Bioorgnic & Medicinl Chemistry 17 (2009) 6824 6831 Contents lists vilble t ScienceDirect Bioorgnic & Medicinl Chemistry journl homepge: www.elsevier.com/locte/bmc Synthesis nd in vitro ctivities of new nticncer duplex drugs linking 2 0 -deoxy-5-fluorouridine (5-dU) with 3 0 -C-ethynylcytidine (ECyd) vi phosphodiester bonding Herbert Schott, *, Srh Schott b, Reto A. Schwendener c Institute of rgnic Chemistry, University Tuebingen, Auf der Morgenstelle 18, D-72076 Tuebingen, Germny b Deprtment of Gynecology nd bstetrics, University of Heidelberg Medicl School, D-69120 Heidelberg, Germny c Institute of Moleculr Cncer Reserch, University of Zuerich, CH-8057 Zuerich, Switzerlnd rticle info bstrct Article history: Received 29 My 2009 Revised 15 August 2009 Accepted 18 August 2009 Avilble online 21 August 2009 Keywords: 2 0 -Deoxy-5-fluorouridine (5-dU) 3 0 -C-Ethynylcytidine (ECyd) Antimetbolites Cytosttic duplex drugs Hydrogenphosphonte method Two isomeric cytosttic duplex drugs 2 0 -deoxy-5-fluorouridylyl-(3 0?5 0 )-3 0 -C-ethynylcytidine [5- du(3 0?5 0 )ECyd] nd 2 0 -deoxy-5-fluorouridylyl-(5 0?5 0 )-3 0 -C-ethynylcytidine [5-dU(5 0?5 0 )ECyd] were designed nd synthesized t grm scle ccording to the hydrogenphosphonte method in n overll yield of bout 40%. The in vitro evlution of the nticncer effects indicted highly vrying sensibilities of the pnel of 60 tested tumor cell lines ginst the duplex drugs. 5-dU(3 0?5 0 )ECyd hd 50% growth inhibition (IC 50 6 10 8 M) in 44/58 cell lines. However, only 25/53 of those cell lines showed corresponding IC 50 vlues when the isomeric 5-dU(5 0?5 0 )ECyd ws tested. Totl growth inhibition ws chieved using micromolr concentrtions of the duplex drugs. The 5-dU residue of the duplex drug cn cuse very different effects like dditive, synergistic, ntgonistic s well s sequence-depending ctivities, which drsticlly chnged efficiency s well s specificity of the nticncer ctivities of the duplex drugs, in comprison to those of the monomeric drugs. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction It is known tht ntitumor chemotherpy is unsuccessful if resistnce ginst the dministered cytosttic drug occurs. Drug monotherpy, using only one nticncer compound, often leds to drug resistnce. The combintion therpy s n lterntive, which is bsed on simultneous or sequentil ppliction of vrious nticncer drugs, cn optimize the therpeutic success nd my prevent resistnce. However, combintion therpy schedules re often complex nd exhusting for the ptients. Another promising but not yet well-evluted possibility of nticncer combintion therpy consists in the chemicl linkge of two different, cliniclly well-chrcterized cytosttic drugs into one molecule, so clled duplex drug. After ppliction of duplex drug s monotherpy the molecule should be degrded into mixture of severl metbolites ech possessing different cytosttic profiles with dditive or synergistic properties. This concept could exploit the dvntge of combintion therpy without dditionl burden for ptients. Cytosttic drugs re suitble for the design of duplex drugs provided tht they vry considerbly in their nticncer mechnisms in order to cuse dditive or synergistic ntitumor ctivities nd, optimlly, simultneous reduction of side effects. * Corresponding uthor. x: +49 7071 65782. E-mil ddress: herbert.schott@uni-tuebingen.de (H. Schott). The chemicl coupling of two single compounds to new duplex drug must go long with justifible synthetic effort. urthermore, the moleculr structure of the creted duplex drug should llow n in vivo metbolism resulting in multiple ctive compounds. ucleoside nlogues 1 3 hve become mjor clss of successful ntimetbolites in cncer therpy over mny yers nd fulfill importnt preconditions for the preprtion of duplex drugs. Two different nucleoside nlogues cn be coupled vi nturl phosphodiester bonding resulting in heterodinucleoside phosphte nlogues tht cn esily be cleved in vivo by phosphodiesterses into the prent nucleosides. Heterodinucleoside phosphtes linking 2 0 -deoxy-5-fluorouridine (5-dU) with thioinosine were mong the first dimers tht inhibited effectively both 6-mercptopurine sensitive s well s resistnt cncer cells in vitro. 4 However, the potentil of such dimers s possible duplex drugs hs not yet been recognized. The direct coupling of 5-dU with the lipophilic 2 0 -deoxy-5-fluoro- 4 - octdecylcytidine resulted in n mphiphilic heterodinucleoside phosphte nlogue. In in vitro clonogenic growth ssys using the humn pncretic denocrcinom cell line MIAPC 2, this duplex drug ws significntly more cytotoxic thn 5-dU. 5 The ntitumor potentil of the duplex drug evluted in p53-mutted nd ndrogen-independent DU-145 humn prostte tumor cells showed 100% erdiction of tumor cells wheres 10% of cells were resistnt to 5-dU. 6 In PC-3 cells the duplex drug exerted 0968-0896/$ - see front mtter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.08.033
H. Schott et l. / Bioorg. Med. Chem. 17 (2009) 6824 6831 6825 stronger cytotoxicity nd induced more S-phse rrest nd poptosis thn 5-dU. 7 The direct linkge of rbinofurnosylcytosine (rc) with 4 -octdecyl-1-b-d-rbinofurnosylcytosine, lipophilic derivtive of the ntitumor drug rc resulted in potent duplex drug. 8 An mphiphilic duplex drug cn lso be stbly incorported in liposomes. This offers new opportunity for trgeted nticncer combintion therpy tht hs been proven in mouse models. 9 The indirect linkge of 5-dU nd rc vi phospholipid bckbone resulted in n mphiphilic duplex drug tht might be n dditionl option for the tretment of 5-dU sensitive nd resistnt colon cncer, humn lymphom nd hemtologicl mlignncies. 10 12 A new derivtive of cytidine, 3 0 -C-ethynylcytidine (ECyd), which is menwhile evluted in clinicl studies, 13 is highly ctive nd suitble for the synthesis of cytosttic duplex drugs. Becuse the ntitumor mechnism of ECyd differs from tht of 5-dU it cn be expected tht the synthesis of dinucleoside phosphtes linking ECyd nd 5-dU, which is described here, will result in new duplex drugs with brod spectrum of ntitumor ctivity bsed on dditive or synergistic effects of 5-dU nd ECyd metbolites rising from enzymtic degrdtion of the duplex drug. As proof of principl to this ssumption two isomeric duplex drugs were synthesized by combining 5-dU nd ECyd either vi 3 0?5 0 or 5 0?5 0 phosphodiester linkge to evlute the influence of the direction of the phosphodiester on the ntitumor ctivity of the dinucleoside phosphte. 2. Results nd discussion 2.1. Chemistry The synthesis of the two isomeric duplex drugs summrized in igure 1 ws performed ccording to the phosphonte method. Prtilly protected ECyd ws used s the 5 0 -hydroxyl compound Bz H H,b,c,b (Me)Tr d,b Bz 2 92% H 1 90% d, b 82 % Bz HBz H P H H TMS (Me)Tr H Bz 3 H 5 TBDMS H P H 4 42 % 1. condenstion: e, b 39 % 2. deprotection: f, g, h H 2 H H H H 2 P H H H H H P 6 7 H H igure 1. Synthesis of the heterodinucleoside phosphte 2 0 -deoxy-5-fluorouridylyl-(5 0?5 0 )-3 0 -C-ethynylcytidine (6) nd 2 0 -deoxy-5-fluorouridylyl-(3 0?5 0 )-3 0 -C-ethynylcytidine (7) ccording to the phosphonte method strting from 5 0 --(4-monomethoxytrityl)-2 0 -deoxy-5-fluorouridine (1) nd 4 -benzoyl-2 0 --(tert-butyldimethylsilyl)-3 0 - (trimethylsilylethynyl)cytidine (5). Regents nd conditions: () Benzoylchloride, pyridine, (b) column chromtogrphy on silic gel, (c) p-toluenesulfonic cid, cetone, (d) slicylchlorophosphite, pyridine/dioxne, (e) (1) pivloylchloride, pyridine, (2) iodine, TH/pyridine/wter, (f) TBA in TH, (g) std H 3 /MeH, (h) column chromtogrphy on RP 18.
