1
(C-1), ethyl acetateetoluene 1:1 (T-1), ethyl acetateeacetonee
ethanolewater 4:1:1:1 (H-1), ethyl acetateeacetoneeethanole
water 12:2:2:1 (H-3), and 2-propanol-conc. aqueous ammoniae
water 7:1:2 (IPAW).
HPLC analyses were performed on an Alliance (Waters) instru-
ment using Luna C18 column (5
under gradient elution with acetonitrile in 0.1 M-triethylammo-
nium acetate buffer (TEAA). The preparative reversed-phase HPLC
mm, 4.6 ꢃ 150 mm, Phenomenex)

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156
was performed on an Axia column (20 ꢃ 200 mm, Luna C18(2),
m, Phenomenex), using a linear gradient of methanol in 0.1 M
4.2.2. TP activity assay and determination of NdUKm and PiKm values
for TP (Table 3)
5
m
triethylammonium hydrogencarbonate buffer (TEAB) or in water.
The MS HR-ESI spectra (m/z) were recorded on an LTQ Orbitrap
XL (Thermo Fischer Scientific) instrument. The IR spectra were
recorded on FTIR spectrometer (Bruker Equinox 55, Germany). The
1H and 13C NMR spectra were measured on a Bruker AVANCE-600
spectrometer (1H at 600.13 MHz, 13C at 150.9 MHz) using cryo-
probe (5 mm CPTCI 1He13C/15N/D Z-GRD) in D2O or CDCl3 at
300 K. Structural assignment of proton and carbon signals was
achieved using 2D-H,H-COSY, 2D-H,H-ROESY, 2D-H,C-HSQC and
2D-H,C-HMBC spectra. NMR data are presented in Tables 5 and 6.
All compounds used in biochemical testing were of proved pu-
rity of over 95% as determined by RP HPLC.
The instruments used in the work on enzyme purification
involved Beckman Coulter Allegra X-22R centrifuge, Beckman
Coulter Optima L-100 XP ultracentrifuge, and Cell ultrasound
disintegrator Soniprep 150 (Sanyo) and, in enzyme assays, the
Infinite F500 Tecan reader was used. The protease inhibitor cocktail
was bought from Sigma. The human recombinant thymidine
phosphorylase hrTP-1 expressed in hamster ovarian cells was ob-
tained from Sigma (this enzyme is not further available), and hu-
man recombinant thymidine phosphorylase expressed in E. coli
A partially purified thymidine phosphorylase (hpTP), hrTP-2, or
hrTP-1 was assayed in 96-well plates filled with 200
m
L of 50 mM
TriseHCl buffer (pH 6.5) containing 300
mM
20-deoxy-5-
nitrouridine (NdU) and 5 mM potassium phosphate. The phos-
phorolysis of NdU was initiated at 37 ꢀC by the addition of TP so-
lution (10 mL z 2e3 mg of protein) and was monitored at 340 nm for
5e7 min using the Infinite F500 Tecan reader. The initial steady-
state rates were calculated from a linear portion of the phospho-
rolytic patterns of NdU over its wide concentration range (10e
500
mM) at a constant 5 mM concentration of inorganic phosphate
(for hrTP-1, 300
m
M phosphate was used), using the extinction
coefficient ε340 ¼ 9200 Mꢁ1 cmꢁ1. The determination of PiKm values
was performed at a constant 300 mM concentration of NdU and at
variable concentrations of inorganic phosphate (from 150 mM to
5 mM). The obtained data fit the MichaeliseMenten equation. For
calculations of NdUKm and PiKm values, the GraphPad Prism 4 and
SigmaPlot software packages were used.
4.2.3. Inhibition study
Initially, all synthesized compounds (Fig. 4) were tested against
hrTP-1 (Sigma) at concentrations of 1, 10, and 50
mixture (200 L) was composed of 300 M NdU and 300
tassium phosphate, with variable concentrations of inhibitor (1, 10,
and 50 M) and 50 mM Tris buffer (pH 6.5). The cleavage was
mM. The reaction
ꢀ
(hrTP-2) was kindly provided by Dr. Jirí Brynda of this Institute.
m
m
mM po-
m
4.2. Biochemistry
started by the addition of w1.2 mU of hrTP-1 (5e10 mL), and the
absorbance change was monitored within 5e7 min at 37 ꢀC.
The human recombinant thymidine phosphorylase (hrTP-1) and
protease inhibitor cocktail were obtained from Sigma (hrTP-1 is no
longer available). Peripheral blood mononuclear cells (PBMC) were
isolated from the buffy coat of healthy donors provided by the 1st
Department of Internal Medicine, Department of Hematooncology,
General Teaching Hospital and by the 1st Faculty of Medicine,
Charles University, Prague.
Compounds which demonstrated inhibitory activity at concentra-
tions less than 50
and PiKi values (the latter only for the most potent inhibitors). For
the determination of NdUKi, the reaction mixture (200
L) was
composed of 50 mM Tris buffer (pH 6.5), 150 or 300 M NdU, and
300
M potassium phosphate. For the determination of PiKi, the
reaction mixture (200 L) was composed of 50 mM Tris buffer (pH
6.5), 300 M NdU, and 150 or 300 M phosphate. In both cases, the
concentrations of the inhibitors were varied from 50 nM to 10 M.
The phosphorolysis of NdU was initiated at 37 ꢀC by the addition of
hrTP-1 (w1.2 mU), hrTP-2, and/or hpTP (approximately 1e3 g of
m
M were subjected to the determination of NdUKi
m
m
m
m
m
m
4.2.1. Partial purification of thymidine phosphorylase (hpTP)
All steps were conducted at 4 ꢀC. Human peripheral blood
mononuclear cells (PBMC) of healthy donors (2 packages,
w300 mL) were sedimented at 4000ꢃg, washed (2 ꢃ 50 mL) with
buffer A (20 mM TriseHCl, 15 mM sodium citrate, 135 mM sodium
chloride, 2.5 mM EDTA, 5.5 mM glucose, pH 7.5), and stored
at ꢁ70 ꢀC.
m
m
protein in the latter two cases) and was monitored within 5e7 min
at 37 ꢀC, as previously described. The type of inhibition and Ki
values were determined from a Dixon plot (1/v versus [I]) and a
Hanes plot, respectively, using the SigmaPlot software. The Ki
values were calculated on the basis of three independent
measurements.
The frozen blood cells were disrupted by three thawinge
freezing cycles, and the homogenate was suspended in 50 mM
TriseHCl buffer (pH 7.5; 15 mL) containing a protease inhibitor
cocktail (0.1 mL). The suspension was sonicated three times for 10 s.
The crude lysate was centrifuged at 11000 ꢃ g for 30 min to remove
cell debris and then at 100 000 ꢃ g for 1 h. The resulting super-
natant (15 mL) was saturated with ammonium sulfate (3.5 g) to
40%. After 30 min of standing, the precipitate was collected by
centrifugation at 9000ꢃg for 40 min and dissolved in 50 mM Trise
HCl buffer (pH 7.5; 7.5 mL). The enzyme solution was dialyzed
overnight against the same buffer (1 L) containing 25 mM DTT and
20% glycerol. The desalted solution was loaded onto a HiTrap Q
column (GE Healthcare) equilibrated with 50 mM TriseHCl buffer
(pH 6.5; 20 mL). The column was eluted with a linear gradient (0e
1 M) of NaCl in the same buffer at a flow rate of 1 mL/min. Twenty
fractions (1 mL each) were collected and analyzed for TP activity
and protein content. The fractions containing TP activity were used
immediately for assays. The two-step purification process resulted
in a partially purified enzyme with a specific activity of approxi-
4.3. Chemistry
Unless stated otherwise, all phosphonic acids were lyophylized
from water as triethylammonium salts and dried at 13 Pa over
phosphorus pentoxide for 48 h.
4.3.1. (R)-(uridin-20,30-di-O-Yl)-methanephosphonic acid (12a) and
(S)-(uridin-20,30-di-O-yl)-methanephosphonic acid (12b) (Scheme
1)
The suspension of 50-O-tert-butyldiphenylsilyluridine 8 (5.0 g,
10.4 mmol) in dichloromethane (100 mL) and trimethyl ortho-
formate (3.4 mL, 31.2 mmol) was acidified with 4 M HCl in DMF
(slightly red color on a wet pH paper), and the resulting mixture
was stirred at rt overnight (TLC in C-1). Triethylamine was added,
and the clear solution was concentrated under reduced pressure.
The product was purified by silica gel chromatography using a
linear gradient of ethanol (0e10%) in chloroform. The obtained
nucleoside orthoester 10 (4.9 g, 9.4 mmol) was treated with
dimethyl chlorophosphite (2.4 g, 18.8 mmol) in dry acetonitrile
mately 13
m
mol hꢁ1 mgꢁ1. The protein concentration was deter-
mined by the Bradford method [57] using bovine serum albumin as
a standard.

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I. Kosiová et al. / European Journal of Medicinal Chemistry 74 (2014) 145e168
157
(90 mL) at rt overnight (TLC in C-1). The reaction mixture was
quenched by addition of 1M TEAB in 50% aqueous ethanol (30 mL)
at 0 ꢀC, and concentrated under reduced pressure. The residue was
co-evaporated with ethanol (2 ꢃ 50 mL), and the phosphonate
diesters of compounds 12a,b were purified as a mixture by silica gel
chromatography using a linear gradient of ethanol (0e10%) in
chloroform. The obtained mixture of protected diesters of com-
pounds 12a,b (4.4 g, 7.3 mmol) was co-evaporated with dry
acetonitrile and treated with bromotrimethylsilane (3.8 mL,
29.2 mmol) and 2,6-lutidine (6.9 mL, 58.4 mmol) in acetonitrile
(70 mL). The resulting solution was left aside at rt overnight and
then concentrated under reduced pressure. The residue was treated
with 0.5 M TBAF in THF (50 mL) at rt for 30 min, the solution was
concentrated under reduced pressure, and the mixture of epimers
12a,b was separated by RP HPLC. Overall yield: 1.4 g (4.3 mmol,
31%), out of which 0.95 g (68%) of (R)-epimer 12a and 0.45 g (32%)
of (S)-epimer 12b. The 1H and 13C NMR spectra were identical with
those reported earlier [45].
rt, stirred overnight (TLC in H-3), then quenched by the addition of
glacial acetic acid (0.4 mL; 7.6 mmol) at 0 ꢀC, and concentrated
under reduced pressure. The product was purified by silica gel
chromatography using a linear gradient of H-1 (0e100%) in ethyl
acetate. This product (2.1 g, 3.2 mmol) was treated with 80% aq.
acetic acid (30 mL) at rt for 30 min (TLC in C-1). The reaction
mixture was concentrated at reduced pressure, the residue was
diluted with chloroform (100 mL), and the solution was washed
with water (3 ꢃ 50 mL). Water layers were combined, evaporated,
and the pure product 19a was co-distilled subsequently with
ethanol and acetonitrile, diluted in dry acetonitrile (10 mL), and
treated with bromotrimethylsilane (1.7 mL, 12.8 mmol) and 2,6-
lutidine (2.9 mL, 25.6 mmol). The resulting mixture was stirred at
rt overnight and concentrated under reduced pressure. The crude
product 23a was purified by RP HPLC.
Overall yield of 23a: 0.41 g (1.2 mmol, 32%). HRMS (M ꢁ H)ꢁ for
C
11H16N2O8P: calcd m/z 335.0645, found 335.0641; IR (KBr, cmꢁ1):
3501, 3404, 3355, 3282, 3065, 1697, 1685, 1064, 915, 563. NMR data
e see Tables 5 and 6
4.3.2. (R)-(5-Chlorouridin-20,30-di-O-Yl)-methanephosphonic acid
(13a) and (S)-(5-chlorouridin-20,30-di-O-yl)-methanephosphonic
acid (13b) (Scheme 1)
4.3.5. (30-Deoxy-5-methyluridin-20-O-yl)-methanephosphonic acid
(23b) (Scheme 2)
50-O-tert-Butyldiphenylsilyluridine 8 (5.0 g, 10.4 mmol) was
treated with N-chlorosuccinimide (2.1 g, 15.6 mmol) in pyridine
(100 mL) at 90 ꢀC for 45 min. The solution was concentrated under
reduced pressure, and the product was purified by silica gel chro-
matography using a linear gradient of ethanol (0e10%) in chloro-
form. The obtained derivative 9 (4.1 g, 7.9 mmol) was suspended in
dichloromethane (80 mL) under stirring, trimethylorthoformate
(2.6 mL, 23.7 mmol) was added, the suspension was acidified with
4 M HCl in DMF (slightly red color on a wet pH paper), and the
resulting mixture was stirred at rt overnight (TLC in C-1). Trie-
thylamine was added, and the clear solution was concentrated
under reduced pressure. The product was purified by silica gel
chromatography using a linear gradient of ethanol (0e10%) in
chloroform. The obtained orthoester 11 (3.3 g, 6.4 mmol) was used
directly for the synthesis of phophonates 13a,b by the procedure
described for compounds 12a and 12b.
