The novel nucleoside 5-iodo-4-thio-2′-deoxyuridine (4) was synthesized and fully characterized by IR, NMR, and MS. Its structure was determined by single-crystal X-ray diffraction. Compound 4 absorbs strongly at 346 nm and is minimally toxic to human tumour cells, but its cytotoxicity is substantially enhanced by low dose UVA radiation. The combined use of 4 and UVA offers a promising route to selectively and effectively kill proliferating cells

Allen F. H.

Giuseppe Trigiante

Hong-Yan Yin

Mateusz B. Pitak

Peter Karran

Reto Brem

Simon J. Coles

Xiao-Hui Zhang

Yao-Zhong Xu

English

Zhang, Xiao-Hui

Yin, Hong-Yan

Trigiante, Joseph

Brem, Reto

Karran, Peter

Pitak, Mateusz B.

Coles, Simon J.

Xu, Yao-Zhong

Open Research Online (The Open University)

Open Research OnlineThe Open University’s repository of research publicationsand other research outputs5-iodo-4-thio-2-deoxyuridine: synthesis, structure, andcytotoxic activityJournal ItemHow to cite:Zhang, Xiao-Hui; Yin, Hong-Yan; Trigiante, Joseph; Brem, Reto; Karran, Peter; Pitak, Mateusz B.; Coles,Simon J. and Xu, Yao-Zhong (2015). 5-iodo-4-thio-2-deoxyuridine: synthesis, structure, and cytotoxic activity.Chemistry Letters, 44(2) pp. 147–149.For guidance on citations see FAQs.c© 2015 Chemical Society of JapanVersion: Version of RecordLink(s) to article on publisher’s website:http://dx.doi.org/doi:10.1246/cl.140965http://www.journal.csj.jp/cl-article/cl-44-2-147Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyrightowners. For more information on Open Research Online’s data policy on reuse of materials please consult the policiespage.oro.open.ac.uk5-Iodo-4-thio-2¤-deoxyuridine: Synthesis, Structure, and Cytotoxic ActivityXiao-Hui Zhang,*1 Hong-Yan Yin,1 Giuseppe Trigiante,2 Reto Brem,3 Peter Karran,3Mateusz B. Pitak,4 Simon J. Coles,4 and Yao-Zhong Xu*21College of Environment and Chemical Engineering, Dalian University, Dalian 116622, P. R. China2Department of Life, Health and Chemical Sciences, the Open University, Walton Hall, Milton Keynes, MK7 6AA, UK3Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK4UK National Crystallography Service, Chemistry, Faculty of Natural and Environmental Sciences,University of Southampton, Highfield Campus, Southampton, SO17 1BJ, UK(E-mail: y.z.xu@open.ac.uk)The novel nucleoside 5-iodo-4-thio-2¤-deoxy-uridine (4) was synthesized and fully charac-terized by IR, NMR, and MS. Its structure wasdetermined by single-crystal X-ray diﬀraction.Compound 4 absorbs strongly at 346 nm and isminimally toxic to human tumour cells, but itscytotoxicity is substantially enhanced by lowdose UVA radiation. The combined use of 4 andUVA oﬀers a promising route to selectively andeﬀectively kill proliferating cells.REPRINTED FROMVol.44 No.2 2015 p.147–149CMLTAGFebruary 5, 2015The Chemical Society of Japan5-Iodo-4-thio-2¤-deoxyuridine: Synthesis, Structure, and Cytotoxic ActivityXiao-Hui Zhang,*1 Hong-Yan Yin,1 Giuseppe Trigiante,2 Reto Brem,3 Peter Karran,3Mateusz B. Pitak,4 Simon J. Coles,4 and Yao-Zhong Xu*21College of Environment and Chemical Engineering, Dalian University, Dalian 116622, P. R. China2Department of Life, Health and Chemical Sciences, the Open University, Walton Hall, Milton Keynes, MK7 6AA, UK3Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK4UK National Crystallography Service, Chemistry, Faculty of Natural and Environmental Sciences,University of Southampton, Highfield Campus, Southampton, SO17 1BJ, UK(E-mail: y.z.xu@open.ac.uk)The novel nucleoside 5-iodo-4-thio-2¤-deoxyuridine (4) wassynthesized and fully characterized by IR, NMR, and MS. Itsstructure