6826 H. Schott et l. / Bioorg. Med. Chem. 17 (2009) 6824 6831 5 wheres two derivtives of 5-dU were used s 5 0 -or3 0 -phosphonte compounds 3, 4. The rtionle for this synthesis concept is tht the necessry 5 0 -hydroxyl compound of the prtilly protected ECyd derivtive 4 -benzoyl-2 0 --(tert-butyldimethylsilyl)- 3 0 -C-(trimethylsilylethynyl)cytidine 5 occurs s n intermedite product of the published six step synthesis of ECyd, which used cytidine s strting mteril. 14 According to the lterntive multistep synthesis of ECyd 13,15,16 which did not use cytidine s strting compound fully protected ECyd ws obtined s n intermedite compound. The selective deprotection of the 5 0 -or 3 0 -hydroxyl function of these fully protected ECyd intermedites cnnot esily be performed in order to obtin the desired prtilly protected hydroxyl compound for the dinucleoside phosphte synthesis. The synthesis of the sequence isomer duplex drugs with 5-dU s the 3 0 nd ECyd s the 5 0 -terminl, which cnnot be obtined ccording to our proposed synthesis route is expected to be more complicted. The postulted selective derivtion of one of three hydroxyl groups of ECyd to obtin 3 0 -hydroxyl or 3 0 -phosphonte compound of ECyd is more difficult s in the cse of 5-dU, which hs only two hydroxyl groups. The 5-dU-3 0 -phosphonte compound 4 cn be obtined from 5- du in two steps, wheres the synthesis of the 5-dU-5 0 -phosphonte 3 needs three step procedure. The dditionl steps render the synthesis of 5-dU-(5 0?5 0 )ECyd in respect to the preprtion of the isomeric compound 5 0 -du(3 0?5 0 )ECyd more difficult. The synthesis of both phosphonte compounds 3, 4 strted with 5 0 --(4-monomethoxytrityl)-2 0 -deoxy-5-fluorouridine (1) which ws obtined from 5-dU ccording to published procedures. 17 The phosphonyltion of the 3 0 -hydroxyl residue of 1 resulted with 90% yield in the desired 3 0 -phosphonte compound 5 0 --(4-monomethoxytrityl)-2 0 -deoxy-5-fluorouridine-3 0 -hydrogenphosphonte (4). The 5 0 -phosphonte compound 3 ws lso obtined from 1 in two steps. In the first step protection of hydroxyl groups of 1 with benzoyl residues followed by removing of the 4 0 -monomethoxytrityl protection from the 5 0 hydroxyl group resulted in 92% yield of 3 0,4-di--benzoyl-2 0 -deoxy-5-fluorouridine (2) which ws phosphonylted t the 5 0 -hydroxyl group without further purifiction t 82% yield resulting in the desired 5 0 -phosphonte compound 3 0-4-di--benzoyl-2 0 -deoxy-5-fluorouridine-5 0 -hydrogenphosphonte (3). After coupling of the free 5 0 -hydroxyl group of 5 with the 5 0 -or 3 0 -phosphonte compounds 3 or 4 ccording to the conditions given in Tble 1 the resulted heterodinucleoside phosphontes were subsequently oxidized with iodine resulting in the protected heterodinucleoside phosphtes. It cnnot be excluded tht the unprotected free 3 0 -hydroxyl group of 5 my be involved in the condenstion rection. In this cse undesired side rections will occur which reduces the yield of the desired condenstion product. The prevlent condenstion rection, however, should be the coupling to the 5 0 -hydroxyl group of 5 becuse this lcohol function Tble 1 Experimentl dt for the synthesis ccording to the hydrogenphosphonte method yielding the fully protected isomeric heterodinucleoside phosphtes of 2 0 -deoxy-5- fluorouridylyl-(5 0?5 0 -)-3 0 -C-ethynylcytidine (6) nd of 2 0 deoxy-5-fluorouridylyl- (3 0?5 0 )-3 0 -C-ethynylcytidine (7) Experimentl dt for the condenstion btined protected crude heterodinucleoside phosphtes 6 7 Hydroxyl compound number, (g/mmol) 5, 9.5/17 5, 18.4/33 Phosphonte compound number, (g/mmol) +3, 11.0/21.2 +4, 19.2/33 Pyridine (ml) 80 100 Pivloylchloride (ml/mmol) 13/106 24/195 H 2 (ml) 10 20 Iodine in TH (ml) + H 2 (ml) 80 + 10 160 + 20 CHCl 3 /MeH (9:1) (ml) 200 300 H 2 (ml) 150 250 hs stericl dvntge for the condenstion rection in respect to the 3 0 -hydroxyl group of 5. After chromtogrphic purifiction the different protection groups of the dimers were removed using the following procedures. At first the cid lbile 4-monomethoxytrityl group of the dimer formed by condenstion of 4 + 5 ws cleved with cetic cid. Trimethylsilyl- nd tert-butyldimethylsilyl protecting groups of both dimers were removed by tretment with tetrbutylmmonium fluoride (TBA) nd the deprotected dinucleoside phosphte nlogues were obtined s tetrbutylmmonium slts. The lkli lbile benzoyl residue ws finlly removed with mmoni. After the chromtogrphic purifiction of the deprotected dinucleoside phosphte nlogues using preprtive reversed phse (RP-18) column nd their lyophiliztion both duplex drugs 2 0 -deoxy-5-fluorouridylyl-(5 0?5 0 )-3 0 -C-ethynylcytidine (6) nd the isomeric 2 0 - deoxy-5-fluorouridylyl-(3 0?5 0 )-3 0 -C-ethynylcytidine (7) were obtined t bout 40% yield. The low yield my be prtilly explined by possible side rections during the condenstion s discussed bove. The quntittive exchnge of the tetrbutylmmonium ctions cnnot be chieved using ction exchnger (H + form). The presence of the tetrbutylmmonium ction in both dinucleoside phosphtes ws detected by elementry nlysis nd mss spectrometry. The course of the synthesis nd purifiction ws controlled by thin-lyer chromtogrphy (TLC) on silic gel pltes. The chemicl structure nd the nlyticl purity of the products were confirmed by MR spectr, elementry nlysis nd high resolution mss spectr. 