Synthetic Strategies for 14C Labelling of Drug Molecules Dr ... - Almac

Introduction to 14 Ccpres/v1b/ca/1998-08/ 4

Discovery of 14 CMartin Kamen & Sam Ruben (27-FEB-1940)T 1/2 ~ 5730 Years

14C Starting Materials

Barium 14 C carbonate staircaseOHCH 314C 6AA14CH 14 C14CHPEPTIDESHNOH 14 CHO14CH 3IMeOC H 3HOMeO14C 614CO 214NCH 3OH H143COHN OCu 14 CNK 14 CN14C 6 14F 3C C OHClOHOMeOMeOHOHBa 14 CO 3Cl

14C Radiotracer• In pharmaceutical research 14 C is used asa tracer to ensure that potential drugs aremetabolized without forming harmful byproducts– ADMET Studies• The 14 C label should ideally form part ofthe compounds molecular skeleton

14C Synthetic Strategies14Clabelling

Target: [ 14 C]XEN-D0401• XEN-D0401 is a novel andselective small moleculeactivator of the largeconductancecalciumactivatedpotassiumchannel (known as the BKchannel)• Currently in Phase 1development for thetreatment of overactivebladder (OAB)F 3CCl*HNOOH* = 14 C LabelOH

14C Starting MaterialF 3CHN*OOHOHClMeO*OOO14CO 2Cl[ 14 C]XEN-D0401 [ 14 C]-4

Generation of ‘Dry’ 14 CO 2HIGH VACUUMPUMPX14CO 2(wet)High Vacuum ManifoldP 2O 5c H 2SO 4Ba 14 CO 314CO 2(dry)14CO 2Liquid Nitrogen

CarboxylationBrOH1. NaH / DMFBrOOn-BuLi/HexaneLiOOCl2. MOM-ClCl(1) (2)THF-78 o C, 20 minsCllithiated intermediateHOOBa 14 CO 3c H 2SO 414CO 2[ 14 C]-3-196 o C to rt>50 mCi/mmol250 mCiRadiochemical yield = 76%*OOCl

O-MethylationHO*OOOK 2CO 3/ AcetoneCH 3IMeO*OOOCl35 o CCl[ 14 C]-3[ 14 C]-4

ClNH 2CF 3OSTEP 1NHCOt-BuCF 3n-BuLi, Hex, THF-78 o CLiNOLi(5)83%(6)CF 3MeO*OOMOMCl[ 14 C]-4F 3CNHCOt-BuO* OMOMColumn chromatographyClSTEP 271%[ 14 C]-7

S L Kitson, S J Jones et al. J Label Compd. Radiopharm 2010, 53, 140-146NaOHEtOHSTEP 368%F 3CClNH 2O* OMOM[ 14 C]-8OOLiHMDS / THF-20 o C to -10 o CSTEP 4F 3CClHN*[ 14 C]-9OOHOMOMIPA, HCl45 o CColumn ChromatographyF 3CClHN*OOHOHOverall Radiochemical Yield ~ 30%STEP 563%[ 14 C]XEN-D0401* = Carbon-14 label

Key Step: A one pot construction of the quinolin-2-onering under strong baseF 3CClHN HO*O[ 14 C]-8OLiHMDS (2eqv)OOcondensationF 3CClOLiOHNLiO*OdianionOLiHMDSF 3CClOLiO-NLiO*OOcyclisationF 3CLiN O*LiOOCltrianionOLiOH 2OF 3CHOHN*OHOOHOLiOH-H 2OdehydrationF 3CClHN*OOOHOCl[ 14 C]-9

Aporphine skeleton‘naturally occurring’213AC4B14C6aN56R11710D89Dicentra formosa'Bleeding heart'S L Kitson. J Label Compd Radiopharm; 2007, 50, 290-294S L Kitson. J Label Compd Radiopharm; 2006, 49, 517-531

