Novel pyrimidine and purine derivatives of L-ascorbic acid: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm991017z

The novel pyrimidine derivatives 1-6 of 2,3-dibenzyl-4,5-didehydro-5,6- dideoxy-L-ascorbic acid were synthesized by the condensation of pyrimidine bases with 5,6-diacetyl-2,3-dibenzyl-L-ascorbic acid (DDA). Both N-9 (7) and N-7 (8) regioisomers were obtai

Full text of DOI:10.1021/jm991017z

J . Med. Chem. 1999, 42, 2673-2678
2673
Novel P yr im id in e a n d P u r in e Der iva tives of L-Ascor bic Acid : Syn th esis a n d
Biologica l Eva lu a tion
Silvana Raic´-Malic´,*,† Antonija Hergold-Brundic´, Ante Nagl,| Mira Grdisˇa,‡ Kresˇimir Pavelic´,‡
Erik De Clercq,§ and Mladen Mintas†
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, Marulic´ev trg 20, P.O. Box 177,
HR-10000 Zagreb, Croatia, Division of Molecular Medicine, Ru[er Bosˇkovic´ Institute, Bijenicˇka cesta 54, P.O. Box 1016,
HR-10001 Zagreb, Croatia, Laboratory of General and Inorganic Chemistry, Faculty of Science, and Laboratory of General and
Inorganic Chemistry, Faculty of Textile Technology, University of Zagreb, Croatia, and Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received February 8, 1999
The novel pyrimidine derivatives 1-6 of 2,3-dibenzyl-4,5-didehydro-5,6-dideoxy-L-ascorbic acid
were synthesized by the condensation of pyrimidine bases with 5,6-diacetyl-2,3-dibenzyl-L-
ascorbic acid (DDA). Both N-9 (7) and N-7 (8) regioisomers were obtained in the reaction of
6-chloropurine with 5-acetyl-6-bromo-2,3-dibenzyl-L-ascorbic acid (ABDA), while the reaction
of 6-(N-pyrrolyl)purine with ABDA afforded exclusively the N-9 isomer 9. Structures of all
1
newly prepared compounds were deduced from the chemical shifts in H and 13C NMR spectra,
as well as connectivities in 2D homo- and heteronuclear correlation spectra. An unambiguous
proof of the structure and conformation of 7 was obtained by X-ray crystallographic analysis.
Compounds 1-9 were found to exert cytostatic activities against malignant cell lines: pancreatic
carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (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 cytomegalovirus (CMV). The compound 6 containing
a trifluoromethyl-substituted uracil ring exhibited marked antitumor activity. The N-7-
substituted purine regioisomer 8 had greater inhibitory effects on the murine L1210/0 and
human CEM/0 cell lines than the N-9 isomer 7. Compound 9 with the 6-purine-substituted
pyrrolo moiety had a more pronounced selective cytostatic activity against human (Molt4/C8
and CEM/0) cell lines than murine (L1210/0 and FM3A/0) and human (MiaPaCa2, MCF7, HeLa,
and Hep2) tumor cell lines and normal fibroblasts (Hef522). The compound 6 exhibited the
most potent antiviral activities against TK+VZV, TK-VZV, and CMV, albeit at concentrations
that were only slightly lower than the cytotoxic concentrations.
In tr od u ction
L-Ascorbic acid and its derivatives have been of
multifold biological and pharmacological interest.1-4
Thus, some derivatives of L-ascorbic acid, e.g., 6-bromo-,
6-amino-, and N,N-dimethyl-6-amino-6-deoxy-L-ascorbic
acid, have been found to inhibit the growth of certain
human malignant tumor cells lines: cervical carcinoma
(HeLa), laryngeal carcinoma (Hep2), and pancreatic
carcinoma (MiaPaCa2).5 Furthermore, several nucleo-
F igu r e 1. Pyrimidine (1-6) and purine (7-9) derivatives of
sides containing a 5-substituted pyrimidine moiety have
been shown to inhibit growth of the murine mammary
carcinoma (FM3A TK-/HSV 1 TK+) cells transformed
with the herpes simplex virus type 1 (HSV-1) TK
gene.6-9 Searching for compounds related to these
classes of biologically and pharmacologically active
molecules, we have prepared new types of molecules
containing pyrimidine (1-6) or purine (7-9) moieties
connected via an acyclic chain to 2,3-dibenzyl-4,5-
didehydro-5,6-dideoxy-L-ascorbic acid (Figure 1). The
2,3-dibenzyl-4,5-didehydro-5,6-dideoxy-L-ascorbic acid.
synthesis, 1H NMR, and X-ray crystal structure, as well
as biological studies, are reported in this paper.
