Use of Pd-catalyzed Suzuki-Miyaura coupling reaction in the rapid synthesis of 5-aryl-6-(phosphonomethoxy)uracils and evaluation of their inhibitory effect towards human thymidine phosphorylase

Article abstract of DOI:10.1016/j.tet.2009.08.039

A number of new 5-aryl substituted pyrimidine acyclic nucleoside phosphonates were synthesized and tested for their ability to inhibit human TP. Their rapid synthesis using Pd-catalyzed Suzuki-Miyaura coupling reactions of various arylboronic acids with 5

Full text of DOI:10.1016/j.tet.2009.08.039

Tetrahedron 65 (2009) 8486–8492
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Use of Pd-catalyzed Suzuki–Miyaura coupling reaction in the rapid synthesis
of 5-aryl-6-(phosphonomethoxy)uracils and evaluation of their inhibitory
effect towards human thymidine phosphorylase
*
´
´
ˇ
´
Karel Pomeisl , Antonın Holy, Radek Pohl, Kvetoslava Horska
Gilead Sciences & IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
´
Centre for New Antivirals and Antineoplastics (IOCB), Flemingovo nam. 2, 166 10, Prague, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 April 2009
Received in revised form 28 July 2009
Accepted 13 August 2009
Available online 20 August 2009
A number of new 5-aryl substituted pyrimidine acyclic nucleoside phosphonates were synthesized and
tested for their ability to inhibit human TP. Their rapid synthesis using Pd-catalyzed Suzuki–Miyaura
coupling reactions of various arylboronic acids with 5-bromo-4-(phosphonomethoxy)-2,6-dibutoxy-
pyrimidine was successfully applied. For a series of 5-aryl-6-phosphonomethoxyuracils, an increased
inhibitory effect was determined. This effect is supported by the results found for 4-fluorophenyl
(Kdi Thd¼4.89ꢀ0.62) and 3-nitrophenyl (Kdi Thd¼3.98ꢀ0.46) substituents.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Acyclic nucleoside phosphonates
Suzuki–Miyaura reaction
Thymidine phosphorylase
Human thymidine phosphorylase
SD-lymphoma
Suzuki coupling
Uracil
Pyrimidine
1. Introduction
In our study, we have focused on the development of synthetic
methods for the rapid preparation of novel ANPs, which were further
Acyclic nucleoside phosphonates (ANPs) are known as com-
pounds possessing various kinds of biological activities.1 Currently,
the biological effects for pyrimidine ANP derivatives could be also
extended in context of their potential inhibitory potency towards
thymidine phosphorylase (TP).2,3 This enzyme catalyzes reversible
phosphorolysis of 20-deoxythymidine (dThd) to thymine and
tested for their ability to inhibit human TP. Recently, we published
a series of ANPs, such as 1-[2-(phosphonomethoxy)ethyl]thy-
mine (PMET),1-[3-hydroxy-2-(phosphonomethoxy)propyl]thymine
(HPMPT) and 1-[3-fluoro-2-(phosphonomethoxy)propyl]thymine
(FPMPT) and many others,6–9 which possess
a considerable
inhibitory effect newly tested on TP isolated from SD-lymphoma.6
Unfortunately, these compounds showed a marginal activity on
human enzymes expressed in V79 hamster cells as well as the
enzyme isolated from human placenta. It seems the differences in
recognition of active sites of both enzymes are probably appreciable
and these findings may therefore exclude the utilization of TP from
SD-lymphoma as a model enzyme.
2-deoxy-
D
-ribose 1-phosphate,4 which is dephosphorylated to
-ribose. Regarding the identical principle of TP to plate-
2-deoxy-
D
let-derived endothelial cell growth factor (PD-ECGF), a potential
inhibition of this enzyme may be important in tumour angiogen-
esis.5 For ANPs designed as ‘multisubstrate inhibitors’ of TP with
various interfered pyrimidine bases and phosphonoalkyl groups at
thymine and phosphate-binding sites2 there are several advantages
compared to most single-substrate inhibitors. These compounds
are catabolically stable and flexible in structure and there could be
more points for interactions with enzyme through their function-
alized acyclic linkage. Therefore, ANP inhibitors may find utility as
efficient suppressors of tumour growth5c in future.
In contrast, it is known that various 5-alkyl and 5-aryl-6-halo-
geno substituted uracils10 as single-substrate inhibitors demon-
strate a considerable impact on the inhibitory activity towards
human TP probably due to their potential hydrophobic interaction
of alkyl and aryl substituents directed by the halogen electron-
withdrawing effect (Fig. 1a).
On the other hand, [5-chloro-6-(2-iminopyrrolidin-1-yl)methyl-
2,4-(1H,3H)-pyrimidine] (TPI)11 represents the most efficient in-
hibitor towards required TP (Fig. 1b). However, its interaction with
active site of enzyme is probably energetically favoured through the
* Corresponding author. Tel.: þ420 220 183 475; fax: þ420 220 183 560.
E-mail address: pomeisl@uochb.cas.cz (K. Pomeisl).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.08.039

K. Pomeisl et al. / Tetrahedron 65 (2009) 8486–8492
8487
O
-4-yl. Thus we have started the synthesis of 5-aryl-6-(phosphono-
methoxy)uracils 8a–i as a new class of ANPs aimed at positive
influence of inhibitory effect towards human TP (Fig. 1c; Table 1).
(a)
(b)
O
Cl
N
R
HN
HN
O
N
H
O
N
H
Cl
NH
2. Results and discussion
R =
R =
R = Ph
For the efficient arylation method of uracil moiety in some
pyrimidine ANPs we utilized Suzuki–Miyaura cross-coupling,13,14
which have generally been used widely due to their low-toxicity. As
the arylation of pyrimidine ANPs has not been studied in detail via
this process14 we decided to introduce various aryl and heteroaryl
groups to pyrimidine ANP derivatives using this synthetic method.
As indicated in our preliminary communication,15 we have
successfully employed the Suzuki–Miyaura cross-coupling for the
preparation of N1-substituted 5-arylpyrimidine ANPs 9a–i (Fig. 1d;
Table 1). We have extended this work by studying the arylation of
a 5-bromo uracil derivative 5 (Scheme 1). In addition, we have
introduced various functionalized aryl groups.
TPI
O
(c)
(d)
O
Ar
O
Ar
HN
HN
O
N
O
N
H
O
P(O)(OH)2
P(O)(OH)2
9
8
Figure 1. Pyrimidine derivatives as investigated inhibitors of human TP.
