Design, synthesis, and enzymatic evaluation of multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase

Article abstract of DOI:10.1021/jm9911377

A series of acyclic phosphonate derivatives of thymine has been synthesized and tested as multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase. The compounds synthesized include 1- (phosphonoalkyl)thymines with six to nine methyl

Full text of DOI:10.1021/jm9911377

J . Med. Chem. 2000, 43, 971-983
971
Design , Syn th esis, a n d En zym a tic Eva lu a tion of Mu ltisu bstr a te An a logu e
In h ibitor s of Esch er ich ia coli Th ym id in e P h osp h or yla se
Antonio Esteban-Gamboa,† J an Balzarini,‡ Robert Esnouf,‡ Erik De Clercq,‡ Mar´ıa-J ose´ Camarasa,† and
Mar´ıa-J esu´s Pe´rez-Pe´rez*,†
Instituto de Quı´mica Me´dica, C.S.I.C., J uan de la Cierva 3, 28006 Madrid, Spain, and Rega Institute for Medical Research,
Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received August 2, 1999
A series of acyclic phosphonate derivatives of thymine has been synthesized and tested as
multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase. The compounds
synthesized include 1-(phosphonoalkyl)thymines with six to nine methylenes (1-4, respectively);
1-[(Z)-4-phosphonomethoxy-2-butenyl]thymine (5) and its butyl and 2,3-cis-dihydroxybutyl
derivatives (6 and 7, respectively); 1-[(Z)-(4-(phosphonomethoxy)methoxy)-2-butenyl]thymine
(8) and also its butyl and 2,3-cis-dihydroxybutyl analogues (9 and 10); and 1-[((Z)-4-
(phosphonomethoxy)-2-butenoxy)methyl]thymine (11). Evaluation of these compounds against
E. coli revealed significant enzymatic inhibition by 2, 3, 4, 6, and 8 at a concentration of 1000
µM, 3 and 4 being the most potent. Replacement of the thymine base in 3 by 6-amino-5-
bromouracil and 7-deazaxanthine afforded compounds 12 and 13, which showed a pronounced
improvement of TPase inhibition, comparable to 7-deazaxanthine. When inorganic phosphate
was used as a variable substrate, compounds 12 and 13 displayed competitive kinetics with
respect to phosphate, indicating a direct interaction of these compounds with the phosphate
binding site. Also compounds 12 and 13 were found to be competitive inhibitors of TPase against
thymidine as a variable substrate. These results are consistent with the compounds being
multisubstrate analogue inhibitors of E. coli TPase, and they represent the first example of
such TPase inhibitors.
Sch em e 1
In tr od u ction
Thymidine phosphorylase (TPase; EC 2.4.2.4) is one
of the key enzymes involved in salvage biosynthesis and
catabolism of pyrimidine 2′-deoxynucleosides. This en-
zyme catalyzes their reversible phosphorolysis with
formation of 2-deoxy-R-D-ribose-1-phosphate and the
pyrimidine, as shown in Scheme 1.
Thymidine phosphorylase recognizes not only thymi-
dine and 2′-deoxyuridine but also a variety of 5-substi-
tuted pyrimidine 2′-deoxynucleoside analogues that are
endowed with potent antiviral (i.e., 5-(E)-(bromovinyl)-
2′-deoxyuridine, 5-trifluoromethyl-2′-deoxyuridine, and
5-iodo-2′-deoxyuridine) and antitumor (i.e., 5-fluoro-2′-
deoxyuridine) activities.1 There has been a long-stand-
ing interest in the development of compounds inhibiting
TPase, since such compounds could be expected to result
in a considerable increase in the therapeutic effective-
ness of the above-mentioned nucleosides.1
However, most interest in TPase in recent years came
from the observation that TPase is identical to platelet-
derived endothelial cell growth factor (PD-ECGF), an
important factor involved in angiogenesis.2-4 Moreover,
it has been demonstrated that the enzymatic activity
of TPase/PD-ECGF is crucial for its angiogenic effect.5,6
Histological analyses of a variety of human tumors
have shown abnormally elevated TPase levels, and in
many cases these levels have been correlated with an
aggressive progression of the tumor.7 TPase levels have
also been correlated with microvessel density in breast,8
colon,9 and renal cell carcinomas,10 indicating that
TPase contributes to tumor vasculature. A very recent
study suggests that TPase may not only be involved in
angiogenesis but also in apoptosis.11 Therefore, there
is a need for TPase inhibitors to help clarify the precise
role of TPase in the progression of a variety of tumors.
Furthermore, such inhibitors could be used alone or in
combination with other antitumoral agents, including
TPase-sensitive nucleoside analogues, as therapeutic
agents.
Very few inhibitors of TPase have been reported, the
most potent being pyrimidine bases such as 6-amino-
5-bromouracil or 6-aminothymine12 and, very recently,
5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil.11 Last
year 7-deazaxanthine was reported by us as the first
purine derivative able to inhibit TPase.13,14
The structure of human TPase has not yet been
determined. To date, structure determinations are
limited to those of two prokaryotic homologues: Es-
cherichia coli TPase15,16 and Bacillus stearothermophilus
* To whom correspondence should be addressed. Tel: 91-562-29-
00. Fax: 34-91-564-48-53. E-mail: mjperez@iqm.csic.es.
† Instituto de Qu´ımica Me´dica.
‡ Rega Institute for Medical Research.
10.1021/jm9911377 CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/11/2000

972 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Esteban-Gamboa et al.
F igu r e 1. A stereo diagram showing the relantionship between the thymine and the phosphate binding sites based on the crystal
structure of E. coli thymidine phosphorylase (Walter et al.15). Thymine and the phosphate ion are shown by thick ball-and-stick
models, and the surrounding protein is represented by thinner ball-and-stick models. Nearby residues and stabilizing interactions
are shown. Figure was drawn using BobScript.39
pyrimidine nucleoside phosphorylase.17 These enzymes
have 40% sequence identity with human TPase and
with each other. However, the folds of the two bacterial
enzymes are similar, and moreover, surrounding the
catalytic site there is a particularly high degree of
sequence and structural conservation. Of the residues
near the catalytic site there is only one significant amino
acid difference between human and bacterial TPases:
residue 210 being either valine (human) or phenylala-
nine (bacterial). In other respects the catalytic sites of
the bacterial enzymes would be expected to provide good
models for human TPase. These bacterial enzyme
structures reveal TPase as a dimer of identical subunits.
Each subunit contains a large mixed R/â domain, where
the phosphate ion (Pi) binds, and a smaller R-helical
domain separated by a cleft which contains the nucleo-
side binding site. The two binding sites are separated
by 8-10 Å.15 It has been suggested that in the phos-
phorolytic reaction a domain movement must occur
causing the cleft to close to an active conformation,
bringing the Pi closer to the anomeric position of the
nucleoside.17 Therefore, there are two binding sites, one
for the phosphate and one for the 2′-deoxynucleoside.
The amino acids defining this second binding site (see
Figure 1) explain the specifity of TPase for thymine and
other 5-substituted uracil 2′-deoxynucleosides, but not
2′-deoxycytidine, based on the recognition pattern for
the 2-CO, 3-NH, and 4-CO of the pyrimidine base.
In our search for TPase inhibitors, in the present
paper it is described the design and synthesis of multi-
substrate inhibitors, i.e., compounds that contain in
their structure covalently linked elements of both
substrates. In particular, these elements are a phos-
phate analogue, able to interact at the phosphate
binding site, and a pyrimidine base, interacting at the
nucleoside binding site. The goal was to obtain com-
pounds able to “freeze” the conformation of the enzyme
in an opened, inactive conformation. The approach of
multisubstrate analogue inhibitors has led, among other
reports, to potent inhibitors of purine nucleoside phos-
phorylase.18 However, to the best of our knowledge, this
strategy has not been previously applied to TPase.
The proposed inhibitors combined three structural
elements: a phosphate analogue, a pyrimidine base, and
an appropiate spacer (Figure 2). As a phosphate ana-
logue, a phosphonate moiety has been chosen, which has
a tetrahedral structure similar to that of phosphate but
increased metabolic stability.19 As spacers, polymeth-
ylene chains of six to nine units were initially used
(compounds 1-4). Although these could be considered
as “too” flexible, they might provide an early test of the
validity of the working hypothesis. Second, spacers of
the same length were constructed by combining frag-
ments (in particular, (Z)-2-butene-1,4-diol and oxy-
methylene units) that impose conformational restriction
and/or can be easily transformed into other entities. The
(Z)-2-butene-1,4-diol, present in compounds 5, 8, and
11, can be easily transformed into the butyl (such as in
6 and 9) and 2,3-cis dihydroxybutyl analogues (com-
pounds 7 and 10). The hydroxyl groups introduced into
the spacers in 7 and 10 could give additional interac-
tions with amino acids present in the channel between
the two binding sites, such as His85 or Ser86. The
incorporation of oxymethylene units in the spacers not
only introduces a certain degree of conformational
restriction but also, being contiguous to the phospho-
nate, can affect its pKa value, making it closer to that
of the phosphate.20 For the pyrimidine base, thymine,
the natural substrate, was initially chosen. The infor-
mation obtained from the evaluation of the thymine-
spacer-phosphonate series (1-11) was used for the
synthesis of new series of compounds where thymine
was replaced by 6-amino-5-bromouracil and 7-deaza-
xanthine (compounds 12 and 13) and that are expected
to show a greater inhibitory potency against TPase. The
different synthetic strategies employed to obtain these
structures and the evaluation of the compounds against
E. coli TPase are described in detail in this paper.
Ch em istr y
The synthesis of the 1-(phosphonoalkyl)thymines (1-
4) was performed in three steps starting from thymine
(Scheme 2). Thus, reaction of the thymine sodium salt
with the corresponding dibromoalkane afforded the N1-

Multisubstrate Analogue Inhibitors of E. coli TPase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 973
F igu r e 2. Chemical structures of the proposed multisubstrate analogue inhibitors.
Sch em e 2a
The general strategy for the synthesis of compounds
5-11 (Figure 2) was the introduction of the oxymeth-
ylphosphonate moiety prior to the incorporation of the
thymine base, to avoid possible alkylations on the N3
position of thymine. The starting material, in all cases,
was (Z)-2-butene-1,4-diol, conveniently monoprotected
(23 and 29 in Schemes 3 and 4).
Compounds 5-7, with a six-membered spacer, were
synthesized as described in Scheme 3. Reaction of 2323
with diisopropyl[(p-toluenesulfonyl)oxy]methanephos-
phonate24 in the presence of NaH gave compound 24,
which was deprotected by treatment with 80% AcOH
to yield the alcohol 25 (44% yield from 23). Mitsunobu
reaction25 of this alcohol with N3-benzoylthymine,26
followed by in situ deprotection with methanolic MeNH2,
afforded the N1-substituted derivative 26 in 62% yield.
Reduction of the double bond in 26 by catalytic hydro-
genation afforded the saturated derivative 27. On the
other hand, treatment of 26 with OsO4 yielded the
enantiomeric mixture of 1,2-cis diols 28. Deprotection
of the phosphonate esters in 26, 27, and 28 was
performed by reaction with TMSBr, to afford compounds
5,27 6, and 7 (73, 61, and 50% yields, respectively).
a
(a) Thymine, NaH, DMF (40-33%); (b) P(OR)3 (86-58%); (c)
1. TMSBr, 2. H2O (81-67%).
substituted products (14-17).21,22 Although the yields
are moderate and lower than those in a previously
described procedure,22 the present method provides the
desired compounds in a “one pot” reaction, avoiding
tedious silylation of thymine. Compounds 14-17 were
heated with triisopropyl- or triethyl-phosphite according
to the Arbuzow reaction to afford the corresponding
alkylphosphonate derivatives 18-22 (58-85% yields).
Deprotection of the phosphonate esters with TMSBr
followed by hydrolysis and purification by ion exchange
chromatography yielded the desired compounds 1-4,
isolated as their ammonium salts (67-81% yields).
The synthesis of compounds 8-10, with an eight-
membered spacer, is shown in Scheme 4. Reaction of
the O-benzoyl derivative 2928 with paraformaldehyde
and HCl(g) afforded the chloromethyl ether 30, which
was transformed into the acetoxymethyl derivative 31
by treatment with sodium acetate in acetone. Conden-
24
sation of 31 with HOCH2P(O)(OiPr)2 in the presence
of TMSTf, followed by deprotection with methanolic
MeNH2, afforded the alcohol 33. Mitsunobu reaction25
of 33 with N3-benzoylthymine,26 followed by in situ

