6-Substituted and 5,6-disubstituted derivatives of uridine: Stereoselective synthesis, interaction with uridine phosphorylase, and in vitro antitumor activity

Article abstract of DOI:10.1021/jm950675q

Stereoselective procedures are described for the synthesis of 6- alkyluridines by Lewis acid-catalyzed condensation of (a) trimethylsilylated 6-alkyl-4-alkylthiouracils with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D- ribofuranose (ABR) and (b) trimethylsilylated 6-alkyl-3-benzyluracils with ABR. The 4-methylthio group was subsequently removed with the use of 1 N trifluoroacetic acid and the 3-benzyl group by a new modified procedure with the use of the complex BBr₃-THF. Furthermore, 6-(hydroxymethyl)uridine (39) and 5-fluoro-6-(hydroxymethyl)uridine (40) were obtained by sequential oxidation with SeO₂ and reduction with tetrabutylammonium borohydride of the 6-methyl group of 6-methyluridine (5) and 5-fluoro-6-methyluridine (35), and their corresponding 6-fluoromethyl congeners 41 and 42 were obtained by DAST treatment of 39 and 40, respectively. For all the foregoing nucleosides in the fixed syn conformation about the glycosyl bond, ¹H NMR spectroscopy further demonstrated that the pentose rings exist predominantly in the conformation N (3'-endo). Most of the nucleosides were weak substrates of Escherichia coli pyrimidine nucleoside phosphorylase. Enhanced susceptibility to phosphorolysis was exhibited by two of them, 39 and 41, with 6-CH₂OH and 6-CH₂F substituents capable of formation of an additional hydrogen bond with the enzyme. The 5-fluoro-6-substituted uridines were the poorest substrates. Cytotoxicities of the nucleosides were examined vs the human tumor cell lines MOLT-3, U-937, K-562, and IM-9, as well as PHA-stimulated human lymphocytes. Two of the analogues, 5-fluoro-6-(fluoromethyl)uridine (42) and 5-fluoro-6- (hydroxymethyl)uridine (40), exhibited cytotoxicities comparable to that of 5-fluorouracil.

Full text of DOI:10.1021/jm950675q

1720
J . Med. Chem. 1996, 39, 1720-1728
6-Su bstitu ted a n d 5,6-Disu bstitu ted Der iva tives of Ur id in e: Ster eoselective
Syn th esis, In ter a ction w ith Ur id in e P h osp h or yla se, a n d in Vitr o An titu m or
Activity
Krzysztof Felczak,† Alicja K. Drabikowska,† J uhani A. Vilpo,‡ Tadeusz Kulikowski,*,† and David Shugar†
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5a Pawin´skiego St., 02-106 Warszawa, Poland, and
Laboratory of Molecular Hematology, Department of Clinical Chemistry, Tampere University Central Hospital,
SF-33520 Tampere, Finland
Received September 12, 1995X
Stereoselective procedures are described for the synthesis of 6-alkyluridines by Lewis acid-
catalyzed condensation of (a) trimethylsilylated 6-alkyl-4-alkylthiouracils with 1-O-acetyl-2,3,5-
tri-O-benzoyl-â-D-ribofuranose (ABR) and (b) trimethylsilylated 6-alkyl-3-benzyluracils with
ABR. The 4-methylthio group was subsequently removed with the use of 1 N trifluoroacetic
acid and the 3-benzyl group by a new modified procedure with the use of the complex BBr3-
THF. Furthermore, 6-(hydroxymethyl)uridine (39) and 5-fluoro-6-(hydroxymethyl)uridine (40)
were obtained by sequential oxidation with SeO2 and reduction with tetrabutylammonium
borohydride of the 6-methyl group of 6-methyluridine (5) and 5-fluoro-6-methyluridine (35),
and their corresponding 6-fluoromethyl congeners 41 and 42 were obtained by DAST treatment
of 39 and 40, respectively. For all the foregoing nucleosides in the fixed syn conformation
1
about the glycosyl bond, H NMR spectroscopy further demonstrated that the pentose rings
exist predominantly in the conformation N (3′-endo). Most of the nucleosides were weak
substrates of Escherichia coli pyrimidine nucleoside phosphorylase. Enhanced susceptibility
to phosphorolysis was exhibited by two of them, 39 and 41, with 6-CH2OH and 6-CH2F
substituents capable of formation of an additional hydrogen bond with the enzyme. The
5-fluoro-6-substituted uridines were the poorest substrates. Cytotoxicities of the nucleosides
were examined vs the human tumor cell lines MOLT-3, U-937, K-562, and IM-9, as well as
PHA-stimulated human lymphocytes. Two of the analogues, 5-fluoro-6-(fluoromethyl)uridine
(42) and 5-fluoro-6-(hydroxymethyl)uridine (40), exhibited cytotoxicities comparable to that of
5-fluorouracil.
In tr od u ction
thesis of a number of C(6)-substituted uridines and
5-fluorouridines, their substrate properties with uridine
phosphorylase from Escherichia coli, and their cytotox-
icities toward a variety of human tumor cell lines.
In both aqueous and nonaqueous media most pyrim-
idine nucleosides exist predominantly in the anti con-
formation about the glycosyl bond, in part due to
repulsive interaction between the C(2) carbonyl and the
furanose ring. One notable exception is the naturally
occurring orotidine (6-carboxyuridine), which is con-
strained to the syn conformation by steric hindrance
between the sugar ring and the bulky 6-carboxy sub-
stituent.1 Pyrimidine nucleosides constrained to the syn
conformation by a C(6)-substituent are of interest (a)
as potential antimetabolites, e.g., 6-thiocarboxamide-
UMP, a structural analogue of orotidine-5′-phosphate
(OMP), is a potent inhibitor of OMP decarboxylase,2 and
(b) as model compounds to determine whether the
parent nonsubstituted nucleoside (or nucleotide) is
involved in a given enzymatic reaction in the syn and/
or anti conformation; e.g., it was long ago shown that
6-methyluridine is a moderate substrate for uridine
phosphorylase from Salmonella typhimurium.3
Ch em istr y
The major obstacle to synthesis of 6-substituted and
5,6-disubstituted pyrimidine nucleosides by condensa-
tion methods is the partial, or exclusive, formation of
the undesired N(3)-isomers, even under the optimal
conditions described by Vorbruggen et al.4 for maximal
yields of the N(1)-isomers. With a view to avoiding
formation of the N(3)-glycosides, we have developed
condensation methods in which the pyrimidine N(3) is
blocked, e.g., N(3)-benzyl or 4-alkylthio derivatives.
Condensation of the TMS derivative 2 of 3-benzyl-6-
methyluracil (1) with 1-O-acetyl-2,3,5-tri-O-benzoylrib-
furanose (ABR), catalyzed by trimethylsilyl triflate
(TMSOTfl) in MeCN (Scheme 1), in our hands led to
2′,3′,5′-tri-O-benzoyl-3-benzyl-6-methyluridine (3) in 76%
yield. Earlier attempts to remove the benzyl group from
3-benzyl-6-methyluridine (4) by catalytic reduction or
with sodium in liquid ammonia were unsuccessful.5 The
use of sodium naphthalene was subsequently found to
be effective,6 and we initially attempted to use it for
removal of the benzyl group from 3-benzyl-6-methyl-
uridine (4). But, since preparation of the reagent is
tedious, requiring the use of oxygen-free medium devoid
of CO2, we made use of the complex of boron tribromide
with THF, formed in situ. This procedure is simpler
Since modification of substrate/inhibitor properties
may be due not only to constrainment to the syn
conformation by a C(6)-substituent but also to steric
effects of the latter, it is clearly desirable to examine
the biological effects of various C(6)-substituents. We
herein describe procedures for the stereospecific syn-
† Polish Academy of Sciences.
‡ Tampere University Central Hospital.
X Abstract published in Advance ACS Abstracts, March 1, 1996.
0022-2623/96/1839-1720$12.00/0 © 1996 American Chemical Society

6- and 5,6-Substituted Uridine Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1721
Sch em e 1a
Sch em e 3a
a
a
Reagents: (i) HMDS/(NH4)2SO4, reflux, 12 h; (ii) ABR, TMSTfl/
Reagents: (i) MeI or EtI, NaOH, room temperature; (ii)
CH3CN, 2 h; (iii) NaOMe/MeOH; (iv) BBr3/THF, room tempera-
ture, 2 h.
HMDS/(NH4)2SO4, reflux, 12 h; (iii) ABR, TMSTfl/MeCN; (iv)
NaOMe/MeOH; (v) 1 N TFA/1,4-dioxane, room temperature.
Sch em e 2a
Sch em e 4a
a
Reagents: (i) Et2NH, TiCl4/hexane, room temperature, 1 h;
a
(ii) ethoxycarbonyl isothiocyanate, Et2O, room temperature, 1 h;
(iii) NH3 (aqueous), room temperature, 48 h.
Reagents: (i) methylpseudoisourea, Ca(OH)2, H2O/EtOH, room
temperature, 72 h; (ii) 2 N H2SO4, 70 °C, 2 h.
and effective; e.g., it permitted conversion of 4 to
6-methyluridine (5) in 67% yield.
from the product of condensation (18) with the use of 1
N TFA and then crystallization of 1-(2,3,5-tri-O-benzoyl-
â-D-ribofuranosyl)-6-propyluracil (22) in 74% yield fol-
lowed by methanolysis with sodium methoxide.
A 4-methylthio group was employed by Mizuno et al.
and Winkley and Robins in the noncatalyzed condensa-
tion of 5-(methylthio)-6-azauracil7a,b and 4-(methylthio)-
6-methyluracil7c with 1-halogeno-2,3,5-tri-O-benzoyl-â-
D-ribofuranose, and the products, without isolation, were
subjected to amination to obtain 6-substituted cytidines.
Despite the low yield of 6-methylcytidine (8%), this
procedure appeared promising if applied in conjunction
with catalyzed condensation methods.
