Synthesis of Isotopically Labelled

Research Article

Received 31 May 2011, Revised 8 July 2011, Accepted 13 July 2011 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1918 Synthesis of isotopically labelled [14C]ZT-1 (Debio-9902), [d3]ZT-1 and ()-[d3]huperzine A, a new generation of acetylcholinesterase inhibitors Loïc Leman,a* Sean L Kitson,a Rodney T Brown,a Jana Cairns,a William Watters,a Austin McMordie,a Victor L Murrell,a and Judith Marfurtb

A method has been developed for the synthesis of two isotopically labelled forms of a pro-drug of the acetylcholinesterase 14 inhibitor ()-huperzine A. These labelled compounds, [ C]ZT-1 (Debio-9902) and [d3]ZT-1, were used in clinical studies to evaluate a potential treatment for Alzheimer’s disease. The pro-drug [14C]ZT-1 was isolated with a radiochemical purity of >98% and a gravimetric speciﬁc activity of 129 mCi/mg in a seven-step synthesis starting from [U-14C]phenol in 7% yield. Subsequently, the deuterium labelled target ()-[d3]huperzine A was achieved in six steps with an overall yield of 15% and gave an isotopic distribution of d2 (1.65% huperzine A) and d3 (97.93% huperzine A) with a chemical purity of 98.5%. Con- densation of the substrate ()-[d3]huperzine A with 5-chloro-o-vanillin gave the Schiff base [d3]ZT-1 in a chemical yield of 80%. Reduction of the Schiff base gave reduced-[d3]ZT-1, which was converted into the hydrochloride salt with an isotopic distribution of d2 (1.60%) and d3 (98.02%).

Keywords: Alzheimer’s disease; ()-huperzine A; acetylcholinesterase; ZT-1; Debio-9902; deuterium; carbon-14

Introduction galanthamine and donepezil, which are both used in the treatment of patients suffering from mild to moderate Alzheimer’s In Chinese medicine, the compound ( )-huperzine A (1), shown disease.10 The structure of the complex of ()-huperzine A with in Figure 1, is known as Chien Tseng Ta and Shuangyiping. This acetylcholinesterase has been elucidated by X-ray naturally occurring sesquiterpene alkaloid is present only in the crystallography.11 ( )-enantiomer form. The alkaloid was originally isolated from Alzheimer’s disease is a neurodegenerative disorder asso- the Chinese club moss Huperzia serrata and is part of the Lycopo- ciated with extracellular deposits of amyloidal plaques in the 1 dium plant family. The Chinese have used this plant for over grey matter that affect the cerebral cortex, amygdale and hippo- several centuries in the treatment of various blood disorders, campus brain regions. Progression of the disease is characterised control of fever, schizophrenia and hypertension. by damage to the brain neural networks, especially within the cho- The synthesis of the alkaloid ( )-huperzine A was achieved inde- linergic (acetylcholine-producing) pre-synaptic neurons, leading to 2–4 5 pendently in 1989 by Kozikowski et al. and Qian et al. This was weak neurotransmission.12 This forms the basis of the ‘cholinergic then followed by the total synthesis of ( )-huperzine A by hypothesis’ to develop drugs such as ()-huperzine A to increase 6 Yamada et al. in 1991. To date, the best synthesis of the racemic the levels of cerebral acetylcholine for the treatment of sympto- 7,8 product is the route published by Campiani et al. in 1993. matic Alzheimer’s disease.13 The chemical structure of ()-huperzine A contains a bicyclo[3.3.1] carbon skeleton fused to a pyridone moiety with an exocyclic ethylidene and a primary amino group. The stereochemistry and chemical structure have been assigned as (5R,9R,11E)-5-amino-11- ethylidene-5,6,9,10-tetrahydro-7-methyl-5,9-methanocycloocta- a [b]-pyridin-2[1H]-one, which contains the embedded pharma- Isotope Chemistry Laboratories, Almac, Almac House, 20 Seagoe Industrial Estate, Craigavon, BT63 5QD, UK cophore 5-substituted aminomethyl-2(1H)-pyridone.1 Pharmacological studies have shown that ()-huperzine A diffuses bDebiopharm S.A., Forum Après-demain, Chemin Messidor 5-7, CP 5911-1002 across the blood-brain barrier and is a selective, reversible inhibitor of Lausanne, Switzerland acetylcholinesterase.9 This cholinesterase inhibitor is 38 times 6 *Correspondence to: Loïc Leman, Isotope Chemistry Laboratories, Almac, Almac more potent than the other enantiomer, (+)-huperzine A. The House, 20 Seagoe Industrial Estate, Craigavon BT63 5QD, UK. mode of action of ()-huperzine A is similar to that of E-mail: [email protected]

12 prescribed against Alzheimer’s disease, such as ()-huperzine CH3 A, donepezil, tacrine and rivastigmine.21 7 H 8 1 The development of this implant was dependent on the chemi- 10 N 6 O cal modifications that can be carried out on ()-huperzine A. These 9 2 14 modifications could include the exocyclic and bridge double 5 H C 3 bonds, pyridin-2-one ring and primary amine group. Various 3 11 4 NH2 13 in vitro studies have shown that when either or both the exocyclic and bridge double bonds are reduced, or pyridin-2-one ring is (1) substituted, the activity towards cholinesterase decreases.14 Figure 1. ()-Huperzine A and numbering. Therefore, the primary amine of ( )-huperzine A was reacted with various aldehydes to form Schiff base derivatives.22 The reaction with the aldehyde 5-chloro-o-vanillin produced the lead compound ZT-1, and its chemical name is [5R-(5a,9b,11E)]-5- ()-Huperzine A was found to protect neuronal and glial [[(5-chloro-2-hydroxy-3-methoxyphenyl)methylene]amino]-11- cells against the cytotoxicity of b-amyloid plaques. Various ethylidene-5,6,9,10-tetrahydro-7-methyl-5,9-methanocycloocta[b] clinical trials of ()-huperzine A have been carried out in pyridin-2(1H)-one.23 The Schiff base ZT-1 was shown to have a China, and have shown its ability to enhance memory func- 14 degree of stability under biological conditions due to the electro- tion in young adults. Hence, these pharmacological observa- nic properties of the 5-chloro-o-vanillin moiety reducing the tions make it a suitable investigational drug in the treatment of electron density around the imine. Another key feature was the progressive neurodegenerative diseases, and it is presently mar- ’ ability to form an intramolecular six-membered ring via hydrogen keted in China as a therapeutic agent to treat Alzheimer s dis- 14 10,14 bonding, which stabilised the Schiff base. ease. In another therapeutic area, (þ)-huperzine A shows The slow release of the pro-drug ZT-1 from the implant into antagonistic behaviour towards the N-methyl-D-aspartic acid the blood stream undergoes in vivo progressive hydrolysis to receptor, which protects the brain against glutamate-induced 15 liberate the active compound ()-huperzine A and the associated damage. 5-chloro-o-vanillin metabolite, as shown in Figure 2.24,25 The use of acetylcholinesterase inhibitors in clinical studies is limited by their short half-life and associated severe side effects 16 caused by the activation of the peripheral cholinergic system. Results and discussion For example, donepezil has adverse toxic effects on the liver.17 To circumvent problems linked to oral administration (variability The convergent syntheses of the carbon-14- and deuterium- in exposure, difficulty of compliance), an injectable and biode- labelled targets of the active metabolite ()-huperzine A and its 14 gradable sustained-release implant formulation of ZT-1 was devel- corresponding pro-drugs [ C]ZT-1 and [d3]ZT-1 are discussed oped by Debiopharm as a potential treatment of symptomatic throughout this paper, and the chemical structures are shown Alzheimer’s disease.18,19 This implant was injected into the in Figure 3. The synthetic route to the carbon-14 ring labelled patient and, over a period of several weeks, slowly released the 5-chloro-o-vanillin will be described, followed by a description 20 pro-drug ZT-1 into the blood stream. Extensive in vitro and of the syntheses of ()-[d3]huperzine A and [d3]ZT-1. in vivo studies have shown that ZT-1 has a good selectivity The retro-synthetic pathway for the incorporation of a carbon-14 between acetylcholinesterase and butyrylcholinesterase, higher ring label (denoted by *) in the 5-chloro-o-vanillin moiety is shown bioavailability, lower toxicity and better restorative effects on in Figure 4. The target, [14C]-7, was formed from the deprotec- cognitive impairments compared with several drugs currently tion and chlorination of A. The protected phenol A was

