Fluorophosphonylated nucleoside derivatives as new series of thymidine phosphorylase multisubstrate inhibitors

Article abstract of DOI:10.1021/jm201694y

The synthesis of new class of potential TPase inhibitors containing a difluoromethylphosphonate function as phosphate mimic is reported. This new series was prepared from a readily available fluorinated building block in few steps. Two series were evaluated as potential inhibitors: a linear series and a conformational constrained series. The activity of these multisubstrate inhibitors depends on the size of the spacer introduced between the pyrimidine ring and the phosphonate function. Best results were observed from triazolyl derivatives, easily obtained from propargylthymine and corresponding azides.

Full text of DOI:10.1021/jm201694y

Article
pubs.acs.org/jmc
Fluorophosphonylated Nucleoside Derivatives as New Series of
Thymidine Phosphorylase Multisubstrate Inhibitors
Sonia Amel Diab,† Coralie De Schutter,† Murielle Muzard,‡ Richard Plantier-Royon,‡ Emmanuel Pfund,†
and Thierry Lequeux*,†
†Laboratoire de Chimie Molec
Basse-Normandie, 6 Boulevard du Marec
‡Institut de Chimie Molec
ulaire de Reims, UMR CNRS 6229, UFR des Sciences Exactes et Naturelles, BP 1039,
́
ulaire et Thioorganique, UMR CNRS 6507 and FR3038, ENSICAEN, Universite
hal Juin, 14050 Caen Cedex, France
́
de Caen
́
́
51687 Reims Cedex 2, France
S
* Supporting Information
ABSTRACT: The synthesis of new class of potential TPase
inhibitors containing a difluoromethylphosphonate function as
phosphate mimic is reported. This new series was prepared
from a readily available fluorinated building block in few steps.
Two series were evaluated as potential inhibitors: a linear
series and a conformational constrained series. The activity of
these multisubstrate inhibitors depends on the size of the
spacer introduced between the pyrimidine ring and the phos-
phonate function. Best results were observed from triazolyl
derivatives, easily obtained from propargylthymine and corresponding azides.
angiogenesis is a chloropyrrolidinyl uracil derivative called
INTRODUCTION
■
TPI (Figure 2).4a,5
Platelet-derived endothelial cell growth factor (PD-ECGF) is a
protein involved in tumor angiogenesis. This protein at low
abundance is overexpressed in many human solid tumors and
might be an attractive cancer chemotherapy target for inhibi-
tion of tumor angiogenesis, subsequent tumor growth, and metas-
tasis. Because of a structural and genetic similarity between the
Escherichia coli thymidine phosphorylase (EC 2.4.2.4) and the
human recombinant PD-ECGF, it has been suggested that PD-
ECGF could be identified as the human thymidine phosphory-
lase.1,2 Thymidine phosphorylase (TPase) catalyzes the reversible
phosphorolysis of pyrimidine 2′-deoxynucleosides (Figure 1). The
Multisubstrate inhibitors, simultaneously bound to the nucle-
oside and phosphate binding sites, are less efficient than TPI.
Their moderate activities could be attributed to the low acidic
character of the phosphonate function (pKa2(phosphonate) =
7.5−8). Indeed, high difference in activities between naturally
occurring phosphates and phosphonates has been already
reported for purine nucleoside phosphorylase and tyrosine
kinase inhibitors.7 As difluoromethylphosphonates are the best
surrogate mimic of naturally occurring phosphates,8 our work is
focused on the synthesis and the evaluation of the inhibitory
activities of new fluorinated multisubstrate compounds. Taking
into account that the distance between the phosphate oxygen
atom and the thymidine N1 atom lies between 4 and 10 Å when
the enzymatic substitution occurred,9 the present study deals
with the synthesis of fluorinated phosphonates as multi-
substrate inhibitors of TPase containing different spacers such
as alkyl chains and heterocycles (Figure 3).
RESULTS AND DISCUSSION
Figure 1. Thymidine phosphorylase EC 2.4.2.4.
■
Chemistry. In connection with our previous studies regard-
ing the synthesis of fluorophosphonate derivatives,10 the prepa-
ration of aliphatic series 7−10 and 14−16 was realized from the
readily available tosylate derivatives 1 and 2 (Scheme 1).11
We recently reported that 6-chloropurine or N3-benzoyl-
thymine alkylation with fluorophosphonylated tosylates 1 and 2
affording compounds 3, 4, and 12 proceeded in good yields and
main product of the conversion, the 2-deoxyribose-1-phosphate,
is transformed into 2-deoxyribose (2dR), which stimulates endo-
thelial cell migration and angiogenesis.3
An important effect on angiogenesis and apoptosis in tumors
has been observed by using thymidine phosphorylase inhibi-
tors.4,5 The known TPase inhibitors are commonly structurally
close to modified nucleosides or acyclonucleosides (Figure 2),
and some of them are well-known as antiviral agents.6
Among these inhibitors, the most efficient on the tumor
Received: December 15, 2011
Published: February 28, 2012
© 2012 American Chemical Society
2758
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
Figure 2. Thymidine phosphorylase inhibitors.6a,b,e,f
obtained in 78% yield after deprotection of the benzoyl group.
Bromination of the 5-position of uracil was realized from 5 to
afford fluorophosphonylated 5-bromouracil 6 in 75% yield.
Cleavage of phosphonic alkyl esters was carried out under
standard conditions. Fluorinated phosphonates 3−6 were treated
with TMSBr (5 equiv) followed by addition of methanol, leading
to phosphonic acids 7−10 in 67−75% yields after precipitation.
From 6-chloropurine derivatives 11−13, an additional step
allowing the chlorine atom substitution by a hydroxyl group led
to phosphonic acids 14−16 in 64−77% yields. Fluorophos-
phonylated acyclonucleosides 27−29, containing longer alkyl
chain spacers (8−11 carbon atoms), were prepared from 17
(Scheme 2).10c
Figure 3. Multisubstrate inhibitors.
a
Scheme 1. Synthesis of Phosphonic Acids 7−10 and 14−16
a
Scheme 2. Synthesis of Phosphonic Acids 27−29
a
Reagents and conditions: (a) (i) N3-benzoylthymine, TMG, DMSO,
rt, 15 h; (ii) MeNH2, MeOH, rt, 15 h to give 3 (75%) and 4 (81%);
(i) N3-benzoyluracil, TMG, DMSO, rt, 15 h; (ii) MeNH2, MeOH, rt,
15 h to give 5 (78%); (b) NBS, AIBN, THF, 60 °C, 1.5 h (75%); (c)
(i) TMSBr, CH2Cl2, 0 °C to rt, 72 h; (ii) MeOH, rt, 2 h to give 7
(67%), 8 (75%), 9 (70%), and 10 (74%); (d) 6-chloropurine, TMG,
DMSO, rt, 15 h to give 12 (67%); 2-amino-6-chloropurine, TMG,
DMSO, rt, 15 h to give 11 (60%) and 13 (68%); (e) (i) TMSBr,
CH2Cl2, 0 °C to rt, 72 h; (ii) H2O, rt, 16 h to give 14 (77%), 15
(75%), and 16 (64%).
a
t
Reagents and conditions: (a) (i) BuLi, THF, −78 °C, 10 min; (ii)
1,8-dibromooctane, THF, 30 min at −78 °C, then 1 h at −10 °C to
give 18 (52%); 1,9-dibromononane, THF, 30 min at −78 °C, then 1 h
at −10 °C to give 19 (51%); 1,11-dibromoundecane, THF, 30 min at
−78 °C, then 1 h at −10 °C to give 20 (50%); (b) N3-
benzoylthymine, TMG, DMSO, rt, 15 h to give 21 (64%), 22
(77%), and 23 (76%); (c) MeNH2, MeOH, rt, 18 h to give 24 (94%),
25 (76%), and 26 (90%); (d) (i) TMSBr, CH2Cl2, 0 °C to rt, 72 h;
(ii) MeOH, rt, 2 h to give 27 (99%), 28 (88%), and 29 (97%).
excellent regioselectivities when the reaction was conducted in
the presence of 1,1,3,3-tetramethylguanidine (TMG).11 These
coupling conditions were extended to other nucleic bases.
Indeed, tosylates 1 and 2 were treated with 2-amino-6-
chloropurine in the presence of TMG. After 15 h of stirring,
corresponding alkylation products 11 and 13 were isolated in
60−68% yields. From N3-benzoyluracil and tosylate 2, the
reaction was also very efficient and uracil derivative 5 was
Alkylation reactions proceeded smoothly when the carbanion
was formed from 17. In this case, the anion trapped with 1,8-
dibromooctane, 1,9-dibromononane, and 1,11-dibromounde-
cane afforded corresponding difluorophosphonylated bromoal-
kanes 18−20 in 50−52% yields. Products 18−20 were reacted
with N3-benzoylthymine, and nucleotide analogues 24−26 were
obtained after nucleobase deprotection. Deprotection of phos-
phonic esters with TMSBr/MeOH afforded analogues 27−29
2759
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
isolated by precipitation in 88−99% yields. In this series, better
yields were obtained when intermediates 21−23 were isolated
prior to debenzoylation reaction. Surprisingly, when the
alkylation step was conducted from an anion formed by
deprotonation of the diethyl difluoromethylphosphonate by
LDA, less than 10% of products were detected in the crude.12
In order to modify the conformation of the alkyl chain and
the enzymatic activity, the introduction of a sulfur atom was
realized (Scheme 3). The presence of a sulfur atom has already
TMSBr followed by methanolysis and precipitation yielded the
targeted phosphonic acids 39−42 with good efficiency.
Functionalization of the spacer by introduction of an iodine
atom was explored from iododifluoromethylphosphonate 43 and
alkenes.14 Free radical addition onto allylthymine was realized
(Scheme 4).
a
Scheme 4. Synthesis of Phosphonic Acid 45
Scheme 3. Synthesis of Sulfur-Containing Nucleotide
a
Analogues 39−42
a
t
Reagents and conditions. (a) (i) BuLi, THF, −78 °C, 5 min; (ii) I2,
THF, −78 °C, 1 h (81%). (b) Method A: allylthymine, dilauroyl
peroxide, C2H4Cl2, 80 °C, 3 h (71%). Method B: allylthymine,
Na2S2O4, NaHCO3, MeCN, H2O, rt, 20 h (17%). Method C:
allylthymine, Et3B, CH2Cl2, rt, 3h (58%). (c) (i) TMSBr, CH2Cl2, rt,
72 h; (ii) MeOH, rt, 2 h (76%).
