Synthesis and antitumor activities of novel pyrimidine derivatives of 2,3-O,O-dibenzyl-6-deoxy-L-ascorbic acid and 4,5-didehydro-5,6-dideoxy-L-ascorbic acid

Article abstract of DOI:10.1021/jm0009540

The new pyrimidine derivatives of 2,3-O,O-dibenzyl-6-deoxy-L-ascorbic acid (8-10) were synthesized by condensation of uracil and its 5-fluoro- and 5-trifluoromethyl-substituted derivatives with 4-(5,6-epoxypropyl)-2,3-O,O-dibenzyl-L-ascorbic acid (7), whi

Full text of DOI:10.1021/jm0009540

4806
J . Med. Chem. 2000, 43, 4806-4811
Syn th esis a n d An titu m or Activities of Novel P yr im id in e Der iva tives of
2,3-O,O-Diben zyl-6-d eoxy-L-a scor bic Acid a n d 4,5-Did eh yd r o-5,6-
d id eoxy-L-a scor bic Acid
Silvana Raic´-Malic´,*,† Drazˇenka Svedruzˇic´,† Tatjana Gazivoda,† Andreja Marunovic´,† Antonija Hergold-Brundic´,
Ante Nagl,‡ J an Balzarini,§ Erik De Clercq,§ and Mladen Mintas†
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, Laboratory of General and Inorganic
Chemistry, Faculty of Science, and Laboratory of General and Inorganic Chemistry, Faculty of Textile Technology,
University of Zagreb, Marulic´ev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia, and Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received J uly 31, 2000
The new pyrimidine derivatives of 2,3-O,O-dibenzyl-6-deoxy-L-ascorbic acid (8-10) were
synthesized by condensation of uracil and its 5-fluoro- and 5-trifluoromethyl-substituted
derivatives with 4-(5,6-epoxypropyl)-2,3-O,O-dibenzyl-L-ascorbic acid (7), while pyrimidine
derivatives of 4,5-didehydro-5,6-dideoxy-L-ascorbic acid (14-17) with free C-2′ and C-3′ hydroxy
groups in the lactone ring were obtained by debenzylation of 11-13 with boron trichloride.
Z-Configuration of the C4′dC5′ double bond and position of the benzyl group in the lactone
ring of 14 were deduced from their 1H and 13C NMR spectra and connectivities in COSY,
ROESY, and HMBC spectra. The exact stereostructure of 13 was confirmed by its X-ray crystal
structure analysis. Of all the compounds in the series, compound 16 containing a 5-fluoro-
substituted uracil ring showed the most significant antitumor activities against murine
leukemia L1210/0 (IC50 ) 1.4 µg/mL), murine mammary carcinoma FM3A/0 (IC50 ) 0.78 µg/
mL), and, to a lesser extent, human T-lymphocyte cells Molt4/C8 (IC50 ) 31.8 µg/mL) and CEM/0
cell lines (IC50 ) 20.9 µg/mL).
In tr od u ction
hydro-5,6-dideoxy-L-ascorbic acid exerted cytostatic ac-
tivities against malignant cell lines: pancreatic carcinoma
(MiaPaCa2), breast carcinoma (MCF7), cervical carci-
noma (HeLa), laryngeal carcinoma (Hep2), murine
leukemia (L1210/0), murine mammary carcinoma
(FM3A), and human T-lymphocytes (Molt4/C8 and
CEM/0), as well as antiviral activities against varicella-
zoster virus (TK+ VZV and TK- VZV) and cytomegalo-
virus (CMV).11 The compound containing a trifluoro-
methyl-substituted uracil ring displayed the most potent
cytostatic activity particularly against human Molt4/
C8 cells with an IC50 of 0.9 µM.11 The synthesis of novel
pyrimidine derivatives of 2,3-O,O-dibenzyl-6-deoxy-L-
ascorbic acid (8-10) and 4,5-didehydro-5,6-dideoxy-L-
ascorbic acid (14-17) (see Figure 1) and their evaluation
for antitumor and antiviral activities are described
herein.
L-Ascorbic acid and its derivatives have been found
to possess antitumor and antiviral activities.1-3 Thus,
L-ascorbic acid inhibited apoptosis induced by oxidative
stress in HL-60 myeloid leukemia cell.4 Some deriva-
tives of L-ascorbic acid, e.g. 6-bromo-, 6-amino-, and N,N-
dimethyl-6-amino-6-deoxy-L-ascorbic acid, inhibited the
growth of certain human malignant tumor cell lines:
cervical carcinoma (HeLa), laryngeal carcinoma (Hep2),
and pancreatic carcinoma (MiaPaCa2).3,5 Furthermore,
vitamin C and vitamin K-3 combination showed syner-
gistic antitumor activity against human prostatic car-
cinoma cell lines (DU145).6 Sodium 5,6-benzylidene-L-
ascorbate and its related compounds induced DNA
fragmentation in human myelogenous leukemic cell
lines.7 In addition, several nucleosides containing a
5-substituted pyrimidine moiety were shown to inhibit
growth of murine mammary carcinoma (FM3A TK-/
HSV 1 TK+) cells transformed with the herpes simplex
virus type 1 (HSV-1) TK gene.8-10 L-Ascorbic acid-2-
phosphate was shown to afford long-lasting antiviral
activity against several human cytomegalovirus (CMV)
strains in human foreskin fibroblasts (HFF) and endo-
thelial cells (EC).1
Ch em istr y
The syntheses of 5,6-O,O-diacetyl-2,3-O,O-dibenzyl-
L-ascorbic acid12 (4) and 4-(5,6-epoxypropyl)-2,3-O,O-
dibenzyl-L-ascorbic acid (7), key intermediates for cou-
pling with pyrimidine bases, are outlined in Scheme 1.