Ch em istr y
Compounds 1-6 were prepared by the general Vor-
bru¨ggen method10,11 involving silylation of the uracil and
its 5-substituted derivatives with 1,1,1,3,3,3-hexameth-
yldisilazane (HMDS) in the presence of (NH4)2SO4 and
subsequent reaction of the intermediates thus obtained
with 5,6-diacetyl-2,3-dibenzyl-L-ascorbic acid (DDA)12,13
and trimethylsilyl triflate as Friedel-Crafts catalyst
(Scheme 1).
* To whom correspondence should be addressed.
† Faculty of Chemical Engineering and Technology.
Faculty of Science.
| Faculty of Textile Technology.
The compounds 7-9 were obtained by an alternative
route which involved direct condensation of 6-chloro-
‡ Ru[er Bosˇkovic´ Institute.
§ Katholieke Universiteit Leuven.
10.1021/jm991017z CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/15/1999

2674 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Raic´-Malic´ et al.
OCH2
Ta ble 1. Chemical Shifts (δ, ppm)a and H-H Coupling Constants (J , Hz)b in 1H NMR Spectra for Compounds 1-9
H-2
compd
H-6
H-8
NH
H-5′
H-6′
C6H5
1c
7.38-7.17 (1H)
9.17 (s, 1H)
5.44 (1H)
J ) 7.69 (t)
5.43 (1H)
J ) 7.69 (t)
5.43 (1H)
7.69 (t)
5.45 (1H)
J ) 7.69 (t)
5.44 (1H)
J ) 7.69 (t)
5.59 (1H)
J ) 6.39 (t)
5.53 (1H)
J ) 7.56 (t)
5.50 (1H)
J ) 7.57 (t)
5.56 (1H)
J ) 7.57 (t)
4.52 (2H)
J ) 7.69 (d)
4.52 (2H)
J ) 7.69 (d)
4.56 (2H)
J ) 7.69 (d)
4.57 (2H)
J ) 7.69 (d)
4.57 (2H)
J ) 7.70 (d)
4.63 (2H)
J ) 6.23 (d)
5.12 (2H)
J ) 7.69 (d)
5.31 (2H)
7.38-7.17
(10H, m)
7.40-7.21
(10H, m)
7.42-7.23
(10H, m)
7.42-7.22
(10H, m)
7.41-7.23
(10H, m)
7.43-7.35
(10H, m)
7.38-7.19
(10H, m)
7.39-7.19
(10H, m)
7.38-7.18
(10H, m)
5.21 (s, 2H)
5.18 (s, 2H)
5.23 (s, 2H)
5.21 (s, 2H)
5.24 (s, 2H)
5.22 (s, 2H)
5.25 (s, 2H)
5.23 (s, 2H)
5.24 (s, 2H)
5.22 (s, 2H)
5.32 (s, 2H)
5.16 (s, 2H)
5.22 (s, 4H)
2
7.40-7.21 (1H)
7.42-7.23 (1H)
7.54 (s, 1H)
9.79 (s, 1H)
9.23 (s, 1H)
9.33 (s, 1H)
9.50 (s, 1H)
11.87 (s, 1H)
3
4
5
7.63 (s, 1H)
6
8.41 (s, 1H)
7
8.75 (s, 1H)
8.15 (s, 1H)
8.30 (s, 1H)
8.88 (s, 1H)
8.69 (s, 1H)
8.03 (s, 1H)
8
5.22 (s, 4H)
5.21 (s, 4H)
J ) 7.18 (d)
5.10 (2H)
J ) 7.44 (d)
9d
a
CDCl3, chemical shifts referred to TMS. Multiplicity of coupling and number of protons are given in parentheses: s, singlet; d, doublet;
t, triplet; m, complex multiplet. Digital resolution ( 0.28 Hz. c Chemical shift for H-5: 5.69 ppm (d, 1H), J ) 7.69 Hz. Chemical shifts
for pyrrolo moiety H-2′′,5′′: 8.31 ppm (2H, t), J ) 2.05 Hz and H-3′′,4′′: 6.42 ppm (2H, t), J ) 2.05 Hz.
b
d
Sch em e 1a
analysis of chemical shifts and H-H coupling constants
in 1H and 13C NMR spectra (see Table 1 and Experi-
mental Section). These data, as well as connectivities
1
in heteronuclear H and 13C NMR spectra, showed that
the lactone ring in the condensation reaction of nucleo-
side bases was preserved, whereas elimination of water
in the 4,5-position occurred. This was also corroborated
with IR spectra which showed a strong absorption band
in the range 1764-1777 cm-1. This band could be
ascribed to the CdO stretching vibration of the lactone
ring. Comparison of the NMR data from Table 1
indicated that strong electron-withdrawing substituents
(F, Cl, Br, I, and CF3) at position C-5 in the pyrimidine
ring caused deshielding of the protons H-6 and NH.