6-Methyl-4-thiouracil (6), 5,6-dimethyl-4-thiouracil
(7), and 6-propyl-4-thiouracil (8) were obtained by
thiation of the parent alkyluracils with the Lawesson
reagent in 1,4-dioxane.8 6-Isopropyl-4-thiouracil (12)
was prepared by pyrimidine ring closure9 (Scheme 2),
whereby the reaction of methyl isopropyl ketone (9) with
diethylamine gave initially the enamine 10. This was
treated with ethoxycarbonyl isothiocyanate to give the
adduct 11 (75%) which was converted with ammonia to
the desired product 12 in 92% yield.
The four (alkylthio)uracils 13-16 were obtained by
treatment of the corresponding 4-thiouracils with the
appropriate alkyl iodide in the presence of base.7c As
shown in Scheme 3, condensation of the TMS deriva-
tives of 4-(alkylthio)uracils 13-16 with ABR was con-
ducted in MeCN. With 0.5 mol equiv of TMSOTfl as
catalyst, the course of the reaction was smooth, with
good yields (80-90%) and no cleavage of the 4-alkylthio
group. The condensation products 17 and 19 were
deblocked with methoxide, and the 4-alkylthio group
was subjected to hydrolysis with 1 N TFA (80% yields).
This procedure led to 6-methyluridine (5) and 6-isopro-
pyluridine (21). 1-â-D-Ribofuranosyl-6-propyluracil (20)
was obtained by first removing the 4-methylthio group
The foregoing high condensation yields were profited
from to select conditions for direct synthesis of 6-meth-
yluridine (5) from 4-(methylthio)-6-methyluracil (69)
and (ABR), by methanolysis of the product of condensa-
tion, followed by hydrolysis of the 4-methylthio group
with 1 N TFA, leading to 6-methyluridine (5) in 80%
yield. The same procedure with 5,6-dimethyl-4-(eth-
ylthio)uracil (14) gave 5,6-dimethyluridine (23) in 60%
yield.
Unfortunately, the foregoing simple and selective
procedures proved inapplicable to the synthesis of
analogues with electrophilic substituents at C(5) or C(6),
since prolongation of the reaction time, or the use of
higher concentrations of catalyst, was inconsistent with
the lability of 4-alkylthio derivatives. Consequently, for
synthesis of 5-fluoro-6-methyluridine (35) and 6-(tri-
fluoromethyl)uridine from 5-fluoro-6-methyluracil (27)
and 6-(trifluoromethyl)uracil (28), respectively, use was
made of the optimal conditions described by Vorbru¨ggen
et al.4 for condensation of 6-methyluracil with the
ribose derivative ABR.
5-Fluoro-6-methyluracil (27)10 and 6-(trifluorometh-
yl)uracil (28)10,11 were prepared from the appropriate
â-keto esters: ethyl R-(fluoroacetyl)acetate (23) and
ethyl (trifluoroacetyl)acetate (24), as shown in Scheme
4, using the procedure hitherto applied for preparation
of 5,6-dialkyl- and 6-alkyluracils.12 Reaction of the
appropriate â-keto ester in aqueous alcohol with meth-
ylpseudoisourea, catalyzed by Ca(OH)2, gave 5-fluoro-
2-methoxy-6-methyluracil (25) in 63% yield and 2-meth-

1722 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Felczak et al.
Sch em e 5a
Sch em e 6a
a
Reagents: (i) with 29, HMDS/TCS, reflux, 6 h, ABR, TMSTfl/
MeCN, room temperature, 2 h; with 33, BSTFA/MeCN, 1,2,3,5-
tetra-O-acetyl-1-â-D-ribofuranose, TMSTfl/MeCN, room tempera-
ture, 30 min; (ii) NaOMe/MeOH.
a
Reagents: (i) Ac2O, DMAP, room temperature; (ii) SeO2, 1,4-
dioxane/AcOH, reflux, 18 h; (iii) TBABH4/MeOH; (iv) DAST/
CH2Cl2, room temperature; (v) NH3/MeOH, 20 °C.
oxy-6-(trifluoromethyl)uracil (26) in 66% yield. Acid
hydrolysis of 25 and 26 led to 85% yields of 27 and 28.
Treatment of 27 with HMDS/TCS gave 2,4-bis-O-
(trimethylsilyl)-5-fluoro-6-methyluracil (29), which was
condensed with ABR in MeCN (Scheme 5) in the
presence of 1.1 equiv of TMSOTfl to give a mixture of
blocked isomers consisting of 1-(2,3,5-tri-O-benzoyl-â-
D-ribofuranosyl)-5-fluoro-6-methyluracil (30; 45%), the
corresponding 3-isomer 31 (21%), and the 1,3-disubsti-
tuted analogue 32 (20%). These were fractionated on a
column of activated Al2O3. Methanolysis catalyzed by
MeONa led to the free nucleosides 35 (80%), 36 (85%),
and 37 (87%). Use of a stronger Lewis acid, SnCl4, as
catalyst resulted in formation of only the N(3)-riboside
31. The UV spectrum of 35 exhibited an increase in
absorbance, relative to that of 5, characteristic for a
5-fluoronucleoside. Spectrophotometric titration gave
a pKa for 35 of 7.75, as compared to 9.80 for 5,
characteristic for 5-fluorouridine.13
In contrast to 29, condensation of di-O-TMS-6-(tri-
fluoromethyl)uracil (33) with 1,2,3,5-tetra-O-acetyl-1-
â-D-ribofuranose gave only the N(3)-isomer 34. The
reaction was conducted in MeCN, with formation of 33
in situ in the reaction mixture, but with replacement
of BSA as the silylating reagent by the more effective
BSTFA. The catalyst in the condensation reaction was
1.5 equiv of TMSOTfl in MeCN. Following 0.5 h one
product (34) appeared (87%) which, following metha-
nolysis catalyzed with MeONa, was identified as 3-â-
D-ribofuranosyl-6-(trifluoromethyl)uracil (38). Replace-
ment of TMSOTfl by SnCl4 and the solvent MeCN by
1,2-dichloroethane did not lead to the desired N(1)-
isomer. The UV spectrum of 38 exhibited, characteristic
for N(3)-substituted uracils,14 a strong bathochromic
shift for the anionic form relative to the neutral species.
As mentioned above, uracils with an electrophilic
substituent at C(6), when used in a condensation
reaction with ABR, lead to formation of only the N(3)-
ribosides. On the other hand, introduction of a fluorine
at C(5) of 6-substituted uridines is technically difficult,
involving use of toxic and explosive F2 or CF3OF.15
Hence, to prepare the biologically interesting 5-fluoro-
6-(fluoromethyl)uridine (42), recourse was made to
modification of an analogue with a 5-fluoro substituent.
Introduction of a fluoromethyl substituent at C(6) was
based on the relatively mild exchange of an alcoholic
hydroxyl with fluorine, e.g., with the use of DAST
((diethylamino)sulfur trifluoride). The key step was the
use of an analogue with a hydroxymethyl group at C(6).
As starting compounds, we employed 5 or 5-fluoro-6-
methyluridine (35), as shown in Scheme 6. These were
acetylated with acetic anhydride in the presence of
(N,N-dimethylamino)pyridine (DMAP) as catalyst and
oxidized in situ with SeO2 followed by reduction with
tetrabutylammonium borohydride, which is very soluble
in organic solvents. The crude products, O′-acetyl
derivatives of 6-(hydroxymethyl)uridine (39) or its
5-fluoro congener 40, were subjected to fluorination with
DAST and, after initial workup, the acetyl groups
removed with MeOH-saturated ammonia. The products
were isolated by preparative layer chromatography to
give 6-(fluoromethyl)uridine (41; 45%) and 5-fluoro-6-
(fluoromethyl)uridine (42; 35%). The crude acetylated
derivatives of 39 and 40 were deblocked with methanolic
ammonia to obtain free 6-(hydroxymethyl)uridine (39;
45%) and 5-fluoro-6-(hydroxymethyl)uridine (40; 48%).
Con for m a tion a l Asp ects
The conformational parameters of the foregoing nu-
cleosides in neutral aqueous medium (D2O), derived
1
from analyses of the vicinal H,1H coupling constants
(see Experimental Section) with the aid of the modified
Karplus relationship,16a are listed in Table 1. It will
be noted that, for all the 6-substituted analogues,
irrespective of the presence of a C(5)-fluoro substituent,
the pentose ring conformation is predominantly N, i.e.,
C(3′)-endo as compared to 50% for the parent uridine
and 60% for 5-fluorouridine. The rotamers Ng+ and Nt
have comparable energies, as in the case of orotidine
and cyanuric acid riboside.1 The 6-substituent on the
uracil ring destabilizes the rotamer Sg+ and stabilizes
the Nt rotamer, a characteristic feature associated with
the conformation syn.16b,c This suggests that repulsive
interaction occurs between the pentofuranose substit-
uents and C(2) oxygen of the uracil moiety. These
results are in accord with previous reports that, for
6-methylribo- and -2′-deoxyribonucleosides in solution,
there is a trend in favor of the sugar ring conformation
N (C(3′)-endo).16d Furthermore, bearing in mind that

6- and 5,6-Substituted Uridine Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1723
Ta ble 1. Solution Conformation of 6-Substituted and 5,6-Disubstituted Pyrimidine Nucleosides Calculated from 500-MHz 1H NMR
Spectraa
conformations
nucleoside
S
g+
t
g-
Ng+
Nt
Sg+
St
Sg-
Urd
5-F-Urd
6-Me-Urd (5)
6-Pr-Urd (20)
0.46
0.40
0.17
0.14
0.13
0.17
0.21
0.17
0.15
0.20
0.17
0.61
0.67
0.42
0.41
0.41
0.43
0.42
0.42
0.43
0.42
0.42
0.32
0.29
0.52
0.52
0.53
0.51
0.52
0.52
0.53
0.52
0.52
0.08
0.04
0.06
0.06
0.05
0.06
0.06
0.06
0.03
0.07
0.06
0.41
0.42
0.39
0.41
0.40
0.40
0.37
0.40
0.39
0.39
0.40
0.19
0.17
0.43
0.45
0.46
0.42
0.41
0.43
0.45
0.41
0.43
0.20
0.24
0.02
0.01
0.01
0.03
0.04
0.02
0.04
0.03
0.02
0.13
0.12
0.09
0.07
0.07
0.09
0.11
0.09
0.08
0.10
0.09
0.08
0.04
0.06
0.06
0.05
0.06
0.06
0.06
0.03
0.07
0.06
6-i-Pr-Urd (21)
5,6-diMe-Urd (23)
6-CH2F-Urd (39)
6-CH2OH-Urd (41)
5-F-6-Me-Urd (35)
5-F-6-CH2OH-Urd (40)
5-F-6-CH2F-Urd (42)
a
Spectra obtained in D2O.