974 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Esteban-Gamboa et al.
Sch em e 3a
a
(a) (iPrO)2P(O)CH2OTs, NaH, DMF (43%); (b) 80%-AcOH(aq) (96%); (c) 1. N3-BzT, PPh3, DIAD, THF, 2. MeNH2, MeOH (62%); (d) H2,
10%-Pd/C, EtOH (80%); (e) OsO4, N-methylmorpholine-N-oxide, acetone-H2O (90:1) (70%); (f) 1. TMSBr, DMF, 2. H2O, 5 (73%), 6 (61%),
7 (50%).
Sch em e 4a
a
(a) HCl(g), (CH2O)n, CH2Cl2; (b) AcONa, acetone; (c) (iPrO)2P(O)CH2OH, TMSTf, CH3CN (91% from 29); (d) MeNH2, MeOH (68%); (e)
1. N3-BzT, PPh3, DIAD, THF, 2. MeNH2, MeOH (76%); (f) H2, Pd/C, EtOH (66%); (g) OsO4, N-methylmorpholine-N-oxide, acetone-H2O
(90:1) (76%); (h) 1. TMSBr, 2,6-lutidine, CH2Cl2 or DMF 2. H2O, 8 (70%), 9 (84%), 10 (46%).
deprotection with methanolic MeNH2, afforded com-
pound 34 (76% yield). Catalytic hydrogenation (Pd/C)
of 34 afforded 35. Treatment of 34 with OsO4 gave the
dihydroxylated derivative 36 in 76% yield. Deprotection
of the phosphonate esters in 34, 35, and 36 with TMSBr,
in the presence of 2,6-lutidine to avoid the breakthrough
of the acetalic bond, afforded the target compounds 8,
9, and 10 in 71, 84, and 46% yields, respectively.
Concerning the synthesis of compound 11 (Figure 2),
the initial strategy was based, as in the previous series,
on the introduction of the phosphonate moiety prior to
the pyrimidine base. Thus, the previously synthesized
synthon 25 (Scheme 5) was transformed into the chlo-
romethyl ether 37, which by reaction with silylated
thymine in the presence of catalytic amounts of BuN4I
afforded only traces of the desired compound 38 (<10%
yield). Alternatively, the chloromethyl ether 37 was
transformed into the acetoxymethyl ether 39 and then
reacted with silylated thymine using SnCl4 as catalyst.
This method afforded a complex reaction mixture, from
which the N3-substituted thymine 40 was the only
compound isolated. These results prompted us to employ
a different strategy based on the sequential incorpora-
tion of the thymine base and then the phosphonate
terminus (Scheme 6). Thus, reaction of the chloromethyl
ether 30 with silylated thymine in the presence of BuN4I
afforded exclusively the N1-substituted derivative 41
(75% yield). The reaction of the acetoxymethyl ether 31
with silylated thymine and SnCl4 was also assayed. This
second procedure afforded mixtures of N1-(41) and N3-

Multisubstrate Analogue Inhibitors of E. coli TPase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 975
Sch em e 5a
(39 and 31) with thymine in the presence of SnCl4. It
should be mentioned that there are very few reports of
obtaining N3-isomers of 5-substituted uracils, including
thymine, in acyclonucleoside synthesis,29,30 but in all
these cases SnCl4 was used as catalyst.
Removal of the benzoyl group in 41, followed by
treatment with NaH and then with TsOCH2P(O)(OiPr)2,
afforded a mixture of the phosphonate derivative 38 and
the monodeprotected derivative 44 together with N3-
alkylated compounds. Deprotection of 38 or 44 with
TMSBr, in the presence of 2,6-lutidine, afforded the
acycloderivative 11.
When compounds 1-11 were evaluated against the
E. coli enzyme (see Biological Results section), several
of them showed moderate inhibition of thymidine deg-
radation, the most potent being compounds 3 and 4.
According to the working plan, the thymine in com-
pound 3 was replaced by 6-amino-5-bromouracil (12)
and 7-deazaxanthine (13), keeping the same anchoring
point of the spacer on the pyrimidine base. Both
compounds 12 and 13 could be obtained from a common
intermediate of N1-substituted 6-aminouracil. Direct
alkylation of 6-aminouracil, as described for the thymine
series, was discarded since this procedure is expected
to afford mixtures of isomers. Even exclusive formation
of the N3-substituted derivative has been described
when the base is silylated.31 The method of choice to
obtain N1-monosubstituted 6-aminouracils is the con-
densation of monosubstituted ureas with ethyl cyano-
acetate.32,33 Therefore, the synthesis of 12 and 13 was
accomplished as described in Scheme 7.
a
(a) HCl(g), (CH2O)n, CH2Cl2; (b) silylated thymine, Bu4NI,
CH2Cl2, rt, 24 h; (c) AcONa, acetone; (d) silylated thymine, SnCl4,
0 °C to rt, 1.5 h, CH3CN.
Sch em e 6a
Reaction of N-(8-bromooctyl)phthalimide34 (45) with
triethyl phosphite, as described for similar analogues,35
yielded the diethylalkylphosphonate 46, which was
deprotected at the amino terminus by reaction with
NH2NH2 in MeOH to give 47 (90% yield). This amine,
47, was transformed into the monoalkyl urea 48 by
treatment with KNCO in acidic medium. The urea 48
was condensed with ethyl cyanoacetate under basic
conditions to yield the N1-substituted 6-aminouracil
(49). Bromination of 49 with N-bromosuccinimide in the
presence of AIBN in THF afforded the 5-bromo-6-
aminouracil derivative 50 (72% yield). Alternatively,
compound 49 was transformed into the 7-deazaxanthine
derivative 51 by reaction with chloroacetaldehyde and
sodium acetate.36 Removal of the phosphonate esters of
50 and 51 was performed by treatment with TMSBr
followed by hydrolysis to give 12 and 13 in 75 and 85%
yields, respectively.
Biologica l Resu lts a n d Discu ssion
a
(a) Silylated thymine, Bu4NI, CH2Cl2, rt, 24 h (72%); (b)
In h ibition of Th ym id in e P h osp h or yla se. The
phosphonate derivatives 1-13, as well as their corre-
sponding dialkyl ester, have been tested for their
inhibitory effect on thymidine phosphorylase from E.
coli (see Experimental Section). 6-Amino-5-bromoura-
cil,36 a well-established TPase inhibitor,12 and 7-deaza-
xanthine,37 recently described by us as a novel TPase
inhibitor,13 are included as reference inhibitors. Com-
pounds 1-4 having a polymethylene spacer of 6-9
atoms between the N1 position of thymine and the
phosphonate moiety, were found to be inhibitory to E.
coli TPase: the longer the spacer, the higher the inhibi-
tion (Table 1). Thus, compound 1, containing six CH2
MeNH2, MeOH (98%); (c) 1. NaH, DMF 2. (iPrO)2P(O)CH2OTs,
DMF 38 (25%) and 44 (31%); (d) 1. TMSBr, CH2Cl2, 2,6-lutidine;
2. H2O (75%); (e) silylated thymine, SnCl4, 0 °C to rt, 4 h, 42 (17%)
+ 41 (9%).
(42) substituted products, the latter predominating.
Unequivocal assignment of the substitution position in
41 and 42 was performed based on 1H and 13C NMR
correlation experiments (HSQC and HMBC) to establish
directly bonded and long distance coupled proton and
carbon atoms, respectively. In our opinion, it is rather
surprising to obtain the N3-derivative as the predomi-
nant isomer in the reaction of the acetoxymethyl ethers

976 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Esteban-Gamboa et al.
Sch em e 7a
a
(a) P(OEt)3 (96%); (b) H2N-NH2‚H2O, EtOH (90%); (c) HCl(aq), KOCN, H2O (73%); (d) EtONa, NC-CH2-COOEt, EtOH (66%); (e)
NBS, AIBN, THF (72%); (f) Cl-CH2-CHO, AcONa, EtOH (47%); (g) 1, TMSBr, CH2Cl2 2. H2O, 12 (75%), 13 (85%).
Ta ble 1. Inhibition of E. coli Thymidine Phosphorylase by Test
55 to 73% inhibition at 1000 µM and 8-17% inhibition
Compounds as a Function of Incubation Time
at 100 µM, whereas 3 and 4, containing eight and nine
inhibition of TPase (%)a
CH2 units, respectively, inhibited TPase by 61 to 81%
at 1000 µM and by 16 to 35% at 100 µM. It should be
pointed out that their corresponding diethyl (19, 20, 22)
or diisopropyl (21) ester derivatives were devoid of anti-
TPase activity. Only 22 showed a marginal inhibitory
effect at 1000 µM (2-4%), indicating the importance of
a free phosphonic acid for anti-TPase activity.
Among the phosphonates carrying a (Z)-butene moi-
ety in the spacer (i.e., 5, 8, and 11), only compound 8
showed an inhibitory effect at the highest concentration
tested (1000 µM: 17-34% inhibition). Interestingly,
whereas 5 was devoid of anti-TPase activity, its butyl
analogue 6 showed significant inhibition at 1000 µM
(12-27%). The corresponding diisopropyl esters of 6 and
8 (viz. 27 and 34) were completely inactive. Among the
remaining thymine phosphonate derivatives synthe-
sized (7, 9, and 10), no inhibition was found at 1000
µM, neither for their dialkyl ester derivatives.
Interestingly, replacement of the thymine base in
compound 3 by 5-bromo-6-aminouracil or 7-deazaxan-
thine (compounds 12 and 13, respectively) led to a
considerable improvement of the inhibitory activity.
Indeed, whereas 3 was inhibitory by 61-75% at 1000
µM and 16-29% at 100 µM, 12 was inhibitory by 89-
94% at 1000 µM, 46-64% at 100 µM, and 13-26% at
20 µM. Compound 13 was even more inhibitory to TPase
than 12 and, thus, showed an inhibition of TPase that
was at least comparable, if not more pronounced, than
previously noted for 7DX (Table 1). The isopropyl esters
of 12 and 13 (viz. 50 and 51) had still an inhibitory
potential at 1000 µM (i.e., 10-21% and 27-43%,
respectively); however, comparison of 50 and 51 with
their deprotected analogues 12 and 13 stresses the
importance of a free phosphonic acid for TPase inhibi-
tion by these series of compounds.
compd
concn (µM)
20 min
40 min
60 min
1
1000
1000
100
1000
100
1000
100
1000
1000
100
11
73
17
75
29
81
35
0
5
2
55
8
61
16
70
18
0
12
1
0
17
0
0
2
3
4
63
13
68
22
75
27
0
5
6
27
5
17
2
7
8
1000
1000
100
0
34
0
0
22
0
9
1000
1000
1000
1000
100
20
1000
100
0
0
0
94
64
26
97
83
48
0
0
0
4
0
0
0
0
0
0
0
0
91
55
19
96
77
38
0
0
0
3
0
0
0
0
0
10
11
12
0
0
89
46
13
94
72
31
0
0
0
2
0
0
0
0
0
0
10
0
27
0
71
44
41
7
13
20
19
20
21
22
26
27
28
34
35
36
50
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
100
0
21
0
0
16
0
51
500
100
43
0
37
0
6A5BU
7DX
100
25
100
25
nd
nd
nd
nd
nd
nd
nd
nd
To clarify whether the inhibition of TPase was really
due to a multisubstrate analogue inhibition (multisub-
strate binding), kinetic experiments with the most
potent compounds 12 and 13, together with the previ-
ously described inhibitor 7DX, were carried out. By
varying the concentrations of inorganic phosphate, a
a
The inhibition data represent the mean of at least two or three
independent experiments.
units between thymine and the phosphonate, was only
marginally active at 1000 µM, and compound 2, con-
taining seven methylene units in the spacer, afforded

