Synthesis of certain 2'-deoxyuridine derivatives containing substituted phenoxy groups attached to C-5'; evaluation as potential dUTP analogues.

Article abstract of DOI:10.1081/NCN-100105905

Derivatives of 2'-deoxyuridine in which the 5'-OH group is replaced by a 2,3,6-trifluoro-5-hydroxy-4-nitrophenoxy or a 4-carboxy-2,3,6-trifluoro-5-hydroxyphenoxy group have been prepared for evaluation as possible dUTP analogues. They showed a weak abilit

Full text of DOI:10.1081/NCN-100105905

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SYNTHESIS OF CERTAIN 2′-DEOXYURIDINE DERIVATIVES
CONTAINING SUBSTITUTED PHENOXY GROUPS
ATTACHED TO C-5′; EVALUATION AS POTENTIAL dUTP
ANALOGUES
Jonathan H. Marriott a , G. Wynne Aherne a , Anthea Hardcastle a & Michael Jarman b
a Cancer Research Campaign Centre for Cancer Therapeutics at the Institute of Cancer
Research , 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UK
b Cancer Research Campaign Centre for Cancer Therapeutics at the Institute of Cancer
Research , 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UK
Published online: 21 Aug 2006.
To cite this article: Jonathan H. Marriott , G. Wynne Aherne , Anthea Hardcastle & Michael Jarman (2001) SYNTHESIS OF
CERTAIN 2′-DEOXYURIDINE DERIVATIVES CONTAINING SUBSTITUTED PHENOXY GROUPS ATTACHED TO C-5′; EVALUATION AS
POTENTIAL dUTP ANALOGUES, Nucleosides, Nucleotides and Nucleic Acids, 20:9, 1691-1704, DOI: 10.1081/NCN-100105905
To link to this article: http://dx.doi.org/10.1081/NCN-100105905
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(9), 1691À1704 (2001)
SYNTHESIS OF CERTAIN 20-DEOXYURIDINE
DERIVATIVES CONTAINING SUBSTITUTED
PHENOXY GROUPS ATTACHED
TO C-50; EVALUATION AS POTENTIAL
dUTP ANALOGUES
Jonathan H.Marriott, G.Wynne Aherne, Anthea Hardcastle,
and Michael Jarman*
Cancer Research Campaign Centre for Cancer Therapeutics
at the Institute of Cancer Research, 15 Cotswold Road, Sutton,
Surrey SM2 5NG, UK
ABSTRACT
Derivatives of 20-deoxyuridine in which the 50-OH group is replaced by a
2,3,6-trifluoro-5-hydroxy-4-nitrophenoxy or a 4-carboxy-2,3,6-trifluoro-
5-hydroxyphenoxy group have been prepared for evaluation as possible
dUTP analogues. They showed a weak ability to displace radiolabelled
dUTP from a dUTP-binding antiserum. The corresponding compounds
lacking the three fluorine substituents were prepared for comparison.
INTRODUCTION
We have speculated that the 2,3,6-trifluoro-5-hydroxy-4-nitrophenoxy
and 4-carboxy-2,3,6-trifluoro-5-hydroxyphenoxy groups (1 and 2) may have
some di- and/or tri-phosphate-like properties, such that compounds corre-
sponding to natural substrates of di- or tri-phosphate-utilising enzymes,
*Corresponding author.
1691
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1692
MARRIOTT ET AL.
with the di=tri-phosphate group replaced by the group 1 or 2, might in
some instances prove to be inhibitors of the relevant enzymes. Following
preliminary reports involving compounds containing the group 1 or 2
attached to isoprenoid groups1, this paper describes the synthesis and
biochemical evaluation of compounds 3 and 4, in which these groups are
attached to C-50 of 20-deoxyuridine, in place of the 50-OH group2a. We
required these compounds for testing 1) for ability to displace radiolabelled
dUTP from a dUTP-binding antiserum, and 2) for ability to inhibit the
activity of the enzyme 20-deoxyuridine 50-triphosphate nucleotidohydrolase
(dUTPase), which catalyses the hydrolysis of dUTP to dUMP and inor-
ganic pyrophosphate. We regarded these assays as a means of evaluating
possible triphosphate mimicking ability associated with the groups 1 and 2;
the dUTPase inhibition assay was carried out for the additional reason that
dUTPase has been suggested as a possible target for design of therapeutic
agents against cancer3. In order to determine the effect of the fluorination
within the groups 1 and 2 on the properties observed in the assays, we also
prepared and evaluated the unfluorinated analogues of 3 and 4, i.e. com-
pounds 5 and 6.
SYNTHESIS
The four desired compounds 3À6 were each prepared from 30-O-(4-
methoxytetrahydropyran-4-yl)-20-deoxyuridine (7)4. The synthesis of com-
pound 3, which contains a 2,3,6-trifluoro-5-hydroxy-4-nitrophenoxy group
replacing the 50-OH of 20-deoxyuridine, is shown in Scheme 1. Compound 7
reacted with pentafluoronitrobenzene under liquid-liquid phase transfer
catalysis conditions5 to give the 50-O-aryl derivative 8. Aqueous sodium
hydroxide (0.1 M at room temperature) effected substitution of one of the
fluorines ortho to the nitro group to give 9, which was deprotected with
aqueous acetic acid, producing 3.
Compound 5, the unfluorinated analogue of 3, was produced
according to Scheme 2. A Mitsunobu reaction between compound 7 and 3-
fluoro-4-nitrophenol gave compound 10, and this was converted to the
phenol 11 with aqueous sodium hydroxide; a longer reaction time and more
concentrated sodium hydroxide (1.5 M) were used in this case than for the
analogous transformation of 8 into 9. Deprotection of 11 gave the desired
compound 5.
The synthesis of compound 4, which contains a 4-carboxy-2,3,6-tri-
fluoro-5-hydroxyphenoxy group replacing the 50-OH of 20-deoxyuridine,
employed compound 13, which may be synthesised from 2,3,5,6-tetrafluoro-
4-hydroxybenzoic acid (12)2b. Mitsunobu etherification between compounds
13 and 7 (Scheme 3), followed by removal of the Mthp group, gave the
crystallisable intermediate 14. Aqueous sodium hydroxide (0.1 M) rapidly