Iododifluoromethylphosphonate 43 was reacted with allylth-
ymine in the presence of three different initiators (sodium
dithionite, triethylborane, dilauroyl peroxide). Best results were
observed in the presence of 0.3 equiv of dilauroyl peroxide.
After 3 h of stirring under refluxed dichloroethane, fluorinated
iodophosphonate 44 was isolated in 71% yield. It is worthy of
note that no protection of the nucleobase is required for the
reaction to proceed. After deprotection the corresponding
phosphonic acid 45 was obtained in 76% yield.
a
Reagents and conditions. (a) 2-mercaptoethanol, TMG, MeCN, rt,
15 h to give 30 (86%); 3-mercaptopropanol, TMG, MeCN, rt, 15 h to
give 31 (77%); 4-mercaptobutanol, TMG, MeCN, rt, 15 h to give 32
(89%); 6-mercaptohexanol, TMG, MeCN, rt, 15 h to give 33 (79%);
(b) (i) N3-benzoylthymine, PPh3, DIAD, THF, rt, 15 h; (ii) MeNH2,
MeOH, rt, 18 h to give 35 (64%), 36 (55%), and 37 (64%); (c) (i)
TMSBr, CH2Cl2, rt, 72 h; (ii) MeOH, rt, 2 h to give 39 (50%), 40
(86%), 41 (94%), and 42 (83%); (d) TsCl, NEt3, CH2Cl2, rt, 15 h
(79%); (e) (i) N3-benzoylthymine, TMG, rt, 15 h; (ii) MeNH2,
MeOH, rt, 15 h (59%).
Finally, phosphonic acids 56−62 containing a triazolyl hetero-
cycle were synthesized from fluorophosphonylated azides
46−48 and propargyl nucleic bases (Scheme 5).15 Azido com-
pounds 46−48 were easily obtained from their corresponding
tosylates and sodium azide, as previously reported.15 They were
reacted with propargyl nucleic bases (1.1 equiv) in the presence
of sodium ascorbate (10 mol %) and copper sulfate (5 mol %).
From propargylthymine, uracil, 6-chloropurine, and 2-amino-6-
chloropurine, corresponding triazolyl derivatives 49−55 were
isolated in 71−96% yields after 24 h of stirring at room tem-
been applied with success to improve the activity of PNP
inhibitors.13
Sulfur-containing spacers of variable length were prepared by
alkylation of thiols using tosylate 2. This latter was treated with
2 equiv of thiol (2-mercaptoethanol, 3-mercaptopropanol,
4-mercaptobutanol, or 6-mercaptohexanol) in the presence of
1.5 equiv of TMG. After 15 h of stirring in acetonitrile at room
temperature, corresponding fluorophosphonylated sulfanyl
alcohols 30−33 were obtained in 77−89% yields. N3-Benzoyl-
thymine was introduced after activation of alcohols 30−33
from the corresponding tosylate or by using the Mitsunobu
conditions. From fluorinated hydroxyphosphonate 33, corre-
sponding tosylate 34 was formed in 79% yield, while tosylation
of 30−32 was unsuccessful. Alternatively, alcohols 30−32 were
treated with N3-benzoylthymine in the presence of PPh3 and
DIAD to afford corresponding thymine derivatives 35−37 in
55−64% yields after deprotection. In this later case, no O-
alkylation product was detected in the crude. Indeed, the
observed chemical shift was in agreement with the formation of
a methylene−nitrogen bond when compared to the starting
alcohols 30−32 (13C NMR analysis). Furthermore, tosylate 34
was easily transformed into its corresponding alkylated thymine
38. Finally, deprotection of phosphonate esters 35−38 with
t
perature in BuOH/H2O (1/1). Phosphonic ester hydrolysis
and chlorine substitution by a hydroxyl group, carried out with
TMSBr followed by hydrolysis steps with MeOH and/or H2O,
afforded pure phosphonic acids 56−62 in 34−87% yields after
purification by precipitation.
To confirm the effect of the fluorine atoms in the series of
targeted acyclic nucleotides, phosphonic acid 65, as non-
fluorinated analogue of 7, was also prepared. The synthesis was
performed in three steps starting from commercially available
1,4-dibromobutane as depicted (Scheme 6).
Biological Studies. The magnitude of the size of the spacer
was evaluated for compound 49 by X-ray analysis16 and
compared to the value obtained by molecular modeling. In this
case, similar values were observed by both methods (Figure 4),
and the molecular modeling approach was extended to all the
prepared compounds to correlate the enzymatic inhibition per-
centage with the phosphorus−nitrogen atoms distance. As ex-
pected, the different spacers introduced in the series permitted
2760
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
a
Scheme 5. Synthesis of Phosphonic Acids 56−62
a
t
Reagents and conditions. (a) propargylthymine, CuSO4, sodium ascorbate, BuOH, H2O, rt, 24 h to give 49 (71%), 50 (96%), and 51 (93%);
t
t
propargyluracil, CuSO4, sodium ascorbate, BuOH, H2O, rt, 24 h to give 52 (85%); propargyl-6-chloropurine, CuSO4, sodium ascorbate, BuOH,
H2O, rt, 24 h to give 53 (85%) and 54 (82%); propargyl-2-amino-6-chloropurine, CuSO4, sodium ascorbate, tBuOH, H2O, rt, 24 h to give 55 (75%);
(b) (i) TMSBr, CH2Cl2, rt, 72 h; (ii) MeOH, rt, 2 h to give 56 (34%), 57 (87%), 58 (73%), and 59 (75%); (c) (i) TMSBr, CH2Cl2, 0 °C to rt, 72 h;
(ii) MeOH, rt, 2 h; (iii) H2O, rt, 16 h to give 60 (56%), 61 (63%), and 62 (81%).
a
Scheme 6. Synthesis of Phosphonic Acid 65
Table 1. TP Inhibition Studies with Phosphonic Acids
Containing a Linear Spacer
phosphorus−nitrogen TP inhibition
TP inhibition
entry compd
distance (Å)
(%) at 1 mM (%) at 100 μM
1
6A5BU
POT
7
100 (100)6d
76 (75)6d
51
77 (71)6d
2
15 (29)6d
3
6.60
4
4
8
7.38
68
15
15
20
4
5
9
7.38
44
6
10
14
15
16
27
28
29
39
40
41
42
45
65
7.38
61
7
7.81
25
8
7.82
25
9
9
7.82
74
34
55
55
56
0
a
Reagents and conditions: (a) P(OiPr)3, 130 °C, 24 h (81%); (b) (i)
10
11
12
13
14
15
16
17
18
12.57
13.84
16.33
10.23
11.72
7.82
85
N3-benzoylthymine, TMG, DMSO, rt, 16 h; (ii) MeNH2, MeOH, rt,
86
16 h (92%); (c) (i) TMSBr, CH2Cl2, rt, 35 h; (ii) MeOH, rt, 2 h (88%).
88
36
90
43
40
38
52
0
78
15.22
5.36
86
95
6.56
23
Inhibitory activity of phosphonic acids 7−10, 14−16, 27−
29, 39−42, 45, 56−62, and 65 against thymidine phosphor-
ylase from E. coli was evaluated using the assay reported by
́ ́
Perez-Per
ez (Tables 1 and 2).6d Typically, to a mixture of
thymidine, orthophosphate, and TPase in Tris buffer was added
phosphonic acid at two concentrations (1 mM, 100 μM). After
20 min of incubation at room temperature followed by 5 min at
90 °C, percentage of inhibition was determined by HPLC after
separation of thymidine from thymine. Results were compared
to those observed from 6-amino-5-bromouracil (6A5BU), a
well-established TPase inhibitor, and 1-phosphonooctylthymine
(POT), a moderate inhibitor.6d Percentages of inhibition of phos-
phonic acids containing a linear spacer are listed in Table 1. As
expected, introduction of fluorine atoms increased the activity
Figure 4. (a) X-ray structure of difluorophosphonate 49. (b) Compu-
tational modeling of 49.
coverage of a large range of distance between 6.5 and 16.3 Å
(Table 1). These distances should cover the two possible
opened and closed conformations adopted by the enzyme.
2761
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
1 mM, while 70% was observed for compound 8 and 85% for
compound 27, both containing, respectively, one and seven
additional carbon atoms in the spacer (entries 3, 4, and 10).
This pattern was even more pronounced at 100 μM (5% for 7,
15% for 8, and 55% for 27). In this series, results were found to
be optimum with 27 and the activity remained unchanged
when more than eight carbon atoms were introduced in the
spacer. Indeed, compounds 28 and 29 displayed about 85%
inhibition at 1 mM and 55% at 100 μM (entries 10, 11, and 12).
These results seems to indicate that thymidine phosphorylase is
locked in its open, inactive conformation, as already demonstrated
by Balzarini and co-workers in a previous study.6b
Substitution of a methylene group in the spacer by a sulfur
atom did not induce any change in the activity in almost every
case. In fact, sulfur-containing phosphonic acids 40−42 showed
the same activity as their corresponding analogues 27−29
(entries 10−12 and 14−16). Surprisingly, the difluorophos-
phonylated nucleoside 39, in which the sulfur atom and the
thymine are separated by only two carbon atoms, was found to
be inactive even at high concentration (entry 13).
PNP inhibition studies realized in the late 1990s revealed
that the distance between the nitrogen atom of the nucleic base
and the phosphorus atom of the difluoromethylphosphonate
function was not the only important factor required for the
design of potent inhibitors. In fact, it was also reported that
introduction of a cycle (phenyl, cyclopropane, or THF) in the
spacer allowed an increase in the activity.18 In addition, to
explore if a click enzymatic reaction could be possible,19 the
tolerance of a triazolyl ring was evaluated. This was confirmed
by the difluorophosphonylated nucleoside analogues 56−60,
containing a triazolyl ring (Table 2). The presence of a triazolyl
ring is tolerated by the enzyme, and phosphonic acids 56−60
exhibited moderate to excellent activities at 1 mM (entries 3−9).