Structure & Function of (R)-(-)-Apomorphine• (R)-(-)-Apomorphine is a potent non-selectiveD 1 /D 2 agonist at the dopamine receptor.• (R)-(-)- Apomorphine (Apokyn) is used tocontrol Parkinson’s disease (D 2 receptor).• (R)-(-)- Apomorphine (Uprima ® ) is used to treaterectile dysfunction (D 1 receptor).• Features:• Contains an ortho-phenol motif.• Tetracyclic carbon skeleton.• Tetrahydroisoquinoline nucleus.• ‘Planar’ structure.• Contains an embedded dopaminepharmacophore.SR

Retrosynthesis of ApomorphineHOHOFGIA BN CH3C *MeODMeO(R/S)-[6a- 14 C]Apomorphine dimethyl ether*= 14 CRING CFORMATIONPschorrcouplingMeOADOMeBN*NO 2CH 3Bischler-NapieralskicyclodehydrationRING BFORMATIONMeOMeONO 2D*ON HATetrahydro[ 14 C]isoquinolineN-[carboxyl- 14 C]Acetamide

Synthesis of N-[carboxyl- 14 C]acetamideK 14 CN55mCi/mmolNO 2MeODMeO(COCl) 2DMFMeOMeOClNO 2D*OClMeOMeOANO 290% 86%86%(2 steps)NOacid2* hydrolysis MeOCN*OMeONONH 2H2 MeO* NDOMeOOHN-[carboxyl- 14 C]AcetamideARadiochemical yield = 67%

Bischler-Napieralski CyclodehydrationMeOMeONO 2D*OHNAP 2O 5/POCl 3TolueneMeOMeONO 2ND *OPimineAN-[carboxyl- 14 C]AcetamideMeOMeONO 2D*N +-OPAnitrilium saltRING BFORMATIONA*BN +ClHbasic work upA*BNMeODOMeNO 2MeODNO 2OMeDihydro[ 14 C]isoquinolineRadiochemical yield = 43%

N-Methylation Reduction StepMeOOMeNN +CH 3I/THFNO 2* * *I -NaBH 4/EtOHCH 3MeO NO MeO2OMeOMeNNO 2CH 3Dihydro[ 14 C]isoquinoline [ 14 C]Methiodide Tetrahydro[ 14 C]isoquinolineRadiochemical yield = 77%Bischler-NapieralskiEndocyclic Product

Reduction-Pschorr CouplingNCH 3N*CHZn/HCl *310% H 2SO 4DDNaNO 2MeONO 2MeONH 2OMeOMeTetrahydro[ 14 C]isoquinolineA BDNCH 3+MeO N + N HSO-OMe4aryldiazonium salt*PSCHORR COUPLINGCu 2ORING C FORMATIONACMeODMeO*NCH3H(R/S)-[6a- 14 C]Apomorphine dimethyl etherB

Chiral SeparationMeO*N CH3HMeO(R/S)-(+/-)-[6a- 14 C]Apomorphine dimethyl etherHPLC Conditions: Chiralcel OD column, eluting withhexane/propan-2-ol/dimethylamine [950:50:5]MeO*N CH3HSMeO*N CH3HRMeOMeO(S)-(+)-[6a- 14 C]Apomorphine dimethyl etherRadiochemical yield = 11% fromTetrahydro[ 14 C]isoquinoline(R)-(-)-[6a- 14 C]Apomorphine dimethyl etherRadiochemical yield = 16% fromTetrahydro[ 14 C]isoquinoline

(R)-(-)-[6a- 14 C]ApomorphineMeO*N CH3HBBr 3CH 2Cl 2HO*N CH3HMeOHO(R)-(-)-[6a- 14 C]Apomorphine dimethyl ether(R)-(-)-[6a- 14 C]Apomorphine• PLRP-S column (CH 3 CN:HCl aq)• Specific Activity: 55mCi/mmol• Radiochemical purity => 98%• Radiochiral purity => 99%