Overall yield of 13a and 13b: 0.77 g (2.1 mmol, 33%). (R)-epimer
13a: 0.56 g (71%); HRMS (M ꢁ H)ꢁ for C10H11N2O9P: calcd m/z
369.0016, found 368.9900; IR (KBr, cmꢁ1): 3422, 3074, 2966, 1699,
1646, 1541, 1111, 1007, 976, 780, 593. NMR data e see Tables 5 and 6
(S)-epimer 13b: 0.21 g (29%); HRMS (M ꢁ H)ꢁ for C10H11N2O9P:
calcd m/z 369.0017, found 368.9896; IR (KBr, cmꢁ1): 3383, 2965,
2877, 1700, 1645, 1447,1541, 1447, 1109, 1008, 975, 780, 758, 591.
NMR data e see Tables 5 and 6
Compound 23b was prepared from 15b (1.2 g, 1.4 mmol) by the
procedure described for compound 23a. Overall yield: 0.219 g
(0.65 mmol, 46%). HRMS (M ꢁ H)ꢁ for C11H16N2O8P: calcd m/z
335.0645, found 335.0652. IR (KBr, cmꢁ1): 3425, 2930, 1697, 1476,
1444, 1271, 1116, 1076, 973, 913, 768, 588. NMR data e see Tables 5
and 6
4.3.6. (5-Methyluridin-30-O-yl)-methanephosphonic acid (24a)
(Scheme 2)
Compound 24a was prepared from 16a (1.0 g, 1.2 mmol) by the
procedure described for compound 23a. Overall yield: 0.197 g
(0.56 mmol, 47%). HRMS (M ꢁ H)ꢁ for C11H16N2O9P: calcd m/z
351.0594, found 351.0591; IR (KBr, cmꢁ1): 3418, 3251, 3062, 2985,
2929, 2680, 2490.1693,1472,1393,1264,1233,1136,1114,1067, 914,
839, 790, 767, 575, 550, 490, 464. NMR data e see Tables 5 and 6
4.3.7. (5-Methyluridin-20-O-yl)-methanephosphonic acid (24b)
(Scheme 2)
Compound 24b was prepared from 16b (1.0 g, 1.8 mmol) by the
procedure described for compound 23a. Overall yield: 0.352 g
(1.0 mmol, 55%). HRMS (M ꢁ H)ꢁ for C11H16N2O9P: calcd m/z
351.0594, found 351.0596; IR (KBr, cmꢁ1): 3420, 3069, 2940, 2909,
2828, 2361, 1696, 1474, 1270, 1093, 1067, 919, 792, 770, 591. NMR
data e see Tables 5 and 6
4.3.3. (R)-(5-Iodouridin-20,30-di-O-Diyl)-methanephosphonic acid
(14a) and (S)-(5-iodouridin-20,30-di-O-diyl)-methanephosphonic
acid (14b) (Scheme 1)
4.3.8. (20-Deoxyuridin-30-O-yl)-methanephosphonic acid (25a)
(Scheme 2)
Compound 25a was prepared from 17a (3.6 g, 6.8 mmol) by the
procedure described for compound 23a. Overall yield: 0.64 g
(1.9 mmol, 28%). HRMS (M ꢁ H)ꢁ for C10H14N2O8P: calcd m/z
321.0488, found 321.0487; IR (KBr, cmꢁ1): 3193, 3057, 2991, 2946,
2834, 2694, 2475, 2361, 1692, 1463, 1384, 1276, 1145, 1121, 1098,
1063, 916, 811, 765, 559, 538, 470, 420. NMR data e see Tables 5 and
6
The solution of epimeric mixture of 12a,b [45] (34 mg;
0.10 mmol) and iodine (25 mg; 0.10 mmol) in 1.5% aq. nitric acid
(5 mL) was stirred at rt for 24 h. The reaction mixture was
neutralized with triethylamine and applied on Axia C18 column.
The elution with a linear gradient of methanol in water (0 / 50%)
yielded single epimers 14a (29 mg; 63%) and 14b (11 mg; 24%). UV
spectrum, lmax ¼ 289.5 nm (in water). HRMS (M ꢁ H)ꢁ for
C
10H12N2O9INaP: calcd m/z 484.92228, found 484.92173;
4.3.9. (5-Chloro-20-deoxyuridin-30-O-yl)-methanephosphonic acid
(26a) (Scheme 2)
4.3.4. (Thymidin-30-O-yl)-methanephosphonic acid (23a) (Scheme
2)
Sodium hydride (0.6 g, 15.2 mmol) was added at 0 ꢀC to a stirred
solution of 50-O-dimethoxytrityl thymidine (15a) (2.0 g, 3.8 mmol)
and methyl tosyloxymethanephosphonate [48] (2.1 g, 7.6 mmol) in
DMF (30 mL). The resulting mixture was left to warm gradually to
N-chlorosuccinimide (1.8 g, 13.5 mmol) was added under stir-
ring to a solution of 20-deoxy-50-O-dimethoxytrityluridine 17a
(4.8 g, 9.0 mmol) in pyridine (90 mL). The resulting mixture was
heated at 90 ꢀC for 45 min and then concentrated under reduced
pressure. The separation on silica gel column using a linear gradient
of ethanol (0 / 10%) in chloroform afforded the product 18a (4.5 g,

Table 5
Proton NMR data.
Compound Solv.
H-10
H-20
H-200/H-300
H-30
H-40
H-50a
H-50b
Base
Substituents
13a
13b
23a
D2O
D2O
D2O
5.95 d (2.5)
5.06 ddd
(2.5; 6.5; 0.5)
4.98 dd (2.9; 6.5)
e
4.84 ddd
(6.5; 3.5; 0.5)
4.86 dd
(6.5; 4.7)
4.28 m
(6.5; 3.2; 3.1)
4.50 m
(3.5; 3.7; 5.5)
4.26 ddd
(4.7; 5.8; 3.7)
4.17 ddd
(3.2; 3.9; 5.0)
3.86 dd
(3.7; 12.4)
3.90 dd
(12.3; 3.7)
3.83 dd
(12.4; 3.9)
3.79 dd
(5.5; 12.4)
3.83 dd
(12.3; 5.8)
3.78
(12.4; 5.0)
H-6: 8.12 s
PeCH<: 5.12 d (20.8)
5.88 d (2.9)
e
H-6: 8.02 s
PeCH<: 5.08 d (16.0)
6.29 dd (7.7; 6.3) 2.32 ddd
(14.2; 7.7; 6.5)
2.52 ddd (14.2;
6.3; 3.1)
H-6: 7.65 q (1.2);
5-CH3: 1.89 d (1.2) (12.9; 9.4) and 3.59 dd
(12.9; 9.2)
PeCH2eO: 3.63 dd
23b
24a
24b
25a
26a
31a
D2O
D2O
D2O
D2O
D2O
D2O
5.96 d (1.3)
5.94 d (4.9)
5.99 d (3.7)
4.28 dt
(1.3; 1.4; 5.7)
2.18 ddd
(14.0; 1.4; 5.4)
2.03 ddd
(14.0; 5.7; 11.0)
4.49 m
3.96 dd
3.76 dd
(12.9; 4.4)
H-6: 7.78 q (1.3);
5-CH3: 1.87 d (1.3) (12.7; 9.5) and 3.64 dd
(12.7; 9.4)
H-6: 7.69 q (1.2);
5-CH3: 1.88 d (1.2) (13.0; 9.1) and 3.72 dd
(13.0; 9.0)
H-6: 7.77 q (1.3);
5-CH3: 1.88 d (1.3) (13.0; 9.4) and 3.75 dd
(13.0; 9.3)
H-5: 5.88 d (8.1);
H-6: 7.88 d (8.1)
PeCH2eO: 3.71 dd
(11.0; 5.4; 2.8; 4.4) (12.9; 2.8)
4.46 dd (4.9; 5.3)
e
e
4.08 t
(5.3; 5.2)
4.23 ddd
(5.2; 3.2; 4.1)
3.90 dd
(12.8; 3.2)
3.83 dd
(12.8; 4.1)
PeCH2eO: 3.76 dd
4.18 dd (3.7; 5.3)
4.33 dd
(5.3; 6.4)
4.12 ddd
(6.4; 2.8; 4.1)
3.93 dd
(12.9; 2.8)
3.82 dd
(12.9; 4.1)
PeCH2eO: 3.88 dd
6.27 dd (7.7; 6.2) 2.31 ddd
(7.7; 14.3; 6.3)
2.56 ddd
(6.2; 14.3; 2.9)
4.27 m
(6.3; 2.9; 3.0)
4.20 ddd
(3.0; 3.8; 5.0)
3.82 dd
(12.4; 3.8)
3.76 dd
(12.4; 5.0)
PeCH2eO: 3.69 dd
(13.1; 9.4) and 3.64 dd
(13.1; 9.4)
PeCH2eO: 3.65 dd
(12.9; 9.3) and 3.60 dd
(12.9; 9.3)
6.26 dd (7.4; 6.1) 2.31 ddd
(7.4; 14.3; 6.4)
2.58 ddd
(6.1; 14.3; 3.1)
4.28 m
(6.4; 3.1; 3.2)
4.21 ddd
(3.2; 3.7; 4.8)
3.84 dd
(3.7; 12.5)
3.79 dd
(4.8; 12.5)
H-6: 8.16 s
6.26 dd (7.5; 2.2) 2.70 m
(7.5; 15.2; 6.2)
6.25 dd (7.5; 2.2)
2.34 dt (15.2; 2.2) 4.24 m
(6.2; 2.2; 1.8)
2.31 dt (15.2; 2.2) 4.22
(6.2; 2.2; 1.8)
2.071 ddd
(5.7; 14.0; 10.8)
4.58 m
(1.8; 4.2; 5.5)
4.61 m
(1.8; 4.2; 5.5)
4.477 m
(10.8; 5.6; 2.8; 4.4) (2.8; 12.8)
3.71 dd
(4.2; 12.3)
3.70 dd (4.2; 12.3)
3.64 dd
(5.5; 12.3)
H-6: 7.76 q (1.2);
5-CH3: 1.91 d (1.2) 3.80e3.88 m and 3.60 m
PeCH(OH)eCH2eO:
31b
33a
D2O
D2O
5.953 d (1.5)
4.317 m
(1.5; 5.7; 1.6)
2.156 ddd
(1.6; 14.0; 5.6)
3.966 dd
3.76 dd
(4.4; 12.8)
H-6: 7.783 q (1.2); PeCH(OH)eCH2eO:
5-CH3: 1.87 d (1.2) w3.94 m, 1H; 3.996 ddd
and 3.694 ddd, 2H
H-6: 7.786 q (1.2); PeCH(OH)eCH2eO:
5-CH3: 1.87 d (1.2) w3.94 m, 1H;
3.91 m and 3.752 m, 2H
5.961 d (1.4)
4.319 m
(1.4; 5.7; 1.6)
2.066 ddd
(5.7; 14.0; 10.8)
2.173 ddd
(1.6; 14.0; 5.6)
4.479 m
(10.8; 5.6; 2.8; 4.4) (2.8; 12.8)
3.969 dd
3.76 dd
(4.4; 12.8)
6.21 dd (7.2; 2.4) 2.68 ddd
(7.2; 15.2; 6.2)
2.29 ddd
(2.4; 15.2; 2.0)
4.22 dt
(6.2; 2.0; 1.9)
4.58 ddd
(1.9; 4.2; 5.5)
3.70 dd
(4.2; 12.4)
3.64 dd
(5.5; 12.4)
H-6: 7.69 q (1.2);
5-CH3: 1.91 d (1.2) 1.82e1.92 m (2H);
PeCH2eCH2eO:
3.71 m (2H)
6.27 dd (7.4; 6.5) 2.36 ddd
(7.4; 14.4; 6.6)
2.49 ddd
(6.5; 14.4; 3.5)
4.26 dt
(6.6; 3.5; 3.7)
4.11 td
(3.7; 3.7; 5.0)
3.83 dd
(3.7; 12.4)
3.77 dd
(5.0; 12.4)
H-6: 7.64 q (1.2);
5-CH3: 1.89 d (1.2) 1.82e1.92 m (2H);
PeCH2eCH2eO:
3.97 m (2H)
33b
45a
45b
50
D2O
D2O
D2O
D2O
D2O
D2O
5.93 d (1.5)
5.94 d (2.7)
5.94 d (2.6)
5.90 d (2.7)