solvatation of its positively charged imino group with phosphate
and/or H2O located near the phosphate binding site. Thus activated
TPI may be similar to the thymidine cleavage.12 From this point of
view, a mechanism of inhibitor binding with TP may be explained by
several ways depending on their structure.2,12 Based on this pro-
posal, our strategy was to mimic a potential solvatation of TPI in
conjunction with hydrophobic effect expected in those of 5-alkyl
and 5-aryl-6-chlorouracils. That means we tried to introduce a cat-
abolically stable phosphonomethoxy group to C-6-position and
various aryl groups bearing electron-withdrawing substituents,
heteroatoms or other conjugated moieties to the position C-5 of
pyrimidine base such as phenyl, naphth-1-yl, (E)-2-(phenyl)vinyl,
4-fluorophenyl, 3-nitrophenyl, 3-furyl, 3-thienyl, pyridin-3-yl and
Ot-Bu
Cl
Ot-Bu
(b)
(a)
N
N
N
t-BuO
N
Cl
O
Cl
N
Cl
t-BuO
N
2
4
1
P(O)(Oi-Pr)2
(c)
6a, (d)
or
6c-i, (e)
Ot-Bu
Ot-Bu
Br
R
N
N
O
O
t-BuO
N
t-BuO
N
P(O)(Oi-Pr)2
P(O)(Oi-Pr)2
7a, 7c-i
5
Table 1
Suzuki-coupling reaction and deprotection for 5-aryl-C-6-subtituted uracil ANPs
(f)
6a or 6b
(e)
Entry
R (Ar)b
Solvent
Reaction Yielda of 7 (%) Yield of 8 (%)
time (h)
O
4
R
O
HN
a
b
c
Toluene
23
45
d
33
d
S
(f)
O
N
H
Toluene (DMF) 12 (7)
P(O)(OH)2
O
O
8a, 8c-h
H
O
HN
DMF
DMF
DMF
6
6
4
77
72
66
23
53
68
O
N
H
P(O)(OH)2
d
e
F
8j
R = a-i see Table 1
O2N
Scheme 1. Reagents and conditions: (a) t-BuONa (2 equiv), THF, 0 ꢁC/reflux, 45%;
(b) NaH, HOCH2P(O)(Oi-Pr)2 (3), THF, 0 ꢁC/rt, 39%; (c) NBS, AIBN, THF, 60 ꢁC, 99%;
(d) Pd(PPh3)4 (0.10 equiv), Na2CO3, toluene, H2O, 130 ꢁC, 45%; (e) Pd(PPh3)4
(0.10 equiv), Na2CO3, DMF, H2O, 130 ꢁC, 43–78%; (f) (CH3)3SiBr, CH3CN, rt, 0 ꢁC, 23–68%.
f
DMF
DMF
4.5
4
32
78
61
29
First we prepared the 5-bromo derivative 5 by a three-step syn-
thesis from commercially available 2,4,6-trichloropyrimidine 1.
In this case, we selectively protected 1 by reaction with 2 equiv of
sodium tert-butoxide to give chloro derivative 2. In a further reaction
step, we chose hydroxymethylphosphonate 3 as a simple phospho-
nate building block with the assumption that the final structure of 8
could largely mimic a leading structure of TPI in interaction with
additional phosphate or water, respectively.12 The reaction of chloro
derivative 2 with 3 converted quantitatively in the presence of
sodium hydride into phosphonate 4 (monitoring by TLC). In contrast
to a previous method of bromination,15 we left a deprotection of
methoxy groups in uracil moiety regarding to possible difficulty with
unfavourable cleavage of phosphonomethoxy substituents. Instead,
g
N
h
i
DMF
5.5
43
57
N
DMF
6
25
d
j
H
d
d
d
58
a
Conditions: 5 (1.0 equiv), 6 (2 equiv), Pd catalyst (0.1 equiv), base (3.3–4.7 equiv
of Na2CO3), 130 ꢁC; solvent/H2O 1:6 (entries a and b) and 8:1 (entries c–i).
b
Aryl subtituents of ANPs 9a–i correspond to entries a–i. The yields of these
compounds are mentioned in Ref. 15.

8488
K. Pomeisl et al. / Tetrahedron 65 (2009) 8486–8492
we brominated C-5-position of the pyrimidine moiety in 4 with
N-bromosuccinimide. Similarly to previous procedure with
inhibitory activity observed for our 5-aryluracil phosphonates
showed lower order values in comparisons, e.g., with most effective
5-chloro-6-cyclopentenyluracil (Ki¼0.20ꢀ0.03) and 6-chloro-5-
phenyluracil (Ki¼0.40ꢀ0.04).10
a
N1-substituted phosphonates, this reaction was initiated with azo-
bisisobutyronitrile and afforded in quantitative yield the 5-bromo-
pyrimidine derivative 5. For coupling reactions we applied a number
of commercial aryl and heteroaryl boronic acids 6a–i. Simple trans-
formation of 5-bromo derivative 5 to aryl derivatives 7a–i took place
in DMF–H2O solution catalyzed by Pd(PPh3)4. Sodium carbonate was
used for activation of the arylboronic acids. Full conversion of 5 to
products proceeded in all cases at w130 ꢁC (monitored by TLC).
However, the application of Suzuki-coupling reaction conditions
to bromo derivative 5 results in some different observations. While
N1-phosphonoalkyl-5-aryluracils as products of arylation were
isolated in 24–58% yields (see Ref.15), the reaction of 5 with a series
of arylboronic acids 6c–i afforded phosphonates 7c–i with higher
preparative yields (43–78%) (Table 1). In addition, we have found
that 2-furyl and 3-thienyl boronic acids 6a and 6b surprisingly
reacted with 5 to give no arylated product. Instead, the major
product 4 of dehalogenation16 in DMF was isolated in good pre-
parative yield. This competitive dehalogenation may proceed in
consequence of energetically unfavourable transmetallation16,17
due to possible steric hindrance of bromo derivative 5. Further-
more, arylboronic acids are disposed to hydrolysis17 in the presence
of base. Therefore, all these factors such as shielding and other
electronic effects of heteroaryl substituents could play an impor-
tant role during those of rate-determining step. The poor reactivity
of thienylboronic acid 6b with 5 confirmed by its treatment in
benzene. After extending the reaction time (14 h), we isolated
a mixture of 5/7a/4 in a ratio 13.6:1 (monitoring by NMR spec-
troscopy). Based on this observation, we carried out the arylation in
toluene at temperature (w130 ꢁC of bath) for an additional 23 h. For
this reaction the hydrodehalogenated product 4 was formed as the
minor component, and the ratio of 5/7a/4¼1:6:1. The correspond-
ing thienyl derivative 7a was isolated in acceptable yield (45%) after
difficulties during purification by preparative TLC followed by
HPLC. In contrast, the reaction of 5 with 2-furylboronic acid did not
proceed in toluene (see Table 1, entry b).
Table 2
Comparison of the inhibitory effect on human TP for C-6-subtituted 5-arylpyr-
imidine ANPs
Entry
R
Inhibition of human TP expressed in V79
Compound 8
a
a
Kdi Thd
(
m
mol Lꢂ1
)
KPi i mol Lꢂ1
(m )
d
4.89ꢀ0.62
3.98ꢀ0.46
5.55ꢀ0.67
F
O2N
e
2.43ꢀ0.33
a
The Km (thymidine) value for V79TP was 124ꢀ23
m
mol Lꢂ1 and 83ꢀ12
m
mol Lꢂ1
for phosphate as a substrate.
In contrast to the above-mentioned 5-alkyl(aryl)-6-chlorour-
acils and other single-substrate inhibitors, the inhibitory potency of
our compounds was carried out with respect to the both substrates
20-deoxythymidine and inorganic phosphate. Our data determined
by KPi i for compounds 8d and 8e also demonstrate a possible
competition of phosphonate unity with inorganic phosphate (Table
2). It may be consequently postulated that these compounds could
resemble the multisubstrate character of previously reported ANP
inhibitors towards TP from Escherichia coli, such as 1-(8-phospho-
nooctyl)-6-amino-5-bromouracil (TP-64) and 1-(8-phosphonooc-
tyl)-7-deazaxanthine (TP-65).3
Despite of the in vitro activity, none of the presented com-
pounds (c¼10
m
mol Lꢂ1) did not exhibit a significant cytostatic ac-
tivity in tissue cultures estimated in mouse lymphocytic leukaemia
L1210 cells (ATCC CCL 219), CCRF-CEM T lymphoblastoid cells
(human acute lymphoblastic leukaemia, ATCC CCL 119), human
promyelocytic leukaemia HL-60 cells (ATCC CCL 240) and human
cervix carcinoma HeLa S3 cells (ATCC CCL 2.2).
Intermediates 4 and 7a, 7c–i obtained by cross-coupling were
deprotected using bromotrimethylsilane followed by hydrolysis at
low temperature (0 ꢁC). Thus, we expected the deprotection of tert-
butoxy groups under mild conditions could avoid a cleavage of ether
group at C-6-position. While a deprotection of phosphonate 7i
afforded a complicated mixture of non-identified compounds and
final phosphonates 8a, 8c, 8d, 8g, 8h were obtained in lower yields
by this hydrolysis; these preparative values are comparable to cor-
responding N1-substituted uracil derivatives 9a–i (see Ref. 15).
