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BY-NC-ND 3.0 license Open Access Published by De Gruyter September 24, 2015

5′-Norcarbocyclic analogues of furano[2,3-d]pyrimidine nucleosides

  • Elena S. Matyugina , Evgeniya B. Logashenko , Marina A. Zenkova , Sergey N. Kochetkov and Anastasia L. Khandazhinskaya EMAIL logo

Abstract

5′-Norcarbocyclic analogues of furano[2,3-d]pyrimidine nucleosides as well as 5-bromo and 5-iodouracil derivatives were synthesized to evaluate their potential antitumor activity. The halogenated derivatives display no cytotoxicity with respect to all tested cells: KB-3-1 (human epidermoid carcinoma), HeLa (human cervical epithelioid carcinoma), HuTu-80 (human duodenal cancer), B16 (mouse melanoma), and MDCK (normal epithelial). The cytotoxicity of the non-halogenated furano[2,3-d]pyrimidine derivatives increases with the lengthening of the alkyl chain of the substituent from 45 to 60 μm for octyl to from 3 to 10 μm for dodecyl.

Bicyclic furano[2,3-d]pyrimidine nucleosides are known primarily for their high inhibitory activity against the herpes virus family. The compounds bearing a 2′-deoxyribose moiety are not toxic and are highly effective inhibitors of varicella-zoster virus, whereas analogues containing 2′,3′-dideoxyribose or acyclic fragments inhibit human cytomegalovirus (Figure 1). Attempts to replace the 2′-deoxyribose sugar of these furano[2,3-d] pyrimidine nucleosides by other carbohydrate fragments (such as ribose, arabinose, or 2′-methylribose) or their carbocyclic analogues led to partial or complete loss of antiviral activity [1–2]. It has been shown that the antiviral activity of bicyclic furano[2,3-d]pyrimidine nucleosides requires the nucleosides to be phosphorylated by viral deoxythymidine kinase; however, the full mechanism of inhibitory action has not been elucidated.

Figure 1 Bicyclic furano[2,3-d]pyrimidine nucleoside analogues.
Figure 1

Bicyclic furano[2,3-d]pyrimidine nucleoside analogues.

Recently, the first papers describing the antitumor potential of bicyclic furano and pyrrolo[2,3-d]pyrimidine nucleosides have been published. According to these reports, some of these analogues have the ability to not only inhibit the growth of various tumor cell lines but also to induce apoptosis [3–5].

Carbocyclic analogues of nucleosides are a separate class of modified nucleosides in which the oxygen atom of the carbohydrate moiety is replaced by a methylene or methine group. Over the past 20 years, many compounds of this class have become the object of intense study both as antiviral, antiparasitic, and anticancer agents [6]. A characteristic feature of the 5′-norcarbocyclic nucleoside analogues is the lack of the 5′-methylene group and, consequently, the failure to undergo intracellular phosphorylation [7]. This makes them a useful tool for determining the necessity of phosphorylation for biological activity.

Compounds 4a–c as well as 5-bromo and 5-iodouracil derivatives 6 and 8 were synthesized to evaluate the potential antitumor activity of 5′-norcarbocyclic nucleoside analogues. The 5-halogenated substituents were selected owing to their widespread use in anticancer therapy [8–10]. Recent reports have indicated that halogens are critical for anticancer activity in similar heterocyclic scaffolds [11, 12]. In this work, compound 6 is an intermediate product in the synthetic route to the targets compounds 4a–c.

The 5-halogenated derivatives 6 and 8 were prepared by reaction of 6-oxabicyclo[3.1.0.]hex-2-ene (5) and 5-bromouracil or 5-iodouracil under Trost conditions of palladium catalysis [13] (Scheme 1). Following standard procedures for isolation and purification, the yields of the target compounds 6 and 8 were in the range of 28–35%. According to published data, the yield of the condensation reaction is typically not high, ranging on average between 30% and 45%. The use of an excess of 6-oxabicyclo[3.1.0.]hex-2-ene 5 increases the yield of the target compounds 6 and 8 up to 35–49%, however, with the concomitant formation of a byproduct 7 in a yield of 36%.