The chemical shifts pattern of pyrimidine, purine, and
lactone moieties in both 1H and 13C NMR spectra are
consistent with those observed for related purine,15,16
pyrimidine,16 and ascorbic acid derivatives.17 Structures
of N-9 (7) and N-7 (8) regioisomers were also confirmed
a
Reagents and conditions: (i) HMDS, (NH4)2SO4/argon atmo-
sphere/reflux/3 h; then trimethylsilyl triflate/dry acetonitrile/
55-70 °C/12 h.
Sch em e 2a
1
by H and 13C NMR. These observations are in accord
with those found earlier.18,19 The most pronounced
differences in N-9 and N-7 isomers were found for the
proton signals H-2 and H-8 of the purine skeleton. Thus,
the proton H-8 in the N-7 isomer (8) was shifted
downfield (8.88 ppm) relative to the corresponding one
(8.15 ppm) in the N-9-substituted molecule (7). On the
contrary, the signal H-2 was shifted upfield (8.30 ppm)
in the N-7 derivative relative to the H-2 signal (8.75
ppm) in the N-9 derivative. Furthermore, the equivalent
N-methylene protons were more deshielded (5.31 ppm)
than the corresponding ones in the N-9 isomer (5.12
ppm). Vicinal coupling constant of protons in the side
chain was greater in the N-9 than in the N-7 regio-
isomer.
a
Reagent and conditions: (i) triethylamine/dry DMF/70 °C/11
h.
purine with 5-acetyl-6-bromo-2,3-dibenzyl-L-ascorbic acid
(ABDA)12,13 using a modified procedure to that for the
preparation of purine and pyrimidine trihydroxyacyclo-
nucleosides14 (Scheme 2). This reaction afforded both
N-9 and N-7 isomers in a ratio of 4:1. On the contrary,
reaction of 6-(N-pyrrolyl)purine with ABDA gave only
the N-9 regioisomer.
X-r a y Cr ysta l Str u ctu r e An a lysis. Stereostructure
of compound 7 was determined by its X-ray crystal-
lographic analysis. Solid-state conformation with atom
numbering of 7 is displayed in Figure 2. The skeleton
of the molecule consists of a planar lactone ring con-
nected with the purine moiety by the acyclic chain which
contains a double bond between atoms C4 and C5. That
bond is conjugated to the lactone ring. The plane defined
by the atoms O2-O3-O4-C5 directly connected to the
1H a n d 13C NMR Stu d ies. The structures of the
novel compounds were determined on the basis of

Pyrimidine and Purine Derivatives of L-Ascorbic Acid
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2675
Ta ble 2. Inhibitory Effects of Compounds 1-9 on the Growth of Malignant Tumor Cell Lines and Normal Fibroblasts (Hef522)
a
IC50 (µM)
compd
L1210/0
FM3A/0
Molt4/C8
CEM/0
MiaPaCa2
MCF7
HeLa
Hep2
Hef522
1
2
3
4
5
6
7
8
9
12.9
5.1
12.2
7.5
7.5
2.0
11.6
4.1
110
17.7
3.4
16.9
17.1
22.6
3.6
4.8
10.9
6.1
3.9
3.9
0.9
14.8
6.8
13.0
15.1
6.0
3.5
3.3
1.6
15.9
4.4
30
40
40
80
80
30
60
40
20
60
60
40
70
50
40
40
30
30
20
30
30
70
40
20
20
90
10
40
60
60
50
40
30
30
40
60
50
20
40
40
16.4
11.4
g200
>100
>100
3.9
5.8
>100
>100
>100
a
50% inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation by 50%.
containing a trifluoromethyl group substituted at posi-
tion C-5 of the uracil ring, showed the most significant
cytostatic activity, particularly against human Molt4/
C8 cells (IC50 ) 0.9 µM, Table 2). The N-9-substituted
purine regioisomer 7 showed slight inhibitory activity
against malignant murine and human cells, whereas its
N-7 isomer 8 displayed more pronounced inhibition on
the majority of examined cell lines. The most pro-
nounced differences in inhibitory effects of the N-9 and
N-7 regioisomers 7 and 8 were observed for murine
L1210/0 and human CEM/0 cell proliferation (for 7: IC50
) 11.6 and 15.9 µM, as compared to 8: IC50 ) 4.1 and
4.4 µM, Table 2). Compound 9, containing a pyrrolyl
moiety at position C-6 of the purine ring, exhibited
marked selectivity in cytostatic activity: This compound
inhibited specifically the human Molt4/C8 (IC50 ) 3.9
µM) and CEM/0 (IC50 ) 5.8 µM) cells but not the other
ones. It was also the most active antitumor agent from
the purine series (7-9).
In comparison with ascorbic acid, all derivatives
exhibited a better inhibitory effect on the growth of
human tumor cell lines. As was shown earlier2,5 ascorbic
acid inhibited the growth of human tumor cell lines
(about 25%) at a much higher concentration (2 × 10-3
M). At the concentrations used in our experiments
(10-6-10-4 M), ascorbic acid did not inhibit the growth
of examined cell lines (results are not shown).