The determination of inhibitory activity in all final compounds
was performed with TP isolated from human enzyme expressed in
V79 Chinese hamster cells. Data obtained from the biochemical
assay did not show any significant effect of phosphonates 8f–j and
9a–i by comparison with FPMPT, HPMPT, PMETand their previously
synthesized analogues,7–9 we have not found any influence of their
aryl substituents on the inhibitory activity.
On the other hand, a series of 5-aryl-6-phosphonomethoxy-
uracils 8c–e exhibits an increase in inhibitory activity against the
enzyme. This effect seems to be supported for 3-nitrophenyl and 4-
fluorophenyl groups (Table 2, compounds 8d and 8e). In this case,
we suppose that a potential hydrophobic effect could be slightly
influenced with electron-withdrawing groups as well as this effect
is probably induced in 5-(alkyl)aryl-6-chlorouracils possessing
a halogen at the C-6-position. The inhibitory effect determined by
Kdi Thd for these two inhibitors with respect to 20-deoxythymidine
was comparable with most of 5-alkyl and 5-aryl-6-chlorouracils
(Kw1.03–5.81),10 5-halo 6-aminouracils (Kiw3.8–11.6)18 and re-
cently published sugar-containing phosphonates mimicking a lead
structure of thymidine (Kiw1.05 and w8.03).19 However, the
3. Conclusions
In summary, we have developed a simple method for the
preparation of new 5-aryl-6-(phosphonomethoxy)uracils for bio-
chemical study bearing some functionalized aryl substituents. In
comparison with N1-substituted uracil ANPs, this arylation
method afforded higher yields of 5-aryluracil derivatives in DMF
and this is limited for thienyl and furyl groups due to unfavourable
hydrodehalogenation. In some cases, we have found that an
incorporation of
a phosphonoalkyl group to C-6-position of
5-aryluracil moiety results in an increase of inhibitory activity
towards human TP. This inhibitory effect is supported for 4-fluo-
rophenyl and 3-nitrophenyl groups. From this point of view, this
new class of uracil ANPs could be promising for other structural
modifications.
4. Experimental
4.1. General comments
Unless stated otherwise, solvents were evaporated at 40 ꢁC/
0.5–2 kPa and compounds were dried at 50 ꢁC/13 Pa. Melting points
were determined on a Kofler block and are uncorrected. IR spectra
were recorded on an FTIR spectrometer Bruker IFS 55 (Equinox) in
KBr and CCl4. Analytical TLC was carried out on Silufol UV254 plates
(Kavalier Votice, Czech Republic). Preparative TLC was carried out on

K. Pomeisl et al. / Tetrahedron 65 (2009) 8486–8492
8489
45ꢃ18ꢃ0.4 cm loose-layer silica gel containing UV indicator (system
was washed with water (20 mL). Organic layer was dried with
magnesium sulfate, filtered and evaporated in vacuo. The residue
was chromatographed on silica gel (chloroform–light petroleum
1:1 followed by 5:1). Yield 5.1 g (45%) of colourless oil; nmax (CCl4)
1575, 1463, 1546, 1478, 1435, 1398, 1388, 1366; dH (400.1 MHz,
CDCl3) 1.60 and 1.61 (2ꢃs, 2ꢃ9H, (CH3)3C), 6,26 (s, 1H, H-5); dC
(100.6 MHz, CDCl3) 28.3 and 28.4 ((CH3)3C), 81.4 and 82.2
(C(CH3)3), 102.2 (CH-5), 160.6 (C-6), 163.5 (C-2), 171.2 (C-4). ESI-
MS m/z 579 [MHþNaþK]þ (100). Anal. Calcd for C12H19ClN2O2: C,
55.70; H, 7.40; N, 10.83. Found: C, 55.67; H, 7.32; N, 10.61.
S1). Reversed-phase HPLC was performed on a Waters Delta 600;
phenomenexÒ, Luna, preparative C18 column 10
mm 21.20ꢃ250 mm,
gradient 0-8 min in 0–50% MeOH–H2O (system S2). Purification of
phosphonates 8a and 8c–h was performed on Sephadex A-25-120
DEAE (Clꢂ form, activated with 0.02M triethylammonium hydro-
gencarbonate) and eluted in 0–0.4M triethylammonium hydro-
gencarbonate buffer (system S3). NMR spectra were recorded on
a Bruker Avance 600, Bruker Avance 500 or Bruker Avance 400 (1H at
600.1, 500.0, 499.8 or 400.1 MHz, 13C at 150.9, 125.7 or 100.6 MHz,
31P at 202.3 or 162.0 and 19F at 470.3 MHz) in CDCl3 (referenced to
TMS), DMSO (referenced to residual solvent signal; 2.50 ppm for 1H
and 39.70 ppm for 13C) or in D2O solutions with dioxane20 as an
4.5. 2,6-Di-tert-butyl-4-[(diisopropoxyphosphoryl)-
methoxy]pyrimidine (4)
internal standard (3.75 ppm for 1H and 69.3 ppm for 13C). 31P and 19
F
NMR spectra were referenced to the signal of H3PO4 (0 ppm) and
hexafluorobenzene (ꢂ163 ppm), respectively, which were used as an
internal standards or as external standards in coaxial 2 mm capillary
A solution of hydroxymethylphosphonate 3 (6 g, 31.4 mmol) in
dioxane (80 mL) was stirred at 10 ꢁC and 60% sodium hydride
(1.2 g, 31.4 mmol) was added. The mixture was allowed to warm to
80 ꢁC and chloropyrimidine 2 (2.6 g, 10 mmol) was added. The
mixture was vigorously refluxed for 14 h until the conversion of 2
finished. The mixture was concentrated in vacuo to a minimum
volume and diluted with chloroform (80 mL). The mixture was
washed with water (10 mL). Organic layer was dried with mag-
nesium sulfate and evaporated in vacuo. The residue was chro-
matographed on silica gel (chloroform–light petroleum 4:1)
followed by ethyl acetate–chloroform 1:1. Yield 1.63 g (39%) of
slightly yellow oil; nmax (CCl4) 2981, 1592, 1586, 1459, 1398, 1365,
1380, 1259, 1173, 1161, 1108, 1009, 990, 890, 546; dH (400.1 MHz,
CDCl3) 1.32 and 1.35 (2ꢃd, 2ꢃ6H, Jvic¼6.2, (CH3)2CH), 1.57 and 1.61
(2ꢃs, 2ꢃ9H, (CH3)3C), 4.55 (d, 2H, JH,P¼8.7, CH2P), 4.79 (dh, 2H,
JH,P¼7.6, Jvic¼6.2, CH(CH3)2), 5.71 (s, 1H, H-5); dC (100.6 MHz,
CDCl3) 23.9 (d, JC,P¼4.7, (CH3)2CH), 24.1 (d, JC,P¼3.9, (CH3)2CH), 28.5
and 28.7 ((CH3)3C), 59.8 (d, JC,P¼172.0, CH2P), 71.5 (d, JC,P¼6.5,
CH(CH3)2), 80.4 and 80.9 (C(CH3)3), 86.6 (CH-5), 163.2 (C-2), 170.9
(d, JC,P¼12.5, C-6), 172.0 (C-4). FABMS m/z 419 [MH]þ (38); HRMS
(FAB) calcd for C19H36N2O6P 419.2311, found 419.2316. Anal. Calcd
for C19H35N2O6P: C, 54.53; H, 8.43; N, 6.69; P, 7.40. Found: C, 54.60;
H, 8.46; N, 6.41; P, 7.27.
tube. Chemical shifts (d, ppm) and coupling constants (J, Hz) were
obtained by first-order analysis of the spectra. Mass spectra were
measured on a ZAB-EQ (VG Analytical) spectrometer using FAB
(Xe ionization, accelerating voltage 8 kV, glycerol matrix) and Q-Tof
micro (Waters, Milford, MA, USA) equipped with an ion source
for atmospheric pressure chemical ionization (APCI probe temper-
ature 400 ꢁC, sample cone voltage 30 V, corona discharge current
5.0 mA).