Treatment of L-ascorbic acid (AA) with acetyl chloride
in acetone afforded the 5,6-ketal of L-ascorbic acid (1).12
Benzylation of the C-2′ and C-3′ hydroxy groups of the
lactone ring in 1 was accomplished using K2CO3 and
benzyl chloride in DMF to provide 2.12,13 Deblocking of
the 5,6-O,O-protected derivative of L-ascorbic acid 2 with
acetic acid in methanol gave 2,3-O,O-dibenzyl-L-ascorbic
acid (3).12 Subsequent acetylation of 3 was carried out
with acetic anhydride in pyridine and CH2Cl2 to give 4
using an analogous procedure for the preparation of 5-O-
In our previous studies we demonstrated that pyri-
midine and purine derivatives of 2,3-dibenzyl-4,5-dide-
* To whom correspondence should be addressed. Tel: 385-1-4597-
214. Fax: 385-1-4597-250. E-mail: Silvana.Raic@pierre.fkit.hr.
† Faculty of Chemical Engineering and Technology, University of
Zagreb.
Faculty of Science, University of Zagreb.
‡ Faculty of Textile Technology, University of Zagreb.
§ Katholieke Universiteit Leuven.
10.1021/jm0009540 CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/17/2000

Synthesis/ Antitumor Activities of Novel Pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4807
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (i) HMDS, (NH4)2SO4/argon atmo-
sphere/reflux/3 h, then trimethylsilyl triflate/dry acetonitrile/55-
70 °C/12 h.
ROESY, and HMBC spectra (see Table 1 and Experi-
mental Section).
The 1H NMR spectra of compounds 8-10 exhibit
signals of the pyrimidine ring and benzyl groups in
positions 2′ and 3′ of the lactone ring and signals for
the protons H-4′, H-5′, H-6′, and 5′-OH of the aliphatic
chain. The H-H coupling patterns in the side chain are
singlet for H-4′, multiplet for H-5′, doublet of doublet
for H-6′, and multiplet for 5′-OH.
a
Reagents and conditions: (i) acetyl chloride/acetone/rt/3-4 h;
(ii) benzyl chloride, K2CO3/dry DMF; (iii) 50% acetic acid/MeOH/
100 °C/5 h; (iv) acetic anhydride/pyridine/CH2Cl2/-10-24 °C/2 h;
(v) toluene-4-sulfonyl chloride/CH2Cl2/pyridine/0-24 °C; (vi) NaBr/
acetone/130 °C/7 h; (vii) Na2CO3/acetonitrile/rt/1 h.
1
In the H NMR spectra of compounds 14-17, besides
signals of the pyrimidine skeleton which are in agree-
ment with those reported for other pyrimidine deriva-
tives,11,14,15 signals for H-5′ and H-6′ in the side chain
as well as for the hydroxy groups at the 2- and
3-positions of the lactone ring were observed. Side chain
H-H coupling patterns in all compounds are doublet
for H-6′ and triplet for H-5′. To determine the position
of substitution of the benzyl group in the lactone ring
in monobenzylated derivative 14, its HMBC spectrum
was recorded. This spectrum showed a cross-peak
arising from three-bond coupling between carbon atom
C-3′ of the lactone ring and methylene protons CH2-Ph
confirming substitution of the benzyl group at position
3. The structure of 14 was also verified by the ROESY
spectrum which displayed interaction for methylene
protons (CH2-Ph) and phenyl protons of the benzyl
group and methine proton H-5′. These observations also
corroborate the Z-stereostructure around the C4′dC5′
double bond. In the hypothetical E-configuration, an
interaction between protons H-6 of the pyrimidine ring
and methylene protons of the benzyl group should have
been seen. The lack of this cross-peak in the ROESY
spectrum of 14 is in agreement with the proposed
Z-geometry. Similarly, in the ROESY spectra of com-
pounds 15-17 with C-2′ and C-3′ free hydroxy groups,
interactions between protons of the C-3′ hydroxy group
and methine proton H-5′ were found indicating Z-
configuration of the C4′dC5′ double bond.
F igu r e 1. Pyrimidine derivatives of 2,3-O,O-dibenzyl-6-deoxy-
L-ascorbic acid (8-10), 3-O-benzyl-4,5-didehydro-5,6-dideoxy-
L-ascorbic acid (14), and 4,5-didehydro-5,6-dideoxy-L-ascorbic
acid (15-17).
acetyl-6-bromo-6-deoxy-2,3-O,O-dibenzyl-L-ascorbic acid.13
2,3-O,O-Dibenzyl-6-O-tosyl-L-ascorbic acid (5) was ob-
tained by tosylation of the 6-hydroxy group in 3 using
toluene-4-sulfonyl chloride in pyridine.12 Bromination
of 5 with sodium bromide in acetone gave 6-bromo-2,3-
O,O-dibenzyl-L-ascorbic acid (6).13 4-(5,6-Epoxypropyl)-
2,3-O,O-dibenzyl-L-ascorbic acid (7) was obtained by
reaction of 6 with sodium carbonate in acetonitrile.
