Syntheses of N3-substituted thymine acyclic nucleoside phosphonates and a comparison of their inhibitory effect towards thymidine phosphorylase

Article abstract of DOI:10.1016/j.bmcl.2008.01.006

A series of N³-substituted thymine acyclic nucleoside phosphonates bearing a number of (phosphonomethoxy)alkyl groups were synthesized and investigated for their ability to inhibit the human thymidine phosphorylase expressed in V79 Chinese hams

Full text of DOI:10.1016/j.bmcl.2008.01.006

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1364–1367
Syntheses of N3-substituted thymine acyclic nucleoside
phosphonates and a comparison of their inhibitory effect
towards thymidine phosphorylase
*
´
´
Karel Pomeisl, Antonın Holy, Ivan Votruba and Radek Pohl
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 na´m. 2, 16610 Prague, Czech Republic
Received 22 November 2007; revised 2 January 2008; accepted 3 January 2008
Available online 8 January 2008
Abstract—A series of N3-substituted thymine acyclic nucleoside phosphonates bearing a number of (phosphonomethoxy)alkyl
groups were synthesized and investigated for their ability to inhibit the human thymidine phosphorylase expressed in V79 Chinese
hamster cells, as well as thymidine phosphorylase from SD-lymphoma, Escherichia coli and human placenta. In comparison to
N1- substituted analogues which possess a considerable inhibitory activity towards thymidine phosphorylase from SD-lymphoma,
the results showed a marginal inhibitory effect of these compounds. None of the presented N3-substituted derivatives possess a
significant cytostatic activity.
ꢀ 2008 Published by Elsevier Ltd.
Pyrimidine acyclic nucleoside phosphonates (ANPs) are
compounds which possess significant antiviral and cyto-
static activity.1 The scale of biological effects for pyrim-
idine ANP derivatives could be also extended in
connection with their potential inhibitory potency to-
wards thymidine phosphorylase (TP).2 This enzyme,
which is identical to platelet-derived endotherial cell
growth factor (PD-ECGF),3 catalyses the reversible
phosphorolysis of thymidine to thymine and 2-deoxy-
D-ribose 1-phosphate.3a The dephosphorylated product
of the latter, 2-deoxy-D-ribose, plays an important role
in tumour angiogenesis.4 Therefore, inhibitors of TP
may find utility as suppressors of tumour growth.4c
results show a considerable selective multisubstrate ef-
fect of a number of side-chain modified pyrimidine
ANPs towards TP isolated from SD-lymphoma6–8
whereas the marginal inhibitory activity on the other en-
zymes has been observed. In contrast, various kinds of
known 5-halogeno 6-amino substituted4c,9 uracils pos-
sess a significant effect on human TP. However, the
low values of their inhibitory activity on the enzyme
purified from spontaneous T-cell lymphoma of an
inbred Sprague–Dawley rat strain were found.9b There-
fore, we guess the structures of mentioned enzymes
could probably be different in recognition of active sites
as well as the interactions of known substituted ura-
cils,10 thymine2, and/or 7-deazaxantyl5 ANPs predicted
on E. coli and human TP.
The aim of our work has been the development of new
ANP multisubstrate inhibitors of this enzyme bearing
pyrimidine base and various phosphonoalkyl groups to
interfere at thymine and phosphate-binding sites.2,5
The determination of inhibitory activity of our previ-
ously reported compounds6–8 was performed with TP
expressed in V79 Chinese hamster cells, human placenta,
Escherichia coli and newly used SD-lymphoma. The
Based on this assumption, we systematically deal with
modifications of structure in catabolically stable pyrimi-
dine ANPs to influence their interaction with human
TP. In this report, we described in particular one of the
possible modifications of structures in known thymine
(phosphonomethoxy)alkyl derivatives6 such as 1-[2-
(phosphonomethoxy)ethyl]thymine (PMET), 1-[3-hy-
droxy-2-(phosphonomethoxy)propyl]thymine (HPMPT)
and 1-[3-fluoro-2-(phosphonomethoxy)-propyl]thymine
(FPMPT) which were found as efficient inhibitors of TP
isolated from SD-lymphoma (Fig. 1). That means we syn-
thesized their new N3-substituted analogues 6, 7, and 10.
Keywords: Acyclic nucleoside phosphonates; Thymidine phosphory-
lase; Fluorination; Pyrimidine; Alkylation.