Scheme 1
Scheme 1

Initially, compounds 4a–c were obtained as byproducts in the synthesis of 5-alkynyl derivatives of 5′-norcarbocyclic uracil by the Sonagashira method [14], but their yields in our hands did not exceed 15%. In this work, the synthesis of 4a–c by the reaction of 6 with an alkyne was carried out in boiling acetonitrile in the presence of 10% palladium on carbon, copper iodide, and triethylamine. The target products 4a–c were obtained as a mixture of enantiomers in 39–47% yields. The structures and purity of the synthesized compounds were confirmed by 1H and 13C NMR spectroscopy, TLC, and mass spectrometry. All values were in accordance with the literature data [14].

We compared the cytotoxic activity of the new derivatives 4a–c, 6, and 8against different type of cell lines. KB-3-1 (human epidermoid carcinoma), HeLa (human cervical epithelioid carcinoma), HuTu-80 (human duodenal cancer), B16 (mouse melanoma), and MDCK (normal epithelial) cells were used. The cytotoxicity was determined using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay, a colorimetric test of cell viability after the treatment with each of the compounds under the study. The IC50 values (the concentration of a compound allowing survival of 50% of the cells in a population) of the tested compounds are listed in Table 1. The well-known 5′-norcarbocyclic nucleoside analogue, 5′-noraristeromycin [15, 16], was used as a control.

Table 1

IC50 values of the compounds with respect to cancer and normal cell lines (μm).a

Cell lines4a4b4c685′-Noraristeromycin
B-164543>>100>>100>>100
HeLa4064>100>100>100
HuTu804066>100>100≥100
KB-3-1504.55>10080>100
MDCK60510>>100>>100>>100

aIC50 was defined as the compound concentration that results in 50% cell survival as measured by the MTT assay.

As can be seen from Table 1, the halogenated derivatives 6 and 8 display no cytotoxicity with respect to all tested cells. Similarly, 5′-noraristeromycin is not toxic to cultivated cancer and normal cells. The cytotoxicity of the furano[2,3-d]pyrimidine derivatives increases with the lengthening of the alkyl chain of the R group at C-5 from 45–60 μm for 4a to 3–10 μm for 4c. A similar relationship has been previously described for the antiviral activity of furano[2,3-d]pyrimidine nucleosides [1, 2], with the optimal substituents for inhibition of the herpes viruses being the octyl- or p-pentylphenyl groups. In our case, the critical point was the transition from C8 to C10 (4a and 4b). Notably, for dodecyl-containing compound 4c (C12), some selectivity was observed between cytotoxicity with respect to the tumor (IC50 from 3 to 6 μm) and normal (IC50 10 μm) cells. In general, however, there was no significant selectivity observed for compounds tested. Further study of the 5′-norcarbocyclic furano [2,3-d]pyrimidine nucleoside analogues will be done for better understanding of the structure- antitumor activity relationship.

Experimental

1-(4′-Hydroxy-2′-cyclopenten-1′-yl)-3-(4′″-hydroxy-2″′-cyclopenten-1″′-yl)-5-iodouracil (7)

This compound has been previously obtained as a side product in the synthesis of 6 [14]. Purification on a silica gel column eluting with CHCl3:MeOH (98:2) gave product 7 as a yellow syrup; yield 120 mg (36%); 1H NMR (CDCl3): δ 7.85 (1H, s, H-6), 6.26 (1H, m, H-2′), 6.11 (1H, m, H-2″), 5.92 (1Н, m, H-1′) 5.78 (1Н, m, H-1″), 5.70 (1Н, m, H-3′), 5.52 (1H, m, H-3″), 4.83 (1H, m, H-4′), 4.68 (1H, m, H-4″), 3.93 (1Н, m, ОН′), 3.39 (1Н, s, ОН″), 2.66 (2H, m, Ha-5′,Ha-5″′), 1.91 (1H, m, Hb-5′), 1.59 (1H, m, Hb-5′″); 13C NMR (DMSO-d6): 162.3, 150.3 (C-4, C-2), 143.3 (C-6), 129.9 (C-5), 129.2 (C-2′, C-2″), 126.8 (C-1″), 116.6 (C-1′), 114,9 (C-3′, C-3″), 40.1 (C-4′), 38.9 (C-1′), 20.1 (C-5′, C-5″). HRMS. Calcd for C14H15IN2O4 ([M+H]+): m/z 403.0145. Found: m/z 403.0142.

