Synthesis of 4′/5′-Spirocyclopropanated Uridine and d -Xylouridine Derivatives and Their Activity against the Human Respiratory Syncytial Virus

Syn hesis o 4’/5’-Spi ocyclop opana ed U idine and D- Xylou idine De i a i es and hei Ac i i y agains he Hu- man Respi a o y Syncy ial Vi us Ch is oph Kllmann,† S enja M. Wieche ,§ Pe e G. Jones,‡ Thomas Pie schmann,§ and Daniel B. We z*,† †Technische Uni e si ä B aunschweig, Ins i u e o O ganic Chemis y and ‡Technische Uni e si ä B aunschweig, Ins i u e o Ino ganic and Analy ical Chemis y, Hagen ing 30, 38106 B aunschweig, Ge many § Ins i u e o Expe imen al Vi ology, TWINCORE, Cen e o Expe imen al and Clinical In ec ion Resea ch; a Join Ven u e Be ween he Medical School Hanno e (MHH) and he Helmhol z Cen e o In ec ion Resea ch (HZI), Hanno e , Feodo - Lynen-S . 7, 30625 Hanno e Suppo ing In o ma ion Placeholde NH O ON O OH HO NH O O N O OH HO HO U idine cpUMP NH O ON O OH HO iP COO (HO)2(O)PO ABSTRACT: The Simmons-Smi h-Fu ukawa eac ion was used o gene a e 4’/5’-spi ocyclop opana ed u idine analogs om elec on- ich exocyclic enol es e s. Du ing syn hesis he na i e hyd oxyla ion pa e n o he nucleoside is p ese ed and o e s he possibili y o a la e s age 5’-phospho yla ion a he spi ocyclop opanol moie y. All syn hesized spi ocyclop opana ed u idine de i - a i es, including he co esponding 5’-monophospha e (cpUMP), we e e alua ed wi h espec o hei an i i al ac i i y in an HRSV assay showing modes , bu p omising ac i i y. The syn hesis o nucleoside de i a i es o medical he apy has been o majo in e es o se e al decades;1,2 nucleosides ha e become a s iking and powe ul g oup o he apeu ic agen s, especially in case o i al diseases and an i-cance he apy. While an i i al agen s agains ce ain i uses such as HIV-1, Hepa i is C i us, CMV o In luenza i us a e a ail- able, ea men op ions o human espi a o y syncy ial i us (HRSV) a e s ill limi ed.3 NH O O N O OH HO iP COO NH O O N O OH HO O O O Base P O O O P O O O F F I II III P e ious Wo k This Wo k Figu e 1. Con o ma ional es ic ed cyclop opana ed nucleoside de i a i es. HRSV is a globally dis ibu ed nega i e s and RNA i us o he amily Pneumo i idae ha causes a se e e disease bu den among in an s, he elde ly and in immunocomp omised pa ien s.4–6 Apa om a p ophylac ic monoclonal an ibody ha is Scheme 1. Syn hesis o Spi ocyclop opana ed U i- dine De i a i e 6. BOMCl (6 equi ) NiP 2E (5 equi ) DCM, 0 °C o 3 d (84%) TBAF·3 H2O (1.5 equi ) THF, , 1 h (87%) 1) IBX (2.5 equi ) ACN, e lux 2) K2CO3 (6 equi ) (iP CO)2O (6 equi ) CH3CN, e lux, 3 h (54%, only Z o e 2 s eps) ZnE 2 (5 equi ) CH2I2 (10 equi ) DCE, 50 °C 2 h (54%, only  acial) NH O ON O OHHO RO NBOM O ON O OBOMBOMO RO NBOM O ON O OBOMBOMO RO NBOM O ON O OBOMBOMO RO NBOM O ON O OBOM BOMO NH O O N O OH HO 1 R =TBDPS 2 R = TBDPS 3 R = H 4 R = iP CO 5 R = iP CO 6 R = iP CO RO RO H2 (1 a m) Pd/C (10%) MeOH, 3.5 h (quan ) gi en o child en a high isk o se e e in ec ion, only Rib- a i in, a guanosine analog wi h b oad an i