Multisubstrate Analogue Inhibitors of E. coli TPase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 977
experiment and was optimized for long-range coupling con-
stants of 7 Hz.
competitive inhibition of 12 and 13 with respect to
phosphate was revealed, while the free base 7DX proved
noncompetitive with respect to phosphate. Whereas the
Km value of the enzyme for inorganic phosphate was
1420 µM, the Ki values of 12, 13, and 7DX for the
enzyme against inorganic phosphate as the variable
substrate were 48 µM, 39 µM, and 27.5 µM, respectively.
Also, 12 and 13 were found to be competitive inhibitors
of TPase against thymidine as the variable substrate
(Km value was 600 µM, Ki values were 123 µM and 68
µM, respectively). The enzyme kinetic data strongly
suggest that, in contrast to 7DX, compounds 12 and 13
indeed must interact with both, the phosphate binding
site and the nucleoside binding site of TPase, as origi-
nally designed.
Analytical TLC was performed on silica gel 60 F254 (Merck)
precoated plates (0.2 mm). Spots were detected under UV light
(254 nm) and/or by charring with phosphomolibdic acid and/
or ninhydrin. Separations on silica gel were performed by
preparative centrifugal circular thin-layer chromatography
(CCTLC) on a Chromatotron (Kiesegel 60 PF
gipshaltig
254
(Merck); layer thickness, 1 or 2 mm; flow rate, 4 or 8 mL/min,
respectively). Flash column chromatography was performed
with silica gel 60 (230-400 mesh) (Merck). Sephadex A-25
(HCO3-form) was used for ion exchange chromatography.
Analytical HPLC was performed on a Waters 484 system using
a µBondapak C18 (3.9 × 300 mm: 10 µm), with UV detection
at 254 nm and a flow rate of 1 mL/min. The systems used were
the following: Mobile phases A: CH3CN, B: H2O (0.05% TFA).
System I: 2% A (0-2 min), 2-20% A (2-4 min), 20% A (4-6
min), 20-95% A (6-16 min), 95-2% A (16-20 min). System
II: 0% A (0-2 min), 0-10% A (2-4 min), 10% A (4-12 min),
10-95% A (12-25 min), 95-0% A (25-30 min). All retention
times are quoted in minutes.
Con clu sion
A series of acyclic phosphonate derivatives of thymine
with a spacer of six to nine atoms have been synthesized
in order to evaluate their potential as multisubstrate
analogue inhibitors of E. coli thymidine phosphorylase.
Multisubstrate analogue inhibitors have yielded ex-
tremely potent inhibitors against other targets, such as
purine nucleoside phosphorylase.18 To the best of our
knowledge, there are no previous reports on multisub-
strate analogue inhibitors of TPase. Among the thymine
derivatives synthesized in this paper, compounds 3 and
4, having a polymethylene chain of eight and nine atoms
between the thymine base and the phosphonic acid,
showed marked inhibition of TPase. Replacement of
thymine in 3 by 6-amino-5-bromouracil or 7-deazaxan-
thine (compounds 12 and 13, respectively) resulted in
a marked increase of the inhibitory potency, being
comparable to that of 7DX. Kinetic experiments in the
presence of variable concentrations of inorganic phos-
phate and variable concentrations of thymidine have
shown that 12 and 13 compete with both substrates to
inhibit TPase. These results strongly indicate that
compounds 12 and 13 behave as multisubstrate ana-
logue inhibitors of TPase. These new leads should be
further explored to improve their inhibitory potency
against TPase.
The deprotected phosphonates were noted to be hygroscopic
and did not give useful microanalytical data but were found
to be pure by high-field multinuclear NMR spectroscopy, mass
spectrometry, and rigorous HPLC analysis.
All experiments involving water-sensitive compounds were
conducted under scrupulously dry conditions. Triethylamine
and acetonitrile were dried by refluxing over calcium hydride.
Tetrahydrofuran was dried by refluxing over sodium/benzo-
phenone. Anhydrous N,N′-dimethylformamide was purchased
from Aldrich.
Thymidine, thymine, and E. coli thymidine phosphorylase
(TPase) (1030 units/mL) were obtained from Sigma Chemical
Co., St. Louis, MO. The synthesis of 7-deazaxanthine was
performed according to West et al.37 6-Amino-5-bromouracil
(6A5BU) was synthesized according to E. F. Schroeder.36
Gen er a l P r oced u r e for th e P r ep a r a tion of 1-(Br om o-
a lk yl)th ym in es. Sodium hydride (349 mg, 60% dispersion in
mineral oil, 8.72 mmol) was added into a suspension of
thymine (1.00 g, 7.93 mmol) in dry DMF (32 mL), and the
mixture was heated at 80 °C for 1 h. After the mixture cooled
to room temperature, the corresponding dibromoalkane (7.93
mmol) was added, and the resulting mixture was heated at
80 °C for 5 h whereupon it was evaporated. The crude was
treated with CH2Cl2 (150 mL) and filtered. The filtrate was
purified by column chromatography using EtOAc:hexane (1:
2) as eluent.
1-(6-Br om oh exyl)th ym in e (14). Yield: 40%. Mp (EtOAc):
113-114 °C (lit.21 109-110 °C; lit.22 112-113.5 °C).
1-(7-Br om oh ep tyl)th ym in e (15). Yield: 39%. Mp (EtOAc):
109-110 °C (lit.21 107-108 °C).
Exp er im en ta l Section
Ch em ica l P r oced u r es. Melting points were obtained on
a Reichert-J ung Kofler apparatus and are uncorrected. Micro-
analyses were obtained with a Heraeus CHN-O-RAPID instru-
ment. Electrospray mass spectra were measured on a quad-
rupole mass spectrometer equipped with an electrospray
1-(8-Br om ooctyl)th ym in e (16). Yield: 39%. Mp (EtOAc):
96-97 °C (lit.21 96-97 °C; lit.22 96-96.5 °C).
1-(9-Br om on on yl)th ym in e (17). Yield: 33%. Mp (EtOAc):
104-105 °C (lit.21 104-105 °C).
source (Hewlett-Packard, LC/MS HP 1100). H and 13C NMR
1
Gen er a l P r oced u r e for th e P r ep a r a tion of 1-[(Dia lk yl-
p h osp h on o)a lk yl]th ym in es. A mixture of the corresponding
1-(bromoalkyl)thymine (5.00 mmol) (14-17) and trialkyl phos-
phite (35 mL) was heated at 130 °C for 24 h. After evaporation,
the crude was purified by column chromatography to yield 18-
22 as syrups.
spectra were recorded on a Varian Gemini operating at 200
MHz (1H) and 50 MHz (13C), respectively, on a Varian INOVA
300 operating at 299 MHz (1H) and 75 MHz (13C), respectively,
and Varian INOVA-400 operating at 400 MHz (1H) and 100
MHz (13C), respectively. Monodimensional H and 13C spectra
1
were obtained using standard conditions. Two-dimensional
inverse proton detected heteronuclear one-bond shift correla-
tion spectra were obtained using the pulsed field gradient
HSQC pulse sequence. Data were collected in a 2048 × 512
matrix with a spectral width of 3460 Hz in the proton domain
and 22500 Hz in the carbon domain and were processed in a
2048 × 1024 matrix. The experiment was optimized for one
bond heteronuclear coupling constant of 150 Hz. Two-
dimensional inverse proton detected heteronuclear long-range
shift correlation spectra were obtained using the pulsed field
gradient HMBC pulse sequence. The HMBC experiment was
acquired in the same conditions as those for the HSQC
1-[6-(Diisop r op ylp h osp h on o)h exyl]th ym in e (18). The
product was obtained from 14 (1.45 g) and triisopropyl-
phosphite, followed by column chromatography (EtOAc: EtOH,
20:1). Yield 60%. 1H NMR (CDCl3) δ: 0.90-1.80 (m, 10H), 1.27
[d, J ) 6.2 Hz, 12H, CH(CH3)2], 1.90 (s, 3H, 5-CH3), 3.65 (t, J
) 7.3 Hz, 2H, CH2N), 4.40-4.80 [m, 2H, CH(CH3)2], 6.95 (s,
1H, H-6), 8.94 (br s, 1H, NH); 13C NMR (CDCl3) δ: 12.2 (5-
CH3), 22.4 (d, J C,P ) 5.2 Hz, CH2CH2P), 24.0 [CH(CH3)2], 25.9,
28.8 (CH2), 26.7 (d, J C,P ) 141.2 Hz, CH2P), 30.0 (d, J C,P
)
16.7 Hz, CH2(CH2)2P), 48.3 (CH2N), 69.8 [d, J C,P ) 6.6 Hz, CH-
(CH3)2], 110.5 (C-5), 140.3 (C-6), 150.9 (C-2), 164.4 (C-4). Anal.
(C17H31N2O5P) C, H, N.