20-DEOXYURIDINE DERIVATIVES
1693
Scheme 1. i) pentafluoronitrobenzene, Bun4NþHSO4 À , 1 M aq. NaOH, CH2Cl2, 37.5%; ii)
0.1 M aq. NaOH, 90.5%; iii) AcOH-H2O, 85%.
removed the methylene protecting group from 14, and compound 4 was
isolated after neutralisation.
For the synthesis of compound 6, the unfluorinated analogue of 4,
the intermediate 17 was required; this was prepared by a reaction6
between phenyl 2,4-dihydroxybenzoate (16)7 and paraformaldehyde using
Dabco (Scheme 4). A Mitsunobu reaction between 17 and 7 gave the
protected intermediate 18. To obtain the desired compound 6 from 18, we
chose to remove the methylene protecting group first, then the Mthp
group (Scheme 4).
BIOCHEMICAL ASSAYS
dUTP Radioimmunoassay. This assay8 may be used to compare a
compound with dUTP, with respect to ability to displace radiolabelled
dUTP from an antiserum that binds dUTP (the antiserum was raised to a
dUTP-protein conjugate). Radiolabelled dUTP was mixed with a limited
amount of dUTP antiserum and various amounts of unlabelled dUTP, and
after incubation, antiserum-bound and unbound ligand were separated by
addition of dextran-coated charcoal and centrifugation. The radioactivity in
the supernatant (bound fraction) was measured by liquid scintillation

1694
MARRIOTT ET AL.
Scheme 2. i) 3-fluoro-4-nitropohenol, DIAD, PPh3, THF, 38%; ii) 1.5 M aq. NaOH, 85%;
iii) AcOH-H2O, 83%.
counting. In the Figure, the black squares (j) plot shows the effect of the
amount of unlabelled dUTP added on B=B0, where B is the amount of
bound radiolabel, and B0 is the amount of bound radiolabel when no
unlabelled dUTP was added. The other plots in the Figure show
corresponding results obtained when compounds 3À6 were added in
place of unlabelled dUTP, and, for comparison, results for addition of
UTP in place of unlabelled dUTP. Percent cross-reactivity for each
compound was calculated as the ratio of the concentration of compound
and unlabelled dUTP, required to reduce the amount of bound radiolabel
(in the absence of unlabelled dUTP) by 50% or by 30%. The Table shows
calculated percentage cross-reactivity figures for compounds 3À6 and UTP
at 50% and 30% displacement, and the percentage B=B0 value in the
presence of 20000 pmol of each compound. The antiserum did not have a
Scheme 3. i) 13, DIAD, PPh3, THF; ii) AcOH-H2O, 61.5% overall from 7; iii) 0.1 M aq.
NaOH; iv) AcOH; v) Bio-Rad AG50W X-4 (Hþ form), 81% overall from 14.

20-DEOXYURIDINE DERIVATIVES
1695
Scheme 4. i) PhOH, POCl3, PhMe, 45%; ii) paraformaldehyde, Dabco, CHCl3, 31.5%; iii)
17, DEAD, PPh3, THF, 33.8%; iv) 0.05 M aq. NaOH; v) AcOH-H2O, 80% overall from 18.
high cross-reactivity with any of the compounds 3À6. Cross-reactivity with
the non-fluorinated compounds was slightly less than with their fluorinated
counterparts. Cross-reactivity with the carboxylic acids was slightly greater
than with the nitro compounds, in each pair of compounds. Cross-reactivity
was greatest with compound 4, but was still 65À100-fold less than with
UTP. The 50-substituents in 4 and 6 may be compared with the salicyl
Figure. Cross-reaction of dUTP-binding antiserum with compounds 3À6 and UTP;
B ¼ amount of bound radiolabel and B0 ¼ amount of bound radiolabel when no unlabelled
dUTP added.

