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Synthesis Antiviral and Cytostatic Evaluation of Unsaturated Exomethylene and Keto span class=smallCapsDspan-Lyxopyranonucleoside Analogues.

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Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360
N. Tzioumaki et al.
353
Full Paper
Synthesis, Antiviral and Cytostatic Evaluation of Unsaturated
Exomethylene and Keto D-Lyxopyranonucleoside Analogues
Niki Tzioumaki1, Evangelia Tsoukala1, Stella Manta1, George Agelis2, Jan Balzarini3,
and Dimitri Komiotis1
1
Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly,
Larissa, Greece
2
Department of Chemistry, Laboratory of Organic Chemistry, University of Patras, Patras, Greece
3
Rega Institute for Medical Research, Katholieke Universtiteit Leuven, Leuven, Belgium
This report describes the synthesis of unsaturated exomethylene lyxopyranonucleoside analogues as potential biologically active agents. Commercially available 1,2,3,4-tetra-O-acetyl-a-Dlyxopyranose 1 was condensed with silylated thymine and uracil, respectively, deacetylated and
acetalated to afford 1-(2,3-O-isopropylidene-a-D-lyxopyranosyl)thymine 4a and 1-(2,3-O-isopropylidene-a-D-lyxopyranosyl)uracil 4b. The new derivatives 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)thymine 8a and 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)uracil 8b were
prepared via two different key intermediates, 7a, b and 13a, b in order to elucidate the influence
of 29,39-unsaturation and to clarify the difference between the keto and exomethylene group on
the biological activity of the target molecules. Compounds 7a, b, 8a, b, and 13a, b were evaluated for their antiviral and cytostatic activity using several virus strains and cell lines. Whereas
no marked antiviral activity was noticed, 13a and 13b showed a cytostatic activity that ranged
between 7 and 23 lM for 13a and 26 and 38 lM for 13b against murine leukemia L1210, human
lymphocyte Molt4/C8 and CEM cells, and human breast carcinoma MCF7 cells.
Keywords: Cytotoxicity / Exomethylene nucleosides / Ketonucleosides / Unsaturated nucleosides /
Received: January 8, 2009; accepted: April 17, 2009
DOI 10.1002/ardp.200900004
Introduction
Nucleosides and their analogues have a very important
place in medicinal chemistry as structural basis for the
development of antitumor and antiviral agents [1 – 6]. A
variety of strategies have been devised to design nucleoside analogues, involving several structural modifications on the sugar moiety of naturally occurring nucleosides [7]. Consequently, over the past two decades, extensive work has been conducted to search for new types of
nucleosides with high activity and less toxicity, and those
that are less prone to trigger drug resistance [8].
Correspondence: Dr. Dimitri Komiotis, Department of Biochemistry & Biotechnology, Laboratory of Organic Chemistry, University of Thessaly,
Ploutonos Str. 26, 41221 Larissa, Greece.
E-mail: dkom@bio.uth.gr
Fax: +30 2410 565-290
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Lately, nucleosides bearing a six-membered carbohydrate moiety have been evaluated for their potential antiviral [9 – 11], antioxidant [12], and antibiotic [13] properties and as building blocks in nucleic acid synthesis
[14, 15]. Insertion of an unsaturation in the 29,39-position
of the sugar moiety of the aforementioned nucleosides,
distinguishes this ring system from common six-membered rings [16] and allows to categorize the cyclohexene
ring as a bioisoster of a saturated furanose ring. Thus,
unsaturated nucleosides are recognized as an important
class of biologically active molecules [7]. Among them,
the unsaturated keto and exocyclic methylene nucleosides exhibit interesting antitumor and antiviral properties [17 – 22] and a number of them proved to be key intermediates in synthetic and biosynthetic processes.
In an attempt to find new antitumor and antiviral
agents, we have previously synthesized a series of unsaturated keto [23, 24] as well as exomethylene pyranonucleo-
354
N. Tzioumaki et al.
side analogues [25, 26], which proved to be efficient as
tumor cell growth inhibitors and showed to have a promising potential in combating rotaviral infections. Our
studies demonstrated that the presence of a primary
hydroxyl group did not seem to be a prerequisite for biological activity, contrary to the 69-protected analogues
that exhibited the most promising cytotoxic and antiviral properties.
In view of these observations, it was of interest to synthesize and evaluate a new class of sugar-modified pyranonucleosides, devoid of the primary hydroxymethyl
function. Therefore, we present herein the synthesis of
29,39-unsaturated-49-exomethylene and 49-keto lyxopyranonucleoside analogues, containing a thymine or an uracil base moiety. This conjugation, reported for the first
time, appeared to us as a challenge and the biological
results confirmed our hypothesis.
Results and discussion
Chemistry
Our synthetic strategy to the target unsaturated exomethylene lyxopyranonucleoside analogues 8a, b was to
design and synthesize the saturated exomethylene compounds 7a, b, as key intermediates in order to elucidate
the influence of the presence of a carbon-carbon double
bond on the biological activity of the target molecules.
Therefore, condensation of commercially available
1,2,3,4-tetra-O-acetyl-D-lyxopyranose 1 with silylated thymine and uracil in the presence of tin (IV) chloride
afforded 1-(2,3,4-tri-O-acetyl-a-D-lyxopyranosyl)thymine
2a and 1-(2,3,4-tri-O-acetyl-a-D-lyxopyranosyl)uracil 2b,
respectively. Removal of all O-acetyl protecting groups of
2a and 2b, with saturated methanolic ammonia [27],
gave 3a, b in excellent yields. Specific acetalation of 3a, b
using 2,2-dimethoxypropane (2,2-dmp) [27] in acetone
led to the 29,39-O-isopropylidene derivatives 4a and 4b,
respectively. Oxidation of the free hydroxyl group with
pyridinium dichromate (PDC) / acetic anhydride (Ac2O)
[28] led to the formation of the desired 49-ketonucleosides
5a, b. Wittig reaction [29] of the keto intermediates 5a
and 5b resulted in 6a and 6b, respectively. Deisopropylidenation of the resulted derivatives 6a and 6b in CH2Cl2 /
HCOOH, 1 : 1, led to the vicinal diol derivatives 7a and
7b, respectively. Olefination of 7a, b with iodine-triphenylphosphine (Ph3P)-imidazole [30-33] afforded 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)thymine 8a in
55% yield and 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)uracil 8b in 52% yield.