CH3 H N O

bicyclo[3.3.1] double bond H3C exocyclic E-double bond NH2

CH 3 active compound, (-)-huperzine A hydrogen bond CH 3 in vivo progressive hydrolysis + H H O N N R CHO MeO H O OH

pyridin-2-one ring Cl OMe Cl

Pro-drug ZT-1 inactive metabolite, 5-chloro-o-vanillin

Figure 2. Slow release of ()-huperzine A.

CH3 CH3 H H N N CH3 O O H N H C D C O 3 N 3 N

D C 3 HO HO NH2 * MeO Cl MeO Cl

(-)-[d3]Huperzine-A [14C]ZT-1 [d3]ZT-1

14 Figure 3. [ C]- and [d3]-labelled targets of ()-huperzine A derivatives. synthesised by lithiation and formylation of B. Substrate B was pro- anhydrous conditions gave MOM-protected anisole [14C]-4,ina duced from protected phenol C, which, in turn, was derived from radiochemical yield of 45% and a radiochemical purity of 99.3%. the carbon-14-labelled phenol [14C]-1. The MOM-protected anisole [14C]-4 was ortho-lithiated, as in The pro-drug [14C]ZT-1 was prepared in seven steps with a the previous step, followed by reaction with anhydrous N,N- radiochemical yield of 7%, starting from [U-14C]phenol with a dimethylformamide to give [14C]-5 in a radiochemical yield of specific activity of 131 mCi/mmol. The final step is shown in Figure 5, 94%. The MOM protecting group was then removed under acidic where condensation of the aldehyde moiety of 5-chloro-o- conditions to give o-vanillin [14C]-6 with a radiochemical yield of [ring U-14C]vanillin with the primary amine of ()-huperzine A 62% and a radiochemical purity of 98.4%. This was shown to be a leads to the subsequent loss of water and formation of the Schiff single regioisomer, as determined by proton-NMR spectroscopy. base [14C]ZT-1. The target was obtained with a gravimetric spe- The o-vanillin [14C]-6 was chlorinated by stirring with iodine cific activity of 129 mCi/mg. monochloride and sodium hydrogen carbonate. A small trace of an unknown impurity was observed, but this was removed by silica gel column chromatography to give pure 5-chloro-o- Synthesis of 14C-labelled compounds vanillin, [14C]-7, in a radiochemical yield of 54%. The radiochemical purity by HPLC area% was 98.3% and the chemical purity was The first step in the synthesis, as illustrated in Figure 6, was 14 fi 14 99.2%. The material [ C]-7 underwent a speci c activity dilution the methoxymethyl (MOM)-ether protection of phenol [ C]-1. fi fi 14 using unlabelled 5-chloro-o-vanillin to give a nal speci c activ- Phenol [ C]-1 was reacted with sodium hydride in anhydrous 14 ity of 54.6 mCi/mmol. This material was coupled to ( )-huperzine N,N-dimethylformamide to produce the [ C]phenoxide anion. A (Figure 7), giving the pro-drug [14C]ZT-1 in a radiochemical The anion was quenched with MOM chloride to give MOM-protected yield of 66% and a radiochemical purity of 98.5%. phenol [14C]-2 in a radiochemical yield of 98% with a radiochemical purity of >97%. Synthesis of d3-labelled compounds The MOM-protected phenol [14C]-2 was able to facilitate a directed ortho-lithiation, which was achieved using 1.2 equiva- The retro-synthetic analysis for the incorporation of deute- lents of n-butyl lithium in hexane, at 0 C.26–28 The ortho-lithiated rium in ()-huperzine A is shown in Figure 8. The main fea- intermediate was then quenched with iodine to give the MOM- tures of this synthesis plan are the introduction of the amino protected ortho-iodophenol [14C]-3, in a radiochemical yield of moiety through a Curtius rearrangement and the generation of 84% and a radiochemical purity of 73.4%.29 This material was the exocyclic olefin using Wittig chemistry on (+)-(5S,9R)methyl used without further purification in the next step where aromatic 9,10-dihydro-2-methoxy-7-methyl-11-oxo-5,9-methanocycloocta nucleophilic substitution of the iodide with methoxide under [b]pyridine-5(6H)-carboxylate [(+)-8].30 The route for incorporation

O OH OP OP OMe lithiation H deprotection OHC OMe OMe * * * formylation halogenation Cl

[14C]-7 A B

Alkoxy- OP OH de-halogenation protection (P)

* *

C [14C]-1

Figure 4. Retro-synthetic pathway of [14C]-7.

CH3 H N O CH3 Schiff base formation H O OH H C N O 3 N OMe + H * HO NH2 H3C * Cl MeO Cl

[14C]ZT-1 (-)-Huperzine A 5-Chloro-o-[ring U-14C]vanillin

Figure 5. Position of the carbon-14 labelling.

OH OMOM OMOM NaH/DMF n-BuLi I * * * MOM-Cl I2

[14C]-1 Step 1 [14C]-2 Step 2 [14C]-3

OMOM O OMOM n-BuLi CuBr/DMF OMe OMe H * * NaOMe/MeOH DMF

Step 3 [14C]-4 Step 4 [14C]-5

O OH O OH OMe ICl / CH Cl HCl/MeOH OMe 2 2 H H * * NaHCO3 Cl Step 5 [14C]-6 Step 6 [14C]-7

Figure 6. Radiosynthesis of 5-chloro-o-[phenyl U-14C]-vanillin, [14C]-7.