i al ac i i y, is ap- p o ed o Scheme 2. Syn hesis o Spi ocyclop opana ed U idine Monophospha e (cpUMP) 16. NH O ON O O O NBOM O ON O O O NH4OH (25 % (w/w)) 1,4-dioxane , 1.5 h (quan ) 1/3 NH4 NBOM O ON O O O N O ON O OH HO NBOM O ON O O O NBOM O ON O OH HO OH NH O ON O OH HO D2O 50 °C, 3 d (quan ) BOMCl (2.5 equi ) NiP 2E (4 equi ) DCM, 0 °C o , 1 d (89%) NaOMe (0.3 equi ) MeOH, 0 °C, 3 h (quan ) NBOM O ON O O O PCl(NiP 2)OCE (2 equi ) NiP 2E (5 equi ) DCM, 0 °C, 0.5 h (55%) NBOM O ON O O O NBOM O ON O O O 1) BnOH (1.2 equi ) e azole (3 equi ) CH3CN, , 6 h 2) mCPBA (1.2 equi ) CH3CN, 0 °C, 0.5 h (69% o e wo s eps) BnB (5 equi ) TBAI (0.2 equi ) Cs2CO3 (2 equi ) DMSO, 50 °C, 12 h (33%) HCl (4 M) 1,4-dioxane MeOH, , 10 h (31%) H2 (1 a m) Pd/C (10%) MeOH, , 5 h (quan ) 10 R = (CEO)(NiP 2)P 7 R = i P CO 8 R = i P CO 9 11 R = (CEO)(BnO)(O)P 12 R = O(BnO)(O)P 13 R = (BnO)2(O)P 14 R = (BnO)2(O)P 15 R = (HO)2(O)P 16 R = (HO)2(O)P DMC (3.5 equi ) pTsOH (0.1 equi ) DCE, 50 °C, 3 h (76%) NH O ON O OH HO 6 R = i P CO 2/3 NH3CH2CH2CN RO RO RO RO RO RO RO HO RO RO RO ea men o RSV-in ec ed pa ien s.7 Ne e heless, side e ec s o es ablished he apeu ics and he e olu ion o i al d ug e- sis ance ha e e ealed a lack o app op ia e new d ug classes. Du ing he las ew decades he cyclop opyl mo i has shown as onishing biological e ec s in d ug de elopmen and was i- nally in oduced as a spi ocyclop opana ion mo i o he D- i- bose sca old by Mula d e al.8–11 Since hen, spi ocyclop opa- na ions ha e been ealized a ca bons 2’, 3’ and 4’ by he addi- ion o di luo ome hylene (I) o me hylene uni s, gene a ed om me al eagen s o diazome hane (Figu e 1).8,12–14 Al- hough 4’/5’-me hylene spi ocyclop opana ed u idine de i a- i es ha e been syn hesized by he g oups o Boye and Robins, one aspec o he na i e subs i u ion pa e n o he nu- cleoside, namely he 5’-hyd oxyl g oup a he spi ocyclo- p opane, has been neglec ed.8,15 In ac , conse a ion o he 5’- hyd oxy mo i o polycyclic (II) o spi ocyclop opana ed sys ems (III) p o es challenging, as li e a u e-known spi ocy- clop opana ion p o ocols o nucleosides equi e elec onically neu al, exocyclic, p ima y alkenes. S a ing ma e ials con ain- ing elec on- ich alkenes a e associa ed wi h low yields unde epo ed eac ion condi ions.16 Recen ly, ou lab success ully in oduced he Simmons-Smi h-Fu ukawa me hod o he spi ocyclop opana ion o elec on- ich enol es e sys ems o a ious monosaccha ides o he pu pose o educing con o - ma ional eedom along he C5/C6-bond o achie ing 5-C- me hyla ion.17–19 Fu he , we ha e shown ha he s abili y o a ee spi ocyclop opanol mo i depends on he ca bohyd a e sca old. As isobu y ic es e s a e widely used in p od ug s a egies, eleasing he inal d ug in i o a e enzyme-ca - alyzed es e clea age, his p o ec ing g oup is sui able o p e- se ing he spi ocyclop opanol mo i .20 Ne e heless, he ac i- a ion o nucleosides in biological sys ems is ealized by en- zyme-p omo ed phospho yla ion gi ing 5’-phospho yla ed nu- cleo ides. The