Synthesis and antiviral and cytostatic evaluations of the new c-5 substituted pyrimidine and furo[2,3-d]pyrimidine 4′,5′-didehydro-L- ascorbic acid derivatives

Article abstract of DOI:10.1021/jm070324z

The novel C-5 alkynyl substituted pyrimidine (1-11) and furo[2,3-d]pyrimidine derivatives (12-22) of L-ascorbic acid were synthesized by coupling of 5-iodouracil-4′,5′-didehydro-5′,6′-dideoxy- L-ascorbic acid with terminal alkynes by using Sonogashira cro

Full text of DOI:10.1021/jm070324z

J. Med. Chem. 2007, 50, 4105-4112
4105
Synthesis and Antiviral and Cytostatic Evaluations of the New C-5 Substituted Pyrimidine and
Furo[2,3-d]pyrimidine 4′,5′-Didehydro-L-ascorbic Acid Derivatives
†
†
‡
‡
‡
§
§
Tatjana Gazivoda, Mario Sˇ ok cˇ evi c´ , Marijeta Kralj, Lidija Sˇ uman, Kre sˇ imir Paveli c´ , Erik De Clercq, Graciela Andrei,
§
§
†
,†
Robert Snoeck, Jan Balzarini, Mladen Mintas, and Silvana Rai c´ -Mali c´ *
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, UniVersity of Zagreb, Maruli c´ eV trg 19, HR-10000
Zagreb, Croatia, DiVision of Molecular Medicine, Rudjer Bo sˇ koVi c´ Institute, Bijeni cˇ ka 54, P.O.B. 1016, HR-10001 Zagreb, Croatia, and
Rega Institute for Medical Research, Katholieke UniVersiteit LeuVen, Minderbroedersstraat 10, B-3000 LeuVen, Belgium
ReceiVed March 21, 2007
The novel C-5 alkynyl substituted pyrimidine (1-11) and furo[2,3-d]pyrimidine derivatives (12-22) of
L-ascorbic acid were synthesized by coupling of 5-iodouracil-4′,5′-didehydro-5′,6′-dideoxy-L-ascorbic acid
with terminal alkynes by using Sonogashira cross-coupling reaction conditions. The new compounds were
evaluated for their cytostatic and antiviral activities. Among all evaluated compounds, the octynyl-substituted
uracil derivative of L-ascorbic acid (3) exhibited the most pronounced cytostatic activities against all examined
tumor cell lines (IC50 ) 2-12 µM). Pyrimidine derivatives of L-ascorbic acid containing p-substituted
phenylacetylene groups (8-11) displayed also a rather pronounced (IC50 ) 3-37 µM) inhibitory effect
toward all tumor cell lines. From the bicyclic series of compounds, 6-butylfuro[2,3-d]pyrimidine derivative
(
)
12) and 6-p-bromophenylfuro[2,3-d]pyrimidine derivative (19) showed the highest cytostatic activity (IC50
4.5-20 µM), particularly against malignant leukemia (L1210) and T-lymphocyte (Molt4/C8 and CEM)
cells. Compounds 3 and 9 showed specific albeit moderate activity against cytomegalovirus (CMV, Davis
strain, EC50 ) 1.8 and 3.8 µM, respectively, for compounds 3 and 9) at a ∼5-fold lower concentration than
that required to show cytotoxicity.
Introduction
L-Ascorbic acid is one of the most important biomolecules.
It acts as an antioxidant and radical scavenger widely distributed
1
in aerobic organisms. Thus, it protects cellular compounds
2
against oxidative damage by free radicals and oxidants. It was
found that L-ascorbic acid derivatives induce apoptosis in tumor
3
4,5
cells, possess immunostimulant activity, and/or protect against
the lipid peroxidation of the biomembrane.6 Drug conjugation
with L-ascorbic acid can improve the delivery of drugs into the
brain, via interaction with the sodium-dependent ascorbate
,7
a
8
transporter (SVCT2 ). Furthermore, many nucleoside analogues
with interesting biological properties have arisen by substitution
at the 5-position of the uracil base in the 2′-deoxyuridine series.
Figure 1. The C-5 alkynylated pyrimidine (1-11) and 6-alkylfuro-
2,3-d]pyrimidine (12-22) derivatives of L-ascorbic acid.
9
[
The 5-(2-substituted) vinyl-2′-deoxyuridines, in particular (E)-
nucleoside analogues represent highly specific and extremely
5
-(2-bromovinyl)-2′-deoxyuridine, have emerged as potent and
17-19
potent antiviral agents.
SAR studies on furo[2,3-d]pyrimi-
selective inhibitors of herpes virus replication, particularly
against HSV-1 (herpes simplex virus type 1) and VZV (vari-
dine nucleoside analogues have shown that the length of the
alkyl chain at the C-6 position of the furopyrimidine ring plays
a crucial role in their anti-VZV potency. Compounds with
shorter alkyl chains (eC6) had little or no activity, while those
1
0-12
cella-zoster virus).
Pyrimidine nucleosides containing C-5
alkynyl groups have been shown to possess significant antiviral
and/or anticancer properties.13 The 5-alkynyluracil nucleosides
with a C7 or C11 side chain had moderate activity, and those
with a longer alkynyl chain at the C-5 position exhibited
appreciable antiviral activity in contrast to the corresponding
alkyl derivatives that showed decreasing antiviral activity with
with C8 to C10 alkyl chains had the highest potency.1
9-21
We
have reported that some pyrimidine and purine derivatives of
4′,5′-didehydro-5′,6′-dideoxy-L-ascorbic acid exerted pro-
nounced cytostatic activities against malignant tumor cell
increasing C-5 side chain length.1
3-16
Moreover, bicyclic
2
2-24
lines.
Recently we have also reported that some C-5
*
Corresponding author: Tel.: +38 51 4597 213; Fax: +38 51 4597
substituted pyrimidine derivatives of L-ascorbic acid exhibited
selective albeit slight inhibitory activity against certain human
tumor cell lines and moderate but not highly specific inhibitory
potential against herpes simplex viruses type 1 and 2, vaccinia
2
24; e-mail: silvana.raic@fkit.hr.
†
University of Zagreb.
Rudjer Bo sˇ kovi c´ Institute.
‡
§
Katholieke Universiteit Leuven.
Abbreviations: CMV, cytomegalovirus; TK VZV, varicella-zoster
a
+
2
5
-
virus, and Punta Toro virus. These results prompted us to
synthesize a series of the novel C-5 pyrimidine derivatives of
L-ascorbic acid containing an acetylene with longer side chain
and p-alkylphenyl acetylene (1-11), and variously C-6 substi-
tuted furo[2,3-d]pyrimidine (12-22) moiety (Figure 1). The
virus (thymidine kinase positive strain); TK VZV, varicella-zoster virus
thymidine kinase deficient strain); HSV-1, herpes simplex virus type 1
(
HSV-2, herpes simplex virus type 2; SVCT2, sodium-dependent transporter
of L-ascorbic acid; IUAA, 5-iodouracil-2′,3′-di-O-benzyl-4′,5′-didehydro-
5
′,6′-dideoxy-L-ascorbic acid; ABAA, 5′,6′-di-O-acetyl-2′,3′-di-O-benzyl-
L-ascorbic acid; S-DHPA, (S)-9-(1,3-dihydroxypropyl)adenine.