In order to provide further information regarding the
correlation between exomethylene unsaturated and keto
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360
unsaturated nucleosides and their biological activity, we
also conducted the synthesis of 49-keto-29,39-unsaturated
analogues 13a and 13b.
The isolated intermediates 4a, b were transformed
into 9a, b by standard acetylation using Ac2O in pyridine.
Deisopropylidenation of the resulted derivatives 9a, b, in
CH2Cl2 / HCOOH, 1 : 1, led to the vicinal diol derivatives
10a, b in high yields (88% and 89%, respectively). Olefination of 1-(4-O-acetyl-a-D-lyxopyranosyl)thymine 10a and 1(4-O-acetyl-a-D-lyxopyranosyl)uracil 10b with iodoformPh3P-imidazole [33 – 36] afforded 11a, b, which after
deacetylation with saturated methanolic ammonia [27]
furnished the unprotected derivatives 12a, b. Oxidation
of the free hydroxyl group in 4'-position of the sugar moiety of 12a, b with PDC / Ac2O [28] led to the formation of
the desired unsaturated 49-ketonucleosides 13a, b, of
which compound 13a has been previously described
through an alternative approach starting from diacetylD-xylal [37]. Wittig condensation [29] of the obtained 49ketonucleosides 13a, b, with NaH and methyl triphenylphosphonium bromide (Ph3PCH3Br) at 08C, in the presence of t-amyl alcohol [37] in tetrahydrofuran (THF),
afforded 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)thymine 8a and 1-(2,3,4-trideoxy-4-methylene-apent-2-enopyranosyl)uracil 8b, respectively.
Biological activity
The newly synthesized compounds 8a, b and the key
intermediates 7a, b and 13a, b were evaluated for their
potential antiviral activity. However, none of the compounds were inhibitory against a wide variety of DNA
and RNA viruses.
The test compounds 7a, b, 8a, b and 13a, b were also
evaluated for their cytostatic activity against murine leukemia L1210, human lymphocyte Molt4/C8 and CEM,
human breast cancer MCF7, and human colon carcinoma
CACO-2 cells in cell culture. Whereas the exomethylene
and the unsaturated exomethylene lyxopyranonucleoside analogues showed marginal, if any, significant cytostatic activity at 147 to A500 lM, the unsaturated keto
lyxopyranonucleoside analogues were endowed with significantly more pronounced cytostatic activity. In particular, the thymine derivative 13a showed 50%-inhibitory
concentrations in the lower micromolar range against
L1210, Molt4/C8, CEM and MCF7 cells (i. e. IC50: 7 to
23 lM). It was less cytostatic against CACO-2 cell cultures.
The corresponding uracil derivative 13b was cytostatic at
26-38 lM against L1210, Molt4/C8, CEM and MCF7 cells.
Alike 13a, 13b was also markedly less cytostatic against
CACO-2 cells (Table 1). Previously, the corresponding hexenopyranosyl derivatives have been evaluated on their
cytostatic activity against CACO-2 cells and found signifiwww.archpharm.com
Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360
Exomethylene and Keto D-Lyxopyranonucleoside Analogues
355
Reactions and conditions: (i) Silylated base, SnCl4, dry CH3CN; (ii) NH3 / MeOH; (iii) CH3COCH3, 2,2-DMP, p-TsOH; (iv) PDC, Ac2O, dry CH2Cl2 / DMF (5 : 3); (v) Ph3PCH3Br,
NaH, t-amyl alcohol in THF, 0–258C; (vi) HCOOH / CH2Cl2 (1 : 1); (vii) dry Tol / DMF (4: 1), I2-imidazole-Ph3P, 15 min; (viii) (CH3CO)2O, pyridine; (ix) dry Tol / DMF (4 : 1), CHI3-imidazole-Ph3P, 1208C, 90 min; (x) PDC, (CH3CO)2O, dry CH2Cl2.
Scheme 1. Synthesis of compounds 8a and 8b.
Table 1. Cytostatic activity of test compounds.
IC501 (lM)
Compound
7a
7b
8a
8b
13a
13b
1
L1210
Molt4/C8
CEM
MCF7
CACO-2
>500
>500
147 € 0
210 € 15
7.0 € 0.4
26 € 4.0
>500
>500
213 € 15
210 € 15
11 € 5.0
38 € 3.0
>500
>500
220 € 55
256 € 64
11 € 5.0
36 € 9.0
220 € 20
209 € 8.0
176 € 11
172 € 25
23 € 7.0
35 € 9.0
500
488 € 16
320 € 27
230 € 9
77 € 7.0
134 € 13
50% Inhibitory concentration.
cantly more inhibitory (IC50 in the lower micromolar
range [26] than the pentenopyranosyluracil derivatives.
However, the CACO-2 cell assay was performed during 10
days of drug exposure [26] whereas in the current study,
only a 3-day exposure period was used for determining
the IC50 value. This can explain the different cytostatic
potential of the compounds. The IC50 values found for the
other tumor murine and human cell lines were comparable for both types of compounds.
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Conclusion
In conclusion, the synthesis of unsaturated exomethylene lyxopyranonucleoside analogues bearing thymine
and uracil was undertaken. The target nucleosides 8a, b
and their key intermediates saturated exomethylene
7a, b and keto unsaturated 13a, b were tested for their
inhibitory effects on the proliferation of murine leukemia cells (L1210), human T-lymphocyte cells (Molt4/C8,
www.archpharm.com
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N. Tzioumaki et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360
CEM), human mammary breast carcinoma (MCF7), and
human colon carcinoma cells (CACO-2).
The unsaturated keto analogues 13a, b were endowed
with pronounced cytostatic activity, indicating that the
presence of the keto group in the sugar moiety is indispensable. Although the exomethylene derivatives 7a, b
showed marginal, if any, significant inhibitory activity,
the 29,39-unsaturation somewhat enhanced their activity,
suggesting that the carbon-carbon double bond has not
much influence on the biological activity compared to
the keto moiety. Conversely to the previously reported
keto and exomethylene mannopyranosyl nucleoside analogues [26], the newly synthesized molecules showed similar inhibitory activity, establishing the non requisiteness of the presence of the primary hydroxyl group.