978 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Esteban-Gamboa et al.
1-[7-(Dieth ylp h osp h on o)h ep tyl]th ym in e (19). The prod-
uct was obtained from 15 (1.52 g) and triethyl phosphite,
followed by column chromatography (CH2Cl2:acetone, 2:1).
negative mode): m/z 317 (M - NH4)-. 1H NMR (D2O) δ: 1.10-
1.75 (m, 14H), 1.79 (s, 3H, 5-CH3), 3.66 (t, J ) 7.0 Hz, 2H,
CH2N), 7.41 (s, 1H, H-6). HPLC: system I, Rt ) 14.40 min
(100%); system II, Rt ) 22.46 min (99.6%).
1
Yield: 82%. H NMR (CDCl3) δ: 1.10-1.85 (m, 12H), 1.31 (t,
J ) 7.1 Hz, 6H, CH2CH3), 1.91 (s, 3H, 5-CH3), 3.67 (t, J ) 7.2
Hz, 2H, CH2N), 3.90-4.30 (m, 4H, CH2CH3), 6.97 (s, 1H, H-6),
8.57 (br s, 1H, NH). Anal. (C16H29N2O5P) C, H, N.
1-(9-P h osp h on on on yl)t h ym in e Am m on iu m Sa lt (4).
Deprotection of 22 (100 mg, 0.26 mmol) according to the
general procedure afforded 4 (55 mg, 67%) as a white lyophi-
1-[8-(Dieth ylp h osp h on o)octyl]th ym in e (20). The prod-
uct was obtained from 16 (1.59 g) and triethyl phosphite,
followed by column chromatography (CH2Cl2:acetone, 2:1).
late. UV (H2O) λ
) 275 (ꢀ ) 9000). MS (ES, negative
max
mode): m/z 331 (M - NH4)-. 1H NMR (D2O) δ: 1.10-1.75 (m,
16H), 1.84 (s, 3H, 5-CH3), 3.71 (t, J ) 7.0 Hz, 2H, CH2N), 7.46
(s, 1H, H-6). 13C NMR (D2O): 12.5 (5-CH3), 24.2 (d, J C,P ) 3.9
Hz, CH2CH2P), 26.7, 29.2, 29.5, 29.6 (CH2), 29.0 (d, J C,P ) 131.4
Hz, CH2P), 31.4 (d, J C,P ) 16.0 Hz, CH2CH2CH2P), 50.0 (CH2N),
111.8 (C-5), 144.7 (C-6), 153.7 (C-2), 168.3 (C-4). HPLC:
system I, Rt ) 15.51 min (96.9%); system II, Rt ) 23.48 min
(97.5%).
1
Yield: 74%. H NMR (CDCl3) δ: 1.10-1.85 (m, 14H), 1.34 (t,
J ) 7.1 Hz, 6H, CH2CH3), 1.92 (s, 3H, 5-CH3), 3.67 (t, J ) 7.3
Hz, 2H, CH2N), 4.0-4.30 (m, 4H, CH2CH3), 6.97 (s, 1H, H-6),
8.67 (br s, 1H, NH). 13C NMR (CDCl3) δ: 12.1 (5-CH3), 16.2
(d, J C,P ) 6.1 Hz, CH2CH3), 22.1 (d, J C,P ) 5.3 Hz, CH2CH2P),
25.4 (d, J C,P ) 141.1 Hz, CH2P), 26.1, 28.8, 28.7 (CH2), 30.2
[d, J C,P ) 16.1 Hz, CH2(CH2)2P], 48.2 (CH2N), 61.2 (d, J C,P
)
(Z)-4-[(Diisop r op ylp h osp h on o)m eth oxy]-1-tr ip h en yl-
m eth oxy-2-bu ten e (24). A solution of 2323 (4.50 g, 13.62
mmol) and diisopropyl[(p-toluensulfonyl)oxy]methanephos-
phonate24 (7.50 g, 20.43 mmol) in dry DMF (81 mL) was cooled
at -20 °C. Then, NaH (1.20 g, 60% dispersion in mineral oil,
30.00 mmol) was added, and the reaction was allowed to reach
room temperature and stirred overnight. Volatiles were re-
moved, and the residue was treated with EtOAc (100 mL) and
brine (100 mL). The organic phase was dried on anhydrous
Na2SO4, filtered, and evaporated. The residue was purified by
column chromatography (CH2Cl2:EtOAc, 20:1) to yield 2.98 g
(43% yield) of 24 as a syrup. 1H NMR (CDCl3) δ: 1.32, 1.35
[2d, J ) 6.0 Hz, 12H, CH(CH3)2], 3.63 (d, J ) 9.2 Hz, 2H,
CH2P), 3.68 (d, J ) 6.2 Hz, 2H, CH2OTr), 4.00 (d, J ) 6.4 Hz,
2H, CH2OCH2P), 4.60-4.90 [m, 2H, CH(CH3)2], 5.56-6.00 (m,
2H, CHdCH), 7.10-7.70 (m, 15H, arom.). Anal. (C30H37O5P)
C, H, N.
6.9 Hz, CH2CH3), 110.3 (C-5), 140.2 (C-6), 151.0 (C-2), 164.5
(C-4). Anal. (C17H31N2O5P) C, H, N.
1-[8-(Diisop r op ylp h osp h on o)octyl]th ym in e (21). The
product was obtained from 16 (1.59 g) and triisopropyl phos-
phite, followed by column chromatography (EtOAc:acetone,
2:1). Yield: 58%.1H NMR (CDCl3) δ: 1.00-1.90 (m, 14H), 1.30
[d, J ) 6.2 Hz, 12H, CH(CH3)2], 1.92 (s, 3H, 5-CH3), 3.67 (t, J
) 7.3 Hz, 2H, CH2N), 4.40-4.80 [m, 2H, CH(CH3)2], 6.97 (s,
1H, H-6), 8,60 (br s, 1H, NH). Anal. (C19H35N2O5P) C, H, N.
1-[9-(Dieth ylp h osp h on o)n on yl]th ym in e (22). The prod-
uct was obtained from 17 (1.66 g) and triethyl phosphite,
followed by column chromatography (CH2Cl2:acetone, 2:1).
1
Yield: 86%. H NMR (CDCl3) δ: 1.10-1.85 (m, 16H), 1.34 (t,
J ) 7.1 Hz, 6H, CH2CH3), 1.92 (s, 3H, 5-CH3), 3.67 (t, J ) 7.2
Hz, 2H, CH2N), 3.90-4.30 (m, 4H, CH2CH3), 6.97 (s, 1H, H-6),
8.70 (br s, 1H, NH). Anal. (C18H33N2O5P) C, H, N.
Gen er a l P r oced u r e for th e Dep r otection of 1-[(Di-
a lk ylp h osp h on o)a lk yl]th ym in es. To a cooled solution of the
corresponding 1-[(dialkylphosphono)alkyl]thymines (18-22)
(1.00 mmol) in dry DMF or CH2Cl2 (5 mL) was added TMSBr
(1.29 mL, 10.00 mmol). The mixture was stirred at room
temperature for 18 h. Volatiles were removed, and the residue
was stirred in concentrated NH4OH (3 mL) for 30 min. The
mixture was evaporated and applied onto an Amberlite XAD-2
column and eluted with H2O and H2O:MeOH (50:50). UV
positive fractions were collected, evaporated, and purified by
(Z)-4-[(Diisop r op ylp h osp h on o)m eth oxy]-2-bu ten -1-ol
(25). A solution of 24 (2.80 g, 5.50 mmol) in 80% AcOH (90
mL) was heated at 60 °C for 20 min. Solvents were removed,
and the residue was purified by column chromatography (CH2-
Cl2:MeOH, 20:1) to yield 261 mg (96% yield) as a syrup. 1H
NMR (CDCl3) δ: 1.30, 1.31 [2d, J ) 6.2 Hz, 12H, CH(CH3)2],
3.34 (br s, 1H, OH), 3.70 (d, J ) 8.2 Hz, 2H, CH2P), 4.17, 4.20
(2d, J ) 6.7, 6.5 Hz, 4H, CH2O), 4.62-4.82 [m, 2H, CH(CH3)2],
5.54-5.92 (m, 2H, CHdCH). 13C NMR (CDCl3) δ: 23.8-24.1
[CH(CH3)2], 58.0 (CH2OH), 64.0 (d, J C,P ) 167.7 Hz, CH2P),
67.9 (d, J C,P ) 11.1 Hz, CH2OCH2P), 71.3 [d, J C,P ) 7.1 Hz,
CH(CH3)2], 126.3, 133.8 (CHdCH). Anal. (C11H23O5P) C, H, N.
-
DEAD-Sephadex A25 (HCO3 form), eluting with a gradient
H2O-0.15 M NH4HCO3. Appropriate fractions were evapo-
rated, coevaporated with H2O, and lyophilized.
1-[(Z)-4-[(Diisop r op ylp h osp h on o)m eth oxy]-2-bu ten yl]-
th ym in e (26). To a solution of 25 (400 mg, 1.50 mmol), Ph3P
(788 mg, 3.00 mmol), and N3-benzoylthymine (692 mg, 3.00
mmol) in dry THF (15 mL) was slowly added a solution of
DIAD (0.61 mL, 3.00 mmol) in dry THF (5 mL). The mixture
was stirred at room temperature for 2 h. Volatiles were
removed, and the residue was treated with MeNH2 in MeOH
(20 mL) overnight. Solvents were evaporated, and the residue
was purified by column chromatography (CH2Cl2:acetone, 1:2)
to yield 350 mg (62%) of 26 as a syrup. 1H NMR (CDCl3) δ:
1.33, 1.34 [2d, J ) 6.2 Hz, 12H, CH(CH3)2], 1.92 (s, 3H, 5-CH3),
3.77 (d, J ) 8.2 Hz, 2H, CH2P), 4.30 (d, J ) 6.1 Hz, 2H, CH2N),
4.44 (d, J ) 6.8 Hz, 2H, CH2OCH2P), 4.60-4.90 [m, 2H,
CH(CH3)2], 5.56-5.92 (m, 1H, CHdCH), 7.15 (s, 1H, H-6), 8.65
(br s, 1H, NH). 13C NMR (CDCl3) δ: 12.1 (5-CH3), 23.8-24.1
[CH(CH3)2], 44.6 (CH2N), 64.9 (d, J C,P ) 168.5 Hz, CH2P), 68.1
(d, J C,P ) 10.9 Hz, CH2OCH2P), 71.1 [d, J C,P ) 6.7 Hz, CH-
(CH3)2], 110.9 (C-5), 127.5, 129.9 (CHdCH), 140.0 (C-6), 151.0
(C-2), 164.4 (C-4). Anal. (C16H27N2O6P) C, H, N.
1-[(Z)-4-P h osp h on om et h oxy-2-b u t en yl]t h ym in e Am -
m on iu m Sa lt (5). Compound 26 (100 mg, 0.27 mmol) was
deprotected following the general procedure for deprotection
of 1-[(dialkylphosphono)alkyl]thymines to yield 60 mg (73%)
of 527 as a white lyophilate. UV (H2O) λmax ) 272 (ꢀ ) 9000).
MS (ES, negative mode): m/z 289 (M - NH4)-. 1H NMR (D2O)
δ: 1.75 (s, 3H, 5-CH3), 3.42 (d, J ) 8.7 Hz, 2H, CH2P), 4.15 (d,
J ) 6.5 Hz, 2H, CH2OCH2P), 4.34 (d, J ) 6.8 Hz, 2H, CH2N),
5.50-5.80 (m, 2H, CHdCH), 7.36 (s, 1H, H-6). 13C NMR
(CDCl3) δ: 12.5 (5-CH3), 46.7 (CH2N), 67.5 (d, J C,P ) 157.7
1-(6-P h osp h on oh exyl)th ym in e Am m on iu m Sa lt (1).
Compound 18 (99 mg, 0.26 mmol) was deprotected and purified
following the general procedure to afford 55 mg (68%) of 1 as
a white lyophilate. UV (H2O) λ max ) 273 (ꢀ ) 8700). MS (ES,
negative mode): m/z 389 (M - NH4)-. 1H NMR (D2O) δ: 1.00-
1.70 (m, 10H), 1.75 (s, 3H, 5-CH3), 3.63 (t, J ) 7.1 Hz, 2H,
CH2N), 7.37 (s, 1H, H-6). 13C NMR (D2O): 12.5 (5-CH3), 24.2
(d, J C,P ) 4.5 Hz, CH2CH2P), 26.4, 29.2 (CH2), 29.0 (d, J C,P
)
132.2 Hz, CH2P), 31.0 (d, J C,P ) 16.5 Hz, CH2CH2CH2P), 50.0
(CH2N), 111.8 (C-5), 144.7 (C-6), 153.7 (C-2), 168.3 (C-4).
HPLC: system I, Rt ) 14.50 min (100%); system II, Rt ) 22.33
min (100%).
1-(7-P h osp h on oh ep tyl)th ym in e Am m on iu m Sa lt (2).
Starting from 19 (100 mg, 0.28 mmol) and following the
general procedure, 65 mg (73%) of 2 was obtained as a white
lyophilate. UV (H2O) λ max ) 274 (ꢀ ) 8600). MS (ES, negative
mode): m/z 303 (M - NH4)-. 1H NMR (D2O) δ: 1.10-1.75 (m,
12H), 1.83 (s, 3H, 5-CH3), 3.70 (t, J ) 7.1 Hz, 2H, CH2N), 7.45
(s, 1H, H-6). 13C NMR (D2O): 12.5 (5-CH3), 24.0 (d, J C,P ) 4.5
Hz, CH2CH2P), 26.6, 29.1, 29.3 (CH2), 28.7 (d, J C,P ) 132.9 Hz,
CH2P), 31.2 (d, J C,P ) 16.6 Hz, CH2CH2CH2P), 49.9 (CH2N),
111.8 (C-5), 144.6 (C-6), 153.6 (C-2), 168.3 (C-4). HPLC:
system I, Rt ) 13.06 min (100%); system II, Rt ) 21.01 min
(100%).
1-(8-P h osph on ooctyl)th ym in e Am m on iu m Salt (3). Com-
pound 20 (100 mg, 0.25 mmol) was deprotected and purified
following the general procedure to afford 68 mg (81%) of 3 as
a white lyophilate. UV (H2O) λ max ) 274 (ꢀ ) 8700). MS (ES,