1
0.1021/jm070324z CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/02/2007

4
106 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
GaziVoda et al.
Scheme 1. Synthesis of the C-5 Substituted Pyrimidine (1-11) and Furo[2,3-d]pyrimidine (12-22) Derivatives of
4
′,5′-Didehydro-5′,6′-dideoxy-L-ascorbic Acida
a
Reagents and conditions: (i) HMDS, (NH4)2SO4, reflux, then TMS-triflate, CH3CN, 60 °C; (ii) R-CtCH, iPr2EtN, (PPh3)4Pd, CuI, DMF, rt; (iii) Et3N,
CuI, 60 °C.
principal aim of this study was to evaluate the cytostatic and
antiviral potencies of this class of novel compounds.
13C NMR spectra in comparison with acetylenic carbons C-1′′
and C-2′′ for 1-11 (e.g., C-1′′ at 89.41 ppm for 1 f C-5 at
9
4.41 ppm for 12, C-2′′ at 97.35 ppm for 1 f C-6 at 138.11
Chemistry
ppm for 12).
5
-Iodouracil-2′,3′-di-O-benzyl-4′,5′-didehydro-5′,6′-dideoxy-
Biological Results
22
L-ascorbic acid (IUAA), the key precursor for cross-coupling
with alkynes, was synthesized by condensation of 5′,6′-di-O-
acetyl-2′,3′-di-O-benzyl-L-ascorbic acid (ABAA)2 and 5-io-
douracil. IUAA was obtained as a mixture of Z and E isomers
in which the Z isomer predominated (in the range 80-90%).
These stereoisomers were separated by column chromatography
and pure Z isomer is applied in subsequent reactions (Scheme
). Different substituents at the C-5 position of the pyrimidine
ring were introduced by reaction of IUAA under optimized
Sonogashira Pd(0)-catalyzed reactions.
note that this reaction must be run at room temperature to avoid
the formation of byproducts produced by heating the reaction
mixture. Reaction of IUAA with various terminal alkynes under
Sonogashira conditions gave 5-substituted uracil derivatives
-11 (Scheme 1). The targeted 6-alkylfuro[2,3-d]pyrimidine-
-one L-ascorbic acid derivatives (12-22) were synthesized by
Sonogashira coupling of terminal alkynes with IUAA and
copper(I)-promoted in situ cyclization.3 These fused bicyclic
pyrimidine analogues were prepared through an O-heteroannu-
lation process using base and CuI as a catalyst (Supporting
Information, Scheme S1). This 5-endo-dig (electrophile) cy-
clization of akynyl derivatives (1-11) involving the C-4
pyrimidine oxygen and acetylenic bond gave the targeted
bicyclic nucleoside compounds (12-22).
Antiviral Activity. The compounds 1-22 were evaluated for
their inhibitory activities against varicella-zoster virus (TK -
VZV, thymidine kinase-positive, and TK VZV, thymidine
6,27
+
-
kinase-deficient strains) and cytomegalovirus (CMV, AD-169
and Davis strains) in human embryonic lung (HEL) cells;
vesicular stomatitis virus in HeLa cell culture; parainfluenza-
3-virus in Vero cell culture; herpes simplex virus type 1 and 2
(HSV-1 and HSV-2) in HEL cell culture; reovirus-1, Sindbis,
and Punta Toro virus in Vero cell cultures; and Coxsackie B4
virus in HeLa cell cultures (Tables 1 and 2, and Supporting
Information). Their activities were compared with those of
ganciclovir [9-[(1,3-dihydroxy-2-propoxy)methyl]guanine], ci-
dofovir [(S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cy-
tosine], acyclovir [9-(3-hydroxyethoxymethyl)guanine], brivudin
[(E)-5-(2-bromovinyl)-2′-deoxyuridine], (S)-DHPA [(S)-9-(1,3-
dihydroxypropyl)adenine], and ribavirin [1-(â-D-ribofuranosyl)-
1H-1,2,4-triazole-3-carboxamide] (Tables 1 and 2).
A variety of derivatives (i.e., the 5-alkynylpyrimidines 2, 3,
7, 9, and 10 and the furo[2,3-d]pyrimidine 14) showed a slight
anti-CMV (Davis strain) activity (EC50 ) 1.8-8.4 µM) in HEL
cell cultures (MCC, minimum cytotoxic concentration, 6-20-
fold higher than the EC50 values). Such activity is within the
same order of magnitude as that of ganciclovir (EC50 ) 2.6
µM) (Table 1). However, it should also be mentioned that they
show cytostatic activities at concentrations that are only 3-5-
fold higher than the antiviral active concentrations. Except for
compound 14, which was able to inhibit the replication of both
laboratory CMV strains with EC50 values of 20 µM (AD-169)
and 6.0 µM (Davis), the compounds had no significant activity
against the AD-169 strain of CMV.
1
28-33
It is important to
1
2
0,34
The structures of the novel C-5 alkynyl substituted pyrimidine
1-11) and furo[2,3-d]pyrimidine derivatives (12-22) were
determined on the basis of analysis of chemical shifts and H-H
coupling constants in H (Experimental Section) and C NMR
spectra (Supporting Information). The cyclization is accompa-
nied by a new vinylic proton H-5 signal ( H NMR, δ 6-7 ppm)
and dramatic downfield shift of C-5 and C-6 for 12-22 in the
(
1
13
1

EValuations of L-Ascorbic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4107
Table 1. Activity of the Compounds 1-22 against Varicella-Zoster Virus (VZV) and Cytomegalovirus (CMV) in Human Embryonic Lung (HEL) Cells
antiviral activity, EC50 (µM)a
cytotoxicity (µM)
CMV
+
-
TK VZV
TK VZV
07/1 strain
AD-169
strain
Davis
strain
cell morphology
(MCC)b
cell growth
(CC50)
c
compd
OKA strain
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
17.5
>4
>1.6
>20
51
>20
10.2
7.3
>20
>4
>1.6
>20
49
>20
11.9
9.3
>20
>4
>20
3.1
g100
20
g68
12.3
10
>1.6
>20
g100
>20
>20
>4
1.8
20
>20
28.4
g20
8.4
100
g100
g20
60
g4
60
60
g4
20
100
100
100
g100
>100
100
g20
100
g20
g20
g84
g33
g86
g28.5
g10.9
g20.4
g9.3
g8.6
g8.3
41
5.2
3.8
3.1
2.4
>4
>4
3.3
>4
10
8.2
g4
>4
>20
20
>20
>100
>100
>20
11
>20
>20
>20
1
1
1
1
1
1
1
1
1
1
2
2
2
>4
>4
>4
>4
g2.9
g14.5
6.0
>20
>100
g69
12
>20
>20
>20
78
>20
>20
>20
>100
80
41
>100
>100
>100
>100
44
>100
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
8.9
g14.5
>20
g20
46
50
50
ganciclovir
cidofovir
acyclovir
brivudin
-
-
-
6.5
0.67
-
2.6
0.67
-
-
g1575
g1270
>1778
g1201
262
133
545
270
-
1.0
0.014
32
168
-
a
b
Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque-forming units (PFU). Minimum cytotoxic
concentration causing a microscopically detectable alteration of normal cell morphology. c Cytostatic concentration required to reduce cell growth by 50%.