2.2. In vitro nticncer ctivities The in vitro ntitumor ctivities of the duplex drugs 5- du(3 0?5 0 )ECyd, 5-dU(5 0?5 0 )ECyd nd of the prent monomeric drugs 5-dU nd ECyd were evluted in the frmework of the nticncer screen progrm of the tionl Cncer Institute (CI, USA). The CI nticncer screen consists of 60 humn tumor cell lines. In those 60 cell lines the drugs re tested t minimum of five concentrtions t 10-fold dilutions. A 48 h drug exposure protocol nd sulforhodmine B (SRB) protein ssy were used to estimte cell vibility or growth. 18 Additionl detils cn be found t http://dtp.nci.nih.gov. The nticncer ctivities were obtined from the dt screening report including the dt sheet, dose response curves nd the men grphs. Men grphs fcilitted visul scnning of dt for the selection of potentil compounds for prticulr cell lines or for prticulr tumor subpnels with respect to suspected response prmeter. The response prmeter GI 50 (log 10 of molr smple concentrtion resulting in 50% growth inhibition) of 5-dU nd ECyd given in the men grphs re listed in Tble 2. The verge of the GI 50 vlues for ll 60 cell lines is indicted by the men grphs midpoint. The comprison of the in vitro tumor cell growth inhibition ctivity which is bsed on the men grphs midpoint of ECyd [log 10 GI 50 (M) = 7.6] nd of 5-dU [log 10 GI 50 (M) = 6.4] shows tht ECyd ws ctive ginst ll 60 tested tumor cell lines t high nnomolr concentrtions nd tht it ws in generl bout 10-times more cytosttic thn 5-dU. In mny cses however, the response of ECyd nd 5-dU to the sme cell line significntly differs s follows. More thn 100-fold difference of the GI 50 -vlues of ECyd nd 5-dU ws observed in 3/9 non smll cell lung; 2/8 melnom; 2/6 ovrin; 3/8 renl; 5/7 colon nd 6/8 brest cncer cell lines. These dt demonstrted pronounced drug specific ntitumor ctivity of ECyd nd 5-dU ginst severl cncer cell lines, predominntly in colon nd brest tumor cell lines. Corresponding high differences of GI 50 vlues were not observed in the 6 leukemis, 6 CS nd 2 prostte tumor cell lines included in the screen. The response prmeter GI 50 ws lso used to compre the ntitumor ctivities of ECyd nd both isomeric duplex drugs. Hlf of
H. Schott et l. / Bioorg. Med. Chem. 17 (2009) 6824 6831 6827 Tble 2 In vitro nticncer ctivities resulting in 50% growth inhibition (log 10 GI 50 ) of the monomeric drugs 5-dU nd ECyd nd the 100% growth inhibition (log 10 TGI) of the duplex drugs 5-dU(3 0?5 0 )ECyd, 5-dU(5 0?5 0 )ECyd nd ECyd which were screened twofold on 60 humn cncer cell lines (pnel/cell line) nd expressed by smple concentrtion (M) Pnel/Cell line Log 10 GI 50 (M) Log 10 TGI (M) 5-dU ECyd ECyd 5-dU(3 0?5 0 )ECyd 5-dU(5 0?5 0 )ECyd Leukemi CCR-CEM 8.2 < 8.0 5.90 5.65 5.76 HL-60 6.7 < 8.0 5.98 A K-562 6.1 < 8.0 MLT-4 7.4 < 8.0 4.99 4.82 7.91 RPMI-8226 6.1 < 8.0 6.89 7.78 SR 7.9 < 8.0 6.85 on-smll cell lung cncer A549/ATCC 7.9 < 8.0 4.00 > 4.53 EKVX 5.0 7.2 5.40 5.20 5.05 HP-62 7.6 < 8.0 6.77 HP-92 6.1 7.2 5.36 5.66 4.67 CI-H226 5.0 7.8 4.89 CI-H23 6.3 7.5 6.59 4.84 6.92 CI-H322M 6.3 7.7 5.94 4.94 A CI-H460 8.7 < 8.0 7.00 4.99 5.34 CI-H522 5.6 < 8.0 7.45 5.94 Colon cncer CL 205 5.8 < 8.0 6.71 6.06 6.02 HCC-2998 9.0 < 8.0 7.40 6.96 A HCT-116 6.9 < 8.0 4.94 HCT-15 5.7 < 8.0 4.82 HT29 5.6 < 8.0 KM12 5.2 < 8.0 6.06 5.08 5.25 SW-620 5.0 < 8.0 5.89 CS cncer S-268 7.9 7.8 5.89 5.11 5.54 S-295 7.4 < 8.0 6.24 5.97 6.06 S-539 8.4 7.4 5.17 6.78 6.86 SB-19 5.7 7.2 5.24 SB-75 6.7 7.8 6.05 A U251 6.9-7.7 5.78 > 4.00 Melnoms LXI MVI 7.6 < 8.0 7.42 6.46 A MALME-3 M 5.1 7.8 6.28 5.89 6.49 M14 6.8 7.4 5.57 6.54 6.14 SK-MEL-2 5.0 7.9 6.60 6.37 5.24 SK-MEL-28 5.7 7.3 5.76 5.85 6.42 SK-MRL-5 6.7 8.0 6.46 6.80 7.39 UACC-257 5.5 7.1 5.27 5.66 5.27 UACC-62 7.4 7.5 6.51 5.89 6.53 vrin cncers IGRV1 5.6 7.9 4.84 4.89 VCAR-3 5.6 7.1 5.37 5.54 6.13 VCAR-4 5.0 6.8 5.81 5.70 VCAR-5 5.2 7.3 4.95 4.46 VCAR-8 6.9 < 8.0 5.54 5.68 SK-V-3 5.7 7.1 Renl cncers 786-0 7.0 < 8.0 A489 5.9 < 8.0 7.03 6.12 7.08 ACH 7.5 < 8.0 6.58 5.82 CAKI-1 7.5 < 8.0 7.53 7.10 A RX 393 5.4 7.6 5.68 5.83 6.36 S12C 6.7 < 8.0 A TK-10 5.3 7.8 6.12 4.74 A U-31 6.9 < 8.0 6.40 Prostte cncers PC-3 6.5 7.5 6.51 4.62 4.32 DU-145 6.6 < 8.0 6.02 6.36 (continued on next pge)