was determined by single-crystal X-ray diﬀraction.Compound 4 absorbs strongly at 346 nm and is minimally toxicto human tumour cells, but its cytotoxicity is substantiallyenhanced by low dose UVA radiation. The combined use of 4and UVA oﬀers a promising route to selectively and eﬀectivelykill proliferating cells.Nucleic acids (DNA and RNA) direct the ﬂow of geneticinformation. In addition to the four canonical bases, structurallysimilar base analogues can be incorporated into DNA. Thesealter its properties and facilitate basic studies of DNA-relatedprocesses such as its interaction with proteins, replication, andtranscription. Site-speciﬁcally modiﬁed bases and nucleosidesare particularly useful in this regard, and thiobases, in whichan oxygen atom in the base is replaced by sulfur,1­3 haveproved invaluable in studies of DNA duplex stability, proteinrecognition, and replication.4­6 We have been exploring thephotochemical properties of thiobases and their derivatives,particularly 6-thioguanine (6-TG) and 4-thiothymidine (4-thioTdR). Unlike canonical DNA bases, both are strong UVAchromophores and are extremely sensitive to wavelengths of315­400 nm (UVA range). Upon exposure to UVA radiation,DNA-embedded 6-thioguanine is oxidized to guanine-6-sulﬁnate and guanine-6-sulfonate in an oxygen-dependentreaction.7­9 UVA irradiation of cells containing DNA 6-TG alsoinduces DNA strand breakage and interstrand crosslinking.10These DNA lesions block replication and transcription andcause cell death.11 DNA 4-thioTdR also displays synergisticlethality with UVA radiation. It undergoes UVA-mediatedconversion to potentially cytotoxic lesions including inter- andintrastrand DNA crosslinks.12 In the absence of UVA, cells canaccumulate signiﬁcant levels of 4-thioTdR without detrimentaleﬀects on proliferation.13,14 The combined use of 4-thioTdRand UVA oﬀers a novel approach to selectively kill rapidlyproliferating cells, such as cancer cells, while causing minimaldamage to normal tissues.15 The advantage this synergisticphotoactivation of DNA thiobases has over conventionaltherapies is that it can target proliferating cells more selective-ly. We have therefore explored the properties of additional 4-thioTdR analogues and have previously reported the synthesisand chemical properties of 5-bromo-4-thio-2¤-deoxyuridine.16Here we report a synthetic route for 5-iodo-4-thio-2¤-deoxy-uridine. We also describe its crystal structure and initial studiesof its cytotoxicity.The synthetic route is shown in Scheme 1 in which thestarting material 1 was ﬁrst acetylated at its 3¤- and 5¤-positionsto give the acetylated nucleoside 2, followed by a thiolation atthe 4-postion to give the protected 4-thio-nucleoside 3. This wasthen deprotected to aﬀord the target product 4. Previously weused triazole/POCl3 and thioacetic acid to produce 3.17a Thecurrent synthesis is straightforward and proceeds with goodyields (see ESI for details). The intermediates 2 and 3 andthe product 4 were fully characterized by MS, UV, IR spectra,1HNMR and 13CNMR measurements (see ESI for details).Some key spectral data of 1 and 4 are summarized in Table 1 forcomparison.The spectral characterization provides a good conﬁrmationfor the structure of 4. High-resolution MS analysis of 4 gavean ion at 392.9382 for the molecular fragment (M + Na+), them/z being within 1 ppm of the calculated m/z (392.9378) forC9H11IN2NaO4S. 4 absorbs strongly at 346 nm, substantiallyshifted from the 290 nm maximum of 1 (Figure S1, see ESI).This red shift reﬂects the presence of the sulfur atom.16 Theobserved band at 1088 cm¹1 in the infrared spectrum is withinthe characteristic range (1070­1150 cm¹1) assigned to the C=Sstretching mode of the thioamide group in 4-thiouridine,18indicating the presence of the C4=S group in 4. NMR dataoﬀer further support. D2O exchange experiments readily identifythe signal in ¤H 12.99 ppm as proton N3­H. The presence of thethioamide in 4 is also evident from the substantial NMRHNNOOOORROIHNNOSOORROI3: R = Ac4: R = H4 41: R = H2: R = Ac(i), (ii), (iii)Scheme 1. Synthesis of 5-iodo-4-thio-2¤-deoxyuridine (4).