*
Corresponding author. Tel.: +420 220183475; fax: +420
220183560; e-mail: pomeisl@uochb.cas.cz
0960-894X/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.01.006

K. Pomeisl et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1364–1367
1365
In this case, we investigated N3-selective and effective
(i-PrO)2(O)P
(HO)2(O)P
alkylation of the thymine moiety with a number of (phos-
phonomethoxy)alkyl groups which have previously never
been tested with ANPs and therefore still remains a syn-
thetic and biochemical challenge. For biological studies
we prepared both the optical isomers to compare their
activity in N3-substituted HPMPT and FPMPT deriva-
tives. In conclusion, the inhibitory activity of obtained
compounds 6, 7, and 10 was compared with those of
our reported N1-substituted pyrimidine ANPs.
O
O
O
O
CH3 1) Me3SiBr, MeCN, rt
2) CF3COOH, H2O, reflux
CH3
N
N
O
N
O
N
H
THP
9
10
Cl
O
P(O)(O-iPr)2
8
°
NaH, DMF, 100
C
OTr
OH
O
TrO
O
O
N
2a,2b
It is well known that the N3-position of pyrimidines
plays an important role in the forming of hydrogen
interactions in various biological systems such as the
base pairing, e.g., in DNA.11 It seems that the hydrogen
at pyrimidine N1 and/or N3-position also interacts with
TP as recently reported by the proposal of inhibitor
binding in E. coli and human TP crystal structures.2,10
Our effort was to evaluate the substitution of N3-posi-
tion with (phosphonomethoxy)alkyl groups while the
hydrogen at N1-position of base would be available for
potential interaction with the enzyme.10 We expected it
could influence the inhibitory effect of our synthesized
ANPs towards TP in different directions. Therefore,
the results of biochemical screening could be helpful in
better understanding of the enzyme function in the thy-
mine active site and to design more efficient ANP inhib-
itors in future.
CH3
CH3
TsO P(O)(Oi-Pr)2
1)
N
HN
°
NaH, DMF, 100
C
°
°
C
NaH, THF, -20 C->40
2) 99% AcOH, H2/Pd/C
MeOH, rt
O
N
THP
O
THP
1
3a,3b
P(O)(i-PrO)2
P(O)(Oi-Pr)2
O
N
O
N
F
O
HO
O
CH3
C4F9SO2F, DBU
CH3
°
toluene, rt->90
C
O
N
THP
O
N
THP
5a,5b
4a,4b
P(O)(OH)2
O
N
F
O
Me3SiBr, MeCN, rt
CH3
CF3COOH, H2O, reflux
a,(R )-derivatives
b,(S )-derivatives
O
N
H
6a,6b
Scheme 1. Syntheses of N3-substituted thymine ANPs.
As demonstrated in the described novel syntheses of
phosphonates 6a, 6b, and 10, for the preparation of re-
quired compounds we used the selective N3-alkylation
of 1-(tetrahydro-2H-pyran-2-yl)thymine (1)12 with chiral
oxiranes 2a, 2b and halogenoalkylphosphonate 8 as a
key step followed by deprotection (Scheme 1). Alkyl-
ation of the protected base proceeded in good prepara-
tive 57–70% yields of corresponding intermediates 3
and 9 in the presence of sodium hydride in dimethyl-
formamide.13 Compounds 3a and 3b were further
converted to 4a and 4b by their reaction with 2-[(diiso-
propoxyphosphoryl)methyl tosylate followed by mild
hydrogenation over 10% palladium on charcoal in the
presence of glacial acetic acid. For the replacement of
hydroxy group with fluorine in the intermediates 4a
and 4b we applied our simple improved method,7 devel-
oped for the preparation of N1-substituted FPMPT
from easily accessible HPMPT intermediates using com-
mercial and weakly corrosive perfluorobutane-1-sulfo-
nyl fluoride as a fluorination agent. All the compounds
obtained (e.g., 5 and 9, see Scheme 1) were deprotected
by their reaction with bromotrimethylsilane followed by
hydrolysis in the presence of trifluoroacetic acid.14
The inhibitory activity of compounds 6a, 6b, 7a, 7b, and
10 was compared with those of N1-phosphonoalkyl thy-
mines. Data listed in Table 1 show that none of the syn-
thesized phosphonates possesses an inhibitory effect on
TP isolated from E. coli, human placenta and TP ex-
pressed in V79 Chinese hamster cells. In contrast to
N1-substituted phosphonoalkyl thymines,6 alkylation
of N3-position even results in the significant decrease
of the inhibitory activity on TP from rat spontaneous
T-cell lymphoma. Furthermore, the influence of chiral-
ity and substitution on the side chain of the phospho-
noalkyl group to inhibitory effect is marginal towards
TP from SD-lymphoma as shown for described N3-
substituted analogues 6a, 6b, and 10 of some efficient
ANP inhibitors6 (see Table 1).