Table 2. Activity of the Compounds 1-22 against Vesicular Stomatitis
Virus in HeLa Cell Culture, Parainfluenza-3-virus in Vero Cell Culture,
and Herpes Simplex Viruses Type 1 and 2 (HSV-1 and HSV-2) in HeL
Cell Culture
a
minimum inhibitory concentration (µM)
vesicular
minimum
cytotoxic
stomatitis Parainfluenza- HSV-1
HSV-2
(G)
b
compd
virus
3-virus
(KOS)
concn (µM)
1
2
3
4
5
6
7
8
9
>16
>8
>3.2
4.8
>16
8
>16
80
40
40
>8
>8
>8
>1.6
>1.6
>4
>4
40
>4
>4
20
Figure 2. Dose-response profiles for compound 3 tested on various
>8
>8
>8
200
200
40
40
40
40
20
40
200
40
200
200
200
200
40
200
200
200
>250
>250
30
human tumor cell lines in vitro.
>40
>8
>8
>8
>8
4.8
>8
>8
chains showed EC50 values against varicella-zoster virus (OKA
and 07/1 strains) in the range of 7-12 µM (Table 1). However,
compound 8 produced an alteration of the cellular morphology
at concentrations similar to those at which the compound
inhibited viral plaque formation (MCC g 4), and both com-
pounds 7 and 8 showed cytostatic effects for HEL cells (CC50
g 28.5 and 10.9, respectively, for compound 7 and 8), resulting
in a poor selectivity. On the contrary, the furo[2,3-d]pyrimidine
derivatives 12-22 did not exhibit an inhibitory effect against
varicella-zoster virus. Furthermore, 5-alkynyluracil (2, 7, 9 and
10) and furo[2,3-d]pyrimidine (13) derivatives displayed also
some specific activity against parainfluenza-3-virus (EC50: 2,
>8
>1.6
8
>8
>4
>1.6
>8
>8
>40
>40
>40
40
>1.6
>1.6
>1.6
>4
>1.6
>1.6
>1.6
>4
>8
10
11
12
13
14
15
16
17
18
19
20
21
22
8
>4
>8
>1.6
>8
>8
>1.6
>8
>8
>40
40
>8
>40
>40
>40
>40
>8
>40
120
>40
>40
>8
120
>40
>40
>8
>8
>40
>40
>40
>250
>250
>250
-
>8
>8
>8
40
>40
>40
0.08
-
>40
>40
10
>40
>250
50
150
-
-
4
.8 µM; 7, 4.8 µM; 9, 8 µM), herpes simplex virus type 1
brivudin
S)-DHPA
ribavirin
acyclovir
ganciclovir
(EC50: 2, 8 µM) and vesicular stomatitis virus (EC50: 10, 8
(
-
250
0.4
0.0064
250
0.4
0.032
µM; 13, 40 µM) (Table 2). None of the compounds were active
against vaccinia virus, respiratory syncytial virus, reovirus-1,
Sindbis virus, Coxsackie virus B4, and Punta Toro virus
>250
>100
-
a
Required to reduce virus-induced cytopathogenicity by 50%. b Required
to cause a microscopically detectable alteration of normal cell morphology.
(Supporting Information).
The antiviral and cytostatic activities observed for several of
the pyrimidine derivatives of L-ascorbic acid are interesting,
since these compounds may represent novel antiviral leads.
Indeed, due to the lack of a free hydroxyl group in the ascorbic
C-5 alkynyl pyrimidine derivatives of L-ascorbic acid con-
taining phenylacetylene (7) and p-bromophenylacetylene (8) side

4
108 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
GaziVoda et al.
Table 3. Inhibitory Effects of Compounds 1-22 on the Growth of Malignant Tumor Cell Lines.
IC50 (µM)a
compd
L1210
Molt4/C8
CEM
HeLa
MiaPaCa-2
SW 620
MCF-7
H-460
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
19 ( 6
10 ( 3
15 ( 2
19 ( 14
3.0 ( 1.1
218 ( 36
167 ( 64
177 ( 18
36 ( 7
9.4 ( 5.0
8.2 ( 0.2
2.0 ( 0.3
81 ( 24
45 ( 1
55 ( 13
9.2 ( 0.4
7.6 ( 0.6
9.6 ( 1.1
8.3 ( 0.2
6.6 ( 0.4
7.7 ( 0.1
25 ( 6
10 ( 0
96 ( 3
106 ( 7
94 ( 40
49 ( 8
8.3 ( 0.4
35 ( 4
38 ( 1
36 ( 6
16 ( 3
14 ( 0.5
12 ( 2
56 ( 35
35 ( 19
38 ( 16
16 ( 0.7
8 ( 6
16 ( 2
13 ( 0.5
3 ( 0.5
>100
23 ( 2
14 ( 3
4 ( 0.3
>100
23 ( 9
g100
18 ( 1
15 ( 3
4 ( 1.4
NTb
15 ( 1
6.8 ( 1.7
220 ( 52
82 ( 42
68 ( 38
24 ( 9
2.4 ( 0.4
b
>100
55 ( 11
NT
37 ( 6
>100
g100
b
g100
NT
15 ( 0
13 ( 0.3
21 ( 3
16 ( 0.3
10 ( 9
15 ( 3
17 ( 2
22 ( 5
>100
78 ( 20
>100
>100
14 ( 2
16 ( 2
13 ( 1.2
11 ( 0.9
16 ( 4
7 ( 0.04
18 ( 2
7 ( 1.5
6 ( 0.8
16 ( 3
15 ( 4
19 ( 2
20 ( 3
21 ( 6
19 ( 7
17 ( 11
9 ( 4
18 ( 0.3
11 ( 2.5
23 ( 2.8
12 ( 1
10 ( 1
15 ( 7
8.7 ( 1.4
8.2 ( 2.3
8.0 ( 0.0
4.5 ( 4.0
38 ( 6
37 ( 6
14 ( 2
4 ( 1
1
1
1
1
1
1
1
1
1
1
2
2
2
9.3 ( 1.0
6.9 ( 0.3
9.0 ( 0.3
45 ( 5
b
3 ( 1
NT
17 ( 2
17 ( 4
20 ( 1.5
16 ( 0.1
19 ( 11
16 ( 2
23 ( 1.6
28 ( 11
>100
64 ( 36
>100
>100
16 ( 0.4
17 ( 1.6
33 ( 4
25 ( 7
41 ( 6
40 ( 8
52 ( 27
b
117 ( 8
164 ( 20
172 ( 19
80 ( 38
9.5 ( 0.6
44 ( 5
105 ( 7
142 ( 6
190 ( 9
120 ( 39
9.6 ( 0.8
41 ( 2
>100
60 ( 39
>100
>100
20 ( 4
14 ( 0.1
11 ( 0.1
14 ( 2
>100
NT
>100
>100
>100
33 ( 21
g100
>100
13 ( 0.2
20 ( 3
16 ( 3
12 ( 0.2
20 ( 9
20 ( 0.6
16 ( 0.5
16 ( 0.5
38 ( 4
40 ( 3
45 ( 2
44 ( 3
a
0% inhibitory concentration or compound concentration required to inhibit tumor cell proliferation by 50%. b NT, not tested.