Multisubstrate Analogue Inhibitors of E. coli TPase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 979
Hz, CH2P), 69.0 (d, J C,P ) 11.6 Hz, CH2OCH2P), 112.2 (C-5),
128.5, 131.1 (CHdCH), 144.0 (C-6), 153.5 (C-2), 168.3 (C-4).
HPLC: system I, Rt ) 9.01 min (98.7%); system II, Rt ) 11.60
min (98.3%).
additional 6 h. Then, MgSO4 was added, filtered, and evapo-
rated. The resultant syrup was used as such in the next steps.
1H NMR (CDCl3) δ: 4.34 (d, J ) 6.3 Hz, 2H, CH2OCH2Cl),
4.86 (d, J ) 6.1 Hz, 2H, CH2OBz), 5.45 (s, 2H, CH2OCH2Cl),
5.60-6.00 (m, 2H, CHdCH), 7.15-8.10 (m, 5H, arom.).
1-[4-[(D iis o p r o p y lp h o s p h o n o )m e t h o x y ]b u t y l]t h y -
m in e (27). A solution of 26 (190 mg, 0.51 mmol) in EtOH (15
mL) was hydrogenated at room temperature in the presence
of 10% Pd/C (50 mg) at 30 psi for 4 h. The mixture was filtered,
and the filtrate was purified in the chromatotron CCTLC (CH2-
Cl2:MeOH, 15:1) to give 150 mg (80% yield) of 27 as a syrup.
1H NMR (acetone-d6) δ: 1.28, 1.30 [2d, J ) 6,2 Hz, 12H, CH-
(CH3)2], 1.45-1.90 (m, 4H, CH2), 1.84 (s, 3H, 5-CH3), 3.62, 3.76
(2t, J ) 6.1, 7.0 Hz, 4H, CH2O, CH2N), 3.71 (d, J ) 8.3 Hz,
CH2P), 4.60-4.80 [m, 2H, CH(CH3)2], 7.48 (s, 1H, H-6), 9.97
(br s, 1H, NH). Anal. (C16H29N2O6P) C, H, N.
1-[(4-P h osph on om eth oxy)bu tyl]th ym in e Disodiu m Salt
(6). Compound 27 (100 mg, 0.27 mmol) was deprotected
following the general procedure for deprotection of 1-[(dialkyl-
phosphono)alkyl]thymines and then transformed into the
disodium salt by eluting through a Dowex 50WX4 (Na+ form)
to yield 50 mg (61%) of 6 as a white lyophilate. UV (H2O) λmax
) 271 (ꢀ ) 8400). MS (ES, negative mode): m/z 291 (M + H -
2Na)-. 1H NMR (D2O) δ: 1.40-1.65 (m, 4H, CH2), 1.71 (s, 3H,
5-CH3), 3.45 (d, J ) 8.5 Hz, 2H, CH2P), 3.46, 3.62 (2t, J ) 7.3,
7.1 Hz, 4H, CH2O, CH2N), 7.35 (s, 1H, H-6). 13C NMR (D2O)
δ: 12.5 (5-CH3), 26.1, 26.6 (CH2), 49.6 (CH2N), 68.0 (d, J )
155.6 Hz, CH2P), 73.6 (d, J ) 10.6 Hz, CH2O), 111.8 (C-5),
144.5 (C-6), 153.6 (C-2), 168.3 (C-4). HPLC: system I, Rt )
10.53 min (99.1%); system II, Rt ) 11.90 min (98.8%).
(Z)-1-Ben zoxy-4-(a cetoxy)m eth oxy-2-bu ten e (31). To a
solution of the crude 30 in acetone (31 mL) was added AcONa
(3.57 g, 43.53 mmol). The mixture was stirred at room
temperature for 18 h. It was filtered, evaporated, and used as
1
such in the next steps. H NMR (CDCl3) δ: 2.10 (s, 3H, CH3-
CO), 4.35 (d, J ) 5.3 Hz, 2H, CH2OCH2OAc), 4.90 (d, J ) 5.3
Hz, 2H, CH2OBz), 5.30 (s, 2H, CH2OCH2OAc), 5.60-6.00 (m,
2H, CHdCH), 7.30-8.20 (m, 5H, arom.).
(Z)-1-Ben zoxy-4-[(d iisop r op ylp h osp h on o)m et h oxy)-
m eth oxy]-2-bu ten e (32). To a solution of the crude 31 and
diisopropyl hydroxymethylphosphonate (7.55 g, 38.52 mmol)
in dry CH3CN (46 mL) at 0 °C, TMSTf (3.5 mL, 3.77 mmol)
was added. The mixture was allowed to reach room temper-
ature and stirred for 4 h. Then, it was poured onto a cooled
NaHCO3 solution and extracted with CH2Cl2 (3 × 50 mL). The
combined organic extracts were dried on Na2SO4, filtered, and
evaporated. The residue was purified by column chromatog-
raphy (EtOAc:hexane, 1:1) to yield 4.66 g (91%) of 32 as a
syrup. 1H NMR (CDCl3) δ: 1.33, 1.34 [2d, J ) 6.1 Hz, 12H,
CH(CH3)2], 3.81 (d, J ) 9.1 Hz, 2H, CH2P), 4.28 (d, J ) 5.1
Hz, 2H, CH2OCH2O), 4.76 (s, 2H, OCH2O), 4.60-4.88 [m, 2H,
CH(CH3)2], 4.90 (d, J ) 5.4 Hz, 2H, CH2OBz), 5.70-6.00 (m,
2H, CHdCH), 7.30-8.20 (m, 5H, arom.). Anal. (C19H29O7P) C,
H, N.
(2S,3S)- a n d (2R,3R)-1-[2,3-Dih yd r oxy-4-[(d iisop r op yl-
p h osp h on o)m eth oxy]bu tyl]th ym in e (28). To a solution of
26 (136 mg, 0.363 mmol) in acetone:H2O (90:10) (8 mL) were
added N-methylmorpholine-N-oxide (0.075 mL of a 60% aque-
ous solution, 0.440 mmol) and OsO4 (0.044 mL of a 4% aqueous
solution, 0.007 mmol). The mixture was stirred at room
temperature for 3 days. A small portion of Na2S2O5 was added,
and the mixture was filtered through Celite. The filtrate was
evaporated and purified by column chromatography (CH2Cl2:
(Z)-4-[((Diisop r op ylp h osp h on o)m eth oxy)m eth oxy]-2-
bu ten -1-ol (33). Compound 32 (960 mg, 2.40 mmol) was
treated with MeNH2 in MeOH (25 mL) overnight. Volatiles
were removed, and the residue was purified by column
chromatography (EtOAc) to yield 504 mg (68%) of 33 as a
syrup. 1H NMR (CDCl3) δ: 1.33, 1.34 [2d, J ) 6.1 Hz, 12H,
CH(CH3)2], 2.90 (br s, 1H, OH), 3.78 (d, J ) 9.5 Hz, 2H, CH2P),
4.10-4.30 (m, 4H, CH2OH and CH2OCH2O), 4.73 (s, 2H,
OCH2O), 4.60-4.90 [m, 2H, CH(CH3)2], 5.50-6.00 (m, 2H,
CHdCH). 13C NMR (CDCl3) δ: 23.8-24.1 [CH(CH3)2], 58.0
(CH2OH), 61.5 (d, J C,P ) 172.2 Hz, CH2P), 63.1 (CH2OCH2O),
71.2 [d, J C,P ) 6.6 Hz, CH(CH3)2], 95.2 (d, J C,P ) 12.5 Hz,
OCH2O), 126.8, 133.1 (CHdCH). Anal. (C12H25O6P) C, H, N.
1-[(Z)-4-[((Diisop r op ylp h osp h on o)m eth oxy)m eth oxy]-
2-bu ten yl]th ym in e (34). Compound 33 (504 mg, 1.70 mmol)
was reacted with N3-benzoylthymine, as described for the
synthesis of 26. After column chromatography (CH2Cl2:
acetone, 2:1), 526 mg (76% yield) of 34 was obtained as a syrup.
1H NMR (CDCl3) δ: 1.30, 1.31 [2d, J ) 6.2 Hz, 12H, CH(CH3)2],
1.90 (s, 3H, 5-CH3), 3.78 (d, J ) 9.3 Hz, 2H, CH2P), 4.24 (d, J
) 6.4 Hz, 2H, CH2N), 4.40 (d, J ) 5.9 Hz, 2H, CH2OCH2O),
4.72 (s, 2H, OCH2O), 4.60-4.90 [m, 2H, CH(CH3)2], 5.50-6.00
(m, 2H, CHdCH), 7.08 (s, 1H, H-6), 9.90 (br s, 1H, NH). 13C
NMR (CDCl3) δ: 12.2 (5-CH3), 23.8-24.1 [CH(CH3)2], 44.5
(CH2N), 61.6 (d, J C,P ) 172.8 Hz, CH2P), 62.4 (CH2OCH2O),
71.1 [d, J C,P ) 6.5 Hz, CH(CH3)2], 95.1 (d, J C,P ) 10.4 Hz,
OCH2O), 111.0 (C-5), 127.4, 130.1 (CHdCH), 139.9 (C-6), 150.7
(C-2), 164.2 (C-4). Anal. (C17H29N2O7P) C, H, N.
1
acetone, 1:2) to yield 118 mg (79%) of 28 as a syrup. H NMR
(CDCl3) δ: 1.32, 1.33 [2d, J ) 6.2 Hz, 12H, CH(CH3)2], 1.89
(s, 3H, 5-CH3), 3.40-3.52 (m, 1H, CHOH), 3.66 (dd, J ) 4.6,
14.5 Hz, 1H, CHP), 3.65-4.04 (m, 5H, CHOH, CHP, CH2-
OCH2P, CHN), 4.11 (dd, J ) 4.4, 14.3 Hz, 1H, CHN), 4.13 (d,
J ) 6.5 Hz, 1H, OH), 4.62-4.84 [m, 2H, CH(CH3)2], 5.01 (d, J
) 4.5 Hz, 1H, OH), 7.25 (s, 1H, H-6), 9.21 (br s, 1H, NH). 13C
NMR (CDCl3) δ: 12.2 (5-CH3), 23.8-24.1 [CH(CH3)2], 50.8
(CH2N), 65,9 (d, J C,P ) 166.7 Hz, CH2P), 70.1, 70,6 (CHOH),
71.5 [d, J C,P ) 6.4 Hz, CH(CH3)2], 74.0 (d, J C,P ) 6.0 Hz, CH2-
OCH2P), 110.0 (C-5), 142.8 (C-6), 152.5 (C-2), 164.6 (C-4). Anal.
(C16H29N2O8P) C, H, N.
(2S,3S)- a n d (2R,3R)-1-[2,3-Dih yd r oxy-4-[(p h osp h on o-
m eth oxy)bu tyl]th ym in e Am m on iu m Sa lt (7). A solution
of 28 (80 mg, 0.20 mmol) in dry DMF (3 mL) was treated with
TMSBr (0.26 mL, 2.00 mmol) overnight and was coevaporated
with H2O. The residue was treated with a mixture of acetone:
H2O (1:6) at 5 °C for 24 h. The solid obtained was filtered and
dissolved in NH4OH to obtain the ammonium salt. The residue
-
was finally purified by DEAD-Sephadex A25 (HCO3 form),
1-[(Z)-4-[(P h osp h on om et h oxy)m et h oxy]-2-b u t en yl]-
th ym in e Am m on iu m Sa lt (8). To a solution of 34 (65 mg,
0.16 mmol) in dry CH2Cl2 (2 mL) and 2,6-lutidine (0.28 mL,
2.45 mmol), cooled at 0 °C, was added TMSBr (0.21 mL, 1.60
mmol). After 18 h, solvents were removed. The residue was
treated with H2O (20 mL) and extracted with CH2Cl2 (2 × 5
mL). The aqueous phase was treated with NH4OH (3 mL) for
1 h and then evaporated. The residue was purified by ion
exchange chromatography DEAD-Sephadex A25 (HCO3- form),
eluting with a gradient of H2O-0.15 M NH4HCO3. Appropriate
fractions were evaporated, coevaporated with H2O, and lyoph-
ilized to give 38 mg (70%) of 8 as a white lyophilate. UV (H2O)
λ max ) 272 (ꢀ ) 8700). MS (ES negative mode): m/z 319 (M -
eluting with a gradient of H2O-0.15 M NH4HCO3. Appropriate
fractions were evaporated, coevaporated with H2O, and lyoph-
ilized to give 24 mg (50%) of 7 as a white lyophilate. UV (H2O)
λ
) 272 (ꢀ ) 9600). MS (ES, negative mode): m/z 323 (M
max
- NH4)-; 1H NMR (D2O) δ: 1.81 (s, 3H, 5-CH3), 3.50-3.78 (m,
6H, CH2P, CH2OCH2P, CHN, CHOH), 3.87 (ddd, J ) 2.7, 7.5,
9.0 Hz, 1H, CHOH), 4.09 (dd, J ) 2.7, 14.3 Hz, 1H, CHN),
7.40 (H-6). 13C NMR (D2O) δ: 12.5 (5-CH3), 52.4 (CH2N), 68.9
(d, J C,P ) 168.0 Hz, CH2P), 70.5, 72.5 (CHOH), 74.6 (d, J C,P
)
11.1 Hz, CH2OCH2P), 111.6 (C-5), 145.3 (C-6), 153.8 (C-2),
168.3 (C-4). HPLC: system I, Rt ) 4.85 min (97.6%); system
II, Rt ) 5.93 min (100%).
(Z)-1-Ben zoxy-4-ch lor om et h oxy-2-b u t en e (30). To a
cooled solution of 2928 (2.46 g, 12.80 mmol) and paraformal-
dehyde (1.93 g, 64.20 mmol) in dry CH2Cl2 (39 mL) was
bubbled HCl(g) for 2 h. The mixture was stored at 4 °C for an
1
NH4)-. H NMR (D2O) δ: 1.68 (s, 3H, 5-CH3), 3.52 (d, J ) 9.1
Hz, 2H, CH2P), 4.14, 4.28 (2d, J ) 6.6, 6.8 Hz, 4H, CH2N, CH2-
OCH2O), 4.62 (s, 2H, OCH2O), 5.46-5.78 (m, 2H, CHdCH),
7.29 (H-6). 13C NMR (CDCl3) δ: 12.5 (5-CH3), 46.5 (CH2N),