5.88 d (2.6)
5.87 d (2.6)
4.30 ddd
(1.5; 5.6; 1.7)
2.13 ddd
(1.7; 14.1; 5.7)
2.07 ddd
(5.6; 14.1; 10.7)
4.44 m
3.96 dd
3.75 dd
(4.5; 12.8)
H-6: 7.77 q (1.2);
5-CH3: 1.87 d (1.2) 1.87 m (2H); 3.84 m
and 3.79 m
PeCH2eCH2eO:
(10.7; 5.7; 2.8; 4.5) (2.8; 12.8)
5.00 dd (2.7; 6.5)
e
e
e
e
e
4.83 dd (6.5; 3.0) 4.54 ddd
(3.0; 3.7; 5.4)
3.84 dd
(3.7; 12.3)
3.79 dd
(5.4; 12.3)
H-6: 8.10 s
PeCH(OH)eCH<:
3.80 dd (2.1; 14.0);
5.40 d (2.1)
PeCH(OH)eCH<:
3.78 dd (1.6; 14.0);
5.42 d (1.6)
5.00 dd (2.6; 6.5)
5.01 dd (2.7; 6.7)
5.01 dd (2.6; 6.6)
4.96 dd (2.6; 6.6)
4.79 dd (6.5; 3.1) 4.50 m
(3.1; 3.6; 5.4)
3.86 dd
(3.6; 12.3)
3.78 dd
(5.4; 12.3)
H-6: 8.05 s
4.78 dd (6.7; 3.6) 4.38 m
(3.6; 3.9; 5.8)
3.83 dd
(3.9; 12.2)
3.77 dd
(5.8; 12.2)
H-6: 7.59 q (1.2);
5-CH3: 1.88 d (1.2) (5.5; 17.4); 5.34 td
(5.5; 5.5; 3.9)
H-5: 5.84 d (8.1);
H-6: 7.78 d (8.1)
PeCH2eCH<: 2.11 dd
51
4.77 dd (6.6; 3.3) 4.43 m
(3.3; 3.8; 5.8)
3.83 dd
(3.8; 12.3)
3.77 dd
(5.8; 12.3)
PeCH2eCH<: 2.11 dd
(5.5; 17.4); 5.33 td
(5.5; 5.5; 3.9)
PeCH2eCH<: 2.10 dd
(5.5; 17.3); 5.33 td
(5.5; 5.5; 3.8)
52
4.77 dd (6.6; 3.0) 4.48 m
(3.0; 3.7; 5.5)
3.86 dd (3.7; 12.3) 3.78 dd (5.5; 12.3) H-6: 8.08 s

53
D2O
D2O
5.85 ss (2.6; 0.8)
5.85 d (2.5)
4.94 dd (2.6; 6.8)
5.02 dd (2.5; 6.6)
4.33 dd (7.6; 5.0)
e
e
e
4.74 dd
(6.8; 3.5)
4.38 m
(3.5; 3.8; 5.6)
3.84 dd
(3.8; 12.2)
3.78 dd
(5.6; 12.2)
H-6: 7.74 d (6.2)
H-6: 7.89 s
PeCH2eCH<: 2.08 dd
(5.4; 16.8); 5.32 td
(5.4; 5.4; 3.8)
PeCH2eCH2eCH<:
1.51 m, 2H; 1.98 m,
2H; 5.23 t (4.5; 4.5)
OeCH2eCH]O: 3.65 dd
(17.2; 0.9); 3.50 dd
(17.2; 0.9); 9.57 t (0.9; 0.9)
56
4.80 dd
(6.6; 3.8)
4.38 td
(3.8; 3.8; 5.6)
3.84 dd
(3.8; 12.4)
3.77 dd
(5.6; 12.4)
60aa
CDCl3 6.36 d (7.6)
CDCl3 6.09 d (3.9)
3.06 dd
(5.0; 1.4)
4.05 m
(1.4; 4.0; 2.4)
3.76 dd
(4.0; 11.6)
3.60 dd
(2.4; 11.6)
NH: 8.38 bd (2.4);
H-5: 5.24 dd
(8.1; 2.4);
H-6: 7.25 d (8.1);
NH: 8.37 bd (2.2);
H-5: 5.16 dd
(8.1; 2.2);
H-6: 7.68 d (8.1);
H-6: 8.14 s
60bb
63aa
3.04 dd (3.9; 4.5)
4.45 dd (7.9; 4.5)
e
e
4.15 dd
(4.5; 5.5)
3.92 dt
(5.5; 2.1; 2.2)
3.94 dd
(2.1; 11.8)
3.25 dd
(2.2; 11.8)
OeCH2eCH]O: 4.12 dd
(17.7; 0.8); 3.89 dd
(17.7; 0.8); 9.67 t
(0.8; 0.8)
D2O
6.58 d (7.9)
4.18 dd
(4.5; 1.2)
4.24 dt
(1.2; 3.6; 3.6)
3.80 d, 2H
(3.6)
PeCH2eCH(O)eCH2eO:
1.82 ddd (4.9; 14.8;
19.2); 1.74 ddd (9.0;
14.8; 17.0); 4.30 m
(2.6; 9.9; 4.9; 9.0; 7.8);
4.17 ddd (12.4; 2.6; 0.9);
3.51 dd (12.4; 9.9)
63ab
D2O
5.79 s
4.39 d (4.5)
e
4.32 dd
(4.5; 9.6)
4.64 ddd
(9.6; 2.4; 3.5)
4.03 dd
(2.4: 13.1)
3.84 dd
(3.5; 13.1)
H-6: 8.28 s
PeCH2eCH(O)eCH2eO:
1.88 ddd (6.1; 15.0;
18.2); 1.75 ddd (7.5;
15.0; 17.6); 3.98 m
(2.6; 10.2; 6.1; 7.5;
8.1); 3.84 dd (12.0; 2.6);
3.62 dd (12.0; 10.2)
PeCH2eCH(O)eCH2eO:
1.81 ddd (14.8; 5.4;
18.7); 1.72 ddd (14.8;
8.3; 17.1); 4.21 m (2.7;
9.5; 5.4; 8.3; 9.7); 4.13 dd
(12.2; 2.7); 3.47 dd (12.2; 9.5);
PeCH2eCH(O)eCH2eO:
1.85 ddd
63ba
63bb
D2O
D2O
5.85 d (1.2)
6.58 (8.2)
4.24 dd (1.2; 4.5)
4.37 dd (8.2; 4.4)
e
e
4.37 dd
(4.5; 9.0)
4.66 ddd
(9.0; 2.4; 3.6)
4.03 dd
(2.4; 13.2)
3.88 dd
(3.6; 13.2)
H-6: 8.26 s
H-6: 8.14 s
4.32 bd
(4.4; <0.5)
4.23 bt
(<0.5; 3.7; 3.4)
3.82 dd
(3.7; 12.6)
3.79 dd
(3.4; 12.6)
(14.8; 5.8; 18.3); 1.73 ddd
(14.8; 7.9; 17.3); 4.00 m
(2.6; 10.2; 5.8; 7.9; 8.0);
3.91 dd (12.0; 2.6);
3.69 dd (12.2; 10.2);
PeCH2eCH(O)eCH2eO:
2.11 ddd (5.7; 15.3; 19.5);
1.94 ddd (7.6; 15.3; 19.6);
4.01 m (2.7; 9.6; 5.7;
7.6; 9.9); 3.76 dd (11.8; 2.7);
3.49 dd (11.8; 9.6);
67c
CDCl3 5.83 d (1.2)
4.21 dd (1.2; 4.6)
e
4.36 dd
(4.6; 8.8)
4.47 ddd
(8.8; 1.9; 3.1)
4.12 dd
(1.9; 12.3
3.85 dd
(3.1; 12.3)
NH: 8.75 bs;
H-6: 7.85 s
P(OEt)2: 4.10e4.16 m;
1.33 t (7.1)
CH3eCOeOeCH(O)eCH2e
O: 2.25 s; 6.17 dd
69ad
CDCl3 6.70 d (8.3)
6.15 d (4.7)
4.24 dd (8.3; 4.6)
4.24 t (4.7; 5.0)
4.30 dd (8.3; 4.3)
e
e
e
4.01 d (4.6)
4.19 t
(1.9; 1.5)
4.00 dd
(1.9; 12.0)
3.78 dd
(1.5; 12.0)
NH: 8.38 bs;
H-6: 7.91 s
(4.1; 4.9); 4.00 dd
(12.2; 4.1); 3.50 dd (12.2; 4.9)
CH3eCOeOeCH(O)eCH2e
O: 2.12 s; 5.90 d (1.5);
3.93 d (12.7); 3.78 dd
(12.7; 1.5)
4.30 t
(5.0; 4.6)
4.25 m
(4.6; 2.0; 2.8)
4.03 dd
(2.0; 12.0)
3.81 dd
(2.8; 12.0)
NH: 8.49 bs;
H-6: 7.78 s
70aae
CDCl3 6.61 d (8.3)
3.97 d (4.3)
4.10 t (2.0; 1.9)
3.95 dd
(2.0; 12.0)
3.75 dd
(1.9; 12.0)
NH: 8.45 bs;
H-6: 7.92 s
PeCH(O)eCH2eO: 4.42 dt
(2.7; 11.3; 11.3);
(continued on next page)

Table 5 (continued)
Compound Solv.
H-10
H-20
H-200/H-300
H-30
H-40
H-50a
H-50b
Base
Substituents
4.09 ddd (12.0; 2.7; 0.7);
3.77 ddd (12.0; 11.3;
3.6); P(OEt)2: 4.20 m;
1.36 t (7.1)
70 abf
71aa
CDCl3 5.81 d (1.0)
4.21 dd (1.0; 4.7)
e
4.43 ddd
(4.7; 9.1; 2.3)
4.51 ddd
(9.1; 1.9; 3.2)
4.12 dd
(1.9; 12.2)
3.86 dd
(3.2; 12.2)
NH: 8.75 bs;
H-6: 7.81 s
PeCH(O)eCH2eO:
4.01 ddd (10.7; 3.1;
12.3); 3.87 ddd (12.0;
10.7; 4.7); 3.78 ddd
(12.0; 3.1; 1.6); P(OEt)2:
4.23 m; 1.35 m
D2O
D2O
6.37 d (8.2)
5.83 s
4.26 dd (8.2; 4.4)
e
4.00 d (4.4)
4.28 m
3.98 t (3.4; 3.4)
3.61 dd
(3.4; 12.6)
3.58 dd
(3.4; 12.6)
H-6: 8.05 s
H-6: 8.26 s
PeCH(O)eCH2eO:
3.85 ddd (2.8; 11.2;
10.9); 3.90 ddd (12.1;
2.8; 0.9); 3.59 ddd
(12.1; 11.2; 3.8)
71ab
69bg
4.27 m
e
e
e
e
4.69 m
(9.0; 2.4; 3.7)
4.04 dd
(2.4; 13.2)
3.84 dd
(3.7; 13.2)
PeCH(O)eCH2eO:
3.74 ddd (11.3; 2.7;
11.7); 3.91 ddd (11.9;
11.3; 3.8); 3.82 dd (11.9; 2.7)
OeCH2eCH<: 4.15 dd
(12.8; 2.9); 3.61 dd
(12.8; 2.4); 6.06 bt
(2.9; 2.4; 0.8); OAc: 2.17 s
OeCH2eCH<: 3.90 dd
(12.2; 5.0); 3.77 dd
(12.2; 2.3); 5.88 dd
(5.0; 2.3); OAc: 2.08 s
PeCH(O)eCH2eO:
4.07 m (1H); 4.10 m
(1H) and 3.84 m (1H);
P(OEt)2: 4.19 m and
4.23 m; 1.37 t (7.2)
and 1.35 t (7.2)
CDCl3 6.56 d (7.7)
6.28 d (4.6)
4.28 ddd
(7.7; 4.7; 0.8)
4.31 dd
(4.7; 1.1)
4.15 td
(1.1; 2.2; 2.1)
3.99 dd
(2.2; 12.0)
3.76 dd
(2.1; 12.0)
NH: 8.97 bs;
H-6: 7.92 s
4.21 t (4.6; 4.5)
4.38 dd
(4.5; 5.0)
4.40 ddd
(5.0; 1.8; 2.3)
4.05 dd
(1.8; 12.1)
3.80 dd
(2.3; 12.1)
NH: 8.95 bs;
H-6: 7.88 s
70bbh
CDCl3 6.66 d (8.4)
4.25 dd (8.4; 4.3)
4.06 d (4.3)
4.19 t (1.9; 1.9)
3.95 dd
(1.9; 12.0)
3.76 dd
(1.9; 12.0)
NH: 8.94 bs;
H-6: 7.94 s
70bai
71ba
71bb
CDCl3 6.02 d (2.8)
4.20 dd (2.8; 4.7)
4.25 dd (0.5; 4.6)
e
e
e
4.65 dd
(4.7; 7.0)
4.37 ddd
(7.0; 1.8; 2.8)
4.08 dd
(1.8; 12.2)
3.83 dd
(2.8; 12.2)
NH: 8.58 bs;
H-6: 7.86 s
PeCH(O)eCH2eO:
4.13 m (2H); 3.86 m
(1H); P(OEt)2: 4.19 m
and 4.17 m; 1.32t (7.2)
PeCH(O)eCH2eO:
4.01 td (2.8; 11.1; 11.1);
4.08 ddd (2.8; 12.1; <1);
3.74 ddd (11.1; 12.1; 3.6)
PeCH(O)eCH2eO:
D2O
D2O
5.79 d (0.5)
6.63 d (8.2)
4.36 dd
(4.6; 9.6)
4.65 ddd
(9.6; 2.4; 3.5)
4.05 dd
(2.4; 13.2)
3.90 dd
(3.5; 13.2)
H-6: 8.30 s
H-6: 8.15 s
4.34 ddd
4.19 bd
4.29 m
3.83 dd
3.80 dd
(8.2; 4.4; 2.1)
(4.4; 0.6)
(0.6; 3.8; 3.5)
(3.8; 12.6)
(3.5; 12.6)
3.79 ddd (11.5; 2.6;
12.0); 4.01 ddd
(11.5; 11.9; 3.7);
3.87 ddd (11.9; 2.6)
a
ODMTr: 7.30 m, 2H (ortho-ArH); 7.28 m, 2H (ortho-ArH); 6.76 m, 2H (meta-ArH); 6.74 m, 2H (meta-ArH); 7.40e7.23 m, 5H (C6H5); 3.755 s, 3H (OCH3); 3.750 s, 3H (OCH3); TBDPS: 1.01 s, 9H (tert-Bu); 7.52 m, 2H (ortho-ArH);
7.48 m, 2H (ortho-ArH); 7.36 m, 2H (meta-ArH); 7.31 m, 2H (meta-ArH); 7.45 m, 1H (ipso-ArH); 7.43 m, 1H (ipso-ArH).