Pyrimidine derivatives of 2,3-O,O-dibenzyl-6-deoxy-
L-ascorbic acid (8-10) were prepared by silylation of the
uracil and its 5-substituted derivatives with 1,1,1,3,3,3-
hexamethyldisilazane and subsequent condensation of
the intermediates thus obtained with 7 (Scheme 2).
Coupling of 4 with uracil and its 5-substituted deriva-
tives gave pyrimidine derivatives of 2,3-O,O-dibenzyl-
4,5-didehydro-5,6-dideoxy-L-ascorbic acid (11-13; Scheme
3).11 Debenzylation of 11-13 was accomplished by
treatment with boron trichloride in CH2Cl2, affording
14-17 (Scheme 3).14,15
The 13C NMR data are given in the Experimental
Section. Generally, the 13C NMR spectra for 14-17
showed four signals for the pyrimidine ring, four signals
for the lactone moiety, and two signals for carbons in
the side chain, and for 8-10 and 14 the spectra
displayed additional peaks for the phenyl and methyl-
ene protons of the benzyl group. The C-3′ benzyl
substitution in 14 caused differences in chemical shifts
for carbons C-2′ (ca. -3 ppm) and C-3′ (ca. 2 ppm) of
the lactone ring. The most pronounced differences in the
13C NMR spectrum of 9 and 16 caused by substitution
1H a n d 13C NMR Stu d ies. The assignment of 1H and
13C NMR spectra was performed on the basis of chemical
shifts and the magnitude and multiplicity of H-H spin-
spin coupling, as well as connectivities in the COSY,

4808 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Ta ble 1. Chemical Shifts (δ, ppm)a and H-H Coupling Constants (J , Hz)b in 1H NMR Spectra for Compounds 4, 7-10, and 14-17
2′-OH,
Raic´-Malic´ et al.
compd
H-5
H-6
NH
H-4′
H-5′
H-6′
3′-OH
4c
4.81 (1H)
5.36-5.32 (1H) 4.33 (1H) J ) 11.53; 6.92 (dd);
4.23 (1H) J ) 11.66; 5.51 (dd)
7d
4.56 (1H)
J ) 3.93 (d) (m)
4.51 (s, 1H) 5.26-5.17 (1H) 3.63 (1H) J ) 13.59; 8.72 (dd);
3.14-3.10 (1H) 2.87-2.81 (2H) (m)
8e
5.66 (1H) J ) 7.7 (d) 7.24-7.14 (1H) 9.59 (s, 1H)
(m)
4.03 (1H) J ) 13.71; 3.97 (dd)
9f
7.44-7.20 (1H) 10.15 (b, 1H) 4.57 (s, 1H) 5.17-5.08 (1H) 3.98 (1H) J ) 13.07 (d);
(m)
3.58 (1H) J ) 13.72; 9.87 (dd)
3.82 (1H) J ) 13.59 (d);
3.46 (1H) J ) 11.92 (dd)
4.47 (2H) J ) 6.96 (d)
10g
14h
15
16
17
7.83 (s, 1H)
10.21 (s, 1H) 4.47 (s, 1H) 4.93 (1H) (m)
5.54 (1H) J ) 7.84;
2.24 (dd)
5.57 (1H) J ) 7.83;
1.78 (dd)
7.60 (1H)
J ) 7.86 (d)
7.63 (1H)
J ) 7.86 (d)
8.08 (1H)
J ) 6.69 (d)
8.39 (s, 1H)
11.28 (1H)
J ) 1.51
5.31 (1H)
J ) 6.95 (t)
5.38 (1H)
J ) 6.98 (t)
5.39 (1H)
J ) 6.81 (t)
5.42 (1H)
10.35
11.29 (s, 1H)
4.50 (2H) J ) 6.99 (d)
4.47 (2H) J ) 6.82 (d)
4.59 (2H) J ) 6.68 (d)
11.20,
9.70
11.29,
9.62
11.29,
9.60
11.82 (s, 1H)
11.85 (s, 1H)
J ) 6.68 (t)
a
CDCl3 except for 14-17 (DMSO-d6); chemical shifts referred to TMS. Multiplicity of coupling and number of protons are given in
b
parentheses: s ) singlet, d ) doublet, t ) triplet, m ) complex multiplet. Digital resolution (0.28 Hz. See Figure 1 and Schemes 1-3
for structures. c Chemical shifts for OCOCH3: 1.94 (3H, s), 2.04 (3H, s); C6H5: 7.22-7.38 (10H, m); OCH2: 5.22-5.16 (4H, m). Chemical
d
shifts for C6H5: 7.46-7.19 (10H, m); OCH2: 5.22-5.06 (4H, m). e Chemical shifts for 5′-OH: 4.28 (1H, m); C6H5: 7.36-7.34 (10H, m);
OCH2: 5.13-5.07 (4H, m). f Chemical shifts for 5′-OH: 4.20 (1H, m); C6H5: 7.44-7.20 (10H, m); OCH2: 5.08 (s, 2H); 5.06 (s, 2H). g Chemical
h
shifts for 5′-OH: 4.06 (1H, m); C6H5: 7.35-7.15 (10H, m); OCH2: 5.09-5.06 (4H, m). Chemical shifts for C6H5: 7.44-7.35 (5H, m);
OCH2: 5.44 (s, 2H).