1696
MARRIOTT ET AL.
Table. Percentage Cross-Reactivity of Compounds 3À6 and UTP with dUTP-Binding
Antiserum; B ¼ Amount of Bound Radiolabel and B0 ¼ Amount of Bound Radiolabel When
No Unlabelled dUTP Added; ne ¼ Not Evaluable
Percentage Cross-reactivity Compared
to dUTP
% B=B0 in Presence of 20000
pmol Added Compound
@ 50% binding
@ 70% binding
Compound
dUTP
UTP
3
4
5
6
100
0.13
ne
0.002
ne
ne
100
0.4
0.001
0.004
ne
0
0
61.4
46.3
85.1
69.5
0.0006
group present in certain EGF receptor-associated protein tyrosine kinase
inhibitors9. The carboxyl together with the hydroxyl group have been
proposed in this context as superimposable on oxygen atoms of the g
and b phosphoryl groups of ATP. It may be speculated that the analogous
substituents in 4 and 6 perform a similar role in relation to dUTP and in
the present context it is noteworthy that the inclusion of the fluorine atoms
appears beneficial.
dUTPase Activity Assay10. None of the compounds 3À6 was found to
inhibit the activity of dUTPase.
EXPERIMENTAL
1H NMR spectra were obtained at 250 MHz using the residual
undeuterated solvent signal as internal standard, on a Bruker spectrometer.
19F spectra were obtained at 235 MHz on the same instrument, and chemical
shifts are relative to FCCl3 (dF ¼ 0). ESI mass spectra were obtained on a
Finnigan MAT TSQ700 triple quadrupole mass spectrometer or a Finnigan
LCQ ion trap mass spectrometer. FAB mass spectra were obtained at the
School of Pharmacy, London University. The IR spectrum of 17 was
obtained with a Perkin–Elmer 1720X spectrometer. Melting points were
determined with a Reichert micro-hot-stage apparatus, and are uncorrected.
Elemental analyses were carried out by C.H.N. Analysis Ltd, Leicester,
England. Preparative reactions were monitored by TLC using silica-coated
glass plates (Merck), visualised under UV light.

20-DEOXYURIDINE DERIVATIVES
1697
30-O-(4-Methoxytetrahydropyran-4-yl)-50-O-(2,3,5,6-tetrafluoro-4-nitro
phenyl)-20-deoxyuridine (8). Aqueous NaOH solution (1 M; 5 ml) was added
to a rapidly stirred, cooled (water bath at 10 ꢀC) mixture of 30-O-(4-
methoxytetrahydropyran-4-yl)-20-deoxyuridine (7)4 (0.344 g, 1 mmol),
pentafluoronitrobenzene
(0.220 g,
1 mmol),
tetra-n-butylammonium
hydrogensulfate (0.342 g, 1 mmol) and CH2Cl2 (5 ml). After 40 min, further
pentafluoronitrobenzene (0.110 g, 0.52 mmol) was added, and after a further
20 min, CH2Cl2 (45 ml) was added. The CH2Cl2 layer was separated and the
aqueous layer diluted with water (15 ml) and extracted with CH2Cl2
(465 ml). The combined CH2Cl2 solution was washed with water
(3610 ml) and dried (MgSO4), then evaporated after addition of silica
(Merck 7734; 2.7 g). The impregnated silica was applied to a column
of silica (Merck 9385). Elution with CH2Cl2-MeOH (95:5) followed
by evaporation of appropriate fractions, crystallisation (MeCN) of the
residue, and recrystallisation gave 8 (0.202 g, 37.5%), mp 163À165 ꢀC;
dH[(CD3)2SO] 1.75 (m, 4H, Mthp 3,5-H), 2.33 (m, 2H, 20-H), 3.15 (s, 3H,
Me), 3.48, 3.62 (26m, each 2H, Mthp 2,6-H), 4.24 (m, 1H) and 4.58 (m, 3H)
(30,40,50-H), 5.63 (d, J ¼ 8.2 Hz, 1H, 5-H), 6.18 (t, J ¼ 6.8 Hz, 1H, 10-H), 7.63
(d, J ¼ 8.1 Hz, 1H, 6-H), 11.36 (s, 1H, N-H); dF[(CD3)2SO]À155.13 (m, 2F),
À147.01 (m, 2F); HRMS (FAB) 536.1270; calcd for C21H22F4N3O9
[(MþH)þ], 536.1292; Found: C, 47.05; H, 3.90; N, 7.95; F, 14.06;
C21H21F4N3O9 requires C, 47.11; H, 3.95; N, 7.85; F, 14.19%.
30-O-(4-Methoxytetrahydropyran-4-yl)-50-O-(2,3,6-trifluoro-5-hydroxy-4-
nitro-phenyl)-20-deoxyuridine (9). Compound 8 (0.122 g, 0.23 mmol) was
stirred with aqueous NaOH solution (0.1 M; 11.5 ml) at room temperature.
The reaction could be monitored by TLC with CH2Cl2-MeOH (4:1) or
PriOH-aq. NH3 (d 0.88)-H2O (7:1:2). After 44 h the solution was cooled in
ice and adjusted to pH 5 with acetic acid whilst being stirred. The resulting
suspension was concentrated to ca. 10 ml then cooled, and the solid which
had separated was collected, washed with cold water, and dried to give 9
(0.110 g, 90.5%), mp 156À158 ꢀC; dH[(CD3)2SO] 1.74 (m, 4H, Mthp 3,5-H),
2.31 (m, 2H, 20-H), 3.15 (s, 3H, Me), 3.48 (m, overlaps HOD peak) and 3.62
(m, 2H) (Mthp 2,6-H), 4.22 (m, 1H) and 4.51 (m, 3H) (30,40,50-H), 5.62 (dd,
J ¼ 2.1, 8.1 Hz, 1H, 5-H), 6.19 (t, J ¼ 6.8 Hz, 1H, 10-H), 7.63 (d, J ¼ 8.1 Hz,
1H, 6-H), 11.35 (s, 1H, N-H); dF[(CD3)2SO] À 163.48 (m, 1F), À 151.85 (m,
2F); ESI MS ( À ve ion mode) m=z 532.1 [(M À H)À ]; HRMS (FAB)
534.1337; calcd for C21H23F3N3O10 [(MþH)þ], 534.1336; Found: C, 47.05;
H, 4.04; N, 7.85; C21H22F3N3O10 requires C, 47.29; H, 4.16; N, 7.88%.
50-O-(2,3,6-Trifluoro-5-hydroxy-4-nitrophenyl)-20-deoxyuridine (3). Com-
pound 9 (0.090 g, 0.169 mmol), acetic acid (2 ml) and water (0.5 ml) were stirred
together at 23 ꢀC. After 15 h the mixture was evaporated and the solid residue
triturated with Et2O to give a solid (0.071 g). Part (0.045 g) of this material was