Compound 56 with only one carbon atom between the
difluorophosphonate group and the triazolyl ring displayed a
weak inhibition activity (entry 3). This result indicates that the
distance between the fluorinated phosphonate and the nucleic
base is crucial and reinforces the idea of an inhibition of the
enzyme in its opened conformation. Furthermore, we noticed a
drastic improvement of the activity when the triazolyl ring was
introduced onto a larger spacer. Indeed, nucleotide analogues
57 and 58 were found to be 3 times more active than 56 at
1 mM and up to 7 times at 100 μM (entries 3−5). As a
high difference in activity was observed from nonfluorinated
(Table 1, POT, 65) and the fluorinated series (Table 1, 27, 7),
the synthesis of nonfluorinated triazolyl derivatives was not
explored. As previously discussed, substitution of the thymine
nucleobase by uracil, hypoxanthine, or guanine had a negative
effect. Derivative 58 presented 68% inhibition at 100 μM, while
compounds 59, 61, and 62 did not exceed 35% at the same
concentration (entries 5, 6, 8, 9). The same pattern was
observed with 57 and 60 (entries 4 and 7).
Table 2. TP Inhibition Studies with Conformational
Constrained Phosphonic Acids
phosphorus−nitrogen TP inhibition
TP inhibition
entry compd
distance (Å)
(%) at 1 mM (%) at 100 μM
1
2
3
4
5
6
7
8
9
6A5BU
POT
56
100 (100)6d
77 (71)6d
76 (75)6d
15 (29)6d
7.82
29
79
90
79
69
76
76
9
57
10.86
10.86
10.86
9.95
36
68
22
29
35
35
58
59
60
61
10.83
10.83
62
as already observed for the inhibition of PNP.7a Indeed,
compound 7 containing a short spacer was found to be twice
more active at 1 mM than its corresponding nonfluorinated
analogue 65 (entries 3 and 18). This difference in activity was
confirmed when the inhibition percentages of compounds 27
and POT were compared. In addition, the substitution of the
pyrimidine ring appeared essential to improve the inhibition of
TPase, as observed for TP64 and POT (Figure 5). Unfortunately,
Figure 5. Influence of the presence of fluorine atoms and the
pyrimidine ring onto the inhibition percentage of TPase at 100 μM.6d
the synthesis of the 6A5BU derivatives was attempted but not
successful in our hands.
Surprisingly, phosphonic acid 45, containing an iodine atom
in the same size spacer, inhibited TPase at 95% inhibition at
1 mM and 52% at 100 μM (entry 17). The introduction of a
supplementary halogen atom into the spacer had a remarkable
positive effect on the inhibition compared to 7 (entry 3). This
effect is difficult to rationalize; we presume that the iodine atom
might occupy the pentose ring pocket of the natural substrate.
This later case suggests that TPase would be inhibited in its
closed active conformation.17
Substitution of thymine by uracil decreased the inhibitory
effect, and compound 9 was consequently less active than 8
at 1 mM (entries 5 and 4). However, phosphonic acid 10 in
which a bromine atom was introduced onto the 5-position
of uracil displayed roughly the same activity as its corres-
ponding thymine derivative 8 (entries 6 and 4), confirming the
presence of lipophilic pocket around the nucleic base.6c Sub-
stitution of thymine by a purine nucleic base such as hypoxan-
thine and guanine induced a decrease of the activity. Com-
pounds 14 and 15 were less active than their thymine analogues
7 and 8 (entries 3, 4, 7, and 8). In contrast, only phosphonic
acid 16 showed a marginal inhibitory effect of 74% at 1 mM
(entry 9).
Difluorophosphonylated analogue 58 combining the triazolyl
ring and a large size spacer appears to be the best inhibitor
revealed by this study. Thus, its inhibition constant was
evaluated (Ki = 58 μM), and 58 acted as a competitive inhibitor
(Figure 6). Note that the other competitive inhibitors TP64
and TP65, evaluated on E. coli TPase, presented 142 and
54 μM respectively,6b for a similar assay, showing again the
superiority of the difluoromethylphosphonates as phosphate
surrogates.
We also noticed that the activity increased with the size of
the spacer. Nucleotide analogue 7 exhibited 50% inhibition at
2762
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
■
◆
▲
Figure 6. (A) Lineweaver−Burk plot and (B) replot of slope versus [58]: ( ) 58, 100 μM; ( ) 58, 50 μM; (×) 58, 20 μM; ( ) 58, 0 μM.
with air pressure, and products were detected by thin layer chro-
CONCLUSION
■
matography, in which the spots were visualized by UV irradiation and/
Novel fluorinated acyclic nucleotides have been synthesized to
or KMnO4 solution. NMR spectra were recorded on a 250 or 400 MHz
apparatus in deuterated solvent at 25 °C. 31P and 19F NMR spectral lines
are with respect to the internal references H3PO4 (capillary) and CFCl3.
All chemical shifts are reported in δ parts per million (ppm), and coupling
constants are in hertz (Hz). High-resolution mass data were recorded on
a high-resolution mass spectrometer in the EI or ESI mode. IR spectra
were recorded on a Perkin-Elmer ATR IR instrument. Analytical
HPLC was performed on a Waters systems (model 600 controller,
model 717plus autosampler, model 996 photodiode array detector)
using reverse phase Phenomenex Gemini C18 110A (5 μm, 4.6 mm ×
250 mm). The mobile phase was 70% methanol, 30% H2O with
0.05% of TFA, and the flow rate was 1.0 mL/min. UV spectra were
recorded using a Waters 996 photodiode array detector. Data were
integrated and reported using Waters Empower software. All com-
pounds submitted to enzymatic assays displayed purity of >95% as
determined by this method, unless stated otherwise.
develop new multisubstrate inhibitors of E. coli thymidine
phosphorylase as potent antiangiogenic factors. The first series
contained a difluoromethylphosphonate group and a nucleic
base that were separated by linear spacers from different size. A
second series of inhibitors functionalized by a triazolyl ring was
developed, and their biological activities were evaluated.
For both series, thymine derivatives were much more active
than their analogues bearing uracil, guanine, and hypoxanthine
nucleic base. It has been confirmed that difluoromethylphospho-
nate derivatives are more active than their nonfluorinated ana-
logues (65 vs 7). However in the fluorinated series, compounds
having a short spacer (6−8 Å) are less active except when the
iodine atom was present on the alkyl chain (compound 45). In
contrast, a long length spacer up to 16 Å dramatically enhanced
the inhibition properties of the compounds. In this series the
optimum activity for a chain length of 14−15 Å suggests an
inhibition of the enzyme in its opened, inactive conformation,
as previously observed in the literature. Substitution of a
methylene group in the spacer by a sulfur atom induced no
change of the activity. The presence of the triazolyl ring in the
spacer is well tolerated by the enzyme, and phosphonic acids
56−60 exhibited moderate to good activities at 1 mM. The
best inhibition was obtained with 58 (90% at 1 mM and 68% at
100 μM) that combined the triazolyl ring and a long spacer.
Because of its large size, it is reasonable to assume that 58 inhi-
bits the thymidine phosphorylase in its opened inactive confor-
mation. Finally, these fluorinated acyclic nucleosides are the
first examples of multisubstrate TPase inhibitors bearing a
difluoromethylphosphonate group as phosphate mimic. These
new series will be further exploited in order to improve the
activity against thymidine phosphorylase to design new anti-
angiogenic agents after derivation into their corresponding
prodrugs.6i−k,20 In addition, their activity toward viruses will be
evaluated and reported elsewhere.
Diisopropyl 1,1-Difluoro-5-(2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)pentylphosphonate (5). Typical Procedure for the Pre-
paration of Alkyl Spacer from Tosylate Derivatives. To a stirred
solution of tosylate 2 (442.1 mg, 1.0 mmol) in DMSO (5 mL),
N3-protected pyrimidine (324 mg, 1.50 mmol) was added
followed by dropwise addition of TMG (0.19 mL, 1.50 mmol).
The mixture was stirred and monitored by TLC. After
completion (15 h), DMSO was evaporated and the residue
diluted in CH2Cl2 (15 mL), washed with water (5 mL) and
brine (5 mL). The aqueous layers were extracted with CH2Cl2
(2 × 10 mL) and the combined organic layers washed with
brine (5 mL) and dried over MgSO4. Solvents were evaporated
under reduced pressure. To a stirred solution of crude product
(382.8 mg, 0.46 mmol) in MeOH (5 mL) was added 40%
N-methylamine in H2O (5 mL, 20.0 mmol), and the solution
was stirred for 15 h at room temperature. The methanol was
evaporated under reduced pressure. The residue was purified by
flash column chromatography on silica using EtOAc−pentane
(8/2) as eluent to give 5 as a light yellow syrup (298.6 mg,
78%). 1H NMR (CDCl3, 400 MHz) δ 9.75 (s, 1H), 7.15 (d, J =
8.0 Hz, 1H), 5.66 (d, J = 8.0 Hz, 1H), 4.80 (dsept, J = 6.0 Hz,
2H), 3.71 (t, J = 6.8 Hz, 2H), 1.95−2.15 (m, 2H), 1.68−1.78
(m, 2H), 1.49−1.65 (m, 2H), 1.32 (dd, J = 6.1, 3.1 Hz, 12H);
19F NMR (CDCl3, 376 MHz) δ −112.83 (dt, J = 108.5, 19.8 Hz,
EXPERIMENTAL SECTION
31
2F); P NMR (CDCl3, 162 MHz) δ 5.1 (t, J = 108.5 Hz, 1P);
■
13C NMR (CDCl3, 63 MHz) δ 164.3, 151.1, 144.7, 121.6 (dt, J =
General. All commercially available reagents were bought from
Aldrich and used as received. For anhydrous conditions, the glassware
was dried in the oven at 120 °C and cooled to room temperature
under a continuous nitrogen flow. THF, CH2Cl2, Et2O, and CH3CN
were dried in a solvent generator from Innovative Technologies Inc.,
which uses an activated alumina column to remove water. DMF and
NEt3 were distilled under CaH2 or 4 Å molecular sieves. Flash column
chromatography was realized on silica gel 60 (40−63 μm) from Merck
260.6, 218.1 Hz), 102.4, 73.9 (d, J = 7.1 Hz), 48.8, 33.6 (dt, J =
21.2, 15.1 Hz), 29.9, 24.3 (d, J = 3.4 Hz), 23.9 (d, J = 4.8 Hz),
18.13 (dt, J = 4.9 Hz). LRMS-ESI (m/z): [M + H]+ 383 (31),
341 (73), 299 (100). HRMS-ESI (m/z): [M + H]+ calcd for
C15H26F2N2O5P 383.1547, found 383.1532.
Diisopropyl 1,1-Difluoro-5-[5-bromo-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl]pentylphosphonate (6). To a suspension of
2763
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
5 (250.0 mg, 0.65 mmol) in dry THF (5 mL) were added
N-bromosuccunimide (115.7 mg, 0.65 mmol) and AIBN (a pinch).