Sch em e 3a
F igu r e 2. ORTEP17 view with the labeling scheme for 13.
for C-4, C-5, and C-6 in 17 were ca. -4, 2, and 1 ppm,
respectively. The magnitude of one-bond (ca. 245 Hz)
and two-bond (ca. 38 Hz) C-F couplings at C-4, C-5,
and C-6 in 9, 16, and 17 are in accord with the literature
data for fluorinated derivatives.16
X-r a y Cr ysta l Str u ctu r e An a lysis. To determine
the exact stereostructure of 13,11 its X-ray crystal
structure analysis was undertaken. The perspective
view with atom numbering and packing of the molecules
in the unit cell of 13 are displayed in Figures 217 and 3.
The skeleton of the molecule consists of a planar lactone
ring connected to the pyrimidine moiety by an acyclic
a
Reagents and conditions: (i) HMDS, (NH4)2SO4/argon atmo-
sphere/reflux/3 h, then trimethylsilyl triflate/dry acetronitrile/55-
70 °C/12 h; (ii) BCl3, CH2Cl2/-78 °C/2 h.
chain which contains a double bond between atoms C-4
and C-5. That bond is conjugated to the lactone ring and
exists in the E-configuration. The planes defined by the
atoms directly bonded to all four moieties - lactone (A),
pyrimidine (B), and two phenyl (C and D) rings - are
coplanar with those rings. Conformation of the molecule
is defined by dihedral angles between the planes of the
lactone ring (A) and other rings (B, C, and D): C4-C5-
C6-N1 [114.2(3)°], C3-O4-C14-C15 [167.7(3)°], C2-
O3-C7-C8 [174.8(3)°]. Details of the conformation are
described as follows. The angles between planes of the
of the fluoro atom at C-5 in the pyrimidine ring were
observed for atoms C-4, C-5, and C-6. Thus, carbon C-5
was deshielded in 9 and 16 by ca. 39 ppm with respect
to that of the unsubstituted uracil derivatives 14 and
15, while carbons C-4 and C-6 in 9 and 16 were shielded
by ca. 6 and 18 ppm, respectively. These differences in
substituent-induced chemical shifts (SCS/ppm) for the
same carbons in 10 and 17 that carry a trifluoromethyl
group at the C-5 position were much smaller. Thus, SCS

Synthesis/ Antitumor Activities of Novel Pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4809
approximately 10-fold less pronounced cytostatic activity
of all the examined tumor cells. 5-Fluorouracil deriva-
tives of 4,5-didehydro-5,6-dideoxy-L-ascorbic acid 1211
and 16 (Scheme 3) expressed better inhibitory effects
on the growth of all tumor cell lines than the corre-
sponding 6-deoxy derivative of L-ascorbic acid (9). Of all
the studied compounds the 5-fluorouracil derivatives of
4,5-didehydro-5,6-dideoxy-L-ascorbic acid (16) exhibited
the most pronounced inhibitory activity against FM3A/0
cells (IC50 ) 0.78 µg/mL). None of the compounds in
Table 2 showed any selectivity for tumor cells, either
human or murine.
An tivir a l Activity. Compounds 8-10 and 14-17
were also evaluated against varicella-zoster virus (VZV),
cytomegalovirus (CMV) in human embryonic lung (HEL)
cells, and human immunodeficiency virus (HIV) in
acutely infected MT-4 cells. None of the compounds
showed appreciable antiviral activity (IC50 > 50 µg/mL)
against any of these viruses (data not shown).
F igu r e 3. Packing diagram in the unit cell of 13.
rings A/B and A/D were 72° and 74°, respectively,
whereas planes B and D were nearly coplanar. A similar
sterical relationship was found for planes B, C, and D.
The angles between rings B/C and C/D were 101.44(10)°
and 101.55(13)°; that is they are almost identical. An
intramolecular hydrogen bond C24-H24‚‚‚F2 [2.676(4)
Å] in the pyrimidine moiety was observed. The mol-
ecules in the crystal structure are linked together by
intermolecular hydrogen bonds of 2.862(4) Å between
N-2 and O-5 atoms of symmetrically related molecules
(symmetry code: -x + 1, -y, -z + 2), as shown in the
packing diagram of Figure 3.18
Con clu sion s
The present work describes the synthesis and anti-
tumor and antiviral evaluation of the novel pyrimidine
derivatives of 2,3-O,O-dibenzyl-6-deoxy-L-ascorbic acid
(8-10) and 4,5-didehydro-5,6-dideoxy-L-ascorbic acid
(14-17). Benzyl substitution at C-3′ of the lactone ring
in 14 and the Z-configuration of the C4′dC5′ double
1
bond in 14-17 were elucidated by both H and 13C NMR
spectra. The stereostructure of 13 was unambiguously
confirmed by its X-ray structural analysis.
The shortening of the σ-bond C23-C25 compared to
the corresponding unfluorinated derivatives resulted
from the perfluoroalkyl effect of the trifluoromethyl
group.19
Compound 16, containing a 5-fluorouracil ring, was
found to exhibit appreciable antitumor cell activity,
particularly against the murine leukemia L1210/0 (IC50
) 1.4 µg/mL) and murine mammary carcinoma FM3A/0
(IC50 ) 0.78 µg/mL) cell lines.
Biologica l Resu lts a n d Discu ssion
The synthesis of new compounds within that class of
molecules guided by the results of biological screening
will be performed shortly.