4.2. Materials and chemicals
Standard chemicals, ion-exchange resin (Dowex 50WX8-200)
and activated charcoal were purchased from Sigma–Aldrich (Czech
Republic). Ion-exchange resin (Sephadex A-25-120 DEAE) was
purchased from Fluka. Dimethylformamide, tetrahydrofuran and
acetonitrile were dried by distillation from calcium hydride. Thy-
midine phosphorylase was used from human, expressed in V79
Chinese hamster cells (0.41 mU, Sigma T 9319). The phosphonates
9a–i were prepared by our method reported in Ref. 15 (see
Supplementary data).
4.3. Enzyme assay
4.6. 5-Bromo-2,6-di-tert-butyl-4-[(diisopropoxyphosphoryl)-
methoxy]pyrimidine (5)
The reaction mixture (50
20 mM bisTris–HCl pH 6.4, 1 mM EDTA and 2 mM DTT, 200
m
L, Kdi Thd estimation) contained
m
M
A mixture of compound 4 (2.38 g, 7.12 mmol), N-bromosucci-
nimide (1.28 g, 7.19 mmol) and azobisisobutyronitrile (30 mg,
0.18 mmol) in THF (30 mL) was heated at 60 ꢁC for 10 h. The
mixture was concentrated in vacuo to a minimum volume. The
residue was purified by chromatography on silica gel (chloroform–
methanol 19:1) to give 2.9 g (99% yield) of 5 as a colourless oil;
nmax (CCl4) 1576, 1550, 1478, 1416, 1397, 1366, 1259, 1177, 1136,
1107, 1006, 987; dH (400.1 MHz, CDCl3) 1.35 and 1.36 (2ꢃd, 2ꢃ6H,
Jvic¼6.2, (CH3)2CH), 1.61 and 1.62 (2ꢃs, 2ꢃ9H, (CH3)3C), 4.63 (d, 2H,
JH,P¼8.5, CH2P), 4.85 (dh, 2H, JH,P¼7.4, Jvic¼6.2, CH(CH3)2); dC
(100.6 MHz, CDCl3) 23.9 (d, JC,P¼4.8, (CH3)2CH), 24.0 (d, JC,P¼3.8,
(CH3)2CH), 28.3 and 28.5 ((CH3)3C), 60.5 (d, JC,P¼171.1, CH2P), 72.2
(d, JC,P¼6.6, CH(CH3)2), 80.9 (C(CH3)3), 82.3 (C-5), 82.9 (C(CH3)3),
161.2 (C-2), 166.0 (d, JC,P¼11.6, C-6), 167.3 (C-4). FABMS m/z 497
[MH]þ (38); HRMS (FAB) calcd for C19H35BrN2O6P 497.1416, found
497.1412. Anal. Calcd for C19H34BrN2O6P: C, 45.88; H, 6.89; Br,
16.07; N, 5.63; P, 6.23. Found: C, 45.86; H, 7.08; Br, 16.08; N, 5.41; P,
6.41.
potassium phosphate pH 6.7, various concentrations of [3H-
methyl]thymidine and tested compounds. KPi i was determined in
the presence of 200 mM [3H-methyl]thymidine and various con-
centrations of phosphate. The reaction was started by the addition
of 44 nU of enzyme, incubated at 37 ꢁC for 8 min and stopped by
spotting a 2
prespotted with 0.01
m
L aliquot onto Silica gel 60 F254 plate that had been
mol of each thymine and thymidine. The
m
plate was developed in the non-aqueous phase of the solvent
system ethyl acetate–water–formic acid (60:35:5). The spots were
visualized under UV light (254 nm) and cut out for radioactivity
determination in the toluene-based scintillation cocktail (4 g
PPOþ0.2 g POPOP/1 L toluene (Sigma)). Kinetic constants Km and
Ki were determined from the Lineweaver–Burk and Dixon plots.
Data based on results from at least four independent experiments
were evaluated by the non-linear regression method using soft-
ware GOSA, Bio-Log, France.
4.4. 2,6-Di-tert-butyl-4-chloropyrimidine (2)
4.7. 2,6-di-tert-Butyl-4-[(diisopropoxyphosphoryl)methoxy]-
A solution of trichloropyrimidine 1 (8 g, 43.6 mmol) in THF
(50 mL) was stirred at 0 ꢁC and sodium tert-butoxide powder (8.38 g,
87.2 mmol) was added. The mixture was allowed to warm to rt and
stirred for 14 h. The mixture was concentrated in vacuo to a mini-
mum volume and diluted with chloroform (100 mL). The mixture
5-thienylpyrimidine (7a)
A mixture of compound 5 (151 mg, 0.30 mmol), arylboronic acid
6a (180 mg, 1.41 mmol), Pd(PPh3)4 (97 mg, 0.08 mmol) and sodium
carbonate (112 mg, 1.06 mmol) in toluene (1 mL) and degassed H2O

8490
K. Pomeisl et al. / Tetrahedron 65 (2009) 8486–8492
(6 mL) was heated (w130 ꢁC, oil bath) under argon for 23 h. The
mixture was then concentrated and codistilled with toluene
(2ꢃ20 mL). The residue was diluted with EtOAc (20 mL) and
washed with aqueous EDTA saturated with NH4Cl (5 mL). The or-
ganic layer was dried over anhydrous MgSO4, filtered and con-
centrated. The residue was purified by preparative TLC on silica gel
(S1) in toluene–EtOAC 1:1 followed by HPLC (S2). Yield 69 mg (45%)
as a colourless oil; nmax (CCl4) 2981, 1593, 1553, 1528, 1478, 1457,
1428, 1400, 1385, 1365, 1253, 1177, 1168, 1132, 1105, 1009, 988, 890,
847, 465; dH (500.0 MHz, CDCl3) 1.14 and 1.22 (2ꢃd, 2ꢃ6H, Jvic¼6.2,
(CH3)2CH), 1.54 and 1.57 (2ꢃs, 2ꢃ9H, (CH3)3C), 4.53 (d, 2H, JH,P¼8.3,
CH2P), 4.64 (dh, 2H, JH,P¼7.6, Jvic¼6.2, CH(CH3)2), 7.16 (dd, 1H,
J5,4¼5.0, J5,2¼3.1, H-5-thienyl), 7.39 (dd, 1H, J4,5¼5.0, J4,2¼1.3, H-4-
thienyl), 7.52 (dd, 1H, J2,5¼3.1, J2,4¼1.3, H-2-thienyl); dC (125.7 MHz,
CDCl3) 23.8 (d, JC,P¼4.6, (CH3)2CH), 24.1 (d, JC,P¼3.5, (CH3)2CH), 28.5
and 28.7 ((CH3)3C), 60.1 (d, JC,P¼170.7, CH2P), 71.5 (d, JC,P¼6.4,
CH(CH3)2), 80.4 and 81.9 (C(CH3)3), 96.3 (C-5), 122.5 (CH-5-thienyl),
124.5 (CH-2-thienyl), 130 (CH-4-thienyl), 130.5 (C-3-thienyl), 160.8
(C-2), 166.8 (d, JC,P¼11.7, C-6), 167.9 (C-4). ESI-MS m/z 523 [MNa]þ
(100); HRMS (ESI) calcd for C23H37N2NaO6PS 523.2002, found
523.2002.