F igu r e 2. ORTEP20,21 view with the labeling scheme for 7.
Displacement ellipsoids are drawn at the 20% probability
level.
An tivir a l Activity. The ability of novel compounds
1-9 to inhibit thymidine kinase-positive (TK+) and
thymidine kinase-deficient (TK-) strains of varicella-
zoster virus (VZV) was compared with the antiviral
activity of acyclovir (ACV) and (E)-5-(2-bromovinyl)-2′-
deoxyuridine (BVDU). Inhibition of cytomegalovirus
(CMV) by the test compounds was compared with
9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG) and
(S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cyto-
sine (HPMPC). The results presented in Table 3 show
that compound 6 containing a trifluoromethyl group at
C-5 of the uracil ring displayed more potent activity
than the other compounds. Moreover, this compound
was 6-10-fold more active than compound 1 with an
unsubstituted uracil moiety. Also, compound 6 exhibited
more potent activity against TK+VZV/YS and TK-VZV
strains than ACV. Compound 6 also showed greater
activity against cytomegalovirus than DHPG. Com-
pounds 3-5 displayed almost identical activity against
VZV and CMV. The N-7 isomer was more active against
TK-VZV (YS/R) than ACV and BVDU, while the N-9
derivative did not show appreciable antiviral activity.
Compound 9, which carries the 6-(N-pyrrolyl)purine
lactone ring is coplanar with the lactone ring. Such
coplanarity is also observed for the plane defined by the
adjacent atoms of the purine and benzene rings. Con-
formation of this molecule was determined by the
dihedral angles between the planes of the lactone and
other rings: C2-O3-C7-C8 [-171,3(2)°], C3-O4-
C14-C15 [171,3(2)°], and C4-C5-C6-N9 [140,8(3)°].
Biologica l Resu lts a n d Discu ssion
An titu m or Activity. Compounds 1-9 were evalu-
ated for their activity against malignant tumor cell
lines: pancreatic carcinoma (MiaPaCa2), breast carci-
noma (MCF7), cervical carcinoma (HeLa), laryngeal
carcinoma (Hep2), murine leukemia (L1210/0), murine
mammary carcinoma (FM3A), and human T-lympho-
cytes (Molt4/C8 and CEM/0).
Inhibitory effects of compounds 1-9 differ from one
type of tumor cell to another. Better growth inhibition
was achieved for the L1210/0, FM3A, Molt4/C8, and
CEM/0 cell lines in comparison with the malignant
MiaPaCa2, MCF7, HeLa, and Hep2 cells. The difference
in IC50 was about 10 times (Table 2). Compound 6,

2676 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Raic´-Malic´ et al.
Ta ble 3. Cytotoxicities and Antiviral Activities of Compounds 1-9 against Varicella-Zoster Virus (YS, OKA, 07/1, and YS/R Strains)
and Cytomegalovirus (AD-169 and Davis Strains) in Human Embryonic Lung Cells
a
antiviral activity IC50 (µM)
TK+VZV
OKA
TK-VZV
CMV
cytotoxicity (µM)
c
compd
YS
3.4
07/1
YS/R
AD-169
Davis
cell morph (MCC)b
cell growth (CC50)
1
2
3
4
5
6
7
8
3.0
2.6
1.3
1.3
1.3
0.3
>5
5.0
3.4
3.5
>2
1.8
2.7
1.0
1.0
1.0
0.3
>5
3.8
0.6
12
3.5
3.5
3.5
3.5
>2
>2
>2
0.4
>5
>5
>0.5
NDd
NDd
5
20
20
5
5
5
2
20
20
g20
>50
>50
>50
>50
5
9
3
3
3
1
8
20
3.8
>2
>2
>2
>2
>2
1.4
>2
>2
0.5
0.5
>5
>0.5
>5
>5
>5
>5
>5
9
1.5
1.2
2.8
17
>0.5
NDd
NDd
1
4
ACV
BVDU
DHPG
HPMPC
0.78
0.005
NDd
NDd
0.16
0.001
NDd
NDd
>100
>100
>50
NDd
>50
NDd
NDd
>50
NDd
NDd
0.11
1
a
b
Inhibitory concentration required to reduce virus plaque formation by 50%. Virus input was 20 plaque forming units (PFU). Minimum
cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. c Cytotoxic concentration required to reduce
d
cell growth by 50%. ND, not determined.
moiety, was 20-fold more potent against TK-VZV
The electron impact mass spectra were recorded with an
EXTREL FT MS 2001 instrument with ionizing energy of 70
(YS/R) than ACV and BVDU.
eV. The IR spectra were recorded on a Nicolet-Magna IR 760
spectrometer and UV spectra on a Hewlett-Packard 8452
spectrometer. Elemental analyses were performed by the
Comparison of the cytotoxicity of 1-9 (Table 3)
indicates that all compounds showed relatively low
selectivity in their antiviral potencies.