Ta ble 2. Kinetic Constants for Phosphorolysis of the Neutral
Forms of 6-Substituted and 5-Fluoro-6-substituted Uridines
the 5-fluoro-substituted analogues are partially in the
monoanionic form at neutral pH (pKa ∼7.9), it appears
also that the state of ionization of the pyrimidine ring
has little effect on the pentose ring conformation.
It may be concluded that the predominant N confor-
mation of the sugar ring in aqueous medium is imposed
by the constrained syn conformation due to the steric
effect of the 6-substituents. It is therefore of interest
that, in the solid state, 6-methyluridine17a and 6-methyl-
2′-deoxyuridine17b both exhibit the pentose ring in the
S conformation, i.e., C(2′)-endo conformation. In 6-meth-
yl-2′-deoxyuridine, with the exocyclic 5′-CH2OH in the
g+ rotameric form, this conformation appears at first
sight to be stabilized by an intramolecular C(5′)-
OH‚‚‚O(2′) hydrogen bond. However 6-methyluridine
exhibits two independent molecules in the asymmetric
unit, both with the pentose ring in the S conformation,
but only one of them possesses the g+ rotamer of the
exocyclic CH2OH group, which is hydrogen bonded to
the O(2) of the pyrimidine ring. In the other molecule
the exocyclic group is in the gt rotameric form, and there
is no bond to O(2). It follows that the S conformation
in the solid state is not necessarily dependent on
intramolecular hydrogen bonding.
An interesting feature displayed by 5-fluoro-6-(hy-
droxymethyl)uridine (40) with respect to 39, and pos-
sibly relevant to its biological activity (see below), is the
reduced half-width of the H signal of the 6-CH2OH
group, indicative of a lower rate of proton exchange.
Furthermore, the 0.35 ppm upfield shift of this proton,
relative to that of 39, is suggestive of electrostatic
interaction between the C(5)-F and 6-CH2OH, resulting
in hindered rotation about the C(6)-CH2 bond, and
reflected by the coalescence of the methylene group
signals. In the parent 6-(hydroxymethyl)uridine (39),
the half-width of the methylene group signals is 2 Hz,
consistent with much more rapid, albeit hindered,
rotation of the C(6)-CH2OH group, and the OH signal
is broadened by rapid exchange with H2O.
Vmax
app Km
(µM)
(µmol/min
efficacy
nucleoside
pKa
× mg of prot) (Vmax/Km)
pH 7.5
122 ( 11
9.50 1030 ( 53
9.50 1200 ( 90
9.50 1150 ( 150
Urd
6-Me-Urd (5)
6-Et-Urd (43)
6-Pr-Urd (20)
6-CH2F-Urd (39)
6-CH2OH-Urd (41)
9.23
340 ( 12
150 ( 5
175 ( 10
40 ( 4
254 ( 27
297 ( 14
2.8a
0.15
0.15
0.03
0.53
0.99
9.80
9.80
476 ( 90
300 ( 27
pH 6.0
152 ( 20
7.70 1355 ( 30
7.90 4000
5-F-Urd
5-F-6-Me-Urd (35)
5-F-6-CH2F-Urd (40)
7.75
323 ( 22
67 ( 1
370
2.1b
0.05
0.09
0.06
5-F-6-CH2OH-Urd (42) 7.30
979 ( 50
57 ( 4
a
At pH 6.0, Km ) 182 ( 25 mM, Vmax ) 312 ( 12, and Vmax/Km
b
) 1.7. At pH 7.5, Km ) 110 ( 4.6 mM, Vmax ) 43 ( 1, and Vmax
Km ) 0.4.
/
parent uridine (for which pKa ) 9.3), it will be noted
that the kinetic constants at pH 6.0 are slightly lower
than those at pH 7.5. By contrast, 5-fluorouridine (pKa
) 7.75) is an appreciably poorer substrate at pH 7.5,
where it consists of a mixture of the neutral and
monoanionic forms, suggesting that the neutral form is
the substrate for phosphorolysis.
It will be seen from Table 2 that 6-methyluridine (5)
is a substrate, albeit a weak one, which is consistent
with the earlier finding that it is a much better
substrate for the analogous enzyme from S. typhimu-
rium.3 As expected, 6-methyluridine (5) proved to be a
competitive inhibitor with respect to uridine (with the
aid of Dixon plots, not shown), with a Ki ∼ 2 mM,
comparable with the Km for phosphorolysis, 1.03 mM.
6-Ethyluridine (43) is not a better substrate, whereas
6-propyluridine (20) is a poorer one, presumably because
of enhanced steric effects. In general the foregoing
results seem to be consistent with the postulate that
the actual substrate in the case of uridine is the syn
conformer, imposed on binding to the enzyme. This
conclusion is supported by the substrate properties of
6-(hydroxymethyl)uridine (39) and 6-(fluoromethyl)-
uridine (41).
Biologica l Resu lts
In ter a ction w ith E. coli Ur id in e P h osp h or yla se.
Table 2 exhibits the kinetic constants for phosphorolysis
of the C(6)-substituted uridines and 5-fluorouridines,
relative to those for the parent uridine and 5-fluorou-
ridine, respectively.
To ensure that the data refer to the behavior of the
neutral forms of both series of analogues, those for the
5-fluorouridines (for which the pKa values are in the
range 7.3-7.9) were measured at pH 6. As regards the
Quite unexpected was the finding that, whereas
5-fluorouridine is as good a substrate as uridine, the
5-fluoro-6-substituted congeners are very poor sub-
strates. This is most striking for compounds 40 and 42,
which are the 5-fluoro analogues of the good substrates
39 and 41, respectively. Although not readily interpret-
able, this may be relevant to the potential chemothera-
peutic activities in vivo of these compounds.

1724 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Felczak et al.
Ta ble 3. Inhibition of Cell Growth by 6-Substituted and
5,6-Disubstituted Uridines
CO, 90:10; (H) benzene-EtOAc, 80:20; (I) CHCl3-MeOH, 85:
15; (J ) EtOAc-n-PrOH-H2O, 75:16:9; (K) benzene-EtOAc, 60:
40; (L) CHCl3-Me2CO, 85:15; and (M) CHCl3-MeOH, 100:10.
a
IC50 (µg/mL)
Biology: (a ) En zym e P r ep a r a tion . Uridine phosphory-
lase from E. coli was purified to homogeneity according to Vita
and Magni.18
MOLT-
3b
U-
K-
IM-
9
PHA-
Ly
compound
5-F-Ura
937
562
1.2
<0.01
>10.0
>10.0
1.2
0.5
<0.01
>10.0
>10.0
3.7
4.3
0.026
>10.0
>10.0
4.9
>10.0
>10.0
0.34
1.4
<0.1
>10.0
98
2.8
>10.0
4.5
(b) En zym e Assa ys. All assays were performed in 50 mM
phosphate buffer at 37 °C. For each potential substrate, the
pH was selected so that the neutral form predominated. Hence
all analogues with a 5-fluoro substituent were assayed at pH
6 and the others at pH 7.5. Phosphorolysis was monitored
spectrophotometrically by the decrease in absorbance at 285
nm (∆ꢀ ) 3.2 × 103) for the 5-fluoro analogues and at 280 nm
(∆ꢀ ) 2.1 × 103) for the remaining compounds, using a Uvicon
860 instrument fitted with a thermostated cuvette compart-
5-F-Urd
<0.01
>10.0
>10.0
3.0
9.9
>10.0
5-F-6-Me-Urd (35)
6-CH2OH-Urd (39)
5-F-6-CH2OH-Urd (40)
6-CH2F-Urd (41)
5-F-6-CH2F-Urd (42)
>10.0
3.5
>10.0
3.7
a
IC50 is the concentration (µg/mL) of compound which caused
a 50% decrease in [14C]leucine incorporation by the cells in culture.
Values were calculated from dose-response curves done in
b
ment and equipped with
a software package for kinetic
triplicate for each analogue. MOLT-3, acute T cell leukemia;
analysis and statistical treatment of the data. Initial reaction
rates were employed for determination of v (20 s). Apparent
Km and Vmax values were evaluated using a computer program
that uses the Wilkinson procedure.19 The program was kindly
written by Dr. Z. Kamin´ski.
For 5-fluoro-6-(hydroxymethyl)uridine (40), with a very high
Km (see Table 2), the reaction was conducted for 1 min and
terminated by addition of 30 µL of 30% NaOH. A control
contained each component, except enzyme, which was added
after alkalization. The difference in absorbance was measured
at 300 nm (∆ꢀ ) 3.5 × 103). Under these conditions, the
enzyme activity was linear with time and enzyme concentra-
tion.
U-937, histiocytic lymphoma; K-562, chronic myelogeneous leu-
kemia; IM-9, myeloma; PHA-Ly, phytohemagglutinin-stimulated
peripheral blood lymphocytes.
In Vitr o An titu m or Activity. The cytotoxicities of
the foregoing 6-substituted and 5,6-disubstituted uri-
dine analogues vs four human leukemic cell lines,
MOLT-3, U-937, IM-9, and K-562, were determined and
compared with those vs phytohemagglutinin-stimulated
human lymphocytes (PHA-Ly) with results shown in
Table 3.
Two of the analogues, 5-fluoro-6-(hydroxymethyl)-
uridine (40) and 5-fluoro-6-(fluoromethyl)uridine (42),
exhibited potent antileukemic activities, comparable to
that of the standard drug 5-fluorouracil (FUra). Fur-
thermore both compounds were less toxic to human
lymphocytes and, in the case of MOLT-3 and K-562 cells,
more selective than FUra.
Protein was determined by the method of Bradford20 with
bovine serum albumin as standard.