1698
MARRIOTT ET AL.
crystallised from MeOH-H2O to give 3 (0.039 g, 85%), mp 178À179 ꢀC;
dH[(CD3)2SO] 2.19 (m, 2H, 20-H), 4.05 and 4.33 (26m, each 1H, 30-H, 40-H),
4.47 (m, 2H, 50-H), 5.48(br, 1H, OH), 5.61(dd, J ¼ 2.1, 8.1 Hz, 1H, 5-H), 6.20 (t,
J ¼ 6.9 Hz, 1H, 10-H), 7.63 (d, J ¼ 8.1 Hz, 1H, 6-H), 11.34 (s, 1H, N-H);
dF[(CD3)2SO] À 163.22 (m, 1F), À 151.76 (m, 2F); ESI MS ( À ve ion mode)
m=z 418.1 [(M-H)À ]; HRMS (FAB) 420.0640; calcd for C15H13F3N3O8
[(MþH)þ], 420.0655; Found: C, 41.85; H, 2.92; N, 9.74; F, 13.33;
C15H12F3N3O8ꢁ0.5H2O requires C, 42.07; H, 3.06; N, 9.81; F, 13.31%.
50-O-(3-Fluoro-4-nitrophenyl)-30-O-(4-methoxytetrahydropyran-4-yl)-20-
deoxyuridine (10). Diisopropyl azodicarboxylate (0.392 g, 1.9 mmol) was
added dropwise during 5 min to a stirred, cooled (ice-water bath) solution
of 30-O-(4-methoxytetrahydropyran-4-yl)-20-deoxyuridine (7)4 (0.516 g,
1.5 mmol), 3-fluoro-4-nitrophenol (0.262 g, 1.67 mmol) and triphenylphos-
phine (0.47 g, 1.8 mmol) in dry THF (9 ml) under N2. The mixture was then
allowed to warm to room temperature. After 19 h the mixture was
concentrated and the residue chromatographed [Merck 7729 silica;
CH2Cl2-EtOH (100:0 to 95:5)]; further purification by
a
second
chromatography column [Merck 7729 silica; CH2Cl2-EtOH (100:0 to 97:3)]
gave a white solid which was crystallised from MeCN to give 10 (0.276 g,
38%), mp 206–208 ꢀC (remelting of resolidified crystals); dH[(CD3)2SO] 1.74
(m, 4H, Mthp 3,5-H), 2.32 (m, 2H, 20-H), 3.14 (s, 3H, Me), 3.47, 3.62 (26m,
each 2H, Mthp 2,6-H), 4.24 (m, 1H, 30-H or 40-H), 4.34 (m, 2H, 50-H), 4.56
(m, 1H, 40-H or 30-H), 5.65 (d, J ¼ 8.1 Hz, 1H, 5-H), 6.19 (t, J ¼ 6.8 Hz, 1H,
10-H), 7.02 (dt, J ¼ 1.2, 9.3 Hz, 1H, aryl H), 7.25 (dd, J ¼ 2.6, 13.6 Hz, 1H,
aryl H), 7.64 (d, J ¼ 8.0 Hz, 1H, 6-H), 8.16 (t, J ¼ 9.2 Hz, 1H, aryl H), 11.31
(s, 1H, N-H); dF[(CD3)2SO] À 113.86 (t, J ¼ 11.7 Hz); HRMS (FAB)
482.1562; calcd for C21H25FN3O9 [(MþH)þ], 482.1575; Found: C, 52.21;
H, 4.97; N, 8.71; F, 3.93; C21H24FN3O9 requires C, 52.39; H, 5.02; N, 8.73; F,
3.95%.
50-O-(3-Hydroxy-4-nitrophenyl)-30-O-(4-methoxytetrahydropyran-4-yl)-
20-deoxyuridine (11). Compound 10 (0.130 g, 0.27 mmol) was stirred with
aqueous NaOH solution (1.5 M; 3.6 ml) at room temperature for 14 days [this
reaction may be followed by TLC with PriOH À aq. NH3 (d 0.88) À H2O
(7:1:2)]. The resulting solution was cooled in ice and acetic acid (0.33 ml) was
added with stirring. The solid which separated was collected, washed with
water, dried, and crystallised from EtOH to give 11 (0.110 g, 85%) as a
yellow solid, mp 172À174 ꢀC; dH[(CD3)2SO-D2O] 1.75 (m, 4H, Mthp 3,5-H),
2.34 (m, 2H, 20-H), 3.13 (s, 3H, Me), 3.48, 3.61 (26m, each 2H, Mthp 2,6-H),
4.25 (m, 3H, 50-H and 30-H or 40-H), 4.55 (m, 1H, 40-H or 30-H), 5.65 (d,
J ¼ 8.1 Hz, 1H, uracil 5-H), 6.18 (t, J ¼ 6.7 Hz, 1H, 10-H), 6.66 (m, 2H, aryl
2,6-H), 7.66 (d, J ¼ 8.1 Hz, 1H, uracil 6-H), 7.98 (d, J ¼ 9.1 Hz, 1H, aryl 5-H);
before addition of D2O, additional signals [10.94 (br s, 1H) and 11.32 (s, 1H)