(i) Ac2O/pyridine (91% for 2); (ii) P4S10/1,4-dioxane (74%for 3); (iii) NH3(g)/MeOH (68% for 4).Table 1. Key spectral data of compounds 1 and 4CpdUV(­max/nm)IR/cm¹11HNMR(N3­H)/ppm13CNMR(C4)/ppm1 290 1677 (C4=O) 11.66 160.484 346 1088 (C4=S) 12.99 189.31Received: October 21, 2014 | Accepted: November 7, 2014 | Web Released: November 18, 2014 CL-140965Chem. Lett. 2015, 44, 147–149 | doi:10.1246/cl.140965 © 2015 The Chemical Society of Japan | 147chemical shifts17 of N3­H (12.99 from 11.66) and of C4 (189.31from 160.48) (Table 1).The molecular structure and absolute conﬁguration of 4(Figure 1) was conﬁrmed by single-crystal X-ray diﬀractionanalysis.The structure belongs to the triclinic system and space groupP1 (Table S1, see ESI). It is worth noting that the unit cell of thecrystal structure of 5-iodo-4-thio-2¤-deoxyuridine contains threeindependent molecules (Figure S2, see ESI). It is not unusual tohave two independent molecules of a nucleoside in a single unitcell, for example deoxyuridine and 5-bromo-2¤-deoxyuridine,19but it is rare for the same unit cell to contain three or morenucleosides.20Figure 2 shows the comparison of the lengths and angles ofindividual bonds in the bases 4 (5-iodo-4-thio-2¤-deoxyuridine)and 1 (5-iodo-2¤-deoxyuridine).21 Overall, these two molecularstructures are very similar, except the bond involving the sulfuratom at the 4-position. The thiocarbonyl (C4=S) bond in 4 issubstantially longer than the carbonyl (C4=O) bond in 1. Theaverage length (from three individual molecules of 4) of C4=Sbond is 1.662¡ and is similar to that of the documented C=Sbond of 4-thiouracil derivatives (1.672¡) and the mean C=Sdistance (1.654­1.665¡) reported by Allen et al.22 This lengthis much shorter than a standard C­S single bond of 1.80¡,demonstrating that the thiocarbonyl group is in fact a doublebond. The angle ¾SC4C5 (126°) is slightly wider than the angle¾OC4C5 (122°), and the C4=S bond tilts away from the iodogroup at the 5-position.In the crystal lattice each of three independent molecules arelinked together by an intermolecular hydrogen-bond network(Figure S3, see ESI). Each independent molecule serves as adonor and acceptor of moderate O­H£O (D£A µ 2.765¡) andN­H£O (D£A µ 2.285¡) type hydrogen bonds. Additionally,each sulfur atom is involved in a weak O­H£S (D£A µ3.394¡) hydrogen bond (Table S2). The C=S bond is knownto have a broader distance range and to be less polar than aCδ+=Oδ¹ bond, thus it is also a weaker hydrogen bond acceptor.For C=S acceptors, the mean hydrogen bond distances are morethan 0.5¡ longer than for C=O acceptors. This is also largerthan the diﬀerence of the S and O van der Waals radii andthe lone-pair directionality at C=S acceptors is very pro-nounced.22,23 Interestingly, systematic studies by Allen et al.22showed that only the longer C=S bonds appear to be involved inhydrogen bonding (the percentage of hydrogen bonds formed byC=S increases systematically from 5% to 75% as the C=S bondlength increases from 1.63 to 1.75¡). Detailed bond lengths andangles for the three individual molecules present in the crystalstructure