An titu m or Activity. Inhibitory effects of 8-10 and
14-17 on the proliferation of murine leukemia (L1210/
0), murine mammary carcinoma (FM3A/0), and human
T-lymphocyte cells (Molt4/C8 and CEM/0) as well as
their cytotoxicity for human embryonic lung (fibroblast)
cells (HEL) and murine embryo fibroblasts (MEF) are
displayed in Table 2. The inhibitory effects are compared
with antitumor activities of 5-fluorouracil (5-FU) and
5-fluorodeoxyuridine (5-FdUrd). A growth experiment
with MEF was performed in the presence of 5-FU,
5-FdUrd, and 16.
Exp er im en ta l Section
Gen er a l Meth od s. New compounds were characterized by
1H and 13C NMR, electron impact mass spectra, and elemental
analysis (C, H, N). Melting points of compounds were deter-
mined with a Kofler micro hot-stage (Reichert, Wien) and are
uncorrected. Precoated Merck silica gel 60F-254 plates were
used for thin-layer chromatography (TLC) and the spots were
detected under UV light (254 nm). Column chromatography
was performed using silica gel (0.05-0.2 mm) Merck; glass
column was slurry-packed under gravity. Solvent system used
for column chromatography was CH2Cl2:MeOH, 30:1. Ad-
In comparison with 2,3-O,O-dibenzyl-6-deoxy-L-ascor-
bic acid derivatives 8-10 the corresponding 5,6-dideoxy-
L-ascorbic acid derivatives11 11-13 (Scheme 3) showed
Ta ble 2. Inhibitory Effects of Compounds 8-10 and 14-17 on the Growth of Malignant Tumor Cell Lines
a
tumor cell growth IC50 (µg/mL)
cytotoxicity (µg/mL)
c
compd
L1210/0
FM3A/0
Molt4/C8
CEM/0
cell morphology (MCC)b
cell growth (CC50
>50
33.4
31.7
)
MEFd
8
9
10
70.8
65.6
26.0
74.1
52.2
38.4
67.8
81.2
47.4
68.6
74.0
19.0
>50
50
50
14
15
16
17
5-FU
5-FdUrd
>200
>200
1.4
76.1
0.04
0.0003
>200
>200
0.78
162
0.02
0.0008
>200
>200
31.8
>200
>200
20.9
>50
>50
>50
>50
>50
>50
23
0.92
>200
>200
>50
2.9
2.6
1.2
0.003
0.20
0.0001
a
b
50% Inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation by 50%. Minimum cytotoxic
concentration that causes a microscopically detectable alteration of normal HEL cell morphology. c Cytotoxic concentration required to
d
reduce normal HEL cell growth by 50%. HEL cells, human embryonic lung (fibroblast) cells. MEF, murine embryo fibroblasts.

4810 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Raic´-Malic´ et al.
ditional purification of compounds 8-10 and 14-17 by re-
crystallization from ethanol afforded their analytical samples.
6′), 135.42, 129.08-128.02 (C6H5), 73.96, 73.56 (CH2Ph); MS
m/z 468 (M+•). Anal. (C24H21N2O7F) C, H, N.
The electron impact mass spectra were recorded with an
EXTREL FT MS 2001 instrument with ionizing energy 70 eV.
Elemental analyses were performed by the Central Analytical
Service, Ru]er Bosˇkovic´ Institute, Zagreb. Results were within
(0.4% of the theoretical values and are indicated by symbols
1-[2,4-Dioxo-5-(tr iflu or om eth yl)-(1H,3H)-p yr im id in -1-
yl]-2-(2,3-O,O-d ib en zyl-2-b u t en -4-olid ylid en e)et h a n -2-
ol (10). 5-(Trifluoromethyl)uracil (60% excess, 283 mg, 1.57
mmol) was treated according to the procedure analogous to
that for the preparation of compound 8 to give 10 (139 mg,
42%, mp 86-89 °C): UV (methanol) λmax 208 (log ꢀ 3.16), λmax
248 (log ꢀ 2.89); 13C NMR (CDCl3) δ 150.59 (C-2), 159.51 (C-
4), 104.17 (C-5), 147.38 (C-6), 121.22 (CF3, J ) 270.39 Hz),
169.71 (C-1′), 156.66 (C-2′), 121.22 (C-3′), 75.76 (C-4′), 66.12
(C-5′), 51.80 (C-6′), 73.96, 73.78 (CH2Ph), 135.26, 129.20-
128.34 (C6H5); MS m/z 518 (M+•). Anal. (C25H21N2O7F3) C, H,
N.
1
of the elements. The H and 13C NMR spectra were recorded
on a Varian Gemini 300 spectrometer, operating at 75.46 MHz
for the 13C resonance. The samples were dissolved in DMSO-
d6 or CDCl3 and measured at 21 °C in 5-mm NMR tubes. The
1H and 13C chemical shift values are in ppm, referred to TMS.
COSY, ROESY, and HMBC digital resolution in 1H spectra
was 0.28 Hz, while in 13C spectra it was 0.65 Hz per point.
5,6-O,O-Isop r op ylid en e-L-a scor bic a cid (1),12 2,3-O,O-
diben zyl-5,6-O,O-isopr opyliden e-L-ascor bic acid (2),12 2,3-
O,O-d iben zyl-L-a scor bic a cid (3), 2,3-O,O-d iben zyl-6-O-
tosyl-L-a scor bic a cid (5),12 a n d 6-br om o-2,3-O,O-d iben zyl-
L-a scor bic a cid (6)13 were synthesized in accord with the
original procedures given in the literature.