None of the presented compounds 6, 7, and 10 possess, at
a concentration of 10 lmol Lꢀ1, a significant cytostatic
activity in tissue cultures estimated in mouse lymphocytic
leukemia L1210 cells (ATCC CCL 219), CCRF-CEM T
lymphoblastoid cells (human acute lymphoblastic leuke-
mia, ATCC CCL 119), human promyelocytic leukemia
HL-60 cells (ATCC CCL 240) and human cervix carci-
noma HeLa S3 cells (ATCC CCL 2.2).16,17
P(O)(OH)2
O
N
CH3
R
R
O
N
HN
O
CH3
O
O
N
H
O
P(O)(OH)2
6, R = CH2F
7, R = CH2OH
10, R = H
PMET, R = H
HPMPT, R = CH2OH
FPMPT, R = CH2F
Based on these biochemical results, we have proved that
the inhibitory activity of pyrimidine ANPs towards TP
from SD-lymphoma is strongly dependent on their thy-
mine substitution with phosphonoalkyl groups. The de-
Figure 1. N1- and N3-substituted pyrimidine ANPs under study.

1366
K. Pomeisl et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1364–1367
Table 1. Inhibition of thymidine phosphorylases by ANPs
References and notes
Compound
PMETb
Inhibition of thymidine phosphorylasea, Vi/V0
E. coli Human, V79 SD- Human
Lymphoma placenta
1. (a) Holy´, A. Curr. Pharm. Design 2003, 9, 2567; (b) Holy´,
A. In Recent Advances in Nucleosides: Chemistry and
Chemotherapy; Chu, C. K., Ed.; Elsevier: Amsterdam,
2002; p 167.
expressed
1.00
1.02
0.84
0.82
1.01
1.05
0.79
0.95
1.12
0.27
0.31
0.11
0.75
n.d.
0.78
n.d.
1.44
0.85
0.84
0.56
n.d.
n.d.
0.91
1.02
0.82
(R)-HPMPTb 0.98
(R)-FPMPTb 0.93
2. Esteban-Gamboa, A.; Balzarini, J.; Esnouf, R.; De Clercq,
´ ´
E.; Camarasa, M.-J.; Perez-Perez, M.-J. J. Med. Chem.
2000, 43, 971.
6a
6b
7a
7b
10
0.98
0.93
1.01
0.94
1.07
3. (a) Friedkin, M.; Roberts, D. W. J. Biol. Chem. 1954, 207,
245; (b) Miyazono, K.; Okabe, T.; Urabe, A.; Takaku, F.;
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4. (a) Furakawa, T.; Yoshimura, A.; Sumizawa, T.; Harag-
uchi, M.; Akiyama, S.; Fukui, K.; Ishizawa, M.; Yamada,
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K.; Akiyama, S.; Yamada, Y. Nature 1994, 368, 198; (c)
Matsushita, S.; Nitanda, T.; Furukawa, T.; Sumizawa, T.;
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a 100 lM [3H]-20-deoxythymidine, 250 lM Pi (pH 6,7), tested com-
pound 10 lmolÆlꢀ1, an appropriate amount of enzyme, 10 min incu-
bation at 37 ꢁC Ref. 6,9b,15. The inhibitory efficacy is expressed by
Vi/V0 (Vi. . .rate of phosphorolysis in the presence of inhibitors, V0. . .
rate of phosphorolysis in the absence of inhibitors).
b The structures of compared N1-substituted thymine ANPs are shown
in Figure 1.
crease of the inhibitory effect could be induced by low
affinity of N3-substituted ANPs to enzyme in contrast
to N1-substituted derivatives. This probably results from
the differences in recognition of the thymine active site
by a variable formation of potential interactions be-
tween heteroatoms of N1- and N3-substituted thymine
moiety and SD-lymphoma TP or by missing of the thy-
mine essential carbonyl group in correct direction which
may exclude an interaction with those of TP. However,
the marginal values of inhibition on human TP could
also indicate the significant diversity in the phosphate
binding site in both enzymes. Therefore, we have as-
sumed that newly used TP from SD-lymphoma is not
an appropriate model enzyme to human TP probably
due to a supposable short length between the thymine
and phosphate binding sites and this is a subject of cur-
rent research. On the other hand, data obtained from
SD-lymphoma TP afford a valuable information and a
more comprehensive view on problems of pyrimidine
multisubstrate inhibitors and their potential utilization
on model and commercial enzymes.