20-DEOXYURIDINE DERIVATIVES
1699
(OH and N-H)] were apparent; ESI MS ( À ve ion mode) m=z 478.1
[(M À H)À ]; HRMS (FAB) 502.1454; calcd for C21H25N3NaO10
[(MþNa)þ], 502.1438; Found: C, 52.40; H, 5.26; N, 8.68; C21H25N3O10
requires C, 52.61; H, 5.26; N, 8.76%.
50-O-(3-Hydroxy-4-nitrophenyl)-20-deoxyuridine (5). Compound 11
(0.072 g, 0.15 mmol), acetic acid (4 ml) and water (1 ml) were stirred
together at room temperature. After 19 h the solution was evaporated to
dryness and the residue triturated with Et2O. The resulting solid was
collected after cooling, washed with further Et2O, and crystallised from
water to give 5 (0.047 g, 83%), mp 120À122 ꢀC; dH[(CD3)2SO] 2.21 (m, 2H,
20-H), 4.07 (m, 1H) and 4.26 (m, 3H) (30,40,50-H), 5.44 (d, J ¼ 4.3 Hz, 1H, 30-
OH), 5.63 (m, 1H, reduced to d, J ¼ 8.1 Hz, 1H, on addition of D2O, uracil 5-
H), 6.20 (t, J ¼ 6.7 Hz, 1H, 10-H), 6.66 (m, 2H, aryl 2,6-H), 7.66 (dd, J ¼ 1.2,
8.2 Hz, 1H, reduced to d, J ¼ 8.1 Hz, 1H, on addition of D2O, uracil 6-H),
7.97 (m, 1H, reduced to d, J ¼ 9.4 Hz, 1H, on addition of D2O, aryl 5-H),
10.93 (s, 1H) and 11.28 (s, 1H) (aryl OH and N-H); ESI MS ( À ve ion mode)
m=z 363.9 [(M À H)À ]; HRMS (FAB) 366.0947; calcd for C15H16N3O8
[(MþH)þ], 366.0937; Found: C, 47.53; H, 4.16; N, 11.08;
C15H15N3O8ꢁ0.75H2O requires C, 47.56; H, 4.39; N, 11.09%.
50-O-(4-Oxo-5,6,8-trifluoro-4H-1,3-benzodioxin-7-yl)-20-deoxyuridine
(14). Diisopropyl azodicarboxylate (DIAD) (0.066 g, 0.33 mmol) was added
dropwise during 5 min to a stirred, cooled (ice-water bath) solution of 30-O-
(4-methoxytetrahydropyran-4-yl)-20-deoxyuridine (7)4 (0.092 g, 0.27 mmol),
5,6,8-trifluoro-7-hydroxy-4H-1,3-benzodioxin-4-one
(13)2b
(0.059 g,
0.27 mmol) and triphenylphosphine (0.087 g, 0.33 mmol) in dry THF (2 ml)
under argon. The mixture was then allowed to warm to room temperature.
After 2 h, further triphenylphosphine (0.028 g, 0.11 mmol) and DIAD
(0.022 g, 0.11 mmol) were added, and after 20 h (total) the mixture was
concentrated. A portion of CH2Cl2 was added and evaporated and the
residue chromatographed [Merck 7729 silica; CH2Cl2-EtOH (100:0, 98:2
and 97:3 in succession)] to give a colourless glass (0.115 g); dH[(CD3)2SO]
1.75 (m, 4H, Mthp 3,5-H), 2.33 (m, 2H, 20-H), 3.15 (s, 3H, Me), 3.48, 3.62
(26m, each 2H, Mthp 2,6-H), 4.23 (m, 1H) and 4.59 (m, 3H) (30,40,50-H),
5.61 (d, J ¼ 8.2 Hz, 1H, 5-H), 5.89 (s, 2H, O-CH2-O), 6.18 (t, J ¼ 6.8 Hz, 1H,
10-H), 7.63 (d, J ¼ 8.2 Hz, 1H, 6-H), 11.31 (s, 1H, N-H); dF[(CD3)2SO]
À 159.17 (d, J ¼ 22.9 Hz, 1F), À 155.07 (d, J ¼ 10.8 Hz, 1F), À 139.38 (dd,
J ¼ 11.8, 22.0 Hz, 1F); HRMS (FAB) 567.1230; calcd for C23H23F3N2NaO10
[(MþNa)þ], 567.1202. A solution of this material (0.105 g) in acetic acid
(2 ml) and water (0.5 ml) was stirred at room temperature. After 23 h the
solution was evaporated and portions of PhMe-EtOH (1:1; 4610 ml) were
added to the residue and evaporated successively. The resulting white solid
was triturated with Et2O, then crystallised from EtOH to give 14 (0.065 g,