of deuterium methyl is the reaction between 2,2,2-[d3]ethyl bro- at 110 C, E-isomer predominated at 93%, as indicated by mide (isotopic enrichment 98 atom% D) and triphenylphosphine proton-NMR analysis. The E-olefin isomer was obtained in 62% (step 8) to produce 2,2,2-[d3]ethyltriphenylphosphonium bromide. yield. In Figure 9, the optically pure starting material [(+)-8] shown is The sterically hindered methyl ester group of [d3]-10 was a versatile intermediate for the synthesis of several analogues hydrolysed using 20% sodium hydroxide in a mixture of tetrahy- of huperzine A.3,4,31,32 This is because the ketone carbonyl drofuran and methanol at an elevated temperature to produce group of (+)-8 can undergo Wittig reactions and the endocyclic the E-isomer carboxylic acid [d3]-11 in a yield of 65%. The more bridge double bond is in the same position as in huperzine A. hindered Z-isomer ester of [d3]-10 did not undergo base hydro- The ketone carbonyl centre in (+)-8 was reacted with 2,2,2-[d3] lysis. The E-isomer carboxylic acid [d3]-11 was converted into the ethyltriphenylphosphonium bromide in the presence of n-butyl carbamate [d3]-12 via the isocyanate intermediate, which was lithium. The absence of the 3H signal at 1.51 ppm indicates com- generated from the reaction of diphenylphosphoryl azide on 33 plete labelling of the olefin-methyl to d3-methyl. The reaction pro- [d3]-11. duced an approximately 75:25 mixture of the (Z)- and (E)- The isocyanate intermediate was heated in methanol to olefins [d3]-9 in a chemical yield of 92%. produce the methyl carbamate [d3]-12 as white foam, in a yield Unfortunately, the olefin mixture of [d3]-9 consisted largely of of 68%. Proton-NMR analysis of [d3]-12 indicated approxi- the incorrect isomer needed to assemble huperzine A. To overcome mately 8% of the acid [d3]-11, which was extracted using a base. this problem, it was necessary to carry out an isomerisation of the The final step in the synthesis was the dual cleavage of the ethylidene moiety [d3]-9 using the radical initiator 2,2′-azobis(2- methyl ether and the methyl carbamate of [d3]-12, by the use 31 methylpropionitrile) and thiophenol as in step 10. The extent of iodotrimethylsilane, to afford ()-[d3]huperzine A as an off- of isomerisation was dependent on the reaction temperature; white solid in a yield of 60%.

O OH OMe H CH3 * CH H 3 N 14 O H [ C]-7 N Cl O H C 3 N H C EtOH 3 NH 2 HO Step 7 * MeO Cl (-)-Huperzine A [14C]-ZT-1

Figure 7. Radiosynthesis of [14C]ZT-1.

CH3 CH3 H Curtius N O rearrangement N OMe

D3C D3C NH2 CO2H

(-)-[d3]Huperzine A

CH3 D C Br Wittig N OMe 3 D C PPh + 3 3 + CO Me Ph P O 2 3

(+)-8

Figure 8. Retro-analysis of ()-[d3]huperzine A.

Comparison of the proton-NMR spectra of ( )-[d3]huperzine A Materials and methods with unlabelled ()-huperzine showed the absence of a signal for the methyl peak on E-olefin. The characteristic resonance The chemical reagents and solvents used in the synthetic proce- signals for ()-huperzine include the methyl at 1.68 ppm (doublet) dures were obtained from Aldrich, Alfa Aesar, Lancaster and Mole- and the E-olefinic proton at 5.49 ppm (quartet).3,31 The carbon- kula and used without further purification. The [U-14C]-phenol 13-coupled spectrum was identical to that of the unla- (specific activity, 131 mCi/mmol) was obtained from a commer- belled compound and showed a discrete quintet centred at cial supplier, and 2,2,2-[d3]ethyl bromide (isotopic enrichment 11.53 ppm for the d3-methyl. Mass spectrometric (MS) analysis 98 atom% D, chemical purity 99.9%) was purchased from CDN of ()-[d3]huperzine A showed an increase of 3 mass units. Isotopes. The chiral substrate [(+)-8] was provided by Debiopharm The isotopic distribution was calculated by comparison of the S.A. Reactions were performed under an inert atmosphere, using relative abundances of the labelled and unlabelled species in the filtered nitrogen gas, unless specified otherwise. 1H-NMR spectra mass spectrum. The observed isotopic distribution of the final were recorded on a Bruker Avance spectrometer operating at product was d2, 1.65% huperzine A, and d3, 97.93% huperzine 500 MHz. Chemical shifts were recorded in parts per million (d) A. Finally, ()-[d3]huperzine A was coupled with 5-chloro-o- from an internal tetramethylsilane (TMS) standard (d = 0.00 ppm) vanillin to give the Schiff base [d3]ZT-1 in a yield of 80%. Conse- in either d6-DMSO (d = 2.50 ppm) or CDCl3 (d = 7.24 ppm), and 1 quently, the imine moiety of [d3]ZT-1 was readily reduced to coupling constants (J) are reported in hertz for H-NMR. Deuter- the benzylamino derivative (reduced-[d3]ZT-1) and converted ated solvent CDCl3 (d = 77.23 ppm) and/or TMS was used as an into the stable hydrochloride salt (Figure 10). The reduced internal standard for 13C-NMR. The chemical shifts are reported form was required for the direct determination of ZT-1 in biolo- in parts per million, downfield from TMS as broad (br), singlets (s), gical matrices, which is hampered by its in vivo hydrolysis to doublets (d), triplets (t), quartets (q) and multiplets (m). The mass huperzine A. spectra were measured on an API 3000 LC-MS/MS spectrometer

D3CCH2Br

Step 8 CH3 Ph3P CH3 N OMe Ph P+CH CD .Br - 3 2 3 N OMe PhSH, AIBN base CO Me O 2 Step 9 Step 10 CO2Me (+)-8 CD3 [d3]-9 Z > E

CH3 CH3 N OMe (PhO) PON NaOH N OMe 2 3 D C MeOH 3 CO Me Step 11 D C 2 3 CO H 2 Step 12 [d ]-10 3 [d ]-11 E > Z 3 CH3 CH 3 H N O N OMe TMS-I

D3C Step 13 NH2 D3C NHCO2Me

[d3]-12 (-)-[d3]huperzine A

Figure 9. Synthesis of ()-[d3]huperzine A.