Whereas, the mode of action of these analogues is still
unclear, the unsaturated keto system might function as
an acceptor in a Michael-addition mechanism. It would
therefore be interesting to further explore the structureactivity relationship of the compounds on their cytostatic potential.
from sodium / benzophenone before use and pyridine stored
over pellets of potassium hydroxide.
We are grateful to Mrs. Lizette van Berckelaer, Leentje Persoons, Frieda De Meyer, Vickie Broeckx, and Leen Ingels for
dedicated technical help. The financial support was provided
by the ”Geconcerteerde Onderzoeksacties” (GOA no. 05/19) of the
K. U. Leuven.
1-(a-D-Lyxopyranosyl)thymine 3a
The authors have declared no conflict of interest.
Synthesis of 1-(2,3,4-trideoxy-4-methylene-a-pent-2enopyranosyl)thymine 8a
1-(2,3,4-Tri-O-acetyl-a-D-lyxopyranosyl)thymine 2a
A mixture of thymine (2.87 g, 22.72 mmol), hexamethyldisilazane (HMDS) (5.94 mL, 28.17 mmol), and saccharin (191.45 mg,
1.05 mmol) in dry CH3CN (85 mL) was refluxed for 30 min under
nitrogen. To this were added 1,2,3,4-tetra-O-acetyl-D-lyxopyranose 1 (5.16 g, 16.23 mmol) and SnCl4 (2.7 mL, 22.72 mmol). The
reaction mixture was refluxed for 1 h, neutralized with saturated sodium bicarbonate, and then extracted with CH2Cl2. The
organic extract was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by
flash chromatography (EtOAc / hexane, 7 : 3) to give compound
2a (4.49 g, 72%, Rf = 0.38) as a clear viscous oil. [a]D22 = 16.6 (c =
0.130, CHCl3). UV (CHCl3) kmax: 260 nm (e = 6926). 1H-NMR (CDCl3)
d: 8.79 (br s, 1H, NH), 7.15 (s, 1H, H-6), 6.03 (d, J19,29 = 9.9 Hz, 1H, H19), 5.49 (t, J29,39 = J39,49 = 3.2 Hz, 1H, H-39), 5.26 (dd, 1H, H-29), 4.89 (m,
1H, H-49), 4.09 (m, 2H, H-5a9, H-5b9), 2.22, 2.21, 1.99 (3s, 9H, 3OAc),
1.95 (s, 3H, 5-CH3). ESI-MS (m/z): 385.04 [M + H+]. Anal. Calcd. for
C16H20N2O9: C, 50.00; H, 5.25; N, 7.29. Found: C, 49.75; H, 5.32; N,
7.51.
Compound 2a (4.49 g, 11.68 mmol) was treated with ammonia /
MeOH (saturated at 08C, 0.48 L). The solution was stirred overnight at room temperature and, then, was concentrated under
reduced pressure to give compound 3a (2.95 g, 98%, Rf = 0.40 in
EtOAc / MeOH, 8 : 2) as a white foam. [a]D22 = – 10.0 (c = 0.500,
MeOH). UV (MeOH) kmax: 261 nm (e = 6302). ESI-MS (m/z): 259.38
[M + H+]. Anal. Calcd. for C10H14N2O6: C, 46.51; H, 5.46; N, 10.85.
Found: C, 46.96; H, 5.32; N, 10.41.
1-(2,3-O-Isopropylidene-a-D-lyxopyranosyl)thymine 4a
Experimental
Chemistry
Melting points were recorded in a Mel-Temp apparatus (Kleinfeld Labortechnik GmbH, Gehrden, Germany) and are uncorrected. Thin-layer chromatography (TLC) was performed on
Merck precoated 60F254 plates (Merck, Germany). Reactions were
monitored by TLC on silica gel, with detection by UV light
(254 nm) or by charring with sulphuric acid. Flash chromatography was performed using silica gel (240 – 400 mesh, Merck). 1HNMR spectra were recorded at room temperature with a Bruker
400 MHz spectrometer (Bruker Bioscience, USA) using chloroform-d (CDCl3) and methanol-d4 (CD3OD). Chemical shifts are
reported in parts per million (d) downfield from tetramethylsilane (TMS) as internal standard. Mass spectra were obtained with
a Micromass Platform LC (ESI-MS; Micromass, Manchester, UK).
Optical rotations were measured using Autopol I polarimeter
(Rudolph Research, Hackettstown, NJ. USA).
All reactions were carried out in dry solvents. CH2Cl2 was distilled from phosphorous pentoxide and stored over 4E molecular sieves. Acetonitrile, toluene (Tol), and N,N-dimethyl formamide (DMF) were distilled from calcium hydride and stored over
3E molecular sieves. THF was freshly distilled under nitrogen
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
To a stirred suspension of 3a (2.95 g, 11.45 mmol) in anhydrous
acetone (215 mL) and 2,2-dmp (11 mL) was added p-toluenesulfonic acid (p-TsOH) monohydrate (0.44 g, 2.29 mmol). After 3 h
the resulting solution was neutralized with triethylamine so
that pH did not exceed 7. The solution was concentrated and the
residue was purified by flash chromatography (EtOAc) to give 4a
(2.77 g, 81%, Rf = 0.34) as a white foam. [a]D22 = 22.0 (c = 0.505,
CHCl3). UV (CHCl3) kmax: 260 nm (e = 1625). 1H-NMR (CDCl3) d: 7.35
(s, 1H, H-6), 5.49 (d, J19,29 = 8.6 Hz, 1H, H-19), 4.30 (m, 1H, H-39), 4.23
(dd, J29,39 = 4.9 Hz, 1H, H-29), 3.93 – 3.89 (m, 3H, H-49, H-5a9, H-5b9),
1.84 (s, 3H, 5-CH3), 1.51, 1.31 (2s, 6H, 2CH3). ESI-MS (m/z): 299.35
[M + H+]. Anal. Calcd. for C13H18N2O6: C, 52.34; H, 6.08; N, 9.39.
Found: C, 52.65; H, 5.84; N, 9.63.