The mixture was heated at 60 °C for 90 min. Then it was diluted with
EtOH (10 mL) and filtered through Celite. The filtrate was evaporated
and purified by flash column chromatography on silica using CH2Cl2−
MeOH (20/1) as eluent to give 6 (224.8 mg, 75%) as a yellow syrup.
1H NMR (CDCl3, 400 MHz) δ 10.09 (s, 1H), 7.56 (s, 1H), 4.80 (2H,
dsept, J = 6.0 Hz), 3.73 (2H, t, J = 7.2 Hz), 1.99−2.16 (m, 2H), 1.64−
1.80 (m, 2H), 1.50−1.62 (m, 2H), 1.31 (dd, J = 6.2, 3.5 Hz, 12H); 19F
16 h and dried under reduced pressure. The residue was diluted
in MeOH, and compound 14 (54.75 mg, 77%) was obtained as a
white solid after precipitation in Et2O−CH2Cl2: mp 230 °C; H
1
NMR (D2O, 400 MHz) δ 8.53 (s, 1H), 4.21 (t, J = 7.5 Hz, 2H),
1.89−2.22 (m, 4H); 19F NMR (D2O, 376 MHz) δ −112.43
(dt, J = 112.3, 21.5 Hz, 2F); 31P NMR (D2O, 162 MHz) δ 3.6
(t, J = 112.3 Hz 1P); 13C NMR (D2O, 101 MHz) δ 166.2,
163.5, 148.6, 141.2, 128.3, 122.4 (m), 46.0, 29.9 (m), 23.9 (m).
LRMS-ESI (m/z): [M − H]− 322 (100), 279 (25), 222 (10).
HRMS-ESI (m/z): [M − H]− calcd for C9H11F2N5O4P 322.0517,
found 322.0512.
NMR (CDCl3, 376 MHz) δ −112.75 (dt, J = 108.6, 19.6 Hz, 2F); 31
P
NMR (CDCl3, 162 MHz) δ 4.9 (t, J = 108.6 Hz, 1P); 13C NMR
(CDCl3, 101 MHz) δ 160.1, 150.6, 144.2, 120.4 (dt, J = 260.3, 218.4
Hz), 96.5, 74.0 (d, J = 7.1 Hz), 49.1, 33.5 (dt, J = 21.2, 14.7 Hz), 28.8,
24.1 (d, J = 3.5 Hz), 23.9 (d, J = 4.8 Hz), 18.1 (dt, J = 4.9 Hz). LRMS-
ESI (m/z): [M + H]+ 461 (58), 419 (100), 377 (75), 229 (2). HRMS-
ESI (m/z): [M + H]+ calcd for C15H25BrF2N2O5P 461.0653, found
461.0633.
Diisopropyl 1,1-Difluoro-9-bromononylphosphonate
(18). General Procedure for the Preparation of Diisopropyl 1,1-
Difluorobromoalkylphosphonate. To a solution of tert-butyllithium
(2.17 mL 1 M in pentane, 3.47 mmol, 1.3 equiv) in anhydrous
THF (40 mL) at −78 °C, difluoromethylphosphonate 17 (700 mg,
2.67 mmol, 1 equiv) was added dropwise. The mixture was
stirred for 10 min at −78 °C, and 1,8-dibromooctane (0.98 mL,
5.34 mmol, 2 equiv) was added slowly. The mixture was stirred
for 30 min at −78 °C and warmed from −78 to −10 °C over
1 h. The reaction mixture was quenched by addition of saturated
NH4Claq (4 mL), and the mixture was extracted with CH2Cl2
(10 mL), washed with saturated aqueous solution of NaHCO3,
dried over MgSO4, filtered, and concentrated under reduce pres-
sure. The residue was purified by flash column chromatography
using EtOAc/pentane (1/9 and 2/8) as eluent to afford com-
1,1-Difluoro-5-(6-hydroxy-9H-purin-9-yl)pentylphosphonic
Acid (9). General Procedure for the Hydrolysis of Difluorophos-
phonylated Pyrimidine Derivatives. To a cooled solution of 5
(84.1 mg, 0.22 mmol) in anhydrous CH2Cl2 (5 mL) was added
TMSBr (0.174 mL, 1.32 mmol). The mixture was stirred at
room temperature for 72 h and then concentrated under
vacuum. The residue was diluted in MeOH (2 mL) and stirred
for 2 h at room temperature. The solvent was removed, and the
crude was diluted in MeOH (0.5 mL). Compound 9 (45.9 mg,
70%) was obtained as a white powder after precipitation in
Et2O−CH2Cl2 (3/1): mp 193 °C; 1H NMR (MeOD, 500 MHz)
δ 7.41 (d, J = 9.5 Hz, 1H), 5.62 (d, J = 9.5 Hz, 1H), 3.79 (t, J =
9.0 Hz, 2H), 2.02−2.22 (m, 2H), 1.71−1.83 (m, 2H), 1.57−1.70
(m, 2H); 31P NMR (MeOD, 202 MHz) δ 5.7 (t, J = 110.1 Hz,
1P); 19F NMR (MeOD, 470 MHz) δ −118.65 (dt, J = 110.1,
23.5 Hz, 2F); 13C NMR (DMSO, 101 MHz) δ 163.9, 152.3,
146.8, 121.3 (dt, J = 260.8, 194.2 Hz), 106.5, 43.9, 31.2 (m),
23.5, 17.0 (m). LRMS-ESI (m/z): [M − H]− 297 (90), 277 (6),
254 (100), 234 (8), 185 (32), 111 (36). HRMS-ESI (m/z):
[M − H]− calcd for C9H12F2N2O5P 297.0452, found 297.0443.
Diisopropyl 4-(2-Amino-6-chloro-9H-purin-9-yl)-1,1-difluor-
obutylphosphonate (11). General Procedure for the Introduc-
tion of Purine Nucleic Bases. To a stirred solution of tosylate 1
(200.0 mg, 0.47 mmol) and 2-amino-6-chloropurine (119.5 mg,
0.71 mmol) in DMSO (5 mL) was added dropwise TMG
(0.089 mL, 0.71 mmol). The mixture was stirred at 20 °C and
monitored by TLC. After completion (15 h), DMSO was
evaporated and the residue diluted in CH2Cl2 (15 mL), washed
with water (5 mL), and brine (5 mL). The aqueous layer was
extracted with CH2Cl2 (2 × 10 mL), and the combined organic
layers were washed with brine (10 mL), dried over MgSO4, and
filtered. Solvents were evaporated under reduced pressure and
crude product was purified by flash column chromatography on
silica using ethyl acetate−pentane (7/3) as eluent to give 11
(120.1 mg, 60%) as a light yellow syrup. 1H NMR (CDCl3, 250
MHz) δ 7.78 (s, 1H), 5.28 (sbr, 2H), 4.83 (dsept, J = 6.3 Hz,
2H), 4.14 (t, J = 6.7 Hz, 2H), 2.02−2.40 (m, 4H), 1.33 (dd, J =
5.9, 3.3 Hz, 12H); 19F NMR (CDCl3, 235 MHz) δ −112.26 (dt,
J = 106.9, 19.0 Hz, 2F); 31P NMR (CDCl3, 101 MHz) δ 4.8 (t,
J = 106.9 Hz, 1P); 13C NMR (CDCl3, 101 MHz) δ 160.1,
154.4, 153.0, 146.7, 131.9, 120.1 (dt, J = 259.8, 216.9 Hz), 73.8
(d, J = 7.0 Hz), 48.1, 32.6 (dt, J = 21.0, 14.9 Hz), 24.4 (d, J =
3.3 Hz), 23.9 (d, J = 4.7 Hz), 22.2 (dt, J = 5.0 Hz). LRMS-ESI
(m/z): [M + H]+ 427 (62), 385 (100), 342 (23). HRMS-ESI
(m/z): [M + H]+ calcd for C15H24ClF2N5O3P 426.1273, found
426.1285.
1
pound 18 (565 mg, 52%) as a colorless oil. H NMR (CDCl3,
400 MHz) δ 4.84 (dsept, J = 6.8 Hz, 2H), 3.40 (t, J = 6.8 Hz,
2H), 2.10−1.95 (m, 2H), 1.85 (quint, J = 6.9 Hz, 2H), 1.60−
1.38 (m, 4H), 1.37 (dd, J = 6.1, 4.1 Hz, 12H), 1.35−1.30 (m,
6H); 19F NMR (CDCl3, 376 MHz) δ −112.83 (dt, J = 20.0,
109.7 Hz, 2F); 31P NMR (CDCl3, 161 MHz) δ 5.7 (t, J = 109.7
Hz, 1P); 13C NMR (CDCl3, 101 MHz) δ 120.6 (dt, J = 260.0,
217.1 Hz), 73.3 (d, J = 7.1 Hz), 33.8 (dt, J = 20.9, 14.2 Hz),
33.9, 32.7, 29.1, 29.0, 28.5, 28.0, 24.1 (d, J = 3.5 Hz), 23.7 (d, J =
4.9 Hz), 20.5 (dd, J = 4.7 Hz). LRMS-ESI (m/z): [M + H]+ 407
(30), 365 (62), 323 (100). HRMS-ESI (m/z): [M + H]+ calcd
for C15H31O3F2PBr 407.1162, found 407.1161.
Diisopropyl 1,1-Difluoro-9-(5-methyl-2,4-dihydropyrimidin-
1,3(2H)-yl)nonylphosphonate (21). General Procedure for
Thymine Introduction from Bromoalkyl Derivatives. To a solution
of 18 (285 mg, 0.70 mmol) and N3-benzoylthymine (320 mg,
1.39 mmol) in CH3CN (6 mL) and DMSO (0.5 mL), was added
dropwise TMG (0.191 mL, 1.53 mmol). After 15 h of stirring at
20 °C the mixture was poured in Et2O (10 mL). The organic
layer was washed with water (2 × 2 mL), brine (5 mL), dried
over MgSO4, filtered, and concentrated under reduce pressure.