Combretastatin A-1MeOOHMeOOMeOMeOH• Combretastatin A-1 is a natural productfirst isolated from the African bush willow treein the 1980s• Chemotherapeutic properties by acting on thetumour vasculature, leading to blood flow shutdown and ultimately tumour death

Target: [methyl- 14 C]Combretastatin A-1 diphosphateMeO14CH 3OOMe• Treatment of solid tumoursOMeO• The pro-drug undergoes in vivo de-phosphorylationto generate the active agent combretastatin A-1OPOOPOHO.2K +OOH

14C Synthetic routeR T Brown et al. J Label Compd. Radiopharm 2009, 52, 567-570MeOO14CH 3ITBAFCH 3CNMeOOTBSOO75%14CH 3OOOMeOMeOMeOMeTiCl 4/ DCMMeOOH(BnO) 2P(O)H27%14CH 3OOHCCl 4/ CH 3CNOMeOMeDIPEA / DMAP72%MeOOP(O)(OBn) 21. TMSBr / MeCN2. KOMe, MeOH3. HCl/H 2O/EtOHMeOOPOOHO14CH 3OOMeOMeOP(O)(OBn) 257%14CH 3OOMeOMeOOP.2K +OOH[methyl- 14 C]-Combretastatin A1 diphosphate

[ 14 C]AcetylenesOH14COTarget: [5- 14 C]Hex-5-ynoic acid

[ 14 C]Acetylenes:Ohira Bestmann ReagentMeOMeOOPON +N -CH 3K 2CO 3MeOHrt, 4-16 hMeOMeOOPON +N -OMeCH 3MeOMeOOP-N +N -Ohira-Bestmann ReagentO14R CHR14CH

14C Acetylene SynthesisBrOHOp-TsOHEtOHBrOTHPK 14 CN, EtOH71 %67 %NC *OTHPDiBAL80 %HO14COTHPON +NOPOMeOMeK 2CO 3, MeOH14COTHPCrO 3, H 2SO 4,H 2O, acetone68 % 74 %Overall Radiochemical Yield 29 % from [ 14 C]KCN14COOH

[ 14 C]ZT-1: a new generation of acetylcholinesterase(AChE) inhibitorsL Leman, S L Kitson et al. J. Label Compd. Radiopharm 2011, 720-730CH 3exocyclic E-double bondbicyclo[3.3.1] double bondHNOH 3CCH 3NH 2hydrogen bondHOMeOCH 3N RHNHOactive metabolite, (-)-huperzine Ain vivoprogressive hydrolysis+CHOOHClpyridin-2-one ringClOMePro-drug (-)ZT-1inactive metabolite, 5-chloro-o-vanillin

14C SynthesisOHOMOMOMOMNaH/DMFn-BuLiI* * *MOM-ClI 2[ 14 C]-1Step 1 [ 14 C]-2 Step 2 [ 14 C]-3CuBr/DMFNaOMe/MeOHOMOMO OMOMOMe n-BuLiOMeH* *DMFStep 3 [ 14 C]-4 Step 4[ 14 C]-5O OHHCl/MeOH ICl / CHOMe2 Cl 2H*NaHCO 3HOOH*ClOMe* = U- 14 CStep 5[ 14 C]-6Step 6[ 14 C]-7

[ 14 C]-ZT-1OOHC H 3CH 3NH 2HNOH*[ 14 C]-7ClEtOHStep 7OMeC H 3HOMeOCH 3N*HNClO(-)-Huperzine A[ 14 C]-ZT-1

ConclusionWhen designing a 14 C labelled synthesis it is important toconsider the following:• Identify simple starting materials from the barium 14 Ccarbonate ‘staircase’ which are commercially available oralternatively easily made• Plan, develop and execute the synthetic methodology tothe final drug substance. This approach can often restrictthe position of the label in the drug and will cause a changein the drug purity profile from the original laboratorysynthesis route• Locate a biologically stable position for the 14 C label

14C Radiochemistry Laboratory