980 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Esteban-Gamboa et al.
63.9 (CH2OCH2O), 64.7 (d, J C,P ) 158.1 Hz, CH2P), 96.5 (d,
J C,P ) 12.6 Hz, OCH2O), 112.1 (C-5), 128.7, 130.6 (CHdCH),
143.9 (C-6), 153.4 (C-2), 168.2 (C-4). HPLC: system I, Rt )
11.15 min (97.8%); system II, Rt ) 12.48 min (97.3%).
1-[4-[((Diisopr opylph osph on o)m eth oxy)m eth oxy]bu tyl]-
th ym in e (35). Compound 34 (210 mg, 0.52 mmol) was
hydrogenated as described for 27, to yield after column
chromatography (EtOAc:MeOH 10:1) 150 mg (66%) of 35 as a
syrup that was used in the next step.
1-[4-[(P h osph on om eth oxy)m eth oxy]bu tyl]th ym in e Am -
m on iu m Sa lt (9). A solution of 35 (85 mg, 0.21 mmol) and
2,6-lutidine (0.36 mL, 3.15 mmol) in dry CH2Cl2 was reacted
with TMSBr (0.27 mL, 2.10 mmol) as described for the
synthesis of 8. After purification, 60 mg (84%) of 9 was
(s, 1H, H-6), 7.30-8.10 (m, 5H, arom.), 8.67 (br s, 1H, NH).
13C NMR (CDCl3) δ: 12.1 (5-CH3), 60.4 (CH2OBz), 64.8 (CH2-
OCH2N), 75.8 (NCH2O), 111.6 (C-5), 127.5, 129.3 (CHd), 128.2
(2C, arom.), 129.4 (2C, arom.), 129.8 (arom.), 132.8 (arom.),
138.9 (C-6), 151.3 (C-2), 164.3 (C-4), 166.0 (CO). Anal.
(C17H18N2O5) C, H, N.
Meth od B. Thymine was silylated as described in method
A. To the syrup containing silylated thymine was added a
solution of 31 (freshly prepared from 500 mg (2.60 mmol) of
29) in dry CH2Cl2 (10 mL). The resulting solution was cooled
to 0 °C, and then SnCl4 (0.15 mL, 1.30 mmol) was added. The
mixture was stirred for 4 h at room temperature. Then it was
worked up as in method A. Purification by column chroma-
tography (EtOAc:hexane, 1:1) afforded 77 mg (9%) of 41 from
the fastest moving fractions; the slowest moving fractions
afforded 146 mg (17%) of the N3 isomer (42). Data for 42: 1H
NMR (CDCl3) δ: 1.90 (s, 3H, 5-CH3), 4.34 (d, J ) 4.0 Hz, 2H,
CH2OCH2N), 4.90 (d, J ) 4.2 Hz, 2H, CH2OBz), 5.44 (s, 2H,
NCH2O), 5.70-6.00 (m, 2H, CHdCH), 7.09 (s, 1H, H-6), 7.30-
8.10 (m, 5H, arom.), 10.27 (br s, 1H, NH). 13C NMR (CDCl3) δ:
12.9 (5-CH3), 60.9 (CH2OBz), 66.0 (CH2OCH2N), 69.9 (NCH2O),
110.4 (C-5), 127.0, 130.4 (CHd), 128.4 (2C, arom.), 129.6 (2C,
arom.), 130.0 (arom.), 133.0 (arom.), 135.2 (C-6), 153.2 (C-2),
164.0 (C-4), 166.4 (CO).
obtained as a white lyophilate. UV (H2O) λ
) 273 (ꢀ )
max
9400). MS (ES, negative mode): m/z 321 (M - NH4)-. 1H NMR
(D2O) δ: 1.40-1.58 (m, 4H, CH2), 1.66 (s, 3H, 5-CH3), 3.42 (d,
J ) 9.1 Hz, 2H, OCH2P), 3.45, 3.57 (2t, J ) 6.1, 7.0 Hz, 4H,
CH2N, CH2OCH2O), 4.54 (s, 2H, OCH2O), 7.30 (s, 1H, H-6).
13C NMR (D2O) δ: 12.5 (5-CH3), 26.2, 26.8 (CH2), 49.5 (CH2N),
65.1 (d, J C,P ) 156.2 Hz, CH2P), 68.7 (CH2OCH2P), 97.0 (d,
J C,P ) 12.0 Hz, OCH2O), 111.8 (C-5), 144.3 (C-6), 153.5 (C-2),
168.2 (C-4). HPLC: system I, Rt ) 11.11 min (100%); system
II, Rt ) 12.53 min (99.9%).
1-[((Z)-4-H yd r oxy-2-b u t en oxy)m et h yl]t h ym in e (43).
Compound 41 (413 mg, 1.25 mmol) was treated with MeNH2
in MeOH (15 mL) overnight. Volatiles were removed, and the
residue was purified by column chromatography (EtOAc) to
yield 279 mg (98% yield) of 43 as a solid. Mp: 88-89 °C (AcOEt:
diethyl ether 1:1) (lit.38 mp 79-81 °C). 1H NMR (CDCl3) δ: 1,95
(s, 3H, 5-CH3), 2.70 (br s, 1H, OH), 4.14 (br t, J ) 5.4 Hz, 2H,
CH2OH), 4.20 (d, J ) 6.7 Hz, 2H, CH2OCH2N), 5.15 (s, 2H,
NCH2O), 5.50-5.94 (m, 2H, CHdCH), 7.16 (s, 1H, H-6), 9.20
(br s, 1H, NH). 13C NMR (CDCl3) δ: 12.1 (5-CH3), 57.7 (CH2-
OH), 64.6 (CH2OCH2N), 75.3 (NCH2O), 111.9 (C-5), 126.4,
133.4 (CHdCH), 139.0 (C-6), 151.8 (C-2), 164.3 (C-4). Anal.
(C10H14N2O4) C, H, N.
(2S,3S)- a n d (2R,3R)-1-[2,3-Dih yd r oxy-4-[((d iisop r op yl-
p h osp h on o)m eth oxy)m eth oxy]bu tyl]th ym in e (36). Start-
ing from 34 (200 mg, 0.49 mmol), a procedure analogous to
that described for the synthesis of 28 was followed. After
column chromatography (CH2Cl2:acetone, 1:2), 167 mg (76%
yield) of 36 was obtained as a syrup. 1H NMR (CDCl3) δ: 1.32,
1.33 [2d, J ) 6.2 Hz, 12H, CH(CH3)2], 1.90 (s, 3H, 5-CH3),
3.30-3.60 (m, 1H, CHOH), 3.71 (dd, J ) 3.2, 9.9 Hz, 1H,
CHOCH2O), 3.86 (d, J ) 8.8 Hz, 2H, CH2P), 3.84-4.04 (m,
2H, CHOH, CHN), 4.10 (dd, J ) 3.1, 10.0 Hz, 1H, CHOCH2O),
4.14 (dd, J ) 3.9, 15.3 Hz, 1H, CHN), 4.32 (d, J ) 5.6 Hz, 1H,
OH), 4.62-4.82 [m, 2H, CH(CH3)2], 4.73 (s, 2H, OCH2O), 5.29
(d, J ) 3.5 Hz, 1H, OH), 7.29 (s, 1H, C-6), 9.00 (br s, 1H, NH).
13C NMR (CDCl3) δ: 12.2 (5-CH3), 23.8-24.1 [CH(CH3)2], 51.1
(CH2N), 62.4 (d, J C,P ) 173.2 Hz, CH2P), 68.8 (CH2OCH2O),
69.7, 70.7 (CHOH), 71.4, 71.8 [2d, J C,P ) 6.9 Hz, CH(CH3)2],
96.6 (d, J C,P ) 9.9 Hz, OCH2O), 110.0 (C-5), 142.9 (C-6), 152.6
(C-2), 164.5 (C-4). Anal. (C17H31N2O9P) C, H, N.
1-[((Z)-4-(Diisopr opylph osph on o)m eth oxy)-2-bu ten oxy)-
m eth yl]th ym in e (38). To a solution of 43 (230 mg, 1.02 mmol)
in dry DMF (5 mL) at -30 °C and under Ar atmosphere was
added NaH (122 mg of 60% dispersion in mineral oil, 3.06
mmol). The mixture was allowed to reach room temperature
and stirred for 1 h. Then it was cooled again to -30 °C, and
diisoproypl[(p-toluensulfonyl)oxy] methanephosphonate (427
mg, 1.22 mmol) was added. The reaction was allowed to reach
room temperature and was stirred for 20 h. Then, it was
neutralized with AcOH and evaporated, and the residue was
purified by column chromatography. Elution with CH2Cl2:
MeOH 25:1 afforded 38 (100 mg, 25%) as a syrup. 1H NMR
(CDCl3) δ: 1.31, 1.32 [2d, J ) 6.2 Hz, 12H, CH(CH3)2], 1.92
(s, 3H, 5-CH3), 3.70 (d, J ) 9.0 Hz, 2H, CH2P), 4.13, 4.17 (2d,
J ) 4.9, 5.0 Hz, 4H, CH2O), 4.60-4.90 [m, 2H, CH(CH3)2], 5.13
(s, 2H, NCH2O), 5.50-5.85 (m, 2H, CHdCH), 7.13 (s, 1H, H-6),
9.37 (br s, 1H, NH). 13C NMR (CDCl3) δ: 12.3 (5-CH3), 23.8-
24.1 [m, CH(CH3)2], 64.8 (d, J C,P ) 169.5 Hz, CH2P), 65.2 (CH2-
(2S,3S)- a n d (2R,3R)-1-[2,3-Dih yd r oxy-4-[(p h osp h on o-
m eth oxy)m eth oxy]bu tyl]th ym in e Am m on iu m Sa lt (10).
Starting from 36 (80 mg, 0.18 mmol) and following an
analogous procedure to that described for the synthesis of 7,
compound 10 was isolated as a white lyophilate (22 mg, 46%
yield). UV (H2O) λ
) 272 (ꢀ ) 9000). MS (ES, negative
max
mode): m/z 353 (M - NH4)-. 1H NMR (D2O) δ: 1.81 (s, 3H,
5-CH3), 3.62 (d, J ) 9.2 Hz, 2H, CH2P), 3.45-3.90 (m, 5H,
CHN, CH2OCH2O, CHOH x 2), 4.10 (dd, J ) 2.0, 14.4 Hz, 1H,
CHN), 4.72 (s, 2H, OCH2O), 7.39 (s, 1H, H-6). 13C NMR (CDCl3)
δ: 12.3 (5-CH3), 52.2 (CH2N), 64.5 (d, J C,P ) 157.0 Hz, CH2P),
69.8 (CH2OCH2O), 70.4, 72.4 (CHOH), 97.3 (d, J C,P ) 11.4 Hz,
OCH2O), 111.3 (C-5), 145.1 (C-6), 153.5 (C-2), 168.1 (C-4).
HPLC: system I, Rt ) 4.88 min (98.8%); system II, Rt ) 6.78
min (100%).
OCH2N), 68.4 (d, J C,P ) 13.8 Hz, CH2OCH2P), 71.1 [d, J C,P
)
6.9 Hz, CH(CH3)2], 76.1 (NCH2O), 111.6 (C-5), 128.7, 129.2
(CHdCH), 138.9 (C-6), 151.2 (C-2), 164.1 (C-4). Anal. (C17H29
-
1-[((Z)-4-Ben zoxy-2-b u t en oxy)m et h yl]t h ym in e (41).
Meth od A. A suspension of thymine (394 mg, 3.12 mmol) and
(NH4)2SO4 (13 mg) in hexamethyldisilazane (3 mL) was
refluxed overnight. The resulting solution was evaporated and
coevaporated with dry toluene. To the resulting syrup were
added a solution of 30 (freshly prepared from 500 mg of 29) in
dry CH2Cl2 (5 mL) and Bu4NI (192 mg, 0.52 mmol). The
mixture was stirred at room temperature for 24 h. Then, CH2-
Cl2 (20 mL) and cooled saturated solution of NaHCO3 (10 mL)
were added. The aqueous phase was further extracted with
CH2Cl2 (2 × 10 mL). The combined organic extracts were dried
on anhydrous Na2SO4, filtered, and evaporated. The residue
obtained was purified by column chromatography (EtOAc:
hexane, 1:1) to yield 615 mg (72%) of 41. Mp: 80-81 °C (EtOAc:
diethyl ether). 1H NMR (CDCl3) δ: 1.93 (s, 3H, 5-CH3), 4.30
(d, J ) 5.2 Hz, 2H, CH2OCH2N), 4.88 (d, J ) 5.8 Hz, 2H, CH2-
OBz), 5.16 (s, 2H, NCH2O), 5.60-6.00 (m, 2H, CHdCH), 7.16
N2O7P) C, H, N.
Then elution with iPrOH-NH4OH:H2O (8:1:1) gave the
monoprotected derivative 44 (116 mg, 31%). 1H NMR (DMSO-
d6) δ: 1.10 [d, J ) 5.0 Hz, 6H, CH(CH3)2], 1.75 (s, 3H, 5-CH3),
3.38 (d, J ) 8.6 Hz, 2H, CH2P), 3.90-4.20 (m, 4H, CH2O),
4.20-4.60 [m, 1H, CH(CH3)2], 5.03 (s, 2H, NCH2O), 5.40-5.80
(m, 2H, CHdCH), 7.57 (s, 1H, H-6), 8.40 (br s, 1H, NH).
1-[((Z)-4-P h osp h on om eth oxy)-2-bu ten oxy)m eth yl]th y-
m in e Am m on iu m Sa lt (11). A solution of 38 (80 mg, 0.20
mmol) and 2,6-lutidine (0.35 mL, 3.06 mmol) in dry CH2Cl2 (3
mL) reacted with TMSBr (0.26 mL, 2.00 mmol) as described
for compound 8. A white lyophilate (44 mg, 75% yield) was
obtained. UV (H2O) λmax ) 265 (ꢀ ) 8400). MS (ES, negative
mode): m/z 319 (M - NH4)-. 1H NMR (D2O) δ: 1.68 (s, 3H,
5-CH3), 3.39 (d, J ) 8.3 Hz, 2H, CH2P), 3.95 (d, J ) 5.1 Hz,
2H, CH2O), 4.02 (d, J ) 5.3 Hz, 2H, CH2O), 4.99 (s, 2H,