Compound 21 (249 mg, 64%) was isolated as colorless oil by
flash column chromatography using EtOAc/pentane (6/4) as
eluent. 1H NMR (CDCl3, 400 MHz) δ 7.89 (dd, J = 8.0, 0.8 Hz,
2H), 7.62 (dd, J = 7.4 Hz, 1H), 7.47 (dd, J = 7.7 Hz, 2H), 7.09
(s, 1H), 4.82 (dsept, J = 6.4 Hz, 2H), 3.70 (t, J = 7.3 Hz, 2H),
2.20−1.99 (m, 2H), 1.94 (s, 3H), 1.70−1.50 (m, 4H), 1.35 (dd,
J = 6.1, 4.5 Hz, 12H), 1.35−1.30 (m, 8H); 19F NMR (CDCl3,
376 MHz) δ −112.79 (dt, J = 20.0, 109.6 Hz, 2F); 31P NMR
(CDCl3, 162 MHz) δ 5.6 (t, J = 109.6 Hz, 1P); 13C NMR
(CDCl3, 101 MHz) δ 169.2, 163.2, 149.8, 140.2, 134.9, 131.7,
130.4, 129.1, 120.6 (dt, J = 259.0, 217.1 Hz), 110.6, 73.93 (d,
J = 7.1 Hz), 48.8, 33.8 (dt, J = 20.9, 14.3 Hz), 29.2, 29.1, 29.0,
28.9, 26.3, 24.1 (d, J = 3.5 Hz), 23.7 (d, J = 4.8 Hz), 20.5 (dd,
J = 4.6 Hz, C3), 12.4. LRMS-ESI (m/z): [M + H]+ 557 (61), 515
(64), 473 (100), 393 (8), 351 (16). HRMS-ESI (m/z): [M + H]+
calcd for C27H40N2O6F2P 557.2592, found 557.2607.
1,1-Difluoro-4-(2-amino-6-hydroxy-9H-purin-9-yl)-
pentylphosphonic Acid (14).21 General Procedure for the
Hydrolysis of Difluorophosphonylated Purine Derivatives. To
a solution of 11 (93.7 mg, 0.22 mmol) in anhydrous CH2Cl2
(3 mL) cooled at 0 °C was added TMSBr (0.174 mL, 1.32 mmol).
The mixture was stirred at room temperature for 72 h. Volatiles
were removed, and the residue was stirred in H2O (1 mL) for
Diisopropyl 1,1-Difluoro-9-(5-methyl-2,4-dihydropyrimidin-
1,3(2H)-yl)nonylphosphonate (24). General Procedure for the
Deprotection of N3-Benzoylthymine. To a stirred solution of 21
(256 mg, 0.46 mmol) in MeOH (5 mL) was added N-methyl-
amine (5 mL, 40% aqueous solution). After 18 h, the mixture
was concentrated under reduced pressure. Flash column chro-
matography using EtOAc/pentane (7/3) afforded 24 (195 mg,
2764
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
1
94%) as a colorless oil. H NMR (CDCl3, 400 MHz) δ 9.77
(t, J = 6.8 Hz, 2H), 2.80 (t, J = 6.8 Hz, 2H), 2.55 (t, J = 6.9 Hz,
2H), 2.10−1.97 (m, 2H), 1.92 (d, J = 1.1 Hz, 3H), 1.67−1.65
(m, 4H), 1.36 (dd, J = 6.2, 4.0 Hz, 12H); 19F NMR (CDCl3, 376
(sbr, 1H), 6.94 (d, J = 1.2 Hz, 1H), 4.77 (dsept, J = 6.2 Hz,
2H), 3.62 (t, J = 7.4 Hz, 2H), 2.05−1.92 (m, 2H), 1.85 (d, J =
1.1 Hz, 3H), 1.65−1.50 (m, 4H), 1.30 (dd, J = 6.2, 4.6 Hz, 12H),
1.27−1.20 (m, 8H); 19F NMR (CDCl3, 376 Mz) δ −112.81
(dt, J = 20.0, 109.7 Hz, 2F); 31P NMR (CDCl3, 162 MHz) δ 5.6
(t, J = 109.7 Hz, 1P); 13C NMR (CDCl3, 101 MHz) δ 164.7,
151.1, 140.4, 120.6 (dt, J = 259.0, 217.0 Hz), 110.5, 73.3 (d, J =
7.1 Hz), 48.4, 33.7 (dt, J = 20.8, 14.2 Hz), 29.1, 29.1, 29.0, 28.9,
26.3, 24.1 (d, J = 3.4 Hz), 23.7 (d, J = 4.9 Hz), 20.5 (dd, J = 4.6
Hz), 12.3. LRMS-ESI (m/z): [M + H]+ 453 (92), 411 (100),
369 (51). HRMS-ESI (m/z): [M + H]+ calcd for C20H36N2O5-
F2P 453.2330, found 453.2333.
31
Mz) δ −112.81 (dt, J = 19.7, 109.0 Hz, 2F); P NMR (CDCl3,
162 MHz) δ 5.4 (t, J = 109.0 Hz, 1P); 13C NMR (CDCl3, 101
MHz) δ 164.4, 150.9, 140.8, 120.4 (dt, J = 259.5, J = 217.3 Hz),
110.4, 73.6 (d, J = 7.1 Hz), 48.6, 33.4 (dt, J = 20.9, 14.5 Hz), 32.1,
30.8, 29.1, 24.1 (d, J = 3.5 Hz), 23.8 (d, J = 4.9 Hz), 19.9 (dd, J =
4.9 Hz), 12.3. LRMS-ESI (m/z): [M + H]+ 457 (75), 415 (100),
373 (70), 331 (7). HRMS-ESI (m/z): [M + H]+ calcd for
C18H32N2O5F2PS 457.1738, found 457.1752.
1,1-Difluoro-5-(2-(5-methyl-2,4-dihydropyrimidin-1,3(2H)-
yl)-1-ethanesulfure)pentylphosphonic Acid (39). Following the
general procedure used for 9 from difluorophosphonate 35 (100 mg,
0.22 mmol), TMSBr (0.174 mL, 1.32 mmol), and CH2Cl2 (5 mL),
compound 39 was isolated as a white solid (41 mg, 50%): mp 155 °C;
1H NMR (DMSO, 400 MHz) δ 11.29 (s, 1H), 7.61 (d, J = 1.2 Hz,
1H), 3.85 (t, J = 6.8 Hz, 2H), 2.79 (t, J = 7.3 Hz, 2H), 2.61 (t, J = 6.7
Hz, 2H), 2.10−1.97 (m, 2H), 1.81 (d, J = 1.0 Hz, 3H), 1.67−1.60 (m,
4H); 19F NMR (DMSO, 376 MHz) δ −112.35 (dt, J = 20.0, 100.8 Hz,
2F); 31P NMR (DMSO, 162 MHz) δ 4.1 (t, J = 100.8 Hz, 1P); 13C
NMR (MeOD, 101 MHz) δ 166.9, 152.8, 143.5, 121.9 (dt, J = 257.1,
210.5 Hz), 110.8, 34.4 (dt, J = 21.2, 14.8 Hz), 32.4, 31.2, 30.3 (4C),
21.2 (dd, J = 4.7 Hz), 12.2. LRMS-ESI (m/z): [M + H]+ 373 (19), 247
(100), 187 (43). HRMS-ESI (m/z): [M + H]+ calcd for C12H20-
N2O5F2PS 373.0799, found 373.0800.
1,1-Difluoro-9-(5-methyl-2,4-dihydropyrimidin-1,3(2H)-yl)-
nonylphosphonic Acid (27). Following the general procedure used
for 9 from difluorophosphonate 24 (99 mg, 0.22 mmol), TMSBr
(0.174 mL, 1.32 mmol), and CH2Cl2 (5 mL), compound 27 was iso-
1
lated as a white solid (80 mg, 99%): mp 175−180 °C; H NMR
(DMSO, 400 MHz) δ 11.19 (s, 1H), 7.53 (d, J = 1.2 Hz, 1H), 3.60
(t, J = 7.2 Hz, 2H), 2.00−1.85 (m, 2H), 1.75 (d, J = 1.0 Hz, 3H),
1.60−1.40 (m, 4H), 1.30−1.20 (m, 8H); 19F NMR (DMSO, 376 Mz)
δ −112.41 (dt, J = 20.1, 101.1 Hz, 2F); 31P NMR (DMSO, 162 MHz)
δ 4.2 (t, J = 101.1 Hz, 1P); 13C NMR (DMSO, 101 MHz) δ 164.4,
151.0, 141.6, 121.5 (dt, J = 257.9, 204.5 Hz), 108.6, 48.7, 47.3, 33.5
(dt, J = 21.0, 14.2 Hz), 28.8, 28.6, 25.9, 20.6 (dt, J = 4.4 Hz), 12.0.
LRMS-ESI (m/z) [M − H]− 367 (7), 347 (10), 324 (100), 304 (15).
HRMS-ESI (m/z): [M − H]− calcd for C14H22N2O5F2P 367.1234,
found 367.1231.
Diisopropyl 1,1-Difluoro-3-iodo-4-(5-methyl-2,4-dioxo-3,4-
dihydropyridin-1(2H)-yl)butylphosphonate (44). To a refluxed
solution of 43 (100.0 mg, 0.29 mmol) and allylthymine (54.0 mg,
0.32 mmol) in 1,2-dichloroethane (2 mL) was added over a period of
1 h via a syringe pump dilauroyl peroxide (35.0 mg, 0.09 mmol) in 1,2-
dichloroethane (1 mL). After 3 h, the mixture was cooled, concen-
trated and the crude product was purified by flash column chromato-
graphy using EtOAc/pentane (6/1) as eluent to give 44 (106.0 mg,
Diisopropyl 1,1-Difluoro-5-(2-hydroxy-1-ethylsulfanyl)-
pentylphosphonate (30). General Procedure for the Preparation
of Difluorophosphonylated Thioethers. To a solution of 2 (845
mg, 1.91 mmol) and 2-mercaptoethanol (0.270 μL, 3.82 mmol)
in anhydrous CH3CN (10 mL) was added TMG (0.350 μL,
2.86 mmol). The mixture was stirred 15 h at room temperature.