1478, 1428, 1420, 1405, 1392, 1386, 1378, 1365, 1254, 1233, 1106,
1009, 1002, 989; dH (400.1 MHz, CDCl3) 1.13 and 1.26 (2ꢃd, 2ꢃ6H,
Jvic¼6.2, (CH3)2CH), 1.53 and 1.65 (2ꢃs, 2ꢃ9H, (CH3)3C), 4.56 (d, 2H,
JH,P¼8.3, CH2P), 4.61 (dh, 2H, JH,P¼7.5, Jvic¼6.2, CH(CH3)2), 7.01 (m,
2H, H-m-C6H4F), 7.36 (m, 2H, H-o-C6H4F); dC (100.6 MHz, CDCl3)
23.7 (d, JC,P¼4.8, (CH3)2CH), 24.0 (d, JC,P¼3.7, (CH3)2CH), 28.5 and
28.6 ((CH3)3C), 60.1 (d, JC,P¼170.7, CH2P), 71.4 (d, JC,P¼6.5,
CH(CH3)2), 80.5 and 81.6 (C(CH3)3), 99.9 (C-5), 114.2 (d, JC,F¼21.3,
CH-m-C6H4F), 127.6 (d, JC,F¼3.4, C-i-C6H4F), 132.5 (d, JC,F¼7.9, CH-o-
C6H4F), 161.5 (C-2), 161.6 (d, JC,F¼245.2, C-p-C6H4F), 167.0 (d,
JC,P¼11.6, C-6), 168.3 (C-4). FABMS m/z 513 [MH]þ (12); HRMS (FAB)
calcd for C25H39FN2O6P 513.2530, found 513.2520.
4.8.4. 2,6-Di-tert-butyl-4-[(diisopropoxyphosphoryl)methoxy]-5-(3-
nitrophenyl) pyrimidine (7e). Column chromatography was per-
formed in toluene–ethyl acetate 1:1. Yield 357 mg (66%) as a yellow
oil; nmax (CCl4) 1594, 1577, 1549, 1533, 1480, 1437, 1428, 1405, 1394,
1381, 1366, 1349, 1254, 1106, 1008, 990; dH (600.1 MHz, CDCl3) 1.15
and 1.23 (2ꢃd, 2ꢃ6H, Jvic¼6.2, (CH3)2CH), 1.56 and 1.66 (2ꢃs, 2ꢃ9H,
(CH3)3C), 4.59 (d, 2H, JH,P¼8.3, CH2P), 4.63 (dh, 2H, JH,P¼7.6, Jvic¼6.2,
CH(CH3)2), 7.50 (dd, 1H, J5,4¼8.3, J5,6¼7.7, H-5-C6H4NO2), 7.81 (ddd,
1H, J6,5¼7.7, J6.2¼1.6, J6,4¼1.1, H-6-C6H4NO2), 8.10 (ddd, 1H, J4,5¼8.3,
J4,2¼2.4, J4,6¼1.1, H-4-C6H4NO2), 8.33 (dd, 1H, J2,4¼2.4, J2,6¼1.6, H-2-
C6H4NO2); dC (150.9 MHz, CDCl3) 23.7 (d, JC,P¼4.6, (CH3)2CH), 23.9
(d, JC,P¼3.7, (CH3)2CH), 28.5 and 28.6 ((CH3)3C), 60.2 (d, JC,P¼170.6,
CH2P), 71.4 (d, JC,P¼6.5, CH(CH3)2), 81.0 and 82.4 (C(CH3)3), 98.3
(C-5), 121.4 (CH-4-C6H4NO2), 125.9 (CH-2-C6H4NO2), 128.1 (CH-5-
C6H4NO2), 133.7 (C-1-C6H4NO2), 137.7 (CH-6-C6H4NO2), 147.6 (C-3-
C6H4NO2), 162.0 (C-2), 166.9 (d, JC,P¼11.2, C-6), 168.2 (C-4). FABMS
m/z 540 [MH]þ (12); HRMS (FAB) calcd for C25H38N3O8P 540.2475,
found 540.2480.
4.8. Pd-catalyzed Suzuki–Miyaura coupling reactions of 5-
bromopyrimidine 5 with boronic acids (compounds
4 and 7c–i). General procedure
A mixture of compound 5 (1 mmol), arylboronic acid 6a–i
(2 mmol), Pd(PPh3)4 (116 mg, 0.10 mmol), sodium carbonate
(352 mg, 3.32 mmol) in DMF (20 mL) and degassed H2O (4 mL) was
heated (w130 ꢁC, oil bath) under argon for 4–6 h (see Table 1). The
mixture was then concentrated and codistilled with toluene
(2ꢃ20 mL). The residue was diluted with EtOAc (20 mL) and washed
with aqueous EDTA saturated with NH4Cl (5 mL). The organic layer
was dried over anhydrous MgSO4, filtered and concentrated. The
residue was purified by preparative TLC on silica gel (S1) in below
mentioned systems (see Sections 4.8.1–4.8.8) to give compounds 4
and 7c–i.
4.8.5. 2,6-Di-tert-butyl-4-[(diisopropoxyphosphoryl)methoxy]-5-(1-
naphthyl)pyrimidine (7f). Column chromatography was performed
in toluene–ethyl acetate 1:1. Yield 176 mg (32%) as a yellow oil; nmax
(CCl4) 1602,1589,1553,1509,1478,1415,1393,1385,1365,1256,1007,
987; dH (600.1 MHz, CDCl3) 0.82, 0.90, 0.96 and 1.01 (4ꢃd, 2ꢃ3H,
Jvic¼6.2, (CH3)2CH), 1.38 and 1.70 (2ꢃs, 2ꢃ9H, (CH3)3C), 4.24 (m, 2H,
CH(CH3)2), 4.54 and 4.57 (2ꢃdd, 2H, Jgem¼14.2, JH,P¼8.4, CH2P), 7.35
(dd, 1H, J2,3¼7.0, J2,4¼1.3, H-2-naphth), 7.39 (ddd, 1H, J7,8¼8.3,
J7,6¼6.8, J7,5¼1.5, H-7-naphth), 7.43 (ddd, 1H, J6,5¼8.1, J6,7¼6.8,
J6,8¼1.4, H-6-naphth), 7.46 (dd, 1H, J3,4¼8.2, J3,2¼7.0, H-3-naphth),
7.54 (m, 1H, J8,7¼8.3, J8,6¼1.4, J8,4¼1.0, J8,5¼0.7, H-8-naphth), 7.78
(ddd, 1H, J4,3¼8.2, J4,2¼1.3, J4,8¼1.0, H-4-naphth), 7.83 (ddd, 1H,
J5,6¼8.1, J5,7¼1.5, J5,8¼0.7, H-5-naphth); dC (150.9 MHz, CDCl3) 23.4
and 23.5 (d, JC,P¼4.5, (CH3)2CH), 23.73 and 23.79 (d, JC,P¼3.7,
(CH3)2CH), 28.5 and 28.6 ((CH3)3C), 60.1 (d, JC,P¼168.9, CH2P), 71.4
and 71.5 (d, JC,P¼6.5, CH(CH3)2), 80.5 and 81.3 (C(CH3)3), 98.9 (C-5),
125.2 (CH-3-naphth), 125.3 (CH-6-naphth), 125.4 (CH-7-naphth),
125.9 (CH-8-naphth), 127.5 (CH-4-naphth), 128.0 (CH-5-naphth),
128.6 (CH-2-naphth),130.0 (C-1-naphth),132.4 (C-8a-naphth),133.5
(C-4a-naphth), 162.3 (C-2), 167.9 (d, JC,P¼11.2, C-6), 169.2 (C-4).
FABMS m/z 545 [MH]þ (15); HRMS (FAB) calcd for C29H42N2O6P
545.2781, found 545.2773.