1700
MARRIOTT ET AL.
61.5% overall from 7), mp 195À197 ꢀC; dH[(CD3)2SO] 2.22 (m, 2H, 20-H),
4.07 and 4.35 (26m, each 1H, 30-H, 40-H), 4.62 (m, 2H, 50-H), 5.51 (br s,
OH), 5.61 (d, J ¼ 8.1 Hz, 1H, 5-H), 5.90 (s, 2H, O-CH2-O), 6.20 (t, J ¼ 6.8 Hz,
1H, 10-H), 7.63 (d, J ¼ 8.1 Hz, 1H, 6-H), 11.35 (s, 1H, N-H); dF[(CD3)2SO]
À 159.00 (d, J ¼ 21.2 Hz, 1F), À 154.90 (d, J ¼ 11.8 Hz, 1F), À 139.31 (dd,
J ¼ 11.8, 21.2 Hz, 1F); HRMS (FAB) 431.0718; calcd for C17H14F3N2O8
[(MþH)þ], 431.0702; Found: C, 47.29; H, 3.03; N, 6.42; F, 13.11;
C17H13F3N2O8 requires C, 47.45; H, 3.05; N, 6.51; F, 13.25%.
50-O-(4-Carboxy-2,3,6-trifluoro-5-hydroxyphenyl)-20-deoxyuridine
(4). Compound 14 (0.048 g, 0.11 mmol) was stirred with aqueous NaOH
solution (0.1 M; 3.3 ml) at room temperature. After 40 min the solution was
diluted with MeOH (5 ml) and water (5 ml), and acetic acid (0.069 g) was
added. The resulting slightly turbid solution was applied to a column (bed
volume 9 ml) of Bio-Rad AG50W-X4 100 À200 mesh cation exchange resin
(Hþ form) that had been packed in MeOH-H2O (1:1). MeOH-H2O (1:1)
was passed through the column until the emerging eluate was no longer
UV-absorbing. The product-containing fractions were combined and
concentrated to ca. 4 ml and the solid which separated was collected after
cooling the suspension, washed with cold water, and dried to give 4 (0.038 g,
81%), mp 240 ꢀC (decomp.); dH[(CD3)2SO-D2O] 2.19 (m, 2H, 20-H), 4.03 and
4.34 (26m, each 1H, 30-H, 40-H), 4.46 (m, 2H, 50-H), 5.60 (d, J ¼ 8.1 Hz, 1H,
5-H), 6.19 (t, J ¼ 6.8 Hz, 1H, 10-H), 7.63 (d, J ¼ 8.1 Hz, 1H, 6-H) [before
addition of D2O, additional signal at 11.33 (s, 1H, N-H) was apparent];
dF[(CD3)2SO] À 165.70 (m, 1F), À 156.84 (m, 1F), À 140.66 (m, 1F); ESI MS
( À ve ion mode) m=z 416.9 [(M À H)À ]; HRMS (FAB) 419.0685; calcd for
C16H14F3N2O8 [(MþH)þ], 419.0702; Found: C, 45.82; H, 3.05; N, 6.64; F,
13.41; C16H13F3N2O8 requires C, 45.94; H, 3.13; N, 6.70; F, 13.63%.
Phenyl 2,4-Dihydroxybenzoate (16)11. Phosphorus oxychloride (3.4 ml,
36.5 mmol) was added to a mixture of 2,4-dihydroxybenzoic acid (10.0 g,
64.9 mmol), phenol (68.0 g, 72.3 mmol), and dry PhMe (50 ml). The mixture
was stirred under Ar at 100 ꢀC (bath temperature) for 2 h, then boiled
under reflux (bath temperature ¼ 150 ꢀC) for 30 min. It was then allowed to
cool to room temperature and partitioned between CHCl3 (200 ml) and
water (200 ml). The aqueous layer was extracted with CHCl3 (4625 ml)
and the combined CHCl3 solution washed successively with water (100 ml)
and half-saturated brine (36100 ml), dried (MgSO4) and concentrated
thoroughly (water pump followed by oil pump). The solid residue was
crystallised from hexane-EtOAc to give 16 (6.769 g, 45%) as pale pink
crystals, mp 148À150 ꢀC (lit7a., 147À149 ꢀC); dH[(CD3)2SO] 6.37 (d,
J ¼ 2.2 Hz, 1H, dihydroxyphenyl 3-H), 6.46 (dd, J ¼ 2.0, 8.8 Hz, 1H,
dihydroxyphenyl 5-H), 7.28 (m, 3H) and 7.47 (m, 2H) (Ph), 7.88 (d,
J ¼ 8.8 Hz, 1H, dihydroxyphenyl 6-H), 10.39 (s, OH), 10.57 (s, OH);