(c) Cytotoxicity Stu d ies. Cytoxicity (IC50) of the com-
pounds was determined by their effects on protein ([14C]-L-
leucine incorporation) synthesis in the cell lines (Table 3)
obtained from the American Type Culture Collection. Test
compounds were added to triplicate cultures in 96-well mi-
croplates containing 2 × 104 cells/200 µL well or 105 peripheral
blood lymphocytes, stimulated with phytohemaglutinin. Cells
were cultured in RPMI 1640 medium containing fetal calf
serum (10%), in a humidified atmosphere containing 5% CO2
at 37 °C. [14C]-L-Leucine (specific activity 1.3 mCi/mmol and
0.5 µCi/mL) was added to the cultures for the final 24 h of the
3-day culture period (4 days with PHA-Ly). After incubation,
the proteins were precipitated with 0.2 N perchloric acid and
collected on glass fiber filters using a multiple cell harvester
(Wallac, Finland). The radioactivity incorporated into proteins
was measured in a scintillation counter (LKB-Wallac, 81.000;
Turku, Finland). The incorporation of [14C]leucine per culture
remained constant during the final 24 h of culture. A good
correlation between cell number and [14C]leucine incorporation
has been demonstrated.21,22
Con clu sion s
A number of 6-substituted uridines, all in the non-
typical syn conformation, are reasonable substrates of
E. coli uridine phosphorylase, two of them comparable
to the parent uridine, and do not exhibit cytotoxicity vs
leukemic cells in vitro. The 5-fluoro derivatives of these
6-substituted uridines are poorer substrates of the
enzyme, and two of them, 40 and 42, exhibit cytotox-
icities comparable to that of 5-fluorouracil vs hemato-
poietic human leukemic cell lines. These uridine phos-
phorylase-resistant compounds are reasonable candidates
as in vivo antitumor agents.
Ch em istr y. 1-â-D-Ribofu r a n osyl-3-ben zyl-6-m eth ylu -
r a cil (4). A suspension of 2.04 g (10 mmol) of 3-benzyl-6-
methyluracil5 (1) in 25 mL of HMDS with 10 mg of (NH4)2SO4
was heated under reflux for 12 h. HMDS was removed under
reduced pressure, 50 mL of anhydrous xylene added, and the
whole evaporated to an oil (2), to which was added 5.04 g (10
mmol) of anhydrous (70 °C, P2O5, 0.1 mmHg) ABR in 20 mL
of anhydrous MeCN. The mixture was cooled in iced H2O, 1.11
g (5 mmol) of TMSOTfl added, and the whole left at room
temperature for 2 h, the course of the reaction being monitored
by TLC on silica gel with solvent A. The reaction was
terminated by adding the mixture to 100 mL of vigorously
stirred, cooled, saturated NaHCO3. After several minutes the
product was extracted with 3 × 50 mL of CHCl3. The
combined extracts were brought to dryness under reduced
pressure, and the product 3 was isolated by preparative TLC
on silica gel with solvent A and dissolved in 100 mL of hot
MeOH. To the cooled solution was added 5 mL of 1 N NaOMe
in MeOH, and the solution was stirred overnight at room
temperature, brought to neutrality with Dowex 50W(H+), and
evaporated to an oil and the product isolated by preparative
TLC on silica gel with solvent B, to give 4 (2.26 g) in the form
of an oil, chromatographically homogeneous, which was dried
over P2O5 in vacuo: UV λmax (pH 7) 265 nm (ꢀ 8.6 × 103);
Exp er im en ta l Section
Gen er a l Meth od s. Melting points (uncorrected) were
measured on a Boetius microscopic hot stage; UV spectra were
recorded on a Cary 3 instrument, using 10-mm path length
cuvettes, acetate buffers in the pH range 3-4, and phosphate
buffers in the range 6-8.4. Extremes of pH made use of
standard solutions of HCl and NaOH. A Cole-Parmer instru-
ment with combination electrode was employed for pH mea-
surements. High-resolution EI mass spectra for pyrimidines
were carried out on a Finigan MAT spectrometer and liquid
matrix secondary ion mass spectra (LSIMS) for nucleosides
with an AMD-604 spectrometer. High-resolution 1H NMR
spectra were recorded on a Bruker 500-MHz spectrometer in
D2O with DSS as internal standard, unless otherwise indi-
cated. All evaporations were under vacuum at 35 °C. Thin-
layer chromatography (TLC) was run on Merck silica gel F254
glass plates (DC, 20 × 20 cm, 0.25 mm, no. 5715), Merck
aluminum oxide F254 glass plates (DC, 20 × 20 cm, 0.1 mm,
no. 5713), and Merck cellulose F glass plates (DC, 20 × 20
cm, 0.1 mm, no. 5718). The following solvents (v/v) were used
(A) benzene-EtOAc, 70:30; (B) CHCl3-MeOH, 80:20; (C)
CHCl3-MeOH, 90:10; (D) CHCl3-EtOAc, 80:20; (E) benzene-
EtOAc, 90:10; (F) benzene-EtOAc, 100:10; (G) CHCl3-Me2-

6- and 5,6-Substituted Uridine Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1725
TLC (silica gel) Rf (B) 0.71; MS m/ z for C17H20N2O6 calcd
1-(2,3,5-Tr i-O-ben zoyl-â-D-r ibofu r an osyl)-4-(m eth ylth io)-
6-p r op ylu r a cil (18). This was obtained from 1.84 g (10
mmol) of 4-(methylthio)-6-propyluracil (15) as described above
for 17 but with prolongation of the reaction time to 90 min.
The product was crystallized from 50 mL of i-PrOH/MeOH to
yield 2.57 g (90%) of large colorless crystals, mp 105-106 °C;
TLC (Al2O3) Rf (F) 0.27. Anal. (C34H32N2O8S) C, H, N.
1-(2,3,5-Tr i-O-b en zoyl-â-D-r ib ofu r a n osyl)-6-p r op ylu -
r a cil (22). A solution of 200 mg (0.32 mmol) of 15 in 10 mL
of a 3:7 (v/v) mixture of 1,4-dioxane and 1 N TFA was kept at
room temperature until disappearance of 15, the reaction being
monitored by TLC on Al2O3 with solvent G. Solvent was
removed under reduced pressure and the residue crystallized
from EtOH to yield 141 mg (74%) of 22 as thin needles: mp
85-87 °C; TLC (silica gel) Rf (H) 0.16. Anal.(C33H30N2O8‚
2H2O) C, H, N.
1-(2,3,5-Tr i-O-ben zoyl-â-D-r ibofu r a n osyl)-6-isop r op yl-
4-(m eth ylth io)u r a cil (19). This was prepared as described
for 17 with slight modifications: 348 mg (1.90 mmol) of 16 in
MeCN was converted to the trimethylsilyl derivative (0.5 mL
of BSTFA, 80 °C, 30 min). To the cooled solution was added
200 µL (1.025 mmol) of TMSOTfl with stirring for 30 min at
room temperature and monitoring the course of the reaction
by TLC on Al2O3 with solvent E. Following termination of the
reaction and preliminary workup, the product was isolated by
preparative TLC on Al2O3 (two 20 × 40 cm plates) with solvent
E. Crystallization from i-PrOH/MeOH gave 1.0 g (88%): mp
79-81 °C; TLC (Al2O3) Rf (F) 0.28. Anal. (C31H32N2O8S) C,
H, N.
384.132 136, found 384.132 145.5
6-Isop r op yl-4-th iou r a cil (12). To a mixture of 10 mL of
anhydrous hexane and 6.2 mL (60 mmol) of diethylamine
(distilled over NaH), cooled in ice, was added, portionwise, a
solution of 986 µL (9 mmol) of TiCl4 in 4 mL of anhydrous
hexane followed by slow addition of 1.064 mL (10 mmol) of
methylisopropyl ketone (9) and stirring for 1 h at room
temperature. The dark brown mixture was diluted with
hexane and, following addition of 2 mL of MeOH, filtered
through Celite. The pale yellow filtrate was concentrated to
an oil (10; l.3 g), which was dissolved in 20 mL of Et2O followed
by slow addition, with cooling, of 4 mL of a solution of 940 µL
(7.8 mmol) of ethoxycarbonyl isothiocyanate in Et2O. The
mixture was stirred for 1 h at room temperature and 10 mL
of hexane added, leading to formation of crystals of 11. The
mixture was stored in the deep freeze for 12 h, and the
resulting deep red crystalline adduct 11 was collected by
filtration (2.0 g, 75%): mp 75-77 °C. Anal. (C13H23N2O2S)
C, H, N.
The adduct 11 (500 mg, 1.84 mmol) was suspended in 10
mL of vigorously stirred concentrated ammonia, partially
dissolved by warming, and left at room temperature for 48 h.
Crystallization from aqueous EtOH gave 287 mg (92%) of long
needles of 12: mp 172-174 °C; UV λmax (pH 2) 333 (ꢀ 18.9 ×
103), (pH 7) 330 (ꢀ 18.9 × 103), (pH 12) 332 nm (ꢀ 20.3 × 103);
TLC (silica gel) Rf (C) 0.72; MS m/ z for C7H10N2OS calcd
170.051 407, found 170.051 38. Anal. (C7H10N2OS) C, H, N.
4-(Meth ylth io)-6-m eth ylu r a cil (13). This was obtained
as described by Winkley and Robins7cand recrystallized from
H2O: mp 152-156 °C (lit.7c mp 150-157 °C); UV λmax (pH 2)
325 (ꢀ 14.5 × 103), 316 (ꢀ 13.8 × 103), 267 (ꢀ 5.3 × 103), (pH 7)
300 (ꢀ 13.9 × 103), 270 (ꢀ 8.0 × 103), (pH 12) 299 (ꢀ 11.4 ×
103), 223 nm (ꢀ 12.0 × 103); TLC (silica gel) Rf (C) 0.53.