1-(4′-Hydroxy-2′-cyclopenten-1′-yl)-5-bromouracil (8)

This compound was obtained from 6-oxabicyclo[3.1.0.]hex-2-ene (5) and 5-bromouracil as described earlier [14]. Purification on silica gel column eluting with CHCl3/MeOH (95:5) gave product 8 as a pale yellow powder; yield 320 mg (42%); 1H NMR (CDCl3): δ 7.83 (1H, s, H-6), 6.17 (1H, m, H-2′), 5.83 (1Н, m, H-1′), 5.39 (1Н, m, H-3′), 5.29 (1Н, m, ОН′), 4.62 (1H, m, H-4′), 2.70 (1H, m, Ha-5′), 1.44 (1H, m, Hb-5′); 13C NMR (DMSO-d6): δ 159.2, 150.2 (C-4, C-2), 141.5 (C-6), 140.4 (C-2′), 130,7 (C-3′), 95.4 (C-5), 73.2 (C-4′), 58.8 (C-1′), 39.5 (C-5′). HRMS. Calcd for C9H9BrN2O3([M+H]+): m/z 272.9869. Found: m/z 272.9869.

Biological assay

Human KB-3-1 epidermoid carcinoma cell line, HeLa cervical epithelioid carcinoma cell line, human HuTu-80 duodenal cancer cells, mouse B16 melanoma cell line, and MDCK epithelial cells (Russian Cell Culture Collection, St. Petersburg) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL), and amphotericin (250 μg/mL). Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C.

Cells growing in the logarithmic phase were seeded in triplicate in 96-well plates at a density of 105 cells per well. The plates were incubated at 37°C in a humidified 5% CO2 atmosphere. Cells were allowed to adhere to the surface for 24 h, and then they were treated with varying doses of the tested compounds over 48 h. Later, aliquots of MTT solution (10 μL, 5 mg/mL) were added to each well, and the incubation was continued for an additional 3 h. Dark blue formazan crystals that formed within the healthy cells were solubilized with DMSO, and the absorbance was measured in a plate reader Multiscan RC (Thermo LabSystems, Finland) at 570 nm. IC50 values were determined as the compound concentration required to decrease the absorption at 570 nm to 50% of the control (no test compounds, DMSO) value and were determined by interpolation from dose-response curves.


Corresponding author: Anastasia L. Khandazhinskaya, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russian Federation, e-mail:

Acknowledgments

This work was supported by the RFBR project No. 13-04-00742 and RAS program Molecular and Cellular Biology.

References

[1] Balzarini, J.; McGuigan, C. Bicyclic pyrimidine nucleoside analogues (BCNAs) as highly selective and potent inhibitors of varicella-zoster virus replication. J. Antimicrob. Chemother. 2002, 50, 5–9.Search in Google Scholar

[2] Jahnz-Wechmann, Z.; Framski, G.; Januszczyk, P.; Boryski, J. Bioactive fused heterocycles: nucleoside analogs with an additional ring. Eur. J. Med. Chem. 2015, 97, 388–396.Search in Google Scholar

[3] Gangjee, A.; Devraj, R. McGuire, J. J.; Kisliuk, R. L.; Queener, S. F.; Barrows, L. R. Classical and nonclassical furo[2,3-d]pyrimidines as novel antifolates: synthesis and biological activities. Med. Chem.1994, 37, 1169–1176.Search in Google Scholar

[4] Romeo, R.; Giofrè, S. V.; Garozzo A.; Bisignano, B.; Corsaro, A.; Chiacchio M. A. Synthesis and biological evaluation of furopyrimidine N,O-nucleosides. Bioorg. Med. Chem. 2013, 21, 5688–5693.Search in Google Scholar