4.8.1. 2,6-Di-tert-butyl-4-[(diisopropoxyphosphoryl)methoxy]pyri-
midine (4). (a) The reaction of 5 with boronic acid 6a afforded
187 mg (42%) of pyrimidine 4; (b) the reaction of 5 with boronic
acid 6b afforded 236 mg (57%) of pyrimidine 4. 1H NMR data were
identical with those of compound prepared in Section 4.5.
4.8.2. 2,6-Di-tert-butyl-4-[(diisopropoxyphosphoryl)methoxy]-5-
phenylpyrimidine (7c). Column chromatography was performed in
toluene–ethyl acetate 1:1. Yield 383 mg (77%) as a colourless oil;
nmax (CCl4) 1602, 1593, 1579, 1550, 1478, 1458, 1426, 1406, 1393,
1382, 1365, 1255, 1177, 1169, 1134, 1106, 1007, 995, 988, 848, 463; dH
(500.0 MHz, CDCl3) 1.12 and 1.21 (2ꢃd, 2ꢃ6H, Jvic¼6.2, (CH3)2CH),
1.53 and 1.65 (2ꢃs, 2ꢃ9H, (CH3)3C), 4.57 (d, 2H, JH,P¼8.3, CH2P),
4.58 (dh, 2H, JH,P¼7.6, Jvic¼6.2, CH(CH3)2), 7.22 (m, 1H, H-p-Ph), 7.31
(m, 2H, H-m-Ph), 7.38 (m, 2H, H-o-Ph); dC (125.7 MHz, CDCl3) 23.7
(d, JC,P¼4.7, (CH3)2CH), 24.0 (d, JC,P¼3.7, (CH3)2CH), 28.5 and 28.6
((CH3)3C), 60.1 (d, JC,P¼170.1, CH2P), 71.5 (d, JC,P¼6.5, CH(CH3)2),
80.4 and 81.4 (C(CH3)3), 100.9 (C-5), 126.5 (CH-p-Ph), 127.3 (CH-m-
Ph), 130.9 (CH-o-Ph), 131.7 (C-i-Ph), 164.3 (C-2), 167.1 (d, JC,P¼11.0,
C-6), 168.3 (C-4). FABMS m/z 497 [MH]þ (12); HRMS (FAB) calcd for
C25H40N2O6P 495.2624, found 495.2636. Anal. Calcd for
C25H39N2O6P: C, 60.71; H, 7.95; N, 5.66; P, 6.26. Found: C, 60.28; H,
7.99; N, 5.50; P, 6.41.
4.8.6. 2,6-Di-tert-Butyl-4-[(diisopropoxyphosphoryl)methoxy]-5-
(pyridin-4-yl)pyrimidine (7g). Column chromatography was per-
formed in ethyl acetate–chloroform–methanol 21:20:1. Yield
385 mg (78%) as a slightly yellow oil; nmax (CCl4) 1599, 1585, 1549,
1478, 1425, 1403, 1366, 1254, 1106, 1008, 996, 986; dH (400.1 MHz,
CDCl3) 1.16 and 1.26 (2ꢃd, 2ꢃ6H, Jvic¼6.2, (CH3)2CH), 1.56 and 1.65
(2ꢃs, 2ꢃ9H, (CH3)3C), 4.57 (d, 2H, JH,P¼8.4, CH2P), 4.65 (dh, 2H,
JH,P¼7.6, Jvic¼6.2, CH(CH3)2), 7.40 (m, 2H, H-3,5-py), 8.55 (m, 2H, H-
2,6-py); dC (100.6 MHz, CDCl3) 23.7 (d, JC,P¼4.7, (CH3)2CH), 24.0 (d,
JC,P¼3.8, (CH3)2CH), 28.5 and 28.6 ((CH3)3C), 60.2 (d, JC,P¼171.0,
CH2P), 71.5 (d, JC,P¼6.5, CH(CH3)2); 80.9 and 82.3 (C(CH3)3); 98.0 (C-
4.8.3. 2,6-Di-tert-butyl-5-(4-fluorophenyl)-4-[(diisopropoxy-
phosphoryl)methoxy]-pyrimidine (7d). Column chromatography
was performed in toluene–ethyl acetate 1:1. Yield 370 mg (72%) as
a white amorphous solid; nmax (CCl4) 1611, 1592, 1551, 1514, 1451,

K. Pomeisl et al. / Tetrahedron 65 (2009) 8486–8492
8491
5); 125.3 (CH-3,5-py); 140.4 (C-4-py); 148.8 (CH-2,6-py); 162.1 (C-
2); 167.0 (d, JC,P¼11.3, C-6); 168.2 (C-4). FABMS m/z 496 [MH]þ (39);
HRMS (FAB) calcd for C24H39N3O6P 496.2577, found 496.2592. Anal.
Calcd for C24H38N3O6P: C, 58.17; H, 7.73; N, 8.48; P, 6.25. Found: C,
57.84; H, 7.85; N, 8.31; P, 6.04.
D2O) 66.6 (d, JC,P¼151.1, CH2P), 91.8 (C-5), 126.5 (CH-2-thienyl),
127.3 (CH-5-thienyl), 132.9 (CH-4-thienyl), 135.1 (C-3-thienyl),
162.0 (C-2), 169.7 (C-4), 173.7 (d, JC,P¼10.8, C-6); dP (202.3 MHz,
D2O) 12.51; ESI-MS m/z 315 [MꢂH]ꢂ (24); HRMS (ESI) calcd for
C9H6Li2N2O6PS 315.0010, found 315.0003.
4.8.7. 2,6-Di-tert-butyl-4-[(diisopropoxyphosphoryl)methoxy]-5-
(pyridin-3-yl)pyrimidine (7h). Column chromatography was per-
formed in ethyl acetate–chloroform–methanol 21:20:2. Yield
212 mg (43%) as a slightly yellow oil; nmax (CCl4) 1598, 1587, 1557,
1539, 1497, 1478, 1438, 1429, 1406, 1394, 1366, 1105, 1009, 993, 987;
dH (400.1 MHz, CDCl3) 1.15 and 1.24 (2ꢃd, 2ꢃ6H, Jvic¼6.2,
(CH3)2CH), 1.55 and 1.66 (2ꢃs, 2ꢃ9H, (CH3)3C), 4.58 (d, 2H, JH,P¼8.3,
CH2P), 4.62 (dh, 2H, JH,P¼7.5, Jvic¼6.2, CH(CH3)2), 7.26 (ddd, 1H,
J5,4¼7.9, J5,6¼4.8, J5,2¼0.9, H-5-py), 7.75 (ddd, 1H, J4,5¼7.9, J4,2¼2.0,
J4,6¼1.6, H-4-py), 8.46 (dd, 1H, J6.5¼4.8, J6,4¼1.6, H-6-py), 8.63 (br d,
1H, J2,4¼2.0, H-2-py); dC (100.6 MHz, CDCl3) 23.7 (d, JC,P¼4.7,
(CH3)2CH), 24.0 (d, JC,P¼3.9, (CH3)2CH), 28.5 and 28.6 ((CH3)3C), 60.1
(d, JC,P¼170.5, CH2P), 71.5 (d, JC,P¼6.6, CH(CH3)2), 80.8 and 82.0
(C(CH3)3), 97.3 (C-5), 122.4 (CH-5-py), 128.1 (C-3-py), 138.1 (CH-4-
py), 147.4 (CH-6-py), 151.6 (CH-2-py), 162.0 (C-2), 167.3 (d, JC,P¼11.1,
C-6), 168.4 (C-4). FABMS m/z 496 [MH]þ (27); HRMS (FAB) calcd for
C24H39N3O6P 496.2577, found 496.2572.