Central Analytical Service, Ru]er Bosˇkovic´ Institute, Zagreb.
Results were within (0.4% of the theoretical values and are
indicated by symbols of the elements. The 1H 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 CDCl3 and measured at 21 °C in 5-mm NMR
tubes. The 1H and 13C chemical shift values are in ppm,
Con clu sion s
New types of compounds consisting of pyrimidine
(1-6) or purine (7-9) moieties connected by an acyclic
chain to 2,3-dibenzyl-4,5-didehydro-5,6-dideoxy-L-ascor-
bic acid were prepared and evaluated for their anti-
tumor and antiviral activities.
1
referred to TMS. Digital resolution in H spectra was 0.28 Hz,
while in 13C spectra it was 0.65 Hz/point.
1-(Ur a cil-1-yl)-2-(2,3-O,O-d ib en zyl-2-b u t en -4-olid yli-
d en e)eth a n e (1). A suspension of anhydrous uracil (60%
excess compared with DDA, 176 mg, 1.57 mmol) and (NH4)2-
SO4 (15 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 was added DDA (433
mg, 0.98 mmol) dissolved in anhydrous acetonitrile (10 mL).
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 (40 mL). The mixture
was extracted several times with CH2Cl2. The organic phase
was dried over Na2SO4 and evaporated. Silica gel column
chromatography (CH2Cl2:MeOH - 40:1) of the oily residue
afforded 1 (179 mg, 42%, mp 95-97 °C): IR (KBr, ν/cm-1) 3033
(dC(5′)-H), 2821-2970 (C-H), 1774 (CdO), 1680 (CdC),
1654-1680 (CdO); UV (methanol) λmax 208 (log ꢀ 4.37), λmax
270 (log ꢀ 4.30); 13C NMR (CDCl3) δ 150.73 (C-2), 163.67 (C-
4), 102.69 (C-5), 144.08 (C-6), 163.77 (C-1′), 147.67 (C-2′),
123.60 (C-3′), 144.98 (C-4′), 100.98 (C-5′), 42.49 (C-6′), 73.93,
73.32 (C-7′,7′′), 135.51, 135.07 (C-8′,8′′), 128.84-127.74 (C-9′,-
10′,11′,9′′,10′′,11′′); MS m/z 432 (M+•). Anal. (C24H20N2O6) C,
H, N.
The stereostructure of compound 7 was unambigu-
ously demonstrated by its X-ray structural analysis. The
compounds 1-9 exhibited greater inhibitory activities
against L1210/0, FM3A, Molt4/C8, and CEM/0 cells
than the MiaPaCa2, MCF7, HeLa, and Hep2 ones.
Compound 6, containing a trifluoromethyl-substituted
uracil ring, was found to be the most active against
human Molt4/C8 cells. The most pronounced differences
in inhibitory effects of the N-9- and N-7-substituted
purine regioisomers 7 and 8 were observed for murine
L1210/0 and human CEM/0 cell proliferation. Com-
pound 9 with the purine substituted at position 6 with
a pyrrolo moiety showed the most selective inhibitory
activity against human Molt4/C8 and CEM/0 cell pro-
liferation as compared to normal fibroblast Hef522 cell
proliferation. The most potent (yet relatively aspecific)
antiviral effects against TK+VZV, TK-VZV, and CMV
were found for compound 6.
Exp er im en ta l Section
Gen er a l Meth od s. All new compounds were fully charac-
terized by 1H and 13C NMR, electron impact mass spectra, and
elemental analysis (C, H, N). Melting points of compounds
were determined with a Kofler micro hot-stage apparatus
(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. Additional purification of compounds 1-9 by recrys-
tallization from ethanol afforded analytical samples of 1-9.
Acetonitrile was first refluxed several hours over P2O5, distilled
from P2O5, and finally refluxed over CaH2 and distilled.
1-(5-F lu or ou r a cil-1-yl)-2-(2,3-O,O-d iben zyl-2-b u t en -4-
olid ylid en e)eth a n e (2). 5-Fluorouracil (60% excess, 237 mg,
1.82 mmol) was treated according to the procedure analogous
to that for the preparation of compound 1, to give 2 (255 mg,
50%, mp 165-167 °C): IR (KBr, ν/cm-1) 3035 (dC(5′)-H),
2828-2970 (C-H), 1777 (CdO), 1693 (CdC), 1659-1693
(CdO); UV (methanol) λmax 208 (log ꢀ 4.23), λmax 276 (log ꢀ 4.09);
13C NMR (CDCl3) δ 149.48 (C-2), 157.19 (C-4, J ) 25.5 Hz),
140.62 (C-5, J ) 240.2 Hz), 127.91 (C-6), 163.68 (C-1′), 147.56
(C-2′), 123.69 (C-3′), 145.35 (C-4′), 105.52 (C-5′), 42.61 (C-6′),
73.95, 73.36 (C-7′,7′′), 135.46, 135.01 (C-8′,8′′), 129.09-127.77
(C-9′,10′,11′,9′′,10′′,11′′); MS m/z 450 (M+•). Anal. (C24H19N2O6F)
C, H, N.