[5] Framski, G.; Wawrzyniak, D.; Jahnz-Wechmann, Z.; Szymańska-Michalak, A.; Barciszewski, J.; Boryski, J.; Kraszewski, A.; Stawiński, J. New Applications of 6-alkyl-2,3-dihydrofurano[2,3-d]pyrimidin-2(1H)-one and 6-Alkyl-2,3-dihydropyrrolo[2,3-d]pyrimidin-2(3H,7H)one nucleosides: anticancer properties. XXI Round Table on Nucleosides, Nucleotides and Nucleic Acids. Chemical Biology of Nucleic Acids, Electronic Abstract Book, Poster 13. IS3NA, 2014.Search in Google Scholar

[6] Matyugina, E.; Khandazhinskaya, A.; Kochetkov, S. Carbocyclic nucleoside analogues: classification, target enzymes, mechanisms of action and synthesis. Russ. Chem. Rev.2012, 81, 729–746.Search in Google Scholar

[7] Wang, J.; Rawa, R. K.; Chu, C. K. Medicinal Chemistry of Nucleic Acids; First Edition; John Wiley & Sons: Hoboken, NJ, USA, 2011.Search in Google Scholar

[8] Cavanagh, B. L.; Walker, T.; Norazit, A.; Meedeniya, A. C. B. Thymidine analogues for tracking DNA synthesis. Molecules2011, 16, 7980–7993.Search in Google Scholar

[9] Galmarini, C. M.; Mackey, J. R.; Dumontet, C. Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol.2002, 3, 415–424.Search in Google Scholar

[10] Li, X.; Patel, R.; Melamed, M. R.; Darzynkiewicz, Z. The cell cycle effects and induction of apoptosis by 5-bromouridine in cultures of human leukaemic MOLT-4 and HL-60 cell lines and mitogen-stimulated normal lymphocytes. Cell proliferation1994, 27, 307–319.Search in Google Scholar

[11] Ross, C. R.; Temburnikar, K. W.; Wilson, G. M.; Seley-Radtke; K. L. Mitotic arrest of breast cancer MDA-MB-231 cells by halogenated thieno[3,2-d]pyrimidines. Bioorg. Med. Chem. Lett. 2015, 25, 1715–1717.Search in Google Scholar

[12] Temburnikar, K.; Zimmermann, S. C.; Kim. N.; Ross, C.; Gelbmann, C.; Salomon, C.; Wilson, G.; Balzarini, J.; Seley-Radtke, K. L. Antiproliferative activity of halogenated thieno[3,2-d]pyrimidines. Bioorg. Med. Chem.2014,22, 2113–2122.Search in Google Scholar

[13] Trost, B. M.; Van Vranken, D. V.; Bingel, C. A modular approach for ligand design for asymmetric allylic alkylations via enantioselective palladium-catalysed ionizations. J. Am. Chem. Soc.1992, 114, 9327–9343.Search in Google Scholar

[14] Matyugina, Е; Khandazhinskaya, А; Chernousova, L; Andreevskaya, S; Smirnova, T; Chizhov, A; Karpenko, I; Kochetkov, S; Alexandrova, L. The synthesis and antituberculosis activity of 5′-norcarbocyclic uracil derivatives. Bioorg. Med. Chem. 2012, 20, 6680–6686.Search in Google Scholar

[15] Matyugina, E. S.; Khandazinskaya, A. L. 5′-Norcarbocyclic nucleoside analogues. Russ. Chem. Bull. 2014, 63, 1069–1080.Search in Google Scholar

[16] Siddiqi, S. M.; Chen, X.; Schneller, S. W.; Ikeda, S.; Snoeck, R.; Andrei, G.; Balzarini, J.; De Clercq, E. Antiviral enantiomeric preference for 5′-noraristeromycin. J. Med. Chem.1994, 37, 551–554.Search in Google Scholar

Received: 2015-7-30
Accepted: 2015-8-20
Published Online: 2015-9-24
Published in Print: 2015-10-1

©2015 by De Gruyter

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