6828 H. Schott et l. / Bioorg. Med. Chem. 17 (2009) 6824 6831 Tble 2 (continued) Pnel/Cell line Log 10 GI 50 (M) Log 10 TGI (M) 5-dU ECyd ECyd 5-dU(3 0?5 0 )ECyd 5-dU(5 0?5 0 )ECyd Brest cncers MC7 8.2 < 8.0 6.86 CI/ADR-RES 5.9 < 8.0 5.53 5.01 MDA-MB-231 5.4 7.4 4.42 6.56 HS 578T 5.4 7.9 4.80 4.90 5.82 MDA-MB-435 5.5 < 8.0 6.77 6.60 6.74 MDA- 5.9 7.9 6.76 BT-549 6.0 7.4 5.23 6.64 6.26 T-47D 5.9 < 8.0 4.75 A Men grphs midpoint 6.4 7.6 5.56 5.28 5.39 Results of one representtive screen re shown. The lowest GI 50 vlue of ECyd nd 5-dU being more thn 100-fold different in the sme cell lines nd the lowest TGI vlues of ECyd nd of the two duplex drugs being more thn 10-fold different in the sme cell lines re shown by bold fce. TGI vlues re not listed when the screening resulted in more thn 100-fold different vlues. the tested tumor cell lines hd GI 50 vlues of 610 8 M fter ECyd tretment. Correspondingly, 44/58 cell lines produced low GI 50 vlues with 5-dU(3 0?5 0 )ECyd. However, GI 50 vlues of 610 8 M were observed only in 25/53 cell lines when the sme cell lines were treted with isomeric 5-dU(5 0?5 0 )ECyd. n the bsis of these results it could be speculted tht the ntitumor ctivities of 5-dU(3 0?5 0 )ECyd linking 5-dU with ECyd vi the nturl 3 0?5 phosphodiester bonding is generlly higher in respect to those of the 5 0?5 0 isomer. This ssumption is in greement with the published results tht 3 0?5 0 linked rbinofurnosylcytidine dimers were found to be more ctive thn 5 0?5 0 linked dimers. 19 or the evlution of the ntitumor ctivities of the duplex drugs the cell growth inhibition ws compred only with tht of ECyd becuse ECyd ws 10-fold more ctive thn 5-dU. or this comprison the too low nd similr GI 50 vlues of ECyd nd the duplex drugs were less suitble. Therefore, insted of the GI 50 the response prmeter TGI (log 10 of molr smple concentrtion resulting in 100% growth inhibition) listed in Tble 2 ws used to demonstrte the different sensibility of the sme tumor cell lines ginst ECyd nd the duplex drugs. The men grphs midpoint of ECyd [log 10 TGI (M) = 5.56 corresponds to the vlues of 5-dU-(3 0?5 0 )ECyd [log 10 TGI (M) = 5.28 nd of 5-dU(5 0?5 0 )ECyd [log 10 TGI (M) = 5.39. In respect to the similr TGI men grphs midpoint of the three compounds it could be ssumed tht the cytosttic potentil of ECyd nd both duplex drugs would be similr. However, the TGI vlues of the drugs obtined fter the prllel tretment of the sme tumor cell line clerly demonstrted tht the min prt of the 60 tested cell lines showed significntly different sensibilities ginst ECyd, 5- du(3 0?5 0 )ECyd nd 5-dU(5 0?5 0 )ECyd becuse their responses ginst single cell lines vried in brod rnge. The following 14 cell lines showed TGI vlues of ECyd (see Tble 2), which re more thn 10-fold lower thn those of one or both duplex drugs: Melnoms (SK-Mel-2); ovrin (VAR4); colon (SW-620); CS (SB-19, U251); renl (ACH, TK-10, U-31); non smll lung (CI-H23, CI-H322M, CI-H460, CI-H522) nd the prostte cncer cell lines (PC-3, DU-145). n the bsis of these TGI vlues it cn be concluded tht the equimolr coupling of 5 0 - du with ECyd results in n ntgonistic effect, thus reducing the cytosttic ctivity of the duplex drug, in respect to tht of ECyd. In contrst to these results the following 9 cell lines were more thn 10-times more sensitive to one or both duplex drugs in comprison to ECyd: HL-60, Molt-4 (leukemi); S-539 (CS); VCAR- 8 (ovrin); MC7, CI/ADR-RES, MDA-MB-231, HS 578T; BT-549 (brest). In these exmples the linked 5-dU cts dditively or synergisticlly when coupled with ECyd. f the tested cell lines 27 showed only little to unchnged growth inhibition in comprison to tht of ECyd independently of tretment with 5-dU(3 0?5 0 )ECyd or 5-dU(5 0?5 0 )ECyd. The double screened TGI vlues of 18 tests of the 60 tested cell lines could not be evluted becuse the vlues obtined differed more thn 100-fold. Besides the chnge of the cytosttic ctivities of ECyd, which ws observed fter coupling with 5-dU it ws surprising tht the direction of the phosphodiester linkge sequence in which 5- du nd ECyd were coupled hs significnt influence on the ntitumor ctivity nd specificity of the synthesized duplex drugs. However, 25 of the tested 60 cell lines demonstrted mrked structure ctivity reltionship ginst the duplex drugs. The sequence dependent ntitumor ctivity cn be observed from the difference of the TGI vlues resulting fter prllel tretment of the sme cell line with 5-dU(3 0?5 0 )ECyd nd 5-dU(5 0?5 0 )ECyd. ne hlf of these cell lines were more sensitive ginst 5- du(3 0?5 0 )ECyd wheres the other hlf showed higher growth inhibition with the isomeric 5-dU(5 0?5 0 )ECyd duplex. The following 7 cell lines showed significnt high sequence ctivity reltionship expressed by n bout 100-fold difference of the TGI vlues obtined with the sme cell line fter prllel tretment with both duplex drugs: Molt-4, SR (leukemi); HP-62, CI-H23 (non smll cell lung cncer), DU-145 (prostte); MC7; MDA-MB- 231(brest). The results demonstrte tht the chemicl coupling of 5-dU nd ECyd cused, depending on the cell lines resulted either in incresed, decresed or unchnged ntitumor ctivities of the corresponding duplex drug compred to monomeric ECyd. Activity nd specificity of the ntitumor effects of the duplex drugs depend on the cytosttic potentil of the coupled monomeric drugs s well s on the structure of the obtined dimer, for exmple the direction of the phosphodiester bonding in dinucleoside phosphte. Thus, the described trnsformtion of cytosttic ntimetbolites to dinucleoside phosphte nlogues represents vluble lterntive to the intensive serch for new nticncer drugs. 