4.9.2. 5-Phenyl-6-(phosphonomethoxy)uracil (8c). Yield 46 mg
(23%) as a slightly yellow amorphous solid; mp >300 ꢁC; nmax (KBr)
1710, 1626, 1601, 1499, 1444, 1130, 1094, 1077, 1035, 996, 937, 755, 699,
573; dH (400.1 MHz, D2O, ref(dioxane)¼3.75 ppm) 4.21 (d, 2H,
JH,P¼7.8, CH2P), 7.35 (m,1H, H-p-Ph), 7.39 (m, 2H, H-o-Ph), 7.45 (m, 2H,
H-m-Ph); dC (100.6 MHz, D2O, ref(dioxane)¼69.3 ppm) 68.3 (d,
JC,P¼149.5, CH2P), 97.9 (C-5), 129.9 (CH-p-Ph), 131.2 (CH-m-Ph), 134.0
(CH-o-Ph), 134.6 (C-i-Ph), 159.2 (C-2), 169.2 (d, JC,P¼7.1, C-6), 170.0
(C-4); dP (162.0 MHz, D2O) 12.62; ESI-MS m/z 311 [MH]þ (70), HRMS
(ESI) calcd for C11H10Li2N2O6P 311.0597, found 311.0605. Anal. Calcd
for C11H9Li2N2O6P$3.5H2O: C, 35.41; H, 4.32; N, 7.51; P, 8.30. Found: C,
35.14; H, 4.21; N, 7.29; P, 7.88.
4.9.3. 5-Fluorophenyl-6-(phosphonomethoxy)uracil (8d). Yield
111 mg (53%) as a white amorphous solid; mp >300 ꢁC; nmax (KBr)
1717, 1624, 1559, 1509, 1406, 995, 934, 577; dH (500.0 MHz, D2O,
ref(dioxane)¼3.75 ppm) 4.22 (d, 2H, JH,P¼7.8, CH2P), 7.17 (m, 2H, H-
m-C6H4F), 7.39 (m, 2H, H-o-C6H4F); dC (125.7 MHz, D2O, ref(diox-
ane)¼69.3 ppm) 68.1 (d, JC,P¼149.3, CH2P), 96.9 (C-5), 117.9 (d,
JC,F¼21.4, CH-m-C6H4F), 130.6 (d, JC,F¼3.2, C-i-C6H4F), 135.7 (d,
JC,F¼8.2, CH-o-C6H4F), 159.5 (C-2), 164.3 (d, JC,F¼243.5, C-p-C6H4F),
169.5 (d, JC,P¼6.8, C-6), 170.0 (C-4), dP (202.3 MHz, D2O) 12.73; dF
(470.3 MHz, D2O) ꢂ112.17. FABMS m/z 351 [MþNa]þ (9); HRMS
(FAB) calcd for C11H8FLi2NaN2O6P 351.0321, found 351.0337. Anal.
Calcd for C11H8FLi2N2O6P$3.0H2O: C, 34.57; H, 2.69; N, 7.33; F, 4.97;
P, 8.11. Found: C, 34.29; H, 2.42; N, 7.10; F, 4.83; P, 8.41.
4.8.8. 2,6-Di-tert-butyl-4-[(diisopropoxyphosphoryl)methoxy]-5-
[(E)-2-phenylvinyl]pyrimidine (7i). Column chromatography was
performed in toluene–ethyl acetate 1:1. Yield 131 mg (25%) as
a yellow oil; nmax (CCl4) 1648, 1583, 1549, 1497, 1450, 1478, 1425,
1402, 1387, 1377, 1365, 1252, 1107, 1009, 996, 991; dH (499.8 MHz,
CDCl3) 1.32 and 1.35 (2ꢃd, 2ꢃ6H, Jvic¼6.2, (CH3)2CH), 1.63 and 1.68
(2ꢃs, 2ꢃ9H, (CH3)3C), 4.65 (d, 2H, JH,P¼8.2, CH2P), 4.83 (dh, 2H,
JH,P¼7.6, Jvic¼6.2, CH(CH3)2), 7.18 (d, 1H, Jtrans¼16.7, CH¼CH-Ph),
7.20 (m, 1H, H-p-Ph), 7.32 (m, 2H, H-m-Ph), 7.46 (m, 2H, H-o-Ph),
7.50 (d, 1H, Jtrans¼16.7, CH]CH–Ph); dC (125.7 MHz, CDCl3) 24.0 (d,
JC,P¼4.7, (CH3)2CH), 24.1 (d, JC,P¼3.9, (CH3)2CH), 28.6 and 28.8
((CH3)3C), 60.2 (d, JC,P¼170.9, CH2P), 71.5 (d, JC,P¼6.5, CH(CH3)2),
80.6 and 82.2 (C(CH3)3), 97.7 (C-5), 117.4 (CH]CH–Ph), 126.1 (CH-o-
Ph), 126.9 (CH-p-Ph), 128.5 (CH-m-Ph), 130.5 (CH]CH–Ph), 139.1
(C-i-Ph), 160.5 (C-2), 167.3 (d, JC,P¼11.9, C-6), 168.3 (C-4). FABMS m/z
521 [MH]þ (11); HRMS (FAB) calcd for C27H42N2O6P 521.2781, found
521.2799.
4.9.4. 5-(3-Nitrophenyl)-6-(phosphonomethoxy)uracil (8e). Yield
150 mg (68%) as a yellow amorphous solid; mp >320 ꢁC; nmax
(KBr) 1710, 1627, 1602, 1580, 1526, 1496, 1423, 1353, 1128, 1085,
990, 905; dH (499.8 MHz, D2O, ref(dioxane)¼3.75 ppm) 4.29 (d,
2H, JH,P¼8.0, CH2P), 7.62 (dd, 1H, J5,4¼8.3, J5,6¼7.8, H-5-C6H4NO2),
7.85 (ddd, 1H, J6,5¼7.8, J6.2¼1.6, J6,4¼1.1, H-6-C6H4NO2), 8.14 (ddd,
1H, J4,5¼8.3, J4,2¼2.4, J4,6¼1.1, H-4-C6H4NO2), 8.30 (dd, 1H, J2,4¼2.4,
J2,6¼1.6, H-2-C6H4NO2); dC (125.7 MHz, D2O, ref(dioxane)
¼69.3 ppm) 66.8 (d, JC,P¼151.8, CH2P), 95.5 (C-5), 124.2 (CH-4-
C6H4NO2), 128.6 (CH-2-C6H4NO2), 131.8 (CH-5-C6H4NO2), 136.9 (C-
1-C6H4NO2), 140.7 (CH-6-C6H4NO2), 150.4 (C-3-C6H4NO2), 160.6
(C-2), 169.7 (C-4), 170.7 (d, JC,P¼7.2, C-6); dP (202.3 MHz, D2O)
13.34. ESI-MS m/z 342 [MHꢂ2Li]þ (100), HRMS (ESI) calcd for
4.9. Deprotection of 5-aryluracils 7a, 7c–h and 7j. General
procedure
A solution of 7 (0.54 mmol) in acetonitrile (10 mL) was cooled to
0 ꢁC. Bromotrimethylsilane (839 mg, 5.44 mmol) was added drop-
wise and the mixture was stirred overnight at room temperature.
The mixture was concentrated and neutralized with 0.5M TEAB
solution at 0 ꢁC. The mixture was evaporated in vacuo and then
codistilled with water (2ꢃ2 mL). The residue was purified on DEAE-
Sephadex (Clꢂ, 0–0.4M TEAB, S3) with subsequent deionization of
the product on activated charcoal with water. The residue was
dissolved in water (3 mL), applied onto a column of Dowex 50ꢃ8
(Liþ form, 30 mL) and then the column was washed with water. The
appropriate UV absorbing fraction containing product 8 was
evaporated to dryness in vacuo. The residue was dissolved in water
and lyophilized. The following compounds were obtained as
dilithium salts:
C11H9N3O8P 342.0127, found 342.0128. Anal. Calcd for C11H8Li2
-
N3O8P$2.75H2O: C, 32.66; H, 3.36; N, 10.38; P, 7.66. Found: C,
32.47; H, 3.40; N, 10.05; P, 8.02.