1-(2,3-O-Isopropylidene-a-L-erythro-pentopyranosyl-4ulose)thymine 5a
A mixture of 4a (1.97 g, 6.60 mmol), PDC (2.98 g, 7.93 mmol),
and Ac2O (1.87 mL, 19.80 mmol) was stirred in 80 mL of dry
CH2Cl2 / DMF (5 : 3) for 1 h under nitrogen at room temperature
and was concentrated in vacuo. Purification by flash chromatography (EtOAc / hexane, 8 : 2) yielded pure 5a (1.64 g, 84%, Rf =
0.36 in EtOAc / hexane, 9 : 1) as a white foam. [a]D22 = 8.0 (c =
0.533, CHCl3). UV (CHCl3) kmax: 260 nm (e = 3879). 1H-NMR (CDCl3)
d: 8.43 (br s, 1H, NH), 7.11 (s, 1H, H-6), 5.51 (d, J19,29 = 5.2 Hz, 1H, Hwww.archpharm.com
Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360
19), 4.81 (dd, J29,39 = 7.2 Hz, 1H, H-29), 4.70 (d, 1H, H-39), 4.36, 4.27 (q,
AB-system, J = 17.9 Hz, 2H, H-59), 1.97 (s, 3H, 5-CH3), 1.55, 1.41 (2s,
6H, 2CH3). ESI-MS (m/z): 297.32 [M + H+]. Anal. Calcd. for
C13H16N2O6: C, 52.70; H, 5.44; N, 9.46. Found: C, 52.92; H, 5.78; N,
9.61.
1-(4-Deoxy-2,3-O-isopropylidene-4-methylene-a-Lerythro-pentopyranosyl)thymine 6a
To a stirred suspension of Ph3PCH3Br (6.53 g, 18.28 mmol) and tamyl alcohol (2.18 mL, 19.94 mmol) in dry THF (56 mL) was
added NaH (0.76 g, 60% in oil, 31.90 mmol) at 08C under nitrogen and the reaction mixture was stirred for 2 h at ambient temperature. To this yellow phosphorous ylide was added a solution
of 5a (1.64 g, 5.54 mmol) in dry THF (9 mL) dropwise, at 08C
under nitrogen. After the mixture was stirred for 30 min at
ambient temperature, the reaction mixture was quenched with
saturated sodium bicarbonate and extracted with EtOAc. The
organic layer was washed with water, dried with sodium sulfate,
and evaporated. The residue was purified by flash chromatography (EtOAc / hexane, 5 : 5) to give 6a (0.99 g, 61%, Rf = 0.3), as a
white foam. [a]D22 = – 6.0 (c = 0.433, CHCl3). UV (CHCl3) kmax:
265 nm (e = 6874). 1H-NMR (CDCl3) d: 8.55 (br s, 1H, NH), 7.10 (s,
1H, H-6), 5.74 (d, J19,29 = 5.0 Hz, 1H, H-19), 5.43, 5.36 (br s, 2H,
methylene), 4.77 (d, J29,39 = 8.9 Hz, 1H, H-39), 4.48, 4.32 (q, AB-system, J = 13.2 Hz, 2H, H-59), 4.18 – 4.15 (m, 1H, H-29), 1.93 (s, 3H, 5CH3), 1.64, 1.43 (2s, 6H, 2CH3). ESI-MS (m/z): 295.26 [M + H+]. Anal.
Calcd. for C14H18N2O5: C, 57.13; H, 6.16; N, 9.52. Found: C, 57.40;
H, 6.42; N, 9.43.
1-(4-Deoxy-4-methylene-a-L-erythropentopyranosyl)thymine 7a
Compound 6a (0.99 g, 3.38 mmol) was dissolved in a mixture of
CH2Cl2 (12 mL) and HCOOH (12 mL, 90%). The solution was
stirred for 3 h at room temperature and then concentrated
under reduced pressure. The residue was purified by flash chromatography (EtOAc / hexane, 9 : 1) to give 7a (0.72 g, 84%, Rf =
0.14) as a white foam. [a]D22 = – 14.0 (c = 0.605, CHCl3). UV (CHCl3)
kmax: 260 nm (e = 6538). 1H-NMR (CDCl3) d: 10.36 (br s, 1H, NH),
7.19 (s, 1H, H-6), 6.03 (d, J19,29 = 9.3 Hz, 1H, H-19), 5.22, 5.12 (br s, 2H,
methylene), 4.61 (d, J29,39 = 1.9 Hz, 1H, H-39), 4.57, 4.14 (q, AB-system, J = 12.6 Hz, 2H, H-59), 3.69 (d, 1H, H-29), 1.85 (s, 3H, 5-CH3).
ESI-MS (m/z): 255.28 [M + H+]. Anal. Calcd. for C11H14N2O5: C; 51.97,
H; 5.55, N; 11.02. Found: C, 52.23; H, 5.76; N, 10.88.
1-(2,3,4-Trideoxy-4-methylene-a-pent-2enopyranosyl)thymine 8a
Ph3P (2.22 g, 8.49 mmol), iodine (1.08 g, 4.25 mmol), and imidazole (0.29 g, 4.25 mmol) were added to the suspension of 7a
(0.72 g, 2.83 mmol) in 78 mL of dry Tol / DMF (4 : 1). The reaction
mixture was heated (808C, oil bath) under nitrogen for 15 min
and concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc / hexane, 9 : 1) and yielded 8a (0.34 g,
55%), as a white foam. [a]D22 = – 8.0 (c = 0.500, CHCl3). UV (CHCl3)
kmax: 260 nm (e = 6389). 1H-NMR (CDCl3) d: 8.44 (br s, 1H, NH), 7.06
(s, 1H, H-6), 6.58 (dd, J19,39 = 1.5 Hz, J29,39 = 10.0 Hz, 1H, H-39), 6.41 (br
s, 1H, H-19), 5.62 (d, 1H, H-29), 5.04, 4.99 (br s, 2H, methylene),
4.39, 4.33 (q, AB-system, J = 13.9 Hz, 2H, H-59), 1.85 (s, 3H, 5-CH3).
ESI-MS (m/z): 221.34 [M + H+]. Anal. Calcd. for C11H12N2O3: C, 59.99;
H, 5.49; N, 12.72. Found: C, 59.82; H, 5.68; N, 12.93.