of 4 are available from the CCDC, depository number:959345.5-Iodo-2¤-deoxyuridine (1) was one of the ﬁrst selectiveantiviral nucleosides.24 It is still used in the treatment of variousdiseases including herpetic keratitis25 and for radiosensitizationof high-grade gliomas.26 Previously we showed 4-thioTdR hasvery similar base-pairing properties to thymidine.14 As 4 and 1diﬀer only slightly in structure, it is highly likely that theirbehavior would be similar in terms of incorporation into viralDNA and base-pairing. However, the strong UVA absorption of4 would enhance its potential as an UVA-mediated antiviralagent or DNA probe. We therefore examined the eﬀects ofphotochemical activation of 4 on human cells growing in culture.Compound 4 was toxic to cultured human cells only at high¯M concentrations. Figure 3 shows the eﬀects of compound 4on the viability of the human oral cell carcinoma CA-1 cells.Continuous 24 h exposure induced a dose-dependent increase incytotoxicity with an approximate LD50 of 320¯M. Clearly 4 wasminimally toxic to the cultured human cells.Figure 1. Molecular structure and absolute conﬁguration of 4.Only one independent molecule present in the crystal structureis shown. Full structural data are deposited at the CambridgeCrystallographic Data Centre, depository number: 959345.Figure 2. Selected bond lengths (¡) and angles (°) of the basesin 4 and 1. For compound 4 the average values of cell bonddistances and angles of three independent molecules present inthe asymmetric part of the unit are shown.Figure 3. Dose-dependent cytotoxicity of 4 in human oral cellcarcinoma CA-1 cells. The solid line is the experimental dataand the dotted line is a ﬁtted curve.148 | Chem. Lett. 2015, 44, 147–149 | doi:10.1246/cl.140965 © 2015 The Chemical Society of JapanThe toxicity of 4 towards human HEK293 embryonickidney cells was similar and treatment with 100 or 200¯M for48 h reduced survival by 10­20% (Figure 4). Direct analysis ofDNA from treated cells conﬁrmed a dose-dependent accumu-lation of 4 in DNA. The presence of DNA-embedded 4 wasassociated with a signiﬁcant sensitization to nontoxic dose ofUVA (340 nm) (Figure 4).UVA doses up to 20 kJm¹2 caused only ¯10% toxicity inthe absence of drug pretreatment, but cell survival wassigniﬁcantly reduced after combined compound 4/UVA treat-ment in both a UVA-dependent and compound 4-dependentfashion. At the highest drug concentration (200¯M) and20 kJm¹2, UVA reduced cell viability by >99.5%. Thesepreliminary results suggest that 5-iodo-4-thio-2¤-deoxyuridinein combination with UVA is more cytotoxic than 5-bromo-4-thio-2¤-deoxyuridine.16 This increased cytotoxicity of 4 mightreﬂect a lower bond enthalpy for the C­I bond27 (e.g. DH298 =241 kJmol¹1 for CH3­I) compared to that for C­Br bond (e.g.DH298 = 302 kJmol¹1 for CH3­Br), suggesting 5-iodo-4-thio-2¤-deoxyuridine would be more susceptible to UVA-inducedactivation than 5-bromo-4-thiodeoxyuridine.16 Studies areongoing to deﬁne the nature of the lethal DNA lesions and themechanism of cytotoxicity of the 5-iodo-4-thio-2¤-deoxyuridine/UVA combination.In conclusion, a UVA-sensitive nucleoside, 5-iodo-4-thio-2¤-deoxyuridine, has been synthesized in a good yield and fullycharacterized by spectroscopy. A single-crystal structure wasalso obtained by X-ray analysis. The thio-nucleoside wasincorporated eﬀectively into DNA of cultured human cells.This caused a modest antiproliferative eﬀect that was greatlyenhanced by low doses of UVA radiation. The combined use of5-iodo-4-thio-2¤-deoxyuridine and UVA oﬀers a promising routeto selectively and eﬀectively kill proliferating cells.The project was sponsored by the Scientiﬁc ResearchFoundation for the Returned Overseas Chinese Scholars, StateEducation Ministry, P. R. China. RB and PK are supported byCancer Research UK. Dr. Xu is grateful to Prof. Peter Taylor andDr. Youssef Elaziz for technical assistance.Supporting Information is available electronically on J-STAGE.References1 A. G. Lezius, K. H. Scheit, Eur. J. Biochem. 