1-â-D-Ribofu r a n osyl-6-m eth ylu r a cil (5). Meth od A: A
solution of 100 mg (0.29 mmol) of 3-benzyl-6-methyl-1-â-D-
ribofuranosyluracil (4) in 5 mL of anhydrous THF was cooled
on an ice bath and 25 µL (0.264 mmol) of BBr3. Stirring for 2
h at room temperature was followed by addition of 200 µL of
MeOH and 100 µL of 25% aqueous ammonia. The solution
was brought to dryness under reduced pressure and the
product isolated by preparative TLC on silica gel with solvent
B. Crystallization from MeOH yielded 50 mg (67%) of 5.
Meth od B: A suspension of 1.56 g (10 mmol) of 4-(meth-
ylthio)-6-methyluracil (13) in 20 mL of HMDS, with a catalytic
amount of (NH4)2SO4, was heated under reflux for 12 h.
HMDS was removed under reduced pressure, 50 mL of xylene
added, and the mixture concentrated to an oily residue, to
which was added 5.04 g (10 mmol) of ABR in 80 mL of
anhydrous MeCN. The mixture was cooled on an ice bath and
1.11 g (5 mmol) of TMSOTfl added. After 2 h the mixture was
diluted with 300 mL of CHCl3 and extracted with 2 × 100 mL
of saturated NaHCO3 and then with 3 × 200 mL of H2O. The
organic phase was dried over anhydrous Na2SO4 and evapo-
rated to dryness. The residue was dissolved in 100 mL of 1 N
NaOMe and, after 12 h, brought to neutrality with Dowex
50W(H+), concentrated under vacuum to a small volume,
dissolved in 30 mL of H2O, and extracted with 3 × 25 mL of
CHCl3. The aqueous layer was brought to dryness, and the
residue was dissolved in 15 mL of 1 N TFA. The mixture was
stirred for 12 h at room temperature, brought to dryness, and
reevaporated from 30 mL of EtOH, and the residue was
crystallized from EtOH/EtOAc to yield 2.06 g (80%) of 5: mp
174-176 °C (lit.7c mp 177-178 °C); TLC (silica gel) Rf (B) 0.34;
5,6-Dim eth yl-4-(eth ylth io)u r a cil (14): prepared as de-
scribed for 13, above, with EtI instead of MeI; crystallization
from EtOH gave the product in 93% yield; mp 195-197 °C;
UV λmax (pH 2) 325 (ꢀ 14.5 × 103), 316 (ꢀ 13.8 × 103), 267 (ꢀ
5.3 × 103), (pH 7) 300 (ꢀ 13.9 × 103), 270 (ꢀ 8.0 × 103), (pH 12)
299 (ꢀ 11.4 × 103), 223 nm (ꢀ 12.0 × 103); TLC (silica gel) Rf
(D) 0.05; MS m/ z for C8H12N2OS calcd184.067 06, found
184.066 912.
4-(Met h ylt h io)-6-p r op ylu r a cil (15): prepared as de-
scribed for 13 (above); rRecrystallization from aqueous EtOH
yielded the product in 65% yield; mp 115-117 °C; UV λmax (pH
2) 325 (ꢀ 14.5 × 103), 316 (ꢀ 13.8 × 103), 267 (ꢀ 5.3 × 103), (pH
7) 300 (ꢀ 13.9 × 103), 270 (ꢀ 8.0 × 103), (pH 12) 299 (ꢀ 11.4 ×
103), 223 nm (ꢀ 12.0 × 103); TLC (silica gel) Rf (C) 0.63; MS
m/ z for C8H12N2OS calcd 184.067 06, found 184.066 91.
6-Isop r op yl-4-(m eth ylth io)u r a cil (16): prepared as de-
scribed for 13 (above); recrystallization from aqueous EtOH
gave 90% yield; mp 164-166 °C. UV λmax (pH 2) 325 (ꢀ 14.5
× 103), 316 (ꢀ 13.8 × 103), 267 (ꢀ 5.3 × 103), (pH 7) 300 (ꢀ 13.9
× 103), 270 (ꢀ 8.0 × 103), (pH 12) 299 (ꢀ 11.4 × 103), 223 nm (ꢀ
12.0 × 103); TLC (silica gel) Rf (C) 0.64; MS m/ z for C8H12N2-
OS calcd 184.067 06, found 184.067 27.
1-(2,3,5-Tr i-O-ben zoyl-â-D-r ibofu r an osyl)-4-(m eth ylth io)-
6-m eth ylu r a cil (17). A suspension of 1.56 g (10 mmol) of
4-(methylthio)-6-methyluracil (13) in 15 mL of HMDS with
10 mg of (NH4)2SO4 was heated under reflux for 12 h. HMDS
was removed under reduced pressure, 100 mL of xylene added,
and the mixture concentrated to an oil, to which was added
5.04 g (10 mmol) of carefully dried ABR in 20 mL of MeCN.
The mixture was cooled on ice, 1.11 g (5 mmol) of TMSOTfl
added, and the whole left at room temperature for 30 min,
with monitoring of the course of the reaction by TLC on Al2O3
with solvent E. The reaction was terminated by pouring the
mixture into 100 mL of cooled and vigorously stirred saturated
NaHCO3. After several minutes the product was extracted
with 3 × 50 mL of CHCl3. The pooled extracts were brought
to dryness and fractionated with a mixture of i-PrOH and
cyclohexane to release a chromatographically homogeneous
oily product, which was dried over P2O5 under vacuum (5.13
g, 85%): mp 91-92 °C; TLC (Al2O3) Rf (F) 0.24. Anal.
(C32H28N2O8S‚1/2H2O) C, H, N.
1H NMR (D2O) δ 5.77 (1H, s, 5-H), 5.66 (1H, d, 1′-H, J 1′2′
)
3.35 Hz), 4.39 (1H, t, 3′-H, J 2′3′ ) 6.39 Hz), 3.97 (1H, m, 4′-H,
J 3′4′ ) 7.22 Hz), 3.89 (1H, dd, 5′-H, J 4′5′ ) 3.01 Hz), 3.75 (1H,
dd, 5′′-H, J 4′5′′ ) 6.26 Hz, J 5′5′′ ) -12.34 Hz), 2.40 (3H, s,
6-CH3).
1-â-D-Ribofu r a n osyl-6-eth ylu r a cil (43). This was pre-
pared as described by Holy:23 mp 115-118 °C (lit.23 mp 119-
121 °C); TLC (silica gel) Rf (B) 0.44; UV spectra as for 5.
1-â-D-Ribofu r a n osyl-6-p r op ylu r a cil (20). 1-(2,3,5-Tri-O-
benzoyl-â-D-ribofuranosyl)-6-propyluracil (22) (100 mg, 0.167
mmol) was dissolved in 100 mL of hot EtOH. To the cooled
solution was added 5 mL of 1 N NaOMe in MeOH, and the
mixture was stirred overnight at room temperature, brought
to neutrality with Dowex 50W(H+), and evaporated to dryness.
The product was isolated by preparative TLC on silica gel with
solvent B to yield 39 mg (82%) as an amorphous powder: mp
173-176 °C (lit.24 mp 177-178 °C); TLC (silica gel) Rf (B) 0.53;

1726 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Felczak et al.
1H NMR (D2O) δ 5.65 (1H, d, 1′-H, J 1′2′ ) 3.34 Hz), 4.39 (1H,
t, 3′-H, J 2′3′ ) 6.38 Hz), 3.96 (1H, m, 4′-H, J 3′4′ ) 7.46 Hz),
5-fluoro-6-methyluracil (27) in 30 mL of HMDS, to which was
added 0.1 mL (0.8 mmol) of TCS, was heated under reflux to
obtain a clear solution (about 6 h). The silylating reagents
were removed under vacuum, and the residue was codistilled
with 3 × 50 mL of anhydrous xylene and taken up in 30 mL
of anhydrous MeCN. To the resulting solution of 2,4-bis-O-
(trimethylsilyl)-5-fluoro-6-methyluracil (29), in a round-bot-
tomed flask with protection against moisture, was added 5.04
g (10 mmol) of anhydrous ABR. To this mixture, cooled on an
ice bath, was added, dropwise, 20 mL of a solution containing
2.18 mL (12 mmol) of TMSOTfl, with stirring for 2 h. The
mixture was brought slowly to room temperature and stirred
for 3 h. Following addition of 300 mL of CHCl3, the mixture
was extracted with 2 × 100 mL of saturated NaHCO3 and 3
× 200 mL of H2O. The organic phase was dried over
anhydrous Na2SO4 and then evaporated to dryness. The
residue was deposited on a 2.5 × 25 cm column of neutral Al2O3
(Merck; 70-230 mesh, activity according to Brockman III) and
eluted with a 9:1 (v/v) mixture of n-hexane/EtOAc, to give 1,3-
bis(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-5-fluoro-6-methylu-
racil (32). Further elution with a linear gradient (10-100%)
of EtOAc in hexane resulted in a component identified as
3-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-5-fluoro-6-methylura-
cil (31). Subsequent elution with EtOAc/MeOH (9:l, v/v)
yielded 1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-5-fluoro-6-
methyluracil (30). This third fraction was brought to dryness
and crystallized from CH2Cl2/Et2O to give 3 g (45%) of 30: mp
3.90 (1H, dd, 5′-H, J 4′5′ ) 3.02 Hz), 3.75 (1H, dd, 5′′-H, J 4′5′′
)
6.26 Hz, J 5′5′′ ) -12.34 Hz), 2.65 (2H, t, 6-CH2CH2CH3), 1.68
(2H, m, 6-CH2CH2CH3), 0.99 (3H, t, 6-CH2CH2CH3).
1-â-D-Ribofu r a n osyl-6-isop r op ylu r a cil (21). 1-(2,3,5-Tri-
O-benzoyl-â-D-ribofuranosyl)-6-isopropyl-4-(methylthio)ura-
cil (19) (500 mg, 0.80 mmol) was dissolved in 10 mL of 0.1 N
NaOMe in MeOH. The solution stirred for 12 h, brought to
neutrality with Dowex 50W(H+), reduced to a small volume
by evaporation, dissolved in 15 mL of H2O, and extracted with
3 × 15 mL of CHCl3. The aqueous layer was brought to
dryness, dissolved in 5 mL of 1 N TFA, stirred for 12 h at room
temperature, and brought to dryness; the residue was evapo-
rated from 30 mL of EtOH and crystallized from EtOH/EtOAc
to give 189 mg (83%) in an amorphous form: mp 202-204 °C
1
(lit.24 mp 204-206 °C); TLC (silica gel) Rf (B) 0.52; H NMR
(D2O) δ 5.81 (1H, 5-H, s), 5.77 (1H, d, 1′-H, J 1′2′ ) 3.17 Hz),
4.40 (1H, t, 3′-H, J 2′3′ ) 6.64 Hz), 3.95 (1H, m, 4′-H, J 3′4′
)
7.53 Hz), 3.90 (1H, dd, 5′-H, J 4′5′ ) 2.94 Hz), 3.76 (1H, dd, 5′′-
H, J 4′5′′ ) 6.35 Hz, J 5′5′′ ) -12.41 Hz), 3.08 (2H, m, 6-CH2-
(CH3)2), 1.28 (6H, d, 6-CH2(CH3)2).