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Exomethylene and Keto D-Lyxopyranonucleoside Analogues
357
1-(4-O-Acetyl-2,3-O-isopropylidene-a-Dlyxopyranosyl)thymine 9a
Compound 4a (0.80 g, 2.68 mmol) was dissolved in a mixture of
pyridine (2.3 mL) and Ac2O (1.2 mL, 12.72 mmol). The reaction
was carried out at room temperature for 2 h, then quenched
with MeOH at 08C and concentrated. The residue was purified by
flash chromatography (EtOAc / hexane, 8 : 2) to give 9a (0.87 g,
95%, Rf = 0.54) as a white solid, m.p.: 158 – 1608C. [a]D22 = 12.0 (c =
0.583, CHCl3). UV (CHCl3) kmax: 265 nm (e = 7065). 1H-NMR (CDCl3)
d: 8.89 (br s, 1H, NH), 7.13 (s, 1H, H-6), 5.65 (d, J19,29 = 8.6 Hz, 1H, H19), 5.17 (d, J39,49 = 1.3 Hz, 1H, H-49), 4.34 (m, 1H, H-39), 4.23 (m, 1H,
H-29), 4.04 – 3.99 (m, 2H, H-5a9, H-5b9), 2.18 (s, 3H, OAc), 1.95 (s, 3H,
5-CH3), 1.62, 1.39 (2s, 6H, 2CH3). ESI-MS (m/z): 341.13 [M + H+].
Anal. Calcd. for C15H20N2O7: C, 52.94; H, 5.92; N, 8.23. Found: C,
53.30; H, 5.38; N, 8.52.
1-(4-O-Acetyl-a-D-lyxopyranosyl)thymine 10a
Compound 9a (0.87 g, 2.55 mmol) was dissolved in a mixture of
CH2Cl2 (8.6 mL) and HCOOH (8.6 mL, 90%). The solution was
stirred overnight at room temperature and then was concentrated under reduced pressure. The residue was purified by flash
chromatography (EtOAc) to give 10a (0.67 g, 88%, Rf = 0.28) as a
white solid, m.p.: 229 – 2318C. [a]D22 = – 20.0 (c = 0.780, MeOH).
UV (MeOH) kmax: 260 nm (e = 5073). 1H-NMR (CD3OD) d: 7.45 (d,
J5b9,6 = 1.1 Hz, 1H, H-6), 5.84 (d, J19,29 = 9.7 Hz, 1H, H-19), 4.85 (d, J29,39 =
3.1 Hz, 1H, H-39), 4.12 (m, 2H, H-49, H-5a9), 3.95 (dd, 1H, H-29), 3.87
(dd, J49,5b9 = 1.1 Hz, J5a9,5b9 = 13.0 Hz, 1H, H-5b9), 2.14 (s, 3H, OAc), 1.91
(s, 3H, 5-CH3). ESI-MS (m/z): 301.12 [M + H+]. Anal. Calcd. for
C12H16N2O7: C, 48.00; H, 5.37; N, 9.33. Found: C, 47.72; H, 5.78; N,
9.79.
1-(4-O-Acetyl-2,3-dideoxy-a-D-glycero-pent-2enopyranosyl)thymine 11a
Imidazole (0.32 g, 4.70 mmol), Ph3P (2.46 g, 9.41 mmol), and
iodoform (1.85 g, 4.70 mmol) were added to the suspension of
10a (0.67 g, 2.24 mmol) in 19 mL of dry Tol / DMF (4 : 1). The reaction mixture was heated (1008C, oil bath) under nitrogen for 2 h,
concentrated in vacuo and the residue diluted with EtOAc,
washed with saturated sodium bicarbonate, sodium thiosulfate
and water. The organic extract was dried over anhydrous
sodium sulfate, filtered, and evaporated to dryness. The residue
was purified by flash chromatography (EtOAc / hexane, 6 : 4) and
yielded 11a (0.33 g, 56%, Rf = 0.45 in EtOAc) as a colorless oil.
[a]D22 = 46.0 (c = 0.330, CHCl3). UV (CHCl3) kmax: 260 nm (e = 4622).
1
H-NMR (CDCl3) d: 8.20 (br s, 1H, NH), 7.15 (s, 1H, H-6), 6.35 (m,
2H, H-19, H-39), 5.92 (dd, J19,29 = 1.3 Hz, J29,39 = 10.0 Hz, 1H, H-29), 5.11
(m, 1H, H-49), 4.09 – 3.99 (m, J = 13.0 Hz, 2H, H-59), 2.15 (s, 3H,
OAc), 1.94 (s, 3H, 5-CH3). ESI-MS (m/z): 267.29 [M + H+]. Anal. Calcd.
for C12H14N2O5: C, 54.13; H, 5.30; N, 10.52. Found: C, 54.58; H,
4.72; N, 10.68.
1-(2,3-Dideoxy-a-D-glycero-pent-2-enopyranosyl)thymine
12a
Compound 11a (0.33 g, 1.24 mmol) was treated with ammonia /
MeOH (saturated at 08C, 17 mL). The solution was stirred overnight at room temperature and then concentrated under
reduced pressure. Purification by flash chromatography (EtOAc)
yielded 12a (0.19 g, 68%, Rf = 0.16) as a white solid, m.p.: 1861888C. [a]D22 = 4.0 (c = 0.230, CHCl3). UV (CHCl3) kmax: 260 nm (e =
8584). 1H-NMR (CDCl3) d: 8.56 (br s, 1H, NH), 7.21 (s, 1H, H-6), 6.40
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(m, 1H, H-39), 6.29 (br s, 1H, H-19), 5.79 (dd, J19,29 = 1.2 Hz, J29,39 =
10.1 Hz, 1H, H-29), 4.15 – 4.13 (m, 1H, H-49), 4.00 (m, 2H, H-5a9, H5b9), 1.92 (s, 3H, 5-CH3). ESI-MS (m/z): 225.25 [M + H+]. Anal. Calcd.
for C10H12N2O4: C, 53.57; H, 5.39; N, 12.49. Found: C, 53.42; H,
5.47; N, 12.58.
in EtOAc / MeOH, 8 : 2) as a white foam, and it was used without
further purification. [a]D22 = – 12.0 (c = 0.500, MeOH). UV (MeOH)
kmax: 258 nm (e = 6869). ESI-MS (m/z): 245.26 [M + H+]. Anal. Calcd.
for C9H12N2O6: C, 44.27; H, 4.95; N, 11.47. Found: C, 44.56; H,
5.12; N, 11.78.