Pyrimidine and Purine Derivatives of L-Ascorbic Acid
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2677
1-(5-Ch lor ou r a cil-1-yl)-2-(2,3-O,O-d iben zyl-2-bu ten -4-
olid ylid en e)eth a n e (3). 5-Chlorouracil (60% excess, 266 mg,
1.82 mmol) was treated according to the procedure analogous
to that for the preparation of compound 1, to give 3 (312 mg,
59%, mp 168-170 °C): IR (KBr, ν/cm-1) 3033 (dC(5′)-H),
2820-2960 (C-H), 1768 (CdO), 1710 (CdC), 1627-1695
(CdO), 835 (C-Cl); UV (methanol) λmax 208 (log ꢀ 4.11), λmax
284 (log ꢀ 3.99); 13C NMR (CDCl3) δ 149.73 (C-2), 159.14
(C-4), 109.29 (C-5), 140.78 (C-6), 163.69 (C-1′), 147.56 (C-2′),
123.74 (C-3′), 145.39 (C-4′), 100.18 (C-5′), 42.84 (C-6′), 73.98,
73.41 (C-7′,7′′), 135.46, 135.00 (C-8′,8′′), 129.45-127.82 (C-9′,-
10′,11′,9′′,10′′,11′′); MS m/z 466 (M+•). Anal. (C24H19N2O6Cl) C,
H, N.
1-(5-Br om ou r a cil-1-yl)-2-(2,3-O,O-d ib en zyl-2-b u t en -4-
olid ylid en e)eth a n e (4). 5-Bromouracil (60% excess, 611 mg,
3.2 mmol) was treated by the procedure analogous to that for
the preparation of compound 1, to give 4 (518 mg, 50%, mp
181-183 °C): IR (KBr, ν/cm-1) 3033 (dC(5′)-H), 2853-2924
(C-H), 1774 (CdO), 1700 (CdC), 1649-1686 (CdO); UV
(methanol) λmax 208 (log ꢀ 4.50), λmax 284 (log ꢀ 4.31); 13C NMR
(CDCl3) δ 150.05 (C-2), 159.36 (C-4), 96.92 (C-5), 143.35 (C-
6), 163.69 (C-1′), 147.58 (C-2′), 123.71 (C-3′), 145.27 (C-4′),
100.31 (C-5′), 42.84 (C-6′), 73.96, 73.36 (C-7′,7′′), 135.46, 135.00
(C-8′,8′′), 128.86-127.79 (C-9′,10′,11′,9′′,10′′,11′′); MS m/z 511
(M+•). Anal. (C24H19N2O6Br) C, H, N.
1-(5-Iod ou r a cil-1-yl)-2-(2,3-O,O-d ib e n zyl-2-b u t e n -4-
olid ylid en e)eth a n e (5). 5-Iodouracil (60% excess, 762 mg,
3.3 mmol) was treated according to the procedure analogous
to that for the preparation of compound 1 to give 5 (473 mg,
42%, mp 171-173 °C): IR (KBr, ν/cm-1) 3028 (dC(5′)-H), 2834
(C-H), 1776 (CdO), 1697 (CdC), 1647 (CdO); UV (methanol)
λmax 208 (log ꢀ 4.39), λmax 290 (log ꢀ 4.23); 13C NMR (CDCl3) δ
150.33 (C-2), 160.36 (C-4), 68.27 (C-5), 148.35 (C-6), 163.69
(C-1′), 147.58 (C-2′), 123.68 (C-3′), 145.15 (C-4′), 100.46 (C-5′),
42.78 (C-6′), 73.95, 73.35 (C-7′,7′′), 135.44, 135.00 (C-8′,8′′),
128.85-127.77 (C-9′,10′,11′,9′′,10′′,11′′); MS m/z 558 (M+•).
Anal. (C24H19N2O6I) C, H, N.
8: IR (KBr, ν/cm-1) 3065 (C-8-H), 3033 (dC(5′)-H), 2957
(C-H), 1775 (CdO), 1697 (CdC); UV (methanol) λmax 208 (log
ꢀ 4.50), λmax 264 (log ꢀ 4.20); 13C NMR (CDCl3) δ 152.62 (C-2),
142.95 (C-4), 122.19 (C-5), 149.16 (C-6), 148.93 (C-8), 163.37
(C-1′), 148.28 (C-2′), 123.80 (C-3′), 145.01 (C-4′), 100.38 (C-5′),
41.45 (C-6′), 73.93, 73.46 (C-7′,7′′), 135.35, 134.92 (C-8′,8′′),
129.34-127.80 (C-9′,10′,11′,9′′,10′′,11′′); MS m/z 474 (M+•).