1-[2,4-Dioxo-(1H ,3H )-p yr im id in -1-yl]-2-(3-O-b en zyl-2-
h yd r oxy-2-bu ten -4-olid ylid en e)eth a n e (14) a n d 1-[2,4-
Dioxo-(1H,3H)-p yr im id in -1-yl]-2-(2,3-d ih yd r oxy-2-bu ten -
4-olid ylid en e)eth a n e (15). To a solution of UDBnAA (11)
(200 mg, 0.46 mmol) in dry CH2Cl2 at -78 °C under argon
was added a 1 M solution of BCl3 in CH2Cl2 (0.63 mL). The
mixture was stirred at -78 °C for 2 h, then the temperature
was raised to 10 °C and the reaction was continued for 2 h. A
mixture of CH2Cl2/MeOH (1:1) was added and the solvent was
then removed under reduced pressure. The crude product,
purified by column chromatography, yielded 14 (41 mg, 25%,
mp 189-190 °C) and 15 (49 mg, 39%, mp 202-204 °C). 14:
UV (methanol) λmax 208 (log ꢀ 4.43), λmax 268 (log ꢀ 4.32); 13C
NMR (DMSO-d6) δ 150.79 (C-2), 163.71 (C-4), 101.24 (C-5),
145.34 (C-6), 164.66 (C-1′), 123.36 (C-2′), 141.10 (C-3′), 143.70
(C-4′), 100.95 (C-5′), 42.59 (C-6′), 136.12, 128.52-127.98 (C6H5),
72.17 (CH2Ph); MS m/z 342 (M+•). Anal. (C17H14N2O6) C, H,
N. 15: UV (methanol) λmax 208 (log ꢀ 3.91), λmax 264 (log ꢀ 3.99);
13C NMR (DMSO-d6) δ 150.79 (C-2), 163.66 (C-4), 101.26 (C-
5), 145.29 (C-6), 164.78 (C-1′), 121.49 (C-2′), 143.21 (C-3′),
144.57 (C-4′), 100.24 (C-5′), 42.60 (C-6′); MS m/z 252 (M+•).
Anal. (C10H8N2O6) C, H, N.
5,6-O,O-Dia cetyl-2,3-O,O-d iben zyl-L-a scor bic Acid (4).
To a cooled (-10 °C) solution of 3 (2 g, 5.6 mmol) in CH2Cl2
(80 mL) and pyridine (28 mL) was added dropwise acetic
anhydride (28 mL, 296 mmol). Reaction mixture was stirred
at room temperature for 2 h and then the solvent was
evaporated. The crude product was submitted to column
chromatography, yielding pure 4 (1.64 g, 67%, mp 83-86 °C):
13C NMR (CDCl3) δ 168.16 (C-1′), 154.77 (C-2′), 120.98 (C-3′);
73.34 (C-4′), 67.28 (C-5′), 61.66 (C-6′), 169.84, 169.06 (COCH3),
20.23, 20.07 (COCH3) 135.17, 128.65-127.71 (C6H5), 73.34,
73.29 (CH2Ph).
4-(5,6-Ep oxy)-2,3-O,O-d iben zyl-L-a scor bic Acid (7). To
a solution of 6 (1.3 g, 3.1 mmol) in acetonitrile (25 mL) was
added an aqueous solution of Na2CO3 (0.33 g, 3.1 mmol) and
reaction mixture was stirred at room temperature for 1 h.
Solvent was then removed under reduced pressure and the
oily residue was extracted with chloroform. Organic layers
were dried over MgSO4. Recrystallization of oily product from
methanol gave white crystals of 7 (0.896 g, 85.5%, mp 85-88
°C): 13C NMR (CDCl3) δ 168.5 (C-1′), 120.46 (C-2′), 156.05 (C-
3′), 74.09 (C-4′), 50.33 (C-5′), 43.68 (C-6′), 135.42, 129.08-
127.64 (C6H5), 73.68, 73.46, (CH2Ph); MS m/z 339 (MH+).
1-[2,4-Dioxo-5-flu or o-(1H,3H)-p yr im id in -1-yl]-2-(2,3-d i-
h yd r oxy-2-bu ten -4-olid ylid en e)eth a n e (16). Compound 12
(290 mg, 0.64 mmol) was treated according to a procedure that
was analogous to that for the preparation of compounds 14
and 15 to give 16 (13 mg, 6.8%, mp 242-244 °C): UV
(methanol) λmax 208 (log ꢀ 4.09), λmax 274 (log ꢀ 4.13); 13C NMR
(DMSO-d6) δ 149.41 (C-2), 157.40 (C-4, J ) 25.77 Hz), 139.63
(C-5, J ) 229.25 Hz), 129.78 (C-6, J ) 33.60 Hz), 164.75 (C-
1′), 121.48 (C-2′), 143.13 (C-3′), 144.46 (C-4′), 100.02 (C-5′),
42.92 (C-6′); MS m/z 270 (M+•). Anal. (C10H7N2O6F) C, H, N.
1-[2,4-Dioxo-(1H,3H)-p yr im id in -1-yl]-2-(2,3-O,O-d iben -
zyl-2-bu ten -4-olid ylid en e)eth a n -2-ol (8). A suspension of
anhydrous uracil (70% excess with respect to the compound
7, 353 mg, 3.15 mmol) and (NH4)2SO4 (30 mg) in HMDS (10
mL) was heated under reflux for 3 h in argon atmosphere.