20-DEOXYURIDINE DERIVATIVES
1701
HRMS (FAB) 231.0646; calcd for C13H11O4 [(MþH)þ], 231.0657. A sample
for analysis was recrystallised with a little silica (Merck 7734) being added to
the hot solution before filtration; Found (for recrystallised material): C,
67.54; H, 4.29; N, 0.00; C13H10O4 requires C, 67.82; H, 4.38; N, 0.00%.
7-Hydroxy-4H-1,3-benzodioxin-4-one (17). CHCl3 (2.75 ml) was added
to a stirred mixture of 16 (3.657 g, 15.9 mmol), paraformaldehyde (2.38 g)
and 1,4-diazabicyclo[2.2.2]octane (Dabco) (1.78 g, 15.9 mmol) at room
temperature. After 30 h, further Dabco (1.78 g) was added. The reaction
was followed by TLC with CH2Cl2-EtOAc (19:1). After 97 h (total time) the
mixture was partitioned between EtOAc (100 ml) and 10% w=w aqueous
citric acid solution (200 ml). The aqueous layer was filtered to remove
insoluble solid material, and extracted with EtOAc (5670 ml). The
combined EtOAc solution was washed successively with 10% w=w aqueous
citric acid solution (70 ml followed by 25 ml) and saturated brine (35 ml), then
dried (MgSO4) and evaporated. The residue was redissolved in EtOAc (50 ml)
and the solution evaporated after addition of silica (Merck 7734; 10 g).
Hexane was added and evaporated and the impregnated silica applied to a
column of silica (Merck 9385). Elution with hexane-EtOAc (3:2 and 1:1
in succession), evaporation of appropriate fractions, and crystallisation
(hexane-EtOAc) of the residue gave 17 (0.832 g, 31.5%), mp 128À130 ꢀC;
dH[(CD3)2SO] 5.73 (s, 2H, 2-H), 6.46 (d, J ¼ 2.2 Hz, 1H, 8-H), 6.66 (dd,
J ¼ 2.2, 8.7 Hz, 1H, 6-H), 7.72 (d, J ¼ 8.7 Hz, 1H, 5-H), 10.90 (s, OH);
n
max(KBr disc) 1614, 1699 cmÀ 1; ESI MS ( À ve ion mode) m=z 165.1
[(M À H)À ]; HRMS (FAB) 167.0342; calcd for C8H7O4 [(MþH)þ],
167.0344; Found: C, 57.63; H, 3.57; N, 0.01; C8H6O4 requires C, 57.84; H,
3.64; N, 0.00%.
30-O-(4-Methoxytetrahydropyran-4-yl)-50-O-(4-oxo-4H-1,3-benzodioxin-
7-yl)-20-deoxyuridine (18). Diethyl azodicarboxylate (0.249 g, 1.4 mmol)12
was added dropwise during 20 min to a stirred, cooled (water bath at
10 ꢀC) solution of 30-O-(4-methoxytetrahydropyran-4-yl)-20-deoxyuridine
(7)4 (0.500 g, 1.46 mmol), 17 (0.244 g, 1.47 mmol) and triphenylphosphine
(0.383 g, 1.46 mmol) in dry THF (8 ml) under argon. After a further 5 min,
the solution was allowed to warm to room temperature. After 27 h the
solution was evaporated and the residue chromatographed on silica (Merck
7729); higher RF materials were eluted with CH2Cl2 followed by CH2Cl2-
EtOH (98.5:1.5 then 98:2), and the desired product with CH2Cl2-EtOH
(97.5:2.5). Appropriate fractions were evaporated and the residual glass
was triturated with hexane to give 18 (0.242 g, 33.8%), which was used in
the next step; dH[(CD3)2SO] 1.76 (m, 4H, Mthp 3,5-H), 2.32 (m, 2H, 20-H),
3.14 (s, 3H, Me), 3.48, 3.62 (26m, each 2H, Mthp 2,6-H), 4.28 (m, 3H) and
4.55 (m, 1H) (30,40,50-H), 5.66 (d, J ¼ 8.1 Hz, 1H, uracil 5-H), 5.79 (s, 2H,
O-CH2-O), 6.20 (t, J ¼ 6.7 Hz, 1H, 10-H), 6.82 (m, 2H, benzodioxin 6,8-H),