´
5. Balzarini, J.; Degreve, B.; Esteban-Gamboa, A.; Esnouf,
R.; De Clercq, E.; Engelborghs, Y.; Camarasa, M.-J.;
´
´
Perez-Perez, M.-J. FEBS Lett. 2000, 483, 181.
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6. Votruba, I.; Pomeisl, K.; Tloust’ova, E.; Holy, A.; Otova,
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B. Biochem. Pharmacol. 2005, 69, 1517.
7. Pomeisl, K.; Pohl, R.; Holy´, A.; Votruba, I. Collect.
Czech. Chem. Commun. 2005, 70, 1465.
8. Pomeisl, K.; Votruba, I.; Holy´, A.; Pohl, R. Collect.
Czech. Chem. Commun. 2006, 71, 595.
9. (a) Klein, R. S.; Lenzi, M.; Lim, T. H.; Hotchkiss, K. H.;
Wilson, P.; Schwartz, E. L. Biochem. Pharmacol. 2001, 62,
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1257; (b) Nencka, R.; Votruba, I.; Hrebabecky, H.;
Tloust’ova, E.; Horska, K.; Masojıdkova, M.; Holy, A.
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10. Norman, R. A.; Barry, S. T.; Bate, M.; Breed, J.; Colls, J.
G.; Ernill, R. J.; Luke, R. W. A.; Minshull, C. A.;
McAlister, M. S. B.; McCall, E. J.; McMiken, H. H. J.;
Paterson, D. S.; Timms, D.; Tucker, J. A.; Pauptit, R. A.
Structure 2004, 12, 75.
11. Voet, D.; Voet, J. G. In Biochemistry; John Wiley & sons
Inc, 1990; p 795, 809.
12. Noell, C. W.; Cheng, C. C. J. Heterocycl. Chem. 1966, 3, 5.
13. A typical N3-alkylation procedure in the synthesis of 9: a
mixture of compound 1 (505 mg, 2.4 mmol) and 60%
sodium hydride dispersion (144 mg, 3.6 mmol) in dimeth-
ylformamide (30 mL) was stirred at room temperature.
After 1 h of stirring, the mixture was allowed to warm to
60 ꢁC and compound 7 (620 mg, 2.4 mmol) in dimethyl-
formamide (20 ml) was added. The resulting mixture was
heated at 100 ꢁC for 9 h until the conversion of 1 finished.
The mixture was concentrated in vacuo to a minimum
volume. The residue was codistilled with toluene (3·
20 mL) and diluted with chloroform (30 mL). The mixture
was filtered through a Celite pad and the filtrate was
concentrated to a minimum volume. The residue was
chromatographed on neutral aluminium oxide (ethyl
acetate/chloroform/methanol = 26:25:1). The crude prod-
uct was purified by preparative TLC (ethyl acetate/
chloroform/methanol = 26:25:1). The relevant fractions
were combined and evaporated in vacuo. Yield 592 mg
(57%) of a colourless liquid: IR mmax (CCl4) 2980, 1707,
1672, 1651, 1462, 1451, 1386, 1375, 1260, 1250, 1181, 1142,
Acknowledgments
The study has been performed as a part of research pro-
ject #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 of the Czech Republic. The financial support
of the Program of target projects of Academy of Sci-
ences of the Czech Republic (Grant #1QS400550501)
and of Gilead Sciences (Foster City, CA, USA) is grate-
fully acknowledged.
Supplementary data
1
1107, 1089, 1063, 1046, 1010, 991, 889, 556, 503 cmꢀ1; H
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.01.006.
NMR (CDCl3) d 1.322, 1.324, 1.346 and 1.35 (4· d, 4· 3H,
Jvic = 6.2, (CH3)2CH), 1.57–1.78 (m, 4H, CH2–THP), 1.87

K. Pomeisl et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1364–1367
1367
(m, 1H, CHaHb–THP), 1.93 (d, 3H, JCH3,6 = 1.2, CH3),
1.99 (m, 1H, CHaHb–THP), 3.77–3.84 (m, 5H, H-20, H-40,
CHaHbO–THP), 4.12 (ddt, 1H, J = 11.70, 4.2, 2.3,
mp > 300 ꢁC: IR mmax (KBr) 3259, 3186, 3123, 1715,
1648, 1489, 1448, 1387, 1330, 1213, 1136, 1112, 1001,
1
913, 805, 771, 723, 581, 564, 480 cmꢀ1; H NMR (D2O) d
1.88 (d, 3H, JCH3,6 = 1.2, CH3), 3.52 (d, 2H, JH,P = 8.5,
0
0
CHaHbO–THP), 4.19 (t, 2H, J1 ,2 = 6.0, H-1 ), 4.78–4.80
(m, 2H, CH(CH3)2), 5.60 (dd, 1H, J = 10.8, 2.4, CHO–
THP), 7.24 (q, 1H, J6,CH3 = 1.2, H-6); 13C NMR (CDCl3) d
13.21 (CH3); 22.74 (CH2–THP), 23.94 and 24.07 (d,
JC,P = 4, (CH3)2CH), 24.90 and 30.96 (CH2–THP), 39.70
(CH2-10), 65.41 (d, JC,P = 167, CH2-40), 69.04 (CH2O–
THP), 69.51 (d, JC,P = 12, CH2-20), 71.02 (d, JC,P = 6,
CH(CH3)2), 82.79 (CH–OTHP), 109.95 (C-5), 133.79 (CH-
6), 150.56 (C-2), 163.34 (C-4); FAB-MS m/z 433 [MH]+
(77); FABHRMS calcd for C19H34N2O7P 433.2103: Found
433.2099.