The solution was diluted in Et2O (10 mL) and washed with
water (2 × 5 mL). The aqueous layers were combined and
extracted with CH2Cl2 (10 mL). The combined organic layers
were washed with brine, dried over MgSO4, filtered, and con-
centrated under reduced pressure. The crude product was
purified by flash column chromatography using EtOAc/pen-
tane (7/3) as eluent to give 30 (572 mg, 86%) as a colorless oil:
1H NMR (CDCl3, 400 MHz) δ 4.81 (dsept, J = 6.4 Hz, 2H),
1
71%) as a white solid: mp 110.5 °C; H NMR (CDCl3, 400 MHz) δ
8.87 (s, 1H), 6.98 (d, J = 1.1 Hz, 1H), 4.79 (dsept, J = 6.2 Hz, 2H),
4.59−4.71 (m, 1H), 4.11 (dd, J = 14.5, 10.0 Hz, 1H), 3.80 (dd, J =
14.5, 10.0 Hz, 1H), 2.67−2.96 (m, 2H), 1.88 (d, J = 1.0 Hz, 3H), 1.32
(dd, J = 6.2, 3.0 Hz, 12H); 19F NMR (CDCl3, 376 MHz) δ −112.29
(dddd, J = 296.1, 104.0, 25.0, 13.0 Hz, 1F), −109.8 (dddd, J = 296.1,
103.0, 27.1, 11.2 Hz, 1F); 31P NMR (CDCl3, 162 MHz) δ 3.5 (dd, J =
104.0, 103.0 Hz, 1P); 13C NMR (CDCl3, 101 MHz) δ 164.7, 149.9,
140.1, 119.2 (dt, J = 262.8, 216.8 Hz), 110.4, 74.2 (d, J = 7.0 Hz), 55.3,
41.9 (ddd, J = 20.6, 15.6 Hz), 24.0 (d, J = 3.5 Hz), 23.6 (d, J = 4.6 Hz),
16.6, 12.2. LRMS-ESI (m/z): [M + H]+ 509 (28), 466 (87), 425
(100). HRMS-ESI (m/z): [M + H]+ calcd for C15H25F2IN2O5P 509.0514,
found 509.0519.
3.69 (t, J = 6.0 Hz, 2H), 2.70 (t, J = 6.0 Hz, 2H), 2.52 (t, J = 6.8
Hz, 2H), 2.10−1.95 (m, 2H), 1.70−1.60 (m, 4H), 1.35 (dd, J =
19
6.0, 3.5 Hz, 12H); F NMR (CDCl3, 376 Mz) δ −112.70 (dt,
J = 19.7, 108.8 Hz, 2F); 31P NMR (CDCl3, 161 Mz) δ 5.4
(t, J = 108.8 Hz, 1P); 13C NMR (CDCl3, 101 MHz) δ 120.4
(dt, J = 259.3, 217.4 Hz), 73.6 (d, J = 7.1 Hz), 60.4, 35.2, 33.4
(dt, J = 35.6, 14.5 Hz), 31.3, 29.3, 24.1 (d, J = 3.5 Hz), 23.7 (d,
J = 4.9 Hz), 20.0 (dd, J = 4.9 Hz). LRMS-ESI (m/z): [M + H]+
349 (15), 331 (100), 289 (38), 271 (2), 247 (10), 229 (2).
HRMS-ESI (m/z): [M + H]+ calcd for C13H28O4F2PS
349.1414, found 349.1412.
1,1-Difluoro-3-iodo-4-(5-methyl-2,4-dioxo-3,4-dihydropyri-
din-1(2H)-yl)butylphosphonic Acid (45). Following the general
procedure used for 9, from 44 (112 mg, 0.22 mmol), TMSBr (0.174 mL,
1.32 mmol), and CH2Cl2 (5 mL), compound 45 was obtained as a white
1
powder (71 mg, 76%): mp 168 °C; H NMR (DMSO, 400 MHz) δ
10.51 (s, 1H), 7.19 (d, J = 1.2 Hz, 1H), 4.25−4.73 (m, 1H), 3.85−4.10
(m, 2H), 2.58−2.89 (m, 2H), 1.96 (d, J = 1.0 Hz, 3H); 31P NMR
(DMSO, 162 MHz) δ 3.6 (dd, J = 104.0, 103.0 Hz, 1P); 19F NMR
(DMSO, 376 MHz) δ −113.05 (dddd, J = 297.0, 104.0, 25.2, 12.6 Hz,
1F), −111.36 (dddd, J = 297.0, 103.0, 26.7, 11.2 Hz, 1F); 13C NMR
(DMSO, 101 MHz) δ 163.7, 150.3, 147.5, 121.2 (dt, J = 263.9, 217.6 Hz),
110.9, 57.2, 41.9 (m), 17.1 (m), 14.2. LRMS-ESI (m/z): [M − H]− 422
(64), 295 (100), 126 (2). HRMS-ESI (m/z): [M − H]− calcd for
C9H11F2IN2O5P 422.9418, found 422.9423.
1-[1-(5-Diisopropoxyphosphono-5,5-difluoropentyl)-1,2,3-
triazolo-4-methyl]uracil (52). General Procedure for the
Preparation of Triazolyl Derivatives. To a stirred solution of 48
(320.0 mg, 1.02 mmol) in tBuOH (2.5 mL) and H2O (2.5 mL)
were added propargyl uracil (168.1 mg, 1.12 mmol), sodium
ascorbate (19.8 mg, 0.10 mmol), and CuSO4·5H2O (8.00 mg,
0.05 mmol). The mixture was stirred for 24 h at room tem-
perature, then concentrated under reduced pressure. The resi-
due was diluted in CH2Cl2 (10 mL) and washed with water
Diisopropyl 1,1-Difluoro-5-(2-(3-benzoyl-5-methyl-2,4-dihy-
dropyrimidin-1(2H)-yl)-1-ethanesulfure)pentylphosphonate
(35). General Procedure for Thymine Introduction from Fluori-
nated Hydroxyphosphonates. Alcohol 30 (570 mg, 1.64 mmol),
N3-benzoylthymine (453 mg, 1.97 mmol), and Ph3P (515 mg,
1.97 mmol) in anhydrous THF (12 mL) were cooled at 0 °C.
DIAD (0.485 μL, 2.46 mmol) was introduced dropwise, and
the reaction mixture was stirred for 15 h at room temperature.
The solvent was evaporated under reduce pressure, and the
crude was filtered through a pad of silica gel using EtOAc/
pentane (6/4 and 7/3). After concentration, the crude product
was diluted with MeOH (17 mL), and N-methylamine (17 mL,
40% aqueous solution) was added slowly. After 18 h, the mix-
ture was concentrated under reduced pressure. Flash column chro-
matography using EtOAc/pentane (8/2) afforded 35 (479.1 mg,
1
64%) as a colorless oil: H NMR (CDCl3, 400 MHz) δ 8.80 (s,
1H), 7.03 (d, J = 1.2 Hz, 1H), 4.84 (dsept, J = 6.2 Hz, 2H), 3.85
2765
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
(10 mL). The aqueous layer was extracted with CH2Cl2 (2 ×
10 mL), and combined organic layers were washed with a
saturated solution of NaCl (5 mL), dried over MgSO4, filtered,
and concentrated. The crude product was purified by flash
column chromatography using CH2Cl2/CH3OH (10/1) as eluent
AUTHOR INFORMATION
Corresponding Author
*Phone: (33)231452854. E-mail: Thierry.Lequeux@ensicaen.fr.
Notes
■
1
to give 52 (401.8 mg, 85%) as a white solid: mp 105 °C; H
The authors declare no competing financial interest.
NMR (CDCl3, 400 MHz) δ 8.69 (s, 1H), 7.59 (s, 1H), 7.23 (d,
J = 1.1 Hz, 1H), 5.60 (s, 1H), 4.80 (s, 2H), 4.76 (dsept, J =
6.2 Hz, 2H), 4.16 (t, J = 6.9 Hz, 2H), 1.81−2.06 (m, 4H), 1.52−
1.68 (m, 2H), 1.33 (dd, J = 6.1, 3.1 Hz, 12H); 19F NMR (CDCl3,
376 MHz) δ −112.90 (dt, J = 106.9, 19.0 Hz, 2F); 31P NMR
(CDCl3, 162 MHz) δ 5.0 (t, J = 106.9 Hz, 1P); 13C NMR
(CDCl3, 101 MHz) δ 164.4, 151.3, 142.1, 139.9, 124.0, 119.6 (dt,
J = 261.3, 218.0 Hz), 110.9, 74.2 (d, J = 7.1 Hz), 50.1, 43.0, 33.2
(dt, J = 20.1, 14.9 Hz), 29.7, 24.3 (d, J = 3.6 Hz), 23.7 (d, J =
5.1 Hz,), 18.0 (dt, J = 4.7 Hz). LRMS-ESI (m/z): [M + H]+ 464
(46), 422 (100), 380 (72). HRMS-ESI (m/z): [M + H]+ calcd for
C18H29F2N5O5P 464.1874, found 464.1868.
ACKNOWLEDGMENTS
■
This work was supported by the CNRS and the Crunch
(Interregional Organic Chemistry Network). Dr. J. F. Lohier is
gratefully thanked for providing crystallographic data.
ABBREVIATIONS USED
■
TPase, thymidine phosphorylase; PD-ECGF, platelet-derived
endothelial cell growth factor; 2dR, 2-deoxyribose; TPI, thymi-
dine phosphorylase inhibitor; TMG, tetramethylguanidine;
LDA, lithium diisopropylamide; DIAD, diethyl diazodicarbox-
ylate; 6A5BU, 6-amino-5-bromouracyl; POT, 1-phosphonoc-
tylthymine; PNP, purine nucleoside phosphorylase; Ki, inhibi-
tion constant; TMSBr, trimethylsilyl bromide
9-[1-(4-Diisopropoxyphosphono-4,4-difluorobutyl)-1,2,3-tri-
azolo-4-methyl]-6-chloropurine (53). General procedure used for
52 was followed with 47 (300.0 mg, 1.00 mmol) and propargyl-6-
chloropurine (211.9 mg, 1.10 mmol) to afford 53 (418.1 mg, 85%) as
1
a white solid: mp 108 °C; H NMR (CDCl3, 400 MHz) δ 8.66 (s,
1H), 8.31 (s, 1H), 7.72 (s, 1H), 5.51 (s, 2H), 4.73 (dsept, J = 6.0 Hz,
2H), 4.35 (t, J = 7.2 Hz, 2H), 2.05−2.18 (m, 2H), 1.89−2.05 (m, 2H),
1.25 (dd, J = 6.4, 4.0 Hz, 12H); 31P NMR (CDCl3, 162 MHz) δ 4.4 (t,
J = 106.4 Hz, 1P); 19F NMR (CDCl3, 376 MHz) δ −112.34 (dt, J =
106.4, 18.8 Hz, 2F); 13C NMR (CDCl3, 63 MHz) δ 156.5, 151.0,
145.4, 131.6, 129.6, 126.2, 123.4, 119.5 (dt, J = 260.8, 218.1 Hz), 74.0
(d, J = 7.2 Hz, 2C), 49.8, 39.1, 31.00 (dt, J = 21.4, 15.1 Hz), 24.1 (d,
J = 3.5 Hz, 2C), 23.8 (d, J = 4.7 Hz, 2C), 22.2 (dt, J = 4.8 Hz). LRMS-
ESI (m/z): [M + H]+ 492 (100), 450 (82), 408 (22), 226 (15), 173
(18). HRMS-ESI (m/z): [M + H]+ calcd for C18H26ClF2N7O3P
492.1491, found 492.1470.