e o e, we en isioned a la e s age phospho yla- ion p o ocol o gene a e 4’/5’-spi ocyclop opana ed u idine monophospha e (cpUMP) om a cyclop opana ed p ecu so . In his wo k, we p esen a syn he ic app oach o 4’/5’-spi ocy- clop opana ed u idine de i a i es bea ing a 5’-oxy subs i u ion pa e n. The seconda y alcohol a he spi ocyclop opane mo i was masked by a ca boxylic es e o a monophospha e. Fi- nally, we in es iga ed he po en ial applica ion o compounds 6, 16, 19, 20 and 21 as an i i al agen s agains in ec ion wi h HRSV. As s a ing ma e ial we used he O5-silyla ed nucleoside 1, which was p epa ed om u idine acco ding o he epo ed p ocedu e by an O e lo e al.21 T ea men wi h benzy- loxyme hyl chlo ide (BOMCl) a o ded he ully p o ec ed nu- cleoside 2 (Scheme 1). BOM was chosen o enable simul ane- ous dep o ec ion o he nucleobase and he 2,3-syn-diol mo i by hyd ogenolysis wi h palladium on cha coal in a single s ep. Use o his hyd ogenoly ically clea able e he p o ed o be su- pe io o o he common p o ec ing s a egies lacking o hogo- nali y owa ds ca boxylic es e s o he spi ocyclop opane. Re- mo al o he O5-silyl g oup by TBAF a o ded compound 3 in 87% yield. A e oxida ion o he p ima y alcohol by IBX, he sensi i e aldehyde was di ec ly subjec ed o u he eac ion wi hou pu i ica ion. Enolisa ion by po assium ca bona e and subsequen es e i ica ion led o compound 4 (54%). Al hough high dias e eoselec i i y was epo ed o he co esponding li e a u e-known enol ace a es, only he (Z)-con igu ed di- as e eome was isola ed wi h he isobu y a es.22,23 Cyclop opa- na ion o he exocyclic enol es e was ca ied ou by applica- ion o he Simmons-Smi h-Fu ukawa p o ocol, p o iding compound 5 exclusi ely as he α- acial cyclop opana ed di- as e eome in 54% yield.24,25 The s iking dias e eoselec i i y is assumed o be con olled by he e he -p o ec ed O2 and O3, di ec ing he zinc ca benoid du ing cyclop opana ion. Follow- ing global dep o ec ion by hyd ogenolysis, using palladium on cha coal, secu ed he spi ocyclop opana ed u idine de i a i e 6 (Figu e 2). The con igu a ion o he isobu y ic es e a he spi ocyclop opane mo i and he p e ious α- acial dias e eose- lec i e cyclop opana ion we e con i med by NOE and sin- gle-c ys al X- ay analysis (Figu e 2 and Suppo ing In o ma- ion). De e mined by X- ay analysis, he D- ibose sca old showed a 3E pucke , wi h ca bon 3’ lying 0.57NÅ ou o he ing plane (5’-O-1’-2’). The χ- alue (-140.40(17)°) o he nu- cleoside de i a i e co esponds o he common an i-con o ma- ion. In compa ison o ee u idine (1.507(3)NÅ), no signi ican e ec o he spi ocyclop opane on he 4’-5’ bond leng h was obse ed (1.502(3)NÅ). The isobu y ic es e could no be eplaced by a phospho ic es- e wi hou ep o ec ion o bo h N5 o he nucleobase and he syn-diol mo i a he u anose. The e o e, we used an acid-ca - alyzed spi oke al o ma ion wi h 1,1-dime hoxycyclohexane (DMC) leading o compound 7 in 76% yield. The ollowing ep o ec ion o N5 wi h BOMCl p oceeded easily, gi ing com- pound 8 (89%). The isobu y ic es e was inally clea ed by al- kaline eac ion condi ions using sodium me hoxide in me hanol a 0N°C; es e clea age a oom empe a u e p o- mo ed decomposi ion o he seconda y alcohol a he spi ocy- clop opane and hampe ed o ma ion o p oduc 9. Addi ion o phospho us (V) species such as phospho yl chlo ides a o ded only aces o he desi ed phospho yla ed u idine de i a i e. Howe e , phospho us (III) species nea ly con e ed he sec- onda y alcohol o he phospho amidi e 10, a e which he di- isop opylamino g oup was subs i u ed by benzylic alcohol in a e azole-media ed eac ion. The eme ging phosphi e was sub- sequen ly oxidized o he dias e eome ic mix u e o he co e- sponding phospha e 11 by he addi ion o mCPBA a 0N°C (69%). An aqueous solu ion o ammonium hyd oxide was used o selec i e clea age o he cyanoe hyl p o ec ing g oup in quan i a i e yield.25 The esul ing phospha e es e was ob- ained as a mixed sal wi h ammonium and ammonium p opi- oni ile ca ions. Column pu i ica ion using ie hylamine as an addi i e in he eluan led o he espec i e ie hylammonium sal bu s ill emained di icul (12a, see he Suppo ing In o - ma ion). The ollowing s eps we e pe o med wi hou u he pu i ica ion as his p o ed o be needless. Benzyla ion o com- pound 12 was applied o easons o pu i ica ion, as pa ially unp o ec ed phospha es p e en ed p ope aqueous wo k-up a - e ke al clea age. T ea men wi h hyd ogen chlo ide in diox- ane deli e ed he dep o ec ed 1,2-syn diol mo i o he spi ocy- clop opana ed u idine monophospha e. In con as o com- pound 6, only he pa ially dep o ec ed nucleo ide 15 was ob- ained a e palladium-media ed hyd ogenolysis o 14, lea ing a hemiaminal a N5. Dissol ing he nucleo ide in D2O a 50 °C u nished comple e hyd olysis o he hemiaminal unde o ma- ion o he desi ed cpUMP nucleo ide 16 (SchemeN2).27 The p ocess o empe a u e-p omo ed hyd olysis, gi ing ise o an inc easing signal se o cpUMP, was moni o ed by 1H NMR spec oscopy (see Suppo ing In o ma ion). Scheme 3. Syn hesis o Spi ocyclop opana ed D-Xylou idine Sca old 19. NH O ON O OTBDMS HO NH O ON O OH HO TBDMSCl (2.2 equi ) AgNO3 (2.2 equi ) Py (5 equi ) THF, , 16 h (81%) DMP (2 equi ) DCM, , 3 h (50%) 6 17 R = OCiP 18 R = OCiP 1) NaBH4 (1.1 equi ) MeOH, 0 °C, 0.5 h (d 5.9:1) 2) TBAF·3 H2O (1.1 equi ) THF, 0 °C, 3 h (67% o e wo s eps) NH O ON O OTBDMS O 19 R = OCiP RO RO RO D-Xylose sca olds a e aluable syn he ic p ecu so s o he p epa a ion o u he 3’-subs i u ed de i a i es.28 S uc u al elucida ion expe imen s by NMR e ealed D-xylose nucleos- ides o adop s ic ly a 3’-endo con o ma ion.29 In o de o in- es iga e he con o ma ional impac o 4’/5’-spi ocyclop o- pana ions on he ing lexibili y o u idine, we submi ed 6, showing a 3E-con o ma ion, o a ou -s ep 3’-epime isa ion. The eby, 17 was p epa ed by a modi ied p ocedu e o an anal- ogous syn hesis by K eu z.30 Epime isa ion o ca bon 3’ was pe o med by p io oxida ion o he hyd oxy g oup using Dess–Ma in pe iodinane (50%).31 The 3-oxonucleoside 18 was dias