Multisubstrate Analogue Inhibitors of E. coli TPase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 981
NCH2O), 5.40-5.70 (m, 2H, CHdCH), 7.34 (s, 1H, H-6). 13C
NMR (D2O) δ: 12.3 (5-CH3), 65.8 (CH2OCH2N), 67.6 (d, J C,P
) 162.3 Hz, CH2P), 69.1 (d, J C,P ) 12.1 Hz, CH2OCH2P), 78.1
(NCH2O), 112.5 (C-5), 129.7, 131.2 (CHdCH), 143.0 (C-6),
153.5 (C-2), 168.1 (C-4). HPLC: system I, Rt ) 10.65 min
(96.6%); system II, Rt ) 12.15 min (96.1%).
3.90-4.20 (m, 4H, CH2CH3), 6.15 (br s, 2H, 6-NH2), 9.47 (br s,
1H, NH-3). 13C NMR (CDCl3) δ: 16.4 (d, J C,P ) 6.1 Hz,
CH2CH3), 22.2 (d, J C,P ) 5.4 Hz, CH2CH2P), 25.2 (d, J C,P
139.6 Hz, CH2P), 26.3, 27.7, 28.8, 28.9 (CH2), 30.2 [d, J C,P
)
)
16.1 Hz, CH2(CH2)2P], 43.6 (CH2N), 61.6 (d, J C,P ) 6.9 Hz, CH2-
CH3), 72.7 (C-5), 150.1, 152.5 (C-2, C-6), 159.4 (C-4). Anal.
(C16H29BrN3O5P) C, H, N.
N-[8-(Dieth ylp h osp h on o)octyl]p h th a lim id e (46). A so-
lution of 4534 (3.00 g, 8.87 mmol) in triethyl phosphite (30 mL)
was heated at 130 °C for 12 h. Solvent was removed, and the
residue was purified by column chromatography (CH2Cl2:
6-Am in o-5-br om o-1-(8-p h osp h on ooctyl)u r a cil Am m o-
n iu m Sa lt (12). A solution of 50 (70 mg, 0.15 mmol) in dry
CH2Cl2 (1.5 mL) was reacted with TMSBr (0.20 mL, 1.52
mmol) in an analogous way to that described for compound 8.
After purification, 47 mg (75% yield) of compound 12 was
1
acetone, 5:1) to yield 3.37 g (96%) of 46 as a syrup. H NMR
(CDCl3) δ: 1.31 (t, J ) 7.1 Hz, 6H, CH2CH3), 1.20-1.80 (m,
14H), 3.66 (t, J ) 7.2 Hz, 2H, CH2N), 3.96-4.20 (m, 4H, CH2-
obtained as a white lyophilate. UV (H2O) λ
) 278 (ꢀ )
max
CH3), 7.65-7.75 (m, 2H, arom.), 7.80-7.90 (m, 2H, arom.). 13
C
12000). MS (ES, negative mode): m/z 396 and 398 [(M - NH4)-
and [(M + 2 - NH4)-]. 1H NMR (D2O) δ: 1.40-2.10 (m, 14H),
4.21 (t, J ) 7.1 Hz, 2H, CH2N). 13C NMR (D2O) δ: 27.7 (d,
J C,P ) 4.6 Hz, CH2CH2P), 30.2, 31.2, 32.7, 32.9 (CH2), 32.5 (d,
J C,P ) 132.8 Hz, CH2P), 34.7 [d, J C,P ) 16.8 Hz, CH2(CH2)2P],
48.3 (CH2N), 72.7 (C-5), 155.9, 158.5 (C-2, C-6), 165.8 (C-4).
HPLC: system I, Rt ) 13.63 min (100%); system II, Rt ) 22.15
min (100%).
NMR (CDCl3) δ: 16.3 (d, J C,P ) 5.4 Hz, CH2CH3), 22.2 (d, J C,P
) 5.4 Hz, CH2CH2P), 25.5 (d, J C,P ) 140.3 Hz, CH2P), 26.6,
28.4, 28.8 (CH2), 30.3 (d, J C,P ) 16.9 Hz, CH2(CH2)2P), 37.8
(CH2N), 61.2 (d, J C,P ) 6.1 Hz, CH2CH3), 123.0, 132.0, 133.7
(arom.), 168.3 (CO). Anal. (C20H30NO5P) C, H, N.
8-(Dieth ylp h osp h on o)octyla m in e (47). To a solution of
46 (455 mg, 1.15 mmol) in EtOH (40 mL) was added H2N-
NH2‚H2O (0.57 mL, 11.50 mmol), and the mixture was stirred
at room temperature for 18 h. It was filtered, and the filtrate
was evaporated. The residue was purified by column chroma-
tography (CH2Cl2:acetone:NH4OH, 5:1:0.6) to afford 275 mg
1-[8-(Dieth ylp h osp h on o)octyl]-2,4-d ioxop yr r olo[2,3-d ]-
p yr im id in e (51). To a stirred suspension of 49 (140 mg, 0.38
mmol) in EtOH (2 mL) was added AcONa (31 mg, 0.38 mmol),
and the mixture was heated at 70 °C. Then, chloroacetalde-
hyde (0.073 mL of a 50% aqueous solution, 0.57 mmol) and
AcONa (31 mg, 0.38 mmol) were added, and heating at 70 °C
was maintained for 3 h. Then, it was cooled and filtered
through a Celite path, and the filtrate evaporated. The residue
was purified in the Chromatotron CCTLC (AcOEt:MeOH, 10:
1
(90% yield) of 47 as a syrup. H NMR (CDCl3) δ: 1.31 (t, J )
7.0 Hz, 6H, CH2CH3), 1.10-1.85 (m, 14H), 2.14 (br s, 2H, NH2),
2.68 (t, J ) 6.8 Hz, 2H, CH2N), 3.90-4.20 (m, 4H, CH2CH3).
13C NMR (CDCl3) δ: 16.1 (d, J C,P ) 5.4 Hz, CH2CH3), 22.1 (d,
J C,P ) 4.6 Hz, CH2CH2P), 25.3 (d, J C,P ) 140.3 Hz, CH2P), 26.5,
28.7, 28.8 (CH2), 30.2 (d, J C,P ) 16.9 Hz, CH2(CH2)2P), 32.7
(CH2CH2N), 41.5 (CH2N), 61.2 (d, J C,P ) 6,1 Hz, CH2CH3).
1
1) to yield 71 mg (47%) of 51 as a syrup. H NMR (CDCl3) δ:
1.00-2.00 (m, 14H), 1.33 (t, J ) 7.1 Hz, 6H, CH2CH3), 3.80-
4.25 (m, 6H, CH2N, CH2CH3), 6.48 (dd, 1H, J ) 1.9, 3.3 Hz,
1H, H-6), 6.62 (dd, J ) 2.3, 3.3 Hz, 1H, H-5), 8.86 (br s, 1H,
N-[8-(Dieth ylp h osp h on o)octyl]u r ea (48). To a stirred
solution of 47 (577 mg, 2.18 mmol) in H2O (3.5 mL) at 10 °C
was added concentrated HCl (0.22 mL, 2.18 mmol). After a
few minutes, KOCN (353 mg, 4.36 mmol) was added, and the
mixture was heated at 70 °C overnight. Then, it was cooled
and extracted with CH2Cl2 (15 mL). The organic phase was
dried on anhydrous Na2SO4, filtered, and evaporated. The
obtained syrup containing 48 (487 mg) was used as such in
the next step. 1H NMR (CDCl3) δ: 1.31 (t, J ) 7.0 Hz, 6H,
CH2CH3), 1.10-1.90 (m, 14H), 3.15 (q, J ) 6.5 Hz, 2H, CH2N),
3.70-4.30 (m, 4H, CH2CH3), 4.63 (br s, 2H, NH2), 5.13 (br s,
1H, NH). 13C NMR (CDCl3) δ: 16.3 (d, J C,P ) 5.4 Hz, CH2CH3),
22.1 (d, J C,P ) 4.6 Hz, CH2CH2P), 25.3 (d, J C,P ) 139.6 Hz,
CH2P), 26.5, 28.7, 29.9 (CH2), 30.1 [d, J C,P ) 18.4 Hz, CH2-
(CH2)2P], 40.0 (CH2N), 61.4 (d, J C,P ) 6.1 Hz, CH2CH3), 159.7
(CO).
NH), 11.33 (br s, 1H, NH). 13C NMR (CDCl3) δ: 16.3 (d, J C,P
)
6.1 Hz, CH2CH3), 22.0 (d, J C,P ) 4.6 Hz, CH2CH2P), 25.0 (d,
J C,P ) 140.3 Hz, CH2P), 26.2, 27.9, 28.6, 28.8 (CH2), 30.1 [d,
J C,P ) 16.1 Hz, CH2(CH2)2P], 43.4 (CH2N), 61.6 (d, J C,P ) 6.9
Hz, CH2CH3), 99.9 (C-4a), 104.1 (C-5), 117.3 (C-6), 140.3 (C-
7a), 150.8 (C-2), 160.2 (C-4). Anal. (C18H30N3O5P) C, H, N.
1-(8-P h osp h on ooct yl)-2,4-d ioxop yr r olo[2,3-d ]p yr im i-
d in e Am m on iu m Sa lt (13). A solution of 51 (100 mg, 0.25
mmol) and 2,6-lutidine (0.51 mL, 3.76 mmol) in dry CH2Cl2
(2.5 mL) was reacted with TMSBr (0.20 mL, 1.52 mmol) as
described for compound 8. Compound 13 was obtained as a
white lyophilate (77 mg, 85% yield). UV (H2O) λ
) 275 (ꢀ
max
) 7600), 243 (ꢀ ) 6500). MS (ES, negative mode): m/z 342 (M
1
- NH4)-. H NMR (D2O) δ: 1.00-1.70 (m, 14H), 3.83 (t, J )
6-Am in o-1-[8-(d ieth ylp h osp h on o)octyl]u r a cil (49). To
a 1.7 M solution of NaEtO in EtOH (0.45 mL) were added
ethylcyanoacetate (0.07 mL, 0.65 mmol) and the urea 48 (200
mg, 0.65 mmol). The mixture was refluxed for 20 h. Then, it
was cooled and neutralized with 1 M HCl, diluted with EtOH
(10 mL), and filtered. The filtrate was purified in the Chro-
matotron CCTLC (CH2Cl2:MeOH, 10:1). Compound 49 (160
6.7 Hz, CH2N), 6.37, 6.71 (2d, J ) 3.5 Hz, 2H, H-6, H-5). 13C
NMR (D2O) δ: 24.6 (d, J C,P ) 5.0 Hz, CH2CH2P), 27.3, 28.6,
29.7, 29.8 (CH2), 29.2 (d, J C,P ) 163.1 Hz, CH2P), 31.6 [d, J C,P
) 15.6 Hz, CH2(CH2)2P], 45.5 (CH2N), 101.4 (C-4a), 105.1 (C-
5), 120.5 (C-6), 142.5 (C-7a), 153.5 (C-2), 163.5 (C-4). HPLC:
system I, Rt ) 14.01 min (100%); system II, Rt ) 21.98 min
(100%).
1
mg, 66% yield) was obtained as a syrup. H NMR (methanol-
TP a se En zym e Assa y. The phosphorolysis of thymidine
(dThd) by E. coli thymidine phosphorylase was measured by
HPLC analysis. One milliliter of the incubation mixture
contained 10 mM Tris‚HCl (pH 7.6), 1 mM EDTA, 2 mM
potassium phosphate, 150 mM NaCl, and 100 µM of thymidine
in the presence or absence of 100 µM 2-deoxyribose-R-1-
phosphate (R-dR-1-P) and 0.025 units of TPase. Incubations
were performed at room temperature. At different time points
(i.e., 0, 20, 40, and 60 min), 100 µL fractions were taken,
transferred to an Eppendorf tube thermo block, and boiled at
95 °C for 5 min. Thereafter, the samples were rapidly cooled
on ice, and thymidine was separated from thymine and
quantified in the samples by HPLC analysis.
d4) δ: 1.51 (t, J ) 7.0 Hz, 6H, CH2CH3), 1.40-2.10 (m, 14H),
4.03 (t, J ) 7.3 Hz, 2H, CH2N), 4.14-4.40 (m, 4H, CH2CH3).
13C NMR (methanol-d4) δ: 17.0 (d, J C,P ) 5.4 Hz, CH2CH3),
23.6 (d, J C,P ) 4.6 Hz, CH2CH2P), 26.0 (d, J C,P ) 139.6 Hz,
CH2P), 27.7, 29.0, 30.3, 30.4 (CH2), 31.6 [d, J C,P ) 16.1 Hz,
CH2(CH2)2P], 43.0 (CH2N), 63.4 (d, J C,P ) 6.9 Hz, CH2CH3),
76.9 (C-5), 153.3 (C-2), 158.9 (C-6), 166.6 (C-4). Anal.
(C16H30N3O5P) C, H, N.
6-Am in o-5-b r om o-1-[8-(d iet h ylp h osp h on o)oct yl]u r a -
cil (50). To a suspension of 49 (120 mg, 0.320 mmol) in dry
THF (1.5 mL) were added N-bromosuccinimide (57 mg, 0.320
mmol) and AIBN (0.6 mg, 0.003 mmol). The mixture was
heated at 60 °C for 90 min. Then, it was diluted with EtOH
(10 mL) and filtered through a Celite path. The filtrate was
evaporated and purified in the Chromatotron CCTLC (CH2-
Cl2:MeOH, 20:1) to yield 104 mg (72%) of 50 as a syrup. 1H
NMR (CDCl3) δ: 1.20-1.45 (m, 8H), 1.32 (t, J ) 7.1 Hz, 6H,
CH2CH3), 1.45-1.90 (m, 6H), 3.97 (t, J ) 6.5 Hz, 2H, CH2N),
In the assays where the inhibitory effect of the compounds
were evaluated, a variety of inhibitor concentrations, including
1 mM, 500 µM, 100 µM, 20 µM, and 0 µM (control) were added
to the reaction mixture that contained 100 µM of dThd.
Aliquots of 100 µL were withdrawn from the reaction mixture