b
ODMTr: 7.31 m, 4H (ortho-ArH); 6.73 m, 4H (meta-ArH); 7.33 m, 2H (ortho-ArH); 7.45 m, 2H (meta-ArH); 7.21 m, 1H (ipso-ArH); 3.75 s, 3H (OCH3); 3.74 s, 3H (OCH3); TBDPS: 0.98 s, 9H (tert-Bu); 7.51 m, 4H (ortho-ArH); 7.21
m, 4H (meta-ArH); 7.43 m, 2H (ipso-ArH).
c
TBDPS: 1.10 s (tert-Bu); 7.67 m, 4H (ortho-ArH); 7.39 m, 4H (meta-ArH); 7.45 m, 2H (ipso-ArH).
TBDPS: 1.12 s and 1.11 s, (tert-Bu); 7.64e7.66 m (ortho-ArH); 7.40e7.43 m (meta-ArH); 7.46e7.49 m (ipso-ArH).
TBDPS: 1.11 s (tert-Bu); 7.65 m, 4H (ortho-ArH); 7.43 m, 4H (meta-ArH); 7.48 m, 2H (ipso-ArH).
TBDPS: 1.10 s (tert-Bu); 7.66 m, 4H (ortho-ArH); 7.39 m, 4H (meta-ArH); 7.45 m, 2H (ipso-ArH).
TBDPS: 1.12 s and 1.10 s (tert-Bu); w7.66 m, 4H (ortho-ArH); w7.42 m, 4H (meta-ArH); w7.47 m, 2H (ipso-ArH).
TBDPS: 1.10 s (tert-Bu); 7.64 m, 4H (ortho-ArH); 7.43 m, 4H (meta-ArH); 7.48 m, 2H (ipso-ArH).
TBDPS: 1.10 s (tert-Bu); 7.66 m, 4H (ortho-ArH); 7.40 m, 4H (meta-ArH); 7.45 m, 2H (ipso-ArH).
d
e
f
g
h
i

ꢀ
I. Kosiová et al. / European Journal of Medicinal Chemistry 74 (2014) 145e168
161
Table 6
Carbon-13 NMR data. Coupling constants J(C,P) are given in brackets.
Compound
Solvent
D2O
C-10
C-20
C-30
C-40
89.02
C-50a
64.01
Base
Substituents
13a
95.64
88.17
d
84.79
(9.2)
C-2: 153.22; C-4: 164.74;
C-5: 111.14; C-6: 143.02
PeCH < O: 106.73 d (181.2)
(9.2)
85.56
d
(8.1)
38.55
13b
23a
23b
24a
24b
D2O
D2O
D2O
D2O
D2O
95.13
87.95
92.84
91.64
90.24
83.15
d
(8.3)
83.50
d
86.89
87.35
84.35
85.06
86.46
64.34
64.31
64.40
63.47
62.98
C-2: 154.66; C-4: 157.01;
C-5: 111.53; C-6: 142.86
PeCH < O: 104.49 d (172.9)
PeCH2eO: 68.67 d (155.1)
PeCH2eO: 68.94 d (155.1)
PeCH2eO: 69.51 d (155.8)
PeCH2eO: 69.46 d (156.7)
C-2: 154.47; C-4: 169.32;
C-5: 114.20; C-6: 140.33;
5-CH3: 14.26
C-2: 154.16; C-4: 169.44;
C-5: 113.50; C-6: 140.32;
5-CH3: 14.28
C-2: 154.52; C-4: 169.26;
C-5: 114.21; C-6: 140.05;
5-CH3: 14.26
C-2: 154.29; C-4: 169.25;
C-5: 114.12; C-6: 140.18;
5-CH3: 14.30
(11.8)
33.32
88.17
d
(12.4)
75.28
81.79
d
(11.3)
71.16
85.78
(12.3)
25a
D2O
D2O
D2O
88.42
88.72
89.46
38.74
39.03
39.74
83.82d
(12.2)
83.45d
(12.0)
82.01
87.51
87.71
89.21
64.33
64.18
64.48
C-2: 154.33; C-4: 169.05;
C-5: 104.95; C-6: 144.78
C-2: 153.52; C-4: 164.72;
C-5: 111.54; C-6: 141.48
C-2: 154.45; C-4: 169.55;
C-5: 113.32; C-6: 141.06;
5-CH3: 14.36
PeCH2eO: 68.00 d (157.4)
PeCH2eO: 68.46 d (156.0)
26a
31a 2 diastereomers
PeCH(OH)eCH2eO: 72.03 d (148.9);
73.13 d (11.3)
89.51
93.01
92.95
89.70
87.88
93.00
39.89
86.54
86.47
39.97
38.82
85.94
82.07
33.48
33.40
81.52
81.28
33.52
89.44
84.32
84.31
89.35
87.42
84.27
64.54
64.44
64.41
64.50
64.16
64.46
C-2: 154.38; C-4: 169.52;
C-5: 113.26; C-6: 141.02;
5-CH3: 14.37
C-2: 154.14; C-4: 169.40;
C-5: 113.50; C-6: 140.24;
5-CH3: 14.29
C-2: 154.14; C-4: 169.40;
C-5: 113.50; C-6: 140.22;
5-CH3: 14.29
C-2: 154.31; C-4: 169.53;
C-5: 113.06; C-6: 140.87;
5-CH3: 14.38
C-2: 154.45; C-4: 169.28;
C-5: 114.20; C-6: 140.26;
5-CH3: 14.27
C-2: 154.16; C-4: 169.42;
C-5: 113.52; C-6: 140.22;
5-CH3: 14.29
PeCH(OH)eCH2eO: 71.99 d (149.2);
73.07 d (11.3)
31b 2 diastereomers
D2O
D2O
D2O
PeCH(OH)eCH2eO: 71.82 d (149.9);
73.78 d (11.1)
PeCH(OH)eCH2eO: 71.73 d (149.9);
73.78 d (11.1)
33a mixture of
C-1 epimers (70:30)
PeCH2eCH2eO: 32.22 d (128.0);
68.00 d (2.9)
PeCH2eCH2eO: 32.22 d (128.0);
68.65 d (2.2)
33b
PeCH2eCH2eO: 32.72 d (125.8);
69.49 d (3.3)
45a
45b
50
D2O
D2O
D2O
95.64
95.69
95.32
86.76
88.11
86.97
84.50
83.47
83.59
88.93
88.84
89.12
64.10
64.15
64.10
C-2: 154.33; C-4: 166.09;
C-5: 111.23; C-6: 142.51
C-2: 156.40; C-4: 168.50;
C-5: 111.37; C-6: 141.97
C-2: 154.18; C-4: 169.52;
C-5: 113.67; 5-CH3: 14.17;
C-6: 141.99
PeCH(OH)eCH<: 71.48 d (144.3);
110.00 d (7.7)
PeCH(OH)eCH<: 71.42 d (143.0);
110.10 d (7.3)
PeCH2eCH<: 36.76 d (127.8); 108.64
51
D2O
96.02
96.09
95.78
96.00
87.03
86.81
84.89
92.29
91.34
84.04
89.11
87.25
87.47
87.40
87.51
75.17
82.56
78.39
79.77
78.45
77.25
77.48
83.80
83.88
83.53
83.65
79.20
70.90
75.74
71.22
72.53
77.23
68.71
86.49
89.53
88.99
89.04
83.22
83.20
86.62
79.20
80.20
87.31
w77.0
64.12
64.07
64.23
64.19
64.38
62.96
64.08
62.18
62.31
64.20
61.95
C-2: 154.10; C-4: 169.29;
C-5: 104.32; C-6: 146.23
C-2: 154.32; C-4: 166.03;
C-5: 111.11; C-6: 142.89
C-2: 159.79; C-4: n.d.;
C-5: n.d; C-6:128.31
PeCH2eCH<: 36.71 d (128.0); 108.45
PeCH2eCH<: 36.84 d (126.9); 108.63
PeCH2eCH<: 37.30 d (125.1); 109.29
52
D2O
53
D2O
56
D2O
C-2: 159.83; C-4: 163.46;
C-5: 111.77; C-6: 141.89
C-2: 150.28; C-4: 162.68;
C-5: 102.61; C-6: 140.45
C-2: 149.85; C-4: 162.66;
C-5: 102.12; C-6: 139.46
C-2: 154.12; C-4: 164.74;
C-5: 112.32; C-6: 140.91
C-2: 153.39; C-4: 165.03;
C-5: 111.15; C-6: 141.42
C-2: 153.67; C-4: 165.35;
C-5: 111.25; C-6: 141.14
C-2: 154.56; C-4: 165.23;
C-5: 112.42; C-6: 140.74
C-2: 148.72; C-4: 158.86;
C-5: 109.40; C-6: 136.19
PeCH2eCH2eCH<: 25.45 d (132.8);
31.04 d (2.9); 111.51 d (19.0)
OeCH2eCH]O: 75.15; 200.18
60aa
60bb
63aa
63ab
63ba
63bb
67c
CDCl3
CDCl3
D2O
OeCH2eCH]O: 75.72; 199.00
PeCH2eCH(O)eCH2eO: 33.70 d (130.2);
70.01 d (2.1); 71.64 d (5.4)
PeCH2eCH(O)eCH2eO: 33.93 d (131.1);
73.80; 67.44 d (9.4)
PeCH2eCH(O)eCH2eO: 33.54 d (130.2);
69.49; 71.48 d (11.3)
PeCH2eCH(O)eCH2eO: 34.21 d (130.2);
74.14; 68.21 d (8.7)
D2O
D2O
D2O
CDCl3
PeCH2eCH(O)eCH2eO: 29.24 d (141.9);
69.04; 65.00 d (9.4); P(OEt)2: 62.23 s (6.3);
61.89 d (6.4); 16.44 d (6.2); 16.40 d (6.2)
(continued on next page)

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I. Kosiová et al. / European Journal of Medicinal Chemistry 74 (2014) 145e168
162
Table 6 (continued )
Compound
Solvent
CDCl3
C-10
C-20
C-30
C-40
83.98
C-50a
64.42
Base
Substituents
69ad 2 diastereomers
84.89
74.84
74.18
C-2: 149.17; C-4: 158.32;
C-5: 110.15; C-6: 135.94
CH3eCOeOeCH(O)eCH2eO: 21.36;
170.54;
87.71; 64.57
87.50
80.30
74.57
72.06
73.59
81.81
84.25
63.21
64.22
C-2: 148.82; C-4: 158.36;
C-5: 109.86; C-6: 136.61
C-2: 149.25; C-4: 158.35;
C-5: 110.02; C-6: 135.89
CH3eCOeOeCH(O)eCH2eO: 20.96;
169.14; 85.94; 66.18
70aae
70abf
71aa
71ab
69bg
CDCl3
CDCl3
D2O
76.67
d
(10.5)
77.44
d
(11.7)
77.82
d
(9.2)
79.59
d
PeCH(O)eCH2eO: 65.70 d (167.2);
64.67 d (4.7); P(OEt)2: 63.24 d (6.8);
63.03 d (6.1); 16.40 d (5.7); 16.37 d (5.8)
PeCH(O)eCH2eO: 70.39 d (169.7);
60.30 d (5.9); P(OEt)2: 63.59 d (6.6);
63.06 d (6.6); 16.48 d (w6.0)
PeCH(O)eCH2eO: 70.76 d (152.0);
68.64 d (5.5)
89.76
84.07
92.55
69.05
76.35
71.50
76.82
87.09
79.22
61.90
64.31
62.21
C-2: 148.60; C-4: 158.65;
C-5: 109.36; C-6: 136.63
C-2: 154.63; C-4: 164.78;
C-5: 112.13; C-6: 140.94
D2O
C-2: 153.33; C-4: 164.96;
C-5: 111.08; C-6: 141.43
PeCH(O)eCH2eO: 75.54 d (150.5);
64.23 d (6.8)
(10.3)
76.64
CDCl3
81.82
84.17
87.36
68.59
71.51
83.79
82.36
79.02
64.07
62.66
62.41
C-2: 149.23; C-4: 158.52;
C-5: 110.19; C-6: 135.87
C-2: 149.53; C-4: 158.57;
C-5: 109.88; C-6: 136.00
C-2: 148.86; C-4: 158.41;
C-5: 109.65; C-6: 136.14