Evaporation of HMDS under reduced pressure gave an oily
product to which 4-(5,6-epoxy)-2,3-O,O-dibenzyl-L-ascorbic acid
(7) (332 mg, 0.92 mmol) dissolved in anhydrous acetonitrile
(5 mL) was added. After the reaction mixture was cooled (-20
°C), trimethylsilyl trifluoromethanesulfonate (0.5 mL, 2.76
mmol) was added dropwise and the mixture was heated at 55-
70 °C for 12 h. The reaction was terminated by diluting with
CH2Cl2 (40 mL) and adding ice-cold NaHCO3 solution (10 mL).
The mixture was extracted several times with CH2Cl2. The
organic phase was dried over Na2SO4 and evaporated. Silica
gel column chromatography of the oily residue afforded 8 (303
mg, 73%, mp 113-116 °C): UV (methanol) λmax 206 (log ꢀ 2.88),
λmax 250 (log ꢀ 2.61); 13C NMR (CDCl3) δ 151.46 (C-2), 164.19
(C-4), 100.59 (C-5), 147.14 (C-6), 169.49 (C-1′), 157.79 (C-2′),
121.01 (C-3′), 75.86 (C-4′), 65.45 (C-5′), 51.21 (C-6′), 73.82,
72.97 (CH2Ph), 136.15, 129.01-128.08 (C6H5); MS m/z 450
(M+•). Anal. (C24H22N2O7) C, H, N.
1-[2,4-Dioxo-(5-tr iflu or om eth yl)-(1H,3H)-p yr im id in -1-
yl]-2-(2,3-d ih yd r oxy-2-bu ten -4-olid ylid en e)eth a n e (17).
Compound 13 (250 mg, 0.5 mmol) was treated according to a
procedure that was analogous to that for the preparation of
compounds 14 and 15 to give 17 (16 mg, 9.1%, mp 292-294
°C): 13C NMR (DMSO-d6) δ 150.01 (C-2), 159.40 (C-4), 102.21
(C-5, J ) 32.04 Hz), 147.28 (C-6), 122.71 (CF3, J ) 269.06 Hz),
164.75 (C-1′), 121.43 (C-2′), 143.24 (C-3′), 144.27 (C-4′), 100.11
(C-5′), 43.86 (C-6′); MS m/z 320.20 (M+•). Anal. (C11H7N2O6F3)
C, H, N.
X-r a y Deter m in a tion . The single crystal of 13 suitable for
X-ray structure analysis was obtained by growth at room
temperature of a very dilute solution of ethanol. The intensities
were measured on a Philips PW1100 diffractometer upgraded
by Stoe20 using Mo KR radiation (λ ) 0.71073 Å) at 20 °C with
the ω scan mode and corrected only for Lorentz polarization
factor. During the data collection crystal decomposition of
21.3% was observed. The structure was solved by direct
methods and refined by full-matric least-squares on F2, using
SHELXL9321 program package. The hydrogen atoms were
either located in a difference Fourier synthesis or generated
and allowed to ride at a fixed distance from the attached atoms
except for the hydrogen atom bonded to the pyrimidine
nitrogen which was refined. A weighting scheme w ) 1/[σ2Fo
1-[2,4-Dioxo-5-flu or o-(1H ,3H )-p yr im id in -1-yl]-2-(2,3-
O,O-d ib en zyl-2-b u t en -4-olid ylid en e)et h a n -2-ol (9). 5-
Fluorouracil (60% excess, 205 mg, 1.58 mmol) was treated
according to the procedure analogous to that for the prepara-
tion of compound 8 to give 9 (169 mg, 57%, mp 85-90 °C):
UV (methanol) λmax 208 (log ꢀ 4.39), λmax 240 (log ꢀ 4.09); 13C
NMR (CDCl3) δ 149.93 (C-2), 157.62 (C-4, J ) 58.1 Hz), 139.86
(C-5, J ) 236.7 Hz), 127.85 (C-6, J ) 32.49 Hz), 169.78 (C-1′),
156.85 (C-2′), 121.25 (C-3′), 75.93 (C-4′), 66.60 (C-5′), 51.47 (C-
+ 0.0525P2], where P ) (Fo + 2Fc2)/3, was assumed for all
2
observations. The final difference map contained no significant
features.

Synthesis/ Antitumor Activities of Novel Pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4811
(7) Kuribayashi, N.; Sakagami, H.; Sakagami, T.; Niimi E.; Shioka-
wa, D.; Ikekita, M.; Takeda, M.; Tanuma, S. Induction of DNA
Fragmentation in Human Myelogenous Leukemic Cell Lines By
Sodium 5,6-Benzylidene-L-Ascorbate and Its Related Com-
pounds. Anticancer Res. 1994, 14, 969-976.
(8) Balzarini, J .; Bohman, C.; De Clercq, E. Differential Mechanism
of Cytostatic Effect of (E)-5-(2-Bromovinyl)-2′-deoxyuridine,
9-(1,3-Dihydroxy-2-propoxymethyl)guanine and Other Antiher-
petic Drugs on Tumor Cells Transfected by the Thymidine
Kinase Gene of Herpes Simplex Virus Type 1 or Type 2. J . Biol.
Chem. 1993, 268, 6332-6337.
(9) Balzarini, J .; Bohman, C.; Walker, R. T.; De Clercq, E. Compara-
tive Cytostatic Activity of Different Antiherpetic Drugs against
Herpes Simplex Virus Thymidine Kinase Gene-Transfected
Tumor Cells. Mol. Pharmacol. 1994, 45, 1253-1258.