1-(2,3-Dideoxy-a-pent-2-enopyranosyl-4-ulose)thymine
13a
1-(2,3-O-Isopropylidene-a-D-lyxopyranosyl)uracil 4b
A mixture of 12a (0.19 g, 0.84 mmol), PDC (0.38 g, 1.01 mmol),
and Ac2O (0.24 mL, 2.52 mmol) was stirred in dry CH2Cl2 (14 mL)
for 2 h under nitrogen at room temperature. Purification by
flash chromatography (EtOAc / hexane, 8 : 2) yielded pure 13a
(0.16 g, 87%, Rf = 0.54 in EtOAc) as a white solid, m.p.: 169 –
1718C. [a]D22 = – 6.0 (c 0.305, CHCl3). UV (CHCl3) kmax: 258 nm (e =
6466). 1H-NMR (CDCl3) d: 10.12 (br s, 1H, NH), 7.36 (s, 1H, H-6),
7.15-7.12 (m, 1H, H-39), 6.59 (br s, 1H, H-19), 6.34 – 6.31 (m, 1H, H29), 4.37, 4.22 (q, AB-system, J = 16.3 Hz, 2H, H-59), 1.98 (s, 3H, 5CH3). ESI-MS (m/z): 223.24 [M + H+]. Anal. Calcd. for C10H10N2O4: C,
54.05; H, 4.54; N, 12.61. Found: C, 54.22; H, 4.38; N, 12.71.
1-(2,3,4-Trideoxy-4-methylene-a-pent-2enopyranosyl)thymine 8a
To a stirred suspension of Ph3PCH3Br (0.86 g, 2.41 mmol) and tamyl alcohol (0.29 mL, 2.63 mmol) in dry THF (6.7 mL) was added
NaH (0.10 g, 60% in oil, 4.19 mmol) at 08C under nitrogen and
the reaction mixture was stirred for 2 h at ambient temperature.
To this yellow phosphorous ylide was added a solution of 13a
(0.16 g, 0.73 mmol) in dry THF (1.1 mL) dropwise, at 08C. After
the mixture was stirred for 30 min at ambient temperature, the
reaction mixture was quenched with saturated sodium bicarbonate and extracted with EtOAc. The organic layer was washed
with water, dried with sodium sulfate, and evaporated. The residue was purified by flash chromatography (EtOAc / hexane, 9 : 1)
to give 8a (0.98 g, 61%, Rf = 0.6) as a white foam.
Synthesis of 1-(2,3,4-trideoxy-4-methylene-a-pent-2enopyranosyl)uracil 8b
1-(2,3,4-Tri-O-acetyl-a-D-lyxopyranosyl)uracil 2b
A mixture of uracil (2.46 g, 21.99 mmol), HMDS (5.75 mL,
27.26 mmol), and saccharin (185.29 mg, 1.01 mmol) in dry
CH3CN (68 mL) was refluxed for 30 min under nitrogen. To this
was added 1,2,3,4-tetra-O-acetyl-D-lyxopyranose 1 (5.0 g,
15.71 mmol) and SnCl4 (2.6 mL, 21.99 mmol). The reaction mixture was refluxed for 2 h, neutralized with saturated sodium
bicarbonate, and then extracted with CH2Cl2. The organic
extract was dried over anhydrous sodium sulfate, filtered, and
evaporated to dryness. The residue was purified by flash chromatography (EtOAc / hexane, 7 : 3) to give compound 2b (3.79 g,
65%, Rf = 0.3) as a white foam. [a]D22 = – 32.0 (c = 0.500, CHCl3). UV
(CHCl3) kmax: 258 nm (e = 9344). 1H-NMR (CDCl3) d: 8.86 (br s, 1H,
NH), 7.30 (d, J5,6 = 8.2 Hz, 1H, H-6), 5.96 (d, J19,29 = 9.9 Hz, 1H, H-19),
5.75 (d, 1H, H-5), 5.41 (t, J29,39 = J39,49 = 3.2 Hz, 1H, H-39), 5.18 (dd, 1H,
H-29), 4.82 (d, 1H, H-49), 4.05, 3.98 (q, AB-system, J = 13.3 Hz, 2H, H59), 2.15, 2.13, 1.92 (3s, 9H, 3OAc). ESI-MS (m/z): 371.36 [M + H+].
Anal. Calcd. for C15H18N2O9: C, 48.65; H, 4.90; N, 7.56. Found: C,
49.14; H, 5.24; N, 7.41.
1-(a-D-Lyxopyranosyl)uracil 3b
Uracil derivative 3b was synthesized from 2b by the similar procedure as described for 3a. 3b was obtained (2.47 g, 99%, Rf = 0.34
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Uracil derivative 4b was synthesized from 3b by the similar procedure as described for 4a. It was purified by flash chromatography (EtOAc) (2.24 g, 78%, Rf = 0.57 in EtOAc / MeOH, 9 : 1) and 4b
was obtained as a white foam. [a]D22 = 6.0 (c = 0.485, MeOH). UV
(MeOH) kmax: 258 nm (e = 4457). 1H-NMR (CD3OD) d: 10.05 (br s,
1H, NH), 7.63 (d, J5,6 = 8.1 Hz, 1H, H-6), 5.61 (d, 1H, H-5), 4.52 (d, J19,29
= 6.4 Hz, 1H, H-19), 4.34 – 4.29 (m, 2H, H-29, H-39), 3.90 – 3.82 (m,
3H, H-49, H-5a9, H-5b9), 1.44, 1.26 (2s, 6H, 2CH3). ESI-MS (m/z):
285.31 [M + H+]. Anal. Calcd. for C12H16N2O6: C, 50.70; H, 5.67; N,
9.85. Found: C, 50.85; H, 5.80; N, 9.98.
1-(2,3-O-Isopropylidene-a-L-erythro-pentopyranosyl-4ulose)uracil 5b
Uracil analogue 5b was synthesized from 4b by the similar procedure as described for 5a. It was purified by flash chromatography (EtOAc / hexane, 8 : 2) and yielded pure 5b (0.85 g, 69%, Rf =
0.45 in EtOAc / hexane, 9 : 1) as a white foam. [a]D22 = 4.0 (c =
0.500, CHCl3). UV (CHCl3) kmax: 258 nm (e = 2444). 1H-NMR (CDCl3)
d: 8.02 (br s, 1H, NH), 7.31 (d, J5,6 = 8.0 Hz, 1H, H-6), 5.85 (d, 1H, H5), 5.52 (d, J19,29 = 5.3 Hz, 1H, H-19), 4.77 (m, 1H, H-29), 4.69 (d, J29,39 =
7.2 Hz, 1H, H-39), 4.37, 4.30 (q, AB-system, J = 17.9 Hz, 2H, H-59),
1.56 (2s, 6H, 2CH3). ESI-MS (m/z): 283.22 [M + H+]. Anal. Calcd. for
C12H14N2O6: C, 51.06; H, 5.00; N, 9.93. Found: C, 51.38; H, 5.11; N,
9.68.