(turbo spray, positive polarity). Flash chromatography was 14C-Radiosynthesis performed using silica gel (200–400 mesh) with the indicated 14 [14C]-2 solvents. Thin-layer chromatography (TLC) was carried out on (Methoxymethoxy)-[U- C]benzene, pre-coated silica plates (Merck Kieselgel 60 F254) and com- Sodium hydride, 60% dispersion in mineral oil (68 mg, 1.69 mmol), pounds were visualised by UV fluorescence or using the Cyclone was suspended in anhydrous N,N-dimethylformamide (1.6 mL) Storage Phosphor System. Radiochemical activity was deter- and the mixture was cooled to 0 C under a nitrogen atmo- mined by liquid scintillation counting using the Perkin-Elmer sphere. A solution of [U-14C]phenol [14C]-1 (200 mCi, 131 mCi/ Tricarb 2900 Scintillation Analyzer. The analytical HPLC systems mmol; 1.53 mmol) in anhydrous N,N-dimethylformamide (4 mL) used were as follows: Generic method for in process analysis: was transferred into a reaction flask. Gas was evolved, and the Agilent 1200 HPLC system with UV detection at 254 nm reaction mixture became grey in colour. The reaction flask was wavelength; Waters Xbridge C18 analytical HPLC column flushed with nitrogen; the mixture was stirred at ambient (250 4.6 mm; porosity 5 mm; ambient temperature; eluent temperature for 0.5 h and then cooled to 0 C. Chloromethyl A: 0.1% TFA in water, eluent B: 0.1% TFA in acetonitrile; flow methyl ether (0.26 mL, 3.1 mmol) was added in one portion. The rate 1.00 mL/min; injection volume 10 mL) and radiochemical reaction mixture was stirred for 50 min at 0 C, allowed to warm detection. HPLC analysis of huperzine A: Agilent 1200 HPLC sys- to ambient temperature and then stirred for a further 40 min. tem with UV detection at 230 nm wavelength and radiochemical The solution was then diluted with diethyl ether (55 mL) and detection; Merck Purospher Star C-18 analytical HPLC column quenched with water (15 mL). The organic layer was separated 75 mm 4.0 mm; porosity 3 mm; column temperature 35 C; and washed with an aqueous solution of sodium hydroxide eluent A: 20 mM ammonium acetate buffer (pH 6), eluent B: (1 M, 2 15 mL) and water (2 15 mL) to give [14C]-2 methanol; flow rate 1.00 mL/min; injection volume 10 mL. HPLC (196.8 mCi, 98% yield). Radiochemical purity HPLC area%: 97.9%; analysis of ZT-1: Agilent 1200 HPLC system with UV detection radiochemical purity by TLC [silica gel, n-hexane/ethyl acetate 1 at 230 nm wavelength and radiochemical detection; Merck (9:1)]: >99%. H NMR (CDCl3, 500 MHz): d = 3.50 (3H, s, OCH3), Purospher Star C-18 analytical HPLC column 75 mm 4.0 mm; 5.21 (2H, s, OCH2), 7.02–7.08 (3H, m), 7.29–7.33 (2H, m) ppm. porosity 3 mm; column temperature 30 C; eluent A, 20 mM ammonium acetate buffer pH 6, eluent B, ammonium acetate 1-Iodo-2-(methoxymethoxy)-[U-14C]benzene, [14C]-3 buffer pH 6/ acetonitrile (60:40); flow rate 1.0 mL/min; injection volume 10 mL. The radiochemical and chemical purity (UV) of A diethyl ether solution of [14C]-2 (196.8 mCi) was dried over final products were described as area%. High-purity HPLC-grade Na2SO4, filtered and concentrated under vacuum. The resulting solvents were used in all methods. oil was dissolved in anhydrous diethyl ether (4.1 mL), stirred

O OH OMe CH H 3 H CH3 N O H Cl N O D C 3 N Step 14 D C 3 NH 2 HO

MeO Cl

[d3]ZT-1 [d3]-huperzine A

CH CH3 3 H H N N O O

D C D3C 3 NaBH4 HN HCl HN .HCl MeOH HO Et2O HO Step 15 Step 16 MeO Cl MeO Cl

reduced-[d3]ZT-1 reduced-[d3]ZT-1.HCl

Figure 10. Synthesis of [d3]ZT-1, reduced-[d3]ZT-1 and reduced-[d3]ZT-1.HCl. under a nitrogen atmosphere and cooled to 0 C. A solution of n- conditions as described above were used. This afforded a solu- butyllithium in hexane (0.72 mL, 1.8 mmol, 2.5 M) was added tion of crude [14C]-4 (621.6 mCi). The two portions were com- slowly, and the reaction mixture was stirred at ambient temperature bined and concentrated under reduced pressure. The residue for 1.5 h. To the resulting cloudy solution was added iodine (457 mg, was purified by silica gel column chromatography using a mix- 1.8 mmol) using anhydrous diethyl ether (3 1 mL). The reaction ture of n-hexane and diethyl ether to give a solution of pure mixture was then stirred for 0.5 h, diluted with diethyl ether [14C]-4 (381.1 mCi, 45% yield). Radiochemical purity by HPLC (40 mL), quenched with a 5% sodium thiosulphate solution area%: 99.3%. (2 30 mL) and washed with brine, and the organic layer was separated. This gave crude [14C]-3 (166.2 mCi, 84% yield). Radio- chemical purity by HPLC area%: 73.4%; chemical purity (UV) by 3-Methoxy-2-(methoxymethoxy)-[ring U-14C]benzaldehyde, [14C]-5 HPLC area%: 80.3%. A portion of the n-hexane/diethyl ether solution was removed and concentrated to dryness under reduced pressure, and the 1-Methoxy-2-(methoxymethoxy)-[U-14C]benzene, [14C]-4 pure product [14C]-4 (170.3 mCi, 1.3 mmol) was re-dissolved in The crude diethyl ether solution [14C]-3 (166.2 mCi, 1.27 mmol) anhydrous diethyl ether. The ether solution was transferred into was dried over Na2SO4, filtered and concentrated under reduced a reaction flask and concentrated, followed by the addition of pressure. The residue was re-dissolved in anhydrous methanol anhydrous diethyl ether (2.4 mL). The solution was cooled to (25 mL), transferred into a reaction flask and concentrated to 0 C under a nitrogen atmosphere, and then n-butyllithium in dryness. The resulting material was dissolved in anhydrous n-hexane (1.3 mL, 2.5 M, 3.25 mmol) was added dropwise to the methanol (0.28 mL) and N,N-dimethylformamide (0.68 mL) under resulting mixture. The solution was stirred at ambient tempera- a nitrogen atmosphere. Sodium methoxide (103 mg, 1.91 mmol) ture for 2 h, after which time it was cooled to 0 C, and anhy- and copper(I) bromide (30 mg, 0.21 mmol) were added. The reac- drous N,N-dimethylformamide (0.32 mL) was added. Stirring tion mixture was heated at 110 C. Heating was continued for was continued for 1 h, and then the reaction was quenched by 2 h, with the reaction profile being monitored by HPLC and adding water (20 mL). Diethyl ether was added and the layers radio-TLC analysis. The reaction was quenched with water were separated. The aqueous layer was re-extracted with diethyl (90 mL) and extracted with dichloromethane (2 100 mL). The ether (50 mL), and the organic layers were combined to give organic layers were combined, affording crude [14C]-4 as a solu- [14C]-5 (160.8 mCi, 94% yield). An additional portion was brought tion in dichloromethane (144.9 mCi, 87% yield). An additional through using [14C]-4 (209.7 mCi) to afford a diethyl ether solu- portion was brought through using [14C]-3 (675.7 mCi). Similar tion of [14C]-5 (183.2 mCi, 87% yield). Radiochemical purity