H-40), 3.77 4.19 (t, 2H, J2 ,1 = 5.8, H-20), 4.14 (t, 2H,
0
0
J1 ,2 = 5.8, H-10), 7.33 (q, 1H, J6,CH3 = 1.2, H-6); 13C
NMR (D2O) d 14.72 (CH3), 42.87 (CH2-10), 71.09 (d,
JC,P = 152, CH2-40), 71.90 (d, JC,P = 10, CH2-20), 112.40
(C-5), 139.84 (CH-6), 155.94 (C-2), 169.51 (C-4); 31P
NMR (202.3 MHz, D2O) d 14.41; FAB-MS m/z 277
[MH]+ (67); FABHRMS calcd for C8H12Li2N2O6P
277.0753: Found 277.0762.
0
0
15. Enzyme assay. The standard reaction mixture (50 ll)
contained 20 lM bisTris–HCl, pH 6.4, 1 mM EDTA and
2 mM DTT, 100 lM [3H-methyl]thymidine, 250 lM
potassium phosphate, pH 6,7, and 25.5 pU of enzyme
according to Ref.6 The reaction was carried out at 37 ꢁC for
10 min and stopped by spotting a 2 ll aliquot onto Silica
gel 60 F254 plate that had been prespotted with 0.01 lmol
of each thymine and thymidine. The plate was developed
in the non-aquaeous phase of the solvent system ethyl
acetate/water/formic acid (60:35:5). The spots were visu-
alised under UV light (254 nm) and cut out for radioac-
tivity determination in the toluene-based scintillation
cocktail.
16. Cytostatic activity assays. L1210 cells, CCRF-CEM cells,
and HL-60 cells were cultivated in RPMI 1640 medium
supplemented with calf foetal serum using 24-well tissue
culture plates. The endpoint of the cell growth was 72 h
following the drug addition. Cells were then counted in
Celtac MEK 5208 (NIHON KOHDEN) haematological
analyzer. HeLa S3 cells were seeded to 24-well dishes in
RPMI 1640 HEPES modification with foetal calf serum.
48 h following the drug addition the cultivation was
stopped and the cell growth was evaluated after methylene
blue addition.
14. A typical deprotection procedure in the synthesis of 10: a
mixture of compound 9 (562 mg, 1.3 mmol), acetonitrile
(20 mL) and bromotrimethylsilane (1.9 g, 12.3 mmol) was
stirred overnight at room temperature. The mixture was
concentrated in vacuo and then codistilled with water (2·
2 mL). The residue was further refluxed in water (3 mL)
and trifluoroacetic acid (10 mL) for 1.5 h. The mixture
was cooled to room temperature and neutralized with
dilute triethylammonium hydrogencarbonate. The mix-
ture was extracted with chloroform. The water layer was
separated and concentrated in vacuo. The residue was
purified on 40 mL of DEAE-Sephadex (Clꢀ form, 0–
0.4 M triethylammonium hydrogencarbonate) with sub-
sequent deionization on activated charcoal. The product
was eluted with 12% aqueous ammonia/methanol = 1:1.
The relevant fractions were combined, evaporated in
vacuo and codistilled with water (3· 5 mL). The residue
was dissolved in water (3 mL), applied onto a column of
Dowex 50 · 8 (Li+ form, 30 mL) and then the column
was washed with water. The appropriate UV absorbing
fraction containing product 10 was evaporated to dryness
in vacuo. The residue was dissolved in water and
lyophilized. The following compounds were obtained as
dilithium salts. Yield 110 mg (32%) of a white solid,
ˇ´ ´
17. Hocek, M.; Holy´, A.; Votruba, I.; Dvorakova, H. J. Med.
Chem. 2000, 43, 1817.