2.2.1. Possible resons of the ntitumor ctivities of the duplex drugs The principl mechnism for the cytosttic effect of 5-dU is the inhibition of thymidylte synthse (TS). 20 The inhibition of TS enzyme ctivity leds to depletion of deoxythymidine triphosphte which is necessry for DA synthesis. ECyd cn contribute to the ntitumor ctivity through its ction mechnisms which re different from those of 5-dU. 21 25 By coupling ECyd nd 5-dU the resulting duplex drug should be potent inhibitor of tumor cell growth by simultneous inhibition of both DA nd RA synthesis. It cn be supposed tht the intct duplex drugs do not ct s ctive compounds. However, the severl cytosttic metbolites, which cn be formed by enzymtic degrdtion of the duplex drugs should initite the
H. Schott et l. / Bioorg. Med. Chem. 17 (2009) 6824 6831 6829 ntitumor effects. Becuse the phosphodiester clevge cn either be initited t the 3 0 - s well s the 5 0 -terminl, mixture of different ctive metbolites cn be expected. The clevge of 5-dU (3 0?5 0 )ECyd produces n equimolr mixture of ECyd-5 0 -monophosphte nd 5-dU if the phosphodiesterse cleves t the 3 0 -terminl. Clevge t the 5 0 -terminl results in mixture of 5- du-3 0 -monophosphte nd ECyd. The degrdtion of the isomeric 5-dU(5 0?5 0 )ECyd results in 1:1 molr mixture of 5-dU-5 0 - monophosphte nd ECyd if clevge occurs t the 5-dU residue. An equimolr mixture of ECyd-5 0 -monophosphte nd 5-dU will be formed if clevge occurs t the ECyd residue of the duplex drugs. However, certin steps of the metbolic pthwy cn be fvored, if for exmple the recognition nd clevge of the ECyd will be hindered by the 3 0 -C-ethynyl group of the crbohydrte residue, wheres the unmodified crbohydrte residue of 5-dU is more ccessible to the enzyme. The ntitumor ctivity of ECyd depends essentilly on its phosphoryltion. The first phosphoryltion step from ECyd to ECyd-5 0 - monophosphte is unnecessry if ECyd-5 0 -monophosphte is produced by metbolic ction. In this exmple the nucleotide sequence of the duplex drug which fvors the metbolic pthwy resulting in ECyd-5 0 -monophosphte insted of ECyd cn result in incresed ntitumor ctivity of the duplex. Under this spect the other sequence isomeric dinucleoside phosphte ECyd(3 0?5 0 )5-dU with ECyd s the 5 0 - nd 5-dU s the 3 0 -terminl coupling prtner is less fvored to be metbolized by phosphodiesterse to ECyd-5 0 -monophosphte in comprison to 5-dU(3 0?5 0 )ECyd. This duplex drug ws therefore not synthesized. In ddition to phosphodiesterse clevge phosphohydrolses cn convert the phosphorylted metbolites to the corresponding nucleosides. The min prt of the duplex drug will be metbolized before reching the cytoplsm becuse the negtively chrged phosphodiester renders the dinucleoside phosphte too hydrophilic to penetrte the lipid rich cell membrne esily. 26 The extrcellulr relese of ctive metbolites my cuse depot effect which does not occur by ppliction of mixture of 5-dU nd ECyd. The suspected extrcellulr metbolism could be disdvntge if duplex drugs re synthesized s hydrophilic heterodinucleoside phosphte nlogues. In cse tht the metbolism of both isomeric duplex drugs would produce n identicl mixture of ctive metbolites the observed structure ctivity reltionship would be difficult to explin. Thus, it cn be speculted tht the sequence dependent metbolic pthwys which produce in time dependent mnner different mounts nd species of cytotoxic metbolites cn modulte their ntitumor ctivity nd tumor cell specificity. The results of the in vitro cytotoxicity test screens provide preliminry orienttion of expected in vivo ntitumor ctivities. However, wide vriety of biochemicl nd physiologicl processes cn drsticlly influence the ptterns of the ntitumor effects in vivo. or exmple, it hs been reported tht duplex drug tested on murine leukemi cells with the highest cytotoxicity in vitro exerted the lowest therpeutic effect in vivo. 12 evertheless, the excellent in vitro ntitumor ctivities of the described new duplex drugs justify further evlution in in vivo tumor models. 3. Experimentl 3.1. Generl chemistry 3.1.1. Regents Pivloyl chloride, benzoyl chloride, tetrbutylmmonium fluoride trihydrte, p-toluenesulfonic cid monohydrte were obtined commercilly. Slicylchlorophosphite 27 5 0 --(4-monomethoxytrityl)-2 0 -deoxy-5-fluorouridine (1), 17 4 -benzoyl-2 0 --(tert-butyldimethylsilyl)-3 0 -(trimethylsilylethynyl) cytidine (5) 14 were prepred s described. All solvents were of technicl grde nd used without further purifiction unless stted otherwise. Dioxne ws dried with sodium, distilled nd stored over 5 Å moleculr sieve nd pyridine ws refluxed over KH, distilled nd stored over 4 Å moleculr sieve. The TBA cleving solution ws obtined by dissolving tetrbutylmmonium fluoride trihydrte (157.8 g) in TH (500 ml). or the oxidtion rection solution of iodine (25 g) in TH (500 ml) ws used. All rections were monitored by TLC on precoted Silic Gel 60 254 pltes (0.25 mm, Merck) using UV light for visuliztion nd spry regents s developing gents. 