J. Label Compd. Radiopharm 2011 Copyright © 2011 John Wiley & Sons, Ltd. www.jlcr.org L. Leman et al. by HPLC area%: 93.9%; radiochemical purity by TLC (silica gel, [5R-(5a,9b,11E)]-5-[[(5-Chloro-2-hydroxy-3-methoxy[phenyl- n-hexane/ether 9:1): 85.7%. U-14C])methylene]amino]-11-ethylidene-5,6,9,10-tetrahydro-7- methyl-5,9-methanocycloocta[b]pyridin-2(1H)-one, [14C]ZT-1 14 [14C]-6 2-Hydroxy-3-methoxy[ring U- C]benzaldehyde, A solution of [14C]-7 (109 mCi, 54.6 mCi/mmol, 2.0 mmol) was A diethyl ether solution of [14C]-5 (183.2 mCi, 1.4 mmol) was concentrated under reduced pressure to afford a yellow residue. concentrated under reduced pressure, and the residue was re- The residue was dissolved in ethanol (8.0 mL), and ()-huperzine dissolved in methanol and then concentrated to a volume of A (416 mg, 1.7 mmol) was added using ethanol (0.5 mL) to completely approximately 2 mL. To the methanolic solution of [14C]-5 was transfer the material into a reaction flask. The reaction mixture was further added methanol (1.3 mL), followed by an aqueous solu- heated at reflux for 2 h and then cooled to ambient temperature. tion of 6 M hydrochloric acid (0.6 mL). The reaction mixture was The mixture was further cooled in an ice/water bath for 20 min. The heated at 80 C under a nitrogen atmosphere for 2.5 h, with resulting precipitate was collected by filtration, washed with ethanol the progress of the reaction being monitored by HPLC, using (4.0 mL) and dried under high vacuum to afford [14C]ZT-1 as a the generic method. Upon completion of the reaction, the mix- yellow solid (634 mg, 81 mCi). The material was slurried in ethanol ture was cooled to ambient temperature and diluted with water (8.0 mL) for 2 h, filtered, washed with ethanol (8.0 mL) and dried (40 mL). The products were then extracted into ethyl acetate under high vacuum to give the desired product, [14C]ZT-1 (4 50 mL). The organic layers were combined to give a solution (72 mCi, 66% yield), as a dark yellow solid. Radiochemical purity of [14C]-6 (172.2 mCi, 94% yield). The remaining batch of [14C]-5 by HPLC area%: 98.5%; chemical purity (UV) by HPLC area%: 1 (160.8 mCi) was reacted under the conditions described above, 99.3%. Specific activity: 129 mCi/mg; H(d6-DMSO, 500 MHz): 14 affording a solution of the desired product [ C]-6 (150 mCi, d = 1.61 (3H, m, CH3), 1.62 (3H, s, CH3), 2.31 (1H, d, J = 16.0 Hz), 93%). Both batches were combined and dried over Na2SO4, 2.62 (1H, d, J = 17.3 Hz), 2.74 (1H, d, J = 17.0 Hz), 2.80 (1H, dd, filtered and concentrated to dryness. The resulting residue was J = 17.3 Hz, J = 4.7 Hz), 3.60–3.70 (1H, m), 3.81 (3H, s, OCH3), 5.02 purified by silica gel column chromatography, eluting with n- (1H, q, J = 6.7 Hz, CHCH3), 5.4–5.5 (1H, m), 6.13 (1H, d, J = 9.6 Hz), hexane/ethyl acetate (9:1) to afford a solution of [14C]-6 7.06–7.08 (2H, m), 7.15–7.25 (1H, m), 8.68 (1H, s), 11.40–11.71 (213.5 mCi, 62% yield). Radiochemical purity by HPLC area%: (1H, br s), 14.40–14.54 (1H, br s, pyridone NH) ppm. 98.4%. The solution was concentrated on a rotary evaporator to 1 afford a solid. H NMR (CDCl3, 500 MHz): d = 3.86 (3H, s, OCH3), d3-Synthesis 6.88–6.92 (1H, m), 7.04–7.06 (1H, m), 7.11–7.13 (1H, m), 9.85 (1H, s, CHO), 11.04 (1H, s, OH) ppm. 2,2,2-[d3]Ethyltriphenylphosphonium bromide

14 Triphenylphosphine (50.0 g, 190.6 mmol) and 2,2,2-[d3]ethyl bro- 14 [ C]-7 5-Chloro-2-hydroxy-3-methoxy[ring U- C]benzaldehyde, mide (9.5 mL, 127.1 mmol, 98 atom% D) were heated at 135 C, The solid [14C]-6 (213.5 mCi, 1.63 mmol) was dissolved in anhy- overnight, in a Parr reactor. The reaction mixture was cooled to drous dichloromethane and transferred into a reaction flask. ambient temperature. The resultant solid was removed from The solution was concentrated to dryness and then re-dissolved the reactor and slurried in toluene to remove excess triphenyl- in anhydrous dichloromethane (2.8 mL). Sodium hydrogen carbo- phosphine. The suspension was filtered under nitrogen to afford nate (777 mg, 9.2 mmol) was added, and the resulting suspension 2,2,2-[d3]ethyltriphenylphosphonium bromide as a white solid was stirred and cooled to approximately 10 Cinanice/acetone (46.8 g, 99% yield), which was dried under high vacuum to constant 1 d bath, under nitrogen atmosphere. Iodine monochloride (1.0 M weight. H NMR (d6-DMSO): = 3.60 (2H, d, J = 13.2 Hz, CH2P), solution, 4.1 mL) was added. The reaction progress was monitored 7.77–7.93 (15H, m) ppm. by HPLC analysis, using the generic method. Upon completion of (+)-(5R,9R,11Z)-11-(2,2,2-[d ]Ethylidene)-9,10-dihydro-2- the reaction, dichloromethane (10 mL) was added, and the reac- 3 methoxy-7-methyl-5,9-methanocycloocta[b]pyridine-5(6H)- tion was carefully quenched by addition of 5% aqueous sodium carboxylic acid methyl ester, [d ]-9 dithionite (10 mL). The dichloromethane layer was separated, and 3 the aqueous layer was diluted with water (15 mL) and re-extracted 2,2,2-[d3]Ethyltriphenylphosphonium bromide (62.5 g, 167.0 mmol) with dichloromethane (2 50 mL). The combined organic layers was charged into a 500-mL round-bottomed flask, followed by were concentrated under reduced pressure. The crude material anhydrous tetrahydrofuran (250 mL). The resulting heterogeneous was purified by silica gel column chromatography, eluting with a mixture was stirred while a solution of n-butyl lithium in hexanes hexane/ethyl acetate gradient from 97:3 to 95:5. The isolated frac- (58 mL, 145.0 mmol, 2.5 M) was added over 20 min. The tempera- tions were analysed by HPLC analysis, using the generic method. ture was maintained at <30 C with a water bath. The reaction The pure fractions were combined to give [14C]-7 (116.2 mCi, mixture was then stirred at ambient temperature for 30 min, dur- 54% yield). Radiochemical purity by HPLC area%: 98.3%; chemi- ing which time it became red in colour. The reaction mixture was cal purity (UV) by HPLC area%: 99.2%. Specific activity dilution: cooled to between 0 and 2 C in an ice/acetone bath. The solution was concentrated to dryness to afford a solid, which The substrate (+)-(5S,9R)-methyl-9,10-dihydro-2-methoxy-7- was dried under high vacuum to constant weight. A number of methyl-11-oxo-5,9-methanocycloocta[b]pyridine-5(6H)-carboxylate aliquots of the solid were used to evaluate the specific activity. [(+)-8] (12.0 g, 41.7 mmol), in anhydrous tetrahydrofuran (38 mL), The high specific activity [14C]-7 (109 mCi/mmol) was diluted was added dropwise. On completion of the addition, the reac- with inactive 5-chloro-o-vanillin (187 mg) to give lower specific tion mixture was stirred at this temperature for 30 min and then activity [14C]-7 (54.6 mCi/mmol). Radiochemical purity by HPLC warmed to ambient temperature. Stirring was continued at area%: 99.4%; chemical purity (UV) by HPLC area%: 98.2%; 1H ambient temperature for 1 h, after which TLC (ethyl acetate/n- NMR (CDCl3, 500 MHz): d = 3.86 (3H, s, OCH3), 6.98 (1H, t, heptane, 30:70) showed the reaction to be complete. The reac- J = 2.5 Hz), 7.10 (1H, t, J = 2.5 Hz), 9.79 (1H, s, CHO), 10.91 (1H, br tion was quenched with water (125 mL) and then concentrated s, OH) ppm. in vacuo to remove tetrahydrofuran. The aqueous residue was