1-â-D-Ribofu r a n osyl-5,6-d im eth ylu r a cil (23). This was
obtained essentially as described for the 6-methyl analogue
5, the starting substance being 4-(ethylthio)-6-methyluracil
(14). The final product was isolated by preparative TLC on
silica gel with solvent I and crystallized from i-PrOH in 60%
yield: mp 180-181 °C (lit.7c mp 82 °C); TLC (silica gel) Rf (I)
0.38; 1H NMR (D2O) δ 5.73 (1H, d, 1′-H, J 1′,2′ ) 3.39 Hz), 4.38
(1H, dd, 3′-H, J 2′,3′ ) 6.43 Hz, J 3′,4′ ) 7.26 Hz), 3.97 (1H, m,
4′-H, J 4′,5′ ) 2.97 Hz, J 4′,5′′ ) 6.16 Hz), 3.90 (1H, dd, 5′-H, J 5′,5′′
) -12.38 Hz), 3.75 (1H, dd, 5′′-H), 2.41 (3H, s, 5-CH3), 2.20
(3H, s, 6-CH3).
5-F lu or o-2-m eth oxy-6-m eth ylu r a cil (25). To a solution
of 1.72 g (10 mmol) of methylpseudoisourea in 30 mL of H2O
was added 616 mg (11 mmol) of CaO followed by portionwise
addition of a solution of 1.46 mL (10 mmol) of ethyl R-(fluo-
roacetyl)acetate (23) in 30 mL of EtOH and stirring for 72 h.
The mixture was then acidified to pH 3 with HCOOH and
filtered through Celite and the latter washed with EtOH, and
the combined filtrates were evaporated to dryness. The
residue was crystallized from toluene/i-PrOH to yield 995 mg
(63%): mp182-185 °C; UV λmax (pH 2) 260 (ꢀ 7.7 × 103), (pH
7) 265 (ꢀ 7.42 × 103), (pH 12) 268 nm (ꢀ 7.57 × 103); TLC (silica
gel) Rf (C) 0.64; MS m/ z for C6H7FN2O2 calcd 158.049 15, found
158.049 15.
2-Meth oxy-6-(tr iflu or om eth yl)u r a cil (26). To a solution
of 1.722 g (10 mmol) of methylpseudoisourea in 30 mL of H2O
was added 616 mg (11 mmol) of CaO followed by portionwise
addition of 1.46 mL (10 mmol) of ethyl(trifluoroacetyl)acetate
(24) in 30 mL of EtOH and then stirring for 72 h, acidification
to pH 3 with HCOOH, filtration through Celite, and evapora-
tion to dryness. Crystallization from toluene/i-PrOH yielded
1.28 g (66%) of 26: mp 106-110 °C; UV λmax (pH 2) 272 (ꢀ 5.8
× 103), (pH 7) 273 (ꢀ 5.05 × 103), (pH 12) 273 nm (ꢀ 5.3 × 103);
TLC (silica gel) Rf (C) 0.74; MS m/ z for C6H5F3N2O2 calcd
194.0303 19, found 194.031 17.
5-F lu or o-6-m eth ylu r a cil (27). A suspension of 1 g (6.3
mmol) of 5-fluoro-2-methoxy-6-methyluracil (25) in 20 mL of
2 N H2SO4 was heated under reflux at 70 °C, and the course
of the reaction was monitored by TLC on silica gel with solvent
C. After about 2 h, the cooled solution was filtered through
Celite, reduced to a small volume, and allowed to crystallize,
yielding 792 mg (87%) of 27: mp 300 °C dec (lit.10 mp 300 °C
dec); UV λmax (pH 2) 270 (ꢀ 7.0 × 103), (pH 7) 271 (ꢀ 6.3 × 103),
(pH 14) 287 nm (ꢀ 6.9 × 103); TLC (silica gel) Rf (J ) 0.63.
6-(Tr iflu or om eth yl)u r a cil (28). A suspension of 1 g (5.1
mmol) of 2-methoxy-6-(trifluoromethyl)uracil (26) in 20 mL of
2 N H2SO4 was heated under reflux at 70 °C for 2 h, with
monitoring of the course of the reaction on silica gel with
solvent C. The solution was cooled, filtered through Celite,
and reduced to a small volume for crystallization, to yield 765
mg (83%) of 28: mp 225-228 °C (lit.11 mp 220-222 °C); TLC
(silica gel) Rf (C) 0.37.
1
97-98 °C; TLC (silica gel) Rf (K) 0.45; H NMR (CDCl3/Me4-
Si) δ 8.25 (15H, m, aromatic), 6.24-6.14 (2H, m, 2′-H, 3′-H),
5.63 (1H, s, 1′-H), 4.84-4.68 (3H, m, 4′-H, 5′-H, 5′′- H), 2.32
(3H, 6-CH3, d, J F,CH ) 3.5 Hz). Anal. (C31H25FN2O9) C, H;
3
N: calcd, 4.76; found, 4.34.
3-(2,3,5-Tr i-O-b en zoyl-â-D-r ib ofu r a n osyl)-5-flu or o-6-
m eth ylu r a cil (31). The second fraction from the foregoing
procedure was brought to dryness and crystallized from EtOH
to obtain 1.4 g (21%) of 31: mp 189-190 °C; TLC (silica gel)
1
Rf (K) 0.38; H NMR (CDCl3/Me4Si) δ 10.45 (1H, br s, 1-NH),
8.30-7.94, 7.75-7.30 (15H, m, aromatic), 6.62 (1H, s, 1′-H),
6.32-6.08 (2H, m, 2′-H, 3′-H), 5.95-5.65 (3H, m, 4′-H, 5′-H,
5′′-H), 2.20 (3H, d, 6-CH3, J F,CH ) 3.5 Hz). Anal. (C31H25
-
3
FN2O9) C, H, N.
1,3-Bis(2,3,5-tr i-O-ben zoyl-â-D-r ibofu r a n osyl)-5-flu or o-
6-m eth ylu r a cil (32). The first fraction from the foregoing
procedure was brought to dryness and crystallized from Et2O
to give 1.33 g (20%) of 32: mp 133-134 °C; TLC (silica gel) Rf
(K) 0.65; 1H NMR (CDCl3/Me4Si) δ 8.30-7.88, 7.70-7.30 (30H,
aromatic), 6.68 (1H, s, 1′-H near N-3), 6.37-6.10 (4H, m, 2′-
H, 3′-H), 5.75 (1H, s, 1′-H near N-1), 5.00-4.50 (6H, m, 4′-H,
5′-H, 5′′-H), 2.33 (3H, 6-CH3, d, J F,Me ) 3.5 Hz). Anal. (C57H45
FN2O16) C, H, N.
-
1-â-D-Ribofu r a n osyl-5-flu or o-6-m eth ylu r a cil (35). Com-
pound 30 (2.5 g, 4.3 mmol) was dissolved in 100 mL of hot
EtOH. To the cooled solution was added 5 mL of a methanolic
solution of 1 N NaOMe; the mixture was stirred overnight at
room temperature and then brought to neutrality with Dowex
50W(H+) and evaporated to dryness. The residue was crystal-
lized from i-PrOH/EtOAc and then from EtOH, to yield 938
mg (80%) of 35: mp 180.5-182 °C; UV λmax (pH 2) 268 (ꢀ 9.6
× 103), (pH 7) 268 (ꢀ 8.6 × 103), (pH 12) 269 nm (ꢀ 7.15 × 103);
TLC (cellulose) Rf (J ) 0.43; MS m/ z for C10H14FN2O6 calcd
277.0836, found 277.0840; 1H NMR (D2O) δ 5.59 (1H, d, 1′-H,
J 1′2′ ) 3.51 Hz), 4.35 (1H, t, 3′-H, J 2′3′ ) 6.38 Hz), 3.97 (1H, m,
4′-H, J 3′4′ ) 7.18 Hz), 3.89 (1H, dd, 5′-H, J 4′5′ ) 2.99 Hz), 3.75
(1H, dd, 5′′-H, J 4′5′′ ) 6.25 Hz, J 5′5′′ ) -12.41 Hz), 2.40 (3H,
6-CH3, d, J F,Me ) 3.19 Hz). Anal. (C10H14FN2O6) C, H, N.
3-â-D-Ribofu r a n osyl-5-flu or o-6-m eth ylu r a cil (36). This
was obtained by deblocking of 1 g of 31, as described in the
previous section for the preparation of 35 from 30, to give 400
mg (85%) of amorphous 36: UV λmax (pH 2) 273 (ꢀ 8.01 × 103),
(pH 7) 272 (ꢀ 8.26 × 103), (pH 12) 300 nm (ꢀ 10.4 × 103); TLC
(cellulose) Rf (J ) 0.53; MS m/ z for C10H14FN2O6 calcd 277.0836,
found 277.0842; 1H NMR (D2O) δ 6.30 (1H, d, 1′-H, J 1′2′ ) 2.85
Hz), 4.50 (1H, t, 3′-H, J 2′3′ ) 6.20 Hz), 4.06 (1H, m, 4′-H, J 3′4′
) 7.22 Hz), 3.96 (1H, dd, 5′-H, J 4′5′ ) 3.02 Hz), 3.82 (1H, dd,
5′′-H, J 4′5′′ ) 6.20 Hz, J 5′5′′ ) -12.41 Hz), 2.29 (3H, 6-CH3, d,
J F,Me ) 3.19 Hz). Anal. (C10H13FN2O6‚1/2H2O) C, H, N.