17 Multi step flsh chromtogrphy ws crried out on self pcked Silic Gel 60 (0.040 0.0063 mm, Merck) columns using eluent mixtures prepred by volume rtios. All rections were performed t room temperture, if not stted differently. The concentrtion of the rection mixtures, solutions, orgnic lyers nd eluted frctions ws done in vcuum t bth temperture of 40 C. 1 H- nd 13 C MR spectr were obtined on Bruker AC 250 spectrometer t 250 MHz nd 62.9 MHz, respectively or on Bruker Avnce 400 spectrometer t 400 MHz nd 100 MHz, respectively. DMS-d 6 ws used s solvents. Me 4 Si ws used s n internl stndrd. 31 P MR spectr were obtined on Bruker Avnce 400 spectrometer t 161 MHz, using H 3 P 4 s n externl stndrd. Mss spectr were mesured on innign TSQ 70 or MAT 95 instrument. or AB mss-spectr, ll compounds were mesured in BA-or glycerine-mtrix. HRMS were mesured on Bruker Apex II T-ICR instrument. 3.2. Generl chemistry methods 3.2.1. Condenstion procedure using the hydrogenphosphonte method The corresponding hydroxyl- nd hydrogenphosphonte compounds were dissolved together in dry pyridine nd rigorously dried by repetitive evportion nd ddition of pyridine before the condenstion rection ws strted by ddition of pivloyl chloride. After drying nd ddition of the required mount of pyridine (see Tble. 1) the solution ws cooled to 0 C before pivloyl chloride ws dded under exclusion of moisture. After stirring for 5 min the rection mixture ws cooled gin to 0 C before the rection ws stopped by ddition of wter. The obtined phosphonic diester ws immeditely oxidized by ddition of iodine in TH nd H 2. After 1 h stirring excess iodine ws reduced by ddition of solid sodium hydrogensulfite before the rection mixture ws concentrted to syrup tht ws dissolved in CHCl 3 /MeH nd extrcted with wter. The orgnic lyer ws concentrted to syrup, which ws co-evported three times with toluene yielding the crude protected heterodinucleoside phosphtes, which were purified nd de-protected s described below. 3.3. Synthesis of 3 0, 4-Di--benzoyl-2 0 -deoxy-5-fluorouridine- 5 0 -hydrogenphosphonte (3) To solution of 5 0 --(4-monomethoxytrityl)-2 0 -deoxy-5-fluorouridine (1) (26 g, 50 mmol) in dry pyridine (150 ml) benzoylchloride (56 g, 400 mmol) ws dded under cooling. The rection vessel ws seled irtight nd shken for 8 h t room temperture before sturted (std) q 2 C 3 (130 ml) ws dded under cooling. The resulting rection mixture ws concentrted to syrup which ws diluted with CHCl 3 (300 ml) nd then extrcted with std q 2 C 3 (130 ml). The CHCl 3 lyer ws concentrted to syrup which ws dissolved in mixture of CHCl 3 /petroleum ether (1:1, 200 ml) followed by chromtogrphy on silic gel column using CHCl 3 /petroleum ether grdient with incresing percentge of CHCl 3. The frctions contining the desired product were concentrted to fom (46 g) which ws dissolved in cetone (100 ml) contining p-toluenesulfonic cid monohydrte (16 g)
6830 H. Schott et l. / Bioorg. Med. Chem. 17 (2009) 6824 6831 nd stirred for 20 min t room temperture, before std q 2 C 3 (50 ml) ws dded. The resulting solution ws concentrted to syrup which ws dissolved in CHCl 3 (500 ml) nd extrcted with H 2 (100 ml). The orgnic lyer ws seprted nd chromtogrphed on silic gel column using two step grdient with step 1; CHCl 3 /petroleum ether with incresing percentge of CHCl 3 nd step 2; ether. The frctions contining the desired product were concentrted, ffording crude 3 0,4-di--benzoyl-2 0 -deoxy-5-fluorouridine (2) s colorless fom (21 g, 46 mmol) t 92% yield. In the following step 2 ws dissolved without further purifiction in dry pyridine (90 ml) nd diluted with dry dioxne (180 ml). To this solution dioxne (75 ml) ws dded in which slicylchlorophosphite (13 g, 64 mmol) ws dissolved. After stirring of the rection mixture t room temperture for 2 h std q HC 3 (12 ml) ws dded nd the solution ws evported to syrup which ws dissolved in CHCl 3 (500 ml) nd extrcted with mixture of H 2 /std q Cl/MeH (1:1:2) (3 100 ml). The seprted CHCl 3 -phse ws concentrted nd the resulting syrup dissolved in CHCl 3 (150 ml). By slow ddition of the obtined solution to vigorously stirred ether (1.5 L) the desired 3 precipitted. The isolted nd dried precipitte ws then extrcted during 70 h with ether. After drying of the remining precipitte, 3 (20 g, 39 mmol) ws obtined t 82% yield s white powder. TLC (CHCl 3 /MeH 70:30) R f = 0.46; MS (AB ) m/z: 517.1 [M H] ; 1 H MR, 400 MHz, DMS-d 6 : d = 2.44 2.75 (m, 2H, H 2 0), 3.48 3.89 (m, 5H, PH, H 2 (DMS-d 6 )), 4.0 4.425 (m, 2H, H 5 0), 4.34 4.47 (m, 1H, H 4 0), 5.47 5.64 (m, 1H, H 3 0), 6.22 6.4 (m, 1H, H 1 0), 7.35 8.27 (m, 10H, H romt. ), 8.51 (s, 1H, H 6 ), 12.0 (s, br, 1H, P-H); 13 C MR, 100 MHz, DMS-d 6 : d = 36.7 (C 2 0), 62.8 (C 5 0), 75.5 (C 3 0), 83.6 (C 1 0), 85.4 (C 4 0), 128.6 147.9 (C romt. ), 155.7 (C 6 ), 156.0 (C 5 ), 165.1 (C@), 168.0 (C@); 31 P MR, 161 MHz, DMS-d 6 : d = 1.23 ppm( PH() H). Anl. Clcd for C 23 H 20 2 9 P1/ 2H 2 : C, 50.28; H, 3.67;, 5.10. ound: C, 50.30; H, 4.21;, 5.35. 