1702
MARRIOTT ET AL.
7.67 (d, J ¼ 8.1 Hz, 1H, uracil 6-H), 7.82 (d, J ¼ 8.7 Hz, 1H, benzodioxin 5-
H), 11.35 (s, 1H, N-H); HRMS (FAB) 491.1651; calcd for C23H27N2O10
[(MþH)þ], 491.1666. Attempted crystallisation (MeOH) of a sample of this
material from another run gave an amorphous white solid, mp 142À144 ꢀC,
which was used for elemental analysis; Found: C, 56.21; H, 5.34; N, 5.69;
C23H26N2O10 requires C, 56.32; H, 5.34; N, 5.71%.
50-O-(4-Carboxy-3-hydroxyphenyl)-20-deoxyuridine (6). Compound 18
(0.168 g, 0.34 mmol) was stirred with aqueous NaOH solution (0.05 M;
34 ml) at room temperature. TLC [CHCl3-MeOH-AcOH (95:5:3)] after
10 min indicated complete conversion to a product of lower RF than 18.
After 25 min (total time), the solution obtained was cooled in ice and acetic
acid (0.1 ml) added; the pH was then found to be ca. 5. The mixture was
concentrated to ca. 3.9 ml , and further acetic acid (7.8 ml) added. The
resulting mixture was stirred at room temperature for 34 h, then
evaporated, and water (2 ml) was added to the residue and evaporated.
The residue was triturated with Et2O and dried. Water (2 ml) was added and
the resulting mixture heated briefly, then cooled in ice. The solid which
separated was collected, washed with cold water, and dried to give 6 (0.100 g,
80%), mp 205 ꢀC (decomp.); dH[(CD3)2SO] 2.20 (m, 2H, 20-H), 4.06 (m, 1H,
30- or 40-H), 4.21 (m, 2H, 50-H), 4.31 (m, 1H, 40- or 30-H), 5.45 (br s, 1H, 30-
OH), 5.63 (dd, J ¼ 2.1, 8.1 Hz, 1H, uracil 5-H), 6.21 (t, J ¼ 6.8 Hz, 1H, 10-H),
6.55 (m, 2H, aryl 2,6-H), 7.69 (m, 2H, aryl 5-H, uracil 6-H), 11.33 (s, 1H, N-
H), 11.51 (br s, 1H, aryl OH), 13.60 (br s, 1H, CO2H); ESI MS ( À ve ion
mode) m=z 363.1 [(M À H)À ]; HRMS (FAB) 365.0992; calcd for
C16H17N2O8 [(MþH)þ], 365.0985; Found: C, 52.50; H, 4.39; N, 7.65;
C16H16N2O8 requires C, 52.75; H, 4.43; N, 7.69%.
dUTP Radioimmunoassay. Compounds 3À6 were dissolved in PBS at
an initial concentration of 1 mg=ml. Various dilutions of these solutions were
included in a radioimmunoassay for dUTP8. The assay was carried out as
previously reported except that whole antiserum was used (R9 1=1000) and
dilutions and dispensing of reagents were carried out using a Multiprobe
(Canberra Packard).
dUTPase Activity Assay. Compounds 3À6 were tested for ability to
inhibit the activity of dUTPase. The dUTPase activity assay was based on a
reported method10. A cellular extract from the human lung carcinoma cell
line MOR, which has been shown to have high dUTPase activity, was
employed13. Tritiated dUTP was incubated with the cellular extract and
the product, tritiated dUMP, separated from substrate by TLC and
quantitated using a plate scanner. Compounds were assayed at 20, 50, 100,
and 200 mM.

20-DEOXYURIDINE DERIVATIVES
ACKNOWLEDGMENTS
1703
This work was supported by grants from the Cancer Research Campaign
(grant reference number SP2330=0201) and from Cancer Research Campaign
Technology. We thank B. Nutley, K. Goddard, M. Mohan, A. Hayes and A. Mirza
for ESI mass spectra and M. Cocksedge of the University of London Intercollegiate
Research Service for FAB mass spectra. We thank Dr S. Webley and Ms S. Welsh
for dUTPase activity assay results.
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1704
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Parker, F.; Mailliet, P.; Commerc¸
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deleterious to the yield of 18.
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Received September 4, 2000
Accepted February 20, 2001