8-Aza-7,9-dideazaxanthine acyclic nucleoside phosphonate inhibitors of thymidine phosphorylase

Article abstract of DOI:10.1016/j.bmcl.2010.12.027

3- and 8-(8-phosphonooctyl)-8-aza-7,9-dideazaxanthine, and 1,8-bis(8-aza-7,9-dideazaxanthin-8-yl)octane were prepared and found to inhibit thymidine phosphorylase from Escherichia coli, human recombinant TP expressed in V79, and TP purified from human pla

Full text of DOI:10.1016/j.bmcl.2010.12.027

Bioorganic & Medicinal Chemistry Letters 21 (2011) 652–654
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Bioorganic & Medicinal Chemistry Letters
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8-Aza-7,9-dideazaxanthine acyclic nucleoside phosphonate inhibitors
of thymidine phosphorylase
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David Marák, Miroslav Otmar , Ivan Votruba, Martin Dracínsky, Marcela Krecmerová
Institute of Organic Chemistry and Biochemistry, v.v.i., Gilead Sciences & IOCB Research Center, Academy of Sciences of the Czech Republic,
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
3- and 8-(8-phosphonooctyl)-8-aza-7,9-dideazaxanthine, and 1,8-bis(8-aza-7,9-dideazaxanthin-8-yl)
octane were prepared and found to inhibit thymidine phosphorylase from Escherichia coli, human recom-
binant TP expressed in V79, and TP purified from human placenta. The IC50 values ranged from 3.5 to
Received 13 October 2010
Revised 2 December 2010
Accepted 6 December 2010
Available online 10 December 2010
27 lM.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
8-Aza-7,9-dideazaxanthine
Pyrrolo[3,4-d]pyrimidine
Nucleotide analog
Thymidine phosphorylase (TP, EC 2.4.2.4) catalyzes a reversible
phosphorolysis of thymidine and other pyrimidine 20-deoxynucle-
osides, except for those bearing an amino group in position 4. Its
main metabolic function appears to be catabolic, although some
bacteria and tumors utilize the reverse reaction anabolically under
the stress of certain gene damage or a dietary deficiency. Thymi-
dine phosphorylase (TP) is identical to the human angiogenic fac-
tor PD-ECGF, which is clearly involved in tumor dependent
angiogenesis. Its high levels correlate with neoplastic growth.
Therefore TP inhibitors are of permanent interest as potential anti-
tumor agents. Particular attention has been paid to the multisub-
strate inhibitors of TP binding to both the nucleoside and the
phosphate binding sites. Their advantage consists mainly in having
more points of interaction with the receptor than single-substrate
inhibitors.1 In a report published a decade ago, a series of acyclic
phosphonate derivatives of thymine was tested as multisubstrate
analog inhibitors of Escherichia coli TP, among which 3-(8-phos-
phonooctyl)-7-deazaxanthine (1, Chart 1) was shown to have a
remarkable inhibitory potency.2
O
N
HN
N
H
O
(CH2)8P(O)(OH)2
1
Chart 1.
1-(8-bromooctyluracil) (3), obtained by the alkylation of uracil by
1,8-dibromooctane, afforded the phosphonate 4. The BOM-pro-
tected phosphonate 5 was treated with toluenesulfonylmethyl
isocyanide (TosMIC) under conditions of a van Leusen pyrrole
synthesis4 to yield the protected 8-aza-7,9-dideazaxanthine 6. A
catalytic hydrogenation of 6 produced compound 7. Finally, the
phosphonate esters of 7 were removed with TMSBr under a forma-
tion of the acyclic 8-aza-7,9-dideazaxanthine nucleotide 2.5
Considering the promising inhibition capability of compound 2
(see below), we decided to extend the SAR study by a preparation
of its positional isomer 8 (Scheme 2). Compound 8 was synthesized
in a manner analogous to that for phosphonate 2. 1,3-Bis(benzyl-
oxymethyl)uracil6 was treated with TosMIC4 to yield the protected
8-aza-7,9-dideazaxanthine 9. Alkylation of 9 with 1,8-dibromooc-
tane afforded compound 10 together with a small amount of the
disubstituted product 11. When the protected nucleobase 9 was
taken into the reaction in a 2:1 ratio to 1,8-dibromooctane, the
yield of 11 amounted to 73%. Compound 11 was deprotected to
the ‘double-headed’ acyclic nucleoside 12. The Michaelis–Arbuzov
reaction of the bromide 10 afforded the protected nucleotide 13.
Our aim3 was to design further multisubstrate inhibitors of TP
by a structural modification of the parent compound 1. At first
we tried to make a slight structural change consisting in a shift
of the pyrrole nitrogen atom from position 9 to position 8, which
led us to the synthesis of 3-(8-phosphonooctyl)-8-aza-7,9-dideaza-
xanthine (2).
Compound 2 was synthesized in six steps from uracil as it is
outlined in Scheme 1. The Michaelis–Arbuzov reaction of
⇑
Corresponding author. Tel.: +420 220183375.
E-mail address: otmar@uochb.cas.cz (M. Otmar).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.027