5
acid moiety of the molecules, it is excluded that the compounds
can be phosphorylated. Therefore, their mechanism of antiviral
action should be different from that of nucleoside analogues
their parent 5-alkynyluracil derivatives (1 and 8). Moreover,
introduction of a p-substituted phenylacetylene group in 8-11
increased the cytostatic effects in comparison with their parent
alkynyl chain analogues (1-2 and 4-6).
+
such as ganciclovir. The equal anti-VZV activity against TK
-
and TK strains is in agreement with these observations. It
By comparison of the antiviral activities of the two series of
compounds, we can infer that the C-5 alkynylpyrimidine
derivatives of L-ascorbic acid showed a better antiviral potential.
Compounds 2, 3, 7, 9, and 10 showed some anti-CMV activity
(Davis strain) with EC50 values similar to those of the reference
anti-CMV compound ganciclovir; however, their selectivities
were relatively limited. Compound 14 was the only derivative
able to inhibit the replication of both CMV laboratory strains.
The mechanism of action of these new derivatives, although
still unclear, is different from that of ganciclovir, making these
compounds novel antiviral leads to be further explored.
would now be imperative to further investigate the mechanism
of antiviral action of these compounds. Also, additional efforts
will be devoted to increase the antiviral selectivity by increasing
the antiviral potency and/or by decreasing their cytostatic/
cytotoxic activity.
Cytostatic Activity. Compounds 1-22 were evaluated for
their cytostatic activities against the following malignant human
tumor cell lines: murine leukemia (L1210), human T-lympho-
cytes (Molt4/C8, CEM), cervical carcinoma (HeLa), pancreatic
carcinoma (MiaPaCa-2), colon carcinoma (SW 620), breast
carcinoma (MCF-7), and non-small cell lung carcinoma (H-
4
60) (Table 3). From C-5 alkynyl uracil series, compounds 3
Experimental Section
(
Figure 2) and 8-11 exhibited pronounced antiproliferative
General Methods. The bicyclic ring has been numbered in
accordance with the recommended IUPAC nomenclature guidelines.
Melting points (uncorrected) were determined with a Kofler micro
hot-stage (Reichert, Wien). High-field one- and two-dimensional
activities against all tumor cell lines (IC50 in the range 3-37
µM). Thus, correlation of the C-5 alkynyl side chains (1-4)
clearly revealed that the cytostatic activity was strongly and
nonlinearly dependent on the side chain length, showing that 3
with a C8 side chain had the strongest inhibitory activity. Among
furo[2,3-d]pyrimidine derivatives, 6-butyl- (12) and 6-p-bro-
mophenylfuro[2,3-d]pyrimidine (19) derivatives of L-ascorbic
acid showed the highest cytostatic activities, particularly against
malignant leukemia (L1210 IC50: 12, 4.5 µM; 19, 9.5 µM) and
T-lymphocytes (Molt4/C8 IC50: 12, 9 µM; 19, 9.6 µM; CEM
IC50: 12, 7.7 µM; 19, 8.3 µM).
1
13
H and C NMR spectra were recorded on a Bruker Avance 300
13
spectrometer, operating at 75.46 MHz for the C resonance. The
samples were dissolved in DMSO-d and measured in 5 mm NMR
6
1
13
tubes. The H and C NMR chemical shift values (δ) are expressed
in ppm referred to TMS and coupling constants (J) in hertz. The
electron impact mass spectra were recorded with an EXTREL FT
MS 2001 instrument with ionizing energy of 70 eV. Elemental
analyses were performed in the Central Analytic Service, Rudjer
Bo sˇ kovi c´ Institute, Zagreb, Croatia. Precoated E. Merck silica gel
6
0F-254 plates were used for thin layer chromatography (TLC) and
for preparative TLC, and the spots were detected under UV light
254 nm). Column chromatography (CLC) was performed using
Conclusions
(
The novel C-5 alkynyl uracil derivatives of L-ascorbic acid
Fluka silica gel (0.063-0.2 mm); glass columns were slurry-packed
under gravity. Compound purity was analyzed by HPLC with DAD
detector. The novel compounds 1-22 are soluble in dichlo-
romethane, acetonitrile, tetrahydrofuran, acetone, and dimethyl
sulfoxide.
General Procedure for the Preparation of 5-Substituted
Uracil Derivatives of L-Ascorbic Acid (1-11). To a stirred
solution of 5-iodouracil-2′,3′-di-O-benzyl-L-ascorbic acid (200 mg,
0.36 mmol) in anhydrous dimethylformamide (10 mL) were added
diisopropylethylamine (0.13 mL, 0.72 mmol), the acetylene deriva-
(
1-11) were prepared by Sonogashira coupling of 5-iodouracil
derivatives of L-ascorbic acid with various terminal alkynes.
Subsequent in situ cyclization of 1-11 catalyzed with base and
copper(I) iodide gave fused bicyclic furo[2,3-d]pyrimidine
derivatives (12-22). Comparison of cytostatic activities of
5
-alkynyluracil (1-11) and furo[2,3-d]pyrimidine (12-22)
series indicated that cyclization of the alkynyl side chain caused
a significant reduction in the inhibitory effects, except for 12
and 19, which showed cytostatic activities similar to those of

EValuations of L-Ascorbic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4109
tive (0.54 mmol), tetrakis(triphenylphosphine)palladium(0) (41.5
mg, 0.036 mmol), and copper(I) iodide (13.4 mg, 0.072 mmol).
The mixture was stirred for 20 h at room temperature under nitrogen
and then evaporated to dryness and purified by column chroma-
1-[5-(p-Bromophenylethyn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-
benzyl-2′-buten-4′-olidylidene)ethane (8). The procedure was
carried out using p-bromophenylacetylene (65 mg, 0.54 mmol),
1
which gave oil 8 (141 mg, 66.7%). H NMR: δ 11.65 (1H, s, NH),
tography (initial eluent CH
).
2
Cl
2
, followed by CH
2
Cl
2
:MeOH ) 60:
8.17 (1H, s, H-6), 7.34-7.62 (14H, m, Ph), 5.51 (1H, t, J ) 6.41
1
2 2
Hz, H-5′), 5.31 (2H, s, OCH ), 5.14 (2H, s, OCH ), 4.52 (2H, m,
+
‚
1-[5-(Hex-1′′-yn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-benzyl-2′-
H-6′) ppm. MS: m/z calcd 610.07, found 611.4 (M ). Anal.
) C, H, N.