OeCH2eCH<: 62.02; 88.13; OAc: 169.46;
21.00
OeCH2eCH<: 63.89; 87.84; OAc: 169.04;
21.03
PeCH(O)eCH2eO: 66.71 d (165.5);
63.02 d (4.2); P(OEt)2: 63.23 d (6.6);
62.74 d (6.6); 16.52 d (5.6); 16.45 d (5.6)
PeCH(O)eCH2eO: 70.98 d (170.3);
60.63 d (5.3); P(OEt)2: 63.53 d (6.4);
62.92 d (6.7); 16.50 d (5.0); 16.43 d (5.7)
PeCH(O)eCH2eO: 70.10 d (152.5);
68.54 d (5.9)
76.16
76.76
70bbh
70bai
71ba
CDCl3
CDCl3
D2O
69.33
d
(7.6)
74.99
d
(12.0)
72.02
d
(9.3)
77.06 d
(10.3)
80.06
91.80
84.26
76.18
78.75
77.64
84.05
79.57
87.50
64.24
62.14
64.24
C-2: 149.49; C-4: 158.60;
C-5: 110.08; C-6: 135.88
C-2: 153.18; C-4: 164.80;
C-5: 111.10; C-6: 141.22
71bb
D2O
C-2: 154.04; C-4: 164.68;
C-5: 112.29; C-6: 140.93
PeCH(O)eCH2eO: 75.65 d (162.7);
64.91 d (7.1)
a
ODMTr: 130.40 (4ꢃ ortho-ArC); 128.36 (2ꢃ ortho-ArC); 129.12 (2ꢃ meta-ArC); 113.24 (2ꢃ meta-ArC); 113.21 (2ꢃ meta-ArC); 158.86 (2ꢃ ipso-ArC); 127.27 (ipso-ArC);
135.45 (ipso-ArC); 135.34 (ipso-ArC); 144.41 (ipso-ArC); 86.92 (>C<); 55.24 (2ꢃ OCH3); TBDPS: 26.99 and 19.22 (t-Bu); 135.37 (2ꢃ ortho-ArC); 135.25 (2ꢃ ortho-ArC); 128.01
(2ꢃ meta-ArC); 128.05 (2ꢃ meta-ArC); 130.23 and 130.15 (2ꢃ ipso-ArC); 132.38 and 132.09 (2ꢃ ipso-ArC).
b
ODMTr: 130.54 (2ꢃ ortho-ArC); 130.42 (2ꢃ ortho-ArC); 127.94 (2ꢃ ortho-ArC); 128.50 (2ꢃ meta-ArC); 113.17 (2ꢃ meta-ArC); 113.13 (2ꢃ meta-ArC); 158.89 (ipso-ArC);
158.86 (ipso-ArC); 127.28 (ipso-ArC); 135.93 (ipso-ArC); 135.75 (ipso-ArC); 144.85 (ipso-ArC); 87.03 (>C<); 55.25 and 55.23 (2ꢃ OCH3); TBDPS: 27.02 and 19.38 (t-Bu); 135.54
(2ꢃ ortho-ArC); 135.25 (2ꢃ ortho-ArC); 127.92 (2ꢃ meta-ArC); 127.86 (2ꢃ meta-ArC); 130.20 and 130.15 (2ꢃ ipso-ArC); 132.74 and 132.21 (2ꢃ ipso-ArC).
c
TBDPS: 27.03 and 19.34 (t-Bu); 135.41 and 135.61 (ortho-ArC); 127.88 (meta-ArC); 129.99 (ipso-ArC); 132.74 and 132.35 (ipso-ArC).
TBDPS: 27.11, 27.03 and 19.27, 19.23 (t-Bu); 135.66, 135.62, 135.39 and 135.36 (ortho-ArC); 128.18, 128.10, 127.98 and 127.97 (meta-ArC); 130.41, 130.29, 130.17 and
d
130.12 (ipso-ArC); 132.33, 132.19, 131.85 and 131.83 (ipso-ArC).
e
TBDPS: 27.08 and 19.19 (t-Bu); 135.62 and 135.38 (ortho-ArC); 128.14 and 128.11 (meta-ArC); 130.36 and 130.26 (ipso-ArC); 131.85 and 131.83 (ipso-ArC).
TBDPS: 27.00 and 19.33 (t-Bu); 135.60 and 135.42 (ortho-ArC); 127.87 and 127.86 (meta-ArC); 129.99 (ipso-ArC); 132.73 and 132.41 (ipso-ArC).
TBDPS e major: 27.11 and 19.24 (t-Bu); 135.61 (2ꢃ ortho-ArC); 135.42 (2ꢃ ortho-ArC); 128.07 (2ꢃ meta-ArC); 128.06 (2ꢃ meta-ArC); 130.34 and 130.26 (2ꢃ ipso-ArC);
f
g
132.03 and 132.00 (2ꢃ ipso-ArC); TBDPS e minor: 27.01 and 19.26 (t-Bu); 135.61 (2ꢃ ortho-ArC); 135.36 (2ꢃ ortho-ArC); 128.05 (2ꢃ meta-ArC); 128.02 (2ꢃ meta-ArC); 130.22
and 130.14 (2ꢃ ipso-ArC); 132.26 and 131.94 (2ꢃ ipso-ArC).
h
TBDPS: 27.01 and 19.31 (t-Bu); 135.57 and 135.37 (ortho-ArC); 127.97 and 127.95 (meta-ArC); 130.08 (ipso-ArC); 132.46 and 132.16 (ipso-ArC).
TBDPS: 27.06 and 19.18 (t-Bu); 135.62 and 135.36 (ortho-ArC); 128.17 and 128.10 (meta-ArC); 130.40 and 130.27 (ipso-ArC); 131.83 and 131.69 (ipso-ArC).
i
8.0 mmol) which was transformed into the title compound 26a by
the procedure described for compound 23a. Overall yield: 0.5 g
(1.4 mmol, 16%). HRMS (M ꢁ H)ꢁ for C10H13ClN2O8P: calcd m/z
355.0098, found 355.0103. IR (KBr, cmꢁ1): 3432, 2924, 2837, 1699,
1631, 1449,1274,1063, 916, 558, 454. NMR data e see Tables 5 and 6
2:1 (v/v) THFewater mixture at rt overnight (TLC in C-1). The re-
action mixture was diluted with chloroform (250 mL); the organic
layer was washed with water (3 ꢃ 100 mL), dried over anhydrous
sodium sulfate and evaporated. The crude vicinal diol 28a was
dissolved in a 1:1 acetoneewater mixture (100 mL), then sodium
periodate (3.9 g, 18.4 mmol) was added, and the resulting mixture
was stirred at room temperature for 1 h (TLC in C-1). Chloroform
(100 mL) was added, the organic layer was washed with water
(3 ꢃ 50 mL), dried over anhydrous sodium sulfate and evaporated.
Purification of the residue by silica gel chromatography using a
linear gradient of ethanol (0e10%) in chloroform afforded 79%
(4.3 g, 7.3 mmol) of the aldehyde 29a which was immediately
reacted with diethyl phosphite (2.8 mL, 21.9 mmol) in dichloro-
methane (50 mL) and in the presence of a 10 mol% of triethylamine
(0.73 mL, 5.7 mmol). The resulting mixture was stirred at room
temperature overnight (TLC in C-1) and then concentrated under
reduced pressure. Oily residue was purified by silica gel chroma-
tography using a linear gradient of ethanol (0e10%) in chloroform.
The obtained epimeric mixture 30a (5.1 g, 7.0 mmol, 76%) was
detritylated in 80% acetic acid (70 mL) for 30 min (TLC in C-1), then
4.3.10. (1RS)-1-hydroxy-2-(thymidin-30-O-yl)-ethanephosphonic
acid (31a) (Scheme 3)
Sodium hydride (1.1 g, 27.6 mmol) was added to a solution of 50-
O-dimethoxytritylthymidine (15a, 5.0 g, 9.2 mmol) in THF (90 mL)
and the mixture was stirred at room temperature (rt) for 15 min.
Then allyl bromide (1.2 mL, 13.8 mmol) was added dropwise to the
stirred solution. The resulting mixture was stirred at room tem-
perature overnight (TLC in C-1), quenched with acetic acid at 0 ꢀC
and concentrated under reduced pressure. The residue was diluted
with chloroform (100 mL), and the solution was washed with water
(3 ꢃ 50 mL). The organic layer was dried over anhydrous sodium
sulfate, evaporated, and the allyl derivative 27a was treated with a
catalytic amount of osmium tetroxide (0.1 mL, 2.5% solution in tert-
butanol) and N-methylmorpholine-N-oxide (1.08 g, 9.2 mmol) in a

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the solution was concentrated under reduced pressure, and the
residue was co-distilled sever times with water, alkalified with
triethylamine, and finally dried by co-evaporation with ethanol and
toluene. The residue was then dissolved in acetonitrile (40 mL), 2,6-
lutidine (2.9 mL, 25.6 mmol) followed with bromotrimethylsilane
(1.7 mL, 12.8 mmol) were added and the solution was stirred at rt
overnight and then concentrated under reduced pressure. The
crude 31a was purified by an RP HPLC.
3262, 3071, 2928, 2820, 2360, 1697, 1518, 1477, 1444, 1389, 1272,
1110, 1079, 973, 903, 789, 769, 587, 566, 495, 421. NMR data e see
Tables 5 and 6
4.3.14. 2(R)-(5-chlorouridin-20,30-di-O-yl)-1(S)-
hydroxyethanephosphonic acid (45a) and 2(R)-(5-chlorouridin-
20,30-di-O-yl)-1(R)-hydroxyethanephosphonic acid (45b) (Scheme 5)
To a stirred suspension of 50-O-tert-butyldiphenylsilyluridine (8)
(8.0 g, 16.6 mmol) in dichloromethane (150 mL) was added acrolein
dimethylacetal (5.1 g, 49.8 mmol) and the mixture was acidified by
4 M HCl in DMF (pH on wet pH paper slightly red). After stirring at
rt (TLC in C-1) for 24 h and neutralization with triethylamine, the
clear reaction mixture was concentrated under reduced pressure.