www.jlcr.org Copyright © 2011 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2011 L. Leman et al. washed with ethyl acetate (3 75 mL), and the combined (6.3 mL, 44.0 mmol) were added, and the mixture was heated at organic layers were washed with 5% sodium chloride solution 90 C for 2.5 h. TLC analysis (silica gel, ethyl acetate/n-hexane, (2 40 mL). The organic layer was adsorbed onto silica gel and 1:1) showed no starting material remaining. The reaction was purified by column chromatography using gradient elution cooled to 65 C and quenched with methanol (190 mL). The (n-heptane to n-heptane/ethyl acetate, 95:5). The pure fractions resulting mixture was refluxed for 24 h, after which TLC analysis were combined and concentrated in vacuo to afford [d3]-9 as a showed the reaction to be complete. The reaction mixture was pale yellow oil, which crystallised on standing (11.6 g, 92% yield). concentrated in vacuo to afford 33 g of crude product as yellow 1 H NMR (CDCl3, 500 MHz): (Z)-olefin d = 1.53 (3H, s, CH3), 2.21 (1H, oil. The oil was adsorbed onto silica gel and purified by column d, J = 17.0 Hz), 2.81 (1H, dd, J = 17.0 Hz, J = 1.6 Hz), 3.02–3.10 (2H, chromatography using gradient elution (n-hexane/ethyl acetate, m), 3.15 (1H, dd, J = 17.0 Hz, J = 5.1 Hz), 3.70 (3H, s, CO2CH3), 95:5 to 70:30). Pure fractions were combined and concentrated 3.89 (3H, s, OCH3), 5.40–5.41 (1H, m), 5.49 (1H, s, CHCD3), 6.53 in vacuo to afford 11.2 g of product. Proton-NMR analysis of this (1H, d, J = 8.6 Hz), 7.09 (1H, d, J = 8.6 Hz) ppm. material showed residual acid (~8%). The mixture was dissolved in ethyl acetate (50 mL) and washed with 1 M sodium hydroxide (+)-(5R,9R,11E)-11-(2,2,2-[d ]Ethylidene)-9,10-dihydro-2- 3 solution (10 mL 2) and brine (10 mL 2). The organic layer methoxy-7-methyl-5,9-methanocycloocta[b]pyridine-5(6H)- was dried over MgSO4, filtered and concentrated in vacuo to carboxylic acid methyl ester, [d3]-10 afford the carbamate [d3]-12 (9.1 g, 68% yield) as white foam. 1 The ethylidene ester [d3]-9 (11.6 g, 38.4 mmol) in toluene (110 mL) H NMR (CDCl3, 500 MHz): (E)-olefin d = 1.51 (3H, s, CH3), 2.23 was heated at 110 C, along with thiophenol (6.3 mL, 61.4 mmol) (1H, d, J = 16.1 Hz), 2.58 (1H, br d, J = 15.6 Hz), 2.82 (1H, dd, and 2,2′-azobis(2-methylpropionitrile) (6.9 g, 42.4 mmol). Reac- J = 16.7 Hz, J = 1.9 Hz), 3.07 (1H, br d, J = 16.7 Hz), 3.57–3.69 tion progression was monitored by proton-NMR analysis. After (3H, m, CO2CH3), 3.88 (3H, s, OCH3), 4.99 (1H, br s, NH), 5.34 fi 24 h, proton-NMR analysis showed the (E)-ole n isomer to be (1H, s, CHCD3), 5.45–5.46 (1H, m), 6.55 (1H, d, J = 8.6 Hz), 7.56 present in 93%. The reaction mixture was cooled to ambient tem- (1H, d, J = 8.6 Hz) ppm. perature, and the solvent was removed in vacuo to afford an oily ()-(5R,9R,11E)-5-Amino-11-(2,2,2-[d ]ethylidene)-5,6,9,10- residue. The residue was dissolved in n-heptane, and the mixture 3 tetrahydro-7-methyl-5,9-methanocycloocta[b]pyridin-2(1H)- stirred at 0 C for 1 h. The resulting precipitate was collected by one, ()-[d ]huperzine A filtration and washed with ice-cold n-heptane. This afforded 3 fi the (E)-ole n isomer [d3]-10 (7.2 g, 62% yield) as an off white The carbamate [d3]-12 (9.1 g, 28.7 mmol) was dissolved in 1 solid. H NMR (CDCl3, 500 MHz): (E)-olefin d = 1.54 (3H, s, CH3), chloroform (170 mL), and the mixture was stirred at ambient 2.15 (1H, d, J = 17 Hz), 2.85 (1H, dd, J = 17.0 Hz, J = 1.9 Hz), 3.04– temperature for 10 min. Trimethylsilyl iodide (34.0 mL, 229 mmol) 3.08 (2H, m), 3.57–3.60 (1H, m), 3.74 (s, 3H, CO2CH3), 3.89 (s, 3H, was added, and the mixture was heated at 50 C overnight. The OCH3), 5.04 (1H, s, CHCD3), 5.40–5.41 (1H, m), 6.53 (1H, d, reaction mixture was cooled and methanol (170 mL) was added, J = 8.6 Hz), 7.08 (1H, d, J = 8.6 Hz) ppm. after which the reaction was heated at 50 C overnight. TLC analysis (dichloromethane/methanol, 90:10) showed no starting

(+)-(5R,9R,11E)-11-(2,2,2-[d3]Ethylidene)-9,10-dihydro-2- material remaining. The reaction mixture was concentrated in methoxy-7-methyl-5,9-methanocycloocta[b]pyridine-5(6H)- vacuo to afford a yellow sticky solid. The solid was adsorbed onto fi carboxylic acid, [d3]-11 silica gel and puri ed by column chromatography using gradient elution (dichloromethane/methanol, 95:5 to 90:10). Pure frac- The (E)-ethylidene ester [d ]-10 (7.2 g, 23.7 mmol) was refluxed 3 tions were combined and concentrated in vacuo to afford 4.6 g in a mixture of 20% aqueous sodium hydroxide/tetrahydro- of product as an orange solid. The product was dissolved in furan/methanol (40:45:62 mL). The reaction progress was moni- dichloromethane and washed with 5% sodium thiosulphate tored by LC-MS. After refluxing for 45 h, the reaction was (2 25 mL) solution. The aqueous layer was extracted with complete. The solution was allowed to cool to ambient tempera- dichloromethane (3 20 mL). The organic layers were combined, ture, and the solvents were removed in vacuo. The aqueous resi- dried (Na SO ) and concentrated in vacuo to afford ()-[d ] due was acidified to pH 6–7 and then extracted with ethyl 2 4 3 huperzine A (4.2 g, 60% yield) as an off-white solid. Chemical acetate (3 100 mL). The combined organic phases were con- purity (UV) by HPLC area%: 98.5%; isotopic distribution of d centrated in vacuo, and the crude material was adsorbed onto 2 (1.65% huperzine A) and d (97.93% huperzine A); HPLC MS: silica gel. The material was purified by silica gel column chroma- 3 [M + 1]+ (246.4); 1H NMR (CDCl , 500 MHz): (E)-olefin d = 1.55 tography using gradient elution [n-hexane to n-hexane/ethyl 3 (3H, s, CH ), 1.30–1.67 (2H, br s, NH ), 2.11 (1H, d, J = 17.0 Hz), acetate (90:10) to ethyl acetate]. Pure fractions were combined 3 2 2.16 (1H, d, J = 17.0 Hz), 2.72 (1H, dd, J = 16.7 Hz, J = 1.6 Hz), 2.89 and concentrated to afford the carboxylic acid [d ]-11 as viscous 3 (1H, dd, J = 16.7 Hz, J = 5.4 Hz), 3.60 (1H, t, J = 4.7 Hz), 5.42 (1H, oil (4.5 g, 65% yield). 1H NMR (CDCl , 500 MHz): (E)-olefin d = 1.54 3 d, J = 5.0 Hz), 5.48 (1H, s, CHCD ), 6.42 (1H, d, J = 9.6 Hz), 7.90 (3H, s, CH ), 2.17 (1H, d, J = 17.3 Hz), 2.87 (1H, dd, J = 17.0 Hz, 3 3 (1H, d, J = 9.6 Hz), 12.7 (1H, br s, pyridone NH) ppm; 13C NMR J = 1.6 Hz), 3.01–3.10 (2H, m), 3.61 (1H, t, J = 4.7 Hz), 3.89 (3H, s, (CDCl , 125 MHz): d = 11.53 (quintet, CD ), 22.61, 32.90, 35.29, OCH ), 5.29 (1H, s, CHCD ), 5.41–5.42 (1H, m), 6.57 (1H, d, 3 3 3 3 49.20, 53.43, 54.33, 111.10, 117.00, 122.86, 124.31, 134.12, J = 8.5 Hz), 7.25 (1H, d, J = 8.5 Hz) ppm. 140.25, 142.64, 143.20, 165.47 ppm.