982 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
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at several time points, as described above, heated at 95 °C to
inactivate the enzyme, and analyzed on HPLC.
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compounds was evaluated against TPase at varying inorganic
phosphate, compounds 12, 13, and 7DX were tested at
concentrations ranging between 200 and 10 µM, in the
presence of 2, 5, 10, 25, 50, and 100 mM inorganic phosphate.
The dThd concentration was kept fixed at 1000 µM. The
reaction mixture was then incubated at room temperature for
20 min, after which an aliquot was withdrawn and analyzed
by HPLC for dThd conversion to Thy. In the kinetic assays
where the inhibitory effect of the test compounds was evalu-
ated against TPase at varying dThd concentrations, com-
pounds 12 and 13 were tested at concentrations ranging
between 200 and 20 µM in the presence of 125, 250, 500, 750,
and 1000 µM dThd. The inorganic phosphate concentration
was kept constant at 25 mM. The analysis of the dThd-to-Thy
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Ack n ow led gm en t. We are grateful to Mrs. Ria Van
Berwaer for excellent technical assistance. This research
was supported by grants from the Biomedical Research
Program of the European Commission, the Spanish
CICYT (SAF Programme), and the Belgische Federatie
teg en kanker. A.E.G. is grateful to GlaxoWellcome-
Spain for an award to young researchers for the present
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