1-(4-Deoxy-2,3-O-isopropylidene-4-methylene-a-Lerythro-pentopyranosyl)uracil 6b
Uracil analogue 6b was synthesized from 5b by the similar procedure as described for 6a. It was purified by flash chromatography (EtOAc / hexane, 7 : 3) to give 6b (0.46 g, 54%, Rf = 0.54 in
EtOAc / hexane, 9 : 1) as a white foam. [a]D22 = – 10.0 (c = 0.500,
CHCl3). UV (CHCl3) kmax: 258 nm (e = 8024). 1H-NMR (CDCl3) d: 8.26
(br s, 1H, NH), 7.26 (d, J5,6 = 8.4 Hz, 1H, H-6), 5.77 (d, J29,39 = 7.3 Hz,
1H, H-39), 5.73 (d, 1H, H-5), 5.53, 5.43 (br s, 2H, methylene), 4.76
(br s, 1H, H-19), 4.47, 4.32 (q, AB-system, J = 12.7 Hz, 2H, H-59), 4.11
(m, 1H, H-29), 1.63, 1.48 (2s, 6H, 2CH3). ESI-MS (m/z): 281.29 [M +
H+]. Anal. Calcd. for C13H16N2O5: C, 55.71; H, 5.75; N, 9.99. Found:
C, 56.12; H, 5.93; N, 9.85.
1-(4-Deoxy-4-methylene-a-L-erythropentopyranosyl)uracil 7b
Uracil analogue 7b was synthesized from 6b by the similar procedure as described for 7a. It was purified by flash chromatography (EtOAc / hexane, 9 : 1) to give 7b (0.32 g, 82%, Rf = 0.13) as a
white foam. [a]D22 = -12.0 (c = 0.480, MeOH). UV (MeOH) kmax:
258 nm (e = 5825). 1H-NMR (CDCl3) d: 7.36 (d, J5,6 = 8.2 Hz, 1H, H-6),
5.94 (d, J19,29 = 9.3 Hz, 1H, H-19), 5.76 (d, 1H, H-5), 5.23, 5.15 (br s,
2H, methylene), 4.58 (d, J29,39 = 2.9 Hz, 1H, H-39), 4.54, 4.17 (q, ABsystem, J = 12.6 Hz, 2H, H-59), 4.60 – 4.54 (m, 2H, H-5a9, H-5b9), 3.62
(dd, 1H, H-29). ESI-MS (m/z): 241.25 [M + H+]. Anal. Calcd. for
C10H12N2O5: C, 50.00; H, 5.04; N, 11.66. Found: C, 50.41; H, 5.22;
N, 11.43.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360
1-(2,3,4-Trideoxy-4-methylene-a-pent-2enopyranosyl)uracil 8b
Uracil analogue 8b was synthesized from 7b by the similar procedure as described for 8a. It was purified by flash chromatography (EtOAc / hexane, 8 : 2) (0.14 g, 52%) and 8b was obtained as a
white foam. [a]D22 = 8.0 (c = 0.425, CHCl3). UV (CHCl3) kmax: 258 nm
(e = 6249). 1H-NMR (CDCl3) d: 8.24 (br s, 1H, NH), 7.35 (d, J5,6 =
8.1 Hz, 1H, H-6), 6.66 (d, J29,39 = 10.0 Hz, 1H, H-39), 6.47 (br s, 1H, H19), 5.72-5.68 (m, 2H, H-29, H-5), 5.11, 5.07 (br s, 2H, methylene),
4.45, 4.38 (q, AB-system, J = 14.0 Hz, 2H, H-59). ESI-MS (m/z): 207.29
[M + H+]. Anal. Calcd. for C10H10N2O3: C, 58.25; H, 4.89; N, 13.59.
Found: C, 58.42; H, 4.63; N, 13.51.
1-(4-O-Acetyl-2,3-O-isopropylidene-a-Dlyxopyranosyl)uracil 9b
Uracil derivative 9b was synthesized from 4b by the similar procedure as described for 9a. It was purified by flash chromatography (EtOAc / hexane, 5 : 5) (0.99 g, 86%, Rf = 0.52 in EtOAc / hexane, 8 : 2) and 9b was obtained as a white solid, m.p.: 185 – 1868C.
[a]D22 = 36.0 (c = 0.505, CHCl3). UV (CHCl3) kmax: 258 nm (e = 8015).
1
H-NMR (CDCl3) d: 8.29 (br s, 1H, NH), 7.32 (d, J5,6 = 8.01 Hz, 1H, H6), 5.80 (d, 1H, H-5), 5.64 (d, J19,29 = 8.6 Hz, 1H, H-19), 5.16 (br s, 1H,
H-49), 4.33 (br s, 1H, H-39), 4.17 (m, 1H, H-29), 4.06, 4.01 (q, AB-system, J = 13.2 Hz, 2H, H-59), 2.16 (s, 3H, OAc), 1.62, 1.39 (2s, 6H,
2CH3). ESI-MS (m/z): 327.33 [M + H+]. Anal. Calcd. for C14H18N2O7: C,
51.53; H, 5.56; N, 8.59. Found: C, 51.79; H, 5.38; N, 8.82.
1-(4-O-Acetyl-a-D-lyxopyranosyl)uracil 10b
Uracil derivative 10b was synthesized from 9b by the similar procedure as described for 10a. It was purified by flash chromatography (EtOAc) (0.77 g, 89%, Rf = 0.17) and 10b was obtained as a
white solid, m.p.: 196 – 1978C. [a]D22 = – 18.0 (c = 0.500, MeOH). UV
(MeOH) kmax: 258 nm (e = 9495). 1H-NMR (CD3OD) d: 10.60 (br s,
1H, NH), 8.07 (d, J5,6 = 8.1 Hz, 1H, H-6), 5.86 (d, J19,29 = 9.1 Hz, 1H, H19), 5.77 (d, 1H, H-5), 4.97 (br s, 1H, H-39), 4.25 (br s, 1H, H-49), 4.14,
3.94 (q, AB-system, J = 13.0 Hz, 2H, H-59), 3.83 (m, 1H, H-29), 1.90 (s,
3H, OAc). ESI-MS (m/z): 287.71 [M + H+]. Anal. Calcd. for
C11H14N2O7: C, 46.16; H, 4.93; N, 9.79. Found: C, 46.32; H, 4.75; N,
9.83.