(+)-(5R,9R,11E)-[11-(2,2,2-[d3]Ethylidene)-9,10-dihydro-2- [5R-(5a,9b,11E)]-5-[[(5-Chloro-2-hydroxy-3-methoxymethoxy-7-methyl-5,9-methanocycloocta[b]pyridine-5(6H)-yl] phenyl)methylene]amino]-11-(2,2,2-[d3]ethylidene)-5,6,9,10-tet- carbamic acid methyl ester, [d3]-12 rahydro-7-methyl-5,9-methanocycloocta[b]pyridin-2(1H)-one, [d3]ZT-1 The carboxylic acid [d3]-11 (12.8 g, 44.0 mmol) was dissolved in toluene (190 mL), and the solution was stirred under nitrogen. A solution of ()-[d3]huperzine A (2.1 g, 8.6 mmol) and 5-chloro- Diphenylphosphoryl azide (9.0 mL, 44.0 mmol) and triethylamine ortho-vanillin (1.92 g, 10.3 mmol) in ethanol (42.0 mL) was

J. Label Compd. Radiopharm 2011 Copyright © 2011 John Wiley & Sons, Ltd. www.jlcr.org L. Leman et al. refluxed for 2.5 h. TLC analysis (silica gel, dichloromethane/ disease. The carbon-14-labelled synthesis was executed from methanol, 90:10) showed no starting material remained. The [U-14C]phenol to give 5-chloro-vanillin [14C]-7 in six radiochemical reaction mixture was cooled in an ice bath for 1 h, and then steps. This material was successfully coupled with ()-huperzine the precipitated product was collected by filtration and washed A to give [14C]ZT-1 in an overall radiochemical yield of 7% and a with ethanol. This afforded [d3]ZT-1 (2.8 g, 80% yield) as an radiochemical purity of 98.5%. The deuterium-labelled ()-[d3] 1 orange solid. H NMR (d6-DMSO, 500 MHz): d = 1.61 (3H, m, huperzine A was achieved in six steps including a Wittig cou- CH3), 2.31 (1H, d, J = 16.0 Hz), 2.62 (1H, d, J = 17.3 Hz), 2.74 pling with 2,2,2-[d3]ethyltriphenylphosphonium bromide to (1H, d, J = 17.0 Hz), 2.80 (1H, dd, J = 17.3 Hz, J = 4.7 Hz) 3.60–3.70 accommodate the insertion of the three deuterium atoms on the (1H, m), 3.81 (3H, s, OCH3), 5.02 (1H, s, CHCD3), 5.40–5.50 (1H, methyl group. The proceeding steps included Z/E-isomerisation, m), 6.13 (1H, d, J = 9.6 Hz), 7.06–7.08 (2H, m), 7.14–7.25 (1H, m), followed by a functional group inter-conversion via base hydro- 8.6 (1H, s), 11.50 (1H, br s), 14.50 (1H, br s, pyridone NH) ppm. lysis, Curtis rearrangement, carbamate hydrolysis and cleavage of methyl ether, giving an overall yield of 15%. ()-[d ]Huperzine a b 3 [5R-(5 ,9 ,11E)]-5-[[(5-Chloro-2-hydroxy-3-methoxy)benzyl] A (chemical purity by HPLC area%, 98.5%) produced a Schiff base amino]-11-(2,2,2-[d3]ethylidene)-5,6,9,10-tetrahydro-7-methyl- with 5-chloro-o-vanillin to afford [d3]ZT-1 in 80% yield. 5,9-methanocycloocta[b]pyridin-2(1H)-one hydrochloride, reduced-[d3]ZT-1.HCl Acknowledgement To a solution of [d3]ZT-1 (2.8 g, 6.8 mmol) in methanol (30 mL) was added sodium borohydride (313 mg, 8.3 mmol), and the The authors and Almac would like to thank Debiopharm S.A. for mixture was stirred under an atmosphere of nitrogen for 2.5 h. their sponsorship of this labelling project. During this time, the reaction mixture changed from an orange colour to colourless. TLC analysis (silica gel, dichloromethane/ References methanol, 90:10) showed the reaction to have gone to completion. The reaction was quenched with water (0.9 mL) and then [1] J.-S. Liu, Y.-L. Zhu, C.-M. Yu, Y.-Z. Zhou, Y.-Y. Han, F.-W. Wu, B.-F. Qi. Canadian J Chem, 1986, 64, 837–839. concentrated to dryness in vacuo. The residue was re-dissolved [2] Y. Xia, A. P. Kozikowski. J. Am. Chem. Soc.; 1989, 111, 4116–4117. in water (10 mL)/dichloromethane (10 mL), and the mixture was [3] A. P. Kozikowski, Y. Xia, E. R. Reddy, W. Tückmantel, I. Hanin, X. C. acidified to pH 5 using concentrated hydrochloric acid. The Tang. J. Org. Chem.; 1991, 56, 4636–4645. [4] A. P. Kozikowski, G. Campiani, P. Aagaard, M. McKinney. J. Chem. Soc. phases were separated, and the aqueous phase was re-extracted – Chem. Commun.; 1993, 860 862. with dichloromethane (2 10 mL). The organic layers were com- [5] L. G. Qian, R. Y. Ji. Tetrahedron Lett; 1989, 30, 2089–2090. bined, dried over Na2SO4, filtered and concentrated in vacuo to [6] F. Yamada, A. P. Kozikowski, E. R. Reddy, Y.-P. Pang, J. H. Miller, M. afford the product as a white solid (3.47 g). To ensure that the McKinney. J. Am. Chem. Soc.; 1991, 113, 4695–4696. product contained no residual salts, the product was purified [7] G. Campiani, L-Q. Sun, A. P. Kozikowski, P. Aagaard, M. McKinney. J. – by silica gel chromatography using dichloromethane/methanol Org. Chem.; 1993, 58, 7660 7669. [8] C. Lucey, S. A. Kelly, J. Mann. Org. Biomol. Chem.; 2007, 5, 301–306 (90:10). This afforded reduced-[d3]ZT-1 (3.06 g) as a white solid. [9] X. C. Tang, X. C. He, D. L. Bai. Drugs Futur, 1999, 24(6), 647–663. 