The obtained acetal 37 was treated with a catalytic amount of
osmium tetroxide (0.1 mL, 2.5% solution in tert-butanol) and so-
dium periodate (7.1 g, 33.2 mmol) in THFewater mixture (7:3,
100 mL) at rt overnight (TLC in C-1). Then the mixture was diluted
with chloroform (150 mL), and the whole was washed with water
(3 ꢃ 50 mL). The organic layer was dried over anhydrous sodium
sulfate and evaporated. The aldehyde 40 (6.2 g,11.8 mmol) obtained
by silica gel chromatography using a linear gradient of ethanol (0e
10%) in chloroform, was treated with diethyl phosphite (4.5 mL,
35.4 mmol) and triethylamine (0.14 mL, 1 mmol) in dichloro-
methane (100 mL) at rt overnight (TLC in C-1). The solution was
concentrated under reduced pressure and the residue was purified
by silica gel chromatography using a linear gradient of ethanol (0e
10%) in chloroform to afford pure hydroxyphosphonate 43 (5.0 g,
7.57 mmol). This compound was treated with acetic anhydride
(1.43 mL, 15.14 mmol) in pyridine (70 mL) at rt overnight (TLC in C-
1). The reaction was quenched with water at 0 ꢀC, and the solution
was concentrated under reduced pressure. The residue was diluted
with chloroform (100 mL), the solution was washed with water
(3 ꢃ 50 mL), and the organic layer was dried over anhydrous so-
dium sulphate and evaporated. The obtained O-acetyl derivative of
compound 43 was heated with N-chlorosuccinimide (5.24 g,
39.30 mmol) in dry pyridine (70 mL) at 90 ꢀC for 45 min and the
solution was concentrated under reduced pressure. The crude
product was purified by silica gel chromatography using a linear
gradient of ethyl acetate (0e50%) in toluene to afford TLC pure 5-
chlorouracil derivative 43a,b (2.76 g, 3.87 mmol) as an epimeric
mixture. This product was treated with a 50% concentrated aq. NH3
in ethanol (100 mL) at rt overnight (TLC in C-1). The solution was
concentrated at reduced pressure, the residue was co-distilled with
ethanol and toluene and the residue was treated with bromo-
trimethylsilane (1.45 mL, 11.06 mmol) and 2,6-lutidine (2.6 mL,
22.12 mmol) in acetonitrile (10 mL) at rt overnight. The mixture was
concentrated in vacuo, the residue was treated with 0.5 M TBAF in
THF (40 mL) at rt for 30 min and the solution was concentrated
under reduced pressure. The oily residue was partitioned between
water and diethyl ether, aqueous layer was washed with ether and
evaporated in vacuo. The product 45a,b was separated by an RP
HPLC to afford both epimers in a pure form.
Overall yield of 31a: 0.269
g (0.736 mmol, 8%). HRMS
(M ꢁ H)ꢁfor C12H18N2O9P: calcd m/z 365.0750, found 365.0745; IR
(KBr, cmꢁ1): 3194, 3065, 2928, 2824, 1699, 1475, 1465, 1277, 1059,
970, 913, 768, 570, 493, 419. NMR data e see Tables 5 and 6
4.3.11. (1RS)-2-(30-deoxy-5-methyluridin-20-O-yl)-1-
hydroxyethanephosphonic acid (31b) (Scheme 3)
30-Deoxy-50-O-dimethoxytrityl-5-methyluridine 15b (2.0 g,
3.7 mmol) was transformed in four steps (/27b/28b/29b/),
according to the procedure described above for 31a, into hydrox-
yphosphonate 30b in 81% yield (2.2 g, 3.0 mmol). This compound
was subsequently detritylated and finally treated with bromo-
trimethylsilane (followed by the procedure for 31a) to afford the
phosphonic acid 31b.
Overall yield of 31b: 0.6 g (1.6 mmol, 44%). HRMS (M ꢁ H)ꢁ for
C
12H18N2O9P: calcd m/z 365.0750, found 365.0748; IR (KBr, cmꢁ1):
3210, 3061, 2984, 2946, 2820, 2683, 2483, 1696, 1471, 1391, 1266,
1122, 1108, 1082, 974, 911, 838, 795, 768, 585, 565, 492, 416. NMR
data e see Tables 5 and 6
4.3.12. 2-(Thymin-30-O-yl)-ethanephosphonic acid (33a) (Scheme
3)
The protected hydroxyphosphonate 30a (3.6 g, 5.0 mmol),
prepared according to the procedure described in 31a, was treated
with 1,10-thiocarbonyldiimidazole (1.98 g, 11.0 mmol) in 1,2-
dichloroethane (50 mL) at rt overnight (TLC in T-1 and C-1). The
solution was concentrated under reduced pressure, and the residue
was purified by silica gel chromatography using a linear gradient of
ethyl acetate (0e50%) in toluene to afford the corresponding imi-
dazoylthiocarbonyl derivative which was immediately deoxygen-
ated. This compound in toluene (50 mL) containing AIBN (33 mg,
0.2 mmol) was heated under stirring at 120 ꢀC, then tribu-
tyltinhydride (2.7 mL, 10 mmol) was added in one portion, the
resulting mixture was heated for 1 h (TLC in T-1), and evaporated.
The residue was purified by silica gel chromatography using a linear
gradient of ethyl acetate (0e50%) in toluene affording 32a (2.5 g,
3.5 mmol). This compound was detritylated in 80% acetic acid
(30 mL) for 30 min (TLC in C-1), then the solution was concentrated
under reduced pressure, and the residue was co-evaporated several
times with water, alkalified with trietylamine, and finally dried by
co-evaporation with ethanol and toluene. The residue was then
dissolved in acetonitrile (40 mL), N,O-bis(trimethylsilyl)acetamide
(8.7 mL, 35 mmol) followed with bromotrimethylsilane (1.9 mL,
14.0 mmol) were added and the solution was stirred at rt overnight,
and then concentrated under reduced pressure. The crude 33a was
purified by an RP HPLC.
Yield 45a 0.44 g (1.1 mmol, 7%, R,S diastereomer); HRMS
(M ꢁ H)ꢁ for C11H13ClN2O10P: calcd m/z 398.9997, found 399.0002;
IR (KBr, cmꢁ1): 3419, 1696, 1447, 1269, 1137, 1082, 976, 781, 587.
NMR data e see Tables 5 and 6
Yield: 0.343
g
(0.98 mmol, 28%). HRMS (M ꢁ H)ꢁ for
C
12H18N2O8P: calcd m/z 349.0801, found 349.0802; IR (KBr, cmꢁ1):
3432, 1695, 1476, 1278, 1072, 1057, 975, 901, 769. NMR data e see
Tables 5 and 6
Yield 45b 0.50 g (1.25 mmol, 8%, R,R diastereomer); HRMS
(M ꢁ H)ꢁ for C11H13ClN2O10P: calcd m/z 398.9997, found 399.0001;
IR (KBr, cmꢁ1): 3396, 2948, 1698, 1448, 1273, 1140, 1074, 973, 905,
759, 668, 579. NMR data e see Tables 5 and 6
4.3.13. 2-(30-Deoxy-5-methyluridin-20-O-yl)-ethanephosphonic
acid (33b) (Scheme 4)
Compound 33b was prepared from 30b (0.44 g, 0.6 mmol) by
the procedure described for compound 33a.
4.3.15. 2(R)-(5-methyluridin-20,30-di-O-yl)-ethanephosphonic acid
(50) (Scheme 5)
Yield: 0.094
g
(0.26 mmol, 43%). HRMS (M ꢁ H)ꢁ for
The 4 M HCl in DMF was carefully added to a stirred suspension
C
12H18N2O8P: calcd m/z 349.0801, found 349.0801; IR (KBr, cmꢁ1):
of 50-O-tert-butyldiphenylsilyl-5-methyluridine (34, 6.0 g,

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164
12 mmol) and acrolein dimethylacetal (3.8 g, 37.4 mmol) in
dichloromethane (100 mL) until pH reached slightly red color using
a wet pH paper. The resulting mixture was stirred at rt overnight
(TLC in C-1), neutralized by addition of triethylamine at 0 ꢀC, and
concentrated under reduced pressure. The obtained acetal 36 was
treated with a catalytic amount of osmium tetroxide (0.2 mL, 2.5%
solution in tert-butanol) and sodium periodate (7.1 g, 33.2 mmol) in
THFewater mixture (7:3, 100 mL) at rt overnight (TLC in C-1). Then
the mixture was diluted with chloroform (150 mL), and the whole
was washed with water (3 ꢃ 50 mL). The organic layer was dried
over anhydrous sodium sulfate and evaporated. The aldehyde 39
(4.3 g, 9.0 mmol) obtained by silica gel chromatography using a
linear gradient of ethanol (0e10%) in chloroform, was treated with
diethyl phosphite (4.5 mL, 35.4 mmol) and triethylamine (0.14 mL,
1 mmol) in dichloromethane (100 mL) at rt overnight (TLC in C-1).
The solution was concentrated under reduced pressure and the
residue was purified by silica gel chromatography using a linear
gradient of ethanol (0e10%) in chloroform to afford TLC pure
hydroxyphosphonate 42 (4.4 g, 6.5 mmol) which was treated
with 1,10-thiocarbonyldiimidazole (3.0 g, 16.9 mmol) in 1,2-
dichloroethane (60 mL) at rt overnight (TLC in T-1 and C-1), and
the solution was concentrated under reduced pressure. The residue
was purified by silica gel chromatography using a linear gradient of
ethyl acetate (0e50%) in toluene to afford the corresponding thio-
ester. The solution of the thioester and AIBN (45 mg, 0.26 mmol) in
toluene (60 mL) was heated under stirring at 120 ꢀC, and then
tributyltin hydride (3.62 mL, 13.46 mmol) was added and heating
continued for the next 1 h (TLC in T-1). Diester 46 (3.5 g, 5.1 mmol)
was obtained by silica gel chromatography using a linear gradient
of ethyl acetate (0e50%) in toluene. This compound was treated
with bromotrimethylsilane (2.8 mL, 20.41 mmol) and 2,6-lutidine
(4.6 mL, 40.8 mmol) in acetonitrile (50 mL) at rt overnight and
then concentrated under reduced pressure. The residue was treated
with 0.5 M TBAF in THF (20 mL), the resulting mixture was stirred
at room temperature for 30 min, and concentrated under reduced
pressure. The oily residue was partitioned between water and
diethyl ether, aqueous layer was washed with ether and evaporated
in vacuo. The product 50 was separated by an RP HPLC. Yield: 1.34 g
(3.7 mmol, 73%). HRMS (M ꢁ H)ꢁ for C12H16N2O9P: calcd m/z
363.0594, found 363.0587; IR (KBr, cmꢁ1): 3190, 2933, 1697, 1472,
1278, 1122, 1066, 974, 769, 582. NMR data e see Tables 5 and 6
cmꢁ1): 3406, 2360, 1697, 1644, 1583, 1445, 1270, 1240, 1125, 2064,
977, 576. NMR data e see Tables 5 and 6
4.3.18. (2R)-(5-fluorouridin-20,30-di-O-yl)-ethanephosphonic acid
(53) (Scheme 5)
The conversion of 50-O-tert-butyldiphenylsilyl-5-fluorouridine
(35) (1.9 g, 3.8 mmol) into the title phosphonate 53 was per-
formed as follows. The compound 35 was converted into 49 by the
procedure described for 5-methyluracil derivative 46. The fully
protected phosphonate 49 was deprotected as described for
phosphonate 50.
Yield of 53: 0.097 g (0.2 mmol, 5%); HRMS (M ꢁ H)ꢁ for
C
11H13FN2O9P: calcd m/z 367.0343, found 367.0345; IR (KBr, cmꢁ1):
3398, 1740, 1672, 1628, 1456, 1395, 1360, 1085, 1051, 977, 834, 701,
685, 577. NMR data e see Tables 5 and 6
4.3.19. (3R)-(5-ChloroUridin-20,30-di-O-yl)-propanephosphonic
acid (56) (Scheme 6)
The stirred suspension of 50-O-tert-butyldiphenylsilyluridine (8)
(1.1 g, 2.4 mmol) and bromopropionaldehyde diethyl acetal (1.0 g,
4.8 mmol) in dichloromethane (20 mL) was acidified with 4 M HCl
in DMF until pH reached slightly red color using a wet pH paper,
and stirred at rt overnight (TLC in C-1). The clear solution was
neutralized with triethylamine and concentrated under reduced
pressure. The obtained product 54 was purified by silica gel chro-
matography using
chloroform.
a linear gradient of ethanol (0e10%) in
Sodium salt of diethyl phosphite prepared from sodium hydride
(0.13 g, 3.3 mmol) and diethyl phosphite (0.42 mL, 3.3 mmol) in THF
(10 mL) under 20 min of stirring was added to the bromo derivative
54 (0.66 g, 1.1 mmol), and the resulting mixture was stirred at room
temperature overnight (TLC in C-1). Then the homogeneous solu-
tion was quenched with 2 M TEAB (1 mL), and concentrated under
reduced pressure. The residue was purified by silica gel chroma-
tography using a linear gradient of ethanol (0e10%) in chloroform
to afford the diethyl ester 55.