3.4. Synthesis of 5 0 --(4-monomethoxytrityl)-2 0 -deoxy-5- fluorouridine-3 0 -hydrogenphosphonte (4) 5 0 --(4-Monomethoxytrityl)-2 0 -deoxy-5-fluorouridine (1) (20 g, 39 mmol) ws dissolved in dry pyridine (50 ml) nd the resulting solution diluted with dry dioxne (90 ml) followed by ddition of slicylchlorophosphite (11 g, 54 mmol). After stirring the rection mixture t room temperture for 1.5 h the formed precipitte ws removed by filtrtion nd wshed with cold ether. To the combined filtrte nd wsh liquid std q 2 C 3 (50 ml) ws dded. The obtined mixture ws concentrted to fom which ws then dissolved in mixture of CHCl 3 /MeH (95:5) nd chromtogrphed on silic gel column using CHCl 3 /MeH elution grdient with incresing percentge of MeH. After the evportion of the product contining frctions compound 4 ws obtined s fom (20 g, 35 mmol) t 90% yield. TLC (CHCl 3 /MeH 80:20) R f = 0.17; MS (AB ) m/z: 581.2 [M H] ; 603.2 [M+]; 1 H MR, 400 MHz, DMS-d 6 : d = 2.20 2.65 (m, 2H, H 2 0), 3.18 3.43 (m, 2H, H 5 0), 3.45 3.68 (m, 3H, H, PH, PH), 3.73 (s, 4H, CH 3, H 2 (DMS-d 6 )), 4.21 (m, 1H, H 4 0), 4.91 (m, 1H, H 3 0), 6.23 (m, 1H, H 1 0), 6.79 7.48 (m, 14H, H romt. ), 7.79 (d, 1H, J = 6.2 Hz, H 6 ); 13 C MR, 100 MHz, DMS-d 6 : d = 23.4 (C 2 0), 60.0 (CH 3 ), 61.6, (C 5 0), 68.4 (C 3 0), 89.9 (C 4 0), 91.8 (C 1 0), 128.9 (C 6 ), 132.0.148.9 (C romt. +C 5 ), 154.2 (C@), 163.5 (C@); 31 P MR, 161 MHz, DMS-d 6 : d = 7.3 ppm, ( PH() H). Anl. Clcd for C 29 H 28 2 8 P2 H 2 : C, 53.96; H, 4.53;, 4.34. ound: C, 53.55; H, 4.44;, 4.17. 3.5. Synthesis of 2 0 -deoxy-5-fluorouridylyl-(5 0?5 0 )-3 0 -Cethynylcytidine (6) The syrup obtined fter the condenstion of 5 with 3 ccording to the experimentl dt of Tble 1 ws dissolved in CHCl 3 nd chromtogrphed on silic gel column using CHCl 3 /MeH grdient with incresing percentges of MeH. The frctions contining the desired protected heterodinucleoside phosphte were concentrted to syrup which chnged to fine solid by vigorously shking fter ddition of ether. To the isolted solid, dissolved in TH (45 ml) solution (22 ml) of TBA in TH ws dded. The seled rection mixture ws stirred for three dys whereby the trimethylsilyl- nd tert-butyldimethylsilyl protecting groups were cleved. After the concentrtion of the rection mixture 33% q mmoni (80 ml) ws dded to the resulting syrup nd the seled solution ws stirred for five dys to cleve the benzoyl protecting groups. When the rection mixture ws concentrted to bout 250 ml, fine solid precipitte ws removed by centrifugtion nd the superntnt liquid concentrted nd lyophilized. The resulting lyophiliste of crude 6 ws dissolved in H 2 (60 ml) nd chromtogrphed on preprtive RP-18 column using H 2 /MeH grdient with incresing percentge of methnol. The desired 6 eluted t 15 40% CH 3 H. The product contining frctions were pooled, the ph vlue djusted to 5.8 by ddition of ction exchnger resin (H + ), tht ws removed before the solution ws concentrted nd lyophilized yielding 6 s white powder (5.8 g, 41.8%). HRMS clcd for C 20 H 22 5 12 P [M H] : 574.09921, found: 574.09907; 1 H MR (DMS-d 6, 250 MHz): d = 11.,8 (br, s, 1H, H 5dU), 8.10 (d, J = 6.64 Hz, 1H, H 6 5dU), 7.85 (d, J = 7.25 Hz, 1H, H 6 ECyd), 6.14 (t, J = 6.42 Hz, 1H, H 1 5dU), 5.85 (d, J = 6.19 Hz), 1H, H 1 ECyd) 5.81 (d, J = 7.07 Hz, 1H, H 5 ECyd), 5.64 5.75 (m, 2H, H 2 0 H, H 3 0 H ECyd), 4.29 (m, 1H, H 3 0 5dU), 4.08 (m, 1H, H 2 0 ECyd), 3.83 3.97 (m, 6H, H 4 0,H 5 0,H 5 00 ECyd/5dU), 3.46 (s, 1H, CH ECyd) 3.16 (m, 8H, CH 2 TBA), 2.10 (m, 2H, H2 0,H2 00-5dU) 1.56 (m, 8H, CH 2 C 2 H 5 TBA), 1.30 (m, 8H, CH 2 CH 2 TBA), 0.92 (m, 12H, CH 3 TBA); 13 C MR (DMS-d 6, 62 MHz): d = 165.3 (C 4 ECyd), 158.0 (C 4 5dU), 156.9 (C 2 ECyd), 149.1 (C 2 5dU), 142.2 (C 6 ECyd), 137.8 (C 5 5dU), 124.6 (C 6 5dU), 94.4 (C 5 ECyd), 87.3 (C 1 0 ECyd), 86.3 (C 4 0 ECyd), 84.6 (C 4 0-5dU), 82.9 ( CH ECyd), 78.7 (C 2 0 ECyd), 77.3 (C ECyd), 72.7 (C 3 0 ECyd), 71.1 (C 3 0 5dU), 64.6 (C 5 0 ECyd, C 5 0 5dU), 57.6 (C 1 TBA), 23.1 (C 2 TBA), 19.2 (C 3 TBA), 13.5 (C 4 TBA). Anl. Clcd for C 20 H 22 5 12 PC 16 H 36 : C, 52.93; H, 7.16;, 10.29. ound: C, 52.58; H, 6.86;, 9.56; 31 P MR, 161 MHz DMS-d 6 : d = 1.5 ppm. 3.6. Synthesis of 2 0 -deoxy-5-fluorouridylyl-(3 0?5 0 )-3 0 Cethynylcytidine (7) The syrup, obtined fter condenstion of 5 with 4 ccording to the experimentl dt of Tble 1 ws dissolved in CHCl 3 (300 ml) nd chromtogrphed on silic gel column using mixtures of CHCl 3 /MeH with incresing percentges of MeH s the eluent. ully protected 7 ws eluted first, followed by frctions contining 7 without the monomethoxytrityl protection group. rctions contining fully protected s well s prtilly deprotected 7 were pooled nd concentrted to fom which ws dissolved in MeH (60 ml). Then 80% q cetic cid (60 ml) ws dded, the rection mixture stirred for 24 h nd then concentrted to syrup. The syrup chnged to fine