-[5-(p-Methylphenylethyn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-
buten-4′-olidylidene)ethane (1). The procedure was carried out
using hexyne (0.06 mL, 0.54 mmol). Additional purification by
preparative thin layer chromatography gave brown oil 1 (76 mg,
2 6
(C32H23BrN O
1
benzyl-2′-buten-4′-olidylidene)ethane (9). The procedure was
carried out using p-methylphenylacetylene (0.05 mL, 0.54 mmol),
1
3
7
5.1%). H NMR: 11.52 (1H, s, NH), 7.94 (1H, s, H-6), 7.22-
.41 (10H, m, Ph), 5.53 (1H, t, J ) 6.46 Hz, H-5′), 5.27 (2H, s,
1
which gave oil 9 (137 mg, 72.9%). H NMR: δ 11.67 (1H, s, NH),
.14 (1H, s, H-6), 7.28-7.42 (14H, m, Ph), 5.55 (1H, t, J ) 6.43
Hz, H-5′), 5.31 (2H, s, OCH ), 5.14 (2H, s, OCH ), 4.56 (2H, d, J
6.42 Hz, H-6′), 2.32 (3H, s, CH ) ppm. MS: m/z calcd 546.18,
) C, H, N.
-[5-(p-Butylphenylethyn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-
2 2
OCH ), 5.19 (2H, s, OCH ), 4.51 (2H, d, J ) 7.01 Hz, H-6′), 2.41
8
(
2H, m, H-3′′), 1.48 (2H, m, H-4′′), 1.27 (2H, m, H-5′′), 0.83 (3H,
2
2
+
‚
m, CH
3
) ppm. MS: m/z calcd 512.19, found 512.6 (M ). Anal.
) C, H, N.
-[5-(Hept-1′′-yn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-benzyl-2′-
)
3
(C
H
30 28
N
2
O
6
+‚
26 2 6
found 546.6 (M ). Anal. (C33H N O
1
1
buten-4′-olidylidene)ethane (2). The procedure was carried out
using heptyne (0.07 mL, 0.54 mmol), which gave oil 2 (92 mg,
benzyl-2′-buten-4′-olidylidene)ethane (10). The procedure was
carried out using p-butylphenylacetylene (0.09 mL, 0.54 mmol),
1
4
7
8.7%). H NMR: δ 11.52 (1H, s, NH), 7.92 (1H, s, H-6), 7.22-
1
which gave oil 10 (151 mg, 71.3%). H NMR: δ 11.54 (1H, s,
.41 (10H, m, Ph), 5.40 (1H, t, J ) 6.61 Hz, H-5′), 5.30 (2H, s,
OCH ), 5.14 (2H, s, OCH ), 4.51 (2H, d, J ) 6.71 Hz, H-6′), 2.35
2 2
NH), 8.08 (1H, s, H-6), 7.24-7.39 (14H, m, Ph), 5.11 (1H, m,
H-5′), 4.52 (2H, s, OCH
.59 (2H, m, CH
3H, m, CH ) ppm. MS: m/z calcd 588.23, found 588.7 (M ).
) C, H, N.
2
), 4.26 (2H, s, OCH
2
), 3.94 (2H, m, H-6′),
(
2H, t, J ) 6.82 Hz, H-3′′), 1.46 (2H, m, H-4′′), 1.26-1.35 (4H,
m, H-5′′, H-6′′), 0.87 (3H, t, J ) 6.87 Hz, CH ) ppm. MS: m/z
) C, H, N.
-[5-(Oct-1′′-yn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-benzyl-2′-
2
(
2
), 1.56 (2H, m, CH
2
), 1.25 (2H, m, CH ), 0.93
2
3
+‚
3
+
‚
30 2 6
calcd 526.21, found 526.6 (M ). Anal. (C31H N O
32 2 6
Anal. (C36H N O
1
1-[5-(p-Pentylphenylethyn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-
buten-4′-olidylidene)ethane (3). The procedure was carried out
using octyne (0.08 mL, 0.54 mmol), and additional purification by
benzyl-2′-buten-4′-olidylidene)ethane (11). The procedure was
carried out using p-pentylphenylacetylene (0.21 mL, 1.08 mmol),
preparative thin layer chromatography afforded oil 3 (71 mg,
1
which gave powder 11 (76 mg, 35.1%, mp 59-61 °C). H NMR:
1
3
7
6.5%). H NMR: δ 11.38 (1H, s, NH), 8.14 (1H, s, H-6), 7.31-
δ 11.67 (1H, s, NH), 8.11 (1H, s, H-6), 7.14-7.42 (14H, m, Ph),
.45 (10H, m, Ph), 5.29 (1H, t, J ) 6.40 Hz, H-5′), 4.99 (2H, s,
5
.53 (1H, t, J ) 6.41 Hz, H-5′), 5.28 (2H, s, OCH
2
), 5.14 (2H, s,
), 1.53-1.59 (6H,
) ppm. MS: m/ z calcd 602.24, found
) C, H, N.
General Procedure for the Preparation of 6-Substituted Furo-
2,3-d]pyrimidin-2-ones of L-Ascorbic Acid (12-22). To a stirred
2 2
OCH ), 4.87 (2H, s, OCH ), 3.98 (2H, d, J ) 7.08 Hz, H-6′), 2.83
OCH
m, CH
34 2 6
02.7 (M ). Anal. (C37H N O
2
), 4.53 (2H, m, H-6′), 2.55 (2H, m, CH
2
(
3
2H, m, H-3′′), 1.21-1.48 (8H, m, H-4′′-7′′), 0.81 (3H, m, CH )
2
), 1.27 (3H, m, CH
3
+‚
32 2 6
ppm. MS: m/z calcd 540.23, found 540.6 (M ). Anal. (C32H N O )
+
‚
6
C, H, N.
1
-[5-(Dec-1′′-yn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-benzyl-2′-
[
buten-4′-olidylidene)ethane (4). The procedure was carried out
using decyne (0.4 mL, 0.54 mmol), which gave oil 4 (39.2 mg,
solution of 5-iodouracil-2′,3′-di-O-benzyl-L-ascorbic acid (200 mg,
0.36 mmol) in anhydrous dimethylformamide (10 mL) were added
diisopropylethylamine (0.13 mL, 0.72 mmol), acetylene derivative
1
1
7
9.1%). H NMR: δ 11.42 (1H, s, NH), 8.43 (1H, s, H-6), 7.24-
.41 (10H, m, Ph), 5.51 (1H, t, J ) 6.48 Hz, H-5′), 5.27 (2H, s,
(
0
0.54 mmol), tetrakis(triphenylphosphine)palladium(0) (41.5 mg,
.036 mmol), and copper(I) iodide (13.7 mg, 0.072 mmol). The
mixture was stirred for 20 h at room temperature, after which time
TLC (CH Cl :MeOH ) 40:1) showed complete conversion of the
OCH
(
9
(
2
), 5.17 (2H, s, OCH
2
), 4.81 (2H, d, J ) 7.16 Hz, H-6′), 2.67
2H, m, H-3′′), 1.66 (2H, m, H-4′′), 1.22-1.27 (10H, m, H-5′′-
′′), 0.87 (3H, m, CH
3
) ppm. MS: m/z calcd 568.26, found 568.7
) C, H, N.