This compound 55 (0.23 g, 0.35 mmol) was treated with N-
chlorosuccinimide (0.052 g, 0.385 mmol) in pyridine (5 mL) under
heating at 90 ꢀC for 45 min, and the solution was concentrated
under reduced pressure. The residue was co-evaporated with
acetonitrile (3 ꢃ 5 mL), dissolved in the same solvent (5 mL), and
treated with bromotrimethylsilane (0.184 mL, 1.4 mmol) and 2,6-
lutidine (0.326 mL, 2.8 mmol) at room temperature overnight.
The solution was concentrated under reduced pressure, the residue
was treated with 0.5 M TBAF in THF (10 mL) at rt for 30 min and the
mixture was concentrated under reduced pressure. The obtained
crude product 56 was purified by an RP HPLC.
4.3.16. (2R)-(uridin-20,30-di-O-yl)-ethanephosphonic acid (51)
(Scheme 5)
Title compound 51 was prepared by the two step procedure
including the deoxygenation of 43 (3.0 g, 4.5 mmol) by the proce-
dure described for compound 46, followed by deprotection of the
obtained diester 47 by the procedure described for compound 50.
Yield of 51: 0.84 g (2.4 mmol, 53%). HRMS (M ꢁ H)ꢁ for
Yield of 56: 0.106 g (0.26 mmol, 11%); HRMS (M ꢁ H)ꢁ for
11H14N2O9P: calcd m/z 349.0437, found 349.0427; IR (KBr, cmꢁ1):
C
12H15ClN2O9P: calcd m/z 397.0204, found 397.0211; IR (KBr, cmꢁ1):
C
3422, 2932, 1697, 1646, 1626, 1540, 1449, 1398, 1537, 1276, 1084,
1049, 995, 834, 705, 689, 522. NMR data e see Tables 5 and 6
3419, 2923,1694,1454,1276,1122,1065, 974, 572, 551. NMRdata e see
Tables 5 and 6
4.3.17. (2R)-(5-chlorouridin-20,30-di-O-yl)-ethanephosphonic acid
(52) (Scheme 5)
4.3.20. (50-O-tert-Butyldiphenylsilyl-20-O-dimethoxytrityluridin-30-
O-yl)-acetaldehyde (60a) (Scheme 7)
The deoxygenation of 43 (1.5 g, 2.1 mmol) by the procedure
described for compound 46 afforded diester 47 (1.08 g, 1.7 mmol)
which was treated with N-chlorosuccinimide (1.8 g, 13.5 mmol) in
pyridine (20 mL) at 90 ꢀC for 45 min. The solution was then
concentrated under reduced pressure and the 5-chlorouracil de-
rivative 48 (1.5 g, 2.1 mmol) obtained by silica gel chromatography
using a linear gradient of ethanol (0e10%) was deprotected by the
procedure described for compound 50.
Sodium hydride (0.9 g, 22.9 mmol) was added to a stirred so-
lution of 50-O-t-butyldiphenylsilyl-20-O-dimethoxytrityluridine
(57a, 6.0 g, 7.6 mmol) in THF (70 mL), and the resulting mixture was
heated at 48 ꢀC for 45 min. Allyl bromide (0.86 mL, 9.9 mmol) was
added to the stirred suspension, and the stirring was continued at rt
overnight (TLC in C-1). On quenching the reaction with acetic acid
(0.9 mL, 15 mmol) at 0 ꢀC, the mixture was concentrated under
reduced pressure, and the residue was dissolved in chloroform
(100 mL) and washed with water (3 ꢃ 50 mL). The organic phase
was dried over anhydrous sodium sulfate and evaporated. The
Yield of 52: 0.26 g (0.69 mmol, 33%). HRMS (M ꢁ H)ꢁ for
C
11H13ClN2O9P: calcd m/z 383.0047, found 383.0044; IR (KBr,

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165
crude product 58a was used to the next reaction without
characterization.
6.4 mmol) was added, and the mixture was stirred at rt overnight
and finally concentrated under reduced pressure. The obtained
crude product 63aa was purified by an RP HPLC.
The product 58a was treated with a catalytic amount of osmium
tetroxide (0.1 mL, 2.5% solution in tert-butanol) in a 2:1 THF/water
mixture (60 mL) and N-methylmorpholine-N-oxide (1.0 g,
8.6 mmol). The resulting mixture was stirred at rt overnight (TLC in
C-1), diluted with chloroform (250 mL), and the whole was washed
with water (3 ꢃ 100 mL). Organic layer was dried over anhydrous
sodium sulfate and evaporated. The obtained dihydroxy derivative
59a and sodium periodate (2.2 g, 10.6 mmol) was stirred in 50%
aqueous acetone (40 mL) at room temperature for 1 h (TLC in C-1).
The mixture was diluted with chloroform (250 mL) and washed
with water (3 ꢃ 100 mL). The organic layer was dried over anhy-
drous sodium sulfate and evaporated. The crude product 60a was
purified by silica gel chromatography using a linear gradient of
ethanol (0e10%) in chloroform).
Yield of 63aa: 0.44 g (1.09 mmol, 19%), R epimer; HRMS (M ꢁ H)ꢁ
for C12H15ClN2O9P: calcd m/z 368.0204, found 397.0211; IR (KBr,
cmꢁ1): 3407, 2892, 2362, 1697, 1632, 1451, 1380, 1279, 1130, 1090,
1050, 803, 780, 754, 667, 572, 555. NMR data e see Tables 5 and 6
4.3.23. (2S),3-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/2; (30-O)/3]-
propanephosphonic acid (63ab) (Scheme 7)
Compound 67 (25 mg; 0.036 mmol) was treated with TBAF
(23 mg; 72 mmol) in THF (5 mL) under stirring at rt for 3 h (TLC in C-
1). Reaction mixture was diluted with chloroform (20 mL) and
poured directly onto a silica gel column. The product was eluted
with a linear gradient of ethanol (0e10%) in chloroform yielding
12 mg (0.026 mmol, 75%) of product which was converted into
63ab according to the procedure described for 63aa. Overall yield,
Yield of 60a: 3.8 g (4.6 mmol, 60%); HRMS (M ꢁ H)ꢁ for
C
48H49N2O9Si: calcd m/z 825.3208, found 825.3228; IR (CHCl3,
9 mg (23
m
mol, 86%). HRMS (M ꢁ H)ꢁ for C12H15O9N2ClP: calcd m/z
cmꢁ1): 3391, 3012, 2933, 2861, 1716, 1694, 1608, 1510, 1463, 1254,
1114, 1106, 1035, 831, 703, 602, 517, 505. NMR data e see Tables 5
and 6
397.02092, found 397.02087; IR (KBr, cmꢁ1): 3433, 3253, 3060,
2805, 1698, 1627, 1450, 1417, 1366, 1345, 1345, 1270, 1147, 1111, 1062,
957, 924, 860, 783, 760, 735. NMR data e see Tables 5 and 6
4.3.21. (50-O-tert-Butyldiphenylsilyl-30-O-dimethoxytrityluridin-20-
O-yl)acetaldehyde (60b) (Scheme 8)
4.3.24. (2S),3-(5-chlorouridin-20,30-di-O-yl)-[(30-O)/2; (20-O)/3]-
propanephosphonic acid (63ba) and (2R)-2,3-(5-chlorouridin-20,30-
di-O-yl)-[(30-O)/2; (20-O)/3]-propanephosphonic acid (63bb)
(Scheme 8)
Compounds 63ba and 63bb were prepared from 60b (1.2 g,
1.45 mmol) by the procedure described for compound 63aa. The
obtained epimeric mixture of 63ba and 63bb was resolved by an RP
HPLC.
The title compound 60b was prepared from 57b (6.0 g,
7.6 mmol) by the procedure as described for 60a.
Yield of 60b: 4.2 g (5.1 mmol, 67%); HRMS (M ꢁ H)ꢁ for
C
48H49N2O9Si: calcd m/z 825.3208, found 825.3217; IR (CHCl3,
cmꢁ1): 3392, 3012, 2962, 2934, 1737, 1692, 1608, 1509, 1463, 1463,
1429, 1392, 1302, 1253, 1179, 1114, 1082, 1036, 986, 830, 788, 783,
738, 704, 602, 585, 506. NMR data e see Tables 5 and 6
Overall yield: 0.055 g (0.014 mmol, w1%). 63ba (S-epimer):
0.035g (0.09 mmol); HRMS (M ꢁ H)ꢁ for C12H15ClN2O9P: calcd m/z
397.0204, found 397.0207; IR (KBr, cmꢁ1): 3404, 2931, 2902, 1700,
1628,1447,1383,1274,1256,1117,1091,1051, 907, 782, 756, 681, 559.
63bb (R-epimer): 0.020 g (0.05 mmol); HRMS (M ꢁ H)ꢁ for
4.3.22. 2(R),3-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/2; (30-O)/
3]-propanephosphonic acid (63aa) (Scheme 7)
Tetraethylmethylenediphosphonate (1.4 mL, 5.7 mmol) was
added dropwise to a stirred mixture of sodium hydride (0.23 g,
5.7 mmol) in dry THF (10 mL) at 0 ꢀC. The stirring was continued at
C
12H15ClN2O9P: calcd m/z 397.0204, found 397.0213; IR (KBr, cmꢁ1):
3432, 3067, 2361,1702,1634,1449,1363,1276,1257,1143,1117,1061,
960, 895, 782, 759, 661, 599, 569. NMR data e see Tables 5 and 6
0
ꢀC for 10 min and then at room temperature for 30 min. After-
wards, the mixture was cooled to 0 ꢀC again and the solution of
aldehyde 60a (2.6 g, 3.1 mmol) in dry THF (10 mL) was added to the
stirred reaction mixture dropwise. The resulting mixture was stir-
red at room temperature for 3 h (TLC in C-1), and concentrated
under reduced pressure. The phosphonate 61a (2.43 g, 2.5 mmol),
obtained by purification by silica gel chromatography using a linear
gradient of ethanol (0e10%) in chloroform, was treated with N-
chlorosuccinimide (0.37 g, 2.78 mmol) in pyridine (25 mL) at 90 ꢀC
for 45 min, and the mixture was concentrated under reduced
pressure. The residue was dissolved in 80% acetic acid (40 mL), the
resulting mixture was stirred at room temperature for 30 min (TLC
in C-1) and concentrated under reduced pressure. The residue was
co-evaporated several times with water, neutralised with triethyl-
amine, and finally co-evaporated with ethanol and toluene. The
residue was treated with 0.5 M TBAF in THF (30 mL), the resulting
mixture was stirred for 30 min at rt and concentrated under
reduced pressure. The product 62a (0.86 g, 1.9 mmol), obtained by
silica gel chromatography using a linear gradient of ethyl acetate
(0e50%) in toluene, was cyclized with sodium methoxide (0.13 g,
2.5 mmol) in dry methanol (20 mL). The mixture was stirred at
room temperature for 6 h (TLC in C-1), neutralized with acetic acid
at 0 ꢀC, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography using a linear gradient of
ethanol (0e10%) in chloroform to afford diester of compound 63aa
(0.71 g, 1.6 mmol). This diester was treated with N,O-bis(-
trimethylsilyl)acetamide (4.0 mL, 16.0 mmol) in dry acetonitrile
(12 mL) at rt for 1 h. Then bromotrimethylsilane (0.84 mL,
4.3.25. Diethyl 3-(50-O-tert-butyldiphenylsilyluridin-20-O-yl)-
trans-2-propenephosphonate (65) and diethyl (2R)-3-(50-O-tert-
butyldiphenylsilyluridin-20,30-di-O-yl)-[(20-O)/2; (30-O)/3]-
propanephosphonate (66a), and diethyl (2S)-3-(50-O-tert-
butyldiphenylsilyluridin-20,30-di-O-yl)-[(20-O)/2; (30-O)/3]-
propanephosphonate (66b) (Scheme 7)
The vinylphosphonate 61a (300 mg, 0.3 mmol) was detritylated
in 80% aq. acetic acid (20 mL) at rt overnight (TLC in C-2). The so-
lution was concentrated under reduced pressure, the residue was
co-evaporated several times with water to remove acetic acid, and
the compound 64 was purified by a silica gel chromatography using
a linear gradient of ethanol (0e10%) in chloroform. The obtained
compound 64 was treated with Cs2CO3 (469 mg, 1.44 mmol) in tert-
butanol (20 mL) under stirring at rt for 3 h. The progress of the