1967, 3, 85.2 M. Sprinzl, K.-H. Scheit, F. Cramer, Eur. J. Biochem. 1973,34, 306.3 R. S. Coleman, E. A. Kesicki, J. Am. Chem. Soc. 1994, 116,11636.4 I. V. Kutyavin, R. L. Rhinehart, E. A. Lukhtanov, V. V. Gorn,R. B. Meyer, Jr., H. B. Gamper, Jr., Biochemistry 1996, 35,11170.5 A. M. Sismour, S. A. Benner, Nucleic Acids Res. 2005, 33,5640.6 H. O. Sintim, E. T. Kool, J. Am. Chem. Soc. 2006, 128, 396.7 P. O’Donovan, C. M. Perrett, X. Zhang, B. Montaner, Y.-Z.Xu, C. A. Harwood, J. M. McGregor, S. L. Walker, F.Hanaoka, P. Karran, Science 2005, 309, 1871.8 X. Zhang, G. Jeﬀs, X. Ren, P. O’Donovan, B. Montaner,C. M. Perrett, P. Karran, Y.-Z. Xu, DNA Repair (Amst.) 2007,6, 344.9 X. Ren, F. Li, G. Jeﬀs, X. Zhang, Y.-Z. Xu, P. Karran, NucleicAcids Res. 2010, 38, 1832.10 R. Brem, P. Karran, Cancer Res. 2012, 72, 4787.11 R. Brem, F. Li, P. Karran, Nucleic Acids Res. 2009, 37, 1951.12 O. Reelfs, P. Macpherson, X. Ren, Y.-Z. Xu, P. Karran, A. R.Young, Nucleic Acids Res. 2011, 39, 9620.13 A. Massey, Y.-Z. Xu, P. Karran, Curr. Biol. 2001, 11, 1142.14 A. Massey, Y.-Z. Xu, P. Karran, DNA Repair (Amst.) 2002, 1,275.15 a) O. Reelfs, Y.-Z. Xu, A. Massey, P. Karran, A. Storey, Mol.Cancer Ther. 2007, 6, 2487. b) S. W. Pridgeon, R. Heer, G. A.Taylor, D. R. Newell, K. O’Toole, M. Robinson, Y.-Z. Xu, P.Karran, A. V. Boddy, Br. J. Cancer 2011, 104, 1869.16 Y.-Z. Xu, X. Zhang, H.-C. Wu, A. Massey, P. Karran, Bioorg.Med. Chem. Lett. 2004, 14, 995.17 a) X. Zhang, Y.-Z. Xu,Molecules 2011, 16, 5655. b) X. Zhang,J. Wang, Y.-Z. Xu, Magn. Reson. Chem. 2013, 51, 523.18 H. Rostkowska, K. Szczepaniak, M. J. Nowak, J.Leszczynski, K. KuBulat, W. B. Person, J. Am. Chem. Soc.1990, 112, 2147.19 a) A. Rahman, H. R. Wilson, Nature 1971, 232, 333. b) J.Iball, C. H. Morgan, H. R. Wilson, Proc. R. Soc. London, Ser.A 1966, 295, 320.20 W. Saenger, J. Am. Chem. Soc. 1972, 94, 621.21 N. Camerman, J. Trotter, Acta Crystallogr. 1965, 18, 203.22 F. H. Allen, C. M. Bird, R. S. Rowland, P. R. Raithby, ActaCrystallogr., Sect. B: Struct. Sci. 1997, 53, 680.23 G. Desiraju, T. Steiner, The Weak Hydrogen Bond inStructural Chemistry and Biology, OUP, Chichester, 1999.24 W. H. Prusoﬀ, Biochim. Biophys. Acta 1959, 32, 295.25 E. De Clercq, Biochem. Pharmacol. 2013, 85, 727.26 S. Kummar, L. Anderson, K. Hill, E. Majerova, D. Allen, Y.Horneﬀer, S. P. Ivy, L. Rubinstein, P. Harris, J. H. Doroshow,J. M. Collins, Clin. Cancer Res. 2013, 19, 1852.27 S. J. Blanksby, G. B. Ellison, Acc. Chem. Res. 2003, 36, 255.Figure 4. UVA sensitization by compound 4 in HEK cells.Cells were treated in growth medium with compound 4 for 48 hat the concentrations shown (see ESI for details).Chem. Lett. 2015, 44, 147–149 | doi:10.1246/cl.140965 © 2015 The Chemical Society of Japan | 149

5-iodo-4-thio-2′-deoxyuridine: synthesis, structure, and cytotoxic activity

Crossref

Chemistry Letters

5-Iodo-4-thio-2′-deoxyuridine: Synthesis, Structure, and Cytotoxic Activity

http://oro.open.ac.uk/42190/1/__userdata_documents2_yzx2_Desktop_Chem-Lett-F_44_147.pdf

5-iodo-4-thio-2′-deoxyuridine: synthesis, structure, and cytotoxic activity

Abstract

Similar works

Full text

Available Versions

Open Research Online (The Open University)

Crossref