-[5-(3′′-Hydroxyoct-1′′-yn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-
+
‚
2
2
36 2 6
M ). Anal. (C34H N O
starting material. Copper(I) iodide (13.7 mg, 0.072 mmol) and
triethylamine (20 mL) were then added to the mixture, which was
subsequently stirred at 60 °C for 6 h. The reaction mixture was
then concentrated in Vacuo to dryness and product was purified by
1
benzyl-2′-buten-4′-olidylidene)ethane (5). The procedure was
carried out using 3-hydroxy-1-octyne (0.08 mL, 0.54 mmol), which
1
gave oil 5 (76 mg, 38%). H NMR: δ 11.61 (1H, s, NH), 7.63
column chromatography (initial eluent: CH
Cl :MeOH ) 60:1). The appropriate fractions were combined, and
the solvent was evaporated to give the pure product.
2 2 2
Cl , followed by: CH -
(
1H, s, H-6), 7.32-7.42 (10H, m, Ph), 5.76 (1H, s, OH), 5.51 (1H,
2
m, H-5′), 5.31 (2H, s, OCH
)
0
2
), 5.14 (2H, s, OCH
2
), 4.49 (2H, d, J
),
6.76 Hz, H-6′), 1.97 (1H, m, CH), 1.24-1.32 (8H, m, CH
2
+‚
1-(6-Butylfuro[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-(2′,3′-di-O-
benzyl-2′-buten-4′-olidylidene)ethane (12). The procedure was
carried out using hexyne (0.12 mL, 1.08 mmol). Additional
.86 (3H, m, CH
3
) ppm. MS: m/z calcd 556.22, found 556.6 (M ).
) C, H, N.
Anal. (C32
H N
32 2
O
7
1-[5-(Hepta-1′′,6′′-diyn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-ben-
purification by preparative thin layer chromatography afforded oil
zyl-2′-buten-4′-olidylidene)ethane (6). The procedure was carried
out using 1,6-heptadiyne (0.07 mL, 0.54 mmol), which gave oil 6
1
1
2 (91 mg, 49.3%). H NMR: δ 8.56 (1H, s, H-4), 7.32-7.42 (10H,
1
m, Ph), 6.56 (1H, s, H-5), 5.56 (1H, t, J ) 6.67 Hz, H-5′), 5.30
2H, s, OCH ), 5.14 (2H, s, OCH ), 4.79 (2H, d, J ) 6.35 Hz,
H-6′), 2.40 (2H, t, J ) 6.80 Hz, H-1′′), 1.30-1.54 (4H, m, CH ),
(
7
(
87 mg, 46.3%). H NMR: δ 11.54 (1H, s, NH), 8.09 (1H, s, H-6),
.28-7.40 (10H, m, Ph), 5.36 (1H, t, J ) 6.42 Hz, H-5′), 5.22
2H, s, OCH ), 5.11 (2H, s, OCH ), 3.87 (2H, d, J ) 6.92 Hz,
H-6′), 2.31 (4H, m, H-3′′, H-5′′), 1.78 (1H, m, H-7′′), 1.66 (2H,
(
2
2
2
2
2
+‚
0
.89 (3H, m, CH
3
) ppm. MS: m/z calcd 512.19, found 512.6 (M ).
+
•
Anal. (C H N O ) C, H, N.
m, H-4′′) ppm. MS: m/z calcd 522.18, found 522.6 (M ). Anal.
) calcd 71.46 (C), 6.18 (H), 5.05 (N), found 71.27 (C),
.00 (H), 5.06 (N).
-[5-(4′′-Phenylbut-1′′-yn-1′′-yl)uracil-1-yl]-2(Z)-(2′,3′-di-O-
30 28
2
6
(C
H N O
31 26 2 6
1-(6-Pentylfuro[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-(2′,3′-di-O-
benzyl-2′-buten-4′-olidylidene)ethane (13). The procedure was
carried out using heptyne (0.05 mL, 0.54 mmol), and additional
purification by preparative thin layer chromatography gave oil 13
7
1
benzyl-2′-buten-4′-olidylidene)ethane (7). The procedure was
1
carried out using 4-phenylbutyne (0.05 mL, 0.54 mmol), which gave
(79 mg, 41.6%). H NMR: δ 8.59 (1H, s, H-4), 7.33-7.41 (10H,
1
oil 7 (134 mg, 66.5%). H NMR: δ 11.63 (1H, s, NH), 8.16 (1H,
m, Ph), 6.44 (1H, s, H-5), 5.30 (1H, t, J ) 6.62 Hz, H-5′), 5.22
s, H-6), 7.18-7.42 (15H, m, Ph), 5.51 (1H, t, J ) 6.18 Hz, H-5′),
(2H, m, OCH
2
), 4.96 (2H, m, OCH
2
), 4.06 (2H, m, H-6′), 2.53
), 1.31 (4H, m, CH ),
5
.31 (2H, s, OCH
2
), 5.14 (2H, s, OCH
2
), 4.51 (2H, d, J ) 6.53 Hz,
(2H, t, J ) 7.33 Hz, H-1′′), 1.59 (2H, m, CH
0.85 (3H, m, CH ) ppm. MS: m/z calcd 526.21, found 526.6 (M ).
Anal. (C31 ) C, H, N.
2
2
+
‚
H-6′), 2.78 (2H, m, H-3′′), 2.64 (2H, m, H-4′′) ppm. MS: m/z calcd
3
+
‚
560.19, found 560.6 (M ). Anal. (C34
H N
28 2
O
6
) C, H, N.
30 2 6
H N O

4
110 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
-(6-Hexylfuro[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-(2′,3′-di-O-
GaziVoda et al.
1
6.63 Hz, H-6′), 2.57 (2H, m, CH
m, CH ), 0.89 (3H, m, CH ) ppm. MS: m/z calcd 588.23, found
588.7 (M ). Anal. (C H N O ) C, H, N.
2 2
), 1.54 (2H, m, CH ), 1.32 (2H,
benzyl-2′-buten-4′-olidylidene)ethane (14). The procedure was
carried out using octyne (0.08 mL, 0.54 mmol), which gave oil 14
2
3
+
‚
36
32
2
6
1
(106 mg, 54.5%). H NMR: δ 8.45 (1H, s, H-4), 7.33-7.41 (10H,
1
-(6-(p-Pentylphenyl)furo[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-
2′,3′-di-O-benzyl-2′-buten-4′-olidylidene)ethane (22). The pro-
cedure was carried out using p-pentylphenylacetylene (0.07 mL,
.54 mmol). Additional purification by preparative thin layer
m, Ph), 6.43 (1H, s, H-5), 5.55 (1H, t, J ) 6.59 Hz, H-5′), 5.30
2H, s, OCH ), 5.14 (2H, s, OCH ), 4.75 (2H, d, J ) 6.81 Hz,
H-6′), 2.26 (2H, t, J ) 7.22 Hz, H-1′′), 1.62 (2H, m, CH ), 1.19-
) ppm. MS: m/z calcd 540.23,
) C, H, N.