reaction was monitored by LCeMS analysis. The reaction was
quenched by DOWEX 50 in Hþ cycle, the suspension was filtered,
and the filtrate was concentrated under reduced pressure. The
residue was separated by an RP HPLC. Yield: 66a, 54 mg
(0.08 mmol, 30%); 66b, 72 mg (0.10 mmol, 40%); and 65, 18 mg
(0.03 mmol, 10%). NMR data e see Tables 5 and 6
4.3.26. Diethyl (2S)-3-(50-O-terc-butyldiphenylsilyl-5-chlorouridin-
20,30-di-O-yl)-[(20-O)/2; (30-O)/3]-propanephosphonate (67)
(Scheme 7)
Compound 66b (29 mg, 0.04 mmol) in dry pyridine (2 mL) was
treated with N-chlorosuccinimide (8 mg, 0.05 mmol) under stirring

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I. Kosiová et al. / European Journal of Medicinal Chemistry 74 (2014) 145e168
166
at 90 ꢀC for 30 min (TLC in C-1). The reaction mixture was diluted
with CHCl3 (50 mL) and the solution was washed with brine
(2 ꢃ 50 mL). The organic layer was dried over anhydrous sodium
sulfate and evaporated. The crude product 67 was purified by silica
gel chromatography using a linear gradient of ethanol (0e10%) in
chloroform. Yield: 24 mg (0.03 mmol, 80%). HRMS (M ꢁ H)ꢁ for
27 mg of 70aa, 48 mg of 70ab. For 70aa HRMS (M ꢁ H)ꢁ for
C
31H40ClN2O9PSiNa: calcd m/z 701.1821, found 701.1820; IR (CHCl3,
cmꢁ1):3383, 3181, 3074, 3054, 2990, 2963, 2932, 2860, 1722, 1701,
1632, 1590, 1566, 1488, 1471, 1465, 1429, 1393, 1370, 1364, 1346,
1329, 1305, 1247, 1162, 1114, 1104, 1084, 1042, 1025, 978, 956, 877,
844, 704, 616, 489. For 70ab HRMS (M ꢁ H)ꢁ for C31H40ClN2O9P-
SiNa: calcd m/z 701.1821, found 701.1819; IR (CHCl3, cmꢁ1): 3381,
3175, 3074, 3053, 3028, 2995, 2930, 2857, 1720, 1702, 1631, 1620,
1590, 1487, 1471, 1465, 1455, 1446, 1429, 1392, 1368, 1270, 1245,
1162,1114,1105,1080,1045,1025, 973, 950, 880, 703, 645, 489. NMR
data e see Tables 5 and 6
C
32H43ClN2O9PSi: calcd m/z 693.21585, found 693.21632; IR (CHCl3,
cmꢁ1): 3382, 3177, 3074, 3054, 3000, 2962, 1720, 1701, 1622, 1590,
1487, 1472, 1447, 1429, 1393, 1363, 1344, 1283, 1269, 1160, 1131, 1115,
1106, 1053, 1029, 998, 962, 864, 709, 703, 489. NMR data e see
Tables 5 and 6
4.3.27. (1RS)-2-(50-O-tert-butyldiphenylsilyl-5-chlorouridin-20,30-
di-O-yl)-[(20-O)/1; (30-O)/2]-ethyliden-1-ylacetate (69a)
(Scheme 9)
4.3.30. (1S),2-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/1; (30-O)/
2]-ethanephosphonic acid (71aa) (Scheme 9)
Compound 70aa (54 mg; 80 mmol) was treated with TBAF (51 mg;
The solution of 60a (1.2 g, 1.4 mmol) in 80% aq. acetic acid
(20 mL) was set aside at rt for 30 min (TLC in C-1) and concentrated
under reduced pressure. The compound 68a (0.58 g, 1.1 mmol),
obtained by silica gel chromatography using a linear gradient of
ethanol (0e10%) in chloroform, was treated with acetic anhydride
(0.21 mL, 2.2 mmol) in pyridine (20 mL). The resulting mixture was
stirred at rt overnight (TLC in C-1), quenched by addition of water
(5 mL) at 0 ꢀC, and concentrated under reduced pressure. The
residue was diluted with chloroform (50 mL), the organic layer was
washed with water (3 ꢃ 20 mL), dried over anhydrous sodium
sulfate and evaporated. The crude product was treated with N-
chlorosuccinimide (0.19 g, 1.4 mmol) in dry pyridine (10 mL) at
90 ꢀC for 45 min (analysis by RP HPLC). The solution was concen-
trated under reduced pressure, and the crude product 69a was
purified by silica gel chromatography using a linear gradient of
ethyl acetate (0e50%) in toluene.
0.16 mmol) in THF (5 mL) under stirring at rt for 10 min (TLC in C-2).
The reaction mixture was diluted with chloroform (20 mL), the so-
lution was then poured directly onto a silica gel column, and the
product was eluted with a linear gradient of ethanol (0e10%) in
chloroform. The obtained product (desilylated derivative of 70aa)
was subsequently treated with N,O-bis(trimethylsilyl)acetamide
(100
ml, 0.4 mmol) in acetonitrile (5 mL) under argon atmosphere at
rt for 12 h. Then bromotrimethylsilane (21
ml, 0.16 mmol) was added
and the mixture was stirred overnight (TLC in IPAW). The resulting
clear solution was concentrated under reduced pressure, the residue
was treated shortly with 2 M triethylammonium hydrogen carbon-
ate buffer (1 mL) and the obtained solution was evaporated to dry-
ness. The residue was co-evaporated with ethanol (2 ꢃ 50 mL) and
purified by an RP HPLC. The product was converted into sodium salt
on Dowex 50 (Naþ). Yield of 71aa: 9 mg (0.02 mmol, 64%). HRMS
(M ꢁ H)ꢁ for C11H14ClN2O9P: calcd m/z 383.0053, found 383.0054; IR
(KBr, cmꢁ1): 1697,1632,1537,1452,1278,1129,1110,1085,1071,1050,
925, 782, 760. NMR data e see Tables 5 and 6
Yield of 69a: 0.65 g (1.08 mmol, 77%). HRMS (M ꢁ H)ꢁ for
C
29H33ClN2O8SiNa: calcd m/z 623.1587, found 623.1587; IR (CHCl3,
cmꢁ1): 3383, 3187, 3074, 3054, 3027, 2963, 1745, 1724, 1703, 1632,
1590, 1488, 1471, 1450, 1429, 1393, 1376, 1365, 1348, 1280, 1230,
1139, 1114, 1106, 1088, 1046, 1023, 999, 964, 862, 710, 703, 623, 616,
602, 505, 489. NMR data e see Tables 5 and 6
4.3.31. (1R)-2-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/1; (30-O)/2]-
ethanephosphonic acid (71ab) (Scheme 9)
Compound 70ab (48 mg, 0.07 mmol) was converted into 71ab
according to the procedure described for 71aa. Yield, 12 mg
(0.02 mmol, 44%). HRMS (M ꢁ H)ꢁ for C11H14ClN2O9P: calcd m/z
383.0053, found 383.0048; IR (KBr, cmꢁ1): 1697, 1643, 1624, 1561,
1537, 1449, 1271, 1142, 1111, 1092, 1069, 1052, 928, 783, 760. NMR
data e see Tables 5 and 6
4.3.28. (1RS),2-(50-O-tert-butyldiphenylsilyl-5-chlorouridin-20,30-
di-O-yl)-[(20-O)/2; (30-O)/1]-ethyliden-1-ylacetate (69b)
(Scheme 10)
Compound 69b was prepared from 60b (2.0 g, 2.4 mmol) by the
same procedure as compound 69a.
Overall yield: 0.88 g (1.46 mmol, 61%); HRMS (M ꢁ H)ꢁ for
C29H32ClN2O8Si: calcd m/z 599.1622, found 599.1628; IR (CHCl3,
cmꢁ1): 3382, 3074, 3029, 2932, 2861, 1722, 1702, 1631, 1451, 1429,
1231, 1227, 1163, 1137, 1113, 1074, 1035, 964, 941, 879, 823, 807, 789,
783, 703, 667, 616, 568, 505. NMR data e see Tables 5 and 6
4.3.32. Diethyl (1S)-2-(50-O-tert-butyldiphenylsilyl-5-chlorouridin-
20,30-di-O-yl)-[(20-O)/2; (30-O)/1]-ethanephosphonate (70ba)
and diethyl (1R)-1,2-(50-O-tert-butyldiphenylsilyl-5-chlorouridin-
20,30-di-O-yl)-[(20-O)/2; (30-O)/1]-ethanephosphonate (70bb)
(Scheme 10)
Compound 69b (200 mg, 0.33 mmol) was converted into 70ba
and 70bb according to the procedure described for 70aa and 70ab.
Overall yield: 85 mg (0.13 mmol, 38%), 54 mg of 70bb, 31 mg of
70ba. For 70bb, HRMS (M ꢁ H)ꢁ for C31H40ClN2O9PSiNa: calcd m/z
701.1821, found 701.1821; IR (CHCl3, cmꢁ1): 3409, 3207, 3074, 2962,
2932, 2860, 1722, 1711, 1632, 1590, 1487, 1471, 1465, 1453, 1431,
1392, 1363,1328, 1303, 1113,1051, 1025, 970, 917, 849, 703, 618, 489.
For 70ba, HRMS (M ꢁ H)ꢁ for C31H40ClN2O9PSiNa: calcd m/z
701.1821, found 701.1819; IR (CHCl3, cmꢁ1): 3381, 3177, 3074, 3054,
2961, 1720, 1702, 1631, 1620, 1590, 1566, 1487, 1471, 1463, 1452,
1429, 1393, 1364, 1329, 1305, 1252, 1161, 1115, 1106, 1049, 1028, 977,
917, 846, 703, 613, 489. NMR data e see Tables 5 and 6
4.3.29. Diethyl (1S)-2-(50-O-tert-butyldiphenylsilyl-5-chlorouridin-
20,30-di-O-yl)-[(20-O)/1; (30-O)/2]-ethanephosphonate (70aa)
and diethyl (1R)-2-(50-O-tert-butyldiphenylsilyl-5-chlorouridin-
20,30-di-O-yl)-[(20-O)/1; (30-O)/2]-ethanephosphonate (70ab)
(Scheme 9)
Compound 69a (150 mg, 0.25 mmol), dried by co-evaporation
with acetonitrile (2 ꢃ 5 mL), was treated in dry acetonitrile
(5 mL) with diethyl trimethylsilyl phosphite (164 mL, 0.75 mmol)
and trimethylsilyl triflate (67 mL, 0.38 mmol) under argon atmo-
sphere at rt for 12 h (TLC in C-2). The reaction was quenched by
addition of 1 M TEAB in 50% aqueous ethanol (10 mL) at 0 ꢀC, and
the solution was concentrated under reduced pressure. The residue
was co-evaporated with ethanol (2 ꢃ 50 mL) and finally with
toluene (1 ꢃ 50 mL). The epimeric phosphonate diesters were
resolved by silica gel chromatography using a linear gradient of
ethanol (0e10%) in chloroform. Yield: 75 mg (0.11 mmol, 44%);
4.3.33. (1R)-2-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/2; (30-O)/
1]-ethanephosphonic acid (71ba) (Scheme 10)
Compound 70ba (16 mg, 0.04 mmol) was converted into 71ba
according to the procedure described for 71aa. Overall yield, 8 mg

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I. Kosiová et al. / European Journal of Medicinal Chemistry 74 (2014) 145e168
167
(0.02 mmol, 57%). HRMS (M ꢁ H)ꢁ for C11H14ClN2O9P: calcd m/z
383.0053, found 383.0050; IR (KBr, cmꢁ1): 1699, 1628, 1540, 1455,
1273, 1112, 1071, 908, 782, 761. NMR data e see Tables 5 and 6
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4.3.34. (1S)-2-(5-chlorouridin-20,30-di-O-yl)-[(20-O)/2; (30-O)/
1]-ethanephosphonic acid (71bb) (Scheme 10)
Compound 70bb (25 mg, 0.06 mmol) was converted into 71bb
according to the procedure described for 71aa. Overall yield, 10 mg
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383.0053, found 383.0055; IR (KBr, cmꢁ1): 1698, 1634, 1539, 1450,
1266, 1137, 1099, 1070, 917, 781, 761. NMR data e see Tables 5 and 6
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Acknowledgements
Financial support by grants 203/09/0820, 202/09/0193, 13-
24880S, and 13-26526S from the Czech Science Foundation under
the IOCB Research Project RVO: 61388963 is gratefully acknowl-
edged. The authors are indebted to Eva Zborníková, MSc. for the
LCeMS measurements and to the staff of the Mass Spectrometry
Department of Institute of Organic Chemistry and Biochemistry AS
ꢀ
CR (Dr. Josef Cvacka, Head) and Pavel Fiedler for the measurements
and interpretations of the HR mass spectra and IR spectra,
respectively.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.026.
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