Anal. (C25H19N4O4Cl) C, H, N.
1-[6-(N-P yr r olyl)pu r in -9-yl]-2-(2,3-O,O-diben zyl-2-bu ten -
4-olid ylid en e)eth a n e (9). 6-(N-Pyrrolyl)purine (153.72 mg,
0.83 mmol) was dissolved in dry DMF (10 mL). To this mixture
were added triethylamine (0.2 mL, 1.43 mmol) and ABDA (383
mg, 0.83 mmol), and the reaction solution was stirred at 70
°C for 5 h. A further quantity of triethylamine (0.2 mL, 1.43
mmol) was added, and heating was continued at 70 °C for 6
h. The solution was filtered and evaporated in vacuo. The
residual oil was purified by column chromatography (CH2Cl2:
MeOH - 40:1) to give 9 (97 mg, 23%, mp 105-107 °C): IR
(KBr, ν/cm-1) 3028 (dC(5′)-H), 2929 (C-H), 1768 (CdO), 1690
(CdC); UV (methanol) λmax 206 (log ꢀ 4.62), λmax 290 (log ꢀ 4.64),
λmax 298 (log ꢀ 4.60); 13C NMR (CDCl3) δ 152.30 (C-2), 147.62
(C-4), 121.90 (C-5), 152.93 (C-6), 142.93 (C-8), 163.75 (C-1′),
147.62 (C-2′), 123.62 (C-3′), 144.73 (C-4′), 100.75 (C-5′), 37.89
(C-6′), 73.93, 73.32 (C-7′,7′′), 135.46 135.03 (C-8′,8′′), 129.12-
127.75 (C-9′,10′,11′,9′′,10′′,11′′); MS m/z 505 (M+•). Anal.
(C29H23N5O4) C, H, N.
X-r a y Deter m in a tion . Single crystals of 7 suitable for
X-ray structure analysis were prepared by growth under slow
evaporation at room temperature of a very dilute solution of
ethanol. The intensities were measured on a Philips PW1100
diffractometer upgraded by Stoe22 using Mo KR radiation
(λ ) 0.71073 Å) at 20 °C with the Θ - 2Θ scan mode and
corrected only for Lorentz polarization factor. During the data
collection crystal decomposition of 6.2% was observed. The
structure was solved by direct methods and refined on the
basis of observed reflections [I g 2σ(I)], using SHELX-8523 and
SHELX-9324 program packages. Hydrogen atoms were located
either in a difference Fourier synthesis or generated and
allowed to ride at a fixed distance from the attached carbon
1-[5-(Tr iflu or om eth yl)u r a cil-1-yl]-2-(2,3-O,O-d iben zyl-
2-bu ten -4-olid ylid en e)eth a n e (6). 5-(Trifluoromethyl)uracil
(60% excess, 500 mg, 2.78 mmol) was treated according to the
procedure analogous to that for the preparation of compound
atoms with
a fixed isotropic temperature factor for two
groupings according to the stereochemical rules for sp2- and
sp3-hybridized carbon atoms. A unit weight was assumed for
all observations. Final difference map contained no significant
features.
1, to give 6 (430 mg, 52%, mp 181-183 °C): IR (KBr, ν/cm-1
)
3032 (dC(5′)-H), 2851-2960 (C-H), 1764 (CdO), 1670
(CdC), 1655-1670 (CdO); UV (methanol) λmax 206 (log ꢀ 4.44),
λmax 270 (log ꢀ 4.39); 13C NMR (CDCl3) δ 150.35 (C-2), 159.81
(C-4), 102.41 (C-5, J ) 31.33 Hz), 147.76 (C-6, J ) 5.8 Hz),
122.14 (CF3, J ) 270.39 Hz), 164.05 (C-1′), 148.46 (C-2′), 123.07
(C-3′), 142.23 (C-4′), 104.05 (C-5′), 44.12 (C-6′), 74.19, 73.17
(C-7′,7′′), 136.02, 135.65 (C-8′,8′′), 129.08-128.30 (C-9′,10′,-
11′,9′′,10′′,11′′); MS m/z 500 (M+•). Anal. (C25H19N2O6F3) C, H,
N.
Crystal data for 7: Mr ) 474.89, space group P21/c, a )
9.653(1), b ) 24.442(4), c ) 6.692(2) Å, â ) 98.79(2)°, V )
2259.9(6) Å3, Z ) 4, F(000) ) 984, dx ) 1.396 g cm-3, µ(Mo
KR) ) 0.210 mm-1, S ) 0.956, R/Rw ) 0.0590/0.1378 for 309
parameters and 2522 reflections, and R/Rw ) 0.1378/0.2023
for all 6572 independent reflections measured in the range 2.13
- 2Θ - 30.00°.