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D. Marák et al. / Bioorg. Med. Chem. Lett. 21 (2011) 652–654
653
Q Sepharose) and the hydroxyapatite fraction was used for the
inhibition experiments. The standard reaction mixture (50 L) con-
tained 100 M potassium phos-
L [3H-methyl]thymidine, 200
phate buffer (pH 6.7), and enzyme (6.7 mU TP from E. coli, Sigma
T 2807; 0.41 mU human TP, expressed in V79 Chinese hamster
cells, Sigma T 9319; 0.45 mU TP purified from human placenta7
in 20 mM bis Tris–HCl, pH 6.4, 1 mM EDTA, and 2 mM DTT). The
reaction was carried out at 37 °C for 10 min and stopped by spot-
O
O
O
l
R
O
ii
iv
i
HN
N
HN
l
l
75%
76%
46%
N
O
N
O
N
H
(CH2)8Br
(CH2)8P(O)(Oi-Pr)2
3
iii
4
R = H
5 R = BOM
60%
ting a 2
prespoted with 0.01
l
L aliquot onto a Silica Gel 60 F254 plate that had been
O
O
N
lmol of each thymine and thymidine. The
R
O
vi
43%
plate was developed in the solvent system ethyl acetate/water/for-
mic acid (60:35:5). The spots were visualized under UV light
(254 nm) and cut out for a radioactivity determination in the tolu-
ene-based scintillation cocktail.
HN
N
NH
NH
O
N
(CH2)8P(O)(OH)2
(CH2)8P(O)(Oi-Pr)2
The results show that all of the above-mentioned compounds
efficiently inhibit at 0.1 mM thymidine and 0.2 mM potassium
phosphate buffer bacterial and/or human TPs with IC50 values in
2
v
6 R = BOM
7 R = H
85%
the concentration range from 3.5 to 27 lM (Table 1). At a higher
phosphate concentration (1 mmol/L) in the reaction mixture the
Scheme 1. Reagents and conditions: (i) BSA, 1,8-dibromooctane, CH3CN, 75 °C, 3 h;
(ii) P(Oi-Pr)3, 160 °C, 6 h; (iii) NaH, BOM-Cl, DMF, 75 °C, 5 h; (iv) TosMIC,
DMSO–dioxane (1:4), 75 °C, 6 h; (v) H2/Pd, MeOH, overnight; (vi) (1) Me3SiBr, CH3CN,
overnight; (2) NH3/H2O.
IC50 are about 50% higher (data not shown).
In the literature,2 the capability of the parent compound 1 to in-
hibit TP from E. coli is reported as a 97%, 83%, and 48% inhibition at
1000, 100, and 20 lM concentrations in 20 min of incubation time.
The final deprotection of 13 by catalytic hydrogenation followed by
a treatment with TMSBr afforded the acyclic nucleoside phospho-
nate 8.
All of the new compounds were fully characterized on the basis
of their 1H and 13C NMR spectra and further spectroscopic and ana-
lytical data.
The inhibitory potency of 8-aza-7,9-dideazaxanthine acyclic
nucleoside phosphonates 2 and 8, ‘double-headed’ nucleoside 12,
and the parent compound 1 on the phosphorolysis of thymidine
was evaluated using two recombinant TPs of a commercial origin
(TP from E. coli and human TP expressed in V79 Chinese hamster
cells) and the enzyme isolated from human placenta. The TP from
the human placenta was purified according to Yoshimura et al.7
The values roughly correspond to the IC50 for compound 1 given in
Table 1. The inhibition of TP from other sources was not reported.
Table 1 shows that, on TP from E. coli, the inhibition capability of
the new compounds 2, 8, and 12 is quite comparable with that
for the parent compound 1. On human enzymes, the IC50 values
of compound 2 are still comparable with those for 1, but the highly
modified derivatives 8 and 12 are less active.
In conclusion, the modification of the parent compound 1 lead-
ing to the acyclic nucleoside 2, which differs by the position of the
pyrrole nitrogen, only slightly influences the degree of TP inhibi-
tion. When the above-mentioned structural change is accompanied
by a different substitution of the nucleobase with the 8-phospho-
nooctyl chain (compound 8), the inhibition potency on the TP from
E. coli still remains, but on the human enzymes it decreases.
with
a minor modification (additional chromatography on
O
O
BOM
O
BOM
O
i
ii
N
N
N
NH
85%
N
BOM
N
BOM
9
O
O
O
N
BOM
O
BOM
BOM
O
N
N
N
N
(CH2)8 Br
65%
(CH2)8
N
+
N
BOM
O
N
10
BOM
11
BOM
14%
89%
O
iv
iii
53%
O
O
BOM
O
O
N
N
HN
NH
(CH2)8
P
Oi-Pr
Oi-Pr
N
(CH2)8
N
N
BOM
N
H
O
N
H
O
12
v
13
O
20%
O
P
OH
HN
N
(CH2)8
OH
O
N
H
8
Scheme 2. Reagents and conditions: (i) TosMIC, NaH, DMSO–Et2O (1:4), 6 h; (ii) NaH, 1,8-dibromooctane, DMF, 95 °C, 3 h; (iii) H2/Pd, MeOH, overnight; (iv) P(Oi-Pr)3, 160 °C,
6 h; (iv) (1) H2/Pd, MeOH, overnight; (2) Me3SiBr, CH3CN, overnight; (3) NH3/H2O.