(10) Balzarini, J .; Morin, K. W.; Knaus, E. E.; Wiebe, L. I.; De Clercq,
E. Novel (E)-5-(2-Iodovinyl)-2′-deoxyuridine Derivatives as Po-
tential Cytostatic Agents against Herpes Simplex Virus Thy-
midine Kinase Gene Transfected Tumors. Gene Ther. 1995, 2,
317-322.
(11) Raic´-Malic´, S.; Hergold-Brundic´, A.; Nagl, A.; Grdisˇa, M.; Pavelic´,
K.; De Clercq, E.; Mintas, M. Novel Pyrimidine and Purine
Derivatives of L-Ascorbic Acid: Synthesis and Biological Evalu-
ation. J . Med. Chem. 1999, 42, 2673-2678.
Crystal data for 13: Mr ) 500.42, space group P-1; a )
9.299(4), b ) 11.760(3), c ) 12.277(4) Å; R ) 109.31(2), â )
97.36(3), γ ) 107.49(3)°; V ) 1169.2(7) Å3, Z ) 2, F(000) )
516, dx ) 1.421 g cm-3; µ(Mo KR) ) 0.118 mm-1, S ) 0.851;
R/Rw ) 0.0440/0.1007 for 328 parameters and 1560 reflections
with I g2σ(I), R/Rw ) 0.1357/0.2164 for all 5609 independent
reflections measured in the range 2.10°-2Θ-27.96°.
Ma ter ia ls for Biologica l Tests. Cell cu ltu r in g: Antitu-
mor activities against L1210/0 (murine leukemia), FM3A/0
(murine mammary carcinoma), Molt4/C8 (human T-lympho-
blast), and CEM/0 (human T-lymphoblast) cell lines as well
as inhibition of the proliferation of human embryonic cell
(HEL) and murine embryo (MEF) fibroblasts were measured
essentially as originally described for the mouse leukemia/
L1210 cell lines.22
An tivir a l a ctivity a ssa ys: Antiviral activities against
thymidine kinase-positive (TK+) and -negative (TK-) strains
of varicella-zoster virus (VZV), cytomegalovirus (CMV),23 and
human immunodeficiency virus (HIV)24 were determined as
described previously.23,24
Ack n ow led gm en t. Support for this study by the
Ministry of Science of Croatia (Project No. 175.003) is
gratefully acknowledged. We thank Lizette van Berck-
elaer for excellent technical assistance in performing the
antitumor cell activity assays, as well as Ann Absillis,
Anita Van Lierde, Frieda De Meyer, Anita Camps, and
Lies Vandenheurck for excellent technical assistance in
performing the antiviral activity assays. We also thank
Dr. Vanja Vela (Pliva Research Institute) for providing
some starting materials and Dr. Oliver Zerbe (Depart-
ment of ETH, Zu¨rich) for recording and helpful discus-
sions concerning one- and two-dimensional NMR spec-
tra.
(12) Von Dallacker, F.; Sanders, J . Derivative der L-Ascorbinsa¨ure,
1, Darstellung und Eigenschaften der O2,O3-Ethandiyl- und der
O2,O3-Dibenzyl-L-ascorbinsa¨uren. Chem. Ztg. 1985, 109, 197-
202.
(13) Von Dallacker, F.; Sanders, J . Derivative der L-Ascorbinsa¨ure,
2, Darstellung von Deoxy-L-ascorbinsa¨uren. Chem. Ztg. 1985,
109, 277-280.
(14) Hsu, L. Y.; Wise, D. S.; Shannon, W. M.; Drach, J . C.; Townsend,
L. B. Synthesis of C-6 Pyrimidine Acyclic Nucleoside Analogues
as Potential Antiviral Agents. Nucleosides Nucleotides 1994, 13,
563-584.
(15) Ogilvie, K. K.; Hamilton, R. G.; Gillen, M. F.; Radatus, B. K.;
Smith, K. O.; Galloway, K. S. Uracil Analogues of the Acyclo-
nucleoside 9-[[2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl]gua-
nine (BIOLF-62). Can. J . Chem. 1984, 62, 16-21.
(16) Vikic´-Topic´, D.; Loncˇar, L.; Lukic´, T.; Mintas, M. Photochemical
Synthesis and NMR Analysis of Novel Regiospecifically Tri-
fluoromethyl-substituted Dibenzosemibullvalene. J . Fluorine
Chem. 1995, 74, 159-164.
(17) J ohanson, C. K. ORTEPII; Report ORNL-5138; Oak Ridge
National Laboratory: Oak Ridge, TN, 1976.
(18) Stek, A. L. The EUCLID Package. Computational Crystal-
lography; Clarendon Press: Oxford, 1982; p 528.
(19) Otocˇan, K.; Mintas, M.; Kastner, F.; Mannschreck, A.; Golen, J .
A.; Williard P. G. Regiospecific Synthesis and Structural Study
of Some Trifluoromethyl Substituted Dibenzosemibullvalenes.
Monatsh. Chem. 1992, 123, 1193-1205.
Su p p or tin g In for m a tion Ava ila ble: Tables of atomic
coordinates and equivalent isotropic displacement parameters,
bond lengths and angles, anisotropic displacement parameters,
hydrogen coordinates, and isotropic displacement parameters.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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