4.9.5. 5-(Naphth-1-yl)-6-(phosphonomethoxy)uracil (8f). Yield
121 mg (61%) as a white amorphous solid; mp >320 ꢁC; nmax (KBr)
1715,1624,1592,1579,1507,1497,1430,1399,1363,1126,1095,1000,
939, 805, 780, 588; dH (600.0 MHz, D2O, ref(dioxane)¼3.75 ppm)
4.05 (dd, 1H, Jgem¼13.7, JH,P¼7.8, CHaHbP), 4.22 (dd, 1H, Jgem¼13.7,
JH,P¼7.8, CHaHbP), 7.49 (dd, 1H, J2,3¼7.0, J2,4¼1.3, H-2-naphth), 7.54
(ddd, 1H, J7,8¼8.3, J7,6¼6.8, J7,5¼1.5, H-7-naphth), 7.57 (ddd, 1H,
J6,5¼8.1, J6,7¼6.8, J6,8¼1.4, H-6-naphth), 7.60 (dd, 1H, J3,4¼8.3,
J3,2¼7.0, H-3-naphth), 7.79 (m,1H, J8,7¼8.3, J8,6¼1.4, J8,4¼1.1, J8,5¼0.7,
H-8-naphth), 7.97 (ddd, 1H, J4,3¼8.3, J4,2¼1.3, J4,8¼1.1, H-4-naphth),
8.99 (ddd,1H, J5,6¼8.1, J5,7¼1.5, J5,8¼0.7, H-5-naphth); dC (150.9 MHz,
D2O, ref(dioxane)¼69.3 ppm) 68.3 (d, JC,P¼149.1, CH2P), 95.6 (C-5),
128.0 (CH-8-naphth), 128.8 (CH-3-naphth), 128.9 (CH-6-naphth),
129.3 (CH-7-naphth), 131.0 (CH-4-naphth), 131.2 (CH-5-naphth),
132.2 (C-1-naphth), 132.7 (CH-2-naphth), 135.2 (C-8a-naphth),
136.3 (C-4a-naphth), 159.8 (C-2), 170.3 (C-4,6); dP (202.3 MHz, D2O)
4.9.1. 6-(Phosphonomethoxy)-5-(3-thienyl)uracil (8a). Yield 15 mg
(33%) as a slightly brown amorphous solid; mp >300 ꢁC; nmax (KBr)
3270, 3110,1718,1708, 1630, 1492, 1426,1354, 1125,1086,1001, 800;
dH (499.8 MHz, D2O) 4.14 (d, 2H, JH,P¼8.6, CH2P), 7.40 (dd, 1H,
J4,5¼5.0, J4,2¼1.3, H-4-thienyl), 7.45 (dd, 1H, J5,4¼5.0, J5,2¼3.0, H-5-
thienyl), 7.54 (dd, 1H, J2,5¼3.0, J2,4¼1.3, H-2-thienyl); dC (125.7 MHz,

8492
K. Pomeisl et al. / Tetrahedron 65 (2009) 8486–8492
16.82. ESI-MS m/z 347 [MHꢂ2Li]ꢂ (100); HRMS (ESI) calcd for
Supplementary data
C15H12N2O6P 347.0433, found 347.0448.
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2009.08.039.
4.9.6. 6-(Phosphonomethoxy)-5-(pyridin-4-yl)uracil (8g). Yield
48 mg (29%) as a white amorphous solid; mp >300 ꢁC; nmax (KBr)
1723, 1630, 1496, 1374, 1287, 1091, 1000, 594; dH (600.0 MHz, D2O,
ref(dioxane)¼3.75 ppm) 4.34 (d, 2H, JH,P¼8.3, CH2P), 8.23 (m, 2H,
H-3,5-py), 8.35 (m, 2H, H-2,6-py); dC (150.9 MHz, D2O, ref(diox-
ane)¼69.3 ppm) 66.0 (d, JC,P¼153.4, CH2P), 93.0 (C-5), 128.4 (CH-
3,5-py), 142.1 (CH-2,6-py), 155.0 (C-4-py), 162.2 (C-2), 169.43 (C-4),
173.6 (d, JC,P¼10.4, C-6); dP (202.3 MHz, D2O) 12.99. ESI-MS m/z 305
[MH-Li]þ (32); HRMS (ESI) calcd for C10H8LiN3O6P 304.0311, found
304.0318.
References and notes
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Pe´rez-Pe´rez, M.-J. J. Med. Chem. 2000, 43, 971–983.
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Yamada, Y. Nature 1994, 368, 198; (c) Matsushita, S.; Nitanda, T.; Furukawa, T.;
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1916.
4.9.7. 6-(Phosphonomethoxy)-5-(pyridin-3-yl)uracil (8h). Yield
96 mg (57%) as a slightly yellow amorphous solid; mp >300 ꢁC; nmax
(KBr) 1635, 1558, 1491, 1465, 1375, 1284, 1090, 810, 710, 791, 625; dH
(500.0 MHz, D2O, ref(dioxane)¼3.75 ppm) 4.30 (d, 2H, JH,P¼8.2,
CH2P), 7.68 (br dd, 1H, J5,4¼8.1, J5,6¼4.9, H-5-py), 8.33 (br d, 1H,
J4,5¼8.1, H-4-py), 8.39 (br s, 1H, H-6-py), 8.73 (br s, 1H, H-2-py); dC
(125.7 MHz, D2O, ref(dioxane)¼69.3 ppm) 66.3 (d, JC,P¼153.4, CH2P),
92.0 (C-5), 127.6 (CH-5-py), 134.4 (C-3-py), 144.1 (CH-6-py), 145.9
(CH-4-py),148.9 (CH-2-py),161.4 (C-2),169.5 (C-4),171.7 (d, JC,P¼8.8,
C-6); dP (202.3 MHz, D2O) 13.25. ESI-MS m/z 304 [MꢂHꢂLi]ꢂ (11);
HRMS (ESI) calcd for C10H8LiN3O6P 304.0311, found 304.0311.
ˇ
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´
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7. Pomeisl, K.; Pohl, R.; Holy, A.; Votruba, I. Collect. Czech. Chem. Commun. 2006, 71,
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4.9.8. 6-(Phosphonomethoxy)uracil (8j). Yield 139 mg (58%) as
a white amorphous solid; mp >300 ꢁC; nmax (KBr) 1720, 1634, 1520,
1372, 1242, 1070, 1031, 925; dH (400.1 MHz, DMSO-d6) 3.78 (d, 2H,
JH,P¼9.1, CH2P), 4.89 (s, 1H, H-5); dC (100.6 MHz, DMSO-d6) 66.9 (d,
JC,P¼151.4, CH2P), 77.0 (CH-5), 150.8 (C-2), 164.3 (d, JC,P¼12.6, C-6),
165.2 (C-4).; dP (162.0 MHz, D2O) 10.54. ESI-MS m/z 223 [MHꢂ2Li]þ
(56); HRMS (ESI) calcd for C5H8N2O6P 223.0120, found 223.0128.
9. Krecˇmerova´, M.; Holy´, A.; Masojı´dkova´, M. Collect. Czech. Chem. Commun. 2007,
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The study has been performed as a part of research project #Z
40550506 of the Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic and the Centre of New
Antivirals and Antineoplastics 1M0508 supported by the Ministry of
Education, Youth and Sports of the Czech Republic. The financial
support of the Program of target projects of Academy of Sciences of
the Czech Republic (Grant #1QS400550501) and of Gilead Sciences
and IOCB Research Centre (Foster City, CA, USA) is gratefully
acknowledged.
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