1-(2,3,5-Tr i-O-b en zoyl-â-D-r ib ofu r a n osyl)-5-flu or o-6-
m eth ylu r a cil (30). A suspension of 1.58 g (11 mmol) of

6- and 5,6-Substituted Uridine Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1727
4.40 (1H, t, 3′-H, J 2′3′ ) 6.37 Hz), 3.97 (1H, m, 4′-H, J 3′4′
7.27 Hz), 3.89 (1H, dd, 5′-H, J 4′5′ ) 3.00 Hz), 3.76 (1H, dd, 5′′-
H, J 4′5′′ ) 6.23 Hz, J 5′5′′ ) -12.41 Hz).
Compound 40 was prepared in an analogous manner to
obtain 422 mg (48%) in an amorphous form, which could not
be crystallized: UV λmax (pH 2) 268 (ꢀ 9.6 × 103), (pH 7) 268 (ꢀ
6.9 × 103), (pH 12) 269 nm (ꢀ 5.7 × 103); TLC (silica gel) Rf (I)
0.17; MS m/ z for C10H14O7N2F calcd 293.0785, found 293.0777;
1H NMR (D2O) δ 5.66-5.59 (2H, m, 6-CH2OH), 5.59 (1H, d,
1′-H, J 1′2′ ) 3.44 Hz), 4.80 (1H, dd, 2′-H, J 2′3′ ) 6.52 Hz), 4.41
(1H, dd, 3′-H), 3.98 (1H, m, 4′-H, J 3′4′ ) 7.04 Hz), 3.89 (1H,
dd, 5′-H, J 4′5′ ) 3.05 Hz), 3.76 (1H, dd, 5′′-H, J 4′5′′ ) 6.22 Hz,
J 5′5′′ ) -12.41 Hz). Anal. (C10H14N2O7F) C, H, N.
1,3-Di-â-D-r ibofu r a n osyl-5-flu or o-6-m eth ylu r a cil (37).
This was prepared by deblocking 1 g of 32, as for 35 and 36,
above, to give 355 mg (87%) of amorphous 37, with no defined
melting point:UV λmax (pH 7) 268 nm (ꢀ 9.5 × 103); TLC
(cellulose) Rf (J ) 0.21; MS m/ z for C15H22FN2O10 calcd
409.125 85, found 409.1262. Anal. (C15H21FN2O10‚H2O) C, H,
N.
)
3-(2,3,5-Tr i-O-a cet yl-â-D-r ib ofu r a n osyl)-6-(t r iflu or o-
m eth yl)u r a cil (34). To a suspension of 360 mg (2 mmol) of
6-(trifluoromethyl)uracil (28) in 10 mL of anhydrous MeCN
was added 500 µL (1.88 mmol) of BSTFA and 650 mg (2 mmol)
of 1,2,3,5-tetra-O-acetyl-1-â-D- ribofuranose, and the mixture
was heated under reflux to obtain a clear solution. After
cooling to room temperature, 340 mg (11.5 mmol) of TMSOTfl
was slowly added, and after 30 min, TLC on silica gel with
solvent L demonstrated quantitative conversion to a single
product. Following addition of 50 mL of CHCl3, the mixture
was extracted with 2 × 10 mL of saturated NaHCO3. The
organic phase was dried on anhydrous Na2SO4 and evaporated
to dryness. The residue was fractionated by preparative TLC
(silica gel) with solvent L. Following 2-fold development, the
reaction product was eluted with EtOH and brought to
dryness, yielding 790 mg (87%) of 34 as a foam, which could
not be crystallized: TLC (silica gel) Rf (L) 0.60; MS m/ z (M -
CH3COOH)+ for C14H13F3N2O7 calcd 378.067 486, found
378.067 526.
1-â-D-Ribofu r a n osyl-6-(flu or om eth yl)u r a cil (41) a n d
1-â-D-Ribofu r a n osyl-5-flu or o-6-(flu or om eth yl)u r a cil (42).
To a solution of 724 mg (3 mmol) of 6-methyluridine (5) in 10
mL of distilled Ac2O was added 15 mg (0.12 mmol) of DMAP,
and the mixture was stirred at room temperature until TLC
(silica gel, solvent M) demonstrated disappearance of 5. The
mixture was evaporated to dryness and, following addition of
25 mL of EtOH, again brought to dryness. The residue was
treated with 30 mL of anhydrous toluene and brought to
dryness, and this was repeated twice more. The residue was
then dissolved in 9 mL of 1,4-dioxane/AcOH (11:1, v/v) and 1
g of SeO2 added. The mixture was then brought to boiling
under reflux for 18 h, with monitoring of the course of the
reaction by TLC (silica gel, solvent M), and then cooled and,
following addition of 50 mL of benzene, filtered. The filtrate
was brought to dryness under reduced pressure, the residue
dissolved in 50 mL of CHCl3, and 50 mL of H2O added. The
organic phase was removed and dried over Na2SO4. The
drying agent was removed by filtration. To the filtrate was
added 25 mL of MeOH followed by portionwise addition of 257
mg (l mmol), tetrabutylammonium borohydride, and the course
of the reaction was monitored by TLC (silica gel with solvent
M). The reaction mixture was filtered, the filtrate concen-
trated to a small volume under reduced pressure, 50 mL of
CHCl3 added, and the solution washed sequentially with 20
mL of H2O, 20 mL of 0.1 N HCl, 30 mL of H2O, and 20 mL of
saturated NaHCO3. The organic phase was dried over Na2-
SO4, brought to dryness under reduced pressure, and then
dried in an Abderhalden pistol. The residue was dissolved in
20 mL of anhydrous CH2Cl2, to which was added 300 µL (2.27
mmol) of DAST in 5 mL of CH2Cl2. The solution was stirred
at room temperature, and the reaction course was monitored
by TLC (silica gel with solvent M). After 13 h, 50 mL of CHCl3
was added, and the mixture was extracted with saturated
NaHCO3. The organic phase was washed with 2 × 30 mL of
H2O, dried over Na2SO4, and brought to dryness under reduced
pressure. The residue was dissolved in 15 mL of ammonia-
saturated MeOH (at 0 °C), left at room temperature for 5 h,
brought to a small volume, and subjected to preparative TLC
on silica gel plates with solvent I to yield 322 mg (37%) of
3-â-D-Ribofu r an osyl-6-(tr iflu or om eth yl)u r acil (38). Ad-
dition of 454.4 mg (1 mmol) of 3-(2′,3′,5′-tri-O-acetyl-â-D-
ribofuranosyl)-6-(trifluoromethyl)uracil (34) to 20 mL of hot
MeOH resulted in a clear solution. After cooling to room
temperature, 1.5 mL of 1 N methanolic NaOMe was added
and the mixture was stirred overnight, neutralized with Dowex
50W(H+), and evaporated to dryness to yield 272 mg (87%) of
38 as a foam, which could not be crystallized: mp 114-117
°C; UV λmax (pH 2) 262 (ꢀ 5.75 × 103), (pH 7) 298 (ꢀ 7.65 ×
103), (pH 12) 298 nm (ꢀ 8.15 × 103); TLC (silica gel) Rf
(C) 0.16; MS m/ z for C10H12F3N2O6 calcd 313.0648, found
313.064 72; 1H NMR (D2O) δ 6.27 (1H, d, 1′-H, J 1′2′ ) 3.35 Hz),
6.23 (1H, br s, 5-H), 4.43 (1H, t, 3′-H, J 2′3′ ) 6.56 Hz), 3.99
(1H, m, 4′-H, J 3′4′ ) 7.02 Hz), 3.89 (1H, dd, 5′-H, J 4′5′ ) 3.02
Hz), 3.85 (1H, dd, 5′′-H, J 4′5′′ ) 6.04 Hz, J 5′5′′ ) -12.10 Hz).
Anal. (C10H12F3N2O6‚1/2MeOH) C, H, N.
1-â-D-Ribofu r a n osyl-6-(h yd r oxym eth yl)u r a cil (39) a n d
1-â-D-Ribofu r an osyl-5-flu or o-6-(h ydr oxym eth yl)u r acil (40).
To a solution of 774 mg (3 mmol) of 6-methyluridine (5) in 10
mL of distilled Ac2O was added 15 mg (0.12 mmol) of DMAP.
The mixture was stirred at room temperature until 5 had
disappeared (TLC on silica gel with solvent M). The mixture
was evaporated to dryness, 25 mL of EtOH added, and the
mixture again brought to dryness. This was repeated with 3
× 30 mL of anhydrous toluene. The residue was then taken
up in 9 mL of 1,4-dioxane/AcOH (11:1, v/v), and 1 g (9 mmol)
of SeO2 was added. The mixture was heated to boiling under
reflux for 18 h, the reaction course being monitored by TLC
(silica gel, solvent M). Following addition of 50 mL of benzene
to the cooled solution, it was filtered, the filtrate brought to
dryness under reduced pressure, the residue dissolved in 50
mL of CHCl3, and 50 mL of H2O added. The organic phase
was separated and dried over Na2SO4. The drying agent was
removed by filtration, 25 mL of MeOH added, and 257 mg (1
mmol) of tetrabutylammonium borohydride added in small
portions, with the reaction course being monitored by TLC
(silica gel with solvent M). The mixture was filtered, brought
to a small volume under reduced pressure, and, after addition
of 50 mL of CHCl3, washed sequentially with 20 mL of H2O,
20 mL of 0.1 N HCl, 30 mL of H2O, and 20 mL of saturated
NaHCO3. The organic phase was concentrated under reduced
pressure and the residue dissolved in 15 mL of ammonia-
saturated MeOH (at 0 °C) and left at room temperature for 5
h. The mixture was brought to dryness, and the product was
isolated by preparative TLC on silica gel plates with solvent
I, yielding 395 mg (48%) of 39: mp 173-175 °C (lit.25 mp 177-
178 °C); TLC (silica gel) Rf (B) 0.17; 1H NMR (D2O) δ 5.99 (1H,
d, 5-H), 5.56 (1H, d, 1′-H, J 1′2′ ) 3.38 Hz), 4.85 (1H, dd, 2′-H),
amorphous 41: TLC (silica gel) Rf (B) 0.33; MS m/ z for C10H13
-
FN2NaO6 calcd 299.065 58, found 299.065 54; 1H NMR δ (D2O)
δ 6.03 (1H, d, 5-H), 5.46 (1H, d, 1′-H, J 1′2′ ) 3.29 Hz), 5.45
(2H, m, 6-CH2OH), 4.40 (1H, dd, 3′-H, J 2′3′ ) 6.63 Hz), 3.97
(1H, m, 4′-H, J 3′4′ ) 6.97 Hz), 3.89 (1H, dd, 5′-H, J 4′5′ ) 2.97
Hz), 3.76 (1H, dd, 5′′-H, J 4′5′′ ) 6.28 Hz, J 5′5′′ ) -12.41 Hz).