REFERENCES
■
(1) Moghaddam, A.; Bicknell, R. Expression of Platelet-Derived
Endothelial Cell Growth Factor in Escherichia coli and Confirmation of
Its Thymidine Phosphorylase Activity. Biochemistry 1992, 31, 12141−
12146.
(2) Barton, G. J.; Ponting, C. P.; Spraggon, G.; Finnis, C.; Sleep, D.
Human Platelet-Derived Endothelial Cell Growth Factor Is Homol-
ogous to Escherichia coli Thymidine Phosphorylase. Protein Sci. 1992,
1, 688−690.
(3) Schwartz, E. L.; Hotchkiss, K. A.; Ashton, A. W. Thymidine
Phosphorylase and 2-Deoxyribose Stimulate Human Endothelial Cell
Migration by Specific Activation of the Integrins α5β1 and αVβ3. J. Biol.
Chem. 2003, 278, 19272−19279.
(4) (a) Matsushita, S.; Nitanda, T.; Furukawa, T.; Sumizawa, T.;
Tani, A.; Nishimoto, K.; Akiba, S.; Miyadera, K.; Fukushima, M.;
Yamada, Y.; Yoshida, H.; Kanzaki, T.; Akiyama, S. The Effect of a
Thymidine Phosphorylase Inhibitor on Angiogenesis and Apoptosis in
Tumors. Cancer Res. 1999, 59, 1911−1916. (b) Takao, S.; Akiyama, S.;
Nakajo, A.; Yoh, H.; Kitazono, M.; Natsgoe, S.; Miyadera, K.;
Fukushima, M.; Yamada, Y.; Aikou, T. Suppression of Metastasis by
Thymidine Phosphorylase Inhibitor. Cancer Res. 2000, 60, 5345−5348.
(c) Sengupta, S.; Sellers, L. A.; Matheson, H. B.; Fan, T. P. D.
Thymidine Phosphorylase Induces Angiogenesis in Vivo and in Vitro:
An Evaluation of Possible Mechanisms. Br. J. Pharmacol. 2003, 139,
219−231.
(5) (a) Uracil Derivatives, and Antitumor Effect Potentiator and
Antitumor Agent Containing the Same. Patent US5744475, 1998;
Taiho Pharmaceutical Co. Ltd.; Chem. Abstr. 1998, 125, 328727.
(b) Angiogenesis Inhibitors. Patent EP1354589, 2003; Japan Science
and Tech Corp.; Chem. Abstr. 2003, 137, 41733
1,1-Difluoro-5-(4-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)methyl)-1H-1,2,3-triazol-1-yl)pentylphosphonic Acid (59).
General procedure used for 9 was followed with 52 (101.9 mg, 0.22
mmol), TMSBr (0.174 mL, 1.32 mmol), and CH2Cl2 (5 mL) to afford
1
59 (62.6 mg, 75%) as a white solid: mp 184 °C; H NMR (DMSO,
400 MHz) δ 8.15 (s, 1H), 8.07 (s, 1H), 7.87 (s, 1H), 5.80 (s, 2H),
4.54 (t, J = 7.2 Hz, 2H), 1.90−2.15 (m, 2H), 1.70−1.85 (m, 2H),
1.43−1.55 (m, 2H); 31P NMR (DMSO, 162 MHz) δ 4.3 (t, J = 97.0 Hz,
1P); 19F NMR (DMSO, 376 MHz) δ −112.82 (dt, J = 97.0, 19.9 Hz,
2F); 13C NMR (DMSO, 101 MHz) δ 164.9, 151.4, 141.9, 139.3, 129.6,
119.9 (m), 104.1, 55.3, 47.8, 33.5 (m), 28.9, 25.3 (m). LRMS-ESI (m/z):
[M − H]− 378 (100), 358 (15), 335 (70), 315 (45). HRMS-ESI (m/z):
[M − H]− calcd for C12H15F2N5O5P 378.1235, found 378.1246.
1,1-Difluoro-4-(4-((6-hydroxy-9H-purin-9-yl)methyl)-1H-
1,2,3-triazol-1-yl)butylphosphonic Acid (60). General procedure
used for 14 was followed with 53 (108.2 mg, 0.22 mmol), TMSBr
(202.1 mg, 1.32 mmol), and CH2Cl2 (5 mL) to afford 60 (47.9 mg,
56%) as a white solid: mp 185 °C; 1H NMR (D2O, 400 MHz) δ 8.45
(s, 1H), 8.23 (s, 1H), 8.12 (s, 1H), 5.62 (s, 2H), 4.49 (t, J = 6.8 Hz,
2H), 2.06−2.25 (m, 2H), 1.85−2.05 (m, 2H); 31P NMR (D2O,
162 MHz) δ 4.5 (t, J = 97.2 Hz, 1P); 19F NMR (D2O, 376 MHz) δ
−112.23 (dt, J = 97.2, 19.9 Hz, 2F); 13C NMR (D2O, 101 MHz) δ
155.8, 155.2, 146.0, 144.4, 142.5, 123.3, 121.3, 114.89 (m), 50.1,
42.1, 32.4 (m), 17.3 (m). LRMS-ESI (m/z): [M − H]− 388 (100),
368 (8), 216 (20), 171 (62), 135 (26). HRMS-ESI (m/z): [M − H]−
calcd for C12H13F2N7O4P 388.0735, found 388.0718.
(6) (a) Focher, F.; Spadari, S. Thymidine Phosphorylase: A Two-
Face Janus in Anticancer Chemotherapy. Curr. Cancer Drug Targets
2001, 1, 141−153. (b) Balzarini, J.; Degrev
Esnouf, R.; De Clercq, E.; Engelborghs, Y.; Camarasa, M. J.; Per
Perez, M. J. Kinetic Analysis of Novel Multisubstrate Analogue
Inhibitors of Thymidine Phosphorylase. FEBS Lett. 2000, 483, 181−
185. (c) Langen, P.; Etzold, G.; Barwolff, D.; Preussel, B. Inhibition of
Thymidine Phosphorylase by 6-Aminothymine and Derivatives of 6-
Aminouracil. Biochem. Pharmacol. 1967, 16, 1833−1837. (d) Esteban-
Gamboa, A.; Balzarini, J.; Esnouf, R.; De Clercq, E.; Camarasa, M. J.;
̀
e, B.; Esteban-Gamboa, A.;
́
ez-
́
̈
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the preparation of compounds 7−65
not described in the manuscript, HPLC data of all tested
compounds (POT, 5BRU, 7−10, 14−16, 27−29, 39−42, 45,
́ ́
Perez-Perez, M. Design, Synthesis, and Enzymatic Evaluation of
Multisubstrate Analogue Inhibitors of Escherichia coli Thymidine
Phosphorylase. J. Med. Chem. 2000, 43, 971−983. (e) Votruba, I.;
Pomeisl, K.; Tloust ova, E.; Holy, A.; Otova, B. Inhibition of
Thymidine Phosphorylase (PD-ECGF) from SD-Lymphoma by
Phosphonomethoxyalkyl Thymines. Biochem. Pharmacol. 2005, 69,
1
56−61, 65), and H and 13C NMR spectra for compounds
5−65. This material is available free of charge via the Internet
at http://pubs.acs.org.
2766
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
1517−1521. (f) Allan, A. L.; Gladstone, P. L.; Price, M. L. P.; Hopkins,
S. A.; Juarez, J. C.; Donate, F.; Fernansky, R. J.; Shaw, D. E.; Ganem,
B.; Lee, Y.; Wang, W.; Ealick, S. Synthesis and Evaluation of
Multisubstrate Bicyclic Pyrimidine Nucleoside Inhibitors of Human
Thymidine Phosphorylase. J. Med. Chem. 2006, 49, 7807−7815.
(g) Casanova, E.; Hernandez, A. I.; Priego, E. M.; Liekens, S.;
Efficient Synthesis of Fluorothiosparfosic Acid Analogues with Potential
Antitumoral Activity. Bioorg. Med. Chem. 2005, 13, 4921−4928.
(11) Diab, S. A.; Sene, A.; Pfund, E.; Lequeux, T. Efficient Synthesis
of Fluorophosphonylated Alkyles by Ring-Opening Reaction of Cyclic
Sulfates. Org. Lett. 2008, 10, 3895−3898.
(12) Jakeman, D. L.; Ivory, A. J.; Williamson, M. P.; Blackburn, G. M.
Highly Potent Bisphosphonate Ligands for Phosphoglycerate Kinase.
J. Med. Chem. 1998, 41, 4439−4452.
(13) Kelly, J. L.; Linn, J. A.; McLean, E. W.; Tuttle, J. V. 9-
[(Phosphonoalkyl)benzyl]guanines. Multisubstrate Analogue Inhib-
itors of Human Erythrocyte Purine Nucleoside Phosphorylase. J. Med.
Chem. 1993, 36, 3455−3463.
(14) (a) Sene, A.; Diab, S. A.; Hienzsch, A.; Cahard, D.; Lequeux, T.
Radical Conjugated Addition: Addition of Dialkyl Phosphonodifluor-
omethyl Radical onto Unsaturated Ketones. Synlett 2009, 981−985.
(b) Yang, Z.; Burton, D. J. A Novel and Practical Preparation of α,α-
Difluoro Functionalized Phosphonates from Iododifluoromethyl-
phosphonate. J. Org. Chem. 1992, 57, 4676−4683.
(15) Diab, S. A.; Hienzch, A.; Lebargy, C.; Guillarme, S.; Pfund, E.;
Lequeux, T. Synthesis of Fluorophosphonylated Acyclic Nucleotide
Analogues via Copper(I)-Catalyzed Huisgen 1−3 Dipolar Cyclo-
addition. Org. Biomol. Chem. 2009, 7, 4481−4490.