(
(
2
2
2
0
1
.28 (6H, m, CH
2
), 0.84 (3H, m, CH
3
1
chromatography gave oil 22 (59 mg, 27.2%). H NMR: δ 7.78
1H, s, H-4), 7.23-7.33 (14H, m, Ph), 6.54 (1H, s, H-5), 5.48 (1H,
d, J ) 6.42 Hz, H-5′), 5.15 (2H, m, OCH ), 4.88 (2H, m, OCH ),
.99 (2H, d, J ) 6.36 Hz, H-6′), 2.48 (2H, m, CH ), 1.89 (2H, m,
CH ), 1.11-1.21 (4H, m, CH ), 0.78 (3H, m, CH ) ppm. MS: m/z
calcd 602.24, found 602.7 (M ). Anal. (C37 ) C, H, N.
+
‚
found 540.6 (M ). Anal. (C32
H N
32 2
O
6
(
1
-(6-Octylfuro[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-(2′,3′-di-O-
2
2
benzyl-2′-buten-4′-olidylidene)ethane (15). The procedure was
carried out using decyne (0.07 mL, 54 mmol), and additional
purification by preparative thin layer chromatography gave oil 15
3
2
2
2
3
+
‚
34 2 6
H N O
1
(64 mg, 32.1%). H NMR: δ 8.59 (1H, s, H-4), 7.21-7.39 (10H,
Antiviral Activity Assays. Antiviral activity against VZV, CMV,
vesicular stomatitis virus, parainfluenza-3-virus, HSV-1, HSV-2,
vaccinia virus, Coxsackie B4 virus, respiratory syncytial virus,
reovirus-1, Sindbis virus, and Punta Toro virus was determined
essentially as described previously.3 Confluent human embryonic
lung (HEL) fibroblasts were grown in 96-well microtiter plates and
infected with the human cytomegalovirus (CMV) strains Davis and
AD-169 at 100 PFU per well. After a 2 h incubation period, residual
virus was removed, and the infected cells were further incubated
with the minimum essential medium (MEM) containing different
concentrations of the compounds. After incubation for 7 days at
37 °C, virus-induced cytopathogenicity was monitored microscopi-
cally before and after ethanol fixation and staining with Giemsa
(for CMV and VZV). Antiviral activity was expressed as the EC50
or concentration required to reduce virus-induced cytopathogenicity
by 50%. EC50 values were calculated from graphic plots of the
percentage of cytopathogenicity as a function of concentration of
the compounds. The cytostatic concentration was calculated as the
CC , the compound concentration required to reduce cell growth
m, Ph), 6.43 (1H, s, H-5), 5.37 (1H, t, J ) 6.61 Hz, H-5′), 5.18
2H, s, OCH ), 4.99 (2H, s, OCH ), 4.22 (2H, m, H-6′), 2.63 (2H,
t, J ) 7.01 Hz, H-1′′), 1.83 (2H, s, CH ), 1.60 (2H, m, CH ), 1.24-
) ppm. MS: m/z calcd 568.26,
) C, H, N.
(
2
2
2
2
1
.28 (8H, m, CH
2
+
), 0.85 (3H, m, CH
3
5,36
‚
found 568.7 (M ). Anal. (C34
H
36
N
2
O
6
1
-(6-(1′′-Hydroxyhex-1′′-yl)furo[2,3-d]pyrimidin-2-on-3-yl)-
2(Z)-(2′,3′-di-O-benzyl-2′-buten-4′-olidylidene)ethane (16). The
procedure was carried out using 3-hydroxy-1-octyne (0.08 mL, 0.54
1
mmol), which gave oil 16 (81 mg, 41.9%). H NMR: δ 7.99 (1H,
s, H-4), 7.32-7.41 (10H, m, Ph), 6.51 (1H, s, H-5), 5.62 (1H, s,
2
OH), 5.33 (1H, t, J ) 6.55 Hz, H-5′), 5.22 (2H, s, OCH ), 5.14
(
1
5
2H, s, OCH
2
), 4.35 (2H, d, J ) 6.70 Hz, H-6′), 1.97 (1H, m, CH),
.24-1.32 (8H, m, CH ), 0.86 (3H, m, CH ) ppm. MS: m/z calcd
) C, H, N.
-(6-(Pent-4′′-yn-1′′-yl)furo[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-
2′,3′-di-O-benzyl-2′-buten-4′-olidylidene)ethane (17). The pro-
2
3
+
‚
32 2 7
56.22, found 556.6 (M ). Anal. (C32H N O
1
(
cedure was carried out using 1,6-heptadyine (0.07 mL, 0.54 mmol),
1
which gave oil 17 (94 mg, 50%). H NMR: δ 8.24 (1H, s, H-4),
7
.30-7.43 (10H, m, Ph), 6.48 (1H, s, H-5), 5.35 (1H, t, J ) 6.42
50
Hz, H-5′), 5.27 (2H, s, OCH
6.42 Hz, H-6′), 2.33 (2H, t, J ) 6.76 Hz, H-1′′), 1.76 (1H, m,
H-5′′), 1.36-1.51 (4H, m, H-2′′, H-3′′) ppm. MS: m/z calcd 522.18,
2
), 5.16 (2H, s, OCH
2
), 3.89 (2H, d, J
by 50% relative to the number of cells in the untreated controls.
CC50 values were estimated from graphic plots of the number of
cells (percentage of control) as a function of the concentration of
the test compounds. Cytotoxicity was expressed as minimum
cytotoxic concentration (MCC), the compound concentration that
causes a microscopically detectable alteration of cell morphology
of the confluent cell cultures that were exposed to the compounds.
)
+
‚
found 522.6 (M ). Anal. (C31
26 2 6
H N O ) C, H, N.
1-(6-Phenylethylfuro[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-(2′,3′-di-
O-benzyl-2′-buten-4′-olidylidene)ethane (18). The procedure was
carried out using 4-phenylbutyne (0.05 mL). Additional purification
by preparative thin layer chromatography afforded oil 18 (43 mg,
Cytostatic Activity Assays. Cytostatic activity against L1210
1
2
6
1.3%). H NMR: δ 8.54 (1H, s, H-4), 7.16-7.33 (15H, m, Ph),
(murine leukemia), Molt4/C8, and CEM (human T-lymphocytes)
.43 (1H, s, H-5), 5.42 (1H, t, J ) 6.46 Hz, H-5′), 5.23 (2H, s,
cells were measured essentially as originally described for the mouse
leukemia (L1210) cell line.37 Cytostatic measurements based on
the inhibition of HEL cell growth were performed as follows: HEL
2 2
OCH ), 5.18 (2H, s, OCH ), 4.37 (2H, d, J ) 6.82 Hz, H-6′), 2.62
(
2H, m, H-1′′), 2.33 (2H, m, H-2′′) ppm. MS: m/z calcd 560.19,
+
‚
found 560.6 (M ). Anal. (C34
-(6-(p-Bromophenyl)furo[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-
2′,3′-di-O-benzyl-2′-buten-4′-olidylidene)ethane (19). The pro-
28 2 6
H N O ) C, H, N.