Anal. (C10H13N2O6F) C, H, N.
Compound 42 was synthesized as above (309 mg, 35%) but
could not be crystallized: UV λmax (pH 2) 268 (ꢀ 9.6 × 103),
(pH 7) 268 (ꢀ 6.9 × 103), (pH 12) 269 nm (ꢀ 5.7 × 103); TLC
(silica gel) Rf (J ) 0.78; MS m/ z for C10H12F2N2O6 calcd
295.0742, found 295.0740; 1H NMR (D2O) δ 5.75-5.40 (2H,
m, 6-CH2F), 5.72 (1H, d, 1′-H, J 1′2′ ) 3.31 Hz), 4.40 (1H, t, 3′-
H, J 2′3′ ) 6.51 Hz), 3.96 (1H, m, 4′-H, J 3′4′ ) 7.23 Hz), 3.87
(1H, dd, 5′-H, J 4′5′ ) 3.04 Hz), 3.74 (1H, dd, 5′′-H, J 4′5′′ ) 6.22
Hz, J 5′5′′ ) -12.41 Hz). Anal. (C10H12F2N2O6‚H2O) C, H, N.
Ack n ow led gm en t. We are indebted to J aros/law
Poznan´ski for assistance with the interpretations of the
NMR spectra and conformation calculations.

1728 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Felczak et al.
(14) Shugar, D.; Fox, J . J . Spectrophotometric Studies of Nucleic Acid
Derivatives and Related Compounds as a Function of pH. II.
Pyrimidine Nucleosides. Biochim. Biophys. Acta 1952, 9, 369-
386.
Refer en ces
(1) Hruska, F. E. Molecular Conformation of Orotidine, Naturally
Occurring Nucleoside, in the Syn Conformation in Aqueous
Solution. J . Am. Chem. Soc. 1971, 93, 1795-1858.
(2) Cody, V.; Kalman, T. J . Conformational Analysis of 6-Substituted
Uridine Analogues: Crystal Structures of Uridine-6-thiocar-
boxamide and 6-Cyanouridine. Nucleosides Nucleotides 1985, 4,
587-594.
(3) Krajewska, E.; Shugar, D. Pyrimidine Ribonucleoside Phospho-
rylase Activity vs 5- and/or 6-Substituted Uracil and Uridine
Analogs, Including Conformational Aspects. Biochem. Pharma-
col. 1982, 31, 1097-1102.
(4) Vorbru¨ggen, H.; Krolikiewicz, K.; Bennua, B. Nucleoside syn-
thesis, XXII. Nucleoside Synthesis with Trimethylsilyl Triflate
and Perchlorate as Catalyst. Chem. Ber. 1981, 114, 1234-1255.
(5) Prystaˇ, S.; Sˇorm, F. Nucleic Acid Components and Their
Analogues CXXVII. Synthesis of Uridine and Cytidine 6-Methyl
and 5,6-Dimethyl Derivatives. Collect. Czech. Chem. Commun.
1969, 35, 2316-2347.
(6) Philips, K. D.; Horwitz, J . P. Nucleosides. XVII. Benzylation -
Debenzylation Studies in Nucleosides J . Org. Chem. 1975, 40,
1856-1858.
(7) (a) Mizuno, Y.; Ikehara, M.; Watanabe, K. A. Potential Antime-
tabolites. I. Selective Thiation of Uracil and 1,2,4-Triazine-3,5-
(2H,4H)-dione (6-Azauracil). Chem. Pharm. Bull. 1962, 10, 647-
652. (b) Mizuno, Y.; Ikehara, M.; Watanabe, K. A. Potential
Antimetabolites. II. Chemical Synthesis of 6-Azacytidine. Chem.
Pharm. Bull. 1962, 10, 653-659. (c)Winkley, M. W.; Robins, R.
K. Pyrimidine Nucleosides. I. The Synthesis of 6-Methylcytidine,
6-Methyluridine and Related 6-Methylpyrimidine Nucleosides.
J . Org. Chem. 1968, 33, 2822-2827.
(8) Felczak, K.; Bretner, M.; Kulikowski, T.; Shugar, D. High-Yield
Regioselective Thiation of Biologically Important Pyrimidinones,
Dihydropyrimidinones and their Ribo, 2′-Deoxyribo and 2′,3′-
Dideoxyribo Nucleosides. Nucleosides Nucleotides 1993, 12, 245-
261.
(15) (a) Cech, D.; Herrmann, G.; Holy, A. Fluorination of 6-Methyl-
uracil and its Nucleosides. Nucleic Acids Res. 1977, 4, 3259-
3266. (b) Felczak, K.; Kulikowski, T.; Vilpo, J . A.; Giziewicz, J .;
Shugar, D. Synthesis and Biological Properties of 6-Substituted
5-Fluorouridines. Nucleosides Nucleotides 1987, 6, 257-260.
(16) (a) De Leeuw, F. A. A. M.; Altona, C. Conformational Analysis
of â-D-Ribo-, â-D-Deoxyribo-, â-D-Arabino-, â-D-Xylo-, and â-D-
Lyxonucleosides from Proton-proton Coupling Constants. J .
Chem. Soc. Perkin Trans. II 1982, 375-384. (b) Uhl, W.; Reiner,
J .; Gassen, H. G. On the Conformation of 5-Substituted Uridines
as Studied by Proton Magnetic Resonance. Nucleic Acids Res.
1983, 11, 1167-1180. (c) Remin, M. Determination of the
Complete Correlation between the Sugar Ring Puckers and 5′-
Exocyclic Group Rotamers in Conformationally Flexible Nucleic
Acid Components from NMR Study. J . Biomol. Struct. Dyn.
1984, 2, 211-220. (d) Saenger, W. Principles of Nucleic Acid
Structure; Springer-Verlag: New York, 1984; pp 71-73.
(17) (a) Suck, D.; Saenger, W. Molecular and Crystal Structure of
6-Methyluridine, a Pyrimidine Nucleoside in the Syn Conforma-
tion. J . Am. Chem. Soc. 1972, 94, 6520-6526. (b) Birnbaum, G.
I.; Hruska, F. E.; Niemczura, W. P. A Pyrimidine Nucleoside
Constrained in the Syn Form. Structure and Conformation of
6-Methyl-2′-deoxyuridine. J . Am. Chem. Soc. 1980, 102, 5586-
5590.
(18) Vita, A.; Magni, G. A. One-Step Procedure for Purification of
Uridine Phosphorylase from Escherichia Coli. Anal. Biochem.
1983, 133, 153-156.
(19) Cleland, W. W. The Statistical Analysis of Enzyme Kinetic Data.
Adv. Enzymol. 1967, 29, 1-32.
(20) Bradford, M. M. A Rapid and Sensitive Method for the Quan-
titation of Microgram Quantities of Protein Utilizing the Prin-
ciple of Protein - Dye Binding. Anal. Biochem. 1976, 72, 248-
254.
(21) Vilpo, J . A.; Vilpo, L. M. Metabolism, Incorporation into DNA,
and Interaction with 1-â-D-Arabinofuranosylcytosine of 5-Hy-
droxymethyl-2′-deoxyuridine in Human Promyleocytic Leukemia
Cells (HL-60). Cancer Res. 1988, 48, 3117-3122.
(9) Lamon, R. W. A New Synthesis of 4-Thiouracils. J . Heterocyc.
Chem. 1968, 5, 837-844.
(10) Bergmann, E.; Cohen, S.; Shahak, I. Organic Fluorine Com-
pounds. Part XI. Ethyl Fluoroacetoacetates and Fluoropyrim-
idines. J . Chem. Soc. 1959, 3278-3285.
(11) Giner-Sorolla, A.; Bendich, A. Fluorine-Containing Pyrimidines
and Purines: Synthesis and Properties of Trifluoromethyl
Pyrimidines and Purines. J . Am. Chem. Soc. 1958, 80, 5744-
5752.
(12) Botta, M.; Cavalieri, M.; Ceci, D.; De Angelis, F.; Finizia, G.;
Nicoletti, R. 6-Alkyl and 5,6-Dialkyl-2-methoxy-4(3H)-pyrimi-
dinones in the Transformations of Pyrimidines. Tetrahedron
1984, 40, 3313-3320.
(22) Kahilainen, L.; Bergstrom, D.; Kangas, L.; Vilpo, J . A. In Vitro
and In Vivo Studies of A Promising Antileukemic Thymidine
Analogue, 5-Hydroxymethyl-2′-deoxyuridine. Biochem. Pharma-
col. 1986, 4211-4215.
(23) Holy, A. Preparation of 6-Methyluridine, 6-Ethyluridine and 2′-
Deoxy-6-ethyluridine. Collect. Czech. Chem. Commun. 1974, 39,
3374-3382.
(24) Tanaka, H.; Nasu, I.; Hayakawa, H.; Miyasaka, T. A Facile and
Regiospecific Preparation of 6-Alkyluridines. Nucleic Acids Res.
Symp. Ser. 1980, 8, 33-37.
(25) Tanaka, H.; Hayakawa, H.; Tadashi, M. “Umpolung” of Reactiv-
ity at the C-6 Position of Uridine: a Simple and General Method
for 6-Substituted Uridines. Tetrahedron 1982, 38, 2635-2642.
(13) Berens, K.; Shugar, D. Ultraviolet Absorption Spectra and
Structure of Halogenated Uracils and Their Glycosides. Acta
Biochim. Polon. 1963, 10, 25-47.
J M950675Q