e eoselec i ely educed by NaBH4 deli e ing he de- si ed D-xylose nucleoside as he majo dias e eome .32,33 Suc- cessi e desilyla ion by applica ion o TBAF a lowe empe a- u e a o ded 19 in 67% yield o e wo s eps (Scheme 3). In e es ingly, NMR in es iga ions showed ha 4’/5’-spi ocy- clop opana ion e ec i ely guided he u idine s uc u e om he na i e 3’-endo con o ma ion o a 3E pucke , as obse ed in X- ay analysis o compound 6.34 Ne e heless, his e ec does no su i e he 3’-epime iza ion. Changing he molecula con- igu a ion a 3’ causes a e ans o ma ion o a 3’-endo pucke , as indica ed by he s ongly educed coupling cons an be- ween H1 and H2 (Scheme 4).35 Con e ing 6 o he espec i e 5’-phospha e 16 led only o a mino e ec on he ing con o - ma ion (3JH1-H2 = 6.9NHz). Fo a mo e de ailed and compa a i e iew on he o sion angles o compound 6 and o he con o - ma ional es ic ed ibose and 2’-deoxy ibose nucleosides in DNA, see he Suppo ing In o ma ion.36,37 Compa ed o he 2’- deoxy ibose backbone o DNA, he δ- alue o compound 6 (137.6N°) i s he egion o he DNA B- o m gi en by he sca - e plo o Dicke son e al.38 Scheme 4. P epa a ion o Es e i ica ed D-Xylo Nucleoside 20 and D-Ribo Nucleoside 21. NH O ON O OH R 1 ( i P CO) 2 O (30 equi ) py , , 16 h R 2 NH O ON O OOC i P R 1 R 2 19 R 1 = OH; R 2 = H 3 J H1-H2 0.9 Hz 6 R 1 = H; R 2 = OH 3 J H1-H2 7.7 Hz 20 R 1 = i P COO; R 2 = H (59%) 21 R 1 = H; R 2 = i P COO (98%) i P COO i P COO Acco ding o he es ablished p od ug s a egy we sough o in- c ease memb ane solubili y and pe meabili y o ou nucleoside Figu e 3. An i i al ac i i y and cell oxici y o compounds 6, 16, 19, 20 and 21 compa ed o he an i i al guanosine analog iba i in. Mean and s anda d de ia ions o h ee o ou independen expe imen s a e shown. de i a i es by dec easing he hyd ophilici y. Fo his pu pose, bo h spi ocyclop opana ed compounds 19 and 6 we e es e i i- ca ed wice by applica ion o isobu y ic anhyd ide in py idine. Compounds 20 and 21 we e ob ained in 59% and 98% yield, espec i ely. Figu e 2. X- ay c ys al s uc u e o compound 6 (50% ellipsoid p obabili y). To de e mine he an i i al ac i i y o he spi ocyclop opana ed u idine nucleosides, hei inhibi o y e ec was exempla ily es ed agains he nega i e s and RNA i us HRSV. The e o e, HEp2 cells we e in ec ed wi h HRSV39 in he p esence o a i- ous compound concen a ions. Riba i in, he only nucleoside analogue licensed as RSV a ge ing an i i al he apeu ic, was used as assay con ol. In he es ed dose ange, compounds 6, 16 and 19 did no show any an i i al e ec (Figu e 3). In con as , compounds 20 and 21 inhibi ed i al in ec ion when used a concen a ions o 100NµM o 33NµM, espec i ely. Al- hough he an i i al e ec was modes , i occu ed indepen- den ly o any o e cy o oxic e ec , sugges ing ha he ob- se ed e ec was a ibu able o di ec inhibi ion o HRSV in- ec ion a he han o indi ec e ec s caused by comp omised cell iabili y. Mo eo e , nucleoside 21 was mo e e ec i e han compound 20, p o iding he i s hin s o op imizing he an