1-(6-Ch lor op u r in -9-yl)-2-(2,3-O,O-d ib en zyl-2-b u t en -4-
olid ylid en e)eth a n e (7) a n d 1-(6-Ch lor op u r in -7-yl)-2-(2,3-
O,O-d iben zyl-2-bu ten -4-olid ylid en e)eth a n e (8). 6-Chloro-
purine (167 mg, 1.08 mmol) was dissolved in dry DMF (10 mL)
and triethylamine (0.2 mL, 1.43 mmol). ABDA (500 mg, 1.08
mmol) was then added, and the reaction mixture was stirred
at 70 °C for 5 h, the course of the reaction being monitored by
TLC. Triethylamine hydrobromide separated gradually. After
a further quantity of triethylamine (0.2 mL, 1.43 mmol) was
added, heating of the mixture was continued at 70 °C for 6 h.
The precipitated triethylamine hydrobromide was filtered off
and the solvent evaporated. The crude oily product was
purified by column chromatography (CH2Cl2:MeOH - 40:1)
providing colorless crystals of 7 (124 mg, 61%, mp 121-122
°C) and the oily compound 8 (34 mg, 17%).
7: IR (KBr, ν/cm-1) 3110 (C-8-H), 3030-3060 (dC(5′)-H),
2923 (C-H), 1775 (CdO), 1693 (CdC); UV (methanol) λmax 206
(log ꢀ 4.52), λmax 264 (log ꢀ 4.23); 13C NMR (CDCl3) δ 152.10
(C-2), 147.45 (C-4), 121.10 (C-5), 151.51 (C-6), 144.89 (C-8),
163.55 (C-1′), 151.11 (C-2′), 123.74 (C-3′), 145.12 (C-4′), 99.91
(C-5′), 38.23 (C-6′), 73.92, 73.36 (C-7′,7′′), 135.41, 134.97
(C-8′,8′′), 129.06-127.75 (C-9′,10′,11′,9′′,10′′,11′′); MS m/z 474
(M+•). Anal. (C25H19N4O4Cl) C, H, N.
Ma ter ia ls for Biologica l Tests. Dulbecco’s modified Ea-
gle’s medium (DMEM) and fetal bovine serum (FBS) were
purchased from Life Techology (Paisley, U.K.). 3-(4,5-Dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
obtained from Sigma (St. Louis, MO). All other chemicals were
of analytical grade (Kemika, Zagreb, Croatia).
Cell Cu ltu r in g. The human cell lines pancreatic carcinoma
(MiaPaCa2), cervical carcinoma (HeLa), laryngeal carcinoma
(Hep2), and breast carcinoma (MCF7) and the normal fibro-
blasts (Hef522) were cultured in DMEM supplemented with
10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/
mL streptomycin in a humidified atmosphere with 5% CO2 at
37 °C. All cells were grown as a monolayer culture. The cells
were plated in 96-microwell plates at a concentration of 1.5 ×
104/mL (MiaPaCa2, HeLa, Hep2, and MCF7) and 2 × 104/mL
(Hef522). Twenty-four hours later, the test compounds were
added and the cells were treated for an additional 72 h. Control
cells were grown under the same conditions, but without
addition of test compounds. The number of MiaPaCa2, HeLa,
Hep2, MCF7, and Hef522 cells was determined by the MTT
test, and percentage of growth was calculated. Each number
was the mean from three parallel samples in three individual
experiments. The variations between the experiments were

2678 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Raic´-Malic´ et al.
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less than 10%. The results are expressed as IC50. The com-
pounds were dissolved in DMSO at a concentration of 10-1
M
and diluted with DMEM to concentrations between 10-6 and
10-4 M. The concentration of DMSO was less than 0.1%, and
at that concentration it did not affect cell growth.
Antitumor activity against L1210/0 (murine leukemia),
FM3A/0 (murine mammary carcinoma), Molt4/C8 (human
T-lymphoblast), and CEM/0 (human T-lymphoblast) cell lines
was measured essentially as originally described for the mouse
leukemia/L1210 cell lines.25
An tivir a l Activity Assa ys. Antiviral activity against thy-
midine kinase-positive (TK+) and -negative (TK-) strains of
varicella-zoster virus (VZV) and against cytomegalovirus
(CMV) was determined as described previously.26
Ack n ow led gm en t. Support for this study by the
Ministry of Science of Croatia (Project No. 175.003) and
“Pliva Research Institute” is gratefully acknowledged.
We thank Lizette van Berckelaer for excellent technical
assistance with the antitumor cell activity assays, as
well as Anita Van Lierde, Frieda De Meyer, Anita
Camps, and Lies Vandenheurck for excellent technical
assistance with the antiviral activity assays.
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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