solid fter dding ether nd vigorous shking. The solid ws isolted by centrifugtion, dissolved in CHCl 3 (250 ml) nd re-chromtogrphed on silic gel column s described bove. After the second chromtogrphic purifiction the resulting solid of the prtilly protected 7 ws dissolved in TH (170 ml), followed by ddition of TBA (8 ml). The seled rection mixture ws stirred for three dys nd concentrted to syrup. The obtined syrup ws treted nother five dys with 33% q mmoni (300 ml) nd chromtogrphed on RP-18 column s described bove for the purifiction of 6 ffording pure 7 (10.6 g, 39.3%). MS (AB ) m/z: 574.0 [M H] ; 815.3 [M+C 16 H 36 ] ; 1 HMR (DMS-d 6, 250 MHz):d = 11.82 (br, s, 1H, H 5dU), 8.20 (d, J = 7.1 Hz, 1H, H 6 5dU), 7.92 (d, J = 7.5 Hz, 1H, H 6 ECyd), 7.83
H. Schott et l. / Bioorg. Med. Chem. 17 (2009) 6824 6831 6831 (d, J = 7.5 Hz, 1H, H 5 ECyd), 6.09 6.14 (m, 2H, 2H 1 0 ECyd/5dU) 5.69 5.85 (m, 6H, H/H ECyd/5dU), 4.65 (m, 1H, H 3 0 5dU), 3.87 4.01 (m, 4H, H 5 0 ECyd/H 3 0 5dU), 3.59 (m, 2H, H 5 0 5dU), 3.48 (s, 1H, C C H), 3.16 (m, 8H, CH 2 TBA), 2.50 (m, 1H, H 2 0 ECyd), 2.28 2.1 (m, 2H, H 2 0 5dU), 1.53 (m, 8H, CH 2 TBA), 1.30 (m, 8H, CH 2 TBA), 0.92 (t, J = 7.4, 12H, CH 3 TBA); 13 C MR (DMS-d 6, 62 MHz): d = 165.0 (C 4 ECyd), 158.0 (C 4-5dU), 157.29 (C 2 ECyd), 149.0 (C 2 5dU), 142.2 (C 6 ECyd), 137.8 (C 5 5dU), 124.6 (C 6-5dU), 94.4 (C 5 ECyd), 87.5 (C 1 0 ECyd), 86.5 (C 4 0 ECyd), 84.5 (C 4 0-5dU), 82.9 ( CH ECyd), 78.7 (C 2 0 ECyd), 77.4 (C ECyd), 74.5 (C 3 0 5dU), 72.6 (C 3 0 ECyd), 65.2 (C 5 0 ECyd), 61.5 (C 5 0 5dU) 57.6 (C 1 TBA), 23.1 (C 2 TBA), 19.2 (C 3 TBA), 13.5 (C 4 TBA); 31 P MR, 161 MHz, DMS-d 6 : d = 1.85 ppm. Anl. Clcd for C 20 H 22 5 12 PC 16 H 36 : C, 52.93; H, 7.16;, 10.29. ound: C, 52.62; H, 7.00;, 9.60. Acknowledgments or the performed MR nlysis we thnk Dr. Ludwig. The uthors thnk the tionl Cncer Institute (Bethesd, USA) for the in vitro nticncer testing of ECyd nd both duplex drugs. References nd notes 1. Krise, J. P.; Stell, V. J. Adv. Drug Delivery Rev. 1996, 19, 287. 2. Wgner, C. R.; Iyer, V. V.; McIntee, E. J. Med. Res. Rev. 2000, 20, 417. 3. Plunkett, W.; Gndhi, V. Cncer Chemother. Biol. Response Modif. 2001, 19, 21. 4. Montgomery, J. A.; Dixon, G. J.; Dulmge, E. A.; Thoms, H. J.; Brockmnn, R. W.; Skipper, H. E. ture 1963, 199, 769. 5. Schott, H.; Ludwig, P. S.; Gnsuge,.; Gnsuge, S.; Schwendener, RA. Liebigs Ann. Recl. 1997, 413. 6. Cttneo-Pngrzzi, R. M. C.; Schott, H.; Schwendener, R. A. Prostte 2000, 45,8. 7. Cttneo-Pngrzzi, R. M. C.; Schott, H.; Wunderli-Allenspch, H.; Derighetti, M.; Schwendener, R. A. Biochem. Phrmcol. 2000, 60, 1887. 8. Horber, D. H.; Cttneo-Pngrzzi, R. M. C.; von Bllmoos, P.; Schott, H.; Ludwig, P. S.; Eriksson, S.; ichtner, I.; Schwendener, R. A. J. Cncer Res. Clin. ncol. 2000, 126, 311. 9. Mrty, C.; dermtt, B.; Schott, H.; eri, D.; Bllmer-Hofer, K.; Klemenz, R.; Schwendener, R. A. Br. J. Cncer 2002, 87, 106. 10. Siko, P.; Horvth, Z.; Buer, W.; Hoechtl, T.; Grusch, M.; Krupitz, G.; Ruko, P.; Mder, R. M.; Jeger, W.; Schott, H.; ovotny, L.; ritzer-szekeres, M.; Szekeres, T. Int. J. ncol. 2004, 25, 357. 11. Siko, P.; Horvth, Z.; Illmer, C.; Mdlener, S.; Buer, W.; Hoechtl, T.; Erlch,.; Grusch, M.; Krupitz, G.; Mder, R. M.; Jeger, W.; Schott, H.; Agrwl, R. P.; ritzer-szekeres, M.; Szekeres, T. Leukocyte Res. 2005, 29, 785. 12. Ruko, P.; ovotny, L.; Mego, M.; Siko, P.; Schott, H.; Szekeres, T. eoplsm 2007, 54, 68. 13. Httori, H.; Tnk, M.; ukushim, M.; Sski, T.; Mtsud, A. J. Med. Chem. 1996, 39, 5005. 14. Ludwig, P. S.; Schwendener, R. A.; Schott, H. Synthesis 2002, 16, 2387. 15. omur, M.; Sto, T.; Wshinosu, M.; Tnk, M.; Aso, T.; Shuto, S.; Mtsud, A. Tetrhedron 2002, 58, 1279. 16. Hrdlick, P. J.; Jepsen, J. S.; ielsen, C.; Wengel, J. Bioorg. Med. Chem. 2005, 13, 1249. 17. Ludwig, P. S.; Schwendener, R. A.; Schott, H. Eur. J. Med. Chem. 2005, 40, 494. 18. Grever, R. M.; Scheprtz, S. A.; Chbner, B. A. Sem. ncol. 1992, 19, 622. 19. Smith, C. G.; Buskirk, H. H.; Lummis, W. L. J. Med. Chem. 1967, 10, 774. 20. Sommer, H.; Snti, D. V. Biochem. Biophys. Res. Commun. 1974, 57, 689. 21. Tktori, S.; Knd, H.; Tkenk, K.; Wty, Y.; Mtsud, A.; ukushim, M.; Shimmoto, Y.; Tnk, M.; Sski, T. Cncer Chemother. Phrmcol. 1999, 44, 97. 22. Azum, A.; Mtsud, A.; Sski, T.; ukushim, M. ucleosides ucleotides ucleic Acids 2001, 20, 609. 23. Murt, D.; Endo, Y.; bt, T.; Skmoto, K.; Syouji, Y.; Kdohir, M.; Mtsud, A.; Sski, T. Drug Metb. Dispos. 2004, 32, 1178. 24. ito, T.; Yokogw, T.; Kim, H. S.; Mtsud, A.; Sski, T.; ukushim, M.; Kitde, Y.; Wty, Y. ucleic Acids Symp. Ser. (xf) 2007, 51, 435. 25. ito, T.; Yokogw, T.; Tktori, S.; God, K.; Hirmoto, A.; Sto, A.; Kitde, Y.; Sski, T.; Mtsud, A.; ukushim, M.; Wty, Y.; Kim, H. S. Cncer Chemother. Phrmcol. 2009, 63, 837. 26. Bijnsdorp, I. V.; Schwendener, R. A.; Schott, H.; Schott, S.; ichtner, I.; Honeywell, R. J.; Losekoot,.; Ln, A. C.; Peters, G. J. ucleic Acids Symp. Ser. (xf) 2008, 52, 651. 27. Anschütz, R.; Emery, W.. Liebigs Ann. Chem. 1887, 239, 301.