1-(4-O-Acetyl-2,3-dideoxy-a-D-glycero-pent-2enopyranosyl)uracil 11b
Uracil derivative 11b was synthesized from 10b by the similar
procedure as described for 11a. It was purified by flash chromatography (EtOAc) (0.40 g, 59%, Rf = 0.42) and 11b was obtained as
a white solid, m.p.: 150 – 1518C. [a]D22 = 48.0 (c = 0.500, CHCl3). UV
(CHCl3) kmax: 258 nm (e = 10696). 1H-NMR (CDCl3) d: 8.72 (br s, 1H,
NH), 7.36 (d, J5,6 = 8.1 Hz, 1H, H-6), 6.36 (m, 2H, H-19, H-39), 5.90 (d,
J29,39 = 10.1 Hz, 1H, H-29), 5.78 (d, 1H, H-5), 5.11 (m, 1H, H-49) 4.063.99 (m, 2H, H-5a9, H-5b9), 2.13 (s, 3H, OAc). ESI-MS (m/z): 253.29 [M
+ H+]. Anal. Calcd. for C11H12N2O5: C, 52.38; H, 4.80, N, 11.11.
Found: C, 52.51; H, 5.02; N, 11.48.
1-(2,3-Dideoxy-a-D-glycero-pent-2-enopyranosyl)uracil
12b
Compound 12b was synthesized from 11b by the similar procedure as described for 12a. It was purified by flash chromatography (EtOAc) (0.23 g, 70%, Rf = 0.12) and 12b was obtained as a
white foam. [a]D22 = 14.0 (c = 0.454, MeOH). UV (MeOH) kmax:
260 nm (e = 2117). 1H-NMR (CDCl3) d: 8.20 (br s, 1H, NH), 7.41 (d,
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Exomethylene and Keto D-Lyxopyranonucleoside Analogues
359
J5,6 = 8.1 Hz, 1H, H-6), 6.40 (m, 1H, H-39), 6.29 (d, J19,29 = 1.7 Hz, 1H,
H-19), 5.79 (dd, J29,39 = 10.1 Hz, 1H, H-29), 5.75 (d, 1H, H-5), 4.15 (br s,
1H, H-49), 3.98 – 3.95 (m, 2H, H-5a9, H-5b9). ESI-MS (m/z): 211.38 [M +
H+]. Anal. Calcd. for C9H10N2O4: C, 51.43; H, 4.80; N, 13.33. Found:
C, 51.52; H, 4.67; N, 13.57.
1-(2,3-Dideoxy-a-pent-2-enopyranosyl-4-ulose)uracil 13b
Compound 13b was synthesized from 12b by the similar procedure as described for 13a. It was purified by flash chromatography (EtOAc) (0.20 g, 89%, Rf = 0.45) and 13b was obtained as a
white solid, m.p.: 158 – 1608C. [a]D22 = -10.0 (c = 0.486, CHCl3). UV
(CHCl3) kmax: 258 nm (e = 3613). 1H-NMR (CDCl3) d: 9.03 (br s, 1H,
NH), 7.29 (d, J5,6 = 8.1 Hz, 1H, H-6), 6.89 (dd, J19,39 = 1.5 Hz, J29,39 =
10.5 Hz, 1H, H-39), 6.67 (br s, 1H, H-19), 6.46 (dd, J19,29 = 1.8 Hz, 1H,
H-29), 5.82 (d, 1H, H-5), 4.43-4.34 (m, 2H, H-5a9, H-5b9). ESI-MS (m/z):
209.21 [M + H+]. Anal. Calcd. for C9H8N2O4: C, 51.93; H, 3.87; N,
13.46. Found: C, 52.24; H, 3.68; N 13.61.
1-(2,3,4-Trideoxy-4-methylene-a-pent-2enopyranosyl)uracil 8b
Uracil analogue 8b was synthesized from 13b by the similar procedure as described for 8a. It was purified by flash chromatography (EtOAc / hexane, 8 : 2) (0.11 g, 57%, Rf = 0.5 in EtOAc) and 8b
was obtained as a white foam.
Antiviral assays
The antiviral assays, other than the anti-HIV assays, were based
on inhibition of virus-induced cytopathicity in HEL (herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus, and
vesicular stomatitis virus), Vero (parainfluenza-3, reovirus-1,
Sindbis, Coxsackie B4, and Punta Toro virus), or HeLa (vesicular
stomatitis virus, Coxsackie virus B4, and respiratory syncytial
virus) cell cultures. Confluent cell cultures in microtiter 96-well
plates were inoculated with 100 CCID50 of virus (1 CCID50 being
the virus dose to infect 50% of the cell cultures). After a 1 h virusadsorption period, residual virus was removed, and the cell cultures were incubated in the presence of varying concentrations
(200, 40, 8, …, lM) of the test compounds. Viral cytopathicity
was recorded as soon as it reached completion in the control
virus-infected cell cultures that were not treated with the test
compounds. The methodology of the anti-HIV assays was as follows: human CEM (l36105 cells/cm3) cells were infected with
100 CCID50 of HIV(IIIB) or HIV-2(ROD)/mL and seeded in 200 lL
wells of a microtiter plate containing appropriate dilutions of
the test compounds. After 4 days of incubation at 378C, HIVinduced CEM giant cell formation was examined microscopically.
Cytostatic assays
Murine leukemia L1210, human lymphocyte Molt4/C8 and CEM,
human breast carcinoma MCF7, and human colon carcinoma
CACO-2 were seeded in 96-well microtiter plates at 50 000 –
75 000 cells/well in RPMI-1640 culture medium in the absence or
presence of serial dilutions of the test compounds. After 2 days
(L1210) or 3 days (other cell lines), the cell numbers were
counted using a Coulter counter. The cytostatic concentration
was calculated as the CC50, or the compound concentration
required reducing cell proliferation 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.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360
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