i-HRSV ac i i y o hese spi ocyclop opana ed u idine nu- cleosides. An i i al ac i i y o nucleoside analogs depends on se e al ac o s. The compounds ha e o pe mea e he cell memb ane40 and be phospho yla ed by he hos cell machine y41,42 in o de o be inco po a ed in o he nascen i al RNA, hus causing chain e mina ion o mu a ions by base mispai ing in genome eplica ion.43 In ou case, ex acellula phospho yla ion o compound 6 did no ende compound 16 an i i al. Howe e , compounds 20 and 21, which a e mo e lipophilic han hei de i a i es, compound 19 and 6, possess an an i i al ac i i y a high doses. Fu he expe imen s a e needed o conclude whe he his is because o imp o ed cell memb ane pene a- ion o hese molecules. Mo eo e , i will be in e es ing o see whe he spi ocyclop opana ed u idine nucleosides ha e b oad spec um an i i al ac i i y o a e HRSV speci ic inhibi o s. In conclusion, we ha e demons a ed an access o 4’/5’-spi o- cyclop opana ed u idine de i a i es while p ese ing he na- i e hyd oxyla ion pa e n o he nucleoside. Fu he , we ha e gene a ed he i s spi ocyclop opana ed nucleo ide o u idine applying he Simmons-Smi h-Fu ukawa p o ocol as he key s ep. NMR analysis o a spi ocyclop opana ed D-xylose sca - old showed a mino impac o he spi ocyclop opane on he ing con o ma ion in compa ison o an axially o ien ed 3’-hy- d oxy g oup. Fo wo u idine de i a i es, we obse ed mode - a e an i i al ac i i y in an HRSV inhibi ion assay. Fo a ans- e o ou de eloped me hodology o cy osine, guanine and adenine nucleosides we expec a p ope p o ec ing s a egy o he nucleobase o be necessa y. ASSOCIATED CONTENT Suppo ing In o ma ion De ailed expe imen al p ocedu es, analy - ical da a o all new compounds, and c ys al da a (CIF) o 6. This ma e ial is a ailable ee o cha ge ia he In e ne a h p://pub- s.acs.o g. AUTHOR INFORMATION Co esponding Au ho *E-mail: [email p o ec ed]. ORCID Daniel B. We z: 0000-0002-3973-2212 No es The au ho s decla e no compe ing inancial in e es . ACKNOWLEDGMENT We hank Ch is ina G e he (TWINCORE) o expe echnical as- sis ance. We hank he S udiens i ung des deu schen Volkes (P o- mo ionss ipendium o C.K.) and he Fonds de Chemischen Indus- ie (P omo ionss ipendium o C.K.) o unding. Wo k in he T.P. labo a o y was suppo ed by a g an om he Helmhol z-Albe a ini ia i e o in ec ious disease esea ch (HAI-IDR). We hank D . Ke s in Ib om (TU B aunschweig) o he kind suppo . HRSV lu- ci e ase epo e i us was a kind gi om Ma ie-Anne Rameix- Wel i (Uni e si é de Ve sailles S . Quen in) and Jean-F ançois Éléue (Uni e si é Pa is Saclay. REFERENCES (1) Cle cq, E. de; Holý, A. Acyclic nucleoside phosphona es: a key class o an i i al d ugs. Na . Re . D ug Disco . 2005, 4, 928–940. (2) Jo dheim, L. P.; Du an el, D.; Zoulim, F.; Dumon e , C. Ad- ances in he de elopmen o nucleoside and nucleo ide analogues o cance and i al diseases. Na . Re . D ug Disco . 2013, 12, 447– 464. (3) Chaudhu i, S.; Symons J. A.; De al J. 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