3
cells were seeded at a rate of 5 × 10 cells/well into 96-well
1
microtiter plates and allowed to proliferate for 24 h. Then, medium
containing different concentrations of the test compounds was
added. After 3 days of incubation at 37 °C, the cell number was
determined with a Coulter counter. The HeLa (cervical carcinoma),
MiaPaCa-2 (pancreatic carcinoma), SW 620 (colon carcinoma),
MCF-7 (breast carcinoma), and H-460 (non-small-cell lung carci-
noma) cells were cultured as monolayers and maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
(
cedure was carried out using p-bromophenylacetylene (97.8 mg),
and additional purification by preparative thin layer chromatography
1
gave oil 19 (76 mg, 34.5%). H NMR: δ 8.40 (1H, s, H-4), 7.27-
7
.43 (14H, m, Ph), 6.45 (1H, s, H-5), 5.24 (1H, t, J ) 6.40 Hz,
H-5′), 5.19 (2H, s, OCH
.90 Hz, H-6′) ppm. MS: m/z calcd 610.07, found 611.4 (M ).
Anal. (C32 ) C, H, N.
-(6-(p-Methylphenyl)furo[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-
2′,3′-di-O-benzyl-2′-buten-4′-olidylidene)ethane (20). The pro-
2 2
), 4.99 (2H, s, OCH ), 3.79 (2H, d, J )
+
‚
6
2 6
H23BrN O
1
0% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL
penicillin, and 100 µg/mL streptomycin in a humidified atmosphere
with 5% CO at 37 °C. The HeLa, MiaPaCa-2, SW 620, MCF-7,
1
(
cedure was carried out using p-methylphenylacetylene (0.05 mL),
2
and H-460 were seeded into a series of standard 96-well microtiter
plates on day 0. Test agents were then added in 5- and 10-fold
and additional purification by preparative thin layer chromatography
1
gave oil 20 (56 mg, 28.4%). H NMR: δ 8.56 (1H, s, H-4), 7.27-
-
8
-4
dilutions (10 -10 M) and incubated for a further 72 h. Working
7
.39 (14H, m, Ph), 6.37 (1H, s, H-5), 5.38 (1H, t, J ) 6.43 Hz,
dilutions were freshly prepared on the day of testing. After 72 h of
H-5′), 5.23 (2H, s, OCH
6
found 546.6 (M ). Anal. (C33H N O
26 2 6
2
), 5.14 (2H, s, OCH
) ppm. MS: m/z calcd 546.18,
) C, H, N.
-(6-(p-Butylphenyl)furo[2,3-d]pyrimidin-2-on-3-yl)-2(Z)-(2′,3′-
2
), 4.47 (2H, d, J )
incubation the cell growth rate was evaluated by the MTT assay,
.42 Hz, H-6′), 2.36 (3H, s, CH
3
+
‚
as described previously.38,39 Each test point was performed in
1
quadruplicate in three individual experiments. The results are
expressed as IC50, the concentration necessary for 50% inhibition.
Each result is a mean value from three separate experiments. The
IC50 values for each compound were calculated from dose-response
curves using linear regression analysis by fitting the test concentra-
tions that gave percentage of growth values above and below the
reference value (i.e., 50%).
di-O-benzyl-2′-buten-4′-olidylidene)ethane (21). The procedure
was carried out using p-butylphenylacetylene (0.06 mL, 0.54 mmol),
and additional purification by preparative thin layer chromatography
1
gave oil 21 (71 mg, 33.5%). H NMR: δ 7.75 (1H, s, H-4), 7.33-
7
.40 (14H, m, Ph), 6.40 (1H, s, H-5), 5.55 (1H, t, J ) 6.39 Hz,
2 2
H-5′), 5.21 (2H, s, OCH ), 4.97 (2H, s, OCH ), 4.07 (2H, d, J )

EValuations of L-Ascorbic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4111
Acknowledgment. Support for this study was provided by
the Ministry of Science of the Republic of Croatia (Projects
125-0982464-2925, #125-0982464-2922, #098-0982464-2514
and #098-0982464-2393). We thank Lizette van Berckelaer for
excellent technical assistance in performing (part of) the
cytostatic cell activity assays, as well as Ann Absillis, Leen
Ingels, Frieda De Meyer, Leentje Persoons, Anita Camps, and
Lies Vandenheurck for excellent technical assistance in per-
forming the antiviral activity assays.
(16) De Clercq, E.; Descamps, J.; Balzarini, J.; Giziewicz, J.; Barr, P. J.;
Robins, M. J. Synthesis and Biological Activities of 5-Alkynyluracil
Nucleosides. J. Med. Chem. 1983, 26, 661-666.
#
(
17) McGuigan, C.; Yarnold, C. J.; Jones, G.; Velazquez;, S.; Barucki,
H.; Brancale, A.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini,
J. Potent and Selective Inhibition of Varicella-Zoster Virus (VZV)
by Nucleoside Analogues with an Unusual Bicyclic Base. J. Med.
Chem. 1999, 42, 4479-4484.
(
18) Loakes, D.; Brown, D. M.; Mahmood, N.; Balzarini, J.; De Clercq,
E. Antiviral Activity of Bicyclic Pyrimidine Nucleosides. AntiVir.
Chem. Chemother. 1995, 6, 371-378.
(
19) McGuigan, C.; Barucki, H.; Blewett, S.; Carangio, A.; Erichsen, J.
T.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. Highly Potent
and Selective Inhibition of Varicella-Zoster Virus (VZV) by Bicyclic
Furo Pyrimidine Nucleosides Bearing an Aryl Side Chain. J. Med.
Chem. 2000, 43, 4993-4997.
Supporting Information Available: Mechanism for the syn-
thesis of 6-alkylfuro[2,3-d]pyrimidine-2-one L-ascorbic acid deriva-
tives (12-22) by Sonogashira coupling of terminal alkynes with
IUAA and subsequent CuI-promoted in situ cyclization; 13C NMR
(20) McGuigan, C.; Brancale, A.; Barucki, H.; Srinivasan, S.; Jones, G.;
Pathirana, R.; Blewet, S.; Alvarez, R.; Yarnold, C. J.; Carangio, A.;
Vel a´ zquez, S.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J.
Fluorescent Bicyclic Furo Pyrimidine Deoxynucleoside Analogues
as Potent and Selective Inhibitors of VZV and Potential Future Drugs
for the Treatment of Chickenpox and Shingles. Drugs Future 2000,
25, 1151-1161.
data for compounds 1-22; activities of compounds 1-22 against
vaccinia virus, respiratory syncytial virus, reovirus-1, Sindbis virus,
Coxsackie virus B4, and Punta Toro virus; and cytotoxicity and
elemental analysis results. This material is available free of charge
via the Internet at http://pubs.acs.org.
(
21) Balzarini, J.; McGuigan, C. Chemotherapy of Varicella-Zoster Virus
by a Novel Class of Highly Specific Anti-VZV Bicyclic Pyrimidine
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