1 H NMR (CDCl3, 500 MHz): d = 1.54 (3H, s, CH3), 2.11 (1H, d, [10] A. R. Desilets, J. J. Gickas, K. C. Dunican. Ann. Pharmacother., 2009, 43, J = 16.7 Hz), 2.28 (1H, d, J = 16.7 Hz), 2.73 (1H, dd, J = 17.0 Hz, 514–518. J = 1.9 Hz), 2.93 (1H, dd, J = 17.0 Hz, J = 5.0 Hz), 3.51 (1H, d, [11] M. L. Raves, M. Harel, Y.-P. Pang, I. Silman, A. P. Kozikowski, J. L. Sussman. Nat. Struct. Biol.; 1997, 4,57–63. J = 14.5 Hz), 3.62 (1H, t, J = 4.4 Hz), 3.81 (1H, d, J = 13.9 Hz), 3.88 [12] J. Ghiso, B. Frangione. Adv. Drug. Deliver Rev; 2002, 54, 1539–1551. (3H, s, OCH3), 5.39–5.40 (1H, m), 5.49 (1H, s, CHCD3), 6.48 (1H, d, [13] P. T. Francis, A. M. Palmer, M. Snape, G. K. Wilcock. J Neurol Neurosurg J = 9.5 Hz), 6.66 (1H, d, J = 2.5 Hz), 6.77 (1H, d, J = 2.5 Hz), 7.63 Psychiatry, 1999, 66, 137–147. (1H, d, J = 9.5 Hz), 13.0 (1H, br s, pyridone NH) ppm. The [14] D.-Y. Zhu, C.-H. Tan, Y.-M. Li. The overview of studies on huperzine A: ’ reduced-[d ]ZT-1 (3.06 g) was stirred in anhydrous diethyl ether a natural drug for the treatment of Alzheimer s disease. Medicinal 3 Chemistry of Bioactive Natural Products (2006), 143–182. Publisher: (10 mL), and ethereal hydrogen chloride (1 M, 20 mL) was added. John Wiley & Sons. The mixture was stirred at ambient temperature for 3 h. The [15] B. R Coleman, R. H. Ratcliffe, S. A. Oguntayo, X. Shi, B. P. Doctor, R. K. product was collected by filtration and dried to a constant Gordon, M. P. Nambiar. Chem Biol Interact, 2008, 175 (1–3): 387–395. – weight at 40 C under vacuum to afford reduced-[d ]ZT-1.HCl [16] J. L. Cummings. Am J Geriatr Psychiatry, 2003, 11, 131 145. 3 [17] R. I. R. Dierckx, M. F. J. Vandewoude. Acta Clin Belg; 2008, 63(5), (3.05 g, 99% yield) as a white powder. Chemical purity 339–342. (UV) by HPLC area%: 98.1%; isotopic distribution (MS): d2 [18] S. Capancioni, F. Pfefferlé, N. Burgi, M.-A. Bardet, L. Bauduin, + 1 (1.60%) and d3 (98.02%); HPLC MS: [M + H] (416.1); H NMR S. Charbon, A. Ménétrey, V. Nicolas, M.-P. Simonin, E. Tamchès, P. Scalfaro, H. Porchet, P. Garrouste. Preparation of a sustained-release (d6-DMSO, 500 MHz): d = 1.55 (3H, s, CH3), 2.49–2.53 (1H, m), 2.60 (1H, dd, J = 17.3 Hz, J = 1.3 Hz), 2.67 (1H, d, J = 16.1 Hz), implant of the acetylcholinesterase inhibitor ZT-1 by hot-melt extrusion – (HME) and evaluation in rats Debiopharm (April 2006). 2.80 (1H, dd, J = 17.3 Hz, J = 5.0 Hz), 3.57 3.67 (2H, m), 3.85 [19] D. Wilkinson, L. Roughan. Futur. Neurol., 2007, 2(4), 379–382. (3H, s, OCH3), 4.02–4.09 (1H, m), 5.45 (1H, d, J = 4.7 Hz), 5.63 [20] X. Ma, C. Tan, D. Zhu, D. R. Gang, P. Xiao. J. Ethnopharmacol., 2007, – (1H, s, CHCD3), 6.33 (1H, d, J = 9.8 Hz), 7.08 (1H, d, J = 2.5 Hz), 113(1), 15 34. 7.36 (1H, d, J = 2.5 Hz), 8.27 (1H, d, J = 9.5 Hz), 9.66 (2H, br t, [21] M.-W. Wang, X. Hao, K. Chen. Phil. Trans. R. Soc. B; 2007, 362, 1093–1105. J = 10.4 Hz), 10.71 (1H, br s) ppm. [22] J. Yan, L. Sun, G. Wu, P. Yi, F. Yang, L. Zhou, X. Zhang, Z. Li, X. Yang, H. Luo, M. Qiu. Bioorg Med Chem; 2009, 17, 6937–6941. Conclusion [23] Debiopharm S.A. URL http://www.debiopharm.com [Accessed on 19 April 2011]. In conclusion, we report the successful synthesis of two isotopi- [24] E. Giacobini. Neurochem. Res.; 2003, 28(3–4), 515–522. [25] X. C. Tang. The studies of ZT-1 on cortical acetylcholine level in rats: cally labelled acetylcholinesterase inhibitors. These labelled com- 14 comparison with huperzine A and donepezil. State Key Laboratory pounds, [ C]ZT-1 (Debio-9902) and [d3]ZT-1, were used in of Drug Research, Shangai Institute of Materia Medica (China) clinical studies to evaluate a potential treatment for Alzheimer’s (2002).

[26] N. S. Narasimhan, F. S. Mali, M. V. Barve, Synthesis, 1979, 906–909. [31] S. Kaneko, T. Yoshino, T. Katoh, S. Terashima, Tetrahedron, 1998, 54, [27] H. A. Bates, J. S. Garelick, J. Org. Chem. 1984, 49, 4552–4555. 5471–5484. [28] C. A. Townsend, S. G. Davis, S. B. Christensen, J. C. Link, C. P. Lewis. J. [32] S. Gemma, E. Gabellieri, P. Huleatt, C. Fattorusso, M. Borriello, B. Am. Chem. Soc.; 1981, 103, 6885–6888. Catalanotti, S. Butini, M. De Angelis, E. Novellino, V. Nacci, T. [29] C. A. Townsend, L. M. Bloom. Tetrahedron Lett; 1981, 22 (40), Belinskaya, A. Saxena, G. Campiani. J. Med. Chem.; 2006, 49, 3923–3924. 3421–3425. [30] G. Underiner, F. Gibson, L. He, H. S. Meera, J. M. Gnanadeepam, R. [33] A. S. Thompson, G. R. Humphrey, A. M. DeMarco, D. J. Mathre, E. J. J. Saiganesh. WO 2009/120774 A2 Debiopharm S.A. (March 2009). Grabowski. J. Org. Chem. 1993, 58, 5886–5888.