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D. Marák et al. / Bioorg. Med. Chem. Lett. 21 (2011) 652–654
654
Table 1
The inhibition of thymidine phosphorylase from different sources by test compounds
Inhibition of TPase (IC50
Compound
[l
M])a
1b
2
8
12
3.5
TP from Escherichia coli
Human recombinant TP expressed in V79
TP purified from human placenta
11.4
7.3
15.1
6.8
12
27
17
NDc
NDc
23
NDc
a
b
c
The inhibition data represent the mean of at least three independent experiments.
Parent compound.2
Not determined. If the compound in a concentration of 10 lM inhibits the enzyme from less than 20% (V/V0), the
IC50 value was not determined.
Paradoxically, when the third modification—replacement of the
phosphonate function by a further 8-aza-7,9-dideazaxanthine
nucleobase—is made (compound 12), the inhibitory capability in-
creases again. The results revealed that there is a considerable
scope for the further development of new multisubstrate inhibitors
of TP possessing an 8-aza-7,9-dideazaxanthine nucleobase.
The in vitro cell growth inhibition by compounds 1, 2, 8, and 12
was evaluated in the following cell lines: CCRF-CEM T-lymphoblas-
toid cells (human acute lymphoblastic leukemia, ATCC CCL 119),
human promyelocytic leukemia HL-60 cells (ATCC CCL 240), hu-
man cervix carcinoma HeLa S3 cells (ATCC CCL 2.2), and mouse
lymphocytic leukemia L1010 cells (ATCC CCL 219); no antitumor
activity was found.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.027.
References and notes
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Acknowledgments
5. 1H NMR (D2O) 7.42 (d, 1H, J(7,5) = 1.8 Hz, H-7), 6.67 (d, 1H, J(5,7) = 1.8 Hz, H-5),
3.70 (t, 2H, J(1,2) = 7.3 Hz, H-10), 1.44–1.62 and 1.20–1.34 (m, H-20–H-80). 13C
NMR (APT, D2O) 165.8 (C-4), 154.8 (C-2), 131.6 (C-4b), 121.30 (C-7), 107.2 (C-
4a), 104.5 (C-5), 47. 7 (C-10), 32.8 (d, J(7,P) = 16.2 Hz, C-70), 31.1, 31.0, 30.9 (C-20,
C-30, C-40, C-50), 29.5 (J(80,P) = 132.9 Hz, C-80), 25.8 (C-60). 31P NMR (D2O): 27.7.
HRMS (ESI) calcd for C14H22N3O5P (M+H): 344.1375, found: 344.1375.
6. Kudu, N. G.; Khatri, S. G. Synthesis 1985, 323.
This work is a part of the research project of the Institute No.
Z40550506. It was supported by the Centre for New Antivirals and
Antineoplastics 1M0508 by the Ministry of Education, Youth and
Sports of the Czech Republic and by Gilead Sciences, Inc. (Foster City,
USA.). The authors’ thanks are due to Dr. P. Fiedler for the IR spectra,
7. Yoshimura, A.; Kuwazuru, Y.; Furukawa, T.; Yoshida, H.; Yamada, K.; Akiyama, S.
Biochim. Biophys. Acta 1990, 1013, 107.
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the staff of the mass spectroscopy (Dr. J. Cvacka, Head) and analytical
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departments (Dr. S. Matejková, Head) of the Institute.