(16) The following crystal structure has been deposited at the
Cambridge Crystallographic Data Centre and allocated the deposition
number CCDC 850861.
(17) Birck, M. R.; Schramm, V. L. Nucleophilic Participation in the
Transition State for Human Thymidine Phosphorylase. J. Am. Chem.
Soc. 2004, 126, 2447−2453.
(18) (a) Yokomatsu, T.; Hayakawa, Y.; Suemune, K.; Kihara, T.;
Soeda, S.; Shimeno, H.; Shibuya, S. Synthesis and Biological
Evaluation of 1,1-Difluoro-2-(tetrahydro-3-furanyl)ethylphosphonic
Acids Possessing a N9-Purinylmethyl Functional Group at the Ring.
A New Class of Inhibitors for Purine Nucleoside Phosphorylases.
Bioorg. Med. Chem. Lett. 1999, 9, 2833−2836. (b) Yokomatsu, T.;
Abe, H.; Sato, M.; Suemune, K.; Kihara, T.; Soeda, S.; Shimeno, H.;
Shibuya, S. Selective α-1A Adrenergic Receptor Antagonists. Effects
of Pharmacophore Regio- and Stereochemistry on Potency and
Selectivity. Bioorg. Med. Chem. 1998, 6, 2495−2505. (c) Yokomatsu,
T.; Hayakawa, Y.; Kihara, T.; Koyanagi, S.; Soeda, S.; Shimeno, H.;
Shibuya, S. Synthesis of the Novel Analogues of Dysidiolide and
Their Structure−Activity Relationship. Bioorg. Med. Chem. 2000, 8,
2571−2579. (d) Halazy, S.; Ehrhard, A.; Eggenspiller, A.; Berges-
Gross, V.; Danzin, C. Fluorophosphonate Derivatives of N9-
Benzylguanine as Potent, Slow-Binding Multisubstrate Analogue
Inhibitors of Purine Nucleoside Phosphorylase. Tetrahedron 1996, 52,
177−184.
́ ́
Camarasa, M. J.; Balzarini, J.; Perez-Perez, M. J. 5′-O-Tritylinosine and
Analogues as Allosteric Inhibitors of Human Thymidine Phosphor-
ylase. J. Med. Chem. 2006, 49, 5562−5570. (h) Reigan, P.; Edwards,
P. N.; Gbaj, A.; Cole, C.; Barry, S. T.; Page, K. M.; Ashton, S. E.; Luke,
R. W. A.; Douglas, K. T.; Stratford, I. J.; Jaffar, M.; Bryce, R. A.;
Freeman, S. Aminoimidazolylmethyluracil Analogues as Potent Inhi-
bitors of Thymidine Phosphorylase and Their Bioreductive Nitro-
imidazolyl Prodrugs. J. Med. Chem. 2005, 48, 392−402. (i) Cole, C.;
Reignan, P.; Gbaj, A.; Edwards, P. N.; Douglas, K. T.; Stratford, I. J.;
Freeman, S.; Jaffar, M. Potential Tumor-Selective Nitroimidazolylme-
thyluracil Prodrug Derivatives: Inhibitors of the Angiogenic Enzyme
Thymidine Phosphorylase. J. Med. Chem. 2003, 46, 207−209.
́
(j) Pradere, U.; Clavier, H.; Roy, V.; Nolan, S. P.; Agrofoglio, L. A.
The Shortest Strategy for Generating Phosphonate Prodrugs by Olefin
Cross-Metathesis: Application to Acyclonucleoside Phosphonates. Eur.
́
J. Org. Chem. 2011, 7324−7330. (k) Montagu, A.; Pradere, U.; Roy,
V.; Nolan, S. P.; Agrofoglio, L. A. Expeditious Convergent Procedure
for the Preparation of Bis(POC) Prodrugs of New (E)-4-Phosphono-
but-2-en-1-yl Nucleosides. Tetrahedron 2011, 67, 5319−5328.
(l) Barral, K.; Weck, C.; Payrot, N.; Roux, L.; Durafour, C.; Zoulim,
F.; Neyts, J.; Balzarini, J.; Canard, B.; Priet, S.; Alvarez, K. Acyclic
Nucleoside Thiophosphonates as Potent Inhibitors of HIV and HBV
Replication. Eur. J. Med. Chem. 2011, 46, 4281−4288. (m) Vakiaeva,
N.; Wyles, D. L.; Schooley, R. T.; Hwu, J. B.; Beadle, J. R.; Prichard,
M. N.; Hostetler, K. Y. Synthesis and Antiviral Evaluation of 9-(S)-[3-
Alkoxy-2-(phosphonomethoxy)-propyl]nucleoside Alkoxyalkyl Esters:
Inhibitors of Hepatitis C Virus and HIV-1 Replication. Bioorg. Med.
Chem. 2011, 19, 4616−4625.
(7) (a) Halazy, S.; Ehrhard, A.; Danzin, C. 9-(Difluorophospho-
noalkyl)guanines as a New Class of Multisubstrate Analog Inhibitors
of Purine Nucleoside Phosphorylase. J. Am. Chem. Soc. 1991, 113,
315−317. (b) Burke, T. R.; Smyth, M. S.; Otaka, A.; Nomizu, M.;
Roller, P. P.; Wolf, G.; Case, R.; Shoelson, S. E. Nonhydrolyzable
Phosphotyrosyl Mimetics for the Preparation of Phosphatase-Resistant
SH2 Domain Inhibitors. Biochemistry 1994, 33, 6490−64941.
(c) Berkowitz, D. B.; Bose, M. α-Monofluoroalkyl)phosphonates: A
Class of Isoacidic and “Tunable” Mimics of Biological Phosphates.
J. Fluorine Chem. 2001, 112, 13−33.
(8) Romanenko, V. D.; Kukhar, V. P. Fluorinated Phosphonates:
Synthesis and Biomedical Application. Chem. Rev. 2006, 106, 3868−
3935.
(9) (a) Birck, M. R.; Schramm, V. L. Nucleophilic Participation in the
Transition State for Human Thymidine Phosphorylase. J. Am. Chem.
Soc. 2004, 126, 2447−2453. (b) Birck, M. R.; Schramm, V. L. Binding
Causes the Remote [5′-3H]Thymidine Kinetic Isotope Effect in
Human Thymidine Phosphorylase. J. Am. Chem. Soc. 2004, 126,
6882−6883.
(19) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier,
P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Click Chemistry in Situ:
Acetylcholinesterase as a Reaction Vessel for the Selective Assembly of
a Femtomolar Inhibitor from an Array of Building Blocks. Angew.
Chem., Int. Ed. 2002, 1053−1057.
(20) (a) Siddiqui, A.; Ballatore, C.; McGuigan, C.; De Clercq, E.;
Balzarini, J. The Presence of Substituents on the Aryl Moiety of the
Aryl Phosphoramidate Derivative of d4T Enhances Anti-HIV
Efficacy in Cell Culture: A Structure−Activity Relationship. J. Med.
(10) (a) Pfund, E.; Lequeux, T.; Vazeux, M; Masson, S. Synthesis of
Thiapyranoside Precursors Using the Building-Block Approach from a
Phosphonodifluorodithioacetate. Tetrahedron Lett. 2002, 43, 2033−
2036. (b) Pfund, E.; Lequeux, T.; Vazeux, M.; Masson, S. Synthesis of
Thiazolines Linked to a Difluoromethylphosphonate Diester via
Dithioester Chemistry. Org. Lett. 2002, 4, 843−846. (c) Henry-dit-
Quesnel, A.; Toupet, L.; Pommelet, J. C.; Lequeux, T. A Difluorosulfide
as a Freon-Free Source of Phosphonodifluoromethyl Carbanion. Org.
Biomol. Chem. 2003, 1, 2486−2491. (d) Pfund, E.; Masson, S.; Vazeux,
M.; Lequeux, T. Syntheses of α-Fluoro-α,β-unsaturated Thioamides and
Thiazolines from a Fluorophosphonodithioacetate. J. Org. Chem. 2004,
69, 4670−4676. (e) Ozouf, P.; Binot, G.; Pommelet, J. P.; Lequeux, T.
Syntheses of ω-Hydroxy-α,α-difluoromethylphosphonates by Oxacycle
Ring-Opening Reactions. Org. Lett. 2004, 6, 3747−3750. (f) Pfund, E.;
́ ́
Chem. 1999, 42, 393−399. (b) Mathe, C.; Perigaud, C.; Gosselin, G.;
Imbach, J. L. Phosphopeptide Prodrug Bearing an S-Acyl-2-thioethyl
Enzyme-Labile Phosphate Protection. J. Org. Chem. 1998, 63, 8547−
8550. (c) Valette, G.; Pompon, A.; Girardet, J. L.; Cappellacci, L.;
́
Franchetti, P.; Grifantini, M.; La Colla, P.; Loi, A. G.; Perigaud, C.;
Gosselin, G.; Imbach, J. L. Decomposition Pathways and in Vitro
HIV Inhibitory Effects of IsoddA Pronucleotides: Toward a Rational
Approach for Intracellular Delivery of Nucleoside 5′-Monophos-
phates. J. Med. Chem. 1996, 39, 1981−1990. (d) Peyrottes, S.;
Coussot, G.; Lefebvre, I.; Imbach, J. L.; Gosselin, G.; Aubertin, A. M.;
́
Perigaud, C. S-Acyl-2-Thioethyl Aryl Phosphotriester Derivatives of
AZT: Synthesis, Antiviral Activity, and Stability Study. J. Med. Chem.
2003, 46, 782−793. (e) Egron, D.; Imbach, J. L.; Gosselin, G.;
Lequeux, T.; Masson, S.; Cordi, A.; Pierre, A.; Herve,
́
G.; Serre, V.
́
Aubertin, A. M.; Perigaud, C. S-Acyl-2-thioethyl Phosphoramidate
2767
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768

Journal of Medicinal Chemistry
Article
Diester Derivatives as Mononucleotide Prodrugs. J. Med. Chem. 2003,
46, 4564−4571.
(21) Kim, C. U.; Luh, B. Y.; Misco, P. F.; Bronson, J. J.; Hitchcock,
M. J. M.; Ghazzouli, I.; Martin, J. C. Acyclic Purine Phosphonate
Analogues as Antiviral Agents. Synthesis and Structure−Activity
Relationships. J. Med. Chem. 1990, 33, 1207−1213.
2768
dx.doi.org/10.1021/jm201694y | J. Med. Chem. 2012, 55, 2758−2768